CN117120447A - Cyclic compounds and methods of use thereof - Google Patents

Cyclic compounds and methods of use thereof Download PDF

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CN117120447A
CN117120447A CN202280022298.9A CN202280022298A CN117120447A CN 117120447 A CN117120447 A CN 117120447A CN 202280022298 A CN202280022298 A CN 202280022298A CN 117120447 A CN117120447 A CN 117120447A
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compound
formula
pharmaceutically acceptable
group
alkyl
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萨扬·蒙达尔
唐海峰
黄贤海
亚当·马克·莱文森
利娅·弗莱
萨蒂什·哈特
彼得·哈姆·博斯
泽夫·康斯特
帕尼·加纳科塔
杰里米·罗伯特·格林伍德
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Schrodinger & Co
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Schrodinger & Co
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Priority claimed from PCT/US2022/020712 external-priority patent/WO2022197898A1/en
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Abstract

The present application relates to compounds of formula (I) as defined herein and pharmaceutically acceptable salts thereof. The application also describes pharmaceutical compositions comprising compounds of formula (I) and pharmaceutically acceptable salts thereof, and methods of using the compounds and compositions to inhibit kinase activity and to treat cancer.

Description

Cyclic compounds and methods of use thereof
Technical Field
The present application relates to tricyclic and other polycyclic compounds useful for treating proliferative disorders such as cancer.
Background
Cancers are characterized by abnormal cell growth and proliferation. Genomic instability is a hallmark of cancer cells, where high mutation rates and genomic rearrangements lead to invasive and therapeutic resistant tumors. See Hanahan and Weinberg, cell 144, pages 646-674 (2011) and McGranahan and Swanton, cell 168, pages 613-628 (2017). Deregulation of DNA replication can promote genomic instability and tumorigenesis. Eukaryotic cells divide through a highly regulated stepwise process called the orientation of the cell cycle. DNA replication is an important component of a highly regulated step-by-step cell cycle, and this tight regulation ensures that DNA replication occurs only once during S-phase and with high fidelity.
During late G1 to S phases, CDC7 kinase (also known as DDK) is activated by binding to its regulatory protein DBF4 (ASK in eukaryotes), which then phosphorylates chromatin-loaded minichromosomes maintenance (MCM) 2, 4 and 6 proteins at multiple phosphorylation sites to initiate DNA synthesis. See Jiang et al, J. European molecular biology institute (EMBO J.)), 18, pages 5703-5713 (1999), cho et al, proc. Natl. Acad. Sci. U.S. A.), 103, pages 11521-11526 (2006) and Masai et al, J. Biol chem.) (281, pages 39249-39261 (2006). CDC7 kinase plays an important role in maintaining DNA replication fork and DNA damage response pathway. See Yamada et al, cell Cycle 13, pages 1859-1866 (2014).
CDC7 is a highly conserved serine/threonine kinase from yeast to humans. Knockout of CDC7 has been shown to cause cell death in cancer cells, but not in normal cells, where p 53-dependent pathways arrest the cell cycle in the G1 phase. CDC7 depletion induces an apoptotic response in cancer cells that is not mediated by p53, but rather by p38 MAPK. See Montagnoli et al, cancer research (Cancer Res.), 64, pages 7110-7116 (2004) and Im and Lee, journal of biochemistry, 283, pages 25171-25177 (2008). Furthermore, CDC7 upregulation is associated with poor prognosis in various cancer types. See, e.g., kulkarni et al, clinical cancer research (clin. Cancer res.), 15, pages 2417-2425 (2009); choschzick et al, human pathology (hum. Pathol.), 41, pages 358-365 (2010); datta et al, european molecular biology tissue report (EMBO rep.), 18, pages 2030-2050 (2017); cheng et al, cancer flash (Cancer Lett.), 337,218-225 (2013).
Disclosure of Invention
It has now been found that certain fused compounds are inhibitors of CDC7 kinase and are useful in the treatment of diseases, such as proliferative diseases, such as cancer.
Accordingly, provided herein is a compound of formula (I):
or a pharmaceutically acceptable salt thereof, wherein R 1 、R 2 、R 3 、R 4 Ring a and m are as defined herein.
Also provided herein is a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
Also provided herein is a method of inhibiting cell proliferation in vitro or in vivo, comprising contacting a cell with an effective amount of a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
Also provided herein is a method of inhibiting CDC7 kinase activity in vitro or in vivo, comprising contacting a cell with an effective amount of a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
Also provided herein is a method of treating cancer in a subject in need of such treatment, the method comprising administering to the subject an effective amount of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.
Also provided herein is a method of treating a CDC7 related disease or disorder in a subject in need of such treatment, the method comprising administering to the subject an effective amount of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.
Also provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to a subject identified as having a cancer associated with CDC7 an effective amount of a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
Also provided herein is a method of treating cancer and/or inhibiting metastasis associated with a particular cancer in a subject in need of such treatment, the method comprising administering to the subject an effective amount of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.
Also provided herein is a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in the treatment of cancer.
Also provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as defined herein, for use in the treatment of a CDC 7-related disease or disorder.
Also provided herein is a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in the treatment of cancer and/or in the inhibition of metastasis associated with a particular cancer.
Also provided herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in inhibiting CDC7 kinase activity.
Also provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as defined herein, for use in the treatment of a CDC 7-related disease or disorder.
Also provided herein is a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of cancer and/or inhibition of metastasis associated with a particular cancer.
Also provided herein is a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for inhibiting CDC7 kinase activity.
Also provided herein is a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of a CDC7 related disease or disorder.
Also provided are methods of treating an individual having a CDC 7-related cancer comprising administering a compound of formula (I), or a pharmaceutically acceptable salt thereof, before, during, or after administration of an additional anti-cancer agent (e.g., a first CDC7 kinase inhibitor or another kinase inhibitor).
Also provided herein is a process for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof.
Also provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, obtained by a process for preparing a compound as defined herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials for the present disclosure are described herein; other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the disclosure will become apparent from the following detailed description, and from the claims.
Detailed Description
Definition of the definition
As used herein, the term "compound" is intended to encompass all stereoisomers, geometric isomers, tautomers and isotopically enriched variants of the depicted structures. Unless otherwise indicated, a compound identified herein by name or structure as one particular tautomeric form is intended to encompass other tautomeric forms.
As used herein, the term "tautomer" refers to a compound whose structure differs significantly in the arrangement of atoms, but exists in an easy and rapid balance, and it is understood that the compounds provided herein may be depicted as different tautomers, and that when the compound has a tautomeric form, all tautomeric forms are intended to be within the scope of the present disclosure, and that the naming of the compound does not exclude any tautomers. Examples of tautomeric forms include the following:
it is to be understood that certain compounds provided herein may contain one or more asymmetric centers and thus may be prepared and isolated in the form of mixtures of isomers (e.g., racemic mixtures) or enantiomerically pure forms.
The term "halo" refers to one of the halogens of group 17 of the periodic table of elements. In particular, this term refers to fluorine, chlorine, bromine and iodine. Preferably, this term refers to fluorine or chlorine.
The term "C1-C6 alkyl" refers to a straight or branched hydrocarbon chain containing 1, 2, 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl. Alkyl groups may be unsubstituted or substituted with one or more substituents as described herein.
The term "C1-C6 haloalkyl" refers to a hydrocarbon chain substituted with at least one halogen atom (e.g., fluorine, chlorine, bromine, and iodine) independently selected at each occurrence. Halogen atoms may be present at any position on the hydrocarbon chain. For example, C1-C6 haloalkyl may refer to chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloroethyl (e.g., 1-chloroethyl and 2-chloroethyl), trichloroethyl (e.g., 1, 2-trichloroethyl, 2-trichloroethyl), fluoroethyl (e.g., 1-fluoromethyl and 2-fluoroethyl), trifluoroethyl (e.g., 1, 2-trifluoroethyl and 2, 2-trifluoroethyl), chloropropyl, trichloropropyl, fluoropropyl, trifluoropropyl.
The term "C1-C6 alkoxy" refers to a C1-C6 alkyl group attached to a molecule through oxygen. This includes moieties where the alkyl moiety may be straight or branched, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and n-hexoxy.
As used herein, the term "hydroxy" refers to an-OH group.
The term "C1-C6 hydroxyalkyl" refers to a hydrocarbon chain substituted with one hydroxyl group. The hydroxyl groups may be present at any position on the hydrocarbon chain. For example, C1-C6 hydroxyalkyl may refer to hydroxymethyl, hydroxyethyl (e.g., 1-hydroxyethyl and 2-hydroxyethyl) and 2-hydroxyisopropyl.
As used herein, the term "aryl" refers to 6-10 all-carbon monocyclic or bicyclic aromatic ring systems. Non-limiting examples of aryl groups include phenyl and naphthyl.
As used herein, the term "heteroaryl" refers to a 5-10 membered monocyclic or bicyclic group wherein each ring in the system is aromatic; wherein one or more carbon atoms in at least one ring in the system are replaced by heteroatoms independently selected from N, O and S. Non-limiting examples of heteroaryl groups include pyridine, pyrimidine, pyrrole, imidazole, and indole.
As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or bicyclic carbon group having 3 to 10 ring atoms; wherein the bicyclic ring system comprises a fused ring system, a spiro ring system (optionally referred to as a "spirocycloalkyl" group), and a bridged ring system. The fused cycloalkyl group may comprise one aromatic ring and another saturated or partially saturated ring, such as 1,2,3, 4-tetrahydronaphthalene and 2, 3-dihydro-1H-indene. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclohexyl, spiro [2.3] hexyl, and bicyclo [1.1.1] pentyl. As a substituent, for example, on an alkyl group, a cycloalkyl group may share a carbon atom with an alkyl chain.
The term "heterocyclyl" refers to a saturated or partially unsaturated hydrocarbon monocyclic or bicyclic ring system which is not fully aromatic, having at least one heteroatom selected from N, O and S within the ring. Bicyclic heterocyclyl groups include fused ring systems, spiro ring systems (optionally referred to as "spiroheterocyclyl" groups), and bridged ring systems. The fused heterocyclic group may contain one aromatic ring and another saturated or partially saturated ring, such as 5,6,7, 8-tetrahydroquinoline and indoline. Heterocyclyl may be represented as a "3 to 10 membered heterocyclyl" which is a ring system containing 3, 4, 5,6,7,8, 9 or 10 atoms, at least one of which is a heteroatom. For example, 1, 2 or 3 heteroatoms may be present, optionally 1 or 2. The heterocyclyl group may be bonded to the remainder of the molecule through any carbon atom or through a heteroatom such as nitrogen. Exemplary heterocyclyl groups include, but are not limited to, piperidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, azetidinyl, oxetanyl, and 2-azaspiro [3.3] heptanyl. As a substituent, for example, on an alkyl group, a heterocyclic group may share a carbon atom with an alkyl chain.
As used herein, the term "geminal" refers to a substituent atom or group attached to the same atom in a molecule.
As used herein, the term "oxo" refers to a "=o" group attached to a carbon atom.
As used herein, a symbolThe point of attachment of an atom or moiety to a designated atom or group in the remainder of the molecule is depicted.
It is to be understood that the A ring included in the compounds of formula (I) does not contain two adjacent oxygen atoms or two adjacent S atoms.
The compounds of formula (I) include pharmaceutically acceptable salts thereof. In addition, the compounds of formula (I) also comprise other salts of such compounds, which are not necessarily pharmaceutically acceptable salts, and can be used as intermediates for the preparation and/or purification of the compounds of formula (I) and/or for the isolation of enantiomers of the compounds of formula (I). Non-limiting examples of pharmaceutically acceptable salts of the compounds of formula (I) include trifluoroacetate salts and hydrochloride salts.
It will be further understood that the compound of formula (I) or a salt thereof may be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present disclosure. For example, the compounds of formula (I) and salts thereof may be present in unsolvated as well as solvated forms in association with pharmaceutically acceptable solvents such as water, ethanol, and the like.
In some embodiments, the compounds of formula (I) include the compounds of examples 1-96 and pharmaceutically acceptable salts and solvates thereof. In some embodiments, the compound of formula (I) is in the free base form. In some embodiments, the compound of formula (I) is in salt form (e.g., a pharmaceutically acceptable salt).
In some embodiments, the compounds of formula (I) include stereoisomers, pharmaceutically acceptable salts, and solvates thereof. In some embodiments, the compound of formula (I) is in the free base form. In some embodiments, the compound of formula (I) is in salt form.
The term "pharmaceutically acceptable" means that the compound or salt or composition thereof is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the subject being treated with the formulation.
The protecting group may be a temporary substituent that protects the potentially reactive functional group from undesired chemical transformations. The choice of the particular protecting group used is well within the skill of one of ordinary skill in the art. Many considerations may dictate the choice of protecting groups, including but not limited to the functional groups to be protected, other functional groups present in the molecule, the reaction conditions for each step of the synthetic sequence, other protecting groups present in the molecule, the tolerance of the functional groups to the conditions required to remove the protecting groups, and the reaction conditions for thermal decomposition of the compounds provided herein. A review of the field of protecting group chemistry has been made (Greene, T.W.; wuts, P.G.M.; protecting group in organic Synthesis (Protective Groups in Organic Synthesis), editorial 2 nd edition; wiley Press (Wiley): new York, 1991).
The nitrogen protecting group may be any temporary substituent that protects the amine moiety from undesired chemical transformations. Examples of moieties that are formed when such protecting groups are bonded to an amine include, but are not limited to, allylamine, benzylamine (e.g., benzylamine, p-methoxybenzylamine, 2, 4-dimethoxybenzylamine, and tritylamine), acetamide, trichloroacetamide, trifluoroacetamide, pent-4-enamide, phthalimide, carbamate (e.g., methyl carbamate, t-butyl carbamate, benzyl carbamate, allyl carbamate, 2-trichloroethyl carbamate, and 9-fluorenylmethyl carbamate), imine, and sulfonamide (e.g., benzenesulfonamide, p-toluenesulfonamide, and p-nitrobenzenesulfonamide).
The oxygen protecting group may be any temporary substituent that protects the hydroxyl moiety from undesired chemical transformations. Examples of moieties that are formed when such protecting groups are bonded to hydroxyl groups include, but are not limited to, esters (e.g., acetyl, t-butylcarbonyl, and benzoyl), benzyl (e.g., benzyl, p-methoxybenzyl, and 2, 4-dimethoxybenzyl, and trityl), carbonates (e.g., methyl carbonate, allyl carbonate, 2-trichloroethyl carbonate, and benzyl carbonate) ketals, acetals, and ethers.
The compounds provided herein may also be useful in constructing one or more of such compoundsThe atoms contain an unnatural proportion of atomic isotopes. That is, an atom (especially when referring to a compound of formula (I)) includes all isotopes and isotopic mixtures of that atom, either naturally occurring or synthetically produced, having natural abundance or in isotopically enriched form. For example, when hydrogen is referred to, it is understood that reference is made to 1 H、 2 H、 3 H or a mixture thereof; when carbon is mentioned, it is understood to mean 11 C、 12 C、 13 C、 14 C or a mixture thereof; when nitrogen is mentioned, it is understood to mean 13 N、 14 N、 15 N or mixtures thereof; when oxygen is mentioned, it is understood to mean 14 O、 15 O、 16 O、 17 O、 18 O or mixtures thereof; and when fluorine is mentioned, it is understood to mean 18 F、 19 F or mixtures thereof. For example, in deuteroalkyl and deuteroalkoxy groups, one or more hydrogen atoms are replaced by deuterium 2 H) Specifically replaced. Because some of the above isotopes are radioactive, the compounds provided herein also include compounds having one or more isotopes of one or more atoms, and mixtures thereof, including radioactive compounds in which one or more non-radioactive atoms have been replaced by one of their radioisotope-rich isotopes. Radiolabeled compounds are useful as therapeutic agents, for example, cancer therapeutic agents, research reagents (e.g., assay reagents), and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations of the compounds provided herein, whether radioactive or non-radioactive, are intended to be encompassed within the scope of the present disclosure.
For illustrative purposes, general methods for preparing compounds are provided herein, as well as key intermediates. For a detailed description of the individual reaction steps, see the "examples" section below. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the compounds of the present invention. Although specific starting materials and reagents are described in the schemes and discussed below, other starting materials and reagents may be readily substituted to provide a wide variety of derivatives and/or reaction conditions. In addition, many of the compounds prepared by the methods described below may be further modified in accordance with the present disclosure using conventional chemical methods well known to those skilled in the art.
The ability of the test compounds to act as CDC7 inhibitors can be demonstrated by the biological and computational assays described herein. IC (integrated circuit) 50 The values are shown in tables 2 and 3.
The compounds of formula (I) (e.g., any of formulas (I-a) to (I-P)) or pharmaceutically acceptable salts thereof are useful in the treatment of diseases and conditions that may be treated with CDC7 kinase inhibitors, such as CDC 7-related cancers, including hematological cancers and solid tumors.
As used herein, the term "treatment" refers to a therapeutic or slowing action. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms associated with a disease or disorder or condition, diminishment of extent of disease, stabilized disease state (i.e., not worsening), delay or slowing of progression of disease, amelioration or palliation of the disease state (e.g., one or more symptoms of the disease), and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean an increase in survival compared to the expected survival in the absence of treatment.
As used herein, the term "subject" refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject has experienced and/or exhibited at least one symptom of the disease or disorder to be treated and/or prevented.
In some embodiments, the subject has been identified or diagnosed as having a cancer (CDC 7-related cancer) in which expression or activity or level of the CDC7 gene, CDC7 protein, or any of them is deregulated (e.g., as determined using a regulatory agency approved (e.g., FDA approved) assay or kit). In some embodiments, the subject has a tumor that is positive for dysregulation of expression or activity or level of the CDC7 gene, CDC7 protein, or any of them (e.g., as determined using regulatory agency-approved assays or kits). The subject may be a subject having a tumor that is positive for a dysregulation of expression or activity or level of the CDC7 gene, CDC7 protein, or any of them (e.g., positive using regulatory agency-approved (e.g., FDA-approved) assays or kits). The subject may be a subject whose tumor has dysregulation of expression or activity or level of the CDC7 gene, CDC7 protein, or any of them (e.g., wherein the tumor is so identified using regulatory agency-approved (e.g., FDA-approved) assays or kits). In some embodiments, the subject is suspected of having CDC 7-related cancer. In some embodiments, the subject has a clinical record that indicates that the subject has a tumor with deregulation of expression or activity or level of CDC7 gene, CDC7 protein, or any of them (and optionally, that the subject should be treated with any of the compositions provided herein). In some embodiments, the subject is a pediatric subject. In some embodiments, the subject has been identified or diagnosed as having a cancer that is determined to be associated with a dysregulation of expression or activity or level of the CDC7 gene, CDC7 protein, or any of them (CDC 7-related cancer) based on a histological examination.
As used herein, the term "pediatric subject" refers to a subject less than 21 years of age at the time of diagnosis or treatment. The term "pediatric" may be further divided into various subgroups, including: neonates (first month from birth to life); infants (1 month to two years); children (two years to 12 years); and teenagers (12 to 21 years of age (up to but not including the twenty-second year of birth)). Berhman RE, kliegman R, arvin AM, nelson WE., nielsen textbook for pediatrics (Nelson Textbook of Pediatrics), 15 th edition, philadelphia: w.b. sanders Company, 1996; rudolph AM et al Rudolph science (Rudolph's Pediatrics), 21 st edition, N.Y.: maglao-Hill Press (McGraw-Hill), 2002; avery MD, first LR., "pediatric medicine (Pediatric Medicine), 2 nd edition, barlmo: williams and Wilkins Press (Williams & Wilkins); 1994. in some embodiments, the pediatric subject is aged from first 28 days of birth to life, from 29 days to under two years, from two years to under 12 years, or from 12 years to 21 years (up to but not including the twenty-two years of birthday). In some embodiments, the pediatric subject is aged 28 days from birth to life, 29 days to under 1 year, one month to under four months, three months to under seven months, six months to under 1 year, 1 year to under 2 years, 2 years to under 3 years, 2 years to under seven years, 3 years to under 5 years, 5 years to under 10 years, 6 years to under 13 years, 10 years to under 15 years, or 15 years to under 22 years.
In some embodiments, the compounds of formula (I), or pharmaceutically acceptable salts thereof, are useful for preventing diseases and conditions defined herein (e.g., autoimmune diseases, inflammatory diseases, and cancers). As used herein, the term "preventing" refers to preventing the onset, recurrence or spread of a disease or condition described herein, or symptoms thereof, either entirely or partially.
As used herein, the term "CDC 7-related cancer" refers to a cancer associated with or having a deregulation (e.g., any type of deregulation of the expression or activity or level of a CDC7 gene, a CDC7 kinase (also referred to herein as a CDC7 kinase protein), or any one thereof (e.g., one or more) of CDC7 genes, CDC7 kinases, CDC7 kinase domains, or any one thereof described herein). Non-limiting examples of CDC7 related diseases or disorders include, for example, cancer and gastrointestinal disorders, such as Irritable Bowel Syndrome (IBS).
As used herein, the term "CDC 7-related cancer" refers to a cancer associated with or having a deregulation of expression or activity or level of a CDC7 gene, CDC7 kinase or any of them. Non-limiting examples of CDC 7-related cancers are described herein.
The phrase "dysregulation of expression or activity or level of a CDC7 gene, CDC7 kinase, or any one thereof" refers to a mutation in a gene of a wild-type CDC7 kinase in a mammalian cell (e.g., as compared to a control non-cancerous cell) (e.g., chromosomal translocation that causes expression of a fusion protein comprising a CDC7 kinase domain and a fusion partner; a mutation in a CDC7 gene that causes expression of a CDC7 protein comprising at least one amino acid deletion as compared to a wild-type CDC7 protein; a mutation in a CDC7 gene that causes expression of a CDC7 protein having one or more point mutations as compared to a wild-type CDC7 protein; a mutation in a CDC7 gene that causes expression of a CDC7 protein having at least one inserted amino acid as compared to a wild-type CDC7 protein; a gene duplication that causes increased levels of a CDC7 protein in a cell; or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) that causes increased levels of a CDC7 protein in a cell); an alternatively spliced form of CDC7mRNA that results in a CDC7 protein having at least one amino acid deletion in the CDC7 protein as compared to the wild-type CDC7 protein; or increased expression (e.g., increased levels) due to abnormal cell signaling and/or deregulated autocrine/paracrine signaling. As another example, a disruption in expression or activity or level of a CDC7 gene, CDC7 protein, or any of them may be a mutation in a CDC7 gene encoding a CDC7 protein, which CDC7 protein is constitutively active or has increased activity compared to the protein encoded by the CDC7 gene not comprising the mutation. As a further example, increased CDC7 gene copy numbers may cause overexpression of CDC7 kinase. For example, a deregulation of the expression or activity or level of a CDC7 gene, CDC7 protein, or any of them, may be the result of a gene or chromosomal translocation that causes expression of a fusion protein containing a first portion of CDC7 (this portion comprising a functional kinase domain) and a second portion of chaperonin (i.e., not CDC 7). In some examples, deregulation of expression or activity or level of a CDC7 gene, CDC7 protein, or any of these may be the result of gene translocation of one CDC7 gene with another non-CDC 7 gene.
The term "wild-type" describes a nucleic acid (e.g., CDC7 gene or CDC7 mRNA) or protein (e.g., CDC7 protein) found in a subject that does not have a CDC 7-related disease (e.g., CDC 7-related cancer) (and optionally, does not increase the risk of having a CDC 7-related disease and/or is not suspected of having a CDC 7-related disease), or a nucleic acid or protein found in cells or tissues from a subject that does not have a CDC 7-related disease (e.g., CDC 7-related cancer) (and optionally, does not increase the risk of having a CDC 7-related disease and/or is not suspected of having a CDC 7-related disease).
The term "regulatory agency" refers to the agency of a country for approval of the medical use of pharmaceutical agents in that country. For example, a non-limiting example of a regulatory agency is the U.S. Food and Drug Administration (FDA).
Provided herein are compounds of formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
R 1 hydrogen or halogen;
R 2 a 5-6 membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl; or a 5-6 membered heterocyclyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl;
R 3 Is hydrogen or C1-C6 alkyl;
ring A is C6-C10 cycloalkyl or 6-10 membered heterocyclyl;
each R 4 Independently selected from the group consisting of: halogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkoxy (C1-C6 alkyl) -, -C (=O) C1-C6 alkyl, C2-C6 alkynyl, C3-C6 cycloalkyl, -NR A R B And 4-6 membered heterocyclyl optionally substituted with 1 or 2 independently selected halo; each R A And R is B Independently hydrogen or C1-C6 alkyl; and is also provided with
m is 0, 1, 2, 3 or 4.
In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.
In some embodiments, R 2 Is a 5 membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.In some embodiments, R 2 Is a 5 membered heteroaryl substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
In some embodiments, R 2 Is a 5-membered heteroaryl selected from the group consisting of: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, isothiazolyl, thiazolyl, furanyl, oxadiazolyl, thiadiazolyl, oxazolyl, and thiatriazolyl. In some embodiments, R 2 Is pyrazolyl or isothiazolyl. In some embodiments, R 2 Is 4-pyrazolyl, 5-pyrazolyl or 5-isothiazolyl.
In some embodiments, R 2 Is a 6 membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl. In some embodiments, R 2 Is a 6 membered heteroaryl substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
In some embodiments, R 2 Is a 6 membered heteroaryl selected from the group consisting of: pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl and triazinyl. In some embodiments, R 2 Is pyridinyl, pyrimidinyl or pyridazinyl. In some embodiments, R 2 Is pyridinyl. In some embodiments, R 2 Is 4-pyridyl. In some embodiments, R 2 Is 4-pyrimidinyl. In some embodiments, R 2 Is 4-pyridazinyl.
In some embodiments, R 2 Is substituted with a substituent selected from the group consisting of halogen and C1-C6 alkyl. In some embodiments, R 2 Is substituted with a substituent selected from the group consisting of fluorine, chlorine and methyl. In some embodiments, R 2 Is substituted with two substituents independently selected from halogen and C1-C6 alkyl. In some embodiments, R 2 Is substituted with two substituents independently selected from fluoro and methyl.
In some embodiments, R 2 The substituents on the above are the same. In some embodimentsIn the example, R 2 The substituents on the two groups are different.
In some embodiments, R 2 Is an unsubstituted 5-6 membered heteroaryl group, such as unsubstituted pyrazolyl, isothiazolyl, pyridinyl or pyridazinyl.
In some embodiments, R 2 A 5-6 membered heterocyclyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl. In some embodiments, R 2 Is unsubstituted 5-6 membered heterocyclic group. In some embodiments, R 2 Morpholinyl, piperidinyl or piperazinyl. In some embodiments, R 2 Is morpholinyl. In some embodiments, R 2 Is 4-morpholinyl.
In some embodiments, ring A is a C6-C10 cycloalkyl group. In some embodiments, ring a is cyclohexyl. In some embodiments, ring a is bicyclo [2.2.2] octyl.
In some embodiments, ring a is a 6-10 membered heterocyclyl. In some embodiments, ring a is tetrahydropyranyl. In some embodiments, ring a is piperidinyl.
In some embodiments, one or two R 4 Independently halogen. In some embodiments, one R 4 Is fluorine or chlorine. In some embodiments, one R 4 Is fluorine. In some embodiments, one R 4 Is chlorine. In some embodiments, two R 4 Is fluorine.
In some embodiments, one R 4 Is hydroxyl.
In some embodiments, one R 4 Independently is a C1-C6 alkyl group. In some embodiments, one or two R 4 C1-C6 alkyl groups are independently selected. In some embodiments, one R 4 Independently selected from the group consisting of: -CH 3 、-CH 2 CH 3 、-CH(CH 3 ) 2 、-CH(CH 3 )CH 2 CH 3 、-C(CH 3 ) 2 CH 2 CH 3 and-C (CH) 3 ) 3 。
In some embodiments, each R 4 Independently is a C1-C6 alkoxy group. At the position ofIn some embodiments, one R 4 Is C1-C6 alkoxy. In some embodiments, each R 4 Independently methoxy, ethoxy or isopropoxy. In some embodiments, one R 4 Methoxy, ethoxy or isopropoxy. In some embodiments, each R 4 Is methoxy. In some embodiments, one R 4 Is methoxy.
In some embodiments, one R 4 Is a C1-C6 hydroxyalkyl group. In some embodiments, one R 4 Is a C1-C6 hydroxyalkyl group. In some embodiments, one R 4 is-CH 2 OH or-C (CH) 3 ) 2 OH. In some embodiments, one R 4 is-CH 2 OH or-C (CH) 3 ) 2 OH。
In some embodiments, one R 4 Is a C1-C6 haloalkyl. In some embodiments, one or two R 4 A C1-C6 haloalkyl group independently selected. In some embodiments, one R 4 is-CF 3 . In some embodiments, one R 4 is-CHF 2 . In some embodiments, one R 4 is-CF 3 . In some embodiments, one R 4 is-CHF 2 . In some embodiments, one R 4 is-CF 2 CH 3 . In some embodiments, one R 4 Is CH 2 CF 2 CH 3 。
In some embodiments, one R 4 Is C1-C6 alkoxy (C1-C6 alkyl) -. In some embodiments, one R 4 Is C1-C3 alkoxy (C1-C3 alkyl) -. In some embodiments, one R 4 Independently methoxy (C1-C6 alkyl) -. In some embodiments, one R 4 is-CH 2 OCH 3 。
In some embodiments, one R 4 is-C (=O) C1-C6 alkyl. In some embodiments, R 4 Is C (=O) CH 3 。
In some embodiments, one R 4 Is a C2-C6 alkynyl group. In some embodiments, one R 4 Is 1-propynyl.
In some embodiments, one R 4 Is a C3-C6 cycloalkyl group. In some embodiments, R 4 Is cyclopentyl.
In some embodiments, one R 4 is-NR A R B . In some embodiments, R A And R is B One of which is hydrogen and R A And R is B The other of (C1-C6 alkyl). In some embodiments, R A And R is B Both are C1-C6 alkyl groups. In some embodiments, R 4 is-NH 2 。
In some embodiments, one R 4 Is a 4-6 membered heterocyclyl optionally substituted with 1 or 2 independently selected halo. In some embodiments, one R 4 Is a 4-6 membered heterocyclyl substituted with 1 or 2 independently selected halogens. In some embodiments, one R 4 Is a 4-6 membered heterocyclic group substituted with 1 or 2 fluorine groups. In some embodiments, one R 4 Is a 4-6 membered heterocyclic group substituted with a geminal difluoro group.
In some embodiments, one R 4 Is azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl or dioxanyl; each optionally substituted with 1 or 2 independently selected halogens. In some embodiments, one R 4 Pyrrolidinyl optionally substituted with 1 or 2 independently selected halogens. In some embodiments, one R 4 Is pyrrolidinyl substituted with 1 or 2 independently selected halogens. In some embodiments, one R 4 Azetidinyl optionally substituted with 1 or 2 independently selected halogens. In some embodiments, one R 4 Azetidinyl substituted with 1 or 2 independently selected halogens. In some embodiments, the 1 or 2 independently selected halogens are fluorine. In some embodiments, the 2 independently selected halogens are geminally difluoro groups.
In some embodiments, one R 4 Is unsubstituted 4-6 membered heterocyclic group. In some embodiments, R 4 Unsubstituted 4-6 membered heterocyclic groups of (2) are azetidinyl, oxygenHeterocycloalkylyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl, or dioxanyl. In some embodiments, one R 4 Is unsubstituted pyrrolidinyl or unsubstituted azetidinyl.
In some embodiments, one R 4 Selected from the group consisting of:
in some embodiments, m is 2, 3, or 4; and 2, 3 or 4R 4 Two of the groups are geminal. In some embodiments, m is 2; and both (i.e. two) R 4 The groups are geminal. In some embodiments, m is 3; and three of R 4 Two of the groups are geminal. In some embodiments, m is 4; and wherein each pair R 4 The groups are geminal.
In some embodiments, R 1 Is halogen. In some embodiments, R 1 Is fluorine or chlorine. In some embodiments, R 1 Is fluorine. In some embodiments, R 1 Is hydrogen.
In some embodiments, R 3 Is a C1-C6 alkyl group. In some embodiments, R 3 Is methyl. In some embodiments, R 3 Is hydrogen.
In some embodiments, m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
In some embodiments, m is 2, and each R 4 Is fluorine.
In some embodiments, m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
In some embodiments, m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C2-C6 alkynyl group.
In some embodiments, m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C3-C6 cycloalkyl group.
In some embodiments of the present invention, in some embodiments,m is 2; r is R 4 Is hydroxyl; and another R 4 Is C1-C6 alkoxy (C1-C6 alkyl) -.
In some embodiments, m is 2; each R 4 C1-C6 alkyl groups are independently selected.
In some embodiments, m is 2; r is R 4 Is C1-C6 alkyl; and another R 4 Is a C1-C6 hydroxyalkyl group.
In some embodiments, m is 2; r is R 4 Is C1-C6 alkyl; and another R 4 Is C1-C6 alkoxy.
In some embodiments, m is 2; r is R 4 Is C1-C6 alkyl; and another R 4 is-NR A R B 。
In some embodiments, m is 2; r is R 4 Is C1-C6 alkyl; and another R 4 Is a 4-6 membered heterocyclyl optionally substituted with 1 or 2 independently selected halo.
In some embodiments, m is 4; two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, m is 4; two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, m is 4; two R 4 Is fluorine; r is R 4 Is hydroxyl; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, m is 4; two R 4 Is fluorine; r is R 4 Is hydroxyl; and one R 4 Is C1-C6 alkoxy (C1-C6 alkyl) -.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyrazolyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyrazolyl; m is 2, and each R 4 Is fluorine.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyrazolyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyrazolyl; m is 4; two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyrazolyl; m is 4; two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyridyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyridyl; m is 2, and each R 4 Is fluorine.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyridyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyridyl; m is 4; two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is unsubstituted pyridyl; m is 4; two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is an unsubstituted isothiazolyl group; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is an unsubstituted isothiazolyl group;m is 2, and each R 4 Is fluorine.
In some embodiments, R 1 Is hydrogen; r is R 2 Is an unsubstituted isothiazolyl group; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
In some embodiments, R 1 Is hydrogen; r is R 2 Is an unsubstituted isothiazolyl group; m is 4; two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, R 1 Is hydrogen; r is R 2 Is an unsubstituted isothiazolyl group; m is 4; two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is cyclohexyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is cyclohexyl; m is 2, and each R 4 Is fluorine.
In some embodiments, ring a is cyclohexyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
In some embodiments, ring a is cyclohexyl; m is 4; two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is cyclohexyl; m is 4; two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is bicyclo [2.2.2]An octyl group; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is bicyclo [2.2.2 ]An octyl group; m is 2, and each R 4 Is fluorine.
In some embodiments, ring a is bicyclo [2.2.2]An octyl group; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
In some embodiments, ring a is bicyclo [2.2.2]An octyl group; m is 4; two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is bicyclo [2.2.2]An octyl group; m is 4; two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is tetrahydrofuranyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is tetrahydrofuranyl; m is 2, and each R 4 Is fluorine.
In some embodiments, ring a is tetrahydrofuranyl; m is 2; r is R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
In some embodiments, ring a is piperidinyl; m is 3; r is R 4 Is hydroxyl; r is R 4 Is C1-C6 alkyl; and one R 4 is-C (=O) C1-C6 alkyl. In some embodiments, when ring A is piperidinyl, one R 4 Attached to the piperidinyl nitrogen atom.
In some embodiments, ring a is tetrahydrofuranyl; m is 4; two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, ring a is tetrahydrofuranyl; m is 4; two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
In some embodiments, the compounds of formula (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-N), (I-O) and (I-P) and pharmaceutically acceptable salts thereof are as described herein, R 1 、R 2 、R 4 And m is as described for formula (I) or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-a):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-B):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-C):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-D):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-E):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-F):
Or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-G):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-H):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-I):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-J):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-K):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-L):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-M):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-N):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-O):
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound of formula (I) is a compound of formula (I-P):
Or a pharmaceutically acceptable salt thereof.
Table 1 depicts the compounds of formula (I). All stereochemistry in table 1 is understood to be arbitrarily specified unless otherwise indicated.
In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is a compound selected from table 1 or a pharmaceutically acceptable salt thereof.
Table 1: exemplary Compounds
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Therapeutic method
Provided herein are methods of treating cancer (e.g., CDC 7-related cancer) in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. For example, provided herein are methods for treating CDC 7-related cancers in a subject in need of such treatment, the methods comprising: a) Detecting a dysregulation in expression or activity or level of a CDC7 gene, CDC7 kinase, or any of them in a sample from the subject; and b) administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the dysregulation of expression or activity or level of the CDC7 gene, CDC7 kinase, or any of them comprises one or more fusion proteins.
In some embodiments of any of the methods or uses described herein, the cancer (e.g., CDC 7-related cancer) is a hematologic cancer. In some embodiments of any of the methods or uses described herein, the cancer (e.g., CDC 7-related cancer) is a solid tumor. In some embodiments of any of the methods or uses described herein, the cancer (e.g., CDC 7-related cancer) is lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), thyroid cancer (e.g., papillary thyroid cancer, medullary thyroid cancer (e.g., sporadic medullary thyroid cancer or hereditary medullary thyroid cancer), differentiated thyroid cancer, recurrent thyroid cancer, or refractory differentiated thyroid cancer), thyroid adenoma, endocrine gland tumor, lung adenocarcinoma, bronchiolar lung cancer, multiple endocrine tumor of type 2A or 2B (MEN 2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, mastadenoma, breast tumor, colorectal cancer (e.g., metastatic colorectal cancer), papillary renal cell carcinoma, gastrointestinal mucosal ganglioneumatosis, inflammatory myofibroblastic tumor, or cervical cancer. In some embodiments of any one of the methods or uses described herein, the cancer (e.g., CDC 7-related cancer) is selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), juvenile cancer, adrenocortical carcinoma, anal carcinoma, appendiceal carcinoma, astrocytoma, atypical malformation tumor/rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumor, burkitt's lymphoma (Burkitt lymphoma), carcinoid tumor, unknown primary cancer, cardiac tumor, cervical cancer, childhood cancer, chordoma, chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), chronic myeloproliferative tumor, tumor distributed by site, tumor, colon cancer, colorectal cancer, craniopharyngeal tumor, cutaneous T cell lymphoma, cutaneous angiosarcoma, cholangiocarcinoma, ductal carcinoma in situ, embryo tumor, endometrial cancer, carcinoma ependymoma, esophageal carcinoma, olfactory neuroblastoma, ewing's sarcoma (Ewing's sarcoma), extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct carcinoma, eye cancer, fallopian tube carcinoma, bone fibroblastic tumor, gall bladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, glioma, hairy cell tumor, hairy cell leukemia, head and neck cancer, thoracic tumor, head and neck tumor, CNS tumor, primary CNS tumor, heart disease, hepatocellular carcinoma, tissue cell hyperplasia, hodgkin's lymphoma, hypopharyngeal carcinoma, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kaposi's sarcoma, renal cancer, langerhans cell histiocytosis (Langerhans cell histiocytosis), laryngeal, leukemia, lip and oral cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, bone malignant fibrous histiocytoma, bone cancer, melanoma, merck cell carcinoma (Merkel cell carcinoma), mesothelioma, metastatic squamous neck cancer, midline cancer, oral cancer, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm tumor, myelogenous leukemia, multiple myeloma, myeloproliferative tumor, nasal and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma non-small cell lung cancer, lung tumor, respiratory tumor, bronchus cancer, bronchus tumor, oral cancer, lip cancer, oropharynx cancer, osteosarcoma, ovarian cancer pancreatic cancer, papillomatosis, paragangliomas, sinus and nasal cancers, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary cancer, plasmacytoma, pleural pneumoblastomas, breast cancer associated with pregnancy, primary central nervous system lymphomas, primary peritoneal cancer, prostate cancer, rectal cancer, colon tumors, renal cell carcinoma, CDC7 inobelastoma, rhabdomyosarcoma salivary gland carcinoma, sarcoma, szebra syndrome (Sezary syndrome), skin carcinoma, pittz tumor (Spitz tumor), small cell lung carcinoma, small intestine carcinoma, soft tissue sarcoma, squamous cell carcinoma, squamous neck carcinoma, stomach carcinoma, T cell lymphoma, testicular carcinoma, laryngeal carcinoma, thymoma and thymus carcinoma, thyroid carcinoma, transitional cell carcinoma of renal pelvis and uCDC7er, unknown primary carcinoma, uCDC7hral carcinoma, uterine sarcoma, vaginal cancer, vulvar cancer and Wilms' tumor.
In some embodiments, the hematologic cancer (e.g., hematologic cancer that is a CDC 7-related cancer) is selected from the group consisting of: leukemias, lymphomas (non-hodgkin lymphomas), hodgkin's disease (also known as hodgkin's lymphoma), and myelomas, such as Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), acute Promyelocytic Leukemia (APL), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), chronic myelogenous leukemia (CMML), chronic Neutrophilic Leukemia (CNL), acute Undifferentiated Leukemia (AUL), anaplastic Large Cell Lymphoma (ALCL), lymphoblastic leukemia (PML), juvenile myelogenous leukemia (JMML), adult T cell ALL, AML with three-line myelodysplasia (AML/TMDS), mixed Lineage Leukemia (MLL), myelodysplastic syndrome (MDS), myelodysplasia (MPD), and Multiple Myeloma (MM). Further examples of hematological cancers include Myelodysplasia (MPD), such as Polycythemia Vera (PV), primary thrombocytopenia (ET), and idiopathic primary myelofibrosis (IMF/IPF/PMF). In some embodiments, the hematologic cancer (e.g., hematologic cancer that is a CDC 7-related cancer) is AML or CMML.
In some embodiments, the cancer (e.g., CDC 7-related cancer) is a solid tumor. Examples of solid tumors (e.g., solid tumors that are CDC 7-related cancers) include, for example, thyroid cancer (e.g., papillary thyroid cancer, medullary thyroid cancer), lung cancer (e.g., lung adenocarcinoma, small cell lung cancer), pancreatic cancer, pancreatic ductal carcinoma, breast cancer, colon cancer, colorectal cancer, prostate cancer, renal cell carcinoma, head and neck tumor, neuroblastoma, and melanoma. See, e.g., cancer Nature comment (Nature Reviews Cancer), 2014,14,173-186.
In some embodiments, the cancer is selected from the group consisting of: lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, multiple endocrine tumors of type 2A or 2B (MEN 2A or MEN 2B), pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglioneuroma, and cervical cancer.
In some embodiments, the subject is a human.
The compounds of formula (I) and pharmaceutically acceptable salts and solvates thereof are also useful in the treatment of CDC 7-related cancers.
Accordingly, also provided herein is a method for treating a subject diagnosed with or identified as having a CDC 7-related cancer (e.g., any of the exemplary CDC 7-related cancers disclosed herein), the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from examples 1-96 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof administered to the subject is a compound of formula (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-N), (I-O), or (I-P) or a pharmaceutically acceptable salt of any of the foregoing.
Deregulation of expression or activity or levels of CDC7 kinase, CDC7 gene, or any one (e.g., one or more) thereof may contribute to tumorigenesis. For example, a fusion protein may have increased kinase activity compared to a wild-type CDC7 protein, and increased expression (e.g., increased levels) of wild-type CDC7 kinase in mammalian cells due to aberrant cell signaling and/or deregulated autocrine/paracrine signaling (e.g., compared to control non-cancerous cells), CDC7 mRNA splice variants may also cause CDC7 deregulation.
In some embodiments, the compounds provided herein exhibit brain and/or Central Nervous System (CNS) permeability. Such compounds are capable of crossing the blood brain barrier and inhibiting CDC7 kinase in the brain and/or other CNS structures. In some embodiments, the compounds provided herein are capable of crossing the blood brain barrier in an effective amount. For example, treatment of a subject with a cancer (e.g., a CDC 7-related cancer, such as a CDC 7-related brain cancer or CNS cancer) may comprise administering (e.g., orally administering) a compound to the subject. In some such embodiments, the compounds provided herein may be used to treat a primary brain tumor or a metastatic brain tumor. For example, the compounds may be used to treat one or more gliomas, such as glioblastoma (also known as glioblastoma multiforme), astrocytomas, oligodendrogliomas, ependymomas and mixed gliomas, meningiomas, medulloblastomas, intracranial gangliogliomas, schwannomas/neurolemomas), and craniopharyngeal tube tumors (see, for example, tumors listed in Louis, d.n. et al, "neurological journal (131 (6), 803-820 (2016, 6)). In some embodiments, the brain tumor is a primary brain tumor. In some embodiments, the subject has been previously treated with another anticancer agent, such as another CDC7 inhibitor (e.g., a compound that is not a compound of formula (I)) or a multi-kinase inhibitor. In some embodiments, the brain tumor is a metastatic brain tumor. In some embodiments, the subject has been previously treated with another anticancer agent, such as another CDC7 inhibitor (e.g., a compound that is not a compound of formula (I)) or a multi-kinase inhibitor.
In some embodiments of any of the methods or uses described herein, an assay for determining whether a subject has deregulation of CDC7 gene or CDC7 kinase or expression or activity or level of either thereof using a sample from the subject may comprise, for example, next generation sequencing, immunohistochemistry, fluorescence microscopy, FISH isolation analysis, southern blotting, western blotting, FACS analysis, northern blotting, and PCR-based amplification (e.g., RT-PCR and quantitative real-time RT-PCR). The assay is typically performed with, for example, at least one labeled nucleic acid probe or at least one labeled antibody or antigen binding fragment thereof, as is well known in the art. The assay may utilize other detection methods known in the art to detect deregulation of expression or activity or level of the CDC7 gene, CDC7 kinase or any of them. In some embodiments, the sample is a biological sample or a biopsy sample from the subject (e.g., a paraffin embedded biopsy sample). In some embodiments, the subject is a subject suspected of having a CDC 7-related cancer, a subject having one or more symptoms of a CDC 7-related cancer, and/or a subject at increased risk of having a CDC 7-related cancer.
In some embodiments, liquid biopsies (variously referred to as liquid biopsies or liquid phase biopsies) may be used to identify dysregulation of expression or activity or level of CDC7 genes, CDC7 kinases, or any of them. Liquid biopsy methods may be used to detect total tumor burden and/or deregulation of expression or activity or level of CDC7 genes, CDC7 kinase or any of them. A biological sample that is relatively easy to obtain from a subject (e.g., by simple blood drawing) may be subjected to a liquid biopsy, and is less invasive than conventional methods for detecting tumor burden and/or deregulation of expression or activity or levels of the CDC7 gene, CDC7 kinase, or any of them. In some embodiments, liquid biopsies may be used at an earlier stage compared to traditional methods to detect the presence or absence of deregulation of expression or activity or level of the CDC7 gene, CDC7 kinase or any of them. In some embodiments, the biological sample for liquid biopsies may comprise blood, plasma, urine, cerebrospinal fluid, saliva, sputum, bronchoalveolar lavage, bile, lymph, cyst fluid, stool, ascites, and combinations thereof. In some embodiments, liquid biopsies can be used to detect Circulating Tumor Cells (CTCs). In some embodiments, liquid biopsies can be used to detect cell-free DNA. In some embodiments, the cell-free DNA detected using liquid biopsy is circulating tumor DNA (ctDNA), which is derived from tumor cells. Analysis of ctDNA (e.g., using sensitive detection techniques such as, but not limited to, next Generation Sequencing (NGS), traditional PCR, digital PCR, or microarray analysis) can be used to identify dysregulation of expression or activity or level of the CDC7 gene, CDC7 kinase, or any of them.
In some embodiments, liquid biopsies may be used to detect ctDNA derived from a single gene. In some embodiments, liquid biopsies can be used to detect ctDNA derived from multiple genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more genes, or any number in between). In some embodiments, ctDNA derived from multiple genes may be detected using any of a variety of commercially available test panels (e.g., commercially available test panels designed to detect dysregulation of expression or activity or levels of CDC7 genes, CDC7 kinase, or any of them). Liquid biopsies can be used to detect deregulation of expression or activity or levels of CDC7 genes, CDC7 kinases, or any of them, including but not limited to point mutations or Single Nucleotide Variants (SNVs), copy Number Variants (CNVs), gene fusions (e.g., translocations or rearrangements), insertions, deletions, or any combination thereof. In some embodiments, liquid biopsies may be used to detect germline mutations. In some embodiments, liquid biopsies can be used to detect somatic mutations. In some embodiments, liquid biopsies can be used to detect primary genetic mutations (e.g., primary mutations or primary fusions associated with the initial development of a disease (e.g., cancer)). In some embodiments, a deregulation of expression or activity or level of a CDC7 gene, CDC7 kinase, or any of them identified using liquid biopsy is also present in cancer cells present in the subject (e.g., in a tumor). In some embodiments, liquid biopsies may be used to detect deregulation of expression or activity or levels of the CDC7 gene, CDC7 kinase, or any of them described herein. In some embodiments, the genetic mutation identified by liquid biopsy may be used to identify a subject as a candidate for a particular treatment. For example, detection of a deregulation of expression or activity or level of a CDC7 gene, CDC7 kinase or any of these in a subject may indicate that the subject will respond to a treatment comprising administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
Liquid biopsies may be performed multiple times during diagnostic, monitoring, and/or therapeutic procedures to determine one or more clinically relevant parameters including, but not limited to, the progression of a disease and/or the efficacy of a treatment. For example, during a diagnostic procedure, monitoring procedure, and/or therapeutic procedure, a first liquid biopsy may be taken at a first point in time, and a second liquid biopsy may be taken at a second point in time. In some embodiments, the first time point may be a time point prior to diagnosing the subject with the disease (e.g., when the subject is healthy), and the second time point may be a time point after the subject has the disease (e.g., the second time point may be used to diagnose the subject with the disease). In some embodiments, the first time point may be a time point prior to diagnosing a subject with a disease (e.g., when the subject is healthy) after which the subject is monitored, and the second time point may be a time point after monitoring the subject. In some embodiments, the first time point may be a time point after diagnosing a subject with a disease, after which the subject is administered a treatment, and the second time point may be a time point after administering a treatment; in this case, the second time point can be used to assess the efficacy of the treatment (e.g., whether the one or more gene mutations detected at the first time point are substantially reduced or undetectable). In some embodiments, the treatment to be administered to the subject may comprise a compound of formula (I) or a pharmaceutically acceptable salt thereof.
In some embodiments, the efficacy of a compound of formula (I) or a pharmaceutically acceptable salt thereof may be determined by assessing the allele frequency of CDC7 gene dysregulation in cfDNA obtained from a subject at different time points (e.g., cfDNA obtained from a subject at a first time point and cfDNA obtained from a subject at a second time point), wherein at least one dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject between the first time point and the second time point. Some embodiments of these methods may further comprise administering at least one dose of a compound of formula (I), or a pharmaceutically acceptable salt thereof, to the subject between the first time point and the second time point. For example, a decrease in the Allele Frequency (AF) of a CDC7 gene disorder in cfDNA obtained from a subject at a second time point compared to the Allele Frequency (AF) of a CDC7 gene disorder in cfDNA obtained from a subject at the first time point (e.g., from 1% to about 99%, from 1% to about 95%, from 1% to about 90%, from 1% to about 85%, from 1% to about 80%, from 1% to about 75%, from 1% to about 70%, from 1% to about 65%, from 1% to about 60%, from 1% to about 55%, from 1% to about 50%, from 1% to about 45%, from 1% to about 40%, from 1% to about 35%, from 1% to about 30%, from 1% to about 25%, from 1% to about 20%, from 1% to about 15a%, from 1% to about 10%, from 1% to about 5%, from about 5% to about 99%, from about 10% to about 99%, from 1% to about 45%, from 1% to about 40%, from 1% to about 20%, from 1% to about 25%, from 1% to about 20%, from 1% to about 5%, from about 5% to about 99%, from about about 15% to about 99%, about 20% to about 99%, about 25% to about 99%, about 30% to about 99%, about 35% to about 99%, about 40% to about 99%, about 45% to about 99%, about 50% to about 99%, about 55% to about 99%, about 60% to about 99%, about 65% to about 99%, about 70% to about 99%, about 75% to about 95%, about 80% to about 99%, about 90% to about 99%, about 95% to about 99%, about 5% to about 10%, about 5% to about 25%, about 10% to about 30%, about 20% to about 40%, about 25% to about 50%, about 70% to about 99%, about 5% to about 10%, about 5% to about 25%, about 10% to about 30%, about 40%, about 25% to about 50%, about, about 35% to about 55% less, about 40% to about 60% less, about 50% to about 75% less, about 60% to about 80% less, or about 65% to about 85% less, indicating that the compound of formula (I) or a pharmaceutically acceptable salt thereof is effective in a subject. In some embodiments, AF is reduced such that the level is below the detection limit of the instrument. Alternatively, an increase in the Allele Frequency (AF) of the CDC7 gene disorder in cfDNA obtained from the subject at the second time point compared to the Allele Frequency (AF) of the CDC7 gene disorder in cfDNA obtained from the subject at the first time point indicates that the compound of formula (I) or a pharmaceutically acceptable salt thereof is not effective in the subject. Some embodiments of these methods may further comprise administering an additional dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject in which the compound of formula (I) or a pharmaceutically acceptable salt thereof is determined to be effective. Some embodiments of these methods may further comprise administering a different treatment (e.g., a treatment that does not comprise administering the compound of formula (I) or a pharmaceutically acceptable salt thereof as monotherapy) to the subject in which the compound of formula (I) or a pharmaceutically acceptable salt thereof is determined to be ineffective.
In some embodiments, the CDC 7-related cancer is a high microsatellite instability (MSI-H) cancer. In other embodiments, the CDC 7-related cancer is not a high microsatellite instability (MSI-H) cancer. In some embodiments, the MSI-H status is determined by detecting a repetitive DNA sequence selected from the group consisting of: a single nucleotide repeat marker, a dinucleotide repeat marker, a quasi-singlet marker, or a combination of any of the foregoing.
In some embodiments, the tumor associated with the cancer comprises a phenotype selected from the group consisting of: chromosome Instability (CIN), spindle checkpoint assembly defects, mitotic defects, gl/S checkpoint defects, and combinations thereof. In some embodiments, the tumor associated with the cancer comprises a Wnt signaling pathway mutation. In some embodiments, the Wnt signaling pathway mutation is selected from the group consisting of: adenomatous Polyposis Coli (APC) gene mutation, FAT1 mutation, FAT4 mutation, or a combination of any of the foregoing.
In some examples of these methods, the process may include, the time difference between the first time point and the second time point may be about 1 day to about 1 year, about 1 day to about 11 months, about 1 day to about 10 months, about 1 day to about 9 months, about 1 day to about 8 months, about 1 day to about 7 months, about 1 day to about 6 months, about 1 day to about 5 months, about 1 day to about 4 months, about 1 day to about 3 months, about 1 day to about 10 weeks, about 1 day to about 2 months, about 1 day to about 6 weeks, about 1 day to about 1 month, about 1 day to about 25 days, about 1 day to about 20 days, about 1 day to about 15 days, about 1 day to about 10 days, about 1 day to about 5 days, about 2 days to about 1 year, about 5 days to about 1 year, about 10 days to about 1 year, about 15 days to about 1 year, about 20 days to about 1 year, about 25 days to about 1 day from about 1 month to about 1 year, from about 6 weeks to about 1 year, from about 2 months to about 1 year, from about 3 months to about 1 year, from about 4 months to about 1 year, from about 5 months to about 1 year, from about 6 months to about 1 year, from about 7 months to about 1 year, from about 8 months to about 1 year, from about 9 months to about 1 year, from about 10 months to about 1 year, from about 11 months to about 1 year, from about 1 day to about 7 days, from about 1 day to about 14 days, from about 5 days to about 10 days, from about 5 days to about 20 days, from about 10 days to about 20 days, from about 15 days to about 1 month, from about 15 days to about 2 months, from about 1 week to about 1 month, from about 2 weeks to about 1 month, from about 1 month to about 3 months, from about 3 months to about 6 months, from about 4 months to about 6 months, from about 5 months to about 8 months, or from about 7 months to about 9 months. In some embodiments of these methods, the subject may have been previously identified as having a cancer with a deregulated CDC7 gene (e.g., any instance of a deregulated CDC7 gene described herein). In some embodiments of these methods, the subject may be previously diagnosed with any of the types of cancers described herein. In some embodiments of these methods, the subject may have one or more metastases (e.g., one or more brain metastases).
In some of the above embodiments, the cfDNA comprises ctDNA, such as CDC 7-related ctDNA. For example, cfDNA is ctDNA, such as CDC 7-related ctDNA. In some embodiments, at least a portion of the cfDNA is determined to be CDC 7-related ctDNA, e.g., determining that a sequenced and/or quantified amount of total cfDNA has CDC7 fusion and/or overexpression of CDC 7.
In the field of medical oncology, it is common practice to treat each subject with cancer using a combination of different forms of treatment. In medical oncology, one or more other components of such combination therapies or treatments may be, for example, surgery, radiation therapy, and chemotherapeutic agents, such as other kinase inhibitors, signal transduction inhibitors, and/or monoclonal antibodies, in addition to the compositions provided herein. For example, the surgical procedure may be an open procedure or a minimally invasive procedure. Thus, the compound of formula (I) or a pharmaceutically acceptable salt thereof may also be used as an adjuvant for cancer treatment, i.e. it may be used in combination with one or more additional therapies or therapeutic agents (e.g. chemotherapeutic agents acting through the same or different mechanisms of action). In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered prior to administration of the additional therapeutic agent or the additional therapy. For example, one or more doses of a compound of formula (I), or a pharmaceutically acceptable salt thereof, may be administered to a subject in need thereof over a period of time, and then the tumor is at least partially resected. In some embodiments, treatment with one or more doses of a compound of formula (I) or a pharmaceutically acceptable salt thereof prior to at least partially resecting the tumor may reduce the size of the tumor (e.g., tumor burden). In some embodiments, one or more doses of a compound of formula (I), or a pharmaceutically acceptable salt thereof, may be administered to a subject in need thereof over a period of time and under one or more rounds of radiation therapy. In some embodiments, treatment with one or more doses of a compound of formula (I), or a pharmaceutically acceptable salt thereof, prior to one or more rounds of radiation therapy, reduces the size of the tumor (e.g., tumor burden).
In some embodiments, the subject has cancer that is refractory or intolerant to standard therapies (e.g., administration of a chemotherapeutic agent (e.g., a first CDC7 inhibitor or a multi-kinase inhibitor), immunotherapy, or radiation (e.g., radioiodine)). In some embodiments, the subject has a cancer that is refractory or intolerant to prior therapies (e.g., administration of a chemotherapeutic agent (e.g., a first CDC7 inhibitor or a multi-kinase inhibitor), immunotherapy, or radiation (e.g., radioiodine)). In some embodiments, the subject has cancer (e.g., locally advanced or metastatic tumor) without standard therapy. In some embodiments, the subject has not used a CDC7 kinase inhibitor. For example, the subject is not treated with a selective CDC7 kinase inhibitor. In some embodiments, the subject has used a CDC7 kinase inhibitor.
In some embodiments, the subject has received a prior therapy. In some embodiments, a subject having NSCLC (e.g., CDC 7-related NSCLS) has been treated with platinum-based chemotherapy, PD-1/PDL1 immunotherapy, or both, prior to treatment with a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, a subject having thyroid cancer (e.g., CDC 7-related thyroid cancer) has been treated with one or more of sorafenib (sorafenib), lenvatinib (lenvatinib), and radioiodine prior to treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a subject having colorectal cancer (e.g., CDC 7-related colorectal cancer) has been treated with fluoropyrimidine-based chemotherapy (with or without anti-VEGF-directed therapy or anti-EGFR-directed therapy) prior to treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a subject having pancreatic cancer (e.g., CDC 7-related pancreatic cancer) has been treated with one or more of fluoropyrimidine-based chemotherapy, gemcitabine-based chemotherapy, and S-1 chemotherapy prior to treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a subject having breast cancer (e.g., CDC 7-related breast cancer) has been treated with one or more of an anthracycline, a taxane, HER2 targeted therapy, and a hormonal therapy prior to treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a subject having MTC (e.g., CDC 7-related MTC cancer) has been treated with one or more of caboxantinib (caboxantinib) and vandetanib (vanretanib) prior to treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof.
In some embodiments of any of the methods described herein, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in combination with an effective amount of at least one additional therapeutic agent selected from one or more additional therapies or therapeutic agents (e.g., chemotherapeutic agents).
Non-limiting examples of additional therapeutic agents include: other CDC7 targeted therapeutic agents (i.e., first or second CDC7 kinase inhibitors), other kinase inhibitors (e.g., therapeutic agents targeting receptor tyrosine kinases (e.g., trk inhibitors or EGFR inhibitors)), signal transduction pathway inhibitors, checkpoint inhibitors, modulators of the apoptotic pathway (e.g., obackra); cytotoxic chemotherapy, angiogenesis-targeted therapies, immune-targeted agents, including immunotherapy and radiotherapy.
In some embodiments, the other CDC7 targeted therapeutic agent is a multi-kinase inhibitor that exhibits CDC7 inhibitory activity. In some embodiments, the additional CDC7 targeted therapy inhibitor is selective for CDC7 kinase. Exemplary CDC7 kinase inhibitors may exhibit inhibitory activity (IC) against CDC7 kinase of less than about 1000nM, less than about 500nM, less than about 200nM, less than about 100nM, less than about 50nM, less than about 25nM, less than about 10nM, or less than about 1nM 50 ) As measured in the assays described herein. In some embodiments, the CDC7 kinase inhibitor may exhibit an inhibitory activity (IC) against CDC7 kinase of less than about 25nM, less than about 10nM, less than about 5nM, or less than about 1nM 50 ) As measured in the assays provided herein.
Non-limiting examples of kinase targeted therapeutic agents (e.g., first CDC7 inhibitor or second CDC7 inhibitor) include TAK931, SRA141, and PHA-767491.
Non-limiting examples of multi-kinase inhibitors include: alternib (aletinib) (9-ethyl-6, 6-dimethyl-8- [4- (morpholin-4-yl) piperidin-1-yl ] -11-oxo-6, 11-dihydro-5H-benzo [ b ] carbazole-3-carbonitrile); a Mu Fa tenib (amuvantinib) (MP 470, HPK 56) (N- (1, 3-benzodioxol-5-ylmethyl) -4- ([ 1] benzofuran [3,2-d ] pyrimidin-4-yl) piperazine-1-carbosulfamide); apatinib (apatinib) (YN 968D 1) (N- [4- (1-cyanocyclopentyl) phenyl-2- (4-picolyl) amino-3-methanesulfonic acid nicotinamide); cabozantinib (cobodinib) (comatriq XL-184) (N- (4- ((6, 7-dimethoxyquinolin-4-yl) oxy) phenyl) -N' - (4-fluorophenyl) cyclopentane-1, 1-dicarboxamide); doratinib (dovitinib) (TKI 258; GFKI-258; CHIR-258) ((3Z) -4-amino-5-fluoro-3- [5- (4-methylpiperazin-1-yl) -1, 3-dihydrobenzimidazol-2-ylidene ] quinolin-2-one); famitinib (5- [2- (diethylamino) ethyl ] -2- [ (Z) - (5-fluoro-2-oxo-1H-indol-3-ylidene) methyl ] -3-methyl-6, 7-dihydro-1H-pyrrolo [3,2-c ] pyridin-4-one); fei Dala Tinib (feldatinib) (SAR 302503, TG 101348) (N- (2-methyl-2-propionyl) -3- { [ 5-methyl-2- ({ 4- [2- (1-pyrrolidinyl) ethoxy ] phenyl } amino) -4-pyrimidinyl ] amino } benzenesulfonamide); foCDC7inib (XL 880, EXEL-2880, gsk1363089, gsk 089) (N1' - [ 3-fluoro-4- [ [ 6-methoxy-7- (3-morpholinopropoxy) -4-quinolinyl ] oxy ] phenyl ] -N1- (4-fluorophenyl) cyclopropane-1, 1-dicarboxamide); fostamaniib (R788) (2H-pyrido [3,2-b ] -1, 4-oxazin-3 (4H) -one, 6- [ [ 5-fluoro-2- [ (3, 4, 5-trimethoxyphenyl) amino ] -4-pyrimidinyl ] amino ] -2, 2-dimethyl-4- [ (phosphoryloxy) methyl ] -, sodium salt (1:2)); ilofacitinib (ilorasertib) (ABT-348) (1- (4- (4-amino-7- (1- (2-hydroxyethyl) -1H-pyrazol-4-yl) thieno [3,2-c ] pyridin-3-yl) phenyl) -3- (3-fluorophenyl) urea); lenvatinib (lenvatinib) (E7080, le Weima (Lenvima)) (4- [ 3-chloro-4- (cyclopropylaminocarbonyl) aminophenoxy ] -7-methoxy-6-quinolinecarboxamide); motesanib (motesanib) (AMG 706) (N- (3, 3-dimethyl-2, 3-dihydro-1H-indol-6-yl) -2- [ (pyridin-4-ylmethyl) amino ] pyridine-3-carboxamide); (nintedanib) (3-Z- [1- (4- (N- ((4-methyl-piperazin-1-yl) -methylcarbonyl) -N-methyl-amino) -anilino) -1-phenyl-methylene ] -6-methoxycarbonyl-2-indolone); panatinib (ponatinib) (AP 24534) (3- (2-imidazo [1,2-b ] pyridazin-3-ylethynyl) -4-methyl-N- [4- [ (4-methylpiperazin-1-yl) methyl ] -3- (trifluoromethyl) phenyl ] benzamide); PP242 (tokini (torkinib)) (2- [ 4-amino-1- (1-methylethyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl ] -1H-indol-5-ol); quezatinib (quinartiinib) (1- (5- (tert-butyl) isoxazol-3-yl) -3- (4- (7- (2-morpholinoethoxy) benzo [ d ] imidazo [2,1-b ] thiazol-2-yl) phenyl) urea); regorafenib (BAY 73-4506, steven's valgo (stivarga)) (4- [4- ({ [ 4-chloro-3- (trifluoromethyl) phenyl ] carbamoyl } amino) -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide hydrate); RXDX-105 (CEP-32496, algeafanil (agerafanib)) (1- (3- ((6, 7-dimethoxyquinazolin-4-yl) oxy) phenyl) -3- (5- (1, 1-trifluoro-2-methylpropan-2-yl) isoxazol-3-yl) urea); semaxanib (SU 5416) ((3Z) -3- [ (3, 5-dimethyl-1H-pyrrol-2-yl) methylene ] -1, 3-dihydro-2H-indol-2-one); west Qu Fa tinib (sitravaninib) (MGCD 516, MG 516) (N- (3-fluoro-4- { [2- (5- { [ (2-methoxyethyl) amino ] methyl } -2-pyridinyl) thieno [3,2-b ] pyridin-7-yl ] oxy } phenyl) -N' - (4-fluorophenyl) -1, 1-cyclopropanedicarboxamide); sorafenib (sorafenib) (BAY 43-9006) (4- [4- [ [ [ [ 4-chloro-3- (trifluoromethyl) phenyl ] amino ] carbonyl ] amino ] phenoxy ] -N-methyl-2-pyridinecarboxamide); varditanib (N- (4-bromo-2-fluorophenyl) -6-methoxy-7- [ (1-methylpiperidin-4-yl) methoxy ] quinazolin-4-amine); varanib (PTK 787, PTK/ZK, ZK 222584) (N- (4-chlorophenyl) -4- (pyridin-4-ylmethyl) phthalazin-1-amine); AD-57 (N- [4- [ 4-amino-1- (1-methylethyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl ] phenyl ] -N' - [3- (trifluoromethyl) phenyl ] -urea); AD-80 (1- [4- (4-amino-1-prop-2-ylpyrazolo [3,4-d ] pyrimidin-3-yl) phenyl ] -3- [ 2-fluoro-5- (trifluoromethyl) phenyl ] urea); AD-81 (1- (4- (4-amino-1-isopropyl-1H-pyrazolo [3,4-d ] pyrimidin-3-yl) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea); ALW-II-41-27 (N- (5- ((4- ((4-ethylpiperazin-1-yl) methyl) -3- (trifluoromethyl) phenyl) carbamoyl) -2-methylphenyl) -5- (thiophen-2-yl) nicotinamide); BPR1K871 (1- (3-chlorophenyl) -3- (5- (2- ((7- (3- (dimethylamino) propoxy) quinazolin-4-yl) amino) ethyl) thiazol-2-yl) urea); CLM3 (1-phenethyl-N- (1-phenethyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine); EBI-907 (N- (2-chloro-3- (1-cyclopropyl-8-methoxy-3H-pyrazolo [3,4-c ] isoquinolin-7-yl) -4-fluorophenyl) -3-fluoropropane-1-sulfonamide); NVP-AST-487 (N- [4- [ (4-ethyl-1-piperazinyl) methyl ] -3- (trifluoromethyl) phenyl ] -N' - [4- [ [6- (methylamino) -4-pyrimidinyl ] oxy ] phenyl ] -urea); NVP-BBT594 (BBT 594) (5- ((6-acetamidopyrimidin-4-yl) oxy) -N- (4- ((4-methylpiperazin-1-yl) methyl) -3- (trifluoromethyl) phenyl) indoline-1-carboxamide); PD173955 (6- (2, 6-dichlorophenyl) -8-methyl-2- (3-methylsulfanylamino) pyrido [2,3-d ] pyrimidin-7-one); PP2 (4-amino-5- (4-chlorophenyl) -7- (dimethylethyl) pyrazolo [3,4-d ] pyrimidine); PZ-1 (N- (5- (tert-butyl) isoxazol-3-yl) -2- (4- (5- (1-methyl-1H-pyrazol-4-yl) -1H benzo [ d ] imidazol-1-yl) phenyl) acetamide); RPI-1 (1, 3-dihydro-5, 6-dimethoxy-3- [ (4-hydroxyphenyl) methylene ] -H-indol-2-one, (3E) -3- [ (4-hydroxyphenyl) methylene ] -5, 6-dimethoxy-1H-indol-2-one); SGI-7079 (3- [2- [ [ 3-fluoro-4- (4-methyl-1-piperazinyl) phenyl ] amino ] -5-methyl-7H-pyrrolo [2,3-d ] pyrimidin-4-yl ] -phenylacetonitrile); SPP86 (1-isopropyl-3- (phenylethynyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-amine); SU4984 (4- [4- [ (E) - (2-oxo-1H-indol-3-ylidene) methyl ] phenyl ] piperazine-1-carbaldehyde); sunitinib (SU 11248) (N- (2-diethylaminoethyl) -5- [ (Z) - (5-fluoro-2-oxo-1H-indol-3-ylidene) methyl ] -2, 4-dimethyl-1H-pyrrole-3-carboxamide); TG101209 (N-tert-butyl-3- (5-methyl-2- (4- (4-methylpiperazin-1-yl) phenylamino) pyrimidin-4-ylamino) benzenesulfonamide); withanosine A (Withaferin A) ((4β,5β,6β, 22R) -4, 27-dihydroxy-5, 6:22, 26-diepoxygsterone-2, 24-diene-1, 26-dione); XL-999 ((Z) -5- ((1-ethylpiperidin-4-yl) amino) -3- ((3-fluorophenyl) (5-methyl-1H-imidazol-2-yl) methylene) indolin-2-one); BPR1J373 (5-phenylthiazol-2-ylamine-pyrimidine derivative); CG-806 (CG' 806); DCC-2157; GTX-186; HG-6-63-01 ((E) -3- (2- (4-chloro-1H-pyrrolo [2,3-b ] pyridin-5-yl) vinyl) -N- (4- ((4-ethylpiperazin-1-yl) methyl) -3- (trifluoromethyl) phenyl) -4-methylbenzamide; SW-01 (cyclobenzaprine hydrochloride); XMD15-44 (N- (4- ((4-ethylpiperazin-1-yl) methyl) -3- (trifluoromethyl) phenyl) -4-methyl-3- (pyridin-3-ylethynyl) benzamide (generated by the structure)); an antibody drug conjugate of Y078-DM1 (composed of CDC7 antibody (Y078) linked to a derivative of the cytotoxic agent maytansine); Y078-DM4 (an antibody drug conjugate consisting of CDC7 antibody (Y078) linked to a derivative of the cytotoxic agent maytansine); ITRI-305 (D0N 5TB, DIB 003599); BLU-667 ((1S, 4 r) -N- ((S) -1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -1-methoxy-4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) cyclohexane-1-carboxamide; BLU6864; DS-5010; GSK3179106; GSK3352589; NMS-E668; TAS0286/HM05; TPX0046; n- (3- (2- (dimethylamino) ethoxy) -5- (trifluoromethyl) phenyl) -2- (4- (4-ethoxy-6-oxo-1, 6-dihydropyridin-3-yl) -2-fluorophenyl) acetamide.
The receptor tyrosine kinase (e.g., non-limiting examples of Trk) targeted therapeutic agents include afatinib (afatinib), cabotinib, cetuximab (cetuximab), crizotinib (crizotinib), dabrafenib (dabrafenib), emtrictinib (entrectinib), erlotinib (erlotinib), gefitinib (gefitinib), imatinib (imatinib), lapatinib (lapatinib), letatinib (lesatinib), nilotinib (nilotinib), gefitinib (gefitinib), and pharmaceutical compositions Pazopanib (pazopanib), panitumumab (panitumumab), pertuzumab (pertuzumab), sunitinib, trastuzumab (trastuzumab), l- ((3S, 4R) -4- (3-fluorophenyl) -l- (2-methoxyethyl) pyrrolidin-3-yl) -3- (4-methyl-3- (2-methylpyrimidin-5-yl) -l-phenyl-lH-pyrazol-5-yl) urea, AG 879, AR-772, AR-786, AR-256, AR-618, AZ-23, AZ623, DS-6051,6976. GNF-5837, GTx-186, GW 441756, LOXO-101, MGCD516, PLX7486, RXDX101, VM-902A, TPX-0005, TSR-011, GNF-4256, N- [3- [ [2, 3-dihydro-2-oxo-3- (1H-pyrrol-2-ylmethylene) -1H-indol-6-yl ]]Amino group]-4-methylphenyl]-N' - [ 2-fluoro-5- (trifluoromethyl) phenyl ]]-urea, AZ623, AZ64, (S) -5-chloro-N2- (1- (5-fluoropyridin-2-yl) ethyl) -N4- (5-isopropoxy-1H-pyrazol-3-yl) pyrimidine-2, 4-diamine, AZD7451, CEP-751, CT327, sunitinib, GNF-8625, (R) -1- (6- (6- (2- (3-fluorophenyl) pyrrolidin-1-yl) imidazo [1, 2-b) ]Pyridazin-3-yl) - [2,4' -bipyridine]-2' -yl) piperidin-4-ol.
Non-limiting examples of BRAF inhibitors include: dabrafenib, vemurafenib (also known as RG7204 or PLX 4032), sorafenib p-toluenesulfonate (sorafenib tosylate), PLX-4720, GDC-0879, BMS-908662 (Bristol-Meyers Squibb), LGX818 (Novartis), PLX3603 (haven michan-LaRoche), RAF265 (norubin), RO5185426 (haven michan) and GSK2118436 (GlaxoSmithKline). Additional examples of BRAF inhibitors are known in the art.
In some embodiments, the receptor tyrosine kinase inhibitor is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR). For example, EGFR inhibitors may comprise octreotide (oseverinib) (merlecitinib, tagriso), erlotinib (Tarceva), gefitinib (Iressa), cetuximab (Erbitux), cetuximab (necitumumab) (portraza), lenatinib (neryinx), lapatinib (Tykerb), panitumumab (vectiix), and vandetanib (calisla).
In some embodiments, the signaling pathway inhibitor comprises a Ras-Raf-MEK-ERK pathway inhibitor (e.g., bimetainib), semetanib (selumetinib), kang Naifei ni (encorafenib), sorafenib, trametanib (trametainib), and vemurafenib), a PI3K-Akt-mTOR-S6K pathway inhibitor (e.g., everolimus, rapamycin, pirtifolicin, temsirolimus) and other kinase inhibitors, such as baratinib, briatinib, capmatinib, dactyltinib, ibrutinib, milcinib, quercetin, regatinib, ruxolitinib, sematinib, AP32788, BLU285, BLU554, INCB39110, INCB40093, INCB50465, INCB52793, INCB54828, MGCD265, NMS-088, NMS-1286937, PF 477736 ((R) -amino-N- [5, 6-dihydro-2- (1-methyl-1H-pyrazol-4-yl) -6-oxo-1H pyrrolo [4,3, 2-f ] [2,3] benzodiazepin-8-yl ] -cyclohexane), PLX3397, PLX7486, x8394, PRN 86, PRN1008, rx1, RXDX-1286937, rx 137106, PLX 9497, and the like, RXDX108 and TG101209 (N-tert-butyl-3- (5-methyl-2- (4- (4-methylpiperazin-1-yl) phenylamino) pyrimidin-4-ylamino) benzenesulfonamide).
Non-limiting examples of checkpoint inhibitors include ipilimumab (ipilimumab), tremelimumab (tremelimumab), nivolumab (nivolumab), pimelimumab (pimelizumab), MPDL3208A, MEDI4736, MSB0010718C, BMS-936559, BMS-956559, BMS-935559 (MDX-1105), AMP-224, and pembrolizumab (pembrolizumab).
In some embodiments, the cytotoxic chemotherapeutic agent is selected from the group consisting of arsenic trioxide (arsenical trioxide), bleomycin (bleomycin), cabazitaxel (cabazitaxel), capecitabine (capecitabine), carboplatin (carboplatin), cisplatin (cispratin), cyclophosphamide (cyclophosphamide), cytarabine (cytarabine), dacarbazine (dacarbazine), daunorubicin (daunorubicin), docetaxel (docetaxel), doxorubicin (doxorubicin), etoposide (etoposide), fluorouracil (fluorouracil), gemcitabine, irinotecan (irinotecan), lomustine (lomustine), methotrexate (methotrexate), mitomycin C (mitomycin C), oxaliplatin (oxaplatin), paclitaxel (paclitaxel), methotrexate (methotrexate), and vincristine (vinblastine).
Non-limiting examples of therapies that target angiogenesis include aflibercept (aflibercept) and bevacizumab (bevacizumab).
In some embodiments, the additional therapy or therapeutic agent can comprise a histidyl-tRNA synthetase (HRS) polypeptide or an expressible nucleotide encoding a HRS polypeptide.
The term "immunotherapy" refers to agents that modulate the immune system. In some embodiments, immunotherapy may increase the expression and/or activity of immune system modulators. In some embodiments, immunotherapy may reduce the expression and/or activity of immune system modulators. In some embodiments, immunotherapy may recruit and/or enhance the activity of immune cells.
In some embodiments, the immunotherapy is a cellular immunotherapy (e.g., adoptive T cell therapy, dendritic cell therapy, natural killer cell therapy). In some embodiments, the cellular immunotherapy is cetylpropoulel-T (APC 8015; provenge) TM The method comprises the steps of carrying out a first treatment on the surface of the Plosker (2011) drug 71 (1): 101-108). In some embodiments, the cellular immunotherapy comprises cells expressing a Chimeric Antigen Receptor (CAR). In some embodiments, the cellular immunotherapy is CAR-T cell therapy. In some embodiments, the CAR-T cell therapy is tisalen (tisallecieucel) (Kymriah TM )。
In some embodiments, the immunotherapy is an antibody therapy (e.g., monoclonal antibodies, conjugated antibodies). In some embodiments, the antibody therapy is bevacizumab (Mvasti) TM ，) Trastuzumab->Avermeab (avelumab)/(Avelumab)>Rituximab (MabThera) TM ，/>) Edeclomab (Panorex), up to Lei Tuoyou mab (daratumuab)>Olympic monoclonal antibody (olarruvo) TM ) Offatumumab (ofatumumab)>Alemtuzumab (alemtuzumab) in/out>Cetuximab->Ago Fu Shan antibody (orenovomab), pembrolizumab +.>Denootuximab>Atozumab (obinutuzumab) in the presence of a drug>Tramadol mab (CP-675, 206), ramucirumab (ramucirumab)Wu Lituo Acximab (ublituximab) (TG-1101), panitumumab ++>Embolizumab (Emplititi) TM ) Avermectin->Xitumumab (Portrazza) TM ) Cetuximab (UC-961), ibutuzumab (ibrituximab)>I Sha Tuo mab (isatuximab) (SAR 650984), nimotuzumab (nimotuzumab), non-hematoxyliumab (fresolimumab) (GC 1008), li Ruilu mab (lirilumab) (INN), mo Geli group mab (mogamulizumab) in the following list>Non-lattuzumab (ficlatuzumab) (AV-299), desulumab (denosumab) or the like>Ganitamab (ganitumab), wu Ruilu mab (urelumab), pidirizumab (pimelizumab) or amatuzumab (amatuximab).
In some embodiments, the immunotherapy is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate is gemtuzumab ozagrel (gemtuzumab ozogamicin) (Mylotarg) TM ) Ottotuzumab ozagrel (inotuzumab ozogamicin)Bentuxi Shan Kangrui statin (brentuximab vedotin)Enmetrastuzumab (ado-trastuzumab emtansine) (TDM-1;)>) Sofoster-Mituximab (mirvetuximab soravtansine) (IMGN 853) or Lei Xing-anetuzumab (anetumab ravtansine)
In some embodiments, the immunotherapy comprises bordetention (AMG 103;) Or midostaurin (Rydapt).
In some embodiments, the immunotherapy comprises a toxin. In some embodiments, the immunotherapy is a dinium interleukin (denileukin diftitox)
In some embodiments, the immunotherapy is cytokine therapy. In some embodiments, the cytokine therapy is interleukin 2 (IL-2) therapy, interferon alpha (IFN alpha) therapy, granulocyte colony-stimulating factor (G-CSF) therapy, interleukin 12 (IL-12) therapy, interleukin 15 (IL-15) therapy, interleukin 7 (IL-7) therapy, or erythropoietin alpha (EPO) therapy. In some embodiments, the IL-2 therapy is aldesleukin (aldeslukin)In some embodiments, the ifnα therapy is +.>In some embodiments of the present invention, in some embodiments, G-CSF therapy is non-filgrastim->
In some embodiments, the immunotherapy is an immune checkpoint inhibitor. In some embodiments, the immunotherapy comprises one or more immune checkpoint inhibitors. In some embodiments The immune checkpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumabOr tremelimumab (CP-675, 206). In some embodiments, the PD-1 inhibitor is pembrolizumab +.>Or NawuzumabIn some embodiments, the PD-L1 inhibitor is atezolizumab (atezolizumab)>Avermectin->Or Devalumab (durvalumab) (Imfinzi) TM )。
In some embodiments, the immunotherapy is mRNA-based immunotherapy. In some embodiments, the mRNA-based immunotherapy is CV9104 (see, e.g., rausch et al (2014) & human vaccine and immunotherapy (Human Vaccin Immunother) & 10 (11): 3146-52; and Kubler et al (2015) & journal of Cancer immunotherapy (J. Immunother Cancer) & 3:26).
In some embodiments, the immunotherapy is Bacillus Calmette Guerin (BCG) therapy.
In some embodiments, the immunotherapy is oncolytic virus therapy. In some embodiments, the oncolytic viral therapy is latamoyl (talimogene alherparepvec) (T-VEC;)。
in some embodiments, the immunotherapy is a cancer vaccine. In some embodiments, the cancer vaccine is a Human Papilloma Virus (HPV) vaccine. In some embodiments HPV vaccine isOr->In some embodiments, the cancer vaccine is a Hepatitis B Virus (HBV) vaccine. In some embodiments, the HBV vaccine is +.>Recombivax/>Or GI-13020->In some embodiments, the cancer vaccine is +.>Or->In some embodiments, the cancer vaccine is +.>GVAX、ADXS11-001、ALVAC-CEA、/>CimaVax-EGF、lapuleucel-T(APC8024；Neuvenge TM )、GRNVAC1、GRNVAC2、GRN-1201、hepcortespenlisimut-L(Hepko-V5)、SCIB 1、BMT CTN 1401、PrCa VBIR、PANVAC、/>DPX-survivinvac or vinenumatucel-L (HS-110).
In some embodiments, the immunotherapy isPeptide vaccine. In some embodiments, the peptide vaccine is nelipepimut-S (E75) (NeuVax TM ) IMA901 or SurVaxM (SVN 53-67). In some embodiments, the cancer vaccine is an immunogenic personal neoantigen vaccine (see, e.g., ott et al (2017) Nature 547:217-221; sahin et al (2017) Nature 547:222-226). In some embodiments, the cancer vaccine is RGSH4K or NEO-PV-01. In some embodiments, the cancer vaccine is a DNA-based vaccine. In some embodiments, the DNA-based vaccine is a lactoglobulin-A DNA vaccine (see, e.g., kim et al (2016) & lt/EN & gt tumor immunology (Oncomelanology) & lt/EN & gt 5 (2): e 1069940).
In some embodiments, the immune targeting agent is selected from the group consisting of aldesleukin, interferon alpha-2 b, ipilimumab, lanrolizumab (lambrolizumab), nivolumab, prednisone (prednisone), and ceteprunopene-T.
Non-limiting examples of radiation therapy include radioiodination therapy, external beam radiation, and radium 223 therapy.
Additional kinase inhibitors include those described in the following patents: for example, U.S. patent No. 7,514,446; 7,863,289; 8,026,247; 8,501,756; 8,552,002; 8,815,901; 8,912,204; 9,260,437; 9,273,051; U.S. publication No. US 2015/0018336; international publication No. WO 2007/002325; WO 2007/002433; WO 2008/080001; WO 2008/079906; WO 2008/079903; WO 2008/079909; WO 2008/080015; WO 2009/007448; WO 2009/012683; WO 2009/143018; WO 2009/143024; WO 2009/014637; 2009/152083; WO 2010/111527; WO 2012/109075; WO 2014/194127; WO 2015/112806; WO 2007/110344; WO 2009/071480; WO 2009/118411; WO 2010/031816; WO 2010/145998; WO 2011/092120; WO 2012/101032; WO 2012/139930; WO 2012/143248; WO 2012/152763; WO 2013/014039; WO 2013/102059; WO 2013/050448; WO 2013/050446; WO 2014/019908; WO 2014/072220; WO 2014/184069; WO 2016/075224; WO 2016/081450; WO 2016/022569; WO 2016/01141; WO 2016/01144; WO 2016/01147; WO 2015/191667; WO 2012/101029; WO 2012/113774; WO 2015/191666; WO 2015/161277; WO 2015/161274; WO 2015/108992; WO 2015/061572; WO 2015/058129; WO 2015/057873; WO 2015/017528; WO/2015/017533; WO 2014/160521 and WO 2014/01900, each of which is incorporated herein by reference in its entirety.
Although the genetic basis of tumorigenesis may vary between different types of cancer, the cellular and molecular mechanisms required for metastasis appear to be similar for all solid tumor types. During the metastatic cascade, cancer cells lose growth inhibition, undergo an adhesive change, and produce enzymes that degrade extracellular matrix components. This results in the separation of tumor cells from the original tumor, infiltration into the circulatory system through the newly formed vasculature, and migration and extravasation of tumor cells at favorable distal sites where colonies may form. Many genes have been identified as promoters or inhibitors of metastasis. For example, the overexpression of glial cell-derived neurotrophic factor (GDNF) and its CDC7 receptor tyrosine kinase has been implicated in the proliferation and metastasis of cancer. See, for example, zeng et al J.International journal of medical research (J.Int. Med. Res.) (2008) 36 (4): 656-64.
Accordingly, also provided herein is a method for inhibiting, preventing, helping to prevent or alleviating the metastatic symptoms of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or pharmaceutical composition thereof. Such methods may be used to treat one or more cancers described herein. See, for example, U.S. publication No. 2013/0029925; international publication No. WO 2014/083567; and U.S. patent No. 8,568,998. See also, e.g., hezam K et al, review of neuroscience (Rev Neurosci), 2018, month 1, day 26; 29:93-98; gao L et al, pancreas (pancrees) 2015, month 1; 44:134-143; ding K et al, journal of biochemistry, 2014, 6; 289:16057-71; and Amit M et al, oncogene, 2017, 6/8; 36:3232-3239. In some embodiments, the cancer is a CDC 7-related cancer. In some embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is used in combination with another therapy or another therapeutic agent (including a chemotherapeutic agent, such as a kinase inhibitor). For example, a first or second CDC7 kinase inhibitor. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is octreotide. In some embodiments, one or more doses of a compound of formula (I), or a pharmaceutically acceptable salt thereof, have been administered to a subject prior to administration of the pharmaceutical composition. In some embodiments, the cancer is lung cancer (e.g., CDC 7-related lung cancer). In some embodiments, the additional therapeutic agent is a PARP inhibitor (e.g., olaparib). In some embodiments, the additional therapeutic agent is an ATR inhibitor (e.g., ceralasertib). In some embodiments, the additional therapeutic agent is a Wee1 inhibitor (e.g., AZD-1775). In some embodiments, the additional therapeutic agent is an EGFR inhibitor (e.g., lapatinib).
The term "metastasis" is a term known in the art and refers to the formation of an additional tumor (e.g., a solid tumor) in a subject at a site distant from the primary tumor, wherein the additional tumor comprises the same or similar cancer cells as the primary tumor.
Also provided is a method of reducing the risk of metastasis or additional metastasis in a subject having a CDC 7-related cancer, the method comprising: selecting, identifying or diagnosing a subject having a CDC 7-related cancer, and administering to the subject selected, identified or diagnosed as having a CDC 7-related cancer an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. Also provided are methods of reducing the risk of metastasis or additional metastasis in a subject having a CDC 7-related cancer, the method comprising administering to a subject having a CDC 7-related cancer an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. The reduction in the risk of metastasis or additional metastasis in a subject having a CDC 7-related cancer may be compared to the risk of metastasis or additional metastasis in a pre-treatment subject, or to a subject or population of subjects having a similar or identical CDC 7-related cancer that have not received any treatment or received a different treatment. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is octreotide. In some embodiments, one or more doses of a compound of formula (I), or a pharmaceutically acceptable salt thereof, have been administered to a subject prior to administration of the pharmaceutical composition. In some embodiments, the cancer is lung cancer (e.g., CDC 7-related lung cancer).
The phrase "risk of developing metastasis" means that a subject having a primary tumor is at risk of developing an additional tumor (e.g., a solid tumor) at a site distant from the primary tumor in the subject within a set period of time, wherein the additional tumor comprises the same or similar cancer cells as the primary tumor. Described herein are methods for reducing the risk of metastasis in a subject having cancer.
The phrase "risk of developing additional metastasis" means that a subject having a primary tumor and having one or more additional tumors at a site distant from the primary tumor (wherein the one or more additional tumors comprise the same or similar cancer cells as the primary tumor) will be at risk of developing one or more additional tumors distant from the primary tumor, wherein the additional tumors comprise the same or similar cancer cells as the primary tumor. Methods of reducing the risk of additional metastasis are described herein.
Treatment of a subject with cancer with a multi-kinase inhibitor (MKI) or a target-specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, RET inhibitor, or RAS inhibitor) may cause a disruption in the expression or activity or level of the CDC7 gene, CDC7 kinase, or any of them in the cancer and/or cause resistance to the CDC7 inhibitor. See, e.g., brange et al, tumor target (Oncostarget) 8:27155-27165,2017; chang et al, J.Yinsei.Med.J.) (58:9-18,2017; and Lopez-Delisle et al, doi 10.1038/s41388-017-0039-5, oncogene 2018.
Treatment of the same or similar subject with a combination of a CDC7 inhibitor and a multi-kinase inhibitor or a target-specific kinase inhibitor in a monotherapy form may have increased therapeutic efficacy compared to treatment of a subject with the same subject with the CDC7 inhibitor or a multi-kinase inhibitor or a target-specific kinase inhibitor in a monotherapy form (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, RET inhibitor, or RAS inhibitor). See, e.g., tang et al, doi 10.1038/modpathol.2017.109, modern pathology (Mod. Pathol.) 2017; andreucci et al, tumor target 7:80543-80553,2017; nelson-Taylor et al, molecular cancer therapy 16:1623-1633,2017; and Kato et al, clinical cancer research 23:1988-1997,2017.
Provided herein are methods of treating a subject having a cancer (e.g., any of the cancers described herein) and having been previously administered a multi-kinase inhibitor (MKI) or a target-specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, RET inhibitor, or RAS inhibitor) (e.g., in monotherapy form), the method comprising: administering to the subject (I) an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in monotherapy form, or (ii) an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and an effective amount of an MKI or a target specific kinase inhibitor previously administered.
Provided herein are methods of treating a subject (e.g., in monotherapy form) having a cancer (e.g., any of the cancers described herein) and having been previously administered an MKI or target-specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, RET inhibitor, or RAS inhibitor), the method comprising: identifying a subject having a cancer cell with a dysregulation of expression or activity or level of CDC7 gene, CDC7 kinase, or any of them; and administering to the identified subject (I) an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or (ii) an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in monotherapy form, and an effective amount of a previously administered MKI or a previously administered target specific kinase inhibitor.
Provided herein are methods of treating a subject having cancer (e.g., any of the cancers described herein), the method comprising: administering to the subject an effective amount of an MKI or target specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, RET inhibitor, or RAS inhibitor) (e.g., in monotherapy form) for a first period of time; after the period of time, identifying a subject having a cancer cell with a dysregulation of expression or activity or level of a CDC7 gene, CDC7 kinase, or any of them; and administering to the identified subject (I) an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or (ii) an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in monotherapy form, and an effective amount of a previously administered MKI or a previously administered target specific kinase inhibitor.
Also provided is a method for inhibiting CDC7 kinase activity in a mammalian cell, the method comprising contacting the mammalian cell with a compound of formula (I). In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vivo, wherein the method comprises administering to a subject having a mammalian cell with CDC7 kinase activity an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the mammalian cell is a mammalian cancer cell. In some embodiments, the mammalian cancer cell is any cancer described herein. In some embodiments, the mammalian cancer cell is a CDC 7-related mammalian cancer cell.
Also provided is a method for inhibiting CDC7 kinase activity in a mammalian cell, the method comprising contacting the mammalian cell with a compound of formula (I). In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vivo, wherein the method comprises administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, to a mammal having a mammalian cell with CDC7 kinase activity. In some embodiments, the mammalian cell is a mammalian cancer cell. In some embodiments, the mammalian cancer cell is any cancer described herein. In some embodiments, the mammalian cancer cell is a CDC 7-related mammalian cancer cell. In some embodiments, the mammalian cell is a gastrointestinal mammalian cell.
As used herein, the term "contacting" refers to bringing together the indicated portions of an in vitro system or an in vivo system. For example, "contacting" a CDC7 kinase with a compound provided herein comprises administering a compound provided herein to a subject (e.g., a human) having a CDC7 kinase, and, for example, introducing a compound provided herein into a sample comprising mammalian cells or a purified preparation comprising a CDC7 kinase.
Also provided herein is a method of inhibiting proliferation of a mammalian cell in vitro or in vivo, the method comprising contacting the mammalian cell with an effective amount of a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
As defined herein, a "CDC7 kinase inhibitor" comprises any compound that exhibits CDC7 inhibitory activity. In some embodiments, the CDC7 kinase inhibitor is selective for CDC7 kinase. Exemplary CDC7 kinase inhibitors may exhibit inhibitory activity (IC) against CDC7 kinase of less than about 1000nM, less than about 500nM, less than about 200nM, less than about 100nM, less than about 50nM, less than about 25nM, less than about 10nM, or less than about 1nM 50 ) As measured in the assays described herein. In some embodiments, the CDC7 kinase inhibitor may exhibit less than about 25nM, less than about 10nM, less than about 5nM for CDC7 kinase Or less than about 1nM inhibitory activity (IC 50 ) As measured in the assays provided herein.
As used herein, a "first CDC7 kinase inhibitor" or "first CDC7 inhibitor" is a CDC7 kinase inhibitor as defined herein, but which does not comprise a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof. As used herein, a "second CDC7 kinase inhibitor" or "second CDC7 inhibitor" is a CDC7 kinase inhibitor as defined herein, but which does not comprise a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof. When both the first and second CDC7 inhibitors are present in the methods provided herein, the first and second CDC7 kinase inhibitors are different.
Exemplary first and second CDC7 kinase inhibitors are described herein. In some embodiments, the first and second CDC7 kinase inhibitors may be selected from the group consisting of: TAK931, SRA141 and PHA-767491.
The phrase "effective amount" refers to an amount sufficient, when administered to a subject in need of such treatment, to (i) treat a CDC 7-related disease or disorder (e.g., a CDC 7-related cancer); (ii) Alleviating, ameliorating, or eliminating one or more symptoms of a particular disease, condition, or disorder; or (iii) an amount of a compound that delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof corresponding to the amount will vary depending on factors such as the particular compound, the disease condition and its severity, the identity (e.g., body weight) of the subject in need of treatment, and the like, but can still be routinely determined by one of ordinary skill in the art.
When used as a medicament, the compounds of formula (I) (including pharmaceutically acceptable salts thereof) may be administered in the form of a pharmaceutical composition. These compositions may be prepared in a manner well known in the pharmaceutical arts and may be administered by a variety of routes, depending on whether local or systemic treatment is desired and on the site to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and mucosal administration, including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer; intratracheal or intranasal administration), oral or parenteral. Oral administration may comprise dosage forms formulated for once-a-day or twice-a-day (BID) administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. Parenteral administration may be in the form of a single bolus dose or may be performed, for example, by a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration may comprise transdermal patches, ointments, emulsions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily matrices, thickeners and the like may be necessary or desirable.
Also provided herein are pharmaceutical compositions containing as an active ingredient a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof present in the composition is a compound of formula (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-N), (I-O) or (I-P) or a pharmaceutically acceptable salt of any of the foregoing.
For example, a pharmaceutical composition prepared using a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the composition is suitable for oral administration. In preparing the compositions provided herein, the active ingredient is typically admixed with, diluted with, or enclosed within such carriers in the form of, for example, capsules, sachets (sachets), paper, or other containers. When the excipient acts as a diluent, it may be a solid, semi-solid, or liquid material that can function as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is a solid oral formulation. In some embodiments, the composition is formulated as a tablet or capsule.
Further provided herein are pharmaceutical compositions comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient may be prepared by thoroughly mixing the compound of formula (I) or a pharmaceutically acceptable salt thereof with a pharmaceutical carrier according to conventional pharmaceutical mixing techniques. The carrier may take a variety of forms depending on the desired route of administration (e.g., oral, parenteral). In some embodiments, the composition is a solid oral composition.
Suitable pharmaceutically acceptable carriers are well known in the art. Descriptions of some of these pharmaceutically acceptable carriers can be found in the handbook of pharmaceutical excipients (The Handbook of Pharmaceutical Excipients), published by the american society of pharmacy and the united kingdom pharmaceutical society.
Methods of formulating pharmaceutical compositions have been described in numerous publications, such as "pharmaceutical dosage forms: tablets, second Edition, revisions and extensions (Pharmaceutical Dosage Forms: tables, second Edition, revised and Expanded), volumes 1-3, lieberman et al; pharmaceutical dosage form: parenteral drug (Pharmaceutical Dosage Forms: parenteral Medications), volume 1-2, edited by Avis et al; pharmaceutical dosage form: dispersion Systems (Pharmaceutical Dosage Forms: disperse Systems), volume 1-2, edited by Lieberman et al; published by Marcel Dekker, inc.
In preparing the composition in oral dosage form, any conventional pharmaceutical medium may be used. Thus, for liquid oral formulations (e.g., suspensions, elixirs and solutions), suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents and the like; for solid oral formulations (e.g., powders, capsules and tablets), suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Suitable binders include, but are not limited to, starch, gelatin, natural sugars (e.g., glucose or beta-lactose), corn sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or sodium oleate), sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Solid oral formulations may also be coated or enteric coated with substances such as sugar to modulate the primary site of absorption. For parenteral administration, the carrier will typically consist of sterile water, and other ingredients may be added to increase solubility or preservability. Injectable suspensions or solutions may also be prepared using aqueous carriers and appropriate additives. The pharmaceutical compositions herein will contain, in each dosage unit (e.g., tablet, capsule, powder, injection, teaspoon, etc.), an amount of the active ingredient necessary to deliver an effective dose as described herein.
Compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof may be formulated in unit dosage forms, each dosage containing from about 1 to about 1,000mg (1 g) of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other subjects, each unit containing a predetermined quantity of active material, i.e., a compound of formula (I) or a pharmaceutically acceptable salt thereof, calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
The active compounds can be effective over a wide dosage range and are generally administered in pharmaceutically effective amounts. The optimal dosage to be administered can be readily determined by one skilled in the art. It will therefore be appreciated that the amount of compound actually administered will generally be determined by the physician and will vary depending on the relevant circumstances, including the mode of administration, the compound actually administered, the strength of the formulation, the condition to be treated and the progress of the disease condition. In addition, factors related to the particular subject being treated, including subject response, age, weight, diet, time of administration, and severity of the subject's symptoms, will result in the need to adjust the dosage.
Those skilled in the art will recognize that both in vivo and in vitro assays using suitable, known and generally accepted cellular and/or animal models are predictive of the ability of a test compound to treat or prevent a given condition.
Those of skill in the art will further recognize that human clinical trials, including first human trials, dose range trials and efficacy trials, in healthy subjects and/or subjects suffering from a given disorder may be accomplished according to methods known in the clinical and medical arts.
Provided herein are pharmaceutical kits useful, for example, in treating CDC 7-related diseases or disorders (e.g., cancer), comprising one or more containers containing a pharmaceutical composition comprising an effective amount of a compound provided herein. As will be apparent to those of skill in the art, such kits may further comprise, if desired, one or more of a variety of conventional pharmaceutical kit components, e.g., containers with one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions (as an insert or as a label) indicating the amount of the component to be administered, instructions for administration, and/or instructions for mixing the components may also be included in the kit.
Examples
Materials and methods
The compounds provided herein (including salts thereof) may be prepared using known organic synthesis techniques and may be synthesized according to any of a variety of possible synthetic pathways.
The reaction for preparing the compounds provided herein may be carried out in a suitable solvent, which may be readily selected by one skilled in the art of organic synthesis. Suitable solvents may be substantially unreactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out (e.g., which may range from the freezing temperature of the solvent to the boiling temperature of the solvent). A given reaction may be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, the appropriate solvent for the particular reaction step may be selected by the skilled artisan.
The preparation of the compounds provided herein may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of the protecting groups can be found, for example, in the following documents: protecting group chemistry (Protecting Group Chemistry), 1 st edition, oxford university press (Oxford University Press), 2000; macchiato organic chemistry: reactions, mechanisms and structures (March's Advanced Organic Chemistry: reactions, mechanisms, and structures), 5 th edition, wiley International science publication (Wiley-Interscience Publication), 2001; and peterssion, s.et al, "protecting group in carbohydrate chemistry (Protecting Groups in Carbohydrate Chemistry)", "journal of chemistry education (j. Chem. Duc.)," 74 (11), 1297 (1997).
The reaction may be monitored according to any suitable method known in the art. For example, the light may be detected by, for example, nuclear magnetic resonance spectroscopy (e.g., 1 h or 13 C) The product formation is monitored by spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or Thin Layer Chromatography (TLC). The compounds can be purified by various methods including High Performance Liquid Chromatography (HPLC) ("Preparative LC-MS Purification: improved compound specific method optimization (Improved Compound Specific Method Optimization)" k.f. boom et al, journal of combinatorial chemistry (j. Combi. Chem.)) "6 (6), 874 (2004)), normal phase silica gel chromatography, and Supercritical Fluid Chromatography (SFC).
The stereochemistry of compounds 1-58 in the following examples should be understood to be arbitrarily specified unless otherwise indicated.
Unless otherwise indicated, all solvents and reagents were obtained from commercial sources and used without further purification. Anhydrous solvents were purchased and used in the supplied form. The reaction was monitored by Thin Layer Chromatography (TLC) using UV lamp (254 nm) and KMnO 4 Visual stainingAnd (5) melting. NMR spectra were obtained on a Bruker Neo400M spectrometer operating at 400 MHz. Chemical shifts in the indicated solvents relative to tetramethylsilane resonance are reported in parts per million (δ). LC mass spectra were acquired using an Agilent 1260-6125B single quadrupole mass spectrometer using a Welch Biomate column (C18, 2.7 μm,4.6 x 50 mm) or waters H-Class SQD2 system. Detection was performed by DAD (254 nm and 210nm and 280 nm). Chiral HPLC was performed on a system Waters acquity UPC, in the presence of alkali, on Daicel chiralpak AD-H (5 μm,4.6×250 mm), daicel chiralpak OD-H (5 μm,4.6×250 mm), daicel chiralpak IG-3 (3 μm,4.6×150 mm), chiral Technologies Europe (Chiral Technologies Europe) AD-3 (3 μm,3.0×150 mm) and Trefoil TM Technology (Trefoil TM Technology) Trefoil AMY1 (2.5 μm,3.0×150 mm). Detection was performed by DAD (254 nm). Preparative HPLC was performed on a GILSON Trilution LC system using Welch XB-C18 column (5 um,21.2 x 150 mm). Flash chromatography was performed on a Biotage Isolera Prime system using a Welch WelFlash flash column (40-63 μm). Unless otherwise indicated, the purity of the synthesized compounds was not less than 95%.
Abbreviations (abbreviations)
* ＝“ * "the amount of the preceding solvent or reagent is used in the technique a number of times equal to" * "indication of the following numbers".
C = degrees celsius
1 H NMR = proton nuclear magnetic resonance spectroscopy
AcOH = acetic acid
Boc 2 O=t-butoxycarbonyl anhydride
con=concentration
d = double peak
Dcm=dichloromethane
DIAD = diisopropyl azodicarboxylate
Dipea=n, N-diisopropylethylamine
Dmf=n, N-dimethylformamide
Ea=ethyl acetate
ESI = electrospray ionization
g=g
h=h
Hatu= (1- [ bis (dimethylamino) dimethyl ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate, azabenzotriazole tetramethyluronium hexafluorophosphate
HPLC = high performance liquid chromatography
LCMS = liquid chromatography-mass spectrometry
M = mass
m/z = mass to charge ratio
Mecn=acetonitrile
Meoh=methanol
MeONa = sodium methoxide
mg = mg
mL = milliliter
mmol = millimoles
mol = mole
Ms=mass spectrum
NBS = N-bromosuccinimide
obsd=observations
PCy 3 =tricyclohexylphosphine
Pd(AcO) 2 =palladium acetate (II)
Pd(dppf)Cl 2 = (1, 1' -bis (diphenylphosphino) ferrocene) palladium (II) dichloride
Pe=petroleum ether
ppm = parts per million
Ptsa=p-toluenesulfonic acid
rt=room temperature
SFC = supercritical fluid chromatography
s = single peak
t=triplet
Tbaf=tetrabutylammonium fluoride
TFA = trifluoroacetic acid
THF = tetrahydrofuran
TLC = thin layer chromatography
Trixiephos = rac-2- (di-tert-butylphosphine) -1,1' -binaphthyl
Example 1-compound 1:2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one
Step A:6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
to a solution of methyl 3-bromothiophene-2-carboxylate (1.00 g,4.52mmol,1.0 eq), (5-diphenylphosphino-9, 9-dimethyl-xanthen-4-yl) -diphenyl-phosphine (787 mg,1.36mmol,0.3 eq), cesium carbonate (3.68 g,11.30mmol,2.5 eq), sodium bisulphite (172 mg,0.90mmol,0.2 eq) and tris (dibenzylideneacetone) dipalladium (254 mg,0.90mmol,0.20 eq) in toluene (40.0 mL) was added cyclohexanone (888 mg,9.04mmol,2.0 eq) at room temperature. The reaction mixture was stirred at 105℃under N 2 Stirred for 16 hours. The solvent was concentrated to dryness and the residue was purified by flash chromatography (SiO 2 0-10% EtOAc/PE) to give 6,7,8, 9-tetrahydro-4H-thieno [2,3-c ]]Chromen-4-one (600 mg, 64%). MS observations (ESI) + )：207.0[(M+H) + ]。
And (B) step (B): 2-iodo-6, 7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
to 6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] at-78 DEG C]To a solution of chromen-4-one (400 mg,1.94mmol,1.0 eq.) in THF (15.0 mL) was added lithium (diisopropylamine) (2M in THF, 1.1mL,2.2mmol,1.1 eq.). The mixture was stirred at-78 ℃ for 30 minutes, then I was added 2 (492 mg,1.94mmol,1.0 eq.). The mixture was stirred at-78 ℃ for 1 hour, then at 0 ℃ with Na 2 SO 3 The aqueous solution (10 mL) was quenched. The mixture was extracted with DCM (10 mL x 3) and the combined organic phases were taken up in water(20 mL) washing over anhydrous Na 2 SO 4 Dried, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 0-10% EtOAc/PE) to give 2-iodo-6, 7,8, 9-tetrahydro-4H-thieno [2,3-c ]]Chromen-4-one (300 mg, 46%). MS observations (ESI) + )：333.1[(M+H) + ]。
Step C:2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
to 2-iodo-6, 7,8, 9-tetrahydro-4H-thieno [2,3-c ] at room temperature]Chromen-4-one (300 mg, 903. Mu. Mol,1.0 eq), 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazole (175 mg, 900. Mu. Mol,1.0 eq), sodium carbonate (287.18 mg,2.71mmol,3.0 eq) and dicyclohexyl- [2- (2, 4, 6-triisopropylphenyl) phenyl]To a solution of phosphine (129 mg, 270.96. Mu. Mol,0.3 eq.) in water (2.0 mL) and 1, 4-dioxane (10.0 mL) was added [1,1' -bis (diphenylphosphino) ferrocene]Palladium (II) dichloride (132 mg, 181. Mu. Mol,0.20 eq). The mixture was stirred in a microwave reactor at 110 ℃ for 2 hours, then concentrated, and the residue was purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2, 3-c)]Chromen-4-one (230 mg, 93%). MS observations (ESI) + )：273.2[(M+H) + ]。
Step D:2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 1):
to 2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]To a solution of chromen-4-one (230 mg, 844.59. Mu. Mol,1.0 eq.) in methanol (2.0 mL) was added aqueous ammonia (6.0 mL,28% w/w). The mixture was stirred in a sealed tube at 120 ℃ for 16 hours. After cooling to room temperature, the mixture was concentrated to dryness. Will beThe residue was dissolved in DMF and purified by preparative HPLC to give 2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]]Quinolin-4 (5H) -one (14 mg, 6.0%). MS observations (ESI) + )：272.1[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δppm:13.19(s,1H),11.15(s,1H),8.28(brs,1H),7.96(brs,1H),7.42(s,1H),2.67–2.62(m,2H),2.53–2.50(m,2H),1.75(s,4H)。
Example 2-compound 2:1,8,8-trifluoro-2- (1H-pyrazol-4-yl) -5,6,7, 9-tetrahydrothieno [2,3-c ] quinolin-4-one
Step A:3, 5-dibromo-4-fluoro-thiophene-2-carboxylic acid methyl ester:
FeBr was added to a solution of methyl 4-fluorothiophene-2-carboxylate (1.00 g,6.24mmol,1.0 eq.) in carbon tetrachloride (5.0 mL) at 25 ℃ 3 (1.29 g,4.37mmol,1.0 eq.) and bromine (39.9 g,250mmol,20.0mL,40.0 eq.) and the mixture stirred at 25℃for 16 hours. The mixture was concentrated and purified by flash column chromatography (SiO 2 0-15% EtOAc/PE) to give methyl 3, 5-dibromo-4-fluoro-thiophene-2-carboxylate (480 mg, 24.0%). 1 H NMR(400MHz,CDCl 3 )δppm:3.91(s,3H)。
And (B) step (B): 3-bromo-4-fluoro-5- [1- (2-trimethylsilyl ethoxymethyl) pyrazol-4-yl ] thiophene-2-carboxylic acid methyl ester:
a solution of palladium (II) acetate (8 mg,0.04mmol,0.05 eq.) and (5-diphenylphosphino-9, 9-dimethyl-xanthen-4-yl) -diphenyl-phosphine (22 mg,0.04mmol,0.05 eq.) in THF (10.0 mL) at 25℃under N 2 Stirred for 5 minutes. Into the mixtureAddition of 3, 5-dibromo-4-fluoro-thiophene-2-carboxylic acid methyl ester (240 mg,0.75mmol,1.0 eq.) trimethyl- [2- [ [4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazol-1-yl]Methoxy group]And]silane (367 mg,1.13mmol,1.5 eq.) and tripotassium orthophosphate (481 mg,2.26mmol,3.0 eq.) and the mixture was reacted at 60℃under N 2 Stirred for 16 hours. The mixture was then concentrated in vacuo and purified by flash column chromatography (SiO 2 0-30% EtOAc/PE) to give 3-bromo-4-fluoro-5- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]Thiophene-2-carboxylic acid methyl ester (168 mg, 51%). MS observations (esi+): 79 Br/ 81 Br435.3/437.3[(M+H) + ]。
step C:1,8,8-trifluoro-2- [1- (2-trimethylsilyl ethoxymethyl) pyrazol-4-yl ] -7, 9-dihydro-6H-thieno [2,3-c ] chromen-4-one
To 3-bromo-4-fluoro-5- [1- (2-trimethylsilyl ethoxymethyl) pyrazol-4-yl]To a solution of thiophene-2-carboxylic acid methyl ester (460 mg,1.07mmol,1.0 eq.) in toluene (100.0 mL) was added tris (dibenzylideneacetone) dipalladium (98 mg,0.11mmol,0.10 eq.), 4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene (93 mg,0.16mmol,0.15 eq.), cesium carbonate (1.0 g,3.20mmol,3.0 eq.), sodium bisulphite (20 mg,0.11mmol,0.1 eq.) and 4, 4-difluorocyclohexanone (429 mg,3.20mmol,3.0 eq.). The mixture was treated with N 2 Purged, then heated to 105 ℃ and stirred for 16 hours. The mixture was concentrated to dryness and the residue was purified by flash chromatography (SiO 2 0-30% EtOAc/PE) to afford the product 1,8,8-trifluoro-2- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]-7, 9-dihydro-6H-thieno [2,3-c]Chromen-4-one (217 mg, 44%). MS observations (esi+): 457.3[ (M+H) + ]。
Step D:1,8,8-trifluoro-2- [1- (2-trimethylsilyl ethoxymethyl) pyrazol-4-yl ] -5,6,7, 9-tetrahydrothieno [2,3-c ] quinolin-4-one:
1,8,8-trifluoro-2- [1- (2-trimethylsilyl ethoxymethyl) pyrazol-4-yl ] at 25 ℃C]-7, 9-dihydro-6H-thieno [2,3-c]To a solution of chromen-4-one (110 mg,0.24mmol,1.0 eq.) in MeOH (5.0 mL) was added ammonium hydroxide (4.5 g,128mmol,5.0mL,320 eq.) and the mixture was stirred at 95℃for 8 hours under microwave radiation. The solution was cooled and concentrated to give 1,8,8-trifluoro-2- [1- (2-trimethylsilyl ethoxymethyl) pyrazol-4-yl ]-5,6,7, 9-tetrahydrothieno [2,3-c]Quinolin-4-one (109 mg, crude) was used in the next step without further purification. MS observations (ESI) + )：456.3[(M+H) + ]。
Step E:1,8,8-trifluoro-2- (1H-pyrazol-4-yl) -5,6,7, 9-tetrahydrothieno [2,3-c ] quinolin-4-one (Compound 2):
to 1,8,8-trifluoro-2- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]-5,6,7, 9-tetrahydrothieno [2,3-c]To a solution of quinolin-4-one (109 mg,0.24mmol,1.0 eq.) in DCM (1.0 mL) was added 2, 2-trifluoroacetic acid (5.9 g,52mmol,4.0mL,220 eq.) and the mixture was stirred at 25℃for 1 h. The mixture was concentrated to dryness and the residue was purified by flash column chromatography (SiO 2 0-8% MeOH/DCM) to give an impure product. The residue was purified by reverse phase column chromatography (C18 SiO 2 Further purification of 0-40% MeCN,0.1% FA/water to give 1,8,8-trifluoro-2- (1H-pyrazol-4-yl) -5,6,7, 9-tetrahydrothieno [2, 3-c)]Quinolin-4-one (22 mg, 28%). MS observations (ESI) + )：326.2[(M+H) + ]。
Examples 3 and 4-compounds 3 and 4: (S) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 3) and (R) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 4) (the stereochemistry of compounds 3 and 4 is arbitrarily specified)
Step A: 2-benzyloxy-cyclohexanol:
BnOH (910 mg,20mmol,2.0 eq.) was added dropwise to a stirred suspension of NaH (820 mg,21.4mmol,60% in mineral oil, 2.0 eq.) in anhydrous DMF (25 mL) at 0deg.C. The mixture was stirred at 0deg.C for 1 hour, then 7-oxabicyclo [4.1.0 ] was added]Heptane (1.0 g,10mmol,1.0 eq). The mixture was heated to 60 ℃ for 2 hours and then cooled. Water was added and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 Dried, filtered and concentrated under vacuum. The residue was purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 2-benzyloxycyclohexanol (1.0 g, 48%). MS observations (ESI) + )：207.3[(M+H) + ]。
And (B) step (B): 2-benzyloxycyclohexanone:
oxalyl chloride (268 mg,5.82mmol,1.2 eq.) was dissolved in anhydrous DCM (5.0 mL) under nitrogen and cooled to-78 ℃. A solution of DMSO (909 mg,11.6mmol,2.0 eq.) in anhydrous DCM (5.0 mL) was added dropwise and the resulting mixture stirred for 20 min. Anhydrous DCM (5.0 mL) containing 2-benzyloxycyclohexanol (1.0 g,4.85mmol,1.0 eq.) was added dropwise to form a white precipitate. The mixture was stirred for 30 min, warmed to-60℃and anhydrous triethylamine (2.5 g,24mmol,3.4mL,1.0 eq.) was added dropwise. The mixture was kept at-60 ℃ for 5 minutes and then warmed to room temperature for 2 hours. Will react with H 2 O was quenched and extracted with EtOAc.The organic layers were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 2-benzyloxycyclohexanone (770 mg, 78%). MS observations (ESI) + )：205.2[(M+H) + ]。
Step C:6- (benzyloxy) -6,7,8, 9-tetrahydrothieno [2,3-c ] chromen-4-one:
to a solution of methyl 3-bromothiophene-2-carboxylate (1.0 g,4.5mmol,1.0 eq.) in toluene (15 mL) was added 2-benzyloxycyclohexanone (1.4 g,6.8mmol,1.5 eq.), tris (dibenzylideneacetone) dipalladium (414 mg,0.45mmol,0.1 eq.), 4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene (785 mg,1.36mmol,0.30 eq.), cesium carbonate (3.0 g,9.1mmol,2.0 eq.) and sodium hydrogen sulfite (170 mg,0.90mmol,0.20 eq.). The reaction was purged with nitrogen and stirred at 105 ℃ for 3 hours. The mixture was concentrated in vacuo and the residue was purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 6- (benzyloxy) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Chromen-4-one (450 mg, 32%). MS observations (ESI) + )：313.3[(M+H) + ]。
Step D:6- (benzyloxy) -2-iodo-6, 7,8, 9-tetrahydrothieno [2,3-c ] chromen-4-one
To 6-benzyloxy-6, 7,8, 9-tetrahydrothieno [2,3-c ] at-65 DEG C]Chromen-4-one (350 mg,1.12mmol,1.0 eq.) and I 2 To a solution of (569 mg,2.24mmol,2.0 eq.) in THF (5.0 mL) was added LDA (2M in THF, 2.2mL,4.0 eq.). The mixture was stirred at-65℃for 1 hour, then with H 2 O was quenched and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo, and the residue was taken up in vacuoThe residue was purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 6- (benzyloxy) -2-iodo-6, 7,8, 9-tetrahydrothieno [2,3-c ]]Chromen-4-one (300 mg, 61%). MS observations (ESI) + )：439.3[(M+H) + ]。
Step E:6- (benzyloxy) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
trimethyl- [2- [ [4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazol-1-yl]Methoxy group]And]silane (444 mg,1.37mmol,1.2 eq.) 6- (benzyloxy) -2-iodo-6, 7,8, 9-tetrahydrothieno [2,3-c ]]Chromen-4-one (500 mg,1.10mmol,1.0 eq.) Pd (dppf) Cl 2 (80 mg,0.11mmol,0.1 eq.), X-Phos (104 mg,0.22mmol,0.2 eq.) and Cs 2 CO 3 (1.08 g,3.30mmol,3.0 eq.) in 1, 4-dioxane (15.0 mL) and H 2 The suspension in the mixture of O (5.0 mL) was irradiated in a microwave reactor at 110℃for 2 hours. The mixture was cooled and extracted with EtOAc. The organic layer was concentrated to dryness and the residue was purified by flash column chromatography (SiO 2 0-70% EtOAc/PE) to give 6- (benzyloxy) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (450 mg, 80%). MS observations (ESI) + )：509.7[(M+H) + ]。
Step F:6- (benzyloxy) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
6- (benzyloxy) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (500 mg,1.32mmol,1.0 eq.) in MeOHA suspension in a mixture of (7.0 mL) and 25% ammonia (7.0 mL) was irradiated in a microwave reactor at 100deg.C for 3 hours. The resulting solid was filtered, washed with MeOH and dried to give 6- (benzyloxy) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (250 mg, 50%). MS observations (ESI) + )：508.7[(M+H) + ]
Step G: (S) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 3) and (R) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 4):
6-benzyloxy-2- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]-6,7,8, 9-tetrahydro-5H-thieno [2,3-c]A solution of quinolin-4-one (250 mg,0.49mmol,1.0 eq.) in trifluoroacetic acid (10.0 mL) was heated at 70℃for 1 h. Removing the solvent and adding NH to the residue 3 (7M in MeOH, 5.0 mL). The mixture was stirred at room temperature for 5 min, concentrated, and the residue was purified by preparative HPLC to give 6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (60 mg, 40%). MS observations (ESI) + )：288.4[(M+H) + ]。
The racemic product was separated by chiral SFC to give each enantiomer. (S) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 3): MS observations (ESI) + )：288.2[(M+H) + ]. (R) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 4): MS observations (ESI) + )：288.2[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δppm:13.20(s,1H),10.82(s,1H),8.30(s,1H),7.96(s,1H),7.47(s,1H),5.22(d,J＝5.6Hz,1H),4.49–4.46(m,1H),2.70–2.65(m,1H),2.51–2.49(m,1H),1.89–1.82(m,2H),1.78–1.69(m,2H)。
Examples 5 and 6-compounds 5 and 6: (R) -6- (3, 3-difluoropyrrolidin-1-yl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 5) and (S) -6- (3, 3-difluoropyrrolidin-1-yl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 6). (the stereochemistry of Compounds 5 and 6 is arbitrarily specified)
Step A: 6-bromo-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
by reacting 6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-5H-thieno [2,3-c]A suspension of quinolin-4-one (120 mg,0.41mmol,1.0 eq.) in TMSBr (8.0 mL) was stirred in a sealed tube at 80℃for 6 hours. The solvent was removed in vacuo and the residue was used directly in the next step without further purification. MS observations (ESI) + )：302.4[(M-Br+OCH 3 )+H) + ]。
And (B) step (B): (R) -6- (3, 3-difluoropyrrolidin-1-yl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 5) and (S) -6- (3, 3-difluoropyrrolidin-1-yl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 6):
a suspension of 3, 3-difluoropyrrolidine hydrochloride (1.9 g,14mmol,20 eq.) and N, N-diisopropylethylamine (1.8 g,13.88mmol,20.0 eq.) in MeCN (10.0 mL) was stirred at room temperature for 2 hours. The solid was filtered off and the filtrate was added to a solution containing 6-bromo-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-5H-thieno [2,3-c]In a sealed vial of quinolin-4-one (200 mg,0.69mmol,1.0 eq). NaI (103 mg,0.69mmol,1.0 eq.) was added to the vial). The mixture was stirred at room temperature for 16 hours, then concentrated in vacuo, and the residue was purified by flash column chromatography (SiO 2 0-100% EtOAc/PE) to give racemic 6- (3, 3-difluoropyrrolidin-1-yl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Quinolin-4 (5H) -one (60 mg, 19%). MS observations (ESI) + )：377.5[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (R) -6- (3, 3-difluoropyrrolidin-1-yl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 5): MS observations (ESI) + )：377.3[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm:13.20 (s, 1H), 10.53 (s, 1H), 8.30 (s, 1H), 7.96 (s, 1H), 7.47 (s, 1H), 3.73 (t, J=5.6 Hz, 1H), 3.06-2.93 (m, 2H), 2.87-2.78 (m, 2H), 2.68-2.62 (m, 2H), 2.30-2.22 (m, 2H), 1.98-1.95 (m, 1H), 1.84-1.77 (m, 2H), 1.74-1.71 (m, 1H). (S) -6- (3, 3-difluoropyrrolidin-1-yl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 6): MS observations (ESI) + )：377.3[(M+H) + ]。
Examples 7 and 8-compounds 7 and 8: 6-methoxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-, 9-ethanoylthiophene [2,3-c ] quinolin-4 (5H) -one (compound 7) and 6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c ] quinolin-4 (5H) -one (compound 8).
Step A: 6-methoxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c ] chromen-4-one:
Cesium carbonate (230 mg, 720. Mu. Mol,3.0 eq.) and Sphos-Pd-G 3 (37 mg, 48. Mu. Mol,0.2 eq.) to 3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-o-fPyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (100 mg, 240. Mu. Mol,1.0 eq.) and 1-methoxybicyclo [2.2.2]Octyl-2-ketone (74 mg, 480. Mu. Mol,2.0 eq, synthesized according to the procedure described in WO 2007070201) in toluene (6.0 mL). The mixture was heated at 105 ℃ under nitrogen for 8 hours and then concentrated in vacuo. The residue obtained was purified by flash column chromatography (SiO 2 0-25% EtOAc/PE) to give 6-methoxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c]Chromen-4-one (32 mg,24%,82% purity). MS observations (ESI) + )：459.4[(M+H) + ]。
And (B) step (B): 6-methoxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c ] quinolin-4 (5H) -one:
to 6-methoxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c]To a solution of chromen-4-one (20 mg, 44. Mu. Mol,1.0 eq.) in isopropanol (3.0 mL) was added aqueous ammonia (17% w/w,12.0 mL). The mixture was stirred at 60 ℃ for 16 hours, then concentrated in vacuo, and purified by flash column chromatography (SiO 2 0-5% MeOH/DCM) to give 6-methoxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c]Quinolin-4 (5H) -one (10 mg,24%,48% purity). MS observations (ESI) + )：458.4[(M+H) + ]。
Step C: 6-methoxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanothieno [2,3-c ] quinolin-4 (5H) -one (compound 7):
6-methoxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c]A solution of quinolin-4 (5H) -one (28 mg, about 50% purity, 29. Mu. Mol,1.0 eq.) in DCM (4.0 mL) was cooled to 0deg.C and trifluoroacetic acid (1.0 mL) was then added. The mixture was stirred at room temperature for 2 hours, then concentrated in vacuo and purified by preparative HPLC to give 6-methoxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanothieno [2,3-c]Quinolin-4 (5H) -one (Compound 7,5mg, 51%). MS observations (ESI) + )：328.1[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δppm:13.16(s,1H),10.52(s,1H),8.10(s,2H),7.59(s,1H),3.39(s,3H),3.40–3.92(m,1H),2.12(t,J＝10.4Hz,2H),1.88(t,J＝10.4Hz,2H),1.49–1.44(m,2H),1.36–1.31(m,2H)。
Step D: 6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanothieno [2,3-c ] quinolin-4 (5H) -one (compound 8):
6-methoxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c]A solution of quinolin-4 (5H) -one (55 mg, 120. Mu. Mol,1.0 eq) in HBr (40% aqueous solution, 5.0 mL) was heated to 100deg.C for 8 hours. The mixture was concentrated in vacuo and purified by reverse phase column chromatography (C18 SiO 2 0-40% MeCN/water, 0.1% NH 4 HCO 3 Water) purification to give 6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c]Quinolin-4 (5H) -one (example 8, 16.2mg, 31%). MS observations (ESI) + )：314.2[(M+H) + ]； 1 HNMR(400MHz,DMSO-d6)δppm:13.17(s,1H),10.22(s,1H),8.14(brs,2H),7.58(s,1H),5.72(s,1H),3.30(s,1H),1.84–1.80(m,4H),1.43(d,J＝6.4Hz,4H)。
Example 9-compound 9: 6-hydroxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c ] quinolin-4 (5H) -one
Step A: 3-bromo-5- (pyridin-4-yl) thiophene-2-carboxylic acid methyl ester:
cesium carbonate (210 mg, 680. Mu. Mol,2.0 eq.) Pd (dppf) Cl 2 (240 mg, 330. Mu. Mol,1.0 eq.) and dicyclohexyl- [2- (2, 4, 6-triisopropylphenyl) phenyl]Phosphine (160 mg, 330. Mu. Mol,1.0 eq) was added to a solution of methyl 3, 5-dibromothiophene-2-carboxylate (100 mg, 330. Mu. Mol,1.0 eq) and 4-pyridylboronic acid (45 mg, 370. Mu. Mol,1.1 eq) in 1, 4-dioxane (10.0 mL). The mixture was treated with N 2 Deaeration was carried out twice and then heated to 80℃for 1 hour. The mixture was concentrated in vacuo, then purified by column chromatography (SiO 2 Purification from 0-25% EtOAc/PE) afforded methyl 3-bromo-5- (pyridin-4-yl) thiophene-2-carboxylate (65 mg, 56%). MS observations (ESI) + )：298.0[(M+H) + ]。
And (B) step (B): 6-methoxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c ] chromen-4-one:
cesium carbonate (330 mg,1.0mmol,3.0 eq.) and spos-Pd-G3 (52 mg, 67. Mu. Mol,0.2 eq.) are added to methyl 3-bromo-5- (4-pyridinyl) thiophene-2-carboxylate (100 mg, 340. Mu. Mol,1.0 eq.) and 1-methoxybicyclo [ 2.2.2.2: ]Octyl-2-one (100 mg, 670. Mu. Mol,2.0 eq) in toluene (4.0 mL). The mixture was stirred under nitrogen at 100 ℃ for 16 hours, concentrated in vacuo and purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give 6-methoxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c]Chromen-4-one (21 mg,16%,82% purity). MS observations (ESI) + )：340.4[(M+H) + ]。
Step C: 6-methoxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c ] chromen-4-one:
to 6-methoxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c]To a solution of chromen-4-one (15 mg, 44.19. Mu. Mol,1.0 eq.) in isopropanol (4.0 mL) was added an aqueous ammonia solution (25% w/w,16.0 mL). The mixture was stirred at 60 ℃ for 16 hours and then concentrated in vacuo to give 6-methoxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-4H-6, 9-ethanoylthiophene [2,3-c ]]Chromen-4-one (15 mg, crude product) was used without further purification. MS observations (ESI) + )：357.3[(M+H) + ]。
Step D: 6-methoxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c ] quinolin-4 (5H) -one:
crude 3- (4-methoxy-3-oxo-bicyclo [2.2.2]Oct-2-yl) -5- (pyridin-4-yl) thiophene-2-carboxamide (30 mg) was dissolved in toluene (4.0 mL) and p-toluenesulfonic acid (3 mg,17 μmol,0.20 eq). The mixture was stirred at 100deg.C for 1 hour, then NaHCO was added 3 (saturated aqueous solution 10 mL). The mixture was extracted with EtOAc (3×15 ml), and the combined organic layers were washed with Na 2 SO 4 Drying and filtering. Concentrated in vacuo to give 6-methoxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanothieno [2,3-c]Quinolin-4 (5H) -one (28 mg, crude product) was used without further purification. MS observations (ESI) + )：339.2[(M+H) + ]。
Step E: 6-hydroxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c ] quinolin-4 (5H) -one (compound 9):
6-methoxy-2- (picolinic acid)Pyridin-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanoylthiophene [2,3-c]A solution of quinolin-4 (5H) -one (30 mg, 89. Mu. Mol) in HBr (48% aqueous solution, 4.0 mL) was heated to 100deg.C for 16 hours, concentrated in vacuo, and purified by preparative HPLC to give 6-hydroxy-2- (pyridin-4-yl) -6,7,8, 9-tetrahydro-6, 9-ethanothieno [2, 3-c)]Quinolin-4 (5H) -one (Compound 9, 11mg, 38%). MS observations (ESI) + )：325.1[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δppm:10.59(s,1H),8.68–8.67(m,2H),8.23(s,1H),7.82–7.81(m,2H),5.76(s,1H),3.43–3.41(m,1H),1.85–1.78(m,4H),1.46–1.43(m,4H)。
Examples 10 and 11-compounds 10 and 11: (R) -4-methoxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 10) and (S) -4-methoxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 11) (stereochemistry is arbitrarily specified)
Step A: 4-methoxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one
To a solution of 3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (300 mg,0.72mmol,1.0 eq, prepared in a similar manner to step A of compound 9) and 3-methoxytetrahydropyran-4-one (190 mg,1.4mmol,2.0 eq, synthesis described in WO 2005014537) in toluene (7.0 mL) was added sodium bisulphite (14 mg, 72. Mu. Mol,0.1 eq), cs 2 CO 3 (700 mg,2.2mmol,3.0 eq.) Pd 2 (dba) 3 (130 mg,0.14mmol,0.2 eq.) and Xantphos (170 mg,0.29mmol,0.4 eq.). The mixture was stirred at 100deg.C for 16 hours, concentrated, and passed throughColumn chromatography (SiO) 2 0-25% etoac/PE) to give 4-methoxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (160 mg, 51%). MS observations (ESI) + )：435.4[(M+H) + ]。
And (B) step (B): 4-methoxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
4-methoxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]A solution of pyran-6-one (160 mg,0.37mmol,1.0 eq.) in 1:4 trifluoroacetic acid/DCM (10 mL) was stirred at room temperature for 2 hours. The mixture was concentrated to give 4-methoxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (112 mg, crude product), which was used without further purification. MS observations (ESI) + )：305.2[(M+H) + ]。
Step C: (R) -4-methoxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 10) and (S) -4-methoxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 11):
4-methoxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (110 mg,1.0 eq.) in NH 4 The solution in OH/meoh=1/1 (10 mL) was stirred in a microwave reactor at 100 ℃ for 6 hours. The mixture was concentrated in vacuo and purified by prep HPLC to give racemic 4-methoxy 1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] yl-8- (1H-pyrazol-4-yl)]Thieno [3,2-d]Pyridin-6-one (75 mg, 77%). MS observations (ESI) + )：304.2[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (R) -4-methoxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 10): MS observations (ESI) + )：304.2[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm:13.23 (s, 1H), 11.53 (s, 1H), 8.30 (brs, 2H), 7.44 (s, 1H), 4.88-4.69 (m, 1H), 4.64-4.46 (m, 1H), 4.18 (d, J=12.0 Hz, 1H), 4.01 (s, 1H), 3.70 (d, J=12.0 Hz, 1H), 3.39 (s, 3H). (S) -4-methoxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 11): MS observations (ESI) + )：304.2[(M+H) + ]。
Examples 12 and 13-compounds 12 and 13: (S) -6- (azetidin-1-yl) -8, 8-difluoro-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 12) and (R) -6- (azetidin-1-yl) -8, 8-difluoro-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 13) (stereochemistry is arbitrarily specified)
Step A:5, 5-difluoro-2, 2-dimethoxy-cyclohexanol:
4, 4-Difluorocyclohexanone (4.0 g,30mmol,1.0 eq.) and KOH (4.0 g,72mmol,3.0 eq.) were dissolved in MeOH (80.0 mL). The mixture was cooled to 0℃and I was added dropwise over 1 hour 2 Solution (8.3 g,33mmol,1.1 eq in 100.0mL MeOH). The reaction was stirred at room temperature for 18 hours, concentrated in vacuo, and the oil was suspended in 80mL DCM. The precipitate was filtered off and the solution was concentrated to give crude 5, 5-difluoro-2, 2-dimethoxy-cyclohexanol (4.2 g,crude product).
And (B) step (B): ((5, 5-difluoro-2, 2-dimethoxycyclohexyl) oxy) methyl) benzene
Sodium hydride (610 mg,15mmol,60% in mineral oil, 1.2 eq.) was added to a solution of BnBr (2.8 g,17mmol,1.3 eq.) in DMF (30.0 mL) at 0deg.C. After 30 minutes, 5-difluoro-2, 2-dimethoxy-cyclohexanol (2.5 g of crude product, 13mmol,1.0 eq.) was added and the mixture was stirred at room temperature for 16 hours. The mixture was poured into water and extracted with EtOAc (15 ml x 2). The organic layer was dried, filtered, concentrated, and purified by column chromatography (Si 2 O,0-12% EtOAc/PE) to give (((5, 5-difluoro-2, 2-dimethoxycyclohexyl) oxy) methyl) benzene (3.2 g, 64%). 1 H NMR(400MHz,CDCl 3 )δppm:7.37–7.25(m,5H),4.72(d,J＝12.0Hz,1H),4.56(d,J＝12.0Hz,1H),3.70(t,J＝8.0Hz,1H),3.24(s,3H),3.22(s,3H),2.38–2.35(m,1H),2.11–1.74(m,5H)。
Step C:2- (benzyloxy) -4, 4-difluorocyclohexane-1-one:
to a solution of (((5, 5-difluoro-2, 2-dimethoxycyclohexyl) oxy) methyl) benzene (2.2 g,7.7mmol,1.0 eq.) in acetone (60.0 mL) was added I 2 (195 mg, 768. Mu. Mol,0.1 eq). After 30 minutes, na was added 2 S 2 O 3 (saturated aqueous) and the aqueous phase was extracted with DCM (15 ml x 3). The combined organic phases were taken up in Na 2 S 2 O 3 (saturated aqueous solution), filtration and concentration. By column chromatography (SiO 2 Purification with 0-8% EtOAc/PE afforded 2- (benzyloxy) -4, 4-difluorocyclohexane-1-one (1.3 g, 70%). 1 H NMR(400MHz,CDCl 3 ):δppm:7.36–7.25(m,5H),4.85(d,J＝11.2Hz,1H),4.51(d,J＝11.2Hz,1H),4.15–4.11(m,1H),2.75–2.14(m,6H)。
Step D:6- (benzyloxy) -8, 8-difluoro-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
to 3-bromo-5- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]To a solution of thiophene-2-carboxylic acid methyl ester (400 mg, 960. Mu. Mol,1.0 eq.) and 2- (benzyloxy) -4, 4-difluorocyclohexane-1-one (460 mg,1.9mmol,2.0 eq.) in toluene (80.0 mL) was added Cs 2 CO 3 (940 mg,2.9mmol,3.0 eq.) Pd 2 (dba) 3 (180 mg, 190. Mu. Mol,0.2 eq.), xantphos (170 mg, 270. Mu. Mol,0.3 eq.) and Na 2 S 2 O 5 (54 mg, 290. Mu. Mol,0.3 eq). The mixture was stirred and heated at 105 ℃ for 16 hours. The mixture was cooled, filtered and concentrated, and the residue was purified by silica gel chromatography (SiO 2 Purification with 0-28% EtOAc/PE) afforded 6- (benzyloxy) -8, 8-difluoro-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ]Chromen-4-one (360 mg, 55%). MS observations (ESI) + )：545.3[(M+H) + ]。
Step E:6- (benzyloxy) -8, 8-difluoro-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
to 6- (benzyloxy) -8, 8-difluoro-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]To a solution of chromen-4-one (150 mg, 200. Mu. Mol,1.0 eq.) in MeOH (8.0 mL) was added aqueous ammonia (30% w/w,8.0 mL). The mixture was heated in a microwave reactor at 95 ℃ for 8 hours and then cooled. The mixture was extracted with DCM (10 ml x 3) and the combined organic phases were dried over sodium sulfate and filteredAnd concentrated. By column chromatography (SiO 2 Purification with 0-4% MeOH/DCM gave 6- (benzyloxy) -8, 8-difluoro-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Quinolin-4 (5H) -one (90 mg, 69%). MS observations (ESI) + )：544.4[(M+H) + ]。
Step F:8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
6- (benzyloxy) -8, 8-difluoro-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]A solution of quinolin-4 (5H) -one (200 mg, 367. Mu. Mol,1.0 eq.) in DCM (12.0 mL) was cooled to 0deg.C and BCl was added 3 (1M in n-hexane, 3.68mL,3.68mmol,10 eq.). After 1 hour at 0 ℃, meOH (3.0 mL) was added and the solution was concentrated to give a solid. Trituration (1:1 n-hexane/MeOH) afforded 8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]]Quinolin-4 (5H) -one (110 mg,92% yield) is pure enough to be used in the next step. MS observations (ESI) + )：324.1[(M+H) + ]。
Step G: (R) -6- (azetidin-1-yl) -8, 8-difluoro-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 12) and (S) -6- (azetidin-1-yl) -8, 8-difluoro-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 13):
to 8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] at room temperature]Slowly add SOCl to quinolin-4 (5H) -one (70 mg, 190. Mu. Mol,1.0 eq) 2 (2.5 g,21mmol,1.5mL,11 eq.). After 4 hours, the mixture was concentrated. Containing 6-chloro-8, 8-difluoro-2- (1H-pyrazol-4-yl) -5,6,7, 9-tetrahydrothieno [2,3-c]A concentrate of quinolin-4-one was dissolved in MeCN (3.0 mL) and KI (241 mg,1.45mmol,10.0 eq.) and azetidine (170 mg,2.9mmol,20 eq.) were added. The mixture was stirred at room temperature for 12 hours, then concentrated and purified by reverse phase chromatography to give 6- (azetidin-1-yl) -8, 8-difluoro-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2, 3-c) ]Quinolin-4 (5H) -one (34 mg,55%,85% purity). MS observations (ESI) + )：363.3[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (R) -6- (azetidin-1-yl) -8, 8-difluoro-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 12): MS observations (ESI) + )：363.4[(M+H) + ]. (S) -6- (azetidin-1-yl) -8, 8-difluoro-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 13): MS observations (ESI) + )：363.3[(M+H) + ]。
Examples 14 and 15-compounds 14 and 15: (S) -8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 14) and (R) -8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 15) (stereochemistry is arbitrarily specified)
Step A:5, 5-difluoro-2-hydroxycyclohex-1-ene-1-carboxylic acid methyl ester:
a solution of 4, 4-difluorocyclohexanone (2.0 g,15mmol,1.0 eq.) in DMF (4.0 mL) was added to a 0℃suspension of NaH (430 mg,18mmol,60% in mineral oil, 1.2 eq.) in DMF (13.0 mL). After 30 minutes at 0deg.C, a solution of dimethyl carbonate (1.6 g,18mmol,1.5mL,1.2 eq.) in DMF (3.0 mL) was added and mixed The mixture was stirred at room temperature for 16 hours, then NH was added 4 Cl (saturated aqueous solution). The mixture was diluted with water (10 mL) and extracted with DCM (20 mL x 3). The combined organic layers were dried (Na 2 SO 4 ) Concentrated in vacuo, and purified by column chromatography (SiO 2 0-8% EtOAc/PE) to give methyl 5, 5-difluoro-2-hydroxycyclohex-1-ene-1-carboxylate (2.0 g, 70%). 1 H NMR(400MHz,CDCl 3 )δppm:12.17(s,1H),3.78(s,3H),2.74(t,J＝14.4Hz,2H),2.55(t,J＝6.8Hz,2H),2.17–2.07(m,2H)。
And (B) step (B): 5, 5-difluoro-1-methyl-2-oxocyclohexane-1-carboxylic acid methyl ester:
to a flask containing acetone (12.0 mL) containing methyl 5, 5-difluoro-2-hydroxycyclohex-1-ene-1-carboxylate (1.0 g,6.4mmol,1.0 eq.) was added K at room temperature 2 CO 3 (2.7 g,19mmol,3.0 eq.) and methyl iodide (2.7 g,19mmol,3.0 eq.). The mixture was heated to 50 ℃ and stirred for 3 hours, then water was added. The mixture was extracted with tert-butyl methyl ether (30 ml x 3) and the combined organic layers were washed with water, brine and dried (Na 2 SO 4 ). Filtered, concentrated in vacuo, and purified by column chromatography (SiO 2 0-15% EtOAc/PE) to give methyl 5, 5-difluoro-1-methyl-2-oxocyclohexane-1-carboxylate (800 mg, 66%). 1 H NMR(400MHz,CDCl 3 )δppm:3.76(s,3H),3.09–2.88(m,2H),2.61–2.54(m,1H),2.46–2.35(m,1H),2.26–1.98(m,2H),1.37(d,J＝0.8Hz,3H)。
Step C: methyl 8, 8-difluoro-6-methyl-4-oxo-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromene-6-carboxylate:
To 3-iodo-5- (1- ((2- (trimethylsilyl) ethoxy)) To a solution of methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate (200 mg, 430. Mu. Mol,1.0 eq.) and methyl 5, 5-difluoro-1-methyl-2-oxocyclohexane-1-carboxylate (180 mg, 860. Mu. Mol,2.0 eq.) in toluene (12.0 mL) was added Cs 2 CO 3 (420 mg,1.3mmol,3.0 eq.) Pd 2 (dba) 3 (79 mg, 86. Mu. Mol,0.2 eq.), xantphos (74 mg, 130. Mu. Mol,0.30 eq.) and Na 2 S 2 O 5 (16 mg, 86. Mu. Mol,0.2 eq). The mixture was heated to 105 ℃ in a microwave reactor for 2 hours, then cooled, concentrated and purified (SiO 2 0-32% EtOAc/PE). This material was further purified by reverse phase chromatography to give 8, 8-difluoro-6-methyl-4-oxo-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromene-6-carboxylic acid methyl ester (100 mg, 46%). MS observations (ESI) + )：511.4[(M+H) + ]。
Step D:8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
equivalent weight) to 8, 8-difluoro-6-methyl-4-oxo-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ]To a cooled (0 ℃) solution of chromene-6-carboxylic acid methyl ester (100 mg, 200. Mu. Mol,1.0 eq.) in EtOH (12.0 mL) CaCl was added 2 (45 mg, 410. Mu. Mol,2.0 eq). After 10 minutes NaBH was added 4 (116 mg,3.08mmol,15.0 eq.) and the ice bath was removed. The mixture was stirred at room temperature for 20 minutes, then cooled to 0 ℃ and NH was added 4 Cl (saturated aqueous solution). The mixture was extracted with DCM (10 ml x 3) and the combined organic phases were dried (Na 2 SO 4 ) Filtered and concentrated. By column chromatography (SiO 2 0-70% etoac/PE) to give 8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno[2,3-c]Chromen-4-one (55 mg, 48%). MS observations (ESI) + )：483.4[(M+H) + ]。
Step E:8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
to 8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]To a solution of chromen-4-one (98 mg, 200. Mu. Mol,1.0 eq.) in MeOH (5.0 mL) was added aqueous ammonia (5.0 mL,30% w/w). The mixture was heated to 95 ℃ in a microwave reactor for 8 hours. The mixture was concentrated and purified by column chromatography (SiO 2 0-7% MeOH/DCM) to give 8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (68 mg, 56%). MS observations (ESI) + )：482.4[(M+H) + ]。
Step F: (S) -8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 14) and (R) -8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 15):
to 8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (43 mg, 89. Mu. Mol) in DCM (4.0 mL) in cooled (0deg.C) solution was added trifluoroacetic acid (2.0 mL). The mixture was warmed to room temperature and stirred for 2 hours, then concentrated in vacuo and purified by reverse phase chromatography to give 8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothiopheneAnd [2,3-c ]]Quinolin-4 (5H) -one (22 mg, 69%). MS observations (ESI) + )：352.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6):δppm 13.22(s,1H),10.86(s,1H),8.28(s,1H),7.98(s,1H),7.53(s,1H),5.23(s,1H),3.84(d,J＝10.8Hz,1H),3.41(d,J＝10.4Hz,1H),3.29–3.13(m,2H),2.58–2.46(m,1H),2.03–1.94(m,1H),1.30(s,3H)。
The individual enantiomers were separated by chiral SFC to give each enantiomer. (S) -8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]Quinolin-4 (5H) -one (compound 14): MS observations (ESI) + )：352.1[(M+H) + ]. (R) -8, 8-difluoro-6- (hydroxymethyl) -6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 15): MS observations (ESI) + )：352.1[(M+H) + ]。
Examples 16 and 17-compounds 16 and 17: (S) -4-hydroxy-4-isopropyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 16) and (R) -4-hydroxy-4-isopropyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 17)
Step A:4- (benzyloxy) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to a solution of 3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (5.0 g,12mmol,1.0 eq) and 3- (benzyloxy) tetrahydro-4H-pyran-4-one (4.9 g,24mmol,2.0 eq) in 1, 4-dioxane (600 mL) was added Na 2 S 2 O 5 (228 mg,1.20mmol,0.1 eq.) Cs 2 CO 3 (12g，36mmol，3.0 eq.) Pd 2 (dba) 3 (2.19 g,2.40mmol,0.20 eq.) and Xantphos (2.8 g,4.8mmol,0.40 eq.). The mixture was treated with N 2 Deaeration was carried out twice, followed by stirring and heating at 100℃for 16 hours. The mixture was filtered and concentrated in vacuo and purified by flash column chromatography (SiO 2 0-60% EtOAc/PE) to give 4- (benzyloxy) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (3.5 g,38% yield, 81% purity). MS observations (ESI) + )：511.5[(M+H) + ]。
And (B) step (B): 4- (benzyloxy) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
4- (benzyloxy) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (1.00 g,1.6mmol,81% purity, 1.0 eq.) and NH 4 A solution of OH (6 mL) in MeOH (6.0 mL) was stirred at 100deg.C under microwave heating for 6 hours. The mixture was concentrated in vacuo to give a residue, which was purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give 4- (benzyloxy) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (560 mg, 68%). MS observations (ESI) + )：510.2[(M+H) + ]。
Step C: 4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
To 4- (benzyloxy) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]To a solution of pyridin-6-one (300 mg, 590. Mu. Mol,1.0 eq.) in DCM (2.0 mL) was added BCl 3 (1M in hexane, 5.9mL,10 eq.). The mixture was stirred at 0deg.C for 15 min and then concentrated in vacuo to give 4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (170 mg, crude product) was used in the next reaction without further purification. MS observations (ESI) + )：290.0[(M+H) + ]。
Step D:4- (4-hydroxy-6-oxo-3, 4,5, 6-tetrahydro-1H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-8-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester:
to 4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]To a suspension of pyridin-6-one (170 mg, crude product) in DMF (2.0 mL) was added trimethylamine (119 mg,1.18mmol,2.0 eq), DMAP (36 mg, 290. Mu. Mol,0.5 eq) and (Boc) 2 O (130 mg, 590. Mu. Mol,1.0 eq). The mixture was stirred at room temperature for 10 min, then poured into water and extracted by EtOAc/THF (10:1, 15ml x 2). The combined organic layers were dried, filtered, concentrated and purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give 4- (4-hydroxy-6-oxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-8-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (130 mg,56%, 2 steps). MS observations (ESI) + )：390.0[(M+H) + ]。
Step E:4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-8-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester:
Dess-Martin periodate (Dess-Martin Periodinane) (425 mg,1.00mmol,3.0 eq) to 4- (4-hydroxy-6-oxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridine-8-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (130 mg, 334. Mu. Mol,1.0 eq.) in DCM (3.0 mL) and DMF (3.0 mL). The mixture was stirred at room temperature for 2 hours, then saturated Na 2 S 2 O 3 (aqueous solution) and NaHCO 3 (aqueous solution) was added together with water (10 mL). The mixture was extracted with DCM/MeOH (10:1, 20ml x 3) and the combined organic layers were dried, filtered, concentrated, and purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give 4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-8-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (70 mg, 54%). MS observations (ESI) + )：388.0[(M+H) + ]。
Step F: (S) -4-hydroxy-4-isopropyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 16) and (R) -4-hydroxy-4-isopropyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 17):
to 4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]To a solution of tert-butyl pyridin-8-yl) -1H-pyrazole-1-carboxylate (1.96 g,5.06mmol,1.0 eq.) in anhydrous THF (6.0 mL) was added i-PrMgCl-LiCl (1.3M in THF, 80mL,20 eq.). The mixture was stirred at 0 ℃ for 30 min, then passed through saturated NH 4 The aqueous Cl solution was quenched and extracted by EtOAc/THF (10:1, 30 mL. Times.2). The combined organic layers were dried, filtered, concentrated, and purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 4-hydroxy-4-isopropyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (680 mg, 41%). MS observations (ESI) + )：332.0[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4-hydroxy-4-isopropyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 16): MS observations (ESI) + )：332.0[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6): delta ppm:13.22 (s, 1H), 10.81 (s, 1H), 8.29 (s, 1H), 7.96 (s, 1H), 7.41 (s, 1H), 5.25 (s, 1H), 4.67 (s, 2H), 3.96 (d, J=11.6 Hz, 1H), 3.53 (d, J=11.6 Hz, 1H), 2.41-2.34 (m, 1H), 1.00 (d, J=6.8 Hz, 3H), 0.76 (d, J=6.8 Hz, 3H). (R) -4-hydroxy-4-isopropyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 17): MS observations (ESI) + )：332.0[(M+H) + ]。
Examples 18 and 19-compounds 18 and 19: (S) -4-ethyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 18) and (R) -4-ethyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 19)
To 4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]To a solution of tert-butyl pyridin-8-yl) -1H-pyrazole-1-carboxylate (180 mg, 460. Mu. Mol) in anhydrous THF (2.0 mL) was added EtMgCl (2M in THF, 12.0mL,52 eq) and the mixture was stirred at 0deg.C for 1 hour. The mixture was passed through saturated NH 4 Cl (aq) was quenched and extracted with EtOAc: THF (10:1, 30 mL. Times.2). The combined organic layers were dried, filtered, concentrated, and purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 4-ethyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (60 mg, 41%). MS observations (ESI) + )：318.1[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4-ethyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 18): MS observations (ESI) + )：318.0[(M+H) + ]； 1 H NMR(400MHz, DMSO-d 6) delta ppm 13.22 (s, 1H), 10.88 (s, 1H), 8.29 (s, 1H), 7.95 (s, 1H), 7.40 (s, 1H), 5.28 (s, 1H), 4.86-4.49 (m, 2H), 3.85 (d, j=11.2 hz, 1H), 3.54 (d, j=11.2 hz, 1H), 2.00-1.79 (m, 2H), 0.85 (t, j=7.6 hz, 3H). (R) -4-ethyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 19): MS observations (ESI) + )：318.0[(M+H) + ]。
Examples 20 and 21-compounds 20 and 21: (S) -4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6 (5H) -one (compound 20) and (R) -4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6 (5H) -one (compound 21). (the stereochemistry of Compounds 20 and 21 is arbitrarily specified)
To 4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4,3-b ] at 0 DEG C]Thieno [3,2-d]To a solution of tert-butyl pyridin-8-yl) -1H-pyrazole-1-carboxylate (50 mg,0.13mmol,1.0 eq.) in anhydrous THF (4.0 mL) was added t-BuMgCl (1M in THF, 3.9mL,30 eq.). The mixture was stirred at 0℃for 0.5 h. The mixture was treated with saturated NH 4 Aqueous Cl (10 mL) was quenched and extracted with EtOAc (20 mL. Times.3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6 (5H) -one (10 mg, 23%). MS observations (ESI) + )：346.5[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6 (5H) -one (compound 20): MS observations (ESI) + )：346.1[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 )δppm:13.23(s,1H),10.02(s,1H),8.29 (s, 1H), 7.97 (s, 1H), 7.40 (s, 1H), 5.32 (s, 1H), 4.80 (m, 2H), 4.14 (d, j=11.6 hz, 1H), 3.46 (d, j=11.6 hz, 1H), 1.03 (s, 9H). (R) -4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H-pyrano [4,3-b ]Thieno [3,2-d]Pyridin-6 (5H) -one (compound 21): MS observations (ESI) + )：346.1[(M+H) + ]。
Examples 22 and 23-compounds 22 and 23: (S) -4- (tert-butyl) -4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (Compound 22) and (R) -4- (tert-butyl) -4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (Compound 23) (the stereochemistry of Compounds 22 and 23 is arbitrarily specified)
Step A:4- (benzyloxy) -8- (pyridin-4-yl) -3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
at room temperature under N 2 Cs was added to a solution of methyl 3-bromo-5- (4-pyridinyl) thiophene-2-carboxylate (1.0 g,3.4mmol,1.0 eq.) and 3-benzyloxytetrahydropyran-4-one (1.4 g,6.7mmol,2.0 eq.) in toluene (50.0 mL) under an atmosphere 2 CO 3 (2.2 g,6.71mmol,2.0 eq.) Pd 2 (dba) 3 (614 mg,0.67mmol,0.2 eq.), xantphos (552 mg,1.01mmol,0.3 eq.) and Na 2 S 2 O 5 (64 mg,0.34mmol,0.1 eq.). The mixture was stirred at 105 ℃ for 16 hours, cooled, filtered and concentrated in vacuo. The residue was purified by flash chromatography (SiO 2 0-10% MeOH/DCM) to give 4- (benzyloxy) -8- (pyridin-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b) ]Thieno [3,2-d]Pyran-6-one (457 mg, 32%). MS observations (ESI) + )：392.1[(M+H) + ]。
And (B) step (B): 4- (benzyloxy) -8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
to 4- (benzyloxy) -8- (pyridin-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]To a solution of pyran-6-one (1.0 g,2.55mmol,1.0 eq.) in MeOH (9.0 mL) NH was added 4 OH (9.0 mL). The mixture was heated in a microwave reactor at 100 ℃ for 3 hours and monitored by LCMS until completion. The mixture was filtered and the filter cake was washed with MeOH (50.0 mL) and dried under vacuum to give 4- (benzyloxy) -8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (512 mg, 50%). MS observations (ESI) + )：391.2[(M+H) + ]。
Step C: 4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
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to 4- (benzyloxy) -8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]To a solution of pyridin-6-one (475 mg,1.22mmol,1.0 eq.) in DCM (12.0 mL) was added BCl 3 (1M in DCM, 12.2mL,10.0 eq). The mixture was stirred at room temperature for 1 hour, then with NaHCO 3 The aqueous solution (24.0 mL) was quenched and diluted with EtOAc (25.0 mL). The mixture was filtered and the filter cake was washed with EtOAc (25.0 mL) and dried under vacuum to give 4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b) ]Thieno [3,2-d]Pyridin-6-one (360 mg, 92%). MS observations (ESI) + )：301.0[(M+H) + ]。
Step D:8- (pyridin-4-yl) -1, 5-dihydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridine-4, 6 (3H) -dione:
to 4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]To a solution of pyridin-6-one (10 mg,0.033mmol,1.0 eq.) in DCM (1.0 mL) and DMF (1.0 mL) was added dess-Martin periodate (140 mg,0.333mmol,10 eq.). The mixture was stirred at room temperature for 2 hours, then saturated NaHCO 3 The aqueous solution (2.0 mL) was quenched and extracted with EtOAc (10 mL. Times.2). The combined organic phases were dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (SiO 2 1-30% MeOH/DCM) to give 8- (pyridin-4-yl) -1, 5-dihydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridine-4, 6 (3H) -dione (6 mg, 60%). MS observations (ESI) + )：299.3[(M+H) + ]。
Step E: (S) -4- (tert-butyl) -4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 22) and (R) -4- (tert-butyl) -4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 23):
to 8- (pyridin-4-yl) -1, 5-dihydro-6H-pyrano [4,3-b ] at 0 DEG C ]Thieno [3,2-d]To a solution of pyridine-4, 6 (3H) -dione (25 mg,0.08mmol,1.0 eq.) in anhydrous THF (1.5 mL) was added t-BuMgBr (1M in THF, 1.7mL,20.0 eq.). The mixture was stirred at 0 ℃ for 2 hours. The mixture was treated with saturated NH 4 Aqueous Cl (2.0 mL) was quenched and extracted with EtOAc (20.0 mL. Times.3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 4- (tert-butyl) -4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (5 mg, 17%). MS observations (ESI) + )：357.1[(M+H) + ]。
Separation by chiral SFCIndividual enantiomers. (S) -4- (tert-butyl) -4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 22): MS observations (ESI) + )：357.2[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 10.35 (s, 1H), 8.70 (d, J=6.0 Hz, 2H), 8.03 (s, 1H), 7.82 (d, J=6.0 Hz, 2H), 5.35 (s, 1H), 4.87 (m, 2H), 4.15 (d, J=11.2 Hz, 1H), 3.47 (d, J=11.2 Hz, 1H), 1.03 (s, 9H). (R) -4- (tert-butyl) -4-hydroxy-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 23): MS observations (ESI) + )：357.2[(M+H) + ]。
Examples 24 and 25-compounds 24 and 25: (S) -4-hydroxy-4-isopropyl-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 24) and (R) -4-hydroxy-4-isopropyl-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 25) (the stereochemistry of compounds 24 and 25 is arbitrarily specified)
Synthesized by a similar pathway to compounds 16 and 17. (S) -4-hydroxy-4-isopropyl-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 24). MS observations (ESI) + )：346.2[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 12.94 (brs, 1H), 10.81 (brs, 1H), 7.96 (brs, 1H), 7.29 (s, 1H), 5.27 (s, 1H), 4.69 (s, 2H), 3.96 (d, J=11.8 Hz, 1H), 3.53 (d, J=11.6 Hz, 1H), 2.48 (s, 3H), 2.40-2.34 (m, 1H), 1.00 (d, J=6.8 Hz, 3H), 0.76 (d, J=6.8 Hz, 3H). (R) -4-hydroxy-4-isopropyl-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 25). MS observations (ESI) + )：346.2[(M+H) + ]。
Examples 26 and 27-compounds 26 and 27: (R) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 26) and (S) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 27) (the stereochemistry of compounds 26 and 27 is arbitrarily specified)
Step A: 4-bromo-3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole:
to a solution of 4-bromo-3-fluoro-1H-pyrazole (1.5 g,9.1mmol,1.0 eq.) in DMF (15 mL) was added sodium hydride (60% in mineral oil, 700mg,18mmol,2.0 eq.) at 0deg.C. The mixture was stirred at 0deg.C for 10 min, then 2- (chloromethoxy) ethyl-trimethyl-silane (2.27 g,13.6mmol,2.41mL,1.5 eq.) was added. The mixture was stirred at room temperature for 2 hours, then by addition of saturated NH 4 Cl (aqueous solution) followed by quenching with the addition of water (200 mL). The mixture was extracted with EtOAc (50 ml x 3) and the combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give a residue which was purified by flash column chromatography (SiO 2 0-4% EtOAc/PE) to give 4-bromo-3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole (2.6 g, 96%); 1 H NMR(400MHz,CDCl 3 )δppm:7.45(d,J＝2.0Hz,1H),5.24(d,J＝0.8Hz,2H),3.58(m,2H),0.92(m,2H),0.00(s,9H)。
and (B) step (B): 3-fluoro-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole:
4-bromo-3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole (850 mg,288mmol,1.0 eq), 4, 5-tetramethyl-2- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1,3, 2-dioxaborolan (804 mg,3.17mmol,1.1 eq.), pd (dppf) Cl 2 A mixture of (426 mg, 576. Mu. Mol,0.2 eq.) and potassium acetate (848 mg,8.64mmol,3.0 eq.) in 1, 4-dioxane (17.0 mL) was heated at 100deg.C under nitrogen for 16 hours. The mixture was cooled and concentrated in vacuo, then water (50 mL) was added and the mixture extracted with EtOAc (40 mL x 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give a residue which was purified by flash column chromatography (SiO 2 0-8% EtOAc/PE) to give 3-fluoro-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole (650 mg, 66%). 1 H NMR(400MHz,CDCl 3 )δppm:7.66(d,J＝2.4Hz,1H),5.26(s,2H),3.59(m,2H),1.34(s,12H),0.92(m,2H),0.00(s,9H)。
Step C: 3-bromo-5- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester:
methyl 3, 5-dibromothiophene-2-carboxylate (1.37 g,4.56mmol,1.2 eq), 3-fluoro-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole (1.3 g,3.80mmol,1.0 eq), xantphos (440 mg, 760. Mu. Mol,0.20 eq), K 3 PO 4 (2.4 g,11mmol,3.0 eq.) and Pd (OAc) 2 (85 mg, 380. Mu. Mol,0.10 eq.) in THF (60.0 mL) with N 2 Deaeration and stirring of the mixture at 60 ℃ for 16 hours. The mixture was filtered and concentrated in vacuo, and the residue was purified by flash column chromatography (SiO 2 0-7% EtOAc/PE) to give 3-bromo-5- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate (810 mg, 49%). MS observations (ESI) + )： 79 Br/ 81 Br 435.2,437.2[(M+H) + ]。
Step D:4- (benzyloxy) -8- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -4-isopropyl-3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to a solution of 3-bromo-5- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate (400 mg, 918.73. Mu. Mol,1.0 eq), 3- (benzyloxy) -3-isopropyltetrahydro-4H-pyran-4-one (460 mg,1.86mmol,2.0 eq, prepared in a similar manner to compound 28, steps B-C) and cesium carbonate (909 mg,2.79mmol,3.0 eq) in toluene (20.0 mL) was added Pd 2 (dba) 3 (170 mg, 186. Mu. Mol,0.20 eq.) and Xantphos (215 mg, 372. Mu. Mol,0.40 eq.). The mixture was treated with N 2 Deaeration was carried out twice and stirred at 105℃for 16 hours. The mixture was filtered and concentrated in vacuo to give a residue, which was purified by flash column chromatography (SiO 2 0-16% EtOAc/PE) to give 4- (benzyloxy) -8- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -4-isopropyl-3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (275 mg, 51%). MS observations (ESI) + )：571.3[(M+H) + ]。
Step E:8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to 4- (benzyloxy) -8- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -4-isopropyl-3, 4-dihydro-1H, 6H-pyrano [4,3-b ] at 0deg.C]Thieno [3,2-d]To a solution of pyran-6-one (275 mg, 482. Mu. Mol,1.0 eq) in DCM (12.0 mL) was added BCl 3 (1M, 1.93mL,4.0 eq.) and stirring the mixture at 0deg.C for 4And 0 minutes. The mixture was concentrated by nitrogen flow and NH was gradually added at 0 c 4 OH (aqueous solution) adjusts the pH to about 9. The mixture was concentrated in vacuo to give 8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (400 mg, crude product), which was used without further purification. MS observations (ESI) + )：351.1[(M+H) + ]。
Step F: (R) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 26) and (S) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 27):
To 8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]NH was added to a solution of pyran-6-one (400 mg, crude product) in isopropanol (6.5 mL) 4 Aqueous OH (6.5 mL). The mixture was stirred at 95 ℃ under microwave heating for 4 hours, then concentrated, and purified by flash column chromatography (SiO 2 0-6% MeOH/DCM) to give 8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-1, 3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (75 mg,31%, 2 steps). MS observations (ESI) + )：350.1[(M+H) + ]。
The racemic mixture was separated by chiral SFC to give each enantiomer. (R) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-isopropyl-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 26): MS observations (ESI) + )：350.0[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm:12.89 (brs, 1H), 10.90 (brs, 1H), 8.32 (d, J=1.2 Hz, 1H), 7.35 (s, 1H), 5.27 (s, 1H), 4.68 (s, 2H), 3.96 (d, J=12.0 Hz, 1H), 3.53 (d, J=12.0 Hz, 1H), 2.41-2.34 (m, 1H), 1.00 (d, J=6.8 Hz, 3H), 0.76 (d, J=6.8 Hz, 3H). (S) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-4-)Isopropyl-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ]]Thieno [3,2-d ]Pyridin-6-one (compound 27): MS observations (ESI) + )：350.0[(M+H) + ]。
Examples 28 and 29-compounds 28 and 29: (S) -4-hydroxy-8- (1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 28) and (R) -4-hydroxy-8- (1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 29) (the stereochemistry of compounds 28 and 29 is arbitrarily specified)
Step A:4, 4-dimethoxy-3- (trifluoromethyl) tetrahydro-2H-pyran-3-ol:
to a solution of 4, 4-dimethoxy-dihydro-2H-pyran-3 (4H) -one (500 mg,3.12mmol,1.0 eq., synthesized according to the procedure described in WO 2013152269) in THF (5.0 mL) was added TBAF (81.6 mg, 312. Mu. Mol, 90.4. Mu.L, 0.10 eq.) at 0deg.C. The mixture was stirred at 0 ℃ for 10 minutes, then trimethyl (trifluoromethyl) silane (666mg,4.68mmol,744 230L,1.5 eq) was added dropwise. The mixture was allowed to warm to room temperature and stirred at this temperature for 16 hours. The mixture was concentrated and purified by flash column chromatography (SiO 2 12-25% EtOAc/PE) to give 4, 4-dimethoxy-3- (trifluoromethyl) tetrahydro-2H-pyran-3-ol (480 mg, 66%). 1 H NMR(400MHz,CDCl 3 )δppm:4.02–3.99(m,2H),3.85–3.82(m,2H),3.37(s,6H),2.03–2.02(m,2H)。
And (B) step (B): 3- (benzyloxy) -4, 4-dimethoxy-3- (trifluoromethyl) tetrahydro-2H-pyran:
to a solution of 4, 4-dimethoxy-3- (trifluoromethyl) tetrahydropyran-3-ol (1 g,4.34mmol,1.0 eq.) in DMF (8.0 mL) was added NaH (333 mg,8.69mmol,60% in mineral oil, 2.0 eq.) at 0deg.C. The mixture was stirred at 0deg.C for 10 min, then bromomethylbenzene (1.49 g,8.69mmol,1.03mL,2.0 eq.) was added dropwise. The mixture was stirred at room temperature for 3 hours, then saturated NH 4 Cl (aq), diluted with water (120 mL) and extracted with EtOAc (50 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give a residue, which was purified by flash column chromatography (SiO 2 0-7% EtOAc/PE) to give 3- (benzyloxy) -4, 4-dimethoxy-3- (trifluoromethyl) tetrahydro-2H-pyran (690 mg, 49%). 1 H NMR(400MHz,CDCl 3 )δppm:7.38–7.20(m,5H),4.87(d,J＝10.5Hz,1H),4.75(d,J＝10.5Hz,1H),4.13–4.06(m,2H),3.77–3.70(m,2H),3.39(s,3H),3.37(s,3H),2.22–2.16(m,1H),1.99–1.93(m,1H)。
Step C:3- (benzyloxy) -3- (trifluoromethyl) tetrahydro-4H-pyran-4-one:
to a solution of 3- (benzyloxy) -4, 4-dimethoxy-3- (trifluoromethyl) tetrahydro-2H-pyran (690 mg,2.15mmol,1.0 eq.) in acetone (15.0 mL) was added iodine (54.68 mg, 215.42. Mu. Mol,0.1 eq.). The reaction mixture was stirred at room temperature for 1 hour, then taken up in saturated Na 2 SO 3 (aqueous) (25 mL) was quenched and extracted into DCM (35 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 0-8% EtOAc/PE) to give 3- (benzyloxy) -3- (trifluoromethyl) tetrahydro-4H-pyran-4-one (550 mg, 93%). 1 H NMR(400MHz,CDCl 3 )δppm:7.39–7.20(m,5H),4.90(d,J＝11.0Hz,1H),4.62(d,J＝11.0Hz,1H),4.33(d,J＝12.8Hz,1H),4.23–4.17(m,1H),3.90(d,J＝12.8Hz,1H),3.88–3.81(m,1H),3.02-2.94(m,1H),2.54–2.48(m,1H)。
Step D:4- (benzyloxy) -4- (trifluoromethyl) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to a solution of 3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (200 mg, 480. Mu. Mol,1.0 eq), 3-benzyloxy-3- (trifluoromethyl) tetrahydropyran-4-one (263 mg, 958. Mu. Mol,2.0 eq) and cesium carbonate (4638 mg,1.44mmol,3.0 eq) in toluene (12.0 mL) was added Pd 2 (dba) 3 (87.8 mg, 95.8. Mu. Mol,0.2 eq.) and Xantphos (111 mg, 192. Mu. Mol,0.4 eq.). The mixture was treated with N 2 Deaeration and stirring at 105℃for 16 hours. The mixture was filtered and concentrated in vacuo, then purified by flash column chromatography (SiO 2 0-20% EtOAc/PE) to give 4- (benzyloxy) -4- (trifluoromethyl) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ]Thieno [3,2-d]Pyran-6-one (185 mg, 66%). MS observations (ESI) + )：579.5[(M+H) + ]。
Step E:4- (benzyloxy) -4- (trifluoromethyl) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
to 4- (benzyloxy) -4- (trifluoromethyl) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]To a solution of pyran-6-one (185 mg, 320. Mu. Mol) in MeOH (5.0 mL) was added ammonium hydroxide (5.0 mL). The mixture was stirred in a microwave reactor at 100 ℃ for 6 hours, then concentrated under vacuum to give a residue, which was purified by flash column chromatography (SiO 2 0-4% MeOH/DCM) to give 4- (benzyloxy) -4- (trifluoromethyl) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (105 mg, 57%). MS observations (ESI) + )：578.3[(M+H) + ]。
Step F: (S) -4-hydroxy-8- (1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 28) and (R) -4-hydroxy-8- (1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 29):
4- (benzyloxy) -4- (trifluoromethyl) -8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] at 0deg.C]Thieno [3,2-d]Pyridin-6-one (105 mg, 181.76. Mu. Mol,1.0 eq) was dissolved in trifluoroacetic acid (2.6 mL). The mixture was stirred at 60 ℃ for 2 hours under microwave irradiation, then concentrated, and purified by gradual addition of NH 3 MeOH (7M) was used to adjust the pH to about 9. The mixture was concentrated in vacuo and purified by flash column chromatography (SiO 2 0-6% MeOH/DCM) to give racemic 4-hydroxy-8- (1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (54 mg, 83%). MS observations (ESI) + )：358.0[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4-hydroxy-8- (1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 28): MS observations (ESI) + )：358.0[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm 13.25 (s, 1H), 10.94 (s, 1H), 8.27 (brs, 2H), 7.48 (s, 1H), 7.13 (s, 1H), 4.89-4.77 (m, 2H), 4.23 (d, J=12.4 Hz, 1H), 3.71 (m, 1H). (R) -4-hydroxy-8- (1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]Thieno [3,2-d]Pyridin-6-one (compound 29): MS observations (ESI) + )：358.0[(M+H) + ]。
Examples 30 and 31-compounds 30 and 31: (S) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 30) and (R) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 31) (stereochemistry of compounds 30 and 31 is arbitrarily specified)
Synthesized by a pathway similar to compounds 28 and 29. (S) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 30). MS observations (ESI) + )：372.1[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 11.21 (brs, 1H), 7.65 (s, 1H), 7.54 (d, j=2.0 hz, 1H), 7.19 (brs, 1H), 6.72 (d, j=2.0 hz, 1H), 4.96 (d, j=15.2 hz, 1H), 4.81 (d, j=15.2 hz, 1H), 4.25 (d, j=12.0 hz, 1H), 4.05 (s, 3H), 3.71 (d, j=12.0 hz, 1H). (R) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 31). MS observations (ESI) + )：372.1[(M+H) + ]。
Examples 32 and 33-compounds 32 and 33: (S) -4-hydroxy-8- (5-methyl-1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 32) and (R) -4-hydroxy-8- (5-methyl-1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 33) (the stereochemistry of compounds 32 and 33 is arbitrarily specified).
Synthesized by a pathway similar to compounds 28 and 29. (S) -4-hydroxy-8- (5-methyl-1H-pyrazol-4-yl) -4- (trifluoromethyl)Phenyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-ones. MS observations (ESI) + )：372.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 12.98 (brs, 1H), 10.90 (brs, 1H), 8.23-7.86 (m, 1H), 7.35 (s, 1H), 7.14 (s, 1H), 4.92 (d, 15.02Hz, 1H), 4.78 (d, 15.02Hz, 1H), 4.24 (d, J=12.0 Hz, 1H), 3.70 (d, J=12.0 Hz, 1H), 2.43 (s, 3H). (R) -4-hydroxy-8- (5-methyl-1H-pyrazol-4-yl) -4- (trifluoromethyl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-ones. MS observations (ESI) + )：372.0[(M+H) + ]。
Examples 34 and 35-compounds 34 and 35: (S) -4- (hydroxymethyl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 34) and (R) -4- (hydroxymethyl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 35) (the stereochemistry of compounds 34 and 35 is arbitrarily specified)
Step A: benzyl 2- (2-hydroxytetrahydrofuran-2-yl) acetate:
a mixture of benzyl 2-bromoacetate (19.2 g,83.6mmol,1.2 eq.) indium (9.60 g,83.6mmol,1.2 eq.), tetrahydrofuran-2-one (6.0 g,69.7mmol,1.0 eq.) and THF (21.0 mL) was stirred at 70deg.C for 16 hours. Saturated NaHCO for reaction 3 Aqueous (20 ml) was quenched and extracted with EtOAc (60 ml x 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. By flash column chromatography (SiO 2 Purification with 10-20% EtOAc/DCM gave benzyl 2- (2-hydroxytetrahydrofuran-2-yl) acetate (4.0 g, 24%). MS observations (ESI) + )：219.2[(M+H-H 2 O) + ]。
And (B) step (B): benzyl 2-diazonium-6-hydroxy-3-oxohexanoate:
to a solution of benzyl 2- (2-hydroxytetrahydrofuran-2-yl) acetate (700 mg,2.96mmol,1.0 eq.) and N-diazo-2, 4, 6-triisopropyl-benzenesulfonamide (1.01 g,3.26mmol, 1.1 eq.) in THF (10.0 mL) was added N, N-diethylamine (900 mg,8.9mmol,3.0 eq.) at 0deg.C. The mixture was stirred at room temperature for 16 hours, then concentrated. The residue was purified by flash column chromatography (SiO 2, 15-30% EtOAc/PE) to give benzyl 2-diazo-6-hydroxy-3-oxo-hexanoate (1.18 g, 81%). MS observations (ESI) + )：263.2[(M+H) + ]。
Step C: 3-oxotetrahydro-2H-pyran-2-carboxylic acid benzyl ester:
a solution of benzyl 2-diazonium-6-hydroxy-3-oxo-hexanoate (1.0 g,3.81mmol,1.0 eq.) in benzene (50.0 mL) was added dropwise to Rh at 100deg.C over 35 min 2 (OAc) 4 (50.0 mg, 113. Mu. Mol,0.030 eq.) in benzene (50.0 mL). The mixture was stirred at 100 ℃ for 1 hour, then filtered and concentrated to give benzyl 3-oxo-tetrahydro-2H-pyran-2-carboxylate (180 mg, crude product), which was used in the next step without purification. MS observations (ESI) + )：235.2[(M+H) + ]。
Step D: 2-methyl-3-oxotetrahydro-2H-pyran-2-carboxylic acid benzyl ester:
to a solution of benzyl 3-oxotetrahydro-2H-pyran-2-carboxylate (890 mg,3.80mmol,1.0 eq.) in DMF (15 mL) was added sodium hydride (247 mg,5.70mmol, 60% in mineral oil, 1.5 eq.) at-20deg.C. The mixture was stirred for 10 minutes, then iodine was addedMethane (1.62 g,11.40mmol,3.0 eq.) and the mixture was stirred at room temperature for 16 hours. The mixture was poured into water and extracted with ethyl acetate (30 ml x 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo, and the residue was purified by flash column chromatography (SiO 2 0-10% EtOAc/PE) to give benzyl 2-methyl-3-oxotetrahydro-2H-pyran-2-carboxylate (474 mg, 50%). MS observations (ESI) + )：271.0[(M+Na) + ]。
Step E: benzyl 4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,2,4, 6-tetrahydropyrano [3,4-b ] thieno [3,2-d ] pyran-4-carboxylate:
3-bromo-5- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]Thiophene-2-carboxylic acid methyl ester (84.1 mg, 201. Mu. Mol,1.0 eq), 2-methyl-3-oxotetrahydro-2H-pyran-2-carboxylic acid benzyl ester (100 mg, 403. Mu. Mol,2.0 eq), sodium bisulphite (11.5 mg, 60.4. Mu. Mol,0.3 eq), cs 2 CO 3 A mixture of (197mg, 604. Mu. Mol,3.0 eq.) Sphos-Pd-G3 (31.4 mg, 40.3. Mu. Mol,0.2 eq.) and toluene (15.0 mL) was stirred at 105℃for 16 hours. The mixture was poured into water and extracted with EtOAc (70 ml x 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 0-25% EtOAc/PE) to give 4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,2,4, 6-tetrahydropyrano [3,4-b]Thieno [3,2-d]Benzyl pyran-4-carboxylate (78 mg, 70%). MS observations (ESI) + )：553.5[(M+H) + ]. Step F:4- (hydroxymethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 4-dihydropyrano [3,4-b]Thieno [3,2-d]Pyran-6 (2H) -one:
4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,2,4, 6-tetrahydropyrano [3,4-b]Thieno [3,2-d]A mixture of benzyl pyran-4-carboxylate (50 mg, 90.5. Mu. Mol,1.0 eq), calcium dichloride (50.2 mg, 452. Mu. Mol,5 eq) and EtOH (5.0 mL) was stirred at room temperature for 20 minutes, then sodium borohydride (51.3 mg,1.36mmol,15.0 eq) was added portionwise to the mixture at 0deg.C, followed by stirring at room temperature for 20 minutes. The mixture was poured into water and extracted with EtOAc (30 ml x 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 50% EtOAc/PE) to give 4- (hydroxymethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 4-dihydropyran [3,4-b]Thieno [3,2-d]Pyran-6 (2H) -one (20 mg, 49%). MS observations (ESI) + )：449.2[(M+H) + ]。
Step G:4- (hydroxymethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one:
4- (hydroxymethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 4-dihydropyrano [3,4-b]Thieno [3,2-d]A mixture of pyran-6 (2H) -one (60 mg, 134. Mu. Mol), ammonium hydroxide (3.5 mL) and MeOH (3.5 mL) was stirred in a microwave reactor at 100deg.C for 3 hours. The mixture was concentrated in vacuo and the residue was purified by flash column chromatography (SiO 2, 15-35% EtOAc/PE) to give 4- (hydroxymethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d]Pyridin-6 (4H) -one (20 mg, 33%). MS observations (ESI) + )：448.5[(M+H) + ]。
Step H: (S) -4- (hydroxymethyl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 34) and (R) -4- (hydroxymethyl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 35):
Trichloroborane (1M, 890. Mu.L, 5.0 eq.) is added to 4- (hydroxymethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b at 0deg.C]Thieno [3,2-d]Pyridin-6 (4H) -one (80 mg, 180. Mu. Mol) was dissolved in DCM (50.0 mL). The mixture was stirred at room temperature for 2 hours, then quenched with MeOH and concentrated in vacuo. The residue was taken up with NaHCO 3 Alkalization and chromatography by column (SiO 2 0-6% MeOH/DCM) to give racemic 4- (hydroxymethyl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3, 4-b)]Thieno [3,2-d]Pyridin-6 (4H) -one (32 mg, 56%). MS observations (ESI) + )：318.5[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4- (hydroxymethyl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d]Pyridin-6 (4H) -one (compound 34): MS observations (ESI) + )：318.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 13.22 (brs, 1H), 10.90 (brs, 1H), 8.31 (s, 1H), 7.97 (s, 1H), 7.48 (s, 1H), 4.87 (s, 1H), 4.09-3.96 (m, 1H), 3.93-3.72 (m, 2H), 3.51 (d, J=11.6 Hz, 1H), 2.86-2.58 (m, 2H), 1.39 (s, 3H). (R) -4- (hydroxymethyl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d ]Pyridin-6 (4H) -one (compound 35): MS observations (ESI) + )：318.0[(M+H) + ]。
Examples 36 and 37-compounds 36 and 37: (S) -6- (difluoromethyl) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 36) and (R) -6- (difluoromethyl) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 37) (the stereochemistry of compounds 36 and 37 is arbitrarily specified)
Step A:2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -8, 9-dihydro-4H-thieno [2,3-c ] chromene-4, 6 (7H) -dione:
3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (200 mg, 480. Mu. Mol,1.0 eq) and cyclohexane-1, 2-dione (1.1G, 9.6mmol,20 eq) were dissolved in anhydrous toluene (40.0 mL), then Sphos-Pd-G3 (56 mg, 96. Mu. Mol,0.20 eq), cs were added 2 CO 3 (470 mg,1.40mmol,3.0 eq.) and Na 2 S 2 O 5 (18 mg,0.096mmol,0.20 eq.). The mixture was treated with N 2 Deaeration and heating to 105 ℃ for 16 hours. The mixture was cooled, concentrated in vacuo, and purified by flash column chromatography (SiO 2 0-45% EtOAc/PE). Further purification (SiO) 2 0-8% MeOH/DCM) to give 2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -8, 9-dihydro-4H-thieno [2,3-c ]Chromene-4, 6 (7H) -dione (20 mg, 10%). MS observations (ESI) + )：417.5[(M+H) + ]。
And (B) step (B): 6- (difluoromethyl) -6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
at N 2 Cesium fluoride (55 mg,0.36mmol,2.5 eq.) was added to 2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -8, 9-dihydro-4H-thieno [2,3-c ] under an atmosphere]Chromene-4, 6 (7H) -dione (60 mg,0.144mmol,1.0 eq.) in anhydrous DMF (4)0 mL) followed by difluoromethyl (trimethyl) silane (90 mg,0.72mmol, 100. Mu.L, 5.0 eq.) was added. The mixture was stirred at room temperature for 24 hours, then diluted with water (50 mL) and extracted with EtOAc (40 mL x 4). The combined organic phases were dried (Na 2 SO 4 ) Filtered and concentrated. Purification (SiO) 2 0-39% EtOAc/PE) afforded 6- (difluoromethyl) -6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (23 mg, 34%). MS observations (esi+): 469.4[ (M+H) + ]。
Step C:6- (difluoromethyl) -6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
6- (difluoromethyl) -6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (65 mg,0.14 mmol) was added to NH 4 OH (5.0 mL, saturated aqueous solution) was then added to MeOH (5.0 mL). The mixture was heated to 95 ℃ in a microwave reactor for 10 hours and then concentrated to give 6- (difluoromethyl) -6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (65 mg, crude product). This material was used in the next step without further purification. MS observations (esi+): 468.5[ (M+H) + ]。
Step D: (S) -6- (difluoromethyl) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 36) and (R) -6- (difluoromethyl) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 37):
will 6- (difluoromethyl)Phenyl) -6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (180 mg, 370. Mu. Mol) was dissolved in DCM (15.0 mL) and cooled to 0deg.C before trifluoroacetic acid (3.0 mL) was added. The reaction mixture was warmed to room temperature and after 4 hours the mixture was concentrated in vacuo. Purification of the residue (C18 SiO) 2 0-25% MeCN/water (0.1% NH) 4 OH)) to give racemic 6- (difluoromethyl) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]]Quinolin-4 (5H) -one (40 mg, 26%). MS observations (ESI) + )：388.4[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -6- (difluoromethyl) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 36): MS observations (ESI) + )：388.1[(M+H) + ]. (R) -6- (difluoromethyl) -6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 37): MS observations (esi+): 388.1[ (M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 )δppm:13.22(s,1H),10.68(s,1H),8.32(s,1H),7.98(s,1H),7.54(s,1H),6.52(t,J＝55.6Hz,1H),6.26(s,1H),2.77–2.73(m,1H),2.68–2.61(m,1H),2.10–2.04(m,1H),1.87–1.85(m,3H)。
Examples 38 and 39-compounds 38 and 39: (S) -9-methoxy-9-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 38) and (R) -9-methoxy-9-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 39) (the stereochemistry of compounds 38 and 39 is arbitrarily specified)
Step A:7, 8-dihydro-4H-thieno [2,3-c ] chromene-4, 9 (6H) -dione:
methyl 3-bromothiophene-2-carboxylate (0.50 g,2.3mmol,1.0 eq.) and cyclohexane-1, 3-dione (1.5 g,14mmol,6.0 eq.) were dissolved in toluene (80.0 mL) and Cs was then added 2 CO 3 (2.2G, 6.8mmol,3.0 eq.) and SPhos-Pd-G3 (180 mg, 230. Mu. Mol,0.10 eq.). The mixture was heated to 105℃for 16 hours, cooled and quenched with H 2 O (50 mL) dilution. The mixture was extracted with EtOAc (50 mL x 3), and the combined organic phases were washed with brine (50 mL), dried (Na 2 SO 4 ) Filtered and concentrated. By column chromatography (SiO 2 0-20% EtOAc/PE) to give 7, 8-dihydro-4H-thieno [2,3-c ]]Chromene-4, 9 (6H) -dione (82 mg, 14%). MS observations (ESI) + )：221.2[(M+H) + ]。
And (B) step (B): 7, 8-dihydrothieno [2,3-c ] quinoline-4, 9 (5 h,6 h) -dione:
7, 8-dihydro-4H-thieno [2,3-c]Chromene-4, 9 (6H) -dione (550 mg,2.50mmol,1.0 eq.) was dissolved in MeOH (8.0 mL) and NH was added 4 OH (8 mL, saturated aqueous). The reaction mixture was heated to 90 ℃ in a microwave reactor for 4 hours, cooled, concentrated in vacuo. By column chromatography (SiO 2 0-25% MeOH/DCM) to give 7, 8-dihydrothieno [2,3-c ]]Quinoline-4, 9 (5H, 6H) -dione (420 mg, 77%). MS observations (ESI) + )：220.2[(M+H) + ]。
Step C:5- ((2- (trimethylsilyl) ethoxy) methyl) -7, 8-dihydrothieno [2,3-c ] quinoline-4, 9 (5 h,6 h) -dione:
7, 8-Dihydrothieno [2,3-c ]]A solution of quinoline-4, 9 (5H, 6H) -dione (1.3 g,5.9mol,1.0 eq.) in anhydrous DMF (60.0 mL) was cooled to 0deg.C, then NaH (360 mg,8.9mmol,60% in mineral oil, 1.5 eq.) was added. At 30 minutes After that, 2- (chloromethoxy) ethyl-trimethyl-silane (2.0 g,12mmol,2.1mL,2.0 eq) was added, and the mixture was warmed to room temperature and stirred at room temperature for 2 hours. Water (150 mL) was added and the mixture was extracted with EtOAc (100 mL. Times.3). The combined organic phases were washed (brine, 100 mL), dried (Na 2 SO 4 ) Filtered and concentrated in vacuo. By column chromatography (SiO 2 Purification of 0-30% EtOAc/PE afforded 5- ((2- (trimethylsilyl) ethoxy) methyl) -7, 8-dihydrothieno [2, 3-c)]Quinoline-4, 9 (5H, 6H) -dione (1.0 g, 48%). MS observations (ESI) + )：350.5[(M+H) + ]。
Step D: 9-hydroxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
5- ((2- (trimethylsilyl) ethoxy) methyl) -7, 8-dihydrothieno [2,3-c]Quinoline-4, 9 (5H, 6H) -dione (1.0 g,2.9mmol,1.0 eq.) was dissolved in THF (30.0 mL) and the solution cooled to 0deg.C, then methylmagnesium bromide (1M, 57.0mL,57mmol,20 eq.) was added. The reaction mixture was warmed to room temperature and stirred for 2 hours, then water (100 mL) was added. The mixture was extracted with EtOAc (100 mL x 3), and the combined organic phases were washed with brine (100 mL), dried (Na 2 SO 4 ) Filtered, and concentrated in vacuo. By column chromatography (SiO 2 Purification of 0-30% EtOAc/PE afforded 9-hydroxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Quinolin-4 (5H) -one (320 mg, 27%). MS observations (ESI) + )：366.5[(M+H) + ]。
Step E: 9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
sodium hydride (145 mg,3.61mmol,60% in mineral oil, 4.0 eq.) and methyl iodide (1.3 g,9.0mmol,0.6mL,10.0 eq.) were added to 9-hydroxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2,3-c ]]A solution of quinolin-4 (5H) -one (330 mg,0.90mmol,1.0 eq.) in dry THF (15.0 mL). After 2 hours, water (50 mL) was added and the mixture was extracted with EtOAc (30 mL x 3). The combined organic phases were washed with brine (30 mL), dried (Na 2 SO 4 ) Filtered and concentrated in vacuo. By column chromatography (SiO 2 0-15% EtOAc/PE) to give 9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Quinolin-4 (5H) -one (190 mg, 55%). MS observations (ESI) + )：380.6[(M+H) + ]。
Step F: 2-iodo-9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (140 mg,0.37mmol,1.0 eq.) was dissolved in THF (8.0 mL) and cooled to-65 ℃. Lithium diisopropylamine (2M, 0.7mL,1.5mmol,4.0 eq.) was added followed by iodine (190 mg,0.74mmol,2.0 eq.) and the mixture stirred at-65℃for 2 hours. Water (10 mL) was added and the mixture was extracted with EtOAc (10 mL. Times.3). The combined organic phases were washed with brine (10 mL), dried (Na 2 SO 4 ) Filtered and concentrated in vacuo. By column chromatography (SiO 2 0-15% EtOAc/PE) to give 2-iodo-9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Quinolin-4 (5H) -one (120 mg, 62%). MS observations (ESI) + )：506.6[(M+H) + ]。
Step G: 9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
pd (dppf) Cl 2 (32 mg, 40. Mu. Mol,0.20 eq), XPhos (42 mg, 90. Mu. Mol,0.40 eq) and Na 2 CO 3 (70 mg,0.65mmol,3.0 eq.) to 2-iodo-9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -6,7,8, 9-tetrahydrothieno [2, 3-c) ]Quinolin-4 (5H) -one (110 mg,0.22mmol,1.0 eq.) and trimethyl- [2- [ [4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazol-1-yl]Methoxy group]And]silane (110 mg,0.35mmol,1.6 eq.) in H 2 In solution in O/1, 4-dioxane (8.0 mL,1:3 mixture). The mixture was heated to 110 ℃ for 2 hours and then concentrated in vacuo. Purification by column chromatography (0-30% EtOAc/PE) afforded 9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (110 mg, 88%). MS observations (ESI) + )：576.9[(M+H) + ]。
Step H: (9S) -9-methoxy-9-methyl-2- (1H-pyrazol-4-yl) -5,6,7, 8-tetrahydrothieno [2,3-c ] quinolin-4-one (compound 38) and (9R) -9-methoxy-9-methyl-2- (1H-pyrazol-4-yl) -5,6,7, 8-tetrahydrothieno [2,3-c ] quinolin-4-one (compound 39):
BCl was added at 0deg.C 3 (1.0M, 1.1mL,1.1mmol,5.0 eq.) to 9-methoxy-9-methyl-5- ((2- (trimethylsilyl) ethoxy) methyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (130 mg,0.22mmol,1.0 eq.) in DCM (13.0 mL). After 10 minutes, NH was added 3 MeOH (to pH 9) and the mixture was put under vacuumConcentrating the mixture. By column chromatography (C18 SiO 2 0-20% MeCN/water (0.1% NH) 4 OH)) to give racemic 9-methoxy-9-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Quinolin-4 (5H) -one (24 mg, 34%). MS observations (ESI) + )：316.2[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (9S) -9-methoxy-9-methyl-2- (1H-pyrazol-4-yl) -5,6,7, 8-tetrahydrothieno [2,3-c]Quinolin-4-one (compound 38): MS observations (ESI) + )：316.2[(M+H) + ]. (9R) -9-methoxy-9-methyl-2- (1H-pyrazol-4-yl) -5,6,7, 8-tetrahydrothieno [2,3-c]Quinolin-4-one (compound 39): MS observations (ESI) + )：316.2[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δppm:13.21(s,1H),11.29(s,1H),8.32(s,1H),7.91(s,1H),7.64(s,1H),2.94(s,3H),2.70–2.52(m,2H),2.15–2.01(m,1H),1.95–1.87(m,1H),1.83–1.67(m,1H),1.55–1.65(m,1H),1.50(m,3H)。
Examples 40 and 41-compounds 40 and 41: (S) -6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 40) and (R) -6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 41) (the stereochemistry of compounds 40 and 41 is arbitrarily specified)
Step A:4- (6-hydroxy-4-oxo-4, 5,6,7,8, 9-hexahydrothieno [2,3-c ] quinolin-2-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester:
triethylamine (280 mg,2.8mmol,2.0 eq.) DMAP (85 mg, 70. Mu. Mol,0.50 eq.) and (Boc) 2 O (300 mg,1.40mmol,1.0 eq.) was added to 6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-5H-thieno [2,3-c]Quinolin-4-one (400 mg,1.40mmol,1.0 eq.) in DMF (20.0 mL)Is added to the suspension of (a). The mixture was stirred at room temperature for 16 hours, then extracted with EtOAc (50 ml x 3). The combined organic phases were washed with brine, dried (Na 2 SO 4 ) Filtered and concentrated. By column chromatography (SiO 2 0-100% EtOAc/PE) to give 4- (6-hydroxy-4-oxo-4, 5,6,7,8, 9-hexahydrothieno [2, 3-c)]Quinolin-2-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (400 mg, 74%). MS observations (ESI) + )：388.5[(M+H) + ]。
And (B) step (B): 4- (4, 6-dioxo-5, 7,8, 9-tetrahydrothieno [2,3-c ] quinolin-2-yl) pyrazole-1-carboxylic acid tert-butyl ester:
4- (6-hydroxy-4-oxo-4, 5,6,7,8, 9-hexahydrothieno [2, 3-c)]A suspension of quinoline-2-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (140 mg,0.36mmol,1.0 eq.) and acetic acid (1, 1-diacetoxy-3-oxo-1, 2-phenyliodi-1-yl) ester (110 mg,3.6mmol,10 eq.) in DCM (14.0 mL) was stirred at room temperature for 1H. The mixture was concentrated, and the residue was purified by column chromatography (SiO 2 0-10% MeOH/DCM) to give 4- (4, 6-dioxo-5, 7,8, 9-tetrahydrothieno [2, 3-c) ]Quinolin-2-yl) pyrazole-1-carboxylic acid tert-butyl ester (120 mg, 87%). MS observations (ESI) + )：386.5[(M+H) + ]。
Step C: (S) -6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 40) and (R) -6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 41):
methyl magnesium bromide (1.0M, 4.8mL,4.8mmol,3.0 eq.) was added to 4- (6-hydroxy-4-oxo-4, 5,6,7,8, 9-hexahydrothieno [2, 3-c)]Quinoline-2-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (60 mg,0.16mmol,1.0 eq.) in THF (24.0 mL). The mixture is put inStirred at room temperature for 2 hours, then MeOH was added, and the resulting mixture was concentrated in vacuo and purified by column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2, 3-c)]Quinolin-4 (5H) -one (39 mg, 62%).
The individual enantiomers were separated by chiral SFC. (S) -6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 40): MS observations (ESI) + )：302.1[(M+H) + ]； 1 HNMR(400MHz,DMSO-d 6 ) Delta ppm 13.10 (s, 1H), 10.51 (s, 1H), 8.12 (s, 2H), 7.46 (s, 1H), 5.12 (s, 1H), 2.32-2.51 (m, 2H), 1.88-1.82 (m, 3H), 1.78-1.67 (m, 1H), 1.46 (s, 3H). (R) -6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]Quinolin-4 (5H) -one (compound 41): MS observations (ESI) + )：302.1[(M+H) + ]。
Examples 42 and 43-compounds 42 and 43: (S) -4- (difluoromethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 42) and (R) -4- (difluoromethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 43) (the stereochemistry of compounds 42 and 43 is arbitrarily specified)
Step A: 4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
sodium hydride (140 mg,3.4mmol,60% in mineral oil, 2.0 eq.) was added to 4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Cooling of pyran-6-one (500 mg,1.7mmol,1.0 eq.) in DMF (20.0 mL) (0 ℃ C.)) In solution. After 10 minutes at 0deg.C, SEMCl (370 μL,350mg,2.0mmol,1.2 eq.) was slowly added to the mixture. The mixture was stirred at room temperature for 2 hours, then quenched with ammonium chloride (saturated aqueous solution) and then extracted with EtOAc (25 ml x 3). The combined organic phases were treated with brine, dried (Na 2 SO 4 ) Filtered and concentrated. By column chromatography (SiO 2 0-40% EtOAc/DCM) to give 4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (400 mg, 52%). MS observations (ESI) + )：421.2[(M+H) + ]。
And (B) step (B): 8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1H, 3H-pyrano [4,3-b ] thieno [3,2-d ] pyran-4, 6-dione:
4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (400 mg, 950. Mu. Mol,1.0 eq) was dissolved in DCM (20.0 mL) and DMF (20.0 mL) and acetic acid (1, 1-diacetoxy-3-oxo-1, 2-phenyliodi-1-yl) ester (1.2 g,2.9mmol,3.0 eq) was added. The mixture was stirred at room temperature for 16 hours, then was taken up in Na 2 S 2 O 3 (saturated aqueous solution) and NaHCO 3 (saturated aqueous solution) quenching. The mixture was extracted with EtOAc (30 ml x 3) and the combined organic phases were dried (Na 2 SO 4 ) Filtered and concentrated. By column chromatography (SiO 2 Purification with 0-30% EtOAc/DCM gave 8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1H, 3H-pyrano [4, 3-b)]Thieno [3,2-d ]Pyran-4, 6-dione (270 mg, 63%). MS observations (ESI) + )：419.2[(M+H) + ]，436.2[(M+NH 4 ) + ]。
Step C:4- (difluoromethyl) -4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
cesium fluoride (210 mg,1.4mmol,2.5 eq.) was added to 8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1H, 3H-pyrano [4,3-b]Thieno [3,2-d]To a solution of pyran-4, 6-dione (270 mg, 560. Mu. Mol,1.0 eq.) in DMF (24.0 mL) was added followed by TMSCF 2 H (350 mg,2.8mmol, 400. Mu.L, 5.0 eq.). The mixture was stirred for 16 hours. Water was added and the mixture was extracted with EtOAc (25 mL. Times.5). The combined organic phases were washed with brine, dried (Na 2 SO 4 ) Filtered and concentrated. By column chromatography (SiO 2 Purification with 0-40% EtOAc/PE) afforded 4- (difluoromethyl) -4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (48 mg, 16%). MS observations (ESI) + )：471.4[(M+H) + ]。
Step D:4- (difluoromethyl) -4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
Ammonia (8.0 mL,25% w/w) was added to 4- (difluoromethyl) -4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (140 mg, 260. Mu. Mol,1.0 eq.) in i-PrOH (8.0 mL). The mixture was heated to 100 ℃ in a microwave reactor for 6 hours, then cooled and concentrated. By column chromatography (SiO 2 Purification with 0-8% MeOH/DCM gave 4- (difluoromethyl) -4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (55 mg, 41%). MS observations (ESI) + )：470.2[(M+H) + ]。
Step E: (S) -4- (difluoromethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 42) and (R) -4- (difluoromethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 43):
4- (difluoromethyl) -4-hydroxy-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (10 mg, 21. Mu. Mol) was dissolved in DCM: trifluoroacetic acid (4.0 mL, 3:1). The mixture was stirred at room temperature for 1 hour, then concentrated, and purified by reverse phase HPLC (C18 SiO 2 0-15% MeCN/water (0.1% NH) 4 OH)) purification. By column chromatography (SiO 2 0-8% MeOH/DCM) to give racemic 4- (difluoromethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (1.3 mg, 17%). MS observations (ESI) + )：340.0[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4- (difluoromethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 42): MS observations (ESI) + )：340.2[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm 13.24 (s, 1H), 11.04 (s, 1H), 8.29 (s, 1H), 7.99 (s, 1H), 7.47 (s, 1H), 6.57 (s, 1H), 6.44 (t, J=55.0 Hz, 1H), 4.76 (s, 2H), 4.11 (d, J=12.0 Hz, 1H), 3.72 (d, J=12.0 Hz, 1H). (R) -4- (difluoromethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 43): MS observations (ESI) + )：340.2[(M+H) + ]。
Examples 44 and 45-compounds 44 and 45: (S) -4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 44) and (R) -4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 45) (stereochemistry of compounds 44 and 45 is arbitrarily specified)
Step A:4, 4-dimethoxy-3- (prop-1-en-2-yl) tetrahydro-2H-pyran-3-ol:
at 0℃under N 2 To a solution of 4, 4-dimethoxy-dihydro-2H-pyran-3 (4H) -one (5.0 g,31mmol,1.0 eq., synthesized according to the procedure described in WO 2013152269) in anhydrous THF (100 mL) was added magnesium bromo (isopropenyl) (1M, 125.0mL,125mmol,4.0 eq.) under atmosphere. After addition, the mixture was stirred at 0 ℃ for 30 minutes, then warmed to 25 ℃ and under N 2 Stirred for an additional 2 hours. The mixture was cooled to 0℃and then saturated NH 4 Cl (aq) (120 mL) was quenched, diluted with water (100 mL), and extracted with EtOAc (100 mL. Times.3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and evaporated in vacuo. By column chromatography (SiO 2 Purification of 0-25% EtOAc/PE afforded 4, 4-dimethoxy-3- (prop-1-en-2-yl) tetrahydro-2H-pyran-3-ol (3.5 g, 55%). 1 H NMR(400MHz,CDCl 3 )δppm:5.37(m,1H),5.08–5.03(m,1H),3.76(d,J＝11.4Hz,1H),3.72–3.60(m,2H),3.36(d,J＝11.4Hz,1H),3.32(d,J＝2.8Hz,6H),2.08–1.99(m,1H),1.98–1.95(m,3H),1.94–1.87(m,1H)。
And (B) step (B): 3-hydroxy-3-isopropyltetrahydro-4H-pyran-4-one:
4, 4-dimethoxy-3- (prop-1-en-2-yl) tetrahydro-2H-pyran-3-ol was added to a 1L pressure vessel under a nitrogen atmosphere(17.5 g,82.20 mmol) in methanol (500.0 mL), 4N aqueous HCl (20.0 mL), and Pd/C (998.34 mg,8.22 mmol). The vessel was sealed and shaken at room temperature under a hydrogen pressure of 20atm for 8 hours. After purging with nitrogen, the vessel was unsealed and the mixture was filtered through a pad of celite. The filtrate was concentrated in vacuo to give 3-hydroxy-3-isopropyltetrahydro-4H-pyran-4-one (10.9 g,65.46mmol,79.63% yield, 95% purity) as a yellow liquid. MS observations (ESI) + )：159.2[(M+H) + ]。
Step C: 4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to a solution of 3-hydroxy-3-isopropyltetrahydro-4H-pyran-4-one (106 mg,0.67mmol,2.0 eq.) in toluene (10.0 mL) was added methyl 3-bromo-5- (pyridin-4-yl) thiophene-2-carboxylate (100 mg,0.34mmol,1.0 eq.), tris (dibenzylideneacetone) dipalladium (61 mg,0.67mmol,0.20 eq.), 4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene (116 mg,0.20mmol,0.60 eq.), cesium carbonate (219 mg,0.67mmol,2.0 eq.) and sodium hydrogen sulfite (13 mg,0.070mmol,0.20 eq.). The mixture was stirred at 105 ℃ for 16 hours and then concentrated. The residue was diluted with water (20 mL) and extracted with EtOAc (20 mL. Times.3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (SiO 2 1-50% etoac/PE) to give racemic 4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -3, 4-dihydro-1 h,6 h-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-ones. MS observations (ESI) + )：344.4[(M+H) + ]。
Step D: (S) -4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 44) and (R) -4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 45):
4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (62 mg,0.18mmol,1.0 eq.) in NH 4 A solution in a mixture of OH (3.0 mL) and MeOH (3.0 mL) was heated in a microwave reactor at 100deg.C for 3 hours. The mixture was concentrated and the residue was purified by column chromatography (SiO 2,1-5% EtOAc/PE) to give racemic 4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (17 mg, 28%). MS observations (ESI) + )：343.5[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 44): MS observations (ESI) + )：343.2[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 11.07 (s, 1H), 8.69 (d, J=6.0 Hz, 2H), 8.04 (s, 1H), 7.81 (d, J=6.0 Hz, 2H), 5.31 (s, 1H), 4.73 (s, 2H), 3.98 (d, J=12.0 Hz, 1H), 3.55 (d, J=11.6 Hz, 1H), 2.39-2.25 (m, 1H), 1.00 (d, J=6.8 Hz, 3H), 0.77 (d, J=7.2 Hz, 3H). (R) -4-hydroxy-4-isopropyl-8- (pyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 45): MS observations (ESI) + )：343.2[(M+H) + ]。
Examples 46 and 47-compounds 46 and 47: (S) -4-hydroxy-4-isopropyl-8- (isothiazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 46) and (R) -4-hydroxy-4-isopropyl-8- (isothiazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 47)
Racemic 4-hydroxy-4-isopropyl-8- (isothiazol-5-yl) -3, 4-dihydro-1H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6 (5H) -one was synthesized by a similar route to compounds 43 and 44.
Each enantiomer was obtained by chiral SFC separation. (S) -4-hydroxy-4-isopropyl-8- (isothiazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 46): MS observations (ESI) + )：349.0[(M+H) + ]. (R) -4-hydroxy-4-isopropyl-8- (isothiazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 47): MS observations (ESI) + )：349.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 )δppm:11.10(s,1H),8.64(d,J＝2.0Hz,1H),7.84(d,J＝2.0Hz,1H),7.81(s,1H),5.31(s,1H),4.71(s,2H),3.97(d,J＝11.6Hz,1H),3.54(d,J＝11.6Hz,1H),2.40–2.37(m,1H),1.01–0.99(d,J＝6.8Hz,3H),0.77–0.75(d,J＝6.8Hz,3H)。
Examples 48 and 49-compounds 48 and 49: (S) -8, 8-difluoro-6-hydroxy-6- (methoxymethyl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 48) and (R) -8, 8-difluoro-6-hydroxy-6- (methoxymethyl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 49)
Step A:5, 5-difluoro-2, 2-dimethoxy-cyclohexane-1-one:
to a solution of 5, 5-difluoro-2, 2-dimethoxy-cyclohexanol (29.4 g,150mmol, synthesized according to the procedure of compound 12 step a) in DCM (800.0 mL) was added, in portions, dess-martin periodate (95.4 g,225 mmol) over 20 minutes. The mixture was stirred for 2 hours and then concentrated. The residue was diluted with water, washed with petroleum ether: ethyl acetate (10:1, 500 mL), and the organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (SiO 2 12% EtOAc/PE) to give 5, 5-difluoro-2, 2-dimethylOxycyclohexane-1-one (16.2 g, 55%). 1 H NMR(400MHz,CDCl 3 ):δppm:3.27(s,6H),3.04(t,J＝14.4Hz,2H),2.28ppm(m,2H),2.02(m,2H)。
And (B) step (B): 5, 5-difluoro-2, 2-dimethoxy-1-vinylcyclohexane-1-ol:
to a solution of 5, 5-difluoro-2, 2-dimethoxycyclohexane-1-one (1.2 g,6.2mmol,1.0 eq.) in anhydrous THF (30.0 mL) was added dropwise magnesium bromide (1 m,18.5mL,3.0 eq.) at 0 ℃. The mixture was stirred at room temperature for 1 hour, then poured into saturated NH 4 Cl (aq) (10 mL) and extracted with ethyl acetate (40 mL. Times.2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 0-10% EtOAc/PE) to give 5, 5-difluoro-2, 2-dimethoxy-1-vinylcyclohexane-1-ol (630 mg, 45%). 1 H NMR(400MHz,CDCl 3 ):δppm:6.19(dd,J＝17.2,10.8Hz,1H),5.47(dd,J＝17.2,1.6Hz,1H),5.13(dd,J＝10.8,1.6Hz,1H),3.24(s,3H),3.23(s,3H),2.54(s,1H),2.16–1.74(m,6H)。
Step C: ((5, 5-difluoro-2, 2-dimethoxy-1-vinylcyclohexyl) oxy) methyl) benzene:
sodium hydride (305 mg,7.63mmol,60% in mineral oil, 1.5 eq.) was added in portions to a solution of 5, 5-difluoro-2, 2-dimethoxy-1-vinylcyclohexane-1-ol (1.13 g,5.08mmol,1.0 eq.) in anhydrous DMF (30.0 mL) at 0deg.C. The mixture was stirred at 0 ℃ for 1 hour, then benzyl bromide (2.61 g,15.2mmol,3.0 eq.) was added and the mixture was stirred at room temperature for another 1 hour. The mixture was poured into ice water and extracted with DCM (30 ml x 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue is led through Column chromatography (SiO) 2 0-10% EtOAc/PE) to give (((5, 5-difluoro-2, 2-dimethoxy-1-vinylcyclohexyl) oxy) methyl) benzene (1.3 g, 81%). 1 H NMR(400MHz,CDCl 3 ):δppm:7.38–7.28(m,5H),6.13–6.04(m,1H),5.44(dd,J＝11.2,0.8Hz,1H),5.29(d,J＝18.0Hz,1H),4.44(dd,J＝34.8,11.2Hz,2H),3.42(s,3H),3.28(s,3H),2.50–1.80(m,6H)。
Step D: (1- (benzyloxy) -5, 5-difluoro-2, 2-dimethoxycyclohexyl) methanol:
ozone was bubbled through a solution of (((5, 5-difluoro-2, 2-dimethoxy-1-vinylcyclohexyl) oxy) methyl) benzene (650 mg,2.08mmol,1.0 eq.) in DCM (20.0 mL) at-78 ℃. The mixture was stirred at-78 ℃ for 30 minutes and then allowed to warm to room temperature. MeOH (5.0 ml) and NaBH were added 4 (236 mg,6.24mmol,3.0 eq.) and the mixture stirred for 5 min. The mixture was poured into water and extracted with ethyl acetate (70 ml x 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (SiO 2 15-30% EtOAc/PE) to give (1- (benzyloxy) -5, 5-difluoro-2, 2-dimethoxycyclohexyl) methanol (1.1 g, 83%). 1 H NMR(400MHz,CDCl 3 ):δppm:7.36–7.19(m,5H),4.53(d,J＝1.6Hz,2H),3.92(d,J＝12.2Hz,1H),3.69–3.57(m,1H),3.38(s,3H),3.25(s,3H),2.35(m,2H),2.08-1.85(m,4H)。
Step E: ((5, 5-difluoro-2, 2-dimethoxy-1- (methoxymethyl) cyclohexyl) oxy) methyl) benzene:
sodium hydride (265 mg,6.64mmol,60% in mineral oil, 1.4 eq.) was added in portions to (1- (benzyloxy) -5, 5-difluoro-2, 2-dimethoxycyclohexyl) methanol (1.5 g,4.74mmol, 1.0 eq.) in anhydrous state at 0deg.C In solution in DMF (30.0 mL). The mixture was stirred at room temperature for 1 hour, then methyl iodide (6.73 g,47.4mmol,10 eq.) was added. The mixture was stirred for 2 hours, then poured into water and extracted with DCM (60 ml x 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (SiO 2 0-10% EtOAc/PE) to give (((5, 5-difluoro-2, 2-dimethoxy-1- (methoxymethyl) cyclohexyl) oxy) methyl) benzene (1.2 g, 76%). 1 H NMR(400MHz,CDCl 3 ):δppm:7.38–7.17(m,5H),4.72(s,2H),3.74(s,2H),3.39(s,3H),3.34(s,3H),3.30(s,3H),2.64–2.25(m,3H),2.22–1.75(m,3H)。
Step F:2- (benzyloxy) -4, 4-difluoro-2- (methoxymethyl) cyclohexan-1-one:
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((5, 5-difluoro-2, 2-dimethoxy-1- (methoxymethyl) cyclohexyl) oxy) methyl) benzene (1.33 g,4.03mmol,1.0 eq.) I 2 A mixture of (102 mg, 402. Mu. Mol,0.1 eq) and acetone (30.0 mL) was stirred at room temperature for 2 hours. The mixture was poured into water, saturated Na 2 S 2 O 3 (aqueous) quench and extract with DCM (60 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 0-5% EtOAc/PE) to give 2- (benzyloxy) -4, 4-difluoro-2- (methoxymethyl) cyclohexan-1-one (1.05 g, 91%). 1 H NMR(400MHz,CDCl 3 ):δppm:7.42–7.26(m,5H),4.60(dd,J＝11.4Hz,1H),4.40(dd,J＝11.4Hz,1H),3.68(m,2H),3.37(s,3H),2.92–2.62(m,2H),2.52–2.23(m,4H)。
Step G:6- (benzyloxy) -8, 8-difluoro-6- (methoxymethyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
3-bromo-5- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]A mixture of thiophene-2-carboxylic acid methyl ester (660 mg,1.6mmol,1.8 eq), 2- (benzyloxy) -4, 4-difluoro-2- (methoxymethyl) cyclohexane-1-one (250 mg, 880. Mu. Mol,1.0 eq), sodium bisulphite (50 mg, 263. Mu. Mol,0.3 eq), cesium carbonate (859 mg,2.64mmol,3.0 eq), sphos-Pd-G3 (137 mg, 175. Mu. Mol,0.20 eq) and toluene (50.0 mL) was stirred under nitrogen at 105℃for 16 hours. The mixture was poured into water and extracted with EtOAc (60 ml x 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (SiO 2 0-35% EtOAc/PE) to give 6- (benzyloxy) -8, 8-difluoro-6- (methoxymethyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (360 mg, 69%). MS observations (ESI) + )：589.5[(M+H) + ]。
Step H:6- (benzyloxy) -8, 8-difluoro-6- (methoxymethyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
6- (benzyloxy) -8, 8-difluoro-6- (methoxymethyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ]A mixture of chromen-4-one (180 mg, 305. Mu. Mol,1.0 eq), ammonium hydroxide (8.0 mL) and 2-propanol (8.0 mL) was stirred in a microwave reactor at 100deg.C for 4 hours. The mixture was concentrated in vacuo and the residue was purified by column chromatography (SiO 2 0-1% MeOH/DCM) to give 6- (benzyloxy) -8, 8-difluoro-6- (methoxymethyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (80 mg, 44%). MS observations (ESI) + )：588.5[(M+H) + ]。
Step I: (S) -8, 8-difluoro-6-hydroxy-6- (methoxymethyl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 48) and (R) -8, 8-difluoro-6-hydroxy-6- (methoxymethyl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 49):
boron trichloride (1M, 4.7mL,15 eq.) was added to 6- (benzyloxy) -8, 8-difluoro-6- (methoxymethyl) -2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] at 0deg.C]Quinolin-4 (5H) -one (190 mg, 320. Mu. Mol,1.0 eq) in DCM (25.0 mL). The mixture was stirred at room temperature for 2 hours, then quenched with MeOH, concentrated and quenched with NH 3 MeOH (7M) neutralization. The residue was purified by column chromatography (SiO 2 0-4% MeOH/DCM) to give racemic 8, 8-difluoro-6-hydroxy-6- (methoxymethyl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]]Quinolin-4 (5H) -one (64 mg, 53%). MS observations (ESI) + )：368.0[(M+H) + ]。
Each enantiomer was isolated by SFC. (S) -8, 8-difluoro-6-hydroxy-6- (methoxymethyl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 48): MS observations (ESI) + )：368.0[(M+H) + ]. (R) -8, 8-difluoro-6-hydroxy-6- (methoxymethyl) -2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 49): MS observations (ESI) + )：368.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δppm:13.23(s,1H),10.70(s,1H),8.30(s,1H),7.97(s,1H),7.58(s,1H),5.69(s,1H),3.60(dd,J＝45.2,10.0Hz,2H),3.29(s,3H),3.21–3.10(m,2H),2.77–2.61(m,1H),2.38–2.19(m,1H)。
Examples 50 and 51-compounds 50 and 51: (S) -6-ethyl-8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 50) and (R) -6-ethyl-8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 51)
Step A: 1-ethyl-5, 5-difluoro-2, 2-dimethoxycyclohexane-1-ol:
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to 5, 5-difluoro-2, 2-dimethoxy-1-vinylcyclohexane-1-ol (1.8 g,8.1mmol,1.0 eq.) in MeOH (90 mL) and H 2 To a solution in O (2.0 mL) was added wetted Pd/C (10%, 540 mg). The mixture was purged with nitrogen and then stirred under a hydrogen atmosphere at 50 ℃ for 16 hours. The reaction mixture was cooled to room temperature, filtered through celite, and the filter cake was washed with MeOH (100 mL). The filtrate was concentrated in vacuo and purified by flash column chromatography (SiO 2 10-50% EtOAc/PE) to give 1-ethyl-5, 5-difluoro-2, 2-dimethoxy-cyclohexane-1-ol (1.5 g, 82%). 1 H NMR(400MHz,CDCl 3 )δppm:3.39(s,3H),3.33(s,3H),1.99–1.62(m,8H),0.99(t,J＝7.6Hz,3H)。
And (B) step (B): 2-ethyl-4, 4-difluoro-2-hydroxycyclohexane-1-one:
to a solution of 1-ethyl-5, 5-difluoro-2, 2-dimethoxycyclohexane-1-ol (1.8 g,8.03mmol,1.0 eq.) in DCM (28.0 mL) was added water (7.0 mL) and trifluoroacetic acid (7.0 mL). The mixture was stirred at room temperature for 1 hour, and then concentrated. By column chromatography (SiO 2 Purification of 10-50% EtOAc/PE afforded 2-ethyl-4, 4-difluoro-2-hydroxycyclohexane-1-one (1.2 g, 85%). 1 H NMR(400MHz,CDCl 3 )δppm:2.84–2.70(m,2H),2.59–2.46(m,2H),2.29–2.11(m,2H),1.97–1.87(m,1H),1.84–1.74(m,1H),0.84(t,J＝7.2Hz,3H)。
Step C: 6-ethyl-8, 8-difluoro-6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
at N 2 To 2-ethyl-4, 4-difluoro-2-hydroxycyclohexane-1-one (205 mg,1.15mmol,2.0 eq.) and 3-bromo-5- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl under an atmosphere]Sodium bisulphite (22 mg,0.12mmol,0.2 eq.) Cs are added to a solution of methyl thiophene-2-carboxylate (240 mg,0.58mmol,1.0 eq.) in toluene (15.0 mL) 2 CO 3 (374 mg,1.15mmol,2.0 eq.) and Pd 2 (dba) 3 (105 mg,0.12mmol,0.2 eq.) Xantphos (100 mg,0.17mmol,0.3 eq.). The mixture was stirred at 105 ℃ for 16 hours, then filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 0-100% EtOAc/PE) to give 6-ethyl-8, 8-difluoro-6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (224 mg, 80%). MS observations (ESI) + )：483.7[(M+H) + ]。
Step D: 6-ethyl-8, 8-difluoro-6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
6-ethyl-8, 8-difluoro-6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (595 mg,1.23mmol,1.0 eq.) in MeOH (9 mL) and NH 4 The solution in the mixture of OH (9.0 mL) was heated in a microwave reactor at 100deg.C for 3 hours. The mixture was concentrated, and the residue was purified by column chromatography (SiO 2 0-10% MeOH/DCM) to give 6-ethyl-8, 8-difluoro-6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (360 mg, 60%). MS observations (ESI) + )：482.7[(M+H) + ]。
Step E: (S) -6-ethyl-8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 50) and (R) -6-ethyl-8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 51):
To 6-ethyl-8, 8-difluoro-6-hydroxy-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]To a solution of quinolin-4 (5H) -one (356 mg,0.74mmol,1.0 eq.) in DCM (5.0 mL) was added trifluoroacetic acid (5.0 mL). The mixture was stirred at room temperature for 1 hour, then concentrated in vacuo, and the residue was purified by column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 6-ethyl-8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ]]Quinolin-4 (5H) -one (140 mg, 54%). MS observations (ESI) + )：352.4[(M+H) + ]。
The individual isomers were separated by chiral SFC. (S) -6-ethyl-8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 50): MS observations (ESI) + )：352.2[(M+H) + ]. (R) -6-ethyl-8, 8-difluoro-6-hydroxy-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 51): MS observations (ESI) + )：352.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 )δppm:13.23(s,1H),10.74(s,1H),8.30(s,1H),8.00(s,1H),7.57(s,1H),5.33(s,1H),3.30–3.27(m,2H),2.68–2.54(m,1H),2.33–2.20(m,1H),1.96–1.87(m,1H),1.83–1.74(m,1H),0.89–0.84(m,3H)。
Examples 52 and 53-compounds 52 and 53: (S) -8, 8-difluoro-6-methoxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 52) and (R) -8, 8-difluoro-6-methoxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 53) (stereoisomers are arbitrarily specified)
Step A:5, 5-difluoro-2, 2-dimethoxy-1-methylcyclohexane-1-ol:
to a dry three-necked flask containing 5, 5-difluoro-2, 2-dimethoxycyclohexane-1-one (5.65 g,29.1mmol,1.0 eq.) in dry THF (270.0 mL) was added methyl magnesium bromide (3M in diethyl ether, 29.1mL,87.4mmol,3.0 eq.) at 0deg.C. The mixture is put under N 2 Stir at room temperature for 2 hours under atmosphere and monitor by TLC until completion. The mixture was treated with saturated NH at 0deg.C 4 Cl (aq), diluted with water (125 mL) and extracted with EtOAc (90 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and the residue was purified by column chromatography (SiO 2 0-20% EtOAc/PE) to give 5, 5-difluoro-2, 2-dimethoxy-1-methylcyclohexane-1-ol (5.98 g, 97%). 1 H NMR(400MHz,CDCl 3 )δppm:3.41(s,3H),3.32(s,3H),2.69(s,1H),2.20–2.06(m,2H),2.03–1.90(m,2H),1.85–1.68(m,2H),1.32(s,3H)。
And (B) step (B): 4, 4-difluoro-1, 2-trimethoxy-2-methylcyclohexane:
to a dry three-necked round bottom flask containing sodium hydride (2.28 g,56.7mmol,60% in mineral oil, 2.0 eq.) was added anhydrous THF (170.0 mL) at 0deg.C followed by 5, 5-difluoro-2, 2-dimethoxy-1-methylcyclohexane-1-ol (5,98 g,28.5mmol,1.0 eq.) in THF (3.0 mL)And (3) a mixture. The mixture was stirred at 0deg.C for 30 min, then methyl iodide (40.4 g, 284 mmol,10.0 eq.) was added and the mixture was stirred for an additional 1.5 h. To the reaction mixture was added additional methyl iodide (40.4 g, 284 mmol,10.0 eq.) at 0deg.C. The mixture was stirred for an additional 1.5 hours, then water at 0 ℃ was added. The mixture was extracted with EtOAc (160 ml x 2), and the combined organic layers were washed with brine (150 ml x 2), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. By column chromatography (SiO 2 Purification of 0-10% EtOAc/PE afforded 4, 4-difluoro-1, 2-trimethoxy-2-methylcyclohexane (5.1 g, 80%). 1 H NMR(400MHz,CDCl 3 )δppm:3.37(s,3H),3.33(s,3H),3.27(s,3H),2.27–2.20(m,1H),2.18–2.05(m,2H),2.00–1.86(m,2H),1.80–1.69(m,1H),1.29(s,3H)。
Step C:4, 4-difluoro-2-methoxy-2-methylcyclohexane-1-one:
to a flask containing DCM (28 mL) containing 4, 4-difluoro-1, 2-trimethoxy-2-methylcyclohexane (2.58 g,11.51 mmol) was added water (7 mL) and trifluoroacetic acid (7 mL) at 0deg.C. The mixture was stirred at room temperature for 5 hours, then extracted with DCM (30 ml x 2). The combined organic layers were concentrated in vacuo. The residue was diluted with DCM (20 mL), washed with aqueous NaHCO3 (10 mL x 2), and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and concentrated in vacuo to give 4, 4-difluoro-2-methoxy-2-methylcyclohexane-1-one (1.9 g,10.1mmol, 88%). 1 H NMR(400MHz,CDCl3)δppm:3.21(s,3H),2.91–2.85(m,1H),2.65–2.57(m,1H),2.48–2.41(m,2H),2.23–2.09(m,2H),1.30(s,3H)。
Step D:8, 8-difluoro-6-methoxy-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
to a solution of 3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (1.0 g,2.4 mmol) and 4, 4-difluoro-2-methoxy-2-methylcyclohexane-1-one (598 mg,3.35 mmol) in toluene (30.0 mL) was added Cs 2 CO 3 (2.34g，7.19mmol)、Pd 2 (dba) 3 (439 mg, 479. Mu. Mol) and Na 2 S 2 O 5 (91.1 mg, 479. Mu. Mol). The mixture was stirred under nitrogen at 105 ℃ for 12 hours. The mixture was cooled to 0℃and H was added 2 O (40 mL) and the resulting mixture was extracted with EtOAc (30 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. By column chromatography (SiO 2 0-10% EtOAc/PE) to give 8, 8-difluoro-6-methoxy-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]Chromen-4-one (385 mg, 33%). MS observations (ESI) + )：483.4[(M+H) + ]。
Step E:8, 8-difluoro-6-methoxy-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
to 8, 8-difluoro-6-methoxy-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]To a solution of chromen-4-one (0.70 g,1.45 mmol) in MeOH (6.0 mL) was added ammonium hydroxide (6.0 mL). The mixture was stirred in a microwave reactor at 95 ℃ for 2 hours. The mixture was cooled to 0℃and H was added 2 O (20 mL). The resulting mixture was extracted with EtOAc (50 ml x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by flash column chromatography (SiO 2 0-30% EtOAc/PE) to give 8, 8-difluoro-6-methoxy-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (170 mg, 24%). MS observations (ESI) + )：482.5[(M+H) + ]。
Step F: (S) -8, 8-difluoro-6-methoxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 52) and (R) -8, 8-difluoro-6-methoxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 53):
to 8, 8-difluoro-6-methoxy-6-methyl-2- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl at 0deg.C]-7, 9-dihydro-5H-thieno [2,3-c]To a solution of quinolin-4-one (170 mg, 353. Mu. Mol,1.0 eq.) in DCM (5.0 mL) was added trifluoroacetic acid (402 mg,3.53mmol, 272. Mu.L, 10.0 eq.). The mixture was stirred at 25℃for 15 hours. The mixture was then cooled to 0deg.C and saturated aqueous NaHCO was added dropwise at 0deg.C 3 (20 mL). The resulting mixture was extracted with EtOAc (20 ml x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 8, 8-difluoro-6-methoxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] ]Quinolin-4 (5H) -one (100 mg, 80%).
The individual enantiomers were separated by chiral SFC. (S) -8, 8-difluoro-6-methoxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 52): MS observations (ESI) + )：352.1[M+H] + . (R) -8, 8-difluoro-6-methoxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 53): MS observations (ESI) + )：352.1[M+H] + ； 1 H NMR(400MHz,DMSO-d6)δppm:13.23(s,1H),11.02(s,1H),8.28(s,1H),8.01(s,1H),7.59(s,1H),3.44–3.37(m,2H),3.07(s,3H)2.82–2.61(m,1H),2.40–2.26(m,1H),1.56(s,3H)。
Examples 54 and 55-compounds 54 and 55: (R) -8, 8-difluoro-6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 54) and (S) -8, 8-difluoro-6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 55) (stereoisomers are arbitrarily specified)
Step A: ((5, 5-difluoro-2, 2-dimethoxy-1-methylcyclohexyl) oxy) methyl) benzene:
to a solution of 5, 5-difluoro-2, 2-dimethoxy-1-methylcyclohexane-1-ol (300 mg,1.43mmol,1.0 eq.) in anhydrous DMF (3.0 mL) was added NaH (109 mg,2.85mmol,60% in mineral oil, 2.0 eq.) at 0 ℃. The mixture was stirred at 0deg.C for 10 min, then benzyl bromide (488.16 mg,2.85mmol,339 μL,2.0 eq.) was added dropwise. The mixture was stirred at room temperature for 1 hour, then saturated NH 4 The aqueous Cl solution was quenched and diluted with water (60 mL). The mixture was extracted with EtOAc (40 ml x 3) and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash column chromatography (SiO 2 0-4% EtOAc/PE) to give (((5, 5-difluoro-2, 2-dimethoxy-1-methylcyclohexyl) oxy) methyl) benzene (370 mg, 86%). 1 H NMR(400MHz,CDCl 3 )δppm:7.36–7.24(m,5H),4.60–4.52(m,2H),3.39(s,3H),3.33(s,3H),2.50–2.35(m,1H),2.26–2.22(m,1H),2.15–2.07(m,1H),2.05–1.89(m,2H),1.83–1.77(m,1H),1.38(s,3H)。
And (B) step (B): 2- (benzyloxy) -4, 4-difluoro-2-methylcyclohexane-1-one:
to (((5, 5-difluoro-2, 2-dimethoxy-1-methylcyclohexyl) oxy) methyl) benzene (370 mg,1.23mmol,1.0 eq.) in acetone (15.0 mL)) Iodine (31.7 mg, 125. Mu. Mol,0.10 eq.) was added to the solution in (B). The mixture was stirred at room temperature for 10 min, then passed through saturated Na 2 SO 3 (aqueous) (20 mL) was quenched and extracted with DCM (30 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 0-10% EtOAc/PE) to give 2- (benzyloxy) -4, 4-difluoro-2-methylcyclohexane-1-one (280 mg, 88%). 1 H NMR(400MHz,CDCl 3 )δppm:7.35–7.26(m,5H),4.56(d,J＝11.6Hz,1H),4.26(d,J＝11.6Hz,1H),2.94–2.91(m,1H),2.79–2.74(m,1H),2.48–2.42(m,2H),2.25–2.18(m,2H),1.41(s,3H)。
Step C:6- (benzyloxy) -8, 8-difluoro-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c ] chromen-4-one:
To a solution of 3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (240 mg, 575. Mu. Mol,1.0 eq) and 2- (benzyloxy) -4, 4-difluoro-2-methylcyclohexane-1-one (292 mg,1.15mmol,2.0 eq) in toluene (18.0 mL) was added Pd 2 (dba) 3 (105 mg, 115. Mu. Mol,0.20 eq.), xantphos (133 mg, 230. Mu. Mol,0.40 eq.) and cesium carbonate (562 mg,1.72mmol,3.0 eq.). The mixture was stirred under nitrogen at 105 ℃ for 16 hours, then cooled and filtered. The filtrate was concentrated in vacuo, and the residue was purified by column chromatography (SiO 2 0-16% EtOAc/PE) to give 6- (benzyloxy) -8, 8-difluoro-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2, 3-c)]Chromen-4-one (220 mg, 68%). MS observations (ESI) + )：559.5[(M+H) + ]。
Step D:6- (benzyloxy) -8, 8-difluoro-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one:
to 6- (benzyloxy) -8, 8-difluoro-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydro-4H-thieno [2,3-c]To a solution of chromen-4-one (220 mg, 394. Mu. Mol) in MeOH (7 mL) was added ammonia (7M in MeOH, 7.0 mL). The mixture was stirred at 95 ℃ for 16 hours, then cooled and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 0-5% MeOH/DCM) to give 6- (benzyloxy) -8, 8-difluoro-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (180 mg, 77%). MS observations (ESI) + )：558.6[(M+H) + ]。
Step E: (R) -8, 8-difluoro-6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 54) and (S) -8, 8-difluoro-6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 55) (stereoisomers are arbitrarily specified):
to 6- (benzyloxy) -8, 8-difluoro-6-methyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] at-5 ℃]To a solution of quinolin-4 (5H) -one (90 mg, 161. Mu. Mol,1.0 eq.) in DCM (10.0 mL) was added boron trichloride (1M, 2.0mL,12 eq.). The mixture was stirred at-5 ℃ for 1.5 hours, quenched with MeOH, and then concentrated under nitrogen flow. By gradual addition of NH 3 MeOH (7M) was used to adjust the pH to about 9. The mixture was then concentrated in vacuo and the residue was purified by flash column chromatography (SiO 2 0-10% MeOH/DCM) to give racemic 8, 8-difluoro-6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] ]Quinolin-4 (5H) -one (23 mg, 42%). MS observations (ESI) + )：338.3[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (R) -8, 8-difluoro-6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 54): MS observations (ESI) + )：338.0[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm:13.24 (s, 1H), 10.88 (s, 1H), 8.13 (s, 2H), 7.56 (s, 1H), 5.57 (s, 1H), 3.33-3.24 (m, 2H), 2.50-2.42 (m, 2H), 1.54 (s, 3H). (S) -8, 8-difluoro-6-hydroxy-6-methyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (compound 55): MS observations (ESI) + )：338.0[(M+H) + ]。
Examples 56 and 57-compounds 56 and 57: (S) -4- (2-hydroxypropan-2-yl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 56) and (R) -4- (2-hydroxypropan-2-yl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 57)
Step A: 4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,4,5, 6-tetrahydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridine-4-carbaldehyde:
4- (hydroxymethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ]Thieno [3,2-d]A mixture of pyridin-6 (4H) -one (100 mg, 223. Mu. Mol,1.0 eq. Synthesized according to the procedure of compounds 34 and 35), IBX (626 mg,2.23mmol,10.0 eq.) and DCM (12 mL) was stirred at 45℃for 24H. The mixture was taken up in saturated Na 2 SO 3 The aqueous solution was quenched and extracted with DCM (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (SiO 2 0-4% MeOH/DCM) to give 4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,4,5, 6-tetrahydro-2H-pyrano [3,4-b]Thieno [3,2-d]Pyridine-4-carbaldehyde (80 mg, 80%). MS observations (ESI) + )：464.3[(M+H 2 O+H) + ]。
And (B) step (B): 4- (1-hydroxyethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one:
to 4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,4,5, 6-tetrahydro-2H-pyrano [3,4-b]Thieno [3,2-d]To a solution of pyridine-4-carbaldehyde (160 mg, 360. Mu. Mol,1.0 eq) in THF (7.5 mL) was added methyl magnesium bromide (3M in diethyl ether, 23.3mL,195 eq). The mixture was stirred at room temperature for 1.5 hours with saturated NH 4 Cl (aq) was quenched, then poured into water and extracted with DCM (50 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (SiO 2 0-4% MeOH/DCM) to give 4- (1-hydroxyethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d]Pyridin-6 (4H) -one (152 mg, 82%). MS observations (ESI) + )：462.3[(M+H) + ]。
Step C: 4-acetyl-4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one:
to 4- (1-hydroxyethyl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] at 0deg.C]Thieno [3,2-d]Pyridin-6 (4H) -onesTo a solution of (126 mg, 273. Mu. Mol,1.0 eq.) in DCM (40.0 mL) was added, in small portions, dess-martin periodate (694 mg,1.64mmol,6.0 eq.). The mixture was stirred at room temperature for 1.5 hours, then saturated Na 2 SO 3 (aqueous) quench and extract with DCM (30 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 0-3% MeOH/DCM) to give 4-acetyl-4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d]Pyridin-6 (4H) -one (85 mg, 67%). MS observations (ESI) + )：460.3[(M+H) + ]。
Step D:4- (2-hydroxypropan-2-yl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one:
to 4-acetyl-4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d]To a solution of pyridin-6 (4H) -one (85 mg, 185. Mu. Mol,1.0 eq.) in THF (2.0 mL) was added methyl magnesium bromide (3M in diethyl ether, 2.0mL,32.4 eq.). The mixture was stirred at room temperature for 2 hours with saturated NH 4 Cl (aq) was quenched, then poured into water and extracted with DCM (30 mL. Times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (SiO 2 0-4% MeOH/DCM) to give 4- (2-hydroxypropan-2-yl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ]Thieno [3,2-d]Pyridin-6 (4H) -one (50 mg, 56%). MS observations (ESI) + )：476.3[(M+H) + ]。
Step E: (S) -4- (2-hydroxypropan-2-yl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 56) and (R) -4- (2-hydroxypropan-2-yl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] thieno [3,2-d ] pyridin-6 (4H) -one (compound 57):
to 4- (2-hydroxypropan-2-yl) -4-methyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b ] at 0deg.C]Thieno [3,2-d]To a solution of pyridin-6 (4H) -one (75 mg, 160. Mu. Mol,1.0 eq.) in DCM (6.0 mL) was added trifluoroacetic acid (2.0 mL). The mixture was allowed to warm to room temperature and stirred for 4 hours. The mixture was concentrated under a stream of nitrogen and NH was gradually added at 0 c 3 MeOH (7M) was used to adjust the pH to about 9. The mixture was concentrated to give a residue, which was purified by column chromatography (SiO 2 0-7% meoh/DCM) to give racemic 4- (2-hydroxypropan-2-yl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3, 4-b)]Thieno [3,2-d]Pyridin-6 (4H) -one (48 mg, 85%). MS observations (ESI) + )：346.0[(M+H) + ]。
The individual enantiomers were separated by chiral SFC. (S) -4- (2-hydroxypropan-2-yl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d]Pyridin-6 (4H) -one (compound 56): MS observations (ESI) + )：346.2[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm:13.22 (s, 1H), 10.21 (s, 1H), 8.31 (s, 1H), 7.97 (s, 1H), 7.51 (s, 1H), 5.98 (s, 1H), 4.02-3.89 (m, 2H), 2.76-2.60 (m, 2H), 1.53 (s, 3H), 1.27 (s, 3H), 1.00 (s, 3H). (R) -4- (2-hydroxypropan-2-yl) -4-methyl-8- (1H-pyrazol-4-yl) -1, 5-dihydro-2H-pyrano [3,4-b]Thieno [3,2-d]Pyridin-6 (4H) -one (compound 57): MS observations (ESI) + )：346.2[(M+H) + ]。
Example 58-compound 58:4, 4-dimethyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one
Step A:4, 4-dimethyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (100 mg, 240. Mu. Mol,1.0 eq) was dissolved in DMF (10.0 mL) and 3, 3-dimethyltetrahydropyran-4-one (61 mg, 480. Mu. Mol,2.0 eq), na was then added 2 S 2 O 5 (9.0 mg, 48. Mu. Mol,0.20 eq.) Cs 2 CO 3 (230 mg, 720. Mu. Mol,3.0 eq.) and BINAP-Pd-G3 (24 mg, 24. Mu. Mol,0.10 eq.). The mixture was stirred at 105 ℃ for 16 hours, cooled, filtered and concentrated. By column chromatography (SiO 2 Purification with 0-4% MeOH/DCM gave 4, 4-dimethyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (20 mg, 19%). MS observations (ESI) + )：433.4[(M+H) + ]。
And (B) step (B): 4, 4-dimethyl-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
4, 4-dimethyl-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]A mixture of pyran-6-one (45 mg, 100. Mu. Mol), DCM (5.0 mL) and trifluoroacetic acid (1.5 mL) was stirred at room temperature for 2 hours. The mixture was concentrated in vacuo to give 4, 4-dimethyl-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (40 mg), which was used without further purification. MS observations (ESI) + )：303.2[(M+H) + ]。
Step C:4, 4-dimethyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
4, 4-dimethyl-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ]Thieno [3,2-d]Pyran-6-one (40 mg, crude product), NH 4 A mixture of OH (5.0 mL) and MeOH (5.0 mL) was heated to 100deg.C in a microwave reactor for 4 hours. The mixture was concentrated in vacuo and the residue was purified by column chromatography (SiO 2 0-5% MeOH/DCM) to give 4, 4-dimethyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (9.0 mg, 36%). MS observations (ESI) + )：302.2[(M+H) + ]； 1 HNMR(400MHz,DMSO-d 6 )δppm:13.21(brs,1H),11.20(s,1H),8.29(s,1H),7.94(s,1H),7.37(s,1H),4.69(s,2H),3.57(s,2H),1.26(s,6H)。
Examples 59 and 60-compounds 59 and 60: (S) -4- (tert-butyl) -8- (3-fluoropyridin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 59) and (R) -4- (tert-butyl) -8- (3-fluoropyridin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 60) (the stereochemistry of compounds 59 and 60 is arbitrarily specified)
Step A:4, 4-dimethoxytetrahydro-2H-pyran-3-ol:
a solution of tetrahydropyran-4-one (40.0 g,400mmol,1.0 eq.) and potassium hydroxide (67.3 g,1.20mol,3.0 eq.) in MeOH (1.7L) was cooled to 0deg.C and slowed downAddition of I 2 (117 g,459mmol,1.15 eq). The reaction mixture was warmed to room temperature and after 4 hours the mixture was concentrated. The crude product was triturated with EtOAc (300 mL) to give 4, 4-dimethoxytetrahydro-2H-pyran-3-ol (50 g, used without further purification). 1 H NMR(400MHz,DMSO-d6)δ4.75(s,1H),3.68–3.54(m,2H),3.52–3.41(m,2H),3.32–3.25(m,1H),3.12(s,3H),3.11(s,3H),1.84–1.76(m,1H),1.62–1.57(m,1H)。
And (B) step (B): 4, 4-dimethoxytetrahydro-2H-pyran-3 (4H) -one:
a solution of 4, 4-dimethoxytetrahydropyran-3-ol (40.0 g,247mmol,1.0 eq.) in DCM (900 mL) was cooled to 0deg.C and DMP (157 g,370mmol,1.5 eq.) was added. The mixture was heated to 30 ℃ for 16 hours, then a mixture of petroleum ether and EtOAc (8:1, 400 mL) was added. The filtrate was concentrated and purified (SiO 2 5% petroleum ether/EtOAc) to give 4, 4-dimethoxytetrahydro-2H-pyran-3 (4H) -one (23 g,45%,2 steps) as a pale yellow solid.
1 H NMR(400MHz,DMSO-d6)δ3.99(s,2H),3.87–3.80(m,2H),3.14(s,6H),2.16–2.10(m,2H)。
Step C:3- (tert-butyl) -3-hydroxytetrahydro-4H-pyran-4-one:
a solution of 4, 4-dimethoxytetrahydropyran-3-one (10.0 g,62.4mmol,1.0 eq.) in anhydrous THF (100 mL) was cooled to-78deg.C and t-BuLi (1.3M in THF, 62mL,1.3 eq.) was added dropwise over 30 minutes. The mixture was stirred at-78 ℃ for 2 hours, then HCl (2M aqueous solution, 94 mL) was added dropwise over 15 minutes. After the addition, the mixture was stirred at 0 ℃ for 2 hours. The mixture was extracted with EtOAc (3 x 100 ml), washed with brine (2 x 100 ml), and the combined organic layers were concentrated and purified (SiO 2 0-10% EtOAc/PE) to give 3- (tert-butyl) -3-hydroxytetrahydro-4H-pyran-4-one (3.6 g, 33%). 1 H NMR(400MHz,DMSO-d6)δ5.05(s,1H),4.06(dd,J＝12.0,1.2Hz,1H),4.03–3.95(m,1H),3.75–3.64(m,1H),3.35(d,J＝12.0Hz,1H),2.60–2.52(m,2H),0.95(s,9H)。
Step D:4- (tert-butyl) -4-hydroxy-3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to a dry three-necked flask containing methyl 3-bromothiophene-2-carboxylate (2 g,9.05mmol,1.0 eq) and 3-tert-butyl-3-hydroxy-tetrahydropyran-4-one (1.56 g,9.05mmol,1.0 eq) was added anhydrous toluene (50 mL). The mixture was degassed and used with N 2 (3 x) purge, then Pd was added 2 (dba) 3 (414 mg, 452. Mu. Mol,0.050 eq.), xantphos (524 mg, 905. Mu. Mol, 0.10 eq.), K 3 PO 4 (3.84 g,18.1mmol,2.0 eq.). Degassing it and re-using N 2 (3 x) purging and stirring at 105 ℃ for 16 hours. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated. By column chromatography (SiO 2 0-30% EtOAc/PE) and purified by reverse phase column chromatography (C18 SiO 2 0-30% MeCN/water (0.1% NH) 4 OH)) to give 4- (tert-butyl) -4-hydroxy-3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] as a white solid]Thieno [3,2-d]Pyran-6-one (600 mg,2.14mmol, 24%). MS observations (ESI) + )：281.2[(M+H) + ]。
Step E: (4- (tert-butyl) -4-hydroxy-6-oxo-4, 6-dihydro-1 h,3 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-8-yl) boronic acid:
4- (tert-butyl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4,3-b ]Thieno [3,2-d]Pyran-6-one (500 mg,1.78mmol,1.0 eq.) 4,a mixture of 4, 5-tetramethyl-1, 3, 2-dioxaborolan (1.37 g,10.7mmol,1.55mL,6.0 eq.) iridium dimer (59.1 mg, 89.2. Mu. Mol,0.050 eq.), 2, 6-diisopropyl-N- (pyridin-2-ylmethylene) aniline (47.5 mg, 178. Mu. Mol,0.10 eq.) and anhydrous DME (10 mL) was stirred at 85deg.C for 24 hours. The mixture was cooled and concentrated in vacuo to give (4- (tert-butyl) -4-hydroxy-6-oxo-4, 6-dihydro-1 h,3 h-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-8-yl) boronic acid (2 g, crude product) which is used without further purification. MS observations (ESI) + )：325.5[(M+H) + ]。
Step F: 10-tert-butyl-4- (3-fluoro-4-pyridinyl) -10-hydroxy-8, 12-dioxa-5-thiatricyclo [7.4.0.0 ] 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one:
to (10-tert-butyl-10-hydroxy-7-oxo-8, 12-dioxa-5-thiotricyclo [ 7.4.0.0) 2,6 ]Tridec-1 (9), 2 (6), 3-trien-4-yl) boronic acid (130 mg, 401. Mu. Mol,1.0 eq.) Na 2 CO 3 (128 mg,1.20mmol,3.0 eq.) in THF (12 mL) and H 2 To a mixture of O (1.2 mL) was added 3-fluoro-4-iodo-pyridine (99 mg, 441. Mu. Mol,1.1 eq.) and Sphos-Pd-G3 (32 mg, 40.1. Mu. Mol,0.10 eq.). The mixture was degassed and used with N 2 (2 x) purging, then stirring at 50℃for 6 hours. The reaction mixture was concentrated and the residue was diluted with DCM/meoh=10/1 (300 mL). The mixture was filtered and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (SiO 2 0-5% MeOH/DCM) to give 10-tert-butyl-4- (3-fluoro-4-pyridinyl) -10-hydroxy-8, 12-dioxa-5-thiatricyclo [7.4.0.0 ] as a yellow solid 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (280 mg, 23%). MS observations (ESI) + )：376.4[(M+H) + ]。
Step G: (S) -4- (tert-butyl) -8- (3-fluoropyridin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 59) and (R) -4- (tert-butyl) -8- (3-fluoropyridin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 60):
10-tert-butyl-4- (3-fluoro-4-pyridinyl) -10-hydroxy-8, 12-dioxa-5-thiatricyclo [7.4.0.0 ] 2 ,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (140 mg, 373. Mu. Mol,1.0 eq.) in NH 3 A solution in MeOH (7.0M, 10 mL) was stirred at 95℃for 3 hours. The reaction mixture was cooled, concentrated in vacuo to give a residue, which was purified by flash column chromatography (SiO 2 0-5% MeOH/DCM) to give 4- (tert-butyl) -8- (3-fluoropyridin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4, 3-b) ]Thieno [3,2-d]Pyridin-6-one (123 mg, 44%). MS observations (ESI) + )：375.0[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4- (tert-butyl) -8- (3-fluoropyridin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyridin-6-one (compound 59): MS observations (ESI) + )：375.0[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm 10.41 (s, 1H), 8.78 (d, J=2.8 Hz, 1H), 8.57 (dd, J=5.2, 0.8Hz, 1H), 8.08-7.99 (m, 2H), 5.36 (s, 1H), 4.82 (dd, J=44.0, 15.2Hz, 2H), 4.14 (d, J=11.2 Hz, 1H), 3.46 (d, J=11.2 Hz, 1H), 1.04 (s, 10H). (R) -4- (tert-butyl) -8- (3-fluoropyridin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyridin-6-one (compound 60): MS observations (ESI) + )：375.0[(M+H) + ]。
Examples 61 and 62-compounds 61 and 62: (S) -4- (tert-butyl) -4-hydroxy-8- (2-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 61) and (R) -4- (tert-butyl) -4-hydroxy-8- (2-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 62) (the stereochemistry of compounds 61 and 62 is arbitrarily specified)
Step A: 10-tert-butyl-10-hydroxy-4- (2-methyl-4-pyridinyl) -8, 12-dioxa-5-thiatricyclo [7.4.0.0 ] 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one:
subjecting 4-iodo-2-methyl-pyridine to conditions similar to those described in step F of compound 59 to give 10-tert-butyl-10-hydroxy-4- (2-methyl-4-pyridinyl) -8, 12-dioxa-5-thiatricyclo [7.4.0.0 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (169 mg, 29%). MS observations (ESI) + )：372.2[M+H] + 。
And (B) step (B): (S) -4- (tert-butyl) -4-hydroxy-8- (2-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 61) and (R) -4- (tert-butyl) -4-hydroxy-8- (2-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 62):
10-tert-butyl-10-hydroxy-4- (2-methyl-4-pyridinyl) -8, 12-dioxa-5-thiatricyclo [7.4.0.0 ] 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one is subjected to conditions similar to those described in step G of compound 59 to give 4- (tert-butyl) -4-hydroxy-8- (2-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyridin-6-one (200 mg, 78%). MS observations (ESI) + )：371.1[M+H] + 。
Enantiomers were separated by chiral SFC. (S) -4- (tert-butyl) -4-hydroxy-8- (2-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d ]Pyridin-6-one (compound 61): MS observations (ESI) + )：371.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δ10.32 (s, 1H), 8.55 (d, j=5.2 hz, 1H), 7.99 (s, 1H), 7.70 (s, 1H), 7.61 (dd, j=5.2, 1.6hz, 1H), 5.35 (s, 1H), 4.80 (dd, j=38.8, 14.8hz, 2H), 4.14 (d, j=11.2 hz, 1H), 3.45 (d, j=11.2 hz, 1H), 2.55 (s, 3H), 1.03 (s, 9H). (R) -4- (tert-butyl) -4-hydroxy-8- (2-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 62): MS observations (ESI) + )：371.0[(M+H) + ]。
Examples 63 and 64-compounds 63 and 64: (S) -4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 63) and (R) -4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 64) (the stereochemistry of compounds 63 and 64 is arbitrarily specified)
Step A: 5-chloro-4-iodopyrimidine:
to 5-chloropyrimidin-4-amine (500 mg,3.86mmol,1.0 eq.) and diiodomethane (2.07 g,7.72mmol, 622. Mu.L, 2.0 eq.) at 0deg.C in CH 3 To a solution of CN (20 mL) was added dropwise CH containing isoamyl nitrite (995 mg,8.49mmol,1.14mL,2.2 eq.) 3 CN (1 mL). The mixture was stirred at 0 ℃ for 30 minutes and then heated to 70 ℃ for 16 hours. The reaction mixture was concentrated in vacuo and the crude residue was purified by flash column chromatography (SiO 2 0-3% EtOAc/PE) to give 5-chloro-4-iodopyrimidine (410 mg,44% yield). MS observations (ESI) + )：241.1[(M+H) + ]。
And (B) step (B): 4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
similar conditions as described in step F of Compound 59 were used for (4- (tert-butyl) -4-hydroxy-6-oxo-4, 6-dihydro-1H, 3H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-8-yl) boronic acid and 5-chloro-4-iodopyrimidine to give 4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyran-6-one (76 mg, 31%). MS observations (ESI) + )：393.4[(M+H) + ]。
Step C: (S) -4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 63) and (R) -4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 64):
similar conditions as described in step G of Compound 59 were used for 4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one to give 4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] as a yellow solid ]Thieno [3,2-d]Pyridin-6-one (53 mg, 70%). MS observations (ESI) + )：392.4[(M+H) + ]。
Enantiomers were separated using chiral SFC. (S) -4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 63): MS observations (ESI) + )：392.0[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm 10.46 (s, 1H), 9.22 (s, 1H), 9.10 (s, 1H), 8.33 (s, 1H), 5.36 (s, 1H), 4.93-4.76 (m, 2H), 4.14 (d, J=11.2 Hz, 1H), 3.46 (d, J=11.6 Hz, 1H), 1.04 (s, 9H). (R) -4- (tert-butyl) -8- (5-chloropyrimidin-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 64): MS observations (ESI) + )：392.0[(M+H) + ]。
Examples 65 and 66-compounds 65 and 66: (R) -4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 65) and (S) -4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 66) (the stereochemistry of compounds 65 and 66 is arbitrarily specified)
Step A:4- (tert-butyl) -8- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
Subjecting 4-bromo-3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole to conditions similar to those described in step D of compound 59 to give 4- (tert-butyl) -8- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyran-6-one (10 mg, 13%). MS observations (ESI) + )：495.5[(M+H) + ]。
And (B) step (B): 4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to 4- (tert-butyl) -8- (3-fluoro-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4,3-b ] at-78deg.C]Thieno [3,2-d]To a solution of pyran-6-one (100 mg, 202. Mu. Mol,1.0 eq.) in DCM (5 mL) was added boron trichloride (1.0M, 1.0mL,5.0 eq.). The mixture was stirred at-78 ℃ for 2 hours, then MeOH was added. The mixture was placed in vacuoConcentrating to give 4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (75 mg, used without further purification). MS observations (ESI) + )：365.5[(M+H) + ]。
Step C: (R) -4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 65) and (S) -4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 66):
4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one is subjected to conditions similar to those described in Compound 59, step G, to give 4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (40 mg, 110. Mu. Mol,45%,2 steps). MS observations (ESI) + )：364.4[(M+H) + ]。
Isomers were separated by chiral HPLC. (R) -4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 65): MS observations (ESI) + )：364.0[(M+H) + ]。 1 H NMR (400 mhz, dmso-d 6) delta ppm:12.83 (brs, 1H), 10.16 (brs, 1H), 8.32 (d, j=1.8 hz, 1H), 7.33 (s, 1H), 5.33 (s, 1H), 4.75 (dd, j=34.2, 14.8hz, 2H), 4.12 (d, j=11.2 hz, 1H), 3.44 (d, j=11.2 hz, 1H), 1.03 (s, 9H). (S) -4- (tert-butyl) -8- (3-fluoro-1H-pyrazol-4-yl) -4-hydroxy-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 66): MS observations (ESI) + )：364.0[(M+H) + ]。
Examples 67 and 68-compounds 67 and 68: (S) -4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 67) and (R) -4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 68) (the stereochemistry of compounds 67 and 68 is arbitrarily specified)
Step A:4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
similar conditions as described in step F of compound 59 were used for 4-bromopyrazine hydrobromide to give 4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyran-6-one (53 mg, 40%). MS observations (ESI) + )：359.4[(M+H) + ]。
And (B) step (B): (S) -4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 67) and (R) -4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 68):
similar conditions as described in step G of Compound 59 were used for 4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one to give 4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyridin-6-one (37 mg, 66%). MS observations (ESI) + )：358.4[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 67): MS observations (ESI) + )：358.0[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm 10.45 (s, 1H), 9.76-9.75 (m, 1H), 9.35-9.33 (m, 1H), 8.22 (s, 1H), 8.08-8.06 (m, 1H), 5.37 (s, 1H), 4.87-4.75 (m, 2H), 4.14 (d, J=11.6 Hz, 1H), 3.46 (d, J=11.2 Hz, 1H), 1.03 (s, 9H). (R) -4- (tert-butyl) -4-hydroxy-8- (pyridazin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 68): MS observations (ESI) + )：358.0[(M+H) + ]。
Examples 69 and 70-compounds 69 and 70: (S) -4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 69) and (R) -4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 70) (the stereochemistry of compounds 69 and 70 is arbitrarily specified)
Step A:4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
similar conditions as described in step F of compound 59 were used for 4-bromo-3-methyl-pyridine hydrochloride to give 4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] as a yellow solid]Thieno [3,2-d]Pyran-6-one (87 mg, 63%). MS observations (ESI) + )：372.4[(M+H) + ]。
And (B) step (B): (S) -4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 69) and (R) -4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 70):
similar conditions as described in step G of Compound 59 were used for 4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one to give 4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (36 mg, 41%). MS observations (ESI) + )：371.4[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 69): MS observations (ESI) + )：371.0[(M+H) + ]。 1 H NMR (400 MHz, DMSO-d 6) delta ppm 10.30 (s, 1H), 8.60 (s, 1H), 8.52 (d, J=4.8 Hz, 1H), 7.60 (s, 1H), 7.55 (d, J=4.8 Hz, 1H), 5.35 (s, 1H), 4.87-4.72 (m, 2H), 4.14 (d, J=11.2 Hz, 1H), 3.45 (d, J=11.2 Hz, 1H), 1.04 (s, 9H). (R) -4- (tert-butyl) -4-hydroxy-8- (3-methylpyridin-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]Thieno [3,2-d]Pyridin-6-one (compound 70) (27.2 mg, 29%). MS observations (ESI) + )：371.0[(M+H) + ]。
Example 71-compound 71: 4-methyl-8- (1H-pyrazol-4-yl) -4- (pyrrolidin-1-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
step A: 4-hydroxy-4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
to a compound containing 4- (4-hydroxy-4-methyl-6-oxo-3, 4,5, 6-tetrahydro-1H-pyrano [4,3-b ]]Thieno [3,2-d]Pyridin-8-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (500 mg,1.29mmol,1.0 eq.) and its use in the preparation of medicamentsTo a dry three-necked flask of (2) was added THF (50 mL), and the mixture was degassed and N was used 2 (3 x) purge. Methyl magnesium bromide (1M, 25.8mL,20.0 eq.) was added at 0deg.C and then warmed to room temperature. After 2 hours, NH was added 4 Cl solution (13 mL) and the mixture was extracted with EtOAc (3X 50 mL). The combined organic layers were dried (MgSO 4 ) Filtered and concentrated to give a residue, which was purified by flash column chromatography (SiO 2 0-8% MeOH/DCM) to give 4-hydroxy-4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (340 mg, 69%). MS observations (ESI) + )：304.1[(M+H) + ]。
And (B) step (B): 4-chloro-4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
oxalyl chloride (15 mL) and N, N-dimethylformamide (0.1 mL) were added to a solution of 4-hydroxy-4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (340 mg,1.12mmol,1.0 eq.) in DCM (50 mL). The reaction mixture was heated to 40 ℃ and stirred for 16 hours. The mixture was concentrated in vacuo to give 4-chloro-4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (280 mg, which was used without further purification).
Step C: 4-methyl-8- (1H-pyrazol-4-yl) -4- (pyrrolidin-1-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 71):
4-chloro-4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]A solution of pyridin-6-one (280 mg, crude product) in acetonitrile (40 mL) was cooled to 0deg.C and then pyrithione was added dropwisePyrrolidine (15 mL). The reaction mixture was warmed to room temperature for 16 hours, then concentrated and purified by reverse phase column chromatography (0-20% MeCN/water (0.1% FA): by preparative TLC (SiO 2 0-10% MeOH/DCM) to give 4-methyl-8- (1H-pyrazol-4-yl) -4- (pyrrolidin-1-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (compound 71) (2.2 mg, 17%). MS observations (ESI) + )：357.2[(M+H) + ]。 1 H NMR(400MHz,DMSO-d6)δppm:8.11(s,2H),7.40(s,1H),4.74(d,J＝14.2Hz,1H),4.61(d,J＝14.2Hz,1H),4.12(d,J＝11.6Hz,1H),3.58(d,J＝11.8Hz,1H),2.80(d,J＝6.4Hz,2H),2.44–2.38(m,1H),2.06–1.92(m,1H),1.65(s,4H),1.42(s,3H)。
Examples 72 and 73-compounds 72 and 73: (S) -4-hydroxy-4- (prop-1-yn-1-yl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 72) and (R) -4-hydroxy-4- (prop-1-yn-1-yl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 73) (stereochemistry of compounds 72 and 73 is arbitrarily specified)
To 4- (7, 10-dioxo-12-oxa-5-thia-8-azatricyclo [7.4.0.0 ] at 0 ℃C 2,6 ]To a solution of trideca-1 (9), 2 (6), 3-trien-4-yl) pyrazole-1-carboxylic acid tert-butyl ester (100 mg, 258. Mu. Mol,1.0 eq.) in 1, 4-dioxane (20 mL) was added prop-1-yn-1-yl magnesium bromide (0.5M, 10mL,20 eq.). The mixture was stirred at 0deg.C for 1 hour, then H was slowly added at 0deg.C 2 O (20 mL). The resulting mixture was extracted with EtOAc (3 x 350 ml) and the combined organic phases were dried (Na 2 SO 4 ) Filtered and concentrated in vacuo. The crude product was purified by flash column chromatography on silica gel (SiO 2 0-10% MeOH/DCM) to give 4-hydroxy-4- (prop-1-yn-1-yl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (110 mg, 43%). MS observations (ESI) + )：328.0[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4-hydroxy-4- (prop-1-yn-1-yl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 72): MS observations (ESI) + )：328.1[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 13.23 (s, 1H), 10.90 (s, 1H), 8.29 (s, 1H), 7.97 (s, 1H), 7.43 (s, 1H), 6.20 (s, 1H), 4.74 (s, 2H), 3.94 (d, J=11.2 Hz, 1H), 3.76 (d, J=11.2 Hz, 1H), 1.86 (s, 3H). (R) -4-hydroxy-4- (prop-1-yn-1-yl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 73): MS observations (ESI) + )：327.8[(M+H) + ]。
Examples 74, 75, 76 and 77-compounds 74, 75, 76 and 77: (S) -4- ((S) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 74), (R) -4- ((R) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 75), (S) -4- ((R) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 76) and (R) -4- ((S) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [ 4-b ] thieno [3,2-d ] pyridin-6-one (compound 75), (S) -4- ((R) -4-hydroxy-6-H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 77), and the stereochemical compound 77 are indicated as compounds of any of (77)
Procedures analogous to step A of compound 72 were performed using tert-amyl magnesium chloride (1M in THF, 7.74mL,30 eq.) to give 4- (sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (35.2 mg, 39%). MS observations (ESI) + )：346.2[(M+H) + ]。
The four diastereomers were separated by chiral SFC. (S) -4- ((S) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 74): MS observations (ESI) + )：346.2[(M+H) + ]。 1 H NMR (400 MHz, DMSO-d 6) delta ppm:13.22 (s, 1H), 10.80 (s, 1H), 8.28 (brs, 1H), 7.96 (brs, 1H), 7.42 (s, 1H), 5.27 (s, 1H), 4.77-4.53 (m, 2H), 3.92 (d, J=11.6 Hz, 1H), 3.56 (d, J=11.6 Hz, 1H), 2.12-2.07 (m, 1H), 1.87-1.80 (m, 1H), 1.09-0.97 (m, 1H), 0.92 (t, J=7.2 Hz, 3H), 0.72 (d, J=7.2 Hz, 3H). (R) -4- ((R) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 75): MS observations (ESI) + )：346.0[(M+H) + ]。 1 H NMR (400 MHz, DMSO-d 6) delta ppm:13.22 (s, 1H), 10.82 (s, 1H), 8.28 (brs, 1H), 7.96 (brs, 1H), 7.41 (s, 1H), 5.24 (s, 1H), 4.73-4.53 (m, 2H), 3.93 (d, J=11.6 Hz, 1H), 3.54 (d, J=11.6 Hz, 1H), 2.13-2.02 (m, 1H), 1.15-1.05 (m, 2H), 0.98 (d, J=7.2 Hz, 3H), 0.77 (t, J=7.2 Hz, 3H). (S) -4- ((R) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]Thieno [3,2-d]Pyridin-6-one (compound 76): MS observations (ESI) + )：346.0[(M+H) + ]。 1 H NMR (400 MHz, DMSO-d 6) delta ppm:13.22 (s, 1H), 10.82 (s, 1H), 8.09 (brs, 2H), 7.41 (s, 1H), 5.25 (s, 1H), 4.72-4.62 (m, 2H), 3.93 (d, J=11.6 Hz, 1H), 3.54 (d, J=11.6 Hz, 1H), 2.13-2.02 (m, 1H), 1.13-1.09 (m, 2H), 0.98 (d, J=7.2 Hz, 3H), 0.76 (d, J=7.2 Hz, 3H). (R) -4- ((S) -sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 77): MS observations (ESI) + )：346.0[(M+H) + ]。 1 H NMR(400MHz,DMSO-d6)δppm:13.22(s,1H),10.81(s,1H),8.27(brs,1H),7.96(brs,1H),7.42(s,1H),5.27(s,1H),4.65(t,J＝9.2Hz,2H),3.92(d,J＝11.6Hz,1H),3.56(d,J＝11.6Hz,1H),2.14–2.03(m,1H),1.91–1.82(m,1H),0.97–1.10(m,1H),0.92(t,J＝7.2Hz,3H),0.72(d,J＝7.2Hz,3H)。
Examples 78 and 79-compounds 78 and 79: (S) -4-cyclopentyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 78) and (R) -4-cyclopentyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 79) (the stereochemistry of compounds 78 and 79 is arbitrarily specified)
Cyclopentylmagnesium bromide (17 mmol,17mL,10 eq.) was added rapidly to 4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b) at room temperature]Thieno [3,2-d]Pyridine-8-yl) -1H-pyrazole-1-carboxylic acid tert-butyl ester (1.0 g,1.73mmol,1.0 eq.) in THF (100 mL). The reaction mixture was degassed and used with N 2 Purged and stirred at room temperature for 2 hours. Adding NH 4 Cl solution (160 mL) and the mixture was extracted with EtOAc (3X 40 mL). The combined organic layers were dried (Na 2 SO 4 ) Filtered and concentrated. Column chromatography (SiO) 2 0-5% MeOH/DCM) followed by reverse phase column chromatography (C18-SiO) 2 0-20% MeCN/water (0.1% FA)) to give 4-cyclopentyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]]Thieno [3,2-d]Pyridin-6-one (130 mg, 14%). MS observations (ESI) + )：358.1[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4-cyclopentyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 78): MS observations (ESI) + )：357.8[(M+H) + ]。 1 H NMR (400 MHz, DMSO-d 6) delta ppm 13.23 (s, 1H), 10.62 (s, 1H), 8.11 (brs, 2H), 7.40 (s, 1H), 5.29 (s, 1H), 4.71 (q, J=14.4 Hz, 2H), 3.90 (d, J=11.2 Hz, 1H), 3.57 (d, J=11.2 Hz, 1H), 1.69-1.37 (m, 9H). (R) -4-cyclopentyl-4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 79): MS observations (ESI) + )：357.8[(M+H) + ]。
Examples 80 and 81-compounds 80 and 81: (S) -4-hydroxy-4- (tert-amyl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 80) and (R) -4-hydroxy-4- (tert-amyl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 81) (the stereochemistry of compounds 80 and 81 is arbitrarily specified)
Procedures analogous to step A of Compound 72 were performed using magnesium chloride (1, 1-dimethylpropyl) to give 4- (sec-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (30 mg, 32%). MS observations (ESI) + )：360.2[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4-hydroxy-4- (tert-amyl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 80): MS observations (ESI) + )：360.2[(M+H) + ]； 1 H NMR (400 mhz, dmso) delta 13.23 (s, 1H), 9.99 (s, 1H), 8.29 (brs, 1H), 7.96 (brs, 1H), 7.40 (s, 1H), 5.31 (s, 1H), 4.74 (q, j=14.8 hz, 2H), 4.17 (d, j=11.4 hz, 1H), 3.44 (d, j=11.4 hz, 1H), 1.63-1.56 (m, 1H), 1.33-1.38 (m, 1H), 0.96 (d, j=6.0 hz, 7H), 0.76 (s, 2H). (R) -4-hydroxy-4- (tert-amyl) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 81): MS observations (ESI) + )：360.2[(M+H) + ]。
Examples 82 and 83-compounds 82 and 83: (S) -4- (dimethylamino) -4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 82) and (R) -4- (dimethylamino) -4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 83) (the stereochemistry of compounds 82 and 83 is arbitrarily specified)
Step A: n- (4, 4-dimethoxy dihydro-2H-pyran-3 (4H) -ylidene) -2-methylpropane-2-sulfinamide:
to 2-methylpropane-2-sulfonylTo a solution of amine (15.2 g,125mmol,2.0 eq.) and 4, 4-dimethoxytetrahydropyran-3-one (10 g,62mmol,1.0 eq.) in THF (150 mL) was added Ti (EtO) 4 (28.6 g,125mmol,2.0 eq.). The mixture was stirred at 70 ℃ for 16 hours and then cooled. Water (100 mL) was added and the biphasic mixture was extracted with EtOAc (100 mL. Times.3). The collected organic phase was washed with brine (100 mL), dried (Na 2 SO 4 ) And concentrated. The residue was purified by flash column chromatography on silica gel (SiO 2 0-60% EtOAc/PE) to give N- (4, 4-dimethoxytetrahydropyran-3-ylidene) -2-methyl-propane-2-sulfonamide (8.4 g, 51%) as a colorless oil. MS observations (ESI) + )：264.1[(M+H) + ]。
And (B) step (B): n- (4, 4-dimethoxy-3-methyltetrahydro-2H-pyran-3-yl) -2-methylpropan-2-sulfinamide:
a solution of N- (4, 4-dimethoxytetrahydropyran-3-ylidene) -2-methyl-propane-2-sulfonamide (8.4 g,32mmol,1.0 eq.) in THF (150 mL) was cooled to 0deg.C and MeMgBr (1.0M, 96mL,96mmol,3.0 eq.) was added dropwise. The mixture was stirred at 0 ℃ for 0.5 hours and at room temperature for 3 hours. Adding saturated NH 4 Cl solution (100 mL) and the biphasic mixture was extracted with EtOAc (100 mL. Times.3) and the combined organic layers were concentrated. The residue was purified by flash column chromatography on silica gel (SiO 2 0-70% EtOAc/PE) to give N- (4, 4-dimethoxy-3-methyl-tetrahydropyran-3-yl) -2-methyl-propane-2-sulfinamide (5.4 g, 61%).
1 H NMR(400MHz,CDCl 3 )δppm:4.10(s,1H),3.82–3.74(m,2H),3.51–3.36(m,2H),3.35(s,3H),3.27(s,3H),1.91–1.77(m,2H),1.46(s,3H),1.21(s,9H)。
Step C: n- (3-methyl-4-oxo-tetrahydropyran-3-yl) carbamic acid tert-butyl ester:
to a solution of N- (4, 4-dimethoxy-3-methyl-tetrahydropyran-3-yl) -2-methyl-propane-2-sulfinamide (660 mg,2.36mmol,1.0 eq.) in 1, 4-dioxane (10 mL) was added HCl/1, 4-dioxane (10 mL), and the mixture was stirred at room temperature for 4 hours. The solvent was removed to give 3-amino-3-methyl-tetrahydropyran-4-one HCl salt (477 mg, crude product). The crude residue was dissolved in THF (5 mL) and added (Boc) 2 O (1.03 g,4.72mmol,2.0 eq.) and triethylamine (955 mg,9.44mmol,1.32mL,4.0 eq.). The mixture was stirred at room temperature for 16 hours, the solvent was removed, and the residue was purified by flash column chromatography on silica gel (SiO 2 0-70% EtOAc/PE) to give tert-butyl N- (3-methyl-4-oxo-tetrahydropyran-3-yl) carbamate (350 mg,1.53mmol, 64%). 1 H NMR(400MHz,CDCl 3 )δppm:5.56(brs,1H),4.30–4.29(m,1H),4.19–4.15(m,1H),3.81–3.75(m,1H),3.66(d,J＝11.2Hz,1H),2.80–2.79(m,1H),2.54–2.50(m,1H),1.55(s,3H),1.43(s,9H)。
Step D: (4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -4, 6-dihydro-1H, 3H-pyrano [4,3-b ] thieno [3,2-d ] pyran-4-yl) carbamic acid tert-butyl ester:
3-bromo-5- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]A solution of thiophene-2-carboxylic acid methyl ester (1.60 g,3.84mmol,2.0 eq.) and tert-butyl N- (3-methyl-4-oxo-tetrahydropyran-3-yl) carbamate (440 mg,1.92mmol,1.0 eq.) in toluene (10 mL) was degassed and then treated with N 2 Purging and then adding Pd 2 (dba) 3 (352 mg, 384. Mu. Mol,0.20 eq.), xantphos (444 mg, 768. Mu. Mol,0.40 eq.), cs 2 CO 3 (1.25 g,3.84mmol,2.0 eq.) and Na 2 S 2 O 5 (73 mg, 380. Mu. Mol,2.0 eq). The mixture was degassed and used with N 2 Purged and stirred at 105 c for 16 hours. The suspension was cooled to room temperature, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (SiO 2 0-50% EtOAc/PE) to give N- [ 10-methyl-7-oxo-4- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl)]-8, 12-dioxa-5-thiatricyclo [7.4.0.0 ] 2,6 ]Tridec-1 (9), 2 (6), 3-trien-10-yl]Tert-butyl carbamate (412 mg, 40%). MS observations (ESI) + )：478.2[(M-55) + ]。
Step E: 4-amino-4-methyl-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
To 2, 2-dimethyl-N- [ 10-methyl-7-oxo-4- [1- (2-trimethylsilylethoxymethyl) pyrazol-4-yl]-12-oxo-5-thia-8-azatricyclo [7.4.0.0 ] 2,6 ]Tridec-1 (9), 2 (6), 3-trien-10-yl]To a suspension of propionamide (400 mg, 770. Mu. Mol) in DCM (15 mL) was added TFA (5 mL) and the mixture was stirred at room temperature for 5 hours. The solvent was removed and the residue was purified by reverse phase column chromatography (0-20% MeCN/water (0.1% NH 4 OH)) to give 10-amino-10-methyl-4- (1H-pyrazol-4-yl) -12-oxa-5-thia-8-azatricyclo [ 7.4.0.0) 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (130 mg, 57%). MS observations (ESI) + )：287.0[(M-16) + ]。
Step F: 4-amino-4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
10-amino-10-methyl-4- (1H-pyrazol-4-yl) -8, 12-dioxa-5-thiatricyclo [7.4.0.0 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (130 mg,428.57 umol) on NH 4 A suspension of OH (8 mL) and IPA (8 mL) was stirred under microwaves at 100deg.C for 5 hours. Removing in vacuumRemoving the solvent to obtain crude 10-amino-10-methyl-4- (1H-pyrazol-4-yl) -12-oxa-5-thia-8-azatricyclo [7.4.0.0 ] 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (110 mg, crude). MS observations (ESI) + )：303.0[(M+H) + ]。
Step G: (S) -4- (dimethylamino) -4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 82) and (R) -4- (dimethylamino) -4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 83):
to 10-amino-10-methyl-4- (1H-pyrazol-4-yl) -12-oxa-5-thia-8-azatricyclo [7.4.0.0 2 ,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (70 mg, 231. Mu. Mol) in methanol (20 mL) was added paraformaldehyde (42 mg,1.4mmol, 39. Mu.L), naBH 3 CN (116 mg,1.85 mmol) and AcOH (70 mg, 1.16 mmol). The mixture was stirred at 60 ℃ for 2 hours, concentrated, and purified by reverse phase column chromatography (C18 SiO 2 0-20% MeCN/water (0.1% NH) 4 OH)) to give 10- (dimethylamino) -10-methyl-4- (1H-pyrazol-4-yl) -12-oxa-5-thia-8-azatricyclo [ 7.4.0.0) 2,6 ]Tridec-1 (9), 2 (6), 3-trien-7-one (60 mg, 78%). MS observations (ESI) + )：330.9[(M+H) + ]。
Enantiomers were separated by SFC. (S) -4- (dimethylamino) -4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 82): MS observations (ESI) + )：330.9[(M+H) + ]； 1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 13.22 (s, 1H), 10.62 (s, 1H), 8.28 (brs, 1H), 7.96 (brs, 1H), 7.25 (s, 1H), 4.74 (d, J=14.0 Hz, 1H), 4.60 (d, J=14.0 Hz, 1H), 4.16 (d, J=12.0 Hz, 1H), 3.50 (d, J=12.0 Hz, 1H), 2.25 (s, 6H), 1.38 (s, 3H). (R) -4- (dimethylamino) -4-methyl-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]Thieno [3,2-d]Pyridin-6-ones (Compounds83): MS observations (ESI) + )：331.0[(M+H) + ]。
Example 84-compound 84: 4-methyl-4- (methylamino) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one
Step A: 3-methyl-3- (methylamino) tetrahydro-2H-pyran-4-ol:
tert-butyl (3-methyl-4-oxotetrahydro-2H-pyran-3-yl) carbamate (2.0 g,8.7mmol,1.0 eq.) is dissolved in THF (40.0 mL) and LiAlH is added 4 (1.0M, 35mmol,35mL,4.0 eq.). The mixture was heated to 65 ℃ for 6 hours, then water (1.0 mL) was added. The resulting suspension was filtered and the filtrate was concentrated to give 3-methyl-3- (methylamino) tetrahydro-2H-pyran-4-ol (700 mg, used without further purification). 1 H NMR(400MHz,CDCl 3 )δppm:3.92-3.90(m,1H),3.87-3.83(m,1H),3.64-3.60(m,3H),3.55-3.52(m,1H),3.42-3.30(m,2H),3.12(d,J＝12Hz,1H),2.29(s,3H),1.04(s,3H)。
And (B) step (B): (4-hydroxy-3-methyltetrahydro-2H-pyran-3-yl) (methyl) carbamic acid tert-butyl ester:
to a solution of 3-methyl-3- (methylamino) tetrahydro-2H-pyran-4-ol (700 mg, crude product) in THF (30 mL) was added (Boc) 2 O (3.2 g,14mmol,3.0 eq.) and triethylamine (2.4 g,24mmol,5.0 eq.). After 16 hours at room temperature, the mixture was concentrated and purified (SiO 2 0-40% EtOAc/PE) to give tert-butyl (4-hydroxy-3-methyltetrahydro-2H-pyran-3-yl) (methyl) carbamate (700 mg, 32%). 1 H NMR(400MHz,CDCl 3 )δppm:4.49-4.02(m,1H),3.96-3.85(m,1H),3.78-3.55(m,1H),3.51 -3.35(m,2H),2.92-2.87(m,3H),2.03-1.68(m,2H),1.48-1.45(m,9H),1.27-1.24(m,3H)。
Step C: methyl (3-methyl-4-oxotetrahydro-2H-pyran-3-yl) carbamic acid tert-butyl ester:
tert-butyl (4-hydroxy-3-methyltetrahydro-2H-pyran-3-yl) (methyl) carbamate (620 mg,2.53mmol,1.0 eq.) was dissolved in DCM (24 mL) and DMP (2.14 g,5.05mmol,2.0 eq.) was added. After 16 hours at room temperature, naHCO was added 3 (saturated aqueous) and the mixture was extracted with EtOAc. The organic layer was dried (Na 2 SO 4 ) Filtered and concentrated. Purification (SiO) 2 0-30% EtOAc/PE) afforded tert-butyl methyl (3-methyl-4-oxotetrahydro-2H-pyran-3-yl) carbamate (530 mg, 86%). 1 H NMR(400MHz,MeOD)δppm:4.20-4.17(m,2H),3.72-3.66(m,1H),3.24(d,J＝8,1Hz),2.99(s,3H),2.92-2.82(m,1H),2.33-2.29(m,1H),1.42(s,9H),1.15(s,3H)。
Step D: tert-butyl methyl (4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 6-tetrahydropyrano [4,3-b ] thieno [3,2-d ] pyran-4-yl) carbamate:
methyl (3-methyl-4-oxotetrahydro-2H-pyran-3-yl) carbamic acid tert-butyl ester (210 mg,0.86mmol,1.0 eq.) 3-bromo-5- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (200 mg,0.48,1.8 eq.), spos-Pd-G3 (75 mg,0.10mmol,0.20 eq.) and Cs 2 CO 3 A mixture of (470 mg,1.4mmol,3.0 eq.) in toluene (10 mL) was heated to 105℃for 16 hours. The reaction mixture was diluted with EtOAc and filtered. The filtrate was concentrated and purified (SiO 2 0-30% EtOAc/PE) to give methyl (4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 6-tetrahydropyrano [4, 3-b)]Thieno [3,2-d]T-butyl pyran-4-yl) carbamate (120 mg, 45%). MS observations (ESI) + )：492.6[(M-56(t-Bu)+H) + ]。
Step E: 4-methyl-4- (methylamino) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 84):
methyl (4-methyl-6-oxo-8- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 6-tetrahydropyrano [4,3-b]Thieno [3,2-d]Tert-butyl pyran-4-yl) carbamate (120 mg) was dissolved in TFA and DCM (1:1, 5 mL). After 2 hours at room temperature, the mixture was concentrated and subjected to conditions similar to those described in step G of compound 82 to give 4-methyl-4- (methylamino) -8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (compound 84) (27 mg, 32%). MS observations (ESI) + )：317.1[(M+H) + ]。 1 H NMR(400MHz,DMSO-d 6 )δppm:13.22(s,1H),11.08(brs,1H),8.27(brs,1H),7.97(brs,1H),8.00–7.92(m,1H),7.40(s,1H),4.68(s,2H),3.82(d,J＝11.2Hz,1H),3.51(d,J＝11.2Hz,1H),2.07(s,3H),1.31(s,3H)。
Example 85-compound 85: (S) -4- (tert-butyl) -4-hydroxy-8-morpholino-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one
Step A: 3-bromo-5-morpholinothiene-2-carboxylic acid methyl ester:
3, 5-Dibromothiophene-2-carboxylic acid methyl ester (200 mg, 670. Mu. Mol,1.0 eq), morpholine (87 mg,1.0mmol,1.5 eq), cs 2 CO 3 (430 mg,1.3mmol,2.0 eq.) in toluene (5 mL), (1E, 4E) -1, 5-diphenylpenta-1, 4-dien-3-one palladium (30 mg, 33. Mu. Mol,0.050 eq.) a mixture of (5-diphenylphosphino-9, 9-dimethyl-xanthen-4-yl) -diphenyl-phosphine (39 mg, 67. Mu. Mol,0.10 eq.) was degassed and N 2 And (5) purging. The mixture was stirred at 80 ℃ for 16 hours, then cooled and filtered. The filtrate was concentrated in vacuo, and the residue was purified by column chromatography (SiO 2 15% EtOAc/PE) to give methyl 3-bromo-5-morpholinothiene-2-carboxylate (110 mg, 54%). MS observations (ESI) + )：306.1,308.1[(M+H) + ]。
And (B) step (B): (R) -3- (tert-butyl) -3-hydroxytetrahydro-4H-pyran-4-one:
3- (tert-butyl) -3-hydroxytetrahydro-4H-pyran-4-one (1.0 g,5.8 mmol) was isolated by chiral HPLC using the following conditions (column: IG-3, column size: 0.46 cm. Times.100 mm,3um, sample introduction: 2.0mL. CO-solvent: CO) 2 EtOH (0.1% DEA), flow rate: 3 ml/min, run time: 6.0 minutes, temperature: 25 ℃) to give (R) -3- (tert-butyl) -3-hydroxytetrahydro-4H-pyran-4-one (370 mg, 37%), first elution peak. Stereochemistry is arbitrarily specified. 1 H NMR(400MHz,DMSO-d6)δ5.05(s,1H),4.06(dd,J＝12.0,1.2Hz,1H),4.03–3.95(m,1H),3.75–3.64(m,1H),3.35(d,J＝12.0Hz,1H),2.60–2.52(m,2H),0.95(s,9H)。
Step C: (R) -4- (tert-butyl) -4-hydroxy-8-morpholino-3, 4-dihydro-1 h,6 h-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
3-bromo-5-morpholinothiene-2-carboxylic acidMethyl ester (110 mg, 360. Mu. Mol,1.0 eq), (R) -3- (tert-butyl) -3-hydroxytetrahydro-4H-pyran-4-one (74 mg, 430. Mu. Mol,1.2 eq), cs 2 CO 3 (350 mg,1.1mmol,3.0 eq.) in toluene (5 mL), (1E, 4E) -1, 5-diphenylpenta-1, 4-dien-3-one palladium (33 mg, 36. Mu. Mol,0.1 eq.) and (5-diphenylphosphino-9, 9-dimethyl-xanth-4-yl) -diphenyl-phosphine (41 mg, 72. Mu. Mol,0.20 eq.) and deaerated with N 2 Purging and then heating to 100 ℃ for 16 hours. The mixture was poured into water and extracted with DCM (20 ml x 3). The organic layers were combined, washed (NaCl), dried (MgSO 4 ) Filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 Purification with 3% MeOH/DCM to give (R) -4- (tert-butyl) -4-hydroxy-8-morpholino-3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (75 mg, 57%). MS observations (ESI) + )：366.4[(M+H) + ]。
Step D: (S) -4- (tert-butyl) -4-hydroxy-8-morpholino-1, 3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 85):
(R) -4- (tert-butyl) -4-hydroxy-8-morpholino-3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (70 mg, 191.55. Mu. Mol,1.0 eq.) NH 4 A mixture of aqueous OH (8 mL) and isopropyl alcohol (8 mL) was stirred at 120℃for 48 hours. The mixture was concentrated in vacuo and the residue was purified by column chromatography (SiO 2 3% meoh/DCM) to give (S) -4- (tert-butyl) -4-hydroxy-8-morpholino-1, 3,4, 5-tetrahydro-6H-pyrano [4, 3-b) as a yellow solid]Thieno [3,2-d]Pyridin-6-one (Compound 85) (30 mg, 43%). MS observations (ESI) + )：365.0[(M+H) + ]。 1 H NMR(400MHz,DMSO-d6)δppm:9.62(s,1H),6.20(s,1H),5.27(s,1H),4.60(q,J＝14.8Hz,2H),4.09(d,J＝11.2Hz,1H),3.83–3.68(m,4H),3.40(d,J＝11.2Hz,1H),3.28–3.19(m,4H),1.00(s,9H)。
Example 86-compound 86:4- (2, 2-difluoropropyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one
Step A:4, 4-dimethoxy-3- (2-methallyl) tetrahydro-2H-pyran-3-ol:
to a solution of 4, 4-dimethoxy-dihydro-2H-pyran-3 (4H) -one (5.0 g,31mmol,1.0 eq.) in THF (5 mL) was added magnesium chloride (2-methallyl) (0.5M in THF, 125mL,2.0 eq.) at room temperature. After 1 hour, NH was added 4 Cl (saturated aqueous, 40 mL). The biphasic mixture was extracted with EtOAc (100 ml x 3) and the organic layer was dried, filtered and concentrated. By flash column chromatography (SiO 2 0-30% EtOAc/PE) to give 4, 4-dimethoxy-3- (2-methallyl) tetrahydro-2H-pyran-3-ol (1.5 g, 55%) as a colorless oil. 1 HNMR(400MHz,CDCl 3 )δppm:4.96(dd,J＝2.4,1.6Hz,1H),4.82(dd,J＝2.4,1.6Hz,1H),3.82–3.72(m,1H),3.68(dd,J＝11.4,1.2Hz,1H),3.43(s,4H),3.31(s,3H),3.10(dd,J＝11.4,1.2Hz,1H),2.53(d,J＝13.8Hz,1H),2.36(d,J＝13.8Hz,1H),1.96–1.87(m,4H),1.80–1.67(m,2H)。
And (B) step (B): 3- (benzyloxy) -4, 4-dimethoxy-3- (2-methallyl) tetrahydro-2H-pyran:
a mixture of 4, 4-dimethoxy-3- (2-methallyl) tetrahydro-2H-pyran-3-ol (4.2 g,19mmol,10 eq.), bnBr (3.14 g,29.1mmol,1.5 eq.) and NaH (893 mg,38.8mmol,2.0 eq.) in DMF (50 mL) was stirred at 0deg.C for 2 hours. Ice was added and the biphasic mixture was extracted with DCM (60 ml x 3). The organic layer was concentrated and the residue was taken upUltra-fast column chromatography (SiO 2 0-30% EtOAc/PE) to give 3- (benzyloxy) -4, 4-dimethoxy-3- (2-methallyl) tetrahydro-2H-pyran (5.6 g, 94%). 1 H NMR(400MHz,CDCl 3 )δppm:7.35–7.21(m,5H),4.95(dd,J＝4.0,2.0Hz,1H),4.84(dd,J＝2.4,1.6Hz,1H),4.76–4.70(m,2H),4.53(d,J＝11.8Hz,1H),4.32(dd,J＝14.8,4.0Hz,1H),4.14(dd,J＝14.8,1.9Hz,1H),4.00(d,J＝12.4Hz,1H),3.79(d,J＝12.4Hz,1H),3.56(s,3H),3.48(s,3H),2.84–2.42(m,3H),1.80(s,3H)。
Step C:3- (benzyloxy) -3- (2-methylallyl) tetrahydro-4H-pyran-4-one:
to a solution of 3- (benzyloxy) -4, 4-dimethoxy-3- (2-methallyl) tetrahydro-2H-pyran (480 mg,3.33mmol,1.0 eq.) in acetone (15 mL) was added I 2 (84 mg,0.33mmol,0.10 eq.). The reaction mixture was stirred at room temperature for 16 hours, then Na was added 2 SO 3 Aqueous (20 mL) and the biphasic mixture was extracted with DCM (30 mL. Times.3). The organic layer was dried (Na 2 SO 4 ) Filtered and concentrated. Column chromatography (SiO) 2 0-10% EtOAc/PE) afforded 3- (benzyloxy) -3- (2-methallyl) tetrahydro-4H-pyran-4-one (310 mg, 37%). 1 H NMR(400MHz,CDCl 3 )δ7.42–7.20(m,5H),4.95–4.84(m,1H),4.81(d,J＝0.8Hz,1H),4.70(d,J＝11.2Hz,1H),4.30(d,J＝11.2Hz,1H),4.10–3.85(m,3H),3.68(d,J＝12.0Hz,1H),2.83–2.83(m,2H),2.56–2.41(m,2H),1.81(s,3H)。
Step D:4- (benzyloxy) -4- (2-methylallyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to 3- (benzyloxy) -3- (2-methylallyl) tetrahydro-4H-pyran-4To a solution of (210 mg,0.81mmol,1.5 eq.) and 3-bromo-5- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (200 mg,0.56mmol,1.0 eq.) in dioxane (10 mL) was added Cs 2 CO 3 (439 mg,1.35mmol,2.0 eq.) and sphos Pd G3 (93 mg,0.11mmol,0.20 eq.). The mixture was degassed and used with N 2 Purging and then heating to 105 ℃ for 16 hours. The mixture was purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 4- (benzyloxy) -4- (2-methylallyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b) as a yellow solid]Thieno [3,2-d]Pyran-6-one (60 mg, 21%). MS observations (ESI) + )：519.3[(M+H) + ]。
Step E:4- (benzyloxy) -4- (2-oxopropyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
4- (benzyloxy) -4- (2-methylallyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]A solution of pyran-6-one (80 mg,0.15 mmol) in DCM (5 mL) in MeOH (5 mL) was cooled to-75deg.C, and ozone was then bubbled into the solution for 20 minutes. Dimethyl sulfide (8 drops) was added at-75℃and the mixture was stirred at-75℃for 30 minutes and then warmed to room temperature. The mixture was concentrated and purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 4- (benzyloxy) -4- (2-oxopropyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (52 mg, 65%). MS observations (ESI) + )：413.2[(M-OBn) + ]。
Step F:4- (benzyloxy) -4- (2, 2-difluoropropyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
4- (benzyloxy) -4- (2-oxopropyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (50 mg,0.10mmol,1.0 eq.) in CHCl 3 The mixture of (0.20 mL) and DAST (10 mL) was stirred at 35℃for 16 h. The mixture was cooled to 0deg.C and quenched with EtOAc (20 mL) and aqueous NaHCO 3 (20 mL) dilution. The organic layer was concentrated and the residue was purified by flash column chromatography (SiO 2 0-50% EtOAc/PE) to give 4- (benzyloxy) -4- (2, 2-difluoropropyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (21 mg, 40%). MS observations (ESI) + )：543.2[(M+H) + ]。
Step G:4- (2, 2-difluoropropyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
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4- (benzyloxy) -4- (2, 2-difluoropropyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]A solution of pyran-6-one (40 mg,0.070mmol,1.0 eq.) in DCE (4 mL) and TFA (1 mL) was stirred at 35℃for 3 hours. NH addition at room temperature 4 OH, and concentrating the mixture to give 4- (2, 2-difluoropropyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (25 mg, crude) which was used in the next step. MS observations (ESI) + )：369.4[(M+H) + ]。
Step H:4- (2, 2-difluoropropyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 86):
4- (2, 2-difluoropropyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (25 mg, crude product), i-PrOH (2.5 mL) and NH 4 The mixture of OH (2.5 mL) was stirred at 95℃for 16 h. The mixture was concentrated and purified by reverse phase column chromatography (C18 SiO 2 0-40% MeCN/water (0.1% NH) 4 OH)) to give 4- (2, 2-difluoropropyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (compound 86) (2.6 mg, 10%). MS observations (ESI) + )：368.0[(M+H) + ]； 1 H NMR(400MHz,DMSO-d6)δppm:13.23(s,1H),11.00(s,1H),8.32(brs,1H),8.26(brs,1H),7.42(s,1H),5.76(s,1H),4.90–4.55(m,2H),4.04(d,J＝11.6Hz,1H),3.65(d,J＝11.6Hz,1H),2.65–2.52(m,2H),1.70(t,J＝19.6Hz,3H)。
Examples 87 and 88-compounds 87 and 88: (S) -6-hydroxy-6-isopropyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 87) and (R) -6-hydroxy-6-isopropyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c ] quinolin-4 (5H) -one (compound 88) (the stereochemistry of compounds 87 and 88 is arbitrarily specified)
To 6-hydroxy-6-isopropyl-2- (1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]To a solution of quinolin-4 (5H) -one (100 mg, 220. Mu. Mol,1.0 eq.) in DCM (7 mL) was added BCl 3 (1.0M, 650. Mu.L, 3.0 eq.). The mixture was stirred at-70℃for 10 min. The reaction mixture was taken up in N 2 Bubbling for 2 min, then adding NH 4 OH (25% aqueous, 1 mL). The mixture was stirred at 0deg.C for 15 min, then MeOH (2 mL) was added. After an additional 10 minutes at 0 ℃, the mixture was concentrated in vacuo to give 6-hydroxy-6-isopropyl-2- (1H-pyrazol-4-yl) -6,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4 (5H) -one (20)mg). MS observations (ESI) + )：330.1[(M+H) + ]。
Enantiomers were separated by chiral SFC. (6S) -6-hydroxy-6-isopropyl-2- (1H-pyrazol-4-yl) -5,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4-one (compound 87): MS observations (ESI) + )：311.9[(M-17) + ]；
1 H NMR(400MHz,DMSO-d 6 ) Delta ppm 13.20 (s, 1H), 10.36 (s, 1H), 8.30 (s, 1H), 7.96 (s, 1H), 7.49 (s, 1H), 5.00 (s, 1H), 2.78-2.65 (m, 1H), 2.54-2.32 (m, 2H), 1.76-1.69 (m, 4H), 0.97 (d, J=6.8 Hz, 3H), 0.59 (d, J=6.8 Hz, 3H). (6R) -6-hydroxy-6-isopropyl-2- (1H-pyrazol-4-yl) -5,7,8, 9-tetrahydrothieno [2,3-c]Quinolin-4-one (compound 88): MS observations (ESI) + )：329.9[(M+H) + ]。
Examples 89 and 90-compounds 89 and 90: (S) -4- (tert-butyl) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 89) and (R) -4- (tert-butyl) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 90) (the stereochemistry of compounds 89 and 90 is arbitrarily specified)
Step A:4- (benzyloxy) -8- (1-methyl-1H-pyrazol-5-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
similar conditions as described in step a of compound 16 were followed using methyl 3-bromo-5- (1-methyl-1H-pyrazol-5-yl) thiophene-2-carboxylate to give 4- (benzyloxy) -8- (1-methyl-1H-pyrazol-5-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one (1.3 g, 50%). MS observations (ESI) + )：395.1[(M+H) + ]。
And (B) step (B): 4- (benzyloxy) -8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
similar conditions to those described in step B of Compound 16 were used to give 4- (benzyloxy) -8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-B)]Thieno [3,2-d]Pyridin-6-one (870 mg, 55%). MS observations (ESI) + )：394.3[(M+H) + ]。
Step C: 4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
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4- (benzyloxy) -8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]A solution of pyridin-6-one (680 mg,1.73mmol,1.0 eq.) in TFA (7 mL) was stirred at 80℃for 3 h. The mixture was concentrated in vacuo and NH was added at 0 ℃ 3 MeOH (7M) to achieve a pH of about 9. The mixture was concentrated in vacuo and purified by flash column chromatography (SiO 2 0-9% MeOH/DCM) to give 4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (45 mg, 87%). MS observations (ESI) + )：304.2[(M+H) + ]。
Step D:8- (1-methyl-1H-pyrazol-5-yl) -1, 5-dihydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridine-4, 6 (3H) -dione:
using the reaction conditions described in compound 16, step E, to give 8- (1-methyl-1H-pyrazol-5-yl) -1, 5-dihydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridine-4, 6 (3H) -dione (330 mg, 73%). MS observations (ESI) + )：302.1[(M+H) + ]。
Step E: (S) -4- (tert-butyl) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 89) and (R) -4- (tert-butyl) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 90):
8- (1-methyl-1H-pyrazol-5-yl) -1, 5-dihydro-6H-pyrano [4,3-b]Thieno [3,2-d]A solution of pyridine-4, 6 (3H) -dione (110 mg, 370. Mu. Mol,1.0 eq.) in THF (6 mL) was cooled to 0deg.C, then t-BuMgCl (1.0M, 6mL,16.5 eq.) was added. The reaction mixture was stirred at 0deg.C for 2 hours, then NH was added 4 Aqueous Cl solution. The mixture was poured into water (20 mL) and extracted with DCM/MeOH (10:1, v/v) (40 mL. Times.3). The organic layers were combined, washed with brine, dried (MgSO 4 ) Filtered and concentrated in vacuo. Purification (SiO) 2 0-6% MeOH/DCM) to give 4- (tert-butyl) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (56 mg, 43%). MS observations (ESI) + )：360.3[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4- (tert-butyl) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 89): MS observations (ESI) + )：360.2[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) delta ppm 10.31 (s, 1H), 7.56 (s, 1H), 7.53 (d, J=2.0 Hz, 1H), 6.70 (d, J=2.0 Hz, 1H), 5.35 (s, 1H), 4.87-4.72 (m, 2H), 4.14 (d, J=11.6 Hz, 1H), 4.04 (s, 3H), 3.45 (d, J=11.2 Hz, 1H), 1.03 (s, 9H). (R) -4- (tert-butyl) -4-hydroxy-8- (1-methyl-1H-pyrazol-5-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 90): MS observations (ESI) + )：360.2[(M+H) + ]。
Examples 91 and 92-compounds 91 and 92: (S) -4- (tert-butyl) -4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 91) and (R) -4- (tert-butyl) -4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 92) (the stereochemistry of compounds 91 and 92 is arbitrarily specified)
Step A: methyl 3-bromo-5- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate and methyl 3-bromo-5- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate:
to a solution of 3-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazole (10 g,48.1mmol,1 eq.) in DMF (150 mL) was added NaH (2.39 g,59.9mmol,60% purity, 1.25 eq.) at 0deg.C. The mixture was stirred at 0deg.C for 1 hour, then 2- (chloromethoxy) ethyl-trimethyl-silane (12.0 g,72.1mmol,12.76mL,1.5 eq.) was added. The reaction was stirred at room temperature for 3 hours and then diluted with water. The biphasic mixture was extracted with EtOAc (80 ml x 3) and washed with brine. The organic layer was dried, filtered and concentrated to give a mixture of 3-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole and 5-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole (16 g, crude product). The crude mixture was dissolved in 1, 4-dioxane (160 mL) and Pd (dppf) Cl was added 2 (3.46 g,4.73mmol,0.1 eq.) K 3 PO 4 (30.1 g,141.88mmol,3 eq.), water (160 mL) and CH 3 CN (160 mL). The reaction mixture was heated to 90 ℃ for 8 hours, cooled and filtered. Concentrating the filtrate in vacuumContracted and passed through a silica gel column (SiO 2 0-10% EtOAc/PE) to give a mixture of 3-bromo-5- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate and methyl 3-bromo-5- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate (12 g,59%,2 steps). 1 H NMR(400MHz,CDCl 3 )δppm:7.66(s,1H),7.05(s,1H),5.47(s,2H),3.92(s,3H),3.64–3.58(m,2H),2.55(s,3H),0.97–0.90(m,2H),0.01(s,9H)。
1 H NMR(400MHz,CDCl 3 )δppm:7.75(s,1H),7.05(s,1H),5.38(s,2H),3.91(s,3H),3.64–3.58(m,2H),2.47(s,3H),0.97–0.90(m,2H),0.02(s,9H)。
And (B) step (B): 4- (benzyloxy) -8- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one and 4- (benzyloxy) -8- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
a mixture (20 g,46mmol,1.0 eq) of 3-bromo-5- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate and methyl 3-bromo-5- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) thiophene-2-carboxylate (20 g,46mmol,1.0 eq) was dissolved in 1, 4-dioxane (600 mL), and 3-benzyloxytetrahydropyran-4-one (19.1 g,92.7mmol,2.0 eq), cs were added 2 CO 3 (45.3 g,139mmol,3.0 eq.) Pd 2 (dba) 3 (2.12 g,2.32mmol,0.050 eq.) Na 2 S 2 O 5 (881 mg,4.64mmol,0.10 eq.) and Xantphos (2.68 g,4.64mmol,0.1 eq.). The reaction mixture was heated to 105 ℃ for 16 hours, cooled, filtered and concentrated. The crude residue was triturated with MeOH to give 4- (benzyloxy) -8- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Pyran-6-one and 4- (benzyloxy) -8- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]Mixture of pyran-6-ones (11.5 g, 46%). MS observations (ESI) + )：525.5[M+H] + 。
Step C:4- (benzyloxy) -8- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one and 4- (benzyloxy) -8- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one:
to 4- (benzyloxy) -8- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ]Thieno [3,2-d]Pyran-6-one and 4- (benzyloxy) -8- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]NH was added to a solution of pyran-6-one (3.0 g,11mmol,1 eq.) in i-PrOH (30 mL) 4 OH (30 mL). The reaction was stirred at 100 ℃ for 20 hours, cooled, concentrated and filtered. Trituration with MeOH afforded 4- (benzyloxy) -8- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one and 4- (benzyloxy) -8- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (3.8 g, 63%). MS observations (ESI) + )：524.5[M+H] + 。
Step D:4- (4-hydroxy-6-oxo-3, 4,5, 6-tetrahydro-1H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-8-yl) -3-methyl-1H-pyrazole-1-carboxylic acid tert-butyl ester:
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4- (benzyloxy) -8- (3-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one and 4- (benzyloxy) -8- (5-methyl-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ]Thieno [3,2-d]A mixture of pyridin-6-one (5.4 g,10.Mmol,1.0 eq.) was dissolved in DCM (150 mL) and cooled to 0deg.C and BCl was added 3 (1.0M, 50mL,5 eq.). The reaction was stirred at 0 ℃ for 1 hour, then MeOH was added. Concentrating the mixture to give 4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (3.2 g, crude product). MS observations (ESI) + )：304.0[M+H] + . The crude residue was dissolved in DMF (90 mL) and Et was added 3 N (4.20 g,41.5 mmol), DMAP (169 mg,1.38 mmol) and (Boc) 2 O (3.63 g,16.6 mmol). The mixture was stirred at room temperature for 6 hours, then concentrated in vacuo. The residue was triturated with DCM to give 4- (4-hydroxy-6-oxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-8-yl) -3-methyl-1H-pyrazole-1-carboxylic acid tert-butyl ester (4.8 g, crude product). MS observations (ESI) + )：404.2[(M+H) + ]。
Step E:4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-8-yl) -3-methyl-1H-pyrazole-1-carboxylic acid tert-butyl ester:
the reaction conditions described in step E of Compound 16 are followed to give 4- (4, 6-dioxo-3, 4,5, 6-tetrahydro-1H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-8-yl) -3-methyl-1H-pyrazole-1-carboxylic acid tert-butyl ester (750 mg, 50%). MS observations (ESI) + )：402.0[(M+H) + ]。
Step F: (S) -4- (tert-butyl) -4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 91) and (R) -4- (tert-butyl) -4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 92):
reaction conditions similar to those described in step E of Compound 89 are carried out to give 4- (tert-butyl) -4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (30 mg, 32%). MS observations (ESI) + )：360.2[M+H] + 。
Enantiomers were separated by SFC. (S) -4- (tert-butyl) -4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 91): MS observations (ESI) + )：360.2[(M+H) + ]； 1 H NMR (400 MHz, DMSO-d 6) ppm 12.96 (s, 1H), 10.02 (s, 1H), 8.17-7.87 (m, 1H), 7.27 (s, 1H), 5.32 (s, 1H), 4.80 (d, J=15.2 Hz, 1H), 4.71 (d, J=15.2 Hz, 1H), 4.13 (d, J=11.6 Hz, 1H), 3.45 (d, J=11.6 Hz, 1H), 2.45 (s, 3H), 1.03 (s, 9H). (R) -4- (tert-butyl) -4-hydroxy-8- (3-methyl-1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 92): MS observations (ESI) + )：360.0[(M+H) + ]。
Example 93-compound 93:4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydrothieno [2,3-c ] [1,6] naphthyridin-6 (2H) -one
Step A:3- (benzoyloxy) -4, 4-dimethoxypiperidine-1-carboxylic acid benzyl ester:
to a solution of potassium hydroxide (7.22 g,129mmol,3.0 eq.) in MeOH (450 mL) was added benzyl 4-oxopiperidine-1-carboxylate (10 g,43mmol,8.5mL,1.0 equivalent), then at N 2 Slowly add I under atmosphere 2 (15.2 g,60.0mmol,1.4 eq.). After addition, the reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was concentrated in vacuo. The crude product was stirred in 150mL DCM and the precipitate was filtered. The filtrate was concentrated in vacuo to give benzyl 3-hydroxy-4, 4-dimethoxypiperidine-1-carboxylate (14.2 g, crude product). The crude residue was dissolved in pyridine (140 mL) and cooled to 0deg.C, then benzoyl chloride (16.9 g,120mmol,13.8mL,2.5 eq.) was added. After the addition, the reaction mixture was stirred at room temperature for 4 hours. The mixture was concentrated and purified in vacuo (SiO 2 0-9% EtOAc/PE) to give benzyl 3- (benzoyloxy) -4, 4-dimethoxypiperidine-1-carboxylate (9.8 g, 57%). 1 H NMR(400MHz,DMSO-d6)δppm:7.96–7.86(m,2H),7.72–7.69(m,1H),7.63–7.50(m,2H),7.49–7.33(m,2H),7.17–7.07(m,2H),7.02–6.93(m,1H),5.07–4.99(m,2H),4.88–4.57(m,1H),4.24–4.18(m,1H),4.07–4.02(m,1H),3.30–3.32(m,1H),3.26(s,3H),3.07(s,3H),2.97–2.82(m,1H),2.02–1.99(m,1H),1.85–1.82(m,1H)。
And (B) step (B): 3- (benzoyloxy) -4-oxopiperidine-1-carboxylic acid benzyl ester:
To a solution of benzyl 3- (benzoyloxy) -4, 4-dimethoxypiperidine-1-carboxylate (9.8 g,24mmol,1.0 eq.) in acetone (100 mL) was added molecular iodine (1.25 g,4.91mmol,0.1 eq.). After 16 hours at room temperature, na was added 2 SO 3 (100 mL, aqueous) and the mixture was extracted with DCM (120 mL. Times.3). The combined organic phases were dried (Na 2 SO 4 ) Filtered and concentrated. By flash column chromatography (SiO 2 Purification of 0-11% EtOAc/PE afforded benzyl 3- (benzoyloxy) -4-oxopiperidine-1-carboxylate (4.7 g, 54%). 1 H NMR(400MHz,CDCl 3 )δppm:8.06(d,J＝7.2Hz,2H),7.61–7.57(m,1H),7.47–7.43(m,2H),7.37(s,5H),5.41–5.37(m,1H),5.20(s,2H),4.73–4.57(m,1H),4.43–4.40(m,1H),3.44–3.26(m,2H),2.65(s,2H)。
Step C:4- (benzoyloxy) -6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -4, 6-dihydro-1H-thieno [3',2':4,5] pyrano [3,2-c ] pyridine-2 (3H) -carboxylic acid benzyl ester:
3-bromo-5- (1-tetrahydropyran-2-ylpyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (500 mg,1.35mmol,1.0 eq), 3- (benzoyloxy) -4-oxopiperidine-1-carboxylic acid benzyl ester (714mg, 2.02mmol,1.5 eq), cs 2 CO 3 (1.32 g,4.04mmol,3.0 eq.) Pd 2 (dba) 3 A mixture of (123 mg, 135. Mu. Mol,0.10 eq.) and Xantphos (156 mg, 269. Mu. Mol,0.20 eq.) in toluene (18 mL) was heated to 105℃for 2 hours. The reaction mixture was filtered, concentrated in vacuo, and purified by flash column chromatography (SiO 2 0-36% EtOAc/PE) to give 4- (benzoyloxy) -6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -4, 6-dihydro-1H-thieno [3',2':4,5]Pyrano [3,2-c]Benzyl pyridine-2 (3H) -carboxylate (450 mg, 55%). MS observations (ESI) + )：612.4[(M+H) + ]。
Step D: 4-hydroxy-6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c ] [1,6] naphthyridine-2 (1H) -carboxylic acid benzyl ester:
4- (benzoyloxy) -6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -4, 6-dihydro-1H-thieno [3',2':4,5]Pyrano [3,2-c]Benzyl pyridine-2 (3H) -carboxylate (900 mg,1.47mmol,1.0 eq.) in NH 3 A solution in MeOH (7M, 10 mL) was stirred at 95℃for 2 hours. The reaction mixture was concentrated in vacuo and purified by flash column chromatography (SiO 2 Purification with 0-4% MeOH/DCM to give 4-hydroxy-6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl)) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c][1,6]Naphthyridine-2 (1H) -carboxylic acid benzyl ester (500 mg, 67%). MS observations (ESI) + )：507.4[(M+H) + ]。
Step E: benzyl 4, 6-dioxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c ] [1,6] naphthyridine-2 (1H) -carboxylate:
using the reaction conditions described in compound 16, step E, to give 4, 6-dioxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c ][1,6]Naphthyridine-2 (1H) -carboxylic acid benzyl ester (399mg, 79%). MS observations (ESI) + )：505.4[(M+H) + ]。
Step F: benzyl 4- (tert-butyl) -4-hydroxy-6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c ] [1,6] naphthyridine-2 (1H) -carboxylate:
to 4, 6-dioxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c][1,6]To a solution of benzyl naphthyridine-2 (1H) -carboxylate (200 mg, 396. Mu. Mol,1.0 eq.) in THF (12 ml) was added ZnCl 2 (0.50M, 1.19mL,1.5 eq.). The mixture was stirred at 0deg.C for 10 min, then t-BuMgCl (1.0M, 3.96mL,10.0 eq.) was added. After 1 hour at 0℃saturated NH was added 4 Cl (aq), and the biphasic mixture was extracted with DCM/MeOH (35 mL. Times.3, 20:1 v/v). The organic layer was dried (Na 2 SO 4 ) Filtered, concentrated, and purified by flash column chromatography (SiO 2 0-3% MeOH/DCM) to give 4- (tert-butyl) -4-hydroxy-6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c][1,6]Naphthyridine-2 (1H) -carboxylic acid benzyl ester (57 mg, 26%). MS observations (ESI) + )：563.5[(M+H) + ]。
Step G:4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydrothieno [2,3-c ] [1,6] naphthyridin-6 (2H) -one (compound 93):
4- (tert-butyl) -4-hydroxy-6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c][1,6]A solution of benzyl naphthyridine-2 (1H) -carboxylate (15 mg, 27. Mu. Mol,1.0 eq.) in TFA (3.00 mL) was heated to 80℃for 1 hour. The resulting mixture was concentrated in vacuo and cooled to 0 ℃ before NH was added 3 MeOH to adjust the pH to about 9. The mixture was concentrated in vacuo and purified by reverse phase column chromatography (SiO 2 Purification of 0-30% MeCN/water (0.1% FA)) to give 4- (tert-butyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydrothieno [2,3-c ]][1,6]Naphthyridin-6 (2H) -one (compound 93) (4.7 mg, 51%). 1 H NMR(400MHz,DMSO-d6)δppm:13.23(s,1H),9.51(s,1H),8.15(brs,2H),7.46(s,1H),5.22(s,1H),3.95–3.80(m,2H),3.32(s,1H),2.78(d,J＝12.8Hz,1H),1.03(s,9H)。
Example 94-compound 94: 2-acetyl-4-tert-butyl-4-hydroxy-8- (1H-pyrazol-4-yl) -3, 5-dihydro-1H-thieno [2,3-c ] [1,6] naphthyridin-6-one
Step A:4- (tert-butyl) -4-hydroxy-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydrothieno [2,3-c ] [1,6] naphthyridin-6 (2H) -one:
to 4- (tert-butyl) -4-hydroxy-6-oxo-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3,4,5, 6-tetrahydrothieno [2,3-c][1,6]Benzyl naphthyridine-2 (1H) -carboxylate (57 mg, 100. Mu. Mol,1.0 eq.) in IPA (6 mL)) Pd/C (13 mg, 10.1. Mu. Mol,10% Pd,0.1 eq.) was added to the solution in (B). The mixture was degassed with H 2 Purged and stirred at room temperature for 1.5 hours. The reaction mixture was filtered and concentrated in vacuo to give 4- (tert-butyl) -4-hydroxy-8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -1,3,4, 5-tetrahydrothieno [2,3-c ] as a yellow solid][1,6]Naphthyridin-6 (2H) -one (42 mg, crude product) was used directly in the next step without further purification. MS observations (ESI) + )：429.5[(M+H) + ]。
And (B) step (B): 2-acetyl-4-tert-butyl-4-hydroxy-8- (1-tetrahydropyran-2-ylpyrazol-4-yl) -3, 5-dihydro-1H-thieno [2,3-c ] [1,6] naphthyridin-6-one:
4-tert-butyl-4-hydroxy-8- (1-tetrahydropyran-2-ylpyrazol-4-yl) -1,2,3, 5-tetrahydrothieno [2,3-c][1,6]A solution of naphthyridin-6-one (47 mg, 110. Mu. Mol,1.0 eq) and triethylamine (33 mg, 330. Mu. Mol,3.0 eq) in DCM (3 mL) was cooled to 0deg.C. Acetyl chloride (8.61 mg, 110. Mu. Mol,1.0 eq) was added dropwise and the reaction mixture was warmed to room temperature. After 2 hours, na was added 2 CO 3 Saturated aqueous (2 mL) and MeCN (3 mL). The mixture was stirred at room temperature for 3 hours, diluted with brine (30 mL), extracted with DCM (30 mL x 3), and washed with brine (30 mL x 2). The combined organic layers were concentrated and purified by flash column chromatography (SiO 2 0-6% MeOH/DCM) to give 2-acetyl-4-tert-butyl-4-hydroxy-8- (1-tetrahydropyran-2-ylpyrazol-4-yl) -3, 5-dihydro-1H-thieno [2,3-c ] ][1,6]Naphthyridin-6-one (20 mg, 39%). MS observations (ESI) + )：471.4[(M+1) + ]。
Step C: 2-acetyl-4-tert-butyl-4-hydroxy-8- (1H-pyrazol-4-yl) -3, 5-dihydro-1H-thieno [2,3-c ] [1,6] naphthyridin-6-one (compound 94):
2-acetyl-4-tert-butyl-4-hydroxy-8- (1-tetrahydropyran-2-ylpyrazol-4-yl) -3, 5-dihydro-1H-thieno [2,3-c][1,6]A solution of naphthyridin-6-one (20 mg, 42. Mu. Mol,1.0 eq.) in HCl/MeOH (4M, 2 mL) was stirred at 0deg.C for 1 hour. The mixture was concentrated in vacuo and purified by addition of NH at 0 ℃ 3 The pH was adjusted to about 9 with MeOH. The mixture was concentrated in vacuo and purified by preparative HPLC (ACN/water/0.1% FA) to give 2-acetyl-4-tert-butyl-4-hydroxy-8- (1H-pyrazol-4-yl) -3, 5-dihydro-1H-thieno [2,3-c][1,6]Naphthyridin-6-one (compound 94) (9.6 mg, 58%). MS observations (ESI) + )：386.1[(M+1) + ]； 1 H NMR(400MHz,DMSO-d 6 )δ13.25(s,1H),10.03(d,J＝37.6Hz,1H),8.32(s,1H),7.98(s,1H),7.58(d,J＝1.2Hz,1H),5.61(d,J＝55.2Hz,1H),4.96–4.05(m,3H),2.68(d,J＝13.2Hz,1H),2.16(d,J＝18.8Hz,3H),0.95(d,J＝12.8Hz,9H)。
Examples 95 and 96-compounds 95 and 96: (S) -4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 95) and (R) -4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 96) (stereochemistry of compounds 95 and 96 is arbitrarily specified)
Step A:3- (benzyloxy) -3- (prop-1-en-2-yl) tetrahydro-2H-pyran-4-ol:
a solution of 3- (benzyloxy) -3- (prop-1-en-2-yl) tetrahydro-4H-pyran-4-one (5.0 g,20.3mmol,1.0 eq.) in methanol (50 mL) was cooled to 0deg.C and then NaBH was added in portions 4 (92mg, 24.4mmol,1.2 eq.). The mixture was warmed to room temperature for 1 hour and NH was added 4 Cl (saturated aqueous solution). Using a biphasic mixtureDCM (50 mL mL x 3) and the combined organic phases were washed with brine and dried Na 2 SO 4 Filtered, and concentrated in vacuo. By flash column chromatography (SiO 2 0-12% EtOAc/PE) to give any of the specified trans and cis diastereomers: (3R, 4S) -3-benzyloxy-3-isopropenyl-tetrahydropyran-4-ol and (3S, 4S) -3-benzyloxy-3-isopropenyl-tetrahydropyran-4-ol (2.4 g, 48%); 1 h NMR (400 MHz, DMSO-d 6) delta ppm 7.39-7.31 (m, 4H), 7.27-7.25 (m, 1H), 5.12 (s, 1H), 5.09 (s, 1H), 4.55 (d, J=6.0 Hz, 1H), 4.41 (d, J=3.2 Hz, 2H), 3.86-3.77 (m, 3H), 3.51 (d, J=12.0 Hz, 1H), 3.47-3.40 (m, 1H), 1.89-1.81 (m, 1H), 1.78 (s, 3H), 1.64-1.60 (m, 1H). (3S, 4R) -3-benzyloxy-3-isopropenyl-tetrahydropyran-4-ol and (3R, 4R) -3-benzyloxy-3-isopropenyl-tetrahydropyran-4-ol (2.3 g, 46%); 1 H NMR(400MHz,DMSO-d6)δppm:7.33–7.27(m,4H),7.26–7.24(m,1H),5.09(s,1H),4.82(d,J＝4.4Hz,1H),4.78(s,1H),4.39(d,J＝11.2Hz,1H),4.09(d,J＝11.2Hz,1H),3.99(d,J＝12.4Hz,1H),3.78–3.72(m,2H),3.63–3.59(m,2H),2.15–2.12(m,1H),1.71(s,3H),1.40–1.36(m,1H)。
And (B) step (B): benzoic acid 3- (benzyloxy) -3- (prop-1-en-2-yl) tetrahydro-2H-pyran-4-yl ester:
a mixture of (3R, 4S) -3-benzyloxy-3-isopropenyl-tetrahydropyran-4-ol and (3S, 4S) -3-benzyloxy-3-isopropenyl-tetrahydropyran-4-ol (2.4 g,9.67mmol, 1.0 eq.) in pyridine (24 mL) was cooled to 0℃and then benzoyl chloride (5.43 g,38.7mmol,4.45mL,4.0 eq.) was added and the reaction mixture warmed to room temperature. The mixture was concentrated in vacuo and the residue was diluted with EtOAc (100 mL). The organic layer was washed with HCl (5 x 60ml,1.0m aqueous) and brine, over Na 2 SO 4 Dried, filtered, and concentrated in vacuo. By flash column chromatography (SiO 2 Purification of 0-5% EtOAc/PE afforded 3- (benzyloxy) -3- (prop-1-en-2-yl) tetrahydro-2H-pyran-4-yl benzoate (3.0 g, 88%). 1 H NMR(400MHz,DMSO-d6)δppm:8.00–7.94(m,2H),7.68–7.64(m,1H),7.56–7.50(m,2H),7.36–7.30(m,4H),7.27–7.25(m,1H),5.50–5.47(m,1H),5.17(s,1H),5.13(s,1H),4.51–4.39(m,2H),4.04–4.01(m,1H),3.92–3.88(m,1H),3.80(d,J＝12.4Hz,1H),3.69–3.64(m,1H),2.16–2.07(m,1H),1.88–1.83(m,1H),1.76(s,3H)。
Step C: benzoic acid 3-acetyl-3- (benzyloxy) tetrahydro-2H-pyran-4-yl ester:
a solution of 3- (benzyloxy) -3- (prop-1-en-2-yl) tetrahydro-2H-pyran-4-yl benzoate (3.0 g,8.51mmol,1.0 eq.) in DCM (60 mL) was cooled to-78deg.C. Ozone was gently bubbled through the stirred solution at-78 ℃ for about 1.5 hours, then N was allowed to stir 2 The solution was passed through for 10 minutes. Five drops of dimethyl sulfide were added and the reaction mixture was slowly warmed to room temperature. The reaction mixture was concentrated in vacuo and purified by flash column chromatography (SiO 2 0-6% EtOAc/PE) to give 3-acetyl-3- (benzyloxy) tetrahydro-2H-pyran-4-yl benzoate (2.5 g, 83%). 1 H NMR(400MHz,DMSO-d6)δppm:7.99–7.97(m,2H),7.70–7.66(m,1H),7.57–7.53(m,2H),7.39–7.27(m,5H),5.57–5.54(m,1H),4.57(s,2H),4.14(d,J＝12.8Hz,1H),3.89–3.85(m,2H),3.70–3.67(m,1H),2.27(s,3H),2.08–2.05(m,1H),1.95–1.92(m,1H)。
Step D: benzoic acid 3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-2H-pyran-4-yl ester:
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to a mixture of 3-acetyl-3- (benzyloxy) tetrahydro-2H-pyran-4-yl benzoate (1.5 g,4.23mmol,1.0 eq.) in chloroform (1.5 mL) was added DAST (18.3 g,114mmol,15mL,26.8 eq.) and the mixture was stirred at 75℃for 16H. The reaction mixture was diluted with DCM (50 mL) and ice water (150 mL), and with DCM (70 mL)x 3) extraction. The organic phase was washed with brine, dried (Na 2 SO 4 ) Filtered and concentrated in vacuo. Column chromatography (SiO) 2 0-5% EtOAc/PE) afforded 3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-2H-pyran-4-yl benzoate (470 mg, 29%). 1 H NMR(400MHz,DMSO-d6)δppm:7.97–7.95(m,2H),7.70–7.66(m,1H),7.57–7.53(m,2H),7.47(d,J＝7.6Hz,2H),7.42–7.38(m,2H),7.34–7.32(m,1H),5.46–5.43(m,1H),4.92(d,J＝11.2Hz,1H),4.70(d,J＝10.8Hz,1H),4.47(d,J＝13.2Hz,1H),3.98–3.95(m,1H),3.66–3.61(m,2H),2.13–2.09(m,1H),1.91–1.89(m,1H),1.68(t,J＝20.4Hz,3H)。
Step E:3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-2H-pyran-4-ol:
3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-2H-pyran-4-yl benzoate (470 mg,1.25mmol,1.0 eq.) in THF (5 mL), H 2 O (5 mL), meOH (5 mL), and LiOH H 2 The mixture in O (157 mg,3.75mmol,3.0 eq.) was stirred at room temperature for 2 hours. The mixture was extracted with DCM (30 ml x 3), the organic phases combined, washed with brine, dried (Na 2 SO 4 ) Filtered and concentrated. By flash column chromatography (SiO 2 Purification with 0-9% EtOAc/PE afforded 3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-2H-pyran-4-ol (305 mg, 89%). 1 H NMR(400MHz,DMSO-d6)δppm:7.43(d,J＝7.2Hz,2H),7.37–7.34(m,2H),7.30–7.26(m,1H),4.84(d,J＝8.0Hz,1H),4.77(d,J＝11.2Hz,1H),4.54–4.51(m,1H),4.35(d,J＝13.2Hz,1H),3.89–3.85(m,1H),3.80–3.77(m,1H),3.43–3.39(m,1H),3.36–3.31(m,1H),1.99–1.90(m,1H),1.81(t,J＝20.4Hz,3H),1.60–1.56(m,1H)。
Step F:3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-4H-pyran-4-one:
to a mixture of 3-benzyloxy-3- (1, 1-difluoroethyl) tetrahydropyran-4-ol (525 mg,1.93mmol,1.0 eq.) in DCM (6 mL) and DMF (6 mL) was added DMP (1.64 g,1.93mmol,2.0 eq.) and the mixture was stirred at 50℃for 2 hours. Adding Na 2 SO 3 Aqueous solution and water (70 mL) and the biphasic mixture was extracted with EtOAc (35 mL x 3). The organic phase was washed with brine, dried (Na 2 SO 4 ) Filtered and concentrated. Column chromatography (SiO) 2 0-6% EtOAc/PE) afforded 3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-4H-pyran-4-one (505 mg, 96%). 1 H NMR(400MHz,DMSO-d6)δppm:7.38–7.34(m,4H),7.33–7.30(m,1H),4.75(d,J＝11.2Hz,1H),4.57(d,J＝11.2Hz,1H),4.31(d,J＝12.8Hz,1H),4.11–4.04(m,2H),3.97–3.94(m,1H),2.84–2.80(m,1H),2.53–2.52(m,1H),1.70(t,J＝20.4Hz,3H)。
Step G:4- (benzyloxy) -4- (1, 1-difluoroethyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b ] thieno [3,2-d ] pyran-6-one:
to a solution of 3- (benzyloxy) -3- (1, 1-difluoroethyl) tetrahydro-4H-pyran-4-one (505 mg,1.87mmol,1.5 eq.) and 3-bromo-5- (1-tetrahydropyran-2-ylpyrazol-4-yl) thiophene-2-carboxylic acid methyl ester (460 mg,1.24mmol,1.0 eq.) in toluene (18 mL) was added Cs 2 CO 3 (1.21G, 3.72mmol,3.0 eq.) Sphos-Pd-G 3 (107 mg, 124. Mu. Mol,0.10 eq). The mixture was heated to 105 ℃ for 2 hours, cooled and filtered. The filtrate was concentrated in vacuo and purified by flash column chromatography (SiO 2 0-27% EtOAc/PE) to give 4- (benzyloxy) -4- (1, 1-difluoroethyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyran-6-one (260 mg, 39%). MS observations (ESI) + )：529.2[(M+H) + ]。
Step H: (S) -4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 95) and (R) -4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b ] thieno [3,2-d ] pyridin-6-one (compound 96):
4- (benzyloxy) -4- (1, 1-difluoroethyl) -8- (1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4,3-b]Thieno [3,2-d]A solution of pyran-6-one (260 mg, 492. Mu. Mol) in TFA (6 mL) was stirred at 50℃for 1 hour. The reaction mixture was concentrated in vacuo to give 4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -3, 4-dihydro-1H, 6H-pyrano [4, 3-b) ]Thieno [3,2-d]Pyran-6-one (305 mg, crude product). The crude residue was dissolved in isopropanol (8 mL) and ammonia (saturated aqueous solution, 8 mL) was added. The mixture was stirred at 95 ℃ for 16 hours, then cooled, concentrated in vacuo and purified by flash column chromatography (SiO 2 0-5% MeOH/DCM) to give 4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4, 3-b)]Thieno [3,2-d]Pyridin-6-one (138 mg, 45%). MS observations (ESI) + )：354.0[(M+H) + ]。
Enantiomers were separated by chiral SFC. (S) -4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 95): MS observations (ESI) + )：354.0[(M+H) + ]。 1 H NMR (400 MHz, DMSO-d 6) delta ppm 13.26 (s, 1H), 10.36 (s, 1H), 8.31 (s, 1H), 7.99 (s, 1H), 7.47 (s, 1H), 6.44 (s, 1H), 4.85-4.74 (m, 2H), 4.13 (d, J=12.0 Hz, 1H), 3.62 (d, J=12.0 Hz, 1H), 1.67 (t, J=19.6 Hz, 3H). (R) -4- (1, 1-difluoroethyl) -4-hydroxy-8- (1H-pyrazol-4-yl) -1,3,4, 5-tetrahydro-6H-pyrano [4,3-b]Thieno [3,2-d]Pyridin-6-one (compound 96): MS observations (ESI) + )：354.0[(M+H) + ]。
Bioassays
CDC7 kinase biochemical assay protocol:
full-length human CDC7 protein co-expressed with DBF4 was purchased from new granaco company (SignalChem) (china). PDKtide (New Genokang Co.) was used as substrate and ADP-Glo was used by following the manufacturer's instructions TM The kinase assay kit (Promega) measures ADP production to determine CDC7 kinase activity. The kinase reaction was performed using the following conditions: buffer solution: 40mM Tris pH 7.5, 20mM MgCl 2 0.1mg/mL BSA and 50. Mu.M DTT. The final reaction mixture contained 0.1nM CDC7/DBF4, 1. Mu.M ATP and 10. Mu.M PDKtide. The kinase reaction time was 4 hours. ADP-Glo signal was measured using an EnVision plate reader (PerkinELmer).
The percent inhibition of CDC7 kinase activity was calculated based on the following formula:
S sample of : signalling of compounds
S High control : high control (DMSO) signal
S Low control : signalling of low control (positive control CDC7 inhibitor)
Phosphorylated MCM2 MSD electrochemiluminescence assay
The effect of CDC7 inhibitors on cell phosphorylation of CDC7 substrate MCM2 was determined using the following protocol:
a total of 40,000 colo205 cells in 100 μl of medium (1640 medium +10% fetal bovine serum +1% penicillin-streptomycin) were plated in 96 well cell culture plates and allowed to attach for 6 hours. 3-fold serial dilutions of test compounds were prepared in complete PBS at 25-fold final concentration and 4. Mu.L each was added to cells and incubated at 37℃with 5% CO 2 Incubate for 20 hours. Each concentration was tested in duplicate. After 20 hours of incubation, the cells were washed with 150 μlpbs and lysed with 40 μl of MSD lysis buffer (obtained from medium-scale diagnostics company (Meso Scale Diagnostics)) provided with 1-fold complete ULTRA mixture (cocktail) inhibitor (obtained from Roche). To detect MCM 2S 53 phosphorylation, 30. Mu.L of capture antibody solution (obtained from the Minodv company (Abnova), catalog number H00004171-M01, 1:500) was added to each well of a multi-array 96-well high-binding plate and incubated overnight. The antibody solution was removed, the wells were blocked with BSA solution and the plates were washed, followed by the addition of 30 μl of cell lysate per well. After 2 hours of incubation, the plates were washed. Then 30. Mu.L of a 1-fold detection antibody solution (obtained from Ai Bokang company (Abcam), catalog number ab109133, 1:1000) was added to each well and incubated for 1 hour. Plates were washed and 30 μl of 1-fold secondary antibody solution (obtained from MSD company (MSD), catalog No. R32AB-1, 1:5000) was added to each well and incubated for 1 hour. Plates were washed and 150 μl of 1-fold read buffer T was added to each well of the MSD plate. Electrochemiluminescence signals were measured on a MESO SECTOR S600 plate reader. The percentage of phosphorylated MCM2 signal remaining was calculated according to the following equation.
HC (high control): cells treated with DMSO
Cpds: cells treated with test compounds
LC (low control): cells treated with positive control CDC7 inhibitors
Table 2: biological data
* A represents IC 50 Less than or equal to 1nM; b represents 1nM<IC 50 Less than or equal to 10nM; c represents IC 50 >10nM
Table 3: electrochemiluminescence assay
** A represents IC 50 Less than or equal to 5nM; b represents 5nM<IC 50 Less than or equal to 20nM; c represents 20nM<IC 50 Less than or equal to 50nM; d represents IC 50 >50nM

Claims (148)

1. A compound of formula (I):
or a pharmaceutically acceptable salt thereof;
wherein:
R 1 hydrogen or halogen;
R 2 a 5-6 membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl; or a 5-6 membered heterocyclyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl;
R 3 is hydrogen or C1-C6 alkyl;
ring A is C6-C10 cycloalkyl or 6-10 membered heterocyclyl;
each R 4 Independently selected from the group consisting of: halogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkoxy (C1-C6 alkyl) -, -C (=O) C1-C6 alkyl, C2-C6 alkynyl, C3-C6 cycloalkyl, -NR A R B And 4-6 membered heterocyclyl optionally substituted with 1 or 2 independently selected halo;
each R A And R is B Independently hydrogen or C1-C6 alkyl; and is also provided with
m is 0, 1, 2, 3 or 4.
2. The compound of claim 1, wherein m is 1.
3. The compound of claim 1, wherein m is 2.
4. The compound of claim 1, wherein m is 3.
5. The compound of claim 1, wherein m is 4.
6. The compound according to any one of claims 1 to 5, wherein R 2 Is a 5 membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
7. The compound according to any one of claims 1 to 5, wherein R 2 Is a 5 membered heteroaryl substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
8. The compound according to any one of claims 1 to 7, wherein R 2 Is pyrazolyl or isothiazolyl.
9. The compound according to any one of claims 1 to 8, wherein R 2 Is 4-pyrazolyl.
10. The compound according to any one of claims 1 to 8, wherein R 2 Is a 5-pyrazolyl group.
11. The compound according to any one of claims 1 to 8, wherein R 2 Is a 5-isothiazolyl group.
12. The compound according to any one of claims 1 to 5, wherein R 2 Optionally taken by 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkylSubstituted 6 membered heteroaryl.
13. The compound according to any one of claims 1 to 5 and 12, wherein R 2 Is a 6 membered heteroaryl substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
14. The compound according to any one of claims 1 to 5 and 12 to 13, wherein R 2 Is pyridinyl, pyrimidinyl or pyridazinyl.
15. The compound according to any one of claims 1 to 5 and 12 to 14, wherein R 2 Is pyridinyl.
16. The compound according to any one of claims 1 to 5 and 12 to 15, wherein R 2 Is 4-pyridyl.
17. The compound according to any one of claims 1 to 5 and 12 to 14, wherein R 2 Is pyrimidinyl.
18. The compound according to any one of claims 1 to 5, 12 to 14 and 17, wherein R 2 Is 4-pyrimidinyl.
19. The compound according to any one of claims 1 to 5 and 12 to 14, wherein R 2 Is a pyridazinyl group.
20. The compound according to any one of claims 1 to 5, 12 to 14 and 19, wherein R 2 Is a 4-pyridazinyl group.
21. The compound according to any one of claims 1 to 20, wherein R 2 Is substituted with a substituent selected from the group consisting of halogen and C1-C6 alkyl.
22. The compound according to any one of claims 1 to 21, wherein R 2 Is substituted with one substituent selected from the group consisting of fluorine, chlorine and methyl.
23. The compound according to any one of claims 1 to 20, wherein R 2 Is substituted with two substituents independently selected from halogen and C1-C6 alkyl.
24. The compound according to any one of claims 1 to 20 and 23, wherein R 2 Is substituted with two substituents independently selected from fluoro and methyl.
25. The compound according to any one of claims 1 to 20 and 23 to 24, wherein R 2 The substituents on the above are the same.
26. The compound according to any one of claims 1 to 20 and 23 to 24, wherein R 2 The substituents on the two groups are different.
27. The compound according to any one of claims 1 to 6 or 11 to 14, wherein R 2 Is unsubstituted 5-6 membered heteroaryl.
28. The compound of claim 27, wherein R 2 Selected from the group consisting of: pyrazolyl, isothiazolyl, pyridinyl and pyridazinyl.
29. The compound of claim 27 or 28, wherein R 2 Is 4-pyrazolyl.
30. The compound of claim 27 or 28, wherein R 2 Is 5-isothiazolyl.
31. The compound according to claim 27 or 28, whichR in (B) 2 Is 4-pyridyl.
32. The compound of claim 27 or 28, wherein R 2 Is 4-pyridazinyl.
33. The compound according to any one of claims 1 to 5, wherein R 2 A 5-6 membered heterocyclyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
34. The compound according to any one of claims 1 to 5 and 33, wherein R 2 Is a 5 membered heterocyclyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
35. The compound according to any one of claims 1 to 5 and 33 to 34, wherein R 2 Is a 5 membered heterocyclyl substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
36. The compound according to any one of claims 1 to 5 and 33, wherein R 2 Is a 6 membered heterocyclyl optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
37. The compound according to any one of claims 1 to 5, 33 and 36, wherein R 2 Is a 6 membered heterocyclyl substituted with 1 or 2 substituents independently selected from the group consisting of halogen and C1-C6 alkyl.
38. The compound according to any one of claims 1 to 5 and 33, wherein R 2 Is unsubstituted 5-6 membered heterocyclic group.
39. The compound according to any one of claims 1 to 5, 33 and 38, wherein R 2 Is morpholinyl.
40. The compound according to any one of claims 1 to 5, 33 and 38 to 39, wherein R 2 Is 4-morpholinyl.
41. The compound of any one of claims 1 to 40, wherein ring a is C6-C10 cycloalkyl.
42. The compound of any one of claims 1 to 41, wherein ring a is cyclohexyl.
43. The compound of any one of claims 1 to 41, wherein ring a is bicyclo [2.2.2] octyl.
44. The compound of any one of claims 1 to 40, wherein ring a is a 6-10 membered heterocyclyl.
45. The compound of any one of claims 1 to 40 and 44, wherein ring a is tetrahydropyranyl.
46. The compound of any one of claims 1 to 40 and 44, wherein ring a is piperidinyl.
47. A compound according to any one of claims 1 to 46, wherein one R 4 Is halogen.
48. The compound of any one of claims 1 to 47, wherein one R 4 Is fluorine.
49. The compound of any one of claims 1 to 47, wherein two R 4 Is fluorine.
50. A compound according to any one of claims 1 to 46, wherein one R 4 Is hydroxyl.
51. According to claims 1 to 46, wherein one R 4 Is a C1-C6 alkyl group.
52. A compound according to any one of claims 1 to 46 and 51, wherein one R 4 Selected from the group consisting of: -CH 3 、-CH 2 CH 3 、-CH(CH 3 ) 2 、-CH(CH 3 )CH 2 CH 3 、-C(CH 3 ) 2 CH 2 CH 3 and-C (CH) 3 ) 3 。
53. A compound according to any one of claims 1 to 46, wherein one R 4 Is C1-C6 alkoxy.
54. A compound according to any one of claims 1 to 46 and 53, wherein one R 4 Is methoxy.
55. A compound according to any one of claims 1 to 46, wherein one R 4 Is a C1-C6 hydroxyalkyl group.
56. The compound of any one of claims 1 to 46 and 55, wherein one R 4 is-CH 2 OH or-C (CH) 3 ) 2 OH。
57. A compound according to any one of claims 1 to 46, wherein one R 4 Is a C1-C6 haloalkyl.
58. The compound of any one of claims 1 to 46 and 57, wherein one R 4 is-CF 3 。
59. The compound of any one of claims 1 to 46 and 57, wherein one R 4 is-CHF 2 。
60. The chemical process of any one of claims 1 to 46 and 57A compound, wherein one R 4 is-CF 2 CH 3 。
61. The compound of any one of claims 1 to 46 and 57, wherein one R 4 is-CH 2 CF 2 CH 3 。
62. A compound according to any one of claims 1 to 46, wherein one R 4 Is C1-C6 alkoxy (C1-C6 alkyl) -.
63. The compound of any one of claims 1 to 46 and 62, wherein one R 4 is-CH 2 OCH 3 。
64. The compound of any one of claims 1 to 46, wherein R 4 One is-C (=o) C1-C6 alkyl.
65. The compound of any one of claims 1 to 46 and 64, wherein one R 4 is-C (=O) CH 3 。
66. A compound according to any one of claims 1 to 46, wherein one R 4 Is a C2-C6 alkynyl group.
67. The compound of any one of claims 1 to 46 and 66, wherein one R 4 Is 1-propynyl.
68. A compound according to any one of claims 1 to 46, wherein one R 4 Is a C3-C6 cycloalkyl group.
69. The compound of any one of claims 1 to 46 and 68, wherein one R 4 Is cyclopentyl.
70. A compound according to any one of claims 1 to 46, wherein one R 4 is-NR A R B 。
71. The compound of claim 70, wherein R is A And R is B One of them is hydrogen, and R A And R is B The other of (C1-C6 alkyl).
72. The compound of claim 70, wherein R is A And R is B Both are C1-C6 alkyl groups.
73. The compound of any one of claims 1 to 46 and 70, wherein R 4 is-NH 2 。
74. A compound according to any one of claims 1 to 46, wherein one R 4 Is a 4-6 membered heterocyclyl optionally substituted with 1 or 2 independently selected halo.
75. The compound of any one of claims 1 to 46 and 74, wherein one R 4 Is a 4-6 membered heterocyclyl substituted with 1 or 2 independently selected halogens.
76. The compound of any one of claims 1 to 46 and 74, wherein one R 4 Pyrrolidinyl optionally substituted with 1 or 2 independently selected halogens.
77. The compound of any one of claims 1 to 46 and 74 to 76, wherein one R 4 Is pyrrolidinyl substituted with 1 or 2 independently selected halogens.
78. The compound of any one of claims 1 to 46 and 74, wherein one R 4 Azetidinyl optionally substituted with 1 or 2 independently selected halogens.
79. A compound according to any one of claims 1 to 46, 74 and 78One of R 4 Azetidinyl substituted with 1 or 2 independently selected halogens.
80. The compound of any one of claims 1 to 46 and 74, wherein one R 4 Is unsubstituted 4-6 membered heterocyclic group.
81. The compound of any one of claims 1 to 46, 74 and 80, wherein one R 4 Is unsubstituted pyrrolidinyl.
82. The compound of any one of claims 1 to 46, 74 and 80 to 81, wherein one R 4 Is 1-pyrrolidinyl.
83. The compound of any one of claims 1 to 46, 74 and 80, wherein one R 4 Is unsubstituted azetidinyl.
84. The compound of any one of claims 1 to 46, 74, 80 and 83, wherein one R 4 Is unsubstituted 1-azetidinyl.
85. The compound of any one of claims 1 to 84, wherein m is 2, 3 or 4; and wherein the two R' s 4 The groups are geminal.
86. The compound of any one of claims 1 to 85, wherein m is 2; and wherein the two R' s 4 The groups are geminal.
87. The compound of any one of claims 1 to 85, wherein m is 3; and wherein the three R' s 4 Two of the groups are geminal.
88. The compound of any one of claims 1 to 85, wherein m is 4; and wherein each pair R 4 The radicals beingAnd geminally.
89. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is hydroxyl; and another R 4 Is a C1-C6 alkyl group.
90. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; wherein each R is 4 Is fluorine.
91. The compound of any one of claims 1 to 46, wherein m is 2; and wherein two of said R 4 The groups are geminal; one of R 4 Is hydroxyl; and another R 4 Is a C1-C6 haloalkyl.
92. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is hydroxyl; and another R 4 Is a C2-C6 alkynyl group.
93. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is hydroxyl; and another R 4 Is a C3-C6 cycloalkyl group.
94. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is hydroxyl; and another R 4 Is C1-C6 alkoxy (C1-C6 alkyl) -.
95. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; wherein each R is 4 To independently selectC1-C6 alkyl of (C1-C6).
96. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is C1-C6 alkyl; and another R 4 Is a C1-C6 hydroxyalkyl group.
97. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is C1-C6 alkyl; and another R 4 Is C1-C6 alkoxy.
98. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is C1-C6 alkyl; and another R 4 is-NR A R B 。
99. The compound of any one of claims 1 to 46, wherein m is 2; and wherein the two R' s 4 The groups are geminal; one of R 4 Is C1-C6 alkyl; and another R 4 Is a 4-6 membered heterocyclyl optionally substituted with 1 or 2 independently selected halo.
100. The compound of any one of claims 1 to 46, wherein m is 4; and wherein the two R' s 4 The groups are geminal; wherein two R 4 Is fluorine; r is R 4 Is C1-C6 alkoxy; and one R 4 Is a C1-C6 alkyl group.
101. The compound of any one of claims 1 to 46, wherein m is 4; and wherein the two R' s 4 The groups are geminal; wherein two R 4 Is fluorine; r is R 4 Is a C1-C6 hydroxyalkyl group; and one R 4 Is a C1-C6 alkyl group.
102. The compound of any one of claims 1 to 46, wherein m is 4; and wherein two R 4 The groups are geminal; wherein two R 4 Is fluorine; r is R 4 Is hydroxyl; and one R 4 Is a C1-C6 alkyl group.
103. The compound of any one of claims 1 to 46, wherein m is 4; and wherein two R 4 The groups are geminal; wherein two R 4 Is fluorine; r is R 4 Is hydroxyl; and one R 4 Is C1-C6 alkoxy (C1-C6 alkyl) -.
104. The compound of claim 1, wherein m is 0.
105. The compound of any one of claims 1 to 104, wherein R 1 Is halogen.
106. The compound of any one of claims 1 to 105, wherein R 1 Is fluorine.
107. The compound of any one of claims 1 to 104, wherein R 1 Is hydrogen.
108. The compound of any one of claims 1 to 107, wherein R 3 Is hydrogen.
109. The compound of any one of claims 1 to 107, wherein R 3 Is a C1-C6 alkyl group.
110. The compound of any one of claims 1 to 40 and 48 to 108, wherein the compound of formula (I) is a compound of formula (I-a):
or a pharmaceutically acceptable salt thereof.
111. The compound of any one of claims 1 to 40 and 48 to 108, wherein the compound of formula (I) is a compound of formula (I-B):
or a pharmaceutically acceptable salt thereof.
112. The compound of any one of claims 1 to 40 and 48 to 108, wherein the compound of formula (I) is a compound of formula (I-C):
or a pharmaceutically acceptable salt thereof.
113. The compound of any one of claims 1 to 40 and 48 to 108, wherein the compound of formula (I) is a compound of formula (I-D):
or a pharmaceutically acceptable salt thereof.
114. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-E):
Or a pharmaceutically acceptable salt thereof.
115. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-F):
or a pharmaceutically acceptable salt thereof.
116. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-G):
or a pharmaceutically acceptable salt thereof.
117. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-H):
or a pharmaceutically acceptable salt thereof.
118. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-I):
or a pharmaceutically acceptable salt thereof.
119. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-J):
or a pharmaceutically acceptable salt thereof.
120. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-K):
or a pharmaceutically acceptable salt thereof.
121. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-L):
Or a pharmaceutically acceptable salt thereof.
122. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-M):
or a pharmaceutically acceptable salt thereof.
123. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-N):
or a pharmaceutically acceptable salt thereof.
124. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-O):
or a pharmaceutically acceptable salt thereof.
125. The compound of any one of claims 1 to 40 and 48 to 85, wherein the compound of formula (I) is a compound of formula (I-P):
or a pharmaceutically acceptable salt thereof.
126. The compound of claim 1, wherein the compound is selected from the group consisting of the compounds in table 1 and pharmaceutically acceptable salts thereof.
127. The compound of claim 1, wherein the compound is selected from the group consisting of the compounds in examples 1-96 and pharmaceutically acceptable salts thereof.
128. A pharmaceutical composition comprising a compound according to any one of claims 1 to 127, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
129. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of any one of claims 1-127, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 128.
130. A method of treating a CDC 7-related cancer in a subject, the method comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 127, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 128, to a subject identified or diagnosed with CDC 7-related cancer.
131. A method for treating cancer in a subject in need thereof, the method comprising:
(a) Determining whether the cancer is associated with a dysregulation of expression or activity or level of a CDC7 gene, CDC7 kinase, or any of them; and
(b) If the cancer is determined to be associated with a deregulation of the expression or activity or level of the CDC7 gene, CDC7 kinase or any one thereof, then administering to the subject an effective amount of a compound according to any one of claims 1 to 127 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 128.
132. The method of claim 131, wherein the step of determining whether the cancer of the subject is a CDC 7-related cancer comprises performing an assay to detect a dysregulation of expression or activity or level of a CDC7 gene, CDC7 kinase protein, or any of them in a sample from the subject.
133. The method of claim 131 or 132, further comprising obtaining a sample from the subject.
134. The method of claim 133, wherein the sample is a biopsy sample.
135. The method of any one of claims 132-134, wherein the assay is selected from the group consisting of: sequencing, immunohistochemistry, enzyme-linked immunosorbent assay and Fluorescence In Situ Hybridization (FISH).
136. The method of claim 135, wherein the sequencing is pyrosequencing or next generation sequencing.
137. The method of any one of claims 129-136, further comprising administering to the subject an additional therapy or therapeutic agent.
138. The method of claim 137, wherein the additional therapy or therapeutic agent is selected from the group consisting of radiation therapy, cytotoxic chemotherapeutic agents, kinase targeted therapeutic agents, apoptosis modulators, signal transduction inhibitors, immune targeted therapies, and angiogenesis targeted therapies.
139. The method of claim 137 or 138, wherein the compound of any one of claims 1 to 126, or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 128 and the additional therapeutic agent are administered simultaneously in separate doses.
140. The method of claim 137 or 138, wherein the compound of any one of claims 1 to 127, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 128 and the additional therapeutic agent are administered sequentially in any order in separate doses.
141. A method for inhibiting proliferation of a mammalian cell, the method comprising contacting the mammalian cell with a compound according to any one of claims 1 to 127, or a pharmaceutically acceptable salt thereof.
142. A method for inhibiting CDC7 kinase activity of a mammalian cell, the method comprising contacting the mammalian cell with a compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 127.
143. The method of claim 141 or 142, wherein the contacting occurs in vivo.
144. The method of claim 141 or 142, wherein the contacting occurs in vitro.
145. The method of any one of claims 141-144, wherein the mammalian cell is a mammalian cancer cell.
146. The method of claim 145, wherein the mammalian cancer cell is a mammalian CDC 7-related cancer cell.
147. The method of any one of claims 141-146, wherein the mammalian cell has a dysregulation of expression or activity or level of a CDC7 gene, CDC7 kinase protein, or any one thereof.
148. A method for inhibiting metastasis in a subject having a particular cancer in need of such treatment, the method comprising administering to the subject an effective amount of a compound of any one of claims 1-127 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 128.
CN202280022298.9A 2021-03-18 2022-03-17 Cyclic compounds and methods of use thereof Pending CN117120447A (en)

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