TREA

BENZO[B][1,8]NAPHTHYRIDINE ACETIC ACID DERIVATIVES AND METHODS OF USE

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Information

  • Patent Application
  • 20220251079
  • Publication Number
    20220251079
  • Date Filed
    May 15, 2020
    4 years ago
  • Date Published
    August 11, 2022
    2 years ago
Information
Abstract
Compounds of Formula I or pharmaceutically acceptable salts or esters thereof capable of binding to and modulating the activity of a stimulator of interferon genes (STING) protein are provided. Methods involving compounds of Formula I as effective modulators of STING are also provided.
Description
BACKGROUND

Modulating innate immune activity by agonizing or antagonizing pattern recognition receptors (PRRs) has vast potential for clinical applications both as monotherapy and in combination with other pharmaceutical or bio-pharmaceutical agents. The applications range from alleviating autoimmune disorders through immunosuppression to treating solid and hematological cancers by stimulating innate anti-tumor immunity, as well as uses in anti-viral therapy or as vaccine adjuvant.


One of the PRRs involved in the effective activation of antigen presenting cells (APCs) is the stimulator of interferon genes (STING) protein. STING is an evolutionarily conserved, cytosolic PRR that is part of the cGAS-CDN-STING axis. Aberrant dsDNA in the cytosol, as a consequence of cell stress, viral or intracellular bacterial infection, failed mitosis, or phagocytosis, is recognized by the cGAS enzyme, which synthesizes the non-canonical cyclic di-nucleotide (CDN), 2′3′cGAMP. 2′3′cGAMP binds to and stabilizes the STING dimer, resulting in IRF3 and NFkB activation and synthesis of type I interferon. STING protein plays an important role in innate cellular responses to viral infection and aberrant cytosolic DNA accumulation in both target cells and responding innate immune cells. The pleiotropic effects of STING activation are cell-type and context dependent. For example, overstimulation of T cells and B cells through STING leads to a pro-apoptotic phenotype, while in myeloid cells STING activation elevates type I IFN and pro-inflammatory cytokines without an increase in apoptosis.


Tumor derived dsDNA is phagocytosed by resident dendritic cells (DCs), which stimulates the cGAS-CDN-STING axis and activates DCs, leading to lymph node migration and, ultimately, proliferation of antigen specific CD4+ and CD8+ T cells. This process and the accompanying type I IFN response are often absent in tumors that lack a T cell infiltrate, highlighting the potential of STING agonization to directly address the mechanism of escape exploited by these tumors. Although several CDN derived ligands have shown pre-clinical promise as STING agonists, their relatively large molecular weight and polarity have limited their application to intratumoral injection. Moreover, binding and activation/inhibition of the cytosolic STING protein is limited in vivo by cell membrane permeability. Furthermore, despite their therapeutic efficacy via systemic administration (i.v. or i.p.), the previously identified small molecule STING agonists DMXAA and CMA exhibit species selectivity, prohibiting their use as human therapeutics. Thus, there is a need for human-active small molecule modulators of STING for use as effective therapeutic agents. The present application addresses this need. The novel compounds of this application overcome the limitations of CDN derived ligands.


SUMMARY

In one aspect, the present application relates to a compound of Formula I:




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or a pharmaceutically acceptable salt or ester thereof, wherein X, Y, Z1, m, and n are each defined herein.


In another aspect, the present application relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the application, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.


Another aspect of the present application relates to a method of modulating (e.g., inhibiting or stimulating) a stimulator of interferon genes (STING) protein. The method comprises administering to a subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application. In one embodiment, the STING protein is a human STING protein.


Another aspect of the present application relates to a method of treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function). The method comprises administering to a subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application.


Another aspect of the present application relates to a method of treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation). The method comprises administering to a subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application.


Another aspect of the present application relates to a kit comprising a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application.


Another aspect of the present application relates to a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, for use in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) a STING protein, for treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function), or for treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation).


Another aspect of the present application relates to use of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) a STING protein, for treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function), or for treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type 1 interferon activation).


Another aspect of the present application relates to a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, for use in modulating (e.g., inhibiting or stimulating) a STING protein, in treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function), or in treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation).


Another aspect of the present application relates to use of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, in modulating (e.g., inhibiting or stimulating) a STING protein, in treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function), or in treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type-1 interferon activation).


The present application provides modulators of a STING protein that are therapeutic agents in the treatment or prevention of diseases such as cancer and immunological disorders.


The details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are now described. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.


The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. The references cited herein are not admitted to be prior art to the application.







DETAILED DESCRIPTION

The present application relates to compounds of Formula I that are shown to potently and selectively activate a STING protein (e.g., the human STING protein). In one embodiment, a compound of the present application is represented by Formula I:




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or a pharmaceutically acceptable salt or ester thereof, wherein


X is C(RX)2, O, S, CH═CH, or absent;


each RX is independently H, CH3, CF3, CF2H, or F, or two RX together form ═O, ═CH2, or ═CF2, or two RX, together with the carbon atom to which they are bonded, form a cyclopropyl;


Z1 is (C(RZ)2)p-T1;


p is 1, 2, 3, 4, 5, or 6;


each RZ is independently H, C1-C4 alkyl, C1-C4 alkyl substituted with one or more halogen, or halogen;


T1 is CHOR1, C(O)R1, C(O)OR1, C(O)N(R1)2, NR1C(O)R1, C(S)R1, C(S)N(R1)2, NR1C(S)R1, C(O)NHS(O)2RS, C(O)NHC(O)R1, C(O)NHOH, or C(O)NHCN;


RS is R1, C3-C8 cycloalkyl, heterocyclyl comprising one 5- or 6-membered rings and 1-2 heteroatoms selected from N, O, and S, or C6-C10 aryl, wherein the cycloalkyl, heterocyclyl, or aryl is optionally substituted with one or more groups independently selected from C1-C4 alkyl;


each R1 is independently H, C1-C4 alkyl, or C1-C4 alkyl substituted with one or more halogen;


m is 0, 1, 2, or 3;


each Y is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, CN, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl), or Q-T, wherein each alkyl, alkenyl, or alkynyl moiety is optionally independently substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2, or two Y, together with the two adjacent carbon atoms to which they are bonded, form a 5- to 7-membered carbocycle or phenyl;


n is 0, 1, 2, 3, or 4;


each Z is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, CN, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl), or Q-T, wherein each alkyl, alkenyl, or alkynyl moiety is optionally independently substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2;


each Q is independently a bond, NH, N(C1-C3 alkyl), O, S, S(O), S(O)2, Q′, NH-Q′, N(C1-C3 alkyl)-Q′, O-Q′, S-Q′, S(O)-Q′, or S(O)2-Q′, wherein each alkyl moiety is optionally independently substituted with one or more groups independently selected from C3-C6 cycloalkyl, OH, NH2, halogen, O—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl); each Q′ is independently a carbon linker comprising one or more C(RQ)2, C(RQ)2—C(RQ)2, CRQ═CRQ, or C≡C;


each RQ is independently H or C1-C3 alkyl;


each T is independently C(O)—C1-C6 alkyl, C(O)O—C1-C6 alkyl, S(O)2—(C1-C6 alkyl), C3-C8 cycloalkyl, C3-C8 cycloalkenyl, heterocyclyl comprising one or two 4- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S, C6-C10 aryl, or heteroaryl comprising one or two 5- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S, wherein each cycloalkyl, cycloalkenyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with one or more RT;


each RT is independently C1-C6 alkyl, C1-C6 haloalkyl, OH, CN, halogen, O—(C1-C6 alkyl), O—(C1-C6 haloalkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, NHS(O)2—(C1-C6 alkyl), (CH2)q—C3-C8 cycloalkyl, (CH2)q-heterocyclyl, (CH2)q-phenyl, or (CH2)q-heteroaryl, wherein the heterocyclyl or heteroaryl comprises one or two 5- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S; and


q is 0, 1, 2, or 3.


In one embodiment, a compound of the present application is represented by Formula I′:




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or a pharmaceutically acceptable salt or ester thereof, wherein


X is C(RX)2, O, S, or CH═CH;


each RX is independently H, CH3, CF3, CF2H, or F, or two RX together form ═O, ═CH2, or ═CF2, or two RX, together with the carbon atom to which they are bonded, form a cyclopropyl;


Z1 is (C(RZ)2)p-T1;


p is 1, 2, 3, 4, 5, or 6;


each RZ is independently H, C1-C4 alkyl, C1-C4 alkyl substituted with one or more halogen, or halogen;


T1 is CHOR1, C(O)R1, C(O)OR1, C(O)N(R1)2, NR1C(O)R1, C(S)R1, C(S)N(R1)2, NR1C(S)R1, C(O)NHS(O)2RS, C(O)NHC(O)R1, C(O)NHOH, or C(O)NHCN;


RS is R1, C3-C8 cycloalkyl, heterocyclyl comprising one 5- or 6-membered rings and 1-2 heteroatoms selected from N, O, and S, or C6-C10 aryl, wherein the cycloalkyl, heterocyclyl, or aryl is optionally substituted with one or more groups independently selected from C1-C4 alkyl;


each R1 is independently H, C1-C4 alkyl, or C1-C4 alkyl substituted with one or more halogen;


m is 1, 2, or 3;


each Y is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, CN, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl), or Q-T, wherein each alkyl, alkenyl, or alkynyl moiety is optionally independently substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2;


n is 1, 2, 3, or 4;


each Z is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, CN, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl), or Q-T, wherein each alkyl, alkenyl, or alkynyl moiety is optionally independently substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2;


each Q is independently a bond, NH, N(C1-C3 alkyl), O, S, S(O), S(O)2, Q′, NH-Q′, N(C1-C3 alkyl)-Q′, O-Q′, S-Q′, S(O)-Q′, or S(O)2-Q′, wherein each alkyl moiety is optionally independently substituted with one or more groups independently selected from C3-C6 cycloalkyl, OH, NH2, halogen, O—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl);


each Q′ is independently a carbon linker comprising one or more C(RQ)2, C(RQ)2—C(RQ)2, CRQ═CRQ, or C≡C;


each RQ is independently H or C1-C3 alkyl;


each T is independently C(O)—C1-C6 alkyl, C(O)O—C1-C6 alkyl, S(O)2—(C1-C6 alkyl), C3-C8 cycloalkyl, C3-C8 cycloalkenyl, heterocyclyl comprising one or two 4- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S, C6-C10 aryl, or heteroaryl comprising one or two 5- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S, wherein each cycloalkyl, cycloalkenyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with one or more RT;


each RT is independently C1-C6 alkyl, C1-C6 haloalkyl, OH, CN, halogen, O—(C1-C6 alkyl), O—(C1-C6 haloalkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, NHS(O)2—(C1-C6 alkyl), (CH2)q—C3-C8 cycloalkyl, (CH2)q-heterocyclyl, (CH2)q-phenyl, or (CH2)q-heteroaryl, wherein the heterocyclyl or heteroaryl comprises one or two 5- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S; and


q is 0, 1, 2, or 3.


In one embodiment, a compound of Formula I is of Formula Ia or Ib:




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or a pharmaceutically acceptable salt or ester thereof.


For any of Formula I, Ia, or Ib, where applicable:


(a1) In one embodiment, X is C(RX)2.


(a2) In one embodiment, X is C(RX)2, O, S, or CH═CH.


(a3) In one embodiment, X is C(RX)2, O, or CH═CH.


(b1) In one embodiment, each RX is H.


(b2) In one embodiment, at least one RX is CH3, CF3, CF2H, or F.


(b3) In one embodiment, one RX is CH3, CF3, CF2H, or F, and the other RX is H.


(b4) In one embodiment, each RX is independently CH3, CF3, CF2H, or F. In one embodiment, each RX is CH3. In one embodiment, each RX is CF3. In one embodiment, each RX is CF2H. In one embodiment, each RX is F.


(b5) In one embodiment, two RX together form ═O, ═CH2, or ═CF2.


(b6) In one embodiment, two RX together form ═O.


(b7) In one embodiment, two RX together form ═CH2.


(b8) In one embodiment, two RX together form ═CF2.


(b9) In one embodiment, two RX, together with the carbon atom to which they are bonded, form a cyclopropyl.


(a2) In one embodiment, X is O.


(a3) In one embodiment, X is S.


(a4) In one embodiment, X is CH═CH.


(a5) In one embodiment, X is absent.


(a6) In one embodiment, X is not absent.


(c1) In one embodiment, p is 1, 2, or 3.


(c2) In one embodiment, p is 1 or 2.


(c3) In one embodiment, p is 1.


(c4) In one embodiment, p is 2, 3, 4, 5, or 6.


(d1) In one embodiment, each RZ is H.


(d2) In one embodiment, at least one RZ is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl), C1-C4 alkyl substituted with one or more halogen (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl, each substituted with one or more halogen (e.g., F, Cl, Br, or I)), or halogen (e.g., F, Cl, Br, or I).


(d3) In one embodiment, at least one RZ is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl).


(d4) In one embodiment, at least one RZ is C1-C4 alkyl substituted with one or more halogen (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl, each substituted with one or more halogen (e.g., F, Cl, Br, or I)).


(d5) In one embodiment, at least one RZ is halogen (e.g., F, Cl, Br, or I).


(e1) In one embodiment, T1 is CHOR1, C(O)R1, C(O)OR1, C(O)N(R1)2, or NR1C(O)R1.


(e2) In one embodiment, T1 is C(O)R1, C(O)OR1, C(O)N(R1)2, or NR1C(O)R1.


(e3) In one embodiment, T1 is C(O)R1, C(O)OR1, or C(O)N(R1)2.


(e4) In one embodiment, T1 is C(O)OR1 or C(O)N(R1)2.


(e5) In one embodiment, T1 is C(O)R1 or C(O)OR1.


(e6) In one embodiment, T1 is C(O)R1, C(O)OR1, C(O)N(R1)2, NR1C(O)R1, C(S)R1, C(S)N(R1)2, or NR1C(S)R1.


(e7) In one embodiment, T1 is C(O)OR1, C(O)N(R1)2, NR1C(O)R1, C(S)N(R1)2, or NR1C(S)R1.


(e8) In one embodiment, T1 is C(O)OR1, C(O)N(R1)2, or C(S)N(R1)2.


(e9) In one embodiment, T1 is C(S)R1, C(S)N(R1)2, or NR1C(S)R1.


(e10) In one embodiment, T1 is C(O)R1, C(O)OR1, C(O)N(R1)2, NR1C(O)R1, C(O)NHS(O)2RS, C(O)NHC(O)R1, C(O)NHOH, or C(O)NHCN.


(e11) In one embodiment, T1 is C(O)OR1, C(O)N(R1)2, C(O)NHS(O)2RS, C(O)NHC(O)R1, C(O)NHOH, or C(O)NHCN.


(f1) In one embodiment, each R1 is H.


(f2) In one embodiment, at least one R1 is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl), C1-C4 alkyl substituted with one or more halogen (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl, each substituted with one or more halogen (e.g., F, Cl, Br, or I)), or halogen (e.g., F, Cl, Br, or I).


(f3) In one embodiment, at least one R1 is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl).


(f4) In one embodiment, at least one R1 is C1-C4 alkyl substituted with one or more halogen (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl, each substituted with one or more halogen (e.g., F, Cl, Br, or I)).


(f5) In one embodiment, at least one R1 is halogen (e.g., F, Cl, Br, or I).


(g1) In one embodiment, m is 0, 1, 2, or 3.


(g2) In one embodiment, m is 0, 1, or 2.


(g3) In one embodiment, m is 0.


(g4) In one embodiment, m is 1 or 2.


(g5) In one embodiment, m is 1.


(g6) In one embodiment, m is 2.


(g7) In one embodiment, m is 1, 2, or 3.


(h1) In one embodiment, at least one Y is C1-C6 straight-chain or C3-C6 branched alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2.


In one embodiment, at least one Y is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl), optionally substituted as described herein. In one embodiment, at least one Y is C1-C6 straight-chain or C3-C6 branched alkyl and is substituted as described herein. In one embodiment, at least one Y is C1-C6 straight-chain or C3-C6 branched alkyl substituted with CN, OH, ═O, NH2, N3, halogen, or O—(C1-C6 alkyl). In one embodiment, at least one Y is methyl, optionally substituted as described herein. In one embodiment, at least one Y is ethyl, optionally substituted as described herein. In one embodiment, at least one Y is i-propyl, optionally substituted as described herein. In one embodiment, at least one Y is t-butyl, optionally substituted as described herein.


(h2) In one embodiment, at least one Y is C2-C6 straight-chain or C3-C6 branched alkenyl (e.g., ethenyl, propenyl, butenyl, pentenyl, or hexenyl), optionally substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is C2-C6 straight-chain or C3-C6 branched alkenyl substituted with OH, NH2, halogen, or O—(C1-C6 alkyl).


(h3) In one embodiment, at least one Y is C2-C6 straight-chain or C4-C6 branched alkynyl (e.g., ethynyl, propynyl, butynyl, pentynyl, or hexynyl), optionally substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is C2-C6 straight-chain or C4-C6 branched alkynyl substituted with CN, OH, ═O, NH2, halogen, or O—(C1-C6 alkyl).


(h4) In one embodiment, at least one Y is halogen (e.g., F, Cl, Br, or I). In one embodiment, at least one Y is F. In one embodiment, at least one Y is Cl. In one embodiment, at least one Y is Br.


(h5-1) In one embodiment, at least one Y is OH or O—(C1-C6 straight-chain or C3-C6 branched alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein the alkyl moiety is optionally substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is O-methyl, optionally substituted as described herein. In one embodiment, at least one Y is O-ethyl, optionally substituted as described herein. In one embodiment, at least one Y is O-i-propyl, optionally substituted as described herein.


(h5-2) In one embodiment, at least one Y is O—(C2-C4 alkenyl) (e.g., ethenyl, propenyl, or butenyl), wherein the alkenyl moiety is optionally substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is O-ethenyl, optionally substituted as described herein. In one embodiment, at least one Y is O-propenyl, optionally substituted as described herein.


(h5-3) In one embodiment, at least one Y is O—(C2-C4 alkynyl) (e.g., ethynyl, propynyl, or butynyl), wherein the alkynyl moiety is optionally substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is O-ethynyl, optionally substituted as described herein. In one embodiment, at least one Y is O-propynyl, optionally substituted as described herein.


(h6-1) In one embodiment, at least one Y is S—(C1-C6 straight-chain or C3-C6 branched alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein the alkyl moiety is optionally substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is S-methyl, optionally substituted as described herein. In one embodiment, at least one Y is S-ethyl, optionally substituted as described herein. In one embodiment, at least one Y is S-i-propyl, optionally substituted as described herein.


(h6-2) In one embodiment, at least one Y is S(O)—(C1-C6 alkyl) or S(O)2—(C1-C6 alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein the alkyl moiety is optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is S(O)2CH3, optionally substituted as described herein.


(h7) In one embodiment, at least one Y is NH2, NH—C1-C6 straight-chain or C3-C6 branched alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), or N—(C1-C6 straight-chain or C3-C6 branched alkyl)2 (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein each alkyl moiety is optionally independently substituted with one or more groups independently selected from CN, OH, ═O, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Y is N—(C1-C6 alkyl)2, optionally substituted as described herein, and the two C1-C6 alkyls are the same.


In one embodiment, at least one Y is N—(C1-C6 alkyl)2, optionally substituted as described herein, and the two C1-C6 alkyls are different. In one embodiment, at least one Y is N(CH3)2, optionally substituted as described herein. In one embodiment, at least one Y is N(CH3)(CH2CH3), optionally substituted as described herein.


(h8) In one embodiment, at least one Y is Q-T.


(h9) In one embodiment, at least one Y is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), or S(O)2—(C1-C6 alkyl), as described herein.


(h10) In one embodiment, at least one Y is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein.


(h11) In one embodiment, at least one Y is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), or S(O)2—(C1-C6 alkyl), as described herein, and at least another Y is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), or S(O)2-(C1-C6 alkyl), as described herein.


(h12) In one embodiment, at least one Y is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein, and at least another Y is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein.


(h13) In one embodiment, at least one Y is Q-T, as described herein, and at least another Y is C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein.


(h14) In one embodiment, at least one Y is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein, and at least another Y is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein.


(h15) In one embodiment, at least one Y is halogen, as described herein, and at least another Y is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein. In one embodiment, at least one Y is Cl, and at least another Y is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein. In one embodiment, at least one Y is Cl, and at least another Y is halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein. In one embodiment, at least one Y is Cl, and at least another Y is Cl, O—(C1-C6 alkyl), or S—(C1-C6 alkyl), as described herein. In one embodiment, at least one Y is Cl, and at least another Y is Cl or O—(C1-C6 alkyl), as described herein.


(h16) In one embodiment, two Y, together with the two adjacent carbon atoms to which they are bonded, form a cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclopentynyl, cyclohexynyl, or cycloheptynyl. In one embodiment, two Y, together with the two adjacent carbon atoms to which they are bonded, form a cyclohexyl.


(h17) In one embodiment, two Y, together with the two adjacent carbon atoms to which they are bonded, form phenyl.


(h18) In one embodiment, at least one Y is at the 6-position, as indicated in Formula I, Ia, or Ib.


(h19) In one embodiment, at least one Y is at the 5-position, as indicated in Formula I, Ia, or Ib.


(h20) In one embodiment, at least one Y is at the 7-position, as indicated in Formula I, Ia, or Ib.


(h21) In one embodiment, at least one Y is at the 5-position and at least one Y is at the 6-position, as indicated in Formula I, Ia, or Ib.


(h22) In one embodiment, at least one Y is at the 7-position and at least one Y is at the 6-position, as indicated in Formula I, Ia, or Ib.


(h23) In one embodiment, one Y is at the 5-position and the other Y is at the 6-position, as indicated in Formula I, Ia, or Ib.


(h24) In one embodiment, one Y is at the 7-position and the other Y is at the 6-position, as indicated in Formula I, Ia, or Ib.


(i1) In one embodiment, n is 0, 1, 2, or 3.


(i2) In one embodiment, n is 0, 1, or 2.


(i3) In one embodiment, n is 0.


(i4) In one embodiment, n is 1 or 2.


(i5) In one embodiment, n is 1.


(i6) In one embodiment, n is 2.


(i7) In one embodiment, n is 1, 2, 3, or 4;


(j1) In one embodiment, at least one Z is C1-C6 straight-chain or C3-C6 branched alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl), optionally substituted as described herein. In one embodiment, at least one Z is C1-C6 straight-chain or C3-C6 branched alkyl and is substituted as described herein. In one embodiment, at least one Z is C1-C6 straight-chain or C3-C6 branched alkyl substituted with OH, NH2, N3, halogen, or O—(C1-C6 alkyl). In one embodiment, at least one Z is methyl, optionally substituted as described herein. In one embodiment, at least one Z is ethyl, optionally substituted as described herein. In one embodiment, at least one Z is i-propyl, optionally substituted as described herein. In one embodiment, at least one Z is t-butyl, optionally substituted as described herein.


(j2) In one embodiment, at least one Z is C2-C6 straight-chain or C3-C6 branched alkenyl (e.g., ethenyl, propenyl, butenyl, pentenyl, or hexenyl), optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is C2-C6 straight-chain or C3-C6 branched alkenyl substituted with OH, NH2, halogen, or O—(C1-C6 alkyl).


(j3) In one embodiment, at least one Z is C2-C6 straight-chain or C4-C6 branched alkynyl (e.g., ethynyl, propynyl, butynyl, pentynyl, or hexynyl), optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is C2-C6 straight-chain or C4-C6 branched alkynyl substituted with OH, NH2, halogen, or O—(C1-C6 alkyl).


(j4) In one embodiment, at least one Z is halogen (e.g., F, Cl, Br, or I). In one embodiment, at least one Z is F. In one embodiment, at least one Z is Z is Cl. In one embodiment, at least one Z is Br.


(j5-1) In one embodiment, at least one Z is OH or O—(C1-C6 straight-chain or C3-C6 branched alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein the alkyl moiety is optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is O-methyl, optionally substituted as described herein. In one embodiment, at least one Z is O-ethyl, optionally substituted as described herein. In one embodiment, at least one Z is O-i-propyl, optionally substituted as described herein.


(j5-2) In one embodiment, at least one Z is O—(C2-C4 alkenyl) (e.g., ethenyl, propenyl, or butenyl), wherein the alkenyl moiety is optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is O-ethenyl, optionally substituted as described herein. In one embodiment, at least one Z is O-propenyl, optionally substituted as described herein.


(j5-3) In one embodiment, at least one Z is O—(C2-C4 alkynyl) (e.g., ethynyl, propynyl, or butynyl), wherein the alkynyl moiety is optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is O-ethynyl, optionally substituted as described herein. In one embodiment, at least one Z is O-propynyl, optionally substituted as described herein.


(j6-1) In one embodiment, at least one Z is S—(C1-C6 straight-chain or C3-C6 branched alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein the alkyl moiety is optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is S-methyl, optionally substituted as described herein. In one embodiment, at least one Z is S-ethyl, optionally substituted as described herein. In one embodiment, at least one Z is S-i-propyl, optionally substituted as described herein.


(j6-2) In one embodiment, at least one Z is S(O)—(C1-C6 alkyl) or S(O)2—(C1-C6 alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein the alkyl moiety is optionally substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is S(O)2CH3, optionally substituted as described herein.


(j7) In one embodiment, at least one Z is NH2, NH—C1-C6 straight-chain or C3-C6 branched alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), or N—(C1-C6 straight-chain or C3-C6 branched alkyl)2 (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl), wherein each alkyl moiety is optionally independently substituted with one or more groups independently selected from OH, NH2, N3, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), and N—(C1-C6 alkyl)2. In one embodiment, at least one Z is N—(C1-C6 alkyl)2, optionally substituted as described herein, and the two C1-C6 alkyls are the same. In one embodiment, at least one Z is N—(C1-C6 alkyl)2, optionally substituted as described herein, and the two C1-C6 alkyls are different. In one embodiment, at least one Z is N(CH3)2, optionally substituted as described herein. In one embodiment, at least one Z is N(CH3)(CH2CH3), optionally substituted as described herein.


(j8) In one embodiment, at least one Z is Q-T.


(j9) In one embodiment, at least one Z is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), or S(O)2—(C1-C6 alkyl), as described herein.


(j10) In one embodiment, at least one Z is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein.


(j11) In one embodiment, at least one Z is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), or S(O)2—(C1-C6 alkyl), as described herein, and at least another Z is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), O—(C2-C4 alkenyl), O—(C2-C4 alkynyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, S(O)—(C1-C6 alkyl), or S(O)2—(C1-C6 alkyl), as described herein.


(j12) In one embodiment, at least one Z is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein, and at least another Z is Q-T, C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein.


(j13) In one embodiment, at least one Z is Q-T, as described herein, and at least another Z is C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or S(O)2—(C1-C6 alkyl), as described herein.


(j14) In one embodiment, at least one Z is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein, and at least another Z is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein.


(j15) In one embodiment, at least one Z is halogen, as described herein, and at least another Z is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein. In one embodiment, at least one Z is Cl, and at least another Z is halogen, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein. In one embodiment, at least one Z is Cl, and at least another Z is halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, as described herein. In one embodiment, at least one Z is Cl, and at least another Z is Cl, O—(C1-C6 alkyl), or S—(C1-C6 alkyl), as described herein. In one embodiment, at least one Z is Cl, and at least another Z is Cl or O—(C1-C6 alkyl), as described herein.


(j16) In one embodiment, at least one Z is at the 3-position, as indicated in Formula I, Ia, or Ib.


(j17) In one embodiment, at least one Z is at the 4-position, as indicated in Formula I, Ia, or Ib.


(j18) In one embodiment, at least one Z is at the 3-position and at least one Z is at the 4-position, as indicated in Formula I, Ia, or Ib.


(j19) In one embodiment, one Z is at the 3-position and the other Z is at the 4-position, as indicated in Formula I, Ia, or Ib.


(k1) In one embodiment, at least one Q is a bond.


(k2) In one embodiment, each Q is independently NH, N(C1-C3 alkyl), O, S, S(O), S(O)2, Q′, NH-Q′, N(C1-C3 alkyl)-Q′, O-Q′, S-Q′, S(O)-Q′, or S(O)2-Q′, wherein each alkyl moiety is optionally independently substituted with one or more groups independently selected from C3-C6 cycloalkyl, OH, NH2, halogen, O—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl).


(k3) In one embodiment, each Q is independently NH, O, S, or Q′.


(k4) In one embodiment, each Q is independently Q′, NH-Q′, O-Q′, or S-Q′.


(k5) In one embodiment, each Q is independently NH, S, Q′, NH-Q′, or S-Q′


(k6) In one embodiment, each Q is independently NH, Q′, or NH-Q′,


(k7) In one embodiment, each Q is independently O, Q′, or O-Q′.


(k8) In one embodiment, each Q is independently S, S(O), S(O)2, Q′, S-Q′, S(O)-Q′, or S(O)2-Q′.


(k9) In one embodiment, at least one Q is Q′.


(k10) In one embodiment, Q is N(C1-C3 alkyl)-Q′, wherein the alkyl moiety is optionally substituted with one or more groups independently selected from C3-C6 cycloalkyl, OH, NH2, halogen, O—(C1-C6 alkyl), S(O)2—(C1-C6 alkyl).


(k11) In one embodiment, Q is N(C1-C3 alkyl)-Q′, wherein the alkyl moiety is substituted with C3-C6 cycloalkyl.


(k12) In one embodiment, Q is N(C1-C3 alkyl)-Q′, wherein the alkyl moiety is substituted with O—(C1-C6 alkyl).


(k13) In one embodiment, Q is N(C1-C3 alkyl)-Q′, wherein the alkyl moiety is substituted with S(O)2—(C1-C6 alkyl).


(k14) In one embodiment, Q is N(C1-C3 alkyl), wherein the alkyl moiety is substituted with C3-C6 cycloalkyl.


(k15) In one embodiment, Q is N(C1-C3 alkyl), wherein the alkyl moiety is substituted with O—(C1-C6 alkyl).


(k16) In one embodiment, Q is N(C1-C3 alkyl), wherein the alkyl moiety is substituted with S(O)2—(C1-C6 alkyl).


(l1) In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2 or C(RQ)2—C(RQ)2. In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2. In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2—C(RQ)2.


(l2) In one embodiment, each Q′ is independently a carbon linker comprising one or more CRQ═CRQ.


(l3) In one embodiment, each Q′ is independently a carbon linker comprising one or more C≡C.


(l4) In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2 or C(RQ)2—C(RQ)2 and CRQ═CRQ. In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2 and CRQ═CRQ. In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2—C(RQ)2 and CRQ═CRQ.


(l5) In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2 or C(RQ)2—C(RQ)2 and C≡C. In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2 and C≡C. In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2—C(RQ)2 and C≡C.


(l6) In one embodiment, each Q′ is independently a carbon linker comprising one or more CRQ═CRQ and C≡C.


(l7) In one embodiment, each Q′ is independently a carbon linker comprising one or more C(RQ)2, C(RQ)2—C(RQ)2, CRQ═CRQ, and C≡C.


(m1) In one embodiment, each RQ is H.


(m2) In one embodiment, at least one RQ is methyl, ethyl, or propyl.


(n1) In one embodiment, at least one T is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, each of which is optionally substituted with one or more RT.


(n1a) In one embodiment, at least one T is C3-C8 cycloalkyl comprising one or two rings optionally substituted with one or more RT.


(n1b) In one embodiment, at least one T is C5-C8 cycloalkyl comprising two rings optionally substituted with one or more RT.


(n1c) In one embodiment, at least one T is a C5-C8 bicyclyl optionally substituted with one or more RT. In one embodiment, at least one T is a C5-C8 spirocyclyl optionally substituted with one or more RT.


(n1d) In one embodiment, T is a spiroheptyl optionally substituted with one or more RT.


(n2) In one embodiment, at least one T is cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, or cyclooctenyl, each of which is optionally substituted with one or more RT.


(n3) In one embodiment, at least one T is heterocyclyl comprising one or two 4- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S (e.g., pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl, piperidinyl, piperazinyl, or morpholinyl), optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one or two 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 4-, 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 5- or 6-membered rings and 1-3 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 5-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 5-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 4-membered ring and 1 heteroatom selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 5-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 6-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 6-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heterocyclyl comprising one 6-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT.


(n4) In one embodiment, at least one T is C6-C10 aryl optionally substituted with one or more RT. In one embodiment, at least one T is phenyl optionally substituted with one or more RT.


(n5) In one embodiment, at least one T is heteroaryl comprising one or two 5- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S (e.g., pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, naphthyridinyl, indolyl, purinyl, indolizinyl, quinoxalinyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzoimidazolyl, benzothiophenyl, or benzofuranyl), optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one or two 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT.


In one embodiment, at least one T is heteroaryl comprising one 5- or 6-membered rings and 1-3 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 5-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 5-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 5-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 5-membered ring and 1-2 N, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 6-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 6-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 6-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein, optionally substituted with one or more RT. In one embodiment, at least one T is heteroaryl comprising one 6-membered ring and 1-2 N, such as those described herein, optionally substituted with one or more RT.


(n6) In one embodiment, at least one T is C(O)—C1-C6 alkyl, C(O)O—C1-C6 alkyl, or S(O)2—(C1-C6 alkyl), each optionally substituted with one or more RT.


(o1) In one embodiment, at least one RT is C1-C6 straight-chain or C3-C6 branched alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl). In one embodiment, at least one RT is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl).


(o2) In one embodiment, at least one RT is C1-C6 haloalkyl, i.e., C1-C6 straight-chain or C3-C6 branched alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl) substituted with one or more halogen (e.g., F, Cl, Br, or I). In one embodiment, at least one RT is C1-C4 haloalkyl, i.e., C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl) substituted with one or more halogen (e.g., F, Cl, Br, or I).


(o3) In one embodiment, at least one RT is OH, CN, halogen, or NH2,


(o4) In one embodiment, at least one RT is halogen (e.g., F, Cl, Br, or I).


(o5) In one embodiment, at least one RT is O—(C1-C6 straight-chain or C3-C6 branched alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl). In one embodiment, at least one RT is O—C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl).


(o6) In one embodiment, at least one RT is O—(C1-C6 haloalkyl) (i.e., wherein the C1-C6 haloalkyl is C1-C6 straight-chain or C3-C6 branched alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl) substituted with one or more halogen (e.g., F, Cl, Br, or I). In one embodiment, at least one RT is O—(C1-C4 haloalkyl) (i.e., wherein the C1-C4 haloalkyl is C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl) substituted with one or more halogen (e.g., F, Cl, Br, or I)).


(o7) In one embodiment, at least one RT is S—(C1-C6 straight-chain or C3-C6 branched alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl). In one embodiment, at least one RT is S—C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl).


(o8) In one embodiment, at least one RT is NH2, NH—(C1-C6 straight-chain or C3-C6 branched alkyl), N—(C1-C6 straight-chain or C3-C6 branched alkyl)2, or NHS(O)2—(C1-C6 alkyl). In one embodiment, at least one RT is NH—(C1-C6 straight-chain or C3-C6 branched alkyl) (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl). In one embodiment, at least one RT is NH—C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl). In one embodiment, at least one RT is NH—(C1-C6 straight-chain or C3-C6 branched alkyl)2 (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, i-pentyl, or hexyl). In one embodiment, at least one RT is NH—(C1-C4 alkyl)2 (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl).


(o9) In one embodiment, at least one RT is (CH2)q—C3-C8 cycloalkyl, (CH2)q-heterocyclyl, (CH2)q-phenyl, or (CH2)q-heteroaryl.


(o10) In one embodiment, at least one RT is (CH2)q—C3-C8 cycloalkyl, wherein the C3-C8 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.


(o11) In one embodiment, at least one RT is (CH2)q-heterocyclyl, wherein the heterocyclyl comprises one or two 5- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S (e.g., pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl, piperidinyl, piperazinyl, or morpholinyl). In one embodiment, the heterocyclyl comprises one or two 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heterocyclyl comprises one 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heterocyclyl comprises one 5- or 6-membered rings and 1-3 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heterocyclyl comprises one 5-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heterocyclyl comprises one 5-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heterocyclyl comprises one 5-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heterocyclyl comprises one 6-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heterocyclyl comprises one 6-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heterocyclyl comprises one 6-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein.


(o12) In one embodiment, at least one RT is (CH2)q-phenyl.


(o13) In one embodiment, at least one RT is (CH2)q-heteroaryl, wherein the heteroaryl comprises one or two 5- or 6-membered rings and 1-4 heteroatoms selected from N, O, and S (e.g., pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, naphthyridinyl, indolyl, purinyl, indolizinyl, quinoxalinyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzoimidazolyl, benzothiophenyl, or benzofuranyl). In one embodiment, the heteroaryl comprises one or two 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heteroaryl comprises one 5- or 6-membered rings and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heteroaryl comprises one 5- or 6-membered rings and 1-3 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heteroaryl comprises one 5-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heteroaryl comprises one 5-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heteroaryl comprises one 5-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heteroaryl comprises one 5-membered ring and 1-2 N, such as those described herein. In one embodiment, the heteroaryl comprises one 6-membered ring and 1-3 heteroatoms selected from N, O, and S, such as those described herein. In one embodiment, the heteroaryl comprises one 6-membered ring and 1-3 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heteroaryl comprises one 6-membered ring and 1-2 heteroatoms selected from N and O, such as those described herein. In one embodiment, the heteroaryl comprises one 6-membered ring and 1-2 N, such as those described herein.


(o14) In one embodiment, at least one RT is C1-C6 alkyl, C1-C6 haloalkyl, OH, CN, halogen, O—(C1-C6 alkyl), O—(C1-C6 haloalkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), N—(C1-C6 alkyl)2, (CH2)q-heterocyclyl, or (CH2)q-phenyl, as described herein.


(o15) In one embodiment, at least one RT is C1-C6 alkyl, C1-C6 haloalkyl, OH, CN, halogen, O—(C1-C6 alkyl), O—(C1-C6 haloalkyl), S—(C1-C6 alkyl), NH2, (CH2)q-heterocyclyl, or (CH2)q-phenyl, as described herein. In one embodiment, at least one RT is methyl, ethyl, propyl, i-propyl, CF3, OH, CN, F, Cl, methoxy, ethoxy, OCF3, SCH3, NH2, (CH2)-morpholinyl, or (CH2)-phenyl.


(p1) In one embodiment, q is 0, 1, or 2.


(p2) In one embodiment, q is 1, 2, or 3.


(p3) In one embodiment, q is 0 or 1.


(p4) In one embodiment, q is 1 or 2.


(p5) In one embodiment, q is 0.


(p6) In one embodiment, q is 1.


(p7) In one embodiment, q is 2.


(p8) In one embodiment, q is 3.


(q1) In one embodiment, RS is R1.


(q2) In one embodiment, RS is C3-C8 cycloalkyl, heterocyclyl comprising one 5- or 6-membered rings and 1-2 heteroatoms selected from N, O, and S, or C6-C10 aryl, wherein the cycloalkyl, heterocyclyl, or aryl is optionally substituted with one or more groups independently selected from C1-C4 alkyl.


(q3) In one embodiment, RS is C3-C8 cycloalkyl or C6-C10 aryl, each optionally substituted with one or more groups independently selected from C1-C4 alkyl.


(q4) In one embodiment, RS is C3-C6 cycloalkyl or phenyl, each optionally substituted with one or more groups independently selected from C1-C4 alkyl.


Any of the groups described herein for any of X, Y, Z, Z1, R1, RQ, RT, RX, RZ, RS, Q, Q′, T1, T, m, n, p, and q can be combined with any of the groups described herein for one or more of the remainder of X, Y, Z, Z1, R1, RQ, RT, RX, RZ, RS, Q, Q′, T1, T, m, n, p, and q.


(A1) In one embodiment, p is as described in any one of (c1)-(c3), and RZ is as described in (d1).


(A2) In one embodiment, p is as described in any one of (c1)-(c3), and RZ is as described in any one of (d2)-(d5).


(A3) In one embodiment, p is as described in (c3), and RZ is as described in (d1).


(A4) In one embodiment, p is as described in (c3), and RZ is as described in any one of (d2)-(d5).


(A5) In one embodiment, p is as described in (c4), and RZ is as described in (d1).


(A6) In one embodiment, p is as described in (c4), and RZ is as described in any one of (d2)-(d5).


(B1) In one embodiment, p and RZ are as described in any one of (A1)-(A6), and T1 is as described in any one of (e1)-(e5).


(B2) In one embodiment, p and RZ are as described in any one of (A1)-(A6), and T1 is as described in any one of (e3)-(e5).


(B3) In one embodiment, p and RZ are as described in any one of (A1)-(A6), and T1 is as described in (e4).


(B4) In one embodiment, p and RZ are as described in any one of (A1)-(A6), and T1 is as described in (e5).


(B5) In one embodiment, p and RZ are as described in any one of (A1)-(A6), and T1 is as described in any one of (e6)-(e9).


(B6) In one embodiment, p and RZ are as described in any one of (A1)-(A6), and T1 is as described in any one of (e10)-(e11).


(B7) In one embodiment, p and RZ are as described in any one of (A3)-(A4), and T1 is as described in any one of (e1)-(e5).


(B8) In one embodiment, p and RZ are as described in any one of (A3)-(A4), and T1 is as described in any one of (e3)-(e5).


(B9) In one embodiment, p and RZ are as described in any one of (A3)-(A4), and T1 is as described in (e4).


(B10) In one embodiment, p and RZ are as described in any one of (A3)-(A4), and Ti is as described in (e5).


(B11) In one embodiment, p and RZ are as described in any one of (A3)-(A4), and Ti is as described in any one of (e6)-(e9).


(B12) In one embodiment, p and RZ are as described in any one of (A3)-(A4), and Ti is as described in any one of (e10)-(e11).


(B13) In one embodiment, p and RZ are as described in (A3), and T1 is as described in any one of (e1)-(e5).


(B14) In one embodiment, p and RZ are as described in (A3), and T1 is as described in any one of (e3)-(e5).


(B15) In one embodiment, p and RZ are as described in (A3), and T1 is as described in (e4).


(B16) In one embodiment, p and RZ are as described in (A3), and T1 is as described in (e5).


(B17) In one embodiment, p and RZ are as described in (A3), and T1 is as described in any one of (e6)-(e9).


(B18) In one embodiment, p and RZ are as described in (A3), and T1 is as described in any one of (e10)-(e11).


(C1) In one embodiment, m is as described in (g1) or (g2), and Y is as described in any one of (h1)-(h7) and (h18)-(h24).


(C2) In one embodiment, m is as described in any one of (g4)-(g6), and Y is as described in any one of (h1)-(h7) and (h18)-(h24).


(C3) In one embodiment, m is as described in (g5), and Y is as described in any one of (h1)-(h7) and (h18)-(h20).


(C4) In one embodiment, m is as described in (g6), and Y is as described in any one of (h1)-(h7) and (h18)-(h24).


(C5) In one embodiment, m is as described in (g1) or (g2), and Y is as described in (h8) and (h18)-(h24).


(C6) In one embodiment, m is as described in any one of (g4)-(g6), and Y is as described in (h8) and (h18)-(h24).


(C7) In one embodiment, m is as described in (g5), and Y is as described in (h8) and (h18)-(h20).


(C8) In one embodiment, m is as described in (g6), and Y is as described in (h8) and (h18)-(h24).


(C9) In one embodiment, m is as described in (g1) or (g2), and Y is as described in any one of (h9)-(h15) and (h18)-(h24).


(C10) In one embodiment, m is as described in any one of (g4)-(g6), and Y is as described in any one of (h9)-(h15) and (h18)-(h24).


(C11) In one embodiment, m is as described in (g5), and Y is as described in any one of (h9)-(h15) and (h18)-(h20).


(C12) In one embodiment, m is as described in (g6), and Y is as described in any one of (h9)-(h15) and (h18)-(h24).


(D1) In one embodiment, m and Y are as described, where applicable, in any one of (C1)-(C12), and n is as described in (i1) or (i2).


(D2) In one embodiment, m and Y are as described, where applicable, in any one of (C1)-(C12), and n is as described in (i3).


(D3) In one embodiment, m and Y are as described, where applicable, in any one of (C1)-(C12), and n is as described in any one of (i4)-(i6).


(D4) In one embodiment, m and Y are as described, where applicable, in any one of (C1)-(C12), and n is as described in (i5).


(D5) In one embodiment, m and Y are as described, where applicable, in any one of (C1)-(C12), and n is as described in (i6).


(E1) In one embodiment, Y is as described in (h16), and n is as described in (i1) or (i2).


(E2) In one embodiment, Y is as described in (h16), and n is as described in (i3).


(E3) In one embodiment, Y is as described in (h16), and n is as described in any one of (i4)-(i6).


(E4) In one embodiment, Y is as described in (h16), and n is as described in (i5).


(E5) In one embodiment, Y is as described in (h16), and n is as described in (i6).


(E6) In one embodiment, Y is as described in (h17), and n is as described in (i1) or


(i2).


(E7) In one embodiment, Y is as described in (h17), and n is as described in (i3).


(E8) In one embodiment, Y is as described in (h17), and n is as described in any one of (i4)-(i6).


(E9) In one embodiment, Y is as described in (h17), and n is as described in (i5).


(E10) In one embodiment, Y is as described in (h17), and n is as described in (i6).


(F1) In one embodiment, n is as described in (i1) or (i2), and Z is as described in any one of (j1)-(j7) and (j16)-(j19).


(F2) In one embodiment, n is as described in any one of (i4)-(i6), and Z is as described in any one of (j1)-(j7) and (j16)-(j19).


(F3) In one embodiment, n is as described in (i5), and Z is as described in any one of (j1)-(j7) and (j16)-(j17).


(F4) In one embodiment, n is as described in (i6), and Z is as described in any one of (j1)-(j7) and (j16)-(j19).


(F5) In one embodiment, n is as described in (i1) or (i2), and Z is as described in any one of (j8) and (j16)-(j19).


(F6) In one embodiment, n is as described in any one of (i4)-(i6), and Z is as described in any one of (j8) and (j16)-(j19).


(F7) In one embodiment, n is as described in (i5), and Z is as described in any one of (j8) and (j16)-(j17).


(F8) In one embodiment, n is as described in (i6), and Z is as described in any one of (j8) and (j16)-(j19).


(F9) In one embodiment, n is as described in (i1) or (i2), and Z is as described in any one of (j9)-(j15) and (j16)-(j19).


(F10) In one embodiment, n is as described in any one of (i4)-(i6), and Z is as described in any one of (j9)-(j15) and (j16)-(j19).


(F11) In one embodiment, n is as described in (i5), and Z is as described in any one of (j9)-(j15) and (j16)-(j17).


(F12) In one embodiment, n is as described in (i6), and Z is as described in any one of (j9)-(j15) and (j16)-(j19).


(G1) In one embodiment, n and Z are as described, where applicable, in any one of (F1)-(F12), and m is as described in (g1) or (g2).


(G2) In one embodiment, n and Z are as described, where applicable, in any one of (F1)-(F12), and m is as described in (g3).


(G3) In one embodiment, n and Z are as described, where applicable, in any one of (F1)-(F12), and m is as described in any one of (g4)-(g6).


(G4) In one embodiment, n and Z are as described, where applicable, in any one of (F1)-(F12), and m is as described in (g5).


(G5) In one embodiment, n and Z are as described, where applicable, in any one of (F1)-(F12), and m is as described in (g6).


(H1) In one embodiment, m, n, and Y are as described, where applicable, in any one of (C1)-(D1), (D3)-(E1), (E3)-(E6), and (E8)-(E10), and Z is as described in any one of (j1)-(j7).


(H2) In one embodiment, m, n, and Y are as described, where applicable, in any one of (C1)-(D1), (D3)-(E1), (E3)-(E6), and (E8)-(E10), and Z is as described in (j8).


(H3) In one embodiment, m, n, and Y are as described, where applicable, in any one of (C1)-(D1), (D3)-(E1), (E3)-(E6), and (E8)-(E10), and Z is as described in any one of (j9)-(j15).


(I1) In one embodiment, m, n, and Z are as described, where applicable, in any one of (F1)-(G1) and (G3)-(G5), and Y is as described in any one of (h1)-(h7).


(I2) In one embodiment, m, n, and Z are as described, where applicable, in any one of (F1)-(G1) and (G3)-(G5), and Y is as described in (h8).


(I3) In one embodiment, m, n, and Z are as described, where applicable, in any one of (F1)-(G1) and (G3)-(G5), and Y is as described in any one of (h9)-(h15).


(I4) In one embodiment, m, n, and Z are as described, where applicable, in any one of (F1)-(G1) and (G3)-(G5), and Y is as described in (h16).


(I5) In one embodiment, m, n, and Z are as described, where applicable, in any one of (F1)-(G1) and (G3)-(G5), and Y is as described in (h17).


(J1) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in (k1).


(J2) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in any one of (k2)-(k9).


(J3) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in (k4).


(J4) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in (k5).


(J5) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in (k6).


(J6) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in (k7).


(J7) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in (k8).


(J8) In one embodiment, Y, Z, RZ, T1, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), and (F5)-(I5), and Q is as described in (k9).


(K1) In one embodiment, Y, Z, RZ, T1, Q, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), and (J2)-(J8), and Q′ is as described in (11).


(K2) In one embodiment, Y, Z, RZ, T1, Q, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), and (J2)-(J8), and Q′ is as described in (12).


(K3) In one embodiment, Y, Z, RZ, T1, Q, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), and (J2)-(J8), and Q′ is as described in (13).


(K4) In one embodiment, Y, Z, RZ, T1, Q, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), and (J2)-(J8), and Q′ is as described in (14).


(K5) In one embodiment, Y, Z, RZ, T1, Q, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), and (J2)-(J8), and Q′ is as described in (15).


(K6) In one embodiment, Y, Z, RZ, T1, Q, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), and (J2)-(J8), and Q′ is as described in (16).


(K7) In one embodiment, Y, Z, RZ, T1, Q, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), and (J2)-(J8), and Q′ is as described in (17).


(L1) In one embodiment, Y, Z, RZ, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), (J1)-(J8), and (K1)-(K7), and T is as described in (n1).


(L2) In one embodiment, Y, Z, RZ, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), (J1)-(J8), and (K1)-(K7), and T is as described in (n2).


(L3) In one embodiment, Y, Z, RZ, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), (J1)-(J8), and (K1)-(K7), and T is as described in (n3).


(L4) In one embodiment, Y, Z, RZ, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), (J1)-(J8), and (K1)-(K7), and T is as described in (n4).


(L5) In one embodiment, Y, Z, RZ, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(B18), (C5)-(D5), (F5)-(I5), (J1)-(J8), and (K1)-(K7), and T is as described in (n5).


(M1) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(L5), and X is as described in (a1).


(M2) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(L5), and X is as described in (a2).


(M3) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(L5), and X is as described in (a3).


(M4) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(L5), and X is as described in (a4).


(M5) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, m, n, and p are as described, where applicable, in any one of (A1)-(L5), and X is as described in (a5).


(N1) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b1).


(N2) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b2).


(N3) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b3).


(N4) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b4).


(N5) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b5).


(N6) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b6).


(N7) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b7).


(N8) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b8).


(N9) In one embodiment, Y, Z, RZ, T, T1, Q, Q′, X, m, n, and p are as described, where applicable, in any one of (A1)-(L5) and (M1), and RX is as described in (b9).


(O1) In one embodiment, m is 1 and n is 0.


(O2) In one embodiment, m is 0 and n is 1.


(O3) In one embodiment, m is 1 and n is 1.


(O4) In one embodiment, m is 2 and n is 0.


(O5) In one embodiment, m is 2 and n is 1.


(O6) In one embodiment, m is 0 and n is 2.


(O7) In one embodiment, m is 1 and n is 2.


(O8) In one embodiment, m is 3 and n is 0.


(O9) In one embodiment, m is 3 and n is 1.


(O10) In one embodiment, m is 3 and n is 2.


(O11) In one embodiment, m is 0 and n is 3.


(O12) In one embodiment, m is 1 and n is 3.


(O13) In one embodiment, m is 2 and n is 3.


(P1) In one embodiment, m and n are as described in any one of (O1), (O3)-(O5), (O7)-(10), (O12), and (O13), and, where applicable, Y is as described in any one of (h1)-(h7) and (h18)-(h24).


(P2) In one embodiment, m and n are as described in any one of (O1), (O3)-(O5), (O7)-(10), (O12), and (O13), and, where applicable, Y is as described in (h8) and (h18)-(h24).


(P3) In one embodiment, m and n are as described in any one of (O1), (O3)-(O5), (O7)-(10), (O12), and (O13), and, where applicable, Y is as described in any one of (h9)-(h15) and (h18)-(h24).


(P4) In one embodiment, m and n are as described in any one of (O1), (O3)-(O5), (O7)-(10), (O12), and (O13), and, where applicable, Y is as described in (h16).


(P5) In one embodiment, m and n are as described in any one of (O1), (O3)-(O5), (O7)-(10), (O12), and (O13), and, where applicable, Y is as described in (h17).


(Q1) In one embodiment, m and n are as described in any one of (O2), (O3), (O5)-(O7), and (O9)-(O13), and, where applicable, Z is as described in any one of (j1)-(j7) and (j16)-(j19).


(Q2) In one embodiment, m and n are as described in any one of (O2), (O3), (O5)-(O7), and (O9)-(O13), and, where applicable, Z is as described in (j8) and (j16)-(j19).


(Q3) In one embodiment, m and n are as described in any one of (O2), (O3), (O5)-(O7), and (O9)-(O13), and, where applicable, Z is as described in any one of (j9)-(j15) and (j16)-(j19).


(R1) In one embodiment, m, n, and Y are as described in any one of (P1)-(P5), and, where applicable, Z is as described in any one of (j1)-(j7) and (j16)-(j19).


(R2) In one embodiment, m, n, and Y are as described in any one of (P1)-(P5), and, where applicable, Z is as described in (j8) and (j16)-(j19).


(R3) In one embodiment, m, n, and Y are as described in any one of (P1)-(P5), and, where applicable, Z is as described in any one of (j9)-(j15) and (j16)-(j19).


(S1) In one embodiment, m, n, and Z are as described in any one of (Q1)-(Q3), and, where applicable, Y is as described in any one of (h1)-(h7) and (h18)-(h24).


(S2) In one embodiment, m, n, and Z are as described in any one of (Q1)-(Q3), and, where applicable, Y is as described in (h8) and (h18)-(h24).


(S3) In one embodiment, m, n, and Z are as described in any one of (Q1)-(Q3), and, where applicable, Y is as described in any one of (h9)-(h15) and (h18)-(h24).


(S4) In one embodiment, m, n, and Z are as described in any one of (Q1)-(Q3), and, where applicable, Y is as described in (h16) and (h18)-(h24).


(S5) In one embodiment, m, n, and Z are as described in any one of (Q1)-(Q3), and, where applicable, Y is as described in (h17) and (h18)-(h24).


(T1) In one embodiment, m, n, Y, and Z are as described in any one of (O1)-(S5), and p, RZ, and T1 are as described, where applicable, in any one of (A1)-(B18).


(U1) In one embodiment, p, RZ, T1, m, n, Y, and Z are as described, where applicable, in any one of (O1)-(T1), and X is as described in (a1).


(U2) In one embodiment, p, RZ, T1, m, n, Y, and Z are as described, where applicable, in any one of (O1)-(T1), and X is as described in (a2).


(U3) In one embodiment, p, RZ, T1, m, n, Y, and Z are as described, where applicable, in any one of (O1)-(T1), and X is as described in (a3).


(U4) In one embodiment, p, RZ, T1, m, n, Y, and Z are as described, where applicable, in any one of (O1)-(T1), and X is as described in (a4).


(U5) In one embodiment, p, RZ, T1, m, n, Y, and Z are as described, where applicable, in any one of (O1)-(T1), and X is as described in (a5).


(V1) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b1).


(V2) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b2).


(V3) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b3).


(V4) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b4).


(V5) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b5).


(V6) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b6).


(V7) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b7).


(V8) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b8).


(V9) In one embodiment, p, RZ, T1, m, n, X, Y, and Z are as described, where applicable, in any one of (O1)-(T1) and (U1), and RX is as described in (b9).


In one embodiment, a compound of Formula I is of Formula I1, I1a1, I1a2, I1a3, I1a4, I1a5, I1a6, I1a7, I1b1, I1b2, I1b3, I1b4, I1b5, I1b6, I1b7, I1c1, I1c2, I1c3, I1c4, I1c5, I1c6, or I1c7:




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or a pharmaceutically acceptable salt or ester thereof, wherein R′ is H or methyl.


For a compound of any one of Formulae I1, I1a1, I1a2, I1a3, I1a4, I1a5, I1a6, I1a7, I1b1, I1b2, I1b3, I1b4, I1b5, I1b6, I1b7, I1c1, I1c2, I1c3, I1c4, I1c5, I1c6, and I1c7, X, Y, Z, RQ, RT, RX, Q, Q′, T, m, and q are each as defined herein in Formula I, and each of the groups described herein for any of X, Y, Z, RQ, RT, RX, Q, Q′, T, m, and q can be combined as described herein above.


For example, for a compound of any one of Formulae I1, I1a1, I1a2, I1a3, I1a4, I1a5, I1a6, I1a7, I1b1, I1b2, I1b3, I1b4, I1b5, I1b6, I1b7, I1c1, I1c2, I1c3, I1c4, I1c5, I1c6, and I1c7, where applicable:


In one embodiment, each Z is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, CN, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, each of which is as described herein.


In one embodiment, each Z is independently C1-C6 alkyl, halogen, CN, OH, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, each of which is as described herein.


In one embodiment, each Z is independently C1-C6 alkyl, halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), NH2, NH—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, each of which is as described herein.


In one embodiment, each Z is independently halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, each of which is as described herein.


In one embodiment, each Z is independently F, Cl, OCH3, OCH2CH3, SCH3, or N(CH3)2.


In one embodiment, one Z is F or Cl, and the other Z is Cl, F, OCH3, OCH2CH3, SCH3, or N(CH3)2. In one embodiment, one Z is F, and the other Z is Cl, OCH3, OCH2CH3, SCH3, or N(CH3)2. In one embodiment, one Z is Cl, and the other Z is F, OCH3, OCH2CH3, SCH3, or N(CH3)2. In a further embodiment, one Z is at the 4-position, and the other Z is at the 3-position. In a further embodiment, one Z is Cl at the 3-position, and the other Z is F, OCH3, OCH2CH3, SCH3, or N(CH3)2 at the 4-position. In a further embodiment, one Z is C1 at the 3-position, and the other Z is OCH3, OCH2CH3, or N(CH3)2 at the 4-position. In a further embodiment, one Z is Cl at the 3-position, and the other Z is N(CH3)2 at the 4-position.


In one embodiment, X is C(RX)2, and each RX is independently H, CH3, CF3, CF2H, or F. In a further embodiment, each RX is independently H or CH3. In another further embodiment, each RX is H. In another further embodiment, each RX is CH3.


In one embodiment, X is C(RX)2, and two RX together form ═O, ═CH2, or ═CF2. In a further embodiment, two RX together form ═O.


In one embodiment, R′ is H.


In one embodiment, R′ is methyl. In a further embodiment, R′ is (S)-methyl. In another further embodiment, R′ is (R)-methyl.


Any of the groups described above for any of X, Z, and R′ can be combined with any of the groups described above for the remainder of X, Z, and R′, and the combination of X, Z, and R′ can be further combined, where applicable, with any of the groups described herein for one or more of Y, Z1, R1, RQ, RT, RX, RZ, RS, Q, Q′, T1, T, m, n, p, and q.


In one embodiment, a compound of Formula I is of Formula I1a1, I1b1, or I1c1:




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or a pharmaceutically acceptable salt or ester thereof, wherein R′ is H or methyl.


For a compound of any one of Formulae Hal, I1b1, and I1c1, X, Y, Z, RQ, RT, RX, Q, Q′, T, m, and q are each as defined herein in Formula I, and each of the groups described herein for any of X, Y, Z, RQ, RT, RX, Q, Q′, T, m, and q can be combined as described herein above.


For any of the formulae described herein, wherein applicable:


In one embodiment, one Z is halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or Q-T, each of which is as described herein, and the other Z is Cl. In a further embodiment, Z is Cl at the 3-position, and the halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), N—(C1-C6 alkyl)2, or Q-T is at the 4-position.


In one embodiment, one Z is halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), or N—(C1-C6 alkyl)2, each of which is as described herein, and the other Z is Cl. In a further embodiment, Z is Cl at the 3-position, and the halogen, O—(C1-C6 alkyl), S—(C1-C6 alkyl), or N—(C1-C6 alkyl)2 is at the 4-position.


In one embodiment, one Z is halogen, and the other Z is Cl. In a further embodiment, Z is Cl at the 3-position. In a further embodiment, Z is Cl at the 3-position, and the halogen is at the 4-position. In a further embodiment, the halogen is Cl, Br, or I. In a further embodiment, the halogen is Cl.


In one embodiment, one Z is O—(C1-C6 alkyl) as described herein, and the other Z is Cl. In a further embodiment, Z is Cl at the 3-position, and the O—(C1-C6 alkyl) is at the 4-position. In a further embodiment, the O—(C1-C6 alkyl) is O-methyl.


In one embodiment, one Z is S—(C1-C6 alkyl) as described herein, and the other Z is Cl. In a further embodiment, Z is Cl at the 3-position, and the S—(C1-C6 alkyl) is at the 4-position. In a further embodiment, the S—(C1-C6 alkyl) is S-methyl.


In one embodiment, one Z is N—(C1-C6 alkyl)2 as described herein, and the other Z is Cl. In a further embodiment, Z is Cl at the 3-position, and the N—(C1-C6 alkyl)2 is at the 4-position. In a further embodiment, the N—(C1-C6 alkyl)2 is N(CH3)2 or N(CH3)(CH2CH3).


In one embodiment, one Z is Q-T as described herein, and the other Z is Cl. In a further embodiment, Z is Cl at the 3-position. In a further embodiment, Z is Cl at the 3-position, and Q-T is at the 4-position. In a further embodiment, Q is a bond and T is cycloalkyl, cycloalkenyl, heterocyclyl, C6-C10 aryl, or heteroaryl, each optionally substituted with one or more RT, as described herein. In a further embodiment, Q is a bond and T is C6-C10 aryl optionally substituted with one or more RT. In a further embodiment, T is phenyl optionally substituted with one or more RT.


In one embodiment, Y is tert-butyl, one Z is S—CH3, and the other Z is Cl. In a further embodiment, one Z is S—CH3 at the 4-position, and the other Z is Cl at the 3-position.


In one embodiment, Y is tert-butyl, one Z is O—CH3, and the other Z is Cl. In a further embodiment, one Z is O—CH3 at the 4-position, and the other Z is Cl at the 3-position.


In one embodiment, Y is methyl, i-propyl, or t-butyl, one Z is phenyl, and the other Z is Cl. In a further embodiment, one Z is phenyl at the 4-position, and the other Z is Cl at the 3-position. In a further embodiment, Y is methyl or i-propyl. In a further embodiment, Y is at the 6-position.


In one embodiment, Y is methyl, i-propyl, or t-butyl, one Z is N(CH3)2, and the other Z is Cl. In a further embodiment, one Z is N(CH3)2 at the 4-position, and the other Z is Cl at the 3-position. In a further embodiment, Y is methyl or t-butyl. In a further embodiment, Y is at the 6-position.


In one embodiment, Y is NH-phenyl, one Z is N(CH3)2, and the other Z is Cl. In a further embodiment, one Z is N(CH3)2 at the 4-position, and the other Z is Cl at the 3-position. In a further embodiment, Y is at the 6-position.


In one embodiment, X is C(RX)2, and each RX is independently H, CH3, CF3, CF2H, or F. In a further embodiment, each RX is independently H or CH3. In another further embodiment, each RX is H. In another further embodiment, each RX is CH3.


In one embodiment, X is C(RX)2, and two RX together form ═O, ═CH2, or ═CF2. In a further embodiment, two RX together form ═O.


In one embodiment, Y is NH-(phenyl) and one Z is N—(CH3)2, and the other Z is halogen. In a further embodiment, the halogen is Cl.


In one embodiment, Y is C1-C6 alkyl and one Z is phenyl, and the other Z is halogen.


In a further embodiment, Z is halogen at the 3-position. In a further embodiment, the halogen is Cl. In another further embodiment, Z is Cl at the 3-position. In another further embodiment, Y is methyl.


In one embodiment, Y is isopropyl and one Z is N—(CH3)2, and the other Z is halogen.


In a further embodiment, Z is halogen at the 3-position. In a further embodiment, the halogen is Cl. In another further embodiment, Z is Cl at the 3-position.


In one embodiment, Y is C1-C6 alkyl and one Z is N—(CH3)2, and the other Z is halogen. In a further embodiment, the halogen is Cl. In another further embodiment, Z is C1 at the 3-position. In another further embodiment, Y is methyl. In another further embodiment, Y is methyl at the 6-position.


In one embodiment, Y is branched C1-C6 alkyl and one Z is phenyl, and the other Z is halogen. In a further embodiment, Z is halogen at the 3-position. In a further embodiment, the halogen is Cl. In another further embodiment, Y is isopropyl.


In one embodiment, R′ is H.


In one embodiment, R′ is methyl. In a further embodiment, R′ is (S)-methyl. In another further embodiment, R′ is (R)-methyl.


In one embodiment, the compound is of Formula (I1c1),




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In one embodiment, the compound is of Formula (I1c1), and Z is N(CH3)2. In one embodiment, the compound is of Formula (I1c1), and Z is N(CH3)2 at the 4-position. In another embodiment, the compound is of Formula (I1c1), and Z is Cl. In another further embodiment, the compound is of Formula (I1c1), and Z is Cl at the 3-position. In a further embodiment, the compound is of Formula (I1c1), and one Z is Cl at the 3-position, and the other Z is N(CH3)2 at the 4-position.


In one embodiment, the compound is of Formula (I1c1), and Y is C1-C6 alkyl as described herein. In a further embodiment, Y is methyl, ethyl, i-propyl, or t-butyl. In a further embodiment, Y is methyl or t-butyl. In a further embodiment, Y is methyl, ethyl, i-propyl, or t-butyl. In a further embodiment, Y is t-butyl. In another embodiment, Y is NH-phenyl.


Any of the groups described above for any of X, Z, and R′ can be combined with any of the groups described above for the remainder of X, Z, and R′, and the combination of X, Z, and R′ can be further combined, where applicable, with any of the groups described herein for one or more of Y, Z1, R1, RQ, RT, RX, RZ, RS, Q, Q′, T1, T, m, n, p, and q.


Representative compounds of the present application are shown in Table 1.










TABLE 1





Cmpd



No.
Structure







 1


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 2


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 3


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 4


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 5


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 6


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 7


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 8


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 9


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10


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11


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12


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13


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14


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15


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16


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17


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18


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20


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21


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22


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24


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25


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26


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28


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Some of the foregoing compounds can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., stereoisomers and/or diastereomers.


Accordingly, compounds of the application may be in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers. In one embodiment, the compounds of the application are enantiopure compounds. In another embodiment, mixtures of stereoisomers or diastereomers are provided.


Another aspect is an isotopically labeled compound of any of the formulae delineated herein and of any of the compounds described herein. Such compounds have one or more isotope atoms which may or may not be radioactive (e.g., 3H, 2H, 14C, 13C, 18F, 35S, 32P, 125I, and 131I) introduced into the compound. Such compounds are useful for drug metabolism studies and diagnostics, as well as therapeutic applications.


Potency can also be determined by IC50 value. A compound with a lower IC50 value, as determined under substantially similar conditions, is more potent relative to a compound with a higher IC50 value. In some embodiments, the substantially similar conditions comprise determining the level of binding of a known STING ligand to a STING protein, in vitro or in vivo, in the presence of a compound of the application.


In one embodiment, the compounds of the present application are useful as therapeutic agents, and thus may be useful in the treatment of a disease caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function) or a disease associated with one or more of the intracellular pathways that STING is involved in (e.g. regulation of intracellular DNA-mediated type I interferon activation), such as those described herein.


A “selective STING modulator” can be identified, for example, by comparing the ability of a compound to modulate STING expression/activity/function to its ability to modulate the other proteins or a STING protein from another species. In some embodiments, the selectivity can be identified by measuring the EC50 or IC50 of the compounds. In some embodiments, the compounds of the present application are “selective human STING modulator”.


In certain embodiments, the compounds of the application are STING modulators (e.g., selective human STING modulator) that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold selectivity over other proteins or a STING protein from another species (e.g., a non human animal, such as mouse). In various embodiments, the compounds of the application exhibit 1000-fold selectivity over other proteins or a STING protein from another species.


The compounds of the application are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.


The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.


In another aspect, the application provides a method of synthesizing a compound disclosed herein. The synthesis of the compounds of the application can be found herein and in the Examples below. Other embodiments are a method of making a compound of any of the formulae herein using any one, or combination of, reactions delineated herein. The method can include the use of one or more intermediates or chemical reagents delineated herein.


The application also provides for a pharmaceutical composition comprising a therapeutically effective amount of a compound of the application, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.


Another aspect of the present application relates to a kit comprising a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application. In another aspect, the application provides a kit comprising a compound capable of modulating STING activity selected from one or more compounds disclosed herein, or a pharmaceutically acceptable salt or ester thereof, optionally in combination with a second agent and instructions for use.


Another aspect of the present application relates to a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, for use in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) a STING protein, for treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression activity, and/or function), or for treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g. deregulation of intracellular dsDNA mediated type I interferon activation).


Another aspect of the present application relates to use of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) a STING protein, for treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity and/or function (e.g., deregulation of STING expression, activity, and/or function), or for treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation).


Another aspect of the present application relates to a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, for use in modulating (e.g., inhibiting or stimulating) a STING protein, in treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function), or in treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation).


Another aspect of the present application relates to use of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application, in modulating (e.g., inhibiting or stimulating) a STING protein, in treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression activity, and/or function (e.g., deregulation of STING expression, activity, and/or function), or in treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation).


Method of Synthesizing the Compounds

Compounds of the present application can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith, M. B., March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; and Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999, incorporated by reference herein, are useful and recognized reference textbooks of organic synthesis known to those in the art. The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present application. The processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt, ester, or prodrug thereof. Suitable synthetic routes are depicted in the schemes below.


Those skilled in the art will recognize if a stereocenter exists in the compounds disclosed herein. Accordingly, the present application includes both possible stereoisomers (unless specified in the synthesis) and includes not only racemic compounds but the individual enantiomers and/or diastereomers as well. When a compound is desired as a single enantiomer or diastereomer, it may be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. Resolution of the final product, an intermediate, or a starting material may be affected by any suitable method known in the art. See, for example, “Stereochemistry of Organic Compounds” by E. L. Eliel, S. H. Wilen, and L. N. Mander (Wiley-Interscience, 1994).


The compounds of the present application can be prepared in a number of ways well known to those skilled in the art of organic synthesis. By way of example, compounds of the present application can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include but are not limited to those methods described below.


Compounds of the present application can be synthesized by following the steps outlined in the following Schemes, which comprise different sequences of assembling intermediates. Starting materials are either commercially available or made by known procedures in the reported literature or as illustrated. As shown in the Schemes below, compounds of the present application may be synthesized from transition-metal catalyzed cross coupling reactions with known compounds. The cross-coupling reactions, as illustrated below, can provide the compounds of the present application or intermediates that can be further hydrogenated or deprotected to provide the target compounds.




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As shown in Schemes 1a-1d, coupling between the requisite aniline (B1, B2, B3, or B4) and the properly substituted (A) yields the corresponding pyridinephenylamines (C1, C2, C3, or C4) (1a). Alternatively, pyridinephenylamines (C1, C2, C3, or C4) can be prepared according to 1b. Cyclodehydration of C1, C2, C3, C4 affords substituted D1, D2a, D2b, D3, or D4 (1c). Alkylation of the D1, D2a, D2b, D3, or D4, followed by saponification, generates the target compounds E1, E2a, E2b, E3, or E4 (1d).




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As shown in Scheme 2, compounds of the present application or precursors thereof can be prepared through bromination of compound F, followed by saponification in the procedure above.




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As shown in Schemes 3a and 3b, compounds of the present application can be prepared through Sonogashira coupling, the product of which may be subject to hydrogenation to produce additional compounds of the present application.




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As shown in Scheme 4, compounds of the present application can be prepared through Suzuki coupling, the product of which may be subject to saponification or hydrogenation to produce additional compounds of the present application.




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Additional compounds of the present application can be prepared using the procedures according to Schemes 2-7.


A compound of the application can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the application can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. The pharmaceutically acceptable salt may include various counterions, e.g., counterions of the inorganic or organic acid, counterions of the inorganic or organic base, or counterions afforded by counterion exchange.


Acids and bases useful in the methods herein are known in the art. Acid catalysts are any acidic chemical, which can be inorganic (e.g., hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic (e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid, ytterbium triflate) in nature. Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. Bases are any basic chemical, which can be inorganic (e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g., triethylamine, pyridine) in nature. Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.


Alternatively, the salt forms of the compounds of the application can be prepared using salts of the starting materials or intermediates. The free acid or free base forms of the compounds of the application can be prepared from the corresponding base addition salt or acid addition salt from, respectively. For example, a compound of the application in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the application in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).


Compounds of the present application that contain nitrogens can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (m-CPBA) and/or hydrogen peroxides) to afford other compounds of the present application. Thus, all shown and claimed nitrogen-containing compounds are considered, when allowed by valency and structure, to include both the compound as shown and its N-oxide derivative (which can be designated as N→O or N+—O−). Furthermore, in other instances, the nitrogens in the compounds of the present application can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m-CPBA. All shown and claimed nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as shown and its N-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R is substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.


Prodrugs of the compounds of the application can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs can be prepared by reacting a non-derivatized compound of the application with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or the like). Specifically, the central N-acetic acid moeity, and other analogous carboxylic acid groups, of the compounds of the present invention can be modified through techniques known in the art to produce effective prodrugs of the present invention.


Protected derivatives of the compounds of the application can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, “Protecting Groups in Organic Chemistry”, 3rd edition, John Wiley and Sons, Inc., 1999.


Compounds of the present application can be conveniently prepared, or formed during the process of the application, as solvates (e.g., hydrates). Hydrates of compounds of the present application can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol.


Optical isomers may be prepared from their respective optically active precursors by the procedures described herein, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981).


The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. In addition, the solvents, temperatures, reaction durations, etc. delineated herein are for purposes of illustration only and one of ordinary skill in the art will recognize that variation of the reaction conditions can produce the desired bridged macrocyclic products of the present application. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.


The compounds of this application may be modified by appending various functionalities via any synthetic means delineated herein to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.


Biological Assays

Biological activities of the compounds of the present application can be measured by various biochemical or cellular assays known to one of ordinary skill in the art. Non-limiting examples of biochemical and cellular assays are listed herein below.


SWAT Protein FP Competition Dose Response Assay

A validated STING ligand, such as c-di-GMP, which is labeled with a read-out signal (e.g., a fluorescence signal such as fluorescein), is mixed with STING-CTD (e.g., mouse STING-CTD or human STING-CTD) with or without the presence of a compound of the present application. Changes in the read-out signal are measured (e.g., by fluorescence anisotropy) to determine the binding of the compound to STING-CTD.


Pharmaceutical Compositions

In another aspect, a pharmaceutical composition is provided. The pharmaceutical composition comprises a therapeutically effective amount of a compound of the application, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.


Compounds of the application may be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, or topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form.


Pharmaceutical compositions including a compound of the present application in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent may be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present application with a carrier. A carrier may include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices may be in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.


The pharmaceutical compositions of the present application comprise a therapeutically effective amount of a compound of the present application formulated together with one or more pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which may serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylenepolyoxy propylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.


The pharmaceutical compositions of this application may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray.


Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.


Injectable preparations, for example, sterile injectable aqueous, or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.


In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.


Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this application with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.


Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.


The active compounds may also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.


Dosage forms for topical or transdermal administration of a compound of this application include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this application.


The ointments, pastes, creams and gels may contain, in addition to an active compound of this application, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.


Powders and sprays can contain, in addition to the compounds of this application, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.


Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.


For any compound, the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.


Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.


The quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a compound of this application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants that are required.


The pharmaceutical compositions containing active compounds of the present application may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.


Techniques for formulation and administration of the disclosed compounds of the application can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds described herein, and the pharmaceutically acceptable salts thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.


Methods of Use

In one aspect, the present application provides a method of modulating (e.g., inhibiting or stimulating) a STING protein. The method comprises administering to a subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application.


In some embodiments, the modulation of a STING protein activity is measured by IC50. In some embodiments, the modulation of a STING protein activity is measured by EC50.


A compound of the present application (e.g., a compound of any of the formulae described herein, or selected from any compounds described herein) is capable of treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function) or a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation).


In one aspect, the present application provides a method of treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function (e.g., deregulation of STING expression, activity, and/or function). The method comprises administering to a subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application. In one aspect, the disease is a STING mediated disorder.


In one aspect, the present application provides a method of treating or preventing a disease associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved (e.g., deregulation of intracellular dsDNA mediated type I interferon activation). The method comprises administering to a subject in need thereof an effective amount of a compound of the application or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition of the application.


In one embodiment, the present application provides a method of treating or preventing any of the diseases, disorders, and conditions described herein, wherein the subject is a human. In one embodiment, the application provides a method of treating. In one embodiment, the application provides a method of preventing.


As modulators of a STING protein, the compounds and compositions of this application are particularly useful for treating or lessening the severity of a disease, condition, or disorder where a STING protein or one or more of the intracellular pathways that STING is involved is implicated in the disease, condition, or disorder. In one embodiment, the present application provides a method for treating or lessening the severity of a disease, condition, or disorder with compounds that modulate binding of a non-canonical cyclic di-nucleotide (CDN), such as 2′3′cGAMP, to a STING protein. In one embodiment, the present application provides a method for treating or lessening the severity of a disease, condition, or disorder with compounds that modulate the synthesis of type I interferon and/or type I IFN response.


In one aspect, the present application also provides a method of treating or preventing cell proliferative disorders such as hyperplasias, dysplasias, or pre-cancerous lesions. Dysplasia is the earliest form of pre-cancerous lesion recognizable in a biopsy by a pathologist. The compounds of the present application may be administered for the purpose of preventing hyperplasias, dysplasias, or pre-cancerous lesions from continuing to expand or from becoming cancerous. Examples of pre-cancerous lesions may occur in skin, esophageal tissue, breast, and cervical intra-epithelial tissue.


In one embodiment, the disease or disorder includes, but is not limited to, immune disorders, autoimmunity a cell proliferative disease or disorder, cancer, inflammation, cachexia, neurodegenerative disease or disorders, neurological diseases or disorders, cardiac disfunction, transplantation, or infection (e.g., viral, bacterial, and/or fungi infection, or infection caused by other microorganism).


In one embodiment, the disease or disorder is a cell proliferative disease or disorder.


As used herein, the term “cell proliferative disorder” refers to conditions in which unregulated or abnormal growth, or both, of cells can lead to the development of an unwanted condition or disease, which may or may not be cancerous. Exemplary cell proliferative diseases or disorders encompass a variety of conditions wherein cell division is deregulated. Exemplary cell proliferative disorder include, but are not limited to, neoplasms, benign tumors, malignant tumors, pre-cancerous conditions, in situ tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells. The term “rapidly dividing cell” as used herein is defined as any cell that divides at a rate that exceeds or is greater than what is expected or observed among neighboring or juxtaposed cells within the same tissue. A cell proliferative disease or disorder includes a precancer or a precancerous condition. A cell proliferative disease or disorder includes cancer.


In one embodiment, the proliferative disease or disorder is a non-cancerous. In one embodiment, the non-cancerous disease or disorder includes, but is not limited to, rheumatoid arthritis; inflammation; autoimmune disease; lymphoproliferative conditions; acromegaly; rheumatoid spondylitis; osteoarthritis; gout; other arthritic conditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma; adult respiratory distress syndrome; chronic obstructive pulmonary disease; chronic pulmonary inflammation; inflammatory bowel disease; Crohn's disease; skin-related hyperproliferative disorders; psoriasis; eczema; atopic dermatitis; hyperpigmentation disorders; eye-related hyperproliferative disorders; age-related macular degeneration; ulcerative colitis; pancreatic fibrosis; hepatic fibrosis; acute and chronic renal disease; irritable bowel syndrome; pyresis; restenosis; cerebral malaria; stroke and ischemic injury; neural trauma; Alzheimer's disease; Huntington's disease; Parkinson's disease; acute and chronic pain; allergic rhinitis; allergic conjunctivitis; chronic heart failure; acute coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter's syndrome; acute synovitis; muscle degeneration, bursitis; tendonitis; tenosynovitis; herniated, ruptures, or prolapsed intervertebral disk syndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonary sarcosis; bone resorption diseases, such as osteoporosis; graft-versus-host reaction; fibroadipose hyperplasia; spinocerebullar ataxia type 1; CLOVES syndrome; Harlequin ichthyosis; macrodactyly syndrome; Proteus syndrome (Wiedemann syndrome); LEOPARD syndrome; systemic sclerosis; Multiple Sclerosis; lupus; fibromyalgia; AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I or II, influenza virus and cytomegalovirus; diabetes mellitus; hemihyperplasia-multiple lipomatosis syndrome; megalencephaly; rare hypoglycemia, Klippel-Trenaunay syndrome; harmatoma; Cowden syndrome; or overgrowth-hyperglycemia.


In one embodiment, the proliferative disease or disorder is cancer. In one embodiment, the cancer is lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemias, lymphomas, myelomas, or solid tumors.


The term “cancer” includes, but is not limited to, the following cancers: breast; ovary; cervix; prostate; testis, genitourinary tract; esophagus; larynx, glioblastoma; neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma; bone; colon; colorectal; adenoma; pancreas, adenocarcinoma; thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma; seminoma; melanoma; sarcoma; bladder carcinoma; liver carcinoma and biliary passages; kidney carcinoma; myeloid disorders; lymphoid disorders, Hodgkin's, hairy cells; buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx; small intestine; colonrectum, large intestine, rectum, brain and central nervous system; chronic myeloid leukemia (CML), and leukemia. The term “cancer” includes, but is not limited to, the following cancers: myeloma, lymphoma, or a cancer selected from gastric, renal, or and the following cancers: head and neck, oropharangeal, non-small cell lung cancer (NSCLC), endometrial, hepatocarcinoma, Non-Hodgkins lymphoma, and pulmonary.


The term “cancer” also refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas and the like. For example, cancers include, but are not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, and multiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodisplastic syndrome, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular), lung cancer (e.g., small-cell and non-small cell), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors related to Gorlin's syndrome (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Additional exemplary forms of cancer which may be treated by the subject compounds include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.


Cancer may also include colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, or melanoma. Further, cancers include, but are not limited to, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.


Cancer may also include colorectal, thyroid, breast, and lung cancer; and myeloproliferative disorders, such as polycythemia vera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mast cell disease. In one embodiment, the compounds of this application are useful for treating hematopoietic disorders, in particular, acute-myelogenous leukemia (AML), chronic-myelogenous leukemia (CML), acute-promyelocytic leukemia, and acute lymphocytic leukemia (ALL).


Exemplary cancers may also include, but are not limited to, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, uringary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer of the tongue, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilm's Tumor.


A “cell proliferative disorder of the hematologic system” is a cell proliferative disease or disorder involving cells of the hematologic system. A cell proliferative disorder of the hematologic system can include lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benign monoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoid papulosis, polycythemia vera, chronic myelocytic leukemia, agnogenic myeloid metaplasia, and essential thrombocythemia. A cell proliferative disorder of the hematologic system can include hyperplasia, dysplasia, and metaplasia of cells of the hematologic system. Compounds and compositions of the present application may be used to treat a cancer selected from the group consisting of a hematologic cancer or a hematologic cell proliferative disorder. A hematologic cancer can include multiple myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia (including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloid neoplasms, and mast cell neoplasms.


A “cell proliferative disorder of the lung” is a cell proliferative disease or disorder involving cells of the lung. Cell proliferative disorders of the lung can include all forms of cell proliferative disorders affecting lung cells. Cell proliferative disorders of the lung can include lung cancer, a precancer or precancerous condition of the lung, benign growths or lesions of the lung, and malignant growths or lesions of the lung, and metastatic lesions in tissue and organs in the body other than the lung. Compounds and compositions of the present application may be used to treat lung cancer or cell proliferative disorders of the lung. Lung cancer can include all forms of cancer of the lung. Lung cancer can include malignant lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer can include small cell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Lung cancer can include “scar carcinoma”, bronchioalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer can include lung neoplasms having histologic and ultrastructual heterogeneity (e.g., mixed cell types).


Cell proliferative disorders of the lung can also include hyperplasia, metaplasia, and dysplasia of the lung. Cell proliferative disorders of the lung can include asbestos-induced hyperplasia, squamous metaplasia, and benign reactive mesothelial metaplasia. Cell proliferative disorders of the lung can include replacement of columnar epithelium with stratified squamous epithelium, and mucosal dysplasia. Individuals exposed to inhaled injurious environmental agents such as cigarette smoke and asbestos may be at increased risk for developing cell proliferative disorders of the lung. Prior lung diseases that may predispose individuals to development of cell proliferative disorders of the lung can include chronic interstitial lung disease, necrotizing pulmonary disease, scleroderma, rheumatoid disease, sarcoidosis, interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathic pulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, and Hodgkin's disease.


A “cell proliferative disorder of the colon” is a cell proliferative disorder involving cells of the colon. A cell proliferative disorder of the colon includes colon cancer. Compounds and compositions of the present application may be used to treat colon cancer or cell proliferative disorders of the colon. Colon cancer can include all forms of cancer of the colon. Colon cancer can include sporadic and hereditary colon cancers. Colon cancer can include malignant colon neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Colon cancer can include adenocarcinoma, squamous cell carcinoma, and adenosquamous cell carcinoma. Colon cancer can be associated with a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis. Colon cancer can be caused by a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome, and juvenile polyposis.


Cell proliferative disorders of the colon can also include colon cancer, precancerous conditions of the colon, adenomatous polyps of the colon and metachronous lesions of the colon. A cell proliferative disorder of the colon can include adenoma. Cell proliferative disorders of the colon can be characterized by hyperplasia, metaplasia, and dysplasia of the colon. Prior colon diseases that may predispose individuals to development of cell proliferative disorders of the colon can include prior colon cancer. Current disease that may predispose individuals to development of cell proliferative disorders of the colon can include Crohn's disease and ulcerative colitis. A cell proliferative disorder of the colon can be associated with a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC. An individual can have an elevated risk of developing a cell proliferative disorder of the colon due to the presence of a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.


A “cell proliferative disorder of the pancreas” is a cell proliferative disorder involving cells of the pancreas. Compounds and compositions of the present application may be used to treat pancreatic cancer or cell proliferative disorders of the pancreas. Cell proliferative disorders of the pancreas can include all forms of cell proliferative disorders affecting pancreatic cells. Cell proliferative disorders of the pancreas can include pancreas cancer, a precancer or precancerous condition of the pancreas, hyperplasia of the pancreas, and dysaplasia of the pancreas, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, and metastatic lesions in tissue and organs in the body other than the pancreas. Pancreatic cancer includes all forms of cancer of the pancreas. Pancreatic cancer can include ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary neoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serous cystadenoma. Pancreatic cancer can also include pancreatic neoplasms having histologic and ultrastructual heterogeneity (e.g., mixed cell types).


A “cell proliferative disorder of the prostate” is a cell proliferative disorder involving cells of the prostate. Compounds and compositions of the present application may be used to treat prostate cancer or cell proliferative disorders of the prostate. Cell proliferative disorders of the prostate can include all forms of cell proliferative disorders affecting prostate cells. Cell proliferative disorders of the prostate can include prostate cancer, a precancer or precancerous condition of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, and metastatic lesions in tissue and organs in the body other than the prostate. Cell proliferative disorders of the prostate can include hyperplasia, metaplasia, and dysplasia of the prostate.


A “cell proliferative disorder of the skin” is a cell proliferative disorder involving cells of the skin. Compounds and compositions of the present application may be used to treat skin cancer or cell proliferative disorders of the skin. Cell proliferative disorders of the skin can include all forms of cell proliferative disorders affecting skin cells. Cell proliferative disorders of the skin can include a precancer or precancerous condition of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma and other malignant growths or lesions of the skin, and metastatic lesions in tissue and organs in the body other than the skin. Cell proliferative disorders of the skin can include hyperplasia, metaplasia, and dysplasia of the skin.


A “cell proliferative disorder of the ovary” is a cell proliferative disorder involving cells of the ovary. Compounds and compositions of the present application may be used to treat ovarian cancer or cell proliferative disorders of the ovary. Cell proliferative disorders of the ovary can include all forms of cell proliferative disorders affecting cells of the ovary. Cell proliferative disorders of the ovary can include a precancer or precancerous condition of the ovary, benign growths or lesions of the ovary, ovarian cancer, malignant growths or lesions of the ovary, and metastatic lesions in tissue and organs in the body other than the ovary. Cell proliferative disorders of the skin can include hyperplasia, metaplasia, and dysplasia of cells of the ovary.


A “cell proliferative disorder of the breast” is a cell proliferative disorder involving cells of the breast. Compounds and compositions of the present application may be used to treat breast cancer or cell proliferative disorders of the breast. Cell proliferative disorders of the breast can include all forms of cell proliferative disorders affecting breast cells. Cell proliferative disorders of the breast can include breast cancer, a precancer or precancerous condition of the breast, benign growths or lesions of the breast, and malignant growths or lesions of the breast, and metastatic lesions in tissue and organs in the body other than the breast. Cell proliferative disorders of the breast can include hyperplasia, metaplasia, and dysplasia of the breast.


In one embodiment, the disease or disorder includes, but is not limited to, a disease or disorders caused by or associated with Entamoeba histolytica, Pneumocystis carinii, Trypnosoma cruzi, Tryanosomo brucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus, foot-and-mouth disease virus, or Crithidia fasciculata, as well as disease or disorder associated with osteoporosis, autoimmunity, schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, or amyotrophy.


Additional examples of the diseases or disorders include, but are not limited to, diseases or disorders caused by or associated with veterinary and human pathogenic protozoa-intracellular active parasites of the phylum Apicomplexa or Sarcomastigophora, Trypanosora, Plasmodia, Leishmania, Babesia and Theileria, Cryptosporidia, Sacrocystida, Amoeba, Coccidia, and Trichononadia. For example, the diseases or disorders include, but are not limited to, Malaria tropica caused by, for example, Plasmodium falciparum; Malaria tertian, caused by Plasmodium vivax or Plasmodium ovale, Malaria quartana, caused by Plasmodium malariae Toxoplasmosis, caused by Toxoplasmium ovale; Coccidiosis, caused for instance by Isospora belli; intestinal Sarcosporidiosis, caused by Sarcocysis suihominis; dysentery caused by Entamoeba histolytica; Cryptosporidiosis, caused by Cryposporidium parvum; Chagas' disease, caused by Trypanosonia cruzi; sleeping sickness, caused by Trypanosoma brucei rhoaesiense or gambiense, the cutaneous and visceral as well as other forms of Leishmaniosis; diseases or disorders caused by veterinary pathogenic protozoa, such as Theileria parva, the pathogen causing bovine East coast fever, Trypanosoma congolense congolense or Typanosomia vivax vivax, Tryanosoma brucei brucei, pathogens causing Nagana cattle disease in Africa, T-Trypanosoma brucei evansi causing Surra, Babesia bagemina, the pathogen causing Texas fever in cattle and buffalos, Babesia bovis, the pathogen causing European bovine Babesiosis as well as Babesiosis in dogs, cats and sheep, Sarcocystis ovicanis and ovifelis pathogens causing Sarcocystiosis in sheep, cattle and pigs, Cryptosporidia, pathogens causing Cryptosporidioses in cattle and birds, Eimeria and Iospora species, pathogens causing Coccidiosis in rabbits, cattle, sheep, goats, pigs and birds, especially in chickens and turkeys. Rickettsia comprise species such as Rickettsia felis, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rickettsia conorii, Rickettsia africae and cause diseases such as typhus, rickettsial pox, Boutonneuse fever. African Tick Bite Fever, Rocky Mountain spotted fever, Australian Tick Typhus, Flinders Island Spotted Fever and Queensland Tick Typhus.


In one embodiment, the disease or disorder is caused by, or associated with, one or more bacteria. Examples of the bacteria include, but are not limited to, the Gram positive organisms (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and E. faecium, Streptococcus pneumonia) and the Gram negative organisms (e.g., Pseudomonas aeruginosa, Burkholdia cepacia, Xanthomonas maltophilia, Escherichia coli, Enterobacter spp, Klebsiella pneumoniae and Salmonella spp).


In one embodiment, the disease or disorder is caused by, or associated with, one or more fungi. Examples of the fungi include, but are not limited to, Candida albicans, Histoplasma neoformans, Coccidioides immitis, and Penicillium marneffei.


In one embodiment, the disease or disorder is a neurological disease or disorder. In one embodiment, the neurological disease or disorder involves the central nervous system (e.g., brain, brainstem and cerebellum), the peripheral nervous system (e.g., cranial nerves) and/or the autononic nervous system (e.g. parts of which are located in both central and peripheral nervous system).


Examples of the neurological disorders include, but are not limited to, acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; age-related macular degeneration; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers' disease; alternating hemiplegia; Alzheimer's disease; Vascular dementia; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Anronl-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia telegiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet's disease; Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme) spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome; causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy; chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's syndrome; cytomegalic inclusion body disease; cytomegalovirus infection; dancing eyes-dancing feet syndrome; Dandy-Walker syndrome; Dawson disease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich's ataxia; fronto-temporal dementia and other “tauopathies”; Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; heredopathia atactica polyneuritiformis herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associated dementia and neuropathy (also neurological manifestations of AIDS); holoprosencephaly; Huntington's disease and other polyglutamine repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis inclusion body myositis; incontinentia pigment; infantile phytanic acid storage disease; infantile refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease Kinsbourne syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kieffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh's disease; Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; Lissencephaly locked-in syndrome; Lou Gehrig's disease (i.e. motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease—neurological sequelae; Machado-Joseph disease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-strokes; nitochondrial nopathies; Mobius syndrome; monomelic anyotrophy; motor neuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with postural hypotension; p muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis myoclonic encephalopathy of infants; myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism sequence; Ohtabara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesia; Parkinson's disease; paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorders; photic sneeze reflex; phytanic acid storage disease; Pick's disease; pinched nerve; pituitary tumors; polymyositis; porencephaly; post-polio syndrome; postherpetic neuralgia; postinfectious encephalomyelitis; postural hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion diseases; progressive hemifacial atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types I and II); Rasmussen's encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; Saint Vitus dance; Sandhoff disease; Schilder's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome; Sjögren's syndrome; sleep apnea; Soto's syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome; Wildon's disease; and Zellweger syndrome.


Examples of neurodegenerative diseases may also include, without limitation, Adrenoleukodystrophy (ALD), Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease), Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, and Toxic encephalopathy.


In one embodiment, the disease or disorder is an autoimmune disease. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases (IBDs) comprising Crohn disease (CD), and ulcerative colitis (UC) which are chronic inflamatory conditions with polygenic susceptibility.


In one embodiment, the disease or disorder is inflammation, arthritis, rheumatoid arthritis, spondyiarthropathies, gouty arthritis, osteoarthritis, juvenile arthritis, and other arthritic conditions, systemic lupus erthematosus (SLE), skin-related conditions, psoriasis, eczema, burns, dermatitis, neuroinflammation, allergy, pain, neuropathic pain, fever, pulmonary disorders, lung inflammation, adult respiratory distress syndrome, pulmonary sarcoisosis, asthma, silicosis, chronic pulmonary inflammatory disease, and chronic obstructive pulmonary disease (COPD), cardiovascular disease, arteriosclerosis, myocardial infarction (including post-myocardial infarction indications), thrombosis, congestive heart failure, cardiac reperfusion injury, as well as complications associated with hypertension and/or heart failure such as vascular organ damage, restenosis, cardiomyopathy, stroke including ischemic and hemorrhagic stroke, reperfusion injury, renal reperfusion injury, ischemia including stroke and brain ischemia, and ischemia resulting from cardiac/coronary bypass, neurodegenerative disorders, liver disease and nephritis, gastrointestinal conditions, inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis, ulcerative diseases, gastric ulcers, viral and bacterial infections, sepsis, septic shock, gram negative sepsis, malaria, meningitis, HIV infection, opportunistic infections, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), pneumonia, herpes virus, myalgias due to infection, influenza, autoimmune disease, graft vs. host reaction and allograft rejections, treatment of bone resorption diseases, osteoporosis, multiple sclerosis, cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bone cancer, epithelial call-derived neoplasia (epithelial carcinoma), basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer, stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous cell and/or basal cell cancers, prostate cancer, renal cell carcinoma, and other known cancers that affect epithelial cells throughout the body, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL), angiogenesis including neoplasia, metastasis, central nervous system disorders, central nervous system disorders having an inflammatory or apoptotic component, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, peripheral neuropathy, or B-Cell Lymphoma.


In one embodiment, the disease or disorder is selected from autoimmune diseases, inflammatory diseases, proliferative and hyperproliferative diseases, immunologically-mediated diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, hormone related diseases, allergies, asthma, and Alzheimer's disease. In one embodiment, the disease or disorder is selected from a proliferative disorder and an immune disorder.


As modulators of a STING protein, the compounds and compositions of this application are also useful in assessing, studying, or testing biological samples. One aspect of the application relates to modulating the activity of a STING protein in a biological sample, comprising contacting the biological sample with a compound or a composition of the application.


The term “biological sample”, as used herein, means an in vitro or an ex vivo sample, including, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof, and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof. Modulation (e.g., inhibition or stimulation) of protein kinase activity in a biological sample is useful for a variety of purposes that are known to one of skill in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ transplantation, and biological specimen storage.


Another aspect of this application relates to the study of a STING protein in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by STING protein. Examples of such uses include, but are not limited to, biological assays such as enzyme assays and cell-based assays.


The activity of the compounds and compositions of the present application as STING modulators may be assayed in vitro, in vivo, or in a cell line. In vitro assays include assays that determine modulation (e.g., inhibition or stimulation) of binding of a STING ligand to a STING protein through competitive binding assay. Alternate in vitro assays quantitate the ability of the modulator (e.g., inhibitor or stimulator) to bind to the protein kinase and may be measured either by radio labelling the modulator (e.g., inhibitor or stimulator) prior to binding, isolating the ligand/protein complex and determining the amount of radio label bound. Detailed conditions for assaying a compound utilized in this application as a modulator or a STING protein are set forth in the Examples below.


In accordance with the foregoing, the present application provides a method for preventing or treating any of the diseases or disorders described herein in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of the application or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the application. For any of the above uses, the required dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired.


Compounds and compositions of the application can be administered in therapeutically effective amounts in a combinational therapy with one or more therapeutic agents (pharmaceutical combinations) or modalities, e.g., anti-proliferative, anti-cancer, immunomodulatory (e.g., CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents), anti-inflammatory, and/or anti-viral agent, and/or non-drug therapies, etc. For example, synergistic effects can occur with anti-proliferative, anti-cancer, immunomodulatory (e.g., CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents), anti-inflammatory, and/or anti-viral substances. Where the compounds of the application are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.


Combination therapy may include the administration of the subject compounds in further combination with one or more other biologically active ingredients (such as, but not limited to, a second STING modulator, a modulator of the cGAS-CDN-STING axis, or a modulator involved in the intracellular dsDNA mediated type-1 interferon activation. Other biologically active ingredients may also include anti-proliferative agents, anti-cancer agents (e.g., chemotherapeutic agents), immunomodulatory (e.g., CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents) agents, antibodies, lipids, liposomes, peptides, etc. For instance, the compounds of the application can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the application. The compounds of the application can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy or treatment modality. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.


In one embodiment, the chemotherapeutic agent is an alkylating agent; an antibiotic; an anti-metabolite; a detoxifying agent; an interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; an MTOR inhibitor; a multi-kinase inhibitor; a serine/threonine kinase inhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule targeting drug, a topoisomerase poison drug, an inhibitor of a molecular target or enzyme (e.g., a kinase inhibitor), a cytidine analogue drug, or any chemotherapeutic, anti-neoplastic or anti-proliferative agent listed in www.cancer.org/docroot/cdg/cdg_0.asp.


Alkylating agents are non-phase specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxy, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide (e.g., CYTOXAN®), melphalan (e.g., ALKERAN®), and chlorambucil (e.g., LEUKERAN®); alkyl sulfonates such as busulfan (e.g., MYLERAN®); nitrosoureas such as carmustine (e.g., BiCNU®); and triazenes such as dacarbazine (e.g., DTIC-Dome®).


Exemplary alkylating agents also include, but are not limited to, busulfan (Busulfex), lomustine (CeeNU), oxaliplatin (Eloxatin), carmustine (Gliadel), ifosfamide (Ifex), mechlorethamine (Mustargen), busulfan (Myleran), carboplatin (PARAPLATIN®), cisplatin (CDDP, PLATINOL®), temozolomide (Temodar), thiotepa (Thioplex), bendamustine (Treanda), streptozocin (Zanosar), 5-azacytidine (e.g., VIDAZA), decitabine (e.g., DECOGEN), temozolomide (e.g., TEMODAR and TEMODAL), dactinomycin (also known as actinomycin-D and sold under the tradename COSMEGEN), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, sold under the tradename ALKERAN), altretamine (also known as hexamethylmelamine (HMM), sold under the tradename HEXALEN), carmustine (e.g., BCNU), bendamustine (e.g., TREANDA), carboplatin (e.g., PARAPLATIN®), lomustine (also known as CCNU, sold under the tradename CEENU®), cisplatin (also known as CDDP, sold under the tradenames PLATINOL® and PLATINOL®-AQ), cyclophosphamide (sold under the tradenames CYTOXAN® and NEOSAR®), dacarbazine (also known as DTIC, DIC and imidazole carboxamide, sold under the tradename DTIC-DOME®), altretamine (also known as hexamethylmelamine (HMM) sold under the tradename HEXALEN®), ifosfamide (e.g., IFEX®), procarbazine (e.g., MATULANE®), mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, sold under the tradename MUSTARGEN®), streptozocin (e.g., ZANOSAR®), and thiotepa (also known as thiophosphoamide, TESPA and TSPA, and sold under the tradename THIOPLEX®).


Antibiotic anti-neoplastics are non-phase specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death. Examples of antibiotic anti-neoplastic agents include, but are not limited to, actinomycins such as dactinomycin (e.g., COSMEGEN®), anthracyclines such as daunorubicin (e.g., as a liposomal injectable form as DAUNOXOME® or as an injectable as CERUBIDINE®) and doxorubicin (e.g., RUBEX® or ADRIAMYCIN RDF®), and bleomycins (e.g., BLENOXANE®).


Exemplary antibiotics also include, but are not limited to, doxorubicin (Adriamycin), doxorubicin liposomal (Doxil), mitoxantrone (Novantrone), bleomycin (Blenoxane), daunorubicin (Cerubidine), daunorubicin liposomal (DaunoXome), dactinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), plicamycin (Mithracin), mitomycin (Mutamycin), pentostatin (Nipent), valrubicin (Valstar), doxorubicin (e.g., ADRIAMYCIN® and RUBEX®), bleomycin (e.g., LENOXANE®), daunorubicin (also known as dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, sold under the tradename CERUBIDINE®), daunorubicin liposomal (daunorubicin citrate liposome, sold under the tradename DAUNOXOME), mitoxantrone (also known as DHAD, sold under the tradename NOVANTRONE®), epirubicin (e.g., ELLENCE™), idarubicin (e.g., IDAMYCIN®, IDAMYCIN PFS®), and mitomycin C (e.g., MUTAMYCIN®).


Anti-metabolite anti-neoplastic agents are phase specific agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows. Examples of antimetabolite anti-neoplastic agents include, but are not limited to, fluorouracil and analogs thereof (e.g., 5-fluoro deoxyuridine (floxuridine), 5-fluorodeoxyuridine monophosphate methotrexate), cytarabine (commonly known as Ara-C, available as CYTOSAR-U®) and analogs thereof (e.g., azacytidine, 2′,2′-difluorodeoxycytidine (gemcitabine)), mercaptopurine (e.g., PURINETHOL®) and analogs thereof (e.g., azathioprine), thioguanine (e.g., TABLOID®) and analogs thereof (e.g., pentostatin, erythrohydroxy-nonyladenine (EHNA), fludarabine phosphate, and cladribine), gemcitabine (e.g., GEMZAR®), and methotrexate.


Exemplary anti-metabolites also include, but are not limited to, fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fludara), nelarabine (Arranon), cladribine (Cladribine Novaplus), clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposomal (DepoCyt), hydroxyurea (Droxia), pralatrexate (Folotyn), floxuridine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX, Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1 or cytarabine (Tarabine PFS), claribine (2-chlorodeoxyadenosine, sold under the tradename LEUSTATIN®), 5-fluorouracil (sold under the tradename ADRUCIL®), 6-thioguanine (sold under the tradename PURINETHOL®), pemetrexed (sold under the tradename ALIMTA®), cytarabine (also known as arabinosylcytosine (Ara-C), sold under the tradename CYTOSAR-U®), cytarabine liposomal (also known as Liposomal Ara-C, sold under the tradename DEPOCYT™) decitabine (sold under the tradename DACOGEN®), hydroxyurea and (sold under the tradenames HYDREA®, DROXIA™ and MYLOCEL™), fludarabine (sold under the tradename FLUDARA®), floxuridine (sold under the tradename FUDR®), cladribine (also known as 2-chlorodeoxyadenosine (2-CdA) sold under the tradename LEUSTATIN™) methotrexate (also known as amethopterin, methotrexate sodium (MTX), sold under the tradenames RHEUMATREX® and TREXALL™), and pentostatin (sold under the tradename NIPENT®).


Exemplary detoxifying agents include, but are not limited to, amifostine (Ethyol), and mesna (Mesnex).


Exemplary interferons include, but are not limited to, interferon alfa-2b (Intron A), and interferon alfa-2a (Roferon-A).


Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin), ofatumumab (Arzerra), bevacizumab (Avastin), rituximab (Rituxan), cetuximab (Erbitux), panitumumab (Vectibix), tositumomab/iodine131 tositumomab (Bexxar), alemtuzumab (Campath), ibritumomab (Zevalin, In-111, Y-90 Zevalin), gemtuzumab (Mylotarg), eculizumab (Soliris), and ordenosumab.


Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa), lapatinib (Tykerb), cetuximab (Erbitux), erlotinib (Tarceva), panitumumab (Vectibix), PKI-166, canertinib (CI-1033), matuzumab (Emd7200), and EKB-569.


Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb), and AC-480.


Exemplary histone deacetylase Inhibitors include, but are not limited to, vorinostat (Zolinza).


Hormones and hormonal analogues are useful compounds for treating cancers in which there is a relationship between the hormone(s) and growth and/or lack of growth of the cancer. Examples of hormones and hormonal analogues useful in cancer treatment include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrozole, vorozole, and exemestane; progestins such as megestrol acetate; estrogens, and anti-estrogens such as fulvestrant, flutamide, nilutamide, bicalutamide, cyproterone acetate and 5-reductases such as finasteride and dutasteride; anti-estrogens such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, as well as selective estrogen receptor modulators (SERMS) such those described in U.S. Pat. Nos. 5,681,835, 5,877,219, and 6,207,716; and gonadotropin-releasing hormone (GnRH) and analogues thereof; and LHRH agonists and antagonists such as goserelin acetate and luprolide.


Exemplary hormones also include, but are not limited to, tamoxifen (Soltamox, Nolvadex), raloxifene (Evista), megestrol (Megace), leuprolide (Lupron, Lupron Depot, Eligard, Viadur), fulvestrant (Faslodex), letrozole (Femara), triptorelin (Trelstar LA, Trelstar Depot), exemestane (Aromasin), goserelin (Zoladex), bicalutamide (Casodex), anastrozole (Arimidex), fluoxymesterone (Androxy, Halotestin), medroxyprogesterone (Provera, Depo-Provera), estramustine (Emcyt), flutamide (Eulexin), toremifene (Fareston), degarelix (Firmagon), nilutamide (Nilandron), abarelix (Plenaxis), or testolactone (Teslac).


Anti-microtubule or anti-mitotic agents or mitotic inhibitors are phase specific agents active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle. Examples of anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids. Examples of diterpenoids include, but are not limited to, paclitaxel (e.g., TAXOL®) and its analog docetaxel (e.g., TAXOTERE®). Examples of vinca alkaloids include, but are not limited to, vinblastine (e.g., VELBAN®), vincristine (e.g., ONCOVIN®), and vinorelbine (e.g., NAVELBINE®).


Exemplary mitotic inhibitors also include, but are not limited to, paclitaxel (Taxol, Onxol, Abraxane), docetaxel (Taxotere), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etoposide (Toposar, Etopophos, VePesid), teniposide (Vumon), ixabepilone (Ixempra), nocodazole, epothilone, vinorelbine (Navelbine), camptothecin (CPT), irinotecan (Camptosar), topotecan (Hycamtin), amsacrine, and lamellarin D (LAM-D).


Exemplary MTOR inhibitors also include, but are not limited to, everolimus (Afinitor), temsirolimus (Torisel), rapamune, ridaforolimus, and AP23573.


Exemplary multi-kinase inhibitors include, but are not limited to, sorafenib (Nexavar), sunitinib (Sutent), BIBW 2992, E7080, Zd6474, PKC-412, motesanib, and AP24534.


Exemplary serine/threonine kinase inhibitors include, but are not limited to, ruboxistaurin, eril/easudil hydrochloride, flavopiridol, seliciclib (CYC202, Roscovitrine), SNS-032 (BMS-387032), Pkc412, bryostatin, KAI-9803, SF1126, VX-680, Azd1152, Arry-142886 (AZD-6244), SCIO-469, GW681323, CC-401, CEP-1347, and PD 332991.


Inhibitors of serine/threonine kinases also include MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracellular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta), IkB kinase family (IKKa, IKKb), PKB family kinases, AKT kinase family members, and TGF beta receptor kinases. Inhibitors of serine/threonine kinases are also described in J. Biochem. 126, 799 (1999), Biochem. Pharmacol. 60, 1101 (2000); Cancer Surveys 27, 41 (1996); Cancer Treatment Res. 78, 3 (1995); Bioorg. Med. Chem. Letters 10, 223 (2000); U.S. Pat. No. 6,268,391; and Int. J. Cancer 88, 44 (2000).


Tyrosine kinases (also nonreceptor tyrosine kinases) include, but are not limited to, cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Inhibitors of non-receptor tyrosine kinase are described in J. Hematotherapy Stem Cell Res. 8, 465 (1999) and Annual Rev. Immunol. 15, 371 (1997).


Exemplary tyrosine kinase inhibitors also include, but are not limited to, erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), trastuzumab (Herceptin), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), everolimus (Afinitor), alemtuzumab (Campath), gemtuzumab (Mylotarg), temsirolimus (Torisel), pazopanib (Votrient), dasatinib (Sprycel), nilotinib (Tasigna), vatalanib (Ptk787, ZK222584), CEP-701, SU5614, MLN518, XL999, VX-322, Azd0530, BMS-354825, SKI-606 CP-690, AG-490, WHI-P154, WHI-P131, AC-220, and AMG888.


Exemplary VEGF/VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, and vandetinib, axitinib, brivanib alaninate ((S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2, 1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate, also known as BMS-582664), motesanib (TST-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide), and pasireotide (also known as SO 230).


Inhibitors of phosphotidyl inositol-3 kinase family members including blockers of Pekinese, ATM, DNA-PK, and Ku may also be combined with the compounds of the present application. Such inhibitors are discussed in Curr. Opinion Immunol. 8, 412 (1996); Oncogene 17, 3301 (1998); Int. J Biochem. Cell Biol. 29, 935 (1997); and Cancer Res. 60, 1541 (2000).


Exemplary kinase inhibitors also include, but are not limited to, Bevacizumab (targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux (targets Erb1), Imatinib/Gleevic (targets Bcr-Abl), Trastuzumab (targets Erb2), Gefitinib/Iressa (targets EGFR), Ranibizumab (targets VEGF), Pegaptanib (targets VEGF), Erlotinib/Tarceva (targets Erb1), Nilotinib (targets Bcr-Abl), Lapatinib (targets Erb1 and Erb2/Her2), GW-572016/lapatinib ditosylate (targets HER2/Erb2), Panitumumab/Vectibix (targets EGFR), Vandetinib (targets RET/VEGFR), E7080 (multiple targets including RET and VEGFR), Herceptin (targets HER2/Erb2), PKI-166 (targets EGFR), Canertinib/CI-1033 (targets EGFR), Sunitinib/SU-11464/Sutent (targets EGFR and FLT3), Matuzumab/Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3), Vatalanib/Ptk787/ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targets JAK), sorafenib/Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-B, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/ABL and Src), AC-220 (targets Flt3), AC-480 (targets all HER proteins, “panHER”), Motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), Denosumab (targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).


Exemplary microtubule targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine.


Exemplary topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.


Additional topoisomerase poison drugs include topoisomerase II inhibitors, such as epipodophyllotoxins. Examples of epipodophyllotoxins include, but are not limited to, etoposide (VP-16, VePESID®) and teniposide (VM-26, VUMON®).


Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.


Antibody antagonists to receptor kinase ligand binding may also serve as inhibitors. Examples include Imclone C225 EGFR specific antibody (see Cancer Treat. Rev. 26, 269 (2000)), Herceptin® erbB2 antibody (see Breast Cancer Res. 2, 176 (2000)), and 2CB VEGFR2 specific antibody (see Cancer Res. 60, 5117 (2000)).


Anti-angiogenic therapeutic agents including non-receptor MEK angiogenesis inhibitors may also be combined with the compounds of the present application. Anti-angiogenic agents such as those which inhibit the effects of vascular endothelial growth factor (for example, bevacizumab [Avastin™]) and compounds that work by other mechanisms (for example, linomide, inhibitors of integrin ανβ3 function, endostatin and angiostatin).


Additional therapeutic agents which may be combined with the compounds of the present application also include SH2/SH3 domain blockers that disrupt SH2 or SH3 domain binding in a variety of enzymes or adaptor proteins including, PI3-K p85 subunit, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2), and Ras-GAP. SH2/SH3 domains blockers as anticancer drugs are discussed in J. Pharm. Toxicol. Methods 34, 125 (1995).


Additional therapeutic agents which may be combined with the compounds of the present application also include Myo-inositol signalling inhibitors such as phospholipase C blockers and Myoinositol analogues. Such signal inhibitors are described in New Molecular Targets for Cancer Chemotherapy ed., Paul Workman and David Kerr, CRC press 1994, London.


Additional therapeutic agents which may be combined with the compounds of the present application also include inhibitors of Ras oncogene, including inhibitors of farnesyltransferase, geranyl-geranyl transferase, and CAAX proteases, as well as anti-sense oligonucleotides, ribozymes and immunotherapy. Such inhibitors are discussed in J Biomed. Sci. 7, 292 (2000); Curr. Opin. Lipidology 9, 99 (1998); and BioChim. Biophys. Acta, 1423, 19 (1989).


Additional exemplary general chemotherapeutic, anti-neoplastic, or anti-proliferative agents which may be combined with the compounds of the present application also include, but are not limited to, altretamine (Hexalen), isotretinoin (Accutane, Amnesteem, Claravis, Sotret), tretinoin (Vesanoid), azacitidine (Vidaza), bortezomib (Velcade) asparaginase (Elspar), levamisole (Ergamisol), mitotane (Lysodren), procarbazine (Matulane), pegaspargase (Oncaspar), denileukin diftitox (Ontak), porfimer (Photofrin), aldesleukin (Proleukin), lenalidomide (Revlimid), bexarotene (Targretin), thalidomide (Thalomid), temsirolimus (Torisel), arsenic trioxide (Trisenox), verteporfin (Visudyne), mimosine (Leucenol), (1M tegafur—0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 M potassium oxonate), and lovastatin.


Additional therapeutic agents which may be combined with the compounds of the present application also include anti-viral agents including, but not limited to, hepatitis B virus (HBV) inhibitors, hepatitis C virus (HCV) protease inhibitors, HCV polymerase inhibitors, HCV NS4A inhibitors, HCV NS5 A inhibitors, HCV NS5b inhibitors, and human immunodeficiency virus (HIV) inhibitors.


Additional therapeutic agents which may be combined with the compounds of the present application also include antigens or adjuvants including, but not limited to, B7 costimulatory molecule, interleukin-2, interferon-y, GM-CSF, CTLA-4 antagonists, OX-40/OX-40 ligand, CD40/CD40 ligand, sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions. In one embodiment, adjuvants, such as aluminum hydroxide or aluminum phosphate, can be added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. In one embodiment, additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, like CpG, a toll-like receptor (TLR) 9 agonist as well as additional agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9, including lipoprotein, LPS, monophosphoryllipid A, lipoteichoic acid, imiquimod, resiquimod, and in addition retinoic acid-inducible gene I (RIG-I) agonists such as poly I:C, can also be used.


Additional therapeutic agents which may be combined with the compounds of the present application also include cytotoxic agents including, but not limited to, arsenic trioxide (TRISENOX®), asparaginase (also known as L-asparaginase, and Erwinia L-asparaginase, sold under the tradenames ELSPAR® and KIDROLASE®).


Additional therapeutic agents which may be combined with the compounds of the present application also include retinoids including, but not limited to, alitretinoin (sold under the tradename PANRETIN®), tretinoin (all-trans retinoic acid, also known as ATRA, sold under the tradename VESANOID®), Isotretinoin (13-c/s-retinoic acid, sold under the tradenames ACCUTANE®, AMNESTEEM®, CLARAVIS®, CLARUS®, DECUTAN®, ISOTANE®, IZOTECH®, ORATANE®, ISOTRET®, and SOTRET®), and bexarotene (sold under the tradename TARGRETIN®).


Additional exemplary chemotherapeutic agents that may be used in combination with the compounds of present application include, but are limited to, abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-1-Lproline-t-butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3′,4′-didehydro-4′deoxy-8′-norvin-caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea andtaxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, taxanes, nilutamide, nivolumab, onapristone, paclitaxel, pembrolizumab, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine.


Additional therapeutic agents which may be combined with the compounds of the present application also include inhibitors of cell cycle signaling, proapoptotic agents, PARP inhibitors, checkpoint therapeutics, and immune modulators.


Cell cycle signalling inhibitors inhibit molecules involved in the control of the cell cycle. A family of protein kinases called cyclin dependent kinases (CDKs) and their interaction with a family of proteins termed cyclins controls progression through the eukaryotic cell cycle. The coordinate activation and inactivation of different cyclin/CDK complexes is necessary for normal progression through the cell cycle. Several inhibitors of cell cycle signalling are under development. For instance, examples of cyclin dependent kinases, including CDK2, CDK4, and CDK6 and inhibitors for the same are described in Exp. Opin. Ther. Patents 10, 215 (2000).


Therapeutic agents used in proapoptotic regimens (e.g., bcl-2 antisense oligonucleotides) may also be used in combination of the compounds of the present application.


As used herein, PARP inhibitors refer to a group of pharmacological inhibitors of the enzyme poly ADP ribose polymerase (PARP). Exemplary PARP inhibitors include, but are not limited to, Olaparib (AZD-2281, Lynparza® by Astra Zeneca), Rucaparib (PF-01367338, Rubraca® by Clovis Oncology), Niraparib (MK-4827, Zejula® by Tesaro), Talazoparib (BMN-673), Veliparib (ABT-888), Olaparib (AZD-2281), Olaparib TOPARP-A, Rucaparib (PF-01367338, AG014699), CEP 9722, E7016 (developed by Eisai), BGB-2901, Iniparib (BSI 201), and 3-aminobezamide.


Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of the present application. Immunotherapy approaches, including ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell energy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine transfected tumor cell lines, and approaches using anti-idiotypic antibodies.


As used herein, checkpoint inhibitor therapy refers to a form of cancer treatment immunotherapy that targets immune checkpoints, key regulators of the immune system that stimulate or inhibit its actions, which may allow tumors to protect themselves from attacks by the immune system. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function.


As used herein, “immune-modulators” or “immunomodulators” refer to any substance including monoclonal antibodies that affect the immune system. Immunomodulators can be used as anti-neoplastic agents for the treatment of cancer. For example, immune-modulators include, but are not limited to, anti-CTLA-4 antibodies such as ipilimumab (YERVOY) and anti-PD-1 antibodies (Opdivo/nivolumab and Keytruda/pembrolizumab) Other immuno-modulators include, but are not limited to, ICOS antibodies, OX-40 antibodies, PD-Li antibodies. LAG3 antibodies, TIM-3 antibodies, 41BB antibodies, and GITR antibodies.


CLTA-4 and PD-1 pathways are important negative regulators of immune response. Activated T-cells up-regulate CTLA-4, which binds on antigen-presenting cells and inhibits T-cell stimulation, IL-2 gene expression, and T-cell proliferation. PD-1 binds to active T-cells and suppresses T-cell activation. PD-1 antagonists have demonstrated anti-tumor effects. CTLA-4 and PD-1 pathway antagonists that may be used in combination with the compounds of the present application include ipilimumab, tremelimumab, nivolumab, pembrolizumab, CT-011, AMP-224, and MDX-1106.


As used herein, PD-1 inhibitors and PD-L1 inhibitors refer to a group of checkpoint inhibitors or immune checkpoint inhibitors useful in the treatment of cancer. PD1 and PD-L1 are both proteins present on the surface of cells. PD-1 and PD-L1 inhibitors act to inhibit the association of the programmed death-ligand (PD-L1) with its receptor, programmed cell death protein 1 (PD-1). Exemplary PD-1 and/or PD-L1 inhibitors include, but are not limited to Nivolumab (Opdivo), Pembrolizumab (MK-3475 or lambrolizumab, Keytruda), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), pidilizumab, AMP-224, AMP-514, PDR001, cemiplimab, BMS-936559, and CK-301.


Anti-PD-L1 antibodies and methods of making the same are known in the art. Such antibodies to PD-L1 may be polyclonal or monoclonal, and/or recombinant, and/or humanized. Exemplary PD-L1 antibodies are disclosed in U.S. Pat. Nos. 8,217,149, 8,383,796, 8,552,154, 9,212,224, and 8,779,108, and US Patent Appln. Pub. Nos. 20110280877, 20140341902, and 20130045201. Additional exemplary antibodies to PD-L1 (also referred to as CD274 or B7-H1) and methods for use are disclosed in U.S. Pat. Nos. 7,943,743, 8,168,179, and 7,595,048; WO2014055897, WO2016007235; and US Patent Appln. Pub. Nos. 20130034559 and 20150274835. In one embodiment, the anti-PD-L1 antibody is BMS-936559 (MDX-1105), MPDL3280A (RG7446), MEDI4736, TECENTRIQ™ (atezolizumab), YW243.55.570, MPDL3280A, BMS-936559, MEDI4736, or MSB0010718C, or an antibody that comprises the VH and VL described in WO2013019906 (e.g., SEQ ID NOs: 21 and 24 therein). Examples of anti-PD-L1 antibodies and methods for making thereof are also described in WO 2010077634, WO 2007005874, WO 2011066389, WO 2013019906, WO 2010077634, U.S. Pat. Nos. 8,217,149 and 8,383,796, and US Patent Appln. Pub. No. 2013034559.


PD-1 antagonists or PD-1 inhibitors refer to any chemical compound or biological molecule that blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-Li; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. Human PD-1 amino acid sequences can be found in NCBI Locus No.: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Locus No.: NP_054862 and NP_079515, respectively.


PD-1 antagonists include a monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody or a chimeric antibody, and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab′-SH, F(ab′)2, scFv and Fv fragments.


Examples of mAbs that bind to human PD-1 are described in U.S. Pat. Nos. 7,488,802, 7,521,051, 8,008,449, 8,354,509, and 8,168,757, WO 2004004771, WO 2004072286, WO 2004056875, and US Patent Appln. Pub. No. 20110271358. In one embodiment, anti-human PD-1 mAbs useful as the PD-1 antagonists include: MK-3475, nivolumab, the humanized antibodies h409All, h409A16 and h409A17, which are described in WO 2008156712, and AMP-514.


Other PD-1 antagonists useful in the any of the aspects and embodiments of the present application include an immunoadhesin that specifically binds to PD-1, and preferably specifically binds to human PD-1, e.g., a fusion protein containing the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region such as an Fc region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in WO 2010027827 and WO 2011066342. In one embodiment, the PD-1 antagonists include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to human PD-1.


In one embodiment, the anti-PD-1 antibody is KEYTRUDA/pembrolizumab, disclosed in U.S. Pat. No. 8,168,757 or Opdivo/nivolumab (also known as BMS-936558, MDX-1106, and ONO-4538, disclosed in U.S. Pat. No. 8,008,449.


In one embodiment, the CTLA-4 antagonist is Yervoy (ipilimumab), described in U.S. Pat. Nos. 6,984,720 and 7,605,238.


Additional examples of other therapeutic agents (anti-neoplastic agent) for use in combination or co-administration with a compound of the present application include antibodies to ICOS. Agonist antibodies to ICOS or ICOS binding proteins are disclosed in WO 2012013004, WO 2014033327, WO 2016120789, US Patent Appln. Pub. Nos. 20160215059 and US20160304610, for example, SEQ ID NOs: 1-6 or a direct equivalent thereof disclosed in WO 2016120789, a VH domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 7 as disclosed in WO 2016120789, and/or a VL domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 8 as disclosed in WO 2016120789.


Additional examples of other therapeutic agents (anti-neoplastic agent) for use in combination or co-administration with a compound of the present application include antibodies to OX040. Such antibodies are described in WO 2012027328, WO 2013028231 (e.g., an antibody comprising a VL having a sequence at least 90% identical to SEQ ID NO: 10 and/or a VH having a sequence at least 90% identical to SEQ ID NO:4 therein) Additional examples of other therapeutic agents for use in combination or co-administered with a compound of the present application include immunostimulatory agents.


As used herein immunostimulatory agent refers to any agent that can stimulate the immune system. As used herein immunostimulatory agents include, but are not limited to, vaccine adjuvants, such as Toll-like receptor agonists, T-cell checkpoint blockers, such as mAbs to PD-1 and CTL4 and T-cell checkpoint agonist, such as agonist mAbs to OX-40 and ICOS.


As used herein “immunostimulatory agent” refers to any agent that can stimulate the immune system. As used herein immunostimulatory agents include, but are not limited to, vaccine adjuvants.


In one embodiment, TLR agonists include, but are not limited to, Pam3Cys, a TLR1/2 agonist; CFA, a TLR2 agonist; MALP2, a TLR2 agonist; Pam2Cys, a TLR2 agonist; FSL-I, a TLR-2 agonist; Hib-OMPC, a TLR-2 agonist; polyinosinic:poly cytidylic acid (Poly I:C), a TLR3 agonist; polyadenosine-polyuridylic acid (poly AU), a TLR3 agonist; Polyinosinic-Polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Hiltonol), a TLR3 agonist; bacterial flagellin a TLR5 agonist; imiquimod, a TLR7 agonist; resiquimod, a TLR7/8 agonist; loxoribine, a TLR7/8 agonist; and unmethylated CpG dinucleotide (CpG-ODN), a TLR9 agonist. Additional TLR agonists include, but are not limited to aminoalkyl glucosaminide phosphates (AGPs). An example of a naturally occurring TLR4 agonist is bacterial LPS. An example of a semisynthetic TLR4 agonist is monophosphoryl lipid A (MPL). Additional AGP derivatives are disclosed in U.S. Pat. Nos. 7,129,219, 6,525,028, and 6,911,434.


In one embodiment, the immunostimulatory agent for use in combination with the compounds of the present application is a TLR4 agonist. In one embodiment, the TLR4 agonist is CRX-601, CRX-527, or CRX 547. Other embodiments include AGPs such as CRX 602 or CRX 526.


In addition, the compounds of the present application may be combined with other therapeutic agents which, because of their adjuvant nature, can act to stimulate the immune system to respond to the cancer antigens present on the inactivated tumor cell(s). Such adjuvants include, but are not limited to, lipids, liposomes, inactivated bacteria which induce innate immunity (e.g., inactivated or attenuated Listeriamonocytogenes), compositions which mediate innate immune activation via, (NOD)-like receptors (NLRs), Retinoic acid inducible gene-based (RIG)-I-like receptors (RLRs), and/or C-type lectin receptors (CLRs). Examples of PAMPs include lipoproteins, lipopolypeptides, peptidoglycans, zymosan, lipopolysaccharide, neisserial porins, flagellin, profillin, galactoceramide, muramyl dipeptide. Peptidoglycans, lipoproteins, and lipoteichoic acids are cell wall components of Gram-positive. Lipopolysaccharides are expressed by most bacteria, with MPL being one example.


Flagellin refers to the structural component of bacterial flagella that is secreted by pathogenic and commensal bacteria, rt-Galactosylceramide (rt.-GalCer) is an activator of natural killer T (NKT) cells. Muramyl dipeptide is a bioactive peptidoglycan motif common to all bacteria.


Additional examples of other therapeutic agents for use in combination or co-administered with a compound of the present application include IDO inhibitors (e.g., Epacadostat, as disclosed in U.S. Pat. No. 8,034,953), CD73 inhibitors, and A2a and A2b adenosine antagonists.


In one embodiment, the compounds may be administered in combination with one or more separate pharmaceutical agents, e.g., a chemotherapeutic agent, an immunotherapeutic agent, or an adjunctive therapeutic agent.


The compounds of the present application may be used in combination with at least one other therapeutic agent useful in the prevention or treatment of bacterial and viral infections. Examples of such agents include, without limitation: polymerase inhibitors such as those disclosed in WO 2004037818 and WO 2006045613; JTK-003, JTK-019, NM-283, HCV-796, R-803, R1728, R1626, as well as those disclosed in WO 2006018725, WO 2004074270, WO 2003095441, US Appl. Pub. No. 20050176701, WO 2006020082, WO 2005080388, WO 2004064925, WO 2004065367, WO 2003007945, WO 2002004425, WO 2005014543, WO 2003000254, EP 1065213, WO 2001047883, WO 2002057287, WO 2002057245; replication inhibitors such as acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine, and similar agents; protease inhibitors such as the HIV protease inhibitors saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir, brecanavir, atazanavir, tipranavir, palinavir, lasinavir, and the HCV protease inhibitors BILN2061, VX-950, SCH503034, and similar agents; nucleoside and nucleotide reverse transcriptase inhibitors such as zidovudine, didanosine, lamivudine, zalcitabine, abacavir, stavudine, adefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine, alovudine, amdoxovir, elvucitabine, tenofovir disproxil fumarate, tenofovir alafenamide fumarate/hemifumarate, and similar agents; non-nucleoside reverse transcriptase inhibitors (including an agent having anti-oxidation activity such as immunocal, oltipraz etc.) such as nevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz, capravirine, TMC-278, TMC-125, etravirine, rilpivirine, and similar agents; entry inhibitors such as enfuvirtide (T-20), T-1249, PRO-542, PRO-140, TNX-355, BMS-806, 5-Helix, and similar agents; integrase inhibitors such as dolutegravir, elvitegravir, raltegravir L-870,180, and similar agents; budding inhibitors such as PA-344 and PA-457, and similar agents; chemokine receptor inhibitors such as vicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK-427,857), TAK449, as well as those disclosed in WO 2002074769, WO 20040054974, WO 2004055012, WO 2004055010, WO 2004055016, WO 2004055011, and WO 2004054581, and similar agents; pharmacokinetic enhancers such as cobicistat; neuraminidase inhibitors such as CS-8958, zanamivir, oseltamivir, peramivir, and similar agents; ion channel blockers such as amantadine or rimantadine, and similar agents; interfering RNA and antisense oligonucleotides and such as ISIS-14803 and similar agents; and antiviral agents of undetermined mechanism of action, for example those disclosed in WO 2005105761, WO 2003085375, WO 2006122011, ribavirin, and similar agents.


The compounds of the present application may be used in combination with at least one other therapeutic agent which may be useful in the treatment of Kaposi's sarcoma-associated herpesvirus infections (KSHV and KSHV-related) including, without limitation, chemotherapeutic agents such as bleomycin, vinblastine, vincristine, cyclophosphamide, prednisone, alitretinoin and liposomal anthracyclines such as doxorubicin, daunorubicin, immunotherapeutics such as Rituximab, Tocilizumab, Siltuximab, and others such as Paclitaxel and Rapamycin.


The compounds of the present application may be used in combination with at least one other therapeutic agent which may be useful in the treatment of TB infection Mycobacterium tuberculosis) and Tularemia (Franciseiia tularensis), including, without limitation, first line oral agents isoniazid, Rifampicin, pyrazinamide, ethambutol, streptomycin, rifabutin; injectable agents including kanamycin, amikacin, capreomycin, streptomycin; fluoroquinolones including levofloxacin moxifloxacin ofloxacin; oral bacteriostatic agents para-aminosalicylic acid cycloserine terizidone thionamide protionamide; SQ-109 PNU-100480, Rifapentine Linezolid, PA-824 AZD5847, Gatifloxacin Moxifloxacin, Sirturo (bedaquiline) Delamanid (OPC-67683) and agents with undetermined mechanism of action in the treatment of drug-resistant TB, including clofazimine, linezolid, amoxicillin/clavulanate thioacetazone imipenem/cilastatin high dose isoniazid clarithromycin, and ciprofloxacin.


The compounds of the present application may be used in combination with at least one antimycobacterial agent (such as isoniazid (INH), ehambutol (Myambutol®), rifampin (Rifadin®), and pyrazinamide (PZA)), a bactericidal antibiotic (such as rifabutin (Mycobutin®) or rifapentine (Priftin®)), an aminoglycoside (Capreomycin®), a fluorquinolone (levofloxacin, moxifloxicin, ofloxacin), thioamide (ehionamide), cyclosporine (Sandimmune®), para-aminosalicyclic acid (Paser®), cycloserine (Seromycin®), kanamycin (Kantrex®), streptomycin, viomycin, capreomycin (Capastat®)), bedaquiline fumarate (Sirturo®), oxazolidinone (Sutezolid®), PNU-100480, or delamanid (OPC-67683).


The compounds of the present application may be used in combination with at least one other therapeutic agent which may be useful in the treatment of Chlamydia including, without limitation, Azithromycin, Doxycycline, Erythromycin, Levofloxacin, and Ofloxacin.


The compounds of the present application may be used in combination with at least one other therapeutic agent which may be useful in the treatment of Plasmodium infection including, without limitation, chloroquine, atovaquone-proguanil, artemether-lumefantrine, mefloquine, quinine, quinidine, doxocycline, cindamycin, artesunate, and primaquine.


In the treatment of amyotrophic lateral sclerosis (ALS), the compounds of the present application may be used in combination with a glutamate blocker (Riluzole (Rilutek®)), quinidine (Nuedexta®), anticholinergics (Amitriptyline®, Artane®, scopolamine patch (Transderm Scop®)), sympathomimetics (pseudoephedrine), mucolytics (guaifenesin), or analgesics (tramadol (Ultram®); ketorolac (Toradol®); morphine; or fentanyl patch (Duragesic®)).


In the treatment of multiple scelrosis, the compounds of the present application may be used in combination with corticosteroids (prednisone, methylprednisolone), Interferon Beta 1-A (Avonex®, Extavia®, Rebif®, Betaseron®), peginterferon beta-IA (Plegridy®), Glatiramer acetate (Copaxone®); glatiramer acetate (Glatopa®-generic equivalent of Copaxone); Dimethyl fumarate (Tecfidera®); Fingolimod (Gilenya®)); teriflunomide (Aubagio®); dalfampridine (Ampyra®); daclizumab (Zinbryta); alemtuzumab (Lemtrada®); natalizumab (Tysabri®); or mitoxantrone hydrochloride (Novantrone®).


The compounds of the present application may be used in combination with one or more vaccines or immugenic antigens useful in the prevention or treatment of viral infections. Such vaccines or immugenic antigens include, without limitation, pathogen derived proteins or particles such as attenuated viruses, virus particles, and viral proteins typically used as immugenic substances. Examples of viruses and viral antigens include, without limitation, Polioviruses, Coronaviridae and Coronaviruses, Rhinovirus (all subtypes), Adenoviruses (all subtypes), Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Human papillomavirus (including all subtypes), Rabies viruses, Human T-cell lympotropic virus (all subtypes), Rubella virus, Mumps virus, Coxsackie virus A (all subtypes), Cosackie virus B (all subtypes), human enteroviruses, herpesviruses including cytomegalovirus, Epstein-Barr virus, human herepesvi ruses (all subtypes), herpes simplex virus, varicella zoster virus, human immunodeficiency virus (HIV) (all subtypes), Epstein-Barr virus, Reoviruses (all subtypes), Filoviruses including Marburg virus and Ebola virus (all stains), Arenaviruses including Lymphocytic choriomeningitis virus, Lassa virus, Junin virus, and Machupo virus, Arboviruses including West Nile virus, Dengue viruses (all serotypes), Zika virus, Colorado tick fever virus, Sindbis virus, Togaviraidae, Flaviviridae, Bunyaviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae, Poxviruses including orthopoxvirus (variola virus, monkypox virus, vaccinia virus, cowpox virus), yatapoxviruses (tanapox virus, Yaba monkey tumor virus), parapoxvirus, molluscipoxvirus, Yellow fever, Hantaviruses including Hantaan, Seoul, Dobrava, Sin Nombre, Puumala, and Dobrava-like Saaremaa, human para influenza viruses and influenza viruses (all types), HIN influenza and swine influenza viruses, respiratory syncytial virus (all subgroups), rotaviruses including human rotaviruses A-E, bovine rotavirus, rhesus monkey rotavirus, Polyomaviruses including simian virus 40, JC virus, BK virus, Coltiviruses, eyach virus, calciviruses, and Parvoviridae including dependovirus, parvovirus and erythrovirus.


The compounds of the present application may be used in combination with at least one other therapeutic agent which may be useful in the prevention or treatment of viral infections for example immune therapies (e.g., interferon or other cytokines/chemokines, cytokine/chemokine receptor modulators, cytokine agonists or antagonists and similar agents); and therapeutic vaccines, anti-fibrotic agents, antiinflammatory agents such as corticosteroids or NSAIDs (non-steroidal anti-inflammatory agents) and similar agents.


The compounds of the present application may be used in combination with other anti-inflammatory agents, including oral or topical corticosteroids, anti-TNF agents, 5-aminosalicyclic acid and mesalamine preparations, hydroxycloroquine, thiopurines, methotrexate, cyclophosphamide, cyclosporine, calcineurin inhibitors, mycophenolic acid, mTOR inhibitors, JAK inhibitors, Syk inhibitors, anti-inflammatory biologic agents, including anti-IL6 biologies, anti-ILl agents, anti-IL17 biologies, anti-CD22, anti-integrin agents, anti-IFNa, anti-CD20 or CD4 biologies and other cytokine inhibitors or biologies to T-cell or B-cell receptors or interleukins.


For example, in the treatment of systemic lupus erythematosus and related lupus disorders, the compounds of the present application may be used in combination with at least one other therapeutic agent, including, a corticosteroid (such as prednisolone (Delatsone®, Orapred, Millipred, Omnipred, Econopred, Flo-Pred), an immunosuppressive agent (such as methotrexate (Rhuematrex®, Trexall®), dexamethasone (Decadron®, Solurex®), Mycophenolate mofetil (Cellcept®), Tacrolimus®, Sirolimus®), B-cell therapy (belimumab (Benlysta®), B-cell inhibitor (Atacicept®, Apratuzumab® (anti-CD22), SBI-087 (anti-CD20), an anti-BAFF antibody (LY2127399, A623), Velcade®), azathioprine (Azasan®, Imuran®), triamcinolone (Clinacort®, Kenalog-10®), hydroxychloroquine (Plaquenil®), thalidomide (Immunoprin®, Contergan®), immunoglobulin therapy (HyQiva®, Flebogamma®, Gamunex®, Privigen®, Gammagard®), anti-interferon-alpha therapy (Rontalizumab®, Sifalimumab®, AGS-009®, IFN Kinoid), TLR7 and TLR9 blockers (IMO-3100), anti-cytokine therapies (anti-IL6 (CNTO-136), anti-interferon-gamma (AMG811), immunomodulatory therapy (Lupuzor™, Abatacept, Orencia®, AMG557, Laquinimod, Paquinimod, Leflunomide, anti-ICOS (Medi-570), anti-CD40 ligand antibody (CDP7657)), and/or a platelet aggregation inhibitor (aspirin).


In treatment of vasculitis and disease with inflammation of small or medium size blood vessels, the compounds of the present application may be used in combination with alkylating agents (cyclophosphamide, Cytoxan®), anti-rheumatic anti-CD20 antibody (Rituxan®, Rituximab®), and anti-TNF inhibitors (Etanrcept®).


In the treatment of psoriasis, the compounds of the present application may be used in combination with ixekizumab, tildrakizumab (MK-3222), or secukinumab (AIN457).


In one embodiment, the at least one other therapeutic agent is selected from an inhaled corticosteroid, a long acting beta agonist, a combination of an inhaled corticosteroid and a long acting beta agonist, a short acting beta agonist, a leukotriene modifier, an anti-IgE, a methylxanthine bronchodilator, a mast cell inhibitor, and a long-acting muscarinic antagonist. For example, in the treatment of asthma, the compounds of the present application may be used in combination with an inhaled corticosteroid ((ICS) such as fluticasone proprionate (Flovent®), beclomethasone dipropionate (QVAR®), budesonide (Pulmicort), trimcinolone acetonide (Azmacort®), flunisolide (Aerobid®), mometasone fuorate (Asmanex® Twisthaler®), or Ciclesonide (Alvesco®)), a long acting beta agonist ((LABA) such as formoterol fumarate (Foradil®), salmeterol xinafoate (Serevent®)), a combination of an ICS and LABA (such as fluticasone furoate and vilanterol (Breo Ellipta®), formoterol/budesonide inhalation (Symbicort®), beclomethasone dipropionate/formoterol (Inuvair®), and fluticasone propionate/salmeterol (Advair®), a short acting beta agonist ((SABA) such as albuterol sulfate (ProAir®, Proventil HFA®, Ventolin HFA®, AccuNeb® Inhalation Solution), levalbuterol tartrate (Xopenex® HFA), ipratropium bromide/albuterol (Combivent® Respimat®), ipratropium bromide (Atrovent® HFA), a leukotriene modifier (such as montelukast sodium (Singulair®), zafirlukast (Accolate®), or zileuton (Zyflo®), and anti-IgE (such as omalizumab (Xolair®)), a methylxanthine bronchodilator (such as theophylline (Accurbron®, Aerolate®, Aquaphyllin®, Asbron®, Bronkodyl®, Duraphyl®, Elixicon®, Elixomin®, Elixophyllin®, Labid®, Lanophyllin®, Quibron-T®, Slo-Bid®, Slo-Phyllin®, Somophyllin®, Sustaire®, Synophylate®, T-Phyll®, Theo-24®, Theo-Dur®, Theobid®, Theochron®, Theoclear®, Theolair®, Theolixir®, Theophyl®, Theovent®, Uni-dur®, Uniphyl®), a mast cell inhibitor (such as cromulyn sodium (Nasalcrom®) and nedocromil sodium (Tilade®)), a long-acting muscarinic antagonist ((LAMA) such as mometasone furoate/formoterol fumarate dihydrate (Dulera®)).


Other agents that may be suitable for use in combination therapy in the treatment of asthma include a protein tyrosine kinase inhibitor (masitinib), CRTH2/D-prostanoid receptor antangonist (AMG 853), indacaterol (Arcapta®Neohaler®), an epinephrine inhalation aerosol (E004), fluticasone furoate/fluticasone proprionate, vinanterol inhalation/fluticasone furoate powder (Relovair™), fluticasone propionate/eformoterol fumarate dehydrate (Flutiform®), reslizumab, salbutamol dry-powder inhalation, tiotropium bromide (Spiriva®HandiHaler®), formoterol/budesonide (Symbicort®SMART®), fluticasone furoate (Veramyst®), Vectura's VR506, lebrikizumab (RG3637), a combination phosphodiesterase (PDE)-3 and (PDE)-4 inhibitor (RPL554).


In one embodiment, the at least one other therapeutic agent is selected from a long acting beta agonist, a long-acting inhaled anticholinergic or muscarinic antagonist, a phosphodiesterase inhibitor, a combination an inhaled corticosteroid long acting beta agonist, a short acting beta agonist, and an inhaled corticosteroid. For example, in the treatment of COPD, the compounds of the present application may be used in combination with a LABA (such as salmeterol xinafoate (Serevent), umeclidinium/vilanterol (Anoro Ellipta®), umeclidinium (Incruse Ellipta®), aformoterol tartrate (Brovana®), formoterol fumarate inhalation powder (Foradil®), indacterol maleate (Arcapta® Neohaler®), or fluticasone propionate/eformoterol fumarate dehydrate (Flutiform®)), a long-acting inhaled anticholinergic (or muscarinic antagonist, such as tiotropium bromide (Spiriva®), and aclidinium bromide (Tudorza® Pressair®), a phosphodiesterase (PDE-r) inhibitor (such as roflumilast, Daliresp®), a combination ICS/LABA (such as fluticasone furoate and vilanterol (Breo Ellipta®), fluticasone propionate/salmeterol (Advair®), budesonide/formoterol (Symbicort®), mometasone/formoterol (Dulera®), ipratropium bromide/albuterol sulfate (Duoneb®, Atrovent®), albuterol/ipratropium (Combivent Respimat®)), a SABA (such as ipratropium bromide (Atrovent®), and albuterol sulfate (ProAir®, Proventil®)), and an ICS (such as budesonide (Pulmicort®) and fluticasone propionate (Flovent®), beclometasone dipropionate (QVAR®).


Other agents that may be suitable for use in combination therapy in the treatment of COPD include SCH527123 (a CXCR2 antagonist), glycoprronium bromide ((NVA237) Seebri® Breezhaler®), glycopyrronium bromide and indacaterol maleate ((QVA149) Ultibro® Breezhaler®), glycopyrrolate and formoterol fumarate (PT003), indacaterol maleate (QVA149), olodaterol (Striverdi® Respimat®), tiotropium (Spiriva®)/olodaterol (Striverdi® Respimat®), and aclidinium/formoterol inhalation.


In one embodiment, the at least one other therapeutic agent is selected from an oral corticosteroid, anti-thymocyte globulin, thalidomide, chlorambucil, a calcium channel blocker, a topical emollient, an ACE inhibitor, a serotonin reuptake inhibitor, an endothelin-1 receptor inhibitor, an anti-fibrotic agent, a proton-pump inhibitor or imatinib, ARG201, and tocilizumab.


For example, in the treatment of systemic scleroderma, the compounds of the present application may be used in combination with an oral corticosteroid (such as prednisolone (Delatsone®, Orapred, Millipred, Omnipred, Econopred, Flo-Pred), an immunosuppressive agent (such as methotrexate (Rhuematrex®, Trexall®), cyclosporine (Sandimmune®), anti-thymocyte globulin (Atgam®), mycophenolate mofetil (CellCept®), cyclophosphamide (Cytoxan®), FK506 (tacrolimus), thalidomide (Thalomid®), chlorambucil (Leukeran®), azathioprine (Imuran®, Azasan®)), a calcium channel blocker (such as nifedipine (Procardia®, Adalat®) or nicardipine (Cardene®), a topical emollient (nitroglycerin ointment), an ACE inhibitor (such as lisinopril (Zestril®, Prinivil®), diltaizem (Cardizem®, Cardizem SR®, Cardizem CD®, Cardia®, Dilacor®, Tiazac®)), a serotonin reuptake inhibitor (such as fluoxetine (Prozac®)), an endothelin-1 receptor inhibitor (such as bosentan (Tracleer®) or epoprostenol (Flolan®, Veletri®, Prostacyclin®)) an anti-fibrotic agent (such as colchicines (Colcrys®), para-aminobenzoic acid (PABA), dimethyl sulfoxide (DMSO), and D-penicillamine (Cuprimine®, Depen®), interferon alpha and interferon gamma (INF-g)), a proton-pump Inhibitor (such as omeprazole (Prilosec®), metoclopramide (Reglan®), lansoprazole (Prevacid®), esomeprazole (Nexium®), pantoprazole (Protonix®), rabeprazole (Aciphex®)) or imatinib (Gleevec®) ARG201 (arGentis Pharmaceutical), belimumab (Benlysta®), tocilizumab (Actema®).


In the treatment of Sjogren's syndrome, the compounds of the present application may be used in combination with anti-rheumatic agents (hydroxychloroquine and Plaquenil®, Ridaura®, Kineret®), cholinergic agonists (Salagen®, Evoxac®), a JAK inhibitor (Xelijanz®, and anti-TNF treatments (Remicade®, Humira®, Enbrel®, Cimzia®, Simponi®).


In one embodiment of this invention, the at least one other therapeutic agent is a ciliary neurotrophic growth factor or a gene transfer agent. For example, in the treatment of retinitis pigmentosa, the compounds of the present application may be used in combination with a ciliary neurotrophic growth factor (NT-501-CNTF) or gene transfer agent, UshStat®.


In one embodiment of this invention, the at least one other therapeutic agent is selected from a trivalent (IIV3) inactivated influenza vaccine, a quadrivalent (IIV4) inactivated influenza vaccine, a trivalent recombinant influenza vaccine, a quadrivalent live attenuated influenza vaccine, an antiviral agent, or inactivated influenza vaccine. For example, in the treatment of influenza, the compounds of the present application may be used in combination with a trivalent (IIV3) inactivated influenza vaccine (such as Afluria®, Fluarix®, Flucelvax®, FluLaval®, Fluvirin®, Fluzone®), a quadrivalent (IIV4) inactivated influenza vaccine (such as Fluarix® Quadrivalent, Flulaval® Quadrivalent, Fluzone® Quadrivalent), a trivalent recombinant influenza vaccine (such as FluBlok®), a quadrivalent live attenuated influenza vaccine (such as FluMist® Quadrivalent), an antiviral agent (such as oseltamivir (Tamiflu®), zanamivir (Relenza®), rimantadine (Flumadine®), or amantadine (Symmetrel®)), or Fluad®, Fludase, FluNhance®, Preflucel, or VaxiGrip®.


In the treatment of a staphylococcus infection, the compounds of the present application may be used in combination with with an antibiotic (such as a β-Lactam cephalosporin (Duricef®, Kefzol®, Ancef®, Biocef®, etc), nafcillin (Unipen®), a sulfonamide (sulfamethoxazole and trimethoprim (Bacrim®, Septra®) sulfasalazine (Azulfidine®), acetyl sulfisoxazole (Gantrisin®), etc), or vancomycin (Vancocin®)).


In one embodiment, the at least one other therapeutic agent is selected from a topical immunomodulator or calcineurin inhibitor, a topical corticosteroid, an oral corticosteroid, an interferon gamma, an antihistamine, or an antibiotic. For example, in the treatment of atopic dermatitis, the compounds of the present application may be used in combination with a topical immunomodulator or calcineurin inhibitor (such as pimecrolimus (Elidel®) or tacrolimus ointment (Protopic®)), a topical corticosteroid (such as hydrocortizone (Synacort®, Westcort®), betamethasone (Diprolene®), flurandrenolide (Cordan®), fluticasone (Cutivate®), triamcinolone (Kenalog®), fluocinonide (Lidex®), and clobetasol (Temovate®)), an oral corticosteroid (such as hydrocortisone (Cortef®), methyl prednisolone (Medrol®), or prednisolone (Pediapred®, Prelone®), an immunosuppressant (such as cyclosporine (Neoral®) or interferon gamma (Alferon N®, Infergen®, Intron A, Roferon-A®)), an antihistamine (for itching such as Atarax®, Vistaril®, Benadryl®), an antibiotic (such as penicillin derivatives flucloxacillin (Floxapen®) or dicloxacillin (Dynapen®), erythromycin (Eryc®, T-Stat®, Erythra-Derm®, etc.)), a non-steroidal immunosuppressive agent (such as azathioprine (Imuran®, Azasan®), methotrexate (Rhuematrex®, Trexall®), cyclosporin (Sandimmune®), or mycophenolate mofetil (CellCept®)).


In one embodiment, the compounds may be administered in combination with one or more separate pharmaceutical agents, e.g., a chemotherapeutic agent, an immunotherapeutic agent, or an adjunctive therapeutic agent, and one or more of the other second agents as described herein.


As used herein, “combination therapy” or “co-therapy” includes the administration of a compound of the present application, or a pharmaceutically acceptable salt or ester thereof, and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected). “Combination therapy” may be, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present application.


“Combination therapy” is intended to embrace administration of these therapeutic agents in a sequential manner, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence in which the therapeutic agents are administered is not narrowly critical.


“Combination therapy” also embraces the administration of the therapeutic agents as described herein in further combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiation treatment). Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.


As used herein, the term “immune response” relates to any one or more of the following: specific immune response, non-specific immune response, both specific and nonspecific response, innate response, primary immune response, adaptive immunity, secondary immune response, memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression. In one embodiment, a compound of the present application, or a pharmaceutically acceptable salt or ester thereof, is administered in conjunction with one or more additional therapeutic agents including anti-viral compounds, vaccines intended to stimulate an immune response to one or more predetermined antigens, adjuvants, CTLA-4 and PD-1 pathway antagonists and other immunomodulatory agents, lipids, liposomes, peptides, anti-cancer agents, and chemotherapeutic agents, etc.


Definitions

Listed below are definitions of various terms used in this application. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.


The term “alkyl,” as used herein, refers to saturated, straight or branched-chain hydrocarbon radicals containing, in certain embodiments, between one and six carbon atoms. Examples of C1-C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, and n-hexyl radicals.


The term “alkenyl,” as used herein, denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six carbon atoms having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, and the like.


The term “alkynyl,” as used herein, denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six carbon atoms having at least one carbon-carbon triple bond. The triple bond may or may not be the point of attachment to another group. Alkynyl groups include, but are not limited to, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like.


The term “alkoxy” refers to an —O-alkyl radical.


The terms “hal,” “halo,” and “halogen,” as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.


The term “cycloalkyl,” as used herein, denotes a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring compound. Examples of C3-C8 cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and examples of C3-C12-cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2] octyl.


The term “cycloalkenyl,” as used herein, denotes a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring compound comprising at least one carbon-carbon double bond. Examples of C4-C8 cycloalkenyl include, but not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclopentenyl and cyclooctenyl.


The term “aryl,” as used herein, refers to a mono- or poly-cyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.


The term “aralkyl,” as used herein, refers to an alkyl residue, such as those described herein, attached to an aryl ring, such as those described herein. Examples include, but are not limited to, benzyl, phenethyl, and the like.


The term “heteroaryl,” as used herein, refers to a mono- or poly-cyclic (e.g., bi-, or tri-cyclic or more) fused or non-fused, radical or ring system having at least one aromatic ring, having from five to ten ring atoms of which one ring atoms is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, indazoyl, cinnolinyl, phthalazinyl, pyridazinyl, indolyl, acridinyl, benzoquinolinyl, pyrimidinyl, a purinyl, pyrrolopyrimidinyl, quinoxalinyl, quinazolinyl, indazolinyl, and phthalazinyl, and the like.


The term “heteroaralkyl,” as used herein, refers to an alkyl residue, such as those described herein, attached to a heteroaryl ring, such as those described herein. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, and the like.


In accordance with the application, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group. Aromatic groups can be substituted or unsubstituted.


The term “heterocyclyl,” as used herein, refers to a non-aromatic mono- or poly-cyclic (e.g., bi-, or tri-cyclic or more) fused or non-fused, radical or ring system having from three to ten ring atoms of which one ring atoms is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl, and the like.


The term “alkylamino” refers to a group having the structure —NH(C1-C12 alkyl), e.g., —NH(C1-C6 alkyl), where C1-C6 alkyl is as previously defined.


The term “dialkylamino” refers to a group having the structure —N(C1-C12 alkyl)2, e.g., —NH(C1-C6 alkyl), where C1-C6 alkyl is as previously defined.


The term “acyl” includes residues derived from acids, including but not limited to carboxylic acids, carbamic acids, carbonic acids, sulfonic acids, and phosphorous acids. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates and aliphatic phosphates. Examples of aliphatic carbonyls include, but are not limited to, acetyl, propionyl, 2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.


The term “ester” includes compounds or moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group. The term “ester” includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.


As described herein, compounds of the application and moieties present in the compounds may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the application. It will be appreciated that the phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” In general, the term “substituted”, whether preceded by the term “optionally” or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The terms “optionally substituted”, “optionally substituted alkyl,” “optionally substituted alkenyl,” “optionally substituted alkynyl”, “optionally substituted cycloalkyl,” “optionally substituted cycloalkenyl,” “optionally substituted aryl”, “optionally substituted heteroaryl,” “optionally substituted aralkyl”, “optionally substituted heteroaralkyl,” “optionally substituted heterocyclyl,” and any other optionally substituted group as used herein, refer to groups that are substituted or unsubstituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to:


—F, —CI, —Br, —I, —OH, protected hydroxy, —NO2, —CN, —NH2, protected amino, —NH—C1-C12-alkyl, —NH—C2-C12-alkenyl, —NH—C2-C12-alkenyl, —NH—C3-C12-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, —O—C1-C12-alkyl, —O—C2-C12-alkenyl, —O—C2-C12-alkenyl, —O—C3-C12-cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocycloalkyl, —C(O)—C1-C12-alkyl, —C(O)—C2-C12-alkenyl, —C(O)—C2-C12-alkenyl, —C(O)—C3-C12-cycloalkyl, —C(O)-aryl, —C(O)— heteroaryl, —C(O)-heterocycloalkyl, —CONH2, —CONH—C1-C12-alkyl, —CONH—C2-C12-alkenyl, —CONH-C2-C12-alkenyl, —CONH—C3-C12-cycloalkyl, —CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl, —OCO2—C1-C12-alkyl, —OCO2—C2-C12-alkenyl, —OCO2—C2-C12-alkenyl, —OCO2—C3-C12-cycloalkyl, —OCO2-aryl, —OCO2-heteroaryl, —OCO2-heterocycloalkyl, —OCONH2, —OCONH—C1-C12-alkyl, —OCONH—C2-C12-alkenyl, —OCONH—C2-C12-alkenyl, —OCONH-C3-C12-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl, —OCONH— heterocycloalkyl, —NHC(O)—C1-C12-alkyl, —NHC(O)—C2-C12-alkenyl, —NHC(O)—C2-C12-alkenyl, —NHC(O)—C3-C12-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl, —NHC(O)— heterocycloalkyl, —NHCO2—C1-C12-alkyl, —NHCO2—C2-C12-alkenyl, —NHCO2—C2-C12-alkenyl, —NHCO2—C3-C12-cycloalkyl, —NHCO2-aryl, —NHCO2-heteroaryl, —NHCO2-heterocycloalkyl, NHC(O)NH2, —NHC(O)NH—C1-C12-alkyl, —NHC(O)NH—C2-C12-alkenyl, —NHC(O)NH—C2-C12-alkenyl, —NHC(O)NH—C3-C12-cycloalkyl, —NHC(O)NH-aryl, —NHC(O)NH-heteroaryl, NHC(O)NH-heterocycloalkyl, —NHC(S)NH2, —NHC(S)NH—C1-C12-alkyl, —NHC(S)NH—C2-C12-alkenyl, —NHC(S)NH—C2-C12-alkenyl, —NHC(S)NH—C3-C12-cycloalkyl, —NHC(S)NH-aryl, —NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH2, —NHC(NH)NH—C1-C12-alkyl, —NHC(NH)NH—C2-C12-alkenyl, —NHC(NH)NH—C2-C12-alkenyl, —NHC(NH)NH—C3-C12-cycloalkyl, —NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NHheterocycloalkyl, —NHC(NH)—C1-C12-alkyl, —NHC(NH)—C2-C12-alkenyl, —NHC(NH)—C2-C12-alkenyl, —NHC(NH)—C3-C12-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl, —NHC(NH)-heterocycloalkyl, —C(NH)NH—C1-C12-alkyl, —C(NH)NH—C2-C12-alkenyl, —C(NH)NH—C2-C12-alkenyl, C(NH)NH—C3-C12-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl, —C(NH)NHheterocycloalkyl, —S(O)—C1-C12-alkyl, —S(O)—C2-C12-alkenyl, —S(O)—C2-C12-alkenyl, —S(O)—C3-C12-cycloalkyl, —S(O)-aryl, —S(O)-heteroaryl, —S(O)-heterocycloalkyl-SO2NH2, —SO2NH—C1-C12-alkyl, —SO2NH—C2-C12-alkenyl, —SO2NH—C2-C12-alkenyl, —SO2NH—C3-C12-cycloalkyl, —SO2NH-aryl, —SO2NH-heteroaryl, —SO2NH-heterocycloalkyl, —NHSO2—C1-C12-alkyl, —NHSO2—C2-C12-alkenyl, —NHSO2—C2-C12-alkenyl, —NHSO2—C3-C12-cycloalkyl, —NHSO2-aryl, —NHSO2-heteroaryl, —NHSO2-heterocycloalkyl, —CH2NH2, —CH2SO2CH3, -aryl, -arylalkyl,-heteroaryl,-heteroarylalkyl,-heterocycloalkyl, —C3-C12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH, —S—C1-C12-alkyl, —S—C2-C12-alkenyl, —S—C2-C12-alkenyl, —S—C3-C12-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, or methylthiomethyl.


The term “cancer” includes, but is not limited to, the following cancers: epidermoid Oral: buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Lung: bronchogenic carcinoma (squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal, rectum; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma) hairy cell; lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, undifferentiated thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma; and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell” as provided herein, includes a cell afflicted by any one of the above-identified conditions.


The term “subject” as used herein refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.


“Treat”, “treating” and “treatment” refer to a method of alleviating or abating a disease and/or its attendant symptoms.


As used herein, “preventing” or “prevent” describes reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder.


The terms “disease(s)”, “disorder(s)”, and “condition(s)” are used interchangeably, unless the context clearly dictates otherwise.


The term “therapeutically effective amount” of a compound or pharmaceutical composition of the application, as used herein, means a sufficient amount of the compound or pharmaceutical composition so as to decrease the symptoms of a disorder in a subject. As is well understood in the medical arts a therapeutically effective amount of a compound or pharmaceutical composition of this application will be at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present application will be decided by the attending physician within the scope of sound medical judgment. The specific modulatory (e.g., inhibitory or stimulatory) dose for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.


As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.


As used herein, the term “pharmaceutically acceptable salt” refers to those salts of the compounds formed by the process of the present application which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the application, or separately by reacting the free base or acid function with a suitable acid or base.


Examples of pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts: salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, 7-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.


As used herein, the term “pharmaceutically acceptable ester” refers to esters of the compounds formed by the process of the present application which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.


The term “pharmaceutically acceptable prodrugs” as used herein, refers to those prodrugs of the compounds formed by the process of the present application which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present application.


“Prodrug”, as used herein, means a compound which is convertible in vivo by metabolic means (e.g., by hydrolysis) to afford any compound delineated by the formulae of the instant application. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). “Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).


“Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.


This application also encompasses pharmaceutical compositions containing, and methods of treating disorders through administering, pharmaceutically acceptable prodrugs of compounds of the application. For example, compounds of the application having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of compounds of the application. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 1-15. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described herein, are also encompassed. Prodrugs of this type are described in J. Med. Chem. 1996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities.


Combinations of substituents and variables envisioned by this application are only those that result in the formation of stable compounds. The term “stable”, as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).


When any variable (e.g., R1) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with one or more R moieties, then R at each occurrence is selected independently from the definition of R. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds within a designated atom's normal valency.


In addition, some of the compounds of this application have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z-double isomeric forms, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. The configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states; thus a carbon-carbon double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion. All such isomeric forms of such compounds are expressly included in the present application.


“Isomerism” means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereoisomers”, and stereoisomers that are non-superimposable mirror images of each other are termed “enantiomers” or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a “racemic mixture”.


A carbon atom bonded to four non-identical substituents is termed a “chiral center”.


“Chiral isomer” means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed “diastereomeric mixture”. When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center, e.g., carbon. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).


“Geometric isomer” means the diastereomers that owe their existence to hindered rotation about double bonds. These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.


Furthermore, the structures and other compounds discussed in this application include all atropic isomers thereof “Atropic isomers” are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques; it has been possible to separate mixtures of two atropic isomers in select cases.


“Tautomer” is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solid form, usually one tautomer predominates. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.


Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs. Ring-chain tautomerism arises as a result of the aldehyde group (—CHO) in a sugar chain molecule reacting with one of the hydroxy groups (—OH) in the same molecule to give it a cyclic (ring-shaped) form as exhibited by glucose. Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings (e.g., in nucleobases such as guanine, thymine and cytosine), amine-enamine and enamine-enamine. The compounds of this application may also be represented in multiple tautomeric forms, in such instances, the application expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the application expressly includes all such reaction products).


In the present application, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present application includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like. In the present specification, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present application includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like.


Additionally, the compounds of the present application, for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.


“Solvate” means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H2O.


EXAMPLES

The purity of all compounds was over 95% and was analyzed with Waters LC/MS system. 1H NMR was obtained at 400 MHz. Chemical shifts are reported relative to dimethyl sulfoxide (δ=2.50) for 1H NMR. Data are reported as (br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet).


Abbreviations used in the following examples and elsewhere herein are:

    • AcOH acetic acid
    • atm atmosphere
    • BOC2O di-tert-butyl dicarbonate
    • br broad
    • CuSO4 copper sulfate
    • CDCl3 deuterated chloroform
    • DCM dichloromethane
    • DIEA N,N-diisopropylethylamine
    • DMA N,N-dimethylacetamide
    • DME dimethoxy ethane
    • DMAP 4-dimethylaminopyridine
    • DMF N,N-dimethylformamide
    • DMSO dimethyl sulfoxide
    • DMSO-d6 deuterated dimethyl sulfoxide
    • EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
    • ESI electrospray ionization
    • EtOAc ethyl acetate
    • HCl hydrochloric acid
    • h hour(s)
    • HATU bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-phosphate
    • HPLC high-performance liquid chromatography
    • KHMDS potassium hexamethylsilazide
    • LCMS liquid chromatography-mass spectrometry
    • m multiplet
    • mL milliliter
    • MeCN acetonitrile
    • MeOH methanol
    • mg milligram
    • mmol millimole
    • MgSO4 magnesium sulfate
    • MHz megahertz
    • min minutes
    • MS mass spectrometry
    • Na2CO3 sodium carbonate
    • NaHCO3 sodium bicarbonate
    • NMR nuclear magnetic resonance
    • Tf triflate
    • Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0)
    • Pd(PPh3)2Cl2 bis(triphenylphosphine)palladium(II) dichloride
    • PE petroleum ether
    • PhN(SO2CF3)2 N-phenyl-bis(trifluoromethanesulfonamide
    • PMe3 trimethylphosphine
    • PPA polyphosphoric acid
    • ppm parts per million
    • PTSA para-toluene sulfonic acid
    • rt room temperature
    • TBAF tetra-n-butylammonium fluoride
    • t-BuOH tert-butanol
    • TFA trifluoroacetic acid
    • TMS trimethylsilane
    • THF tetrahydrofuran
    • TLC thin layer chromatography
    • μL microliter
    • Xphos 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl
    • Z′ Z-factor statistical value


Example 1: Synthesis of Compound 1



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Step 1


To a solution of methyl 2,6-dichloropyridine-3-carboxylic acid (5.00 g, 26.04 mmol, 1.0 equiv) and 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (4.59 g 27.34 mmol, 1.0 equiv) in dioxane (30 mL) and H2O (3 mL) were added K2CO3 (7.20 g, 52.08 mmol, 210 equiv) and Pd(dppf)Cl2 (0.95 g. 1.3 mmol, 0.05 equiv) at room temperature. The resulting mixture was stirred for 2 h at 95° C. under argon atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM/MeOH (20:1) to afford 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylic acid (a, 3.5 g, 68%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=198.0.


Step 2


To a solution of 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylic acid (3.50 g, 17.71 mmol, 1.0 equiv) in DCM (15 mL) were added (COCl)2 (8.99 g, 70.84 mmol, 4.0 equiv) and DMF (12.95 mg, 0.17 mmol, 0.01 equiv) at 0° C. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under vacuum, then was added MeOH (15 mL). The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylate (b, 3.0 g, 80%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=212.0.


Step 3


To a solution of methyl 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylate (3.00 g, 14.17 mmol, 1.0 equiv) and 3-chloro-2-methoxy aniline (2.46 g, 15.60 mmol, 1.1 equiv) in DME (10 mL) were added Cs2CO3 (9.24 g, 28.34 mmol, 2.0 equiv), X-Phos (1.35 g, 2.83 mmol, 0.2 equiv) and Pd2(dba)3 (1.30 g, 1.41 mmol, 0.1 equiv) at room temperature. The resulting mixture was stirred for 2 h at 85° C. under argon atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 2-[(3-chloro-2-methoxyphenyl)amino]-6-(prop-1-en-2-yl)pyridine-3-carboxylate (c, 3.0 g, 64%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=333.1.


Step 4


To a solution of methyl 2-[(3-chloro-2-methoxyphenyl)amino]-6-(prop-1-en-2-yl)pyridine-3-carboxylate (2.00 g, 6.01 mmol, 1.0 equiv) in EtOAc (5 mL) was added PtO2 (395.78 mg, 1.74 mmol, 0.3 equiv) at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (4:1) to afford methyl 2-[(3-chloro-2-methoxyphenyl)amino]-6-isopropylpyridine-3-carboxylate (d, 2.0 g, 66%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=335.1


Step 5


A solution of methyl 2-[(3-chloro-2-methoxyphenyl)amino]-6-isopropylpyridine-3-carboxylate (1.80 g, 5.37 mmol, 1.0 equiv) and LiOH (643.82 mg, 26.88 mmol, 5.0 equiv) in THF (5 mL), MeOH (5 mL) and H2O (5 mL) was stirred for 1 h at room temperature. The mixture was acidified to pH=2 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×50 mL). This resulted in 2-[(3-chloro-2-methoxyphenyl)amino]-6-isopropylpyridine-3-carboxylic acid (e, 1.5 g, 85%) as a white solid. LCMS (ESI, m/z): [M+H]+=321.1.


Step 6


A solution of 2-[(3-chloro-2-methoxyphenyl)amino]-6-isopropylpyridine-3-carboxylic acid (1.50 g, 4.6 mmol, 1.0 equiv) in PPA (5 mL) was stirred for 10 min at 110° C. The mixture was basified to pH=12 with sat. Na2CO3 (aq.). The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (1:1) to afford 8-chloro-9-methoxy-2-(propan-2-yl)-5H,10H-benzo[b]1,8-naphthyridin-5-one (f, 1 g, 77%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=303.1


Step 7


To a solution of 8-chloro-9-methoxy-2-(propan-2-yl)-5H,10H-benzo[b]1,8-naphthyridin-5-one (500.00 mg, 1.65 mmol, 1.0 equiv) and Cs2CO3 (1.61 g, 4.95 mmol, 3.0 equiv) in MeCN (5 mL) was added ethyl iodoacetate (1.41 g, 6.60 mmol, 4.0 equiv) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (1:1) to afford ethyl 2-[8-chloro-9-methoxy-5-oxo-2-(propan-2-yl)-5H,10H-benzo[b]1,8-naphthyridin-10-yl]acetate (g, 1 g, 71%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=389.1.


Step 8


To a solution of ethyl 2-[8-chloro-9-methoxy-5-oxo-2-(propan-2-yl)-5H,10H-benzo[b]1,8-naphthyridin-10-yl]acetate (100.00 mg, 0.25 mmol, 1.0 equiv) in THF (2 mL)/H2O (2 mL)/MeOH (2 mL) was added LiOH (30.79 mg, 1.28 mmol, 5.0 equiv) at room temperature. The reaction solution was stirred for 1 h at room temperature. The mixture was acidified to pH=2 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×5 mL) to afford Compound 1 (60.2 mg, 65%) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 8.54 (d, J=8.0 Hz, 1H), 8.13 (d, J=8.8 Hz, 1H), 7.50 (d, J=8.8 Hz, 1H), 7.40 (d, J=8.0 Hz, 1H), 5.62 (s, 2H), 3.84 (s, 3H), 3.24-3.15 (m, 1H), 1.31 (d, J=6.8 Hz, 6H); LCMS (ESI, m/z): [M+H]+=361.1


Example 2: Synthesis of Compound 2



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Step 1


To a solution of 2-chloro-6-methylpyridine-3-carboxylic acid (10 g, 58.282 mmol, 1.0 equiv) in DCM (150 mL) was added (COCl)2 (22 g, 174.846 mmol, 3.0 equiv) at 0° C. To the above mixture was added DMF (1 mL) dropwise over 2 min at 0° C. The resulting mixture was stirred for 1 h at room temperature. To the above mixture was added CH30H (40 mL) dropwise over 5 min at 0° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (10:1) to afford methyl 2-chloro-6-methylpyridine-3-carboxylate (a, 9 g, 83%) as white oil. LCMS (ESI, m/z): [M+H]+=186.0.


Step 2


To a solution of methyl 2-chloro-6-methylpyridine-3-carboxylate (500.00 mg, 2.694 mmol, 1.0 equiv), 3-chloro-2-ethoxyaniline (554.78 mg, 3.233 mmol, 1.2 equiv) and Cs2CO3 (1755.40 mg, 5.388 mmol, 2.0 equiv) in DME (6.00 mL) were added Pd2(dba)3 (246.68 mg, 0.269 mmol, 0.1 equiv) and X-Phos (256.84 mg, 0.539 mmol, 0.2 equiv) at room temperature. The reaction mixture was stirred for 2 h at 80° C. under argon atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (10:1) to afford methyl 2-[(3-chloro-2-ethoxyphenyl)amino]-6-methylpyridine-3-carboxylate (b, 700 mg, 81%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=321.1.


Step 3


To a solution of methyl 2-[(3-chloro-2-ethoxyphenyl)amino]-6-methylpyridine-3-carboxylate (700.00 mg, 2.182 mmol, 1.0 equiv) and LiOH (261.32 mg, 10.911 mmol, 5.0 equiv) in THF (3 mL) were added MeOH (3 mL) and H2O (3 mL) at room temperature. The reaction solution was stirred for 2 h at room temperature. The mixture was acidified to pH=4 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×10 mL). This resulted in 2-[(3-chloro-2-ethoxyphenyl)amino]-6-methylpyridine-3-carboxylic acid (c, 500 mg, 75%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=307.1


Step 4


The solution of 2-[(3-chloro-2-ethoxyphenyl)amino]-6-methylpyridine-3-carboxylic acid (500.00 mg) in diphosphooxidane-methanesulfonic acid (6 mL) was stirred for 10 min at 100° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeOH in water, 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in 8-chloro-9-ethoxy-2-methyl-5H,10H-benzo[b]1,8-naphthyridin-5-one (d, 130 mg, 28%) as a yellow oil. LCMS (ESI, m/z): [M+H]+=289.1.


Step 5


To a solution of 8-chloro-9-ethoxy-2-methyl-5H,10H-benzo[b]1,8-naphthyridin-5-one (130.00 mg, 0.450 mmol, 1.0 equiv) in MeCN (3 mL) were added ethyl 2-iodoacetate (144.53 mg, 0.675 mmol, 1.5 equiv) and Cs2CO3 (440.10 mg, 1.351 mmol, 3.0 equiv) at room temperature. The reaction mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (1:1) to afford ethyl 2-[8-chloro-9-ethoxy-2-methyl-5-oxo-5H,10H-benzo[b]1,8-naphthyridin-10-yl]acetate (e, 130 mg, 77%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=375.1


Step 6


To a solution of ethyl 2-[8-chloro-9-ethoxy-2-methyl-5-oxo-5H,10H-benzo[b]1,8-naphthyridin-10-yl]acetate (130.00 mg, 0.347 mmol, 1.0 equiv) in THF (2 mL), MeOH (2 mL) and H2O (2 mL) was added LiOH (41.53 mg, 1.734 mmol, 5.0 equiv) at room temperature. The reaction solution was stirred for 30 min at 80° C. The mixture was acidified to pH=4 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×5 mL).


This resulted in Compound 2 (59.4 mg, 49%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 13.40 (brs, 1H), 8.34 (d, J=7.6 Hz, 1H), 8.11 (d, J=9.2 Hz, 1H), 7.84-7.76 (m, 2H), 7.67 (d, J=9.2 Hz, 1H), 7.61-7.55 (m, 1H), 5.54 (s, 2H), 4.21 (s, 3H); LCMS (ESI, m/z): [M+H]+=347.1.


Example 3: Synthesis of Compound 3



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Step 1


To a stirred solution of 2,6-dichloropyridine-3-carboxylic acid (5 g, 26.042 mmol, 1.0 equiv) and (COCl)2 (1.65 g, 130.208 mmol, 5.0 equiv) in DCM (50 mL) was added DMF (1 mL) dropwise portions at 0° C. under air atmosphere. The mixture was stirred for 2 h at room temperature. To the above mixture was added methanol (5 mL) dropwise portions at 0° C. The resulting mixture was stirred for additional 5 min at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (9:1) to afford methyl 2,6-dichloropyridine-3-carboxylate (a, 5 g, 93%) as a white solid. LCMS (ESI, m/z): [M+H]+=205.9.


Step 2


To a solution of methyl 2,6-dichloropyridine-3-carboxylate (3.00 g, 14.562 mmol, 1.00 equiv), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (2.69 g, 16.018 mmol, 1.10 equiv) in dioxane (30 mL) and H2O (5 mL) were added Pd(dppf)Cl2 (1.07 g, 1.456 mmol, 0.1 equiv) and K2CO3 (4025.01 mg, 29.123 mmol, 2 equiv) at room temperature. The reaction mixture was stirred for 2 h at 80° C. under argon atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (10:1), to afford methyl 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylate (b, 2.1 g, 68%) as a yellow oil. LCMS (ESI, m/z): [M+H]+=212.0


Step 3


To a solution of methyl 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylate (500.00 mg, 2.362 mmol, 1.0 equiv), 3-chloro-2-ethoxyaniline (486.52 mg, 2.835 mmol, 1.2 equiv) in DME (6 mL) were added Pd2(dba)3 (216.33 mg, 0.236 mmol, 0.1 equiv), X-Phos (225.24 mg, 0.472 mmol, 0.2 equiv) and Cs2CO3 (1.54 g, 4.725 mmol, 2.0 equiv) at room temperature. The reaction mixture was stirred for 2 h at 80° C. under argon atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (10:1) to afford methyl 2-[(3-chloro-2-ethoxyphenyl)amino]-6-(prop-1-en-2-yl)pyridine-3-carboxylate (c, 560 mg, 68%) as a yellow oil. LCMS (ESI, m/z): [M+H]+=347.1


Step 4


The solution of methyl 2-[(3-chloro-2-ethoxyphenyl)amino]-6-(prop-1-en-2-yl)pyridine-3-carboxylate (560.00 mg, 1.615 mmol, 1.0 equiv) and PtO2 (73.33 mg, 0.323 mmol, 0.2 equiv) in ethyl acetate (6 mL) was stirred for 1 h at room temperature under hydrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 2-[(3-chloro-2-ethoxyphenyl)amino]-6-(propan-2-yl)pyridine-3-carboxylate (d, 520 mg, 92%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=349.1.


Step 5


To a solution of methyl 2-[(3-chloro-2-ethoxyphenyl)amino]-6-(propan-2-yl)pyridine-3-carboxylate (520.00 mg, 1.491 mmol, 1.0 equiv) in THF (3 mL), MeOH (3 mL) and H2O (3 mL) was added LiOH (178.51 mg, 7.453 mmol, 5.0 equiv) at room temperature. The reaction mixture was stirred for 1 h at room temperature. The mixture was acidified to pH=5 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×6 mL). This resulted in 2-[(3-chloro-2-ethoxyphenyl)amino]-6-(propan-2-yl)pyridine-3-carboxylic acid (e, 400 mg, 80%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=335.1.


Step 6


The solution of 2-[(3-chloro-2-ethoxyphenyl)amino]-6-(propan-2-yl)pyridine-3-carboxylic acid (340.00 mg, 1.016 mmol, 1.00 equiv) in diphosphooxidane-methanesulfonic acid (5 mL) was stirred for 10 min at 100° C. The resulting solution was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeOH in water, 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in 8-chloro-9-ethoxy-2-(propan-2-yl)-5H,10H-benzo[b]1,8-naphthyridin-5-one (f, 220 mg, 68%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=317.1.


Step 7


To a solution of 8-chloro-9-ethoxy-2-(propan-2-yl)-5H, 10H-benzo[b]1,8-naphthyridin-5-one (220.00 mg, 0.694 mmol, 1.0 equiv) in MeCN (4.00 mL) were added ethyl 2-iodoacetate (222.93 mg, 1.042 mmol, 1.5 equiv) and Cs2CO3 (678.81 mg, 2.083 mmol, 3.0 equiv) at room temperature. The reaction mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (1:1) to afford ethyl 2-[8-chloro-9-ethoxy-5-oxo-2-(propan-2-yl)-5H,10H-benzo[b]1,8-naphthyridin-10-yl]acetate (g, 250 mg, 89%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=403.1.


Step 8


To a solution of ethyl 2-[8-chloro-9-ethoxy-5-oxo-2-(propan-2-yl)-5H,10H-benzo[b]1,8-naphthyridin-10-yl]acetate (270.00 mg, 0.670 mmol, 1.0 equiv) in THF (3 mL), MeOH (3 mL) and H2O (3 mL) was added LiOH (80.25 mg, 3.351 mmol, 5.0 equiv) at room temperature. The reaction mixture was stirred for 1 h at 80° C. The resulting mixture was acidified to pH=4 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×5 mL). This resulted in Compound 3 (63.2 mg, 25%) as a brown solid. 1H NMR (400 MHz, DMSO-d6): δ 12.92 (brs, 1H), 8.53 (d, J=8.0 Hz, 1H), 8.11 (d, J=8.8 Hz, 1H), 7.50 (d, J=8.8 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H), 5.63 (s, 2H), 3.99 (q, J=6.8 Hz, 2H), 3.19-3.12 (m, 1H), 1.40 (t, J=6.8 Hz, 3H), 1.30 (d, J=6.8 Hz, 6H); LCMS (ESI, m/z): [M+H]+=375.1


Example 4: Synthesis of Compound 4



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Step 1


To a solution of a (2.0 g, 11.4 mmol, 1.0 eq) and b (9.1 mL, 2 M in THF, 1.6 eq) in DCM (50 mL) was added Et3N (1.9 mL, 1.2 eq) at rt. The mixture was stirred at rt overnight, diluted with DCM (50 mL), washed with 1N HCl (aq, 25 mL) and brine (25 mL). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by chromatography on silica gel (PE) to afford c (1.85 g, 81%).


Step 2


A mixture of c (1.85 g, 9.2 mmol, 1.0 eq), Fe (1.55 g, 27.6 mmol, 3.0 eq) and NH4Cl (495.0 mg, 9.2 mmol, 1.0 eq) in EtOH/H2O (30 mL/12 mL) was stirred at 90° C. for 2 h. The mixture was cooled to rt and filtered. The filtrate was diluted with water (20 mL), extracted with EtOAc (100 mL×2). The combined organic layers were washed with brine (25 mL), dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by chromatography on silica gel (PE) to afford d (1.3 g, 83%). LC/MS: 171.2 [M+H]+.


Step 3


A mixture of e (1.1 g, 6.5 mmol, 1.0 eq), d (1.2 g, 7.1 mmol, 1.2 eq), potassium acetate (1.27 g, 13.0 mmol, 2.0 eq), cupric acetate (390.0 mg, 1.95 mmol, 0.3 eq) and copper powder (125.0 mg, 1.95 mmol, 0.3 eq) in 2-pentanol (30.0 mL) was stirred at 130° C. overnight under nitrogen atmosphere. The mixture was cooled to rt, diluted with water (30 mL) and EtOAc (50 mL), acidified to pH=2 with 1N HCl (aq). The mixture was filtered through celite. The filtrate was extracted with EtOAc (50 mL×2). The combined organic layers were washed with brine (25 mL×2), dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by trituration with PE (25 mL) to afford f (1.17 g, 60%). LC/MS: 306.3 [M+H]+.


Step 4


A mixture of f (1.07 g, 3.5 mmol) and Eaton's Reagent (10 mL) was heated at 110° C. for 5 h. The mixture was cooled to rt, poured into ice-water (30 mL) slowly, extracted with EtOAc (50 mL×2). The combined organic layers were washed with brine (25 mL×2), dried over anhydrous Na2SO4, filtered and evaporated to afford crude g (830.0 mg, 83%), which was used into the next step without further purification. LC/MS: 288.1 [M+H]+.


Step 5


To a solution of g (150.0 mg, 0.52 mmol, 1.0 eq) and Cs2CO3 (509.0 mg, 1.56 mmol, 3.0 eq) in acetonitrile (5 mL) was added h (334.0 mg, 1.56 mmol, 3.0 eq). The mixture was stirred at 40° C. for 1 h. The mixture was cooled to rt, diluted with water (20 mL), extracted with EtOAc (25 mL×2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by trituration with MeOH (3 mL) to afford i (63.0 mg, 32%) as a yellow solid. LC/MS: 374.1 [M+H]+.


Step 6


A mixture of i (63.0 mg, 0.17 mmol, 1.0 eq) and LiOH.H2O (35.0 mg, 0.85 mmol, 5.0 eq) in THF (2.0 mL), MeOH (2.0 mL) and H2O (2.0 mL) was stirred at 35° C. for 2 h. The mixture was diluted with water (10.0 mL), acidified with 1N HCl (aq). The precipitate was collected by filtration, washed with water and dried to afford Compound 4 (27.0 mg, yield: 47%) as a yellow solid. LC/MS: 346.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.57 (brs, 1H), 8.45 (d, J=8.0 Hz, 1H), 8.16 (d, J=8.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 5.44 (brs, 2H), 2.74 (s, 6H), 2.62 (s, 3H).


Example 5: Synthesis of Compound 5



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Step 1


To a solution of 2,6-dibromopyridine-3-carboxylic acid (3.00 g, 10.680 mmol, 1.0 equiv) in MeOH (20 mL) were added TMSCHN2 (15 mL) in portions at 0° C. The resulting mixture was stirred for 2 h at room temperature. The resulting solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 2,6-dibromopyridine-3-carboxylate (a, 2.5 g, 79%) as a white solid. LCMS (ESI, m/z): [M+H]+=295.9


Step 2


To a solution of tert-butyl(chloro)magnesium (713.27 mg, 6.103 mmol, 1.2 equiv) in THF (20 mL) was added CuCN (911.03 mg, 10.172 mmol, 2.0 equiv) at room temperature. The reaction mixture was stirred for 20 min at −78° C. under nitrogen atmosphere. To the above mixture was added methyl 2,6-dibromopyridine-3-carboxylate (1.50 g, 5.086 mmol, 1.0 equiv) in THF (5 mL) in portions at −78° C. The resulting mixture was stirred for additional 3 h at −78° C. Then the reaction mixture was stirred overnight at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 2-bromo-6-tert-butylpyridine-3-carboxylate (b, 1.2 g, 87%) as a colorless oil. LCMS (ESI, m/z): [M+H]+=272.0


Step 3


To a solution of methyl 2-bromo-6-tert-butylpyridine-3-carboxylate (600.00 mg, 2.205 mmol, 1.00 equiv) in Dioxane (10 mL) were added 6-chloro-N1,N1-dimethylbenzene-1,2-diamine (451.46 mg, 2.646 mmol, 1.2 equiv), Xantphos Pd 4G (212.18 mg, 0.220 mmol, 0.1 equiv), Xantphos (127.57 mg, 0.220 mmol, 0.1 equiv) and Cs2CO3 (1.44 g, 4.409 mmol, 2.0 equiv) at room temperature. The reaction mixture was stirred for 2 h at 90° C. under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 6-tert-butyl-2-[[3-chloro-2-(dimethylamino)phenyl]amino]pyridine-3-carboxylate (c, 700 mg, 88%) as a yellow oil. LCMS (ESI, m/z): [M+H]+=362.2.


Step 4


To a solution of methyl 6-tert-butyl-2-[[3-chloro-2-(dimethylamino)phenyl]amino]pyridine-3-carboxylate (700.00 mg, 1.934 mmol, 1.0 equiv) in THF (5 mL), MeOH (5 mL) and H2O (5 mL) was added LiOH (231.62 mg, 9.672 mmol, 5.0 equiv) at room temperature. The reaction solution was stirred for 1 h at 80° C. The mixture was acidified to pH=5 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×5 mL). This resulted in 6-tert-butyl-2-[[3-chloro-2-(dimethylamino)phenyl]amino]pyridine-3-carboxylic acid (d, 500 mg, 74%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=348.1.


Step 5


A solution of 6-tert-butyl-2-[[3-chloro-2-(dimethylamino)phenyl]amino]pyridine-3-carboxylic acid (500.00 mg, 1.437 mmol, 1.0 equiv) in diphosphooxidane-methanesulfonic acid (10 mL) was stirred for 1 h at 120° C. The resulting solution was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeOH in water, 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in 2-tert-butyl-8-chloro-9-(dimethylamino)-10H-benzo[b]1,8-naphthyridin-5-one (e, 120 mg, 25%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=330.1.


Step 6


To a solution of 2-tert-butyl-8-chloro-9-(dimethylamino)-10H-benzo[b]1,8-naphthyridin-5-one (120.00 mg, 0.364 mmol, 1.0 equiv) in MeCN (2 mL) were added Cs2CO3 (355.62 mg, 1.091 mmol, 3.0 equiv) and ethyl iodoacetate (116.79 mg, 0.546 mmol, 1.5 equiv) at room temperature. The reaction mixture was stirred for 2 h at 60° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (4:1) to afford ethyl 2-[2-tert-butyl-8-chloro-9-(dimethylamino)-5-oxobenzo[b]1,8-naphthyridin-10-yl]acetate (f, 110 mg, 73%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=416.2.


Step 7


To a solution of ethyl 2-[2-tert-butyl-8-chloro-9-(dimethylamino)-5-oxobenzo[b]1,8-naphthyridin-10-yl]acetate (110.00 mg, 0.264 mmol, 1.0 equiv) in THF (2 mL), MeOH (2 mL) and H2O (2 mL) was added LiOH (31.67 mg, 1.322 mmol, 5.0 equiv) at room temperature. The reaction solution was stirred for 1 h at 60° C. The resulting mixture was acidified to pH=4 with HCl (aq.). The precipitated solids were collected by filtration and washed with MeOH (3×5 mL). This resulted in [2-tert-butyl-8-chloro-9-(dimethylamino)-5-oxobenzo[b]1,8-naphthyridin-10-yl]acetic acid Compound 6 (36.1 mg, 34%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.51 (d, J=8.0 Hz, 1H), 8.18 (d, J=8.4 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 5.46 (brs, 2H), 2.75 (s, 6H), 1.38 (s, 9H); LCMS (ESI, m/z): [M+H]+=388.1. 1.


Example 6: Synthesis of Compound 6



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Step 1


A mixture of a (500.0 mg, 2.30 mmol, 1.0 eq), b (970.0 mg, 3.46 mmol, 1.5 eq), K2CO3 (635.0 mg, 4.60 mmol, 2.0 eq), CuI (135.0 mg, 0.69 mmol, 0.3 eq), and copper powder (45.0 mg, 0.69 mmol, 0.3 eq) in DMAC (20 mL) was stirred at 140° C. overnight under N2 atmosphere. Then cooled to room temperature, water (30 mL) was added. The filtrate was acidified to pH=2 with 2 N HCl (aq.), extracted with EtOAc (25 mL×2). The combined organic layers were washed with brine (25 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to afford the crude which was purified by trituration with PE (20 mL) to afford c (300.0 mg, 35.0%). LCMS: 370.0 [M+H]+.


Step 2


A mixture of c (150.0 mg, 0.41 mmol) and Eaton's Reagent (10 mL) was heated at 110° C. for 1 h, then cooled and poured into a mixture of ice water. The suspension was extracted with EtOAc (20 mL×2). The combined organic layers were washed with water (15 mL) and brine (15 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated. The residue was purified by chromatography on silica gel (PE/EtOAc=20/1) to afford d (30.0 mg, 21.0%). LCMS: 352.0 [M+H]+


Step 3


A mixture of d (30.0 mg, 0.085 mmol, 1.0 eq) and Cs2CO3 (83.0 mg, 0.26 mmol, 3.0 eq) in MeCN (10 mL) was added e (37.0 mg, 0.17 mmol, 2.0 eq). The mixture was stirred at 35° C. overnight. The resulting mixture was extracted with EtOAc (25 mL×2). The combined organic layers were washed with brine (25 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated, and the residue was purified by Prep-HPLC to afford f (30.0 mg, 80.0%) as a yellow solid. LCMS: 438.0 [M+H]+.


Step 4


A mixture of f (30.0 mg, 0.069 mmol, 1.0 eq), g (16.0 mg, 0.172 mmol, 2.5 eq), Pd2(dba)3 (7.0 mg, 0.0069 mmol, 0.1 eq), Xantphos (4.0 mg, 0.0069 mmol, 0.1 eq) and Cs2CO3 (68.0 mg, 0.207 mmol, 3.0 eq) in toluene (5 mL) was stirred at 80° C. in sealed-tube overnight. The mixture was cooled to room temperature, concentrated in vacuo. The residue was purified by Prep-HPLC to afford h (20.0 mg, 64.7%), LCMS: 451.1 [M+H]+.


Step 5


A mixture of h (20.0 mg, 0.045 mmol, 1.0 eq), LiOH.H2O (8.0 mg, 0.18 mmol, 4.0 eq) in THF/H2O (5/2 mL) was stirred at 50° C. overnight. The aqueous layer was acidified to pH=3 with 2 N HCl (aq) and extracted with EtOAc (15 mL×2). The combined organic layers were washed with brine (10 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to afford Compound 6 (12.0 mg, 64.0%) as a yellow solid. LCMS: 423.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.99 (s, 1H), 8.28 (d, J=8.4 Hz, 1H), 8.17 (d, J=8.4 Hz, 1H), 7.75 (d, J=8.0 Hz, 2H), 7.40-7.32 (m, 3H), 7.07 (t, J=7.2 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H), 5.59 (s, 1H), 4.92 (s, 1H), 2.91 (s, 3H), 2.57 (s, 3H).


Example 7: Synthesis of Compound 7



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Step 1


To a solution of methyl 2,6-dichloropyridine-3-carboxylate (900.00 mg, 4.369 mmol, 1.0 equiv) in H2O (2 mL) and dioxane (8 mL) were added 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (880.90 mg, 5.242 mmol, 1.2 equiv), Pd(dppf)Cl2 (319.64 mg, 0.437 mmol, 0.1 equiv) and K2CO3 (1811.26 mg, 13.106 mmol, 3.0 equiv) at room temperature. The reaction mixture was stirred for 2 h at 80° C. under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeOH in water, 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in methyl 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylate (a, 420 mg, 45%) as a white solid. LCMS (ESI, m/z): [M+H]+=212.0


Step 2


To a solution of methyl 2-chloro-6-(prop-1-en-2-yl)pyridine-3-carboxylate (300.00 mg, 1.417 mmol, 1.0 equiv) in DME (3 mL) were added 6-chloro-N1,N1-dimethylbenzene-1,2-diamine (290.25 mg, 1.701 mmol, 1.2 equiv), Pd2(dba)3 (129.80 mg, 0.142 mmol, 0.1 equiv), X-Phos (135.14 mg, 0.283 mmol, 0.2 equiv) and Cs2CO3 (923.66 mg, 2.835 mmol, 2 equiv) at room temperature. The reaction mixture was stirred for 2 h at 80° C. under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 2-[[3-chloro-2-(dimethylamino)phenyl]amino]-6-(prop-1-en-2-yl)pyridine-3-carboxylate (b, 360 mg, 73%) as a red oil. LCMS (ESI, m/z): [M+H]+=346.1.


Step 3


The mixture of methyl 2-[[3-chloro-2-(dimethylamino)phenyl]amino]-6-(prop-1-en-2-yl)pyridine-3-carboxylate (360.00 mg, 1.041 mmol, 1.0 equiv) and PtO2 (47.28 mg, 0.208 mmol, 0.2 equiv) in EtOAc (5 mL) was stirred for 2 h at room temperature under hydrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford methyl 2-[[3-chloro-2-(dimethylamino)phenyl]amino]-6-isopropylpyridine-3-carboxylate (c, 270 mg, 75%) as a yellow oil. LCMS (ESI, m/z): [M+H]+=348.1.


Step 4


To a solution of methyl 2-[[3-chloro-2-(dimethylamino)phenyl]amino]-6-isopropylpyridine-3-carboxylate (270.00 mg, 0.776 mmol, 1.0 equiv) in THF (2 mL), MeOH (2 mL) and H2O (2 mL) was added LiOH (92.95 mg, 3.881 mmol, 5.0 equiv) at room temperature. The reaction solution was stirred for 2 h at room temperature. The mixture was acidified to pH=4 with HCl (aq.). The precipitated solids were collected by filtration and washed with water (3×5 mL) to afford 2-[[3-chloro-2-(dimethylamino)phenyl]amino]-6-isopropylpyridine-3-carboxylic acid (d, 220 mg, 85%) as a yellow oil. LCMS (ESI, m/z): [M+H]+=334.1.


Step 5


The solution of 2-[[3-chloro-2-(dimethylamino)phenyl]amino]-6-isopropylpyridine-3-carboxylic acid (10.00 mg, 0.030 mmol, 1.0 equiv) in diphosphooxidane-methanesulfonic acid (14.26 mg, 0.060 mmol, 2.0 equiv) was stirred for 10 min at 80° C. The resulting solution was concentrated under reduced pressure. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeOH in water, 10% to 50% gradient in 10 min; detector, UV 254 nm. This resulted in 8-chloro-9-(dimethylamino)-2-isopropyl-10H-benzo[b]1,8-naphthyridin-5-one (e, 130 mg, 62%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=316.1.


Step 6


To a solution of 8-chloro-9-(dimethylamino)-2-isopropyl-10H-benzo[b]1,8-naphthyridin-5-one (130.00 mg, 0.412 mmol, 1.0 equiv) in MeCN (4 mL) were added ethyl iodoacetate (132.14 mg, 0.617 mmol, 1.5 equiv) and Cs2CO3 (402.37 mg, 1.235 mmol, 3.0 equiv) at room temperature. The reaction mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (5:1) to afford ethyl 2-[8-chloro-9-(dimethylamino)-2-isopropyl-5-oxobenzo[b]1,8-naphthyridin-10-yl]acetate (f, 50 mg, 30%) as a yellow solid. LCMS (ESI, m/z): [M+H]+=402.2.


Step 7


To a solution of ethyl 2-[8-chloro-2-isopropyl-9-(methylamino)-5-oxobenzo[b]1,8-naphthyridin-10-yl]acetate (50.00 mg, 0.129 mmol, 1.0 equiv) in THF (1 mL), MeOH (1 mL) and H2O (1 mL) was added LiOH (15.44 mg, 0.645 mmol, 5.0 equiv) at room temperature. The reaction solution was stirred for 1 h at 60° C. The mixture was acidified to pH=5 with HCl (aq.). The precipitated solids were collected by filtration to afford the crude product. The crude product was re-crystallized from MeOH to afford Compound 7 (10.4 mg, 20%) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 12.59 (s, 1H), 8.50 (d, J=8.0 Hz, 1H), 8.20 (d, J=8.6 Hz, 1H), 7.42 (d, J=8.6 Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 5.48 (s, 2H), 3.17 (m, 1H), 2.76 (s, 6H), 1.30 (d, J=6.9 Hz, 6H); LCMS (ESI, m/z): [M+H]+=374.1.


Example 8: Synthesis of Compound 8



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Step 1


To a solution of a (1.73 g, 10.0 mmol, 1.0 eq) in MeOH (30 mL) was added b (15 mL, 30.0 mmol, 3.0 eq). The mixture was stirred at 0° C. for 1 h, then diluted with water (30 mL), and extracted with EtOAc (30 mL×2). The combined organic layers were washed with water (30 mL) and brine (30 mL), then dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel (PE/EtOAc=5/1) to afford c (1.3 g, 70%). LC/MS: 187.0 [M+H]+.


Step 2


A mixture of the c (1.3 g, 6.99 mmol, 1.0 eq), d (2.15 g, 13.98 mmol, 2.0 eq), Pd(dppf)Cl2 (510.0 mg, 0.699 mmol, 0.1 eq) and K2CO3 (2.41 g, 17.475 mmol, 2.5 eq) in 1,4-dioxane/H2O (50 mL/5 mL) was stirred at 70° C. overnight under nitrogen atmosphere. The mixture was diluted with water (30 mL), extracted with EtOAc (30 mL×2). The combined organic layers were washed with water (30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel (PE/EtOAc=5/1) to afford e (540.0 mg, 43.4%).


Step 3


A mixture of e (540.0 mg, 3.03 mmol, 1.0 eq) and Pd/C (300.0 mg) in MeOH (10 mL) was stirred at 35° C. overnight under H2 atmosphere (1 atm), then filtered. The filtrate was concentrated in vacuo to afford f (520.0 mg, 99%). LC/MS: 181. 1 [M+H]+.


Step 4


A mixture of f (520.0 mg, 2.89 mmol, 1.0 eq), KOH (325.0 mg, 5.78 mmol, 2.0 eq) in MeOH (20.0 mL) and H2O (3.0 mL) was stirred at 50° C. overnight. The mixture was concentrated in vacuo to afford g (480.0 mg, ca. 100%). LC/MS: 167. 1 [M+H]+.


Step 5


A mixture of g (250.0 mg, 1.5 mmol, 1.0 eq), h (840.0 mg, 3.0 mmol, 2.0 eq), K2CO3 (420.0 mg, 3.0 mmol, 3.0 eq), CuI (90.0 mg, 0.45 mmol, 0.3 eq), and copper powder (20.0 mg, 0.45 mmol, 0.3 eq) in DMAC (10 mL) was stirred at 155° C. overnight under nitrogen atmosphere, then cooled to room temperature. Water (30 mL) was added. The mixture was filtered through celite. The filtrate was acidified to pH=2 with 2N HCl (aq). The resulting mixture was extracted with EtOAc (25 mL×2). The combined organic layers were washed with brine (25 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to afford crude which was purified by trituration with PE (20 mL) to afford i (220.0 mg, 45.8%). LC/MS: 320. 1 [M+H]+.


Step 6


A mixture of i (220.0 mg, 0.69 mmol) and Eaton's Reagent (20 mL) was heated at 110° C. for 2.5 h, then cooled and poured into a mixture of ice and water. The solid was collected by filtration and dried to afford crude j (132.0 mg, 63.6%). LC/MS: 302.1 [M+H]+


Step 7


A mixture of j (132.0 mg, 0.439 mmol, 1.0 eq) and Cs2CO3 (430.0 mg, 1.317 mmol, 3.0 eq) in acetonitrile (20 mL) was added k (188.0 mg, 0.877 mmol, 2.0 eq). The mixture was stirred at 35° C. overnight and then diluted with EtOAc (25 mL×2), washed with brine (25 mL×2). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford l (100.0 mg, 98%) as a yellow solid. LC/MS: 388.1 [M+H]+.


Step 8


A mixture of 1 (100.0 mg, 0.26 mmol, 1.0 eq), LiOH.H2O (43.0 mg, 1.03 mmol, 4.0 eq) in THF (5 mL) and H2O (2 mL) was stirred at 40° C. overnight, then diluted with water (30 mL), extracted with EtOAc (20 mL×2). The aqueous layer was acidified to pH=3 with 2N HCl (aq) and extracted with EtOAc (15 mL×2). The combined organic layers were washed with brine (10 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to afford Compound 8 (78.0 mg, 84%) as a yellow solid. LC/MS: 360.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.59 (brs, 1H), 8.50 (d, J=8.0 Hz, 1H), 8.21 (d, J=8.8 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 7.34 (d, J=8.0 Hz, 1H), 5.48 (s, 2H), 2.94-2.88 (m, 2H), 2.76 (s, 6H), 1.30 (t, J=7.6 Hz, 3H).


Example 9: Synthesis of Compound 9



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Step 1


A mixture of a (8.0 g, 45.6 mmol), Cs2CO3 (52 g, 159.6 mmol) and propan-2-ol (8.2 g, 136.8 mmol) in DMF (200 mL) was stirred at 40° C. for 8 h. The reaction mixture was added with water (500 mL) and extracted with EtOAc (500 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered and concentrated in vacuo to afford crude b (9.5 g, 97%).


Step 2


A mixture of b (9.5 g, 44.2 mmol), Fe (7.4 g, 132.6 mmol) and NH4Cl (7.1 g, 132.6 mmol) in EtOH (100 mL)/H2O (10 mL) was stirred at 80° C. for 2 h. The reaction mixture was concentrated in vacuo to afford the crude which was purified by chromatography column on silica gel (PE/EtOAc=20/1) to afford c (8.0 g, 98%). LCMS: 186.0 [M+H]+.


Step 3


To a mixture of c (8.0 g, 43.2 mmol) in DCM (400 mL) was added Br2 (6.92 g, 43.2 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 1 h, washed with Na2CO3 (sat. aq.) and water successively. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel (PE/EtOAc=100/1) to afford d (4.8 g, 42%). LCMS: 265.9 [M+H]+.


Step 4


A mixture of d (4.8 g, 18.25 mmol), Mel (26 g, 182.5 mmol), and Cs2CO3 (17.8 g, 54.75 mmol) in DMF (72 mL) was stirred at 85° C. for 16 h, then cooled to room temperature. The resulting mixture was concentrated, and the residue was purified by chromatography on silica gel (PE/EtOAc=100/1) to afford e (5.0 g, 94%). LCMS: 294.0 [M+H]+.


Step 5


A mixture of e (2.4 g, 9.16 mmol), f (1.4 g, 9.16 mmol), Cu (0.17 g, 2.75 mmol), CuI (0.52 g, 2.75 mmol) and K2CO3 (2.5 g, 18.32 mmol) in DMA (20 mL) was stirred at 165° C. for 8 h under argon atmosphere. After filtration, the filtrate was concentrated, and the residue purified by Prep-HPLC to afford g (1.4 g, 41%). LCMS: 364.2 [M+H]+.


Step 6


A mixture of g (800 mg) and Eaton's Reagent (40 mL) was heated at 105° C. for 6 h under argon atmosphere, then cooled and poured into a mixture of ice and water. The solid was collected by filtration and purified by trituration with MeCN (5 mL) to afford h (150 mg, 22%). LCMS: 304.0 [M+H]+.


Step 7


To a mixture of h (100 mg, 0.33 mmol) and Cs2CO3 (430 mg, 1.32 mmol) in DMF (30 mL) was added 2-iodopropane (280 mg, 1.65 mmol) at rt. The mixture was then stirred at 90° C. for 16 h under argon atmosphere, filtered. The filtrate was concentrated, and the residue was purified by Prep-HPLC to afford j (50 mg, 44%). LCMS: 346.1 [M+H]+.


Step 8


To a mixture of j (30 mg, 0.087 mmol) and Cs2CO3 (85 mg, 0.261 mmol) in DMF (10 mL) was added ethyl iodoatetate (47 mg, 0.217 mmol) at rt. The mixture was then stirred at 30° C. for 1 h under argon atmosphere. The resulting mixture was diluted with water (50 mL), extracted with EtOAc (30 mL×2). The organic layers were concentrated, and the residue was purified by Prep-HPLC to afford k (65 mg, ca 100%). LCMS: 432.1 [M+H]+.


Step 9


A mixture of k (65 mg) and Eaton's Reagent (5 mL) was heated at 50° C. for 16 h under argon atmosphere. The resulting mixture was diluted with water (50 mL), extracted with EtOAc (30 mL×2). The organic layers were concentrated, and the residue was purified by Prep-HPLC to afford l (9.0 mg, 26.6%). LCMS: 390.1 [M+H]+.


Step 10


A mixture of l (9.0 mg, 0.023 mmol) and LiOH.H2O (10 mg, 0.23 mmol) in THF (5 mL) and H2O (5 mL) was stirred at 50° C. overnight. The mixture was diluted with water (10.0 mL), acidified with conc. HCl to pH=1, extracted with EtOAc (30 mL×2). The organic layer was concentrated, and the residue was purified by Prep-HPLC to afford Compound 9 (5.0 mg, 60%). LCMS: 362.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 14.78 (brs, 1H), 8.46 (d, J=8.8 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 6.75 (s, 1H), 5.63 (s, 2H), 2.73 (s, 6H), 2.60 (s, 3H).


Example 10: Synthesis of Compound 10



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A mixture of 4 (30.0 mg, 0.09 mmol, 1.0 eq), a (10.0 mg, 0.11 mmol, 1.2 eq), EDCI (21.0 mg, 0.11 mmol, 1.2 eq), HOBt (15.0 mg, 0.11 mmol, 1.2 eq) and Et3N (18.0 mg, 0.18 mmol, 2.2 eq) in anhydrous DCM (3 mL) was stirred at rt overnight under nitrogen atmosphere. The solvent was removed under reduced pressure. The residue was purified by Prep-HPLC to afford Compound 10 (3.9 mg, 11%) as a yellow solid. LC/MS: 423.2 [M+H]+, 1H NMR (400 MHz, DMSO-d6): δ 8.45 (d, J=8.0 Hz, 1H), 8.14 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 7.27 (d, J=8.0 Hz, 1H), 5.63 (brs, 2H), 2.80 (s, 6H), 2.72 (s, 3H), 2.61 (s, 3H).


Example 11: Synthesis of Compound 11



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A mixture of 4 (30.0 mg, 0.09 mmol, 1.0 eq), a (5.0 mg, 0.11 mmol, 1.2 eq), EDCI (21.0 mg, 0.11 mmol, 1.2 eq), HOBt (15.0 mg, 0.11 mmol, 1.2 eq) and TEA (18.0 mg, 0.18 mmol, 2.2 eq) in anhydrous DCM (3 mL) was stirred at rt overnight under nitrogen atmosphere. The mixture was diluted with water (10 mL), extracted with EtOAc (15 mL). The organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by Prep-HPLC to afford Compound 11 (18 mg, purity: 90%) which was further purified by Prep-TLC to afford Compound 11 (12 mg, 38%) as a yellow solid. LC/MS: 370.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.46 (d, J=7.6 Hz, 1H), 8.15 (d, J=8.4 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 7.29 (d, J=7.6 Hz, 1H), 5.51 (brs, 2H), 2.76 (s, 6H), 2.62 (s, 3H).


Example 12: Synthesis of Compound 12



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Step 1


To a mixture of a (180.0 mg, 0.59 mmol, 1.0 eq), Cs2CO3 (579.0 mg, 1.78 mmol, 3.0 eq) in MeCN (10.0 mL) was added b (254.0 mg, 1.18 mmol, 2.0 eq). Then the mixture was stirred at 35° C. for 4 h. When the starting material was consumed completely, the reaction mixture was diluted with EtOAc (20 mL) and washed with H2O (10 mL×3). The organic phase was dried over anhydrous Na2SO4, and then filtered. The filtrate was concentrated and purified by chromatography on silica gel (PE/EtOAc=6/1) to afford c (185.0 mg, 80.0%). LCMS: 391.2 [M+H]+.


Step 2


To a mixture of d (78.0 mg, 0.2 mmol, 1.0 eq) in DMF (6 mL) was added NaSMe (16.8 mg, 0.24 mmol, 1.2 eq). The mixture was stirred overnight at 40° C. When the starting material was consumed completely, the reaction mixture was diluted with EtOAc (20 mL) and washed with H2O (10 mL×3). The organic phase was dried over anhydrous Na2SO4, and then filtered. The filtrate was concentrated and purified by chromatography on silica gel (PE/EtOAc=6/1) to afford d (37.0 mg, 44.0%). LCMS: 419.3 [M+H]+.


Step 3


To a mixture of d (37.0 mg, 0.089 mmol, 1.0 eq) in THF/H2O (2.0/0.5 mL) was added LiOH.H2O (37.0 mg, 0.89 mmol, 10.0 eq). The mixture was stirred overnight at 50° C., evaporated to remove THF and then added with H2O (10 mL). The resulting mixture was acidified to pH=5-6 with 1 N HCl (aq.), extracted with EtOAc (15 mL×5). The combined organic phase was washed with brine (20 mL) and dried over anhydrous Na2SO4, then filtered. The filtrate was concentrated and purified by chromatography on silica gel (PE/EtOAc=2/1) to afford Compound 12 (5.7 mg, 16.0%). LCMS: 391.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.47 (d, J=8.4 Hz, 1H), 8.22 (d, J=8.8 Hz, 1H), 7.53 (d, J=8.8 Hz, 1H), 7.47 (d, J=8.0 Hz, 1H), 5.64 (s, 2H), 2.32 (s, 3H), 1.37 (s, 9H).


Example 13: Synthesis of Compound 13



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To a solution of a (100.0 mg, 0.33 mmol, 1.0 eq) in DMF (5.0 mL) was added NaSMe (28.0 mg, 0.39 mmol, 1.2 eq). The mixture was stirred for 24 h at 80° C. then diluted with EtOAc (20 mL) washed with water (25.0 mL×2). The aqueous phase was acidified to pH=5 with 1 N HCl (aq.) and extracted with EtOAc (20 mL×2). The combined organic layers were concentrated. The residue was purified by chromatography on silica gel (PE/EtOAc=2/1) to afford Compound 13 (41.4 mg, 51%). LCMS: 403.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.72 (brs, 1H), 8.49 (d, J=8.0 Hz, 1H), 8.30 (d, J=8.8 Hz, 1H), 7.49 (d, J=8.0 Hz, 1H), 8.49 (d, J=8.4 Hz, 1H), 5.52 (s, 2H), 2.53 (s, 3H), 2.21 (s, 3H), 1.37 (s, 9H).


Example 14: Synthesis of Compound 14



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To a mixture of a (20.0 mg, 0.051 mmol, 1.0 eq) in THF/H2O (5.0/1.0 mL) was added LiOH.H2O (11.0 mg, 0.26 mmol, 10.0 eq). The mixture was stirred at 50° C. overnight, acidified to pH=5 with 1 N HCl (aq.) and extracted with EtOAc (20 mL×2). The combined organic layers were concentrated. The residue was purified by chromatography on silica gel (PE/EtOAc=2/1) to afford Compound 14 (9.8 mg, 53%). LCMS: 363.2 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 13.07 (brs, 1H), 8.58 (d, J=8.4 Hz, 1H), 8.17 (d, J=8.8 Hz, 1H), 7.60-7.53 (m, 2H), 5.54 (s, 2H), 1.38 (s, 9H).


Example 15. Preparation of ethyl 2-(2,8-dichloro-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (Intermediate A)



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Step 1: A heterogeneous mixture of 2-amino-6-chloronicotinic acid (a) (10.0 g, 57.94 mmol), (2-chloro-6-iodophenyl)(methyl)sulfane (b) (21.4 g, 75.32 mmol), K2CO3 (16.0 g, 115.88 mmol), copper powder (1.1 g, 17.38 mmol), and CuI (3.4 g, 17.38 mmol) in DMA (250 mL) was stirred at 140° C. for 16 h under N2 atmosphere, then cooled to room temperature. The mixture was filtered through celite. The filtrate was acidified to pH=2 with conc. HCl and extracted with ethyl acetate (200 mL×3). The combined organic layers were concentrated. The residue was purified by trituration with petroleum ether to afford c (18.0 g, 94.7%). LC/MS: 329.0 [M+H]+.


Step 2: To a mixture of c (18.0 g, 54.9 mmol) in Eaton's Reagent (150 mL) was added P205 (50 g). The mixture was stirred at 60° C. for 2 hours, poured into ice-water and filtered. The solid obtained was further purified by trituration with petroleum ether to afford d (14.0 g, 82.0%). LC/MS: 311.0 [M+H]+.


Step 3: A mixture of d (9.0 g, 29.0 mmol), ethyl 2-iodoacetate (e) (12.5 g, 58.0 mmol), and Cs2CO3 (28.4 g, 87.0 mmol) in acetonitrile (100 mL) was stirred at room temperature for 16 h. The reaction mixture was poured into water (200 mL), then extracted with ethyl acetate (100 mL×3). The extracts were combined and washed with brine. The organic layer dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=10:1) to afford Intermediate A (5.2 g, 45.2%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.57 (d, 1H), 8.27 (d, 1H), 7.67 (d, 1H), 7.52 (d, 1H), 5.52 (s, 2H), 4.13 (q, 2H), 2.31 (s, 3H), 1.16 (t, 3H). LC/MS: 397.1 [M+H]+.


Example 16. Preparation of ethyl 2-(2,8-dichloro-9-ethoxy-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (Intermediate B)



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Step 1: A heterogeneous mixture of 2-amino-6-chloronicotinic acid (a) (14.5 g, 0.084 mmol), 1-chloro-2-ethoxy-3-iodobenzene (a′) (30.8 g, 0.109 mmol), K2CO3 (23.2 g, 0.168 mmol), copper powder (1.57 g, 0.025 mmol), and CuI (4.8 g, 0.025 mmol) in DMA (225 mL) was stirred at 140° C. for 16 h under N2 atmosphere, then cooled to room temperature. The mixture was filtered through celite. The filtrate was acidified to pH=2 with conc. HCl and extracted with ethyl acetate (200 mL×3). The combined organic layers were concentrated. The residue was purified by trituration with petroleum ether to afford b (22.0 g, 80.1%). LC/MS: 327.0 [M+H]+.


Step 2: To a mixture of b (18.4 g, 0.056 mmol) in Eaton's Reagent (500 mL) was added P205 (120 g). The mixture was stirred at 50° C. for 5 hours, poured into ice-water and filtered. The solid was collected by filtration and dried to afford c (13.6 g, crude 78.3%). LC/MS: 309.0 [M+H]+.


Step 3: A mixture of c (13.6 g, 0.044 mmol), ethyl 2-bromoacetate (d) (18.3 g, 0.110 mmol), Cs2CO3 (43.2 g, 0.132 mmol) in acetonitrile (200 mL) was stirred at 35° C. for 16 h. The reaction mixture was poured into water (300 mL), then extracted with ethyl acetate (100 mL×3). The extracts were combined and washed with brine. The organic layer dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=10:1) to afford Intermediate B (7.0 g, 40.5%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.61 (d, 1H), 8.10 (d, 1H), 7.57 (d, 1H), 7.53 (d, 1H), 5.57 (s, 2H), 4.20 (q, 2H), 3.99 (q, 2H), 1.37 (t, 3H), 1.23 (t, 3H). LC/MS: 395.1 [M+H]+.


Example 17. Preparation of ethyl 2-(2,8-dichloro-9-(dimethylamino)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (Intermediate C)



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Step 1: A mixture of 2-amino-6-chloronicotinic acid (la) (10.0 g, 0.058 mmol), 2-chloro-6-iodo-N,N-dimethylaniline (3a) (24.4 g, 0.087 mmol), K2CO3 (16.0 g, 0.116 mmol), copper powder (1.1 g, 0.017 mmol), and CuI (3.23 g, 0.017 mmol) in DMA (100 mL) was stirred at 140° C. for 16 h under N2 atmosphere and cooled to room temperature. The mixture was filtered through celite. The filtrate was acidified to pH=2 with conc. HCl and extracted with ethyl acetate (200 mL×3). The combined organic layers were concentrated. The residue was purified by trituration with petroleum ether to afford 3b (15.0 g, 79.3%). LC/MS: 326.0 [M+H]+.


Step 2: To a solution of 3b (15.0 g, 0.046 mmol) in Eaton's Reagent (300 mL) was added P205 (100 g). The mixture was stirred at 105° C. for 16 h, poured into ice-water and filtered. The solid was collected by filtration to afford 3c (7.0 g, crude 49.4%). LC/MS: 308.0 [M+H]+.


Step 3: A mixture of 3c (7.0 g, 0.023 mmol), ethyl 2-bromoacetate (2d) (18.0 g, 0.108 mmol), and Cs2CO3 (35.1 g, 0.108 mmol) in acetonitrile (200 mL) was stirred at 35° C. for 16 h. The reaction mixture was poured into water (300 mL), then extracted with ethyl acetate (100 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=10:1) to afford Intermediate C (3.3 g, 36.8%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.58 (d, 1H), 8.19 (d, 1H), 7.52-7.49 (m, 2H), 5.28 (s, 2H), 4.24 (q, 2H), 2.72 (s, 6H), 1.26 (t, 3H). LC/MS: 394.0 [M+H]+.


Example 18. Preparation of 2-(3,8-dichloro-9-(dimethylamino)-2-ethyl-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (17)



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Step 1: To a mixture of a (2.0 g, 9.2 mmol) in DMF (40 mL) was added NCS (1.4 g, 10 mmol). The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was subsequently poured into ice water. After the ice had melted, the resulting slurry was filtered through a fritted glass funnel. The collected solids were dried to afford crude b (2.2 g, 95%). LC/MS: 250.9 [M+H]+.


Step 2: A heterogeneous mixture of b (1.0 g, 4.0 mmol), 3a (1.7 g, 6.0 mmol), Cu (77 mg, 1.2 mmol), CuI (230 mg, 1.2 mmol) and K2CO3 (1.1 g, 8 mmol) in DMA (20 mL) was stirred at 140° C. for 16 h under N2 atmosphere and then cooled to room temperature. The mixture was filtered through celite. The filtrate was acidified to pH=2 with conc. HCl and extracted with ethyl acetate (50 mL×3). The extracts were combined and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=20/1) to afford c (950 mg, crude 58.9%). LC/MS: 406.0 [M+H]+.


Step 3: A mixture of c (600 mg, 1.5 mmol) and PPA (25 mL) was stirred at 120° C. for 2 h. The reaction mixture was poured into ice water and then extracted with ethyl acetate. The extracts were combined and dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=40/1) to afford d (100 mg, 17.5%). LC/MS: 387.9 [M+H]+.


Step 4: A heterogeneous mixture of d (100 mg, 0.26 mmol), iodoester (112 mg, 52 mmol), and Cs2CO3 (254 mg, 0.78 mmol) in acetonitrile (20 mL) was stirred at 35° C. for 16 h. The reaction was monitored by LCMS. The reaction mixture was poured into water (10 mL) and then extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=40/1 to 20/1) to afford e (80 mg, 65.5%). LC/MS: 474.0 [M+H]+.


Step 5: A mixture of e (35.0 mg, 0.074 mmol), vinylboronic acid pinacol ester (f) (28.5 mg, 0.185 mmol), Pd(dppf)Cl2. CH2Cl2 (6.1 mg, 0.0074 mmol), K2CO3 (25.5 g, 0.185 mmol) in 1,4-dioxane/H2O (10 mL/2 mL) was stirred at 70° C. for 16 h under nitrogen atmosphere. The mixture was diluted with water (5 mL) and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with water (10 mL), brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel (petroleum ether/ethyl acetate=40/1) to afford g (25.0 mg, 80.6%). LC/MS: 420.1 [M+H]+.


Step 6: A mixture of g (25.0 mg, 0.059 mmol) and Raney-Ni (50 mg) in MeOH (5 mL) was stirred at room temperature for 1 h under H2 atmosphere (1 atm) and then filtered. The filtrate was concentrated in vacuo to afford h (20.0 mg, crude) which was used directly for the next reaction without purification. LC/MS: 422.1 [M+H]+.


Step 7: A mixture of h (20.0 mg, crude, 0.047 mmol), LiOH.H2O (19.9 mg, 0.474 mmol) in a solvent mixture of THF (5 mL), EtOH (2 mL) and H2O (2 mL) was stirred at 40° C. for 16 h. The reaction mixture was diluted with water (5 mL) and washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (5 mL×2). The combined organic layers were washed with brine (5 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford i (5.0 mg, 26.7%) as a yellow solid. LC/MS: 394.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.47 (s, 1H), 8.20 (d, J=8.4 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 5.44 (s, 2H), 3.06-3.00 (m, 2H), 2.76 (s, 6H), 1.31 (t, J=7.2 Hz, 3H).


Example 19. Preparation of sodium 2-(8-chloro-2,9-bis(dimethylamino)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (19)



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Step 1: To a mixture of Intermediate C (250.0 mg, 0.64 mmol) in a mixed solvent of THF (24 mL) and water (6 mL) was added dimethylamine (2 M in THF, 4 mL, 8.0 mmol) and LiOH.H2O (269.0 mg, 6.4 mmol), successively. The reaction mixture was heated to 50° C. and stirred for 16 h in a sealed tube, before evaporating in vacuo to remove the THF. The suspension was acidified to pH=1 with 2 N HCl. The precipitate that formed was collected by filtration and dried to afford 5a (220.0 mg, 92%). LC/MS: 375.1 [M+H]+.


Step 2: To a mixture of 19 (220.0 mg, 0.59 mmol) in a mixed solvent of THF (10 mL) and water (5 mL) was added NaOH (21.0 mg, 0.53 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 19-Na (180.0 mg, 77.0%) as a yellow solid. LC/MS: 375.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.27 (d, J=8.8 Hz, 1H), 8.20 (s, J=7.6 Hz, 1H), 7.41 (d, J=7.6 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H), 5.42 (s, 2H), 3.24 (s, 6H), 2.80 (s, 6H).


Example 20. Preparation of sodium 2-(8-chloro-2-(dimethylamino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (22)



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Step 1: The Intermediate A (100 mg, 0.25 mmol) was dissolved in a mixed solvent of THF (2 mL) and water (1 mL), and added with Dimethylamine (2 N in THF, 1.65 mL, 3.3 mmol) and LiOH.H2O (52.5 mg, 1.25 mmol) successively. The reaction mixture was heated to 50° C. and stirred for 16 h in a sealed tube, and then evaporated to remove THF. The suspension was acidified to pH=1 with 2 N HCl. The precipitate that formed was collected by filtrate and dried to afford 22 (85.0 mg, 89%). LC/MS: 378.0 [M+H]+.


Step 2: To a mixture of 6a (85.0 mg, 0.225 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (8.0 mg, 0.20 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 22-Na (80.0 mg, 88.9%) as a yellow solid. LC/MS: 378.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (d, J=8.8 Hz, 1H), 8.10 (d, J=8.0 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 6.66 (d, J=8.8 Hz, 1H), 5.46 (s, 2H), 3.18 (s, 6H), 2.29 (s, 3H).


Example 21. Preparation of sodium 2-(8-chloro-2-(dimethylamino)-9-ethoxy-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (23)



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Step 1: The Intermediate B (100 mg, 0.25 mmol) was dissolved in a mixed solvent of THF (2 mL) and water (1 mL) where dimethylamine (2 N in THF, 1.65 mL, 3.3 mmol) and LiOH.H2O (52.5 mg, 1.25 mmol) were added successively. The reaction mixture was heated to 50° C. and stirred for 16 h in a sealed tube, before evaporating to remove the THF. The remaining suspension was extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford 23 (30.0 mg, 32%). LC/MS: 376.1 [M+H]+.


Step 2: To a mixture of 23 (30.0 mg, 0.08 mmol) in a mixed solvent of THF (2 mL) and water (2 mL) was added NaOH (2.9 mg, 0.072 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 23-Na (21.1 mg, 67%) as a yellow solid. LC/MS: 376.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.20 (d, J=8.8 Hz, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.32 (d, J=8.4 Hz, 1H), 6.69 (d, J=8.8 Hz, 1H), 5.43 (brs, 2H), 3.93 (q, J=6.8 Hz, 2H), 3.18 (s, 6H), 1.38 (t, J=6.8 Hz, 3H).


Example 22. Preparation of Sodium 2-(8-chloro-2-(((3,3-difluorocyclobutyl)methyl)amino)-9-(dimethylamino)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (26) and 26-sodium salt



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Step 1: A mixture of Intermediate C (100.0 mg, 0.254 mmol), a (80.0 mg, 0.507 mmol) and triethylamine (256.5 mg, 2.54 mmol) in THF (2 mL) was heated to 90° C. and stirred for 16 h in a sealed tube. The reaction mixture was cooled to room temperature and diluted with water (15 mL). The precipitate that formed was filtered and dried to afford b (110.0 mg, 90%). LC/MS: 479.1 [M+H]+.


Step 2: A mixture of b (110.0 mg, 0.23 mmol), LiOH.H2O (77.0 mg, 1.84 mmol) in a mixed solvent of THF (3 mL), EtOH (2 mL) and H2O (1 mL) was stirred at 40° C. for 16 h. The reaction mixture was diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (10 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 26 (89.0 mg, 86%) as a yellow solid. LC/MS: 451.1 [M+H]+.


Step 3: To a mixture of 8c (89.0 mg, 0.20 mmol) in a mixed solvent of THF (3 mL) and water (3 mL) was added NaOH (7.2 mg, 0.18 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 26-Na (57.0 mg, 61%) as a yellow solid. LC/MS: 451.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.04 (d, J=8.8 Hz, 2H), 7.90 (s, 1H), 7.24 (d, J=8.8 Hz, 1H), 6.44 (d, J=8.4 Hz, 1H), 5.45 (brs, 2H), 3.52 (s, 2H), 2.79 (s, 6H), 2.71-2.60 (m, 3H), 2.43-2.32 (m, 2H).


Example 23. Preparation of Sodium 2-(8-chloro-2-(((3,3-difluorocyclobutyl)methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (54)



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Step 1: A mixture of Intermediate C (200.0 mg, 0.51 mmol), a (240.0 mg, 1.52 mmol) and triethylamine (515.0 mg, 5.10 mmol) in THF (3 mL) was heated to 90° C. and stirred for 16 h in a sealed tube. The reaction mixture was cooled to room temperature and diluted with water (15 mL). The precipitate that formed was collected by filtrate to afford b (230.0 mg, 94.6%). LC/MS: 482.1 [M+H]+.


Step 2: A mixture of b (60.0 mg, 0.125 mmol) and LiOH.H2O (21.0 mg, 0.5 mmol) in a mixed solvent of THF (5 mL), EtOH (5 mL) and H2O (2 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (10 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 54 (55.0 mg, 97.3%) as a yellow solid. LC/MS: 454.0 [M+H]+.


Step 3: To a mixture of 54 (55.0 mg, 0.12 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (4.4 mg, 0.11 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 54-Na (48.0 mg, 83.2%) as a yellow solid. LC/MS: 454.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.09 (d, J=8.4 Hz, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.87 (s, 1H), 7.37 (d, J=8.4 Hz, 1H), 6.42 (d, J=8.4 Hz, 1H), 5.48 (brs, 2H), 3.53 (s, 2H), 2.71-2.60 (m, 2H), 2.44-2.31 (m, 3H), 2.29 (s, 3H).


Example 24. Preparation of sodium 2-(8-chloro-9-(methylthio)-2-((oxetan-3-ylmethyl)amino)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (55) and 55-sodium salt



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Step 1: A mixture of Intermediate A (100 mg, 0.250 mmol), a (55 mg, 0.63 mmol), TEA (253 mg, 2.5 mmol), and THF (3 mL) was stirred at 90° C. in a sealed tube for 16 h. LCMS showed Intermediate A was completely consumed and one major peak with the desired mass was detected. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (5 mL×3). The combined organic phases were concentrated and then purified by Prep-HPLC to afford b (90 mg, 80%). LC/MS: 448.1 [M+H]+.


Step 2: To a mixture of b (40 mg, 0.09 mmol) in ethanol (5 mL) was added LiOH (0.8 mL, 0.1 M in water, 0.08 mmol) and the resultant mixture was heated to 90° C. for 48 h. LCMS showed 77% of b remained with 24% of 55. To the reaction mixture was added more LiOH (0.8 mL, 0.1 M in water, 0.08 mmol) and the resulting mixture was heated to 90° C. for 2 h. LCMS showed b was completely consumed. The reaction mixture was diluted with water (15 mL) and washed with ethyl acetate (3 mL). The aqueous layer was acidified with AcOH to pH=4-5 and then extracted with ethyl acetate (5 mL×3). The combined organic phases were concentrated to give 55 (30 mg, 83%). LC/MS: 420.2 [M+H]+.


Step 3: To a mixture of 5 (70 mg, 0.067 mmol) in THF (3 mL) was added NaOH (0.01 M, 6 mL, 0.137 mmol). The resulting mixture was stirred at room temperature for 1 h. The mixture was washed with ethyl acetate and lyophilized to afford 55-Na (58.3 mg, 79%, >99% purity by LCMS). LCMS: 420.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.10 (d, J=8.4 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H), 7.89 (s, 1H), 7.37 (d, J=8.4 Hz, 1H), 6.42 (d, J=9.2 Hz, 1H), 5.47 (s, 2H), 4.68-4.64 (m, 2H), 4.38-4.35 (m, 2H), 3.67-3.66 (m, 2H), 3.29-3.24 (m, 1H), 2.29 (s, 3H).


Example 25. Preparation of sodium 2-(8-chloro-2-((3-cyanocyclobutyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (56)



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Step 1: A mixture of a (2.0 g, 1.7 mmol) and TEA (2.0 mL, 15.0 mmol) in DCM at 0° C. was added methanesulfonyl chloride (1.0 mL, 12.8 mmol). The reaction was stirred at room temperature for 16 h under N2. A saturated, aqueous mixture of sodium bicarbonate was added and then extracted with DCM. The organic layer was washed with brine and then concentrated to afford b (crude, 3.0 g, >99%).


Step 2: A mixture of b (3.0 g, 14.3 mmol), NaCN (2.22 g, 45.2 mmol) and TBAB (2.2 g, 6.8 mmol) in DMF were heated to 120° C. for 16 h under N2. Water was added and then extracted with ethyl acetate. The organic layer was washed with brine and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=2/1) to afford c (crude, 2.0 g, 91%).


Step 3: To a mixture of c (2.0 g, 10 mmol) in dioxane (10 mL) was added a mixture of HCl in dioxane (4 mL, 4 M). The mixture was stirred at room temperature for 16 h under N2. The reaction was concentrated to afford d (crude, 0.8 g, 80%).


Step 4: A mixture of Intermediate A (300.0 mg, 0.75 mmol), d (800.0 mg, 8.33 mmol) and triethylamine (757.5 mg, 7.5 mmol) in THF (10 mL) was heated to 110° C. for 16 h in a sealed tube. The reaction mixture was evaporated and the residue was by Prep-HPLC to afford e (50.0 mg, 14.4%). LC/MS: 457.1 [M+H]+.


Step 5: A mixture of e (35.0 mg, 0.07 mmol) and LiOH.H2O (29.0 mg, 0.7 mmol) in a mixed solvent of THF (2 mL), EtOH (2 mL) and H2O (1 mL) was stirred at room temperature for 2 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (5 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to afford 56 (33.0 mg, 99%) as a yellow solid. LC/MS: 429.0 [M+H]+.


Step 6: To a mixture of 56 (33.0 mg, 0.07 mmol) in a mixed solvent of THF (2 mL) and water (1 mL) was added NaOH (2.5 mg, 0.06 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 56-Na (5.0 mg, 14.4%) as a yellow solid. LC/MS: 429.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.12-8.06 (m, 3H), 7.41 (d, J=8.4 Hz, 1H), 6.42 (d, J=8.8 Hz, 1H), 5.51 (s, 2H), 3.39-3.35 (m, 1H), 3.18-3.11 (m, 1H), 2.71-2.67 (m, 2H), 2.46-3.39 (m, 2H), 2.30 (s, 3H).


Example 26. Preparation of sodium 2-(8-chloro-9-ethoxy-5-oxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)benzo[b][1,8]naphthyridin-10(5H)-yl)acetate (60)



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Step 1: A mixture of Intermediate B (200.0 mg, 0.51 mmol), a (117.5 mg, 1.02 mmol) and Na2CO3 (268.2 mg, 2.53 mmol) in 1,4-dioxane (10 mL)/water (5 mL) was heated to 80° C. for 16 h. The reaction mixture was poured into water (200 mL) and then extracted with ethyl acetate (100 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford b (150.0 mg, 62.7%). LC/MS: 474.2 [M+H]+.


Step 2: A mixture of b (130.0 mg, 0.21 mmol) and LiOH.H2O (44.1 mg, 1.05 mmol) in a mixed solvent of THF (4 mL), EtOH (2 mL) and H2O (1 mL) was stirred at 50° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl. The precipitate was collected by filtration, washed with water and dried under vacuum to afford 60 (112.0 mg, 91.8%) as a yellow solid. LC/MS: 446.1 [M+H]+.


Step 3: To a mixture of 60 (80.0 mg, 0.18 mmol) in a mixed solvent of THF (10 mL) and water (4 mL) was added NaOH (6.9 mg, 0.17 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 60-Na (82.0 mg, 97%) as a yellow solid. LC/MS: 446.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.05 (d, J=8.4 Hz, 1H), 8.0 (d, J=8.4 Hz, 1H), 7.79 (brs, 1H), 7.29 (d, J=8.8 Hz, 1H), 6.46 (d, J=8.8 Hz, 1H), 5.61-5.21 (m, 2H), 3.93-3.82 (m, 4H), 3.32-3.28 (m, 4H), 1.88-1.82 (m, 1H), 1.67-1.64 (m, 2H), 1.37 (t, J=6.8 Hz, 3H), 1.28-1.17 (m, 2H).


Example 27. Preparation of 2-(8-chloro-9-(dimethylamino)-2-methoxy-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl) acetic acid (21)



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A mixture of Intermediate C (50.0 mg, 0.13 mmol), LiOH.H2O (27.3 mg, 0.65 mmol) in a mixed solvent of THF (3 mL), EtOH (1 mL) and H2O (1 mL) was stirred at 35° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (5 mL×2). The combined organic layers were washed with brine (5 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 21 (21.0 mg, 46.7%) as a yellow solid. LC/MS: 362.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.45 (d, J=8.8 Hz, 1H), 8.20 (d, J=8.4 Hz, 1H), 7.45 (d, J=8.8 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 5.40 (s, 2H), 4.00 (s, 3H), 2.76 (s, 6H).


Example 28. Preparation of 2-(3,8-dichloro-9-(dimethylamino)-2-ethoxy-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl) acetic acid (18)



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A mixture of a (35.0 mg, 0.087 mmol) Na2CO3 (28.0 mg, 0.261 mmol) in a mixed solvent of EtOH (5 mL) and H2O (1 mL) was stirred at 90° C. for 16 h. The reaction mixture was diluted with water (5 mL) and washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (5 mL×2). The combined organic layers were washed with brine (5 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 18 (2.0 mg, 6.6%) as a yellow solid. LC/MS: 410.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.47 (s, 1H), 8.19 (d, J=8.8 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 5.33 (s, 2H), 4.58-4.52 (m, 2H), 2.75 (s, 6H), 1.42 (t, J=7.2 Hz, 3H)


Example 29. Preparation of sodium 2-(8-chloro-2-(((3,3-difluorocyclobutyl) methyl) (methyl)amino)-9-(dimethylamino)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (26)



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Step 1: To a mixture of a (85.0 mg, 0.178 mmol) in DMF (5 mL) was added sodium hydride (60% dispersion in mineral oil, 11.0 mg, 0.275 mmol) at 0° C. and stirred for 30 min. Iodomethane (39.0 mg, 0.275 mmol) dissolved in DMF (1 mL) was added dropwise. The resulting mixture was stirred at room temperature for 30 min and quenched by dropwise addition of water at 0° C. The reaction mixture was extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (10 mL), dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5/1) to afford b (80.0 mg, 90%).


Step 2: A mixture of b (80.0 mg, 0.162 mmol) and NaOH (26.0 mg, 0.65 mmol) in a mixed solvent of THF (5 mL), EtOH (5 mL) and H2O (2 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (10 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 26 (70.0 mg, 92.7%) as a yellow solid. LC/MS: 465.1 [M+H]+.


Step 3: To a mixture of 26 (70.0 mg, 0.151 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (5.4 mg, 0.136 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 26-Na (55.0 mg, 75%) as a yellow solid. LC/MS: 465.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J=9.2 Hz, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.24 (d, J=8.8 Hz, 1H), 6.70 (d, J=7.6 Hz, 1H), 5.35 (brs, 2H), 3.80 (d, J=2.4 Hz, 2H), 3.16 (s, 3H), 2.78 (s, 6H), 2.68-2.61 (m, 3H), 2.44-2.38 (m, 2H).


Example 30. Preparation of 2-(8-chloro-2-(methyl((tetrahydro-2H-pyran-4-yl)methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (27)



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Step 1: A mixture of a (100.0 mg, 0.25 mmol), b (86.0 mg, 0.75 mmol) and Et3N (252.0 mg, 2.5 mmol) in 1,4-dioxane (2 mL) was stirred at 100° C. for 12 h. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5/1) to afford c (51.0 mg, 43%). LC/MS: 476.1 [M+H]+.


Step 2: To a mixture of c (51.0 mg, 0.107 mmol) in DMF (1 mL) was added sodium hydride (60% dispersion in mineral oil, 5.2 mg, 0.13 mmol) at 0° C. and the reaction was stirred for 30 min. Iodomethane (17.0 mg, 0.12 mmol) dissolved in DMF (1 mL) was added dropwise. The resulting mixture was stirred at room temperature for 30 min and quenched by dropwise addition of water at 0° C. The reaction mixture was extracted with ethyl acetate (5 mL×2). The combined organic layers were washed with brine (10 mL), dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5/1) to afford d (48.0 mg, 91%). LC/MS: 490.4 [M+H]+.


Step 3: A mixture of d (48.0 mg, 0.098 mmol) and LiOH.H2O (22.7 mg, 0.54 mmol) in a mixed solvent of EtOH (6 mL) and H2O (2 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (10 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 26 (44.0 mg, 99%) as a yellow solid. LC/MS: 462.2 [M+H]+.


Step 4: To a mixture of 26 (44.0 mg, 0.095 mmol) in a mixed solvent of THF (1 mL) and water (1 mL) was added NaOH (3.4 mg, 0.085 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 26-Na (28.0 mg, 60.9%) as a yellow solid. LC/MS: 462.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.13 (d, J=8.8 Hz, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 6.66 (brs, 1H), 5.43 (s, 2H), 3.83 (dd, J=2.4 Hz, 2H), 3.53 (brs, 2H), 3.33 (s, 2H), 3.16 (s, 3H), 2.29 (s, 3H), 2.04 (brs, 1H), 1.55 (d, J=7.6 Hz, 2H), 1.26-1.35 (m, 2H).


Example 31. Preparation of sodium 2-(8-chloro-2-(cyclopropyl(2-methoxyethyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (28)



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Step 1: A mixture of Intermediate A (150.0 mg, 0.38 mmol), a (66.0 mg, 1.14 mmol) and Et3N (383.0 mg, 3.8 mmol) in THF (3 mL) was stirred at 100° C. for 16 h. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5/1) to afford b (131.5 mg, 83%). LC/MS: 418.1 [M+H]+.


Step 2: To a mixture of b (104.0 mg, 0.25 mmol) in DMF (2.5 mL) was added sodium hydride (60% dispersion in mineral oil, 60 mg, 1.5 mmol) at 0° C. and stirred for 30 min. 1-iodo-2-methoxyethane (c) (93.0 mg, 0.5 mmol) dissolved in DMF (1 mL) was added dropwise. The resulting mixture was stirred at room temperature for 16 h, poured into water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 2 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (5 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford afford 28 (63.8 mg, 57%). LC/MS: 448.1 [M+H]+.


Step 3: To a mixture of 28 (63.8 mg, 0.143 mmol) in a mixed solvent of THF (10 mL) and water (30 mL) was added NaOH (5.14 mg, 0.129 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 28-Na (52.0 mg, 77.5%) as a yellow solid. LC/MS: 448.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.20 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 6.98 (d, J=9.2 Hz, 1H), 5.42 (s, 2H), 3.85-3.83 (m, 2H), 3.61-3.59 (m, 2H), 3.27 (s, 3H), 2.77-2.72 (m, 1H), 2.29 (s, 3H), 0.98-0.95 (m, 2H), 0.76-0.73 (m, 2H).


Example 32. Preparation of sodium 2-(8-chloro-2-(cyclopropyl(2-methoxyethyl)amino)-9-ethoxy-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (29)



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Step 1: A mixture of Intermediate B (200.0 mg, 0.51 mmol), a (72.0 mg, 1.27 mmol) and Et3N (515 mg, 5.1 mmol) in THF (3 mL) was stirred at 100° C. for 16 h. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (5 mL×3). The combined organic layers were concentrated to afford b (210 mg, crude, ca. 100%). LC/MS: 416.2 [M+H]+.


Step 2: To a mixture of b (100 mg, 0.24 mmol) in DMF (2 mL) was added sodium hydride (60% dispersion in mineral oil, 96.0 mg, 2.4 mmol) at 0° C. and stirred for 30 min. Then 1-iodo-2-methoxyethane (c) (89.0 mg, 0.48 mmol) was added. The reaction mixture was stirred at room temperature for 30 min, quenched by Sat. NH4Cl at 0° C. The resulting mixture was acidified to pH=3-4 with 1 N HCl and then extracted with ethyl acetate (10 mL×2). The combined organics were concentrated to afford d (100 mg, 87%). LC/MS: 474.2 [M+H]+.


Step 3: A mixture of d (100 mg, 0.21 mmol) and LiOH.H2O (89.0 mg, 2.11 mmol) in a mixed solvent of EtOH (3 mL), THF (3 mL) and H2O (1 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (10 mL) and washed with ethyl acetate (3 mL×2). The aqueous layer was acidified to pH=3-4 with 1 N HCl and extracted with ethyl acetate (10 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 29 (88.0 mg, 94%) as a yellow solid. LC/MS: 446.2 [M+H]+.


Step 4: To a mixture of 29 (88.0 mg, 0.197 mmol) in THF (5 mL) was added NaOH (0.01 M in H2O, 17.7 mL). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 29-Na (56.7 mg, 62%) as a yellow solid. LC/MS: 446.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): (8.26 (d, J=8.8 Hz, 1H), 8.02 (d, J=8.4 Hz, 1H), 7.32 (d, J=8.8 Hz, 1H), 7.01 (d, J=8.8 Hz, 1H), 5.33 (brs, 2H), 3.80-3.94 (m, 4H), 3.58-3.61 (m, 2H), 3.26 (s, 3H), 2.72-2.77 (m, 1H), 1.37 (t, J=7.2 Hz, 3H), 0.97-0.98 (m, 2 h), 0.75 (s, 2H).


Example 33. Preparation of sodium 2-(8-chloro-2-{methyl[2-(2-methylimidazol-1-yl)ethyl]amino}-9-(methylsulfanyl)-5-oxobenzo[b]1,8-naphthyridin-10-yl)acetate (81)



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Compound 81-Na was prepared from Intermediate A and a according to the following procedure. Intermediate A (40 mg, 0.1 mmol, 1.0 eq) and a (70 mg, 0.5 mmol, 5.0 eq) in THF (1 mL)/H2O (1 mL) was added lithium hydroxide monohydrate (21 mg, 0.5 mmol, 5.0 eq). The resulting mixture was stirred at 50° C. for 16 h. After completion of the reaction, as monitored by LCMS, the reaction mixture was acidified with 1N HCl (aq) to pH=5. The resulting mixture was concentrated under vacuum to remove THF. The crude residue was purified by Prep-HPLC (water/acetonitrile=95/5 to 5/95) to afford 81 as a free acid. To 81 was added one equivalent of sodium hydroxide (0.1 M in water) and stirred for 15 min at 25° C. to form 81-Na. LCMS: 472.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) (8.21 (d, J=8.5 Hz, 2H), 7.53 (d, J=8.5 Hz, 1H), 7.12 (d, J=1.4 Hz, 1H), 6.77 (d, J=1.3 Hz, 1H), 6.70-6.62 (m, 1H), 5.46 (s, 2H), 4.17 (t, J=5.9 Hz, 2H), 3.94 (s, 2H), 2.87 (s, 3H), 2.28 (s, 3H), 2.14 (s, 3H).


Example 34. Preparation of sodium 2-(8-chloro-2-(ethyl(oxetan-3-yl)amino)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (30)



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Step 1: A mixture of Intermediate A (200.0 mg, 0.51 mmol), oxetan-3-amine (150.0 mg, 2.05 mmol) and Et3N (2.7 mL, 5.0 mmol) in THF (10 mL) was stirred at 110° C. for 16 h in a sealed tube. The reaction mixture concentrated in vacuo. The residue was purified by Prep-HPLC to afford b (100.0 mg, 45.6%). LC/MS: 434.1 [M+H]+.


Step 2: To a mixture of b (100.0 mg, 0.23 mmol) in DMF (3 mL) was added sodium hydride (60% dispersion in mineral oil, 36 mg, 0.90 mmol) at 0° C. and stirred for 5 min. Bromoethane (50.0 mg, 0.46 mmol) dissolved in DMF (1 mL) was added dropwise. The resulting mixture was stirred at room temperature for 2 h and quenched by dropwise addition of water at 0° C. The reaction mixture was washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=1 with 1 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 30 (90.0 mg, 90%). LC/MS: 434.0 [M+H]+.


Step 3: To a mixture of 20b (90.0 mg, 0.21 mmol) in a mixed solvent of THF (10 mL) and water (2 mL) was added NaOH (72 mg, 0.18 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 30-Na (50.0 mg, 53%) as a yellow solid. LC/MS: 434.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 6.68 (d, J=9.2 Hz, 1H), 5.33 (s, 2H), 5.21 (t, J=7.2 Hz, 1H), 4.87 (t, J=7.2 Hz, 2H), 4.70 (t, J=6.8 Hz, 2H), 3.67-3.62 (m, 2H), 2.29 (s, 3H), 1.17 (t, J=7.2 Hz, 3H).


Example 35. Preparation of 2-(8-chloro-9-ethoxy-2-(methyl((tetrahydro-2H-pyran-4-yl)methyl)amino)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (33)



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Step 1: To a mixture of a (96.0 mg, 0.20 mmol) in DMF (2 mL) was added sodium hydride (60% dispersion in mineral oil, 10.4 mg, 0.26 mmol) at 0° C. and stirred for 30 min. Iodomethane (34.0 mg, 0.24 mmol) was added. The resulting mixture was stirred at room temperature for 30 min and quenched by dropwise addition of water at 0° C. The reaction mixture was extracted with ethyl acetate (5 mL×2). The organic layer was washed with brine (10 mL), dried over MgSO4, and concentrated in vacuo to afford b (85.0 mg, 86%). LC/MS: 488.2 [M+H]+.


Step 2: A mixture of b (80.0 mg, 0.164 mmol) and LiOH.H2O (34.5 mg, 0.82 mmol) in a mixed solvent of EtOH (6 mL) and H2O (1 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 1 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were washed with brine (10 mL×2), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 33 (73.0 mg, 97%) as a yellow solid. LC/MS: 460.2 [M+H]+.


Step 3: To a mixture of 33 (73.0 mg, 0.159 mmol) in a mixed solvent of THF (1 mL) and water (1 mL) was added NaOH (5.7 mg, 0.143 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 33-Na (48.0 mg, 63%) as a yellow solid. LC/MS: 460.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J=8.4 Hz, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.31 (d, J=8.4 Hz, 1H), 6.70 (brs, 1H), 5.40 (brs, 2H), 3.93 (d, J=6.4 Hz, 2H), 3.84-3.81 (m, 2H), 2.36 (d, J=11.2 Hz, 2H), 3.16 (s, 3H), 2.04 (brs, 1H), 1.54 (d, J=12 Hz, 2H), 1.24-1.38 (m, 5H).


Example 36. Preparation of Sodium 2-(8-chloro-2-(((3,3-difluorocyclobutyl) methyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (36)



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Step 1: To a mixture of a (80.0 mg, 0.166 mmol) in DMF (2 mL) was added sodium hydride (60% dispersion in mineral oil, 13 mg, 0.332 mmol) at 0° C. and stirred for 5 min. Iodomethane (47.0 mg, 0.332 mmol) was added. The resulting mixture was stirred at room temperature for 30 min and quenched by dropwise addition of water at 0° C. The reaction mixture was extracted with ethyl acetate (5 mL×2). The organic layer was washed with brine (10 mL), dried over MgSO4, and concentrated in vacuo to afford b (78.0 mg, 94.8%). LC/MS: 496.1 [M+H]+.


Step 2: A mixture of b (78.0 mg, 0.158 mmol) and NaOH (25 mg, 0.632 mmol) in a mixed solvent of EtOH (5 mL), THF (5 mL) and H2O (2 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 1 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 36 (68.0 mg, 92.4%) as a yellow solid. LC/MS: 468.1 [M+H]+.


Step 3: To a mixture of 36 (68.0 mg, 0.146 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (5.2 mg, 0.131 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 36-Na (60.0 mg, 85%) as a yellow solid. LC/MS: 468.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.15 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 6.68 (d, J=8.0 Hz, 1H), 5.41 (brs, 2H), 3.83 (s, 2H), 3.16 (s, 3H), 2.70-2.61 (m, 2H), 2.58-2.54 (m, 1H), 2.46-2.42 (m, 2H), 2.29 (s, 3H).


Example 37. Preparation of sodium 2-(8-chloro-2-((Cyclopropylmethyl)((3,3-difluorocyclobutyl)methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (37)



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Step 1: To a mixture of a (80.0 mg, 0.166 mmol) in DMF (2.5 mL) was added sodium hydride (60% dispersion in mineral oil, 13 mg, 0.332 mmol) at 0° C. and stirred for 5 min. (Bromomethyl)cyclopropane (b) (45.0 mg, 0.332 mmol) was added. The resulting mixture was stirred at room temperature for 30 min and quenched by dropwise addition of water at 0° C. The reaction mixture was extracted with ethyl acetate (5 mL×2). The combined organic layers were washed with brine (10 mL), dried over MgSO4, and concentrated in vacuo to afford c (75.0 mg, 84.3%). LC/MS: 536.2 [M+H]+.


Step 2: A mixture of c (75.0 mg, 0.14 mmol) and NaOH (22.4 mg, 0.56 mmol) in a mixed solvent of EtOH (5 mL), THF (5 mL) and H2O (2 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 1 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 37 (67.0 mg, 94.2%) as a yellow solid. LC/MS: 508.1 [M+H]+.


Step 3: To a mixture of 37 (67.0 mg, 0.132 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (5.0 mg, 0.119 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 37-Na (56.0 mg, 80%) as a yellow solid. LC/MS: 508.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 6.75 (d, J=9.2 Hz, 1H), 5.43 (brs, 2H), 3.84 (s, 2H), 3.49 (s, 2H), 2.70-2.57 (m, 5H), 2.28 (s, 3H), 1.18-1.11 (m, 1H), 0.48 (d, J=6.8 Hz, 2H), 0.37 (d, J=3.6 Hz, 2H).


Example 38. Preparation of sodium 2-(8-chloro-2-((cyclopropylmethyl)(oxetan-3-ylmethyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (38)



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Step 1: To a mixture of a (80.0 mg, 0.179) in DMF (2.5 mL) at 0° C. was added NaH (14.0 mg, 0.358 mmol). The resulting mixture was stirred at 0° C. for 10 minutes. b (48 mg, 0.358 mmol) was added. The resulting mixture was then stirred at room temperature for 1 h and quenched by dropwise addition of water at 0° C. The reaction mixture was extracted with ethyl acetate (5 mL×2). The combined organic layers were washed with brine (10 mL), dried over MgSO4, and concentrated in vacuo to afford c (80.0 mg, 89.2%). LC/MS: 502.1 [M+H]+.


Step 2: To a mixture of c (80 mg, 0.16 mmol) in THF/Ethanol/water (5 mL/5 mL/2 mL) was added LiOH.H2O (26.0 mg, 0.64 mmol). The resulting mixture was stirred at room temperature for 16 h under N2, then diluted with water (5 mL) and washed with ethyl acetate (10 mL×2). The aqueous layer was acidified to pH=3 with 1 N HCl and extracted with ethyl acetate (10 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 38 (72.0 mg, 95.4%). LC/MS: 474.1 [M+H]+.


Step 3: To a mixture of 38 (72 mg, 0.152 mmol) in THF/water (10/10 mL) was added NaOH (5.5 mg, 0.137 mmol). The resulting mixture was stirred at room temperature for 1 h under N2 and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 38-Na (66 mg, 88%) as a yellow solid. LCMS: 474.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 6.74 (d, J=9.2 Hz, 1H), 5.46 (brs, 2H), 4.64 (t, J=6.8 Hz, 2H), 4.49 (t, J=5.6 Hz, 2H), 3.98 (s, 2H), 3.47-3.38 (m, 3H), 2.29 (s, 3H), 1.19-1.10 (m, 1H), 0.48 (d, J=6.8 Hz, 2H), 0.37 (d, J=4.0 Hz, 2H).


Example 39. Preparation of sodium 2-[8-chloro-9-(dimethylamino)-2-{methyl[2-(2-methylimidazol-1-yl)ethyl]amino}-5-oxobenzo[b]1,8-naphthyridin-10-yl]acetate (82)



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Compounds 82 and 82-Na were prepared from Intermediate C and a according to the following procedure. To a mixture of Intermediate C (40 mg, 0.1 mmol, 1.0 eq) and a (70 mg, 0.5 mmol, 5.0 eq) in THF (1 mL)/H2O (1 mL) was added lithium hydroxide monohydrate (21 mg, 0.5 mmol, 5.0 eq). The resulting mixture was stirred at 50° C. for 16 h. After completion of the reaction, as monitored by LCMS, the reaction mixture was acidified with 1N HCl (aq) to pH=5. The resulting mixture was concentrated under vacuum to remove THF. The crude residue was purified by Prep-HPLC (water/acetonitrile=95/5 to 5/95) to afford 82 as a free acid. To 82 was added one equivalent of sodium hydroxide (0.1 M in water) and stirred for 15 min at 25° C. The resulting mixture was lyophilized to provide 82-Na. LCMS: 469.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J=8.8 Hz, 1H), 8.05 (d, J=8.6 Hz, 1H), 7.25 (d, J=8.6 Hz, 1H), 7.20 (s, 1H), 6.68 (d, J=1.3 Hz, 1H), 6.59 (d, J=8.9 Hz, 1H), 5.34 (s, 2H), 4.16 (d, J=6.8 Hz, 2H), 3.89 (s, 2H), 2.89 (s, 3H), 2.79 (s, 6H), 2.15 (s, 3H).


Example 40. Preparation of Sodium 2-(8-chloro-2-(((3-methoxycyclobutyl)methyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (39)



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Step 1: A mixture of Intermediate B (200.0 mg, 0.51 mmol), a (116.0 mg, 1.0 mmol) and Et3N (1.0 mL, 7.1 mmol) in THF (3 mL) was stirred in a sealed tube at 100° C. for 16 h, diluted with water (10 mL) and extracted with ethyl acetate (3 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated to afford b (239 mg, ca. 100%). LC/MS: 476.1 [M+H]+.


Step 2: To a mixture of b (70 mg, 0.15 mmol) in DMF (2 mL) was added sodium hydride (60% dispersion in mineral oil, 12.0 mg, 0.30 mmol) at 0° C. and stirred for 30 min. Then iodoethane (0.1 mL) was added. The reaction mixture was stirred at room temperature for 1 h and quenched with ice water. The resulting mixture was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (10 mL×2). The combined organics were concentrated to afford a mixture of c and 39-Na. The mixture was dissolved in a mixed solvent of EtOH (6 mL), THF (30 mL) and H2O (10 mL) and NaOH (120.0 mg, 3.0 mmol) was added. The mixture was stirred at 50° C. for 2 h, then diluted with water (10 mL) and washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=1-2 with 1 N HCl and extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 39 (64.0 mg, 95.5%) as a yellow solid. LC/MS: 462.1 [M+H]+


Step 3: To a mixture of 39 (64.0 mg, 0.14 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (5.0 mg, 0.13 mmol). The mixture was stirred at room temperature for 0.5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 39-Na (39.0 mg, 58.2%) as a yellow solid. LC/MS: 462.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.13 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 6.65 (d, J=8.4 Hz, 1H), 5.50 (s, 2H), 3.72-3.61 (m, 1H), 3.35 (m, 2H), 3.14 (s, 3H), 3.11 (s, 3H), 2.34-2.29 (m, 5H), 2.17-2.12 (m, 1H), 1.68-1.65 (m, 2H).


Example 41. Preparation of sodium 2-(8-chloro-2-((cyclopropylmethyl)((3-methoxycyclobutyl)methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (40)



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Step 1: To a mixture of a (70 mg, 0.15 mmol) in DMF (2 mL) was added sodium hydride (60% dispersion in mineral oil, 12.0 mg, 0.30 mmol) at 0° C. and stirred for 30 min. Then (bromomethyl)cyclopropane (b) (0.1 mL) was added. The reaction mixture was stirred at room temperature for 1 h, quenched with ice water at 0° C. The resulting mixture was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (10 mL×2). The combined organics were concentrated to afford a mixture of c and 40. The mixture was dissolved in a mixed solvent of EtOH (6 mL), THF (30 mL) and H2O (10 mL) and NaOH (120.0 mg, 3.0 mmol) was added. The mixture was stirred at 50° C. for 2 h, then diluted with water (10 mL) and washed with ethyl acetate (5 mL×2). The aqueous layer was acidified to pH=1-2 with 1 N HCl and extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford c (35.0 mg, 47.3%) as a yellow solid. LC/MS: 502.2 [M+H]+


Step 2: To a mixture of 40 (35.0 mg, 0.07 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (2.5 mg, 0.06 mmol). The mixture was stirred at room temperature for 0.5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 40-Na (27.4 mg, 75%) as a yellow solid. LC/MS: 502.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.12 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 6.69 (d, J=8.8 Hz, 1H), 5.53-5.51 (m, 2H), 3.67-3.60 (m, 3H), 3.47 (m, 2H), 3.10 (s, 3H), 2.33-2.28 (m, 5H), 2.11-2.18 (m, 1H), 1.7-1.63 (m, 2H), 1.09-1.22 (m, 1H), 0.48-0.46 (m, 2H), 0.38-0.36 (m, 2H).


Example 42. Preparation of Sodium 2-(8-chloro-2-((3,3-difluorocyclobutyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (41)



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Step 1: A mixture of Intermediate A (220.0 mg, 0.55 mmol), 3,3-difluorocyclobutan-1-amine hydrochloride (a) (236.0 mg, 165 mmol) and Et3N (555.5 mg, 5.5 mmol) in 1,4-dioxane (2 mL) was stirred in a sealed tube at 100° C. for 16 h. The reaction mixture was concentrated in vacuo. The residue was purified by Prep-HPLC to afford b (110 mg, 42%). LC/MS: 468.1 [M+H]+.


Step 2: To a mixture of b (55.0 mg, 0.13 mmol) in DMF (1 mL) was added sodium hydride (60% dispersion in mineral oil, 7.0 mg, 0.175 mmol) at 0° C. and stirred for 30 min. Then iodoethane (22.2 mg, 0.156 mmol) was added. The reaction mixture was stirred at room temperature for 30 min and quenched with ice water at 0° C. The resulting mixture was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×2). The combined organics were concentrated and the residue was purified by Prep-HPLC to afford c (40.0 mg, 71%). LC/MS: 482.1 [M+H]+.


Step 3: A mixture of c (36 mg, 0.075 mmol) and LiOH.H2O (18.0 mg, 0.43 mmol) in a mixed solvent of EtOH (6 mL), and H2O (1 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and extracted with ethyl acetate (5 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 41 (33.0 mg, 97%) as a yellow solid. 454.1 [M+H]+


Step 4: To a mixture of 41 (33.0 mg, 0.073 mmol) in a mixed solvent of THF (2 mL) and water (8 mL) was added NaOH (2.64 mg, 0.066 mmol). The mixture was stirred at room temperature for 0.5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 41-Na (31.0 mg, 89.5%) as a yellow solid. LC/MS: 454.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.19 (d, J=9.2 Hz, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 6.74 (d, J=8.8 Hz, 1H), 5.42 (s, 2H), 4.79 brs, 1H), 3.11 (s, 3H), 3.03 (brs, 2H), 2.89 (brs, 2H), 2.30 (s, 3H).


Example 43. Preparation of sodium 2-(8-chloro-2-((cyclopropylmethyl)(3,3-difluorocyclobutyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (36)



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Step 1: To a mixture of a (55.0 mg, 0.13 mmol) in DMF (1 mL) was added sodium hydride (60% dispersion in mineral oil, 6.76 mg, 0.169 mmol) at 0° C. and stirred for 30 min. Then (bromomethyl)cyclopropane (b) (21.0 mg, 0.156 mmol) was added. The reaction mixture was stirred at room temperature for 30 min, quenched with ice water at 0° C. The resulting mixture was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×2). The combined organics were concentrated and the residue was purified by Prep-HPLC to afford c (49.0 mg, 72%). LC/MS: 522.4 [M+H]+.


Step 2: A mixture of c (49 mg, 0.094 mmol) and LiOH.H2O (22.0 mg, 0.537 mmol) in a mixed solvent of EtOH (6 mL) and H2O (1 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (5 mL) and extracted with ethyl acetate (5 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 36 (41.0 mg, 88%) as a yellow solid. 494.3 [M+H]+


Step 3: To a mixture of 36 (41.0 mg, 0.083 mmol) in a mixed solvent of THF (1 mL) and water (1 mL) was added NaOH (3.0 mg, 0.075 mmol). The mixture was stirred at room temperature for 0.5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 36-Na (35.0 mg, 82%) as a yellow solid. LC/MS: 494.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J=9.2 Hz, 1H), 8.10 (d, J=8.0 Hz, 1H), 7.39 (d, J=8.0 Hz, 1H), 6.81 (d, J=9.2 Hz, 1H), 5.44 (s, 2H), 4.49 brs, 1H), 3.13 (brs, 2H), 2.88 (brs, 2H), 3.00 (s, 3H), 1.66 (s, 2H), 0.99 (s, 1H), 0.46 (d, J=7.6 Hz, 2H), 0.32 (d, J=4.8 Hz, 2H).


Example 44. Preparation of 2-(8-chloro-2-(((1s,3s)-3-methoxycyclobutyl) (methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (44) and 2-(8-chloro-2-(((1r,3r)-3-methoxycyclobutyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (70)



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Step 1: A mixture of Intermediate A (500.0 mg, 1.26 mmol), 3-methoxycyclobutan-1-amine hydrochloride (a) (431.0 mg, 3.15 mmol) and Et3N (1.27 g, 12.6 mmol) in THF (10 mL) was stirred in a sealed tube at 120° C. for 16 h. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated to afford b (crude, 700.0 mg, ca. 100%). LC/MS: 462.1 [M+H]+.


Step 2: To a mixture of b (700.0 mg, 1.5 mmol) in DMF (5 mL) was added sodium hydride (60% dispersion in mineral oil, 600.0 mg, 15.0 mmol) at 0° C. and stirred for 10 min. Then iodoethane (858.0 mg, 6.0 mmol) was added. The reaction mixture was stirred at room temperature for 20 min, quenched with Sat. NH4Cl (5 mL). The resulting mixture was acidified to pH-=3-4 with 1 N HCl and then extracted with ethyl acetate (10 mL×3). The combined organics were concentrated to afford crude c (500.0 mg, 69%). LC/MS: 476.1 [M+H]+.


Step 3: A mixture of c (500.0 mg, 1.05 mmol) and LiOH.H2O (441.0 mg, 10.50 mmol) in a mixed solvent of EtOH (10 mL), THF (10 mL) and H2O (5 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (20 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=3-4 with 1 N HCl and then extracted with ethyl acetate (15 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to afford d (450.0 mg, 96%) as a yellow solid. 448.1 [M+H]+


Step 4: d was separated by Chiral-HPLC to afford 44 (361.9 mg, Peak 1, Room temperature: 3.667 min) and 70 (39.6 mg, Peak 2, Room temperature: 5.070 min). LC/MS: 448.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.15 (d, J=8.8 Hz, 1H), 8.12 (d, J=8.4 Hz, 1H), 7.41 (d, J=8.4 Hz, 1H), 6.65 (d, J=8.8 Hz, 1H), 5.42 (s, 2H), 5.00 (brs, 1H), 3.98-3.96 (m, 1H), 3.21 (s, 3H), 3.11 (s, 3H), 2.43-2.28 (m, 4H), 1.88 (s, 3H).


Example 45. Preparation of sodium 2-(8-chloro-2-((cyclopropylmethyl)(3-methoxycyclobutyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (43)



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Step 1: To a mixture of a (100.0 mg, 0.22 mmol) in DMF (2 mL) was added sodium hydride (60% dispersion in mineral oil, 88.0 mg, 2.2 mmol) at 0° C. and stirred for 10 min. Then (Bromomethyl)cyclopropane (b) (119.0 mg, 0.88 mmol) was added. The reaction mixture was stirred at room temperature for 20 min and quenched with a saturated aqueous mixture of NH4Cl. The resulting mixture was acidified to pH=3-4 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organics were concentrated to afford crude c (100.0 mg, 89%). LC/MS: 516.2 [M+H]+.


Step 2: A mixture of c (100.0 mg, 0.19 mmol) and LiOH.H2O (80.0 mg, 1.9 mmol) in a mixed solvent of EtOH (3 mL), THF (3 mL) and H2O (1 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (10 mL) and washed with ethyl acetate (3 mL). The aqueous phase was acidified to pH=3-4 with 1 N HCl and then extracted with ethyl acetate (10 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo to afford 43 (95.0 mg, 94%) as a yellow solid. LC/MS: 488.1 [M+H]+


Step 3: To a mixture of 43 (95.0 mg, 0.195 mmol) in THF (5 mL) was added NaOH (0.01 M in H2O, 17.5 mL, 0.175 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 43-Na (58.3 mg, 59%) as a yellow solid. LC/MS: 488.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.16 (d, J=9.2 Hz, 1H), 8.11 (d, J=8.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 6.72 (d, J=8.8 Hz, 1H), 5.45 (s, 2H), 4.21 (brs, 1H), 3.66-3.71 (m, 1H), 3.52-3.57 (m, 2H), 3.24 (s, 3H), 2.75-2.81 (m, 2H), 2.33 (s, 3H), 1.92-1.99 (m, 2 h), 1.03-1.07 (m, 1H), 0.42-0.48 (m, 2H), 0.36-0.41 (m, 2H).


Example 46. Preparation of Sodium 2-(8-chloro-2-((3-(difluoromethoxy)cyclobutyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (45)



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Step 1: To a mixture of a (2.0 g, 10.7 mmol) and CuI (407 mg, 2.1 mmol) in acetonitrile (40 mL) was added b (2.3 g, 12.8 mmol) at 50° C. The resulting mixture was stirred at 50° C. for 2.1 h, poured into water (20 mL), and then extracted with ethyl acetate (20 mL×3). The extracts were combined and washed with brine. The organic layer dried over anhydrous Na2SO4, filtered and concentrated to afford c (crude, 2.0 g, 79%).


Step 2: To a mixture of c (crude 1.0 g, 4.2 mmoL) in DCM (30 mL) was added TFA (3 mL). The mixture was stirred at room temperature for 4 h and then evaporated to afford crude d (690 mg, ca. 100%).


Step 3: A mixture of Intermediate A (220.0 mg, 0.556 mmol), 3-(difluoromethoxy)cyclobutan-1-amine (d) (759.0 mg, 5.54 mmol) and Et3N (561.0 mg, 5.55 mmol) in 1,4-dioxane (20 mL) was stirred in a sealed tube at 120° C. for 16 h. The reaction mixture was concentrated in vacuo. The residue was purified by Prep-HPLC to afford e (120.0 mg, 44%). LC/MS: 498.1 [M+H]+.


Step 4: To a mixture of e (40.0 mg, 0.08 mmol) in DMF (6 mL) was added sodium hydride (60% dispersion in mineral oil, 32.0 mg, 0.80 mmol) at 0° C. and stirred for 30 min. Then iodoethane (114.0 mg, 0.80 mmol) was added. The reaction mixture was stirred at room temperature for 30 min and quenched with saturated, aqueous NH4Cl (5 mL). The resulting mixture was acidified to pH-=3-4 with 1 N HCl and then extracted with ethyl acetate (10 mL×3). The combined organics were concentrated to afford crude f (47.0 mg, ca. 100%). LC/MS: 512.1 [M+H]+.


Step 5: A mixture of f (47.0 mg, 0.092 mmol) and LiOH.H2O (39.0 mg, 0.92 mmol) in a mixed solvent of THF (4 mL) and H2O (4 mL) was stirred at 50° C. for 16 h. The reaction mixture was then diluted with water (20 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 45 (31.0 mg, 70%) as a yellow solid. 484.1 [M+H]+


Step 6: To a mixture of 45 (31.0 mg, 0.064 mmol) in a mixed solvent of THF (4 mL) and water (4 mL) was added NaOH (2.3 mg, 0.058 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 45-Na (28.3 mg, 87%) as a yellow solid. LC/MS: 484.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J=8.8 Hz, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.41 (d, J=8.4 Hz, 1H), 6.72 (s, 1H), 6.70 (t, J=75.6 Hz, 1H), 5.54 (brs, 2H), 4.55-4.54 (m, 1H), 4.49-4.42 (m, 1H), 3.33 (s, 3H), 2.78-2.72 (m, 2H), 2.40-2.24 (m, 5H).


Example 47. Preparation of sodium 2-(8-chloro-2-((cyclopropylmethyl)(3-(difluoromethoxy)cyclobutyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (46)



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Step 1: To a mixture of a (80.0 mg, 0.16 mmol) in DMF (10 mL) was added sodium hydride (60% dispersion in mineral oil, 64.0 mg, 1.6 mmol) at 0° C. and stirred for 10 min. (Bromomethyl)cyclopropane (b) (429.0 mg, 3.2 mmol) was added. The resulting mixture was stirred at room temperature for 30 min and quenched by dropwise addition of water at 0° C. The reaction mixture was washed with ethyl acetate (15 mL×2). The aqueous layer was acidified to pH=2-3 with 1 N HCl and extracted with ethyl acetate (20 mL×2). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 46 (29.0 mg, 35%). LC/MS: 524.1 [M+H]+.


Step 2: To a mixture of 46 (29.0 mg, 0.055 mmol) in a mixed solvent of THF (4 mL) and water (4 mL) was added NaOH (2.0 mg, 0.050 mmol). The mixture was stirred at room temperature for 16 h and washed with ethyl acetate (5 mL×2). The aqueous layer was separated and evaporated to provide 46-Na (26.3 mg, 87%) as a yellow solid. LC/MS: 524.0 [M+H]+. 1H NMR (400 MHz, D20): δ 8.17 (d, J=8.8 Hz, 1H), 8.13 (d, J=8.4 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 6.75 (d, J=8.4 Hz, 1H), 6.70 (t, J=75.2 Hz, 1H), 5.44 (brs, 2H), 4.47-4.43 (m, 1H), 4.32-4.25 (in, 1H), 3.58-3.56 (m, 2H), 2.87-2.85 (m, 2H), 2.28-2.21 (m, 5H), 1.06-1.02 (m, 1H), 0.48-0.46 (m, 2H), 20.35-0.33 (m, 2H).


Example 48. Preparation of sodium 2-(8-chloro-2-(methyl(spiro[3.3]heptan-2-yl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (47)



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Step 1: A mixture of Intermediate A (100.0 mg, 0.25 mmol), a (75.0 mg, 0.50 mmol) and Et3N (0.5 mL, 3.6 mmol) in THF (3 mL) was stirred in a sealed tube at 100° C. for 16 h. The reaction mixture was concentrated in vacuo. The residue was purified by Prep-HPLC to afford b (80.0 mg, 67.2%). LC/MS: 472.1 [M+H]+.


Step 2: To a mixture of b (50.0 mg, 0.11 mmol) in DMF (3 mL) was added sodium hydride (60% dispersion in mineral oil, 6.4 mg, 0.16 mmol) at 0° C. and stirred for 30 min. Then iodoethane (3 drops) was added. The reaction mixture was stirred at room temperature for 30 min and quenched with water (10 mL). The resulting mixture was acidified to pH=2-3 with 1 N HCl and then extracted with ethyl acetate (10 mL×3). The combined organics were concentrated and the residue was purified by Prep-HPLC to afford 47 (45.0 mg, 93.2%). LC/MS: 458.1 [M+H]+.


Step 3: To a mixture of 47 (45.0 mg, 0.10 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (3.6 mg, 0.09 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 47-Na (38.3 mg, 82.6%) as a yellow solid. LC/MS: 458.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.13 (d, J=9.2 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 6.66 (d, J=9.2 Hz, 1H), 5.48-5.45 (m, 2H), 4.65-4.63 (m, 1H), 3.06 (s, 3H), 2.41-2.35 (m, 2H), 2.3 (s, 3H), 2.19-2.11 (m, 4H), 1.99-1.94 (m, 2H), 1.88-1.8 (m, 2H).


Example 49. Preparation of sodium 2-(8-chloro-2-((3,3-dimethylcyclobutyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (48)



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Step 1: A mixture of Intermediate A (100.0 mg, 0.25 mmol), a (101.0 mg, 0.75 mmol) and Et3N (0.35 mL, 2.5 mmol) in THF (3 mL) was stirred in a sealed tube at 100° C. for 16 h, diluted with ethyl acetate (10 mL) and washed with water. The organic layer was separated and dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to afford b (85.0 mg, 74%). LC/MS: 460.1 [M+H]+.


Step 2: To a mixture of b (85.0 mg, 0.185 mmol) in DMF (3 mL) was added sodium hydride (60% dispersion in mineral oil, 74.0 mg, 1.85 mmol) at 0° C. and stirred for 20 min. Then iodoethane (132.0 mg, 0.93 mmol) was added. The reaction mixture was stirred at room temperature for 16 h, quenched with water (10 mL). The resulting mixture was acidified to pH=5 with 1 N HCl and then extracted with ethyl acetate (10 mL×3). The combined organics were concentrated and the residue was purified by Prep-HPLC to afford 48 (17.3 mg, 20%). LC/MS: 446.1 [M+H]+.


Step 3: To a mixture of 48 (17.3 mg, 0.039 mmol) in a mixed solvent of THF (5 mL) and water (5 mL) was added NaOH (1.4 mg, 0.035 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 48-Na (10.6 mg, 58.2%) as a yellow solid. LC/MS: 446.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.12 (d, J=8.8 Hz, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 6.69 (d, J=8.8 Hz, 1H), 5.42 (s, 2H), 4.88 (s, 1H), 3.10 (s, 3H) 2.29 (s, 3H), 2.15-2.10 (m, 2H), 2.04-1.99 (m, 2H), 1.26 (s, 3H), 1.16 (s, 3H).


Example 50. Preparation of sodium 2-(8-chloro-2-((3-cyanocyclobutyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (49)



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Step 1: To a mixture of a (50.0 mg, 0.11 mmol) in DMF (3 mL) was added sodium hydride (60% dispersion in mineral oil, 18.0 mg, 0.45 mmol) at 0° C. and stirred for 5 mm. Then iodoethane (31.0 mg, 0.22 mmol) was added. The reaction mixture was stirred at room temperature for 16 h, quenched with water and extracted with ethyl acetate (5 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford crude b (25.0 mg, 67%). LC/MS: 471.1 [M+H]+.


Step 2: A mixture of b (25.0 mg, 0.053 mmol) and LiOH.H2O (20.0 mg, 0.476 mmol) in a mixed solvent of MeOH (1 mL), THF (1 mL) and H2O (0.5 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (20 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 49 (18.0 mg, 76.6%) as a yellow solid. 443.1 [M+H]+


Step 3: To a mixture of 49 (18.0 mg, 0.041 mmol) in a mixed solvent of THF (2 mL) and water (1 mL) was added NaOH (1.46 mg, 0.036 mmol). The mixture was stirred at room temperature for 5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 49-Na (15.0 mg, 79.3%) as a yellow solid. LC/MS: 442.9 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J=8.8 Hz, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 6.71 (d, J=8.8 Hz, 1H), 5.43 (brs, 2H), 3.33-3.10 (m, 1H), 3.10 (s, 3H), 2.75-2.61 (m, 5H), 2.30 (s, 3H).


Example 51. Preparation of sodium 2-(8-chloro-2-((2-methoxyethyl)(2,2,2-trifluoroethyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (50)



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Step 1: A mixture of Intermediate A (100.0 mg, 0.25 mmol), a (50.0 mg, 0.50 mmol), Pd (OAc)2 (28.0 mg, 0.125 mmol), Xantphos (72.0 mg, 0.125 mmol) and Cs2CO3 (170.0 mg, 0.52 mmol) in 1,4-dioxane (2 mL) was heated to 90° C. for 16 h. The reaction mixture was cooled, diluted with ethyl acetate (10 mL) and washed with water (5 mL) and brine (5 mL) successively. The organic layer was dried over MgSO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford b (85.0 mg, 73%). LC/MS: 460.1 [M+H]+.


Step 2: To a mixture of b (85.0 mg, 0.185 mmol) in DMF (3 mL) was added sodium hydride (60% dispersion in mineral oil, 15.0 mg, 0.37 mmol) at 0° C. and stirred for 15 min. Then 1-bromo-2-methoxyethane (51.0 mg, 0.37 mmol) was added. The reaction mixture was stirred at room temperature for 2 h, quenched with water, then extracted with ethyl acetate (5 mL×3). The extracts were combined and washed with brine. The organic layer dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford c (57.0 mg, 59.5%). LC/MS: 518.1 [M+H]+.


Step 3: A mixture of c (57.0 mg, 0.11 mmol) and LiOH.H2O (20.0 mg, 0.48 mmol) in a mixed solvent of EtOH (5 mL), THF (5 mL) and H2O (2 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 50 (42.0 mg, 77%) as a yellow solid. 490.0 [M+H]+


Step 4: To a mixture of 50 (42.0 mg, 0.086 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (3.1 mg, 0.077 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 50-Na (28.5 mg, 64.9%) as a yellow solid. LC/MS: 490.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.23 (d, J=8.8 Hz, 1H), 8.12 (d, J=8.4 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H), 6.89 (d, J=8.8 Hz, 1H), 5.38 (s, 2H), 4.59 (s, 2H), 3.85 (s, 2H), 3.61 (t, J=5.6 Hz, 2H), 3.29 (s, 3H), 2.29 (s, 3H).


Example 52. Preparation of sodium 2-(8-chloro-2-((2,2-difluoroethyl)(2-methoxyethyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (51)



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Step 1: A mixture of Intermediate A (300.0 mg, 0.63 mmol), 2-methoxyethan-1-amine (172.0 mg, 1.89 mmol) and Et3N (1 mL) in THF (2 mL) was stirred in a sealed tube at 100° C. for 16 h. The reaction mixture was concentrated in vacuo. The residue was purified by Prep-HPLC to afford a (325.0 mg, 98.6%). LC/MS: 436.2 [M+H]+.


Step 2: To a mixture of a (60.0 mg, 0.138 mmol) in DMF (2.5 mL) was added sodium hydride (60% dispersion in mineral oil, 11.0 mg, 0.276 mmol) at 0° C. and stirred for 20 min. Then 2,2-difluoroethyl trifluoromethanesulfonate (b) (59.0 mg, 0.276 mmol) was added. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was poured into water (5 mL), then extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford 38c (60.0 mg, 87.2%). LC/MS: 500.1 [M+H]+.


Step 3: A mixture of c (60.0 mg, 0.12 mmol) and LiOH.H2O (20.0 mg, 0.48 mmol) in a mixed solvent of EtOH (5 mL), THF (5 mL) and H2O (2 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (10 mL), washed with ethyl acetate (5 mL).


The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 51 (45.0 mg, 79.5%) as a yellow solid. 472.1 [M+H]+


Step 4: To a mixture of 51 (45.0 mg, 0.095 mmol) in a mixed solvent of THF (10 mL) and water (10 mL) was added NaOH (3.6 mg, 0.09 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 51-Na (38.0 mg, 80.7%) as a yellow solid. LC/MS: 472.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.19 (d, J=8.8 Hz, 1H), 8.11 (d, J=8.4 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 6.81 (d, J=9.2 Hz, 1H), 6.53-6.25 (m, 1H), 5.28 (brs, 2H), 4.04 (t, J=13.2 Hz, 2H), 3.79 (s, 2H), 3.58 (t, J=4.4 Hz, 2H), 3.28 (s, 3H), 2.30 (s, 3H).


Example 53. Preparation of sodium 2-(8-chloro-2-((2,2-difluoroethyl)(2-methoxyethyl)amino)-9-ethoxy-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (52)



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Step 1: A mixture of Intermediate B (300.0 mg, 0.63 mmol), 2-methoxyethan-1-amine (172.0 mg, 1.89 mmol), TEA (1 mL), and THF (2 mL) was stirred at 100° C. for 16 h under N2. The reaction solvent was evaporated in vacuo. The residue was purified by Prep-HPLC to afford a (320 mg, 97%). LC/MS: 434.2 [M+H]+.


Step 2: To a mixture of a (60.0 mg, 0.139 mmol) in DMF (2.5 mL) was added sodium hydride (60% dispersion in mineral oil, 11.0 mg, 0.277 mmol) at 0° C. and stirred for 10 min. Then 2,2-difluoroethyl trifluoromethanesulfonate (b) (59.0 mg, 0.277 mmol) was added. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was poured into water (5 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford c (64.0 mg, 92.9%). LC/MS: 498.1 [M+H]+.


Step 3: A mixture of c (64.0 mg, 0.129 mmol) and LiOH.H2O (22.0 mg, 0.515 mmol) in a mixed solvent of EtOH (5 mL), THF (5 mL) and H2O (2 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 52 (60.0 mg, 99%) as a yellow solid. 470.1 [M+H]+


Step 4: A mixture of 52 (60.0 mg, 0.128 mmol) in THF/water (10 mL/10 mL) was added NaOH (4.6 mg, 0.115 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 52-Na (45.0 mg, 73%). LCMS: 470.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.24 (d, J=8.8 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 6.83 (d, J=9.2 Hz, 1H), 6.53-6.24 (m, 1H), 5.29 (brs, 2H), 4.08-4.01 (m, 2H), 3.97-3.92 (m, 2H), 3.80 (s, 2H), 3.58 (t, J=4.8 Hz, 2H), 3.28 (s, 3H), 1.37 (t, J=6.8 Hz, 3H).


Example 54. Preparation of sodium 2-(8-chloro-2-(((1s,3s)-3-methoxycyclobutyl)(2-methoxyethyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (74)



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Step 1: To a mixture of a (40 mg, 0.086 mmol, previously isolated from SFC) in dry DMF (3 mL) at 0° C. was added NaH (60%, 34 mg, 0.86 mmol). The resulting mixture was stirred at 0° C. for 10 minutes. 1-bromo-2-methoxyethane (60 mg, 0.43 mmol) was added and the reaction was further stirred at room temperature for 3 h. LCMS showed a was completely consumed. The reaction was quenched with saturated aqueous NH4Cl (3 mL) and then acidified with 1 M HCl to a pH between 3 and 4. The mixture was extracted with ethyl acetate (10 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated to give 74 (34 mg, 81%) as a yellow solid. LC/MS: 492.2 [M+H]+.


Step 2: To a mixture of 74 (34 mg, 0.069 mmol) in THF (3 mL) was added NaOH (0.1 M, 0.62 mL, 0.062 mmol). The resulting mixture was stirred at room temperature for 1 h. Water (10 mL) was added and the reaction mixture was washed with ethyl acetate (10 mL).


The aqueous phase was lyophilized to afford 74-Na (4.5 mg, 13%). LCMS: 492.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (d, J=8.8 Hz, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 6.68 (d, J=8.8 Hz, 1H), 5.38 (s, 2H), 4.21-4.14 (m, 1H), 3.67 (t, J=7.2 Hz, 2H), 3.66-3.64 (m, 1H), 3.52 (t, J=7.2 Hz, 2H), 3.26 (s, 3H), 3.19 (s, 3H), 2.65-2.69 (m, 2H), 2.29 (s, 3H), 2.02-2.07 (m, 2H).


Example 55. Preparation of sodium 2-(3,8-dichloro-2-(ethyl(2-methoxyethyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (59)



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Step 1: N-Chlorosuccinimide (4.30 g, 32.2 mmol) was added to a mixture of 2-amino-6-chloronicotinic acid (a, 4.30 g, 25.0 mmol) in acetonitrile (200 mL), and the resulting mixture was heated at 80° C. for 1.5 h. The reaction mixture was subsequently poured into ice water. After the ice had melted, the resulting slurry was filtered through a fritted glass funnel. The collected solids were dried to afford (b) (4.7 g, 91.3%). LC/MS: 207.0 [M+H]+.


Step 2: A heterogeneous mixture of 2-amino-6-chloronicotinic acid (b) (1.0 g, 4.83 mmol), (2-chloro-6-iodophenyl)(methyl)sulfane (c) (1.65 g, 5.83 mmol), K2CO3 (2.0 g, 14.55 mmol), copper powder (93 mg, 1.46 mmol), and CuI (280.0 mg, 1.46 mmol) in DMA (30 mL) was stirred at 140° C. for 16 h under N2 atmosphere and then cooled to room temperature. The mixture was filtered through celite. The filtrate was acidified to pH=2 with conc. HCl and extracted with ethyl acetate (20 mL×3). The combined organic layers were concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10/1) to afford d (1.3 g, 73.9%). LC/MS: 363.0 [M+H]+.


Step 3: To a mixture of d (1.3 g, 3.59 mmol) in Eaton's Reagent (20 mL) was added P205 (10 g). The mixture was stirred at 65° C. for 2 h, poured into ice-water and then extracted with ethyl acetate (20 mL×3). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=20/1) to afford e (0.35 g, 28.3%). LC/MS: 345.0 [M+H]+.


Step 4: A heterogeneous mixture of e (200.0 mg, 0.58 mmol), ethyl 2-bromoacetate (193.0 mg, 1.16 mmol) and Cs2CO3 (570.0 mg, 1.74 mmol) in acetonitrile (20 mL) was stirred at 35° C. for 16 h. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (20 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=10:1) to afford f (100.0 mg, 38%) as a yellow solid. LC/MS: 431.0 [M+H]+.


Step 5: A mixture of f (50 mg, 0.116 mmol), g (120 mg, 1.16 mmol) and LiOH.H2O (25 mg, 0.58 mmol) in THF/water (2/1 mL) was stirred at 50° C. for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 59 (45 mg, 82%). LC/MS: 470.1 [M+H]+.


Step 6: A mixture of 59 (45 mg, 0.096 mmol) in THF/water (10 mL/10 mL) was added NaOH (3.5 mg, 0.086 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 59-Na (43 mg, 91%). LCMS: 470.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (s, 1H), 8.10 (d, J=8.4 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H), 5.32 (s, 2H), 3.80-3.65 (m, 6H), 3.30 (s, 3H), 2.31 (s, 3H), 1.25 (t, J=6.8 Hz, 3H).


Example 56. Preparation of sodium 2-(3,8-dichloro-2-(dimethylamino)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (58)



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Step 1: A mixture of a (50 mg, 0.116 mmol), dimethylamine (0.58 mL, 2 M in THF, 1.16 mmol) and LiOH.H2O (25 mg, 0.58 mmol) in THF/water (2 mL/1 mL) was stirred at 50° C. for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 58 (38 mg, 80%). LCMS: 412.1 [M+H]+.


Step 2: A mixture of 58 (38 mg, 0.09 mmol) in THF/water (10 mL/10 mL) was added NaOH (3.3 mg, 0.08 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 58-Na (36.2 mg, 89%). LCMS: 412.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.16 (s, 1H), 8.11 (d, J=8.4 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 5.34 (s, 2H), 3.20 (s, 6H), 2.31 (s, 3H).


Example 57. Preparation of sodium 2-(3,8-dichloro-2-((2-methoxyethyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (75)



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Step 1: A mixture of a (50 mg, 0.116 mmol), b (103 mg 1.16 mmol) and LiOH.H2O (24 mg, 0.58 mmol) in THF/water (4 mL/2 mL) was stirred at 50° C. for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 75 (43 mg, 81%). LCMS: 456.1 [M+H]+.


Step 2: A mixture of 75 (43 mg, 0.0945 mmol) in THF/water (10/10 mL) was added NaOH (3.6 mg, 0.0898 mmol) and stirred at room temperature for 1 h. The reaction mixture was washed with ethyl acetate and lyophilized to afford 75-Na (36 mg, 80%). LCMS: 456.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.16 (d, J=10.4 Hz, 2H), 7.49 (d, J=8.4 Hz, 1H), 5.42 (brs, 2H), 3.83 (t, J=6.0 Hz, 2H), 3.66 (t, J=5.6 Hz, 2H), 3.28 (s, 3H), 3.27 (s, 3H), 2.30 (s, 3H).


Example 58. Preparation of sodium 2-(3,8-dichloro-2-(methyl((tetrahydro-2H-pyran-4-yl)methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (76)



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Step 1: A mixture of a (70 mg, 0.163 mmol), b (56 mg, 0.468 mmol) and TEA (0.5 mL) in THF (3 mL) was stirred in a sealed tube at 110° C. for 16 h under N2. The reaction solvent was concentrated in vacuo. The crude residue was purified by Prep-HPLC to afford c (74 mg, 89%). LCMS: 510.2 [M+H]+.


Step 2: To a mixture of c (74 mg, 0.145 mmol) in DMF (5 mL) at 0° C. was added NaH (12 mg, 0.3 mmol). The resulting mixture was stirred at 0° C. for 10 minutes. Mel (42 mg, 0.29 mmol) was added and the mixture was further stirred at room temperature for 1 h under N2. The reaction mixture was poured into water (5 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The crude residue was purified by Prep-HPLC to afford d (64 mg, 84%). LCMS: 524.2 [M+H]+.


Step 3: A mixture of d (64 mg, 0.12 mmol) and LiOH.H2O (21 mg, 0.49 mmol) in THF/ethanol/water (5 mL/5 mL/2 mL) was stirred at room temperature for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 76 (50 mg, 83%). LCMS: 496.1 [M+H]+.


Step 4: A mixture of 76 (50 mg, 0.1 mmol) in THF/water (10 mL/10 mL) was added NaOH (3.8 mg, 0.095 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 76-Na (36 mg, 80%). LCMS: 496.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.15 (s, 1H), 8.11 (d, J=8.4 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 5.43 (brs, 2H), 3.82 (d, J=8.8 Hz, 2H), 3.62 (s, 2H), 3.29-3.25 (m, 5H), 2.32 (s, 3H), 2.10-2.03 (m, 1H), 1.57-1.54 (m, 2H), 1.28-1.20 (m, 2H).


Example 59. Preparation 2-(3,8-dichloro-2-(((1s,3s)-3-methoxycyclobutyl)(methyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (77)



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Step 1: A mixture of a (70 mg, 0.16 mmol), b (44 mg, 0.32 mmol) and TEA (162 mg) in THF (5 mL) was stirred in a sealed tube at 100° C. for 16 h under N2. LCMS showed the desired mass and a was consumed. The mixture was cooled to room temperature, diluted with water (5 mL), and then extracted with ethyl acetate (20 mL). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to afford c (80 mg, 99%) as a yellow solid. LCMS: 496.4 [M+H]+.


Step 2: To a mixture of c (80 mg, 0.16 mmol) in DMF (5 mL) at 0° C. was added NaH (12.8 mg, 0.32 mmol). The resulting mixture was stirred at 0° C. for 10 minutes. Mel (45 mg, 0.32 mmol) was added. The resulting mixture was stirred at room temperature for 2 h, then diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=3-4 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 77 (25.1 mg, 32%). LCMS: 482.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.90 (s, 1H), 8.26 (s, 1H), 8.23 (d, J=8.8 Hz, 1H), 7.59 (d, J=8.4 Hz, 1H), 5.48 (s, 2H), 4.26-4.18 (m, 1H), 3.70-3.63 (m, 1H), 3.18 (s, 3H), 3.10 (s, 3H), 2.67-2.64 (m, 2H), 2.29 (s, 3H), 2.05-1.98 (m, 2H).


Example 60. Preparation of 2-(8-chloro-2-(2-cyanopropan-2-yl)-9-(dimethylamino)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (15) and 2-(2-(1-amino-2-methyl-1-oxopropan-2-yl)-8-chloro-9-(dimethylamino)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (20)



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Step 1: To a mixture of a (300 mg, 0.974 mmol), Pd(OAc)2 (20 mg, 0.089 mmol), BINAP (55 mg, 0.088) and isobutyronitrile (2 mL) in THF (100 mL) was added dropwise NaHMDS (2 M in THF, 2.5 mL, 5.0 mmol) at room temperature. When the addition was complete, the mixture was further stirred at 50° C. for 10 min. The reaction solvent was evaporated in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate/THF=2:1:1) to afford b (234 mg, 63.9%). LC/MS: 341.1 [M+H]+.


Step 2: A heterogeneous mixture of b (234 mg, 0.69 mmol), ethyl 2-iodoacetate (le) (294 mg, 1.37 mmol) and Cs2CO3 (673 g, 2.06 mmol) in acetonitrile (40 mL) was stirred at 35° C. for 16 h. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated to afford crude c. A mixture of crude c and LiOH.H2O (600 mg, 14.3 mmol) in a mixed solvent of EtOH (20 mL), THF (20 mL) and H2O (10 mL) was stirred at 40° C. for 16 h. The reaction mixture was then diluted with water (10 mL) and washed with ethyl acetate (10 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (10 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 20 (26 mg, 75% purity) and Compound 15 (112 mg, 41%).


Compound 15: LCMS: 399.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.69 (brs, 1H), 8.65 (d, J=8.0 Hz, 1H), 8.19 (d, J=8.8 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 5.43 (brs, 2H), 2.76 (s, 6H), 1.77 (s, 6H).


20 was further purified by Prep-HPLC and pure 2.5 mg was obtained. LCMS: 417.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.61 (brs, 1H), 8.53 (d, J=8.0 Hz, 1H), 8.20 (d, J=8.4 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.41 (d, J=8.4 Hz, 1H), 7.07 (brs, 2H), 5.43 (brs, 2H), 2.76 (s, 6H), 1.54 (s, 6H).


Example 61. Preparation of sodium 2-(8-chloro-2-(2-cyanopropan-2-yl)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (24)



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Step 1: To a mixture of a (400 mg, 1.29 mmol), Pd(OAc)2 (30 mg, 0.13 mmol), BINAP (40 mg, 0.13) and isobutyronitrile (890 mg, 12.9 mmol) in THF (100 mL) was added dropwise NaHMDS (2 M in THF, 3.2 mL, 6.4 mmol) at room temperature. When the addition was complete, the mixture was further stirred at 50° C. for 10 min and then evaporated in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate/THF=2:1:1) to afford b (242 mg, 54.8%). LC/MS: 344.0 [M+H]+.


Step 2: A mixture of b (242 mg, 0.71 mmol), ethyl 2-bromoacetate (589 mg, 3.52 mmol), Cs2CO3 (1.2 g, 3.68 mmol) in acetonitrile (40 mL) was stirred at 40° C. for 16 h. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=10:1) to afford c (254 mg, 84%) as a yellow solid. LC/MS: 430.1 [M+H]+.


Step 3: A mixture of c (254 mg, 0.59 mmol) and LiOH.H2O (249 mg, 5.93 mmol) in a mixed solvent of THF (30 mL) and H2O (10 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (20 mL) and washed with ethyl acetate (20 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (15 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 24 (109 mg, 46%) as a yellow solid. 402.1 [M+H]+


Step 4: A mixture of 24 (40 mg, 0.10 mmol) in THF/water (10 mL/30 mL) was added NaOH (3.6 mg, 0.09 mmol). The mixture was stirred at room temperature for 0.5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 24-Na (33 mg, 78.6%). LCMS: 402.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.60 (d, J=8.0 Hz, 1H), 8.19 (d, J=8.8 Hz, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.53 (d, J=8.8 Hz, 1H), 5.64 (brs, 2H), 2.36 (s, 3H), 1.77 (s, 6H).


Example 62. Preparation of sodium 2-(8-chloro-2-(2-cyanopropan-2-yl)-9-ethoxy-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (25)



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Step 1: To a mixture of a (600 mg, 1.94 mmol), Pd(OAc)2 (44 mg, 0.196 mmol), BINAP (121 mg, 0.194) and isobutyronitrile (1.34 g, 19.4 mmol) in THF (30 mL) was added dropwise NaHMDS (2 M in THF, 5 mL, 10 mmol) at room temperature. After the addition was complete, the mixture was further stirred at 50° C. for 10 min. The reaction solvent was evaporated in vacuo. The crude residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate/THF=2:1:1) to afford b (275 mg, 41%). LC/MS: 342.0 [M+H]+.


Step 2: A mixture of b (275 mg, 0.81 mmol), ethyl 2-bromoacetate (673 mg, 4.03 mmol) and Cs2CO3 (1.3 g, 4.0 mmol) in acetonitrile (40 mL) was stirred at room temperature for 16 h. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The crude residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=10:1) to afford c (310 mg, 90%) as a yellow solid. LC/MS: 428.2 [M+H]+.


Step 3: A mixture of c (310 mg, 0.73 mmol) and LiOH.H2O (757 mg, 18.0 mmol) in a mixed solvent of THF (20 mL), EtOH (20 mL) and H2O (10 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (20 mL) and washed with ethyl acetate (20 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (15 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 25 (257 mg, 89%) as a yellow solid. LCMS: 400.1 [M+H]+


Step 4: A mixture of 25 (60 mg, 0.15 mmol) in THF/water (10 mL/30 mL) was added NaOH (5.4 mg, 0.135 mmol). The mixture was stirred at room temperature for 0.5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 25-Na (50 mg, 79%). LCMS: 400.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.64 (d, J=8.0 Hz, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.58 (d, J=8.4 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H), 5.53 (brs, 2H), 3.96 (q, J=6.8 Hz, 2H), 1.77 (s, 6H), 1.4 (t, J=6.8 Hz, 3H).


Example 63. Preparation of sodium 2-(2-acetyl-8-chloro-9-ethoxy-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (31)



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Step 1: A mixture of Intermediate B (150 mg, 0.381 mmol), a (206 mg, 0.571 mmol) and Pd(PPh3)2Cl2 (53 mg, 0.076 mmol) in DMF (5 mL) was heated at 100° C. for 16 h. The reaction mixture was cooled to room temperature. 2 N HCl (5 mL) and ethyl acetate (5 mL) was added that reaction mixture was further stirred at 45° C. for 1 h. The mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (15 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (ethyl acetate) to afford b (149.4 mg, 97.5%) as a yellow solid. LCMS: 403.1 [M+H]+.


Step 2: A mixture of b (149.4 mg, 0.372 mmol) and LiOH.H2O (156 mg, 3.72 mmol) in a mixed solvent of THF (15 mL), EtOH (15 mL) and H2O (1.5 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (20 mL) and washed with ethyl acetate (20 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (15 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 31 (73.1 mg, 53%) as a yellow solid. LCMS: 375.0 [M+H]+


Step 3: A mixture of 31 (18.1 mg, 0.048 mmol) in THF/water (5 mL/15 mL) was added NaOH (1.7 mg, 0.044 mmol). The mixture was stirred at room temperature for 0.5 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 31-Na (16.7 mg, 79%). LCMS: 375.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.71 (d, J=8.0 Hz, 1H), 8.09 (d, J=8.8 Hz, 1H), 7.81 (d, J=7.6 Hz, 1H), 7.46 (d, J=8.4 Hz, 1H), 5.54 (s, 2H), 3.98 (t, J=7.2 Hz, 2H), 2.72 (s, 3H), 1.41 (t, J=7.2 Hz, 3H), Example 64. Preparation of sodium 2-(2-acetyl-8-chloro-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (32)




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Step 1: A mixture of Intermediate A (150 mg, 0.38 mmol), a (206 mg, 0.57 mmol) and Pd(PPh3)2Cl2 (53 mg, 0.076 mmol) in DMF (5 mL) was heated to 100° C. for 16 h. The mixture was cooled to room temperature, acidified with 2 N HCl and further stirred for 1 h. The mixture was extracted with ethyl acetate, washed with brine, dried with Na2SO4, filtered, and then evaporated. The residue was purified by Prep-HPLC to afford b (56 mg, 37%). LCMS: 405.3 [M+H]+.


Step 2: A mixture of b (56 mg, 0.14 mmol) and LiOH.H2O (47 mg, 1.12 mmol) in THF/MeOH/water (3/3/1 mL) was stirred at room temperature for 2 h. The reaction was acidified with 1 M HCl to a pH between 2 to 3 then extracted with ethyl acetate. The combined organics were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to afford 32 (52 mg, 99.6%). LCMS: 377.3 [M+H]+.


Step 3: To a mixture of 32 (52 mg, 0.138 mmol) in THF/water (3/3 mL) was added NaOH (5.0 mg, 0.125 mmol). The resulting mixture was stirred at room temperature for 16 h. The mixture was washed with ethyl acetate. The aqueous layer was lyophilized to afford 32-Na (47.1 mg, 86%) LCMS: 377.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.67 (d, J=8.0 Hz, 1H), 8.20 (d, J=8.4 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.54 (d, J=8.4 Hz, 1H), 5.57 (s, 2H), 2.72 (s, 3H), 2.38 (s, 3H).


Example 65. Preparation of sodium 2-(8-chloro-2-((2-fluorophenyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (57)



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Step 1: A mixture of Intermediate A (100 mg, 0.25 mmol), a (41.9 mg, 0.38 mmol), XantPhos (21.7 mg, 0.038 mmol), Pd (OAc)2 (2.8 mg, 0.0125 mmol) and Cs2CO3 (179.2 mg, 0.55 mmol) in 1,4-dioxane (5 mL) was heated to 90° C. for 16 h. LCMS showed the reaction was complete. The mixture was poured into water then extracted with ethyl acetate. The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford b (85 mg, 71%). LCMS: 471.9 [M+H]+.


Step 2: A mixture of b (166 mg, 0.35 mmol) and LiOH.H2O (73 mg, 1.8 mmol) in THF/MeOH/water (10 mL, 4:2:1) was stirred at 50° C. for 16 h. LCMS showed the reaction was complete. The reaction was poured into water then acidified with conc. HCl to a pH of ˜1. The aqueous layer was extracted with ethyl acetate. The combined extracts were washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford 57 (117.1 mg, 71%). LCMS: 444.1 [M+H]+.


Step 3: To a mixture of 57 (66 mg, 0.15 mmol) in THF/water (10 mL/4 mL) was added NaOH (5.66 mg, 0.142 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and lyophilized to afford 57-Na (68 mg, 100%). LCMS: 444.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.56 (s, 1H), 8.32-8.28 (m, 1H), 8.23 (d, J=8.8 Hz, 1H), 8.13 (d, J=8.8 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H), 6.70 (t, J=8.4 Hz, 1H), 7.15-7.11 (m, 1H), 6.89 (d, J=8.0 Hz, 1H), 5.45-5.39 (m, 2H), 2.28 (s, 3H).


Example 66. Preparation of sodium 2-(8-chloro-2-(methyl(phenyl)amino)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (73)



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Step 1: A mixture of Intermediate A (100 mg, 0.25 mmol), a (53 mg, 0.50 mmol), XantPhos (15 mg, 0.02 mmol), Pd2(dba)3 (20 mg, 0.02 mmol) and Cs2CO3 (240 mg, 0.75 mmol) in toluene (10 mL) was heated to 110° C. for 16 h. The mixture was poured in water and extracted with ethyl acetate. The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford b (50 mg, 42%). LCMS: 468.2 [M+H]+.


Step 2: A mixture of b (50 mg, 0.11 mmol) and LiOH.H2O (42 mg, 1.0 mmol) in THF/MeOH/water (5 mL, 2:2:1) was stirred at room temperature for 16 h. The reaction was poured into water and acidified with conc. HCl to a pH of −1. The aqueous layer was extracted with ethyl acetate. The combined extracts were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated to afford 73 (45 mg, 97%). LCMS: 440.2 [M+H]+.


Step 3: To a mixture of 73 (45 mg, 0.10 mmol) in THF/water (2 mL/1 mL) was added NaOH (3.6 mg, 0.09 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and lyophilized to afford 73-Na (40 mg, 84%). LCMS: 440.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.11 (d, J=8.4 Hz, 1H), 8.07 (d, J=8.8 Hz, 1H), 7.52 (t, J=7.6 Hz, 2H), 7.42-7.37 (m, 4H), 6.29 (d, J=8.8 Hz, 1H), 5.44 (s, 2H), 3.53 (s, 3H), 2.31 (s, 3H).


Example 67. Preparation of sodium 2-(2,9-bis(dimethylamino)-8-methyl-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (61)



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Step 1: A mixture of a (50 mg, 0.13 mmol), methylboronic acid (23 mg, 0.39 mmol), NHC—Pd(II)-1-methylimidazole (8.2 mg, 0.013 mmol, for catalyst preparation see DOI: 10.1039/C6OB02704K) and KOH (23 mg, 0.39 mmol) in 1,4-dioxane (5 mL) was heated to 110° C. for 16 h. The reaction mixture was then diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 61 (15 mg, 33%) as a yellow solid. LC/MS: 355.3 [M+H]+


Step 2: A mixture of 61 (15 mg, 0.042 mmol) in water (15 mL) was added NaOH (1.7 mg, 0.042 mmol). The mixture was stirred at room temperature for 1 h and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 61-Na (7.1 mg, 47%). LCMS: 355.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.15 (d, J=8.8 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.01 (d, J=8.4 Hz, 1H), 6.60 (d, J=8.8 Hz, 1H), 5.35 (brs, 2H), 3.16 (s, 6H), 2.77 (s, 6H), 2.36 (s, 3H).


Example 68. Preparation of sodium 2-(2-(dimethylamino)-8-methyl-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (62)



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Step 1: A mixture of a (35 mg, 0.088 mmol), methylboronic acid (16 mg, 0.263 mmol), NHC—Pd(II)-Im (5.6 mg, 0.009 mmol, for catalyst preparation see DOI: 10.1039/C6OB02704K), KOH (15 mg, 0.263 mmol), and dioxane (5 mL) was heated at 85° C. for 16 h. The reaction mixture was then diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to yield 62 (28 mg, 52%) as a solid. LCMS: 358.2 [M+H]+.


Step 2: A mixture of 62 (28 mg, 0.078 mmol) in water (5 mL) was added NaOH (2.8 mg, 0.070 mmol) was stirred at room temperature for 16 hours, washed with ethyl acetate (3 mL). The aqueous layer was separated and was lyophilized to afford 62-Na (25.2 mg, 84%). LCMS: 358.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (d, J=9.2 Hz, 1H), 8.03 (d, J=8.0 Hz, 1H), 7.18 (d, J=8.0 Hz, 1H), 6.62 (d, J=8.8 Hz, 1H), 5.58 (brs, 2H), 3.16 (s, 6H), 2.57 (s, 3H), 2.17 (s, 3H).


Example 69. Preparation of sodium 2-(2-(tert-butyl)-8-methyl-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetate (63)



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Step 1: A mixture of Compound 12 (50 mg, 0.12 mmol), methylboronic acid (72 mg, 1.2 mmol), NHC—Pd(II)Im (8 mg, 0.012 mmol, for catalyst preparation see DOI: 10.1039/C6OB02704K), KOH (34 mg, 0.6 mmol) and 1,4-dioxane (10 mL) was heated to 85° C. for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 63 (33 mg, 75%). LC/MS: 371.2 [M+H]+.


Step 2: A mixture of 63 (33 mg, 0.089 mmol) in THF/water (10/10 mL) was added NaOH (3.4 mg, 0.085 mmol). The reaction was stirred at room temperature for 1 h and then washed with ethyl acetate (3 mL). The aqueous layer was separated and was lyophilized to afford 63-Na. LC/MS: 371.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.40 (d, J=8.4 Hz, 1H), 8.09 (d, J=8.0 Hz, 1H), 7.35 (d, J=8.0 Hz, 1H), 7.24 (d, J=8.0 Hz, 1H), 5.65 (brs, 2H), 2.22 (s, 3H), 1.37 (s, 9H).


Example 70. Preparation of sodium 2-(2-(2-cyanopropan-2-yl)-9-(dimethylamino)-8-methyl-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (64)



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Step 1: A mixture of Compound 15 (40 mg, 0.10 mmol), methylboronic acid (60 mg, 1.0 mmol), NHC—Pd(II)Im (6.5 mg, 0.01 mmol, for catalyst preparation see DOI: 10.1039/C6OB02704K) and KOH (17 mg, 0.3 mmol) in 1,4-dioxane (10 mL) was heated to 85° C. for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 64 (30.4 mg, 80%). LC/MS: 379.3 [M+H]+.


Step 2: A mixture of 64 (30.4 mg, 0.08 mmol) in THF/water (10/10 mL) was added NaOH (3.0 mg, 0.076 mmol). The reaction was stirred at room temperature for 1 h and then washed with ethyl acetate (3 mL). The aqueous layer was separated and was lyophilized to afford 64-Na (25.4 mg, 78.9%). LC/MS: 379.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.56 (d, J=8.0 Hz, 1H), 7.98 (d, J=8.0 Hz, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.14 (d, J=8.0 Hz, 1H), 5.42 (brs, 2H), 2.82 (s, 6H), 2.40 (s, 3H), 1.76 (s, 6H).


Example 71. Preparation of 2-(9-(dimethylamino)-2-(((1s,3s)-3-methoxycyclobutyl)(methyl)amino)-8-methyl-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid-67 and 2-(9-(dimethylamino)-2-(((1r,3r)-3-methoxycyclobutyl)(methyl)amino)-8-methyl-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid-66



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Step 1: A mixture of Intermediate C (300 mg, 0.763 mmol), a (261 mg, 1.91 mmol) and TEA (1.06 mL, 7.63 mmol) in THF (10 mL) was heated to 120° C. in a sealed tube for 16 h. The reaction was cooled, poured into water (30 mL) then extracted with ethyl acetate (40 mL×2). The combined organics were washed with water (20 mL×2), brine (20 mL), dried over Na2SO4, filtered, and then evaporated. The residue was purified by Prep-HPLC to afford b (342.7 mg, 98%). LC/MS: 459.2 [M+H]+.


Step 2: To a mixture of b (342.7 mg, 0.75 mmol) in DMF (10 mL) at 0° C. was added NaH (60 wt. %, 300 mg, 7.5 mmol). The reaction was stirred for 30 min before adding Iodomethane (536 mg, 3.75 mmol). The mixture was further stirred at room temperature for 16 h. The reaction was poured into water (30 mL) and acidified with 1 N HCl to a pH of 5. The aqueous layer was extracted with ethyl acetate (100 mL×2). The combined extracts were washed with water (20 mL×2), saturated aqueous NH4Cl (20 mL×2) and brine successively. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by Prep-HPLC to afford c (180 mg, 47%). LC/MS: 445.1 [M+H]+.


Step 3: A mixture of c (157.9 mg, 0.356 mmol), methylboronic acid (214 mg, 3.56 mmol), NHC—Pd(II)-Im (23 mg, 0.0356 mmol, for catalyst preparation see DOI: 10.1039/C6OB02704K), KOH (100 mg, 1.78 mmol) and 1,4-dioxane (20 mL) were stirred at 100° C. for 16 h under N2. The reaction was cooled, diluted with water (40 mL), the pH adjusted to 4 with 1 N HCl, then extracted with ethyl acetate (50 mL×2). The combined organics were washed with water (20 mL×2), dried over Na2SO4, evaporated and filtered. The filtrate was concentrated in vacuo and the residue was purified to afford d (40.7 mg, a mixture of 66 and 67, ration of 1:9) which was further separated by SFC to afford 66 (1.6 mg, 1%) and 67 (28.4 mg, 19%).


57e: LC/MS: 425.4 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (d, J=9.2 Hz, 1H), 8.00 (d, J=7.6 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 6.66 (d, J=8.0 Hz, 1H), 5.40-5.32 (m, 2H), 5.02-4.98 (m, 1H), 3.98 (s, 1H), 3.21 (s, 3H), 3.11 (s, 3H), 2.71 (s, 6H), 2.43 (s, 3H), 2.37-2.34 (m, 2H), 2.03-1.97 (m, 2H).


57d: LC/MS: 425.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.58 (s, 1H), 8.19 (d, J=8.8 Hz, 1H), 8.02 (d, J=8.4 Hz, 1H), 7.11 (d, J=8.0 Hz, 1H), 6.72 (d, J=9.2 Hz, 1H), 5.35 (s, 2H), 4.52-4.34 (m, 1H), 3.69-3.62 (m, 1H), 3.19 (s, 3H), 3.08 (s, 3H), 2.70 (s, 6H), 2.67-2.60 (m, 2H), 2.45 (s, 3H), 2.07-2.00 (m, 2H).


Example 72. Preparation of 2-(2-(((1s,3s)-3-methoxycyclobutyl)(methyl)amino)-8-methyl-9-(methylthio)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (69)



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A mixture of Compound 44 (100 mg, 0.22 mmol), methylboronic acid (132 mg, 2.2 mmol), NHC—Pd(II)-Im (14 mg, 0.022 mmol, for catalyst preparation see DOI: 10.1039/C6OB02704K) and KOH (62 mg, 1.10 mmol) in 1,4-dioxane (4 mL) were heated to 90° C. under N2 for 16 h. LCMS showed the starting material was completely consumed and one major peak with the desired mass detected. The reaction mixture was diluted with water (15 mL) then filtered through Celite. The filtrate was concentrated and the residue was purified by Prep-HPLC to afford 69 (45.3 mg, 47%) as a yellow solid. LCMS: 428.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.63 (brs, 1H), 8.19 (d, J=8.8 Hz, 1H), 8.13 (d, J=8.0 Hz, 1H), 7.32 (d, J=8.0 Hz, 1H), 6.75 (d, J=8.8 Hz, 1H), 5.51 (s, 2H), 4.54 (brs, 1H), 3.70-3.65 (m, 1H), 3.19 (s, 3H), 3.09 (s, 3H), 2.66 (s, 3H), 2.64-2.62 (m, 2H), 2.14 (s, 3H), 2.06-2.04 (m, 2H)


Example 73. Preparation of sodium 2-(8-chloro-2-(1-methylcyclopropyl)-9-(methylthio)-5-oxobenzo[b][1,8] naphthyridin-10(5H)-yl)acetate (68)



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Step 1: A mixture of Intermediate A (100 mg, 0.25 mmol), a (85 mg, 0.51 mmol), Pd(PPh3)2Cl2 (18 mg, 0.025 mmol) and Cs2CO3 (165 mg, 0.51 mmol) in THF (10 mL) and water (2 mL) was heated to 60° C. for 16 h then evaporated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10/1) to afford b (95 mg, 93.1%) as a yellow solid. LC/MS: 403.2 [M+H]+


Step 2: Trifluoroacetic acid (41 mg, 0.36 mmol) was added to a mixture of diethylzinc (1 M in toluene, 0.6 mL, 0.6 mmol) in DCM (15 mL) at −20° C. The mixture was stirred at −20° C. for 20 min. Diiodomethane (322 mg, 1.2 mmol) was added and the reaction was stirred for an additional 20 min. b (95 mg, 0.24 mmol) in DCM (5 mL) was added. The resulting mixture was stirred at room temperature for 16 h, poured into water (10 mL), then extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Prep-HPLC to afford c (21 mg, 20.4%). LC/MS: 417.2 [M+H]+.


Step 3: A mixture of c (21 mg, 0.05 mmol) and LiOH.H2O (42 mg, 1.0 mmol) in a mixed solvent of EtOH (3 mL), THF (10 mL) and H2O (3 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 68 (12 mg, 60%) as a yellow solid. LCMS: 389.2 [M+H]+.


Step 4: A mixture of 68 (12 mg, 0.03 mmol) in water (15 mL) was added NaOH (1.2 mg, 0.03 mmol). The mixture was stirred at room temperature for 30 min and washed with ethyl acetate (3 mL). The aqueous layer was separated and evaporated to provide 68-Na (10.0 mg, 83.3%). LCMS: 389.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.38 (d, J=8.0 Hz, 1H), 8.14 (d, J=8.4 Hz, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.27 (d, J=8.0 Hz, 1H), 5.47 (s, 2H), 2.32 (s, 3H), 1.54 (s, 3H), 1.35-1.37 (m, 2H), 0.94-0.96 (m, 2H).


Example 74. Preparation of 2-(8-chloro-9-(methylthio)-2-(oxetan-3-ylidenemethyl)-5-oxobenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (65)



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Step 1: A mixture of Intermediate A (50 mg, 0.126 mmol), a (26 mg, 0.15 mmol), Pd(PPh3)2Cl2 (8.6 mg, 0.012 mmol) and Cs2CO3 (79 mg, 0.24 mmol) in THF (10 mL) and water (2 mL) was heated to 60° C. for 16 h. The reaction solvent was evaporated in vacuo and the crude residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to afford b (45 mg, 83.3%) as a yellow solid. LC/MS: 431.2 [M+H]+


Step 2: A mixture of b (78 mg, 0.18 mmol) and LiOH.H2O (76 mg, 1.8 mmol) in a mixed solvent of THF (10 mL) and H2O (3 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (5 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 65 (31.2 mg, 41%). LCMS: 403.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.48 (d, J=8.4 Hz, 1H), 8.22 (d, J=8.8 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.26 (d, J=8.0 Hz, 1H), 6.46 (s, 1H), 5.68 (s, 2H), 5.42 (s, 2H), 5.34 (s, 2H), 2.30 (s, 3H).


Example 75. Preparation of 2-(8-chloro-2-(dimethylamino)-5-oxo-9-vinylbenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (78)



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Step 1: A mixture of a (1.72 g, 10 mmol), b (3.8 g, 12 mmol), Cu (192 mg, 3 mmol), CuI (576 mg, 3 mmol) and K2CO3 (2.76 g, 20 mmol) in DMA (30 mL) was stirred at 130° C. for 16 h. The reaction mixture was concentrated and the remaining residue was purified by column chromatography on silica gel (petroleum ether) to afford c (3.4 g, 94%). LC/MS: 362.9 [M+H]+.


Step 2: A mixture of c (1.4 g, 4.46 mmol), Eaton's Reagent (30 mL), and P205 (30 g) was stirred at 60° C. for 4 h under N2. The reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (100 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to afford crude d (1.3 g, 98%). LC/MS: 345.0 [M+H]+.


Step 3: A mixture of d (1.3 g, 3.81 mmol), dimethylamine (2 M in THF, 19 mL, 38 mmol) and LiOH.H2O (800 mg, 19.05 mmol) in THF/H2O (10 mL/1 mL) was stirred at 55° C. for 16 h under N2. The reaction mixture was concentrated and the remaining residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=10:1) to provide e (180 mg, 14%). LC/MS: 354.0 [M+H]+.


Step 4: A mixture of e (70 mg, 0.2 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (50 mg, 0.4 mmol), K2CO3 (55 mg, 0.4 mmol) and Pd(PPh3)4 (23 mg, 0.02 mmol) in dioxane/water (10 mL/1 mL) was stirred at 90° C. for 16 h. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer dried over anhydrous Na2SO4, filtered and concentrated to afford crude f (30 mg, 50%). LC/MS: 300.1 [M+H]+.


Step 5: A mixture of f (30 mg, 0.1 mmol), g (43 mg, 0.2 mmol) and Cs2CO3 (98 mg, 0.3 mmol) in ACN (5 mL) was stirred at room temperature for 2 h under N2. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to afford crude h (20 mg, 52%). LC/MS: 386.1 [M+H]+.


Step 6: A mixture of h (20 mg, 0.052 mmol) in THF/water (5 mL/2 mL) was added LiOH.H2O (5 mg, 0.104 mmol) and was stirred at room temperature for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH-=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 78 (9.8 mg, 52%). LC/MS: 358.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.69 (s, 1H), 8.20 (d, J=8.8 Hz, 1H), 8.19 (d, J=8.8 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H), 7.10 (q, J=11.2 Hz, 1H), 6.74 (d, J=9.2 Hz, 1H), 5.63 (dd, J=11.2 Hz, J=1.2 Hz, 1H), 5.25 (d, J=16.8 Hz, 1H), 5.05 (s, 2H), 3.16 (s, 6H).


Example 76. Preparation of 2-(8-chloro-2-(dimethylamino)-5-oxo-9-phenylbenzo[b][1,8]naphthyridin-10(5H)-yl)acetic acid (79)



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Step 1: A mixture of a (70 mg, 0.2 mmol), phenylboronic acid (50 mg, 0.4 mmol), K2CO3 (55 mg, 0.4 mmol) and Pd(PPh3)4 (23 mg, 0.02 mmol) in dioxane/water (10/1 mL) was stirred at 90° C. for 16 h. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to afford b (60 mg, 86%). LC/MS: 350.2 [M+H]+.


Step 2: A mixture of b (60 mg, 0.17 mmol), c (57 mg, 0.34 mmol), Cs2CO3 (170 mg, 0.51 mmol), and in ACN (10 mL) was stirred at room temperature for 2 h under N2. The reaction mixture was poured into water (10 mL), then extracted with ethyl acetate (10 mL×3). The extracts were combined and washed with brine. The organic layer dried over anhydrous Na2SO4, filtered and concentrated to afford crude d (35 mg, 47%). LC/MS: 436.2 [M+H]+.


Step 3: To a mixture of d (35 mg, 0.08 mmol) in ethanol/water (10 mL/2 mL) was added LiOH.H2O (40 mg, 0.32 mmol), which was stirred at room temperature for 16 h under N2. The reaction mixture was diluted with water (10 mL) and washed with ethyl acetate (5 mL). The aqueous phase was acidified to pH=1-2 with 1 N HCl and then extracted with ethyl acetate (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by Prep-HPLC to afford 79 (28 mg, 85%). LCMS: 408.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.45 (s, 1H), 8.33 (d, J=8.4 Hz, 1H), 8.23 (d, J=8.8 Hz, 1H), 7.55-7.48 (m, 4H), 7.33 (d, J=7.6 Hz, 2H), 6.74 (d, J=8.8 Hz, 1H), 4.55 (s, 2H), 3.09 (s, 6H).


Example 77. Preparation of Sodium 2-{8-chloro-2-[(2-methanesulfonylethyl)(methyl)amino]-9-(methylsulfanyl)-5-oxobenzo[b]1,8-naphthyridin-10-yl}acetate (34)



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To a mixture of Intermediate A (40 mg, 0.1 mmol, 1.0 eq) and a (70 mg, 0.5 mmol, 5.0 eq) in THF (1 mL)/H2O (1 mL) was added lithium hydroxide monohydrate (21 mg, 0.5 mmol, 5.0 eq). The resulting mixture was stirred at 50° C. for 16 h. After completion of the reaction, as monitored by LCMS, the reaction mixture was acidified with 1N HCl (aq) to pH=5. The resulting mixture was concentrated under vacuum to remove THF. The crude residue was purified by Prep-HPLC (water/acetonitrile=95/5 to 5/95) to afford 34 as a free acid. To 34 was added one equivalent of sodium hydroxide (0.1 M in water) and stirred for 15 min at 25° C. The resulting mixture was lyophilized to provide the 34-Na sodium salt. LCMS: 470.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.79 (s, 1H), 8.22 (t, J=8.5 Hz, 2H), 7.54 (d, J=8.2 Hz, 1H), 6.78 (d, J=8.7 Hz, 1H), 5.52 (s, 2H), 4.06 (d, J=7.2 Hz, 2H), 3.48 (t, J=6.9 Hz, 2H), 3.18 (s, 3H), 3.05 (s, 3H), 2.28 (s, 3H).


Example 78. Preparation of sodium 2-(8-chloro-2-{methyl[2-(propane-2-sulfonyl)ethyl]amino}-9-(methylsulfanyl)-5-oxobenzo[b]1,8-naphthyridin-10-yl)acetate (35)



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To a mixture of Intermediate A (40 mg, 0.1 mmol, 1.0 eq) and a (70 mg, 0.5 mmol, 5.0 eq) in THF (1 mL)/H2O (1 mL) was added lithium hydroxide monohydrate (21 mg, 0.5 mmol, 5.0 eq). The resulting mixture was stirred at 50° C. for 16 h. After completion of the reaction, as monitored by LCMS, the reaction mixture was acidified with 1N HCl (aq) to pH=5. The resulting mixture was concentrated under vacuum to remove THF. The crude residue was purified by Prep-HPLC (water/acetonitrile=95/5 to 5/95) to afford 35 as a free acid. To 35 was added one equivalent of sodium hydroxide (0.1 M in water) and stirred for 15 min at 25° C. The resulting mixture was lyophilized to provide the 35-Na sodium salt. LCMS: 498.7 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.74 (s, 1H), 8.22 (dd, J=10.4, 8.7 Hz, 2H), 7.54 (d, J=8.4 Hz, 1H), 6.77 (d, J=8.9 Hz, 1H), 5.55 (s, 2H), 4.07 (s, 2H), 3.44 (d, J=6.7 Hz, 2H), 3.19 (s, 3H), 2.28 (s, 3H), 1.25 (d, J=6.8 Hz, 6H).


Example 79. Preparation of Sodium 2-(8-chloro-2-{[2-(imidazole-1-yl)ethyl](methyl)amino}-9-(methylsulfanyl)-5-oxobenzo[b]1,8-naphthyridin-10-yl)acetate (71)



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To a mixture of Intermediate A (40 mg, 0.1 mmol, 1.0 eq) and a (70 mg, 0.5 mmol, 5.0 eq) in THF (1 mL)/H2O (1 mL) was added lithium hydroxide monohydrate (21 mg, 0.5 mmol, 5.0 eq). The resulting mixture was stirred at 50° C. for 16 h. After completion of the reaction, as monitored by LCMS, the reaction mixture was acidified with 1N HCl (aq) to pH=5. The resulting mixture was concentrated under vacuum to remove THF. The crude residue was purified by Prep-HPLC (water/acetonitrile=95/5 to 5/95) to afford 71 as a free acid. To 71 was added one equivalent of sodium hydroxide (0.1 M in water) and stirred for 15 min at 25° C. The resulting mixture was lyophilized to provide the 71-Na sodium salt. LCMS: 458.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J=8.5 Hz, 2H), 7.60 (s, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.22 (d, J=1.2 Hz, 1H), 6.87 (s, 1H), 6.69 (d, J=9.0 Hz, 1H), 5.44 (s, 2H), 4.23 (t, J=6.2 Hz, 2H), 3.96 (s, 2H), 2.96 (s, 3H), 2.28 (s, 3H).


Example 80. Preparation of sodium 2-(8-chloro-9-ethoxy-2-{[2-(imidazole-1-yl)ethyl](methyl)amino}-5-oxobenzo[b]1,8-naphthyridin-10-yl)acetate (72)



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To a mixture of Intermediate B (40 mg, 0.1 mmol, 1.0 eq) and a (70 mg, 0.5 mmol, 5.0 eq) in THF (1 mL)/H2O (1 mL) was added lithium hydroxide monohydrate (21 mg, 0.5 mmol, 5.0 eq). The resulting mixture was stirred at 50° C. for 16 h. After completion of the reaction, as monitored by LCMS, the reaction mixture was acidified with 1N HCl (aq) to pH=5. The resulting mixture was concentrated under vacuum to remove THF. The crude residue was purified by Prep-HPLC (water/acetonitrile=95/5 to 5/95) to afford 72 as a free acid. To 72 was added one equivalent of sodium hydroxide (0.1 M in water) and stirred for 15 min at 25° C. The resulting mixture was lyophilized to provide the 72-Na sodium salt. LCMS: 456.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.21 (s, 1H), 8.04 (d, J=8.6 Hz, 1H), 7.62 (d, J=1.1 Hz, 1H), 7.42 (d, J=8.6 Hz, 1H), 7.22 (d, J=1.3 Hz, 1H), 6.88 (d, J=1.1 Hz, 1H), 6.70 (d, J=8.9 Hz, 1H), 5.45 (s, 2H), 4.24 (t, J=6.2 Hz, 2H), 3.96 (q, J=7.2, 6.7 Hz, 4H), 2.97 (s, 3H), 1.37 (t, J=7.0 Hz, 3H).


Example 81. Preparation of sodium 2-[8-chloro-9-(dimethylamino)-2-{[2-(imidazole-1-yl)ethyl](methyl)amino}-5-oxobenzo[b]1,8-naphthyridin-10-yl]acetate (80)



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To a mixture of Intermediate B (40 mg, 0.1 mmol, 1.0 eq) and a (70 mg, 0.5 mmol, 5.0 eq) in THF (1 mL)/H2O (1 mL) was added lithium hydroxide monohydrate (21 mg, 0.5 mmol, 5.0 eq). The resulting mixture was stirred at 50° C. for 16 h. After completion of the reaction, as monitored by LCMS, the reaction mixture was acidified with iN HCl (aq) to pH=5. The resulting mixture was concentrated under vacuum to remove THF. The crude residue was purified by Prep-HPLC (water/acetonitrile=95/5 to 5/95) to afford 80 as a free acid. To 80 was added one equivalent of sodium hydroxide (0.1 M in water) and stirred for 15 min at 25° C. The resulting mixture was lyophilized to provide the 80-Na sodium salt. LCMS: 455.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J=8.3 Hz, 1H), 8.13 (d, J=8.6 Hz, 1H), 7.66 (s, 1H), 7.35 (d, J=8.6 Hz, 1H), 7.23 (d, J=1.4 Hz, 1H), 6.91 (t, J=1.1 Hz, 1H), 6.70 (d, J=8.9 Hz, 1H), 5.29 (s, 2H), 4.25 (t, J=6.4 Hz, 2H), 3.95 (s, 2H), 2.99 (s, 3H), 2.73 (s, 6H).


Example 82: Radiometric Filtration Binding Competition Assay

A radiometric filtration binding competition assay was performed to quantify the binding affinity of compounds of the present application for the human or mouse STING protein, or associated HAQ human isoform. The competition format filtration binding assay detects the ability of small molecule compounds from binding to and inhibiting the subsequent binding of a tritiated [H3] 2′3′cGAMP high affinity STING ligand. The resulting dose response was fit by non-linear regression to determine an IC50 value, from which a Ki value for each compound was extrapolated.


Briefly, the recombinant C-terminal ligand binding domain of the human STING protein (140-379) or mouse STING protein (139-328) [0.5 μM] was incubated in the presence of a compound of the present application (8 point, 2 fold dilution starting at 300 μM) for 30 min or under control conditions. H3 labeled 2′3′cGAMP [25 nM] was then added and allowed to reach binding equilibrium (1 hour). The resulting complexes were then filtered, dried, and scintillation fluid was added and the remaining radio signal was measured to determine the degree of compound ligand inhibition. This assay format was optimized for both the WT and HAQ isoforms of the human protein and the mouse orthologue protein.


Assay Development

Parameters optimized:

    • a. Form of protein: 6×HIS-SUMO tag or untagged
    • b. Concentration of target protein: Minimum concentration that achieved >80 max signal
    • c. Assay DMSO concentration: 10%-0.1%
    • d. Assay Buffer: Base (Tris or phosphate), Salt concentration, +/−Tween (0.1-2%)
    • e. Synthesis of Probe: (1) Enzymatic incorporation of a S35-ATP and cold GTP into a 2′3′cGAMP product using a mouse cGAS enzyme with subsequent purification. (2) Tritium incorporation into 2′3′cGAMP
    • f. Probe Concentration: Minimum concentration that achieved max assay window
    • g. Assay Format: Scintillation Proximity Bead or Filtration Binding Plate
    • h. Assay Plate: 384 vs 96 well format
    • i. Assay incubation times for successive incubation steps
















2′3′cGAMP control












Target Protein
Kd [nM]
Ki [nM]
Z-factor
Assay Window














hSTING-WT
11 ± 4

 61 ± 9
0.75
21.5


hSTING-HAQ
10 ± 4
50.5 ± 8
0.78
22.3


mSTING
16 ± 5
362.5 ± 21
0.65
27





















Assay
Assay



Parameter
Component
STX Assay







Principle

Filtration Based Direct Competition




Assay


Recombinant
Expression
pET


Target
Vector


Protein
Expression
6XHIS-SUMO-hSTING (140-379)



Construct



Protein

E. Coli Expression Codon Optimized



coding gene
hSTING (140-379)



Expression
BL-21(DE3) E. coli



Cell Line



Purification
(1) Talon Metal Affinity Resin



Strategy
(2) Desalting (HiTrap)




(3) Concentrate




(6) Gel Filtration (Superdex 200)




(7) Concentrate and Store




(8) QC by Analytical SEC, SDS-PAGE



Storage
20 mM PBS, pH 8.0, 150 mM NaCl,



Buffer
0.2% tween-20 and 10% glycerol




(hSTING)



Working Assay
0.5 μM



Concentration


Probe
Probe Radio
H3



Label



Probe
 25 nM



concentration


Controls
ZPE
+Buffer



HPE
+10 μM 2′3′cGAMP



Positive
2′3′cGAMP (20 μM-310 nM)



Control



Negative
DMXAA (150 μM-150 nM)



Control


Buffer
Assay
1x PBS + 1% DMSO



Buffer



Plate
96 Well GF/C unifilter Plate



Instrument
PE Microbeta


Readout
Signal
Scintillation (CPM)



Normalized
% inhibition = (ϕ χ-ϕ ZPE))/(ϕ



Signal
HPE-ϕ ZPE)*100










Biological activities of the compounds of the application are shown in Table 2.


WT-hSWAT: WT_FBA_HIS-SUMO-hSWAT IC50 (μM) (“WT”=Wild-type); and WT-mSWAT: MOUSE_FBA_HIS-SUMO-mSWAT C50 (μM) which is the mouse isoform of STING protein.













TABLE 2








WT-
WT-


Cmpd

Mass
hSWAT
mSWAT


No.
NMR Peak listings
found
IC50 (μM)
IC50 (μM)



















1

1H NMR (400 MHz, DMSO-d6): δ 8.54
361.3
C




(d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.8 Hz,
[M + H]



1H), 7.50 (d, J = 8.8 Hz, 1H), 7.40 (d, J =



8.0 Hz, 1H), 5.62 (s, 2H), 3.84 (s, 3H),



3.24-3.15 (m, 1H), 1.31 (d, J = 6.8 Hz,



6H)


2

1H NMR (400 MHz, DMSO-d6): δ 12.88
347.05
C
C



(brs, 1H), 8.52 (d, J = 8.0 Hz, 1H), 8.12
[M + H]



(d, J = 8.8 Hz, 1H), 7.51 (d, J = 8.8 Hz,



1H), 7.35 (d, J = 8.0 Hz, 1H), 5.67 (s,



2H), 4.00 (q, J = 7.0 Hz, 2H), 2.63 (s,



3H), 1.41 (t, J = 7.0 Hz, 3H).


3

1H NMR (400 MHz, DMSO-d6): δ 12.92
375.1
B
B



(brs, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.11
[M + H]



(d, J = 8.8 Hz, 1H), 7.50 (d, J = 8.8 Hz,



1H), 7.38 (d, J = 8.0 Hz, 1H), 5.63 (s,



2H), 3.99 (q, J = 6.8 Hz, 2H), 3.19-3.12



(m, 1H), 1.40 (t, J = 6.8 Hz, 3H), 1.30 (d,



J = 6.8 Hz, 6H).


4

1H NMR (400 MHz, DMSO-d6): δ 12.57
346.1
B



(brs, 1 H), 8.45 (d, J = 8.0 Hz, 1 H), 8.16
[M + 1]



(d, J = 8.4 Hz, 1 H), 7.39 (d, J = 8.4 Hz, 1



H), 7.30 (d, J = 8.0 Hz, 1 H), 5.44 (brs, 2



H), 2.74 (s, 6 H), 2.62 (s, 3 H).


5

1H NMR (400 MHz, DMSO-d6): δ 8.51
388.2
A



(d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.4 Hz,
[M + 1]



1H), 7.51 (d, J = 8.0 Hz, 1H), 7.39 (d, J =



8.4 Hz, 1H), 5.46 (brs, 2H), 2.75 (s, 6H),



1.38 (s, 9H).


6

1H NMR (400 MHz, DMSO-d6): δ 9.99
423.2
A



(s, 1 H), 8.28 (d, J = 8.4 Hz, 1 H), 8.17
[M + 1]



(d, J = 8.4 Hz, 1 H), 7.75 (d, J = 8.0 Hz, 2



H), 7.40-7.32 (m, 3 H), 7.07 (t, J = 7.2



Hz, 1 H), 6.82 (d, J = 8.8 Hz, 1 H), 5.59



(s, 1 H), 4.92 (s, 1 H), 2.91 (s, 3 H), 2.57



(s, 3 H).


7

1H NMR (400 MHz, DMSO-d6): δ 12.59
374.1
A



(s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.20 (d,
[M + H]



J = 8.6 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H),



7.37 (d, J = 8.0 Hz, 1H), 5.48 (s, 2H),



3.17 (m, 1H), 2.76 (s, 6H), 1.30 (d, J =



6.9 Hz, 6H).


8

1H NMR (400 MHz, DMSO-d6): δ 12.59
360.1
A



(brs, 1 H), 8.50 (d, J = 8.0 Hz, 1 H), 8.21
[M + 1]



(d, J = 8.8 Hz, 1 H), 7.44 (d, J = 8.8 Hz, 1



H), 7.34 (d, J = 8.0 Hz, 1 H), 5.48 (s, 2 H),



2.94-2.88 (m, 2 H), 2.76 (s, 6 H), 1.30 (t,



J = 7.6 Hz, 3 H)


9

1H NMR (400 MHz, DMSO-d6): δ 14.78
362
C



(brs, 1 H), 8.46 (d, J = 8.8 Hz, 1 H), 7.29
[M + H]



(d, J = 8.0 Hz, 1 H), 6.75 (s, 1 H), 5.63 (s,



2 H), 2.73 (s, 6 H), 2.60 (s, 3 H).


10

1H NMR (400 MHz, DMSO-d6): δ 8.45
423.2
D



(d, J = 8.0 Hz, 1 H), 8.14 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.37 (d, J = 8.4 Hz, 1 H), 7.27 (d, J =



8.0 Hz, 1 H), 5.63 (brs, 2 H), 2.80 (s, 6



H), 2.72 (s, 3 H), 2.61 (s, 3 H).


11

1H NMR (400 MHz, DMSO-d6): δ 8.46
370.3
D



(d, J = 7.6 Hz, 1 H), 8.15 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.40 (d, J = 8.4 Hz, 1 H), 7.29 (d, J =



7.6 Hz, 1 H), 5.51 (brs, 2 H), 2.76 (s, 6



H), 2.62 (s, 3 H).


12

1H NMR (400 MHz, DMSO-d6): δ 8.47
391.2
A



(d, J = 8.4 Hz, 1 H), 8.22 (d, J = 8.8 Hz, 1
[M + H]



H), 7.53 (d, J = 8.8 Hz, 1 H), 7.47 (d, J =



8.0 Hz, 1 H), 5.64 (s, 2 H), 2.32 (s, 3 H),



1.37 (s, 9 H),


13

1H NMR (400 MHz, DMSO-d6): δ 12.72
403.2
D



(brs, 1 H), 8.49 (d, J = 8.0 Hz, 1 H), 8.30
[M + H]



(d, J = 8.8 Hz, 1 H), 7.49 (d, J = 8.0 Hz, 1



H), 8.49 (d, J = 8.4 Hz, 1 H), 5.52 (s, 2



H), 2.53 (s, 3 H), 2.21 (s, 3 H), 1.37 (s, 9



H).


14

1H NMR (400 MHz, DMSO-d6): δ 13.07
363.2
D



(brs, 1 H), 8.58 (d, J = 8.4 Hz, 1 H), 8.17
[M + H]



(d, J = 8.8 Hz, 1 H), 7.60-7.53 (m, 2 H),



5.54 (s, 2 H), 2.32 (s, 3 H), 1.38 (s, 9 H),


17

1H NMR (400 MHz, DMSO-d6): δ 8.47
394.1
B



(s, 1 H), 8.20 (d, J = 8.4 Hz, 1 H), 7.47
[M + 1]



(d, J = 8.8 Hz, 1 H), 5.44 (s, 2 H), 3.06-



3.00 (m, 2 H), 2.76 (s, 6 H), 1.31 (t, J =



7.2 Hz, 3 H).


18

1H NMR (400 MHz, DMSO-d6): δ 8.47
410.1
A



(s, 1 H), 8.19 (d, J = 8.8 Hz, 1 H), 7.48 (d,
[M + 1]



J = 8.8 Hz, 1 H), 5.33 (s, 2 H), 4.58-4.52



(m, 2H), 2.75 (s, 6 H), 1.42 (t, J = 7.2 Hz,



3 H)


19

1H NMR (400 MHz, DMSO-d6): δ 12.50
375.3
B



(s, 1 H), 8.20 (d, J = 8.8 Hz, 1 H), 8.14 (d,
[M + 1]



J = 8.8 Hz, 1 H), 7.34 (d, J = 8.8 Hz, 1 H),



6.76 (d, J = 8.8 Hz, 1 H), 5.36 (s, 2 H),



3.18 (s, 6 H), 2.74 (s, 6 H)


20

1H NMR (400 MHz, DMSO-d6): δ 12.61
417.2
C



(brs, 1 H), 8.53 (d, J = 8.0 Hz, 1 H), 8.20
[M + 1]



(d, J = 8.4 Hz, 1 H), 7.43 (d, J = 8.8 Hz, 1



H), 7.41 (d, J = 8.4 Hz, 1 H), 7.07 (brs, 2



H), 5.43 (brs, 2 H), 2.76 (s, 6 H), 1.54 (s,



6 H).


21

1H NMR (400 MHz, DMSO-d6): δ 8.45
362.3
B



(d, J = 8.8 Hz, 1 H), 8.20 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.45 (d, J = 8.8 Hz, 1 H), 6.85 (d,



J = 8.4 Hz, 1 H), 5.40 (s, 2 H), 4.00 (s, 3



H), 2.76 (s, 6 H).


22

1H NMR (400 MHz, DMSO-d6): δ 8.14
378.0
B



(d, J = 8.8 Hz, 1 H), 8.10 (d, J = 8.0 Hz, 1
[M + 1]



H), 7.38 (d, J = 8.4 Hz, 1 H), 6.66 (d, J = 8.8



Hz, 1 H), 5.46 (s, 2 H), 3.18 (s, 6 H),



2.29 (s, 3 H).


23

1H NMR (400 MHz, DMSO-d6): δ 8.20
376.1
C



(d, J = 8.8 Hz, 1 H), 8.01 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.32 (d, J = 8.4 Hz, 1 H), 6.69 (d,



J = 8.8 Hz, 1 H), 5.43 (brs, 2 H), 3.93 (q,



J = 6.8 Hz, 2H), 3.18 (s, 6 H), 1.38 (t,



J = 6.8 Hz, 3 H).


24

1H NMR (400 MHz, DMSO-d6): δ 8.60
402.1
B



(d, J = 8.0 Hz, 1 H), 8.19 (d, J = 8.8 Hz, 1
[M + 1]



H), 7.57 (d, J = 8.0 Hz, 1 H), 7.53 (d,



J = 8.8 Hz, 1 H), 5.64 (brs, 2 H), 2.36 (s, 3



H), 1.77 (s, 6 H).


25

1H NMR (400 MHz, DMSO-d6): δ 8.64
400.1
B



(d, J = 8.0 Hz, 1 H), 8.08 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.58 (d, J = 8.4 Hz, 1 H), 7.45 (d,



J = 8.4 Hz, 1 H), 5.53 (brs, 2 H), 3.96 (q,



J = 6.8 Hz, 2 H), 1.77 (s, 6 H), 1.4 (t, J = 6.8



Hz, 3 H).


26

1H NMR (400 MHz, DMSO-d6): δ 8.18
465.1
A



(d, J = 9.2 Hz, 1 H), 8.05 (d, J = 8.8 Hz, 1
[M + 1]



H), 7.24 (d, J = 8.8 Hz, 1 H), 6.70 (d,



J = 7.6 Hz, 1 H), 5.35 (brs, 2 H), 3.80 (d,



J = 2.4 Hz, 2 H), 3.16 (s, 3 H), 2.78 (s, 6



H), 2.68-2.61(m, 3 H), 2.44-2.38 (m, 2



H).


27

1H NMR (400 MHz, DMSO-d6): δ 8.13
462.2
B



(d, J = 8.8 Hz, 1 H), 8.08 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.41 (d, J = 8.0 Hz, 1 H), 6.66 (brs, 1



H), 5.43 (s, 2 H), 3.83 (dd, J = 2.4 Hz, 2



H), 3.53 (brs, 2 H), 3.33 (s, 2 H), 3.16 (s,



3 H), 2.29 (s, 3 H), 2.04 (brs, 1 H), 1.55



(d, J = 7.6 Hz, 2 H), 1.26-1.35 (m, 2 H),


28

1H NMR (400 MHz, DMSO-d6): δ 8.20
448.1
A



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 1H NMR (400 MHz, DMSO-d6): δ



7.39 (d, J = 8.4 Hz, 1 H), 6.98 (d, J = 9.2



Hz, 1 H), 5.42 (s, 2 H), 3.85-3.83 (m, 2



H), 3.61-3.59 (m, 2 H), 3.27 (s, 3 H),



2.77-2.72 (m, 1 H), 2.29 (s, 3 H), 0.98-



0.95 (m, 2 H), 0.76-0.73 (m, 2 H).


29

1H NMR (400 MHz, DMSO-d6): δ 8.26
446.1
B



(d, J = 8.8 Hz, 1 H), 8.02 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.32 (d, J = 8.8 Hz, 1 H), 7.01 (d, J =



8.8 Hz, 1 H), 5.33 (brs, 2 H), 3.80-3.94



(m, 4 H), 3.58-3.61 (m, 2 H), 3.26 (s, 3



H), 2.72-2.77 (m, 1 H), 1.37 (t, J = 7.2



Hz, 3 H), 0.97-0.98 (m, 2 h), 0.75 (s, 2 H)


30

1H NMR (400 MHz, DMSO-d6): δ 8.18
434.0
A



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.38 (d, J = 8.4 Hz, 1 H), 6.68 (d,



J = 9.2 Hz, 1 H), 5.33 (s, 2 H), 5.21 (t,



J = 7.2 Hz, 1 H), 4.87 (t, J = 7.2 Hz, 2 H),



4.70 (t, J = 6.8 Hz, 2 H), 3.67-3.62 (m, 2



H), 2.29 (s, 3 H), 1.17 (t, J = 7.2 Hz, 3 H).


31

1H NMR (400 MHz, DMSO-d6): δ 8.71
375.0
C



(d, J = 8.0 Hz, 1 H), 8.09 (d, J = 8.8 Hz, 1
[M + 1]



H), 7.81 (d, J = 7.6 Hz, 1 H), 7.46 (d, J =



8.4 Hz, 1 H), 5.54 (s, 2 H), 3.98 (t, J = 7.2



Hz, 2 H), 2.72 (s, 3 H), 1.41 (t, J = 7.2 Hz,



3 H),


32

1H NMR (400 MHz, DMSO-d6): δ 8.67
377.3
C



(d, J = 8.0 Hz, 1 H), 8.20 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.81 (d, J = 8.0 Hz, 1 H), 7.54 (d,



J = 8.4 Hz, 1 H), 5.57 (s, 2 H), 2.72 (s, 3



H), 2.38 (s, 3 H).


33

1H NMR (400 MHz, DMSO-d6): δ 8.18
460.2
B



(d, J = 8.4 Hz, 1 H), 8.01 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.31 (d, J = 8.4 Hz, 1 H), 6.70 (brs, 1



H), 5.40 (brs, 2 H), 3.93 (d, J = 6.4 Hz, 2



H), 3.84-3.81 (m, 2 H), 2.36 (d, J = 11.2



Hz, 2 H), 3.16 (s, 3 H), 2.04 (brs, 1 H),



1.54 (d, J = 12 Hz, 2 H), 1.24-1.38 (m, 5



H)


34

1H NMR (400 MHz, DMSO-d6): δ 12.79
470.5
B



(s, 1H), 8.22 (t, J = 8.5 Hz, 2H), 7.54 (d,
[M + 1]



J = 8.2 Hz, 1H), 6.78 (d, J = 8.7 Hz, 1H),



5.52 (s, 2H), 4.06 (d, J = 7.2 Hz, 2H),



3.48 (t, J = 6.9 Hz, 2H), 3.18 (s, 3H),



3.05 (s, 3H), 2.28 (s, 3H).


35

1H NMR (400 MHz, DMSO-d6): δ 12.74
498.7
B



(s, 1H), 8.22 (dd, J = 10.4, 8.7 Hz, 2H),
[M + 1]



7.54 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 8.9



Hz, 1H), 5.55 (s, 2H), 4.07 (s, 2H), 3.44



(d, J = 6.7 Hz, 2H), 3.19 (s, 3H), 2.28 (s,



3H), 1.25 (d, J = 6.8 Hz, 6H).


36

1H NMR (400 MHz, DMSO-d6): δ 8.15
468.0
B



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.38 (d, J = 8.4 Hz, 1 H), 6.68 (d,



J = 8.0 Hz, 1 H), 5.41 (brs, 2 H), 3.83 (s, 2



H), 3.16 (s, 3 H), 2.70-2.61 (m, 2 H),



2.58-2.54 (m, 1 H), 2.46-2.42 (m, 2



H), 2.29 (s, 3 H).


37

1H NMR (400 MHz, DMSO-d6): δ 8.14
508.1
B



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.37 (d, J = 8.4 Hz, 1 H), 6.75 (d,



J = 9.2 Hz, 1 H), 5.43 (brs, 2 H), 3.84 (s, 2



H), 3.49 (s, 2 H), 2.70-2.57 (m, 5 H),



2.28 (s, 3 H), 1.18-1.11 (m, 1 H), 0.48 (d,



J = 6.8 Hz, 2 H), 0.37 (d, J = 3.6 Hz, 2 H).


38

1H NMR (400 MHz, DMSO-d6): δ 8.14
474.1
B



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.38 (d, J = 8.4 Hz, 1 H), 6.74 (d,



J = 9.2 Hz, 1 H), 5.46 (brs, 2 H), 4.64 (t,



J = 6.8 Hz, 2 H), 4.49 (t, J = 5.6 Hz, 2 H),



3.98 (s, 2 H), 3.47-3.38 (m, 3 H), 2.29 (s,



3 H), 1.19-1.10 (m, 1 H), 0.48 (d, J = 6.8



Hz, 2 H), 0.37 (d, J = 4.0 Hz, 2 H).


39

1H NMR (400 MHz, DMSO-d6): δ 8.13
462.1
B



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.37 (d, J = 8.4 Hz, 1 H), 6.65 (d,



J = 8.4 Hz, 1 H), 5.50 (s, 2 H), 3.72-3.61



(m, 1 H), 3.35 (m, 2 H), 3.14 (s, 3 H),



3.11 (s, 3 H), 2.34-2.29 (m, 5 H), 2.17-



2.12 (m, 1 H), 1.68-1.65 (m, 2 H).


40

1H NMR (400 MHz, DMSO-d6): δ 8.12
502.2
B



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.37 (d, J = 8.4 Hz, 1 H), 6.69 (d,



J = 8.8 Hz, 1 H), 5.53-5.51 (m, 2 H), 3.67-



3.60 (m, 3 H), 3.47 (m, 2 H), 3.10 (s, 3



H), 2.33-2.28 (m, 5 H), 2.11-2.18 (m, 1



H), 1.7-1.63 (m, 2 H), 1.09-1.22 (m, 1



H), 0.48-0.46 (m, 2 H), 0.38-0.36 (m, 2



H).


41

1H NMR (400 MHz, DMSO-d6): δ 8.19
454.1
B



(d, J = 9.2 Hz, 1 H), 8.10 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.41 (d, J = 8.0 Hz, 1 H), 6.74 (d,



J = 8.8 Hz, 1 H), 5.42 (s, 2 H), 4.79 brs, 1



H), 3.11 (s, 3 H), 3.03 (brs, 2 H), 2.89



(brs, 2 H), 2.30 (s, 3 H).


42
8.18 (d, J = 9.2 Hz, 1 H), 8.10 (d, J = 8.0
494.3
B



Hz, 1 H), 7.39 (d, J = 8.0 Hz, 1 H), 6.81
[M + 1]



(d, J = 9.2 Hz, 1 H), 5.44 (s, 2 H), 4.49 brs,



1 H), 3.13 (brs, 2 H), 2.88 (brs, 2 H),



3.00 (s, 3 H), 1.66 (s, 2 H), 0.99 (s, 1 H),



0.46 (d, J = 7.6 Hz, 2 H), 0.32 (d, J = 4.8



Hz, 2 H)


43

1H NMR (400 MHz, DMSO-d6): δ 8.16
488.1
A



(d, J = 9.2 Hz, 1 H), 8.11 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.39 (d, J = 8.4 Hz, 1 H), 6.72 (d, J =



8.8 Hz, 1 H), 5.45 (s, 2 H), 4.21 (brs, 1



H), 3.66-3.71 (m, 1 H), 3.52-3.57 (m, 2



H), 3.24 (s, 3 H), 2.75-2.81 (m, 2 H),



2.33 (s, 3 H), 1.92-1.99 (m, 2 h), 1.03-



1.07 (m, 1 H), 0.42-0.48 (m, 2 H), 0.36-



0.41 (m, 2 H)


44

1H NMR (400 MHz, DMSO-d6): δ 12.82
448.1
B



(brs, 1 H), 8.21 (d, J = 8.4 Hz, 1 H), 8.19
[M + 1]



(d, J = 8.0 Hz, 1 H), 7.52 (d, J = 8.4 Hz, 1



H), 6.78 (d, J = 8.8 Hz, 1 H), 5.47 (s, 2



H), 4.53 (brs, 1 H), 3.68-3.65 (m, 1 H),



3.19 (s, 3 H), 3.09 (s, 3 H), 2.66-2.60 (m,



2 H), 2.28 (s, 3 H), 2.05-2.00 (m, 2 H)


45

1H NMR (400 MHz, DMSO-d6): δ 8.17
484.0
B



(d, J = 8.8 Hz, 1 H), 8.10 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.41 (d, J = 8.4 Hz, 1 H), 6.72 (s, 1



H), 6.70 (t, J = 75.6 Hz, 1 H), 5.54 (brs, 2



H), 4.55-4.54 (m, 1 H), 4.49-4.42 (m, 1



H), 3.33 (s, 3 H), 2.78-2.72 (m, 2 H),



2.40-2.24 (m, 5 H),


46

1H NMR (400 MHz, DMSO-d6): δ 8.17
524.0
A



(d, J = 8.8 Hz, 1 H), 8.13 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.43 (d, J = 8.4 Hz, 1 H), 6.75 (d,



J = 8.4 Hz, 1 H), 6.70 (t, J = 75.2 Hz, 1 H),



5.44 (brs, 2 H), 4.47-4.43 (m, 1 H), 4.32-



4.25 (m, 1 H), 3.58-3.56 (m, 2 H), 2.87-



2.85 (m, 2 H), 2.28-2.21 (m, 5 H), 1.06-



1.02 (m, 1 H), 0.48-0.46 (m, 2 H), 0.35-



0.33 (m, 2 H),


47

1H NMR (400 MHz, DMSO-d6): δ 8.13
458.1
A



(d, J = 9.2 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.37 (d, J = 8.4 Hz, 1 H), 6.66 (d,



J = 9.2 Hz, 1 H), 5.48-5.45 (m, 2 H), 4.65-



4.63 (m, 1 H), 3.06 (s, 3 H), 2.41-2.35



(m, 2 H), 2.3 (s, 3 H), 2.19-2.11 (m, 4 H),



1.99-1.94 (m, 2 H), 1.88-1.8 (m, 2 H).


48

1H NMR (400 MHz, DMSO-d6): δ 8.12
446.1
A



(d, J = 8.8 Hz, 1 H), 8.08 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.36 (d, J = 8.0 Hz, 1 H), 6.69 (d, J =



8.8 Hz, 1 H), 5.42 (s, 2 H), 4.88 (s, 1 H),



3.10 (s, 3 H) 2.29 (s, 3 H), 2.15-2.10 (m,



2 H), 2.04-1.99 (m, 2 H), 1.26 (s, 3



H), 1.16 (s, 3H).


49

1H NMR (400 MHz, DMSO-d6): δ 8.17
442.9
B



(d, J = 8.8 Hz, 1 H), 8.10 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.39 (d, J = 8.4 Hz, 1 H), 6.71 (d,



J = 8.8 Hz, 1 H), 5.43 (brs, 2 H), 3.33-3.10



(m, 1 H), 3.10 (s, 3 H), 2.75-2.61 (m, 5



H), 2.30 (s, 3 H).


50

1H NMR (400 MHz, DMSO-d6): δ 8.23
490.2
B



(d, J = 8.8 Hz, 1 H), 8.12 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.42 (d, J = 8.4 Hz, 1 H), 6.89 (d,



J = 8.8 Hz, 1 H), 5.38 (s, 2 H), 4.59 (s, 2



H), 3.85 (s, 2 H), 3.61 (t, J = 5.6 Hz, 2 H),



3.29 (s, 3 H), 2.29 (s, 3 H).


51

1H NMR (400 MHz, DMSO-d6): δ 8.19
472.1
B



(d, J = 8.8 Hz, 1 H), 8.11 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.40 (d, J = 8.4 Hz, 1 H), 6.81 (d,



J = 9.2 Hz, 1 H), 6.53-6.25 (m, 1 H), 5.28



(brs, 2 H), 4.04 (t, J = 13.2 Hz, 2 H), 3.79



(s, 2 H), 3.58 (t, J = 4.4 Hz, 2 H), 3.28 (s,



3 H), 2.30 (s, 3 H).


52

1H NMR (400 MHz, DMSO-d6): δ 8.24
470.1
C



(d, J = 8.8 Hz, 1 H), 8.03 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.34 (d, J = 8.4 Hz, 1 H), 6.83 (d,



J = 9.2 Hz, 1 H), 6.53-6.24 (m, 1 H), 5.29



(brs, 2 H), 4.08-4.01 (m, 2 H), 3.97-3.92



(m, 2 H), 3.80 (s, 2 H), 3.58 (t, J = 4.8 Hz,



2 H), 3.28 (s, 3 H), 1.37 (t, J = 6.8 Hz, 3



H).


53

1H NMR (400 MHz, DMSO-d6): δ 8.04
451.0
A



(d, J = 8.8 Hz, 2 H), 7.90 (s, 1 H), 7.24 (d,
[M + 1]



J = 8.8 Hz, 1 H), 6.44 (d, J = 8.4 Hz, 1 H),



5.45 (brs, 2 H), 3.52 (s, 2 H), 2.79 (s, 6



H), 2.71-2.60 (m, 3 H), 2.43-2.32 (m, 2



H).


54

1H NMR (400 MHz, DMSO-d6): δ 8.09
454.0
B



(d, J = 8.4 Hz, 1 H), 8.03 (d, J = 8.8 Hz, 1
[M + 1]



H), 7.87 (s, 1 H), 7.37 (d, J = 8.4 Hz, 1 H),



6.42 (d, J = 8.4 Hz, 1 H), 5.48 (brs, 2 H),



3.53 (s, 2 H), 2.71-2.60 (m, 2 H), 2.44-



2.31 (m, 3 H), 2.29 (s, 3 H).


55

1H NMR (400 MHz, DMSO-d6): δ 8.10
420.2
B



(d, J = 8.4 Hz, 1 H), 8.03 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.89 (s, 1 H), 7.37 (d, J = 8.4 Hz, 1



H), 6.42 (d, J = 9.2 Hz, 1 H), 5.47 (s, 2



H), 4.68-4.64 (m, 2 H), 4.38-4.35 (m, 2



H), 3.67-3.66 (m, 2 H), 3.29-3.24 (m, 1



H), 2.29 (s, 3 H)


56

1H NMR (400 MHz, DMSO-d6): δ 8.12-
428.9
A



8.06 (m, 3 H), 7.41 (d, J = 8.4 Hz, 1 H),
[M + 1]



6.42 (d, J = 8.8 Hz, 1 H), 5.51 (s, 2 H),



3.39-3.35 (m, 1 H), 3.18-3.11 (m, 1 H),



2.71-2.67 (m, 2 H), 2.46-3.39 (m, 2 H),



2.30 (s, 3 H).


57

1H NMR (400 MHz, DMSO-d6): δ 9.56
444.0
A



(s, 1 H), 8.32-8.28 (m, 1 H), 8.23 (d,
[M + 1]



J = 8.8 Hz, 1 H), 8.13 (d, J = 8.8 Hz, 1 H),



7.42 (d, J = 8.4 Hz, 1 H), 6.70 (t, J = 8.4 Hz,



1 H), 7.15-7.11 (m, 1 H), 6.89 (d, J = 8.0



Hz, 1 H), 5.45-5.39 (m, 2 H), 2.28 (s, 3



H),


58

1H NMR (400 MHz, DMSO-d6): δ 8.16
412.1
A



(s, 1 H), 8.11 (d, J = 8.4 Hz, 1 H), 7.43 (d,
[M + 1]



J = 8.4 Hz, 1 H), 5.34 (s, 2 H), 3.20 (s, 6



H), 2.31 (s, 3 H).


59

1H NMR (400 MHz, DMSO-d6): δ 8.14
470.2
B



(s, 1 H), 8.10 (d, J = 8.4 Hz, 1 H), 7.42 (d,
[M + 1]



J = 8.4 Hz, 1 H), 5.32 (s, 2 H), 3.80-3.65



(m, 6 H), 3.30 (s, 3 H), 2.31 (s, 3 H), 1.25



(t, J = 6.8 Hz, 3 H).


60

1H NMR (400 MHz, DMSO-d6): δ 8.05
446.1
B



(d, J = 8.4 Hz, 1 H), 8.0 (d, J = 8.4 Hz, 1 H),
[M + 1]



7.79 (brs, 1 H), 7.29 (d, J = 8.8 Hz, 1 H),



6.46 (d, J = 8.8 Hz, 1 H), 5.61-5.21 (m, 2



H), 3.93-3.82 (m, 4 H), 3.32-3.28 (m, 4



H), 1.88-1.82 (m, 1 H), 1.67-1.64 (m, 2



H), 1.37 (t, J = 6.8 Hz, 3 H), 1.28-1.17 (m,



2 H).


61

1H NMR (400 MHz, DMSO-d6): δ 12.41
355.3
B



(s, 1 H), 8.20 (d, J = 8.8 Hz, 1 H), 8.03 (d,
[M + 1]



J = 8.4 Hz, 1 H), 7.11 (d, J = 8.4 Hz, 1 H),



6.71 (d, J = 8.8 Hz, 1 H), 5.39 (s, 2 H),



3.17 (s, 6 H), 2.67 (s, 6 H), 2.45 (s, 3 H).


62

1H NMR (400 MHz, DMSO-d6): δ 8.14
358.2
B



(d, J = 9.2 Hz, 1 H), 8.03 (d, J = 8.0 Hz, 1
[M + 1]



H), 7.18 (d, J = 8.0 Hz, 1 H), 6.62 (d,



J = 8.8 Hz, 1 H), 5.58 (brs, 2 H), 3.16 (s, 6



H), 2.57 (s, 3 H), 2.17 (s, 3 H).


63

1H NMR (400 MHz, DMSO-d6): δ 8.40
371.2
B



(d, J = 8.4 Hz, 1 H), 8.09 (d, J = 8.0 Hz, 1
[M + 1]



H), 7.35 (d, J = 8.0 Hz, 1 H), 7.24 (d,



J = 8.0 Hz, 1 H), 5.65 (brs, 2 H), 2.22 (s, 3



H), 1.37 (s, 9 H).


64

1H NMR (400 MHz, DMSO-d6): δ 8.56
379.3
B



(d, J = 8.0 Hz, 1 H), 7.98 (d, J = 8.0 Hz, 1
[M + 1]



H), 7.46 (d, J = 8.4 Hz, 1 H), 7.14 (d,



J = 8.0 Hz, 1 H), 5.42 (brs, 2 H), 2.82 (s, 6



H), 2.40 (s, 3 H), 1.76 (s, 6 H).


65
8.48 (d, J = 8.4 Hz, 1 H), 8.22 (d, J = 8.8
403.2
B



Hz, 1 H), 7.57 (d, J = 8.4 Hz, 1 H), 7.26
[M + 1]



(d, J = 8.0 Hz, 1 H), 6.46 (s, 1 H), 5.68 (s,



2 H), 5.42 (s, 2 H), 5.34 (s, 2 H), 2.30 (s,



3 H).


66

1H NMR (400 MHz, DMSO-d6): δ 8.18
425.4
C



(d, J = 9.2 Hz, 1 H), 8.00 (d, J = 7.6 Hz, 1
[M + 1]



H), 7.09 (d, J = 7.6 Hz, 1 H), 6.66 (d,



J = 8.0 Hz, 1 H), 5.40-5.32 (m, 2 H), 5.02-



4.98 (m, 1 H), 3.98 (s, 1 H), 3.21 (s, 3 H),



3.11 (s, 3 H), 2.71 (s, 6 H), 2.43 (s, 3 H),



2.37-2.34 (m, 2 H), 2.03-1.97 (m, 2 H).


67

1H NMR (400 MHz, DMSO-d6): δ 12.58
425.3
A



(s, 1 H), 8.19 (d, J = 8.8 Hz, 1 H), 8.02 (d,
[M + 1]



J = 8.4 Hz, 1 H), 7.11 (d, J = 8.0 Hz, 1 H),



6.72 (d, J = 9.2 Hz, 1 H), 5.35 (s, 2 H),



4.52-4.34 (m, 1 H), 3.69-3.62 (m, 1 H),



3.19 (s, 3 H), 3.08 (s, 3 H), 2.70 (s, 6 H),



2.67-2.60 (m, 2 H), 2.45 (s, 3 H), 2.07-



2.00 (m, 2 H).


68

1H NMR (400 MHz, DMSO-d6): δ 8.38
389.2
A



(d, J = 8.0 Hz, 1 H), 8.14 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.44 (d, J = 8.4 Hz, 1 H), 7.27 (d,



J = 8.0 Hz, 1 H), 5.47 (s, 2 H), 2.32 (s, 3



H), 1.54 (s, 3 H), 1.35-1.37 (m, 2 H),



0.94-0.96 (m, 2 H).


69

1H NMR (400 MHz, DMSO-d6): δ 12.63
428.3
A



(brs, 1 H), 8.19 (d, J = 8.8 Hz, 1 H), 8.13
[M + 1]



(d, J = 8.0 Hz, 1 H), 7.32 (d, J = 8.0 Hz, 1



H), 6.75 (d, J = 8.8 Hz, 1 H), 5.51 (s, 2



H), 4.54 (brs, 1 H), 3.70-3.65 (m, 1 H),



3.19 (s, 3 H), 3.09 (s, 3 H), 2.66 (s, 3 H),



2.64-2.62 (m, 2 H), 2.14 (s, 3 H), 2.06-



2.04 (m, 2 H)


70

1H NMR (400 MHz, DMSO-d6): δ 8.15
448.2
A



(d, J = 8.8 Hz, 1 H), 8.12 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.41 (d, J = 8.4 Hz, 1 H), 6.65 (d, J =



8.8 Hz, 1 H), 5.42 (s, 2 H), 5.00 (brs, 1



H), 3.98-3.96 (m, 1 H), 3.21 (s, 3 H),



3.11 (s, 3 H), 2.43-2.28 (m, 4 H), 1.88 (s,



3 H)


71

1H NMR (400 MHz, DMSO-d6): δ 8.21
458.3
B



(d, J = 8.5 Hz, 2H), 7.60 (s, 1H), 7.53 (d,
[M + 1]



J = 8.5 Hz, 1H), 7.22 (d, J = 1.2 Hz, 1H),



6.87 (s, 1H), 6.69 (d, J = 9.0 Hz, 1H),



5.44 (s, 2H), 4.23 (t, J = 6.2 Hz, 2H),



3.96 (s, 2H), 2.96 (s, 3H), 2.28 (s, 3H).


72

1H NMR (400 MHz, DMSO-d6): δ 8.21
456.2
C



(s, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.62 (d,
[M + 1]



J = 1.1 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H),



7.22 (d, J = 1.3 Hz, 1H), 6.88 (d, J = 1.1



Hz, 1H), 6.70 (d, J = 8.9 Hz, 1H), 5.45 (s,



2H), 4.24 (t, J = 6.2 Hz, 2H), 3.96 (q, J =



7.2, 6.7 Hz, 4H), 2.97 (s, 3H), 1.37 (t, J =



7.0 Hz, 3H).


73

1H NMR (400 MHz, DMSO-d6): δ 8.11
440.2
A



(d, J = 8.4 Hz, 1 H), 8.07 (d, J = 8.8 Hz, 1
[M + 1]



H), 7.52 (t, J = 7.6 Hz, 2 H), 7.42-7.37 (m,



4 H), 6.29 (d, J = 8.8 Hz, 1 H), 5.44 (s, 2



H), 3.53 (s, 3 H), 2.31 (s, 3 H).


74

1H NMR (400 MHz, DMSO-d6): δ 8.14
492.2
C



(d, J = 8.8 Hz, 1 H), 8.09 (d, J = 8.4 Hz, 1
[M + 1]



H), 7.37 (d, J = 8.4 Hz, 1 H), 6.68 (d, J =



8.8 Hz, 1 H), 5.38 (s, 2 H), 4.21-4.14 (m,



1 H), 3.67 (t, J = 7.2 Hz, 2 H), 3.66-3.64



(m, 1 H), 3.52 (t, J = 7.2 Hz, 2 H), 3.26



(s, 3 H), 3.19 (s, 3 H), 2.65-2.69 (m, 2



H), 2.29 (s, 3 H), 2.02-2.07 (m, 2 H)


75

1H NMR (400 MHz, DMSO-d6): δ 8.16
456.1
B



(d, J = 10.4 Hz, 2 H), 7.49 (d, J = 8.4 Hz, 1
[M + 1]



H), 5.42 (brs, 2 H), 3.83 (t, J = 6.0 Hz, 2



H), 3.66 (t, J = 5.6 Hz, 2 H), 3.28 (s, 3 H),



3.27 (s, 3 H), 2.30 (s, 3 H).


76

1H NMR (400 MHz, DMSO-d6): δ 8.15
496.2
A



(s, 1 H), 8.11 (d, J = 8.4 Hz, 1 H), 7.44 (d,
[M + 1]



J = 8.8 Hz, 1 H), 5.43 (brs, 2 H), 3.82 (d,



J = 8.8 Hz, 2 H), 3.62 (s, 2 H), 3.29-3.25



(m, 5 H), 2.32 (s, 3 H), 2.10-2.03 (m, 1



H), 1.57-1.54 (m, 2 H), 1.28-1.20 (m, 2



H).


77

1H NMR (400 MHz, DMSO-d6): δ 12.90
482.3
A



(s, 1 H), 8.26 (s, 1 H), 8.23 (d, J = 8.8 Hz,
[M + 1]



1 H), 7.59 (d, J = 8.4 Hz, 1 H), 5.48 (s, 2



H), 4.26-4.18 (m, 1 H), 3.70-3.63 (m, 1



H), 3.18 (s, 3 H), 3.10 (s, 3 H), 2.67-2.64



(m, 2 H), 2.29 (s, 3 H), 2.05-1.98 (m, 2



H).


78

1H NMR (400 MHz, DMSO-d6): δ 12.69
358.2
B



(s, 1 H), 8.20 (d, J = 8.8 Hz, 1 H), 8.19 (d,
[M + 1]



J = 8.8 Hz, 1 H), 7.42 (d, J = 8.4 Hz, 1 H),



7.10 (q, J = 11.2 Hz, 1 H), 6.74 (d, J = 9.2



Hz, 1 H), 5.63 (dd, J = 11.2 Hz, J = 1.2 Hz,



1 H), 5.25 (d, J = 16.8 Hz, 1 H), 5.05 (s, 2



H), 3.16 (s, 6 H).


79

1H NMR (400 MHz, DMSO-d6): δ 12.45
408.2
B



(s, 1 H), 8.33 (d, J = 8.4 Hz, 1 H), 8.23 (d,
[M + 1]



J = 8.8 Hz, 1 H), 7.55-7.48 (m, 4 H), 7.33



(d, J = 7.6 Hz, 2 H), 6.74 (d, J = 8.8 Hz, 1



H), 4.55 (s, 2 H), 3.09 (s, 6 H).


80

1H NMR (400 MHz, DMSO-d6): δ δ 8.20
455.3
B



(d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.6 Hz,
[M + 1]



1H), 7.66 (s, 1H), 7.35 (d, J = 8.6 Hz,



1H), 7.23 (d, J = 1.4 Hz, 1H), 6.91 (t, J =



1.1 Hz, 1H), 6.70 (d, J = 8.9 Hz, 1H),



5.29 (s, 2H), 4.25 (t, J = 6.4 Hz, 2H),



3.95 (s, 2H), 2.99 (s, 3H), 2.73 (s, 6H).


81

1H NMR (400 MHz, DMSO-d6): δ 8.21
472.3
B



(d, J = 8.5 Hz, 2H), 7.53 (d, J = 8.5 Hz,
[M + 1]



1H), 7.12 (d, J = 1.4 Hz, 1H), 6.77 (d, J =



1.3 Hz, 1H), 6.70-6.62 (m, 1H), 5.46 (s,



2H), 4.17 (t, J = 5.9 Hz, 2H), 3.94 (s,



2H), 2.87 (s, 3H), 2.28 (s, 3H), 2.14 (s,



3H).


82

1H NMR (400 MHz, DMSO-d6): δ 8.18
469.2
C



(d, J = 8.8 Hz, 1H), 8.05 (d, J = 8.6 Hz,
[M + 1]



1H), 7.25 (d, J = 8.6 Hz, 1H), 7.20 (s,



1H), 6.68 (d, J = 1.3 Hz, 1H), 6.59 (d, J =



8.9 Hz, 1H), 5.34 (s, 2H), 4.16 (d, J = 6.8



Hz, 2H), 3.89 (s, 2H), 2.89 (s, 3H), 2.79



(s, 6H), 2.15 (s, 3H).





WT_FBA_HIS-SUMO-hSWAT IC50 (μM); and MOUSE_FBA_HIS-SUMO-mSWAT IC50 (μM): “A” < 20 μM; “B” = 20-100 μM; “C” = 100-300 μM; “D” > 300 μM.






Example 83: STING Differential Scanning Fluorimetry (DSF) Assay

A STING DSF assay was performed to determine protein thermostability shift in response to small molecule ligand binding. A STING thermostability shift assay based on differential scanning fluorimetry was used to measure (1) the net shift in melting temperature (ΔTm)between the apo and ligand-bound states of the STING protein and (2) the concentration of the thermal denaturation transition (FC50) as a surrogate measure of binding affinity (Kd). Briefly, recombinant mouse or human STING protein (aa140-379) comprising the C-terminal binding domain (R232-WT) were purified by affinity chromatography and further processed to remove the purification tag, followed by gel filtration to achieve purity >95%. Protein in assay buffer was transferred to a 384 well plate and incubated with the hydrophobic fluorescent dye SYBR Orange. A temperature ramp was applied to the plate and the resulting dye fluorescence was plotted as a function of temperature. The first derivative of this function (dF/dT) was then used to extrapolate the melting temperature of the protein at various concentrations of compound and determine the thermal denaturation profile of a protein/compound complex.


Assay Parameters Optimized

1. Protein construct (affinity tag removal vs SUMO-fusion construct)


2. Protein concentration


3. Dye concentration


4. Buffer


5. Thermal ramp velocity


6. DMSO tolerance


7. Compound titration parameters


8. HPE and ZPE controls


Final Assay Parameters

1. hSTING-R232, mSTING-R232 (aa139-378)


2. 5 μM purified Target STING Protein


3. 8 μM Sypro Orange Dye


4. Buffer: 100 mM NaCl, 30 mM HEPES, pH 7.5


5. Thermal Ramp: 0.5° C./min


6. <1% DMSO


7. 11 point, 2 fold dilution, duplicate, 300 μM high concentrations


8. HPE (100 μM 2′3′cGAMP), ZPE (DMSO 1%)


Biological activities based on DSF assay of the compounds of the present application are depicted in Table 3 below.











TABLE 3





Cmpd
WT_DSF_hSWAT
MOUSE DSF_6xHIS-


No.
Tm Shift (° C.)
SUMO Tm Shift (° C.)

















1
D



2
C
C


3
D
C


4
D


5
D


6
D


7
D


8
D


9
C


10
C


11
B


12
D


13
B


14
B


17
C


18
C


19
D


20
C


21
C


22
D


23
C


24
D


25
D


26
D


27
D


28
D


29
D


30
D


31
C


32
C


33
D


34
D


35
D


36
D


37
D


38
D


39
D


40
D


41
D


42
D


43
D


44
D


45
D


46
D


47
D


48
D


49
D


50
D


51
D


52
C


53
D


54
D


55
D


56
D


57
D


58
D


59
D


60
D


61
D


62
D


63
D


64
D


65
D


66
C


67
D


68
D


69
D


70
D


71
D


72
D


73
D


74
D


75
D


76
D


77
D


78
D


79
D


80
D


81
D


82
D





WT_DSF_hSWAT Tm Shift (° C.): “A” < 0° C.; “B” = 0-2° C.; “C” = 2-7° C.; “D” > 7° C.


MOUSE DSF_6xHIS-SUMO Tm Shift (° C.): “A” < 0° C.; “B” = 0-5° C.; “C” = 5-15° C.; “D” > 15° C.






Example 84. Cell Based Reporter Assays of IRF or IFN-Beta Activation

Cell reporter assays were developed to measure the ability of compounds of the application to activate the STING protein and activate both IRF/IFN and the NFkB signaling pathways in the myeloid derived cell line THP-1, which is commercially available from several suppliers. Briefly, THP-1 monocytes were transfected and selected for stable integration of 2 reporter constructs. Clones incorporating stably integrated vectors were further selected by antibiotic resistance to blastocidin and zeocin. The reporter constructs included: (1) firefly luciferase reporter gene under the control of an ISG54 promoter fused to five interferon (IFN)-stimulated response elements to measure IRF transcription factor binding activity and (2) secreted embryonic alkaline phosphatase reporter gene under transcription control of an IFN-beta minimal promoter fused to 5 copies of the NFkB consensus transcriptional response element and 3 copies of the c-Rel binding site.


The resulting THP-1 cell line was further modified to incorporate 3 amino acid point mutations (H71R A230G Q293R). This modification of the native TMEM173 gene in THP-1 cells (HAQ) was designed to produce a cell reporter that expressed the most common allelic variant in the human population (THP-1 R232, available from InvivoGen). Alternatively, the TMEM173 gene was knocked out to provide a congenic control to counter-screen for compound activation that was independent of the STING protein (THP-1 STING KO).


Once cell lines were established, reporter assay protocols were developed and optimized to screen small molecule compounds for agonist activity. Assay optimization for efficacy and Z′ value included titration of the following parameters:


Assay Optimization Parameters

1. Cell pre-activation with PMA


2. Cell seeding density


3. DMSO tolerance


4. Media selection


5. FBS concentration


6. Incubation time


7. Co-Assessment of cell viability by CTG assay


8. Appropriate HPE and ZPE controls


9. Dose Response Titration


Final Assay SOP Conditions

1. No pre-activation with PMA


2. 5×10∝cells/well in 96 well plate format


3. <0.5% DMSO


4. RPMI 1640+2.5 mg/ml glucose


5. 10% FBS


6. 14-18 hr incubation


7. Cell Titer Glo reagent (Promega) following supermatant removal


8. HPEIRF (100 μM 2′3′cGAMP), HPENFkB (50 nM PM3CSK4), ZPE (DMSO 0.5%)


9. 8 point, 2 fold, duplicate starting at 150 μM PP28T


Assay Performance
















Z′ Value (IRF-luc)
Z′ Value (NFkB-SEAP)











Cell Line
Z′
Assay Window
Z′
Assay Window





THP-1
0.91
>200
0.82
>15


THP-1 R232
0.86
>120
0.96
>30


THP-1 STING KO
N/A
N/A
0.91
>30









Biological activities based on the cell reporter assays for the compounds of the present application are depicted in Table 4 below.











TABLE 4






THP1-dual-KI
THP1-dual-KI


Cmpd
R232_IRF(Luciferase)
R232_IRF(Luciferase)_Activity


No.
IC50 (uM)
Maximum measured (%)

















1
A
D


2
A
D


3
A
D


4
A
D


5
A
D


6
A
D


7
A
D


8
A
D


9
B
D


10
A
D


11
D
A


12
A
D


13
B
D


14
B
D


17
A
D


18
A
D


19
A
D


20
A
D


21
A
D


22
A
D


23
A
D


24
A
D


25
A
D


26
A
D


27
A
D


28
A
D


29
A
D


30
A
D


31
A
D


32
A
D


33
A
D


34
D
C


35
A
D


36
A
D


37
A
D


38
A
D


39
A
D


40
A
D


41
A
D


42
A
D


43
A
D


44
A
D


45
A
D


46
A
D


47
A
D


48
A
D


49
A
D


50
A
D


51
A
D


52
A
D


53
A
D


54
A
D


55
A
D


56
A
D


57
A
D


58
A
D


59
A
D


60
A
D


61
A
D


62
A
D


63
A
D


64
A
D


65
A
D


67
A
D


68
A
D


69
A
D


70
A
D


71
B
B


72
D
B


73
A
D


74
A
D


75
A
D


77
A
D


78
A
D


79
A
D


80
A
D


81
D
B


82
B
B





THP1-dual-KI R232_IRF(Luciferase) IC50 (μM): “A” < 50 μM; “B” = 50-100 μM; “C” = 100-150 μM; “D” > 150 μM.


THP1-dual-KI R232_IRF(Luciferase)_Activity Maximum measured (%): “A” < 5%; “B” = 5-25%; “C” = 25-50%; “D” > 50%.






EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the present application.


All patents, patent applications, and literature references cited herein are hereby expressly incorporated by reference.

Claims
  • 1. A compound of Formula I:
  • 2. The compound of claim 1, wherein X is C(RX)2, O, or S.
  • 3-12. (canceled)
  • 13. The compound of claim 1, wherein T1 is C(O)OR1, C(O)NHS(O)2RS, C(O)NHOH, or C(O)NHCN.
  • 14. The compound of claim 1, of Formula Ia or Ib:
  • 15. The compound of claim 1, wherein m is 0, 1, or 2.
  • 16. (canceled)
  • 17. The compound of claim 1, wherein n is 0, 1, or 2.
  • 18. (canceled)
  • 19. The compound of claim 1, wherein at least one Y is at the 6-position, optionally wherein at least one Y is at the 6-position and at least one Y is at the 7-position.
  • 20. (canceled)
  • 21. The compound of claim 1, wherein: at least one Z is at the 3-position;at least one Z is at the 4-position; orat least one Z is at the 3-position and at least one Z is at the 4-position.
  • 22-23. (canceled)
  • 24. The compound of claim 1, of Formula I1, I1a1, I1a2, I1a3, I1a4, I1a5, I1a6, I1a7, I1b1, I1b2, I1b3, I1b4, I1b5, I1b6, I1b7, I1c1, I1c2, I1c3, I1c4, I1c5, I1c6, or I1c7:
  • 25. The compound of claim 1, wherein; at least one Y is optionally substituted C1-C6 straight-chain or C3-C6 branched alkyl;at least one Y is halogen;at least one Y is OH, O—(C1-C6 straight-chain or C3-C6 branched alkyl), O—(C2-C4 alkenyl), or O—(C2-C4alkynyl), wherein the alkyl, alkenyl, or alkynyl moiety is optionally substituted;at least one Y is S—(C1-C6 straight-chain or C3-C6 branched alkyl), S(O)—(C1-C6 straight-chain or C3-C6 branched alkyl), or S(O)2—(C1-C6 straight-chain or C3-C6 branched alkyl), wherein the alkyl moiety is optionally substituted;at least one Y is NH2, NH—(C1-C6 straight-chain or C3-C6 branched alkyl), or N—(C1-C6 straight-chain or C3-C6 branched alkyl)2, wherein the alkyl moiety is optionally substituted; orat least one Y is Q-T.
  • 26-32. (canceled)
  • 33. The compound of claim 1, wherein: at least one Z is optionally substituted C1-C6 straight-chain or C3-C6 branched alkyl;at least one Z is optionally substituted C2-C6 straight-chain or C3-C6 branched alkenyl;at least one Z is optionally substituted C2-C6 straight-chain or C4-C6 branched alkynyl;at least one Z is halogen;at least one Z is OH, O—(C1-C6 straight-chain or C3-C6 branched alkyl), O—(C2-C4 alkenyl), or O—(C2-C4alkynyl), wherein the alkyl, alkenyl, or alkynyl moiety is optionally substituted;at least one Z is S—(C1-C6 straight-chain or C3-C6 branched alkyl), S(O)—(C1-C6 straight-chain or C3-C6 branched alkyl), or S(O)2—(C1-C6 straight-chain or C3-C6 branched alkyl), wherein the alkyl moiety is optionally substituted;at least one Z is NH2, NH—C1-C6 straight-chain or C3-C6 branched alkyl, or N—(C1-C6 straight-chain or C3-C6 branched alkyl)2, wherein the alkyl moiety is optionally substituted;at least one Z is Q-T.
  • 34-40. (canceled)
  • 41. The compound of claim 1, wherein: at least one Q is a bond;each Q is independently NH, N(C1-C3 alkyl), O, S, S(O), S(O)2, Q′, NH-Q′, N(C1-C3 alkyl)-Q′, O-Q′, S-Q′, S(O)-Q′, or S(O)2-Q′;each Q is independently NH, O, S, or Q′;each Q is independently Q′, NH-Q′, O-Q′, or S-Q′;each Q is independently NH, S, Q′, NH-Q′, or S-Q′;each Q is independently NH, Q′, or NH-Q′;each Q is independently O, Q′, or O-Q′; oreach Q is independently S, S(O), S(O)2, Q′, S-Q′, S(O)-Q′, or S(O)2-Q′.
  • 42-48. (canceled)
  • 49. The compound of claim 1, wherein at least one Q is Q′, optionally wherein: each Q′ is independently a carbon linker comprising one or more C(RQ)2 or C(RQ)2—C(RQ)2;each Q′ is independently a carbon linker comprising one or more CRQ═CRQ;each Q′ is independently a carbon linker comprising one or more C≡C;each Q′ is independently a carbon linker comprising one or more C(RQ)2 or C(RQ)2—C(RQ)2 and CRQ═CRQ;each Q′ is independently a carbon linker comprising one or more C(RQ)2 or C(RO)2—C(RO)2 and C≡C;each Q′ is independently a carbon linker comprising one or more CRQ═CRQ and C≡C; oreach Q′ is independently a carbon linker comprising one or more C(RQ)2, C(RQ)2—C(RQ)2, CRQ═CRQ, and C≡C.
  • 50-56. (canceled)
  • 57. The compound of claim 1, wherein: at least one T is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, each of which is optionally substituted;at least one T is cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, or cyclooctenyl, each of which is optionally substituted;at least one T is optionally substituted heterocyclyl;at least one T is optionally substituted aryl;at least one T is optionally substituted heteroaryl; orat least one T is C(O)—C1-C6 alkyl or C(O)O—C1-C6 alkyl.
  • 58-62. (canceled)
  • 63. The compound of claim 1, selected from Table 1, or a pharmaceutically acceptable salt or ester thereof.
  • 64. The compound of claim 1, selected from
  • 65. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier.
  • 66. A method of modulating a stimulator of interferon genes (STING) protein, comprising administering to a subject in need thereof an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt or ester thereof.
  • 67. A method of treating or preventing a disease, wherein the diseases is caused by, or associated with, STING expression, activity, and/or function, or is associated with deregulation of one or more of the intracellular pathways in which a STING protein is involved, comprising administering to a subject in need thereof an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt or ester thereof.
  • 68-72. (canceled)
RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Application No. 62/848,744, filed on May 16, 2019, the content of which is incorporated by reference in its entirety.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2020/033198 5/15/2020 WO
Provisional Applications (1)
Number Date Country
62848744 May 2019 US