Patent Application
20250206738
The invention provides bicyclic compounds and compositions, the use thereof and methods using the compounds, for inhibiting Werner Syndrome RecQ DNA helicase (WRN) and methods of treating disease using said compounds, in particular the use in treating cancer, and in particular the treatment of cancer characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR), including colorectal, gastric and endometrial cancer. The invention also provides the use of said compounds as research chemicals, intermediate compounds, combinations, processes and formulations.
This application contains a Sequence Listing which has been submitted in .xml format via EFS and is hereby incorporated by reference. The ST.26 copy, created on Dec. 19, 2024, is named 407274-87WRUS_215144_SL.xml and is 9,769 bytes in size.
Loss of DNA mismatch repair is a common initiating event in cancer development occurring in 10-30% of colorectal, endometrial, ovarian and gastric cancers (Aaltonen, L. A. et al. Clues to the pathogenesis of familial colorectal cancer, Science 260, 812-816 (1993), Bonneville R et al., Landscape of Microsatellite Instability Across 39 Cancer Types. JCO Precis Oncol. 1: PO.17.00073 (2017)). Cancers that are deficient in mismatch repair (dMMR) have a high mutational burden, and frequent deletion and insertion events in repetitive DNA tracts, a phenotype known as microsatellite instability (MSI). While progress has been made in the treatment of microsatellite instability high (MSI-H) cancers, and the demonstration that pembrolizumab (anti-PD1) treatment led to significantly longer progression-free survival than chemotherapy when received as first-line therapy for MSI-H-dMMR metastatic colorectal cancer (CRC) which resulted in the recent approval of pembrolizumab as first-line treatment of these cancers, there is still a significant unmet medical need in CRC and other MSI-H indications (Andre T., et al. Pembrolizumab in Microsatellite-Instability-High Advanced Colorectal Cancer. N Engl J Med 383(23):22072218 (2020)). Several large-scale functional genomics screens across large panels of cell lines, including Novartis with 398 cell lines from the Cancer Cell Line Encyclopedia (CCLE) (McDonald E. R. et al., Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170(3):577-592 (2017)), have identified the Werner Syndrome RecQ helicase (WRN) as being selectively required for the survival of cell lines with defective mismatch repair that have become MSI-H (Behan, F. M. et al. Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens. Nature 568, 511-516 (2019), Chan, E. M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568, 551-556 (2019). Kategaya, L., Perumal, S. K., Hager, J. H. & Belmont, L. D. Werner syndrome helicase is required for the survival of cancer cells with microsatellite instability. iScience 13, 488-497 (2019), Lieb, S. et al. Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells. eLife 8, e43333 (2019)). WRN is synthetically lethal with MSI cancers. Depletion of WRN leads to anti-proliferative effects and results in activation of multiple DNA damage signaling markers, induction of cell cycle arrest and apoptosis in MSI-H cancer models but not cancer cells with an intact MMR pathway (otherwise known as microsatellite stable or MSS). The anti-proliferative effects of WRN depletion could not be rescued with a helicase deficient WRN construct, demonstrating that helicase activity of WRN is required for MSI-H viability. These findings indicate that WRN helicase provides a DNA repair and maintenance function that is essential for cell survival in MSI cancers. Recently, the mechanism of WRN dependence has been elucidated. It has been shown that dinucleotide TA repeats are selectively unstable in MSI cells and undergo large scale expansions. These expanded TA repeats form secondary DNA structures that require the WRN helicase for unwinding (van Wietmarschen, N. et al. Repeat expansions confer WRN dependence in microsatellite-unstable cancers. Nature 586, 292-298, 2020). In the absence of WRN (or upon WRN helicase inhibition), expanded TA repeats in MSI cells are subject to nuclease cleavage and chromosome breakage. Thus, inhibiting the WRN helicase is an attractive strategy for the treatment of MSI-H cancers.
There remains a need for new treatments and therapies for the treatment of cancer, and in particular cancers characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR), including colorectal, gastric or endometrial cancer. The invention provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, said compounds being inhibitors of Werner Syndrome RecQ DNA Helicase (WRN). The invention further provides methods of treating, preventing, or ameliorating a disease or condition, comprising administering to a subject in need thereof an effective amount of a WRN inhibitor. The invention also provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, said compounds being useful for the treatment of cancer, in particular cancers characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR). Also provided are compounds that bind to, and/or inhibit WRN, and are therefore useful as research chemicals, e.g., as a chemical probe, and as tool compounds. Various embodiments of the invention are described herein.
In one aspect, the disclosure provides a compound of Formula I, or a pharmaceutically acceptable salt thereof:
wherein bicyclic Ring BC, linker L, R4, and Ring A are as described and defined herein.
In another aspect, the invention provides a pharmaceutical composition comprising a compound of Formula I of the present invention and one or more pharmaceutically acceptable carriers.
In another aspect, the invention provides a combination, in particular a pharmaceutical combination, comprising a compound of Formula I of the present invention and one or more therapeutically active agents.
In another aspect, the invention provides a compound of Formula I of the present invention for use as a medicament, in particular for the treatment of a disorder or disease which can be treated by WRN inhibition.
In another aspect, the invention provides a compound of Formula I of the present invention for use in the treatment of cancer, particularly wherein the cancer is characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR).
In another aspect, the invention provides a method of treating a disorder or disease which can be treated by WRN inhibition in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formula I of the present invention.
In another aspect, the invention provides a method of treating cancer in a subject, more particularly wherein the cancer is characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR), comprising administering to the subject a therapeutically effective amount of a compound of Formula I of the present invention.
In another aspect, the invention provides the use of a compound of Formula I of the present invention in the manufacture of a medicament for the treatment of a disorder or disease which can be treated by WRN inhibition.
In another aspect, the invention provides a compound of Formula I of the present invention for use as a research chemical, for example as a chemical probe or as a tool compound.
In another aspect, the invention provides a solid form, process or intermediate as described herein.
In one aspect, the disclosure provides a compound of Formula I that is a compound of Formula I′, or a pharmaceutically acceptable salt thereof:
In one aspect, the disclosure provides a compound of Formula I that is a compound of Formula I″, or a pharmaceutically acceptable salt thereof:
In one embodiment, the disclosure provides a compound of Formula I′, or a pharmaceutically acceptable salt thereof, wherein bicyclic Ring BC is selected from the group consisting of:
In one embodiment, the disclosure provides a compound of Formula I″, or a pharmaceutically acceptable salt thereof, wherein bicyclic Ring BC is selected from the group consisting of:
In another aspect, the invention provides a method of treating a disorder or disease which can be treated by WRN inhibition in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formula I of the present invention.
Compounds of the present invention include those described generally herein, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001.
Compound structures shown throughout the present specification and in the examples or claims contain designations at certain stereocenters. Stereocenters marked with “abs” intend to cover material wherein the marked stereocenter is of the stereochemistry shown in the diagram. Stereocenters marked with “& 1” or “and1” indicate that the compound material has a mixture of R and S-configured stereoisomers with respect to the marked stereocenter and is in the same relative configuration to each other if they share the same label such as “and1” or “&1” as in Example I-2a.
The term “aliphatic” or “aliphatic group,” as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 5-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. The term “alkyl” refers to a C1-12 straight or branched saturated aliphatic group. In certain instances, alkyl refers to a C1-8 straight or branched saturated aliphatic group or a C1-6 straight or branched saturated aliphatic group. The term “lower alkyl” refers to a C1-4 straight or branched alkyl group.
Exemplary lower alkyl groups are methyl (—CH3), ethyl (—CH2CH3), propyl, isopropyl (also referred to interchangeably herein as 2-propyl, iPr, iPr and i-Pr), butyl, isobutyl (also referred to interchangeably herein as 2-butyl, iBu, iBu and i-Bu) and tert-butyl (also referred to interchangeably herein as 2-methyl-2-butyl, tBu, tBu and t-Bu).
The term “alkenyl” refers to a C2-12 straight or branched partially unsaturated aliphatic group comprising at least one unsaturated carbon carbon double bond. In certain instances, alkenyl refers to a C2-8 or a C2-6 straight or branched partially unsaturated aliphatic group comprising at least one unsaturated carbon carbon double bond. The term “lower alkenyl” refers to a C2-4 straight or branched partially unsaturated aliphatic group comprising at least one unsaturated carbon carbon double bond. Alkenyl groups include both cis (Z) and trans (E) regioisomers. Exemplary lower alkenyl groups are vinyl, allyl, 2-propenyl, and butenyl isomers (—CH2CH2CH═CH2, —CH2CH═CHCH3 and —CH═CHCH2CH3).
The term “alkynyl” refers to a C2-12 straight or branched partially unsaturated aliphatic group comprising at least one unsaturated carbon carbon triple bond. In certain instances, alkynyl refers to a C2-8 or a C2-6 straight or branched partially unsaturated aliphatic group comprising at least one unsaturated carbon carbon triple bond. The term “lower alkynyl” refers to a C2-4 straight or branched partially unsaturated aliphatic group comprising at least one unsaturated carbon carbon triple bond. Exemplary lower alkynyl groups are ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
The term “haloalkyl” refers to a straight or branched alkyl group that is substituted with one or more halogen atoms. The term “lower haloalkyl” refers to a C1-4 straight or branched alkyl group that is substituted with one or more halogen atoms.
The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl).
The term “unsaturated,” as used herein, means that a moiety has one or more units of unsaturation.
The term “cubanyl” refers to a substituent of cubane as shown below.
The substituent -Me, as used herein refers to a methyl group, —CH3.
As used herein, the term “bivalent C1-8 (or C1-6 i.e., C1-C6) saturated or unsaturated, straight or branched, hydrocarbon chain,” refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.
As used herein, the term “bivalent,” to describe a cyclic (and noncyclic) group refers to, for example, bivalent carbocyclylene, phenylene, heterocyclylene, and heteroarylene that are bivalent moieties of carbocycles, phenyls, heterocycles, and heteroaryls described herein. Non-limiting examples include
“Carbocyclylene” as used herein refers to a carbocyclic or cycloalkyl moiety that is bivalent as described above (i.e., attached at two different points to the rest of the compound). Non-limiting examples include cyclopropylene, cyclobutylene, cyclopentylene, or cyclohexylene as shown below.
A carbocyclylene may be saturated as in the examples shown above or partially unsaturated as in the examples shown below.
A carbocyclylene may be multi-cyclic, for example, bicyclic or tricyclic. Such multi-cyclic carbocyclylene systems may be saturated or partially unsaturated (while one ring of the bicyclic system may be aromatic it is to be understood that multi-cyclic ring systems that are not in their entirety aromatic may also fall under the definition of carbocyclylene). The rings may form bridged, fused, or spiro systems. Non-limiting examples are shown below.
“Heterocyclylene” as used herein refers to a heterocyclic or heterocyclyl moiety that is bivalent as described above (i.e., attached at two different points to the rest of the compound) and may also be saturated or partially unsaturated. Non-limiting examples include those shown below. Heterocyclylene is understood to include bicyclic heterocyclylene systems. Non-limiting examples of bicyclic heterocyclylene moieties are also shown below and said bicyclic systems may be spirocyclic, fused, or bridged and may be saturated or partially unsaturated.
“Phenylene” as used herein refers to a phenyl moiety that is bivalent as described above (i.e., attached at two different points to the rest of the compound). Examples are shown below.
“Arylene” as used herein refers to a mono or multi-cyclic aryl (i.e., phenyl or a multi-cyclic aryl) moiety that is bivalent as described above (i.e., attached at two different points to the rest of the compound), wherein the arylene group contains no heteroatoms. Examples are shown below.
“Heteroarylene,” as used herein refers to a mono or multi-cyclic aryl ring system that contains at least one heteroatom wherein the ring system is bivalent as described above (i.e., attached at two different points to the rest of the compound). Examples are shown below.
A “saturated or partially unsaturated bridged, fused or spirocyclic 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, wherein said saturated or partially unsaturated bridged, fused or spirocyclic ring comprises 0, 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur” refers to bicyclic, tricyclic, or tetracyclic bridged, fused or spirocyclic 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered rings, wherein one ring of said multicyclic ring system may be an aryl ring and the other ring(s) of the multicyclic ring system may be unsaturated or partially unsaturated. Representative examples include:
The term “alkylene” refers to a bivalent alkyl group. An “alkylene chain” is a polymethylene group, i.e., —(CH2)n—, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
“Carbocyclyl (or heterocyclyl, aryl, phenyl, or heteroaryl) fused to” another phenyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl, for example, a “phenyl or pyridyl” as used herein, may be referred to as “partially unsaturated” without said “carbocyclyl (or heterocyclyl, aryl, phenyl, or heteroaryl) fused to” the other ring requiring further unsaturation besides the carbon-carbon bond which it shares with the ring to which it is fused (i.e., the “phenyl or pyridyl”). This is illustrated below.
A further example below shows a carbocyclyl moiety fused to a Ring E as defined in the embodiments herein. Said carbocyclyl does not explicitly require a descriptor of “partially unsaturated” to describe said carbocyclyl because it shares two carbons with the aromatic pyridine to which it is fused. Such language is used herein to describe such systems, for example, “R4A and R4B, along with their intervening atoms, join to form 4-7 membered carbocyclyl that is fused to Ring E” as shown in the image below. As such, “Ring E” may refer to a monocyclic ring (i.e., the pyridine shown below and its substituents which do not form a fused ring), without any further fused rings created by its substituents (i.e., R4A and R4B). Any further fused ring created by the substituents of Ring E is described as being “fused to Ring E.” Likewise, R4A and R4B, along with their intervening atoms, join to form 4-7 membered carbocyclyl or heterocyclyl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur) that is fused to Ring E (not pictured), is subject to the same interpretation.
The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
The term “halogen” means F, Cl, Br, or I.
The term “aryl” used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic or bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system comprises 3 to 7 ring members. The term “aryl” may be used interchangeably with the term “aryl ring.” In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
The terms “heteroaryl” and “heteroar-,” used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to groups having 5 to 10 ring atoms, preferably 5, 6, 9 or 10 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, triazinyl, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl (i.e., 1,2,3-triazolyl), 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and “heteroar-,” as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where unless otherwise specified, the radical or point of attachment is on the heteroaromatic ring or on one of the rings to which the heteroaromatic ring is fused. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, indolizinyl, isoindolin-1-only, 1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3-onyl, 2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-1-onyl, imidazo[1,2-a]pyridyl, imidazo[1,5-a]pyridyl, pyrazolo[1,5-a]pyridyl, pyrrolo[1,2-b]pyridazinyl, pyrrolo[1,2-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrimidinyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, and tetrahydroisoquinolinyl. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. Said 7-10-membered bicyclic heterocyclic moiety that is partially unsaturated may include an aryl or heteroaryl ring fused to a non-aromatic ring. For example, said 7-10-membered bicyclic heterocyclic moiety may include a bicyclic heterocyclyl as shown below:
When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).
A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, 2-oxa-6-azaspiro[3.3]heptane, and quinuclidinyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
“Arylene” or “heteroarylene,” as used herein (i.e., phenylene), refers to any bivalent aryl or heterocyclyl described herein, that is a bisradical substituted at each of two substitutable positions of the ring system as described in detail supra.
“Heterocyclyloxy,” as used herein, refers to an —OR group wherein the R is a heterocyclyl. Nonlimiting examples are shown below.
As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH2)0-4B(OR∘)2; —(CH2)0-4R∘; —(CH2)0-4O R∘; —O(CH2)0-4R; —O—(CH2)0-4C(O)OR∘; —(CH2)0-4CH(OR∘)2; —(CH2)0-4SR∘; —(CH2)0-4Ph, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1Ph which may be substituted with R∘; —CH═CHPh, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1-pyridyl which may be substituted with R∘; —NO2; —CN; —N3; —(CH2)0-4N(R∘)2; —(CH2)0-4N(R∘)C(O)R∘; —N(R∘)C(S)R∘; —(CH2)0-4N(R∘)C(O)NR02; —N(R∘)C(S)NR∘2; —(CH2)0-4N(R∘)C(O)OR∘; —N(R∘)N(R∘)C(O)R∘; —N(R∘)N(R∘)C(O)NR∘2; —N(R∘)N(R∘)C(O)OR∘; —N(R∘)C(NR∘)N(R∘)2; —(CH2)0-4C(O)R∘; —C(S)R∘; —(CH2)0-4C(O)OR∘; —(CH2)0-4C(O)SR∘; —(CH2)0-4C(O)OSiR∘3; —(CH2)0-4O C(O)R∘; —OC(O)(CH2)0-4SR∘; —(CH2)0-4SC(O)R∘; —(CH2)0-4C(O)NR02; —C(S)NR∘2; —C(S)SR∘; —SC(S)SR∘; —(CH2)0-4O C(O)NR02; —C(O)N(OR∘)R∘; —C(O)C(O)R∘; —C(O)CH2C(O)R∘; —C(NOR∘)R∘; —(CH2)0-4SSR∘; —(CH2)0-4S(O)2R∘; —(CH2)0-4S(O)2OR∘; —(CH2)0-4O S(O)2R∘; —S(O)2NR∘2; —(CH2)0-4S(O)R∘; —N(R∘)S(O)2NR∘2; —N(R∘)S(O)2R∘; —N(OR∘)R∘; —C(NH)NR∘2; —(CH2)0-4P(O)2R∘; —(CH2)0-4P(O)R∘2; —(CH2)0-4O P(O)R∘2; —(CH2)0-4O P(O)(OR∘)2; —SiR∘3; —(C1-4 straight or branched alkylene)O—N(R∘)2; or —(C1-4 straight or branched alkylene)C(O)O—N(R∘)2, wherein each R∘ may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, —SO2—C1-4 aliphatic (i.e., —SO2CH3)—CH2Ph, —O(CH2)0-1Ph, —CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R∘, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.
Suitable monovalent substituents on R∘ (or the ring formed by taking two independent occurrences of R∘ together with their intervening atoms), are independently halogen, —(CH2)0-2R●,-(haloR●), —(CH2)0-2OH, —(CH2)0-2OR●, —(CH2)0-2CH(OR●)2; —O(haloR●), —CN, —N3, —(CH2)0-2C(O)R●, —(CH2)0-2C(O)OH, —(CH2)0-2C(O)OR●, —(CH2)0-2SR●, —(CH2)0-2SH, —(CH2)0-2NH2, —(CH2)0-2NHR●, —(CH2)0-2NR●2, —NO2, —SiR●3, —OSiR●3, —C(O)SR●, —(C1-4 straight or branched alkylene)C(O)OR●, or —SSR● wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-6 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R∘ include ═O and ═S.
Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group, which includes instances of R∘ (or the ring formed by taking two independent occurrences of R∘ together with their intervening atoms), include the following: ═O, ═S, ═NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, —O(C(R*2))2-3O—, or —S(C(R*2))2-3S—, wherein each independent occurrence of R* is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*2)2-3O—, wherein each independent occurrence of R* is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of R* include halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R†, —NR†2, —C(O)R†, —C(O)OR†, —C(O)C(O)R†, —C(O)CH2C(O)R†, —S(O)2R†, —S(O)2NR†2, —C(S)NR†2, —C(NH)NR†2, or —N(R†)S(O)2R†; wherein each R† is independently hydrogen, C1-6aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of RT are independently halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, Z and E conformational isomers and Ra (or M) and Sa (or P) atropisomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention. In certain embodiments, Ring A of a provided compound may be substituted with one or more deuterium atoms.
The structures as drawn represent relative configurations, unless labeled as absolute configurations. The invention contemplates individual enantiomers and racemic mixtures.
In one aspect, the disclosure provides a compound of Formula I that is a compound of Formula I′:
In one aspect, the disclosure provides a compound of Formula I that is a compound of Formula I″:
In one embodiment, the disclosure provides a compound of Formula I′:
In one embodiment, the disclosure provides a compound of Formula I″:
In some embodiments, Ring A is a 4-7 membered saturated or partially unsaturated bivalent monocyclic carbocyclylene or 4-7 membered saturated or partially unsaturated bivalent heterocyclylene ring (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 0 or 1 nitrogen atoms in addition to the 1-4 heteroatoms). In some embodiments, Ring A is a 4-7 membered saturated or partially unsaturated bivalent monocyclic carbocyclylene, wherein Ring A is substituted with 0-4 independently selected RB substituents. In some embodiments, Ring A is a 4-7 membered saturated or partially unsaturated bivalent monocyclic heterocyclylene (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 0 or 1 nitrogen atoms in addition to the 1-4 heteroatoms), wherein Ring A is substituted with 0-4 independently selected RB substituents.
In some embodiments, Ring A is a 4-12 membered saturated or partially unsaturated bivalent bicyclic ring system that is fused, bridged, or spirocyclic selected from carbocyclylene or heterocyclylene (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur). In some embodiments, Ring A is a 4-12 membered saturated or partially unsaturated bivalent bicyclic ring system that is fused, bridged, or spirocyclic and is a carbocyclylene, wherein Ring A is substituted with 0-4 independently selected RB substituents. In some embodiments, Ring A is a 4-12 membered saturated or partially unsaturated bivalent bicyclic ring system that is fused, bridged, or spirocyclic and is a heterocyclylene (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), wherein Ring A is substituted with 0-4 independently selected RB substituents.
In some embodiments, Ring A is a 4-12 membered saturated or partially unsaturated bivalent bicyclic ring system comprising 2 fused rings. In some embodiments, Ring A is a 4-12 membered saturated or partially unsaturated bivalent bicyclic ring system comprising a spirocyclic ring system. In some embodiments, Ring A is a 4-12 membered saturated or partially unsaturated bivalent bicyclic ring system comprising a bridged ring system.
In some embodiments, Ring A is
In some embodiments, Ring A is
In some embodiments, Ring A is
In some embodiments, Ring A is
In some embodiments, Ring A is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, L is a linker selected from —C(O)—, —S(O)—, —S(O)2—, and
In some embodiments, L is —C(O)C(R)2— or —C(R)2C(O)—.
In some embodiments, linker L is —C(O)—.
In some embodiments, linker L is —S(O)—.
In some embodiments, linker L is —S(O)2—.
In some embodiments, linker L is
In some embodiments, linker L is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, R1a is selected from:
In some embodiments, R1a is a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur) optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy, wherein said 5-6 membered heteroaryl is further substituted with 0-3 independently selected RB. In some embodiments, R1a is a 4-6 membered saturated or partially unsaturated heterocyclyl (having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur), said heterocyclyl substituted with 0-2 RB groups independently selected from halogen, oxo, —NR2, optionally substituted C1-4aliphatic, —OR, azetidinyl optionally substituted with 1 or 2 independently selected halogen, and pyrrolidinyl optionally substituted with 1 or 2 independently selected halogen. In some embodiments, R1a is a 6-8 membered saturated or partially unsaturated bridged bicyclic heterocyclyl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), said heterocyclyl substituted with 0-2 RB groups independently selected from halogen, oxo, —NR2, optionally substituted C1-4aliphatic, —OR, azetidinyl optionally substituted with 1 or 2 independently selected halogen, and pyrrolidinyl optionally substituted with 1 or 2 independently selected halogen. In some embodiments, R1a is a 3-7 membered optionally substituted carbocyclyl. In some embodiments, R1a is an optionally substituted C2-C4alkenyl. In some embodiments, R1a is cyclopropyl substituted C2-C4alkenyl. In some embodiments, R1a is methyl substituted C2-C4alkenyl.
In some embodiments, R1a is a 6-membered partially unsaturated heterocyclyl (having 1 oxygen atom). In some embodiments, R1a is a 4-8 membered saturated heterocyclyl (having 1 oxygen atom). In some embodiments, R1a is a 6-membered heteroaryl (having 1 nitrogen atom), said heteroaryl may be optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy, wherein said heteroaryl is further substituted with 0-1 RB, wherein RB is an optionally substituted C1-6aliphatic group. In some embodiments, R1a is a 6-membered heteroaryl (having 2 nitrogen atoms), said heteroaryl may be optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy, wherein said heteroaryl is further substituted with 0-1 RB, wherein RB is an optionally substituted C1-6aliphatic group. In some embodiments, R1a is —NR10R11 wherein R10 is a 5-6 membered heteroaryl (having 1 or 2 nitrogen atoms) optionally substituted with 1 or 2 groups independently selected from halogen, CH3, OCH3, C3-C6cycloalkyl, and C3-C6cycloalkoxy and wherein R11 is H or CH3. In some embodiments, R1a is —CH2NR10R11 wherein R10 is a 5-6 membered heteroaryl (having 1 or 2 nitrogen atoms) optionally substituted with 1 or 2 groups independently selected from halogen, CH3, OCH3, C3-C6cycloalkyl, and C3-C6cycloalkoxy and wherein R11 is H or CH3. In some embodiments, R1a is C2-C4alkene wherein said alkene is optionally substituted with OCH3 or 1, 2, or 3 fluorine. In some embodiments, R1a is C2-C4alkyne wherein said alkyne is optionally substituted with OCH3 or 1, 2, or 3 fluorine. In some embodiments, R1a is —SO2R12 wherein R12 is selected from CH3 or a 5-6 membered heteroaryl having 1-2 nitrogen heteroatoms optionally substituted with 1 or 2 groups independently selected from halogen and CH3. In some embodiments, R1a is cyclopropyl optionally substituted with 1-2 fluorine. In some embodiments, R1a is C1-C6alkyl optionally substituted with OH or 1-2 fluorine. In some embodiments, R1a is —C(O)NR10R11 wherein R10 is H or CH3 and wherein R11 is H or CH3.
In some embodiments, R1a is a 5-membered heteroaryl (having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur) optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy, wherein said 5-membered heteroaryl is optionally further substituted with 0-3 independently selected RB. In some embodiments, R1a is a 5-membered heteroaryl (having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur) optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy. In some embodiments, R1a is a 5-membered heteroaryl (having 2 nitrogen atoms) optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy, wherein said 5-membered heteroaryl is optionally further substituted with 0-1 RB, wherein RB is hydroxyl substituted C1-C4alkyl.
In some embodiments, R1a is a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur) optionally substituted with one group of C1-C6alkoxy or C3-C6cycloalkyl, wherein said 5-6 membered heteroaryl is optionally further substituted with 0-3 independently selected RB.
In some embodiments, R1a is pyridyl substituted with C1-C4alkoxy and further substituted with 0-2 RB.
In some embodiments, R1a is 5-membered heteroaryl (having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur, and 0 or 1 additional ring nitrogen atoms), wherein said 5-membered heteroaryl is optionally substituted with C1-C6alkyl, or C3-C5cycloalkyl and further substituted with 0-2 RB.
In some embodiments, R1a is selected from groups a-d:
In some embodiments, R1a is a 4-7 membered saturated or partially unsaturated heterocyclyl (having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur), optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C3-C6cycloalkyl, C1-C6alkoxy, and C3-C6cycloalkoxy, and —OR, wherein said 4-7 membered saturated or partially unsaturated heterocyclyl is further substituted with 0-3 independently selected RB.
In some embodiments, R1a is a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur) optionally substituted with 1 or 2 groups independently selected from C1-C6alkyl, C3-C6cycloalkyl, C1-C6alkoxy, and C3-C6cycloalkoxy, wherein said 5-6 membered heteroaryl is further substituted with 0-3 independently selected RB.
In some embodiments, R1a is selected from the group consisting of:
wherein * is the point of attachment to Ring B.
In some embodiments, R1a is
In some embodiments, R11 is
In some embodiments, R11 is
In some embodiments, R1a is
In some embodiments, R1a is
In some embodiments, R11 is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, each R1b is independently selected from H, halogen, CN, OH, C1-C6aliphatic, C1-C6alkoxy, C3-C6cycloalkyl, C1-C6alkylene-O—C1-C6alkyl, haloC1-C6alkyl, haloC1-C6alkoxy, and C3-C6cycloalkoxy, wherein said C1-C6aliphatic, C1-C6alkoxy, C3-C6cycloalkyl, C1-C6alkylene-O—C1-C6alkyl, haloC1-C6alkyl, haloC1-C6alkoxy, and C3-C6cycloalkoxy are each independently and optionally substituted with 1-5 halogen, OH, CN, C1-C6alkyl, or C3-C6cycloalkyl groups.
In some embodiments, R1a and one R1b on adjacent atoms of Ring B, taken together with the adjacent Ring B atoms to which they are attached, form a cyclic group fused to Ring B selected from phenyl, a 5-6 membered heteroaryl (having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur), a 4-7 membered saturated or partially unsaturated carbocyclyl, or a 4-7 membered saturated or partially unsaturated heterocyclyl (having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur), wherein said cyclic group fused to Ring B is substituted with 0-3 independently selected RB.
In some embodiments, R1a and one R1b on adjacent atoms of Ring B, taken together with the adjacent Ring B atoms to which they are attached, form a cyclic group fused to Ring B of phenyl, wherein said cyclic group fused to Ring B is substituted with 0-3 independently selected RB. In some embodiments, R1a and one R1b on adjacent atoms of Ring B, taken together with the adjacent Ring B atoms to which they are attached, form a cyclic group fused to Ring B of a 5-6 membered heteroaryl (having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur), wherein said cyclic group fused to Ring B is substituted with 0-3 independently selected RB. In some embodiments, R1a and one R1b on adjacent atoms of Ring B, taken together with the adjacent Ring B atoms to which they are attached, form a cyclic group fused to Ring B of a 4-7 membered saturated or partially unsaturated carbocyclyl, wherein said cyclic group fused to Ring B is substituted with 0-3 independently selected RB. In some embodiments, R1a and one R1b on adjacent atoms of Ring B, taken together with the adjacent Ring B atoms to which they are attached, form a cyclic group fused to Ring B of a 4-7 membered saturated or partially unsaturated heterocyclyl (having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur), wherein said cyclic group fused to Ring B is substituted with 0-3 independently selected RB.
As described generally above, R2 is C(RC)2C(O)N(R)R2A. In some embodiments, R2 is C(RC)2C(RC)2C(O)N(R)R2A. In some embodiments, R2 is C(RC)2C(RC)2N(R)C(O)N(R)R2A. In some embodiments, R2 is C(RC)2C(RC)2N(R)C(O)R2A. In some embodiments, R2 is CH2C(O)N(H)R2A. In some embodiments, R2 is CH2CH2C(O)N(H)R2A. In some embodiments, R2 is CH2CH2N(R)C(O)N(R)R2A. In some embodiments, R2 is CH2CH2N(H)C(O)R2A. In some embodiments, R2 is C(RC)2C(O)N(H)R2A, wherein R2A is bicyclo[1.1.1]pentyl optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, or haloC1-C4alkyl. In some embodiments, R2 is C(RC)2C(O)N(H)R2A, wherein R2A is bicyclo[1.1.1]pentyl optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, or haloC1-C4alkyl.
In some embodiments, R2 is ,
In some embodiments, R2 is
In some embodiments R2 is
In some embodiments R2 is
In some embodiments, R2 is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, R2A is cubanyl, a saturated or partially unsaturated 4-8 membered monocyclic ring, a saturated or partially unsaturated bridged, fused or spirocyclic 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, wherein said saturated or partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring comprises 0, 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and wherein said cubanyl, saturated or partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring are each optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4aliphatic, haloC1-C4alkyl, C3-C6. cycloalkyl, haloC3-C6cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6cyclalkoxy and —SF5, and wherein two optional substituents on the same atom of said saturated or partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring form a cyclic group selected from:
As described generally above, R2A is cubanyl, a saturated or partially unsaturated 4-8 membered monocyclic ring, a saturated or partially unsaturated bridged, fused or spirocyclic 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, wherein said saturated or partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring comprises 0, 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and wherein said cubanyl, saturated or partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring are each optionally substituted with 1, 2, or 3 substituents independently selected from optionally substituted phenyl, halogen, optionally substituted C1-C4aliphatic, haloC1-C4alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6cyclalkoxy and —SF5, and wherein two optional substituents on the same atom of said saturated or partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring form a cyclic group selected from:
In some embodiments, there are 1-6 respective instances of wherein 2 substituents on the same 1st, 2nd, 3rd, 4th, 5th, or 6th atom of said saturated or partially unsaturated monocyclic ring, or said saturated or partially unsaturated bridged, fused, or spirocyclic ring form 1-6 of said cyclic groups. In some embodiments, there is one instance wherein 2 substituents on the same atom of said saturated or partially unsaturated monocyclic ring, or said saturated or partially unsaturated bridged, fused, or spirocyclic ring form one of said cyclic groups. In some embodiments, there 2 respective instances of wherein 2 substituents on the same 1st and 2nd atoms of said saturated or partially unsaturated monocyclic ring, or said saturated or partially unsaturated bridged, fused, or spirocyclic ring form both of said cyclic groups. In some embodiments, there are 3 respective instances of wherein 2 substituents on the same 1st, 2nd and 3rd, atoms of said saturated or partially unsaturated monocyclic ring, or said saturated or partially unsaturated bridged, fused, or spirocyclic ring form the three of said cyclic groups. In some embodiments, there are 4 respective instances of wherein 2 substituents on the same 1st, 2nd, 3rd and 4th atoms of said saturated or partially unsaturated monocyclic ring, or said saturated or partially unsaturated bridged, fused, or spirocyclic ring form the four of said cyclic groups. In some embodiments, there are 5 respective instances of wherein 2 substituents on the same 1st, 2nd, 3rd, 4th and 5th atoms of said saturated or partially unsaturated monocyclic ring, or said saturated or partially unsaturated bridged, fused, or spirocyclic ring form the five of said cyclic groups. In some embodiments, there 6 respective instances of wherein 2 substituents on the same 1st, 2nd, 3rd, 4th, 5th, and 6th atoms of said saturated or partially unsaturated monocyclic ring, or said saturated or partially unsaturated bridged, fused, or spirocyclic ring form the six of said cyclic groups.
In some embodiments, R2A is cubanyl, a saturated or partially unsaturated 4-8 membered monocyclic ring, a saturated or partially unsaturated bridged, fused or spirocyclic 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, wherein said saturated or partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring which comprises 0, 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and wherein said partially unsaturated monocyclic ring, or saturated or partially unsaturated bridged, fused or spirocyclic ring are each optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, haloC1-C4alkyl, C3-C6cycloalkyl, haloC3-C6. cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6cyclalkoxy and —SF5. In some embodiments, R2A is bicyclo[1.1.1]pentyl optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, and haloC1-C4alkyl.
In some embodiments, R2A is a saturated or partially unsaturated bridged 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, which comprises 0, 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and wherein said bridged ring is optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, haloC1-C4alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6cyclalkoxy and —SF5.
In some embodiments, R2A is a saturated or partially unsaturated fused 5-, 6-, 7-, 8-, 9, 10-, 11-, or 12-membered ring, which comprises 0, 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and wherein said fused ring is optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, haloC1-C4alkyl, C3-C6. cycloalkyl, haloC3-C6cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6cyclalkoxy and —SF5.
In some embodiments, R2A is a saturated or partially unsaturated spirocyclic 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, which comprises 0, 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; and wherein said spirocyclic ring is optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, haloC1-C4alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6. cycloalkoxy, haloC4-C6cyclalkoxy and —SF5.
In some embodiments, R2A is bicyclo[1.1.1]pentyl optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, haloC1-C4alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6-cyclalkoxy and —SF5. In some embodiments, R2A is bicyclo[1.1.1]pentyl optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C4alkyl, and haloC1-C4alkyl. In some embodiments, R2A is bicyclo[1.1.1]pentyl optionally substituted with a halogen, C1-C4alkyl, or haloC1-C4alkyl. In some embodiments, R2A is bicyclo[1.1.1]pentyl optionally substituted with 2 substituents independently selected from halogen, C1-C4alkyl, and haloC1-C4alkyl. In some embodiments, R2A is bicyclo[1.1.1]pentyl optionally substituted with 3 substituents independently selected from halogen, C1-C4alkyl, and haloC1-C4alkyl.
In some embodiments, R2A is Ring F selected from the group consisting of:
wherein x, y, and q are independently selected from 1, 2, or 3, Y1 is independently selected from O, NR15, CHR15 or CR15R15, wherein R2A comprises 0, 1, 2, or 3, instances of R15 independently selected from H, halogen, C1-C4aliphatic, haloC1-C4alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6cyclalkoxy and —SF5.
In some embodiments, R2A is Ring F of the following structure
wherein R15 is selected from halogen, C1-C4aliphatic, haloC1-C4alkyl, C3-C6cycloalkyl, haloC3-C6. cycloalkyl, —OH, —CN, C1-C4alkoxy, haloC1-C4alkoxy, C3-C6cycloalkoxy, haloC4-C6cyclalkoxy and —SF5.
In some embodiments, R2A is bicyclo[1.1.1]pentyl comprising a —CF3 substituent or bicyclo[1.1.1]pentyl comprising a —CHF2 substituent.
In some embodiments, R2A is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, R3 is hydrogen, C1-C4alkyl, C3-C5cycloalkyl, C1-C4alkoxy, —NHR3A, —N(R3A)2 or C1-C4alkylthio each of which, besides hydrogen, is optionally substituted with —OH, 1-5 independently selected halogen, —OR, —C(O)N10R11, or N(R)C(O)R.
In some embodiments, R3 is hydrogen. In some embodiments, R3 is C1-C4alkyl optionally substituted with —OH, 1-5 independently selected halogen, or C1-C4alkoxy. In some embodiments, R3 is C1-C4alkyl. In some embodiments, R3 is —CH2CH3. In some embodiments, R3 is —CH3. In some embodiments, R3 is C3-C5cycloalkyl, C1-C4alkoxy, —NHR3A, —N(R3A)2 or C1-C4alkylthio optionally substituted with —OH, 1-5 independently selected halogen, or C1-C4alkoxy. In some embodiments, R3 is C3-C5cycloalkyl optionally substituted with —OH, 1-5 independently selected halogen, or C1-C4alkoxy. In some embodiments, R3 is C1-C4alkoxy optionally substituted with —OH, 1-5 independently selected halogen, or C1-C4alkoxy. In some embodiments, R3 is —NHR3A optionally substituted with —OH, 1-5 independently selected halogen, or C1-C4alkoxy. In some embodiments, R3 is —N(R3A)2 optionally substituted with —OH, 1-5 independently selected halogen, or C1-C4alkoxy. In some embodiments, R3 is C1-C4alkylthio optionally substituted with —OH, 1-5 independently selected halogen, or C1-C4alkoxy. In some embodiments, R3 is selected from the group consisting of C1-C4alkyl and C3-C5cycloalkyl.
In some embodiments, R3 is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, each R3A is independently selected at each occurrence from C1-C4alkyl. In some embodiments, R3A is —CH3. In some embodiments, R3A is —CH2CH3. In some embodiments, R3A is propyl. In some embodiments, R3A is butyl.
In some embodiments, R3A is as selected from one of the substituents of Table 1 or Table 1a.
In some embodiments, R4 is selected from one of a), b), and c):
wherein * is a point of attachment to L; and
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is Ring E of the following structure:
In some embodiments, R4 is a 5-membered heteroaryl (having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur and 0, 1, 2, or 3 additional ring nitrogen atoms), wherein said heteroaryl is substituted with 0-4 groups independently selected from halogen, —OH, —CN, C1-C4alkyl, haloC1-C4alkyl, C3-C6cycloalkyl, and C1-C4alkoxy.
In some embodiments, R4 is a 5-membered heteroaryl (having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur and 0, 1, 2, or 3 additional ring nitrogen atoms), wherein said heteroaryl is substituted with 0-4 groups independently selected from OH, —CH3, —CHF2, cyclopropyl, and —OCH3.
In some embodiments, R4 is a C1-C4alkyl, C1-C4alkoxy, or C3-C6cycloalkyl, each of which is substituted with 0-3 groups independently selected from halogen, —CN, —OH, C1-C4alkyl, C1-C4alkoxy, optionally substituted 5-6 membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and optionally substituted 5-6 membered heterocyclyloxy having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R4 is a C1-C4alkyl, substituted with 0-3 independently selected halogen, —CN, —OH, C1-C4alkyl, and C1-C4alkoxy. In some embodiments, R4 is a C1-C4alkoxy, substituted with 0-3 independently selected halogen, —CN, —OH, C1-C4alkyl, and C1-C4alkoxy. In some embodiments, R4 is a C3-C6cycloalkyl, substituted with 0-3 independently selected halogen, —CN, —OH, C1-C4alkyl, and C1-C4alkoxy.
In some embodiments, R4 is an isoxazolyl substituted with —OH or C1-C4alkoxy.
In some embodiments, R4 is a 5-membered heteroaryl (having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur and 0, 1, 2, or 3 additional ring nitrogen atoms) selected from the group consisting of thiophenyl, imidazolyl, pyrazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, oxazolyl, isoxazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, wherein said heteroaryl is optionally substituted with 0-4 groups independently selected from halogen, —OH, —CN, C1-C4alkyl, haloC1-C4alkyl, C3-C6cycloalkyl, and C1-C4alkoxy.
In some embodiments, R4 is selected from the group consisting of.
In some embodiments:
In some embodiments, R4 is
In some embodiments, R4 is
In some embodiments, R4 is
In some embodiments, R4 is as shown in a substituent of Table 1 or Table 1a.
As described generally above, R10 is H, C1-C6aliphatic, haloC1-C6alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, —C(O)C1-C6alkyl, or a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur); each R10 except H being optionally substituted with 1 or 2 independently selected RB.
In some embodiments, R10 is H. In some embodiments, R10 is C1-C6aliphatic, haloC1-C6alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, —C(O)C1-C6alkyl, or a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur); each R10 being optionally substituted with 1 or 2 independently selected RB. In some embodiments, R10 is C1-C6aliphatic, haloC1-C6alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, or —C(O)C1-C6alkyl; each R10 being optionally substituted with 1 or 2 independently selected RB. In some embodiments, R10 is a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur); R10 being optionally substituted with 1 or 2 independently selected RB.
In some embodiments, R10 is as shown in a substituent of Table 1 or Table 1a.
As described generally above, R11 is H, C1-C6aliphatic, or C3-C6cycloalkyl, or R10 and R11 are taken together with the nitrogen atom to which they are attached to form a 5-6 membered ring optionally substituted with 1, 2, or 3 substituents independently selected from halogen, —OH, —CN, C1-C4alkoxy, and haloC1-C4alkoxy.
In some embodiments, R11 is H, C1-C6aliphatic, or C3-C6cycloalkyl. In some embodiments, R11 is H. In some embodiments, R11 is C1-C6aliphatic. In some embodiments, R11 is C3-C6cycloalkyl. In some embodiments, R10 and R11 are taken together with the nitrogen atom to which they are attached to form a 5-6 membered ring optionally substituted with 1, 2, or 3 substituents independently selected from halogen, —OH, —CN, C1-C4alkoxy, and haloC1-C4alkoxy.
In some embodiments, R11 is as shown in a substituent of Table 1 or Table 1a.
As described generally above, R12 is C1-C6aliphatic, C3-C6cycloalkyl, or a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur); each R12 optionally substituted with 1 or 2 groups independently selected from halogen, C1-C6aliphatic, haloC1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy.
In some embodiments, R12 is C1-C6aliphatic optionally substituted with 1 or 2 groups independently selected from halogen, C1-C6aliphatic, haloC1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy. In some embodiments, R12 is C1-C6aliphatic optionally substituted with 1 or 2 groups independently selected from halogen, C1-C6aliphatic, haloC1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy. In some embodiments, R12 is C3-C6cycloalkyl optionally substituted with 1 or 2 groups independently selected from halogen, C1-C6aliphatic, haloC1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy. In some embodiments, R12 is a 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur) optionally substituted with 1 or 2 groups independently selected from halogen, C1-C6aliphatic, haloC1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, and C3-C6cycloalkoxy.
As described generally above, RB is independently selected at each occurrence from the group consisting of optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), optionally substituted 4-7 membered saturated or partially unsaturated heterocyclyl (having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur), halogen, optionally substituted C1-C6aliphatic, haloC1-C6alkyl, C3-C6cycloalkyl, halo-C3-C6cycloalkyl, C1-C6alkoxy, halo-C1-C6alkoxy, C3-C6cycloalkoxy, halo-C3-C6cycloalkoxy, C1-C6alkylene-O—C1-C6alkyl, —CN, —NO2, oxo, —OR, —SR, NR2, S(O)2R, S(O)2NR2, S(O)R, S(O)NR2, C(O)R, C(O)OR, —C(O)NR2, C(O)N(R)OR, OC(O)R, OC(O)NR2, —N(R)C(O)OR, N(R)C(O)R, N(R)C(O)NR2, N(R)C(NR)NR2, N(R)S(O)2NR2, and —N(R)S(O)2R.
As also described generally above, in some embodiments, RB is independently selected at each occurrence from the group consisting of optionally substituted phenyl, optionally substituted 5-6 membered heteroaryl (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), optionally substituted 4-7 membered saturated or partially unsaturated heterocyclyl (having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur), halogen, optionally substituted C1-C6aliphatic, haloC1-C6alkyl, C3-C6cycloalkyl, haloC3-C6cycloalkyl, C1-C6alkoxy, haloC1-C6alkoxy, C3-C6cycloalkoxy, haloC3-C6cycloalkoxy, C1-C6alkylene-O—C1-C6alkyl, —B(OR)2, —CN, —NO2, oxo, —OR, —SR, NR2, S(O)2R, S(O)2NR2, S(O)R, S(O)NR2, C(O)R, C(O)OR, —C(O)NR2, C(O)N(R)OR, OC(O)R, OC(O)NR2, —N(R)C(O)OR, N(R)C(O)R, N(R)C(O)NR2, N(R)C(NR)NR2, N(R)S(O)2NR2, and —N(R)S(O)2R; or two RB taken together with the carbon to which they are attached form a 3-7 membered saturated carbocyclic ring.
In some embodiments, RB is independently selected at each occurrence from the group consisting of halogen, —OR, or an optionally substituted C1-6aliphatic group. In some embodiments, RB is independently selected at each occurrence from a halogen. In some embodiments, RB is independently selected at each occurrence from —OR. In some embodiments, RB is independently selected at each occurrence from an optionally substituted C1-6aliphatic group.
In some embodiments, RB is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, RC is independently selected at each occurrence from hydrogen, —CH3, and —CH2CH3, or two RC taken together with the carbon to which they are attached form a cyclopropyl ring. In some embodiments, RC is independently selected at each occurrence from hydrogen, —CH3, and —CH2CH3. In some embodiments, RC is hydrogen. In some embodiments, one RC is —CH3, and the other RC is hydrogen. In some embodiments, two RC taken together with the carbon to which they are attached form a cyclopropyl ring.
In some embodiments, RC is as selected from one of the substituents of Table 1 or Table 1a.
As described generally above, each R is independently hydrogen, or an optionally substituted C1-6aliphatic group, an optionally substituted phenyl, an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring, an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring (having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur), or an optionally substituted 5-6 membered heteroaryl ring (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur); or two R groups on the same atom are taken together with the same atom to form a cyclic group selected from an optionally substituted 4-7 membered saturated ring, a 4-7 membered partially unsaturated ring, or a 5-6 membered heteroaryl ring (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur); wherein said cyclic group has 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur).
In some embodiments, each R is hydrogen. In some embodiments, each R is independently an optionally substituted C1-6aliphatic group, an optionally substituted phenyl, an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring, an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring (having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur), or an optionally substituted 5-6 membered heteroaryl ring (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur).
In some embodiments, two R groups on the same atom are taken together with the same atom to form a cyclic group selected from an optionally substituted 4-7 membered saturated ring, a 4-7 membered partially unsaturated ring, or a 5-6 membered heteroaryl ring (having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur); wherein said cyclic group has 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur). In some embodiments, each R is independently hydrogen or a C1-6 alkyl.
In some embodiments, each R is as selected from one or more of the substituents of Table 1 or Table 1a.
In some embodiments, the compound of Formula I is a compound of Formula II-a-Formula II-z″:
In some embodiments, the compound of Formula I is a compound of Formula IIa-Formula IIs:
In some embodiments, the compound of Formula I is a compound of Formula IIa-Formula IIs:
and R2a is selected from,
In some embodiments, the compound of Formula I is a compound of Formula III-a:
In some embodiments, the compound of Formula I is a compound of Formula III-b:
In some embodiments, the compound of Formula I is a compound of III-c:
In some embodiments, the compound of Formula I is a compound of Formula III-d:
In some embodiments, the compound of Formula I is a compound of Formula III-e:
In some embodiments, the compound of Formula I is a compound of Formula III-f:
In some embodiments, the compound of Formula I is a compound of Formula III-g:
In some embodiments, the compound of Formula I is a compound of Formula III-h:
In some embodiments, the compound of Formula I is a compound of Formula III-i:
In some embodiments, the compound of Formula I is a compound of Formula III-j:
In some embodiments, the compound of Formula I is a compound of Formula III-k:
In some embodiments, the compound of Formula I is a compound of Formula III-1:
In some embodiments, the compound of Formula I is a compound of Formula III-m:
In some embodiments, the compound of Formula I is a compound of Formula III-n:
In some embodiments, the compound of Formula I is a compound of Formula III-o:
In some embodiments, the compound of Formula I is a compound of Formula III-p:
In some embodiments, the compound of Formula I is a compound of Formula III-q:
In some embodiments, the compound of Formula I is a compound of Formula III-r:
In some embodiments, the compound of Formula I is a compound of Formula III-s:
In some embodiments, the compound of Formula I is a compound of Formula III-t:
In some embodiments, the compound of Formula I is a compound of Formula III-u′:
In some embodiments, the compound of Formula I is a compound of Formula III-u″:
In some embodiments, the compound of Formula I is a compound of Formula III-v′:
In some embodiments, the compound of Formula I is a compound of Formula III-v″:
In some embodiments, the compound of Formula I is a compound of Formula III-w′:
In some embodiments, the compound of Formula I is a compound of Formula III-w″:
In some embodiments, the compound of Formula I is a compound of Formula III-x′:
In some embodiments, the compound of Formula I is a compound of Formula III-x″:
In some embodiments, the compound of Formula I is a compound of Formula III-y′:
In some embodiments, the compound of Formula I is a compound of Formula III-y″:
In some embodiments, the compound of Formula I is a compound of Formula III-z′:
In some embodiments, the compound of Formula I is a compound of Formula III-z″:
In some embodiments, the compound of Formula I is a compound of Formula III-a:
In some embodiments, the compound of Formula I is a compound of Formula III-b:
In some embodiments, the compound of Formula I is a compound of III-c:
In some embodiments, the compound of Formula I is a compound of Formula III-d:
In some embodiments, the compound of Formula I is a compound of Formula III-e:
In some embodiments, the compound of Formula I is a compound of Formula III-f:
In some embodiments, the compound of Formula I is a compound of Formula III-g:
In some embodiments, the compound of Formula I is a compound of Formula III-h:
In some embodiments, the compound of Formula I is a compound of Formula III-i:
In some embodiments, the compound of Formula I is a compound of Formula III-j:
In some embodiments, the compound of Formula I is a compound of Formula III-k:
In some embodiments, the compound of Formula I is a compound of Formula III-l:
In some embodiments, the compound of Formula I is a compound of Formula III-m:
In some embodiments, the compound of Formula I is a compound of Formula III-n:
In some embodiments, the compound of Formula I is a compound of Formula III-o:
In some embodiments, the compound of Formula I is a compound of Formula III-p:
In some embodiments, the compound of Formula I is a compound of Formula III-q:
In some embodiments, the compound of Formula I is a compound of Formula III-r:
In some embodiments, the compound of Formula I is a compound of Formula III-s:
In some embodiments, the compound of Formula I is a compound of Formula III-t:
In some embodiments, the compound of Formula I is a compound of Formula III-u′:
In some embodiments, the compound of Formula I is a compound of Formula III-u″:
In some embodiments, the compound of Formula I is a compound of Formula III-v′:
In some embodiments, the compound of Formula I is a compound of Formula III-v″:
In some embodiments, the compound of Formula I is a compound of Formula III-w′:
In some embodiments, the compound of Formula I is a compound of Formula III-w″:
In some embodiments, the compound of Formula I is a compound of Formula III-x′:
In some embodiments, the compound of Formula I is a compound of Formula III-x″:
In some embodiments, the compound of Formula I is a compound of Formula III-y′:
In some embodiments, the compound of Formula I is a compound of Formula III-y″:
In some embodiments, the compound of Formula I is a compound of Formula III-z′:
In some embodiments, the compound of Formula I is a compound of Formula III-z″:
In some embodiments, the compound of Formula I is a compound of Formula IV-a-IV-i:
In some embodiments, the compound of Formula I is a compound of Formula V-a:
In some embodiments, the compound of Formula I is a compound of Formula V-b:
In some embodiments, the compound of Formula I is a compound of Formula V-c:
In some embodiments, the compound of Formula I is a compound of Formula V-d:
In some embodiments, the compound of Formula I is a compound of Formula V-e:
In some embodiments, the compound of Formula I is a compound of Formula V-f:
In some embodiments, the compound of Formula I is a compound of Formula V-g:
In some embodiments, the compound of Formula I is a compound of Formula V-h:
In some embodiments, the compound of Formula I is selected from one of those depicted in Table 1 or Table 1a, or a pharmaceutically acceptable salt thereof. Table 1 or Table 1a, identifies compounds by their IUPAC name and Table 2 or Table 2a lists the same compounds and shows their chemical structure. In the event of any discrepancy between Table 1's or Table 1a's name for a compound and Table 2's or Table 2a's structure for that same compound, Table 2's or Table 2a's compound structures will dominate and identify the compound corresponding to each respective compound number (I—#) in Table 1 or Table 1a.
In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration (e.g. by injection, infusion, transdermal or topical administration), and rectal administration, in particular oral administration. Topical administration may also pertain to inhalation or intranasal application. The pharmaceutical compositions of the present invention can be made up in a solid form (including, without limitation, capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including, without limitation, solutions, suspensions or emulsions). Tablets may be either film coated or enteric coated according to methods known in the art. Typically, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of:
Typical approaches to solubilize compounds for parenteral administration are the optimization of the pH or the use of co-solvents (e.g. PEG300, PEG400, propylene glycol, or ethanol). If these approaches are, for any reason, not feasible, the use of surfactants may be considered (e.g. Tween® 80 or Cremophor EL®). Cyclodextrins are established as safe solubilizing agents. Compounds with a high solubility in natural oils may be solubilized in parenteral fat emulsions.
There is also provided a pharmaceutical composition comprising a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.
The compounds of Formula I of the present invention in free form or in pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, e.g. WRN inhibiting properties, e.g. as indicated in vitro tests as provided in the next sections, and are therefore indicated for therapy, or for use as research chemicals, e.g. as a chemical probe, and as tool compounds.
Also provided is a compound of Formula I, as described herein. Said compound can be used as a research chemical, a compound herein comprising an added biotin moiety, for example a tool compound or chemical probe, in particular for research on WRN. In another embodiment there is provided the use of a compound of Formula I, as described herein, as a research chemical, for example tool compound or chemical probe, in particular for research on WRN.
There is also provided a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. Cancers that may be treated by WRN inhibition include cancers that are characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR). In particular, a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, may be useful in the treatment of a cancer that is characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR).
There is also provided a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, for use as a medicament. In particular, said use is:
There is also provided a method of:
There is also provided the use of a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof:
In some embodiments, the subject has or is identified as having a microsatellite instable (MSI-H) cancer, e.g., in reference to a control, e.g., a normal, subject. In one embodiment, the subject has MSI-H advanced solid tumors, a colorectal cancer (CRC), endometrial, uterine, stomach or other MSI-H cancer. In some embodiments, the subject has a colorectal (CRC), endometrial or stomach cancer, which cancer has or is identified as having a microsatellite instability (MSI-H), e.g., in reference to a control, e.g., a normal, subject. Such identification techniques are known in the art.
Depending on the choice of the starting materials and procedures, the compounds can be present in the form of one of the possible stereoisomers or as mixtures thereof, for example as pure optical isomers, or as stereoisomer mixtures, such as racemates and diastereoisomer mixtures, depending on the number of asymmetric carbon atoms. The present invention is meant to include all such possible stereoisomers, including racemic mixtures, diasteriomeric mixtures and optically pure forms. Optically active (R)- and (S)-stereoisomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have a cis- or trans-configuration. All tautomeric forms are also intended to be included.
Any formula given herein is intended to represent unlabeled forms as well as isotopically labeled forms of the compounds, in addition to the deuteration specifically claimed in Formula I. Isotopically labeled compounds have structures depicted by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Isotopes that can be incorporated into compounds of the invention include, for example, isotopes of hydrogen.
Further, incorporation of certain isotopes, particularly deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index or tolerability. It is understood that deuterium in this context is regarded as a substituent of a compound of the present invention. The concentration of deuterium, may be defined by the isotopic enrichment factor. The term “isotopic enrichment factor” as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this invention is denoted as being deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). It should be understood that the term “isotopic enrichment factor” can be applied to any isotope in the same manner as described for deuterium.
Other examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 3H, 11C, 13C, 14C, 15N, 18F 31P, 32P, 35S, 36Cl, 123I, 124I, and 125I, respectively. Accordingly it should be understood that the invention includes compounds that incorporate one or more of any of the aforementioned isotopes, including for example, radioactive isotopes, such as 3H and 14C, or those into which non-radioactive isotopes, such as 2H and 13C are present. Such isotopically labelled compounds are useful in metabolic studies (with 14C), reaction kinetic studies (with, for example 2H or 3H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an 18F or labeled compound may be particularly desirable for PET or SPECT studies. Isotopically-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.
A “compound of the present invention” or a “compound of Formula I” includes a zwitterion thereof, a non-zwitterion thereof (non-charged form), or a pharmaceutically acceptable salt of said zwitterionic or non-zwitterionic form thereof. “Zwitterion” or “zwitterionic form” means a compound containing both positive and negatively charged functional groups.
The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to colorectal, gastric, endometrial, prostate, adrenocortical, uterine, cervical, esophageal, breast, kidney, ovarian cancer and the like.
The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors. [00264]“WRN inhibitor” or “WRN helicase inhibitor” as used herein means a compound that inhibits Werner Syndrome RecQ DNA helicase (WRN). The term “WRN” as used herein refers to the protein of Werner Syndrome RecQ DNA helicase. The term “WRN” includes mutants, fragments, variants, isoforms, and homologs of full-length wild-type WRN. In one embodiment, the protein is encoded by the WRN gene (Entrez gene ID 7486; Ensembl ID ENSG00000165392). Exemplary WRN sequences are available at the Uniprot database under accession number Q14191.
“Disease or condition mediated by WRN” includes a disease or condition, such as cancer, which is treated by WRN inhibition. In particular this can include cancers characterized as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR).
“Microsatellite unstable cancer,” “microsatellite instability-high cancer,” “microsatellite high cancer” and “MSI-high cancer,” “MSIhi” and “MSI-H” when used herein, are used interchangeably, and describe cancers that have a high number of alterations in the length of simple repetitive genomic sequences within microsatellites.
The determination of MSI-H or dMMR tumor status for patients can be performed using, e.g., polymerase chain reaction (PCR) tests for MSI-H status or immunohistochemistry (IHC) tests for dMMR. Methods for identification of MSI-H or dMMR tumor status are described, e.g., in Ryan et al. Crit Rev Oncol Hematol. 2017; 116:38-57; Dietmaier and Hofstadter. Lab Invest 2001, 81:1453-1456; and Kawakami et al. Curr Treat Options Oncol. 2015; 16(7): 30).
Microsatellite instability can be found in colorectal cancer, gastric cancer and endometrial cancer in particular, but also in adrenocortical, uterine, cervical, esophageal, breast, kidney, prostate and ovarian cancers. Examples of microsatellite high cancers include uterine corpus endometrial carcinoma, colon adenocarcinoma, stomach adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, esophageal carcinoma, breast carcinoma, kidney renal clear cell carcinoma and ovarian serous cystadenocarcinoma.
A cancer that has “defective mismatch repair” (dMMR) or “dMMR character” includes cancer types associated with documented MLH1, PMS2, MSH2, MSH3, MSH6, MLH3, and PMS1 mutations or epigenetic silencing, microsatellite fragile sites, or other gene inactivation mechanisms, including but not limited to cancers of the lung, breast, kidney, large intestine, ovary, prostate, upper aerodigestive tract, stomach, endometrium, liver, pancreas, haematopoietic and lymphoid tissue, skin, thyroid, pleura, autonomic ganglia, central nervous system, soft tissue, pediatric rhabdoid sarcomas, melanomas and other cancers. A cell or cancer with “defective” mismatch repair has a significantly reduced (e.g., at least about 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% decrease) amount of mismatch repair. In some cases, a cell or cancer which is defective in mismatch repair will perform no mismatch repair.
As used herein, the term “pharmaceutical composition” refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier, in a form suitable for oral or parenteral administration.
As used herein, the term “pharmaceutically acceptable carrier” refers to a substance useful in the preparation or use of a pharmaceutical composition and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffering agents, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegration agents, lubricants, wetting agents, sweetening agents, flavoring agents, dyes, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22nd Ed. Pharmaceutical Press, 2013, pp. 1049-1070).
The terms “synthetic lethality,” and “synthetically lethal” are used to refer to reduced cell viability and/or a reduced rate of cell proliferation caused by a combination of mutations or approaches to cause loss of function (e.g., RNA interference or protein function inhibition) in two or more genes but not by the loss of function of only one of these genes.
The term “a therapeutically effective amount” of a compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In some embodiments, the methods of the invention comprise administration of a therapeutically effective amount of a compound herein.
In one embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviate, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by WRN, or (ii) associated with WRN activity, or (iii) characterized by activity (normal or abnormal) of WRN; or (2) reduce or inhibit the activity of WRN.
In another embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of WRN, or reducing WRN protein levels.
As used herein, the term “subject” refers to primates (e.g., humans, male or female), dogs, rabbits, guinea pigs, pigs, rats and mice. In certain embodiments, the subject is a primate, a rat or a mouse. In yet other embodiments, the subject is a human.
As used herein, the term “inhibit,” “inhibition” or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
As used herein, the term “treat,” “treating” or “treatment” of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient.
As used herein, the term “prevent,” “preventing” or “prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.
As used herein, a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
“May join” means joins or does not join.
“May be replaced by deuterium” means is replaced by deuterium, or is not replaced by deuterium.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 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”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.
Any asymmetric atom (e.g., carbon or the like) of the compound(s) of the present invention can be present in racemic or enantiomerically enriched, for example the (R)-, (S)- or (R, S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)-configuration. Substituents at atoms with unsaturated double bonds may, if possible, be present in cis-(Z)— or trans-(E)-form.
Accordingly, as used herein a compound of the present invention can be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) stereoisomers, diastereomers, optical isomers (antipodes), racemates or mixtures thereof.
Any resulting mixtures of stereoisomers can be separated on the basis of the physicochemical differences of the constituents, into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and/or fractional crystallization.
Any resulting racemates of compounds of the present invention or of intermediates can be resolved into the optical antipodes by known methods, e.g., by separation of the diastereomeric salts thereof, obtained with an optically active acid or base, and liberating the optically active acidic or basic compound. In particular, a basic moiety may thus be employed to resolve the compounds of the present invention into their optical antipodes, e.g., by fractional crystallization of a salt formed with an optically active acid, e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di-O,O′-p-toluoyl tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. Racemic compounds of the present invention or racemic intermediates can also be resolved by chiral chromatography, e.g., high pressure liquid chromatography (HPLC) using a chiral adsorbent.
Compounds of the invention, i.e. compounds of Formula I that contain groups capable of acting as donors and/or acceptors for hydrogen bonds may be capable of forming co-crystals with suitable co-crystal formers. These co-crystals may be prepared from compounds of Formula I by known co-crystal forming procedures. Such procedures include grinding, heating, co-subliming, co-melting, or contacting in solution compounds of Formula I with the co-crystal former under crystallization conditions and isolating co-crystals thereby formed. Suitable co-crystal formers include those described in WO 2004/078163. Hence the invention further provides co-crystals comprising a compound of Formula I.
Furthermore, the compounds of the present invention, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.
The compounds of the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, it is intended that the invention embrace both solvated and unsolvated forms. The term “solvate” refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g., water, ethanol, and the like. The term “hydrate” refers to the complex where the solvent molecule is water.
The pharmaceutical composition or combination of the present invention may, for example, be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg.
“Combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a combination partner (e.g. another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non-fixed combinations of the therapeutic agents. The term “fixed combination” means that the therapeutic agents, e.g. a compound of the present invention and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage.
The term “non-fixed combination” means that the therapeutic agents, e.g. a compound of the present invention and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more therapeutic agents.
The combinations described herein can include a compound of Formula I and one or more additional therapeutic agents, e.g., one or more anti-cancer agents, cytotoxic or cytostatic agents, hormone treatment, vaccines, and/or other immunotherapies. In other embodiments, the combination is further administered or used in combination with other therapeutic treatment modalities, including surgery, radiation, cryosurgery, and/or thermotherapy. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the treatment.
There is also provided a combination comprising a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, as described herein, and one or more additional therapeutically active agents. The additional therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment or nucleic acid, which is therapeutically active or enhances the therapeutic activity when administered to a patient in combination with a compound of the present disclosure. In particular, an additional therapeutically active agent is:
In a further embodiment, the additional therapeutically active agent is the chemotherapy irinotecan (Camptosar®).
In another embodiment, the additional therapeutically active agent is an inhibitor of PD-1, e.g., human PD-1. In another embodiment, the immunomodulator is an inhibitor of PD-L1, e.g., human PD-L1. In one embodiment, the inhibitor of PD-1 or PD-L1 is an antibody molecule to PD-1 or PD-L1. In another embodiment, the additional therapeutically active agent is an anti-PD-1 antibody molecule.
In a further embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule as described in US 2015/0210769, published on Jul. 30, 2015, entitled “Antibody Molecules to PD-1 and Uses Thereof.”
In another embodiment, there is provided a combination of a compound of Formula I or a pharmaceutically acceptable salt thereof, and a chemotherapy, and a PD-1 inhibitor. In particular, the chemotherapy and PD-1 inhibitor are selected from those described above. In some embodiments, the PD-1 inhibitor is pembrolizumab, nivolumab, cemiplimab, dostarlimab, or retifanlimab.
The above-mentioned compounds, which can be used in combination with a compound of the present invention, can be prepared and administered as described in the art, such as in the documents cited above.
In one embodiment, the invention provides a product comprising a compound of the present invention and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a disease or condition mediated by WRN. Products provided as a combined preparation include a composition comprising the compound of Formula I and the other therapeutic agent(s) together in the same pharmaceutical composition, or the compound of the present invention and the other therapeutic agent(s) in separate form, e.g. in the form of a kit.
In one embodiment, the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of the present invention. In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like.
The kit of the invention may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the invention typically comprises directions for administration.
In the combination therapies of the invention, the compound of the present invention and the other therapeutic agent may be manufactured and/or formulated by the same or different manufacturers. Moreover, the compound of the present invention and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the compound of the present invention and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the compound of the present invention and the other therapeutic agent.
Accordingly, the invention provides the use of a compound of the present invention for treating a disease or condition mediated by WRN, wherein the medicament is prepared for administration with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition mediated by WRN, wherein the medicament is administered with a compound of the present invention.
The invention also provides a compound of the present invention for use in treating a disease or condition mediated by WRN, wherein the compound of the present invention is prepared for administration with another therapeutic agent. The invention also provides another therapeutic agent for use in treating a disease or condition mediated by WRN, wherein the other therapeutic agent is prepared for administration with a compound of the present invention. The invention also provides a compound of the present invention for use in treating a disease or condition mediated by WRN, wherein the compound of the present invention is administered with another therapeutic agent. The invention also provides another therapeutic agent for use in a method of treating a disease or condition mediated by WRN, wherein the other therapeutic agent is administered with a compound of the present invention.
The invention also provides the use of a compound of the present invention for treating a disease or condition mediated by WRN, wherein the patient has previously (e.g. within 24 hours) been treated with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition mediated by WRN, wherein the patient has previously (e.g. within 24 hours) been treated with compound of the present invention.
Compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying synthetic schemes.
Those having ordinary skill in the art will be able to adapt such synthetic procedures to afford variably substituted compounds of Formula I for synthesis of the compounds of the disclosure.
As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the procedures provided herein. It will be appreciated that, although the methods depict the synthesis of certain compounds of the present disclosure, the methods, and other methods known to one of ordinary skill in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein.
Synthesis of 2-(2-(dimethylamino)-6-ethyl-7-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-8-oxopyrido[2,3-b]pyrazin-5(8H)-yl)-N-(3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl)acetamide (I-1)
To a mixture of 2-(2-(dimethylamino)-6-ethyl-7-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-8-oxopyrido[2,3-b]pyrazin-5(8H)-yl)acetic acid (Intermediate-91) (20 mg, 40 mol, 1.0 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine hydrochloride (15 mg, 81 mol, 2.0 eq) in DMF (1 mL) was added HATU (31 mg, 81 mol, 2.0 eq) and DIEA (16 mg, 121 mol, 3.0 eq), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (5 mL*3). The combined organic layers were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by reverse phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
LCMS: 630.3 [M+H]+.
1H NMR (400 MHz, CDCl3) δ ppm 12.07 (s, 1H), 8.58 (s, 1H), 8.23 (s, 1H), 6.84-6.52 (m, 1H), 5.64-5.37 (m, 1H), 5.15 (br d, 2H), 4.86-4.60 (m, 1H), 4.06-3.87 (m, 2H), 3.62-3.42 (m, 1H), 3.24 (s, 6H), 3.18-3.04 (m, 3H), 2.87-2.75 (m, 2H), 2.57 (s, 3H), 2.29 (s, 6H), 1.28 (br t, 3H).
Synthesis of rel-2-(2-(dimethylamino)-6-ethyl-7-((1S,6S)-5-(5-hydroxy-6-methylpyrimidine-4-carbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl)-8-oxopyrido[2,3-b]pyrazin-5(8H)-yl)-N-(3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl)acetamide (I-2)
To a solution of rel-2-(2-(dimethylamino)-6-ethyl-7-((1S,6S)-5-(5-hydroxy-6-methylpyrimidine-4-carbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl)-8-oxopyrido[2,3-b]pyrazin-5(8H)-yl)acetic acid (rel-Intermediate-62) (17 mg, 33 mol, 1.0 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine hydrochloride (12 mg, 65 mol, 2.0 eq) in DMF (1 mL) was added HATU (25 mg, 65 mol, 2.0 eq) and DIEA (13 mg, 98 mol, 3.0 eq), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (5 mL*3). The combined organic layers were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
LCMS: 656.5 [M+H]+.
1H NMR (400 MHz, CDCl3) δ ppm 12.60 (s, 1H), 8.74-8.49 (m, 1H), 8.32 (br s, 1H), 6.90-6.43 (m, 1H), 5.75-4.79 (m, 3H), 4.39-4.08 (m, 2H), 4.00-3.51 (m, 2H), 3.40 (br s, 1H), 3.31 (s, 6H), 3.28-3.22 (m, 1H), 3.13 (br s, 1H), 2.55 (s, 3H), 2.29 (s, 8H), 1.56-1.36 (m, 2H), 1.30 (br t, 3H).
To a solution of 2-(7-ethyl-2-methyl-5-oxo-6-(piperazin-1-yl)pyrido[2,3-b]thieno[3,2-e]pyrazin-8(5H)-yl)-N-(3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl)acetamide trifluoroacetate (Intermediate-83) (120 mg, 189 mol, 1 eq) and sodium 5-hydroxy-6-methylpyrimidine-4-carboxylate (Intermediate-55) (155 mg, 880 mol, 4.6 eq) in pyridine (3 mL) was added EDCI (181 mg, 946 mol, 5 eq). The mixture was stirred at 60° C. for 6 h. The mixture was concentrated in vacuum directly to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) and reverse Phase HPLC (C18 column, water (10 mmol/L FA-ACN) to afford the title compound.
1H NMR (400 MHz, CD3OD) δ ppm 8.57 (s, 1H), 7.36 (s, 1H), 5.32 (s, 2H), 4.76-4.65 (m, 1H), 4.19-3.88 (m, 3H), 3.53-3.39 (m, 1H), 3.23 (q, 2H), 3.19-3.11 (m, 1H), 2.96-2.85 (m, 1H), 2.78-2.74 (m, 4H), 2.53 (s, 3H), 2.29 (s, 6H), 1.33 (t, 3H).
LCMS: 657.3 [M+H]+.
To a stirred mixture of [2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-{4-hydroxy-2H,3H-furo[2,3-c]pyridine-5-carbonyl}piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]acetic acid (Intermediate-81) (40 mg, 73 mol, 1.0 eq) and 3-cyclopropyl bicyclo[1.1.1]pentan-1-amine (11 mg, 88 mol, 1.2 eq) in THE (4 mL) were added HATU (33 mg, 88 mol, 1.2 eq) and DIEA (28 mg, 219 mol, 3.0 eq) at room temperature under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred for 3 h at room temperature under N2 atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) and concentrated under reduced pressure to give a residue. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.92 (s, 1H), 8.85 (s, 1H), 7.70 (s, 1H), 6.87-6.74 (m, 1H), 4.86 (s, 2H), 4.65 (t, 2H), 4.54 (s, 1H), 4.30-4.25 (m, 2H), 4.20 (s, 1H), 3.81 (t, 2H), 3.55-3.43 (m, 2H), 3.19 (t, 2H), 3.05-2.64 (m, 6H), 2.50 (s, 2H), 1.75 (s, 6H), 1.14 (t, 3H), 0.93-0.86 (m, 1H), 0.40-0.34 (m, 2H), 0.11-0.06 (m, 2H).
LCMS: 657.3 [M+H]+.
To a stirred mixture of 2-[5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]-N-[3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl]acetamide hydrochloride (Intermediate-80) (40 mg, 69 mol, 1.0 eq) and 6-hydroxy-1,3-benzoxazole-7-carboxylic acid (Intermediate-26) (25 mg, 138 mol, 2.0 eq) in THE (2.5 mL) were added DIEA (27 mg, 207 mol, 3.0 eq) and HATU (32 mg, 83 mol, 1.2 eq) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) and concentrated under reduced pressure to give a residue. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.31 (s, 1H), 9.23 (s, 1H), 8.59 (s, 1H), 8.35 (d, 1H), 7.66-7.59 (m, 2H), 7.37 (d, 1H), 6.97 (d, 1H), 4.98 (s, 2H), 4.63 (d, 1H), 3.93 (s, 3H), 3.54-3.40 (m, 3H), 3.33 (s, 1H), 3.04-2.79 (m, 4H), 2.64 (s, 1H), 2.25 (s, 6H), 1.16 (t, 3H).
LCMS: 708.3 [M+H]+.
To a stirred solution of [2-(1-acetyl-3,6-dihydro-2H-pyridin-4-yl)-5-ethyl-6-(4-{4-hydroxy-2H,3H-furo[2,3-c]pyridine-5-carbonyl}piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]acetic acid (Intermediate-36) (35 mg, 59 mol, 1.0 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine (10 mg, 71 mol, 1.2 eq) in THE (2 mL) were added DIEA (38 mg, 295 mol, 5.0 eq) and HATU (34 mg, 88 mol, 1.5 eq) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) and concentrated under reduced pressure to give a residue. The residue was purified by reverse Phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 11.12 (s, 1H), 9.18 (d, 1H), 7.75 (s, 1H), 6.78 (m, 1H), 4.91 (s, 2H), 4.67 (t, 2H), 4.55 (s, 1H), 4.26-4.06 (m, 3H), 3.66 (d, 2H), 3.50-3.46 (m, 3H), 3.22 (t, 3H), 3.05-2.83 (m, 3H), 2.70 (d, 3H), 2.25 (s, 6H), 2.07 (d, 3H), 1.15 (t, 3H).
LCMS: 726.2 [M+H]+.
To a solution of 2-[5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-6-piperazin-1-yl-oxazolo[5,4-b]pyridin-4-yl]-N-[3-(trifluoromethyl)-1-bicyclo[1.1.1]pentanyl]acetamide hydrochloride (Intermediate-13b) (20 mg, 34 mol, 1 eq) and sodium 5-hydroxy-6-methylpyrimidine-4-carboxylate (Intermediate-55) (34 mg, 172 mol, 5 eq) in pyridine (0.2 mL) was added EDCI (26 mg, 137 mol, 4 eq). The reaction mixture was stirred at 25° C. for 12 h. The reaction mixture was poured into water (10 mL) and extracted with DCM (10 mL*3). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO2, MeOH/DCM=10:1) to afford the title compound.
1H NMR (400 MHz, CD3OD) δ ppm 8.29 (d, 1H), 8.14 (s, 1H), 7.57 (d, 1H), 7.40 (s, 1H), 5.07 (s, 2H), 4.66-4.63 (m, 1H), 3.97 (s, 3H), 3.94-3.79 (m, 2H), 3.72-3.59 (m, 1H), 3.48-3.44 (m, 1H), 3.19-3.08 (m, 1H), 3.04 (q, 2H), 2.86-2.82 (m, 1H), 2.70-2.66 (m, 1H), 2.42 (s, 3H), 2.32 (s, 6H), 1.24 (t, 3H).
LCMS: 683.3 [M+H]+.
To a solution of 2-[5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-6-piperazin-1-yl-oxazolo[4,5-b]pyridin-4-yl]-N-[3-(trifluoromethyl)-1 bicyclo[1.1.1]pentanyl]acetamide hydrochloride (Intermediate-15) (80 mg, 137 mol, 1 eq) and sodium 5-hydroxy-6-methylpyrimidine-4-carboxylate (Intermediate-55) (54 mg, 274 mol, 2 eq) in pyridine (1 mL) was added EDCI (79 mg, 412 mol, 3 eq). The mixture was stirred at 25° C. for 4 h. To the reaction mixture was added H2O (10 mL). Then the mixture was extracted with EtOAc (10 mL*3). The combined organic phase was washed with brine (15 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by Prep-TLC (SiO2, DCM:MeOH=10:1) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 11.82 (s, 1H), 8.57 (s, 1H), 8.31 (d, 1H), 7.54 (d, 1H), 7.43 (s, 1H), 6.81 (s, 1H), 5.63-5.60 (m, 1H), 5.00 (s, 2H), 4.84-4.73 (m, 1H), 4.04 (q, 2H), 3.99 (s, 3H), 3.55-3.42 (m, 1H), 3.04-3.02 (m, 3H), 2.89-2.75 (m, 2H), 2.57 (s, 3H), 2.34 (s, 6H), 1.26 (t, 3H).
LCMS: 683.2 [M+H]+.
To a stirred mixture of 2-(6-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl)-N-(3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl)acetamide hydrochloride (Intermediate-20) (60 mg, 105 mol, 1.0 eq) and 6-hydroxy-1,3-benzoxazole-7-carboxylic acid (Intermediate-26) (23 mg, 126 mol, 1.2 eq) in pyridine (5 mL) was added EDCI (40 mg, 210 mol, 2.0 eq) at room temperature under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred for 5 h at 60° C. The mixture was cooled to room temperature and concentrated under vacuum. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) and concentrated under reduced pressure to give a residue. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
LCMS: 734.4 [M+H]+.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.18 (s, 1H), 9.28 (s, 1H), 8.59 (s, 1H), 8.42 (d, 1H), 7.74-7.68 (m, 1H), 7.62 (d, 1H), 7.43 (d, 1H), 6.95 (t, 1H), 4.94 (s, 2H), 4.48 (d, 1H), 3.96 (s, 3H), 3.94-3.83 (m, 1H), 3.75 (d, 1H), 3.53 (d, 1H), 3.42 (s, 1H), 3.19 (s, 1H), 3.00-2.90 (m, 2H), 2.25 (s, 6H), 1.48 (d, 1H), 1.43-1.33 (m, 1H), 1.24 (s, 2H), 1.16 (d, 3H).
To a stirred solution of 2-(5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-6-(piperazin-1-yl)thiazolo[4,5-b]pyridin-4(7H)-yl)-N-(3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl) acetamide hydrochloride (Intermediate-14) (80 mg, 142 mol, 1.0 eq) and 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (Intermediate-13) (26 mg, 170 mol, 1.2 eq) in pyridine (5 mL) was added EDCI (82 mg, 426 mol, 3.0 eq) at room temperature under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred for 4 h at room temperature under N2 atmosphere. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) and concentrated under reduced pressure to give a residue. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.27 (s, 0.577H, partially deuterated), 9.32 (s, 1H), 8.53 (s, 1H), 8.40 (d, 1H), 7.59 (d, 1H), 7.44 (s, 1H), 5.16 (s, 2H), 4.51 (d, 1H), 3.95 (s, 3H), 3.78-3.65 (m, 2H), 3.48 (d, 1H), 3.27-3.20 (m, 1H), 3.04-2.90 (d, 3H), 2.77 (d, 1H), 2.58 (d, 1H), 2.44 (s, 3H), 2.25 (s, 6H), 1.18 (t, 3H).
LCMS: 699.2 [M+H]+.
To a stirred mixture of 2-[2-(dimethylamino)-5-ethyl-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]-N-[3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl]acetamide hydrochloride (Intermediate-28) (50.0 mg, 97 mol, 1.0 eq) and 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (Intermediate-13) (19 mg, 116 mol, 1.2 eq) in THF (2.5 mL) were added DIEA (38 mg, 289 mol, 3.0 eq) and HATU (44 mg, 116 mol, 1.2 eq) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) and concentrated under reduced pressure to give a residue. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.03 (s br, 1H), 9.18 (s, 1H), 8.52 (s, 1H), 4.80 (s, 2H), 4.59-4.46 (m, 1H), 3.55-3.43 (m, 3H), 3.23 (d, 1H), 2.96 (s, 7H), 2.85-2.69 (m, 3H), 2.57 (d, 1H), 2.43 (s, 3H), 2.24 (s, 6H), 1.11 (t, 3H).
LCMS: 619.4 [M+H]+.
Step 1 to step 8 can be performed as described in the synthesis of Intermediates 16, 17, 19 of the current document.
To a stirred solution of tert-butyl (1S,6S)-5-(4-(2-ethoxy-2-oxoethyl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4,7-dihydro-2H-[1,2,3]triazolo[4,5-b]pyridin-6-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (Intermediate-19) (200 mg, 0.35 mmol, 1.00 eq) in DCM (10.0 mL) was added 4 M HCl in 1,4-dioxane (2.00 mL, 8.00 mmol, 22.9 eq) at room temperature. After stirring for 1 h at room temperature, the reaction mixture was concentrated under reduced pressure to afford the title compound, which was used in the next step directly without purification. LCMS: 468.0[M+H]+.
To a mixture of sodium 5-hydroxy-6-methylpyrimidine-4-carboxylate (Intermediate-55) (156 mg, 0.89 mmol, 2.60 eq) and pyridine hydrochloride (117 mg, 1.02 mmol, 3.00 eq) in DCM (3.00 mL) were added a mixture of ethyl 2-(6-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl)acetate hydrochloride (170 mg, 0.34 mmol, 1.00 eq) and DIEA (131 mg, 1.02 mmol, 3.00 eq) in DCM (3.00 mL), followed by the addition of EDCI (390 mg, 2.03 mmol, 6.00 eq) at room temperature. After stirring for 1 h at 40° C., the reaction mixture was allowed to cool down to room temperature. The resulting mixture was filtered and the filter cake was washed with DCM. The combined filtrate was concentrated under reduced pressure to afford the title compound, which was used in the next step directly without purification. LCMS: 604.0[M+H]+.
To a solution of ethyl 2-(5-ethyl-6-((1S,6S)-5-(5-hydroxy-6-methylpyrimidine-4-carbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl)-2-(2-methoxypyridin-4-yl)-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl)acetate (500 mg, 0.22 mmol, 1.00 eq, 26% purity) in MeOH (5.00 mL) was added a solution of NaOH (167 mg, 4.17 mmol, 19.4 eq) in H2O (1.00 mL). After stirring for 2 h at room temperature, the reaction mixture was diluted with H2O (5 mL) and acidified to pH 3 with 1 N HCl (aq.) at 0° C. The resulting mixture was extracted with EtOAc (5×10 mL). The combined organic layers were washed with brine (1×25 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase HPLC (C18 column, water (0.1% FA)-ACN) to afford the title compound. LCMS: 576.0[M+H]+.
A mixture of 3-cyclopropylbicyclo[1.1.1]pentan-1-amine hydrochloride (33 mg, 0.21 mmol, 1.20 eq) in pyridine (5.00 mL) was stirred for 5 min at room temperature. To the above mixture was added 2-(5-ethyl-6-((1S,6S)-5-(5-hydroxy-6-methylpyrimidine-4-carbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl)-2-(2-methoxypyridin-4-yl)-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl)acetic acid (100 mg, 0.17 mmol, 1.00 eq) and EDCI (67 mg, 0.35 mmol, 2.00 eq) at room temperature. After stirring for additional 1 h at room temperature, the resulting mixture was concentrated under reduced pressure. The residue was purified twice by reverse phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN). After lyophilization, the resulting mixture was purified by Prep-TLC (Eluent of MeOH/DCM) to afford the title compound. LCMS: 681.2 [M+H]*. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.36 (br s, 1H), 8.95 (s, 1H), 8.60-8.56 (m, 1H), 8.42 (d, 1H), 7.71 (dd, 1H), 7.42 (d, 1H), 4.99-4.80 (m, 2H), 4.48-4.45 (m, 1H), 3.96 (s, 3H), 3.88-3.82 (m, 1H), 3.56-3.29 (m, 3H), 3.25-3.16 (m, 1H), 3.05-2.85 (m, 2H), 2.44 (s, 3H), 1.76 (s, 6H), 1.61-1.53 (m, 1H), 1.40-1.28 (m, 2H), 1.27-1.11 (m, 4H), 0.94-0.85 (m, 1H), 0.41-0.33 (m, 2H), 0.11-0.05 (m, 2H).
To a solution of tert-butyl (1S,6S)-5-[2-(3,6-dihydro-2H-pyran-4-yl)-4-(2-ethoxy-2-oxoethyl)-5-ethyl-7-oxo-[1,2,3]triazolo[4,5-b]pyridin-6-yl]-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (Intermediate-105) (140 mg, 258 μmol, 1.00 eq) in DCM (1.00 mL) was added 4 M HCl solution in 1,4-dioxane (1.00 mL, 4.00 mmol, 15.5 eq) and the reaction mixture was stirred at room temperature for 1h. The resulting mixture was concentrated under reduced pressure to afford the title compound, which was used in the next step without further purification. LCMS: 443[M+H]+.
To a stirred mixture of ethyl 2-(6-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl) acetate hydrochloride (80 mg, 167 μmol, 1.00 eq) and sodium 5-hydroxy-6-methylpyrimidine-4-carboxylate (Intermediate-55) (59 mg, 334 μmol, 2.00 eq) in Py (1.60 mL) was added HATU (127 mg, 334 μmol, 2.00 eq) at room temperature. The resulting mixture was stirred at room temperature for 30 min. The resulting mixture was concentrated under reduced pressure. The residue was diluted with water (10 mL), and then was extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 579[M+H]+.
To a stirred mixture of ethyl 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-((1S,6S)-5-(5-hydroxy-6-methylpyrimidine-4-carbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl)-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl) acetate (70 mg, 121 μmol, 1.00 eq) in THE (0.70 mL) and H2O (0.70 mL) was added NaOH (10 mg, 242 μmol, 2.00 eq) at room temperature. The resulting mixture was stirred at room temperature for 1h. The resulting mixture was diluted with water (5 mL), and then was acidified to pH 5 with 1N HCl (aq.). The resulting mixture was extracted with EtOAc (3×5.00 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure, which was used in the next step without further purification. LCMS: 551[M+H]+.
To a stirred mixture of 2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-((1S,6S)-5-(5-hydroxy-6-methylpyrimidine-4-carbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl)-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl) acetic acid (50 mg, 91 μmol, 1.00 eq) and 3-cyclopropylbicyclo[1.1.1]pentan-1-amine hydrochloride (17 mg, 107 μmol, 1.18 eq) in THE (1.00 mL) were added DIEA (47 mg, 364 μmol, 4.00 eq) and HATU (52 mg, 137 μmol, 1.50 eq) at room temperature. The resulting mixture stirred at room temperature for 1h. The resulting mixture was poured into water (5 mL) and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase HPLC (C18 Column, (10 mmol/L aqueous NH4HCO3)-ACN) to afford the title compound. LCMS: 656 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.34 (br s, 1H), 8.89 (s, 1H), 8.56 (s, 1H), 6.73 (s, 1H), 4.82 (q, 2H), 4.49-4.33 (m, 3H), 3.94-3.82 (m, 3H), 3.55-3.17 (m, 4H), 2.97-2.75 (m, 4H), 2.44 (s, 3H), 1.75 (s, 6H), 1.59-1.50 (m, 1H), 1.35-1.11 (m, 6H), 0.94-0.82 (m, 1H), 0.40-0.34 (m, 2H), 0.10-0.06 (m, 2H).
To a stirred solution of 2-(6-((1S,6S)-5-(tert-butoxycarbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl)acetic acid (Intermediate-106) (120 mg, 0.23 mmol, 1.00 eq) and 3-isopropylbicyclo[1.1.1]pentan-1-amine hydrochloride (29 mg, 0.18 mmol, 0.78 eq) in THE (1.00 mL) were added HATU (177 mg, 0.47 mmol, 2.00 eq) and DIEA (121 mg, 0.93 mmol, 4.00 eq). The mixture was stirred for 1 h at room temperature. The resulting mixture was diluted with water (5 mL) and extracted with EtOAc (3×5 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 622[M+H]+.
To the solution of tert-butyl (1S,6S)-5-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-4-(2-((3-isopropylbicyclo[1.1.1]pentan-1-yl)amino)-2-oxoethyl)-7-oxo-4,7-dihydro-2H-triazolo[4,5-b]pyridin-6-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (120 mg, 0.19 mmol, 1.00 eq) in DCM (2.00 mL) was added 4M HCl solution in 1,4-dioxane (0.40 mL, 1.60 mmol, 8.29 eq) and the reaction mixture was stirred for 2 h at room temperature. The mixture was concentrated under reduced pressure to afford the title compound, which was used in the next step directly without further purification. LCMS: 522[M+H]+.
To a stirred solution of 2-(6-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-2,7-dihydro-4H-[1,2,3]triazolo[4,5-b]pyridin-4-yl)-N-(3-isopropylbicyclo[1.1.1]pentan-1-yl)acetamide hydrochloride (40 mg, 72 mol, 1.00 eq) and sodium 5-hydroxy-6-methylpyrimidine-4-carboxylate (Intermediate 55) (18 mg, 102 mol, 1.42 eq) in Py (0.50 mL) was added HATU (44 mg, 116 mol, 1.61 eq). The mixture was stirred for 1 h at room temperature and concentrated under reduced pressure. The residue was diluted with water (5 mL) and extracted with EtOAc (3×5 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by reverse phase HPLC (C18 Column, (10 mmol/L aqueous NH4HCO3)-ACN) to afford the title compound. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.89 (s, 1H), 8.50 (s, 1H), 6.74 (s, 1H), 4.83 (s, 2H), 4.45-4.43 (m, 1H), 4.34 (d, 2H), 3.93-3.84 (m, 3H), 3.53-3.42 (m, 1H), 3.38-3.30 (m, 2H), 3.21-3.19 (m, 1H), 2.95-2.85 (m, 2H), 2.80 (s, 2H), 2.42 (s, 3H), 1.76-1.69 (m, 7H), 1.58-1.53 (m, 1H), 1.32-1.25 (m, 2H), 1.17-1.13 (m, 4H), 0.80 (d, 6H).LCMS: 658[M+H]+.
To a solution of PPh3 (64.79 g, 247.0 mmol, 3.0 eq) in 1,4-dioxane (200 mL) was added NCS (33.53 g, 251.1 mmol, 3.05 eq) and the mixture was stirred at 10° C. for 1 h. Then ethyl 3-hydroxy-5-methylpyrazine-2-carboxylate (15.00 g, 82.34 mmol, 1.0 eq) was added and the resulting mixture was stirred at 100° C. overnight. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 201.1 [M+H]-.
To a solution of ethyl 3-chloro-5-methylpyrazine-2-carboxylate (11.70 g, 58.32 mmol, 1.0 eq) in MeOH (40 mL) and H2O (40 mL) was added LiOH—H2O (3.92 g, 93.3 mmol, 1.6 eq) and the reaction was stirred at 5° C. for 20 mins. The reaction mixture was diluted with H2O (40 mL), and then adjusted to pH 3 with aq. 1 N HCl solution. The resulting mixture was extracted with DCM (50 mL*2). The combined organic phase was washed with brine (40 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
To a solution of 3-chloro-5-methylpyrazine-2-carboxylic acid (10.00 g, 57.95 mmol, 1 eq) in DCM (80 mL) was added (COCl)2 (11.03 g, 86.92 mmol, 1.5 eq) and DMF (85 mg, 1.2 mmol, 0.02 eq). The reaction was stirred at room temperature for 2 h under N2 atmosphere. The resulting mixture was concentrated under reduced pressure to afford the title compound, it was used into next step without further purification.
To a solution of butan-2-one (6.23 g, 86.4 mmol, 1.5 eq) in THE (40 mL) was added LDA (2 M in THF, 43.2 mL, 86.4 mmol, 1.5 eq) at −65° C. The reaction mixture was stirred for 5 mins, 3-chloro-5-methylpyrazine-2-carbonyl chloride (11.0 g, 57.6 mmol, 1.0 eq) in THE (40 mL) was added dropwise to the mixture at −65° C. The resulting mixture was warmed to room temperature and stirred at room temperature for 1 h. The reaction was quenched with water (10 mL), adjusted to pH 4 with aq. 1 N HCl solution, and then extracted with EtOAc (50 mL*2). The organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 15.45 (br s, 1H), 8.42 (s, 1H), 6.39 (s, 1H), 2.63 (s, 3H), 2.49 (q, 2H), 1.23 (t, 3H).
To a solution of (Z)-1-(3-chloro-5-methylpyrazin-2-yl)-3-hydroxypent-2-en-1-one (3.00 g, 13.2 mmol, 1.0 eq) in 1,4-dioxane (30 mL) was added DIEA (2.57 g, 19.9 mmol, 1.5 eq) and tert-butyl 2-aminoacetate (1.74 g, 13.2 mmol, 1.0 eq). The reaction was stirred at 100° C. for 2 h. The mixture was diluted with H2O (40 mL), extracted with EtOAc (20 mL*2). The organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 11.23 (br s, 1H), 9.57 (br s, 1H), 7.68 (s, 1H), 6.47 (s, 1H), 4.17 (br d, 2H), 4.02 (d, 2H), 2.39 (s, 3H), 2.33 (q, 2H), 1.51 (s, 9H), 1.48 (s, 9H), 1.23 (t, 3H).
To a solution of tert-butyl (Z)-(1-(3-((2-(tert-butoxy)-2-oxoethyl)amino)-5-methylpyrazin-2-yl)-1-oxopent-2-en-3-yl)glycinate (1.70 g, 3.91 mmol, 1.0 eq) in DCM (15 mL) was added TsOH-H2O (135 mg, 710 μmol, 0.18 eq) and NBS (627 mg, 3.52 mmol, 0.9 eq). The mixture was stirred at room temperature for 0.5 h. The reaction mixture was diluted with H2O (10 mL) and then extracted with DCM (30 mL*2). The organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure to afford the title compound, which was used in the next step without further purification. LCMS: 400.2 [M+H]+.
To a solution of Intermediate-27: tert-butyl (3-(2-bromo-3-oxopentanoyl)-6-methylpyrazin-2-yl)glycinate (1.50 g, 3.75 mmol, 1.0 eq) in THE (9 mL) was added tert-butyl piperazine-1-carboxylate (698 mg, 3.75 mmol, 1.0 eq) and DIEA (969 mg, 7.50 mmol, 2.0 eq) and the mixture was stirred at room temperature for 0.5 h. The reaction mixture was diluted with H2O (10 mL) and then extracted with EtOAc (10 mL*2). The organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound, which was used in the next step without further purification. LCMS: 506.3 [M+H]+.
To a solution of tert-butyl 4-(1-(3-((2-(tert-butoxy)-2-oxoethyl)amino)-5-methylpyrazin-2-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (1.20 g, 2.37 mmol, 1.0 eq) in EtOH (10 mL) was added H3PO4 (465 mg, 4.75 mmol, 2.0 eq) and the resulting mixture was stirred at 60° C. overnight. The reaction mixture was concentrated under reduced pressure. The residue was diluted with H2O (10 mL) and then extracted with EtOAc (10 mL*2). The organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 488.3 [M+H]+.
To a solution of tert-butyl 4-(5-(2-(tert-butoxy)-2-oxoethyl)-6-ethyl-3-methyl-8-oxo-5,8-dihydropyrido[2,3-b]pyrazin-7-yl)piperazine-1-carboxylate (Intermediate-1) (550 mg, 1.13 mmol, 1.0 eq) in DCM (1 mL) was added TFA (5 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used in the next step without further purification.
LCMS: 332.2 [M+H]+.
To a solution of 2-(6-ethyl-3-methyl-8-oxo-7-(piperazin-1-yl)pyrido[2,3-b]pyrazin-5(8H)-yl)acetic acid trifluoroacetate (500 mg, 1.12 mmol, 1.0 eq) in DCM (8 mL) was added DIEA (725 mg, 5.61 mmol, 5.0 eq) and Boc2O (245 mg, 1.12 mmol, 1.0 eq). The mixture was stirred at room temperature for 1 h. The reaction mixture was poured into H2O (10 mL) and extracted with DCM (10 mL*2). The organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure to afford Intermediate-2a: the title compound was used in the next step without further purification.
LCMS: 432.2 [M+H]+.
To a solution of methyl 6-bromo-3-chloropyrazine-2-carboxylate (21.45 g, 85.30 mmol, 1.0 eq) in THE (30 mL) was added MeOH (60 mL), H2O (60 mL) and LiOH—H2O (7.16 g, 171 mmol, 2.0 eq) at 0° C. and the resulting mixture was stirred at 0° C. for 1 h. The reaction mixture was diluted with H2O (100 mL) and then adjusted to pH 2˜3 with aq. 6 N HCl solution. The resulting mixture was extracted with DCM (100 mL*3). The combined organic layers were washed with brine (50 mL*3), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
To a solution of 6-bromo-3-chloropyrazine-2-carboxylic acid (9.50 g, 40.0 mmol, 1.0 eq) in DCM (95 mL) was added (COCl)2 (7.62 g, 60.0 mmol, 5.25 mL, 1.5 eq) and DMF (58 mg, 0.80 mmol, 0.02 eq) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used in the next step without further purification.
To a solution of butan-2-one (3.95 g, 54.7 mmol, 2.0 eq) in THE (70 mL) was added a 2 M solution of LDA (27.4 mL, 54.7 μmol, 2.0 eq) in THE at −65° C. and the reaction mixture was stirred at −65° C. for 0.5 h. 6-bromo-3-chloropyrazine-2-carbonyl chloride (7.00 g, 27.4 mmol, 1.0 eq) was added at −65° C. and the mixture was stirred at −65° C. for 1 h. The reaction mixture was quenched by addition of saturated aq. NH4Cl solution (20 mL) at −65° C. and allowed to warm to room temperature slowly. The resulting mixture was diluted with H2O (100 mL) and extracted with EtOAc (100 mL*2). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 15.32 (s, 1H), 8.56 (s, 1H), 6.36 (s, 1H), 2.51 (q, 2H), 1.23 (br t, 3H).
To a solution of tert-butyl glycinate (450 mg, 3.43 mmol, 2.0 eq) in 1,4-dioxane (10 mL) was added (Z)-1-(6-bromo-3-chloropyrazin-2-yl)-3-hydroxypent-2-en-1-one (1.00 g, 1.72 mmol, 1.0 eq) in 1,4-dioxane (10 mL) dropwise over 10 min at 100° C. The mixture was stirred at 100° C. for 10 min after addition. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 330.2 [M-55]+.
To a solution of tert-butyl (Z)-(5-bromo-3-(3-hydroxypent-2-enoyl)pyrazin-2-yl)glycinate (330 mg, 854 μmol, 1.0 eq) in DCM (5 mL) was added TsOH-H2O (15 mg, 85 μmol, 0.1 eq), NBS (152 mg, 854 μmol, 1.0 eq) at 0° C. and it was stirred at 0° C. for 0.5 h. The reaction mixture was quenched with H2O (10 mL) and extracted with DCM (10 mL*2). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 410.0 [M-55]+.
To a solution of tert-butyl (5-bromo-3-(2-bromo-3-oxopentanoyl)pyrazin-2-yl)glycinate (397 mg, 853 μmol, 1.0 eq) in THE (5 mL) was added tert-butyl piperazine-1-carboxylate (238 mg, 1.28 mmol, 1.5 eq), DIEA (110 mg, 853 μmol, 1.0 eq) and the mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with H2O (10 mL) and extracted with DCM (10 mL*2). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 572.2 [M+H]+.
To a solution of tert-butyl 4-(1-(6-bromo-3-((2-(tert-butoxy)-2-oxoethyl)amino)pyrazin-2-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (350 mg, 613 μmol, 1.0 eq) in EtOH (4 mL) was added H3PO4 (120 mg, 1.23 mmol, 2.0 eq) and the resulting mixture was stirred at 60° C. for 12 h. The reaction mixture was diluted with H2O (10 mL) and then basified with 1 N aq. NaOH solution to pH 8. The resulting mixture was extracted with EtOAc (10 mL*2). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 554.1 [M+H]+.
The solution of tert-butyl 4-(2-bromo-5-(2-(tert-butoxy)-2-oxoethyl)-6-ethyl-8-oxo-5,8-dihydropyrido[2,3-b]pyrazin-7-yl)piperazine-1-carboxylate (Intermediate-3) (100 mg, 181 μmol, 1.0 eq) in DCM (1 mL) was added TFA (3 mL), and it was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 396.0 [M+H]+.
To a solution of 2-(2-bromo-6-ethyl-8-oxo-7-(piperazin-1-yl)pyrido[2,3-b]pyrazin-5(8H)-yl)acetic acid trifluoroacetate (92 mg, 0.18 mmol, 1.0 eq) in DCM (2 mL) was added Et3N (182 mg, 1.80 mmol, 10.0 eq) and Boc2O (39 mg, 0.18 mmol, 1.0 eq). The reaction was stirred at room temperature for 0.5 h. The reaction mixture was diluted with H2O (10 mL) and basified with 1 N aq. NaOH solution to pH 9. The resulting mixture was extracted with DCM (10 mL*2). The DCM phase was discarded. The aq. phase was adjusted to pH 6 with 1 N aq. HCl solution and then extracted with DCM (10 mL*2), the organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under vacuum to afford the title compound, which was used into the next step without further purification.
LCMS: 496.1 [M+H]+.
To a solution of methyl 3-methylfuran-2-carboxylate (5.00 g, 35.7 mmol, 1.00 eq) in CCl4 (50.0 mL) were added NBS (6.68 g, 37.5 mmol, 1.05 eq) and AIBN (2.35 g, 14.3 mmol, 0.40 eq) at room temperature. The mixture was degassed three times with N2 and stirred at 50° C. for 16 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford the title compound.
LCMS: 219/221 [M+H]+.
To a solution of methyl 3-(bromomethyl)furan-2-carboxylate (4.70 g, 21.5 mmol, 1.00 eq) and K2CO3 (5.93 g, 43.0 mmol, 2.00 eq) in ACN (47.0 mL) was added methyl 2-(4-methylbenzenesulfonamido)acetate (5.23 g, 21.5 mmol, 1.00 eq) and the mixture was stirred at room temperature for 16 h. The reaction was filtered, and the filtrate was concentrated in vacuum. The residue was purified by column chromatography on silica (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 382 [M+H]+.
To a solution of methyl 3-(((N-(2-methoxy-2-oxoethyl)-4-methylphenyl)sulfonamido)methyl)furan-2-carboxylate (1.80 g, 4.72 mmol, 1.00 eq) in THE (18.0 mL) was added a 1 M solution of LiHMDS (14.2 mL, 14.2 mmol, 3.00 eq) in THE dropwise at −78° C. under N2 atmosphere. After addition, the reaction mixture was allowed to warm to 0° C. and stirred for 5 h under N2 atmosphere. A saturated NH4Cl (aq.) solution was added to the reaction mixture and the aq. phase was extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 194 [M+H]+.
To a mixture of methyl 7-hydroxyfuro[3,2-c]pyridine-6-carboxylate (760 mg, 3.93 mmol, 1.00 eq) in MeOH (10.0 mL) was added Pd/C (152 mg, 20%). The mixture was degassed and purged with H2 gas (40 psi). Then it was stirred at 50° C. for 16 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) to afford the title compound.
LCMS: 196 [M+H]+.
To a mixture of methyl 7-hydroxy-2,3-dihydrofuro[3,2-c]pyridine-6-carboxylate (680 mg, 3.48 mmol, 1.00 eq) in H2O (3.00 mL) and MeOH (3.00 mL) was added NaOH (557 mg, 13.9 mmol, 4.00 eq) at room temperature and the resulting mixture was stirred at 60° C. for 16 h. After completion, the reaction mixture was concentrated under reduced pressure. The residue was diluted with H2O and acidified by 3 N HCl. The precipitated solids were collected by filtration and dried to afford the title compound, which was used in the next step directly without further purification.
LCMS: 182 [M+H]+.
To a stirred solution of methyl 2-methylfuran-3-carboxylate (10.0 g, 71.4 mmol, 1.00 eq) in CCl4 (55.0 mL) were added NBS (15.2 g, 85.6 mmol, 1.20 eq) and AIBN (586 mg, 3.57 mmol, 0.05 eq) at room temperature. The resulting mixture was degassed three times with N2 and stirred overnight at 50° C. under N2. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.86 (d, 1H), 6.80 (d, 1H), 4.95 (s, 2H), 3.82 (s, 3H).
To a stirred solution of methyl 2-(bromomethyl)furan-3-carboxylate (12.0 g, 54.8 mmol, 1.00 eq) and methyl 2-(4-methylbenzenesulfonamido)acetate (13.3 g, 54.8 mmol, 1.00 eq) in ACN (100 mL) was added K2CO3 (15.1 g, 110 mmol, 2.00 eq) at room temperature. The resulting mixture was degassed three times with N2 and then stirred overnight at room temperature. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford the title compound.
LCMS: 382.1 [M+H]+.
To a stirred solution of methyl 2-([N-(2-methoxy-2-oxoethyl)4-methylbenzenesulfonamido]methylfuran-3-carboxylate (9.00 g, 23.6 mmol, 1.00 eq) in THE (50.0 mL) was added a 1 M solution of LiHMDS (70.0 mL, 70.0 mmol, 3.00 eq) in THE at −78° C. under N2. The resulting mixture was stirred for 1h at room temperature under N2. The reaction mixture was quenched with saturated NH4Cl solution at 0° C. and diluted with H2O (200 mL). The resulting mixture was extracted with EtOAc (2×200 mL). The combined organic layers were washed with brine (3×100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford a crude product, which was purified by trituration with PE (250 mL) to afford the title compound.
LCMS: 194.0 [M+H]+.
To a solution of methyl 4-hydroxyfuro[2,3-c]pyridine-5-carboxylate (1.00 g, 5.18 mmol, 1.00 eq) in AcOH (1.00 mL) and MeOH (10.0 mL) was added Pd/C (1.65 g, 10%). The mixture was degassed three times with H2, and then stirred at room temperature for 1 h under H2. The reaction mixture was filtered, and the filter cake was washed with MeOH. The filtrate was concentrated under reduced pressure and the residue was purified by reverse phase HPLC (C18 column, H2O (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
LCMS: 195.9 [M+H]+.
To a stirred solution of methyl 4-hydroxy-2H,3H-furo[2,3-c]pyridine-5-carboxylate (300 mg, 1.54 mmol, 1.00 eq) in MeOH (3.00 mL) was added NaOH (246 mg, 6.15 mmol, 4.00 eq) and H2O (3.00 mL) at room temperature. The reaction mixture was stirred overnight at 60° C. The mixture was acidified to pH=3 with 1 N HCl. The resulting mixture was extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (2×20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound, which was used in the next step directly without further purification.
LCMS: 181.9 [M+H]+.
To a mixture of 4,6-dichloro-5-methoxypyrimidine (30.00 g, 167.6 mmol, 1.0 eq) in THE (300 mL) was added a 3 M solution of MeMgBr (61.45 mL, 184.4 mmol, 1.1 eq) in diethyl ether dropwise at 0° C. and then the mixture was stirred at 5° C. for 1 h. The resulting mixture was poured into H2O (200 mL) and extracted with EtOAc (100 mL*3). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuum. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford the title compound.
LCMS: 159.1 [M+H]+.
To a mixture of 4-chloro-5-methoxy-6-methylpyrimidine (22.00 g, 138.7 mmol, 1.0 eq) in MeOH (250 mL) was added Pd(dppf)Cl2—CH2Cl2 (6.80 g, 8.32 mmol, 0.06 eq) and TEA (28.1 g, 278 mmol, 2.0 eq). The reaction was purged with CO (50 psi) and stirred at 50° C. overnight. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford the title compound.
LCMS: 183.1 [M+H]+.
A mixture of methyl 5-methoxy-6-methylpyrimidine-4-carboxylate (Intermediate-56) (16.00 g, 87.83 mmol, 1.0 eq) in HBr solution (aq.) (68.5 mL, 68%) was stirred at 50° C. overnight. Then HI solution (aq.) (67.2 mL, 56%) was added and stirred at 50° C. for 6 h. The reaction mixture was cooled to room temperature and basified with 50% NaOH solution (aq.) to pH 9 at 0° C., then adjusted to pH 7 with 2 M HCl solution (aq.) at 0° C. The mixture was filtered, the filter cake was dried in vacuum to afford the title compound, which was used in the next step without further purification. LCMS: 155.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ ppm 15.46 (br s, 1H), 8.37 (s, 1H), 2.34 (s, 3H).
To a solution of methyl 6-bromo-3-chloropyrazine-2-carboxylate (100.00 g, 397.67 mmol, 1.0 eq) in 1,4-dioxane (1000 mL) was added DIEA (77.09 g, 596.5 mmol, 1.5 eq) and bis(4-methoxybenzyl)amine (112.56 g, 437.44 mmol, 1.1 eq), the resulting mixture was stirred at 100° C. for 16 h. The reaction mixture was diluted with H2O (800 mL) and extracted with EtOAc (400 mL*3). The combined organic layers were washed with brine (200 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.26 (s, 1H), 7.05 (d, 4H), 6.83 (d, 4H), 4.55 (s, 4H), 3.84 (s, 3H), 3.82-3.74 (m, 6H).
To a solution of methyl 3-(bis(4-methoxybenzyl)amino)-6-bromopyrazine-2-carboxylate (161 g, 340 mmol, 1.0 eq) in MeOH (1500 mL), THE (1500 mL) and H2O (1500 mL) was added LiOH—H2O (57.2 g, 1.36 mol, 4.0 eq), the resulting mixture was stirred at room temperature overnight. To the mixture was added H2O (1600 mL) and the pH adjusted to 2-3 with 1 N HCl. The reaction mixture was concentrated under reduced pressure to remove organic solvent, the aqueous solution was extracted with DCM (600 mL*3). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
1H NMR (400 MHz, CDCl3) δ ppm 8.26 (s, 1H), 7.05 (d, 4H), 6.83 (d, 4H), 4.55 (s, 4H), 3.84 (s, 3H), 3.82-3.74 (m, 6H).
To a solution of 3-(bis(4-methoxybenzyl)amino)-6-bromopyrazine-2-carboxylic acid (140.00 g, 305.47 mmol, 1.0 eq) and DMF (233 mg, 3.05 mmol, 0.01 eq) in DCM (1400 mL) was added oxalyl dichloride (46.50 g, 366.6 mmol, 1.2 eq) dropwise at room temperature and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used directly in the next step without further purification.
To a solution of butan-2-one (44.49 g, 616.9 mmol, 2.2 eq) in THE (160 mL) was added LDA (2 M in THF, 308.5 mL, 616.9 mmol, 2.2 eq) at −65° C. and it was stirred at −65° C. for 0.5 h. Then 6-bromo-3-((4-methoxybenzyl)amino)pyrazine-2-carbonyl chloride (100.00 g, 280.42 mmol, 1.0 eq) in THE (1000 mL) was added dropwise to the mixture at −65° C. and the resulting mixture was stirred at −65° C. for 1 h. The reaction mixture was slowly poured into aqueous HCl solution (1 M, 1 L), and extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 15.01 (s, 1H), 8.96 (br s, 1H), 8.27 (s, 1H), 7.33-7.28 (m, 1H), 6.93-6.85 (m, 3H), 6.78 (s, 1H), 4.65 (d, 2H), 3.81 (s, 3H), 2.40 (q, 2H), 1.23 (t, 3H).
To a solution of (Z)-1-(6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)-3-hydroxypent-2-en-1-one (Intermediate-78) (19.00 g, 48.44 mmol, 1.0 eq) in DCM (200 mL) was added TsOH-H2O (1.70 g, 39.7 mmol, 0.2 eq) and NBS (8.61 g, 48.4 mmol, 1.0 eq) at 0° C. and the resulting mixture was stirred at 0° C. for 1 h. The reaction mixture was diluted with H2O (300 mL) and extracted with EtOAc (100 mL*3). The combined organic layers were washed with brine (50 mL*3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used in the next step without further purification.
LCMS: 472.0 [M+H]+.
To a solution of 2-bromo-1-(6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)pentane-1,3-dione (Intermediate-40) (23.00 g, 48.82 mmol, 1.0 eq) in THE (250 mL) was added DIEA (12.61 g, 50.93 mmol, 2.0 eq) and tert-butyl piperazine-1-carboxylate (9.09 g, 48.8 mmol, 1.0 eq) and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (200 mL) and extracted with EtOAc (100 mL*3). The combined organic layers were washed with brine (50 mL*3), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.80 (br t, 1H), 8.30 (s, 1H), 7.25 (s, 1H), 6.88 (d, 2H), 5.32 (s, 1H), 4.63 (dq, 2H), 3.81 (s, 3H), 3.53-3.35 (m, 4H), 3.02-2.83 (m, 3H), 2.72-2.59 (m, 2H), 1.59 (s, 2H), 1.46 (s, 9H), 1.14 (t, 3H).
Step 7: Synthesis of 2-bromo-6-ethyl-7-(piperazin-1-yl)pyrido[2,3-b]pyrazin-8(5H)-one trifluoroacetate tert-butyl 4-(1-(6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (21.00 g, 36.43 mmol, 1.0 eq) was dissolved into TFA (40 mL) and then it was stirred at 50° C. for 1 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used in the next step without further purification.
LCMS: 338.1 [M+H]+.
To a solution of 2-bromo-6-ethyl-7-(piperazin-1-yl)pyrido[2,3-b]pyrazin-8(5H)-one trifluoroacetate (12.00 g, 35.48 mmol, 1.0 eq) in DCM (150 mL) was added DIEA (22.92 g, 177.4 mmol, 5.0 eq) and Boc2O (7.68 g, 35.5 mmol, 1.0 eq), and then the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (200 mL) and extracted with DCM (50 mL*3). The combined organic layers were washed with brine (50 mL*3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated in a mixed solvent of DCM/PE (DCM:PE=2:1, 30 mL) at room temperature for 10 mins and then filtered. The filter cake was dried under reduced pressure to afford the title compound, which was used in the next step without further purification.
LCMS: 440.1 [M+H]+.
To a solution of methyl 5-bromo-6-chloro-pyrazin-2-amine (20.00 g, 95.95 mmol, 1 eq) in THE (20 mL) was added Et3N (33.98 g, 335.8 mmol, 46.74 mL, 3.5 eq), Pd(PPh3)2Cl2 (6.73 g, 9.59 mmol, 0.1 eq), CuI (914 mg, 4.80 mmol, 0.05 eq) and prop-1-yne (1 M in THF, 191.90 mL, 2 eq). The mixture was stirred at 50° C. for 2 h. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to give the residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.76 (s, 1H), 7.18 (s, 2H), 2.07 (s, 3H).
LCMS: 168.0 [M+H]+.
To a solution of 6-chloro-5-prop-1-ynyl-pyrazin-2-amine (10.00 g, 59.67 mmol, 1 eq) in DMF (100 mL) and H2O (15 mL) was added Na2S (13.97 g, 179.0 mmol, 3 eq). The mixture was stirred at 90° C. for 3 h. The reaction mixture was diluted with H2O (150 mL) and extracted with EtOAc (80 mL*3). The combined organic layers were washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.98 (s, 1H), 6.97 (s, 1H), 4.58 (br s, 2H), 2.57 (s, 3H).
LCMS: 166.0 [M+H]+.
To a solution of 6-methylthieno[2,3-b]pyrazin-3-amine (3.60 g, 21.79 mmol, 1 eq) in THF (80 mL) was added t-BuOK (7.34 g, 65.4 mmol, 3 eq) and 1-(chloromethyl)-4-methoxy-benzene (8.19 g, 52.3 mmol, 7.10 mL, 2.4 eq). The mixture was stirred at 60° C. for 1 h. The reaction mixture was diluted by water (80 mL) and extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine (60 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 8.06 (s, 1H), 7.21-7.19 (m, 4H), 7.00 (s, 1H), 6.90-6.83 (m, 4H), 4.77 (s, 4H), 3.72-3.69 (s, 6H), 2.49 (s, 3H).
LCMS: 406.2 [M+H]+.
To a solution of N,N-bis[(4-methoxyphenyl)methyl]-6-methyl-thieno[2,3-b]pyrazin-3-amine (6.50 g, 12.0 mmol, 1eq) in DMF (65 mL) was added NCS (1.77 g, 13.2 mmol, 1.1 eq). The mixture was stirred at 80° C. for 0.5 h. The reaction mixture was diluted by H2O (80 mL) and extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine (60 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.23-7.21 (m, 4H), 7.17 (s, 1H), 6.89-6.82 (m, 4H), 4.43 (s, 4H), 3.69 (s, 6H), 2.57 (s, 3H).
LCMS: 440.1 [M+H]+.
To a solution of 2-chloro-N,N-bis[(4-methoxyphenyl)methyl]-6-methyl-thieno[2,3-b]pyrazin-3-amine (2.30 g, 5.23 mmol, 1 eq) and Pd(dppf)Cl2 (382 mg, 523 mol, 0.1 eq) in MeOH (50 mL) was added Et3N (1.59 g, 15.7 mmol, 2.18 mL, 3 eq) under N2 atmosphere. The suspension was degassed under vacuum and purged with CO three times. The mixture was stirred at 50° C. for 12 h under CO (50 Psi). The reaction solution was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.14 (s, 1H), 7.14-7.12 (m, 4H), 6.87-6.85 (m, 4H), 4.51 (s, 4H), 3.82 (s, 3H), 3.70 (s, 6H), 2.57 (s, 3H).
LCMS: 464.3 [M+H]+.
To a solution of methyl 3-[bis[(4-methoxyphenyl)methyl]amino]-6-methyl-thieno[2,3-b]pyrazine-2-carboxylate (1.80 g, 3.88 mmol, 1 eq) in THE (12 mL), MeOH (4 mL) and H2O (4 mL) was added LiOH—H2O (1.63 g, 38.83 mmol, 10 eq). The mixture was stirred at 25° C. for 16 h. The reaction mixture was concentrated in vacuum, the resulting residue was diluted with H2O (150 mL), saturated critic acid aqueous solution was added until the pH of mixture was acidified to 4-5. Then the mixture was extracted with EtOAc (100 mL*3). The combined organic layer was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.18 (s, 1H), 7.12-7.10 (m, 4H), 6.78-6.76 (m, 4H), 4.41 (s, 4H), 3.75 (s, 6H), 2.69 (s, 3H).
To a solution of 3-[bis[(4-methoxyphenyl)methyl]amino]-6-methyl-thieno[2,3-b]pyrazine-2-carboxylic acid (1.60 g, 3.56 mmol, 1 eq) in DCM (20 mL) was added (COCl)2 (678 mg, 5.34 mmol, 467 μL, 1.5 eq) and DMF (5 mg, 71 mol, 5 μL, 0.02 eq) at 0° C., The mixture was stirred at 25° C. for 1 h. The reaction mixture was concentrated in vacuum, the resulting residue was diluted with THF (8 mL) and concentrated under reduced pressure, this process was repeated three times to obtain the title compound, which was used in the next step directly without further purification.
To a solution of butan-2-one (407 mg, 5.65 mmol, 505 μL, 1.5 eq) in THE (8 mL) was added LDA (2 M, 2.82 mL, 1.5 eq) at −78° C. under N2 atmosphere, then the mixture was stirred at −78° C. for 0.5 h. A solution of 3-[(4-methoxyphenyl)methylamino]-6-methyl-thieno[2,3-b]pyrazine-2-carbonyl chloride (1.31 g, 3.77 mmol, 1 eq) in THE (8 mL) was added dropwise into the butan-3-one solution. The mixture was stirred at 25° C. for another 1 h. The reaction mixture was poured into saturated NH4Cl aqueous solution (100 mL) and extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 384.1 [M+H]+.
To a solution of (Z)-3-hydroxy-1-[3-[(4-methoxyphenyl)methylamino]-6-methyl-thieno[2,3-b]pyrazin-2-yl]pent-2-en-1-one (230 mg, 600 mol, 1 eq) in DCM (4 mL) was added NBS (107 mg, 600 mol, 1 eq). The mixture was stirred at −10° C. for 1 h. The reaction mixture was quenched with saturated Na2SO3 aqueous solution (10 mL) and extracted with DCM (5 mL*3). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.71-8.69 (m, 1H), 7.32-7.30 (m, 2H), 6.91-6.87 (m, 2H), 6.82 (s, 1H), 6.31 (s, 1H), 4.77-4.64 (m, 2H), 3.81 (s, 3H), 2.95 (q, 2H), 2.57 (s, 3H), 1.18 (t, 3H).
LCMS: 464.0 [M+H]+.
To a solution of 3,5-dibromopyrazin-2-amine (10.00 g, 39.54 mmol, 1 eq) and DMAP (4.83 g, 39.5 mmol, 1 eq) in ACN (100 mL) was added acetyl chloride (9.31 g, 118 mmol, 8.43 mL, 3 eq), then the mixture was stirred at 80° C. for 1.5 h. The mixture was concentrated to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.38 (s, 1H), 7.91 (s, 1H), 2.45 (s, 3H).
LCMS: 296.0 [M+H]+.
To a solution of N-(3,5-dibromopyrazin-2-yl)acetamide (6.80 g, 23.1 mmol, 1 eq) in toluene (136 mL) was added P2S5 (3.07 g, 13.8 mmol, 1.47 mL, 0.6 eq), then the mixture was stirred at 110° C. for 1 h. The mixture was concentrated under reduced pressure and neutralized with saturated NaHCO3 to pH=−7. Then the mixture was extracted with EtOAc (150 mL*3). Then the organic phase was washed with brine (300 mL), dried over anhydrous Na2SO4 and concentrated under reduce pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.71 (s, 1H), 2.94 (s, 3H).
LCMS: 232.1 [M+H]+.
To a solution of 6-bromo-2-methylthiazolo[4,5-b]pyrazine (10.00 g, 43.46 mmol, 1 eq) and 1-(4-methoxyphenyl)-N-[(4-methoxyphenyl)methyl]methanamine (13.42 g, 52.15 mmol, 1.2 eq) in NMP (100 mL) was added DIEA (8.43 g, 65.2 mmol, 11.4 mL, 1.5 eq), the mixture was stirred at 140° C. for 16 h. The mixture was poured to water (500 mL). Then the mixture was extracted with EtOAc (150 mL*3). The organic phase was washed with brine (500 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) and reversed-phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.01 (s, 1H), 7.17 (d, 4H), 6.86 (d, 4H), 4.77 (s, 4H), 3.80 (s, 6H), 2.81 (s, 3H).
LCMS: 407.3 [M+H]+.
To a solution of N,N-bis(4-methoxybenzyl)-2-methylthiazolo[4,5-b]pyrazin-6-amine (10.00 g, 24.60 mmol, 1 eq) in DMF (100 mL) was added NBS (4.38 g, 24.6 mmol, 1 eq), then the mixture was stirred at 25° C. for 1 h. The mixture was poured into water (500 mL). Then the mixture was extracted with EtOAc (200 mL*3). Then the organic phase was washed with brine (500 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was triturated with (EtOH/EtOAc=3/1, 100 mL) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.22 (d, 4H), 6.83 (d, 4H), 4.48 (s, 4H), 3.79 (s, 6H), 2.84 (s, 3H).
LCMS: 487.2 [M+H]+.
To a solution of 5-bromo-N,N-bis(4-methoxybenzyl)-2-methylthiazolo[4,5-b]pyrazin-6-amine (1.10 g, 2.27 mmol, 1 eq) and Et3N (688 mg, 6.80 mmol, 946 μL, 3 eq) in MeOH (20 mL) and THE (20 mL) was added Pd(dppf)Cl2 (166 mg, 227 mol, 0.1 eq) under N2. The mixture was degassed and purged with CO several times. The mixture was stirred under CO (50 psi) at 50° C. for 16 h. Then it was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.09 (d, 4H), 6.83 (d, 4H), 4.54 (s, 4H), 3.91 (s, 3H), 3.79 (s, 6H), 2.84 (s, 3H).
LCMS: 465.3 [M+H]+.
To a solution of methyl 6-(bis(4-methoxybenzyl)amino)-2-methylthiazolo[4,5-b]pyrazine-5-carboxylate (920 mg, 1.98 mmol, 1 eq) in THF (9 mL), H2O (3 mL) and MeOH (3 mL) was added LiOH—H2O (415 mg, 9.90 mmol, 5 eq), then the mixture was stirred at 25° C. for 3 h. Acidified the mixture to pH=−3 with saturated citric acid aqueous solution. The mixture was extracted with EtOAc (100 mL*3). Then the organic phase was washed with brine (200 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 451.3 [M+H]+.
To a solution of 6-(bis(4-methoxybenzyl)amino)-2-methylthiazolo[4,5-b]pyrazine-5-carboxylic acid (700 mg, 1.55 mmol, 1 eq) in DCM (7 mL) was added (COCl)2 (237 mg, 1.86 mmol, 163 μL, 1.2 eq) and DMF (2 mg, 31 mol, 2 μL, 0.02 eq) at 0° C., the mixture was stirred at 25° C. for 1 h. The mixture was concentrated under reduced pressure to obtain the title compound, which was used into next step directly without further purification.
To a solution of butan-2-one (167 mg, 2.32 mmol, 208 μL, 1.5 eq) in THF (5 mL) was added LDA (2 M in THF, 1.16 mL, 1.5 eq) at −78° C., then the mixture was stirred at −78° C. for 0.5 h. A solution of 6-((4-methoxybenzyl)amino)-2-methylthiazolo[4,5-b]pyrazine-5-carbonyl chloride (540 mg, 1.55 mmol, 1 eq) in THF (5 mL) was added, the mixture was stirred at 25° C. for 1 h. The mixture was poured into a saturated NH4Cl (20 mL) aqueous solution. Then the mixture was extracted with EtOAc (20 mL*3). Then the organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 385.3 [M+H]+.
To a solution of 3-hydroxy-1-(6-((4-methoxybenzyl)amino)-2-methylthiazolo[4,5-b]pyrazin-5-yl)pent-2-en-1-one (380 mg, 988 mol, 1 eq) in DCM (7 mL) was added NBS (176 mg, 988 mol, 1 eq), then the mixture was stirred at 25° C. for 1 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 463.1 [M+H]+.
To a solution of 2-bromo-1-(6-((4-methoxybenzyl)amino)-2-methylthiazolo[4,5-b]pyrazin-5-yl)pentane-1,3-dione (Intermediate-52) (450 mg, 971 mol, 1 eq) and tert-butyl piperazine-1-carboxylate (181 mg, 971 mol, 1 eq) in THE (10 mL) was added DIEA (251 mg, 1.94 mmol, 338 μL, 2 eq), the mixture was stirred at 25° C. for 1 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 569.3 [M+H]+.
To a solution of tert-butyl 4-(1-(6-((4-methoxybenzyl)amino)-2-methylthiazolo[4,5-b]pyrazin-5-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (130 mg, 229 mol, 1 eq) in TFA (3 mL) was stirred at 50° C. for 1 h. The reaction mixture was concentrated in vacuum to obtain the title compound, which was used into next step directly without further purification.
LCMS: 331.2 [M+H]+.
To a solution of 6-ethyl-2-methyl-7-(piperazin-1-yl)pyrido[2,3-b]thiazolo[4,5-e]pyrazin-8(5H)-one trifluoroacetate (101 mg, 227 mol, 1 eq) and DIEA (88 mg, 681 mol, 119 L, 3 eq) in DCM (2 mL) was added (Boc)2O (99 mg, 454 mol, 104 μL, 2 eq), then the mixture was stirred at 25° C. for 1 h. The reaction mixture was concentrated in vacuum to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 431.3 [M+H]+.
To a solution of methyl 5-methoxy-6-methyl-pyrimidine-4-carboxylate (Intermediate-56) (210 g, 1.15 mol, 1 eq) was added HBr (850 mL) at 20° C. After addition, the mixture was stirred at 50° C. for 16 hr, and then was added HI (600 mL) at 50° C. The resulting mixture was stirred at 50° C. for 6 hr. The reaction mixture was filtered. The filtrate was adjusted to pH 8-9 with aqueous NaOH solution (30% in water) at 0-5° C. The mixture was filtered, the filter cake was dried under reduced pressure to afford the title compound, which was used into next step directly without further purification.
1H NMR (400 MHz, CD3OD) δ ppm 8.47 (s, 1H), 2.48 (s, 3H).
To a solution of 2-bromo-5-fluoro-pyridin-3-amine (10.00 g, 52.36 mmol, 1 eq) in MeOH (350 mL) was added Et3N (10.60 g, 104.71 mmol, 14.57 mL, 2 eq) and Pd(dppf)Cl2 (1.92 g, 2.62 mmol, 0.05 eq). The mixture was stirred at 80° C. for 16 h under CO (50 psi). 50 mL H2O was added to the mixture and concentrated under reduced pressure to remove MeOH. Then the mixture was diluted with 100 mL H2O and extracted with EtOAc (80 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.81 (s, 1H), 7.03 (d, 1H), 6.94 (br s, 2H), 3.81 (s, 3H).
To a solution of methyl 3-amino-5-fluoro-pyridine-2-carboxylate (27.00 g, 158.69 mmol, 1 eq) in ACN (500 mL) was added NBS (31.07 g, 174.56 mmol, 1.1 eq). The mixture was stirred at 20° C. for 2.5 h. 200 mL saturated NaHCO3 aqueous solution was added to the reaction solution and the mixture was extracted with EtOAc (300 mL*3). The combined organic layers were washed with brine (200 mL*2), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was added dropwise into DCM (80 mL) and stirred for 10 min, filtered and the filter cake was dried under reduced pressure to give the title compound, which was used in next step directly without further purification.
1H NMR (400 MHz, CDCl3) δ ppm 6.81 (d, 1H), 6.00 (br s, 2H), 3.95 (s, 3H).
LCMS: 250.8 [M+H]+.
To a solution of methyl 3-amino-6-bromo-5-fluoro-pyridine-2-carboxylate (35.00 g, 118.05 mmol, 1 eq) in DCM (350 mL) was added CSA (13.71 g, 59.03 mmol, 0.5 eq) and 4-methoxybenzyl 2,2,2-trichloroacetimidate (50.03 g, 177.08 mmol, 1.5 eq). The mixture was stirred at 20° C. for 1 h. The reaction mixture was quenched by 200 mL saturated NaHCO3 aqueous solution and extracted with DCM (100 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by trituration (MeOH, 30 mL) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 8.21 (br s, 1H), 7.33-7.26 (m, 3H), 6.96-6.85 (m, 2H), 4.41 (d, 2H), 3.83 (s, 3H), 3.72 (s, 3H).
LCMS: 371.0 [M+H]+.
To a solution of butan-2-one (10.01 g, 138.82 mmol, 12.42 mL, 2.5 eq) in THE (30 mL) was added LiHMDS (1 M, 138.82 mL, 138.82 mmol, 2.5 eq) at 0° C. under N2 atmosphere. The reaction mixture was stirred at 25° C. for 0.5 h. Then a solution of methyl 6-bromo-5-fluoro-3-[(4-methoxyphenyl) methylamino]pyridine-2-carboxylate (20.50 g, 55.53 mmol, 1 eq) in THE (150 mL) was added to the mixture. Then the reaction mixture was stirred at 60° C. for 1.5 h under N2 atmosphere. The reaction mixture was added into 250 mL saturated NH4Cl aqueous solution, extracted with EtOAc (150 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 15.06 (s, 1H), 9.14 (br s, 1H), 7.31-7.28 (m, 1H), 6.98-6.88 (m, 2H), 6.82-6.71 (m, 2H), 4.38 (d, 2H), 3.85 (s, 3H), 2.43 (q, 2H), 1.27 (t, 3H).
LCMS: 409.0 [M+H]+.
To a solution of 1-[6-bromo-5-fluoro-3-[(4-methoxyphenyl) methylamino]-2-pyridyl]-3-hydroxy-pent-2-en-1-one (18.20 g, 44.47 mmol, 1 eq) in DCM (360 mL) was added NBS (6.73 g, 37.80 mmol, 0.85 eq). The mixture was stirred at 0° C. for 1 h. The reaction mixture was diluted with H2O (300 mL) and extracted with DCM (100 mL*3). The combined organic layer was washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used in next step directly without further purification.
LCMS: 489.0 [M+H]+.
A mixture of 2-bromo-1-[6-bromo-5-fluoro-3-[(4-methoxyphenyl) methylamino]-2-pyridyl]pentane-1,3-dione (Intermediate-58) (23.90 g, 48.96 mmol, 1 eq), tert-butyl piperazine-1-carboxylate (9.12 g, 48.96 mmol, 1 eq) and DIEA (12.66 g, 97.92 mmol, 17.06 mL, 2 eq) in THF (250 mL) was stirred at 25° C. for 16 h. The reaction mixture was diluted with H2O (300 mL) and extracted with EtOAc (100 mL*3), the combined organic layer was washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was triturated with EtOAc (40 mL) and purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.99 (br s, 1H), 7.22 (d, 2H), 6.89 (d, 2H), 6.74 (d, 1H), 5.32 (s, 1H), 4.33 (d, 2H), 3.81 (s, 3H), 3.56-3.37 (m, 4H), 3.08-2.96 (m, 1H), 2.95-2.86 (m, 2H), 2.76-2.57 (m, 3H), 1.46 (s, 9H), 1.14 (t, 3H).
LCMS: 595.2 [M+H]+.
Tert-butyl 4-[1-[6-bromo-5-fluoro-3-[(4-methoxyphenyl) methylamino]pyridine-2-carbonyl]-2-oxobutyl]piperazine-1-carboxylate (10.00 g, 16.85 mmol, 1 eq) was added to TFA (80 mL) and stirred at 50° C. for 1 h. Then it was cooled to room temperature naturally and concentrated to dryness. Then it was dissolved in DCM (60 mL). DIEA (8.73 g, 67.57 mmol, 11.77 mL, 4 eq) and (Boc)2O (11.06 g, 50.67 mmol, 11.64 mL, 3 eq) were added to the solution. The reaction mixture was stirred at 20° C. for 16 h and quenched by H2O (100 mL) then extracted with DCM (30 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was and purified by silica gel chromatography (Eluent of DCM/MeOH) and triturated with EtOAc (10 mL) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 11.80 (s, 1H), 7.83 (d, 1H), 4.08-3.75 (m, 2H), 3.69-3.39 (m, 2H), 3.07-2.75 (m, 4H), 2.60-2.50 (m, 2H), 1.43 (s, 9H), 1.23 (t, 3H).
LCMS: 455.0 [M+H]+.
To a solution of tert-butyl 4-(6-bromo-2-ethyl-7-fluoro-4-oxo-1H-1,5-naphthyridin-3-yl) piperazine-1-carboxylate (Intermediate-59) (4.00 g, 8.79 mmol, 1 eq) in 1,4-dioxane (120 mL) was added DIEA (6.82 g, 52.72 mmol, 9.18 mL, 6 eq), KI (1.46 g, 8.79 mmol, 1 eq) and ethyl 2-bromoacetate (8.80 g, 52.72 mmol, 5.84 mL, 6 eq). The reaction mixture was stirred at 110° C. for 16 h. The reaction mixture was quenched by brine 100 mL and extracted with EtOAc (100 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.36 (d, 1H), 4.86 (s, 2H), 4.74-4.41 (m, 2H), 4.31 (q, 2H), 4.16-4.00 (m, 2H), 3.83 (q, 2H), 3.36-2.80 (m, 2H), 2.61-2.59 (m, 2H), 1.49 (s, 9H), 1.32 (t, 3H), 1.26 (t, 3H).
LCMS: 543.1 [M+H]+.
To a solution of tert-butyl 4-[6-bromo-1-(2-ethoxy-2-oxo-ethyl)-2-ethyl-7-fluoro-4-oxo-1,5-naphthyridin-3-yl]piperazine-1-carboxylate (Intermediate-60) (1.00 g, 1.85 mmol, 1 eq) and dimethylamine hydrochloride (602 mg, 7.39 mmol, 4 eq) in 1,4-dioxane (6 mL) was added DIEA (1.91 g, 14.78 mmol, 2.57 mL, 8 eq). The mixture was stirred at 100° C. for 16 h. The reaction mixture was quenched by brine (50 mL) and extracted with EtOAc (40 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by reversed-phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.16 (d, 1H), 4.81 (s, 2H), 4.29 (q, 2H), 4.18-3.96 (m, 2H), 3.94-3.80 (m, 2H), 3.21 (s, 6H), 3.13-2.81 (m, 4H), 2.68-2.50 (m, 2H), 1.49 (s, 9H), 1.30 (t, 3H), 1.24 (t, 3H).
LCMS: 506.3 [M+H]+.
To a solution of tert-butyl 4-[6-(dimethylamino)-1-(2-ethoxy-2-oxo-ethyl)-2-ethyl-7-fluoro-4-oxo-1,5-naphthyridin-3-yl]piperazine-1-carboxylate (80 mg, 158 mol, 1 eq) in H2O (1 mL) and THE (2 mL) was added LiOH—H2O (13 mg, 316 mol, 2 eq). The mixture was stirred at 20° C. for 1 h. The reaction mixture was diluted by H2O (5 mL) and pH was adjusted to about 4 by 0.5 M HCl aqueous solution. Then the mixture was extracted with EtOAc (30 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used in next step directly without further purification.
LCMS: 478.2 [M+H]+.
To a solution of 2-bromo-1-(6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)pentane-1,3-dione (Intermediate-40) (24.00 g, 50.94 mmol, 1.0 eq) in THF (250 mL) was added DIEA (13.17 g, 101.88 mmol, 2.0 eq) and tert-butyl (1S,6S)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (CAS: 2920219-11-8) (8.65 g, 40.75 mmol, 0.8 eq), and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with H2O (200 mL) and extracted with EtOAc (60 mL*3). The combined organic layers were washed with brine (50 mL*3), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 602.52 [M+H]+.
Tert-butyl (1S,6S)-5-(1-(6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)-1,3-dioxopentan-2-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (24.30 g, 40.33 mmol, 1.0 eq) was dissolved into TFA (120 mL) and it was stirred at 60° C. for 2 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 364.24 [M+H]+.
To a solution of 7-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-2-bromo-6-ethylpyrido[2,3-b]pyrazin-8(5H)-one trifluoroacetate (14.50 g, 39.81 mmol, 1.0 eq) in DCM (150 mL) was added DIEA (25.73 g, 199.04 mmol, 5.0 eq) and Boc2O (9.56 g, 43.79 mmol, 1.1 eq), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM (60 mL*3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound intermediate-66a.
LCMS: 464.36 [M+H]+.
To a solution of POCl3 (5.70 g, 37.17 mmol, 2.0 eq) in ACN (40 mL) was added ethyl 3-(methylthio)-6-oxo-1,6-dihydro-1,2,4-triazine-5-carboxylate (Intermediate-92) (4.0 g, 18.58 mmol, 1.0 eq), and the resulting mixture was stirred at 90° C. for 3 h. The reaction mixture was concentrated under reduced pressure, the residue was quenched with saturated NaHCO3 aqueous solution (20 mL), and then extracted with DCM (20 mL*3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 234.2[M+H]+.
To a solution of ethyl 6-chloro-3-(methylthio)-1,2,4-triazine-5-carboxylate (2.9 g, 12.41 mmol, 1.0 eq) and bis(4-methoxybenzyl)amine (3.83 g, 14.89 mmol, 1.2 eq) in 1,4-dioxane (15 mL) was added DIEA (4.81 g, 37.23 mmol, 3.0 eq), and the resulting mixture was stirred at 110° C. for 32 h. The reaction mixture was concentrated under reduced pressure and then purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 455.2[M+H]+.
1H NMR (400 MHz, CDCl3) δ=7.02 (d, 4H), 6.79-6.70 (m, 4H), 4.50 (s, 4H), 4.23 (q, 2H), 3.72 (s, 6H), 2.59 (s, 3H), 1.23 (t, 3H).
To a solution of butan-2-one (1.43 g, 19.80 mmol, 3.0 eq) and ethyl 6-(bis(4-methoxybenzyl)amino)-3-(methylthio)-1,2,4-triazine-5-carboxylate (3.00 g, 6.60 mmol, 1.0 eq) in 2-methylfuran (30 mL) was added a solution of LiHMDS (1 M in THF, 19.80 mL, 3.0 eq), and the resulting mixture was stirred at 80° C. for 2 h. The reaction mixture was quenched with saturated NH4Cl aqueous solution (100 mL), and then extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 481.2[M+H]+.
To a solution of 1-(6-(bis(4-methoxybenzyl)amino)-3-(methylthio)-1,2,4-triazin-5-yl)pentane-1,3-dione (2.3 g, 4.79 mmol, 1.0 eq) and TsOH-H2O (165 mg, 957 mol, 0.2 eq) in DCM (20 mL) was added NBS (767 mg, 4.31 mmol, 0.9 eq) at 0° C. and the resulting mixture was stirred at 0° C. for 1 h. The reaction mixture was poured into ice-water (20 mL) and extracted with DCM (20 mL*3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 559.2[M+H]+.
To a solution of 1-(6-(bis(4-methoxybenzyl)amino)-3-(methylthio)-1,2,4-triazin-5-yl)-2-bromopentane-1,3-dione (Intermediate-66) (2.60 g, 4.65 mmol, 1.0 eq) and tert-butyl (1S,6S)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (CAS: 2920219-11-8) (987 mg, 4.65 mmol, 1.0 eq) in THE (26 mL) was added DIEA (1.20 g, 9.29 mmol, 2.0 eq), and the resulting mixture was stirred at 50° C. for 1.5 h. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (20 mL*3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 691.2 [M+H]+.
A solution of tert-butyl (1S,6S)-5-(1-(6-(bis(4-methoxybenzyl)amino)-3-(methylthio)-1,2,4-triazin-5-yl)-1,3-dioxopentan-2-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (1.89 g, 2.74 mmol, 1.0 eq) in TFA (20 mL) was stirred at 50° C. for 1 h, and then it was concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 333.2[M+H]+.
To a solution of 6-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-7-ethyl-3-(methylthio)pyrido[3,2-e][1,2,4]triazin-5(8H)-one trifluoroacetate (914 mg, 2.75 mmol, 1.0 eq) and DIEA (1.78 g, 13.75 mmol, 5.0 eq) in DCM (10 mL) was added Boc2O (600 mg, 2.75 mmol, 1.0 eq), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was poured into H2O (20 mL), extracted with EtOAc (20 mL*3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with a mixed solvent of PE and DCM (PE/DCM=4/1, 5 mL) to afford the title compound.
LCMS: 433.2 [M+H]+.
To a solution of methyl 5-bromo-6-chloro-pyrazin-2-amine (10.00 g, 47.97 mmol, 1 eq) in THF (10 mL) was added Et3N (16.99 g, 167.91 mmol, 23.37 mL, 3.5 eq), Pd(PPh3)2Cl2 (3.37 g, 4.80 mmol, 0.1 eq), CuI (457 mg, 2.40 mmol, 0.05 eq) and prop-1-yne (1 M, 95.95 mL, 2 eq). The mixture was stirred at 50° C. for 12 h. The resulting mixture was filtered to remove the insoluble and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.83 (s, 1H), 4.89 (s, 2H), 2.13 (s, 3H).
LCMS: 168.0 [M+H]+.
To a solution of 6-chloro-5-prop-1-ynyl-pyrazin-2-amine (6.50 g, 38.78 mmol, 1 eq) in DMSO (65 mL) and H2O (65 mL) was added KOH (4.35 g, 77.57 mmol, 2 eq). The mixture was stirred at 100° C. for 16 h. The reaction mixture was poured into saturated NH4Cl aqueous solution (400 mL) and extracted with EtOAc (200 mL*3). The combined organic layer was washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.76 (s, 1H), 6.54 (s, 1H), 6.42 (s, 2H), 2.38 (s, 3H).
LCMS: 150.2 [M+H]+.
To a solution of 6-methylfuro[2,3-b]pyrazin-3-amine (1.50 g, 10.06 mmol, 1 eq) in THF (30 mL) was added t-BuOK (4.51 g, 40.24 mmol, 2.01 mL, 4 eq) and 1-(chloromethyl)-4-methoxy-benzene (5.36 g, 34.19 mmol, 4.64 mL, 3.4 eq). The mixture was stirred at 25° C. for 1 h. The mixture was poured into H2O (80 mL). Then the mixture was extracted with EtOAc (30 mL*3). Then the combined organic phase was washed with brine (40 mL), dried over Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.87 (s, 1H), 7.17 (d, 4H), 6.84 (d, 4H), 6.44 (s, 1H), 4.75 (s, 4H), 3.80 (s, 6H), 2.47 (s, 3H).
LCMS: 390.2 [M+H]+.
To a solution of N,N-bis[(4-methoxyphenyl)methyl]-6-methyl-furo[2,3-b]pyrazin-3-amine (2.10 g, 5.39 mmol, 1 eq) in ACN (25 mL) was added NCS (720 mg, 5.39 mmol, 1 eq). The mixture was stirred at 90° C. for 3 h. Then it was poured into H2O (20 mL). The mixture was extracted with EtOAc (30 mL*3) and the combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 424.2 [M+H]+.
To a solution of 2-chloro-N,N-bis[(4-methoxyphenyl)methyl]-6-methyl-furo[2,3-b]pyrazin-3-amine (1.00 g, 2.36 mmol, 1 eq) in THE (10 mL) and MeOH (10 mL) was added Et3N (716 mg, 7.08 mmol, 985 μL, 3 eq) and Pd(dppf)Cl2 (173 mg, 236 mol, 0.1 eq). The mixture was degassed and purged with N2 for 3 times then degassed and purged with CO for 3 times. The reaction was stirred under CO (50 psi) atmosphere at 50° C. for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 448.2 [M+H]+.
To a solution of methyl 3-[bis[(4-methoxyphenyl)methyl]amino]-6-methyl-furo[2,3-b]pyrazine-2-carboxylate (950 mg, 2.12 mmol, 1 eq) in DCM (10 mL) was added TFA (726 mg, 6.37 mmol, 473 μL, 3 eq). The mixture was stirred at 25° C. for 0.5 h. The mixture was adjust pH to 7 by saturated NaHCO3 aqueous solution (20 mL), then extracted with DCM (15 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give the title compound, which was used into next step without further purification.
LCMS: 328.0 [M+H]+.
To a solution of butan-2-one (611 mg, 8.48 mmol, 758 μL, 3 eq) in THE (5 mL) was added LiHMDS (1 M, 8.48 mL, 3 eq) at 0° C. The mixture was stirred at 0° C. for 0.5 h. Then to the mixture was added a solution of methyl 3-((4-methoxybenzyl) amino)-6-methylfuro[2,3-b]pyrazine-2-carboxylate (925 mg, 2.83 mmol, 1 eq) in THE (5 mL) dropwise at 0° C. The reaction mixture was stirred at 60° C. for 1 h. Then it was quenched by saturated NH4Cl aqueous solution (20 mL) and extracted with EtOAc (20 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 368.2 [M+H]+.
To a solution of 3-hydroxy-1-(3-((4-methoxybenzyl)amino)-6-methylfuro[2,3-b]pyrazin-2-yl)pent-2-en-1-one (330 mg, 898 mol, 1 eq) in DCM (5 mL) was added NBS (160 mg, 898 mol, 1 eq). The mixture was stirred at 25° C. for 1 h. The reaction was poured into H2O (20 mL) and extracted with DCM (10 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 446.1 [M+H]+.
To a solution of 2-bromo-1-(3-((4-methoxybenzyl)amino)-6-methylfuro[2,3-b]pyrazin-2-yl)pentane-1,3-dione (Intermediate-70) (350 mg, 784 mol, 1 eq) and tert-butyl piperazine-1-carboxylate (190 mg, 1.02 mmol, 1.3 eq) in THF (5 mL) was added DIEA (152 mg, 1.18 mmol, 205 μL, 1.5 eq). The mixture was stirred at 25° C. for 2 h. The reaction was poured into H2O (20 mL) and extracted with DCM (10 mL*3). The combined organic layer was concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 552.3 [M+H]+.
A solution of tert-butyl 4-(1-(3-((4-methoxybenzyl)amino)-6-methylfuro[2,3-b]pyrazin-2-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (360 mg, 653 mol, 1 eq) in TFA (5 mL) was stirred at 60° C. for 1 h. The mixture was concentrated in vacuum directly to afford the title compound, which was used into next step without further purification.
LCMS: 314.1 [M+H]+.
To a solution of 7-ethyl-2-methyl-6-(piperazin-1-yl)furo[2,3-b]pyrido[3,2-e]pyrazin-5(8H)-one trifluoroacetate (350 mg, 1.12 mmol, 1 eq) in DCM (5 mL) was added Boc2O (366 mg, 1.68 mmol, 385 μL, 1.5 eq) and DIEA (433 mg, 3.35 mmol, 584 μL, 3 eq). The mixture was stirred at 25° C. for 1 h. The reaction was poured into H2O (20 mL) and extracted with DCM (10 mL*3), the combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 414.2 [M+H]+.
To a solution of methyl 3-(bis(4-methoxybenzyl)amino)-5-chloropyrazine-2-carboxylate (Intermediate-90) (5.00 g, 11.69 mmol, 1 eq) in THF (50 mL) was added prop-1-yne (1 M THF solution, 35.06 mL, 3 eq), Pd(PPh3)4 (2.70 g, 2.34 mmol, 0.2 eq), CuI (445 mg, 2.34 mmol, 0.2 eq) and Et3N (5.91 g, 58.43 mmol, 8.13 mL, 5 eq). The mixture was stirred at 50° C. for 5 h. The resulting mixture was filtered to remove the insoluble and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 432.2 [M+H]+.
To a solution of methyl 3-(bis(4-methoxybenzyl) amino)-5-(prop-i-yn-1-yl) pyrazine-2-carboxylate (5.00 g, 11.59 mmol, 1 eq) in DMF (100 mL) was added NCS (1.55 g, 11.59 mmol, 1.0 eq). The mixture was stirred at 80° C. for 1 h. Then it was diluted with H2O (50 mL) and extracted with EtOAc (50 mL*2). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.03 (d, 4H), 6.82 (d, 4H), 4.52 (s, 4H), 3.85 (s, 3H), 3.79 (s, 6H), 2.19 (s, 3H).
LCMS: 466.2 [M+H]+.
To a solution of methyl 3-(bis(4-methoxybenzyl) amino)-6-chloro-5-(prop-i-yn-1-yl) pyrazine-2-carboxylate (4.50 g, 9.66 mmol, 1 eq) in DMF (45 mL) was added Na2S·9H2O (11.60 g, 48.29 mmol, 5 eq). The mixture was stirred at 90° C. for 0.5 h. Then it was diluted with H2O (100 mL) and filtered to remove the insoluble. The filtrate was extracted with DCM (100 mL*2). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give the title compound, which was used into next step directly without further purification.
LCMS: 450.2 [M+H]+.
To a solution of 2-(bis(4-methoxybenzyl) amino)-6-methylthieno[2,3-b]pyrazine-3-carboxylic acid (2.40 g, 5.34 mmol, 1 eq) in DCM (48 mL) was added (COCl)2 (814 mg, 6.41 mmol, 561 μL, 1.2 eq) and DMF (39 mg, 534 mol, 41 μL, 0.1 eq). The mixture was stirred at 25° C. for 20 min. Then the mixture was concentrated in vacuum to give the title compound, which was used into next step directly without further purification.
To a solution of butan-2-one (622 mg, 8.63 mmol, 772 μL, 3 eq) in THE (16 mL) was added LiHMDS (1 M, 8.63 mL, 3 eq) at −65° C. and stirred for 20 min. Then the solution of 2-((4-methoxybenzyl) amino)-6-methylthieno[2,3-b]pyrazine-3-carbonyl chloride (1.00 g, 2.88 mmol, 1 eq) in THF (8 mL) was added into the solution at −65° C. dropwise and the mixture was stirred at 60° C. for 1 h. The mixture was quenched by saturated NH4Cl aqueous solution (50 mL) and extracted with EtOAc (50 mL*2). The combined organic phase was washed with brine (40 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 384.1 [M+H]+.
To a solution of 3-hydroxy-1-(2-((4-methoxybenzyl) amino)-6-methylthieno[2,3-b]pyrazin-3-yl) pent-2-en-1-one (340 mg, 887 mol, 1 eq) in DCM (6 mL) was added the solution of NBS (142 mg, 798 mol, 0.9 eq) in DCM (1 mL) dropwise at 0° C. The mixture was stirred at 25° C. for 0.5 h. The residue was diluted with H2O (20 mL) and extracted with DCM (25 mL*2). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.32 (d, 2H), 6.93 (s, 1H), 6.88 (d, 2H), 6.32 (s, 1H), 4.71-4.67 (m, 2H), 3.81 (s, 3H), 2.94 (q, 2H), 2.66 (s, 3H), 1.18 (t, 3H).
LCMS: 464.0 [M+H]+.
To a solution of 2-bromo-1-(2-((4-methoxybenzyl) amino)-6-methylthieno[2,3-b]pyrazin-3-yl) pentane-1,3-dione (Intermediate-73) (270 mg, 584 mol, 1 eq) and tert-butyl piperazine-1-carboxylate (163 mg, 876 mol, 1.5 eq) in THE (4 mL) was added DIEA (226 mg, 1.75 mmol, 305 μL, 3 eq). The mixture was stirred at 25° C. for 1 h. The mixture was diluted with H2O (20 mL) and was extracted with EtOAc (25 mL*2). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was and purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 568.3 [M+H]+.
A mixture of tert-butyl 4-(1-(2-((4-methoxybenzyl) amino)-6-methylthieno[2,3-b]pyrazin-3-yl)-1,3-dioxopentan-2-yl) piperazine-1-carboxylate (270 mg, 476 mol, 1 eq) in TFA (2 mL) was stirred at 60° C. for 0.5 h. The mixture was concentrated in vacuum directly to afford the title compound, which was used into next step directly without further purification.
LCMS: 330.2 [M+H]+.
To a solution of 6-ethyl-2-methyl-7-(piperazin-1-yl) pyrido[2,3-b]thieno[2,3-e]pyrazin-8(5H)-one trifluoroacetate (145 mg, 440 mol, 1 eq) in DCM (4 mL) was added DIEA (228 mg, 176 mol, 307 μL, 4 eq) and Boc2O (192 mg, 880 mol, 202 μL, 2 eq). The mixture was stirred at 25° C. for 0.5 h. The mixture was diluted with H2O (20 mL) and was extracted with DCM (25 mL*2). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was and purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 8.45 (s, 1H), 7.01 (s, 1H), 4.08-4.02 (m, 2H), 3.79-3.78 (m, 2H), 2.93 (q, 2H), 2.91-2.75 (m, 2H), 2.67 (s, 3H), 2.59-2.55 (m, 2H), 1.42 (s, 9H), 1.29 (t, 3H).
LCMS: 430.3 [M+H]+.
To a solution of 2-bromo-1-(6-((4-methoxybenzyl) amino)-2-methylthiazolo[4,5-b]pyrazin-5-yl) pentane-1,3-dione (Intermediate-52) (830 mg, 1.79 mmol, 1 eq) and tert-butyl (1S,6S)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (399 mg, 1.88 mmol, 1.05 eq) in THF (16 mL) was added DIEA (463 mg, 3.58 mmol, 624 μL, 2 eq). The mixture was stirred at 25° C. for 1 h. The reaction mixture was poured into H2O (20 mL) and extracted with DCM (30 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 595.3 [M+H]+.
A solution of tert-butyl (1S,6S)-5-(1-(6-((4-methoxybenzyl) amino)-2-methylthiazolo[4,5-b]pyrazin-5-yl)-1,3-dioxopentan-2-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (750 mg, 1.26 mmol, 1 eq) in TFA (8 mL) was stirred at 60° C. for 4 h. The mixture was concentrated in vacuum directly to afford the title compound.
LCMS: 357.1 [M+H]+.
To a solution of 7-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-6-ethyl-2-methylpyrido[2,3-b]thiazolo[4,5-e]pyrazin-8(5H)-one trifluoroacetate (700 mg, 1.96 mmol, 1 eq) in DCM (7 mL) was added (Boc)20 (471 mg, 2.16 mmol, 496 μL, 1.1 eq) and DIEA (1.27 g, 9.82 mmol, 1.71 mL, 5 eq). The mixture was stirred at 25° C. for 1 h. Then it was poured into H2O (20 mL) and extracted with DCM (30 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 457.1 [M+H]+.
To a solution of methyl 6-bromo-3-chloropyrazine-2-carboxylate (52.00 g, 206.79 mmol, 1.0 eq) in 1,4-dioxane (500 mL) was added DIEA (40.09 g, 310.18 mmol, 1.5 eq) and bis(4-methoxybenzyl)amine (31.20 g, 227.47 mmol, 1.1 eq), the resulting mixture was stirred at 100° C. overnight. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (1500 mL) and extracted with EtOAc (1000 mL*2). The combined organic layers were washed with brine (1000 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was triturated with (PE:EtOAc=10:1, 440 mL) to afford the title compound.
LCMS: 352.0 [M+H]+.
1H NMR (400 MHz, CDCl3) δ ppm 8.35 (s, 1H), 8.31 (br s, 1H), 7.29 (d, 2H), 6.90 (d, 2H), 4.65 (d, 2H), 3.97 (s, 3H), 3.82 (s, 3H).
To a solution of methyl 6-bromo-3-((4-methoxybenzyl)amino)pyrazine-2-carboxylate (61.50 g, 166.94 mmol, 1.0 eq) and butan-2-one (26.48 g, 367.27 mmol, 2.2 eq) in toluene (1500 mL) was added LDA (2 M in THF, 183.6 mL, 2.2 eq) dropwise at 0° C. After addition, the mixture was stirred at 60° C. for 2 h. The reaction mixture was slowly poured into aqueous HCl solution (0.5 M, 1500 mL), and then extracted with EtOAc (1000 mL*3). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 392.0 [M+H]+.
To a solution of tert-butyl 4-(7-ethyl-2-methyl-5-oxo-5,8-dihydropyrido[2,3-b]thieno[3,2-e]pyrazin-6-yl)piperazine-1-carboxylate (Intermediate-47) (350 mg, 815 mol, 1 eq) and ethyl 2-bromoacetate (272 mg, 1.63 mmol, 180 μL, 2 eq) in 1,4-dioxane (7 mL) was added DIEA (316 mg, 2.44 mmol, 426 μL, 3 eq). The mixture was stirred at 80° C. for 16 h. Then the second batch of ethyl 2-bromoacetate (272 mg, 1.63 mmol, 180 μL, 2 eq) was added into the mixture and stirred at 80° C. for another 16 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.51 (s, 1H), 5.35 (s, 2H), 4.22 (q, 2H), 4.03-3.92 (m, 2H), 3.70-3.61 (m, 2H), 3.18 (q, 2H), 3.07-2.85 (m, 2H), 2.75 (s, 3H), 2.66-2.64 (m, 2H), 1.48 (s, 9H), 1.26-1.20 (m, 6H).
LCMS: 516.4 [M+H]+.
To a solution of tert-butyl 4-(8-(2-ethoxy-2-oxoethyl)-7-ethyl-2-methyl-5-oxo-5,8-dihydropyrido[2,3-b]thieno[3,2-e]pyrazin-6-yl)piperazine-1-carboxylate (292 mg, 566 mol, 1 eq) in THE (3 mL), MeOH (1 mL) and H2O (1 mL) was added LiOH—H2O (238 mg, 5.66 mmol, 10 eq). Then the mixture was stirred at 25° C. for 1 h. The reaction mixture was added saturated citric acid until pH achieve around 3. Then it was extracted with EtOAc (10 mL*3) and the combined organic phase was washed with brine (40 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford the title compound, which was used into next step directly without further purification.
LCMS: 488.1 [M+H]+.
To a solution of 2-(6-(4-(tert-butoxycarbonyl)piperazin-1-yl)-7-ethyl-2-methyl-5-oxopyrido[2,3-b]thieno[3,2-e]pyrazin-8(5H)-yl)acetic acid (Intermediate-82) (276 mg, 566 mol, 1 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine hydrochloride salt (212 mg, 1.13 mmol, 2 eq) in DMF (5 mL) was added HATU (430 mg, 1.13 mmol, 2 eq) and DIEA (219 mg, 1.70 mmol, 296 μL, 3 eq). The mixture was stirred at 25° C. for 1 h. 30 mL brine was added into the mixture and extracted with EtOAc (30 mL). Then the organic layer was washed with brine (20 mL*3), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 7.95 (s, 1H), 7.46 (s, 1H), 3.93 (s, 2H), 3.68-3.54 (m, 2H), 3.05-3.03 (m, 2H), 2.89 (s, 3H), 2.73 (q, 2H), 2.72-2.71 (m, 2H), 2.62-2.59 (m, 2H), 2.22 (s, 6H), 1.44 (s, 9H), 1.16 (t, 3H).
LCMS: 621.2 [M+H]+.
To a solution of tert-butyl 4-(7-ethyl-2-methyl-5-oxo-8-(2-oxo-2-((3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl)amino)ethyl)-5,8-dihydropyrido[2,3-b]thieno[3,2-e]pyrazin-6-yl)piperazine-1-carboxylate (100 mg, 161 mol, 1 eq) in DCM (3 mL) was added TFA (1 mL). The mixture was stirred at 25° C. for 1 h. Then the mixture was concentrated in vacuum to afford the title compound, which was used into next step directly without further purification.
LCMS: 521.2 [M+H]+.
To a solution of sodium 5-hydroxy-6-methylpyrimidine-4-carboxylate (Intermediate-55) (90 mg, 584 mol, 1.0 eq) in DCM (1 mL) was added oxalyl dichloride (148 mg, 1.17 mmol, 2.0 eq) and one drop of DMF. The resulting mixture was stirred at room temperature for 30 min and then concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
To a solution of 5-chloro-3-((4-methoxybenzyl)amino)pyrazine-2-carboxylic acid (10.00 g, 34.05 mmol, 1 eq) in t-BuOH (100 mL) was added DMAP (5.41 g, 44.26 mmol, 1.3 eq), then Boc2O (14.86 g, 68.10 mmol, 2.0 eq) was added to the mixture dropwise at room temperature, and the reaction was stirred at room temperature for 2 h. The reaction mixture was diluted with H2O (100 mL), extracted with EtOAc (100 mL*2), the organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE), then triturated with PE (40 mL) to afford the title compound.
LCMS: 350.1[M+H]+.
To a solution of tert-butyl 5-chloro-3-((4-methoxybenzyl)amino)pyrazine-2-carboxylate (5.50 g, 15.72 mmol, 1.0 eq), potassium vinyltrifluoroborate (3.16 g, 23.58 mmol, 1.5 eq) in 1,4-dioxane (50 mL) and H2O (10 mL) was added Pd(dppf)Cl2 (575 mg, 786 mol, 0.05 eq), K2CO3 (4.35 g, 31.45 mmol, 2.0 eq), and the reaction was stirred at 80° C. overnight under N2 atmosphere. The reaction mixture was concentrated under reduced pressure and then purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 342.2 [M+H]+.
To a solution of tert-butyl 3-((4-methoxybenzyl)amino)-5-vinylpyrazine-2-carboxylate (1.00 g, 2.93 mmol, 1.0 eq) in DMSO (20 mL) was added methylamine hydrochloride (989 mg, 14.65 mmol, 5.0 eq) and DIEA (3.79 g, 29.29 mmol, 10.0 eq), the resulting mixture was stirred at 90° C. for 0.5 h. The reaction mixture was diluted with H2O (200 mL), extracted with EtOAc (100 mL*2), the organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 373.2[M+H]+.
To a solution of tert-butyl 3-((4-methoxybenzyl)amino)-5-(2-(methylamino)ethyl)pyrazine-2-carboxylate (5.00 g, 13.42 mmol, 1.0 eq) in DCM (50 mL) was added Et3N (2.72 g, 26.85 mmol, 2.0 eq) and CbzCl (2.75 g, 16.11 mmol, 1.2 eq), and the resulting mixture was stirred at room temperature for 0.5 h. The reaction mixture was diluted with H2O (30 mL), extracted with DCM (50 mL*2), the organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 507.4[M+H]+.
To a solution of tert-butyl 5-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)-3-((4-methoxybenzyl)amino)pyrazine-2-carboxylate (5.60 g, 11.05 mmol, 1.0 eq) in acetone (30 mL) was added aqueous HCl solution (6 M, 36.85 mL, 20.0 eq) dropwise at 5-10° C., and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (50 mL), extracted with DCM (50 mL*2), and the organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced to afford the title compound, which was used into the next step without further purification.
LCMS: 451.3[M+H]+.
To a solution of 5-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)-3-((4-methoxybenzyl)amino)pyrazine-2-carboxylic acid (4.60 g, 10.21 mmol, 1.0 eq) in DCM (50 mL) was added two drop of DMF and (COCl)2 (1.94 g, 15.32 mmol, 1.5 eq), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
To a solution of butan-2-one (1.41 g, 19.62 mmol, 2.0 eq) in THE (46 mL) was added LDA (2 M in THF, 9.81 mL, 2.0 eq) at −60° C., and the mixture was stirred at −60° C. for 0.5 h under N2 atmosphere. Then benzyl (2-(5-(chlorocarbonyl)-6-((4-methoxybenzyl)amino)pyrazin-2-yl)ethyl)(methyl)carbamate (4.60 g, 9.81 mmol, 1.0 eq) was added to the mixture, and the reaction was stirred at −60° C. for another 0.5 h under N2 atmosphere. The reaction mixture was quenched with aqueous HCl solution (1N, 100 mL), extracted with EtOAc (50 mL*2), the organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 505.4[M+H]+.
To a solution of benzyl (2-(5-(3-hydroxypent-2-enoyl)-6-((4-methoxybenzyl)amino)pyrazin-2-yl)ethyl)(methyl)carbamate (1.80 g, 3.57 mmol, 1.0 eq) in DCM (20 mL) was added TsOH·H2O (123 mg, 713 mol, 0.2 eq) and NBS (762 mg, 4.28 mmol, 1.2 eq) at 0° C., then the reaction was stirred at 0° C. for 0.5 h. The reaction mixture was quenched with H2O (20 mL), extracted with DCM (20 mL*2), the organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 583.3[M+H]+.
To a solution of benzyl (2-(5-(2-bromo-3-oxopentanoyl)-6-((4-methoxybenzyl)amino)pyrazin-2-yl)ethyl)(methyl)carbamate (2.08 g, 3.56 mmol, 1 eq) in DMF (20 mL) was added NBS (634 mg, 3.56 mmol, 1.0 eq), and the reaction was stirred at room temperature for 2 h. The reaction mixture was quenched with H2O (30 mL), extracted with EtOAc (20 mL*2), the organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 663.0[M+H]+.
To a solution of benzyl (2-(3-bromo-5-(2-bromo-3-oxopentanoyl)-6-((4-methoxybenzyl)amino)pyrazin-2-yl)ethyl)(methyl)carbamate (Intermediate-94) (1.50 g, 2.26 mmol, 1.0 eq) in THE (15 mL) was added DIEA (585 mg, 4.53 mmol, 2.0 eq) and tert-butyl piperazine-1-carboxylate (506 mg, 2.72 mmol, 1.2 eq), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with H2O (10 mL), extracted with EtOAc (10 mL*2), the organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 767.4[M+H]+.
A solution of tert-butyl 4-(1-(5-(2-(((benzyloxy)carbonyl)(methyl)amino)ethyl)-6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (1.00 g, 1.30 mmol, 1.0 eq) in AcOH (8 mL) was stirred at 55° C. for 6 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (20 mL), then the pH was adjusted to 7-8 with saturated NaHCO3 aqueous solution. The resulting mixture was extracted with DCM (20 mL*2) and the organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 749.2[M+H]+.
To a solution of tert-butyl 4-[3-[2-[benzyloxycarbonyl(methyl)amino]ethyl]-2-bromo-6-ethyl-5-[(4-methoxyphenyl)methyl]-8-oxo-pyrido[2,3-b]pyrazin-7-yl]piperazine-1-carboxylate (700 mg, 934 mol, 1.0 eq) in DCM (5 mL) was added HBr solution (768 μL, 33% in water), and the resulting mixture was stirred at room temperature for 0.5 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used into the next without further purification.
LCMS: 515.2[M+H]+.
To a solution of 2-bromo-6-ethyl-5-[(4-methoxyphenyl)methyl]-3-[2-(methylamino)ethyl]-7-piperazin-1-ylpyrido[2,3-b]pyrazin-8-one dihydrobromide (500 mg, 970 mol, 1.0 eq) in DCM (5 mL) was added DIEA (627 mg, 4.85 mmol, 5.0 eq), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and then purified by reverse phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
LCMS: 435.3[M+H]+.
A solution of 11-ethyl-10-[(4-methoxyphenyl)methyl]-4-methyl-12-piperazin-1-yl-2,4,8,10-tetrazatricyclo[7.4.0.03,7]trideca-1(9),2,7,11-tetraen-13-one (60 mg, 138 mol, 1.0 eq) in TFA (0.5 mL) was stirred at 60° C. for 2.5 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 315.2[M+H]+.
To a solution of 11-ethyl-4-methyl-12-piperazin-1-yl-2,4,8,10-tetrazatricyclo[7.4.0.03,7]trideca-1(9),2,7,11-tetraen-13-one trifluoroacetate (43 mg, 137 mol, 1.0 eq) in DCM (0.5 mL) was added DIEA (53 mg, 410 mol, 3.0 eq) and Boc2O (45 mg, 205 mol, 1.5 eq), and the resulting mixture was stirred at room temperature for 20 min. The reaction mixture was diluted with H2O (10 mL), extracted with DCM (10 mL*2), the organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by pre-TLC (SiO2, DCM:MeOH=10:1) to afford the title compound.
LCMS: 415.3[M+H]+.
To a solution of 2-bromo-1-(6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)pentane-1,3-dione (Intermediate-40) (3.8 g, 8.07 mmol, 1.0 eq) in THE (40 mL) was added DIEA (2.08 g, 16.13 mmol, 2.0 eq) and tert-butyl 3-methylpiperazine-1-carboxylate (1.62 g, 8.07 mmol, 1.0 eq), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with H2O (100 mL) and then extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine (40 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 592.2 [M+H]+.
To a TFA (10 mL) solution was added tert-butyl 4-(1-(6-bromo-3-((4-methoxybenzyl)amino)pyrazin-2-yl)-1,3-dioxopentan-2-yl)-3-methylpiperazine-1-carboxylate (1.4 g, 2.37 mmol, 1.0 eq) and it was stirred at 55° C. for 0.5 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 354.1 [M+H]+.
To a solution of 2-bromo-6-ethyl-7-(2-methylpiperazin-1-yl)pyrido[2,3-b]pyrazin-8(5H)-one trifluoroacetate (800 mg, 2.27 mmol, 1.0 eq) and DIEA (1.47 g, 11.36 mmol, 5.0 eq) in DCM (15 mL) was added (Boc)2O (496 mg, 2.27 mmol, 1.0 eq), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (20 mL) and then extracted with DCM (15 mL*3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 454.1 [M+H]+.
To a solution of 2,2,6,6-tetramethylpiperidine (508 mg, 3.60 mmol, 1.2 eq) in THE (2 mL) was added n-BuLi (2.5 M in THF, 1.32 mL, 1.1 eq) at −30° C. under N2 atmosphere. After stirred at −30° C. for 0.5 h, the mixture was cooled to −78° C., and then a solution of bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methane (803 mg, 3.00 mmol, 1.0 eq) in THE (2 mL) was added dropwise, and it was stirred at −78° C. for 0.5 h after addition. Then, a solution of 3-methoxycyclobutan-1-one (300 mg, 3.00 mmol, 1.0 eq) in THE (0.2 mL) was added dropwise at −78° C., and the resulting mixture was slowly warmed to room temperature and stirred at room temperature overnight. The reaction mixture was poured into saturated NH4Cl aqueous solution (80 mL) and extracted with EtOAc (70 mL*3). The combined organic layers were washed with brine (80 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 225.2 [M+H]+.
To a solution of 3,5-dichloropyrazine-2-carboxylic acid (50.00 g, 259.08 mmol, 1 eq), 1-(4-methoxyphenyl)-N-[(4-methoxyphenyl) methyl]methanamine (66.67 g, 259.08 mmol, 1 eq) in 1,4-dioxane (1000 mL) was added DIEA (83.71 g, 647.71 mmol, 113 mL, 2.5 eq). The mixture was stirred at 50° C. for 16 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was suspended into DCM:EtOAc=1:5 (500 mL) and stirred for 30 min. Then filtered, the filtrate was collected and concentrated under reduced pressure to give the title compound, which was used into next step without further purification.
LCMS: 412.1 [M−H]−.
To a solution of 3-(bis(4-methoxybenzyl) amino)-5-chloropyrazine-2-carboxylic acid (90.00 g, 217.47 mmol, 1eq) in DMF (900 mL) was added CH3I (92.60 g, 652.40 mmol, 40 mL, 3 eq) and NaHCO3 (21.92 g, 260.96 mmol, 1.2 eq). Then the mixture was stirred at 25° C. for 5h. It was poured into saturated NH4Cl aqueous solution (2.00 L) and extracted with EtOAc (800 mL*3). The combined organic layer was washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ ppm 7.91 (s, 1H), 7.05 (d, 4H), 6.83 (d, 4H), 4.56 (s, 4H), 3.84 (s, 3H), 3.78 (s, 6H).
LCMS: 428.1 [M+H]+.
To a solution of methyl hydrazinecarbimidothioate hydroiodide hydroiodide (35.13 g, 150.73 mmol, 1.05 eq) and TEA (15.25 g, 150.73 mmol, 1.05 eq) in THF (400 mL) was added dropwise a solution of diethyl 2-oxomalonate (25 g, 143.55 mmol, 1.0 eq) at 0° C. under N2. The resulting mixture was stirred at 45° C. under N2 atmosphere for 15 h. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compounds.
Analytical data of ethyl 3-(methylthio)-6-oxo-1,6-dihydro-1,2,4-triazine-5-carboxylate (Intermediate-92):
LCMS: 216.2 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 4.41 (q, 2H), 2.41 (s, 3H), 1.35 (t, 3H).
Analytical data of ethyl 3-(methylthio)-5-oxo-4,5-dihydro-1,2,4-triazine-6-carboxylate (Intermediate-93):
LCMS:216.1 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 4.48 (q, 2H), 2.63 (s, 3H), 1.45-1.40 (m, 3H).
To a solution of 1-(6-(bis(4-methoxybenzyl)amino)-3-(methylthio)-1,2,4-triazin-5-yl)-2-bromopentane-1,3-dione (Intermediate-66) (1.8 g, 3.22 mmol, 1.0 eq) and tert-butyl (S)-3-methylpiperazine-1-carboxylate (709 mg, 3.54 mmol, 1.1 eq) in THE (20 mL) was added DIEA (832 mg, 6.43 mmol, 2.0 eq), and the resulting mixture was stirred at 50° C. overnight. The reaction mixture was poured into ice-water (20 mL) and extracted with DCM (20 mL*3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 679.2 [M+H]+.
A solution of tert-butyl (3S)-4-(1-(6-(bis(4-methoxybenzyl)amino)-3-(methylthio)-1,2,4-triazin-5-yl)-1,3-dioxopentan-2-yl)-3-methylpiperazine-1-carboxylate (650 mg, 958 mol, 1.0 eq) in TFA (7 mL) was stirred at 50° C. for 1 h and then concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 321.2[M+H]+.
To a solution of (S)-7-ethyl-6-(2-methylpiperazin-1-yl)-3-(methylthio)pyrido[3,2-e][1,2,4]triazin-5(8H)-one trifluoroacetate (305 mg, 952 mol, 1.0 eq) and DIEA (369 mg, 2.86 mmol, 3.0 eq) in DCM (4 mL) was added Boc2O (208 mg, 952 mol, 1.0 eq), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was poured into H2O (10 mL), and then extracted with DCM (10 mL*3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The filtrate was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 421.2 [M+H]+.
To a solution of 2-methoxypyridin-4-ol (5.00 g, 39.96 mmol, 1.0 eq) in ACN (80 mL) was added NBS (7.11 g, 39.96 mmol, 1.0 eq) at 0° C., and the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with H2O (100 mL), and then extracted with EtOAc (100 mL*3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 203.9 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 7.92 (d, 1H), 6.63 (d, 1H), 4.03 (s, 3H).
To a mixture of 3-bromo-2-methoxypyridin-4-ol (3.84 g, 18.82 mmol, 1.0 eq) in MeOH (20 mL) and DMF (20 mL) was added Pd(dppf)Cl2·CH2Cl2 (1.54 g, 1.88 mmol, 0.1 eq) and TEA (5.71 g, 56.46 mmol, 3.0 eq), and the resulting mixture was stirred at 80° C. overnight under CO (50 psi) atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was dissolved into DCM (100 mL), washed with saturated NH4Cl (70 mL*3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, CDCl3) δ 12.26 (s, 1H), 8.05 (d, 1H), 6.57 (d, 1H), 4.01 (d, 6H).
To a solution of methyl 4-hydroxy-2-methoxynicotinate (Intermediate-99) (1.40 g, 7.64 mmol, 1.0 eq) in ACN (14 mL) was added NBS (1.36 g, 7.64 mmol, 1.0 eq), and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was filtered, the filter cake was washed with cold ACN (5 mL) and then dried in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 262.1 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 12.90 (s, 1H), 8.15 (s, 1H), 3.92 (d, 6H).
To a mixture of methyl 5-bromo-4-hydroxy-2-methoxynicotinate (300 mg, 1.14 mmol, 1.0 eq) and tetramethylstannane (409 mg, 2.29 mmol, 2.0 eq) in DMF (2 mL) was added Pd2(dba)3 (105 mg, 114 mol, 0.1 eq) and XPhos (109 mg, 229 mol, 0.2 eq), and the resulting mixture was stirred at 120° C. overnight under N2 atmosphere. The reaction mixture was diluted with H2O (20 mL), extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by reverse phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
LCMS: 198.3 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 12.48 (s, 1H), 7.91 (s, 1H), 3.99 (d, 6H), 2.14 (s, 3H).
To a solution of methyl 4-hydroxy-2-methoxy-5-methylnicotinate (30 mg, 152 mol, 1.0 eq) in H2O (0.2 mL), THE (0.2 mL) and MeOH (0.2 mL) was added aqueous LiOH—H2O solution (1 M, 608 μL, 4.0 eq) and it was stirred at 40° C. overnight. The reaction mixture was acidified by addition of aqueous HCl solution (1 M) to pH 5-6, and then extracted with EtOAc (5 mL*10). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used into the next step without further purification.
LCMS: 184.1 [M+H]+.
To a solution of methyl 4-hydroxy-2-methoxy-pyridine-3-carboxylate (Intermediate-99) (1.00 g, 5.46 mmol, 1.0 eq) in THE (5 mL), H2O (5 mL) and MeOH (10 mL) was added LiOH—H2O (1.37 g, 32.76 mmol, 6.0 eq). The reaction mixture was diluted with H2O (3 mL) and then separated. The aqueous phase was washed with EtOAc 6 mL (3 mL*2) and then adjusted to pH=6 with aqueous HCl solution (2 M). The resulting solution was concentrated under reduced pressure and then purified by reverse phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
LCMS: 170.1 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 12.60 (s, 1H), 11.28-10.86 (m, 1H), 8.08 (d, 1H), 6.68 (d, 1H), 4.19 (s, 3H).
To a solution of 1H-1,2,4-triazole-3,5-diamine (50.00 g, 504.6 mmol, 1.0 eq) in HBr (aq.) solution (40% in H2O, 500 mL) was slowly added a NaNO2 (41.78 g, 605.5 mmol, 1.2 eq) solution in H2O (125 mL) at 0° C. After addition, the mixture was warmed to room temperature and stirred at room temperature for 1 h. Then, the reaction temperature was raised to 40° C. and stirred at 40° C. for 1 h. Then, the reaction temperature was raised to 60° C. and stirred for another 1 h. The reaction mixture was cooled to 0° C. and adjusted to pH=2 by 10% NaOH (aq.) solution. The resulting mixture was extracted with EtOAc (1000 mL) and the organic layer was discarded. The aqueous phase was adjusted to pH=7 by 10% NaOH (aq.) solution. The resulting mixture was extracted with EtOAc (1000 mL*3). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuum to afford the title compound, which was used in next step without further purification.
1H NMR (400 MHz, DMSO-d6) δ ppm 12.27 (br s, 1H), 6.29 (br s, 2H).
Intermediate-4 is then used in the synthetic sequence shown in Scheme 8 below to establish the bicyclic [1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one core seen in Intermediate-32 and in the final compounds derived from Intermediate-32. Ethyl 3-oxopentanoate B is subjected to bromination with N-bromosuccinimide (NBS) to produce C. Nucleophilic displacement of bromide from C using nucleophile D, under basic conditions, produces E. E is reacted with Intermediate-4 to create an in-situ species (not shown) that intramolecularly cyclizes to establish the bicyclic core in Intermediate-32.
As shown in Scheme 9, Intermediate-32 may be cross-coupled with F to attach the R1amoiety and form G.
To a solution of tert-butyl 4-(2-bromo-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylate (Intermediate-32) (2.00 g, 4.68 mmol, 1.0 eq), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.47 g, 7.02 mmol, 1.5 eq) and Pd(dppf)Cl2—CH2Cl2 (382 mg, 468 mol, 0.1 eq) in 1,4-dioxane (20 mL) and H2O (5 mL) was added Na2CO3 (992 mg, 9.36 mmol, 2.0 eq), the resulting mixture was stirred at 100° C. overnight under N2 atmosphere. The reaction mixture was cooled to room temperature, diluted with H2O (30 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford the title compound.
LCMS: 431.2 [M+H]+.
To a stirred solution of tert-butyl 4-{2-bromo-5-ethyl-7-oxo-4H-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl}piperazine-1-carboxylate (Intermediate-2) (1.00 g, 2.34 mmol, 1.0 eq) and ethyl iodoacetate (1.00 g, 4.68 mmol, 2.0 eq) in ACN (20 mL) was added K2CO3 (970 mg, 7.02 mmol, 3.0 eq) at room temperature and stirred at 60° C. for 3.5 h. The mixture was cooled to room temperature. The resulting mixture was diluted with water (60 mL). The resulting mixture was extracted with EtOAc (3*20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 513.1/515.1 [M+H]+.
To a stirred solution of tert-butyl 4-[2-(1-acetyl-3,6-dihydro-2H-pyridin-4-yl)-4-(2-ethoxy-2-oxoethyl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]piperazine-1-carboxylate (Intermediate-37) (150 mg, 269 mol, 1.0 eq) in DCM (5 mL) was added a 4 M solution of HCl/1,4-dioxane (5 mL) dropwise at 0° C. The resulting mixture was stirred for 40 min at 0° C. The resulting mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 458.2 [M+H]+.
A solution of ethyl 2-[2-(1-acetyl-3,6-dihydro-2H-pyridin-4-yl)-5-ethyl-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]acetate hydrochloride (Intermediate-35) (100 mg, 219 mol, 1.0 eq) 4-hydroxy-2H,3H-furo[2,3-c]pyridine-5-carboxylic acid (Intermediate-12) (40 mg, 219 mol, 1.0 eq) in DMF (3 mL) were treated with DIEA (141 mg, 1.10 mmol, 5.0 eq) HATU (125 mg, 329 mol, 1.5 eq) for 1 h at room temperature, followed by the addition of NaOH (aq., 10 M) (250 μL, 6 mol, 0.03 eq) dropwise at 0° C. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse Phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
LCMS: 593.2 [M+H]+.
To a stirred solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,6-tetrahydropyridine (1.00 g, 4.78 mmol, 1.0 eq) and DIEA (1.85 g, 14.3 mmol, 3.0 eq) in DCM (12 mL) was added was added acetyl chloride (563 mg, 7.17 mmol, 1.5 eq) dropwise at 0° C. under N2 atmosphere. The resulting mixture was stirred for 40 min at 0° C. The resulting mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 252.2 [M+H]-.
To a stirred solution of 1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridin-1-yl]ethanone (734 mg, 2.92 mmol, 3.0 eq) and tert-butyl 4-[2-bromo-4-(2-ethoxy-2-oxoethyl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]piperazine-1-carboxylate (Intermediate-34) (500 mg, 974 mol, 1.0 eq) in 1,4-dioxane (12.5 mL) and H2O (2.5 mL) were added K2CO3 (404 mg, 2.92 mmol, 3.0 eq) and Pd(DtBPF)Cl2 (63 mg, 97 mol, 0.1 eq) at room temperature under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred for 1 h at 100° C. under N2 atmosphere. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse Phase HPLC (C18 column, water (0.1% FA-ACN) to afford the title compound.
LCMS: 558.3 [M+H]+.
To a stirred mixture of tert-butyl 4-[2-bromo-4-(2-ethoxy-2-oxoethyl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]piperazine-1-carboxylate (Intermediate-34) (600 mg, 1.17 mmol, 1.0 eq) and 2-methoxypyridin-4-ylboronic acid (214 mg, 1.40 mmol, 1.2 eq) in 1,4-dioxane (6 mL) were added H2O (0.6 mL), K2CO3 (485 mg, 3.51 mmol, 3.0 eq) and Pd(DtBPF)Cl2 (152 mg, 234 mol, 0.2 eq) at room temperature under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred for 1.5 h at 100° C. under N2 atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
LCMS: 542.3 [M+H]+.
To a stirred mixture of tert-butyl 4-[4-(2-ethoxy-2-oxoethyl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]piperazine-1-carboxylate (500 mg, 923 mol, 1.0 eq) in THE (15 mL) were added H2O (5 mL) and LiOH (111 mg, 4.62 mmol, 5.0 eq) at room temperature. The resulting mixture was stirred for 2 h at room temperature. The residue was acidified to pH 4 with conc. HCl at 0° C. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with EtOAc (3*80 mL). The combined organic layers were washed with brine (80 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 514.2 [M+H]+.
To a stirred solution of tert-butyl 4-[2-bromo-4-(2-ethoxy-2-oxoethyl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]piperazine-1-carboxylate (Intermediate-34) (548 mg, 1.07 mmol, 1.0 eq) and 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (897 mg, 4.27 mmol, 4.0 eq) in 1,4-dioxane (8 mL) and H2O (640 L) were added Cs2CO3 (1.04 g, 3.20 mmol, 3.0 eq) and cataCXium-A-Pd-G3 (78 mg, 107 mol, 0.1 eq) at room temperature under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred at 100° C. for 2 h under N2 atmosphere. The resulting mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
LCMS: 517.3 [M+H]+.
To a stirred solution of tert-butyl 4-[2-(3,6-dihydro-2H-pyran-4-yl)-4-(2-ethoxy-2-oxoethyl)-5-ethyl-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]piperazine-1-carboxylate (486 mg, 941 mol, 1.0 eq) in 1,4-dioxane (6 mL) was added a 4 M solution of HCl/1,4-dioxane (6 mL) at room temperature and stirred for 1 h. The resulting mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 417.2 [M+H]+.
To a stirred mixture of {6-[4-(tert-butoxycarbonyl)piperazin-1-yl]-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl}acetic acid (Intermediate-38) (150 mg, 292 mol, 1.0 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine (53 mg, 350 mol, 1.2 eq) in THE (3 mL) were added DIEA (113 mg, 876 mol, 3.0 eq) and HATU (133 mg, 350 mol, 1.2 eq) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) to afford the title compound.
LCMS: 647.3 [M+H]+.
To a stirred mixture of tert-butyl 4-[5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4-({[3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl]carbamoyl}methyl)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]piperazine-1-carboxylate (275 mg, 425 mol, 1.0 eq) in dioxane (5 mL) were added a 4 M solution of HCl/1,4-dioxane (5 mL) at room temperature. The resulting mixture was stirred for 50 min at room temperature. The resulting mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 547.2 [M+H]+.
To a stirred solution of ethyl 2-[2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]acetate hydrochloride (Intermediate-23) (269 mg, 646 mol, 1.0 eq) and 4-hydroxy-2H,3H-furo[2,3-c]pyridine-5-carboxylic acid (Intermediate-12) (105 mg, 581 mol, 0.9 eq) in THE (3 mL) were added DIEA (250 mg, 1.94 mmol, 3.0 eq) and HATU (295 mg, 775 mol, 1.2 eq) at room temperature and stirred for 2 h. The resulting mixture was concentrated under reduced. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
LCMS: 580.2 [M+H]+.
To a stirred solution of ethyl 2-[2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-6-(4-{4-hydroxy-2H,3H-furo[2,3-c]pyridine-5-carbonyl}piperazin-1-yl)-7-oxo-[1,2,4]triazolo[1,5-a]pyrimidin-4-yl]acetate (167 mg, 288 mol, 1.0 eq) in THF (3 mL) and H2O (1 mL) was added LiOH (35 mg, 1.44 mmol, 5.0 eq) at room temperature and stirred for 2 h. The mixture was acidified to pH 3 with HCl (aq., 1M). The resulting mixture was diluted with water (80 mL). The resulting mixture was extracted with EtOAc (3*50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 552.2 [M+H]+.
To a solution of ethyl 5-bromothiazole-4-carboxylate (50.00 g, 211.8 mmol, 1.0 eq) in NMP (500 mL) was added DBU (48.36 g, 317.7 mmol, 1.5 eq) and the mixture was stirred at 80° C. for 2 h. The reaction mixture was cooled to room temperature, and saturated aq. NH4Cl solution (1000 mL) was added. The mixture was extracted with EtOAc (500 mL*3), and the combined organic layers were washed with brine 300 mL, dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 413.2 [M+H]+.
To a solution of butan-2-one (437 mg, 6.06 mmol, 2.5 eq) in THE (3 mL) was added a 1 M solution of LiHMDS (6.06 mL, 6.06 mmol, 2.5 eq) in THE at 0° C. and the solution was stirred at 30° C. for 0.5 h. A solution of ethyl 5-(bis(4-methoxybenzyl)amino)thiazole-4-carboxylate (1.00 g, 2.42 mmol, 1.0 eq) in THF (6 mL) was added dropwise at 30° C. The mixture was heated to 60° C. and stirred at 60° C. for 1 h. The reaction mixture was quenched by saturated aq. NH4Cl solution (200 mL) and extracted with EtOAc (50 mL*2). The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 439.1 [M+H]+.
To a solution of (Z)-1-(5-(bis(4-methoxybenzyl)amino)thiazol-4-yl)-3-hydroxypent-2-en-1-one (4.60 g, 10.5 mmol, 1.0 eq) in DCM (50 mL) was added TsOH-H2O (18 mg, 0.11 mmol, 0.01 eq) and NBS (3.73 g, 21.0 mmol, 2.0 eq) at 0° C. and the mixture was stirred at room temperature for 0.5 h. H2O (50 mL) was added and the organic phase was separated and washed with H2O (50 mL). The organic phase was dried over anhydrous Na2SO4, filtered and the filtrate was concentrated to afford the title compound, which was used into the next step without further purification.
LCMS: 596.9 [M+H]+.
To a solution of 1-(5-(bis(4-methoxybenzyl)amino)-2-bromothiazol-4-yl)-2-bromopentane-1,3-dione (6.00 g, 10.1 mmol, 1.0 eq) in THE (60 mL) was added tert-butyl piperazine-1-carboxylate (5.62 g, 30.2 mmol, 3.0 eq) and DIEA (3.90 g, 30.2 mmol, 3.0 eq), and the mixture was stirred at room temperature for 0.5 h. The reaction mixture was diluted with H2O (50 mL), extracted with EtOAc (50 mL*2). The combined organic layers were washed with H2O (50 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 703.1 [M+H]+.
A solution of tert-butyl 4-(1-(5-(bis(4-methoxybenzyl)amino)-2-bromothiazol-4-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (4.60 g, 6.56 mmol, 1.0 eq) in TFA (23 mL) was stirred at 55° C. for 0.5 h. The reaction mixture was concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 345.1 [M+H]+.
To a solution of 2-bromo-5-ethyl-6-(piperazin-1-yl)thiazolo[5,4-b]pyridin-7(4H)-one trifluoroacetate (2.20 g, 6.41 mmol, 1.0 eq) in DCM (25 mL) was added DIEA (828 mg, 6.41 mmol, 1.0 eq) and (Boc)2O (2.80 g, 12.8 mmol, 2.0 eq) at 0° C. The mixture was stirred at 0° C. for 1 h and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 445.1 [M+H]+.
To a solution of tert-butyl 4-(2-bromo-5-ethyl-7-oxo-4,7-dihydrothiazolo[5,4-b]pyridin-6-yl)piperazine-1-carboxylate (2.40 g, 5.41 mmol, 1.0 eq) and tert-butyl 2-bromoacetate (1.58 g, 8.12 mmol, 1.5 eq) in 1,4-dioxane (24 mL) was added DIEA (2.10 g, 16.2 mmol, 3.0 eq), and the mixture was stirred at 80° C. overnight. The reaction mixture was cooled to room temperature, diluted with H2O (30 mL), and extracted with EtOAc (30 mL*2). The combined organic layers were washed with H2O (10 mL*3), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 557.2 [M+H]+.
To a solution of tert-butyl 4-(2-bromo-4-(2-(tert-butoxy)-2-oxoethyl)-5-ethyl-7-oxo-4,7-dihydrothiazolo[5,4-b]pyridin-6-yl)piperazine-1-carboxylate (Intermediate-5) (460 mg, 825 μmol, 1.0 eq) and (2-methoxypyridin-4-yl)boronic acid (379 mg, 2.48 mmol, 3.0 eq) in 1,4-dioxane (10 mL) and H2O (2 mL) was added K3PO4 (525 mg, 2.48 mmol, 3.0 eq) and Pd(dppf)Cl2—CH2Cl2 (202 mg, 247 mol, 0.3 eq) in a glovebox. The resulting mixture was heated to 80° C. and stirred at 80° C. overnight under nitrogen atmosphere. The reaction mixture was cooled to room temperature and filtered through a pad of celite. The filtrate was concentrated in vacuo, and the residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 586.2 [M+H]+.
To a solution of tert-butyl 4-(4-(2-(tert-butoxy)-2-oxoethyl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4,7-dihydrothiazolo[5,4-b]pyridin-6-yl)piperazine-1-carboxylate (130 mg, 221 μmol, 1.0 eq) in DCM (0.5 mL) was added TFA (2 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 430.2 [M+H]+.
To a solution of 2-(5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-6-(piperazin-1-yl)thiazolo[5,4-b]pyridin-4(7H)-yl)acetic acid trifluoroacetate (50 mg, 0.12 mmol, 1.0 eq) in DCM (2 mL) was added DIEA until pH 8 at 0° C. (Boc)2O (38 mg, 0.17 μmol, 1.5 eq) was added and the reaction mixture was stirred at 0° C. for 0.5 h. The mixture was diluted with H2O (10 mL) and adjusted to pH 5 with aq. 1 N HCl solution. The resulting mixture was extracted with DCM (10 mL*3), the combined organic layers were concentrated in vacuo. The residue was suspended into a mixed solvent of PE/EtOAc (2 mL, PE/EtOAc=10:1) and stirred at room temperature for 10 min. The resulting suspension was filtered, the filter cake was dried in vacuo to afford the title compound, which was used into the next step without further purification. LCMS: 530.3 [M+H]+.
To a solution of 2-methoxyisonicotinonitrile (15.10 g, 112.6 mmol, 1.0 eq) in DMF (400 mL) was added MgCl2 (10.72 g, 112.6 mmol, 1.0 eq) and the mixture was stirred at room temperature for 15 mins. NaHS (25.02 g, 337.7 mmol, 3.0 eq) was added to the mixture and the resulting mixture was stirred at room temperature for 14 h. The reaction mixture was poured into water (1.5 L) and extracted with EtOAc (1 L*3). The combined organic layers were washed with brine (800 mL*5), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 169.0 [M+H]+.
To a solution of 2-methoxypyridine-4-carbothioamide (20.00 g, 118.9 mmol, 1.0 eq) in EtOH (400 mL) was added pyridine (28.21 g, 356.7 mmol, 3.0 eq) and diethyl 2-bromomalonate (28.42 g, 118.9 mmol, 1.0 eq), and then the mixture was stirred at 80° C. for 1 h. The reaction mixture was cooled to room temperature and then filtered. The filtrate was concentrated to approximately half its volume and filtered again. The combined filter cake was dried in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 281.0 [M+H]+.
To a solution of ethyl 4-hydroxy-2-(2-methoxypyridin-4-yl)thiazole-5-carboxylate (16.62 g, 59.28 mmol, 1.0 eq) and pyridine (14.07 g, 177.8 mmol, 3.0 eq) in DCM (100 mL) was added Tf2O (25.09 g, 88.92 mmol, 1.5 eq) at 0° C. Then the mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with H2O (100 mL), extracted with DCM (100 mL*2). The combined organic layers were washed with aqueous HCl solution (80 mL, 0.5 N), brine (100 mL*2), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 413.1 [M+H]+.
To a solution of ethyl 2-(2-methoxypyridin-4-yl)-4-(((trifluoromethyl)sulfonyl)oxy)thiazole-5-carboxylate (25.00 g, 60.63 mmol, 1.0 eq) and bis(4-methoxybenzyl)amine (23.40 g, 90.94 mmol, 1.5 eq) in 1,4-dioxane (400 mL) was added DIEA (23.51 g, 181.9 mmol, 3.0 eq), and the mixture was stirred at 100° C. overnight. The reaction mixture was diluted water (300 mL) and extracted with EtOAc (300 mL*2). The combined organic layers were washed with brine (100 mL*3), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 520.3 [M+H]+.
To a mixture of ethyl 4-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)thiazole-5-carboxylate (500 mg, 962 μmol, 1.0 eq), butan-2-one (278 mg, 3.85 mmol, 4.0 eq) in 2-methyltetrahydrofuran (4 mL) was added LiHMDS (1 M in THF, 3.85 mL, 4.0 eq) at room temperature and then the mixture was stirred at 80° C. for 2 h. The reaction mixture was poured into saturated NH4Cl aqueous solution (200 mL) and extracted with EtOAc (200 mL). The organic layer was concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 546.2 [M+H]+.
To a solution of 1-(4-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)thiazol-5-yl)pentane-1,3-dione (2.47 g, 4.53 mmol, 1.0 eq) in DCM (25 mL) was added TsOH-H2O (78 mg, 453 μmol, 0.1 eq) and NBS (806 mg, 4.53 mmol, 1.0 eq) and then the mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with water (35 mL) and extracted with DCM (30 mL*2). The combined organic layers were washed with brine (25 mL*3), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 626.1 [M+H]+.
To a solution of 1-(4-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)thiazol-5-yl)-2-bromopentane-1,3-dione (Intermediate-8a) (3.00 g, 4.80 mmol, 1.0 eq) in THE (30 mL) was added DIEA (1.86 g, 14.4 mmol, 3.0 eq) and tert-butyl piperazine-1-carboxylate (2.68 g, 14.4 mmol, 3.0 eq), then the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with H2O (30 mL), extracted with EtOAc (30 mL*2). The combined organic layers were washed with brine (20 mL*2), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/PE) to afford the title compound.
LCMS: 730.3 [M+H]+.
A solution of tert-butyl 4-(1-(4-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)thiazol-5-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (1.00 g, 1.37 mmol, 1.0 eq) in TFA (10 mL) was stirred at 70° C. for 4 h, and then the reaction mixture was concentrated in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 372.1 [M+H]+.
To a solution of 5-ethyl-2-(2-methoxypyridin-4-yl)-6-(piperazin-1-yl)thiazolo[4,5-b]pyridin-7(4H)-one trifluoroacetate (600 mg, 1.62 mmol, 1.0 eq) in DCM (10 mL) was added DIEA (626 mg, 4.85 mmol, 3.0 eq) and Boc2O (1.06 g, 4.85 mmol, 3.0 eq). The mixture was stirred at room temperature for 0.5 h. The reaction mixture was diluted with H2O (30 mL), extracted with DCM (30 mL*2). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue suspended in a mixture solvent of DCM and PE (DCM:PE=1:3, 10 mL) and stirred for 10 mins at room temperature. The resulting suspension was filtered, and the filter cake was dried in vacuo to afford the title compound, which was used into the next step without further purification.
LCMS: 472.2 [M+H]+.
To a stirred solution of tert-butyl 4-(5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4,7-dihydrothiazolo [4, 5-b]pyridin-6-yl) piperazine-1-carboxylate (Intermediate-2b) (500 mg, 1.06 mmol, 1.0 eq) in THF (5 mL) was added NaH (31 mg, 1.27 mmol, 1.2 eq) at 0° C. under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred for 0.5 h at 0° C. under N2 atmosphere. To the above mixture was added ethyl 2-iodoacetate (454 mg, 2.12 mmol, 2.0 eq) at 0° C. The resulting mixture was stirred for additional 4 h at 60° C. The mixture was allowed to cool down to room temperature. The reaction was quenched by the addition of Ice Water (2 mL) at 0° C. The resulting mixture was diluted with water (80 mL). The resulting mixture was extracted with EtOAc (3*80 mL). The combined organic layers were washed with brine (80 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse Phase HPLC (C18 column, water (10 mmol/L NH4HCO3)-ACN) to afford the title compound.
LCMS: 558.2 [M+H]+.
To a stirred solution of tert-butyl 4-(4-(2-ethoxy-2-oxoethyl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4, 7-dihydrothiazolo[4,5-b]pyridin-6-yl) piperazine-1-carboxylate (100 mg, 179 mol, 1.0 eq) in THF (3 mL) and H2O (1 mL) were added LiOH (13 mg, 537 mol, 3.0 eq) at 0° C. The resulting mixture was stirred for 2 h at room temperature. The mixture was acidified to pH=5 with 1 M solution aq. HCl. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with EtOAc (3*20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 530.2 [M+H]+.
To a stirred solution of 2-(6-(4-(tert-butoxycarbonyl) piperazin-1-yl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxothiazolo [4, 5-b]pyridin-4(7H)-yl) acetic acid (Intermediate-13a) (90 mg, 170 mol, 1.0 eq) and 3-(trifluoromethyl) bicycle [1.1.1]pentan-1-amine (31 mg, 204 mol, 1.2 eq) in THF (5 mL) was added HATU (97 mg, 255 mol, 1.5 eq) and DIEA (66 mg, 510 mol, 3.0 eq) at room temperature. The resulting mixture was stirred for 4 h at room temperature. The resulting mixture was diluted with water (10 mL). The resulting mixture was extracted with EtOAc (3*10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) to afford the title compound.
LCMS: 663.3 [M+H]+.
To a stirred solution of tert-butyl 4-(5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4-(2-oxo-2-((3-(trifluoromethyl) bicycle [1.1.1]pentan-1-yl) amino) ethyl)-4,7-dihydrothiazolo [4, 5-b]pyridin-6-yl) piperazine-1-carboxylate (90 mg, 136 mol, 1.0 eq) in 4 M solution of HCl/1,4-dioxane (3 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 599.1 [M+H]+.
To a solution of ethyl oxazole-5-carboxylate (10.00 g, 70.86 mmol, 1 eq) in THF (100 mL) was added LiHMDS (1 M, 248.01 mL, 3.5 eq) at −30° C. and the mixture was stirred at −30° C. for 1 h. Then I2 (53.95 g, 212.58 mmol, 3 eq) was added into the mixture and stirred at 25° C. for 15 h. The mixture was poured into H2O (500 mL). Then extracted with EtOAc (200 mL*3). The organic phase was washed with brine (500 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) and triturated with EtOH (10 mL) to afford the title compound as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 4.30 (q, 2H), 1.30 (t, 3H).
LCMS: 394.0 [M+H]+.
To a solution of ethyl 2,4-diiodooxazole-5-carboxylate (Intermediate-6) (4.90 g, 12.47 mmol, 1 eq), (2-methoxy-4-pyridyl)boronic acid (1.91 g, 12.47 mmol, 1 eq) and K2CO3 (3.45 g, 24.94 mmol, 2 eq) in dioxane (100 mL) and H2O (20 mL) was added Pd(dppf)Cl2 (913 mg, 1.25 mmol, 0.1 eq). The mixture was degassed and purged with N2 for 3 times, then stirred at 60° C. for 16 h under N2 atmosphere. The mixture was poured into H2O (200 mL), extracted with EtOAc (100 mL*3). The organic phase was washed with brine (200 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) and recrystallize with EtOH (50 mL) to afford the title compound as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.32 (d, 1H), 7.54 (d, 1H), 7.43 (s, 1H), 4.46 (q, 2H), 4.00 (s, 3H), 1.46 (t, 3H).
LCMS: 375.1 [M+H]+.
To a solution of ethyl 4-iodo-2-(2-methoxy-4-pyridyl)oxazole-5-carboxylate (1.00 g, 2.67 mmol, 1 eq) and 1-(4-methoxyphenyl)-N-[(4-methoxyphenyl)methyl]methanamine (1.03 g, 4.01 mmol, 1.5 eq) in dioxane (20 mL) was added Pd(OAc)2 (60 mg, 267 mol, 0.1 eq), Xantphos (155 mg, 267 mol, 0.1 eq) and Cs2CO3 (1.31 g, 4.01 mmol, 1.5 eq). The mixture was degassed and purged with N2 for 3 times, and stirred at 110° C. for 12 h under N2 atmosphere. It was quenched with H2O (150 mL), then the mixture was extracted with EtOAc (150 mL*3). The combined organic phase was washed with brine (150 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.30 (d, 1H), 7.53 (d, 1H), 7.42 (s, 1H), 7.19 (d, 4H), 6.86 (d, 4H), 4.77 (s, 4H), 4.31 (q, 2H), 4.02 (s, 3H), 3.81 (s, 6H), 1.33 (t, 3H).
LCMS: 504.3 [M+H]+.
A mixture of ethyl 4-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazole-5-carboxylate (500 mg, 993 mol, 1 eq) and N-methoxymethanamine hydrochloride (484 mg, 4.96 mmol, 5.0 eq) in THE (12 mL) was added LiHMDS (1 M, 7.94 mL, 8 eq) at 0° C. under N2 atmosphere and the mixture was stirred at 0° C. for 1 h under N2 atmosphere. The reaction mixture was quenched with saturated NH4Cl aqueous solution (50 mL) at 0° C. Then the mixture was extracted with EtOAc (50 mL*3). The combined organic phase was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to afford the title compound as a yellow gum, which was used into the next step directly without further purification. 1H NMR (400 MHz, CDCl3) δ ppm 8.28 (d, 1H), 7.47 (d, 1H), 7.32 (s, 1H), 7.18 (d, 4H), 6.83 (d, 4H), 4.66 (s, 4H), 4.00 (s, 3H), 3.79 (s, 6H), 3.74 (s, 3H), 3.20 (s, 3H).
LCMS: 519.2 [M+H]+.
A mixture of butan-2-one (556 mg, 7.71 mmol, 690 μL, 5.0 eq) and 4-[bis[(4-methoxyphenyl)methyl]amino]-N-methoxy-2-(2-methoxy-4-pyridyl)-N-methyl-oxazole-5-carboxamide (800 mg, 1.54 mmol, 1 eq) in THE (8 mL) was added LiHMDS (1 M, 7.71 mL, 5.0 eq) at 0° C. under N2 atmosphere. The mixture was stirred at 60° C. for 1 h. The reaction mixture was quenched with saturated NH4Cl aqueous solution (100 mL) at 0° C. Then the mixture was extracted with EtOAc (100 mL*3). The combined organic phase was washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound as a yellow gum.
LCMS: 530.2 [M+H]+.
A mixture of 1-[4-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazol-5-yl]pentane-1,3-dione (320 mg, 604 mol, 1 eq) in DCM (10 mL) was added NBS (86 mg, 483 mol, 0.8 eq) at 0° C. The reaction was stirred at 0° C. for 1 h. Then TsOH·H2O (21 mg, 121 mol, 0.2 eq) was added into the mixture and stirred at 25° C. for another 1 h. The reaction mixture was quenched with H2O (20 mL) at 25° C. Then the mixture was extracted with DCM (15 mL*3). The combined organic phase was washed with brine (20 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to afford the title compound as a yellow oil, which was used into the next step directly without purification.
LCMS: 608.1 [M+H]+.
To a solution of tert-butyl piperazine-1-carboxylate (107 mg, 575 mol, 1 eq) in THE (7 mL) was added DIEA (149 mg, 1.15 mmol, 200 μL, 2 eq) and 1-[4-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazol-5-yl]-2-bromo-pentane-1,3-dione (350 mg, 575 mol, 1 eq). The mixture was stirred at 25° C. for 16 h. It was quenched with H2O (30 mL), then extracted with EtOAc (30 mL*3). The combined organic phase was washed with brine (30 mL*2), dried over anhydrous Na2SO4, filtered and concentrated reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound as a yellow oil.
LCMS: 714.3 [M+H]+.
A solution of tert-butyl 4-[1-[4-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazole-5-carbonyl]-2-oxo-butyl]piperazine-1-carboxylate (300 mg, 420 mol, 1 eq) in TFA (6 mL) was stirred at 50° C. for 1 h. The reaction was cooled to room temperature naturally and concentrated under reduced pressure to afford the title compound as a yellow oil, which was used into the next step directly without purification.
LCMS: 356.1 [M+H]+.
To a solution of 5-ethyl-2-(2-methoxy-4-pyridyl)-6-piperazin-1-yl-4H-oxazolo[4,5-b]pyridin-7-one (150 mg, 422 mol, 1 eq) in DCM (5 mL) was added DIEA (55 mg, 422 mol, 74 μL, 1 eq) and (Boc)2O (184 mg, 844 mol, 194 μL, 2 eq) at 0° C. The mixture was stirred at 0° C. for 1 h. Then the reaction mixture was diluted with H2O (20 mL). Then the mixture was extracted with DCM (15 mL*3). The combined organic phase was washed with brine (15 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.26 (s, 1H), 7.52 (d, 1H), 7.39 (s, 1H), 4.16-4.01 (m, 2H), 3.94 (s, 3H), 3.91-3.80 (m, 2H), 3.02-2.93 (m, 4H), 2.66 (q, 2H), 1.50 (s, 9H), 1.35 (t, 3H).
LCMS: 456.2 [M+H]+.
To a solution of 2-methoxypyridine-4-carboxylic acid (60.00 g, 391.8 mmol, 1 eq) in toluene (500 mL) was added SOCl2 (140 g, 1.18 mol, 85.4 mL, 3 eq) at 20° C., then the reaction mixture was stirred at 80° C. for 2 h. The reaction mixture was filtered and washed with DCM (25 ml*2), the combined filtrate was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
To a solution of ethyl amino(cyano)acetate 4-methylbenzenesulfonate (40.61 g, 135.2 mmol, 1 eq) in pyridine (70 mL) and DCM (100 mL) was added 2 methoxypyridine-4-carbonyl chloride (23.20 g, 135.2 mmol, 1 eq) in DCM (40 mL) at 0° C. The mixture was stirred at 40° C. for 1 h. The reaction mixture was quenched by H2O (100 mL) and extracted with DCM (100 mL*3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOH/DCM) to afford the title compound.
LCMS: 264.1 [M+H]+.
To ethyl 2-cyano-2-[(2-methoxypyridine-4-carbonyl)amino]acetate (18.00 g, 68.38 mmol, 1 eq) was added TFA (125 g, 1.09 mol, 81.3 mL, 16 eq). The mixture was stirred at 40° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was triturated with EtOH (40 ml) at 0° C. for 5 min and filtered, the filter cake was washed with EtOH (20 ml*2) and MTBE (20 ml) and then dried under reduced pressure to afford the title compound.
1H-NMR (400 MHz, DMSO-d6) δ ppm 8.26 (d, 1H), 7.67 (s, 2H), 7.31 (d, 1H), 7.01 (s, 1H), 4.24 (q, 2H), 3.90 (s, 3H), 1.28 (t, 3H).
LCMS: 264.0 [M+H]+.
To a solution of ethyl 5-amino-2-(2-methoxy-4-pyridyl)oxazole-4-carboxylate (5.00 g, 19.0 mmol, 1 eq) in DMF (50 mL) was added Cs2CO3 (27.85 g, 85.47 mmol, 4.5 eq), KI (315 mg, 1.90 mmol, 0.1 eq) and 4-methoxybenzyl chloride (8.92 g, 57.0 mmol, 7.73 mL, 3 eq). The reaction mixture was stirred at 110° C. for 1 h. The reaction mixture was poured into H2O (300 mL) and was extracted with EtOAc (150 mL*3). The combined organic layers were washed with brine (100 mL*2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) and reverse Phase HPLC (C18 column, water (FA-ACN) to afford the title compound.
1H-NMR (400 MHz, CDCl3) δ ppm 8.20 (d, 1H), 7.39 (d 1H), 7.22-7.14 (m, 5H), 6.87 (d, 4H), 4.70 (s, 4H), 4.38 (q, 2H), 3.96 (s, 3H), 3.81 (s, 6H), 1.39 (t, 3H).
LCMS: 504.2 [M+H]+.
To a solution of butan-2-one (752 mg, 10.4 mmol, 933 μL, 2.5 eq) in THE (10 mL) was added LiHMDS (1 M in THF, 10.43 mL, 2.5 eq) at 0° C. under N2 atmosphere. The reaction mixture was stirred at 25° C. for 0.5 h. Then ethyl 5-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazole-4-carboxylate (2.10 g, 4.17 mmol, 1 eq) dissolved in THE (10 mL) was added into the mixture. The reaction mixture was stirred at 60° C. for 1 h under N2 atmosphere. The reaction mixture was poured into saturated NH4Cl solution (200 mL) and extracted with EtOAc (80 mL*3). The combined organic layers were washed with brine (50 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 530.2 [M+H]+.
To a solution of 1-[5-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazol-4-yl]-3-hydroxypent-2-en-1-one (1.00 g, 1.89 mmol, 1 eq) in DCM (10 mL) was added NBS (269 mg, 1.51 mmol, 0.8 eq) at 0° C. The reaction mixture was stirred at 25° C. for 1h. The reaction mixture was poured into H2O (100 mL) and was extracted with DCM (60 mL*3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound, which was used into the next step directly without purification.
LCMS: 610.1 [M+H]+.
To a solution of 1-[5-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazol-4-yl]-2-bromo-pentane-1,3-dione (Intermediate-7b) (700 mg, 1.15 mmol, 1 eq) and tert-butyl piperazine-1-carboxylate (214 mg, 1.15 mmol, 1 eq) in THE (7 mL) was added DIEA (298 mg, 2.30 mmol, 401 μL, 2 eq). The reaction mixture was stirred at 25° C. for 12 h. The reaction mixture was poured into H2O (100 mL) and extracted with EtOAc (60 mL*3). The combined organic layers was washed with brine (50 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H-NMR (400 MHz, CDCl3) δ ppm 8.22 (d, 1H), 7.26 (d, 1H), 7.17 (d, 4H), 7.06 (s, 1H), 6.88 (d, 4H), 5.28 (s, 1H), 4.97-4.86 (m, 2H), 4.84-4.70 (m, 2H), 3.98 (s, 3H), 3.81 (s, 6H), 3.48-3.44 (m, 4H), 2.90-2.54 (m, 6H), 1.44 (s, 9H), 1.10 (t, 3H).
LCMS: 714.3 [M+H]+.
To a solution of tert-butyl 4-[1-[5-[bis[(4-methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazole-4-carbonyl]-2-oxo-butyl]piperazine-1-carboxylate (700 mg, 981 mol, 1 eq) in TFA (7 mL) was stirred at 50° C. for 1 h. The reaction was cooled to room temperature and concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 494.2 [M+H]+.
To a solution of 1-(5-((4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)oxazol-4-yl)-2-(piperazin-1-yl)pentane-1,3-dione trifluoroacetate (590 mg, 971 mol, 1 eq) in DCM (6 mL) was added DIEA (377 mg, 2.91 mmol, 507 μL, 3 eq) and Boc2O (254 mg, 1.17 mmol, 267 μL, 1.2 eq). The reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was poured into H2O (50 mL) and was extracted with DCM (50 mL*3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H-NMR (400 MHz, CDCl3) δ ppm 8.23 (d, 1H), 7.76 (s, 1H), 7.36-7.28 (m, 2H), 7.14 (s, 1H), 6.92 (d, 2H), 5.00 (s, 1H), 4.62 (d, 2H), 3.99 (s, 3H), 3.82 (s, 3H), 3.45-3.49 (m, 4H), 2.79-2.47 (m, 6H), 1.43 (s, 9H), 1.10 (t, 3H).
LCMS: 594.3 [M+H]+.
To a solution of tert-butyl 4-(1-(5-((4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)oxazol-4-yl)-1,3-dioxopentan-2-yl)piperazine-1-carboxylate (460 mg, 775 mol, 1 eq) and tert-butyl 2-bromoacetate (151 mg, 775 mol, 114 μL, 1 eq) in THE (4.6 mL) was added t-BuOK (348 mg, 3.10 mmol, 4 eq). The reaction mixture was stirred at 50° C. for 0.5 h. Then second batch of tert-butyl 2-bromoacetate (151 mg, 775 mol, 114 μL, 1 eq) was added to the mixture. The reaction mixture was stirred at 50° C. for another 0.5 h. Then the third batch of tert-butyl 2-bromoacetate (151 mg, 775 mol, 114 μL, 1 eq) was added into the mixture and stirred at 50° C. for another 0.5 h. The reaction mixture was diluted with H2O (50 mL) and extracted with DCM (40 mL*3). The combined organic phase was washed with brine (30 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H-NMR (400 MHz, CDCl3) δ ppm 8.23 (d, 1H), 7.29 (s, 1H), 7.25 (d, 2H), 7.09 (s, 1H), 6.90 (d, 2H), 5.28 (s, 2H), 4.85 (s, 1H), 4.58-4.29 (m, 2H), 3.98 (s, 3H), 3.82 (s, 3H), 3.48-3.44 (m, 4H), 2.76-2.69 (m, 4H), 2.55 (q, 2H),1.44 (s, 9H), 1.42 (s, 9H), 1.09 (t, 3H).
LCMS: 708.4 [M+H]+.
To a solution of tert-butyl 4-[1-[5-[(2-tert-butoxy-2-oxo-ethyl)-[(4 methoxyphenyl)methyl]amino]-2-(2-methoxy-4-pyridyl)oxazole-4-carbonyl]-2-oxo-butyl]piperazine-1-carboxylate (400 mg, 565 mol, 1 eq) in TFA (4 mL) was stirred at 120° C. for 3 h. The reaction mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 414.1 [M+H]+.
To a solution of 2-(5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-6-(piperazin-1-yl)oxazolo[5,4-b]pyridin-4(7H)-yl)acetic acid Trifluoroacetic acid salt (290 mg, 550 mol, 1 eq) in DCM (3 mL) was added DIEA (355 mg, 2.75 mmol, 479 μL, 5 eq) and (Boc)2O (180 mg, 825 mol, 190 μL, 1.5 eq). The reaction mixture was stirred at 25° C. for 0.5 h. The reaction mixture was poured into H2O (3 mL) and was extracted with DCM (4 mL*3). The combined organic layers were washed with brine (5 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of MeOH/DCM) to afford the title compound.
LCMS: 514.1 [M+H]+.
To a mixture of 2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-oxazolo[5,4-b]pyridin-4-yl]acetic acid (Intermediate-7a) (50 mg, 97 mol, 1 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine hydrochloride (37 mg, 195 mol, 2 eq) in DMF (0.5 mL) were added HATU (74 mg, 195 mol, 2 eq) and DIEA (38 mg, 292 mol, 51 μL, 3 eq). The mixture was stirred for 1 h at 25° C. The reaction mixture was poured into water (20 mL) and was extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) to afford the title compound.
LCMS: 647.3 [M+H]+.
A solution of tert-butyl 4-[5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-4-[2-oxo-2-[[3-(trifluoromethyl)-1-bicyclo[1.1.1]pentanyl]amino]ethyl]oxazolo[5,4-b]pyridin-6-yl]piperazine-1-carboxylate (25 mg, 39 mol, 1 eq) in HCl/1,4-dioxane (2 M, 0.2 mL) was stirred at 25° C. for 0.5 h. The reaction mixture was concentrated under reduced pressure directly to afford the title compound, which was used into the next step without further purification.
LCMS: 547.3 [M+H]+.
To a solution of tert-butyl 4-[5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-4H-oxazolo[4,5-b]pyridin-6-yl]piperazine-1-carboxylate (Intermediate-6a) (1.00 g, 2.20 mmol, 1 eq) and ethyl 2-bromoacetate (440 mg, 2.63 mmol, 292 μL, 1.2 eq) in dioxane (10 mL) was added DIEA (851 mg, 6.59 mmol, 1.15 mL, 3 eq). The mixture was stirred at 100° C. for 0.5 h. The reaction mixture was diluted with H2O (200 mL) and extracted by EtOAc (250 mL*2). The combined organic phase was washed with brine (100 mL*1), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
1H NMR (400 MHz, DMSO-d6) δ ppm 8.43 (d, 1H), 7.60 (d, 1H), 7.37 (s, 1H), 5.23 (s, 2H), 4.21 (q, 2H), 3.94-3.85 (m, 5H), 3.63 (q, 2H), 2.95-2.58 (m, 4H), 2.59-2.56 (m, 2H), 1.43 (s, 9H), 1.23 (t, 3H), 1.11 (t, 3H).
LCMS: 542.2 [M+H]+.
To a solution of tert-butyl 4-[4-(2-ethoxy-2-oxo-ethyl)-5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-oxazolo[4,5-b]pyridin-6-yl]piperazine-1-carboxylate (180 mg, 332 mol, 1 eq) in EtOH (2 mL), THF (2 mL) and H2O (2 mL) was added LiOH·H2O (21 mg, 499 mol, 1.5 eq). The mixture was stirred at 25° C. for 0.5 h. The mixture was adjusted pH to about 4 by addition of HCl (1M) dropwise at 25° C. and the mixture was extracted with EtOAc (5 mL*3). The combined organic phase was washed with brine (15 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to afford the title compound, which was used into next step directly without further purification.
LCMS: 514.2 [M+H]+.
To a solution of 2-[6-(4-tert-butoxycarbonylpiperazin-1-yl)-5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-oxazolo[4,5-b]pyridin-4-yl]acetic acid (Intermediate-14a) (180 mg, 294 mol, 1 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine hydrochloride salt (110 mg, 589 mol, 2 eq) in DMF (2 mL) was added DIEA (114 mg, 883 mol, 154 μL, 3 eq) and HATU (224 mg, 589 mol, 2 eq). The mixture was stirred at 25° C. for 1 h. To the reaction mixture was added H2O (15 mL). Then the mixture was extracted with EtOAc (15 mL*3). The combined organic phase was washed with brine (20 mL*2), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 647.3 [M+H]+
A solution of tert-butyl 4-[5-ethyl-2-(2-methoxy-4-pyridyl)-7-oxo-4-[2-oxo-2-[[3-(trifluoromethyl)-1-bicyclo[1.1.1]pentanyl]amino]ethyl]oxazolo[4,5-b]pyridin-6-yl]piperazine-1-carboxylate (180 mg, 278 mol, 1 eq) in HCl/dioxane (2 M, 3.6 mL, 26 eq) was stirred at 25° C. for 0.2 h. The mixture was concentrated in vacuum directly to afford the title compound, which was used into next step directly without further purification.
LCMS: 547.2 [M+H]+.
To a stirred solution of 2-methoxypyridin-4-amine (1.00 g, 8.06 mmol, 1.0 eq) in HCl (4 mL, conc.) and H2O (4 mL) was added a solution of NaNO2 (560 mg, 8.06 mmol, 1 eq) in H2O (0.5 mL) dropwise at 0° C. under N2 atmosphere. The resulting mixture was stirred for 30 min at 0° C. under N2 atmosphere. To the above mixture was added ethyl 2-cyanoacetate (910 mg, 8.06 mmol, 1.0 eq) in EtOH (4 mL) and NaOAc (3.96 g, 48.3 mmol, 6.0 eq) in H2O (10 mL) at 0° C. The resulting mixture was stirred for additional 5 h at room temperature. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with EtOAc (3*200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) to afford the title compound.
LCMS: 249.1 [M+H]+.
A solution of ethyl 2-cyano-2-(2-(2-methoxypyridin-4-yl)hydrazineylidene) acetate (10.0 g, 40.3 mmol, 1.0 eq) and bis[(4-methoxyphenyl)methyl]amine (15.6 g, 60.4 mmol, 1.5 eq) in ACN (400 mL) was stirred for 10 min at 80° C. To the above mixture was added Cu(OAc)2 (3.66 g, 20.1 mmol, 0.5 eq) at 80° C. and stirred for additional 1 h at 80° C. To the above mixture was added Cu(OAc)2 (3.66 g, 20.1 mmol, 0.5 eq) at 80° C. The resulting mixture was stirred for additional 5 h at 80° C. The mixture was cooled to room temperature. The mixture was filtered, and the filtrate was concentrated. The residue was purified directly by reverse phase HPLC (C18 column, H2O (10 mmol/L NH4HCO3)-ACN) to afford the title compound. 1H NMR (400 MHz, DMSO-d6) δ 8.29-8.27 (m, 1H), 7.47-7.45 (m, 1H), 7.26-7.19 (m, 4H), 7.14-7.12 (m, 1H), 6.91-6.83 (m, 4H), 4.51 (s, 4H), 4.35-4.29 (m, 2H), 3.91 (s, 3H), 3.71 (s, 6H), 1.30-1.25 (m, 3H).
LCMS: 504.2 [M+H]+.
To a stirred solution of butan-2-one (2.18 g, 30.3 mmol, 2.5 eq) in THE (200 mL) was added a 1 M solution of LiHMDS (30.3 mL, 30.3 mmol, 2.5 eq) dropwise at 0° C. under N2 atmosphere. The resulting mixture was stirred for 30 min at room temperature under N2 atmosphere. To the above mixture was added ethyl 5-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)-2H-1,2,3-triazole-4-carboxylate (6.10 g, 12.1 mmol, 1.0 eq) in THF (50 mL) dropwise at 60° C. The resulting mixture was stirred for additional 1 h at 60° C. The mixture was cooled to room temperature and quenched with sat. NH4Cl (aq.) at 0° C. The resulting mixture was extracted with EtOAc (3*500 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 530.2 [M+H]+.
To a stirred solution of 1-(5-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)-2H-1,2,3-triazol-4-yl)-3-hydroxypent-2-en-1-one (7.36 g, 13.9 mmol, 1.0 eq) in DCM (500 mL) was added NBS (4.95 g, 27.8 mmol, 2.0 eq) and TsOH-H2O (30 mg, 0.14 mmol, 0.01 eq) at 0° C. under N2 atmosphere. The resulting mixture was stirred for 2 h at room temperature under N2 atmosphere. The resulting mixture was diluted with water (400 mL). The resulting mixture was extracted with DCM (3*400 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification. LCMS: 608.1 [M+H]+.
A solution of methyl(ethyl) ketone (4.30 g, 59.6 mmol, 3.00 eq) in THE (360 mL) was degassed with N2 three times, followed by the addition of 1 M LiHMDS in THE (59.6 mL, 59.6 mmol, 3.00 eq) dropwise at 0° C. The resulting mixture was stirred for 30 min at room temperature. To the above mixture was added a solution of ethyl 5-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)-2H-1,2,3-triazole-4-carboxylate (can be obtained according to the procedure described above) (10.0 g, 19.9 mmol, 1.00 eq) in THE (40.0 mL) dropwise at 60° C. The resulting mixture was stirred for additional 1 h at 60° C. The reaction was allowed to cool down to 0° C. and then was quenched with sat. NH4Cl (aq.). The resulting mixture was extracted with EtOAc (3×500 mL). The combined organic layers were washed with brine (1×500 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 530.0[M+H]+.
To a solution of 1-(5-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)-2H-1,2,3-triazol-4-yl)-3-hydroxypent-2-en-1-one (6.00 g, 11.3 mmol, 1.00 eq) in DCM (300 mL) was added NBS (1.81 g, 10.2 mmol, 0.90 eq). The resulting mixture was degassed with N2 three times, and then was stirred for 1 h at room temperature. The resulting mixture was used in the next step directly without further purification. LCMS: 608.0[M+H]+.
To the solution of Intermediate 16 was added THE (300 mL), tert-butyl (15,65)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (1.73 g, 8.15 mmol, 0.9 eq) and DIEA (2.34 g, 18.1 mmol, 2.00 eq) at room temperature. The resulting mixture was degassed with N2 three times, and was stirred for 16 h at room temperature. The resulting mixture was diluted with H2O (300 mL) and extracted with DCM (3×500 mL). The combined organic layers were washed with brine (1×500 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound, which was used in the next step directly without purification. LCMS: 740.0[M+H]+.
A solution of tert-butyl (1S,6S)-5-(1-(5-(bis(4-methoxybenzyl)amino)-2-(2-methoxypyridin-4-yl)-2H-1,2,3-triazol-4-yl)-1,3-dioxopentan-2-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (15.5 g, 11.5 mmol, 1.00 eq, 69%) in TFA (100 mL) was stirred for 30 min at 60° C. The resulting mixture was allowed to cool down to room temperature, and then was concentrated under reduced pressure. The residue was purified by trituration with Et2O (150 mL). The precipitated solids were collected by filtration and washed with Et2O to afford the title compound. LCMS: 382.0[M+H]+.
To a stirred mixture of 6-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-5-ethyl-2-(2-methoxypyridin-4-yl)-2,4-dihydro-7H-[1,2,3]triazolo[4,5-b]pyridin-7-one trifluoroacetate (5.40 g, 11.3 mmol, 1.00 eq) in DCM (100 mL) was added DIEA (2.91 g, 22.5 mmol, 2.00 eq) and (Boc)2O (2.46 g, 11.3 mmol, 1.00 eq) at 0° C. After stirring for 30 min at room temperature, the resulting mixture was diluted with H2O (200 ml). And then was extracted with DCM (3×200 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 482.0[M+H]+.
To a stirred solution of tert-butyl (1S,6S)-5-(5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4,7-dihydro-2H-[1,2,3]triazolo[4,5-b]pyridin-6-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (Intermediate-17) (270 mg, 0.56 mmol, 1.00 eq) in THF (8.00 mL) was added NaH (112 mg, 2.81 mmol, 5.00 eq, 60% dispersion in mineral oil) in portions at 0° C. The resulting mixture was degassed with N2 for three times, and then was stirred for 30 min at 0° C. To the above mixture was added a solution of ethyl 2-bromoacetate (562 mg, 3.37 mmol, 6.00 eq) in THF (2.00 mL) at 0° C. After stirring for 30 min at 60° C., the resulting mixture was allowed to cool down to 0° C., and then was quenched by sat. NH4Cl (aq.). The resulting mixture was extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (1×25 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (Eluent of EtOAc/PE) to afford the title compound. LCMS: 568.0[M+H]+.
To a stirred mixture of tert-butyl (1S,6S)-5-[4-(2-ethoxy-2-oxoethyl)-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-[1,2,3]triazolo[4,5-b]pyridin-6-yl]-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (Intermediate-19) (117 mg, 206 mol, 1.0 eq) and LiOH (15 mg, 618 mol, 3.0 eq) in THF (6 mL) was added H2O (2 mL) at room temperature under air atmosphere. The resulting mixture was stirred for 1 h at room temperature. The residue was acidified to pH 5 with HCl (4 mL, conc.). The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with EtOAc (3*20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound, which was used in the next step directly without purification. LCMS: 540.3 [M+H]+.
To a stirred mixture of {6-[(1S,6S)-5-(tert-butoxycarbonyl)-2,5-diazabicyclo[4.2.0]octan-2-yl]-5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-[1,2,3]triazolo[4,5-b]pyridin-4-yl}acetic acid (Intermediate-18) (100 mg, 185 mol, 1.0 eq) and 3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-amine (34 mg, 222 mol, 1.2 eq) in pyridine (5 mL) was added EDCI (71 mg, 370 mol, 2.0 eq) at room temperature under N2 atmosphere. The resulting mixture was degassed with N2 for three times, then stirred for 2 h at 60° C. under N2 atmosphere. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of DCM/MeOH) to afford the title compound. LCMS: 673.3 [M+H]+.
A mixture of tert-butyl (1S,6S)-5-(5-ethyl-2-(2-methoxypyridin-4-yl)-7-oxo-4-(2-oxo-2-((3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl)amino)ethyl)-4,7-dihydro-2H-[1,2,3]triazolo[4,5-b]pyridin-6-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (60 mg, 89 mol, 1.0 eq) in 4 M solution of HCl in 1,4-dioxane (2.5 mL) was stirred for 1 h at room temperature. The resulting mixture was concentrated under reduced pressure to afford the title compound, which was used into the next step directly without purification.
LCMS: 609.2 [M+H]+.
To a stirred solution of ethyl 6-hydroxy benzo[d]oxazole-7-carboxylate (200 mg, 966 mol, 1 eq) in MeOH (3 mL) and H2O (0.6 mL) was added NaOH (193 mg, 4.83 mmol, 5 eq) at room temperature. The resulting mixture was stirred for overnight at 60° C. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under vacuum. H2O (6 mL) was added and the mixture was acidified to pH=3 with aq. HCl (2N) at 0° C. The precipitated solids were collected by filtration and washed with H2O (3×3 mL) to afford the title compound.
LCMS: 178.0 [M−H]−.
To a solution of 3,5-dichloropyrazine-2-carboxylic acid (200 mg, 1.04 mmol, 1.0 eq) and (4-methoxyphenyl)methanamine (142 mg, 1.04 mmol, 1.0 eq) in 1,4-dioxane (2 mL) was added DIEA (335 mg, 2.59 mmol, 2.5 eq), and the resulting mixture was stirred at 100° C. for 2 h. The reaction mixture was acidized to pH 2 with aqueous HCl solution (1 M), and then extracted with EtOAc (30 mL*2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound.
LCMS: 294.1[M+H]+.
1H NMR (400 MHz, CDCl3) δ ppm 8.37 (br s, 1H), 7.72 (s, 1H), 7.32-7.27 (m, 2H), 6.91-6.86 (m, 2H), 4.66 (d, 2H), 3.81 (s, 3H).
A mixture of methyl methyl 2H-1,2,3-triazole-4-carboxylate (5.00 g, 39.3 mmol, 1.00 eq), oxone (7.28 g, 43.3 mmol, 1.10 eq) and KBr (5.15 g, 43.3 mmol, 1.10 eq) in ACN (50.0 mL) and H2O (50.0 mL) was stirred for overnight at room temperature. The resulting mixture was filtered, the filter cake was washed with ACN. The organic layer of the filtrate was separated. The aqueous layer was extracted with ACN. The combined organic layers were concentrated under reduced pressure. The residue was diluted with EtOAc, filtered, the filter cake was washed with EtOAc. The combined filtrate was concentrated under reduced pressure to afford the title compound. LCMS: 206 [M+H]+.
A mixture of methyl 5-bromo-2H-1,2,3-triazole-4-carboxylate (1.00 g, 4.85 mmol, 1.00 eq), (3,6-dihydro-2H-pyran-4-yl)boronic acid (intermediate-107) (312 mg, 2.43 mmol, 0.50 eq), Py (1.54 g, 19.5 mmol, 4.00 eq) and Cu(OAc)2 (971 mg, 4.85 mmol, 1.00 eq) in DCM (10 mL) was stirred for overnight at 40° C. The resulting mixture was filtered, the filter cake was washed with DCM. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (eluent of EtOAc/PE) to afford the title compound. LCMS: 288[M+H]+.
To a mixture of methyl 5-bromo-2-(3,6-dihydro-2H-pyran-4-yl)-2H-1,2,3-triazole-4-carboxylate (8.10 g, 28.1 mmol, 1.00 eq), (4-methoxyphenyl)methanamine (5.79 g, 42.2 mmol, 1.50 eq), XantPhos (6.51 g, 11.2 mmol, 0.40 eq) and Cs2CO3 (18.3 g, 56.2 mmol, 2.00 eq) in 1,4-dioxane (324 mL) was added Pd2(dba)3 (5.15 g, 5.62 mmol, 0.20 eq). The mixture was degassed three times with N2 and stirred for overnight at 100° C. The resulting mixture was cooled to room temperature and filtered, the filter cake was washed with DCM. The combined filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 345[M+H]+.
To a solution of butan-2-one (942 mg, 13.1 mmol, 5.00 eq) in THE (5.00 mL) was added dropwise 1M LiHMDS in THE (13.1 mL, 13.1 mmol, 5.00 eq) at 0° C. under N2. The reaction mixture was stirred at room temperature for 30 min. Then a solution of methyl 2-(3,6-dihydro-2H-pyran-4-yl)-5-((4-methoxybenzyl)amino)-2H-1,2,3-triazole-4-carboxylate (900 mg, 2.61 mmol, 1.00 eq) in THE (13.0 mL) was added dropwise and the mixture was stirred for another 60 mins at 60° C. The reaction was quenched with sat. NH4C1, and then the mixture was extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 385[M+H]+.
To a stirred solution of 1-(2-(3,6-dihydro-2H-pyran-4-yl)-5-((4-methoxybenzyl)amino)-2H-1,2,3-triazol-4-yl)-3-hydroxypent-2-en-1-one (Intermediate-103) (2.50 g, 6.50 mmol, 1.00 eq) in DCM (25.0 mL) was added NBS (1.16 g, 6.50 mmol, 1.00 eq) at 0° C. under N2 atmosphere. The mixture was stirred for 1 h at 0° C. The resulting mixture was used in the next step directly without further purification. LCMS: 463/465[M+H]+.
To the solution of step 1 were added tert-butyl (1S,6S)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (916 mg, 4.32 mmol, 0.80 eq) and DIEA (2.09 g, 16.19 mmol, 3.00 eq) at room temperature. The mixture was stirred for 2 h at room temperature. The mixture was diluted with water (50 mL) and extracted with DCM (2×50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound, which was used to the next step directly without further purification. LCMS: 595[M+H]+.
A solution of tert-butyl (1S,6S)-5-(1-(2-(3,6-dihydro-2H-pyran-4-yl)-5-((4-methoxybenzyl)amino)-2H-1,2,3-triazol-4-yl)-1,3-dioxopentan-2-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (2.70 g, 4.54 mmol, 1.00 eq) in TFA (20.0 mL) was stirred for 30 min at 60° C. The resulting mixture was allowed to cool down to room temperature, and then was concentrated under reduced pressure. The residue was purified by trituration with Et2O (30 mL). The solids were collected by filtration and washed with Et2O to afford the title compound. LCMS: 357[M+H]+.
To a stirred mixture of 6-((1S,6S)-2,5-diazabicyclo[4.2.0]octan-2-yl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-2,4-dihydro-7H-[1,2,3]triazolo[4,5-b]pyridin-7-one, TFA salt (2.30 g, 4.89 mmol, 1.00 eq) in DCM (20.0 mL) were added DIEA (2.50 g, 19.36 mmol, 3.96 eq) and (Boc)2O (1.55 g, 7.10 mmol, 1.45 eq). The mixture was stirred at room temperature for 30 mins and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of PE/EA) to afford the title compound. LCMS: 457[M+H]+.
To a solution of tert-butyl (1S,6S)-5-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-4,7-dihydro-2H-[1,2,3]triazolo[4,5-b]pyridin-6-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (Intermediate-104) (550 mg, 1.21 mmol, 1.00 eq) in THE (6.00 mL) was added NaH (193 mg, 4.82 mmol, 4.00 eq, 60% dispersion in mineral oil) at 0° C. The mixture was stirred for 30 min. Ethyl bromoacetate (402 mg, 2.41 mmol, 2.00 eq) was added and the mixture was allowed to warm to 60° C. and stirred for 2 h. The mixture was cooled down to room temperature and quenched with sat. aq. NH4Cl (10 mL), then the mixture was extracted with EtOAc (3×20 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 543[M+H]+.
To a stirred solution of tert-butyl (1S,6S)-5-(2-(3,6-dihydro-2H-pyran-4-yl)-4-(2-ethoxy-2-oxoethyl)-5-ethyl-7-oxo-4,7-dihydro-2H-[1,2,3]triazolo[4,5-b]pyridin-6-yl)-2,5-diazabicyclo[4.2.0]octane-2-carboxylate (Intermediate-105) (150 mg, 0.28 mmol, 1.00 eq) in MeOH (1.00 mL) and H2O (1.00 mL) was added LiOH (23 mg, 0.55 mmol, 2.00 eq). The mixture was stirred for 1 h at room temperature. The mixture was acidified to pH 3-4 with 1N HCl (aq.). The resulting mixture was extracted with EtOAc (3×10 mL). The combined organic layer was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound. LCMS: 515[M+H]+.
To a stirred solution of NaIO4 (61.1 g, 286 mmol, 3.00 eq) in H2O (600 mL) was added a solution of NH4OAc (22.0 g, 286 mmol, 3.00 eq) in H2O (600 mL) at room temperature. To the above mixture was added a solution of 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (20.0 g, 95.2 mmol, 1.00 eq) in ACN (1.20 L) at 0° C. The resulting mixture was stirred for additional 4 h at room temperature. The resulting mixture was filtered, the filter cake was washed with ACN. The resulting mixture was concentrated under reduced pressure to remove ACN, the residue was extracted with EtOAc (3×3 L). The combined organic layers were washed with brine (2×500 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was diluted in EtOAc (1 L). The resulting mixture was filtered, the filter cake was washed with EtOAc. The combined filtrate was concentrated under reduced pressure to afford the title compound. LCMS: 129[M+H]+.
To a suspension of 3-aminobicyclo[1.1.1]pentan-1-ol hydrochloride (530 mg, 3.91 mmol, 1 eq) in THE (5.4 mL) and H2O (2.7 mL) was added NaHCO3 (985 mg, 11.73 mmol, 3 eq) at 20° C. The mixture was cooled to 0° C., then CbzCl (733 mg, 4.30 mmol, 613 μL, 1.1 eq) was added dropwise at 0° C. under N2 atmosphere. The resulting mixture was stirred at 0° C. for 15 min, then warmed to 20° C. and stirred at 20° C. for 16 h under N2 atmosphere. The mixture was diluted with brine (10 mL) and then extracted with EtOAc (3×10 ml). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 234.0 [M+H]+.
To a solution of benzyl N-(3-hydroxy-1-bicyclo[1.1.1]pentanyl)carbamate (610 mg, 2.62 mmol, 1 eq) and Zn(NTf2)2 (1.96 g, 3.14 mmol, 1.2 eq) in CHCl3 (5 mL) was added 1-(trifluoromethyl)-1,2-benziodoxol-3-one (992 mg, 3.14 mmol, 1.2 eq). The mixture was stirred at 25° C. for 16 h. The mixture was filtered and the filter cake was washed with DCM (2×5 mL). The combined filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of EtOAc/PE) to afford the title compound. LCMS: 324.0 [M+Na]+
A solution of benzyl N-[3-(trifluoromethoxy)-1-bicyclo[1.1.1]pentanyl]carbamate (100 mg, 330 mol, 1eq) in TFA (3 mL) was stirred at 70° C. for 5 h. The mixture was concentrated under reduced pressure to afford the title compound, which was used directly in the next step without further purification. NMR (400 MHz, DMSO-d6) δ ppm 8.96 (s, 2H), 2.40 (s, 6H). LCMS: 168.0 [M+H]+.
The compounds of the disclosure are shown below in Table 2 or Table 2a's along with the LCMS method (see below table for method conditions), mass observed, and retention time of compound.
Further compounds of the disclosure are shown below in Table 2a along with the theoretical mass.
Bovine skin gelatin (BSG), dimethyl sulfoxide (DMSO), Pluronic F-127 and tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO) at the highest level of purity possible. Bicine buffer solution was purchased from Alfa Aesar (Tewksbury, MA) and compound NSC-617145 was purchased from Tocris (Minneapolis, MN). DNA duplex was synthesized at BGI (Shenzhen, China) and was composed of strand 1 with the sequence 5′-GCACTGGCCGTCGTTTTACGGTCG-3′ (SEQ ID NO.: 1) and strand 2 with the sequence 5′-TCCAAGTAAAACGACGGCCAGTGC-3′ (SEQ ID NO.: 2). DNA strands were annealed by heating to 95° C. for 5 minutes followed by slow cooling to room temperature. Compounds in 100% DMSO (0.1 ml) were spotted into a 384-well white polystyrene Optiplate-384 (Perkin Elmer; Waltham, MA) assay plate using a LabCyte Echo 550 (Agilent; Santa Clara, CA). DMSO (0.1 ml) was added to columns 12, rows A-H and column 24, rows I-P for the maximum signal control. Compound NSC-617145 (0.1 ml) was added to columns 12, rows I-P and 24, rows A-H for the minimum signal control (100% inhibition). Compounds/DMSO were preincubated for 15 minutes at 25° C. with 5 ml 2×WRN (BV08), prepared as described below, in assay buffer containing 20 mM Bicine (pH=7.5), 1 mM MgCl2, 10 mM KCl, 0.1% Pluronic F-127, 0.005% BSG, 1 mM TCEP. The reaction was initiated by the addition of 5 ml 2× substrate mixture in assay buffer and incubated for 60 minutes at 25° C. The final concentrations of the assay components were 0.15 nM WRN, 5 mM ATP, and 0.1 nM DNA duplex. The final DMSO concentration was 1% and the reference compound concentration (NSC-617145) used for the minimal signal control was 20 mM. The reaction was stopped by the addition of the ADP-Glo Kit components (Promega; Madison, WI) as directed and the relative luminescence units (RLU) were read on an Envision 2104 (Perkin Elmer; Waltham, MA).
Where RLU=relative luminescence units, sample=signal in sample well, and MIN and MAX are the respective minimum and maximum signal controls.
Where top and bottom are normally allowed to float but may be fixed at 100 or 0 respectively in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Molecular Biology and virus production. The DNA encoding human Werner helicase (Uniprot Q14191, amino acids 517-1235 with L1074F point mutation) was generated with codon-optimization for E. coli expression and subcloned into the pFastBac vector with a TEV cleavable 8×His tag (SEQ ID NO: 5) (WRN—BV08). The baculovirus from the expression plasmid WRN—BV08 was generated from transfection and amplification following the manufacturer's instructions.
Gene sequence of WRN—BV08 [pFastBac1-WRN-(517-1235 L1074F)-TEV-8His](SEQ ID NO.: 3)
Protein sequence of WRN—BV08 [pFastBac1-WRN-(517-1235 L1074F)-TEV-8His](SEQ ID NO.: 4)
Sf9 cells grown in SF900II media were infected with 1:200 WRN—BV08 P2 virus and incubated for protein expression for 72 h at 27° C. The WRN protein was purified using the following protocol. The cell pellets were thawed and resuspended in buffer A (50 mM Tris, pH 7.5, 500 mM NaCl, 1 mM TCEP, 10% Glycerol) supplemented with 0.5% CHAPS, 1 mM PMSF, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin, and the Pierce Universal Nuclease and cocktail tablet. Cleared lysates were loaded onto a Ni Sepharose™ excel column and washed with buffer A and bound protein was eluted with buffer A supplemented with 300 mM imidazole. The eluted protein was dialyzed against buffer A and digested by His-tagged TEV (1:5 ratio) overnight at 4° C. ZnCl2 was added into the sample at final 15 μM before loading onto a second Ni Sepharose™ excel column. Untagged WRN protein was eluted from the column with buffer A supplemented with 20 mM imidazole, dialyzed overnight into buffer B (50 mM Tris, pH 7.5, 1 mM TCEP, 10% Glycerol) supplemented with 150 mM NaCl and loaded onto a Heparin column. Proteins were eluted with a step gradient of buffer B supplemented with 150 mM, 200 mM, 300 mM and 500 mM NaCl. WRN containing fractions were pooled and concentrated prior to loading on to size exclusion chromatography using a HiLoad 16/600 Superdex™ 200 μg column (GE Healthcare) in buffer C (20 mM HEPES, pH 7.5, 250 mM NaCl, 0.25 mM TCEP, 2.5% Glycerol).
The resultant IC50 results obtained for the tested compounds are shown below in Table 3. Compounds with an IC50 less than or equal to 0.005 μM are designated as “A.” Compounds with an IC50 greater than 0.005 μM and less than or equal to 0.05 μM are designated as “B.” Compounds with an IC50 greater than 0.05 μM and less than or equal to 0.1 μM are designated as “C.” Compounds with an IC50 greater than 0.1 μM or equal to 0.5 μM are designated as “D.” Compounds with an IC50 greater than 0.5 μM are designated as “E.”
The colon carcinoma cell line HCT 116 was obtained from ATCC and cultured in growth medium consisting of Mccoy's 5A Medium (Gibco 16600108) supplemented with 10% FBS (Transgene FS201-02) and 100 units/mL penicillin-streptomycin (Gibco 15140122) and maintained at 37° C. under 5% CO2. On the day of seeding, 2,000 cells in 30 μL of culture media were plated per well to Poly-D-Lysine 384 Well Black Clear Plates (Biocoat 356663) and incubated overnight at 37° C. under 5% CO2. The following day, compounds were serially diluted in DMSO for a total of 11 test concentrations. The typical starting concentration of compounds was 10 μM with 2-fold dilutions. Next, 150 nL of diluted compound was added in duplicate to the assay plate, using an Echo 655 (Labcyte). The plate was centrifuged at 500 RPM for 1 min and then incubated at 37° C. under 5% CO2 for 24 h. After 24h, medium was removed, and cells were fixed by adding 40 μL of 4% paraformaldehyde solution to each well and incubated for 20 min at room temperature. The plate was then washed 4 times with 100 μL per well of wash buffer (PBS with 0.1% Tween-20) using a microplate washer. Next, 30 μL of ice-cold methanol was added to each well and the plate was incubated at −20° C. for 10 min. The plate was washed 4 times with 100 μL per well of wash buffer by a microplate washer, then 30 μL per well of blocking buffer (Intercept PBS blocking buffer (LI-COR 927-70001) with 0.05% Tween-20) was added and the plate was incubated at room temperature with shaking for 2h. Next, to each test well, 20 μL of primary antibody solution (p21 Waf1/CIP (12D1) RabbitmAb (Cell Signaling Technologies 2947) diluted 1:1000 and GAPDH (D4C6R) Mouse mAb (Cell Signaling Technologies 97166) diluted 1:2000 in blocking buffer) was added and the plate was placed at 4° C., overnight. The following day, the plate was washed 5 times with 100 μL per well of wash buffer using a microplate washer for 5 min. 20 μL per well of secondary antibody (IRDye 680CW Goat anti-Mouse IgG (H+L) (LI-COR 926-68070) diluted 1: 2000 in Blocking Buffer and IRDye 800CW Goat anti-Rabbit IgG (H+L) (LI-COR 926-32211) diluted 1: 2000 in Blocking Buffer) was then added and the plate was stored for 2h in the dark at room temperature with shaking. The plate was then washed 4 times with 100 μL per well of wash buffer again using a microplate washer. Finally, the p21 signal and the GAPDH signal were quantified using a LI-COR Odyssey CLx Imager machine reading at 800 nm and 700 nm, respectively. Each plate contained DMSO control (low control) and an internal reference WRN inhibitor (high control) respectively. For quantitation, the 800 nm/700 nm ratio was calculated for each well to give fold p21 induction and then percent activation for each compound well was calculated as follows (100×(ratio cpd well−ratio low control)/(ratio high control−ratio low control)). EC50 values for each compound were generated after non-linear regression curve fitting using commercially available software. The resultant EC50 results obtained for the tested compounds are shown below in Table 4. Compounds with an EC50 less than or equal to 0.50 μM are designated as “A.” Compounds with an EC50 greater than 0.50 μM and less than or equal to 2.00 μM are designated as “B.” Compounds with an EC50 greater than 2.00 μM and less than or equal to 5.00 μM are designated as “C.” Compounds with an EC50 greater than 5.00 μM are designated as “D.”
This application claims the benefit of Provisional Application No. 63/613,656, filed Dec. 21, 2023; and Provisional Application No. 63/660,951, filed Jun. 17, 2024; the entireties of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63660951 | Jun 2024 | US | |
| 63613656 | Dec 2023 | US |
