WO2024160058A1 - 草酸二酰胺化合物、包含其的药物组合物及其制备方法和用途 - Google Patents

草酸二酰胺化合物、包含其的药物组合物及其制备方法和用途 Download PDF

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WO2024160058A1
WO2024160058A1 PCT/CN2024/072949 CN2024072949W WO2024160058A1 WO 2024160058 A1 WO2024160058 A1 WO 2024160058A1 CN 2024072949 W CN2024072949 W CN 2024072949W WO 2024160058 A1 WO2024160058 A1 WO 2024160058A1
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compound
alkyl
independently selected
pharmaceutically acceptable
stereoisomer
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French (fr)
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陈忠辉
梅红江
田强
宋宏梅
葛均友
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四川科伦博泰生物医药股份有限公司
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Publication of WO2024160058A1 publication Critical patent/WO2024160058A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4995—Pyrazines or piperazines forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to an oxalic acid diamide compound, a pharmaceutical composition comprising the same, a preparation method thereof and use thereof for preventing or treating diseases or conditions associated with PRMT5 activity.
  • PRMT5 Protein Arginine Methyltransferase 5
  • PRMT5 is an epigenetic enzyme and a member of the PRMT family (the human body has PRMT1-11). It can catalyze the methylation modification of arginine residues of histones and certain non-histone substrates.
  • PRMT5 is widely present in the nucleus and cytoplasm of human cells, including tissues such as the heart, muscle and testis. According to the different ways of catalyzing arginine methylation, it is divided into types I, II and III.
  • PRMT5 belongs to type II symmetric dimethylation (sDMA) PRMT, and its methyl donor is S-adenosylmethionine (SAM).
  • sDMA symmetric dimethylation
  • SAM S-adenosylmethionine
  • PRMT5 regulates the expression of multiple target proteins by catalyzing the arginine methylation of substrates, participates in multiple physiological functions, and plays an important role in tumor cell proliferation, metastasis, and malignant transformation.
  • PRMT5 methylation modification of histones leads to the silencing of tumor suppressor genes such as p53, ST7, NM23, and Rb, thereby promoting the occurrence and development of tumors.
  • PRMT5 regulation of non-histone proteins is mainly reflected in affecting the localization and expression of transcription factors (NF- ⁇ B/P65, E2F1, HoxA ⁇ GATA4), programmed cell death protein 4 (PDCD4), cell cycle and survival-related regulatory proteins E2F1, hypoxia-inducible factor 1 (HIF-1), cyclin-dependent kinases (CDKs), PI3K/Akt, etc. (Koh CM, Bezzi M, Guccione E. Curr Mol Bio Rep, 2015, 1(1):19-28).
  • transcription factors NF- ⁇ B/P65, E2F1, HoxA ⁇ GATA4
  • PDCD4 programmed cell death protein 4
  • E2F1 hypoxia-inducible factor 1
  • CDKs cyclin-dependent kinases
  • PI3K/Akt PI3K/Akt
  • PRMT5 can inhibit the transcription of miR-99 family, increase FGFR3 expression, activate Erk1/2 and Ak pathways, leading to tumor cell growth and metastasis (Pengyu Jing, Nan Zhao, et al. Cancer Letters, 2018, 427, 38-48).
  • PRMT5 can methylate Eif4e and FGFR3, promoting tumor cell growth (ZHANG B, DONG S, ZHU R, et al. Oncotarget, 2015, 6(26): 22799-22811.).
  • MTAP is the gene encoding methylthioadenosine phosphorylase, located on chromosome 9p21, close to the tumor suppressor gene CDKN2A (which often undergoes homozygous deletion). Therefore, MTAP is often co-deleted with CDKN2A in tumors (Marjon K, Kalev P, Marks K. Annual Review of Cancer Biology, 2021, 5(1)). Approximately 15% of solid tumors suffer from MTAP deletion.
  • PRMT5 has two cofactors, namely the activating cofactor (SAM) and the inhibitory cofactor (MTA; 5'-methylthioadenosine).
  • SAM activating cofactor
  • MTA inhibitory cofactor
  • MTAP is responsible for converting MTA to Met (methionine)
  • PRMT5 is responsible for converting SAM to SAH (S-adenosyl-L-homocysteine).
  • SAM activating cofactor
  • MTA inhibitory cofactor
  • MTA 5'-methylthioadenosine
  • MTA is an endogenous competitive inhibitor of PRMT5-SAM.
  • the loss of MTAP leads to the accumulation of MTA, which partially inhibits the activity of PRMT5 and makes tumor cells more dependent on PRMT5.
  • inhibiting PRMT5 can further block the methylation function of PRMT5 and cause tumor cell death.
  • the invention provides a new oxalic acid diamide compound, which has a good inhibitory effect on MTAP-deficient tumor cells and has good pharmacokinetic properties.
  • One aspect of the present invention provides a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled compound, polymorph, solvate, N-oxide, metabolite or prodrug thereof:
  • X 1 , X 2 and X 3 are each independently selected from -CH 2 -, -CH-, -O-, -N-, -NH- and -S-;
  • R 1 is independently selected at each occurrence from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 hydroxyalkyl and C 3-8 cycloalkyl;
  • Ring A is a 5-7 membered saturated or partially saturated heterocyclic ring containing at least one N atom and optionally 1 or 2 other heteroatoms which are the same or different and are each independently selected from O or S;
  • R 2 is independently selected at each occurrence from H, -OH, halogen, -CN, -NR 4 R 5 , -COCH 3 , -NHCOCH 3 , C 1-6 alkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 2-6 heteroalkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 3-8 cycloalkyloxy, C 6-10 aryl, and 5-10 membered heteroaryl, each of said alkyl, alkoxy, hydroxyalkyl, heteroalkyl, cycloalkyl, heterocyclyl, cycloalkyloxy, aryl or heteroaryl being optionally substituted with one or more R 3 ;
  • R 3 is each independently selected at each occurrence from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 2-6 heteroalkyl, C 3-6 cycloalkyl, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl, said alkyl, alkoxy, heteroalkyl, cycloalkyl, cycloalkoxy, heterocyclyl, aryl or heteroaryl being each optionally substituted with one or more substituents each independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy , C 1-6 haloalkoxy, C 3-6 cycloalky
  • R 4 , R 5 , R 6 and R 7 are each independently selected from H and C 1-6 alkyl; or
  • R4 and R5 , R6 and R7 together with the nitrogen atom to which they are attached form a 3-8 membered heterocyclic group, which is optionally substituted with one or more substituents each independently selected from halogen, OH, CN, -NH2 and C1-6 alkyl;
  • n 0, 1, 2 or 3;
  • n 0, 1, 2, 3 or 4.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled compound, polymorph, solvate, N-oxide, metabolite or prodrug thereof and one or more pharmaceutically acceptable carriers.
  • Another aspect of the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotope-labeled compound, polymorph, solvate, N-oxide, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention in the preparation of a medicament for preventing or treating a disease or condition associated with PRMT5 activity.
  • Another aspect of the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled compound, polymorph, solvate, N-oxide, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention, for use in preventing or treating a disease or condition associated with PRMT5 activity.
  • Another aspect of the present invention provides a method for preventing or treating a disease or condition associated with PRMT5 activity, the method comprising administering to an individual in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled compound, polymorph, solvate, N-oxide, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention.
  • Another aspect of the present invention provides a method for preparing a compound of the present invention, comprising the steps of:
  • Step 1 Compound IA-1 and acyl chloride Compound IA-2 is generated through condensation reaction;
  • Step 2 Compound IA-2 is hydrolyzed to generate compound IA-3;
  • Step 3 Compound IA-3 and compound IA-4 undergo condensation reaction to generate compound IA-5;
  • Step 4 The condensation product IA-5 is deprotected to generate compound I;
  • PG is an amino protecting group
  • X 1 , X 2 , X 3 , Ring A, R 1 , R 2 , m and n are as defined above; R 6 and R 7 are H.
  • alkyl is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl has 1 to 12, such as 1 to 6 carbon atoms, 1 to 4 carbon atoms.
  • C 1-6 alkyl and C 1-4 alkyl refer to a linear or branched group (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or n-hexyl) having 1-6 carbon atoms and 1-4 carbon atoms, respectively, which is optionally substituted by one or more (such as 1 to 3) suitable substituents such as halogen (in this case, the group is referred to as "haloalkyl”) (such as CH 2 F, CHF 2 ,
  • C 1-4 alkyl refers to a linear or branched aliphatic hydrocarbon chain having 1 to 4 carbon atoms (ie, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl).
  • heteroalkyl refers to an alkyl group having one or more backbone chain atoms independently selected from atoms other than carbon, such as oxygen, nitrogen, sulfur, phosphorus, or a combination thereof, in the main chain carbon atoms of the alkyl group.
  • Numerical ranges may be given (e.g., C2-6 heteroalkyl) referring to the number of carbons in the chain, which in this example includes 2-6 carbon atoms.
  • a -CH2OCH2CH3 group is referred to as a C3 heteroalkyl group
  • a -CH2OCH2CH2NHCH3 group is referred to as a C4 heteroalkyl group. Connection to the rest of the molecule may be through a heteroatom or a carbon atom in the heteroalkyl chain.
  • haloalkyl refers to an alkyl group substituted by one or more (such as 1 to 3) the same or different halogen atoms
  • C 1-8 haloalkyl refers to haloalkyl groups having 1 to 8 carbon atoms, 1 to 6 carbon atoms and 1-4 carbon atoms, respectively, for example, -CF 3 , -C 2 F 5 , -CHF 2 , -CH 2 F, -CH 2 CF 3 , -CH 2 Cl or -CH 2 CH 2 CF 3 , etc.
  • hydroxyalkyl refers to a group formed by replacing the hydrogen atoms in an alkyl group with one or more hydroxy groups, such as a C1-6 hydroxyalkyl group or a C1-4 hydroxyalkyl group, examples of which include but are not limited to hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, -CH(OH) CH3 , and the like.
  • alkoxy means -O-alkyl, wherein alkyl is as defined above, for example, C 1-8 alkoxy, C 1-6 alkoxy, C 1-4 alkoxy or C 1-3 alkoxy.
  • Representative examples of C 1-6 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, etc.
  • the alkoxy is optionally substituted with one or more (such as 1 to 3) identical or different substituents.
  • haloalkoxy means that the hydrogen atoms of the alkoxy are substituted with one or more (such as 1 to 3) identical or different halogen atoms.
  • paracyclic ring or “fused ring” refers to a ring system formed by two or more cyclic structures sharing two adjacent atoms with each other.
  • spirocycle refers to a ring system formed by two or more cyclic structures that share one ring atom with each other.
  • bridged ring refers to a ring system formed by two or more cyclic structures sharing two atoms that are not directly connected to each other.
  • cycloalkyl refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring group, including but not limited to monocyclic alkyl (such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, etc.) and bicyclic alkyl, including spirocyclic, cyclocyclic (condensed) or bridged ring systems (i.e., spirocyclic alkyl, cyclocyclic (condensed) alkyl and bridged cycloalkyl, such as bicyclo [1.1.1] pentyl, bicyclo [2.2.1] heptyl, etc.).
  • monocyclic alkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclo
  • cycloalkyl is optionally substituted with one or more (such as 1 to 3) identical or different substituents.
  • C3-8 cycloalkyl refers to a cycloalkyl group having 3 to 8 ring-forming carbon atoms, for example, a C3-6 cycloalkyl group, which may be a monocyclic alkyl group, for example, a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl group, or a bicyclic alkyl group, for example, a C5-8 spirocycloalkyl group, a C5-8 bridged cycloalkyl group, a C5-8 fused cycloalkyl group, a C5-6 spirocycloalkyl group, a C5-6 bridged cycloalkyl group or a C5-6 fused cycloalkyl group.
  • a C3-6 cycloalkyl group which may be a monocyclic alkyl group, for example, a cyclopropyl
  • cycloalkoxy means -O-cycloalkyl, wherein cycloalkyl is as defined above.
  • Representative examples of cycloalkoxy include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • substituents independently selected from halogen and C 1-3 alkyl.
  • saturated heterocycle refers to a fully saturated heterocycle, such as a tetrahydrofuran ring, a piperidine ring, a morpholine ring, a tetrahydropyran ring, a piperazine ring, and the like.
  • partially saturated heterocycle refers to a heterocycle containing both saturated single bonds and unsaturated double bonds in the heterocycle, for example, 3,4-dihydro-2H-pyran, 1,2,3,4-tetrahydropyridine, 4,5-dihydroisoxazolyl, 4,5-dihydrooxazolyl, 2,5-dihydrooxazolyl, 2,3-dihydrooxazolyl, etc.
  • the term "4-11 membered heterocyclyl” means a heterocyclyl containing 4-11 ring atoms, including but not limited to 4-10 membered heterocyclyl, 4-9 membered heterocyclyl, 4-8 membered heterocyclyl, 4-7 membered heterocyclyl, 5-6 membered heterocyclyl, 3-8 membered heterocyclyl, 3-7 membered heterocyclyl, 4-7 membered nitrogen-containing heterocyclyl, 4-7 membered oxygen-containing heterocyclyl, 4-7 membered sulfur-containing heterocyclyl, 5-6 membered nitrogen-containing heterocyclyl, 5-6 membered oxygen-containing heterocyclyl, 5-6 membered sulfur-containing heterocyclyl, etc., wherein the "nitrogen-containing heterocyclyl", “oxygen-containing heterocyclyl” and “sulfur-containing heterocyclyl” each optionally further contain one or more other heteroatoms independently selected from oxygen, nitrogen and sulfur.
  • Examples of 4-11 membered heterocyclyls include but are not limited to oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, pyrrolidonyl (such as ), imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl.
  • 3-8 membered heterocyclic radical means a heterocyclic radical containing 3-8 ring atoms, including but not limited to 3-8 membered heterocyclic radicals, 3-7 membered heterocyclic radicals, 3-6 membered heterocyclic radicals, 4-8 membered heterocyclic radicals, 4-7 membered heterocyclic radicals, 4-6 membered heterocyclic radicals, 5-6 membered heterocyclic radicals, 4-7 membered nitrogen-containing heterocyclic radicals, 4-7 membered oxygen-containing heterocyclic radicals, 4-7 membered sulfur-containing heterocyclic radicals, 5-6 membered nitrogen-containing heterocyclic radicals, 5-6 membered oxygen-containing heterocyclic radicals, 5-6 membered sulfur-containing heterocyclic radicals, etc., and the "nitrogen-containing heterocyclic radicals", "oxygen-containing heterocyclic radicals" and "sulfur-containing heterocyclic radicals" each optionally further contain one or more other heteroatoms independently selected
  • 3-8 membered heterocyclic groups include, but are not limited to, oxirane, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, pyrrolidonyl (e.g. ), imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl.
  • the heterocyclic group can form a parallel ring structure with a heterocyclic group or a cycloalkyl group, and the connection point of the parallel ring structure with the other group can be on any heterocyclic group or on the cycloalkyl group.
  • the heterocyclic group of the present invention also includes (but is not limited to) heterocyclic groups and heterocyclic groups, heterocyclic groups and cycloalkyl groups, monoheterocyclic groups and monoheterocyclic groups, and monoheterocyclic groups and monocycloalkyl groups, such as 3-7 membered (mono) heterocyclic groups and 3-7 membered (mono) heterocyclic groups, 3-7 membered (mono) heterocyclic groups and (mono) cycloalkyl groups, and 3-7 membered (mono) heterocyclic groups and C4-6 (mono) cycloalkyl groups.
  • heterocyclic groups and heterocyclic groups such as 3-7 membered (mono) heterocyclic groups and 3-7 membered (mono) heterocyclic groups, 3-7 membered (mono) heterocyclic groups and (mono) cycloalkyl groups, and 3-7 membered (mono) heterocyclic groups and C4-6 (mono) cycloalkyl groups
  • Examples thereof include but are not limited to pyrrolidinyl and cyclopropyl, cyclopentyl and aziridine, pyrrolidinyl and cyclobutyl, pyrrolidinyl and pyrrolidinyl, pyrrolidinyl and piperidinyl, pyrrolidinyl and piperazinyl, piperidinyl and morpholinyl,
  • the heterocyclic group also includes a bridged heterocyclic group and a spiro heterocyclic group.
  • bridged heterocycle refers to a cyclic structure containing one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen atoms, nitrogen atoms, and/or sulfur atoms) formed by two saturated rings sharing two ring atoms that are not directly connected, including but not limited to 7-10 membered bridged heterocycles, 8-10 membered bridged heterocycles, 7-10 membered nitrogen-containing bridged heterocycles, 7-10 membered oxygen-containing bridged heterocycles, 7-10 membered sulfur-containing bridged heterocycles. etc., for example
  • the "nitrogen-containing bridged heterocycle", “oxygen-containing bridged heterocycle” and “sulfur-containing bridged heterocycle” optionally further contain one or more other heteroatoms independently selected from oxygen, nitrogen and sulfur.
  • spiroheterocycle refers to a cyclic structure containing one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen atoms, nitrogen atoms, sulfur atoms) formed by two or more saturated rings sharing one ring atom, including but not limited to 5-10 membered spiroheterocycle, 6-10 membered spiroheterocycle, 6-10 membered nitrogen-containing spiroheterocycle, 6-10 membered oxygen-containing spiroheterocycle, 6-10 membered sulfur-containing spiroheterocycle, etc., for example
  • the "nitrogen-containing spiro heterocycle", “oxygen-containing spiro heterocycle” and “sulfur-containing spiro heterocycle” optionally further contain one or more other heteroatoms independently selected from oxygen, nitrogen and sulfur.
  • 6-10 membered nitrogen-containing spiro heterocyclic group refers to a spiro heterocyclic group containing a total of 6-10 ring atoms and at least one of the ring atoms being a nitrogen atom.
  • Examples of the group obtained by condensing a heterocyclic group with an aryl group include, but are not limited to:
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic group having a conjugated ⁇ electron system.
  • C 6-10 aryl (aromatic ring) means an aryl (aromatic ring) containing 6 to 10 carbon atoms, preferably a phenyl (benzene ring) or a naphthyl (naphthalene ring).
  • the aryl group is optionally substituted with one or more (such as 1 to 3) identical or different substituents (e.g., halogen, OH, CN, NO 2 , C 1 -C 6 alkyl, etc.).
  • heteroaryl or “heteroaromatic ring” refers to a monocyclic or polycyclic aromatic group containing one or more identical or different heteroatoms, including monocyclic heteroaryl groups and bicyclic or polycyclic ring systems containing at least one heteroaromatic ring (an aromatic ring system containing at least one heteroatom), which may have 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, such as 5, 6, 7, 8, 9 or 10 ring atoms, and in each case may be benzo-fused.
  • the heteroatom may be oxygen, nitrogen or sulfur.
  • 5-10 membered heteroaryl or "5-10 membered heteroaromatic ring” means a heteroaromatic group (heteroaromatic ring) containing 5 to 10 (e.g., 5 to 6) ring atoms, including 5-10 membered nitrogen-containing heteroaryl, 5-10 membered oxygen-containing heteroaryl, 5-10 membered sulfur-containing heteroaryl, 5-6 membered nitrogen-containing heteroaryl, 5-6 membered oxygen-containing heteroaryl, 5-6 membered sulfur-containing heteroaryl, etc.
  • 5-10 membered heteroaryl or "5-10 membered heteroaromatic ring” means a heteroaromatic group (heteroaromatic ring) containing 5 to 10 (e.g., 5 to 6) ring atoms, including 5-10 membered nitrogen-containing heteroaryl, 5-10 membered oxygen-containing heteroaryl, 5-10 membered sulfur-containing heteroaryl, 5-6 membered nitrogen-containing heteroaryl, 5-6 membered sulfur
  • nitrogen-containing heteroaryl each optionally contain one or more other heteroatoms independently selected from oxygen, nitrogen and sulfur.
  • examples include, but are not limited to, thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl, etc., or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and 5-10 membered cyclic groups containing these groups.
  • a heteroaryl group (e.g., a monoheteroaryl group) can share two adjacent atoms with an aryl group (e.g., a monocyclic aryl group, such as a phenyl group), a heterocyclic group (e.g., a monoheterocyclic group), a cycloalkyl group (e.g., a monocycloalkyl group) or another heteroaryl group (e.g., another monoheteroaryl group) to form a parallel ring structure, and the connection point can be on any heteroaryl ring or on other rings, including but not limited to (mono)heteroaryl and (mono)heteroaryl, (mono)heteroaryl and (monocyclic) aryl, (mono)heteroaryl and (mono)heterocyclic group and (mono)heteroaryl and (mono)cycloalkyl, such as a 5-6 membere
  • halo or halogen group is defined to include F, Cl, Br, or I.
  • substituted means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that the normal valence of the designated atom in the present context is not exceeded and the substitution forms a stable compound. Combinations of substituents and/or variables are permitted only if such combinations form stable compounds.
  • substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and/or together, with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.
  • each substituent is selected independently of the other.
  • each substituent may be the same as or different from another (other) substituent.
  • one or more means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.
  • the point of attachment of a substituent may be from any suitable position of the substituent.
  • the present invention also includes all pharmaceutically acceptable isotopically labeled compounds, which are identical to the compounds of the present invention except that one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature.
  • isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium ( 2H ), tritium ( 3H )); isotopes of carbon (e.g., 11C , 13C , and 14C ); isotopes of chlorine (e.g., 36Cl ); isotopes of fluorine (e.g., 18F ); isotopes of iodine (e.g., 123I and 125I ); isotopes of nitrogen (e.g., 13N and 15N ); isotopes of oxygen (e.g., 15O , 17O , and 18O ); isotopes of phosphorus (e.g., 32P ); and isotopes of sulfur (e.g., 35S ).
  • isotopes of hydrogen e.g., deuterium ( 2H ), tritium ( 3H
  • Certain isotopically labeled compounds of the invention are useful in drug and/or substrate tissue distribution studies (e.g., assays).
  • the radioisotopes tritium (i.e., 3 H) and carbon-14 (i.e., 14 C) are particularly useful for this purpose because they are easily incorporated and easily detected.
  • Substitution with positron emitting isotopes e.g., 11 C, 18 F, 15 O, and 13 N
  • PET positron emission tomography
  • Isotopically labeled compounds of the invention can be prepared by methods similar to those described in the accompanying routes and/or in the examples and preparations by using appropriate isotopically labeled reagents in place of the non-labeled reagents previously employed.
  • Pharmaceutically acceptable solvates of the invention include those in which the crystallization solvent may be isotopically substituted, for example, D 2 O, acetone-d 6 or DMSO-d 6 .
  • stereoisomer refers to an isomer formed due to at least one asymmetric center.
  • racemic mixtures single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced.
  • Specific individual molecules can also exist as geometric isomers (cis/trans).
  • the compounds of the present invention can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium.
  • tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc.
  • nitroso-oxime can exist in the following tautomeric form equilibrium in solution:
  • solid lines can be used Solid wedge Virtual wedge Depicting chemical bonds of compounds of the invention.
  • the use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.).
  • the use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that There are stereoisomers as shown. When present in a racemic mixture, the real and dashed wedges are used to define relative stereochemistry, rather than absolute stereochemistry.
  • the compounds of the invention are intended to exist as stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof.
  • the compounds of the invention may exhibit more than one type of isomerism and consist of mixtures thereof, such as racemic mixtures and diastereomeric pairs.
  • the present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.
  • Cocrystal refers to a drug active molecule and other physiologically acceptable acid, base, salt, non-ionic compound molecules combined in the same crystal lattice by hydrogen bonds, ⁇ - ⁇ stacking, van der Waals forces and other non-covalent bonds.
  • compositions of the present invention may exist in free form for treatment, or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof.
  • pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites or prodrugs, which, after being administered to a patient in need thereof, can directly or indirectly provide a compound of the present invention or a metabolite or residue thereof. Therefore, when referring to "compounds of the present invention” herein, it is also intended to cover the above-mentioned various derivative forms of the compound.
  • Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.
  • Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof, such as hexafluorophosphate, meglumine salt, etc.
  • acid addition salts and base addition salts thereof such as hexafluorophosphate, meglumine salt, etc.
  • esters means an ester derived from the compounds of the general formulae herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in free acid or alcohol form).
  • physiologically hydrolyzable esters which can be hydrolyzed under physiological conditions to release the compounds of the present invention in free acid or alcohol form.
  • the compounds of the present invention themselves may also be esters.
  • the compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain polar solvents as structural elements of the crystal lattice of the compounds, in particular water, methanol or ethanol.
  • polar solvents as structural elements of the crystal lattice of the compounds, in particular water, methanol or ethanol.
  • the amount of polar solvents, in particular water may exist in a stoichiometric or non-stoichiometric ratio.
  • nitrogen-containing heterocycles can form N-oxides.
  • nitrogen-containing heterocycles that can form N-oxides.
  • tertiary amines can form N-oxides.
  • the synthetic method for preparing the N-oxide of heterocycles and tertiary amines is well known to those skilled in the art, including but not limited to oxidizing heterocycles and tertiary amines with peroxyacids such as peracetic acid and metachloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate and dioxirane such as dimethyl dioxirane.
  • peroxyacids such as peracetic acid and metachloroperbenzoic acid (MCPBA)
  • MCPBA metachloroperbenzoic acid
  • hydrogen peroxide alkyl hydroperoxides
  • sodium perborate and dioxirane such as dimethyl dioxirane.
  • metabolites of the compounds of the present invention i.e., substances formed in vivo upon administration of the compounds of the present invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound.
  • the present invention includes metabolites of the compounds of the present invention, including compounds prepared by contacting the compounds of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.
  • the present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have less pharmacological activity or no pharmacological activity, which when administered into or onto the body can be converted into compounds of the present invention having the desired activity by, for example, hydrolytic cleavage.
  • prodrugs will be functional group derivatives of the compounds that are easily converted into the desired therapeutically active compounds in vivo. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella).
  • Prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain moieties known to those skilled in the art as "pro-moieties” (e.g., as described in “Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).
  • the present invention also encompasses compounds of the present invention containing protecting groups.
  • protecting groups In any process for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example, those described in T.W.Greene & P.G.M.Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Protecting groups may be removed at an appropriate subsequent stage using methods known in the art.
  • the present invention provides a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled compound, polymorph, solvate, N-oxide, metabolite or prodrug thereof:
  • X 1 , X 2 and X 3 are each independently selected from -CH 2 -, -CH-, -O-, -N-, -NH- and -S-;
  • R 1 is independently selected at each occurrence from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 hydroxyalkyl and C 3-8 cycloalkyl;
  • Ring A is a 5-7 membered saturated or partially saturated heterocyclic ring containing at least one N atom and optionally 1 or 2 other heteroatoms which are the same or different and are each independently selected from O or S;
  • R 2 is independently selected at each occurrence from H, -OH, halogen, -CN, -NR 4 R 5 , -COCH 3 , -NHCOCH 3 , C 1-6 alkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 2-6 heteroalkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 3-8 cycloalkyloxy, C 6-10 aryl, and 5-10 membered heteroaryl, each of said alkyl, alkoxy, hydroxyalkyl, heteroalkyl, cycloalkyl, heterocyclyl, cycloalkyloxy, aryl or heteroaryl being optionally substituted with one or more R 3 ;
  • R 3 is each independently selected at each occurrence from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 2-6 heteroalkyl, C 3-6 cycloalkyl, C 3-8 cycloalkyloxy, 3-8 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl, said alkyl, alkoxy, heteroalkyl, cycloalkyl, cycloalkoxy, heterocyclyl, aryl or heteroaryl being each optionally substituted with one or more substituents each independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy , C 1-6 haloalkoxy, C 3-6 cycloalky
  • R 4 , R 5 , R 6 and R 7 are each independently selected from H and C 1-6 alkyl; or
  • R4 and R5 , R6 and R7 together with the nitrogen atom to which they are attached form a 3-8 membered heterocyclic group, which is optionally substituted with one or more substituents each independently selected from halogen, OH, CN, -NH2 and C1-6 alkyl;
  • n 0, 1, 2 or 3;
  • n 0, 1, 2, 3 or 4.
  • X 1 , X 2 and X 3 are each independently selected from -CH 2 -, -CH-, -O-, -N- and -NH-.
  • the present invention provides compounds of formula I wherein X 1 is -CH-, and X 2 and X 3 are -N-.
  • the present invention provides compounds of formula I in which X1 and X2 are -N-, and X3 is -CH-.
  • the present invention provides compounds of formula I in which X1 and X3 are -CH2- , and X2 is -O-.
  • R 1 at each occurrence is independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl and C 3-8 cycloalkyl.
  • the present invention provides compounds of formula I wherein R 1 at each occurrence is independently selected from H and C 1-6 alkyl.
  • the present invention provides compounds of formula I in which R 1 is methyl.
  • ring A is a 5-7 membered saturated heterocyclic ring, wherein the heterocyclic ring contains at least one N atom and optionally 1 or 2 other heteroatoms independently selected from O or S that are the same or different.
  • the present invention provides compounds of formula I, wherein ring A is a 5-7 membered saturated nitrogen heterocycle and a 5-7 membered saturated N- and O-containing heterocycle;
  • the present invention provides compounds of formula I, wherein ring A is a 6-membered saturated nitrogen heterocycle and a 6-membered saturated N- and O-containing heterocycle;
  • ring A is a piperidine ring or a morpholine ring.
  • ring A is a 5-7 membered saturated nitrogen heterocycle.
  • Ring A is a 6-membered saturated nitrogen heterocycle.
  • ring A is a piperidine ring.
  • ring A is a morpholine ring.
  • R 2 is independently selected from H, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl, C 2-6 heteroalkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 3-8 cycloalkyloxy, C 6-10 aryl and 5-10 membered heteroaryl at each occurrence, and the alkyl, alkoxy, hydroxyalkyl, heteroalkyl, cycloalkyl, heterocyclyl, cycloalkyloxy, aryl or heteroaryl are each optionally substituted with one or more R 3 .
  • R 2 is independently selected from H, halogen, C 1-6 alkyl, C 6-10 aryl and 5-10 membered heteroaryl at each occurrence, and each of the alkyl, aryl or heteroaryl is optionally substituted with one or more R 3 .
  • R 2 is independently selected from C 1-6 alkyl, C 6-10 aryl and 6-9 membered heteroaryl at each occurrence, and each of the alkyl, aryl or heteroaryl is optionally substituted with one or more R 3 .
  • R 2 is independently selected from C 1-6 alkyl, phenyl, pyridinyl and benzothiazolyl at each occurrence, and the phenyl, pyridinyl or benzothiazolyl is each optionally substituted with one or more R 3 .
  • R 2 is independently selected from C 1-6 alkyl and 6-9 membered heteroaryl at each occurrence, and each of the alkyl or heteroaryl is optionally substituted with one or more R 3 .
  • R 2 is independently selected from C 1-6 alkyl, pyridyl and benzothiazolyl at each occurrence, and the pyridyl or benzothiazolyl is each optionally substituted with one or more R 3 .
  • R2 is independently selected from C1-6 alkyl, pyridyl and benzothiazolyl at each occurrence, and the pyridyl or benzothiazolyl is each optionally substituted by C1-6 alkyl, C1-6 haloalkyl and 5-6 membered heterocyclyl, and the heterocyclyl is optionally substituted by C1-6 alkyl.
  • the present invention provides compounds of formula I, R2 is independently selected from methyl, The wavy line It indicates the point of attachment of the group to the rest of the molecule.
  • R 3 is independently selected from halogen, -CN, -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl , C 1-6 haloalkoxy, C 2-6 heteroalkyl, 3-8 membered heterocyclyl, C 6-10 aryl and 5-6 membered heteroaryl at each occurrence, and the alkyl, heteroalkyl, heterocyclyl, aryl or heteroaryl is each optionally substituted with one or more independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-6 cycloalkyl, C 3-8 cycloalkoxy and 3-8 membered heterocyclyl.
  • R 3 is independently selected from halogen, -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 2-6 heteroalkyl, 4-7 membered heterocyclyl, phenyl and 5-6 membered heteroaryl at each occurrence, and the alkyl, heteroalkyl, heterocyclyl, phenyl or heteroaryl are each optionally substituted with one or more substituents independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl.
  • R 3 is independently selected from -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, 4-7 membered heterocyclyl and 5-6 membered heteroaryl at each occurrence, and the alkyl, heterocyclyl and heteroaryl are optionally substituted with one or more -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl substituents.
  • R 3 is independently selected from halogen, -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 heteroalkyl, 5-6 membered heterocyclyl, phenyl and 5-6 membered heteroaryl at each occurrence, and the alkyl, heteroalkyl, heterocyclyl, phenyl or heteroaryl are each optionally substituted with one or more substituents independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 3-6 cycloalkyl and C 1-6 haloalkyl.
  • R 3 is independently selected from C 1-6 alkyl, C 1-6 haloalkyl and 5-6 membered heterocyclyl at each occurrence, and the heterocyclyl is optionally substituted with one or more C 1-6 alkyl.
  • the present invention provides compounds of formula I, R 3 at each occurrence is independently selected from methyl, trifluoromethyl, ethyl, amino, methylamino, trifluoromethoxy, The wavy line It indicates the point of attachment of the group to the rest of the molecule.
  • the present invention provides compounds of formula I, wherein R 4 and R 5 are each independently selected from H and methyl.
  • R 6 and R 7 are each independently selected from H and methyl.
  • the present invention provides compounds of formula I wherein R 6 and R 7 are H.
  • the present invention provides compounds of formula I in which m is 0 or 1.
  • the present invention provides compounds of formula I wherein n is 2.
  • the compound of formula I provided by the present invention is a compound of formula II:
  • X1 is -CH-, and X2 and X3 are -N-;
  • R1 is methyl
  • the compound of formula I provided by the present invention is a compound of formula III:
  • X1 is -CH-, and X2 and X3 are -N-;
  • R1 is methyl
  • n 1;
  • R3 is selected from methyl, trifluoromethyl, ethyl, amino, methylamino, trifluoromethoxy, The wavy line It indicates the point of attachment of the group to the rest of the molecule.
  • the compound of formula I provided by the present invention is a compound of formula IV:
  • X1 is -CH-, and X2 and X3 are -N-;
  • R1 is methyl
  • n 1;
  • R3 is selected from methyl, trifluoromethyl, ethyl, amino, methylamino, trifluoromethoxy,
  • the wavy line It indicates the point of attachment of the group to the rest of the molecule.
  • the compound of formula I provided by the present invention is a compound of formula V:
  • X 1 , X 2 , X 3 , R 1 , R 3 , and m are as defined above for the compound of formula I; and Y 1 and Y 2 are independently selected from N and CH.
  • the compound of formula V provided by the present invention is a compound of formula V-1:
  • R 3 is as defined above for the compound of formula I; Y 1 and Y 2 are independently selected from N and CH.
  • the compound of formula V-1 provided herein is a compound of formula V-1a:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula V-1 provided herein is a compound of formula V-1b:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula V-1 provided herein is a compound of formula V-1c:
  • R 3 is as defined above for compounds of formula I.
  • R 3 is selected from halogen, -CN, -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 2-6 heteroalkyl, 3-8 membered heterocyclyl, C 6-10 aryl and 5-6 membered heteroaryl, each of which alkyl, heteroalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more substituents independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-6 cycloalkyl, C 3-8 cycloalkoxy and 3-8 membered heterocyclyl;
  • R 3 is selected from halogen, -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 2-6 heteroalkyl, 4-7 membered heterocyclyl, phenyl and 5-6 membered heteroaryl, each of which alkyl, heteroalkyl, heterocyclyl, phenyl or heteroaryl is optionally substituted with one or more substituents independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl;
  • R 3 is selected from -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, 4-7 membered heterocyclyl and 5-6 membered heteroaryl, wherein the alkyl, heterocyclyl and heteroaryl are optionally substituted with one or more substituents of -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl;
  • R3 is selected from methyl, trifluoromethyl, ethyl, amino, methylamino, trifluoromethoxy,
  • the wavy line It indicates the point of attachment of the group to the rest of the molecule.
  • R3 is selected from C1-6 haloalkyl and C1-6 haloalkoxy
  • R 3 is selected from trifluoromethyl and trifluoromethoxy.
  • the compound of formula I provided by the present invention is a compound of formula VI:
  • p 0 or 1.
  • the compound of formula VI provided by the present invention is a compound of formula VI-1:
  • R 3 is as defined above for compounds of formula I;
  • p 0 or 1.
  • the compound of formula VI-1 provided by the present invention is a compound of formula VI-1a:
  • R 3 is as defined above for compounds of formula I;
  • p 0 or 1.
  • the compound of formula VI-1 provided by the present invention is a compound of formula VI-1b:
  • R 3 is as defined above for compounds of formula I;
  • p 0 or 1.
  • R 3 is selected from halogen, -CN, -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 2-6 heteroalkyl, 3-8 membered heterocyclyl, C 6-10 aryl and 5-6 membered heteroaryl, each of which alkyl, heteroalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with one or more substituents independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-6 cycloalkyl, C 3-8 cycloalkoxy and 3-8 membered heterocyclyl;
  • R 3 is selected from halogen, -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 2-6 heteroalkyl, 4-7 membered heterocyclyl, phenyl and 5-6 membered heteroaryl, each of which alkyl, heteroalkyl, heterocyclyl, phenyl or heteroaryl is optionally substituted with one or more substituents independently selected from halogen, -OH, -CN, -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl;
  • R 3 is selected from -NR 4 R 5 , C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 haloalkoxy, 4-7 membered heterocyclyl and 5-6 membered heteroaryl, wherein the alkyl, heterocyclyl and heteroaryl are optionally substituted with one or more substituents of -NR 4 R 5 , -COCH 3 , C 1-6 alkyl, C 1-6 haloalkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl;
  • R3 is selected from methyl, trifluoromethyl, ethyl, amino, methylamino, trifluoromethoxy,
  • the wavy line It indicates the point of attachment of the group to the rest of the molecule.
  • R3 is selected from 4-7 membered heterocyclyl and 5-6 membered heteroaryl, each of which is optionally substituted by a substituent selected from C1-6 alkyl and C3-6 cycloalkyl;
  • R3 is selected from The wavy line It indicates the point of attachment of the group to the rest of the molecule.
  • the compound of formula I provided by the present invention is a compound of formula III-1a:
  • the compound of formula I provided by the present invention is a compound of formula V-1a-1:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula V-1a-2:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula V-1b-1:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula V-1b-2:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula V-1c-1:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula V-1c-2:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula VI-1a-1:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula VI-1a-2:
  • R 3 is as defined above for compounds of formula I.
  • the compound of formula I provided by the present invention is a compound of formula VI-1a-3:
  • R 3 is as defined above for compounds of formula I.
  • the present invention covers any combination of the above embodiments.
  • compounds of the present invention include, but are not limited to:
  • the compounds of the present invention may be prepared by any method known in the art. Reagents and starting materials are readily available to one of ordinary skill in the art. Individual isomers, enantiomers and diastereomers may be separated or resolved at any convenient point in the synthesis by methods such as selective crystallization techniques or chiral chromatography (See for example, J. Jacques, et al., “Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc., 1981, and E. L. Eliel and S. H. Wilen).
  • the present invention provides a method for preparing a compound of formula I, comprising the following steps:
  • Step 1 Compound IA-1 and acyl chloride Compound IA-2 is generated through condensation reaction;
  • Step 2 Compound IA-2 is hydrolyzed to generate compound IA-3;
  • Step 3 Compound IA-3 and compound IA-4 undergo condensation reaction to generate compound IA-5;
  • Step 4 The condensation product IA-5 is deprotected to generate compound I;
  • PG is an amino protecting group
  • X 1 , X 2 , X 3 , Ring A, R 1 , R 2 , m and n are as defined above; R 6 and R 7 are H.
  • the amino protecting group is p-methoxybenzyl or 2,4-dimethoxybenzyl.
  • the condensation reaction in the first step is carried out in the presence of a base, preferably in the presence of a base such as triethylamine or diisopropylethylamine.
  • the first step reaction is carried out in a solvent such as THF.
  • the hydrolysis reaction in the second step is carried out in an aqueous solution of a base, preferably in an aqueous solution of a base such as LiOH, NaOH, etc.
  • the hydrolysis reaction in the second step is carried out in a solvent such as THF, methanol or ethanol.
  • the condensation reaction in the third step is carried out in the presence of a condensation agent, preferably in the presence of one or more condensation agents such as HATU, PyBOP, T 3 P, EDCI, PyBrOP, etc.
  • the condensation reaction in the third step is carried out in the presence of a base, preferably in the presence of a base such as triethylamine or diisopropylethylamine.
  • the condensation reaction in the third step is carried out in a solvent such as DMF or NMP.
  • the deprotection reaction in the fourth step is preferably carried out under the action of an acid, preferably an aqueous hydrochloric acid solution, a hydrochloric acid-ethyl acetate solution, a hydrochloric acid-1,4-dioxane solution or trifluoroacetic acid or the like, or oxidation is used to remove the protecting group, preferably under the action of an oxidant such as DDQ.
  • an acid preferably an aqueous hydrochloric acid solution, a hydrochloric acid-ethyl acetate solution, a hydrochloric acid-1,4-dioxane solution or trifluoroacetic acid or the like, or oxidation is used to remove the protecting group, preferably under the action of an oxidant such as DDQ.
  • one or more steps in the above-described preparation method may be omitted, and the order of the reaction steps may be appropriately adjusted as well as protection/deprotection reaction steps may be added or omitted as needed.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a preventively or therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotope-labeled compound thereof and one or more pharmaceutically acceptable carriers.
  • the present invention provides a pharmaceutical preparation comprising a preventive or therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotope-labeled compound thereof and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical preparation is preferably a solid preparation, a semisolid preparation, a liquid preparation or a gaseous preparation.
  • the pharmaceutical composition or pharmaceutical formulation may further comprise one or more additional therapeutic agents.
  • the pharmaceutical composition or pharmaceutical formulation is preferably administered orally, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or transdermally.
  • the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention, or a pharmaceutical preparation of the present invention in the preparation of a medicament for preventing or treating a disease or condition associated with PRMT5 activity.
  • the present invention provides use of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention, or a pharmaceutical preparation of the present invention in the preparation of a medicament for modulating (e.g., reducing or inhibiting) PRMT5 activity.
  • the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention, or a pharmaceutical formulation of the present invention, for use in preventing or treating a disease or condition associated with PRMT5 activity.
  • the present invention provides a method for preventing or treating a disease or condition associated with PRMT5 activity, the method comprising administering to an individual in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer or isotope-labeled compound thereof, or a pharmaceutical composition of the present invention, or a pharmaceutical preparation of the present invention.
  • the disease or condition associated with PRMT5 activity is preferably a cancer or tumor with MTAP deficiency.
  • the cancer or tumor is preferably esophageal cancer, lung cancer, pancreatic cancer, glioblastoma, bile duct cancer, bladder cancer, breast cancer, ovarian cancer, hepatocellular carcinoma, prostate cancer, melanoma, gastric cancer, colon cancer, leukemia (especially chronic B-lymphocytic leukemia (B-CLL)), lymphoma, etc.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient or vehicle that is administered together with a therapeutic agent and is suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit/risk ratio within the scope of reasonable medical judgment.
  • Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids. Examples of suitable pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1990).
  • compositions of the invention can act systemically and/or locally. For this purpose, they can be administered by a suitable route.
  • the pharmaceutical composition of the present invention can be administered in suitable dosage forms.
  • the term "effective amount” refers to that amount of a compound which, when administered, relieves to some extent one or more of the symptoms of the condition being treated.
  • the dosage regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering the composition or supervising the administration of the composition.
  • the amount of the compound of the present invention administered will depend on the severity of the individual, disease or condition treated, the rate of administration, the disposal of the compound and the judgment of the prescribing physician. Generally speaking, the effective dose is about 0.0001 to about 50 mg per kg body weight per day. In some cases, the dosage level not higher than the lower limit of the aforementioned range may be sufficient, and in other cases, a larger dose may still be used without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses to be administered throughout the day.
  • the content or dosage of the compound of the present invention in the pharmaceutical composition or pharmaceutical preparation may be about 0.01 mg to about 1000 mg.
  • treating and “treating” mean reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • “individual” includes human or non-human animals.
  • Exemplary human individuals include human individuals (referred to as patients) suffering from diseases (e.g., diseases described herein) or normal individuals.
  • Non-human animals in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).
  • the pharmaceutical composition or pharmaceutical preparation of the present invention may also include one or more additional therapeutic or preventive agents (e.g., other drugs for treating cancer or tumor diseases).
  • the treatment methods of the present invention may also include administering one or more additional therapeutic or preventive agents (e.g., other drugs for treating cancer or tumor diseases).
  • the compounds of the present invention are separated and purified by preparative TLC, silica gel column chromatography, Prep-HPLC and/or flash column chromatography, and their structures are confirmed by 1 H NMR and/or MS. Reaction monitoring is performed by TLC or LC-MS.
  • LC/MS uses Aglient 1260 Infinity/Aglient 6120 Quadrupole.
  • TLC used silica gel GF 254 as the stationary phase.
  • Flash column chromatography was performed using a Biotage flash column chromatograph.
  • Prep-HPLC used Agilent 1260 and Waters 2489.
  • Microwave reactions were performed using a BiotageInitiator microwave reactor.
  • reaction temperature is room temperature (15-30°C).
  • the reagents used in this application were purchased from companies such as Acros Organics, Aldrich Chemical Company or Teber Chemical.
  • Step 1 Synthesis of dimethyl pyridine-3,4-dicarboxylate (Compound Int A-2)
  • Step 3 Synthesis of methyl 2-chloropyridine-3,4-dicarboxylate (Compound Int A-4)
  • Step 6 Synthesis of N-(2,4-dimethoxybenzyl)-1,3-dihydrofuro[3,4-c]pyridin-4-amine (Compound Int A-8)
  • Int A-6 200 mg, 1.29 mmol
  • Int A-7 430 mg, 2.57 mmol
  • toluene 5 mL
  • tBuONa 494 mg, 5.14 mmol
  • BINAP 80 mg, 128.55 ⁇ mol
  • Pd 2 (dba) 3 59 mg, 64.28 ⁇ mol
  • Step 7 Synthesis of 1,3-dihydrofurano[3,4-c]pyridin-4-amine (Compound Int A-9)
  • Int A-8 (230 mg, 803.28 ⁇ mol) was dissolved in TFA (10 mL) and reacted at 25°C for 1 hour. After the reaction was completed, the reaction solution was dried by spin drying, diluted with saturated NaHCO 3 aqueous solution, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure to obtain compound Int A-9 (96 mg).
  • Step 8 Synthesis of 7-bromo-1,3-dihydrofurano[3,4-c]pyridin-4-amine (Compound Int A-10)
  • Step 9 Synthesis of 7-bromo-N,N-bis(tert-butyloxycarbonyl)-1,3-dihydrofurano[3,4-c]pyridin-4-amine (Compound Int A-11)
  • Step 10 Synthesis of tert-butyl (7-((diphenylmethylene)amino)-1,3-dihydrofuro[3,4-c]pyridin-4-yl)carbamate (Compound Int A-13)
  • Step 11 Synthesis of tert-butyl (7-amino-1,3-dihydrofuro[3,4-c]pyridin-4-yl)carbamate (Compound Int A-14)
  • Step 12 Synthesis of ethyl 2-((4-((tert-butyloxycarbonyl)amino)-1,3-dihydrofuro[3,4-c]pyridin-7-yl)amino)-2-oxoacetate (Compound Int A-16)
  • Step 13 Synthesis of 2-((4-((tert-Butyloxycarbonyl)amino)-1,3-dihydrofuro[3,4-c]pyridin-7-yl)amino)-2-oxoacetic acid (Compound Int A)
  • Step 2 Synthesis of 4-bromo-N-(2,4-dimethoxybenzyl)-1-methyl-1H-pyrazolo[3,4-c]pyridin-7-amine (Compound Int B-3)
  • Step 3 Synthesis of N-(2,4-dimethoxybenzyl)-4-((diphenylmethylene)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-7-amine (Compound Int B-4)
  • Step 4 Synthesis of N 7 -(2,4-dimethoxybenzyl)-1-methyl-1H-pyrazolo[3,4-c]pyridine-4,7-diamine (Compound Int B-5)
  • Step 5 Synthesis of ethyl 2-((7-((2,4-dimethoxybenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)amino)-2-oxoacetate (Compound Int B-6)
  • Step 6 Synthesis of 2-((7-((2,4-dimethoxybenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)amino)-2-oxoacetic acid (Compound Int B)
  • Int C-1 (1 g, 6.39 mmol) was dissolved in anhydrous DCE (20 mL), and NBS (1.25 g, 7.03 mmol) was added. The temperature was raised to 60°C under N2 protection and the reaction was continued for 2 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, water was added to dissolve, and the mixture was extracted with ethyl acetate. The organic layer was dried and concentrated to obtain the intermediate Int C-2 (1.4 g).
  • Step 3 Synthesis of 7-bromo-N-(4-methoxybenzyl)-1-methyl-1H-pyrazolo[4,3-c]pyridin-4-amine (Int C-5)
  • Step 4 Synthesis of 7-((diphenylmethylene)amino)-N-(4-methoxybenzyl)-1-methyl-1H-pyrazolo[4,3-c]pyridin-4-amine (Compound Int C-6)
  • Step 5 Synthesis of N 4 -(4-methoxybenzyl)-1-methyl-1H-pyrazolo[4,3-c]pyridine-4,7-diamine (Compound Int C-7)
  • Step 6 Synthesis of ethyl 2-((4-((4-methoxybenzyl)amino)-1-methyl-1H-pyrazolo[4,3-c]pyridin-7-yl)amino)-2-oxoacetate (Compound Int C-8)
  • Step 7 Synthesis of 2-((4-((4-methoxybenzyl)amino)-1-methyl-1H-pyrazolo[4,3-c]pyridin-7-yl)amino)-2-oxoacetic acid (Compound Int C)
  • Step 2 Synthesis of tert-butyl 5-methyl-2-oxopiperidine-1-carboxylate (Compound Int D-3)
  • Step 3 Synthesis of tert-butyl 3-methyl-6-(((trifluoromethyl)sulfonyl)oxy)-3,4-dihydropyridine-1(2H)-carboxylate (Compound Int D-5)
  • Int D-3 (5 g, 23.44 mmol) was dissolved in THF (50 mL), cooled to -78 °C, and then LiHMDS (35.17 mmol, 1M THF solution, 35.17 mL) was slowly added. After the addition was completed, the mixture was kept at -78 °C and stirred for 0.5 hr. Then, a THF (20 mL) solution of Int D-4 (10.05 g, 28.13 mmol) was added. After the addition was completed, the temperature was naturally raised to 25 °C and the reaction was continued for 16 hr.
  • Step 4 Synthesis of tert-butyl 3-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydropyridine-1(2H)-carboxylate (Compound Int D-7)
  • Int D-5 (7.5 g, 21.72 mmol), Int D-6 (8.27 g, 32.58 mmol), PPh 3 (341.78 mg, 1.30 mmol), Pd(PPh 3 ) 4 (752.90 mg, 651.54 ⁇ mol) and K 2 CO 3 (7.50 g, 54.30 mmol) were weighed into a two-necked bottle, and then 1,4-dioxane (50 mL) was added. After nitrogen was replaced, the reaction temperature was raised to 90°C and stirred for 20 hours. The solvent was removed by concentration, and then extracted with dichloromethane three times.
  • Step 5 Synthesis of tert-butyl 5-methyl-5'-(trifluoromethyl)-5,6-dihydro-[2,2'-bipyridine]-1(4H)-carboxylate (Compound Int D-9)
  • Int D-8 (2.17 g, 9.59 mmol), Int D-7 (1.55 g, 4.80 mmol), Pd(dppf)Cl 2 ⁇ DCM (391.60 mg, 479.53 ⁇ mol) were dissolved in 1,4-dioxane (50 mL), and then a solution of Na 2 CO 3 (1.02 g, 9.59 mmol) in H 2 O (10 mL) was added. After replacing the nitrogen, the reaction system was heated to 90°C and reacted for 2 hours. The reaction mixture was cooled to room temperature, directly concentrated, and extracted with dichloromethane three times.
  • Step 6 Synthesis of 5-methyl-5'-(trifluoromethyl)-3,4,5,6-tetrahydro-2,2'-bipyridine (Compound Int D-10)
  • Step 1 Synthesis of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazole (Compound Int E-2)
  • Step 2 Synthesis of tert-butyl 6-(benzo[d]thiazol-5-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (Compound Int E-3)
  • Step 2 Synthesis of tert-butyl 3-methyl-5-oxomorpholine-4-carboxylate (Compound Int F-4)
  • Step 3 Synthesis of tert-butyl 5-((diphenoxyphosphoryl)oxy)-3-methyl-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound Int F)
  • Step 4 Synthesis of tert-butyl 3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound Int G)
  • Step 1 Synthesis of tert-butyl 5-(benzo[d]thiazol-5-yl)-3-methyl-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound Int H-1)
  • Step 4 Synthesis of (3S,5R)-3-(Benzo[d]thiazol-5-yl)-5-methylmorpholine (Compound Int H Peak 1) and (3R,5S)-3-(Benzo[d]thiazol-5-yl)-5-methylmorpholine (Compound Int H Peak 2)
  • Step 2 Synthesis of tert-butyl 3-methyl-5-oxomorpholine-4-carboxylate (Compound Int I-4)
  • Step 3 Synthesis of tert-butyl 5-((diphenoxyphosphoryl)oxy)-3-methyl-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound Int I-5)
  • Step 4 Synthesis of tert-butyl 3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound Int I-6)
  • Step 5 Synthesis of tert-butyl 3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound Int I-7)
  • Step 6 Synthesis of 3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-3,6-dihydro-2H-1,4-oxazine (Compound Int I-8)
  • Step 1 Synthesis of 2-(2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-N-(7-((2,4-dimethoxybenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-oxoacetamide (Compound 1-1)
  • Step 2 Synthesis of N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxo-acetamide (Compound 1)
  • Compound 1 was separated by SFC (alkaline conditions; column: DAICEL CHIRALPAK AS (250 mm*30 mm, 10 um); mobile phase: [CO 2 -ACN/EtOH (0.1% NH 3. H 2 O)]; B%: 60%, isocratic elution mode) to obtain a mixture 1 of compound 1 isomer 1 and compound 1 isomer 2 (102 mg) and a mixture 2 of compound 1 isomer 3 and compound 1 isomer 4 (105 mg).
  • SFC alkaline conditions; column: DAICEL CHIRALPAK AS (250 mm*30 mm, 10 um); mobile phase: [CO 2 -ACN/EtOH (0.1% NH 3. H 2 O)]; B%: 60%, isocratic elution mode
  • Step 1 Synthesis of N-(7-((2,4-dimethoxybenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(5-methyl-2-(5-(trifluoromethyl)pyridin-2-yl)piperidin-1-yl)-2-oxoacetamide (Compound 2-1)
  • Step 2 Synthesis of N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(5-methyl-2-(5-(trifluoromethyl)pyridin-2-yl)piperidin-1-yl)-2-oxoacetamide (Compound 2)
  • Example 2 The following compounds were prepared by the method and general steps described in Example 2. Other required raw materials can be purchased commercially or synthesized by experienced synthesizers in the field of organic synthesis using conventional reactions from commercially purchased reagents.
  • Step 1 Synthesis of 2-[2-(Benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl]-N-(4-((4-methoxyphenyl)methyl)amino)-1-methyl-1H-pyrazolo[4,3-c]pyridin-7-yl]-2-oxo-acetamide (Compound 3-1)
  • Step 2 Synthesis of N-(4-amino-1-methyl-1H-pyrazolo[4,3-c]pyridin-7-yl)-2-(2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl]-2-oxo-acetamide (Compound 3)
  • Step 1 Synthesis of tert-butyl (7-(2-(2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxoacetamido)-1,3-dihydrofurano[3,4-c]pyridin-4-yl)carbamate (Compound 4-1)
  • Step 2 Synthesis of N-(4-amino-1,3-dihydrofurano[3,4-c]pyridin-7-yl)-2-(2-(benzo[d]thiazol-5-yl)-5-methylpiperidin-1-yl)-2-oxoacetamide (Compound 4)
  • Step 1 Synthesis of N-(7-((3,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-((3R,5S)-3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)morpholinyl)-2-oxoacetamide (Compound 35-1)
  • Int B (1.02 g, 2.65 mmol) was dissolved in CH 2 Cl 2 (48.0 mL) and THF (8.00 mL), and then DMF (38.7 mg, 529 ⁇ mol) and (COCl) 2 (840 mg, 6.62 mmol) were added, and the reaction was stirred at 20° C. for 1 hr.
  • Step 2 Synthesis of N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-((3R,5S)-3-methyl-5-(5-(trifluoromethyl)pyridin)-2-yl)morpholino)-2-oxoacetamide (Compound 35)
  • Step 1 Synthesis of tert-butyl 3-methyl-5-(6-(trifluoromethyl)pyridin-3-yl)-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound 46-2)
  • Step 2 Synthesis of 3-methyl-5-(6-(trifluoromethyl)pyridin-3-yl)-3,6-dihydro-2H-1,4-oxazine (Compound 46-3)
  • Step 4 Synthesis of ethyl 2-(3-methyl-5-(6-(trifluoromethyl)pyridin-3-yl)morpholino)-2-oxoacetate (Compound 46-5)
  • Step 5 Synthesis of 2-(3-methyl-5-(6-(trifluoromethyl)pyridin-3-yl)morpholino)-2-oxoacetic acid (Compound 46-6)
  • Step 6 Synthesis of N-(7-((2,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(3-methyl-5-(6-(trifluoromethyl)pyridin-3-yl)morpholino)-2-oxoacetamide (Compound 46-7)
  • Step 7 Synthesis of N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(3-methyl-5-(6-(trifluoromethyl)pyridin-3-yl)morpholino)-2-oxoacetamide (Compound 46)
  • Example 47 ( ⁇ )-N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(cis-3-methyl-5-(4-(trifluoromethyl)phenyl))morpholino)-2-oxoacetamide (Compound 47) and ( ⁇ )-N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(trans-3-methyl-5-(4-(trifluoromethyl)phenyl))morpholino)-2-oxoacetamide (Compound 48)
  • Step 1 Synthesis of tert-butyl 3-methyl-5-(4-(trifluoromethyl)phenyl)-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound 47-2)
  • Step 2 Synthesis of 3-methyl-5-(4-(trifluoromethyl)phenyl)-3,6-dihydro-2H-1,4-oxazine (Compound 47-3)
  • Step 4 Synthesis of ethyl 2-(3-methyl-5-(4-(trifluoromethyl)phenyl)morpholino)-2-oxoacetate (Compound 47-5)
  • Step 6 Synthesis of N-(7-((2,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(3-methyl-5-(4-(trifluoromethyl)phenyl)morpholinyl)-2-oxoacetamide (Compound 47-7)
  • Step 7 Synthesis of ( ⁇ )-N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(cis-3-methyl-5-(4-(trifluoromethyl)phenyl))morpholino)-2-oxoacetamide (Compound 47) and ( ⁇ )-N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(trans-3-methyl-5-(4-(trifluoromethyl)phenyl))morpholino)-2-oxoacetamide (Compound 48)
  • Example 47 The following compounds were prepared by the method and general steps described in Example 47. Other required raw materials can be purchased commercially or synthesized by experienced synthesizers in the field of organic synthesis using conventional reactions from commercially purchased reagents.
  • Step 1 Synthesis of N-(7-((2,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-((2R,5S)-5-methyl-2-(2-(1-methylpiperidin-4-yl)benzo[d]thiazol-5-yl)piperidin-1-yl)-2-oxoacetamide (Compound 49-2)
  • Step 2 Synthesis of N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-((2R,5S)-5-methyl-2-(2-(1-methylpiperidin)-4-yl)benzo[d]thiazol-5-yl)piperidin-1-yl)-2-oxoacetamide (Compound 49)
  • Step 1 Synthesis of tert-butyl 3-methyl-5-(5-(trifluoromethoxy)pyridin-2-yl)-2,3-dihydro-4H-1,4-oxazine-4-carboxylate (Compound 50-2)
  • Step 2 Synthesis of 3-methyl-5-(5-(trifluoromethoxy)pyridin-2-yl)-3,6-dihydro-2H-1,4-oxazine (Compound 50-3)
  • Step 4 Synthesis of ethyl 2-(3-methyl-5-(5-(trifluoromethoxy)pyridin-2-yl)morpholino)-2-oxoacetate (Compound 50-5)
  • Step 5 Synthesis of 2-(3-methyl-5-(5-(trifluoromethoxy)pyridin-2-yl)morpholino)-2-oxoacetic acid (Compound 50-6)
  • Step 6 Synthesis of N-(7-((2,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(3-methyl-5-(5-(trifluoromethoxy)pyridin-2-yl)morpholino)-2-oxoacetamide (Compound 50-7)
  • Step 7 Synthesis of N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-(3-methyl-5-(5-(trifluoromethoxy)pyridin-2-yl)morpholino)-2-oxoacetamide (Compound 50)
  • Step 1 Synthesis of ethyl 2-((3S,5R)-3-(benzo[d]thiazol-5-yl)-5-methylmorpholino)-2-oxoacetate (Compound 51-1)
  • Step 3 Synthesis of 2-((3S,5R)-3-(benzo[d]thiazol-5-yl)-5-methylmorpholino)-N-(7-((2,4-dimethylbenzyl)amino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-oxoacetamide (Compound 51-3)
  • Step 4 Synthesis of N-(7-amino-1-methyl-1H-pyrazolo[3,4-c]pyridin-4-yl)-2-((3S,5R)-3-(benzo[d]thiazol-5-yl)-5-methylmorpholino)-2-oxoacetamide (Compound 51)
  • Example 51 The following compounds were prepared by the method and general steps described in Example 51.
  • Other required raw materials can be purchased commercially or synthesized by experienced synthesizers in the field of organic synthesis using conventional reactions from commercially purchased reagents.
  • Prep-HPLC purification of the compounds in the examples was carried out using Aglient 1260 or Waters 2489 HPLC, the separation column model was Waters SunFire Prep C 18 OBD (19 mm ⁇ 150 mm ⁇ 5.0 ⁇ m), Waters Xbridge Prep C 18 OBD (19 mm ⁇ 150 mm ⁇ 5.0 ⁇ m) or YMC Actus Triart C 18 (20 mm ⁇ 150 mm ⁇ 5.0 ⁇ m), the column temperature was 25°C, the detection wavelength was 214 nm, 254 nm or 280 nm, the mobile phase A was acetonitrile, the mobile phase B was 0.05% formic acid aqueous solution or 0.05% ammonium bicarbonate aqueous solution or 0.05% TFA aqueous solution, the volume ratio of the mobile phase was adjusted according to the polarity of the compound; the mobile phase flow rate was 28 mL/min.
  • the separation column model was Waters SunFire Prep C 18 OBD (19 mm ⁇ 150 mm ⁇ 5.0 ⁇ m),
  • the prepared protein solution (PRMT5/MEP50 and MTA mixture) was pre-incubated with different concentrations of test compounds (500 nM starting, 5-fold dilution, 7 points; or 100 nM starting, 5-fold dilution, 7 points) at 25°C for 30 min, and then the prepared substrate solution (biotinylated histone H4 peptide) was added and incubated at 25°C for 90 min.
  • the prepared detection reagent mixture Protein A-Eu, anti-histone H4 antibody and Streptavidin-D2
  • the fluorescence signal ratio (Ratio) was detected using a BMG microplate reader.
  • the solvent group (DMSO) was used as the negative control and the reaction buffer group (without PRMT5 ⁇ MTA enzyme) was used as the blank control.
  • the percentage inhibition rate of compounds at different concentrations was calculated according to the following formula:
  • Percent inhibition rate (negative control Ratio - compound Ratio) / (negative control Ratio - blank control Ratio) ⁇ 100%.
  • Y is the relative inhibitory activity percentage
  • Top and Bottom are the maximum and minimum values of the fitting curve
  • X is the logarithmic concentration of the compound
  • Hillslope is the slope of the curve.
  • the inhibitory effect of the compound on PRMT5-MTA methyltransferase was determined according to the above method, and the results are shown in Table 1.
  • MTAP-deficient/parental HCT116 cells were selected and purchased from HORIZON.
  • MTAP-deficient/parental HCT116 cells were cultured in vitro in a monolayer in RPMI6140 medium containing 10% FBS + 1% P/S at 37°C and 5% CO 2 .
  • the cells in the logarithmic growth phase were digested and the concentration was adjusted. 250 cells per well were inoculated in a 96-well plate and cultured overnight. Pre-diluted compounds (5000 nM starting, 4-fold dilution, 9 points) were added.
  • DMSO was added to the negative control group and cultured in the blank control group. After culturing for 8 days in a 37°C, 5% CO 2 incubator, 50 ⁇ l of CellTiter-Glo was added to each well and lysed for 10 minutes at room temperature in the dark. The test solution was transferred to a 96-well opaque white plate, and then the relative chemiluminescence unit value was read in the chemiluminescence detection mode of the microplate reader.
  • Percent inhibition rate (1 - (chemiluminescent signal value of the test compound - chemiluminescent signal value of the blank control) / (chemiluminescent signal value of the negative control - chemiluminescent signal value of the blank control)) x 100%.
  • y Min+(Max-Min)/(1+(x/ IC50 ) ⁇ (-Hillslope)), wherein: y is the percentage inhibition rate; Max and Min are the maximum and minimum values of the fitting curve, respectively; x is the logarithmic concentration of the compound; and Hillslope is the slope of the curve.
  • the proliferation inhibitory activity of the compounds on MTAP-deficient/parental HCT116 cells was determined according to the above method. The results are shown in Table 2.
  • the experimental results show that the compound of the present invention has a strong inhibitory effect on MTAP-deficient HCT116 cells and has a certain selectivity relative to MTAP parent HCT116 cells.
  • the compounds of the present invention were administered to female Balb/c mice by oral gavage (PO) to investigate their pharmacokinetic characteristics.
  • the compound of the present invention and TNG462 were administered intravenously (1 mg/kg) and intragastrically (10 mg/kg).
  • the IV solvent was 5% DMSO + 5% Solutol + 90% Saline, and the PO solvent was 10% Solutol + 90% H 2 O.
  • Blood was collected from the eye sockets at 0 h before administration and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10 and 24 h after administration (intravenous), and at 0 h before administration and 0.25, 0.5, 1, 2, 4, 6, 8, 10 and 24 h after administration (oral administration) 40-50 ⁇ l was placed in a K2-EDTA anticoagulant tube.
  • TNG462 The samples of TNG462 were stored on wet ice, centrifuged at 4000 rpm for 10 min (4°C), and plasma was separated (1 ⁇ l of 30% formic acid solution was added to the plasma tube of TNG462 in advance), and stored at -80°C for testing. 10 ⁇ l of plasma sample was added to 90 ⁇ l of acetonitrile containing internal standard, shaken and mixed, and centrifuged at 4000 rpm and 4°C for 10 min. The supernatant was taken for LC-MS/MS analysis. WinNonlin 6.3 software was used to calculate the pharmacokinetic parameters using the non-compartmental model. The results are shown in Table 3.
  • TNG462 structure:
  • the above experimental results show that the compound of the present invention has a low body clearance rate, high blood drug concentration and body exposure, high bioavailability, and good oral absorption effect.

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Abstract

本发明涉及式（I）所示的草酸二酰胺化合物、包含其的药物组合物、其制备方法及其用于预防或治疗与PRMT5活性相关的疾病或病况的用途。

Description

草酸二酰胺化合物、包含其的药物组合物及其制备方法和用途 技术领域
本发明涉及一种草酸二酰胺化合物、包含其的药物组合物、其制备方法及其用于预防或治疗与PRMT5活性相关的疾病或病况的用途。
背景技术
PRMT5(蛋白质精氨酸甲基转移酶5，Protein Arginine Methyltransferase 5)属于表观遗传酶，是PRMT家族(人体具有PRMT1-11)成员之一，可催化组蛋白和某些非组蛋白底物的精氨酸残基甲基化修饰。PRMT5广泛存在于人体细胞的细胞核和细胞质中，包括心脏、肌肉和睾丸等组织。根据催化精氨酸甲基化方式的不同分为I、II、III型，PRMT5属于II型对称双甲基化(sDMA)PRMT，其甲基供体为S-腺苷蛋氨酸(SAM)。
PRMT5通过催化底物的精氨酸甲基化，调控多种靶蛋白的表达程度，参与多种生理功能，对肿瘤细胞增殖、转移、恶性转化具有重要作用。PRMT5对组蛋白的甲基化修饰导致p53、ST7、NM23及Rb等抑癌基因的沉默，进而促进肿瘤的发生发展。PRMT5对非组蛋白的调节主要体现在影响转录因子(NF-κB/P65、E2F1、HoxA\GATA4)、程序性细胞死亡蛋白4(PDCD4)、细胞周期及存活相关调节蛋白E2F1、缺氧诱导因子1(HIF-1)、周期蛋白依赖性激酶(CDKs)、PI3K/Akt等的定位及表达(Koh CM，Bezzi M，Guccione E.Curr Mol Bio Rep,2015,1(1):19-28)。肺癌细胞中，PRMT5可抑制miR-99家族转录，增加FGFR3表达，活化Erk1/2和Ak通路，导致肿瘤细胞生长和转移(Pengyu Jing,Nan Zhao,et al.Cancer Letters,2018,427,38-48)。结肠癌中PRMT5可甲基化Eif4e和FGFR3，促进肿瘤细胞生长(ZHANG B,DONG S,ZHU R,et al.Oncotarget,2015,6(26):22799-22811.)。
MTAP是甲硫腺苷磷酸化酶的编码基因，位于染色体9p21上，与抑癌基因CDKN2A(常发生纯合性缺失)位置相近，因此MTAP常与CDKN2A在肿瘤中发生共缺失现象(Marjon K,Kalev P,Marks K.Annual Review of Cancer Biology,2021,5(1))，大约有15％的实体瘤发生MTAP缺失。
PRMT5有2个共因子(cofactor)，分别为激活性共因子(SAM)和抑制性共因子(MTA；5'-甲硫腺苷)。在正常细胞中，MTAP负责将MTA转化为Met(甲硫氨酸)，PRMT5负责将SAM转化为SAH(S-腺苷-L-高半胱氨酸)。MTA为PRMT5-SAM的内源性竞争性抑制剂，在肿瘤细胞中，MTAP的缺失导致MTA蓄积，部分抑制PRMT5活性，使得肿瘤细胞更加依赖PRMT5。在上述情况下，抑制PRMT5可进一步阻断PRMT5的甲基化功能，造成肿瘤细胞死亡。
综上所述，在MTAP缺失的情况下，抑制PRMT5显示出合成致死作用。目前尚未有PRMT5靶点的抑制剂上市。因此，需要开发新的、高效低毒的PRMT5抑制剂来满足临床需求。
发明内容
本发明提供一种新的草酸二酰胺化合物，其对MTAP缺失的肿瘤细胞具有良好的抑制作用，并且具有良好的药物代谢动力学等性质。
本发明的一个方面提供式I的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药：
其中：
代表单键或双键；
X1、X2和X3各自独立地选自-CH2-、-CH-、-O-、-N-、-NH-和-S-；
R1在每次出现时各自独立地选自H、卤素、C1-6烷基、C1-6卤代烷基、C1-6羟烷基和C3-8环烷基；
环A为5-7元饱和或部分饱和杂环，所述杂环包含至少一个N原子以及任选存在的1或2个各自独立地选自O或S的相同或不同的其它杂原子；
R2在每次出现时各自独立地选自H、-OH、卤素、-CN、-NR4R5、-COCH3、-NHCOCH3、C1-6烷基、C1-6烷氧基、C1-6羟烷基、C2-6杂烷基、C3-8环烷基、3-8元杂环基、C3-8环烷氧基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、羟烷基、杂烷基、环烷基、杂环基、环烷氧基、芳基或杂芳基各自任选地被一个或多个R3取代；
R3在每次出现时各自独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6杂烷基、C3-6环烷基、C3-8环烷氧基、3-8元杂环基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、杂烷基、环烷基、环烷氧基、杂环基、芳基或杂芳基各自任选地被一个或多个各自独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C3-6环烷基、C3-8环烷氧基和3-8元杂环基的取代基取代；
R4、R5、R6和R7各自独立地选自H和C1-6烷基；或者
R4和R5、R6和R7连同其所连接的氮原子共同形成3-8元杂环基，所述杂环基任选地被一个或多个各自独立地选自卤素、OH、CN、-NH2和C1-6烷基的取代基取代；
m为0、1、2或3；
n为0、1、2、3或4。
本发明的另一方面提供一种药物组合物，其包含预防或治疗有效量的本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药以及一种或多种药学上可接受的载体。
本发明的另一方面提供本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药或者本发明的药物组合物在制备用于预防或治疗PRMT5活性相关的疾病或病况的药物中的用途。
本发明的另一方面提供本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，或者本发明的药物组合物，其用于预防或治疗与PRMT5活性相关的疾病或病况。
本发明的另一方面提供预防或治疗与PRMT5活性相关的疾病或病况的方法，所述方法包括向需要其的个体给药有效量的本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，或者本发明的药物组合物。
本发明的另一方面提供制备本发明的化合物的方法，其包括以下步骤：
第一步：化合物I-A-1与酰氯经缩合反应生成化合物I-A-2；
第二步：化合物I-A-2经水解反应生成化合物I-A-3；
第三步：化合物I-A-3和化合物I-A-4经缩合反应生成化合物I-A-5；
第四步：缩合产物I-A-5脱除保护基后生成化合物I；
其中
PG为氨基保护基；并且
X1、X2、X3、环A、R1、R2、m、n如上文所定义，R6和R7为H。
定义
除非在下文中另有定义，本文中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。
术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，其不排除其它未列举的元素或方法步骤，尽管其它未列举的元素或方法步骤不一定存在(即，这些术语也涵盖术语“基本上由……组成”和“由……组成”)。
如本文中所使用，术语“烷基”定义为线性或支化饱和脂肪族烃。在一些实施方案中，烷基具有1至12个，例如1至6个碳原子、1至4个碳原子。例如，如本文中所使用，术语“C1-6烷基”和“C1-4烷基”分别指具有1-6个碳原子和1-4个碳原子的线性或支化的基团(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、新戊基或正己基)，其任选地被一个或多个(诸如1至3个)适合的取代基如卤素取代(此时该基团被称作“卤代烷基”)(例如CH2F、CHF2、CF3、CCl3、C2F5、C2Cl5、CH2CF3、CH2Cl或-CH2CH2CF3等)。术语“C1-4烷基”指具有1至4个碳原子的线性或支化的脂肪族烃链(即甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基)。
如本文中所使用，术语“杂烷基”指烷基的主链碳原子中具有一个或多个独立地选自除碳以外的原子的骨架链原子，例如氧、氮、硫、磷或其组合。可以给出数值范围(例如C2-6杂烷基)是指链中的碳数目，在此实例中包括2-6个碳原子。例如，-CH2OCH2CH3基团被称为C3杂烷基，-CH2OCH2CH2NHCH3基团被称为C4杂烷基。与分子其余部分的连接可以通过杂烷基链中的杂原子或碳原子进行。
如本文中所使用，术语“卤代烷基”是指被一个或多个(诸如1至3个)相同或不同的卤素原子取代的烷基，术语“C1-8卤代烷基”、“C1-6卤代烷基”和“C1-4卤代烷基”分别指具有1至8个碳原子、1至6个碳原子和1-4个碳原子的卤代烷基，例如-CF3、-C2F5、-CHF2、-CH2F、-CH2CF3、-CH2Cl或-CH2CH2CF3等。
如本文中所使用，术语“羟烷基”是指烷基中的氢原子被一个或多个羟基取代所形成的基团，例如C1-6羟烷基或C1-4羟烷基，其实例包括但不限于羟甲基、羟乙基、羟丙基、羟丁基、-CH(OH)CH3等。
如本文中所使用，术语“烷氧基”意指-O-烷基，其中烷基如上文所定义，例如C1-8烷氧基、C1-6烷氧基、C1-4烷氧基或C1-3烷氧基。C1-6烷氧基的代表性实例包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基、己氧基等，所述烷氧基任选地被一个或多个(诸如1至3个)相同或不同的取代基取代。如术语“卤代烷氧基”是指所述烷氧基的氢原子被一个或多个(诸如1至3个)相同或不同的卤素原子取代。
如本文中所使用，术语“并环”或“稠环”指由两个或两个以上环状结构彼此共用两个相邻的原子所形成的环系。
如本文中所使用，术语“螺环”指由两个或两个以上环状结构彼此共用一个环原子所形成的环系。
如本文中所使用，术语“桥环”指由两个或两个以上环状结构彼此共用两个不直接相连的原子所形成的环系。
如本文中所使用，术语“环烷基”指饱和或不饱和的非芳族单环或多环(诸如双环)烃环基，包括但不限于单环烷基(诸如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基等)和双环烷基，包括螺环、并环(稠环)或桥环系统(即，螺环烷基、并环(稠环)烷基和桥环烷基，诸如双环[1.1.1]戊基、双环[2.2.1]庚基等)。本发明中，环烷基任选地被一个或多个(诸如1至3个)相同或不同的取代基取代。环烷基上的碳原子任选地被氧代(oxo)基团取代(即形成C＝O)。术语“C3-8环烷基”指具有3至8个成环碳原子的环烷基，例如C3-6环烷基，其可以是单环烷基，例如环丙基、环丁基、环戊基、环己基、环庚基或环辛基，也可以是双环烷基，例如C5-8螺环烷基、C5-8桥环烷基、C5-8稠环烷基、C5-6螺环烷基、C5-6桥环烷基或C5-6稠环烷基。
如本文中所使用，术语“环烷氧基”意指-O-环烷基，其中环烷基如上文所定义。环烷氧基的代表性实例包括但不限于环丙氧基、环丁氧基、环戊氧基、环己氧基等。
如本文中所使用，术语“杂环基”或“杂环”指具有2个或2个以上(例如3、4、5、6、7、8、9、10、11、12、13或14个)碳原子，以及一个或多个(例如1个、2个、3个或4个)杂原子的脂肪族的单环或多环(例如并环、螺环或桥环)基团，所述杂原子包括但不限于氧原子、氮原子和硫原子，所述杂环基上的碳原子和杂原子任选地被氧代基团取代(例如形成C＝O、S(＝O)或S(＝O)2)，或者任选地被一个或多个(诸如1至3个)独立地选自卤素和C1-3烷基的取代基取代。术语“饱和杂环”指完全饱和的杂环，例如四氢呋喃环、哌啶环、吗啉环、四氢吡喃环、哌嗪环等等。术语“部分饱和杂环”指杂环中既包含有饱和单键也包含有不饱和双键的杂环，例如3,4-二氢-2H-吡喃、1,2,3,4-四氢吡啶、4,5-二氢异噁唑基、4,5-二氢噁唑基、2,5-二氢噁唑基、2,3-二氢噁唑基等。如本文中所使用，术语“4-11元杂环基”意指含有4-11个环原子的杂环基，包括但不限于4-10元杂环基、4-9元杂环基、4-8元杂环基、4-7元杂环基、5-6元杂环基、3-8元杂环基、3-7元杂环基、4-7元含氮杂环基、4-7元含氧杂环基、4-7元含硫杂环基、5-6元含氮杂环基、5-6元含氧杂环基、5-6元含硫杂环基等，所述“含氮杂环基”、“含氧杂环基”和“含硫杂环基”各自任选地还含有一个或多个独立地选自氧、氮和硫的其他杂原子。4-11元杂环基的实例包括但不限于环氧乙烷基、氮丙啶基、氮杂环丁基、氧杂环丁基、四氢呋喃基、吡咯烷基、吡咯烷酮基(如)、咪唑烷基、吡唑烷基、四氢吡喃基、哌啶基、吗啉基、二噻烷基(dithianyl)、硫吗啉基、哌嗪基、三噻烷基(trithianyl)。如本文中所使用，术语“3-8元杂环基”意指含有3-8个环原子的杂环基，包括但不限于3-8元杂环基、3-7元杂环基、3-6元杂环基、4-8元杂环基、4-7元杂环基、4-6元杂环基、5-6元杂环基、4-7元含氮杂环基、4-7元含氧杂环基、4-7元含硫杂环基、5-6元含氮杂环基、5-6元含氧杂环基、5-6元含硫杂环基等，所述“含氮杂环基”、“含氧杂环基”和“含硫杂环基”各自任选地还含有一个或多个独立地选自氧、氮和硫的其他杂原子。3-8元杂环基的实例包括但不限于环氧乙烷基、氮丙啶基、氮杂环丁基、氧杂环丁基、四氢呋喃基、吡咯烷基、吡咯烷酮基(如)、咪唑烷基、吡唑烷基、四氢吡喃基、哌啶基、吗啉基、二噻烷基(dithianyl)、硫吗啉基、哌嗪基、三噻烷基(trithianyl)。
本发明中，杂环基可以与杂环基或环烷基形成并环结构，所述并环结构与其他基团的连接点可以在任一杂环基上或环烷基上，因此，本发明的杂环基还包括(但不限于)杂环基并杂环基、杂环基并环烷基、单杂环基并单杂环基、单杂环基并单环烷基，例如3-7元(单)杂环基并3-7元(单)杂环基、3-7元(单)杂环基并(单)环烷基、3-7元(单)杂环基并C4-6(单)环烷基，其实例包括但不限于吡咯烷基并环丙基、环戊基并氮杂环丙基、吡咯烷基并环丁基、吡咯烷基并吡咯烷基、吡咯烷基并哌啶基、吡咯烷基并哌嗪基、哌啶基并吗啉基、
本发明中，杂环基还包括桥杂环基和螺杂环基。
如本文中所使用，术语“桥杂环”是指两个饱和环共用两个不直接相连的环原子形成的含有一个或多个(例如1个、2个、3个或4个)杂原子(例如氧原子、氮原子和/或硫原子)的环状结构，包括但不限于7-10元桥杂环、8-10元桥杂环、7-10元含氮桥杂环、7-10元含氧桥杂环、7-10元含硫桥杂环 等，例如 等。所述“含氮桥杂环”、“含氧桥杂环”、“含硫桥杂环”任选地还含有一个或多个独立地选自氧、氮和硫的其他杂原子。
如本文中所使用，术语“螺杂环”是指由两个或两个以上饱和环共用一个环原子形成的含有一个或多个(例如1个、2个、3个或4个)杂原子(例如氧原子、氮原子、硫原子)的环状结构，包括但不限于5-10元螺杂环、6-10元螺杂环、6-10元含氮螺杂环、6-10元含氧螺杂环、6-10元含硫螺杂环等，例如 所述“含氮螺杂环”、“含氧螺杂环”、“含硫螺杂环”任选地还含有一个或多个独立地选自氧、氮、硫的其他杂原子。术语“6-10元含氮螺杂环基”是指含有共计6-10个环原子并且其中至少一个环原子为氮原子的螺杂环基。
杂环基与芳基稠合所得基团的实例包括但不限于：
如本文中所使用，术语“芳基”、“苯基”或“芳环”指具有共轭π电子系统的全碳单环或稠合多环芳族基团。如本文中所使用，术语“C6-10芳基(芳环)”意指含有6至10个碳原子的芳基(芳环)，优选为苯基(苯环)或萘基(萘环)。芳基任选地被一个或多个(诸如1至3个)相同或不同的取代基(例如卤素、OH、CN、NO2、C1-C6烷基等)取代。
如本文中所使用，术语“杂芳基”或“杂芳环”指含有一个或多个相同或不同杂原子的单环或多环芳族基团，包括单环的杂芳基和含有至少一个杂芳环(至少含有一个杂原子的芳族环系)的双环或多环环系，其可以具有5、6、7、8、9、10、11、12、13或14个环原子，例如5、6、7、8、9或10个环原子，并且另外在每一种情况下可为苯并稠合的。所述杂原子可以是氧、氮或硫。所述杂芳基上的碳原子和杂原子任选地被氧代基团取代(例如形成C＝O、S(＝O)或S(＝O)2)。
如本文中所使用，术语“5-10元杂芳基”或“5-10元杂芳环”意指含有5至10个(例如5至6个)环原子的杂芳基(杂芳环)，包括5-10元含氮杂芳基、5-10元含氧杂芳基、5-10元含硫杂芳基、5-6元含氮杂芳基、5-6元含氧杂芳基、5-6元含硫杂芳基等。所述“含氮杂芳基”、“含氧杂芳基”和“含硫杂芳基”各自任选地含有一个或多个独立地选自氧、氮和硫的其他杂原子。其实例包括但不限于噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、三唑基、四唑基、噁二唑基、噻二唑基等，或吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基等，以及包含这些基团的5-10元并环基团。
本发明中，杂芳基(例如单杂芳基)可以与芳基(例如单环芳基，例如苯基)、杂环基(例如单杂环基)、环烷基(例如单环烷基)或另一杂芳基(例如另一单杂芳基)彼此共用两个相邻的原子形成并环结构，其连接点可以在任一杂芳环上或其它环上，包括但不限于(单)杂芳基并(单)杂芳基、(单)杂芳基并(单环)芳基、(单)杂芳基并(单)杂环基和(单)杂芳基并(单)环烷基，例如5-6元(单)杂芳基并5-6元(单)杂芳基、 5-6元(单)杂芳基并苯基、5-6元(单)杂芳基并5-6元(单)杂环基或5-6元(单)杂芳基并C4-6(单)环烷基(例如5-6元杂芳基并环丁基、5-6元杂芳基并环戊基或5-6元杂芳基并环己基)，其实例包括但不限于苯并噻唑基、吲哚基、异吲哚基、吲唑基、苯并咪唑、喹啉基、异喹啉基、 等。
如本文中所使用，术语“卤代”或“卤素”基团定义为包括F、Cl、Br或I。
术语“取代”指所指定的原子上的一个或多个(例如一个、两个、三个或四个)氢被从所指出的基团的选择代替，条件是未超过所指定的原子在当前情况下的正常原子价并且所述取代形成稳定的化合物。取代基和/或变量的组合仅仅当这种组合形成稳定的化合物时才是允许的。
如果取代基被描述为“任选地被一个或多个……取代”，则取代基可(1)未被取代或(2)被取代。如果取代基的碳被描述为任选地被取代基列表中的一个或多个取代，则碳上的一个或多个氢(至存在的任何氢的程度)可单独和/或一起被独立地选择的任选的取代基替代。如果取代基的氮被描述为任选地被取代基列表中的一个或多个取代，则氮上的一个或多个氢(至存在的任何氢的程度)可各自被独立地选择的任选的取代基替代。
如果取代基被描述为“独立地选自”一组，则各取代基独立于另一者被选择。因此，各取代基可与另一(其他)取代基相同或不同。
如本文中所使用，术语“一个或多个”意指在合理条件下的1个或超过1个，例如2个、3个、4个、5个或10个。
除非指明，否则如本文中所使用，取代基的连接点可来自取代基的任意适宜位置。
当取代基的键显示为穿过环中连接两个原子的键时，则这样的取代基可键连至该可取代的环中的任一成环原子。
本发明还包括所有药学上可接受的同位素标记的化合物，其与本发明的化合物相同，除了一个或多个原子被具有相同原子序数但原子质量或质量数不同于在自然界中占优势的原子质量或质量数的原子替代。适合包含于本发明的化合物中的同位素的实例包括(但不限于)氢的同位素(例如氘(2H)、氚(3H))；碳的同位素(例如11C、13C及14C)；氯的同位素(例如36Cl)；氟的同位素(例如18F)；碘的同位素(例如123I及125I)；氮的同位素(例如13N及15N)；氧的同位素(例如15O、17O及18O)；磷的同位素(例如32P)；及硫的同位素(例如35S)。某些同位素标记的本发明的化合物(例如掺入放射性同位素的那些)可用于药物和/或底物组织分布研究(例如分析)中。放射性同位素氚(即3H)及碳-14(即14C)因易于掺入且容易检测而特别可用于该目的。用正电子发射同位素(例如11C、18F、15O及13N)进行取代可在正电子发射断层显像术(PET)研究中用于检验底物受体占据情况。被同位素标记的本发明的化合物可通过与描述于随附路线和/或实施例及制备中的那些类似的方法通过使用适当的被同位素标记的试剂代替之前采用的非标记的试剂来制备。本发明的药学上可接受的溶剂合物包括其中结晶溶剂可被同位素取代的那些，例如，D2O、丙酮-d6或DMSO-d6。
术语“立体异构体”表示由于至少一个不对称中心形成的异构体。在具有一个或多个(例如一个、两个、三个或四个)不对称中心的化合物中，其可产生外消旋混合物、单一对映异构体、非对映异构体混合物和单独的非对映异构体。特定个别分子也可以几何异构体(顺式/反式)存在。类似地，本发明的化合物可以两种或更多种处于快速平衡的结构不同的形式的混合物(通常称作互变异构体)存在。互变异构体的代表性实例包括酮-烯醇互变异构体、苯酚-酮互变异构体、亚硝基-肟互变异构体、亚胺-烯胺互变异构体等。例如，亚硝基-肟在溶液中可以下列互变异构形式平衡存在：
要理解，本申请的范围涵盖所有这样的以任意比例(例如60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％)的异构体或其混合物。
本文中可使用实线实楔形或虚楔形描绘本发明的化合物的化学键。使用实线以描绘键连至不对称碳原子的键欲表明，包括该碳原子处的所有可能的立体异构体(例如，特定的对映异构体、外消旋混合物等)。使用实或虚楔形以描绘键连至不对称碳原子的键欲表明， 存在所示的立体异构体。当存在于外消旋混合物中时，使用实及虚楔形以定义相对立体化学，而非绝对立体化学。除非另外指明，否则本发明的化合物意欲可以立体异构体(其包括顺式及反式异构体、光学异构体(例如R及S对映异构体)、非对映异构体、几何异构体、旋转异构体、构象异构体、阻转异构体及其混合物)的形式存在。本发明的化合物可表现一种以上类型的异构现象，且由其混合物(例如外消旋混合物及非对映异构体对)组成。
本发明涵盖本发明的化合物的所有可能的结晶形式或多晶型物，其可为单一多晶型物或多于一种多晶型物的任意比例的混合物。
共晶是指药物活性分子与其它生理上可接受的酸、碱、盐、非离子化合物分子以氢键、π-π堆积作用、范德华力和其它非共价键相连而结合在同一晶格中。
还应当理解，本发明的某些化合物可以游离形式存在用于治疗，或适当时，以其药学上可接受的衍生物形式存在。在本发明中，药学上可接受的衍生物包括但不限于，药学上可接受的盐、酯、溶剂合物、N-氧化物、代谢物或前药，在将它们向需要其的患者给药后，能够直接或间接提供本发明的化合物或其代谢物或残余物。因此，当在本文中提及“本发明的化合物”时，也意在涵盖化合物的上述各种衍生物形式。
本发明的化合物的药学上可接受的盐包括其酸加成盐及碱加成盐。
本发明的化合物的药学上可接受的盐包括其酸加成盐及碱加成盐。例如六氟磷酸盐、葡甲胺盐等。适合的盐的综述参见Stahl及Wermuth的“Handbook of Pharmaceutical Salts:Properties,Selection,and Use”(Wiley-VCH,2002)。
如本文中所使用，术语“酯”意指衍生自本申请中各个通式化合物的酯，其包括生理上可水解的酯(可在生理条件下水解以释放游离酸或醇形式的本发明的化合物)。本发明的化合物本身也可以是酯。
本发明的化合物可以溶剂合物(优选水合物)的形式存在，其中本发明的化合物包含作为所述化合物晶格的结构要素的极性溶剂，特别是例如水、甲醇或乙醇。极性溶剂特别是水的量可以化学计量比或非化学计量比存在。
本领域技术人员会理解，由于氮需要可用的孤对电子来氧化成氧化物，因此并非所有的含氮杂环都能够形成N-氧化物。本领域技术人员会识别能够形成N-氧化物的含氮杂环。本领域技术人员还会认识到叔胺能够形成N-氧化物。用于制备杂环和叔胺的N-氧化物的合成方法是本领域技术人员熟知的，包括但不限于用过氧酸如过氧乙酸和间氯过氧苯甲酸(MCPBA)、过氧化氢、烷基过氧化氢如叔丁基过氧化氢、过硼酸钠和双环氧乙烷(dioxirane)如二甲基双环氧乙烷来氧化杂环和叔胺。这些用于制备N-氧化物的方法已在文献中得到广泛描述和综述，参见例如：T.L.Gilchrist,Comprehensive Organic Synthesis,vol.7,pp 748-750；A.R.Katritzky和A.J.Boulton,Eds.,Academic Press；以及G.W.H.Cheeseman和E.S.G.Werstiuk，Advances in Heterocyclic Chemistry,vol.22,pp 390-392,A.R.Katritzky和A.J.Boulton,Eds.,Academic Press。
在本发明的范围内还包括本发明的化合物的代谢物，即在给药本发明的化合物时体内形成的物质。这样的产物可由例如被给药的化合物的氧化、还原、水解、酰胺化、脱酰胺化、酯化、酶解等产生。因此，本发明包括本发明的化合物的代谢物，包括通过使本发明的化合物与哺乳动物接触足以产生其代谢产物的时间的方法制得的化合物。
本发明在其范围内进一步包括本发明的化合物的前药，其为自身可具有较小药理学活性或无药理学活性的本发明的化合物的某些衍生物当被给药至身体中或其上时可通过例如水解裂解转化成具有期望活性的本发明的化合物。通常这样的前药会是所述化合物的官能团衍生物，其易于在体内转化成期望的治疗活性化合物。关于前药的使用的其他信息可参见“Pro-drugs as Novel Delivery Systems”，第14卷，ACS Symposium Series(T.Higuchi及V.Stella)。本发明的前药可例如通过用本领域技术人员已知作为“前-部分(pro-moiety)(例如“Design of Prodrugs”，H.Bundgaard(Elsevier,1985)中所述)”的某些部分替代本发明的化合物中存在的适当官能团来制备。
本发明还涵盖含有保护基的本发明的化合物。在制备本发明的化合物的任何过程中，保护在任何有关分子上的敏感基团或反应基团可能是必需的和/或期望的，由此形成本发明的化合物的化学保护的形式。这可以通过常规的保护基实现，例如，在T.W.Greene&P.G.M.Wuts,Protective Groups in Organic Synthesis,John Wiley&Sons,1991中所述的那些保护基，这些参考文献通过援引加入本文。使用本领域已知的方法，在适当的后续阶段可以移除保护基。
术语“约”是指在所述数值的±10％范围内，优选±5％范围内，更优选±2％范围内。
化合物
在一些实施方案中，本发明提供式I的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药：
其中：
代表单键或双键；
X1、X2和X3各自独立地选自-CH2-、-CH-、-O-、-N-、-NH-和-S-；
R1在每次出现时各自独立地选自H、卤素、C1-6烷基、C1-6卤代烷基、C1-6羟烷基和C3-8环烷基；
环A为5-7元饱和或部分饱和杂环，所述杂环包含至少一个N原子以及任选存在的1或2个各自独立地选自O或S的相同或不同的其它杂原子；
R2在每次出现时各自独立地选自H、-OH、卤素、-CN、-NR4R5、-COCH3、-NHCOCH3、C1-6烷基、C1-6烷氧基、C1-6羟烷基、C2-6杂烷基、C3-8环烷基、3-8元杂环基、C3-8环烷氧基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、羟烷基、杂烷基、环烷基、杂环基、环烷氧基、芳基或杂芳基各自任选地被一个或多个R3取代；
R3在每次出现时各自独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6杂烷基、C3-6环烷基、C3-8环烷氧基、3-8元杂环基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、杂烷基、环烷基、环烷氧基、杂环基、芳基或杂芳基各自任选地被一个或多个各自独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C3-6环烷基、C3-8环烷氧基和3-8元杂环基的取代基取代；
R4、R5、R6和R7各自独立地选自H和C1-6烷基；或者
R4和R5、R6和R7连同其所连接的氮原子共同形成3-8元杂环基，所述杂环基任选地被一个或多个各自独立地选自卤素、OH、CN、-NH2和C1-6烷基的取代基取代；
m为0、1、2或3；
n为0、1、2、3或4。
在某些实施方案中，本发明提供的式I化合物中，X1、X2和X3各自独立地选自-CH2-、-CH-、-O-、-N-和-NH-。
在某些实施方案中，本发明提供的式I化合物中，X1为-CH-，且X2和X3为-N-。
在某些实施方案中，本发明提供的式I化合物中，X1和X2为-N-，且X3为-CH-。
在某些实施方案中，本发明提供的式I化合物中，X1和X3为-CH2-，且X2为-O-。
在某些实施方案中，本发明提供的式I化合物中，R1在每次出现时各自独立地选自H、C1-6烷基、C1-6卤代烷基和C3-8环烷基。
在某些实施方案中，本发明提供的式I化合物中，R1在每次出现时各自独立地选自H和C1-6烷基。
在某些实施方案中，本发明提供的式I化合物中，R1为甲基。
在某些实施方案中，本发明提供的式I化合物中，环A为5-7元饱和杂环，所述杂环包含至少一个N原子以及任选存在的1或2个各自独立地选自O或S的相同或不同的其它杂原子。
在某些实施方案中，本发明提供的式I化合物中，环A为5-7元饱和氮杂环和5-7元饱和含N和O杂环；
在某些实施方案中，本发明提供的式I化合物中，环A为6元饱和氮杂环和6元饱和含N和O杂环；
在某些实施方案中，本发明提供的式I化合物中，环A为哌啶环或吗啉环。
在某些实施方案中，本发明提供的式I化合物中，环A为5-7元饱和氮杂环。
在某些实施方案中，本发明提供的式I化合物中，环A为6元饱和氮杂环。
在某些实施方案中，本发明提供的式I化合物中，环A为哌啶环。
在某些实施方案中，本发明提供的式I化合物中，环A为吗啉环。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自H、卤素、C1-6烷基、C1-6烷氧基、C1-6羟烷基、C2-6杂烷基、C3-8环烷基、3-8元杂环基、C3-8环烷氧基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、羟烷基、杂烷基、环烷基、杂环基、环烷氧基、芳基或杂芳基各自任选地被一个或多个R3取代。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自H、卤素、C1-6烷基、C6-10芳基和5-10元杂芳基，所述烷基、芳基或杂芳基各自任选地被一个或多个R3取代。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自C1-6烷基、C6-10芳基和6-9元杂芳基，所述烷基、芳基或杂芳基各自任选地被一个或多个R3取代。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自C1-6烷基、苯基、吡啶基和苯并噻唑基，所述苯基、吡啶基或苯并噻唑基各自任选地被一个或多个R3取代。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自C1-6烷基和6-9元杂芳基，所述烷基或杂芳基各自任选地被一个或多个R3取代。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自C1-6烷基、吡啶基和苯并噻唑基，所述吡啶基或苯并噻唑基各自任选地被一个或多个R3取代。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自C1-6烷基、吡啶基和苯并噻唑基，所述吡啶基或苯并噻唑基各自任选地被C1-6烷基、C1-6卤代烷基和5-6元杂环基取代，所述杂环基任选地被C1-6烷基取代。
在某些实施方案中，本发明提供的式I化合物中，R2在每次出现时各自独立地选自甲基、 其中波浪线表示所述基团与分子其余部分的连接点。
在某些实施方案中，本发明提供的式I化合物中，R3在每次出现时各自独立地选自卤素、-CN、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、3-8元杂环基、C6-10芳基和5-6元杂芳基，所述烷基、杂烷基、杂环基、芳基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C3-6环烷基、C3-8环烷氧基和3-8元杂环基取代。
在某些实施方案中，本发明提供的式I化合物中，R3在每次出现时各自独立地选自卤素、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、4-7元杂环基、苯基和5-6元杂芳基，所述烷基、杂烷基、杂环基、苯基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代。
在某些实施方案中，本发明提供的式I化合物中，R3在每次出现时各自独立地选自-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、4-7元杂环基和5-6元杂芳基，所述烷基、杂环基、杂芳基任选地被一个或多个-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代。
在某些实施方案中，本发明提供的式I化合物中，R3在每次出现时各自独立地选自卤素、-NR4R5、C1-6烷基、C1-6卤代烷基、C2-6杂烷基、5-6元杂环基、苯基和5-6元杂芳基，所述烷基、杂烷基、杂环基、苯基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C3-6环烷基和C1-6卤代烷基的取代基取代。
在某些实施方案中，本发明提供的式I化合物中，R3在每次出现时各自独立地选自C1-6烷基、C1-6卤代烷基和5-6元杂环基，所述杂环基任选地被一个或多个C1-6烷基取代。
在某些实施方案中，本发明提供的式I化合物中，R3在每次出现时各自独立地选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、 其中波浪线表示所述基团与分子其余部分的连接点。
在某些实施方案中，本发明提供的式I化合物中，R4和R5各自独立地选自H和甲基。
在某些实施方案中，本发明提供的式I化合物中，R6和R7各自独立地选自H和甲基。
在某些实施方案中，本发明提供的式I化合物中，R6和R7为H。
在某些实施方案中，本发明提供的式I化合物中，m为0或1。
在某些实施方案中，本发明提供的式I化合物中，n为2。
在某些实施方案中，本发明提供的式I化合物为式II的化合物：
其中X1、X2、X3、R1、R3、m如上文对式I化合物所定义。
在某些实施方案中，在式II的化合物中，
X1为-CH-，且X2和X3为-N-；
R1为甲基；且
m为1。
在某些实施方案中，本发明提供的式I化合物为式III的化合物：
其中X1、X2、X3、R1、R3、m如上文对式I化合物所定义。
在某些实施方案中，在式III的化合物中，
X1为-CH-，且X2和X3为-N-；
R1为甲基；
m为1；且
R3选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、 其中波浪线表示所述基团与分子其余部分的连接点。
在某些实施方案中，本发明提供的式I化合物为式IV的化合物：
其中X1、X2、X3、R1、R3、m如上文对式I化合物所定义。
在某些实施方案中，在式IV的化合物中，
X1为-CH-，且X2和X3为-N-；
R1为甲基；
m为1；且
R3选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、
其中波浪线表示所述基团与分子其余部分的连接点。
在某些实施方案中，本发明提供的式I化合物为式V的化合物：
其中X1、X2、X3、R1、R3、m如上文对式I化合物所定义；Y1、Y2独立地选自N和CH。
在某些实施方案中，本发明提供的式V化合物为式V-1的化合物：
其中R3如上文对式I化合物所定义；Y1、Y2独立地选自N和CH。
在某些实施方案中，本发明提供的式V-1化合物为式V-1a的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式V-1化合物为式V-1b的化合物：

其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式V-1化合物为式V-1c的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，在式V、V-1、V-1a、V-1b或V-1c的化合物中，
R3选自卤素、-CN、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、3-8元杂环基、C6-10芳基和5-6元杂芳基，所述烷基、杂烷基、杂环基、芳基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C3-6环烷基、C3-8环烷氧基和3-8元杂环基的取代基取代；
优选地，R3选自卤素、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、4-7元杂环基、苯基和5-6元杂芳基，所述烷基、杂烷基、杂环基、苯基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代；
优选地，R3选自-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、4-7元杂环基和5-6元杂芳基，所述烷基、杂环基、杂芳基任选地被一个或多个-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代；
优选地，R3选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、 其中波浪线表示所述基团与分子其余部分的连接点。
在某些实施方案中，在式V、V-1、V-1a、V-1b或V-1c的化合物中，
R3选自C1-6卤代烷基和C1-6卤代烷氧基；
优选地，R3选自三氟甲基和三氟甲氧基。
在某些实施方案中，本发明提供的式I化合物为式VI的化合物：
其中X1、X2、X3、R1、R3、m如上文对式I化合物所定义；且
p为0或1。
在某些实施方案中，本发明提供的式VI化合物为式VI-1的化合物：
其中R3如上文对式I化合物所定义；且
p为0或1。
在某些实施方案中，本发明提供的式VI-1化合物为式VI-1a的化合物：
其中R3如上文对式I化合物所定义；且
p为0或1。
在某些实施方案中，本发明提供的式VI-1化合物为式VI-1b的化合物：
其中R3如上文对式I化合物所定义；且
p为0或1。
在某些实施方案中，在式VI、VI-1、VI-1a或VI-1b的化合物中，
R3选自卤素、-CN、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、3-8元杂环基、C6-10芳基和5-6元杂芳基，所述烷基、杂烷基、杂环基、芳基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C3-6环烷基、C3-8环烷氧基和3-8元杂环基的取代基取代；
优选地，R3选自卤素、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、4-7元杂环基、苯基和5-6元杂芳基，所述烷基、杂烷基、杂环基、苯基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代；
优选地，R3选自-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、4-7元杂环基和5-6元杂芳基，所述烷基、杂环基、杂芳基任选地被一个或多个-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代；
优选地，R3选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、 其中波浪线表示所述基团与分子其余部分的连接点。
在某些实施方案中，在式VI、VI-1、VI-1a或VI-1b的化合物中，
R3选自4-7元杂环基和5-6元杂芳基，所述杂环基或芳基各自任选地被选自C1-6烷基和C3-6环烷基的取代基取代；
优选地，R3选自其中波浪线表示所述基团与分子其余部分的连接点。
在某些实施方案中，本发明提供的式I化合物为式III-1a的化合物：
其中X1、X2、X3、R1、R3、m如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式V-1a-1的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式V-1a-2的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式V-1b-1的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式V-1b-2的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式V-1c-1的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式V-1c-2的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式VI-1a-1的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式VI-1a-2的化合物：
其中R3如上文对式I化合物所定义。
在某些实施方案中，本发明提供的式I化合物为式VI-1a-3的化合物：
其中R3如上文对式I化合物所定义。
本发明涵盖以上各实施方案的任意组合。
在一些实施方案中，本发明的化合物包括但不限于：


制备方法
本发明的化合物可以通过本领域已知的任何方法制备。试剂和起始材料对于本领域普通技术人员来说是容易获得的。单独的异构体、对映异构体和非对映异构体可以在合成中的任何方便点通过诸如选择性结晶技术或手性色谱法的方法进行分离或拆分(参见例如selective crystallization techniques or chiral chromatography(See for example,J.Jacques,et al.,"Enantiomers,Racemates,and Resolutions",John Wiley and Sons,Inc.,1981,and E.L.Eliel and S.H.Wilen)。
在某些实施方案中，本发明提供式I的化合物的制备方法，其包括以下步骤：
第一步：化合物I-A-1与酰氯经缩合反应生成化合物I-A-2；
第二步：化合物I-A-2经水解反应生成化合物I-A-3；
第三步：化合物I-A-3和化合物I-A-4经缩合反应生成化合物I-A-5；
第四步：缩合产物I-A-5脱除保护基后生成化合物I；
其中
PG为氨基保护基；
X1、X2、X3、环A、R1、R2、m、n如上文所定义，R6和R7为H。
在本发明的一些实施方案中，所述氨基保护基为对甲氧基苄基或2,4-二甲氧基苄基。
第一步
在本发明的一些实施方案中，上述第一步中的缩合反应在碱的存在下进行，优选在诸如三乙胺或二异丙基乙胺等碱的存在下进行。
在本发明的一些实施方案中，上述第一步反应在诸如THF等溶剂中进行。
第二步
在本发明的一些实施方案中，上述第二步中的水解反应在碱的水溶液中进行，优选在诸如LiOH、NaOH等碱的水溶液中进行。
在本发明的一些实施方案中，上述第二步中的水解反应在诸如THF、甲醇或乙醇等溶剂中进行。
第三步
在本发明的一些实施方案中，上述第三步中的缩合反应在缩合剂的存在下进行，优选在诸如HATU、PyBOP、T3P、EDCI、PyBrOP等缩合剂中的一种或多种的存在下进行。
在本发明的一些实施方案中，上述第三步中的缩合反应在碱的存在下进行，优选在诸如三乙胺或二异丙基乙胺等碱的存在下进行。
在本发明的一些实施方案中，上述第三步中的缩合反应在诸如DMF或NMP等溶剂中进行。
第四步
在本发明的一些实施方案中，上述第四步中的脱除保护基反应优选在酸的作用下进行，优选在盐酸水溶液、盐酸-乙酸乙酯溶液、盐酸-1,4-二氧六环溶液或三氟醋酸等酸的作用下进行，或者使用氧化脱除保护基，优选在DDQ等氧化剂作用下进行。
本领域技术人员应当理解，根据期望获得的产物结构，可以省略上述制备方法中的一个或多个步骤，也可根据需要适当地调整反应步骤的顺序以及增加或省略保护/脱保护反应步骤。
药物组合物、制剂和治疗方法
在一些实施方案中，本发明提供一种药物组合物，其包含预防或治疗有效量的本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体或同位素标记的化合物以及一种或多种药学上可接受的载体。
在一些实施方案中，本发明提供一种药物制剂，其包含预防或治疗有效量的本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体或同位素标记的化合物以及一种或多种药学上可接受的载体。所述药物制剂优选为固体制剂、半固体制剂、液体制剂或气态制剂。
在一些实施方案中，所述药物组合物或药物制剂还可包含一种或多种其它治疗剂。
在一些实施方案中，所述药物组合物或药物制剂优选通过口服、静脉内、动脉内、皮下、腹膜内、肌内或经皮途径给药。
在一些实施方案中，本发明提供本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体或同位素标记的化合物或者本发明的药物组合物或者本发明的药物制剂在制备用于预防或治疗与PRMT5活性相关的疾病或病况的药物中的用途。
在一些实施方案中，本发明提供本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体或同位素标记的化合物或者本发明的药物组合物或者本发明的药物制剂在制备用于调节(例如降低或抑制)PRMT5活性的药物中的用途。
在一些实施方案中，本发明提供本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体或同位素标记的化合物，或者本发明的药物组合物，或者本发明的药物制剂，其用于预防或治疗与PRMT5活性相关的疾病或病况。
在一些实施方案中，本发明提供预防或治疗与PRMT5活性相关的疾病或病况的方法，所述方法包括向需要其的个体给药有效量的本发明的化合物或其药学上可接受的盐、立体异构体、互变异构体或同位素标记的化合物，或者本发明的药物组合物，或者本发明的药物制剂。
在一些实施方案中，所述与PRMT5活性相关的疾病或病况优选为MTAP缺失的癌症或肿瘤。
在一些实施方案中，所述癌症或肿瘤优选为食管癌、肺癌、胰腺癌、胶质母细胞瘤、胆管癌、膀胱癌、乳腺癌、卵巢癌、肝细胞癌、前列腺癌、黑色素瘤、胃癌、结肠癌、白血病(特别是慢性B淋巴细胞白血病(B-CLL))、淋巴瘤等。
本发明中“药学上可接受的载体”是指与治疗剂一同给药的稀释剂、辅剂、赋形剂或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险比相应的其它问题或并发症。
在本发明的药物组合物中可使用的药学上可接受的载体包括但不限于无菌液体。适合的药学上可接受的载体的实例如在Remington’s Pharmaceutical Sciences(1990)中所述。
本发明的药物组合物可以系统地作用和/或局部地作用。为此目的，它们可以适合的途径给药。
对于这些给药途径，可以适合的剂型给药本发明的药物组合物。
如本文中所使用的术语“有效量”指被给药后会在一定程度上缓解所治疗病症的一或多种症状的化合物的量。
可调整给药方案以提供最佳所需响应。例如，可给药单次推注，可随时间给药数个分剂量，或可如治疗情况的急需所表明而按比例减少或增加剂量。要注意，剂量值可随要减轻的病况的类型及严重性而变化，且可包括单次或多次剂量。要进一步理解，对于任何特定个体，具体的给药方案应根据个体需要及给药组合物或监督组合物的给药的人员的专业判断来随时间调整。
所给药的本发明的化合物的量会取决于所治疗的个体、病症或病况的严重性、给药的速率、化合物的处置及处方医师的判断。一般而言，有效剂量在每日每kg体重约0.0001至约50mg。在一些情况下，不高于前述范围的下限的剂量水平可以是足够的，而在其它情况下，仍可在不引起任何有害副作用的情况下采用较大剂量，条件是首先将所述较大剂量分成数个较小剂量以在一整天中给药。
本发明的化合物在药物组合物或药物制剂中的含量或用量可以是约0.01mg至约1000mg。
除非另外说明，否则如本文中所使用，术语“治疗(treating)”意指逆转、减轻、抑制这样的术语所应用的病症或病况或者这样的病症或病况的一或多种症状的进展，或预防这样的病症或病况或者这样的病症或病况的一或多种症状。
如本文所使用的“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本发明中“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
在一些实施方案中，本发明的药物组合物或药物制剂还可以包含一种或多种另外的治疗剂或预防剂(例如其它用于治疗癌症或肿瘤疾病的药物)。在一些实施方案中，本发明的治疗方法还可以包括给药一种或多种另外的治疗剂或预防剂(例如其它用于治疗癌症或肿瘤疾病的药物)。
具体实施方式
实施例
以下结合实施例进一步描述本发明，但提供这些实施例并非意图限制本发明的范围。
本文中所用的缩写具有以下含义：
本发明的化合物通过制备TLC、硅胶柱色谱法、Prep-HPLC和/或快速柱色谱法(Flash柱色谱法)来分离纯化，其结构通过1H NMR和/或MS来确证。反应监测采用TLC或LC-MS进行。
1H NMR波谱法采用Bruker超导核磁共振波谱仪(型号AVACE III HD 400MHz)。
LC/MS采用Aglient 1260 Infinity/Aglient 6120 Quadrupole。
TLC采用硅胶GF 254作为固定相。
柱色谱法一般使用200～300目硅胶(青岛海洋)作为固定相。
快速柱色谱法使用Biotage快速柱色谱仪。
Prep-HPLC采用Agilent 1260型和Waters 2489型。
微波反应使用BiotageInitiator微波反应器进行。
在以下实施例中，如无特殊说明，反应的温度为室温(15-30℃)。
本申请中所使用的试剂购自Acros Organics、Aldrich Chemical Company或特伯化学等公司。
合成实施例：
中间体Int A：2-((4-((叔丁氧羰基)氨基)-1,3-二氢呋喃并[3,4-c]吡啶-7-基)氨基)-2-氧代乙酸
第一步：吡啶-3,4-二羧酸二甲酯(化合物Int A-2)的合成
将Int A-1(30g,179.51mmol)及MeOH(300mL)加入反应瓶中，再缓慢加入SOCl2(42.71g,359.03mmol,26.04mL)，升温至80℃反应3小时。将反应液旋干，补加SOCl2(42.71g,359.03mmol,26.04mL)及MeOH(300mL)进入反应瓶中，80℃反应3小时。待反应结束后，将反应液旋干，乙酸乙酯(500ml)溶解，用碳酸氢钠水溶液水洗两次，有机相用硫酸钠干燥，过滤，经减压浓缩得到化合物Int A-2(32.46g)。MS(ESI,m/z):196.1[M+H]+。
第二步：3,4-双(甲氧羰基)吡啶-1-氧化物(化合物Int A-3)的合成
将Int A-2(4.4g,22.54mmol)溶解于DCM(50mL)中，分批加入mCPBA(7.29g,33.82mmol)，25℃搅拌过夜。待反应结束后，向反应液中加入硫代硫酸钠水溶液淬灭，搅拌2小时，二氯甲烷萃取(200mlx3)，合并有机相，用硫酸钠干燥，过滤，经减压浓缩后得到粗品，粗品经硅胶柱层析(DCM:MeOH＝10:1)纯化得到化合物Int A-3(4.7g)。MS(ESI,m/z):212.1[M+H]+。
第三步:2-氯吡啶-3,4-二羧酸甲酯(化合物Int A-4)的合成
将Int A-3(4.7g,22.26mmol)及POCl3(20mL)加入反应瓶中，100℃反应过夜。待反应结束后，减压蒸馏将反应液旋干，缓慢将其倒入水中，碳酸氢钠调节pH＝7-8，乙酸乙酯萃取，合并有机相，用无水硫酸钠干燥，过滤后减压浓缩得到粗品。粗品经硅胶柱层析(EA:PE＝1:10)纯化，得到化合物Int A-4(2.2g)。MS(ESI,m/z):230.0[M+H]+。
第四步：(2-氯吡啶-3,4-二基)二甲醇(化合物Int A-5)的合成
将Int A-4(1.5g,6.53mmol)溶解于EtOH(20mL)中，分批加入NaBH4(1.48g,39.20mmol)，加毕后，25℃反应过夜。待反应结束后，甲酸调节pH～5，减压蒸除溶剂，经硅胶柱层析(PE:EA＝25:75)纯化得到化合物Int A-5(800mg)。MS(ESI,m/z):174.1[M+H]+。
第五步：4-氯-1,3-二氢呋喃并[3,4-c]吡啶(化合物Int A-6)的合成
将Int A-5(1.15g,6.62mmol)溶解于DCM(25mL)中，分批加入MnO2(1.15g,13.25mmol)，滴加Et3SiH(5mL)和三氟乙酸(9mL)，25℃反应过夜。反应结束后减压蒸除溶剂，粗品经硅胶柱层析(EA:PE＝20:80)纯化，得到化合物Int A-6(200mg)。MS(ESI,m/z):156.1[M+H]+。
第六步：N-(2,4-二甲氧基苄基)-1,3-二氢呋喃并[3,4-c]吡啶-4-胺(化合物Int A-8)的合成
将Int A-6(200mg,1.29mmol)、Int A-7(430mg,2.57mmol)、甲苯(5mL)、tBuONa(494mg,5.14mmol)、BINAP(80mg,128.55μmol)、Pd2(dba)3(59mg,64.28μmol)依次加入反应瓶中，氮气置换三次，100℃反应过夜。待反应结束后，将反应液旋干，加入水稀释，乙酸乙酯萃取三次，合并有机相，用硫酸钠干燥有机相，减压蒸馏除去溶剂，经硅胶柱层析(PE:EA＝75:25)纯化，得到化合物Int A-8(230mg)。MS(ESI,m/z):287.1[M+H]+。
第七步：1,3-二氢呋喃并[3,4-c]吡啶-4-胺(化合物Int A-9)的合成
将Int A-8(230mg,803.28μmol)溶解于TFA(10mL)中，25℃反应1小时。待反应结束后，将反应液旋干，加入饱和NaHCO3水溶液稀释，乙酸乙酯萃取，合并有机相，用无水硫酸钠干燥，减压蒸馏除去溶剂，得到化合物Int A-9(96mg)。
第八步：7-溴-1,3-二氢呋喃并[3,4-c]吡啶-4-胺(化合物Int A-10)的合成
将Int A-9(96mg,705.10μmol)、MeCN(3mL)加入反应瓶中，再分批加入NBS(138mg,775.61μmol)，25℃反应2小时。待反应结束后，减压蒸馏除去溶剂，经硅胶柱层析(PE:EA＝70:30)纯化，得到化合物Int A-10(110mg)。MS(ESI,m/z):215.0[M+H]+。
第九步：7-溴-N,N-双(叔丁氧羰基)-1,3-二氢呋喃并[3,4-c]吡啶-4-胺(化合物Int A-11)的合成
将Int A-10(100mg,465.01μmol)、DCM(5mL)、TEA(141mg,1.40mmol)、DMAP(11.36mg,93.00μmol)加入反应瓶中，分批加Boc2O(254mg,1.16mmol)，25℃反应2小时。待反应结束后，反应液加水稀释，用DCM(30ml x3)萃取，合并有机相，用盐水洗涤，无水硫酸钠干燥，过滤，滤液经减压浓缩得到粗产物。粗品经硅胶柱层析纯化(EA:PE＝10:90)，得到化合物Int A-11(83mg)。MS(ESI,m/z):415.1[M+H]+。
第十步：(7-((二苯基亚甲基)氨基)-1,3-二氢呋喃并[3,4-c]吡啶-4-基)氨基甲酸叔丁酯(化合物Int A-13)的合成
将Int A-11(90mg,216.72μmol)、Int A-12(79mg,433.44μmol)、tBuONa(63mg,650.17μmol)、BINAP(14mg,21.67μmol)、Pd2(dba)3(10mg,10.84μmol)、1,4-二氧六环(5mL)依次加入反应瓶中，氮气置换三次，80℃反应过夜。待反应结束后，减压蒸除溶剂，经硅胶柱层析(EA:PE＝15:85)纯化,得到化合物Int A-13(50mg)。MS(ESI,m/z):416.2[M+H]+。
第十一步：(7-氨基-1,3-二氢呋喃并[3,4-c]吡啶-4-基)氨基甲酸叔丁酯(化合物Int A-14)的合成
反应瓶中加入Int A-13(50mg,120.34μmol)、Pd/C(14.62mg,120.34μmol)、MeOH(3mL)、THF(1mL)，氢气球置换三次，60℃反应5hr。待反应结束后，用滤头过滤反应液并加水稀释，用乙酸乙酯(30ml x3)萃取，合并有机相，用盐水洗涤，无水硫酸钠干燥，过滤，滤液经减压浓缩得到化合物Int A-14(30mg)。MS(ESI,m/z):252.2[M+H]+。
第十二步：2-((4-((叔丁氧羰基)氨基)-1,3-二氢呋喃并[3,4-c]吡啶-7-基)氨基)-2-氧代乙酸乙酯(化合物Int A-16)的合成
反应瓶中加入Int A-14(30mg,119.39μmol)、DIPEA(38.57mg,298.47μmol)、THF(3mL)再缓慢加入Int A-15(17mg,119.39μmol)，氮气保护，25℃反应1hr。待反应结束后加水稀释，用乙酸乙酯(30ml x3)萃取，合并有机相，用盐水洗涤，无水硫酸钠干燥，过滤，滤液经减压浓缩得到粗产物。粗品经硅胶柱层析纯化(EA:PE＝35:65)，得到化合物Int A-16(23mg)。MS(ESI,m/z):352.2[M+H]+。
第十三步：2-((4-((叔丁氧羰基)氨基)-1,3-二氢呋喃并[3,4-c]吡啶-7-基)氨基)-2-氧代乙酸(化合物Int A)的合成
反应瓶中加入Int A-16(23mg,65.46μmol)及THF(1mL)，溶解后再加入LiOH·H2O(6mg,130.92μmol)的H2O(0.5mL)溶液，氮气保护，25℃反应2hr。待反应结束后，用2M盐酸调节pH至6-7，直接将反应液旋干，得到化合物Int A(20mg)。MS(ESI,m/z):324.1[M+H]+。
中间体Int B：2-((7-((2,4-二甲氧基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)氨基)-2-氧代乙酸
第一步：4-溴-7-氯-1-甲基-1H-吡唑并[3,4-c]吡啶(化合物Int B-2)的合成
将Int B-1(5g,21.51mmol)溶于DMF(30mL)中，在冰浴下缓慢加入NaH(1.72g,43.02mmol)，加完后反应30min，然后加入MeI(3.66g,25.81mmol)，加完后在25℃持续搅拌2hr。待反应结束后，向反应液中加水淬灭，用乙酸乙酯(50mL x 3)萃取，合并有机相，有机相依次经清水、饱和食盐水洗涤，用无水硫酸钠干燥，过滤并将滤液减压浓缩。粗品经硅胶柱层析纯化(PE:EA＝70:30)，得到化合物Int B-2(3.0g)。MS(ESI,m/z):245.9[M+H]+。
第二步：4-溴-N-(2,4-二甲氧基苄基)-1-甲基-1H-吡唑并[3,4-c]吡啶-7-胺(化合物Int B-3)的合成
将Int B-2(3.0g,12.17mmol)、Int A-7(3.05g,18.26mmol)和DIPEA(2.36g,18.26mmol，3.2mL)溶解于NMP(10mL)中，置换氮气后，在140℃搅拌5hr。待反应结束后，向反应液中加水淬灭，用乙酸乙酯(50mL x3)萃取，合并有机相，有机相依次经清水、饱和食盐水洗涤，用无水硫酸钠干燥，过滤并将滤液减压浓缩。经硅胶柱层析纯化(PE:EA＝70:30)，得化合物Int B-3(4.0g)。MS(ESI,m/z):377.0[M+H]+。
第三步：N-(2,4-二甲氧基苄基)-4-((二苯基亚甲基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-7-胺(化合物Int B-4)的合成
将Int B-3(4.0g,10.60mmol)、Int A-12(3.84g,21.21mmol)、tBuONa(3.06g,31.81mmol)、BINAP(660mg,1.06mmol)和Pd2(dba)3(485mg,0.53mmol)溶解于甲苯(10mL)中，置换氮气后，反应温度升高至80℃，搅拌16hr。待反应结束后，减压浓缩至干得到粗品。粗品经硅胶柱层析纯化(EA:PE＝90:10)，得化合物Int B-4(4.68g)。MS(ESI,m/z):478.2[M+H]+。
第四步：N7-(2,4-二甲氧基苄基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4,7-二胺(化合物Int B-5)的合成
将Int B-4(4.68g,9.80mmol)溶解于MeOH(30mL)中，然后加入4NHCl的1,4-二氧六环溶液(3mL)，置换氮气后，反应在25℃搅拌1hr。待反应结束后，减压浓缩至干得到粗品。粗品经硅胶柱层析纯化(DCM:MeOH＝90:10)，得化合物Int B-5(3g)。MS(ESI,m/z):314.1[M+H]+。
第五步：2-((7-((2,4-二甲氧基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)氨基)-2-氧代乙酸乙酯(化合物Int B-6)的合成
将Int B-5(3g,9.57mmol)和DIPEA(3.71g,28.72mmol)溶解于THF(25mL)中，再缓慢加入草酰氯单乙酯(1.57g,11.49mmol)，加完后25℃搅拌0.5hr。待反应结束后，向反应液中加水淬灭，用乙酸乙酯(50mL x3)萃取，合并有机相，有机相依次经清水、饱和食盐水洗涤，用无水硫酸钠干燥，过滤并将滤液减压浓缩至干得到粗品。粗品经硅胶柱层析纯化(EA:PE＝50:50)，得化合物Int B-6(1.38g)。MS(ESI,m/z):414.1[M+H]+。
第六步：2-((7-((2,4-二甲氧基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)氨基)-2-氧代乙酸(化合物Int B)的合成
将Int B-6(1.38g,3.34mmol)和LiOH·H2O(280mg,6.68mmol)加入到THF(15mL)和H2O(3mL)中，加完25℃搅拌2hr。待反应结束后，直接将反应液减压浓缩除去溶剂，得化合物Int B(1g)。MS(ESI,m/z):386.1[M+H]+。
中间体Int C：2-((4-((4-甲氧基苄基)氨基)-1-甲基-1H-吡唑并[4,3-c]吡啶-7-基)氨基)-2-氧代乙酸
第一步：2-氨基-5-溴-4-氯烟醛(化合物Int C-2)的合成
将Int C-1(1g,6.39mmol)溶于无水DCE(20mL)中，加入NBS(1.25g,7.03mmol)，N2保护后升温至60℃，反应2hr。反应完成后减压浓缩除去溶剂，加水溶解，用乙酸乙酯萃取，有机层干燥浓缩得中间体Int C-2(1.4g)。
第二步：7-溴-1-甲基-1H-吡唑并[4,3-c]吡啶-4-胺(化合物Int C-4)的合成
将Int C-2(1.5g,6.37mmol)、甲基肼硫酸盐(Int C-3,1.38g,9.56mmol)溶于EtOH(30mL)中，加入DIPEA(4.12g,31.85mmol)，N2保护后升温至80℃，反应24hr。反应完成后减压浓缩，粗品经硅胶柱层析(DCM:MeOH＝80:20)分离纯化得中间体Int C-4(320mg)。MS(ESI,m/z):226.9[M+H]+。
第三步：7-溴-N-(4-甲氧基苄基)-1-甲基-1H-吡唑并[4,3-c]吡啶-4-胺(Int C-5)的合成
将Int C-4(280mg,1.23mmol)、DIPEA(318.74mg,2.47mmol)溶于无水NMP(10mL)中，加入PMBCl(289.68mg,1.85mmol)，加毕，N2保护后升温至140℃，反应5hr。反应完成后加水稀释，用乙酸乙酯萃取，用饱和食盐水洗，用无水硫酸钠干燥，过滤，粗品经正相快速色谱法分离纯化(PE:EA＝92:8)分离纯化得中间体Int C-5(150mg)。MS(ESI,m/z):347.0[M+H]+。
第四步：7-((二苯基亚甲基)氨基)-N-(4-甲氧基苄基)-1-甲基-1H-吡唑并[4,3-c]吡啶-4-胺(化合物Int C-6)的合成
将Int C-5(200mg,576.02μmol)、Int E-5(208.79mg,1.15mmol)、tBuONa(166.07mg,1.73mmol)、BINAP(35.87mg,57.60μmol)及Pd2(dba)3(26.37mg,28.80μmol)溶于甲苯(10mL)中，N2保护后，升温至80℃，反应16hr。反应完成后减压浓缩除去甲苯，粗品经正相快速色谱法分离纯化(PE:EA＝79:21)分离纯化得中间体Int C-6(80mg)。MS(ESI,m/z):448.2[M+H]+。
第五步：N4-(4-甲氧基苄基)-1-甲基-1H-吡唑并[4,3-c]吡啶-4,7-二胺(化合物Int C-7)的合成
将Int C-6(80mg,178.76μmol)溶解于甲醇(3mL)中，滴加4N HCl的1,4-二氧六环溶液(0.3mL),反应体系于25℃反应1hr。反应完成后减压浓缩除去溶剂，然后加适量甲醇溶解后，加入几滴TEA调pH至7-8，减压浓缩，粗品经正相快速色谱法(DCM:MeOH＝93:7)分离纯化得中间体Int C-7(50mg)。MS(ESI,m/z):284.1[M+H]+。
第六步：2-((4-((4-甲氧基苄基)氨基)-1-甲基-1H-吡唑并[4,3-c]吡啶-7-基)氨基)-2-氧代乙酸乙酯(化合物Int C-8)的合成
将Int C-7(90mg,317.65μmol)、DIPEA(41.05mg,317.65μmol)溶解于THF(10mL)中，缓慢加入草酰氯单乙酯(52.04mg,381.18μmol)，加毕，反应体系于25℃反应1hr。反应完成后减压浓缩除去溶剂，粗品经正相快速色谱法(DCM:MeOH＝90:10)分离纯化得中间体Int C-8(110mg)。MS(ESI,m/z):384.2[M+H]+。
第七步：2-((4-((4-甲氧基苄基)氨基)-1-甲基-1H-吡唑并[4,3-c]吡啶-7-基)氨基)-2-氧代乙酸(化合物Int C)的合成
将Int C-8(120mg,312.99μmol)溶解于THF(5mL)和H2O(1mL)中，加入NaOH(25.04mg,625.98μmol)，加毕反应体系升温至25℃，反应2hr。反应完成后减压浓缩除去溶剂得中间体Int C(115mg)。MS(ESI,m/z):356.1[M+H]+。
中间体Int D：2-(5-甲基哌啶-2-基)-5-(三氟甲基)吡啶
第一步：5-甲基哌啶-2-酮(化合物Int D-2)的合成
将Int D-1(5g,45.82mmol)溶于MeOH(40mL)中，然后加入Pd(OH)2/C(6.43g,4.58mmol,10％纯度)，置换氢气三次，高压釜升温至80℃，搅拌12hr，反应结束后抽滤，滤液浓缩后得到化合物Int D-2(5g)。MS(ESI,m/z):114.2[M+H]+。
第二步：5-甲基-2-氧代哌啶-1-羧酸叔丁酯(化合物Int D-3)的合成
将Int D-2(1g,8.84mmol)和DMAP(107.97mg,883.73μmol)加入到THF(10mL)中，再缓慢加入(Boc)2O(2.12g,9.72mmol)，加完于25℃搅拌16hr。反应结束后用氯化铵淬灭，用乙酸乙酯萃取，用无水硫酸钠干燥，过滤，浓缩，经硅胶快速柱层析(PE:EA＝9:1)纯化后得到化合物Int D-3(1.72g)。
第三步：3-甲基-6-(((三氟甲基)磺酰基)氧基)-3,4-二氢吡啶-1(2H)-羧酸叔丁酯(化合物Int D-5)的合成
将Int D-3(5g,23.44mmol)溶于THF(50mL)中，降温至-78℃，然后缓慢滴入LiHMDS(35.17mmol，1M THF溶液,35.17mL)，滴加完成后，保持在-78℃下持续搅拌0.5hr，然后加入Int D-4(10.05g,28.13mmol)的THF(20mL)溶液，加完后自然升至25℃，反应16hr。反应结束后，冰浴下加入饱和氯化铵水溶液淬灭反应，用乙酸乙酯萃取三次，合并有机相，用无水硫酸钠干燥，抽滤，浓缩，经硅胶快速柱层析(PE:EA＝9:1)纯化后得到化合物Int D-5(7.5g)。MS(ESI,m/z):363.1[M+18]+。
第四步：3-甲基-6-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)-3,4-二氢吡啶-1(2H)-羧酸叔丁酯(化合物Int D-7)的合成
将Int D-5(7.5g,21.72mmol)、Int D-6(8.27g,32.58mmol)、PPh3(341.78mg,1.30mmol)、Pd(PPh3)4(752.90mg,651.54μmol)和K2CO3(7.50g,54.30mmol)称入两口瓶中，然后加入1,4-二氧六环(50mL)，置换氮气后，反应升温至90℃后，持续搅拌20hr。浓缩除去溶剂，然后用二氯甲烷萃取三次，合并有机相，浓缩后经反相柱分离纯化(H2O[0.05％NH4HCO3]:ACN＝10:90)，浓缩后经乙酸乙酯萃取，干燥，抽滤，浓缩后得到化合物Int D-7(1.55g)。MS(ESI,m/z):324.2[M+H]+。
第五步：5-甲基-5'-(三氟甲基)-5,6-二氢-[2,2'-联吡啶]-1(4H)-羧酸叔丁酯(化合物Int D-9)的合成
将Int D-8(2.17g,9.59mmol)、Int D-7(1.55g,4.80mmol)、Pd(dppf)Cl2·DCM(391.60mg,479.53μmol)溶于1,4-二氧六环(50mL)中，然后再加入Na2CO3(1.02g,9.59mmol)的H2O(10mL)溶液，置换氮气后，反应体系加热至90℃，反应2hr。反应降温至室温，直接浓缩后，用二氯甲烷萃取三次，合并有机相，经无水硫酸钠干燥，抽滤，浓缩得化合物Int D-9(1.6g)直接用于下一步反应。MS(ESI,m/z):343.1[M+H]+。
第六步：5-甲基-5'-(三氟甲基)-3,4,5,6-四氢-2,2'-联吡啶(化合物Int D-10)的合成
将Int D-9(1.6g,4.67mmol)加入到4N HCl的1,4-二氧六环溶液(10mL)中，于25℃反应2hr。直接浓缩得到化合物Int E-3(1g)，直接用于下一步反应。MS(ESI,m/z):243.1[M+H]+。
第七步：2-(5-甲基哌啶-2-基)-5-(三氟甲基)吡啶(化合物Int D)的合成
将Int D-10(1g,4.13mmol)加入到THF(10mL)中，置换氮气后，加入NaBH4(312.36mg,8.26mmol)，反应在25℃搅拌0.5hr。反应结束后滴加饱和氯化铵淬灭反应，浓缩后经反相柱分离纯化 (H2O[0.05％HCO2H]:ACN＝70:30)，冻干后得到目标化合物Int D(500mg)。MS(ESI,m/z):245.1[M+H]+。
中间体Int E：5-(5-甲基哌啶-2-基)苯并[d]噻唑
第一步：5-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)苯并[d]噻唑(化合物Int E-2)的合成
将Int E-1(500mg,2.34mmol)、Int D-6(771.01mg,3.04mmol)、Pd(dppf)Cl2·DCM(190.73mg,233.56μmol)和KOAc(687.63mg,7.01mmol)加入到1,4-二氧六环(30mL)中，置换氮气后，反应体系加热至80℃反应2hr。反应液直接硅胶柱层析(PE:EA＝98:2)分离纯化，浓缩后得到化合物Int E-2(580mg)。MS(ESI,m/z):262.1[M+H]+。
第二步：6-(苯并[d]噻唑-5-基)-3-甲基-3,4-二氢吡啶-1(2H)-羧酸叔丁酯(化合物Int E-3)的合成
将Int E-2(100mg,382.93μmol)、Int D-5(132.24mg,382.93μmol)、Pd(dppf)Cl2·DCM(31.27mg,38.29μmol)和Na2CO3(81.17mg,765.85μmol)加入到1,4-二氧六环(10mL)和H2O(1mL)的混合溶液中，反应体系加热至90℃，反应2hr。反应体系降温后直接浓缩干，粗品经硅胶柱层析纯化(PE:EA＝20:1)，浓缩后得到目标化合物Int E-3(580mg)。MS(ESI,m/z):331.1[M+H]+。
第三步：5-(5-甲基-3,4,5,6-四氢吡啶-2-基)苯并[d]噻唑(化合物Int E-4)的合成
将Int E-3(600mg,1.82mmol)加入到4N HCl的1,4-二氧六环溶液(10mL)中，于25℃反应2hr。直接浓缩至干，加入甲醇和三乙胺再次浓缩至干，硅胶柱层析(DCM:MeOH＝98:2)分离，纯化，浓缩后得到目标化合物Int E-4(385mg)。MS(ESI,m/z):231.1[M+H]+。
第四步：5-(5-甲基哌啶-2-基)苯并[d]噻唑(化合物Int E)的合成
将Int E-4(440mg,1.91mmol)加入到THF(10mL)中，置换氮气后，加入NaBH4(144.54mg,3.82mmol)，反应在25℃搅拌0.5hr。反应结束后滴加饱和氯化铵淬灭反应，浓缩至干后硅胶柱层析(DCM:MeOH＝97:3)分离纯化，浓缩后得到目标化合物Int E(400mg)。MS(ESI,m/z):233.1[M+H]+。
中间体Int F和G：5-((二苯氧基磷酰基)氧基)-3-甲基-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int F)和3-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧环戊硼烷-2-基)-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int G)
第一步：5-甲基吗啉-3-酮(化合物Int F-3)的合成
在冰浴下，将NaH(120g,3.00mol)缓慢加入到THF(1.00L)中，然后保持在0℃下将Int F-1(100g,1.33mol)缓慢滴加到反应液中,持续搅拌30mins后滴加Int F-2(204g,1.66mol)，滴加完成和升温至25℃反应16hrs。TLC(PE/EA＝1/1,Rf＝0.15)和LCMS检测反应完全。降温至0℃后缓慢滴加饱和NH4Cl(1.00L)淬灭反应，经过乙酸乙酯(6.00L)萃取后，合并有机相，无水硫酸钠干燥，抽滤，浓缩得到化合物Int F-3(158g)。MS(ESI,m/z):116.3[M+H]+。
第二步：3-甲基-5-氧代吗啉-4-羧酸叔丁酯(化合物Int F-4)的合成
将化合物Int F-3(158g,1.37mol)溶于THF(1.00L)中，然后加入DMAP(16.8g,137mmol)和Boc2O(329g,1.51mol)。反应保持在25℃搅拌16hrs。LCMS检测反应物完全，反应液中加入水(200mL)淬灭后，减压蒸馏浓缩除去THF，然后用乙酸乙酯(900mL)萃取，合并有机相，饱和食盐水(300mL)洗涤，无水硫酸钠干燥，抽滤，浓缩，经过硅胶柱层析(PE/EA＝1/0到6/1)分离纯化，浓缩得到化合物Int F-4(55.0g)。MS(ESI,m/z):159.9[M-tBu+H]+。
第三步：5-((二苯氧基磷酰基)氧基)-3-甲基-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int F)的合成
将化合物Int F-4(54.9g,255mmol)溶于THF(800mL)中，置换氮气后降温至-70℃，然后缓慢滴加LiHMDS(1M,281mL)，反应液保持在-70℃持续搅拌1hr后加入Int F-5(71.9g,268mmol)，加料完成后反应液保持在-60～-30℃搅拌3hrs。LCMS检测反应完全，在冰浴下加水(200mL)淬灭，然后用乙酸乙酯(300mL x 3)萃取，合并有机相，饱和食盐水(600mL)洗涤，无水硫酸钠干燥，抽滤，浓缩后经硅胶柱层析(PE/EA＝1/0到9/1)分离纯化，浓缩得到化合物Int F(98.9g)。348.1[M+H-Boc]+。
第四步：3-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧环戊硼烷-2-基)-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int G)的合成
将化合物Int F(98.9g,221mmol)、B2Pin2(84.2g,332mmol)、KOAc(65.1g,663mmol)、XPhos Pd G3(18.7g,22.1mmol)溶于1,4-二氧六环(1000mL)中，置换氮气后，反应温度升高至100℃搅拌2hrs。TLC(PE/EtOAc＝5/1,Rf＝0.47)和LCMS检测反应完全后，反应降温至室温，抽滤除去固体，然后滤饼用CH2Cl2(1.0L)洗涤，合并有机相，然后减压蒸馏浓缩，经过硅胶柱层析(PE/EtOAc＝1/0到9/1)分离纯化，浓缩后得到化合物Int G(61.5g)。MS(ESI,m/z):269.7[M+H-tBu]+。
中间体Int H：3-(苯并[d]噻唑-5-基)-5-甲基吗啉和(3S,5R)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉(Int H峰1)、(3R,5S)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉(Int H峰2)
第一步：5-(苯并[d]噻唑-5-基)-3-甲基-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int H-1)的合成
将化合物Int F(6.00g,13.4mmol)和Int E-2(4.20g,16.1mmol)溶于MeCN(80.0mL)和H2O(20.0mL)中，然后在氮气氛围下依次加入K3PO4(8.54g,40.2mmol)、Pd(t-Bu3P)2(685mg,1.34mmol)，加料完成后，反应在65℃搅拌12hrs。LCMS检测反应完全后直接减压蒸馏浓缩，经过硅胶柱层析(PE/EA＝100/1到5/1,Rf＝0.43)分离纯化，浓缩得到化合物Int H-1(2.06g,6.20mmol)。MS(ESI,m/z):333.2[M+H]+。
第二步：5-(苯并[d]噻唑-5-基)-3-甲基-3,4-二氢-2H-1,4-噁嗪(化合物Int H-2)的合成
将化合物Int H-1(2.06g,6.20mmol)溶于CH2Cl2(10.0mL)中，然后加入TFA(7.68g,67.3mmol,5.00mL)，反应保持在25℃搅拌0.5hr。LCMS检测反应完全后直接浓缩得到化合物Int H-2(1.44g)。MS(ESI,m/z):232.9[M+H]+。
第三步：3-(苯并[d]噻唑-5-基)-5-甲基吗啉(化合物Int H)的合成
将化合物Int H-2(1.44g,6.20mmol)溶于MeOH(10.0mL)中，然后在0℃下加入NaBH4(1.23g,32.5mmol)，加料完成后反应在25℃搅拌0.5hr。LCMS检测原料完全转化，反应液中加入NaHCO3(20.0mL)然后用CH2Cl2(20.0mL x 3)萃取，合并有机相，无水硫酸钠干燥，抽滤浓缩得到化合物Int H(1.25g,4.80mmol)。MS(ESI,m/z):234.9[M+H]+。
第四步：(3S,5R)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉(化合物Int H峰1)和(3R,5S)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉(化合物Int H峰2)的合成
化合物Int H经SFC(分离条件；手性柱:DAICEL CHIRALPAKAD(250mm*30mm,10um)；流动相:[CO2-EtOH(0.1％NH3H2O)]；B％:45％)拆分得到化合物Int H峰1(374mg，Rt＝1.934min)和化合物Int H峰2(423mg,Rt＝2.130min)。MS(ESI,m/z):235.0[M+H]+。
Int H峰1：1H NMR(400MHz,CD3OD)δ9.24(s,1H),8.15(s,1H),8.04(d,J＝8.4Hz,1H),7.56(dd,J＝8.4,1.1Hz,1H),4.13(dd,J＝10.4,3.1Hz,1H),3.88(dd,J＝11.2,3.1Hz,1H),3.81(dd,J＝10.8,2.7Hz,1H),3.35–3.41(m,1H),3.18–3.23(m,1H),3.10–3.14(m,1H),1.08(d,J＝6.4Hz,3H)。
Int H峰2：1H NMR(400MHz,CD3OD)δ9.25(s,1H),8.16(d,J＝0.6Hz,1H),8.05(d,J＝8.3Hz,1H),7.57(dd,J＝8.4,1.5Hz,1H),4.13(dd,J＝10.4,3.2Hz,1H),3.89(dd,J＝11.1,3.3Hz,1H),3.79–3.84(m,1H),3.39(s,1H),3.18–3.25(m,1H),3.09–3.16(m,1H),1.09(d,J＝6.4Hz,3H)。
中间体Int I：(3R,5S)-3-甲基-5-(5-(三氟甲基)吡啶-2-基)吗啉
第一步：5-甲基吗啉-3-酮(化合物Int I-3)的合成
将NaH(120g,3.00mol)缓慢加入到THF(1.00L)中,然后在冰浴下缓慢滴加Int I-1(100g,1.33mol)，在0℃反应30mins后升温至25℃，将Int I-2(204g,1.66mol)加入反应液中，并保持在25℃继续反应16hrs。TLC(PE/EtOAc＝1/1,Rf＝0.15)和LCMS检测反应完全，缓慢加入饱和NH4Cl溶液(1.00L)淬灭，用乙酸乙酯(6.00L)萃取没合并有机相，无水Na2SO4干燥，抽滤，浓缩，得到化合物Int I-3(158g)。MS(ESI,m/z):116.3[M+H]+。
第二步：3-甲基-5-氧代吗啉-4-羧酸叔丁酯(化合物Int I-4)的合成
将化合物Int I-3(158g,1.37mol)溶于THF(1L)中，然后加入DMAP(16.8g,137mmol)和Boc2O(329g,1.51mol)。在25℃反应16hrs，TLC(PE/EtOAc＝5/1,Rf＝0.35)和LCMS检测反应完全，直接减压蒸馏浓缩，经过硅胶柱层析(PE/EtOAc＝1/0到4/1)分离纯化得化合物Int I-4(54.9g)。MS(ESI,m/z):160.0[M-55]+。
1H NMR(400MHz,CDCl3)δ4.31-4.14(m,3H),3.81-3.79(m,2H),1.56(s,9H),1.40(d,J＝6.4Hz,3H)。
第三步：5-((二苯氧基磷酰基)氧基)-3-甲基-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int I-5)的合成
化合物Int I-4(54.9g,255mmol)溶于THF(800mL)中，置换氮气后降温至-70℃，然后保持在-70℃下缓慢滴加LiHMDS(1M,281mL)，滴加完成后保持在-70℃搅拌1hr。然后将Int F-5(71.9g,268mmol)滴加到反应液中，并保持在-60～-30℃搅拌3hrs。TLC(PE/EtOAc＝3/1,Rf＝0.34)检测反应发生，冰浴下加入H2O(200mL)淬灭反应，然后用EtOAc(300mL x 3)萃取，合并有机相，饱和食盐水(600mL)洗涤，然后无水硫酸钠干燥，抽滤，浓缩，经过硅胶柱层析(PE/EtOAc＝1/0到9/1)分离纯化，浓缩得到化合物Int I-5(98.9g)。
第四步：3-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧环戊硼烷-2-基)-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int I-6)的合成
化合物Int I-5(98.9g,221mmol)、B2Pin2(84.2g,332mmol)、KOAc(65.1g,663mmol)、XPhos-Pd-G3(18.7g,22.1mmol)溶于1,4-二氧六环(1000mL)中，置换氮气后，反应温度升高至100℃搅拌2hr。TLC(PE/EtOAc＝5/1,Rf＝0.47)检测反应转化完全，降温至室温后抽滤，滤饼用DCM(1L)洗涤，合并滤液，减压蒸馏浓缩后经过硅胶柱层析(PE/EtOAc＝1/0到9/1)分离纯化，浓缩得到化合物Int I-6(61.5g)。
第五步：3-甲基-5-(5-(三氟甲基)吡啶-2-基)-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物Int I-7)的合成
化合物Int I-6(30.0g,92.3mmol)、Int D-8(20.9g,92.3mmol)、LiCl(7.04g,166mmol)、Na2CO3((2M,415mL)和Pd(PPh3)4(10.7g,9.22mmol)溶于DME(750mL)中，置换氮气后反应温度升高至80℃搅拌12hrs。TLC(PE/EtOAc＝5/1,Rf＝0.33)和LCMS检测到产物生成，降至室温后减压蒸馏浓缩，然后用水(300mL)和CH2Cl2(300mLx3)萃取，有机相用饱和食盐水(300mL)洗涤，无水硫酸钠 干燥，抽滤，浓缩后经硅胶柱层析(PE/EtOAc＝1/0到85/15)分离纯化，浓缩得到化合物Int I-7(11.9g)。MS(ESI,m/z):344.9[M+H]+。
1H NMR(400MHz,CDCl3)δ8.73(s,1H),7.81(dd,J＝8.4,2.0Hz,1H),7.23(d,J＝8.4Hz,1H),6.86(s,1H),4.73-4.69(m,1H),4.16-4.13(m,1H),4.04-4.01(m,1H),1.26(d,J＝7.2Hz,3H),1.18(s,9H)。
第六步：3-甲基-5-(5-(三氟甲基)吡啶-2-基)-3,6-二氢-2H-1,4-噁嗪(化合物Int I-8)的合成
化合物Int I-5(8.40g,24.4mmol)溶于TFA(55.6g,488mmol,36.2mL)中，在25℃反应1hr。LCMS检测反应完全，直接浓缩得到化合物Int I-8(5.96g)。MS(ESI,m/z):244.9[M+H]+。
第七步：(3R,5S)-3-甲基-5-(5-(三氟甲基)吡啶-2-基)吗啉(化合物Int I)的合成
化合物Int I-8(70.0mg,287μmol)溶于THF(5mL)中，然后加入NaBH3CN(72.1mg,1.15mmol)，保持在25℃搅拌5hrs。LCMS检测反应转化完全，加入H2O(40mL)淬灭，用CH2Cl2(600mL)萃取后，合并有机相，用饱和食盐水(400mL)洗涤，无水硫酸钠干燥，抽滤，浓缩，经硅胶柱层析(CH2Cl2/MeOH＝100/0到95/5,含0.1％TFA,Rf＝0.33&0.24)分离纯化，浓缩后经prep-SFC(手性柱:DAICEL CHIRALPAK AD(250mm*30mm,10um)；流动相:[CO2-MeOH]；B％:15％)分离得到化合物Int I(3.67g，Rt＝0.609min)。MS(ESI,m/z):246.9[M+H]+。
1H NMR(400MHz,CDCl3)δ8.92-8.86(m,1H),8.09-8.00(m,1H),7.58-7.48(m,1H),4.53-4.38(m,1H),4.24-4.02(m,2H),3.68-3.42(m,4H),1.50-1.38(m,3H)。
实施例1：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-2-氧代-乙酰胺(化合物1)
第一步：2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-N-(7-((2,4-二甲氧基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-氧代乙酰胺(化合物1-1)的合成
反应瓶中加入化合物Int B(300mg,1.37mmol)、Int E(440mg,1.14mmol)溶于无水DMF(4mL)中，加入DIPEA(110mg,1.71mmol)、HATU(520mg,1.37mmol)，加毕反应体系于25℃反应2hr。反应完成后加水稀释，用乙酸乙酯萃取，用饱和食盐水洗，用无水硫酸钠干燥，过滤，粗品经Prep-TLC(DCM:MeOH＝10:1)分离纯化得化合物1-1(300mg)。MS(ESI,m/z):600.2[M+H]+。
第二步：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-2-氧代-乙酰胺(化合物1)的合成
将化合物1-1(300mg,500μmol)溶于DCM(2mL)中，加入4N HCl的1,4-二氧六环溶液(2mL)中，反应体系在25℃下反应1hr，反应完成后减压浓缩，然后加适量甲醇溶解后，加入几滴NH3.H2O调pH至7-8，减压浓缩，粗品经Prep-HPLC分离纯化得化合物1(266mg)。MS(ESI,m/z):450.1[M+H]+。
1H NMR(400MHz,DMSO-d6)δ10.83–10.66(m,1H),9.42–9.32(m,1H),8.27–8.07(m,2H),7.99–7.72(m,2H),7.64–7.48(m,1H),6.31–6.14(m,2H),5.77–5.28(m,1H),4.32–4.20(m,3H),3.52–3.45(m,1H),2.04–1.68(m,3H),1.47–1.18(m,3H),1.14–1.02(m,3H)。
化合物1经过SFC(碱性条件；柱:DAICEL CHIRALPAK AS(250mm*30mm,10um)；流动相:[CO2-ACN/EtOH(0.1％NH3.H2O)]；B％:60％,等度洗脱模式)。拆分得到化合物1异构体1和化合物1异构体2的混合物1(102mg)以及化合物1异构体3和化合物1异构体4的混合物2(105mg)。
化合物1异构体1和化合物1异构体2的混合物1(102mg,226μmol)再次经过SFC(碱性条件；柱:DAICEL CHIRALCEL OJ(250mm*30mm,10um)；流动相:[CO2-i-PrOH]；B％:40％,等度洗脱模式)分离得到化合物1异构体1(10.1mg，Rt＝1.390min)和化合物1异构体2(53.2mg，Rt＝1.475min)。
化合物1异构体11H NMR(400MHz,CD3OD)δ9.31–9.29(m,1H),8.17–8.10(m,2H),8.04–8.00(m,1H),7.94–7.90(m,1H),7.84–7.78(m,1H),7.65–7.54(m,1H),6.07–5.65(m,1H),4.38–4.30(m,3H),3.88–3.81(m,1H),2.84–2.67(m,2H),2.45–2.09(m,2H),1.89–1.77(m,2H),1.41–1.28(m,2H),0.92–0.82(m,3H)。MS(ESI,m/z):450.2[M+H]+。
化合物1异构体2：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((2R,5S)-2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-2-氧代乙酰胺
1H NMR(400MHz,MeOD)δ9.32–9.23(m,1H),8.17–7.80(m,4H),7.77–7.52(m,2H),5.97–5.50(m,1H),4.43–4.27(m,3H),3.85–3.74(m,2H),3.57–3.48(m,1H),2.46–2.31(m,2H),2.12–1.87(m,3H),1.56–1.48(m,1H),1.24–1.17(m,3H)。MS(ESI,m/z):450.1[M+H]+。
化合物1异构体3和化合物1异构体4的混合物2(105mg,233μmol)再次经过SFC(碱性条件；柱:DAICEL CHIRALCEL OJ-H(250mm*30mm,5um)；流动相:[CO2-i-PrOH(0.1％NH3.H2O)]；B％:30％,等度洗脱模式)分离得到化合物1异构体3(2.39mg，Rt＝1.820min)和化合物1异构体4(46.0mg，Rt＝2.305min)。
化合物1异构体3
1H NMR(400MHz,MeOD)δ9.21–9.12(m,1H),8.06–7.98(m,2H),7.93–7.67(m,2H),7.54–7.41(m,1H),7.30–7.11(m,1H),5.95–5.45(m,1H),4.30–4.19(m,3H),3.73–3.69(m,1H),3.57–3.52(m,1H),3.39–3.34(m,1H),2.72–2.57(m,2H),1.76–1.67(m,2H),1.57–1.44(m,2H),1.32–1.22(m,3H)。MS(ESI,m/z):450.2[M+H]+。
化合物1异构体4
1H NMR(400MHz,MeOD)δ9.31–9.23(m,1H),8.16–7.80(m,4H),7.75–7.50(m,2H),5.96–5.53(m,1H),4.43–4.29(m,3H),3.87–3.74(m,1H),3.56–3.48(m,1H),2.47–2.28(m,2H),2.09–1.89(m,3H),1.57–1.47(m,1H),1.37–1.27(m,1H),1.25–1.15(m,3H)。MS(ESI,m/z):450.1[M+H]+。
实施例2：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(5-甲基-2-(5-(三氟甲基)吡啶-2-基)哌啶-1-基)-2-氧代乙酰胺(化合物2)
第一步：N-(7-((2,4-二甲氧基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(5-甲基-2-(5-(三氟甲基)吡啶-2-基)哌啶-1-基)-2-氧代乙酰胺(化合物2-1)的合成
将化合物Int B(40mg,72.66μmol,70％纯度)、Int D(17.75mg,72.66μmol)溶于无水DMF(1mL)中，加入DIPEA(18.78mg,145.31μmol)、HATU(33.15mg,87.19μmol)，加毕反应体系于25℃反应1hr。反应完成后加水稀释，用乙酸乙酯萃取，用饱和食盐水洗，用无水硫酸钠干燥，过滤，粗品经Prep-TLC(DCM:MeOH＝10:1)分离纯化得化合物2-1(9mg)。MS(ESI,m/z):612.3[M+H]+。
第二步：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(5-甲基-2-(5-(三氟甲基)吡啶-2-基)哌啶-1-基)-2-氧代乙酰胺(化合物2)的合成
将化合物2-1(10mg,16.35μmol)溶于DCM(3mL)中，加入4N HCl的1,4-二氧六环溶液(1mL)中，反应体系在25℃下反应1hr，反应完成后减压浓缩，然后加适量甲醇溶解后，加入几滴TEA调pH至7-8，减压浓缩，粗品经Prep-HPLC分离纯化得化合物2(3mg)。MS(ESI,m/z):462.1[M+H]+。
1H NMR(400MHz,DMSO-d6)δ10.79–10.70(m,1H),9.02–8.98(m,1H),8.30–8.26(m,1H),7.95–7.86(m,1H),7.77–7.57(m,1H),6.27–6.23(m,2H),5.68–5.31(m,1H),4.28–4.24(m,3H),3.54–5.50(m,1H),2.45–2.44(m,1H),2.21–1.86(m,4H),1.64–1.62(m,1H),1.37–1.32(m,1H),1.08–1.06(m,3H)。
以下化合物通过实施例2所述方法和一般步骤制备得到，所需其它原料可通过商业购买，或由有机合成领域有经验的合成人员从商业购买试剂使用常规反应合成获得。










实施例3：N-(4-氨基-1-甲基-1H-吡唑并[4,3-c]吡啶-7-基)-2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基]-2-氧代-乙酰胺
第一步：2-[2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基]-N-(4-((4-甲氧基苯基)甲基)氨基)-1-甲基-1H-吡唑[4,3-c]吡啶-7-基]-2-氧代-乙酰胺(化合物3-1)的合成
将Int C(20mg,42.06μmol)、Int E(9.77mg,42.06μmol)溶于无水DMF(1mL)中，加入HATU(19.19mg,50.47μmol)、DIPEA(8.15mg,63.09μmol)，加毕反应体系于25℃反应1hr。反应完成后加水稀释，用乙酸乙酯萃取，用饱和食盐水洗，用无水硫酸钠干燥，过滤，粗品经Prep-TLC(DCM:MeOH＝10:1)分离纯化得中间体3-1(15mg)。MS(ESI,m/z):690.3[M+H]+。
第二步：N-(4-氨基-1-甲基-1H-吡唑并[4,3-c]吡啶-7-基)-2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基]-2-氧代-乙酰胺(化合物3)的合成
将3-1(10mg,16.14μmol)溶于TFA(2mL)中，升温至90℃下反应2hr，反应完成后减压浓缩，粗品经Prep-HPLC分离纯化得化合物3(8mg)。MS(ESI,m/z):450.1[M+H]+。
1H NMR(400MHz,DMSO-d6)δ10.53–10.48(m,1H),9.43–9.41(m,1H),8.27–8.03(m,3H),7.48–7.47(m,1H),6.92–6.81(m,2H),5.78–5.40(m,1H),4.13–4.08(m,1H),4.04–3.88(m,3H),3.61–3.46(m,1H),2.39–2.13(m,2H),1.99–1.73(m,2H),1.79–1.73(m,1H),1.45–1.38(m,1H),1.14–1.00(m,3H)。
实施例4：N-(4-氨基-1,3-二氢呋喃并[3,4-c]吡啶-7-基)-2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-2-氧代乙酰胺
第一步：(7-(2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-2-氧代乙酰胺基)-1,3-二氢呋喃并[3,4-c]吡啶-4-基)氨基甲酸叔丁酯(化合物4-1)的合成
反应瓶中加入Int E(15mg,64.56μmol)、Int A(20mg,64.56μmol)、HATU(30mg,77.47μmol)及DMF(2mL)，搅拌溶解后再加入DIPEA(13mg,96.84μmol)，氮气保护，25℃反应1hr。待反应结束后，反应液加水稀释，用乙酸乙酯(30ml x3)萃取，合并有机相，用盐水洗涤，无水硫酸钠干燥，过滤，滤液经减压浓缩得到粗产物。粗品经硅胶柱层析纯化(DCM:MeOH＝95:5)，得到化合物4-1(10mg)。MS(ESI,m/z):538.3[M+H]+。
第二步：N-(4-氨基-1,3-二氢呋喃并[3,4-c]吡啶-7-基)-2-(2-(苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-2-氧代乙酰胺(化合物4)的合成
反应瓶中加入4-1(10mg,18.60μmol)、TFA(1.5mL)、DCM(1.5mL)，氮气保护，25℃反应2hr。待反应结束后将反应液旋干，饱和NaHCO3溶液稀释，用乙酸乙酯(30ml x3)萃取，合并有机相，用盐水洗涤，无水硫酸钠干燥，过滤，滤液经减压浓缩得到粗产物。粗品经Pre-HPLC纯化，得到化合物4(4.26mg)。MS(ESI,m/z):438.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ10.62–10.47(m,1H),9.47–9.38(m,1H),8.25–8.15(m,1H),8.09–8.00(m,1H),7.91–7.76(m,1H),7.57–7.41(m,1H),6.07–5.95(m,2H),5.77–5.24(m,1H),4.94–4.67(m,4H),3.47–3.39(m,1H),3.32–3.29(m,1H),2.40–2.31(m,1H),2.22–2.04(m,1H),1.96–1.85(m,1H),1.79–1.67(m,1H),1.44–1.31(m,1H),1.10–1.01(m,3H)。
实施例25：2-(2-(2-(1-乙酰基哌啶-4-基)苯并[d]噻唑-5-基)-5-甲基哌啶-1-基)-N-(7-氨基-1-甲基-1H-)吡唑并[3,4-c]吡啶-4-基)-2-氧代乙酰胺
将化合物25-1(51.0mg,70.4μmol)溶于DCM(5.00mL)中，然后加入DDQ(20.8mg,91.5μmol)。反应在20℃搅拌2小时。LCMS监测反应完全后，抽滤，滤饼用二氯甲烷洗涤两次。合并有机相后浓缩，经过prep-HPLC分离纯化冻干得到化合物25(12.0mg)。MS(ESI,m/z):575.2[M+H]+。
1H NMR(400MHz,CDCl3)δ9.43(s,1H),8.08(s,1H),7.96–7.86(m,3H),7.39–7.33(m,1H),5.35–4.91(m,3H),4.40–4.33(m,3H),4.32–3.94(m,1H),3.47–3.09(m,3H),2.87–2.80(m,1H),2.34–2.03(m,2H),2.14(s,3H),2.05–1.84(m,7H),1.49–1.36(m,2H),1.14(d,J＝6.4Hz,3H)。
实施例35：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((3R,5S)-3-甲基-5-(5-(三氟甲基)吡啶)-2-基)吗啉代)-2-氧代乙酰胺
第一步：N-(7-((3,4-二甲基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((3R,5S)-3-甲基-5-(5-(三氟甲基)吡啶-2-基)吗啉基)-2-氧代乙酰胺(化合物35-1)的合成
将Int B(1.02g,2.65mmol)溶于CH2Cl2(48.0mL)和THF(8.00mL)中，然后加入DMF(38.7mg,529μmol)和(COCl)2(840mg,6.62mmol)，反应保持在20℃搅拌1hr。将Int I(931mg,3.78mmol)溶于CH2Cl2(40.0mL)中，加入DIEA(1.95g,15.1mmol)后加入上述反应液并在25℃搅拌2hrs。TLC(CH2Cl2/MeOH＝15/1,Rf＝0.47)和LCMS检测到产物生成，浓缩后经硅胶柱层析(CH2Cl2/MeOH＝100/0到90/10)纯化得到化合物35-1(1.11g)。MS(ESI,m/z):614.1[M+H]+。
第二步：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((3R,5S)-3-甲基-5-(5-(三氟甲基)吡啶)-2-基)吗啉代)-2-氧代乙酰胺(化合物35)的合成
化合物35-1(1.11g,1.81mmol)溶于4M HCl的1,4-二氧六环溶液(13.6mL)中，在25℃反应1hr后，LCMS检测转化完全，用饱和NaHCO3溶液将pH调至8左右，然后用DCM(120mL)萃取，合并有机相，饱和食盐水(50.0mL)洗涤，无水硫酸钠干燥，抽滤，浓缩，经硅胶柱层析(CH2Cl2/MeOH＝1/0到19/1)分离纯化后，再经过prep-SFC(手性柱:DAICEL CHIRALPAK IK(250mm*30mm,10um)；流动相:[CO2-EtOH(0.1％NH3H2O)]；B％:30％)拆分得化合物35(284mg，Rt＝1.763min)。MS(ESI,m/z):464.1[M+H]+。
1H NMR(400MHz,CDCl3)δ9.42–9.33(m,1H),8.92–8.83(m,1H),8.13–7.89(m,3H),7.73–7.55(m,1H),6.59–5.76(m,1H),5.23–4.86(m,3H),4.46–4.37(m,4H),4.10–3.92(m,1H),3.89–3.80(m,2H),1.14–0.80(m,3H)。
实施例46：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(3-甲基-5-(6-(三氟甲基)吡啶-3-基)吗啉代)-2-氧代乙酰胺
第一步：3-甲基-5-(6-(三氟甲基)吡啶-3-基)-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物46-2)的合成
将化合物Int F(700mg,1.56mmol)和化合物46-1(427mg,1.56mmol)溶于1,4-二氧六环(6mL)和H2O(2mL)的混合溶剂中，然后在氮气氛围下依次加入Pd(PPh3)4(180mg,156μmol)和K3PO4(996mg,4.69mmol)。反应保持在90℃搅拌2hrs。LCMS检测到产物生成。反应液浓缩后，经硅胶柱层析(PE/EtOAc＝10/1)分离纯化，浓缩得到化合物46-2(506mg)。MS(ESI,m/z):345.0[M+H]+。
第二步：3-甲基-5-(6-(三氟甲基)吡啶-3-基)-3,6-二氢-2H-1,4-噁嗪(化合物46-3)的合成
将化合物46-2(510mg,1.49mmol)溶于CH2Cl2(5mL)中，然后加入2M HCl的1,4-二氧六环溶液(2mL)，反应保持在25℃搅拌12hrs。LCMS检测反应完全，在25℃下减压蒸馏浓缩得到化合物46-3(361mg)。
第三步：3-甲基-5-(6-(三氟甲基)吡啶-3-基)吗啉(化合物46-4)的合成
将化合物46-3(350mg,1.43mmol)溶于THF(5mL)中，然后在0℃下加入NaBH3CN(360mg,5.73mmol)，加料完成后反应保持在25℃搅拌0.5hr。LCMS检测原料完全转化，减压蒸馏浓缩后得到化合物46-4(160mg)。MS(ESI,m/z):247.0[M+H]+。
第四步：2-(3-甲基-5-(6-(三氟甲基)吡啶-3-基)吗啉代)-2-氧代乙酸乙酯(化合物46-5)的合成
将化合物46-4(150mg,609μmol)溶于CH2Cl2(1mL)中，然后在0℃下加入DIEA(393mg,3.05mmol,530μL)和草酰氯单乙酯(249mg,1.83mmol,204μL)，反应保持在25℃搅拌0.5hr。LCMS和TLC(CH2Cl2/MeOH＝10/1,Rf＝0.45)检测反应完全。反应液浓缩后经过硅胶柱层析(CH2Cl2/MeOH＝100/1到10/1,Rf＝0.45)分离纯化，浓缩得到化合物46-5(210mg)。MS(ESI,m/z):347.0[M+H]+。
第五步：2-(3-甲基-5-(6-(三氟甲基)吡啶-3-基)吗啉代)-2-氧代乙酸(化合物46-6)的合成
将化合物46-5(210mg,594μmol)溶于THF(3mL)和H2O(1mL)的混合溶剂中，然后加入LiOH·H2O(49.8mg,1.19mmol)，反应在25℃搅拌1hr。LCMS检测反应完全后用1M的盐酸水溶液将pH调至2-3，然后用CH2Cl2(20mL x 3)萃取，有机相经过无水Na2SO4干燥后，抽滤浓缩得到化合物46-6(163mg)。MS(ESI,m/z):319.0[M+H]+。
第六步：N-(7-((2,4-二甲基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(3-甲基-5-(6-(三氟甲基)吡啶-3-基)吗啉代)-2-氧代乙酰胺(化合物46-7)的合成
将化合物46-6(68.0mg,207μmol)和化合物Int B-5(50.0mg,159μmol)溶于CH2Cl2(2mL)中，然后依次加入EDCI(45.8mg,239μmol)、HOBt(32.3mg,239μmol)和DIEA(61.8mg,478μmol,83.3μL)。反应在25℃搅拌1hr。LCMS检测反应完全，反应液中加入H2O(20mL)淬灭后，用CH2Cl2(20mL x 3)萃取，收集有机相，然后用无水Na2SO4干燥，抽滤，浓缩，经硅胶柱层析(CH2Cl2/MeOH＝100/1到10/1,Rf＝0.40)分离纯化得到化合物46-7(43.0mg)。MS(ESI,m/z):614.1[M+H]+。
第七步：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(3-甲基-5-(6-(三氟甲基)吡啶-3-基)吗啉代)-2-氧代乙酰胺(化合物46)的合成
将化合物46-6(43.0mg,70.0μmol)溶于CH2Cl2(1mL)中，然后加入2M HCl的1,4-二氧六环溶液(2mL)，反应保持在25℃搅拌2hrs。LCMS检测反应完全后减压蒸馏浓缩，经prep-HPLC分离纯化，冻干得到化合物46(4.86mg)。MS(ESI,m/z):463.9[M+H]+。
1H NMR(400MHz,CD3OD)δ8.99(s,1H),8.27–8.35(m,1H),7.99(s,1H),7.78–7.92(m,2H),5.63–5.83(m,1H),4.79(s,1H),4.36(s,3H),4.18(d,J＝1.0Hz,1H),3.84–3.97(m,3H),1.02(s,3H)。
实施例47：(±)-N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(顺式-3-甲基-5-(4-(三氟甲基)苯基))吗啉代)-2-氧代乙酰胺(化合物47)和(±)-N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(反式-3-甲基-5-(4-(三氟甲基)苯基))吗啉代)-2-氧代乙酰胺(化合物48)
第一步：3-甲基-5-(4-(三氟甲基)苯基)-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物47-2)的合成
将化合物Int F(2.00g,4.47mmol)和化合物47-1(1.22g,4.47mmol))溶于1,4-二氧六环(12mL)和H2O(4.00mL)的混合溶剂中，然后在氮气氛围下依次加入Pd(PPh3)4(516mg,447μmol)和K3PO4(2.85g,13.4mmol)。反应保持在100℃搅拌12hrs。TLC(PE/EtOAc＝10/1,Rf＝0.29)检测到产物生成。反应液浓缩后，经硅胶柱层析(PE/EtOAc＝10/1,Rf＝0.29)分离纯化，浓缩得到化合物47-2(1.50g)。MS(ESI,m/z):287.8[M+H-tBu]+。
第二步：3-甲基-5-(4-(三氟甲基)苯基)-3,6-二氢-2H-1,4-噁嗪(化合物47-3)的合成
将化合物47-2(1.50g,4.37mmol)溶于CH2Cl2(20mL)中，然后加入2M HCl的1,4-二氧六环溶液(10mL)，反应保持在25℃搅拌1hrs。LCMS检测反应完全，在25℃下减压蒸馏浓缩得到化合物46-3(361mg)。MS(ESI,m/z):262.0[M+18]+。
第三步：3-甲基-5-(4-(三氟甲基)苯基)吗啉(化合物47-4)的合成
将化合物46-3(1.00g,4.11mmol)溶于MeOH(2.00mL)中，然后在0℃下加入NaBH4(516mg,8.22mmol)，加料完成后反应保持在25℃搅拌0.15hr。LCMS检测原料完全转化，反应液中NaHCO3(20.0mL)然后用CH2Cl2(20.0mL x 3)萃取，合并有机相后，加入无水硫酸钠干燥，抽滤，浓缩经过硅胶柱层析(CH2Cl2/MeOH＝10/1,Rf＝0.32)分离纯化，浓缩得到化合物47-7(270mg)。MS(ESI,m/z):245.9[M+H]+。
第四步：2-(3-甲基-5-(4-(三氟甲基)苯基)吗啉代)-2-氧代乙酸乙酯(化合物47-5)的合成
将化合物47-4(270mg,671μmol)溶于CH2Cl2(0.5mL)中，然后在0℃下加入DIEA(433mg,3.36mmol,584μL)和草酰氯单乙酯(275mg,2.01mmol,225μL)，反应保持在25℃搅拌0.5hr。LCMS和TLC(CH2Cl2/MeOH＝10/1,Rf＝0.45)检测反应完全。反应液浓缩后经过硅胶柱层析(CH2Cl2/MeOH＝100/1到10/1,Rf＝0.45)分离纯化，浓缩得到化合物47-5(380mg)。MS(ESI,m/z):346.0[M+H]+。
第五步：2-(3-甲基-5-(4-(三氟甲基)苯基)吗啉代)-2-氧代乙酸(化合物47-6)的合成
将化合物47-5(380mg,550μmol)溶于THF(3mL)和H2O(1mL)的混合溶剂中，然后加入LiOH·H2O(46.1mg,1.10mmol)，反应在25℃搅拌1hr。LCMS检测反应完全后用1M的盐酸水溶液将pH调至2-3，然后用CH2Cl2(20.0mL x 3)萃取，有机相经过无水Na2SO4干燥后，抽滤，浓缩得到化合物47-6(349mg)。MS(ESI,m/z):317.8[M+H]+。
第六步：N-(7-((2,4-二甲基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(3-甲基-5-(4-(三氟甲基)苯基)吗啉基)-2-氧代乙酰胺(化合物47-7)的合成
将化合物47-6(87.0mg,191μmol)和化合物Int B-5(50.0mg,159μmol)溶于CH2Cl2(2mL)中，然后依次加入EDCI(45.8mg,239μmol)、HOBt(32.3mg,239μmol)和DIEA(61.8mg,478μmol,83.3μL)。反应在25℃搅拌1hr。LCMS检测反应完全，反应液中加入H2O(20mL)淬灭后，用CH2Cl2(20.0mL x 3)萃取，收集有机相，然后用无水Na2SO4干燥，抽滤，浓缩，经硅胶柱层析(CH2Cl2/MeOH＝100/1到10/1,Rf＝0.45)分离纯化得到化合物47-7(65.0mg)。MS(ESI,m/z):613.0[M+H]+。
第七步：(±)-N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(顺式-3-甲基-5-(4-(三氟甲基)苯基))吗啉代)-2-氧代乙酰胺(化合物47)和(±)-N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(反式-3-甲基-5-(4-(三氟甲基)苯基))吗啉代)-2-氧代乙酰胺(化合物48)的合成
将化合物47-6(65.0mg,50.9μmol)溶于CH2Cl2(4mL)中，然后加入2M HCl的1,4-二氧六环溶液(2mL)，反应保持在25℃搅拌2hrs。LCMS检测反应完全后减压蒸馏浓缩，经prep-HPLC分离纯化，冻干得到化合物(±)47(3.83mg)(Rt＝1.466min)和化合物(±)48(3.56mg)(Rt＝1.246min)。MS(ESI,m/z):462.9[M+H]+。
化合物(±)47：1H NMR(400MHz,CD3OD)δ8.39–8.49(m,1H),7.43–8.02(m,6H),5.68(d,J＝1.2Hz,1H),4.79(s,1H),4.58(s,1H),4.35(s,3H),3.83–4.13(m,3H),1.00(d,J＝5.6Hz,3H)。
化合物(±)48：1H NMR(400MHz,CD3OD)δ8.29–8.44(m,1H),7.73–7.90(m,1H),7.34–7.70(m,5H),5.17–5.23(m,1H),4.55–4.57(m,1H),4.27–4.36(m,3H),3.91–4.00(m,2H),3.68–3.84(m,1H),3.50–3.64(m,1H),1.43–1.59(m,3H)。
以下化合物通过实施例47所述方法和一般步骤制备得到，所需其它原料可通过商业购买，或由有机合成领域有经验的合成人员从商业购买试剂使用常规反应合成获得。

实施例49：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((2R,5S)-5-甲基-2-(2-(1-甲基哌啶)-4-基)苯并[d]噻唑-5-基)哌啶-1-基)-2-氧代乙酰胺
第一步：N-(7-((2,4-二甲基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((2R,5S)-5-甲基-2-(2-(1-甲基哌啶-4-基)苯并[d]噻唑-5-基)哌啶-1-基)-2-氧代乙酰胺(化合物49-2)的合成
将化合物49-1(90.0mg,273.14μmol)和Int B(105mg,273μmol)溶于DMF(5mL)中，然后加入DIEA(106mg,819μmol,143μL)和HATU(156mg,410μmol)，反应保持在25℃搅拌1hr。LCMS检测反应完全，然后加入H2O(30.0mL)淬灭反应EtOAc(30mL x 3)萃取，合并有机相，无水硫酸钠干燥，抽滤浓缩得到化合物49-2(150mg)。MS(ESI,m/z):697.2[M+H]+。
第二步：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((2R,5S)-5-甲基-2-(2-(1-甲基哌啶)-4-基)苯并[d]噻唑-5-基)哌啶-1-基)-2-氧代乙酰胺(化合物49)的合成
将化合物49-2(150mg,215μmol)溶于TFA(5mL)中，然后升温至80℃搅拌0.5hr，LCMS检测反应完全。反应液中加入饱和NaHCO3(50mL)然后用CH2Cl2(50mL x 3)萃取，合并有机相，无水硫酸钠干燥，抽滤浓缩，然后经过prep-HPLC分离纯化，冻干后得到化合物49(43.57mg)。MS(ESI,m/z):547.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ10.65–10.89(m,1H),8.04–8.13(m,1H),7.95–8.01(m,1H),7.92(br s,2H),7.36–7.52(m,1H),6.16–6.29(m,2H),5.22–5.81(m,1H),4.20–4.37(m,3H),3.42–3.50(m,2H),2.81–3.14(m,4H),2.20(s,3H),1.98–2.15(m,5H),1.71–1.94(m,4H),1.33–1.44(m,1H),1.08(d,J＝7.1Hz,3H)。
实施例50：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)吗啉代)-2-氧代乙酰胺
第一步：3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)-2,3-二氢-4H-1,4-噁嗪-4-羧酸叔丁酯(化合物50-2)的合成
将化合物Int G(699mg,2.15mmol)和化合物50-1(400mg,1.65mmol)溶于1,4-二氧六环(30mL)和H2O(10mL)的混合溶剂中，然后在氮气氛围下依次加入XPhos-Pd-G3(140mg,165μmol)和K3PO4(1.05g,4.96mmol)。反应保持在80℃搅拌12hrs。LCMS检测到产物生成。反应液浓缩后，经硅胶柱层析(PE/EtOAc＝100/0到92/8,Rf＝0.49)分离纯化，浓缩得到化合物50-2(358mg)。MS(ESI,m/z):360.9[M+H]+。
第二步：3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)-3,6-二氢-2H-1,4-噁嗪(化合物50-3)的合成
将化合物50-2(318mg,883μmol)溶于CH2Cl2(1mL)中，然后加入2M HCl的1,4-二氧六环溶液(1mL)，反应保持在25℃搅拌12hrs。LCMS检测反应完全，在25℃下减压蒸馏浓缩得到化合物46-3(229mg)。
第三步：3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)吗啉(化合物50-4)的合成
将化合物50-3(229mg,880μmol)溶于MeOH(5mL)和THF(20mL)中，然后在0℃下加入NaBH3CN(221mg,3.52mmol)，加料完成后反应保持在25℃搅拌1.5hr。LCMS检测原料完全转化，反应液中加入H2O(10mL),然后CH2Cl2(20mL x 3)萃取，合并有机相，无水Na2SO4干燥，抽滤，浓缩，经过硅胶柱层析(CH2Cl2/MeOH＝100/0到85/15,Rf＝0.35)分离纯化，浓缩得到化合物50-4(158mg)。MS(ESI,m/z):262.9[M+H]+。
第四步：2-(3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)吗啉代)-2-氧代乙酸乙酯(化合物50-5)的合成
将化合物50-4(158mg,603μmol)溶于CH2Cl2(10mL)中，然后在0℃下加入DIEA(311mg,2.41mmol,420μL)和草酰氯单乙酯(123mg,904μmol,101μL)，反应保持在25℃搅拌1hr。LCMS检测反应完全。反应液浓缩后经过硅胶柱层析(CH2Cl2/MeOH＝100/0到90/10,Rf＝0.65)分离纯化，浓缩得到化合物50-5(193mg)。MS(ESI,m/z):363.0[M+H]+。
第五步：2-(3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)吗啉代)-2-氧代乙酸(化合物50-6)的合成
将化合物50-5(193mg,533μmol)溶于THF(12mL)和H2O(4mL)的混合溶剂中，然后加入LiOH·H2O(44.7mg,1.07mmol)，反应在25℃搅拌1hr。LCMS检测反应完全后用1M的盐酸水溶液将pH调至2-3，然后用CH2Cl2(20mL x 3)萃取，有机相经过无水Na2SO4干燥后，抽滤浓缩得到化合物50-6(186mg)。MS(ESI,m/z):335.1[M+H]+。
第六步：N-(7-((2,4-二甲基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)吗啉代)-2-氧代乙酰胺(化合物50-7)的合成
将化合物50-6(186mg,556μmol)和化合物Int B-5(174mg,556μmol)、HATU(423mg,1.11mmol)溶于DMF(2mL)中，然后加入DIEA(216mg,1.67mmol,291μL)。反应在25℃搅拌1hr。LCMS检测反应完全，反应液中加入H2O(20mL)淬灭后，用EA(20mL x 3)萃取，收集有机相，然后用无水Na2SO4干燥，抽滤，浓缩，经硅胶柱层析(CH2Cl2/MeOH＝100/0到86/14,Rf＝0.37)分离纯化得到化合物50-7(239mg)。MS(ESI,m/z):630.2[M+H]+。
第七步：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-(3-甲基-5-(5-(三氟甲氧基)吡啶-2-基)吗啉代)-2-氧代乙酰胺(化合物50)的合成
将化合物50-6(239mg,380μmol)溶于CH2Cl2(3mL)中，然后加入2M HCl的1,4-二氧六环溶液(1mL)，反应保持在25℃搅拌2hrs。LCMS检测反应完全后减压蒸馏浓缩，经prep-HPLC分离纯化，冻干得到化合物50(96.83mg)。MS(ESI,m/z):479.9[M+H]+。
1H NMR(400MHz,CDCl3)δ9.32–9.47(m,1H),8.31–8.61(m,1H),7.82–8.19(m,2H),7.45–7.68(m,2H),5.65–6.48(m,2H),4.76–5.21(m,1H),4.40(br s,3H),3.75–4.09(m,3H),0.76–1.14(m,3H)。
实施例51：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((3S,5R)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉代)-2-氧代乙酰胺
第一步：2-((3S,5R)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉代)-2-氧代乙酸乙酯(化合物51-1)的合成
将化合物Int H峰1(364mg,1.46mmol)溶于CH2Cl2(5mL)中，然后在0℃下加入DIEA(943mg,7.30mmol,1.27mL)和草酰氯单乙酯(598mg,4.38mmol,489μL)，反应保持在25℃搅拌1hr。LCMS检测反应完全。反应液浓缩后经过硅胶柱层析(CH2Cl2/MeOH＝10/1,Rf＝0.50)分离纯化，浓缩得到化合物51-1(495mg)。MS(ESI,m/z):335.0[M+H]+。
第二步：2-((3S,5R)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉代)-2-氧代乙酸(化合物51-2)的合成
将化合物51-1(495mg,1.20mmol)溶于THF(3mL)和H2O(1mL)的混合溶剂中，然后加入LiOH·H2O(100mg,2.40mmol)，反应在25℃搅拌1hr。LCMS检测反应完全后用1M的盐酸水溶液将pH调至2-3，然后用CH2Cl2(20mL x 3)萃取，有机相经过无水Na2SO4干燥后，抽滤浓缩得到化合物51-2(367mg)。MS(ESI,m/z):307.0[M+H]+。
第三步：2-((3S,5R)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉代)-N-(7-((2,4-二甲基苄基)氨基)-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-氧代乙酰胺(化合物51-3)的合成
将化合物51-2(333mg,1.08mmol)和化合物Int B-5(260mg,829μmol)溶于CH2Cl2(4mL)中，然后依次加入EDCI(238mg,1.24mmol)、HOBt(168mg,1.24mmol)和DIEA(321mg,2.49mmol,433μL)。反应在25℃搅拌1hr。LCMS检测反应完全，直接浓缩，经硅胶柱层析(CH2Cl2/MeOH＝10/1,Rf＝0.54)分离纯化得到化合物51-3(400mg)。MS(ESI,m/z):602.0[M+H]+。
第四步：N-(7-氨基-1-甲基-1H-吡唑并[3,4-c]吡啶-4-基)-2-((3S,5R)-3-(苯并[d]噻唑-5-基)-5-甲基吗啉代)-2-氧代乙酰胺(化合物51)的合成
将化合物51-3(400mg,602μmol)溶于CH2Cl2(10mL)中，然后加入2M HCl的1,4-二氧六环溶液(5mL)，反应保持在25℃搅拌1hr。LCMS检测反应完全后减压蒸馏浓缩，经prep-HPLC分离纯化，冻干得到化合物51(163mg)。MS(ESI,m/z):452.0[M+H]+。
1H NMR(400MHz,CD3OD)δ9.26(s,1H),8.42(s,1H),8.08(d,J＝8.4Hz,1H),7.75–8.02(m,3H),5.76–5.84(m,1H),4.34(s,3H),3.81–4.03(m,5H),1.00(d,J＝5.6Hz,3H)。
以下化合物通过实施例51所述方法和一般步骤制备得到，所需其它原料可通过商业购买，或由有机合成领域有经验的合成人员从商业购买试剂使用常规反应合成获得。

分离方法
实施例中化合物的Prep-HPLC纯化均采用Aglient 1260型或Waters 2489型HPLC进行，分离柱型号为Waters SunFire Prep C18OBD(19mm×150mm×5.0μm)、Waters Xbridge Prep C18OBD(19mm×150mm×5.0μm)或YMC Actus Triart C18(20mm×150mm×5.0μm)，柱温均为25℃，检测波长为214nm、254nm或280nm，流动相A为乙腈，流动相B为0.05％甲酸水溶液或者0.05％碳酸氢铵水溶液或者0.05％TFA水溶液，流动相的体积比根据化合物的极性不同而进行调节；流动相流速为28mL/min。
生物学评价
实验例1：测试化合物对PRMT5-MTA甲基转移酶活性的抑制作用
将配置好的蛋白溶液(PRMT5/MEP50和MTA混合液)分别与不同浓度的测试化合物(500nM起始，5倍稀释，7个点；或100nM起始，5倍稀释，7个点)于25℃预孵育30min后，加入配置好的底物溶液(生物素化组蛋白H4肽(Biotinylated histone H4 peptide))，于25℃孵育反应90min；反应结束后，加入配好的检测试剂混合液(Protein A-Eu、抗组蛋白H4抗体(Anti-Histone H4 antibody)和Streptavidin-D2)，于25℃孵育60min，使用BMG酶标仪检测荧光信号比值(Ratio)。
以溶剂组(DMSO)为阴性对照、反应缓冲液组(不含PRMT5·MTA酶)为空白对照，按照下式计算不同浓度化合物的百分比抑制率：
百分比抑制率＝(阴性对照Ratio-化合物Ratio)/(阴性对照Ratio-空白对照Ratio)×100％。
使用GraphPad Prism 8中“log(inhibitor)vs.response--Variable slope”四参数方程Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*Hillslope))对检测信号值进行拟合，计算IC50值。其中Y为相对抑制活性百分比，Top和Bottom分别为拟合曲线的最大值与最小值，X为化合物的对数浓度，Hillslope为曲线斜率。按照上述方法测定化合物对PRMT5-MTA甲基转移酶的抑制作用，结果如表1中所示。
表1.本发明的化合物对PRMT5-MTA的抑制活性
实验结果表明，本发明化合物对PRMT5-MTA酶均有较强的抑制作用。
实验例2：化合物对MTAP缺失(Deleted)/亲代(Parental)HCT116细胞增殖抑制试验
本实施例中选用了MTAP缺失/亲代HCT116细胞，购自HORIZON。
将MTAP缺失/亲代HCT116细胞体外单层培养，培养条件为10％FBS+1％P/S的RPMI6140培养基，37℃,5％CO2。消化对数生长期细胞并调整浓度，每孔250个接种于96孔板培养过夜，加入预先稀释的化合物(5000nM起始，4倍稀释，9个点)，阴性对照组加DMSO，空白对照组加培养 基，于37℃，5％CO2孵箱中培养8天后，每孔加入50μl CellTiter-Glo室温避光裂解10分钟,将待测液转至96孔不透光白板中，然后在酶标仪化学发光检测模式下读取相对化学发光单位值。
按照下式计算不同浓度化合物的百分比抑制率：
百分比抑制率＝(1-(测试化合物的化学发光信号值-空白对照的化学发光信号值)/(阴性对照的化学发光信号值-空白对照的化学发光信号值))×100％。
将不同浓度化合物的百分比抑制率相对于化合物浓度作图，按照四参数模型拟合曲线，通过下式计算IC50值：
y＝Min+(Max-Min)/(1+(x/IC50)^(-Hillslope))，其中：y为百分比抑制率；Max和Min分别为拟合曲线的最大值与最小值；x为化合物的对数浓度；且Hillslope为曲线斜率。
按照上述方法测定化合物对MTAP缺失/亲代HCT116细胞的增殖抑制活性，结果如表2所示。
表2.本发明的化合物对MTAP缺失/亲代HCT116细胞的增殖抑制活性

实验结果表明，本发明化合物对MTAP缺失的HCT116细胞有较强的抑制作用，且相对于MTAP亲代的HCT116细胞具有一定的选择性。
实验例3：化合物在balb/c小鼠体内药代动力学测试
通过灌胃(PO)向雌性Balb/c小鼠给予本发明的化合物，考察药代动力学特点。
将本发明的化合物和TNG462(根据专利WO2022026892A1制备)进行静脉(1mg/kg)和灌胃(10mg/kg)给药。IV溶媒为：5％DMSO+5％Solutol+90％Saline，PO溶媒为：10％Solutol+90％H2O。于给药前0h及给药后0.083、0.25、0.5、1、2、4、6、8、10和24h(静脉)，给药前0h及给药后0.25、0.5、1、2、4、6、8、10和24h(灌胃)，经眼眶取血40-50μl，置K2-EDTA抗凝试管 中(TNG462样本于湿冰上暂存)，4000rpm离心10min(4℃)，分离血浆(TNG462的血浆管中预先加入1μl 30％甲酸溶液)，-80℃保存待测。取10μl血浆样品加入90μl含内标的乙腈，振摇混匀后，于4000rpm，4℃下离心10min，取上清液进行LC-MS/MS分析。应用WinNonlin 6.3软件，采用非房室模型计算药代动力学参数，结果见表3。
表3：化合物在小鼠体内的药代动力学参数
TNG462结构：
结论：
以上实验结果表明，本发明的化合物体内清除速率低、血药浓度和体内暴露量高，生物利用度高，具有良好的口服吸收效果。
除本文中描述的那些外，根据前述描述，本发明的多种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (28)

  1. 式I的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药：
    其中：
    代表单键或双键；
    X1、X2和X3各自独立地选自-CH2-、-CH-、-O-、-N-、-NH-和-S-；
    R1在每次出现时各自独立地选自H、卤素、C1-6烷基、C1-6卤代烷基、C1-6羟烷基和C3-8环烷基；
    环A为5-7元饱和或部分饱和杂环，所述杂环包含至少一个N原子以及任选存在的1或2个各自独立地选自O或S的相同或不同的其它杂原子；
    R2在每次出现时各自独立地选自H、-OH、卤素、-CN、-NR4R5、-COCH3、-NHCOCH3、C1-6烷基、C1-6烷氧基、C1-6羟烷基、C2-6杂烷基、C3-8环烷基、3-8元杂环基、C3-8环烷氧基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、羟烷基、杂烷基、环烷基、杂环基、环烷氧基、芳基或杂芳基各自任选地被一个或多个R3取代；
    R3在每次出现时各自独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C2-6杂烷基、C3-6环烷基、C3-8环烷氧基、3-8元杂环基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、杂烷基、环烷基、环烷氧基、杂环基、芳基或杂芳基各自任选地被一个或多个各自独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C3-6环烷基、C3-8环烷氧基和3-8元杂环基的取代基取代；
    R4、R5、R6和R7各自独立地选自H和C1-6烷基；或者
    R4和R5、R6和R7连同其所连接的氮原子共同形成3-8元杂环基，所述杂环基任选地被一个或多个各自独立地选自卤素、OH、CN、-NH2和C1-6烷基的取代基取代；
    m为0、1、2或3；
    n为0、1、2、3或4。
  2. 权利要求1的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    X1、X2和X3各自独立地选自-CH2-、-CH-、-O-、-N-和-NH-；
    优选地，X1为-CH-，且X2和X3为-N-；或者
    优选地，X1和X2为-N-，且X3为-CH-；或者
    优选地，X1和X3为-CH2-，且X2为-O-。
  3. 权利要求1或2的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    R1在每次出现时各自独立地选自H、C1-6烷基、C1-6卤代烷基和C3-8环烷基；
    优选地，R1在每次出现时各自独立地选自H和C1-6烷基；
    优选地，R1为甲基。
  4. 权利要求1-3中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    环A为5-7元饱和杂环，所述杂环包含至少一个N原子以及任选存在的1或2个各自独立地选自O或S的相同或不同的其它杂原子；
    优选地，环A为5-7元饱和氮杂环和5-7元饱和含N和O杂环；
    优选地，环A为6元饱和氮杂环和6元饱和含N和O杂环；
    优选地，环A为哌啶环和吗啉环。
  5. 权利要求1-4中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    R2在每次出现时各自独立地选自H、卤素、C1-6烷基、C1-6烷氧基、C1-6羟烷基、C2-6杂烷基、C3-8环烷基、3-8元杂环基、C3-8环烷氧基、C6-10芳基和5-10元杂芳基，所述烷基、烷氧基、羟烷基、杂烷基、环烷基、杂环基、环烷氧基、芳基或杂芳基各自任选地被一个或多个R3取代；
    优选地，R2在每次出现时各自独立地选自H、卤素、C1-6烷基、C6-10芳基和5-10元杂芳基，所述烷基、芳基、杂芳基各自任选地被一个或多个R3取代；
    优选地，R2在每次出现时各自独立地选自C1-6烷基、C6-10芳基和6-9元杂芳基，所述烷基、芳基或杂芳基各自任选地被一个或多个R3取代；
    优选地，R2在每次出现时各自独立地选自C1-6烷基、苯基、吡啶基和苯并噻唑基，所述苯基、吡啶基或苯并噻唑基各自任选地被一个或多个R3取代；
    优选地，R2在每次出现时各自独立地选自甲基、 其中波浪线表示所述基团与分子其余部分的连接点。
  6. 权利要求1-5中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    R3在每次出现时各自独立地选自卤素、-CN、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、3-8元杂环基、C6-10芳基和5-6元杂芳基，所述烷基、杂烷基、杂环基、芳基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C1-6卤代烷氧基、C3-6环烷基、C3-8环烷氧基和3-8元杂环基的取代基取代；
    优选地，R3在每次出现时各自独立地选自卤素、-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、C2-6杂烷基、4-7元杂环基、苯基和5-6元杂芳基，所述烷基、杂烷基、杂环基、苯基或杂芳基各自任选地被一个或多个独立地选自卤素、-OH、-CN、-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代；
    优选地，R3在每次出现时各自独立地选自-NR4R5、C1-6烷基、C1-6卤代烷基、C1-6卤代烷氧基、4-7元杂环基和5-6元杂芳基，所述烷基、杂环基、杂芳基任选地被一个或多个-NR4R5、-COCH3、C1-6烷基、C1-6卤代烷基、C3-6环烷基和4-6元杂环基的取代基取代；
    优选地，R3在每次出现时各自独立地选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、 其中波浪线表示所述基团与分子其余部分的连接点。
  7. 权利要求1-6中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    R6和R7各自独立地选自H和甲基；
    优选地，R6和R7为H。
  8. 权利要求1-7中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    m为0或1。
  9. 权利要求1-8中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    n为2。
  10. 权利要求1-9中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式II的化合物：
  11. 权利要求10的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    X1为-CH-，且X2和X3为-N-；
    R1为甲基；且
    m为1。
  12. 权利要求1-9中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式III的化合物：
  13. 权利要求12的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    X1为-CH-，且X2和X3为-N-；
    R1为甲基；
    m为1；且
    R3选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、 其中波浪线表示所述基团与分子其余部分的连接点。
  14. 权利要求1-9中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式IV的化合物：
  15. 权利要求14的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中，
    X1为-CH-，且X2和X3为-N-；
    R1为甲基；
    m为1；且
    R3选自甲基、三氟甲基、乙基、氨基、甲氨基、三氟甲氧基、 其中波浪线表示所述基团与分子其余部分的连接点。
  16. 权利要求1-9中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式V的化合物：
    其中，
    Y1、Y2各自独立地选自-CH-和-N-。
  17. 权利要求16的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式V-1的化合物：
  18. 权利要求17的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式V-1a的化合物：
  19. 权利要求17的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式V-1b的化合物：
  20. 权利要求17的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式V-1c的化合物：
  21. 权利要求1-9中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式VI的化合物：
    其中，
    p为0或1。
  22. 权利要求21的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式VI-1的化合物：
  23. 权利要求22的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式VI-1a的化合物：
  24. 权利要求22的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物为式VI-1b的化合物：
  25. 权利要求1-24中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药，其中所述化合物选自：


  26. 药物组合物，其包含预防或治疗有效量的权利要求1-25中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药以及一种或多种药学上可接受的载体。
  27. 权利要求1-25中任一项的化合物或其药学上可接受的盐、立体异构体、互变异构体、同位素标记的化合物、多晶型物、溶剂化物、N-氧化物、代谢物或前药或者权利要求26的药物组合物在制备用于预防或治疗与PRMT5活性相关的疾病或病况的药物中的用途，
    优选地，所述与PRMT5活性相关的疾病或病况优选为MTAP缺失的癌症或肿瘤；
    优选地，所述癌症或肿瘤为食管癌、肺癌、胰腺癌、胶质母细胞瘤、胆管癌、膀胱癌、乳腺癌、卵巢癌、肝细胞癌、前列腺癌、黑色素瘤、胃癌、结肠癌、白血病(特别是慢性B淋巴细胞白血病)或淋巴瘤。
  28. 权利要求1-25中任一项的化合物的制备方法，其包括以下步骤：
    第一步：化合物I-A-1与酰氯经缩合反应生成化合物I-A-2；
    第二步：化合物I-A-2经水解反应生成化合物I-A-3；
    第三步：化合物I-A-3和化合物I-A-4经缩合反应生成化合物I-A-5；
    第四步：缩合产物I-A-5脱除保护基后生成化合物I；
    其中
    PG为氨基保护基，优选为对甲氧基苄基或2,4-二甲氧基苄基；并且
    X1、X2、X3、环A、R1、R2、m、n如权利要求权利要求1-25中任一项所定义，R6和R7为H。
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