Patent Application
20230056497
The present invention is directed to immunotherapy drugs, and more particularly to compounds that modulate CD206 as well as their use and methods for preparation.
Pancreatic cancer is a disease in which malignant (cancerous) cells form in the tissues of the pancreas. Pancreatic cancer often has a poor prognosis, even when diagnosed early. Pancreatic cancer typically spreads rapidly and is seldom detected in its early stages, which is a major reason why it's a leading cause of cancer death. Pancreatic cancer is the fourth leading cause of cancer death in both men and women in the United States of America (U.S.), with more than 44,000 deaths annually. Pancreatic cancer is expected to rank second in all cancer-related deaths in the United States by 2030. Furthermore, the 5-year survival rate of pancreatic cancer in the U.S. ranks lowest among solid organ tumors. There is no reliable screening test for the early detection of pancreatic cancer. Signs and symptoms may not appear until pancreatic cancer is quite advanced, and complete surgical removal isn't possible.
Standard treatment of pancreatic cancer, including surgery, radiation therapy, and chemotherapy largely show limited efficacy. Indeed, approved treatments including gemcitabine, folfirinox, the combination of gemcitabine and abraxane, and the combination of gemcitabine and erlotinib, improve survival by a few to several months, at best. Newer therapies have not demonstrated much more success, possibly due to the thick stroma, a unique immune infiltrate characterized by a paucity of cytotoxic tumor-infiltrating T cells, a high number of immune suppressive pro-tumor myeloid cells, and the relative absence of abundant vessels in the pancreas. Pancreatic ductal adenocarcinoma (PDA) accounts for >90% of pancreatic cancer cases, with a five-year survival rate of 6%.
Recent advances in immunotherapy have transformed the care of many cancer patients. However, these positive findings are limited to immunologically ‘hot’ cancers whereas in the greater majority of solid organ cancers, like pancreatic cancer, which are classified as immunologically ‘cold’, the promise of immunotherapy via T cell activation has so far largely evaded patients. These tumors create an immune milieu which excludes cytotoxic T cells or induces an exhausted T cell phenotype through an abundance of immune evasive cues frequently involving innate immune cells. Strategies that reinvigorate innate immune cells are underrepresented within current immuno-oncology therapies.
Tumor cells attract and reprogram innate immune cells including tumor-associated macrophages (TAMs) to support tumor growth and metastatic spread. While the dichotomous M1 versus M2 classification omits to capture the ontogeny and tissue-specific cues of TAMs, in general terms, M1-like TAMs are proposed the more common phenotype in early tumor stages, while M2 TAMs are more prominent in more evolved cancers. CD206high M2 TAMs harness tumor growth via the excretion of cancer-promoting factors or via promotion of angiogenesis, nurturing of cancer stem cells, or the generation of an immune-evasive microenvironment.
CD206 is a member of the large C-type lectin receptor family which can target and modulate the M2 macrophages. CD206 via its eight carbohydrate recognition domains is involved in recognition and binding of mannan and fucose carbohydrate residues from microbial organisms, or via its fibronectin domain II as a scavenger receptor in the phagocytosis of collagen fragments generated during tissue injury and wound healing. Ligand binding or low pH induces ‘rolling-in’ (via multiple Ca+-dependent intramolecular interactions between the carbohydrate recognition domains) and the closed (‘active’) form of the receptor which triggers in M2 macrophages, among other signaling cascades, via GRB2-mediated activation of small Rho-GTPases NF-kB signaling activation and induction of phagocytosis and autophagy.
TAMs express scavenger receptors such as CD206 which facilitate tumor angiogenesis, tumor cell migration, maintenance of an EMT-like phenotype of cancer cells, and metastasis. CD206high expression has been associated with poor clinical outcomes in pancreatic cancer and other solid organ cancers. Selective depletion of M2 tumor associated macrophages may improve anti-tumor immunity and cancer outcome.
Using a synthetic host defense peptide design known to regulate innate immune function via binding to C-type lectin receptors (RP-182) it was previously shown that binding to amino acid carbohydrate recognition domain 5 (CRD5) sequence NFGDLVSIQSESEKK of the CD206 receptor: (1) activates a program of phagocytosis and autophagy in M2 macrophages leading to metabolic reprogramming and a M1-like phenotype of these cells as well as, (2) intracellular signaling activation of NF-kB leading to selective killing via autocrine TNFalpha-mediated caspase 8 and 3 activation of CD206high M2 macrophages (U.S. Pat. No. 10,016,480). However, the unfavorable pharmacokinetic (PK) properties of the peptide-based innate immune regulator hinder clinical prospects of synthetic peptides like RP-182. Therefore, small molecule modulators of CD206 are highly desired.
Described herein are small molecule modulators targeting the CD206 receptor, their methods of manufacture, compositions containing the described compounds, and methods of using the described compounds.
In a first aspect, a compound of Formula I and the pharmaceutically acceptable salts of a compound of Formula I is provided.
Within Formula I the following conditions are met.
Each bond shown as a solid line and a dashed line together, , can be a single bond, double, or aromatic bond.
R1 is hydrogen, halogen, hydroxyl, cyano, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)NR8R9, —(C0-C6alkyl)NR5R6, —CO2R6, —C6H4—R7, and a monocyclic or bicyclic heterocycle of 4 to 10 ring atoms having 1, 2, or 3 ring atoms independently chosen from N, S and O.
R2, R3, and R4 are each independently chosen at each occurrence from hydrogen, halogen, hydroxyl, cyano, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)NR5R6, (C0-C6alkyl)NR8R9, —CO2R6, and —C6H4—R7.
a, b, c, d, and X are each independently chosen at each occurrence from N, C, and CH.
R5, and R6 are each independently chosen at each occurrence from hydrogen, halogen, hydroxy, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6alkoxy, a substituted or unsubstituted —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)(C0-C6alkyl)phenyl, —(C0-C6alkyl)NR8R9, —C(O)(C0-C6alkyl)aryl, —C(O)(C0-C6alkyl)heteroaryl, and a 4- to 7-membered heterocycloalkyl ring having 1, 2, or 3 ring atoms independently chosen from N, O, and S.
Any R5 and R6 bound to the same nitrogen atom may be taken together to form a 4- to 7-membered monocyclic heterocycloalkyl ring or 6- to 11-membered bridged bicyclic heterocycloalkyl ring, which heterocycloalkyl ring contains 0, 1, or 2 additional heteroatoms chosen from N, O, S, S(O), and SO2 which heterocycloalkyl ring is optionally substituted at any carbon or hetero ring atom with halogen, hydroxyl, cyano, oxo, dioxo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)CO2R8, —(C0-C6alkyl)C(O)NR8R9, —(C1-C6alkyl)OR8, —C(O)C1-C6alkyl, —(C0-C6alkyl)NR8R9, or —C(O)(C0-C6alkyl)NR8R9.
R7 is hydrogen, halogen, hydroxyl, cyano, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —CO2R8, —C(O)C1-C6alkyl, —C(O)C2-C6alkenyl, —C(O)C2-C6alkynyl, —C(O)C1-C6alkoxy, —C(O)C1-C6hydroxyalkyl, —C(O)—(C0-C6alkyl)cycloalkyl, —C(O)—(C0-C6alkyl)phenyl, —C(O)—(C0-C6alkyl)aryl, —C(O)—(C0-C6alkyl)heteroaryl, —C(O)NR8R9, —C(O)NR5R6, —C(O)—(C0-C6alkyl)NR5R6, —C(O)—NRB—(C0-C6alkyl)NR5R6, or (C0-C6alkyl)NR5R6.
R8 and R9 are each independently chosen at each occurrence from hydrogen, halogen, C1-C6 alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)NR5R6, —CO2R6, —C(O)C1-C6alkyl, and —(C0-C6alkyl)cycloalkyl.
In a second aspect, a compound of Formula II and the pharmaceutically acceptable salts of a compound of Formula II is provided.
Within Formula II the following conditions are met.
Each bond shown as a solid line and a dashed line together, , can be a single or double bond.
R10, R11, and R13 are each independently chosen at each occurrence from hydrogen, hydroxyl, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)heteroaryl, and —CO2R16.
R12, R14, and R15 are each independently chosen at each occurrence from hydrogen, halogen, hydroxyl, and cyano.
X is O or S.
R16 is hydrogen, halogen, hydroxy, an amino group, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, —C(O)C1-C6alkyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —(C0-C6alkyl)phenyl, or a monocyclic or bicyclic heterocycle of 4 to 10 ring atoms having 1, 2, or 3 ring atoms independently chosen from N, S and O.
In a third aspect, a compound of Formula III and the pharmaceutically acceptable salts of a compound of Formula III is provided.
Within Formula III the following conditions are met.
R17, R18, and R21 are each independently chosen at each occurrence from hydrogen, halogen, hydroxyl, cyano, an amidino group, —NR23R24, a sulfonic acid group or a salt thereof, a phosphoric acid group or a salt thereof, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)(C0-C6alkyl)phenyl, —C(O)(C0-C6alkyl)aryl, —C(O)(C0-C6alkyl)heteroaryl, —C(O)NR23R24, —(C0-C6alkyl)NR23R24, —CO2R23, and a monocyclic or bicyclic heterocycle of 4 to 10 ring atoms having 1, 2, or 3 ring atoms independently chosen from N, S and O.
X is chosen at each occurrence from 0 and S.
R19, R20, and R22 are each independently chosen at each occurrence from hydrogen, halogen, hydroxy, cyano, and an amino group.
R23 and R24 are each independently chosen at each occurrence from hydrogen, halogen, C1-C6 alkyl, C1-C6alkoxy, C1-C6haloalkyl, C1-C6haloalkoxy, C1-C6alkoxy, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)(C0-C6alkyl)phenyl, —C(O)(C0-C6alkyl)aryl, —C(O)(C0-C6alkyl)heteroaryl, —S(O)phenyl, —S(O)aryl, —S(O)heteroaryl, —SO2phenyl, —SO2aryl, —SO2heteroaryl, —(C0-C6alkyl)cycloalkyl, and —CO2R25.
R25 is hydrogen, halogen, hydroxy, an amino group, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, —C(O)C1-C6alkyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —(C0-C6alkyl)phenyl, or a monocyclic or bicyclic heterocycle of 4 to 10 ring atoms having 1, 2, or 3 ring atoms independently chosen from N, S and O.
Pharmaceutical compositions comprising a compound or salt of Formula I or Formula II or Formula III with a pharmaceutically acceptable carrier are also disclosed.
Methods for the treatment of cancer which may involve selective targeting of M2 macrophages and the reprogramming of M2 macrophages towards a M1 phenotype in a patient, comprising the step of administering to the patient in need thereof a compound of Formula I or Formula II or Formula III or a salt thereof, are also disclosed.
In some embodiments targeting CD206 M2 macrophages with compound or salt of Formula I or Formula II or Formula III may have a dual effect: it may reprogram CD206 M2 macrophages into a M1 macrophage and it may directly kill a M2 macrophage.
Methods for the treatment of cancer characterized by the presence of CD206 positive tumor-associated macrophages (TAM), such as glioma (glioblastoma), sarcoma, astrocytoma, melanoma, non-small cell lung cancer, cholangiocarcinomas, colon cancer, hepatocellular, breast, prostate, gastric, renal cell, endometrial, or pancreatic cancer comprising administering a therapeutically effective amount of a compound or salt of Formula I or Formula II or Formula III to a patient in need of such treatment are also disclosed.
The following Detailed Description, given by way of Examples, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying figures, in which:
Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term “or” means “and/or.” The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”).
Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended for illustration and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art of this disclosure.
Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.
All compounds are understood to include all possible isotopes of atoms occurring in the compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include 11C, 13C, and 14C.
Formula I includes all pharmaceutically acceptable salts of Formula I.
Formula II includes all pharmaceutically acceptable salts of Formula II.
Formula III includes all pharmaceutically acceptable salts of Formula III.
The opened ended term “comprising” includes the intermediate and closed terms “consisting essentially of” and “consisting of.”
The term “substituted” means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded. When the substituent is oxo (i.e., ═O), then 2 hydrogens on the atom are replaced. When aromatic moieties are substituted by an oxo group, the aromatic ring is replaced by the corresponding partially unsaturated ring. For example a pyridyl group substituted by oxo is a pyridone. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent.
Suitable groups that may be present on an “optionally substituted” position include, but are not limited to, e.g., halogen, cyano, hydroxyl, amino, nitro, oxo, azido, alkanoyl (such as a C2-C6 alkanoyl group such as acyl or the like (—(C═O)alkyl)); carboxamido; alkylcarboxamide; alkyl groups, alkoxy groups, alkylthio groups including those having one or more thioether linkages, alkylsulfinyl groups including those having one or more sulfinyl linkages, alkylsulfonyl groups including those having one or more sulfonyl linkages, mono- and di-aminoalkyl groups including groups having one or more N atoms, all of the foregoing optional alkyl substituents may have one or more methylene groups replaced by an oxygen or —NH—, and have from about 1 to about 8, from about 1 to about 6, or from 1 to about 4 carbon atoms, cycloalkyl; phenyl; phenylalkyl with benzyl being an exemplary phenylalkyl group, phenylalkoxy with benzyloxy being an exemplary phenylalkoxy group. Alkylthio and alkoxy groups are attached to the position they substitute by the sulfur or oxygen atom respectively.
A dash (“-”) and (“”) that is not between two letters or symbols is used to indicate a point of attachment for a substituent.
“Alkyl” includes both branched and straight chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms, generally from 1 to about 8 carbon atoms. The term C1-C6alkyl as used herein indicates an alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms. Other embodiments include alkyl groups having from 1 to 8 carbon atoms, 1 to 4 carbon atoms or 1 or 2 carbon atoms, e.g. C1-C8alkyl, C1-C4alkyl, and C1-C2alkyl. When C0-Cn alkyl is used herein in conjunction with another group, for example, —C0-C2alkyl(phenyl), the indicated group, in this case phenyl, is either directly bound by a single covalent bond (C0alkyl), or attached by an alkyl chain having the specified number of carbon atoms, in this case 1, 2, 3, or 4 carbon atoms. Alkyls can also be attached via other groups such as heteroatoms as in —O—C0-C4alkyl(C3-C7cycloalkyl). Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, and sec-pentyl.
“Alkenyl” is a branched or straight chain aliphatic hydrocarbon group having one or more carbon-carbon double bonds that may occur at any stable point along the chain, having the specified number of carbon atoms. Examples of alkenyl include, but are not limited to, ethenyl and propenyl.
“Alkynyl” is a branched or straight chain aliphatic hydrocarbon group having one or more double carbon-carbon triple bonds that may occur at any stable point along the chain, having the specified number of carbon atoms.
“Alkoxy” is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes by an oxygen bridge (—O—). Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy. Similarly an “Alkylthio” or a “thioalkyl” group is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes by a sulfur bridge (—S—).
“Aryl” is a substituted stable monocyclic or polycyclic aromatic ring having 1 to 60 ring carbon atoms. Aryl groups include, but are not limited to, tolyl, xylyl, naphthyl, phenanthryl, and anthracenyl.
“Cycloalkyl” is a saturated hydrocarbon ring group, having the specified number of carbon atoms, usually from 3 to about 7 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl as well as bridged or caged saturated ring groups such as norborane or adamantane. “—(C0-Cn alkyl)cycloalkyl” is a cycloalkyl group attached to the position it substitutes either by a single covalent bond (C0) or by an alkylene linker having 1 to n carbon atoms.
“Halo” or “halogen” means fluoro, chloro, bromo, or iodo.
“Heteroaryl” is a stable monocyclic aromatic ring having the indicated number of ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered aromatic ring which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms. In some embodiments bicyclic heteroaryl groups are 9- to 10-membered heteroaryl groups, that is, groups containing 9 or 10 ring atoms in which one 5- to 7-member aromatic ring is fused to a second aromatic or non-aromatic ring. When the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. It is particularly preferred that the total number of S and O atoms in the aromatic heterocycle is not more than 1. Heteroaryl groups include, but are not limited to, oxazolyl, piperazinyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl, pyridizinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzo[d]oxazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl, dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, isothiazolyl, and isoxazolyl.
“Heterocycle” is a saturated, unsaturated, or aromatic cyclic group having the indicated number of ring atoms containing from 1 to about 3 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Examples of heterocycle groups include piperazine and thiazole groups.
“Heterocycloalkyl” is a saturated cyclic group having the indicated number of ring atoms containing from 1 to about 3 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Examples of heterocycloalkyl groups include tetrahydrofuranyl and pyrrolidinyl groups.
“Haloalkyl” means both branched and straight-chain alkyl groups having the specified number of carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.
“Haloalkoxy” is a haloalkyl group as defined above attached through an oxygen bridge (oxygen of an alcohol radical).
“Pharmaceutical compositions” means compositions comprising at least one active agent, such as a compound or salt of Formula (I), and at least one other substance, such as a carrier. Pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturing practice) standards for human or non-human drugs.
“Carrier” means a diluent, excipient, or vehicle with which an active compound is administered. A “pharmaceutically acceptable carrier” means a substance, e.g., excipient, diluent, or vehicle, that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable carrier” includes both one and more than one such carrier.
A “patient” means a human or non-human animal in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder or diagnostic treatment. In some embodiments the patient is a human patient.
“Providing” means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.
“Treatment” or “treating” means providing an active compound to a patient in an amount sufficient to measurably reduce any cancer symptom, slow cancer progression or cause cancer regression. In certain embodiments treatment of the cancer may be commenced before the patient presents symptoms of the disease.
A “therapeutically effective amount” of a pharmaceutical composition means an amount effective, when administered to a patient, to provide a therapeutic benefit such as an amelioration of symptoms, decrease cancer progression, or cause cancer regression.
A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p<0.05.
Compounds of Formula I or Formula II or Formula III may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. These compounds can be, for example, racemates or optically active forms. For compounds with two or more asymmetric elements, these compounds can additionally be mixtures of diastereomers. For compounds having asymmetric centers, all optical isomers in pure form and mixtures thereof are encompassed. In these situations, the single enantiomers, i.e., optically active forms can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column. All forms are contemplated herein regardless of the methods used to obtain them.
All forms (for example solvates, optical isomers, enantiomeric forms, tautomers, polymorphs, free compound and salts) of an active agent may be employed either alone or in combination.
The term “chiral” refers to molecules, which have the property of non-superimposability of the mirror image partner.
“Stereoisomers” are compounds, which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
A “diastereomer” is a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis, crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.
“Enantiomers” refer to two stereoisomers of a compound, which are non-superimposable mirror images of one another. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory.
A “racemic mixture” or “racemate” is an equimolar (or 50:50) mixture of two enantiomeric species, devoid of optical activity. A racemic mixture may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
“Tautomers” or “tautomeric forms” are constitutional isomers that readily interconvert, commonly by the migration of a hydrogen atom combined with a switch of a single bond and a double bond.
“Pharmaceutically acceptable salts” include derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.
Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC—(CH2)n—COOH where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in G. Steffen Paulekuhn, et al., Journal of Medicinal Chemistry 2007, 50, 6665 and Handbook of Pharmaceutically Acceptable Salts: Properties, Selection and Use, P. Heinrich Stahl and Camille G. Wermuth Editors, Wiley-VCH, 2002.
Molecules which modulate CD206 are disclosed herein.
In addition to compounds of Formula I, Formula II, and Formula III shown in the SUMMARY section, the disclosure also includes compounds in which the variables, e.g. X and R1 to R25 carry the following definitions. The disclosure includes all combinations of these definitions so long as a stable compound results.
The disclosure includes the following particular embodiments of Formula I
(A) In an embodiment, R1 is hydrogen, halogen, hydroxyl, cyano, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)NR8R9, —(C0-C6alkyl)NR5R6, —CO2R6, —C6H4—R7, and a monocyclic or bicyclic heterocycle of 4 to 10 ring atoms having 1, 2, or 3 ring atoms independently chosen from N, S and 0.
R2 and R4 are H.
R3 is hydrogen, halogen, hydroxyl, cyano, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)NR5R6, (C0-C6alkyl)NR8R9, —CO2R6, and —C6H4—R7.
a, b, c, and d are each independently chosen at each occurrence from N, C and CH.
X is N.
R5, and R6 are each independently chosen at each occurrence from hydrogen, halogen, hydroxy, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6alkoxy, a substituted or unsubstituted —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —C(O)C1-C6alkyl, —C(O)(C0-C6alkyl)phenyl, —(C0-C6alkyl)NR8R9, —C(O)(C0-C6alkyl)aryl, —C(O)(C0-C6alkyl)heteroaryl, and a 4- to 7-membered heterocycloalkyl ring having 1, 2, or 3 ring atoms independently chosen from N, O, and S.
Any R5 and R6 bound to the same nitrogen atom may be taken together to form a 4- to 7-membered monocyclic heterocycloalkyl ring or 6- to 11-membered bridged bicyclic heterocycloalkyl ring, which heterocycloalkyl ring contains 0, 1, or 2 additional heteroatoms chosen from N, O, S, S(O), and SO2 which heterocycloalkyl ring is optionally substituted at any carbon or hetero ring atom with halogen, hydroxyl, cyano, oxo, dioxo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)CO2R8, —(C0-C6alkyl)C(O)NR8R9, —(C1-C6alkyl)OR8, —C(O)C1-C6alkyl, —(C0-C6alkyl)NR8R9, or —C(O)(C0-C6alkyl)NR8R9.
R7 is hydrogen, halogen, hydroxyl, cyano, —CO2H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)heteroaryl, —CO2R8, —C(O)C1-C6alkyl, —C(O)C2-C6alkenyl, —C(O)C2-C6alkynyl, —C(O)C1-C6alkoxy, —C(O)C1-C6hydroxyalkyl, —C(O)—(C0-C6alkyl)cycloalkyl, —C(O)—(C0-C6alkyl)phenyl, —C(O)—(C0-C6alkyl)aryl, —C(O)—(C0-C6alkyl)heteroaryl, —C(O)NR8R9, —C(O)NR5R6, —C(O)—(C0-C6alkyl)NR5R6, —C(O)—NRB—(C0-C6alkyl)NR5R6, or (C0-C6alkyl)NR5R6.
R8 and R9 are each independently chosen at each occurrence from hydrogen, halogen, C1-C6 alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)NR5R6, —CO2R6, —C(O)C1-C6alkyl, and —(C0-C6alkyl)cycloalkyl.
(B) In an embodiment, R1 is —C6H4—R7.
R2 and R4 are H.
R3 is —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, or —(C0-C6alkyl)heteroaryl.
a, c, and X are N.
b is C.
d is CH.
R7 is —C(O)NR5R6 or —C(O)—NR8—(C0-C6alkyl)NR5R6.
R5 and R6 are each independently chosen at each occurrence from hydrogen, a substituted or unsubstituted —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)heteroaryl, C1-C6hydroxyalkyl, C1-C6alkoxy, —(C0-C6alkyl)NR8R9, and a 4- to 7-membered heterocycloalkyl ring having 1, 2, or 3 ring atoms independently chosen from N, O, and S.
Any R5 and R6 bound to the same nitrogen atom may be taken together to form a 4- to 7-membered monocyclic heterocycloalkyl ring or 6- to 11-membered bridged bicyclic heterocycloalkyl ring, which heterocycloalkyl ring contains 0, 1, or 2 additional heteroatoms chosen from N, O, S, S(O), and SO2 which heterocycloalkyl ring is optionally substituted at any carbon or hetero ring atom with halogen, hydroxyl, cyano, oxo, dioxo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)CO2R8, —(C0-C6alkyl)C(O)NR8R9, —(C1-C6alkyl)OR8, —CO2R8, —C(O)C1-C6alkyl, —(C0-C6alkyl)NR8R9, or —C(O)(C0-C6alkyl)NR8R9.
R8 and R9 are each independently chosen at each occurrence from hydrogen, halogen, C1-C6 alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, —(C0-C6alkyl)NR5R6, —CO2R6, —C(O)C1-C6alkyl, and —(C0-C6alkyl)cycloalkyl.
(C) In an embodiment, the compound of Formula I is a compound represented by at least one of Compound 1, and Compound 4 to Compound 29:
or a pharmaceutically acceptable salt thereof.
(D) In an embodiment, R1 is —C6H4—R7.
R2 and R4 are hydrogen.
R3 is —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, or —(C0-C6alkyl)heteroaryl.
a, c, d, and X are N.
b is C.
R7 is —C(O)—NRB—(C0-C6alkyl)NR5R6.
R5 and R6 bound to the same nitrogen atom may be taken together to form a 4- to 7-membered monocyclic heterocycloalkyl ring or 6- to 11-membered bridged bicyclic heterocycloalkyl ring, which heterocycloalkyl ring contains 0, 1, or 2 additional heteroatoms chosen from N, O, S, S(O), and SO2 which heterocycloalkyl ring is optionally substituted at any carbon or hetero ring atom with halogen, hydroxyl, cyano, oxo, dioxo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, or —(C0-C6alkyl)aryl.
R8 is hydrogen.
(E) In an embodiment, the compound of Formula I is a compound represented by at least one of Compound 30 and Compound 31:
or a pharmaceutically acceptable salt thereof.
(F) In an embodiment, R1 is —C6H4—R7.
R2 and R4 are hydrogen.
R3 is —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, or —(C0-C6alkyl)heteroaryl.
a is C.
b, d, and X are N.
c is CH.
R7 is —C(O)—NR8—(C0-C6alkyl)NR5R6.
R5 and R6 bound to the same nitrogen atom may be taken together to form a 4- to 7-membered monocyclic heterocycloalkyl ring or 6- to 11-membered bridged bicyclic heterocycloalkyl ring, which heterocycloalkyl ring contains 0, 1, or 2 additional heteroatoms chosen from N, O, S, S(O), and SO2 which heterocycloalkyl ring is optionally substituted at any carbon or hetero ring atom with halogen, hydroxyl, cyano, oxo, dioxo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, or —(C0-C6alkyl)aryl.
R8 is hydrogen.
(G) In an embodiment, the compound of Formula I is a compound represented by Compound 32:
or a pharmaceutically acceptable salt thereof.
(H) In an embodiment, R1 is —C6H4—R7.
R2 and R4 are hydrogen.
R3 is —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, or —(C0-C6alkyl)heteroaryl.
a is C.
b and X are N.
c and d are CH.
R7 is —C(O)—NRB—(C0-C6alkyl)NR5R6.
R5 and R6 bound to the same nitrogen atom may be taken together to form a 4- to 7-membered monocyclic heterocycloalkyl ring or 6- to 11-membered bridged bicyclic heterocycloalkyl ring, which heterocycloalkyl ring contains 0, 1, or 2 additional heteroatoms chosen from N, O, S, S(O), and SO2 which heterocycloalkyl ring is optionally substituted at any carbon or hetero ring atom with halogen, hydroxyl, cyano, oxo, dioxo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)phenyl, or —(C0-C6alkyl)aryl.
R8 is hydrogen.
(I) In an embodiment, the compound of Formula I is a compound represented by Compound 33:
or a pharmaceutically acceptable salt thereof.
The disclosure includes the following particular embodiments of Formula II
In some embodiments the compound of Formula II is a compound of Formula IIA
(A) In an embodiment, R10 and R11 are each independently chosen at each occurrence from —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, and —(C0-C6alkyl)heteroaryl.
R12, R14 and R15 are hydrogen.
R13 is —C(O)heteroaryl.
(B) In an embodiment, R10 is —(C0-C6alkyl)phenyl.
R11 is —(C0-C6alkyl)heteroaryl.
R12, R14 and R15 are hydrogen.
R13 is —C(O)heteroaryl.
(C) In an embodiment, the compound of Formula IIA is Compound 2:
or a pharmaceutically acceptable salt thereof.
The disclosure includes the following particular embodiments of Formula III
(A) In an embodiment, R17 is —C(O)C1-C6alkyl, —C(O)(C0-C6alkyl)phenyl, —C(O)(C0-C6alkyl)aryl, or —C(O)(C0-C6alkyl)heteroaryl.
R18 is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, —(C0-C6alkyl)cycloalkyl, C1-C6haloalkyl, —(C0-C6alkyl)phenyl, —(C0-C6alkyl)aryl, or —(C0-C6alkyl)heteroaryl.
R19, R20 and R22 are hydrogen.
R21 is —NR23R24.
X is chosen at each occurrence from O and S.
R23 and R24 are each independently chosen at each occurrence from —S(O)phenyl, —S(O)aryl, —S(O)heteroaryl, —SO2phenyl, —SO2aryl, —SO2heteroaryl, —(C0-C6alkyl)cycloalkyl, and —CO2R25.
R25 is C1-C6alkyl, —(C0-C6alkyl)cycloalkyl, —(C0-C6alkyl)aryl, or —(C0-C6alkyl)phenyl.
(B) In an embodiment, R17 is —C(O)C1-C6alkyl.
R18 is C1-C6alkyl.
R19, R20 and R22 are hydrogen.
R21 is —NR23R24.
X is oxygen.
R23 and R24 are each independently chosen at each occurrence from a substituted or unsubstituted aryl sulfonyl, —CO2R25, —SO2phenyl, —SO2aryl, and —SO2R25.
R25 is phenyl.
In an embodiment, the compound of Formula III is Compound 3:
or a pharmaceutically acceptable salt thereof.
The compounds of Formula I, Formula II, or Formula III or a salt thereof, as well as pharmaceutical compositions comprising the compounds, are useful for treating cancer, including effecting tumor regression in vivo. The method of treating cancer or effecting tumor regression comprises providing to a patient an effective amount of a compound of Formula I, Formula II, or Formula III. In an embodiment the patient is a mammal, and more specifically a human. The disclosure also provides methods of treating non-human patients such as companion animals, e.g. cats, dogs, and livestock animals. An effective amount of a pharmaceutical composition may be an amount sufficient to inhibit the progression of cancer or a cancerous tumor; or cause a regression of a cancer or a cancerous tumor.
An effective amount of a compound or pharmaceutical composition described herein will also provide a sufficient concentration of a compound of Formula I, Formula II, or Formula III when administered to a patient. A sufficient concentration is a concentration of the compound in the patient's body necessary to combat the disorder. Such an amount may be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability.
Methods of treatment include providing certain dosage amounts of a compound of Formula I, Formula II, or Formula III to a patient. Dosage levels of each compound of from about 20 milligram (mg) or less per kilogram of body weight per day are useful in the treatment of the above-indicated conditions Frequency of dosage may also vary depending on the compound used and the particular disease treated.
The compounds of Formula I, Formula II, or Formula III may be used to treat cancers and effect regression of tumors, including cancerous tumors. In certain embodiments, the patient is suffering from a cell proliferative disorder or disease. The cell proliferative disorder can be cancer, tumor (cancerous or benign), neoplasm, neovascularization, or melanoma. Cancers for treatment include both solid and disseminated cancers. Exemplary solid cancers (tumors) that may be treated by the methods provided herein include e.g. cancers of the lung, prostate, breast, liver, colon, breast, kidney, pancreas, brain, skin including malignant melanoma and Kaposi's sarcoma, testes or ovaries, carcinoma, kidney cancer (renal cell), and sarcoma.
Cancers that may be treated with a compound of Formula I, Formula II, or Formula III also include bladder cancer, breast cancer, colon cancer, endometrial cancer, lung cancer, bronchial cancer, melanoma, Non-Hodgkins lymphoma, cancer of the blood, pancreatic cancer, prostate cancer, thyroid cancer, brain or spinal cancer, and leukemia. Exemplary disseminated cancers include leukemias or lymphoma including Hodgkin's disease, multiple myeloma and mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), T-cell leukemia, multiple myeloma, and Burkitt's lymphoma. Particularly included herein are methods of treating cancer by providing a compound of Formula I, Formula II, or Formula III to a patient wherein the cancer is a solid tumor or disseminated cancer.
Further included are methods of treating cancer by providing a compound of Formula I, Formula II, or Formula III to a patient wherein the cancer is selected from glioma (glioblastoma), acute myelogenous leukemia, acute myeloid leukemia, myelodysplastic/myeloproliferative neoplasms, sarcoma, chronic myelomonocytic leukemia, non-Hodgkin lymphoma, astrocytoma, melanoma, non-small cell lung cancer, cholangiocarcinomas, chondrosarcoma, or colon cancer.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
A compound of Formula I, Formula II, or Formula III may be administered singularly (i.e., sole therapeutic agent of a regime) to treat diseases and conditions such as undesired cell proliferation, cancer, and/or tumor growth or may be administered in combination with another active agent. One or more compounds of Formula I, Formula II, or Formula III may be administered in coordination with a regime of one or more other chemotherapeutic agents such as an antineoplastic drug, e.g., an alkylating agent (e.g., mechloroethamine, chlorambucil, cyclophosamide, melphalan, or ifosfamide), an antimetabolite such as a folate antagonist (e.g., methotrexate), a purine antagonist (e.g. 6-mercaptopurine) or a pyrimidine antagonist (e.g., 5-fluorouracil). Other, non-limiting examples of chemotherapeutic agents that might be used in coordination with one or more compounds of Formula I, Formula II, or Formula III include taxanes and topoisomerase inhibitors. In addition, other non-limiting examples of active therapeutics include biological agents, such as monoclonal antibodies or IgG chimeric molecules, that achieve their therapeutic effect by specifically binding to a receptor or ligand in a signal transduction pathway associated with cancer (e.g. therapeutic antibodies directed against CD20 (e.g. rituximab) or against VEGF (e.g. bevacizumab)).
Methods of treatment provided herein are also useful for treatment of mammals other than humans, including for veterinary applications such as to treat horses and livestock e.g. cattle, sheep, cows, goats, swine and the like, and pets (companion animals) such as dogs and cats.
For diagnostic or research applications, a wide variety of mammals will be suitable subjects including rodents (e.g. mice, rats, hamsters), rabbits, primates and swine such as inbred pigs and the like. Additionally, for in vitro applications, such as in vitro diagnostic and research applications, body fluids (e.g., blood, plasma, serum, cellular interstitial fluid, saliva, feces and urine) and cell and tissue samples of the above subjects will be suitable for use.
In an embodiment, the invention provides a method of treating a cancer disorder in a patient identified as in need of such treatment, the method comprising providing to the patient an effective amount of a compound of Formula I, Formula II, or Formula III. The compounds and salts of Formula I, Formula II, or Formula III provided herein may be administered alone, or in combination with one or more other active agent.
In an embodiment, the cancer to be treated is characterized by the selective targeting M2 macrophages and the reprogramming of M2 macrophages towards a M1 phenotype in a patient.
As shown in
Tumor growth was suppressed during in vivo testing of Compound 1 in fully immune-competent transgenic Kras(G12D)/Trp53(R172H)/Pdx-1-Cre (KPC) mice (murine pancreatic cancer model) compared to vehicle as shown in
As shown in
As shown in
All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylformamide (DMF), acetonitrile (ACN), methanol (MeOH) and triethylamine (Et3N) were purchased from Sigma-Aldrich (St. Louis, Mo.). Preparative purification was performed on a Waters semi-preparative HPLC system (Waters Corp., Milford, Mass.). The column used was a Phenomenex Luna C18 (5 micron, 30×75 mm; Phenomenex, Inc., Torrance, Calif.) at a flow rate of 45.0 m/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 min was used during the purification. Fraction collection was triggered by UV detection at 220 nm. Analytical analysis was performed on an Agilent LCMS (Agilent Technologies, Santa Clara, Calif.). Method 1: A 7-min gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8-min run time at a flow rate of 1.0 m/min. Method 2: A 3-min gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5-min run time at a flow rate of 1.0 m/min. A Phenomenex Luna C18 column (3 micron, 3×75 mm) was used at a temperature of 50° C. Purity determination was performed using an Agilent diode array detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers (Agilent Technologies, Santa Clara, Calif.). Chemical shifts are reported in ppm with undeuterated solvent (DMSO at 2.50 ppm, CHCl3 at 7.26 ppm) as internal standard for DMSO-d6 and CDCl3 solutions respectively. All of the analogs tested in the biological assays have a purity of greater than 95% based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight (TOF) LCMS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (Version B.02). Starting materials were purchased from Combi-Blocks (San Diego, Calif.) or Sigma-Aldrich (St. Louis, Mo.), and were used as received, without further purification.
Thionyl chloride (505 μL, 6.92 mmol) was added to a solution of (5-chloropyrazin-2-yl)methanol (500 mg, 3.46 mmol) and catalytic DMF in DCM (20.0 mL). The resulting reaction mixture was stirred at room temperature for 1 hour (h), after which LCMS and TLC (20% EtOAc in HEX) analysis showed completion. Reaction mixture was concentrated to dryness, taken up in DCM and concentrated to dryness again. Residue was taken up in DMF (10.0 mL) and potassium carbonate (478 mg, 3.46 mmol) added, followed by sodium azide (270 mg, 4.15 mmol). The resulting reaction mixture was stirred at room temperature for 2 h, after which LC-MS analysis showed completion. Reaction mixture was taken up in H2O, extracted twice with EtOAc, the combined organic layers washed twice with brine, dried over anhydrous MgSO4, filtered and concentrated to afford 2-(azidomethyl)-5-chloropyrazine (587 mg, 3.46 mmol, 100% yield) as a golden oil, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 8.84 (d, J=1.4 Hz, 1H), 8.57 (d, J=1.2 Hz, 1H), 4.64 (s, 2H). LCMS retention time (RT) (Method 2)=2.644 min, m/z 170.6 [M+H+].
To a solution of 2-(azidomethyl)-5-chloropyrazine (587 mg, 3.46 mmol) in MeOH (40.0 mL) was added triphenylphosphine (1.36 grams (g), 5.19 mmol). The resulting reaction mixture was fitted with a condenser and stirred at 80° C. for 1.5 h, after which LCMS and TLC (20% EtOAc in Hex) analysis showed completion. Reaction mixture was concentrated to dryness and residue taken up in toluene (25.0 mL), treated with 4.0 molar (M) HCl in dioxane (2.00 mL, 8.00 mmol), as product precipitated as the HCl salt. The solid was filtered, rinsed with toluene and air dried to afford crude (5-chloropyrazin-2-yl)methanamine, HCl (550 mg, 3.05 mmol, 88% yield) as a tan solid, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (d, J=1.4 Hz, 1H), 8.68 (d, J=1.4 Hz, 1H), 8.65 (s, 3H), 4.26 (s, 2H).
A mixture of 4-(methoxycarbonyl)benzoic acid (605 mg, 3.36 mmol) and HATU (1394 mg, 3.67 mmol) in DMF (10.0 mL) was stirred for 10 minutes (min). (5-Chloropyrazin-2-yl)methanamine, HCl (550 mg, 3.05 mmol) was added and the mixture allowed to stir for 5 min, after which was added DIPEA (1.87 mL, 10.7 mmol) and the resulting reaction mixture stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with H2O and extracted twice with EtOAc. The combined organic layers where washed twice with brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 20-60% EtOAc in Hex to afford methyl 4-(((5-chloropyrazin-2-yl)methyl)carbamoyl)benzoate (897 mg, 2.93 mmol, 96% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.37 (t, J=5.7 Hz, 1H), 8.75 (d, J=1.4 Hz, 1H), 8.53 (d, J=1.4 Hz, 1H), 8.07-8.04 (m, 2H), 8.03-7.99 (m, 2H), 4.63 (d, J=5.7 Hz, 2H), 3.88 (s, 3H). LCMS RT (Method 2)=2.886 min, m/z 635.6 [2M+Na+].
A 1 molar (M) in DCM solution of triflicanhydride (3.52 mL, 3.52 mmol) was added slowly to a solution of methyl 4-(((5-chloropyrazin-2-yl)methyl)carbamoyl)benzoate (897 mg, 2.93 mmol) and 2-methoxypyridine (339 μL, 3.23 mmol) in DCE (10.0 mL). The resulting reaction mixture was then placed in a 45° C. preheated reaction block and allowed to stir for 2 h, after which LCMS analysis showed completion. Reaction mixture was allowed to cool to room temperature and quenched by addition of saturated sodium carbonate solution, stirred for 5 min, diluted with DCM and H2O, the layers separated and the organic phase washed with brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was triturated in EtOH with 10% hexanes, filtered, rinsed with hexanes and allowed to air dry to afford methyl 4-(6-chloroimidazo[1,5-a]pyrazin-3-yl)benzoate (671 mg, 2.33 mmol, 79% yield) as a light tan-colored solid, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (d, J=1.4 Hz, 1H), 8.66 (t, J=1.2 Hz, 1H), 8.17 (d, J=1.0 Hz, 1H), 8.14 (d, J=8.8 Hz, 2H), 8.11 (d, J=8.9 Hz, 2H), 3.91 (s, 3H). LCMS RT (Method 2)=3.071 min, m/z 287.8 [M+].
A mixture of methyl 4-(6-chloroimidazo[1,5-a]pyrazin-3-yl)benzoate (100 mg, 0.348 mmol), (3-fluorophenyl)boronic acid (58.4 mg, 0.417 mmol), XPhosPd(crotyl)Cl (11.71 mg, 0.017 mmol) and K3PO4 (148 mg, 0.695 mmol) was placed in a vial and purged with N2 for 2 min. 4:1 dioxane-H2O (2.50 mL) was added and degassing continued for 2 min, after which the reaction vessel was placed in a preheated block at 90° C. After stirring for 30 min at 90° C. LCMS analysis showed completion. Reaction mixture was allowed to cool to room temperature diluted with EtOAc and H2O, filtered through celite and the layers separated. The organic phase was washed with brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was triturated in EtOH with 10% hexanes, filtered, rinsed with hexanes and allowed to air dry to afford the intermediate methyl ester compound, which was taken up in 1:1 EtOH-THF (5.00 mL) and treated with 2M sodium hydroxide (2.00 mL, 4.00 mmol). The resulting reaction mixture was stirred at room temperature for 2 h, after which LCMS analysis showed completion. Reaction mixture was concentrated to a slurry, residue taken up in H2O and the pH adjusted with AcOH to ˜5 as product precipitated. Product was then collected by filtration, rinsed generously with H2O and allowed to air dry to afford 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (96.0 mg, 0.289 mmol, 83% yield) as an off-white solid, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 13.15 (s, 1H), 9.30 (d, J=1.6 Hz, 1H), 8.89-8.84 (m, 1H), 8.15 (s, 4H), 8.10 (d, J=0.9 Hz, 1H), 7.98-7.90 (m, 2H), 7.53 (td, J=8.2, 6.3 Hz, 1H), 7.30-7.20 (m, 1H). 19F NMR (376 MHz, DMSO-d6) δ −112.98 (td, J=9.9, 6.3 Hz). LCMS RT (Method 2)=3.144 min, m/z 334.8 [M+H+].
A solution of LiOH (125 mg, 5.21 mmol) in H2O (1.00 mL) was added to a solution of methyl 4-(6-chloroimidazo[1,5-a]pyrazin-3-yl)benzoate (300 mg, 1.043 mmol) in THF (4.00 mL). The resulting reaction mixture was stirred at room temperature for 1 h, after which LCMS analysis showed completion. Reaction mixture was concentrated to a slurry, residue taken up in H2O and the pH adjusted with AcOH to ˜5 as product precipitated. Product was then collected by filtration, rinsed generously with H2O and allowed to air dry to afford the intermediate acid, which was used without further purification.
A mixture of the intermediate 4-(6-chloroimidazo[1,5-a]pyrazin-3-yl)benzoic acid (203 mg, 0.742 mmol) and HATU (310 mg, 0.816 mmol) in DMF (5.00 mL) were stirred for 10 min, after which was added 1-(3-aminopropyl)pyrrolidin-2-one (105 mg, 0.742 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (259 μL, 1.48 mmol) and the reaction stirred for 2 h, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 0-30% MeOH in EtOAc to afford 4-(6-chloroimidazo[1,5-a]pyrazin-3-yl)-N-(3-(2-oxopyrrolidin-1-yl)propyl)benzamide (207 mg, 0.520 mmol, 70.1% yield) as an off-white solid. 1H NMR (400 MHz, Chloroform-d) δ 8.87 (d, J=1.4 Hz, 1H), 8.21 (t, J=1.2 Hz, 1H), 8.17 (d, J=8.4 Hz, 2H), 8.08 (s, 1H), 8.01 (d, J=1.0 Hz, 1H), 7.91 (d, J=8.4 Hz, 2H), 3.49-3.44 (m, 6H), 2.50 (t, J=8.1 Hz, 2H), 2.17-2.07 (m, 2H), 1.86-1.77 (m, 2H). LCMS RT (Method 2)=2.772 min, m/z 398.8 [M+H+].
A mixture of 4-(6-chloroimidazo[1,5-a]pyrazin-3-yl)-N-(3-(2-oxopyrrolidin-1-yl)propyl)benzamide (10.0 mg, 0.025 mmol), pyridin-3-ylboronic acid (3.71 mg, 0.030 mmol), XPhosPd(crotyl)Cl (0.847 mg, 1.26 μmol) and K3PO4 (10.7 mg, 0.050 mmol) were placed in a vial and purged with N2 for 2 min. 4:1 Dioxane-H2O (2.50 mL) was added and degassing continued for 2 min, after which reaction vessel was placed in a preheated block at 90° C. After stirring for 30 min at 90° C., LCMS analysis showed completion. Reaction mixture was allowed to cool to room temperature and loaded directly to a silica gel column and purified by flash column chromatography: silica gel with a gradient of 5-50% MeOH in EtOAc to afford N-(3-(2-oxopyrrolidin-1-yl)propyl)-4-(6-(pyridin-3-yl)imidazo[1,5-a]pyrazin-3-yl)benzamide (9.3 mg, 0.021 mmol, 84% yield) as an off-white crystalline solid. 1H NMR (400 MHz, Chloroform-d) δ 9.14 (d, J=1.6 Hz, 1H), 9.13 (dd, J=2.4, 0.9 Hz, 1H), 8.66 (dd, J=4.8, 1.6 Hz, 1H), 8.50 (dd, J=1.6, 1.0 Hz, 1H), 8.23 (ddd, J=8.0, 2.4, 1.7 Hz, 1H), 8.20-8.16 (m, 2H), 8.08 (t, J=6.3 Hz, 1H), 8.00 (d, J=0.9 Hz, 1H), 7.98-7.93 (m, 2H), 7.42 (ddd, J=8.0, 4.8, 0.9 Hz, 1H), 3.46 (tt, J=7.4, 2.7 Hz, 6H), 2.49 (dd, J=8.7, 7.6 Hz, 2H), 2.17-2.06 (m, 2H), 1.87-1.77 (m, 2H). LCMS RT (Method 1)=3.232 min, m/z 441.9 [M+H+].
A mixture of 4-(6-chloroimidazo[1,5-a]pyrazin-3-yl)-N-(3-(2-oxopyrrolidin-1-yl)propyl)benzamide (10.0 mg, 0.025 mmol), (3-(trifluoromethyl)phenyl)boronic acid (5.73 mg, 0.030 mmol), XPhosPd(crotyl)Cl (0.847 mg, 1.26 μmol) and K3PO4 (10.7 mg, 0.050 mmol) were placed in a vial and purged with N2 for 2 min. 4:1 dioxane-H2O (2.50 mL) was added and degassing continued for 2 min, after which reaction vessel was placed in a preheated block at 90° C. After stirring for 30 min at 90° C., LCMS analysis showed completion. Reaction mixture was allowed to cool to room temperature and loaded directly to a silica gel column and purified by flash column chromatography: silica gel with a gradient of 0-30% MeOH in EtOAc to afford N-(3-(2-oxopyrrolidin-1-yl)propyl)-4-(6-(3-(trifluoromethyl)phenyl)imidazo[1,5-a]pyrazin-3-yl)benzamide (10.2 mg, 0.020 mmol, 80% yield) as an off-white crystalline solid. 1H NMR (400 MHz, Chloroform-d) δ 9.14 (d, J=1.6 Hz, 1H), 8.50 (dd, J=1.7, 1.0 Hz, 1H), 8.23 (dd, J=2.0, 1.1 Hz, 1H), 8.21-8.17 (m, 2H), 8.10-8.02 (m, 2H), 7.99 (d, J=0.9 Hz, 1H), 7.98-7.94 (m, 2H), 7.70-7.65 (m, 1H), 7.59 (dt, J=7.8, 0.7 Hz, 1H), 3.46 (tt, J=7.5, 2.7 Hz, 6H), 2.53-2.44 (m, 2H), 2.17-2.06 (m, 2H), 1.83 (qd, J=7.7, 6.9, 5.1 Hz, 2H). 19F NMR (376 MHz, CDCl3) δ −62.60 (s, 3F). LCMS RT (Method 1)=5.058 min, m/z 508.8 [M+H+].
A mixture of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (50.0 mg, 0.159 mmol) and HATU (72.3 mg, 0.190 mmol) in DMF (2.00 mL) were stirred for 10 min, after which was added 2-morpholinoethan-1-amine (22.7 mg, 0.174 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (69.2 μL, 0.396 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was triturated in EtOH, filtered and air dried to afford N-(2-morpholinoethyl)-4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzamide (49.3 mg, 0.115 mmol, 72.7% yield) as a light golden solid. 1H NMR (400 MHz, Chloroform-d) δ 9.14 (d, J=1.6 Hz, 1H), 8.47 (dd, J=1.6, 1.0 Hz, 1H), 8.03-7.92 (m, 5H), 7.92-7.85 (m, 2H), 7.52-7.46 (m, 2H), 7.45-7.40 (m, 1H), 6.87 (s, 1H), 3.79-3.72 (m, 4H), 3.66-3.57 (m, 2H), 2.65 (t, J=6.0 Hz, 2H), 2.54 (t, J=4.6 Hz, 4H). LCMS RT (Method 1)=3.645 min, m/z 428.1 [M+H+].
A mixture of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (50.0 mg, 0.159 mmol) and HATU (72.3 mg, 0.190 mmol) in DMF (2.00 mL) were stirred for 10 min, after which was added N-(2-aminoethyl)acetamide (17.8 mg, 0.174 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (69.2 μL, 0.396 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 0-20% MeOH in EtOAc to afford N-(2-acetamidoethyl)-4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzamide (41.3 mg, 0.103 mmol, 65.2% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (d, J=1.5 Hz, 1H), 8.74 (dd, J=1.6, 1.0 Hz, 1H), 8.67 (t, J=5.6 Hz, 1H), 8.15-8.03 (m, 7H), 8.00 (t, J=5.9 Hz, 1H), 7.54-7.46 (m, 2H), 7.46-7.38 (m, 1H), 3.40-3.28 (m, 2H), 3.28-3.19 (m, 2H), 1.83 (s, 3H). LCMS RT (Method 1)=3.515 min, m/z 400.1 [M+H+].
A mixture of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (50.0 mg, 0.159 mmol) and HATU (72.3 mg, 0.190 mmol) in DMF (2.00 mL) were stirred for 10 min, after which was added thiomorpholine 1,1-dioxide (21.4 mg, 0.159 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (69.2 μL, 0.396 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was triturated in EtOH, filtered and air dried to afford (1,1-dioxidothiomorpholino)(4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)phenyl)methanone (54.6 mg, 0.126 mmol, 80% yield) as a light golden solid. 1H NMR (400 MHz, Chloroform-d) δ 9.15 (d, J=1.6 Hz, 1H), 8.44 (dd, J=1.6, 1.0 Hz, 1H), 8.01-7.96 (m, 3H), 7.92-7.86 (m, 2H), 7.69-7.64 (m, 2H), 7.53-7.47 (m, 2H), 7.46-7.41 (m, 1H), 4.16 (s, 4H), 3.11 (s, 4H). LCMS RT (Method 1)=3.845 min, m/z 433.1 [M+H+].
A mixture of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (50.0 mg, 0.159 mmol) and HATU (72.3 mg, 0.190 mmol) in DMF (2.00 mL) were stirred for 10 min, after which was added 3-aminopropan-1-ol (13.1 mg, 0.174 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (69.2 μL, 0.396 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 0-20% MeOH in EtOAc to afford N-(3-hydroxypropyl)-4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzamide (41.3 mg, 0.111 mmol, 69.9% yield) as an off-white foam. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (d, J=1.5 Hz, 1H), 8.74 (dd, J=1.6, 1.0 Hz, 1H), 8.60 (t, J=5.6 Hz, 1H), 8.15-8.00 (m, 7H), 7.54-7.45 (m, 2H), 7.45-7.38 (m, 1H), 4.49 (t, J=5.2 Hz, 1H), 3.49 (td, J=6.3, 5.2 Hz, 2H), 3.36 (q, J=6.6 Hz, 2H), 1.71 (dq, J=7.6, 6.4 Hz, 2H). LCMS RT (Method 1)=3.920 min, m/z 373.1 [M+H+].
A mixture of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (50.0 mg, 0.159 mmol) and HATU (72.3 mg, 0.190 mmol) in DMF (2.00 mL) were stirred for 10 min, after which was added 7 Normal (N) ammonia in MeOH (0.200 mL, 1.40 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (0.069 mL, 0.396 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was triturated in EtOH, filtered and air dried to afford 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzamide (25.0 mg, 0.080 mmol, 50.2% yield) as a light yellow-golden solid. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (d, J=1.5 Hz, 1H), 8.75 (dd, J=1.6, 0.9 Hz, 1H), 8.15-8.03 (m, 8H), 7.49 (tq, J=6.2, 1.4 Hz, 3H), 7.45-7.39 (m, 1H). LCMS RT (Method 1)=4.038 min, m/z 651.7 [2M+Na+], 315.9 [M+H+].
Hydrazine (0.211 ml, 6.71 mmol) was added to a solution of 2,5-dichloropyrazine (1.00 g, 6.71 mmol) in EtOH (20.0 mL). The resulting reaction mixture was stirred at 80° C. for 2 h, after which LC-MS analysis showed completion. Reaction mixture was allowed to cool to room temperature and product precipitated. Mixture was poured over ice H2O, stirred vigorously for 5 min, filtered, rinsed with H2O and allowed to air dry to afford 2-chloro-5-hydrazinylpyrazine (885 mg, 6.12 mmol, 91% yield) as a white powder, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.04 (s, 1H), 7.93 (s, 1H), 4.32 (s, 2H).
To a solution of 2-chloro-5-hydrazinylpyrazine (260 mg, 1.80 mmol), 4-(methoxycarbonyl)benzoic acid (405 mg, 2.25 mmol) and DIPEA (0.942 mL, 5.40 mmol) in DMF (5.00 mL) was added a 50% solution of propylphosphonic anhydride (T3P) in DMF (1.58 mL, 2.70 mmol). The resulting reaction mixture was allowed to stir at room temperature for 1 h, after which LC-MS analysis showed completion. Reaction mixture was poured over ice H2O, stirred for 10 min, product collected by filtration, rinsed generously with H2O and allowed to air dry to afford methyl 4-(2-(5-chloropyrazin-2-yl)hydrazine-1-carbonyl)benzoate as a light yellow solid, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 9.33 (s, 1H), 8.21 (d, J=1.4 Hz, 1H), 8.12-8.06 (m, 2H), 8.06-7.99 (m, 2H), 7.94 (d, J=1.4 Hz, 1H), 3.90 (s, 3H). LCMS RT (Method 2)=2.784 min, m/z 306.8 [M+].
Perchloroethane (232 mg, 0.978 mmol) was added to a suspension of methyl 4-(2-(5-chloropyrazin-2-yl)hydrazine-1-carbonyl)benzoate (150 mg, 0.489 mmol), triphenylphosphine (257 mg, 0.978 mmol) and DIPEA (0.342 mL, 1.96 mmol) in ACN (5.00 mL) with 4 Å molecular sieves (MS). The resulting reaction mixture was stirred at 80° C. for 2 h, after which LCMS analysis showed completion. Reaction mixture was cooled to room temperature, filtered through celite and the filter cake rinsed generously with EtOAc. The filtrated was concentrated under reduced pressure and residue purified by flash column chromatography: silica gel with a gradient of 20-60% EtOAc in Hex to afford methyl 4-(6-chloro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)benzoate (105 mg, 0.364 mmol, 74.4% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.46 (d, J=1.5 Hz, 1H), 8.94 (d, J=1.5 Hz, 1H), 8.19 (d, J=2.5 Hz, 2H), 8.17 (d, J=2.7 Hz, 2H), 3.93 (s, 3H). LCMS RT (Method 2)=2.972 min, m/z 600.6 [2M+Na+], 289.9 [M+].
A mixture of methyl 4-(6-chloro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)benzoate (40.0 mg, 0.139 mmol), phenylboronic acid (21.1 mg, 0.173 mmol), XPhosPd(crotyl)Cl (4.67 mg, 6.93 μmol) and K3PO4 (58.8 mg, 0.277 mmol) were placed in a vial and purged with N2 for 2 min. 4:1 Dioxane:H2O (2.50 mL) was added and degassing continued for 2 min, after which reaction vessel was placed in a preheated block at 100° C. After stirring for 30 min at 100° C. LCMS analysis showed completion. Reaction mixture was allowed to cool to room temperature, partitioned between brine and EtOAc, filtered through celite and the layers separated. The organic phase was washed with brined, dried over anhydrous MgSO4, filtered and concentrated to afford crude methyl 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)benzoate (38.0 mg, 0.115 mmol, 83% yield), which was used without further purification. LCMS RT (Method 2)=3.319 min, m/z 683.7 [2M+Na+].
2M sodium hydroxide (1.00 mL, 2.00 mmol) was added to a solution of methyl 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)benzoate (46.0 mg, 0.139 mmol) in EtOH (5.00 mL). The resulting reaction mixture was stirred at room temperature for 1 h, after which LCMS analysis showed completion. Reaction mixture was concentrated to a slurry and residue partitioned between 1M HCl and EtOAc, the layers separated and the organic phase washed with brine, dried over anhydrous MgSO4, filtered and concentrated to afford crude 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)benzoic acid (44.0 mg, 0.139 mmol, 100% yield), which was used without further purification. LCMS RT (Method 2)=2.893 min, m/z 317.0 [M+H+].
A mixture of 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)benzoic acid (44.0 mg, 0.139 mmol) and HATU (63.5 mg, 0.167 mmol) in DMF (1.50 mL) were stirred for 10 min, after which was added 1-(3-aminopropyl)pyrrolidin-2-one (21.5 μL, 0.153 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (60.7 μL, 0.348 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 0-20% MeOH in EtOAc to afford N-(3-(2-oxopyrrolidin-1-yl)propyl)-4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)benzamide (24.0 mg, 0.054 mmol, 39.2% yield) as a faint yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.63 (d, J=1.6 Hz, 1H), 8.92 (d, J=1.6 Hz, 1H), 8.68 (t, J=5.7 Hz, 1H), 8.20-8.15 (m, 2H), 8.14-8.09 (m, 4H), 7.56-7.50 (m, 2H), 7.49-7.43 (m, 1H), 3.37 (t, J=7.1 Hz, 2H), 3.32-3.23 (m, 4H), 2.24 (dd, J=8.3, 7.8 Hz, 2H), 1.98-1.89 (m, 2H), 1.75 (p, J=7.0 Hz, 2H). LCMS RT (Method 1)=4.130 min, m/z 882.3 [2M+H+], 441.1 [M+H+].
A solution of 5-bromo-2-fluoropyridine (1.00 mL, 9.72 mmol) and hydrazine (1.52 mL, 48.6 mmol) in EtOH (10.0 mL) was stirred at 100° C. for 1 h, after which LCMS analysis showed completion. Reaction volume was reduced to half and mixture allowed to cool to room temperature as product precipitated. Slurry was poured into ice H2O and stirred for 5 min, product was filtered and rinsed with H2O and allowed to air dry to afford 5-bromo-2-hydrazinylpyridine (1.60 g, 8.51 mmol, 88% yield) as an off-white fluffy off-white solid which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 8.02 (dd, J=2.6, 0.7 Hz, 1H), 7.65 (s, 1H), 7.58 (dd, J=8.9, 2.5 Hz, 1H), 6.69 (dd, J=9.0, 0.7 Hz, 1H), 4.15 (s, 2H). LCMS RT (Method 2)=1.150 min, m/z 189.3 [M+H+].
To a solution of 5-bromo-2-hydrazinylpyridine (500 mg, 2.66 mmol), 4-(methoxycarbonyl)benzoic acid (599 mg, 3.32 mmol) and DIPEA (1.39 mL, 7.98 mmol) in DMF (5.00 mL) was added a 50% solution of propylphosphonic anhydride (T3P) in DMF (2.33 mL, 3.99 mmol). The resulting reaction mixture was allowed to stir at room temperature for 1 h, after which LCMS analysis showed completion. Reaction mixture was poured over ice H2O, stirred for 10 min, product collected by filtration, rinsed generously with H2O and allowed to air dry to afford methyl 4-(2-(5-bromopyridin-2-yl)hydrazine-1-carbonyl)benzoate (906 mg, 2.59 mmol, 97% yield) as a tan solid, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 10.61 (d, J=1.9 Hz, 1H), 8.80 (d, J=1.9 Hz, 1H), 8.15 (dd, J=2.5, 0.7 Hz, 1H), 8.10-8.05 (m, 2H), 8.05-8.00 (m, 2H), 7.71 (dd, J=8.9, 2.5 Hz, 1H), 6.66 (dd, J=8.9, 0.7 Hz, 1H), 3.89 (d, J=2.5 Hz, 3H). LCMS RT (Method 2)=2.863 min, m/z 352.3 [M+H+].
Perchloroethane (946 mg, 4.00 mmol) was added to a suspension of methyl 4-(2-(5-bromopyridin-2-yl)hydrazine-1-carbonyl)benzoate (700 mg, 1.99 mmol), triphenylphosphine (1.05 g, 4.00 mmol) and DIPEA (1.39 mL, 8.00 mmol) in ACN (10.0 mL) with 4 Å MS. The resulting reaction mixture was stirred at 80° C. for 2 h, after which LCMS analysis showed completion. Reaction mixture was cooled to room temperature, filtered through celite and the filter cake rinsed generously with EtOAc. The filtrated was concentrated under reduced pressure and residue purified by flash column chromatography: silica gel with a gradient of 20-80% EtOAc in Hex to afford methyl 4-(6-bromo-[1,2,4]triazolo[4,3-a]pyridin-3-yl)benzoate (604 mg, 1.818 mmol, 91% yield). LCMS RT (Method 2)=3.004 min, m/z 333.7 [M+H+].
A mixture of methyl 4-(6-bromo-[1,2,4]triazolo[4,3-a]pyridin-3-yl)benzoate (300 mg, 0.903 mmol), phenylboronic acid (138 mg, 1.13 mmol), XPhos Pd(crotyl)Cl (30.4 mg, 0.045 mmol) and K3PO4 (383 mg, 1.81 mmol) were placed in a vial and purged with N2 for 2 min. 4:1 Dioxane:H2O (10.0 mL) was added and degassing continued for 2 min, after which reaction vessel was placed in a preheated block at 100° C. After stirring for 30 min at 100° C. LC-MS analysis showed completion. Reaction mixture was allowed to cool to room temperature, partitioned between brine and EtOAc, filtered through celite and the layers separated. The organic phase was washed with brined, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 40-100% EtOAc in Hex to afford methyl 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl)benzoate (290 mg, 0.880 mmol, 97% yield) as an off-white solid. LCMS RT (Method 2)=3.156 min, m/z 330.1 [M+H+].
A suspension of methyl 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl)benzoate (290 mg, 0.880 mmol) in EtOH (8.00 mL) was treated with 2M sodium hydroxide (2.00 mL, 4.00 mmol). The resulting reaction mixture was allowed to stir at room temperature for 30 min, after which solution became clear and LCMS analysis showed completion. Reaction mixture was concentrated to a slurry and poured into cold 1M HCl solution and stirred vigorously for 10 min. Insoluble product was filtered, rinsed with H2O and air dried to afford 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl)benzoic acid (248 mg, 0.786 mmol, 89% yield) as an off-white solid, which was used without further purification. LCMS RT (Method 2)=2.956 min, m/z 316.8 [M+H+].
A mixture of 4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl)benzoic acid (100 mg, 0.317 mmol) and HATU (145 mg, 0.381 mmol) in DMF (2.00 mL) were stirred for 10 min, after which was added 1-(3-aminopropyl)pyrrolidin-2-one (0.049 mL, 0.349 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (0.138 mL, 0.793 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 0-30% MeOH in EtOAc to afford N-(3-(2-oxopyrrolidin-1-yl)propyl)-4-(6-phenyl-[1,2,4]triazolo[4,3-a]pyridin-3-yl)benzamide (33.0 mg, 0.075 mmol, 23.68% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.68-8.61 (m, 2H), 8.14-8.10 (m, 2H), 8.08 (d, J=8.8 Hz, 2H), 7.99 (dd, J=9.6, 1.0 Hz, 1H), 7.85-7.74 (m, 3H), 7.54-7.48 (m, 2H), 7.47-7.41 (m, 1H), 3.40-3.34 (m, 2H), 3.27 (q, J=6.9 Hz, 4H), 2.23 (dd, J=8.6, 7.5 Hz, 2H), 2.01-1.87 (m, 2H), 1.74 (p, J=7.0 Hz, 2H). LCMS RT (Method 1)=4.071 min, m/z 440.1 [M+H+].
A mixture of 2-chloro-5-nitropyridin-4-amine (200 mg, 1.152 mmol), methyl 4-iodobenzoate (302 mg, 1.152 mmol), copper(I) iodide (32.9 mg, 0.173 mmol) and cesium carbonate (563 mg, 1.73 mmol) were placed in a vial, sealed and purged with N2 for 3 min. DMF (4.00 mL) was added and the reaction mixture purged by bubbling N2 through the mixture for 3 min. The resulting reaction mixture was placed in a preheated reaction block at 120° C. and stirred for 16 h, after which LCMS analysis showed product formation. Reaction mixture was partitioned between EtOAc and H2O, filtered through celite, the layers separated, and the organic phase washed with brine, dried over anhydrous MgSO4, filtered and concentrated. Crude residue was purified by flash column chromatography: silica gel with a gradient of 10-30% EtOAc in Hex to afford methyl 4-((2-chloro-5-nitropyridin-4-yl)amino)benzoate (84.0 mg, 0.273 mmol, 23.69% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.99 (s, 1H), 8.08-8.00 (m, 2H), 7.57-7.49 (m, 2H), 7.03 (s, 1H), 3.87 (s, 3H). LCMS RT (Method 2)=3.334 min, m/z 308.0 [M+H+].
A mixture of methyl 4-((2-chloro-5-nitropyridin-4-yl)amino)benzoate (80.0 mg, 0.260 mmol), iron powder (72.6 mg, 1.30 mmol) and ammonium chloride (278 mg, 5.20 mmol) in 1:1 EtOH—H2O (10.0 mL) was stirred at 70° C. for 1 h, after which LCMS analysis showed completion. Reaction mixture was allowed to cool to room temperature and partition between brine and EtOAc, filtered through celite and the layers separated. The organic phase was washed with brine, dried over anhydrous MgSO4, filtered and concentrated to afford crude methyl 4-((5-amino-2-chloropyridin-4-yl)amino)benzoate (70.0 mg, 0.252 mmol, 97% yield) as a tan solid, which was used without further purification. LCMS RT (Method 2)=2.573 min, m/z 278.0 [M+H+].
A solution of methyl 4-((5-amino-2-chloropyridin-4-yl)amino)benzoate (65.0 mg, 0.234 mmol), triethyl orthoformate (0.100 mL, 0.601 mmol) and catalytic p-toluenesulfonic acid (p-TsOH) (6.68 mg, 0.035 mmol) in THF (5.00 mL) was stirred at 60° C. overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc and washed with saturated NaHCO3, brine, dried over anhydrous MgSO4, filtered and concentrated. Crude residue was purified by flash column chromatography: silica gel with a gradient of 20-80% EtOAc in HEX to afford methyl 4-(6-chloro-1H-imidazo[4,5-c]pyridin-1-yl)benzoate (41.0 mg, 0.143 mmol, 60.9% yield) as a white powder. 1H NMR (400 MHz, Chloroform-d) δ 8.97 (d, J=0.9 Hz, 1H), 8.34-8.28 (m, 2H), 8.22 (s, 1H), 7.61-7.58 (m, 2H), 7.54 (d, J=0.9 Hz, 1H), 4.00 (s, 3H). LCMS RT (Method 2)=3.034 min, m/z 287.8 [M+].
A mixture of methyl 4-(6-chloro-1H-imidazo[4,5-c]pyridin-1-yl)benzoate (35.0 mg, 0.122 mmol), phenylboronic acid (18.54 mg, 0.152 mmol), XPhos Pd(crotyl)Cl (4.10 mg, 6.08 μmol) and K3PO4 (51.6 mg, 0.243 mmol) were placed in a vial and purged with N2 for 2 min. 4:1 Dioxane:H2O (2.50 mL) was added and degassing continued for 2 min, after which reaction vessel was placed in a preheated block at 100° C. After stirring for 30 min at 100° C. LCMS analysis showed completion. Reaction mixture was then treated with 2M sodium hydroxide (0.500 mL, 1.00 mmol) and stirring continued at 100° C. for 30 min, after which LCMS analysis showed complete saponification of ester. Reaction mixture was allowed to cool to room temperature and diluted with EtOAc and H2O. pH was adjusted to ˜4-5 with AcOH and biphasic mixture filtered through celite, the layers separated and the organic phase washed with brine, dried over anhydrous MgSO4, filtered and concentrated to afford crude 4-(6-phenyl-1H-imidazo[4,5-c]pyridin-1-yl)benzoic acid (35.0 mg, 0.111 mmol, 91% yield), which was used without further purification. LCMS RT (Method 2)=2.601 min, m/z 315.8 [M+].
A mixture of 4-(6-phenyl-1H-imidazo[4,5-c]pyridin-1-yl)benzoic acid (40.0 mg, 0.127 mmol) and HATU (57.9 mg, 0.152 mmol) in DMF (1.50 mL) were stirred for 10 min, after which was added 1-(3-aminopropyl)pyrrolidin-2-one (19.57 μL, 0.140 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (55.4 μL, 0.317 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 0-20% MeOH in EtOAc to afford N-(3-(2-oxopyrrolidin-1-yl)propyl)-4-(6-phenyl-1H-imidazo[4,5-c]pyridin-1-yl)benzamide (32.0 mg, 0.073 mmol, 57.4% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.17 (d, J=1.0 Hz, 1H), 8.82 (s, 1H), 8.65 (t, J=5.7 Hz, 1H), 8.19-8.08 (m, 5H), 7.97-7.88 (m, 2H), 7.51-7.44 (m, 2H), 7.43-7.37 (m, 1H), 3.37 (t, J=7.0 Hz, 2H), 3.28 (dt, J=15.9, 6.9 Hz, 4H), 2.24 (dd, J=8.6, 7.4 Hz, 2H), 1.94 (ddd, J=15.4, 13.1, 6.4 Hz, 2H), 1.75 (p, J=7.1 Hz, 2H). LCMS RT (Method 1)=3.523 min, m/z 440.8 [M+H+].
A mixture of 6-chloro-1H-pyrrolo[3,2-c]pyridine (200 mg, 1.31 mmol), methyl 4-iodobenzoate (343 mg, 1.31 mmol), copper(I) iodide (37.4 mg, 0.197 mmol) and cesium carbonate (641 mg, 1.97 mmol) were placed in a vial, sealed and purged with N2 for 3 min. DMF (4.00 mL) was added and the reaction mixture purged by bubbling N2 through the mixture for 3 min. The resulting reaction mixture was placed in a preheated reaction block at 120° C. and stirred for 16 h, after which LCMS analysis showed product formation. Reaction mixture was partitioned between EtOAc and H2O, filtered through celite, the layers separated, and the organic phase washed with brine, dried over anhydrous MgSO4, filtered and concentrated. Crude residue was purified by flash column chromatography: silica gel with a gradient of 5-35% EtOAc in Hex to afford methyl 4-(6-chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)benzoate (213 mg, 0.743 mmol, 56.7% yield). LCMS RT (Method 2)=3.247 min, m/z 287.0 [M+H+].
A mixture of methyl 4-(6-chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)benzoate (100 mg, 0.349 mmol), phenylboronic acid (53.2 mg, 0.436 mmol), XPhosPd(crotyl)Cl (11.75 mg, 0.017 mmol) and K3PO4 (148 mg, 0.698 mmol) were placed in a vial and purged with N2 for 2 min. 4:1 Dioxane:H2O (2.50 mL) was added and degassing continued for 2 min, after which reaction vessel was placed in a preheated block at 100° C. After stirring for 30 min at 100° C. LCMS analysis showed completion. Reaction mixture was allowed to cool to room temperature, partitioned between brine and EtOAc, filtered through celite and the layers separated. The organic phase was washed with brined, dried over anhydrous MgSO4, filtered and concentrated. Crude product was purified by flash column chromatography: silica gel with a gradient of 10-35% EtOAc in Hex to afford methyl 4-(6-phenyl-1H-pyrrolo[3,2-c]pyridin-1-yl)benzoate (110 mg, 0.335 mmol, 96% yield). LCMS RT (Method 2)=2.795 min, m/z 329.1 [M+H+].
2M sodium hydroxide (2.00 mL, 4.00 mmol) was added to a solution of methyl 4-(6-phenyl-1H-pyrrolo[3,2-c]pyridin-1-yl)benzoate (100 mg, 0.305 mmol) in EtOH (5.00 mL). The resulting reaction mixture was stirred at room temperature for 2 h, after which LCMS analysis showed completion. Reaction mixture was concentrated to a slurry and residue partitioned between 1M HCl and EtOAc, the layers separated and the organic phase washed with brine, dried over anhydrous MgSO4, filtered and concentrated to afford crude 4-(6-phenyl-1H-pyrrolo[3,2-c]pyridin-1-yl)benzoic acid (55.0 mg, 0.175 mmol, 57.5% yield), which was used without further purification. LCMS RT (Method 2)=2.664 min, m/z 314.9 [M+].
A mixture of 4-(6-phenyl-1H-pyrrolo[3,2-c]pyridin-1-yl)benzoic acid (25.0 mg, 0.080 mmol) and HATU (36.3 mg, 0.095 mmol) in DMF (1.50 mL) were stirred for 10 min, after which was added 1-(3-aminopropyl)pyrrolidin-2-one (12.3 μL, 0.087 mmol). The resulting reaction mixture was stirred for 20 min, after which was added DIPEA (34.7 μL, 0.199 mmol) and the reaction stirred overnight, after which LCMS analysis showed completion. Reaction mixture was diluted with EtOAc, washed with H2O and brine, dried over anhydrous MgSO4, filtered and concentrated. Residue was purified by flash column chromatography: silica gel with a gradient of 0-20% MeOH in EtOAc to afford N-(3-(2-oxopyrrolidin-1-yl)propyl)-4-(6-phenyl-1H-pyrrolo[3,2-c]pyridin-1-yl)benzamide (17.0 mg, 0.039 mmol, 48.7% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.62 (t, J=5.6 Hz, 1H), 8.10 (t, J=7.4 Hz, 4H), 8.02 (s, 1H), 7.88 (d, J=3.4 Hz, 1H), 7.83 (d, J=8.4 Hz, 2H), 7.46 (t, J=7.5 Hz, 2H), 7.37 (t, J=7.2 Hz, 1H), 6.93 (d, J=3.3 Hz, 1H), 3.37 (t, J=7.0 Hz, 2H), 3.27 (q, J=7.1, 6.6 Hz, 4H), 2.23 (t, J=8.1 Hz, 2H), 1.93 (p, J=7.5 Hz, 2H), 1.74 (p, J=7.1 Hz, 2H). LCMS RT (Method 1)=3.521 min, m/z 439.1 [M+H+].
A heterogeneous solution consisting of (5-phenylpyrazin-2-yl)methanamine (Key Organics) (3.3 g, 17.82 mmol), 4-(methoxycarbonyl)benzoic acid (3.53 g, 19.60 mmol), HOBt (3.55 g, 23.16 mmol), DIPEA (9.33 ml, 53.4 mmol) in DMF (100 ml) was stirred at 65° C. under N2 for 1 minute. To the solution was added EDC (4.10 g, 21.38 mmol). The solution was stirred at 65° C. under N2 for 2.5 hrs. The solution was cooled to room temperature. To the solution was added water (500 ml). The solution was cooled for 18 hrs. The solution was filtered. The solid was washed with water (3×), dried in air then in vacuo to give the desired compound (5.4 g, 87%). (LCMS, ESI pos.) Calculated for C20H17N3O3: 348.4 (M+H), Measured: 348.1. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (t, J=5.7 Hz, 1H), 9.23 (d, J=1.5 Hz, 1H), 8.76 (d, J=1.5 Hz, 1H), 8.19-8.14 (m, 2H), 8.13-8.05 (m, 4H), 7.61-7.50 (m, 3H), 4.72 (d, J=5.7 Hz, 2H), 3.93 (s, 3H).
A heterogeneous solution of methyl 4-(((5-phenylpyrazin-2-yl)methyl)carbamoyl)benzoate (2.5 g, 7.20 mmol) and pyridine (3.49 ml, 43.2 mmol) in DCE (72.0 ml) was treated dropwise with POCl3 (2.68 ml, 28.8 mmol) over 1 min. The heterogeneous solution was stirred at 70° C. under N2. The solution was stirred at 70° C. for 5 h. The reaction was cooled to room temperature. The solution was cooled (ice bath). To the solution was added slowly MeOH (10 ml). The solution was concentrated to a small volume that was chromatographed using gradient silica gel chromatography (5% EtOAc in hexanes to 100% EtOAc over 20 min). Desired fractions were pooled, concentrated and dried in vacuo to give desired compound (1.8 g, 76%). (LCMS, ESI pos.) Calculated for C20H15N3O2: 330.4 (M+H), Measured: 330.1. 1H NMR (400 MHz, DMSO-d6) δ 9.34 (d, J=1.5 Hz, 1H), 8.82 (t, J=1.3 Hz, 1H), 8.21 (d, J=1.1 Hz, 4H), 8.15-8.08 (m, 3H), 7.59-7.49 (m, 2H), 7.49-7.42 (m, 1H), 3.95 (d, J=1.2 Hz, 3H).
To a solution of methyl 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoate (1.8 g, 5.47 mmol) in MeOH/THF 1:1 (40 ml) was added sodium hydroxide (10.93 ml, 10.93 mmol). The solution was stirred at room temperature under N2. After 3 h the reaction solution was concentrated to a small volume. The solution was cooled using an ice/water bath. The pH was adjusted to 2 (litmus) using 1N HCl (slow addition). The solution was placed in the refrigerator overnight. The solution was filtered. The solid was washed with water (3×). The solid was dried in air then in vacuo to give desired product (1.0 g, 58%). (LCMS, ESI pos.) Calculated for C19H13N3O2: 316.3 (M+H), Measured: 316.1. 1H NMR (400 MHz, DMSO-d6) δ 13.23 (s, 1H), 9.27 (s, 1H), 8.75 (s, 1H), 8.07 (m, J=21.4 Hz, 7H), 7.43 (m, J=23.8 Hz, 3H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (1 g, 3.17 mmol) in DMF (10.57 ml) was treated with DIPEA (1.108 ml, 6.34 mmol). To the solution was added HATU (1.326 g, 3.49 mmol). The solution was stirred at room temperature under N2. After 30 min histamine (0.388 g, 3.49 mmol) was added to the solution. The reaction solution was stirred at room temperature under N2 for 18 hrs. To the reaction solution was added 1N NaOH (1.9 mmol). After 30 minutes the solution was concentrated to a small volume. The solution was partitioned between EtOAc and water. The EtOAc layer was separated and washed successively with water (2×), brine (1×), dried over anhydrous MgSO4, filtered and concentrated. The residue was chromatographed using C18 reversed-phase chromatography to give the desired compound (0.7 g, 54%). (LCMS, ESI pos.) Calculated for C24H20N6O: 409.5 (M+H), Measured: 409.2. 1H NMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 9.33 (d, J=1.5 Hz, 1H), 8.84-8.71 (m, 2H), 8.22-8.03 (m, 7H), 7.69-7.40 (m, 4H), 6.90 (s, 1H), 3.57 (td, J=7.5, 5.5 Hz, 2H), 2.83 (s, 2H).
As shown in
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.255 g, 0.809 mmol) and HATU (0.369 g, 0.970 mmol) in DMF (2.70 ml) was treated with DIPEA (0.282 ml, 1.617 mmol). The solution was stirred at room temperature under N2. After 20 min a solution of 1-(3-aminopropyl)pyrrolidin-2-one (0.126 g, 0.890 mmol) in DMF (0.1 ml) was added to the solution. The reaction solution was stirred at room temperature under N2. After 18 h the reaction solution was introduced into a C18 column (15.5 g, equilibrated with water) and purified using a gradient (0-30% CH3CN over 20 min) to give the desired compound (0.142 g, 40%). (LCMS, ESI pos.) Calculated for C26H25N5O2: 440.5 (M+H), Measured: 440.2. 1H NMR (400 MHz, DMSO-d6) δ 9.34 (d, J=1.5 Hz, 1H), 8.79 (t, J=1.3 Hz, 1H), 8.66 (t, J=5.7 Hz, 1H), 8.19-8.05 (m, 7H), 7.53 (dd, J=8.3, 6.6 Hz, 2H), 7.49-7.43 (m, 1H), 3.41 (t, J=7.0 Hz, 2H), 3.32 (dt, J=14.0, 6.9 Hz, 4H), 2.27 (t, J=8.1 Hz, 2H), 2.05-1.91 (m, 2H), 1.78 (p, J=7.1 Hz, 2H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.317 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). After 10 min azetidin-3-ol (0.012 g, 0.159 mmol) was added to the solution. The solution was stirred at room temperature overnight. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.04 g, 68%). (LCMS, ESI pos.) Calculated for C22H18N4O2: 371.4 (M+H), Measured: 371.2. 1H NMR (400 MHz, DMSO-d6) δ 9.33 (d, J=1.5 Hz, 1H), 8.81-8.76 (m, 1H), 8.57 (dd, J=8.4, 1.4 Hz), 8.16-8.06 (m, 4H), 7.91-7.84 (m, 2H), 7.57-7.49 (m, 2H), 7.49-7.43 (m, 1H), 5.83 (s, 1H), 4.57 (d, J=5.1 Hz, 2H), 4.33 (s, 1H), 4.16 (s, 1H), 3.94-3.84 (m, 1H), 1.29 (td, J=7.1, 5.1 Hz, 3H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). The solution was stirred at room temperature for 10 min. To the solution was added piperidin-4-ol (0.016 g, 0.159 mmol). The solution was stirred at room temperature overnight. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.03 g, 48%). (LCMS, ESI pos.) Calculated for C24H22N4O2: 399.5 (M+H), Measured: 399.2. 1H NMR (400 MHz, DMSO-d6) δ 9.32 (d, J=1.5 Hz, 1H), 8.80 (t, J=1.2 Hz, 1H), 8.16-8.05 (m, 5H), 7.70-7.58 (m, 2H), 7.58-7.42 (m, 3H), 4.86 (s, 1H), 3.86-3.76 (m, 1H), 3.63 (s, 1H), 3.28 (s, 3H), 1.82 (s, 2H), 1.44 (s, 3H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). After 10 min N1,N1,N2-trimethylethane-1,2-diamine (0.021 ml, 0.159 mmol) was added. The solution was stirred at room temperature overnight. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.03 g, 47%). (LCMS, ESI pos.) Calculated for C24H25N5O: 400.5 (M+H), Measured: 400.2. 1H NMR (400 MHz, DMSO-d6) δ 9.10 (d, J=1.5 Hz, 1H), 8.41 (t, J=1.2 Hz, 1H), 7.93 (d, J=1.0 Hz, 1H), 7.87 (td, J=6.1, 2.8 Hz, 4H), 7.66-7.59 (m, 2H), 7.52-7.44 (m, 2H), 7.43-7.37 (m, 1H), 3.68 (s, 1H), 3.40 (d, J=10.2 Hz, 1H), 3.09 (d, J=29.0 Hz, 3H), 2.67-2.38 (m, 2H), 2.32 (s, 3H), 2.10 (s, 3H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). After 10 minutes N-(4-aminophenyl)acetamide (0.024 g, 0.159 mmol) was added to the solution. The solution was stirred at room temperature. After 18 h the solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.04 g, 56%). (LCMS, ESI pos.) Calculated for C27H21N5O2: 448.5 (M+H), Measured: 448.2. 1H NMR (400 MHz, Chloroform-d) δ 9.10 (d, J=1.5 Hz, 1H), 8.41 (t, J=1.2 Hz, 1H), 7.93 (d, J=1.0 Hz, 1H), 7.87 (td, J=6.1, 2.8 Hz, 4H), 7.66-7.59 (m, 2H), 7.52-7.44 (m, 2H), 7.43-7.37 (m, 1H), 3.68 (s, 1H), 3.40 (d, J=10.2 Hz, 1H), 3.09 (d, J=29.0 Hz, 3H), 2.67-2.38 (m, 2H), 2.32 (s, 3H), 2.10 (s, 3H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). The solution was stirred for 15 minutes. To the solution was added 3-(1H-imidazol-1-yl)propan-1-amine (0.020 g, 0.159 mmol). The solution was stirred at room temperature. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.03 g, 45%). (LCMS, ESI pos.) Calculated for C25H22N6O: 423.5 (M+H), Measured: 423.1. 1H NMR (400 MHz, Chloroform-d) δ 9.11 (d, J=1.6 Hz, 1H), 8.43 (dd, J=1.6, 0.9 Hz, 1H), 7.93 (s, 5H), 7.89-7.83 (m, 2H), 7.51 (t, J=1.1 Hz, 1H), 7.50-7.43 (m, 2H), 7.43-7.37 (m, 1H), 7.06 (d, J=1.1 Hz, 1H), 6.98 (t, J=1.3 Hz, 1H), 6.60-6.49 (m, 1H), 4.09 (dt, J=11.4, 7.0 Hz, 2H), 3.51 (q, J=6.5 Hz, 2H), 2.15 (p, J=6.8 Hz, 2H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). To the solution was added N1,N1-dimethylethane-1,2-diamine (0.017 ml, 0.159 mmol). The solution was stirred at room temperature overnight. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.045 g, 74%). (LCMS, ESI pos.) Calculated for C23H23N5O: 386.5 (M+H), Measured: 385.9. 1H NMR (400 MHz, Chloroform-d) δ 9.11 (d, J=1.6 Hz, 1H), 8.44 (dd, J=1.7, 0.9 Hz, 1H), 8.00 (d, J=8.4 Hz, 2H), 7.96-7.91 (m, 3H), 7.90-7.85 (m, 2H), 7.50-7.44 (m, 2H), 7.44-7.37 (m, 1H), 6.96 (s, 1H), 3.61-3.51 (m, 2H), 2.54 (t, J=5.9 Hz, 2H), 2.28 (s, 6H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). To the solution was added pyrazin-2-ylmethanamine (0.017 g, 0.159 mmol). The solution was stirred at room temperature for 18 h. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.045 g, 74%). (LCMS, ESI pos.) Calculated for C24H18N6O: 407.5 (M+H), Measured: 407.2. 1H NMR (400 MHz, Chloroform-d) δ 9.10 (d, J=1.6 Hz, 1H), 8.68 (d, J=1.5 Hz, 1H), 8.56-8.48 (m, 2H), 8.42 (t, J=1.3 Hz, 1H), 8.08-8.01 (m, 2H), 7.96-7.90 (m, 3H), 7.88-7.81 (m, 2H), 7.53 (t, J=5.3 Hz, 1H), 7.48-7.34 (m, 3H), 4.84 (d, J=5.1 Hz, 2H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with 1-(piperazin-1-yl)ethan-1-one (0.020 g, 0.159 mmol). The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.045 g, 74%). (LCMS, ESI pos.) Calculated for C25H23N5O2: 426.5 (M+H), Measured: 426.1. 1H NMR (400 MHz, Chloroform-d) δ 9.12 (d, J=1.6 Hz, 1H), 8.49-8.36 (m, 1H), 7.96-7.90 (m, 3H), 7.90-7.84 (m, 2H), 7.65-7.60 (m, 2H), 7.51-7.44 (m, 2H), 7.44-7.38 (m, 1H), 3.93-3.33 (m, 8H), 2.13 (s, 3H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). The solution was stirred for 15 minutes. To the solution was added 2-methoxyethan-1-amine (0.014 ml, 0.159 mmol). The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.040 g, 68%). (LCMS, ESI pos.) Calculated for C22H20N4O2: 373.4 (M+H), Measured: 372.9. 1H NMR (400 MHz, Chloroform-d) δ 9.12 (d, J=1.6 Hz, 1H), 8.44 (t, J=1.2 Hz, 1H), 8.02-7.92 (m, 5H), 7.90-7.85 (m, 2H), 7.47 (dd, J=8.3, 6.5 Hz, 2H), 7.44-7.38 (m, 1H), 6.60 (s, 1H), 3.69 (q, J=5.2 Hz, 2H), 3.59 (t, J=5.0 Hz, 2H), 3.40 (s, 3H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). The solution was stirred at room temperature for 15 minutes. N-methylpiperidine-4-carboxamide (0.023 g, 0.159 mmol) was added to the solution. The reaction solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.033 g, 47%). (LCMS, ESI pos.) Calculated for C26H25N5O2: 440.5 (M+H), Measured: 439.9. 1H NMR (400 MHz, Chloroform-d) δ 9.08 (d, J=1.5 Hz, 1H), 8.39 (p, J=0.7 Hz, 1H), 7.90 (d, J=0.9 Hz, 1H), 7.88-7.80 (m, 4H), 7.61-7.52 (m, 2H), 7.44 (dd, J=8.3, 6.5 Hz, 2H), 7.41-7.34 (m, 1H), 5.84 (q, J=4.9 Hz, 1H), 4.67 (s, 1H), 3.84 (s, 1H), 3.17-2.81 (m, 2H), 2.78 (d, J=4.8 Hz, 3H), 2.35 (tt, J=11.1, 4.1 Hz, 1H), 1.83 (d, J=51.1 Hz, 4H).
A solution of 4-(6-phenylimidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.05 g, 0.159 mmol) and HATU (0.066 g, 0.174 mmol) in DMF (0.5 ml) was treated with DIPEA (0.033 ml, 0.190 mmol). The solution was stirred at room temperature. After 10 minutes 4-aminocyclohexan-1-ol (0.018 g, 0.159 mmol) was added. The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.032 g, 49%). (LCMS, ESI pos.) Calculated for C25H24N4O2: 413.5 (M+H), Measured: 412.9. 1H NMR (400 MHz, Chloroform-d) δ 9.11 (d, J=1.6 Hz, 1H), 8.42 (t, J=1.3 Hz, 1H), 7.94 (dd, J=3.4, 1.1 Hz, 5H), 7.89-7.83 (m, 2H), 7.50-7.43 (m, 2H), 7.43-7.37 (m, 1H), 6.00 (d, J=7.9 Hz, 1H), 4.00 (tdt, J=11.5, 8.0, 4.1 Hz, 1H), 3.66 (tt, J=10.3, 4.1 Hz, 1H), 2.22-2.10 (m, 2H), 2.04 (dd, J=12.0, 3.8 Hz, 2H), 1.58-1.41 (m, 2H), 1.34 (qd, J=12.8, 3.1 Hz, 2H).
A mixture of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.022 g, 0.066 mmol) and HATU (0.028 g, 0.073 mmol) in DMF (0.220 ml) was treated with DIPEA (0.014 ml, 0.079 mmol). The solution was stirred at room temperature. After 10 min 1-(3-aminopropyl)pyrrolidin-2-one (9.39 mg, 0.066 mmol) was added. The solution was stirred at room temperature. The solution was stirred at room temperature for 3 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.007 g, 23%). (LCMS, ESI pos.) Calculated for C26H24FN5O2: 458.5 (M+H), Measured: 458.1. 1H NMR (400 MHz, Chloroform-d) δ 9.11 (d, J=1.6 Hz, 1H), 8.46 (t, J=1.3 Hz, 1H), 8.24-8.14 (m, 2H), 8.06 (t, J=6.4 Hz, 1H), 8.02-7.91 (m, 3H), 7.72-7.60 (m, 2H), 7.43 (td, J=8.2, 5.9 Hz, 1H), 7.10 (tdd, J=8.3, 2.6, 1.0 Hz, 1H), 3.46 (ddt, J=9.2, 6.1, 2.9 Hz, 6H), 2.57-2.42 (m, 2H), 2.23-2.05 (m, 2H), 1.90-1.77 (m, 2H).
A mixture of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.02 g, 0.060 mmol) and HATU (0.025 g, 0.066 mmol) in DMF (0.200 ml) was treated with DIPEA (0.013 ml, 0.072 mmol). The solution was stirred at room temperature for 10 min. To the solution was added ammonia (8.57 μl, 0.060 mmol). The solution was stirred at room temperature for 18 hrs. The solution was filtered. The solid was triturated with EtOAc/MeOH 1:1. The solution was decanted. The solid was dried in vacuo to give desired compound (7.7 mg, 39%). (LCMS, ESI pos.) Calculated for C19H13FN4O: 333.3 (M+H), Measured: 333.1. 1H NMR (400 MHz, Chloroform-d) δ 8.96 (t, J=1.2 Hz, 1H), 8.38 (d, J=1.5 Hz, 1H), 7.90 (d, J=8.1 Hz, 2H), 7.82 (d, J=1.0 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.70 (d, J=5.7 Hz), 7.53-7.47 (m, 2H), 7.20 (td, J=7.9, 5.8 Hz, 1H), 6.91-6.82 (m, 1H), 6.76 (s, 1H).
A solution of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.02 g, 0.060 mmol) and HATU (0.025 g, 0.066 mmol) in DMF (0.200 ml) was treated with DIPEA (0.013 ml, 0.072 mmol). The solution was stirred at room temperature for 10 min. To the solution was added 3-(1H-imidazol-1-yl)propan-1-amine (7.51 mg, 0.060 mmol). The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.005 g, 19%). (LCMS, ESI pos.) Calculated for C25H21FN6O: 441.5 (M+H), Measured: 441.1. 1H NMR (400 MHz, Chloroform-d) δ 9.08 (t, J=1.2 Hz, 1H), 8.40 (dt, J=1.6, 1.0 Hz, 1H), 8.02 (dd, J=7.5, 1.3 Hz, 2H), 7.99-7.85 (m, 4H), 7.78 (s, 1H), 7.61 (dt, J=8.8, 1.6 Hz, 2H), 7.48-7.36 (m, 1H), 7.09 (tdd, J=6.4, 2.9, 1.5 Hz, 2H), 7.01 (s, 1H), 4.20-4.05 (m, 2H), 3.51 (q, J=6.4 Hz, 2H), 2.18 (p, J=6.6 Hz, 2H).
A solution of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.02 g, 0.060 mmol) and HATU (0.025 g, 0.066 mmol) in DMF (0.200 ml) was treated with DIPEA (0.013 ml, 0.072 mmol). The solution was stirred at room temperature for 10 min. To the solution was added 2-methoxyethan-1-amine (5.22 μl, 0.060 mmol). The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.009 g, 39%). (LCMS, ESI pos.) Calculated for C22H19FN4O2: 391.4 (M+H), Measured: 391.2. 1H NMR (400 MHz, Chloroform-d) δ 9.12 (d, J=1.6 Hz, 1H), 8.44 (dd, J=1.6, 1.0 Hz, 1H), 8.04-7.97 (m, 3H), 7.97-7.88 (m, 2H), 7.67-7.59 (m, 2H), 7.43 (td, J=8.2, 6.0 Hz, 1H), 7.16-7.05 (m, 1H), 6.60 (s, 1H), 3.69 (td, J=5.6, 4.3 Hz, 2H), 3.63-3.55 (m, 2H), 3.40 (d, J=0.9 Hz, 3H).
A mixture of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.02 g, 0.060 mmol) and HATU (0.025 g, 0.066 mmol) in DMF (0.200 ml) was treated with DIPEA (0.013 ml, 0.072 mmol). The solution was stirred at room temperature for 10 min. To the solution was added 3-aminopropan-1-ol (4.56 μl, 0.060 mmol). The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.002 g, 9%). (LCMS, ESI pos.) Calculated for C22H19FN4O2: 391.4 (M+H), Measured: 391.2. 1H NMR (400 MHz, Chloroform-d) δ 9.14 (d, J=1.6 Hz, 1H), 8.43 (t, J=1.3 Hz, 1H), 8.04-7.97 (m, 3H), 7.97-7.89 (m, 2H), 7.68-7.59 (m, 2H), 7.43 (td, J=8.2, 5.9 Hz, 1H), 7.15-7.06 (m, 1H), 6.96 (d, J=10.7 Hz, 1H), 3.78 (t, J=5.5 Hz, 2H), 3.68 (q, J=6.0 Hz, 2H), 1.85 (p, J=5.6 Hz, 2H).
A mixture of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.02 g, 0.060 mmol) and HATU (0.025 g, 0.066 mmol) in DMF (0.200 ml) was treated with DIPEA (0.013 ml, 0.072 mmol). The solution was stirred at room temperature for 10 min. To the solution was added tert-butyl (3-aminopropyl)carbamate (10.45 mg, 0.060 mmol). The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.002 g, 7%). (LCMS, ESI pos.) Calculated for C27H28FN5O3: 490.6 (M+H), Measured: 490.3. 1H NMR (400 MHz, Chloroform-d) δ 9.10 (d, J=1.6 Hz, 1H), 8.45 (t, J=1.3 Hz, 1H), 8.08 (d, J=8.1 Hz, 2H), 7.97-7.90 (m, 3H), 7.68-7.61 (m, 2H), 7.51 (d, J=12.0 Hz, 1H), 7.42 (td, J=8.2, 6.0 Hz, 1H), 7.09 (tdd, J=8.3, 2.5, 1.1 Hz, 1H), 4.86 (s, 1H), 3.54 (q, J=6.1 Hz, 2H), 3.28 (q, J=6.4 Hz, 2H), 1.74 (p, J=6.1 Hz, 2H), 1.45 (s, 9H).
A solution of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.02 g, 0.060 mmol) and HATU (0.025 g, 0.066 mmol) in DMF (0.200 ml) was treated with DIPEA (0.013 ml, 0.072 mmol). The solution was stirred at room temperature. After 10 min the solution was treated with N-(2-aminoethyl)acetamide (6.13 mg, 0.060 mmol). The solution was stirred at room temperature. The solution was filtered. The solid was triturated with EtOAc/MeOH 1:1. The solution was decanted. The solid was dried in vacuo to give desired compound (2.4 mg, 10%). (LCMS, ESI pos.) Calculated for C23H20FN5O2: 418.4 (M+H), Measured: 417.8. 1H NMR (400 MHz, Chloroform-d) δ 9.19 (d, J=1.5 Hz, 1H), 8.43-8.39 (m, 1H), 8.26 (s, 1H), 8.11-8.05 (m, 2H), 7.90 (d, J=8.1 Hz, 2H), 7.64-7.52 (m, 3H), 7.40-7.33 (m, 1H), 7.04 (td, J=8.4, 2.2 Hz, 1H), 3.51 (d, J=5.7 Hz, 2H), 3.39 (d, J=8.1 Hz, 2H), 1.96-1.91 (m, 3H).
A mixture of 4-(6-(3-fluorophenyl)imidazo[1,5-a]pyrazin-3-yl)benzoic acid (0.04 g, 0.120 mmol) and HATU (0.050 g, 0.132 mmol) in DMF (0.400 ml) was treated with DIPEA (0.025 ml, 0.144 mmol). The solution was stirred at room temperature for 10 min. To the solution was added 2-(1H-imidazol-5-yl)ethan-1-amine (0.013 g, 0.120 mmol). The solution was stirred at room temperature for 18 hrs. The solution was introduced into a 24 g silica gel column equilibrated with EtOAc. Elution was done with a gradient (EtOAc to 10% MeOH/EtOAc). Desired fractions were combined, concentrated and dried in vacuo to give the desired compound (0.005 g, 10%). (LCMS, ESI pos.) Calculated for C24H19FN6O: 427.5 (M+H), Measured: 427.1. 1H NMR (400 MHz, Chloroform-d) δ 9.33 (d, J=1.5 Hz, 1H), 8.87 (t, J=1.3 Hz, 1H), 8.78 (t, J=5.6 Hz, 1H), 8.52 (dd, J=4.3, 1.4 Hz, 1H), 8.34 (dd, J=8.4, 1.4 Hz, 1H), 8.18-8.06 (m, 4H), 8.01-7.93 (m, 1H), 7.66 (d, J=1.3 Hz, 1H), 7.57 (td, J=8.2, 6.2 Hz, 1H), 7.34 (dd, J=8.4, 4.3 Hz, 1H), 7.29 (ddd, J=10.4, 8.1, 2.6 Hz, 1H), 6.91 (s, 1H), 3.58 (td, J=7.4, 5.5 Hz, 1H), 2.84 (t, J=7.4 Hz, 1H).
Assays were conducted in a 96-well black solid-bottom plate with a final assay volume of 100 μL. As shown in Table 2, Compounds 1-3 show IC50 values that activate CD206 and selectively target M2 macrophages.
When recombinant CD206 was incubated with Compound 1 the electron microscopy studies showed that Compound 1 binds to CD206 and induces a conformational switch of the receptor.
Similar to the activity of M2 macrophage selective synthetic peptide RP-182, the anti-M2 macrophage activity of Compounds 1-3 is also CD206 dependent.
Cell-based 2HG quantification assays were conducted in 96-well clear plates with a final assay volume of 100 μL.
Induction of phagocytosis, autophagy, and apoptosis was studied in two in vitro models using M1 and M2 macrophages. First in a Bone marrow derived macrophages (BMDM) in vitro model, Compound 1 showed excellent selectivity to induce phagocytosis, autophagy, and apoptosis in M2 but not in M1 macrophages.
Compound 1 selectively increases cancer cell phagocytosis in M2 but not in M1 macrophages.
As shown in
This application claims priority to U.S. Provisional Application Ser. No. 62/950,488 filed 19 Dec. 2019, and is incorporated herein by reference in its entirety.
This invention was made in part with government support from the National Institutes of Health under Grant No. ZIA-BC011267. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/065238 | 12/16/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62950488 | Dec 2019 | US |
