WO2017198050A1 - 作为btk抑制剂的5-氨基吡唑甲酰胺衍生物及其制备方法和药物组合物 - Google Patents

作为btk抑制剂的5-氨基吡唑甲酰胺衍生物及其制备方法和药物组合物 Download PDF

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WO2017198050A1
WO2017198050A1 PCT/CN2017/081906 CN2017081906W WO2017198050A1 WO 2017198050 A1 WO2017198050 A1 WO 2017198050A1 CN 2017081906 W CN2017081906 W CN 2017081906W WO 2017198050 A1 WO2017198050 A1 WO 2017198050A1
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compound
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PCT/CN2017/081906
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吴予川
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浙江予川医药科技有限公司
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Priority to KR1020187036605A priority Critical patent/KR102404759B1/ko
Priority to JP2018560949A priority patent/JP6953445B2/ja
Priority to AU2017265219A priority patent/AU2017265219B2/en
Priority to EP17798613.0A priority patent/EP3459940B8/en
Priority to US16/302,036 priority patent/US10882843B2/en
Publication of WO2017198050A1 publication Critical patent/WO2017198050A1/zh

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    • 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
    • C07D401/14—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 three or more hetero rings
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
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    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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
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    • 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
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • 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
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    • 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
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    • 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
    • C07D401/04—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 directly linked by a ring-member-to-ring-member bond
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the embodiment of the invention belongs to the technical field of medicinal chemistry, and relates to a novel high-efficiency, selective and good pharmacokinetic property, 5-aminopyrazolecarboxamide derivative derivative as a BTK inhibitor and preparation thereof Methods and pharmaceutical compositions.
  • Protein kinases are the largest family of biological enzymes in the human body, including over 500 proteins.
  • the phenolic function on the tyrosine residue can be phosphorylated to exert important biosignaling effects.
  • the tyrosine kinase family has members that control cell growth, migration, and differentiation. Abnormal kinase activity has been elucidated in close association with many human diseases, including cancer, autoimmune diseases, and inflammatory diseases.
  • Bruton's tyrosine kinase is a cytoplasmic non-receptor tyrosine kinase belonging to the TEC kinase family (a total of five members BTK, TEC, ITK, TXK, BMX).
  • the BTK gene is located on Xq21.33-Xq22 of the X-chromosome and shares 19 exons spanning 37.5 kb of genomic DNA.
  • BTK expression plays an essential role in almost all hematopoietic cells, especially in the development, differentiation, signaling and survival of B lymphocytes.
  • B cells are activated by the B cell receptor (BCR), and BTK plays a decisive role in the BCR signaling pathway.
  • BCR B cell receptor
  • Activation of BCR on B cells causes activation of BTK, which in turn leads to an increase in downstream phospholipase C (PLC) concentration and activates the IP3 and DAG signaling pathways. This signaling pathway promotes cell proliferation, adhesion and survival.
  • PLC phospholipase C
  • This signaling pathway promotes cell proliferation, adhesion and survival.
  • Mutations in the BTK gene result in a rare hereditary B cell-specific immunodeficiency disease known as X-Iinked agammaglobulinemia (XLA).
  • XLA X-Iinked agammaglobulinemia
  • BTK In this disease, the function of BTK is inhibited, resulting in the production or maturation of B cells. Men with XLA disease have almost no B cells in their bodies, and there are few circulating antibodies. Heavy or even fatal infections. This strongly proves that BTK plays an extremely important role in the growth and differentiation of B cells.
  • BTK inhibitors bind to BTK, inhibit BTK autophosphorylation, and prevent BTK activation. This can block the signal transduction of the BCR pathway, inhibit the proliferation of B lymphoma cells, destroy the adhesion of tumor cells, and promote the apoptosis of tumor cells. And induce apoptosis.
  • B-cell lymphomas and leukemias such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and Recurrent or refractory mantle cell lymphoma (MCL) and the like.
  • BTK inhibitors In addition to fighting against B-cell lymphoma and leukemia, BTK inhibitors also inhibit B cell autoantibodies and cytokine production.
  • B cells present autoantigens, promote the activation and secretion of T cells, cause inflammatory factors, cause tissue damage, and activate B cells to produce a large number of antibodies, triggering autoimmune responses.
  • the interaction of T and B cells forms a feedback regulatory chain, leading to uncontrolled autoimmune response and aggravation of histopathological damage. Therefore, BTK can be used as a drug target for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and allergic diseases (such as diseases such as esophagitis).
  • BTK inhibitors have been reported to be useful in combination with chemotherapeutic agents or immunological checkpoint inhibitors, and have shown superior therapeutic effects in a variety of solid tumors in clinical trials.
  • Ibrutinib is an irreversible BTK inhibitor developed by Pharmacyclics and Johnson & Johnson, and was approved by the FDA in November 2013 and February 2014 for the treatment of mantle cell lymphocytes.
  • Ibrutinib has been designated by the FDA as a "breakthrough" new drug that works by reacting with the thiol group of cysteine in BTK and forming a covalent bond that inactivates the BTK enzyme.
  • ibrutinib is easily metabolized during metabolism (digested by metabolic enzymes to be dihydroxylated or inactivated by other thiol-containing enzymes, cysteine, glutathione, etc.) Affect the efficacy.
  • the clinically administered dose reached 560 mg per day, which increased the burden on the patient.
  • Ibrutinib also has a certain inhibitory effect on some kinases other than BTK, especially the inhibition of EGFR can lead to more serious rash, diarrhea and other adverse reactions. Therefore, there is still a need in the art to develop a new class of BTK inhibitors that are more efficient, selective, and have good pharmacokinetic properties for the treatment of related diseases.
  • the present inventors have developed a novel 5-aminopyrazole carboxamide derivative which is an effective, safe and highly selective inhibitor of protein kinase BTK.
  • Embodiments of the invention provide a novel 5-aminopyrazolecarboxamide derivative. It is a new covalent bond inhibitor that improves its affinity with the target by altering its reactivity with cysteine to improve efficacy, selectivity and safety.
  • Embodiments of the invention also provide methods of preparing the above derivatives.
  • Embodiments of the invention also provide pharmaceutical compositions containing the above derivatives.
  • Embodiments of the invention also provide the use of the above derivatives.
  • an embodiment of the present invention provides a novel 5-aminopyrazolecarboxamide compound, as shown in formula (I), a stereoisomer thereof, a tautomer thereof, Or a pharmaceutically acceptable salt, or solvate, or prodrug:
  • n, m are independently taken from 0, 1 or 2;
  • L is O, -C(O)NH-, -CH 2 -, S, S(O), NH or S(O) 2 ;
  • A is derived from a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site to the mother nucleus and L may be optionally selected;
  • B is independently taken from a substituted or unsubstituted aliphatic ring, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and the linking site with L may be optionally selected ;
  • R 1 and R 2 are each independently selected from hydrogen, C1-C4 alkyl, halogen, cyano, or R 1 and R 2 together with the carbon atom to which they are attached form a ternary carbocyclic or quaternary carbocyclic ring, or R 1 And R 2 are combined into an oxo group;
  • Y is selected from cyano group
  • R 3 , R 4 , R 5 and R 6 are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, halogen, a cyano group, or -(CH 2 ) q N(R a R b ), wherein q is 1, 2, 3, or 4, and R a and R b are each independently selected from hydrogen, unsubstituted C1-C4 alkane base;
  • R 1 and R 2 are hydrogen and the other is methyl, and Y is a cyano group
  • A is a benzene ring, L is O, m is 1 and n is 2
  • B is not substituted or unsubstituted a benzene ring
  • R 1 and R 2 are hydrogen, and A is a benzene ring, m is 1 and n is 2
  • B is not a substituted or unsubstituted benzene ring, and a substituted or unsubstituted pyridine
  • R 1 And R 2 is hydrogen, and A is a pyridine ring, m is 1 and n is 2, B is not a substituted or unsubstituted benzene ring;
  • R 1 and R 2 are both hydrogen, and A is a benzene ring, L is When O, m is 1 and n is 1, B is not a substituted or unsubstituted benzene ring.
  • an embodiment of the present invention provides a 5-aminopyrazolecarboxamide compound of the formula (I), wherein n, m are independently taken from 0, 1 or 2 ; L is O, -C(O)NH-, -CH 2 -, NH or S, more preferably O, -C(O)NH-, NH.
  • an embodiment of the present invention provides a 5-aminopyrazolecarboxamide compound represented by the formula (I), wherein A is derived from a substituted or unsubstituted heterocyclic ring, substituted Or an unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site to the parent nucleus and L may be optionally selected; B is independently derived from a substituted or unsubstituted aliphatic ring, substituted or unsubstituted a heterocyclic ring, a substituted or unsubstituted benzene ring, or a substituted or unsubstituted heteroaryl ring, and a linking site with L may be optionally selected; wherein:
  • the substituted benzene ring means that any position on the phenyl group is substituted with an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, trifluoromethyl.
  • An oxy or cyano group preferably, the substituted benzene ring is a fluoro substituted phenyl group, or a chloro substituted phenyl group, more preferably a 2,4-difluorophenyl group, or a 4-chlorophenyl group;
  • the unsubstituted heteroaryl ring means furan, pyrrole, thiophene, oxazole, isoxazole, pyrazole, imidazole, thiazole, isothiazole, oxadiazole, triazole, thiadiazole, tetrazolium, pyridine , pyrimidine, pyrazine, pyridazine, triazine; said substituted heteroaryl ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy a radical, a fluorine, a chlorine, a trifluoromethyl group, a trifluoromethoxy group or a cyano group; more preferably, the substituted pyridine is a chloropyridine, particularly preferably a 4-chloro-pyridin-2-yl group;
  • the unsubstituted aliphatic ring means cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane; the substituted aliphatic ring means any position on the above group is optionally selected Substituted by a substituent selected from hydrogen, methyl, methoxy, fluoro, chloro, trifluoromethyl, trifluoromethoxy or cyano;
  • the unsubstituted heterocyclic ring means tetrahydrofuran, tetrahydropyran, tetrahydropyrrole, piperidine, Wherein w is taken from 0, 1 or 2; the substituted heterocyclic ring means that any position on the above group is substituted by an optional substituent selected from the group consisting of hydrogen, methyl, methoxy, and fluorine. , chlorine, trifluoromethyl, trifluoromethoxy or cyano.
  • an embodiment of the present invention provides a 5-aminopyrazolecarboxamide compound represented by the formula (I), wherein, preferably, both R 1 and R 2 are hydrogen, or One of them is hydrogen and the other is a C1-C4 alkyl group (methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or t-butyl), or R 1 and R 2 are The carbon atoms to which they are attached together form a cyclopropyl group; more preferably, both R 1 and R 2 are hydrogen, or one of them is hydrogen and the other is methyl, or R 1 and R 2 are formed together with the carbon atom to which they are attached Cyclopropyl.
  • R 1 and R 2 are hydrogen, or One of them is hydrogen and the other is a C1-C4 alkyl group (methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or
  • an embodiment of the present invention provides a 5-aminopyrazolecarboxamide compound represented by formula (II), a stereoisomer, tautomer thereof, or pharmaceutically acceptable An acceptable salt, or solvate, or prodrug:
  • L, A, B and Y are as defined above for formula (I);
  • a 5-aminopyrazolecarboxamide compound represented by formula (II) provided by an embodiment of the present invention is one of the following compounds:
  • L, B and Y in the formula (II-1) or (II-2) are as defined in the above formula (I);
  • B is not a substituted or unsubstituted benzene ring, and a substituted or unsubstituted pyridine.
  • an embodiment of the present invention provides a 5-aminopyrazolecarboxamide compound represented by formula (III), which is a stereoisomer, tautomer, or pharmaceutically acceptable An acceptable salt, or solvate, or prodrug:
  • L, A, B and Y are as defined above for formula (I);
  • a 5-aminopyrazolecarboxamide compound represented by formula (III) provided by an embodiment of the present invention is one of the following compounds:
  • L, B and Y in the formulae (III-1), (III-2), (III-3) and (III-4) are as defined in the above formula (I);
  • an embodiment of the present invention provides a 5-aminopyrazolecarboxamide compound represented by the formula (IV), which is a stereoisomer, a tautomer, or a pharmaceutically acceptable An acceptable salt, or solvate, or prodrug:
  • L, A, B and Y are as defined in the above formula (I).
  • a 5-aminopyrazolecarboxamide compound represented by the formula (IV) provided by an embodiment of the present invention is one of the following compounds:
  • L, B and Y in the formula (IV-1) or (IV-2) are as defined in the above formula (I).
  • Y is -CN
  • R 3 , R 4 , R 5 and R 6 are each independently selected from hydrogen, unsubstituted C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, C1-C4 alkoxy C1-4 alkyl, Halogen, cyano, or -(CH 2 ) q N(R a R b ), wherein q is 1, 2, 3, or 4, and R a and R b are each independently selected from hydrogen, unsubstituted C1- C4 alkyl.
  • the 5-aminopyrazole carboxamide compound provided by the embodiment of the invention is selected from one of the following compounds:
  • the "pharmaceutically acceptable salt” refers to a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt:
  • the "pharmaceutically acceptable acid addition salt” refers to a salt formed with an inorganic or organic acid capable of retaining the bioavailability of the free base without other side effects.
  • Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, phosphates, and the like; organic acid salts include, but are not limited to, formate, acetate, propionate, glycolate, gluconate , lactate, oxalate, maleate, succinate, fumarate, tartrate, citrate, glutamate, aspartate, benzoate, methanesulfonate , p-toluenesulfonate and salicylate. These salts can be prepared by methods known in the art.
  • the "pharmaceutically acceptable base addition salt” refers to a salt capable of maintaining the bioavailability of the free acid without other side effects. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, calcium and magnesium salts and the like. Salts derived from organic bases include, but are not limited to, ammonium salts, triethylamine salts, lysine salts, arginine salts, and the like. These salts can be prepared by methods known in the art.
  • the "solvate” refers to a complex of a compound of an embodiment of the invention with a solvent. They either react in a solvent or precipitate out of the solvent or crystallize out.
  • a complex formed with water is referred to as a "hydrate.”
  • the compounds of the embodiments of the invention may contain one or more chiral centers and exist in different optically active forms.
  • the compound contains a chiral center, the compound contains the enantiomer.
  • Embodiments of the invention include mixtures of the two isomers and isomers, such as racemic mixtures. Enantiomers can be resolved by methods known in the art, such as crystallization and chiral chromatography. When the compound of formula (I) contains more than one chiral center, diastereomers may be present.
  • Embodiments of the invention include resolved optically pure specific isomers as well as mixtures of diastereomers. Diastereomers can be resolved by methods known in the art, such as crystallization and preparative chromatography.
  • the prodrug refers to a known amino protecting group and a carboxy protecting group which are hydrolyzed under physiological conditions or released via an enzymatic reaction to give the parent compound.
  • Specific prodrug preparation methods can be referred to (Saulnier, MG; Frennesson, DB; Deshpande, MS; Hansel, SB and Vysa, DM Bioorg. Med. Chem Lett. 1994, 4, 1985-1990. Greenwald, RB; Choe, YH Conover, CD; Shum, K.; Wu, D.; Royzen, MJ Med. Chem. 2000, 43, 475.).
  • an embodiment of the present invention provides a method for preparing the 5-aminopyrazolecarboxamide compound represented by the above formula (I), comprising the steps of:
  • PG is an amino protecting group (suitable amino protecting groups include acyl (eg acetyl), carbamate (eg 2', 2', 2'- Trichloroethoxycarbonyl, Cbzbenzyloxycarbonyl or BOC-tert-butoxycarbonyl) and arylalkyl (for example Bnbenzyl) which may be hydrolyzed, if appropriate (for example using diacids such as hydrogen chloride) Solution or trifluoroacetic acid in dichloromethane) or by reduction (eg hydrogenolysis of benzyl or benzyloxycarbonyl, or reduction of 2', 2', 2'-trichloroethoxy by zinc in acetic acid
  • suitable amino protecting groups include acyl (eg acetyl), carbamate (eg 2', 2', 2'- Trichloroethoxycarbonyl, Cbzbenzyloxycarbonyl or BOC-tert-butoxycarbonyl) and aryl
  • R 4 in the above compound is a C1-C4 alkyl group (preferably a methyl group);
  • X is chlorine or bromine, preferably chlorine; and
  • R 3 is a C1-C4 alkyl group (preferably B) Base;)
  • L, A and B are as defined for the compound of formula (I);
  • the above 5-aminopyrazolecarboxamide compound provided by the embodiment of the present invention is a production method represented by the formula (I), wherein the compound of the formula (V) can be produced by referring to the following method:
  • embodiments of the invention also provide intermediate compounds for the synthesis of the above 5-aminopyrazolecarboxamide compounds, including but not limited to:
  • Boc is a tert-butoxycarbonyl group and Cbz is a benzyloxycarbonyl group.
  • an embodiment of the present invention provides a 5-aminopyrazolecarboxamide compound, or a stereoisomer, tautomer, solvate thereof, comprising an effective amount of one or more of the above embodiments of the present invention.
  • a pharmaceutical composition of a pharmaceutically acceptable salt thereof, the pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition of the embodiment of the present invention may be formulated into a solid, semi-solid, liquid or gaseous preparation such as a tablet, a capsule, a powder, a granule, a plaster, a solution, a suppository, an injection, an inhalant, a gel, and a micro Balls and aerosols.
  • a solid, semi-solid, liquid or gaseous preparation such as a tablet, a capsule, a powder, a granule, a plaster, a solution, a suppository, an injection, an inhalant, a gel, and a micro Balls and aerosols.
  • compositions of the embodiments of the present invention can be prepared by methods well known in the pharmaceutical art.
  • practical methods for preparing pharmaceutical compositions are known to those skilled in the art, for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • Routes of administration of the pharmaceutical compositions of the embodiments of the invention include, but are not limited to, oral, topical, transdermal, intramuscular, intravenous, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal.
  • dosage forms suitable for oral administration include capsules, tablets, granules, and syrups and the like.
  • the compound of formula (I) of the embodiments of the invention included in these formulations may be a solid powder or granule; a solution or suspension in an aqueous or non-aqueous liquid; a water-in-oil or oil-in-water emulsion, and the like.
  • the above dosage forms can be prepared from the active compound with one or more carriers or excipients via conventional pharmaceutical methods.
  • the above carriers need to be compatible with the active compound or other excipients.
  • commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like.
  • Carriers for liquid preparations include, but are not limited to, water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like.
  • the active compound can form a solution or suspension with the above carriers. The particular mode of administration and dosage form will depend on the physicochemical properties of the compound itself, as well as the severity of the disease being applied.
  • the pharmaceutical compositions of the embodiments of the invention may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose.
  • Preferred unit dosage compositions are those containing a daily or sub-dose, or an appropriate fraction thereof, of the active ingredient.
  • Such unit doses can be administered more than once a day.
  • Optimal The unit dose composition is those containing the daily or sub-dose (more than one dose per day) as described above, or an appropriate fraction thereof, of the active ingredient.
  • compositions of the embodiments of the invention are formulated, quantified, and administered in a manner consistent with medical practice.
  • a "therapeutically effective amount" of a compound of an embodiment of the invention is determined by the particular condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • the dose for parenteral administration may be 1-200 mg/kg
  • the dose for oral administration may be 1-1000 mg/kg.
  • the present invention provides the above 5-aminopyrazolecarboxamide compound, or a stereoisomer, tautomer, solvate thereof or pharmaceutically acceptable salt thereof, for use in the preparation or prevention of Use in BTK-mediated diseases.
  • An embodiment of the present invention provides a method for inhibiting BTK activity, comprising administering to a biological system a 5-aminopyrazolecarboxamide compound of the above embodiment of the present invention, or a stereoisomer, tautomer, solvate thereof or pharmaceutically thereof thereof
  • An acceptable salt or a pharmaceutical composition comprising the 5-aminopyrazolecarboxamide compound of the above embodiment of the present invention or a stereoisomer, tautomer, solvate thereof or a pharmaceutically acceptable salt thereof.
  • the biological system is an enzyme, a cell, or a mammal.
  • Embodiments of the present invention also provide a method of preventing or treating a disease mediated by BTK comprising administering a therapeutically effective amount of one or more 5-aminopyrazoles of the above-described embodiments of the present invention to a patient in need thereof.
  • the BTK mediated diseases include autoimmune diseases, inflammatory diseases, xenogeneic immune conditions or diseases, thromboembolic diseases, and cancer.
  • the cancer comprises B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma Multiple myeloma, mantle cell lymphoma, small lymphocytic lymphoma, Walsh macroglobulin Blood, solid tumors.
  • the autoimmune disease and inflammatory disease are selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile arthritis, chronic obstructive pulmonary disease, multiple sclerosis, systemic lupus erythematosus, psoriasis , psoriatic arthritis, Crohn's disease, ulcerative colitis, and irritable bowel syndrome.
  • the xenogeneic immune condition or disease comprises graft versus host disease, transplantation, blood transfusion, allergic reaction, allergy, type I hypersensitivity, allergic conjunctivitis, allergic rhinitis or atopy dermatitis.
  • Figure 1 shows that the compound of the present invention significantly inhibited the growth of the diffuse large B-cell lymphoma cell line TMD-8 in vivo and showed the same antitumor effect as the control compound Ibrutinib.
  • the unit of temperature is Celsius (°C); the definition of room temperature is 18-25 ° C;
  • 200-300 mesh silica gel is used as a carrier for rapid column chromatography, and thin layer chromatography is used for thin layer chromatography;
  • the progress of the reaction is monitored by thin layer chromatography or LC-MS;
  • the identification of the final product was performed by nuclear magnetic resonance (Bruker AVANCE 300, 300 MHz) and LC-MS (Bruker esquine 6000, Agilent 1200 series).
  • 6-Methylpiperidin-3-ol was dissolved in dichloromethane (100 mL), then triethylamine (7.5 eq) was added dropwise, then benzyloxycarbonyl chloride (2 g, 11.2 mmol). The reaction was completed after stirring for 16 hours. After the reaction mixture was dried, EtOAc mjjjjjj
  • Benzyl 5-hydroxy-2-methylpiperidine-l-carboxylate (1 g, 3.96 mmol) was dissolved in dichloromethane (10 mL), cooled to EtOAc. The reaction was stirred at zero for 1 hour and then at room temperature for 5 hours. The reaction was carefully quenched with a saturated aqueous solution of sodium thiosulfate and then used Dilute with water and dichloromethane. The organic layer was taken, washed with brine and dried over anhydrous sodium sulfate. After the organic layer was concentrated, the obtained crude product, 2-methyl-5-oxopiperidine-l-carboxylate, was used in the next step without purification.
  • Benzyl (5E)-5-[(tert-butoxycarbonyl)hydrazinyl]-2-methylpiperidine-1-carboxylate (1.2 g, 4 mmol) was dissolved in tetrahydrofuran (10 mL) then Sodium cyanoborohydride (1 eq) and a solution of p-toluenesulfonic acid monohydrate (1 eq) in tetrahydrofuran (2 mL). The reaction solution was stirred at room temperature for 20 hours. After the reaction mixture was concentrated under reduced pressure, ethyl acetate (100 mL) was evaporated and evaporated. After concentration of the solution, benzyl 5-[2-(tert-butyloxycarbonyl)indenyl]-2-methylpiperidine-1-carboxylate was obtained as a white solid.
  • Example 2 The product of Example 2 was obtained after chiral resolution of the product of the fourteenth step of Example 1.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min). MS: m/z 452.2 [M+1]
  • Example 3 The product of Example 3 was obtained after chiral resolution of the product of the fourteenth step of Example 1.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min). MS: m/z 452.2 [M+1]
  • Example 4 The product of Example 4 was obtained after chiral resolution of the product of the fourteenth step of Example 1.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min). MS: m/z 452.2 [M+1]
  • Example 5 The product of Example 5 was obtained after chiral resolution of the product of the fourteenth step of Example 1.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min). MS: m/z 452.2 [M+1]
  • the third step is the preparation of methyl 4-hydroxy-5-nitrovalerate
  • the fourth step is the preparation of 5-hydroxypiperidin-2-one
  • Example 7 was prepared by the following steps:
  • Example 8 The product of the reaction of the nineteenth step of Example 6 was used as a starting material, and Example 8 was prepared by the following steps:
  • Example 9 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 10 was prepared in a similar manner to Example 7 starting from the corresponding starting material.
  • Example 11 was prepared in a similar manner to Example 8 starting from the corresponding starting material.
  • Example 9 The product of Example 9 was obtained by chiral resolution to give the compound of Example 12.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min).
  • Example 13 The product of Example 13 was obtained after chiral resolution of the product of Example 6.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min).
  • Example 14 was prepared in a similar manner to Example 8 starting from the corresponding starting material.
  • Example 15 was obtained by chiral resolution of the product of Example 14.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min).
  • Example 8 The product of Example 8 was obtained by chiral resolution to give the compound of Example 16.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min).
  • Example 17 was prepared in a similar manner to Example 8 starting from the corresponding starting material.
  • Example 17 The product of Example 17 was obtained by chiral resolution to give the compound of Example 18.
  • the resolution conditions were: Supercritical fluid chromatography (ChiralPak AD 5 ⁇ , 21x250mm col, 27% methanol, 70 mL/min).
  • Example 19 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 20 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 21 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 22 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 23 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 24 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 25 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • Example 26 was prepared in a similar manner to Example 6 starting from the corresponding starting material.
  • the compounds of the structures shown in Examples 27 to 40 can be produced by a method similar to the inventive examples 1 to 26.
  • BTK kinase activity was tested in a test based on a time resolved fluorescence resonance energy transfer method.
  • Recombinant Btk and the compounds disclosed herein are tested at room temperature in a test buffer containing 50 mM Tris pH 7.4, 10 mM MgCl 2 , 2 mM MnCl 2 , 0.1 mM EDTA, 1 mM DTT, 20 nM SEB, 0.1% BSA, 0.005% tween-20
  • the liquid was pre-incubated for 1 hour. The reaction was initiated by the addition of ATP (at ATP Km concentration) and the peptide substrate (Biotin-AVLESEEELYSSARQ-NH2).
  • the kinase inhibitory activity levels are classified into A, B, C, specifically A (IC 50 ⁇ 100 nM), B (100 nM ⁇ IC 50 ⁇ 1000 nM), C (IC 50 >1000 nM).
  • the detection platform of EGFR and ITK kinase activity was established by time-resolved fluorescence resonance energy transfer method.
  • the detection platform of LCK, SRC and LYN kinase activity was established by Z'-Lyte method.
  • the detection platform of TEC and JAK3 kinase activity was established by Lance Ultra method.
  • the compounds disclosed herein were tested for inhibition of different kinase activities, respectively. Each compound activity data were determined at 11 concentrations of the compound IC 50 value calculated using Graphpad Prism software.
  • the kinase inhibitory activity levels are classified into A, B, C, specifically A (IC 50 ⁇ 100 nM), B (100 nM ⁇ IC 50 ⁇ 1000 nM), C (IC 50 >1000 nM)
  • B cells were purified from healthy donor blood by negative selection using the RosetteSep Human B Cell Enrichment Mix. Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 8-fold dilution in growth medium. The cells were then stimulated with 10 ⁇ g/mL IgM F(ab') 2 for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions.
  • the preferred compound of the present application has a strong inhibitory activity against B cells and has an IC 50 value of less than 10 nM.
  • T cells were purified from healthy donor blood by negative selection using the RosetteSep Human T Cell Enrichment Mix.
  • Cells were plated in growth medium (10% RPMI + 10% fetal bovine serum) and inhibitors of the indicated concentrations were added. After incubating for 1 hour at 37 ° C, the cells were washed three times, and each wash was used for 10-fold dilution in growth medium. The cells were then challenged with anti-CD3/CD28 coated beads (bead/cell ratio of 1:1) for 18 hours at 37 °C. Cells were subsequently stained with anti-CD69-PE antibody and analyzed by flow cytometry using standard conditions.
  • the preferred compound in the present application has a weak inhibitory activity or no inhibition on T cells, and its IC 50 value is greater than 4000 nM.
  • Human whole blood was obtained from healthy volunteers and blood was collected by venipuncture into a Vacutainer tube that was anticoagulated with sodium heparin. Test compounds were diluted to 10 times the required initial drug concentration in PBS), followed by three-fold serial dilutions in 10% DMSO in PBS to give a 9 point dose response curve. 5.5 ⁇ L of each compound dilution was added to the aiil 96-well V-bottom plate in duplicate; 5.5 ⁇ L of 10% DMSO in PBS was added to the control and non-stimulated wells. Human whole blood (100 ⁇ L) was added to each well, and after mixing, the plates were incubated for 30 minutes at 37 C, 5% CO 2 , 100% humidity.
  • the sample was then lysed with 1 ml of IX Pharmingen Lyse Buffer (BD Pharmingen) and the plate was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed by aspiration, and the remaining pellet was again lysed with an additional 1 ml of IX Pharmingen Lyse Buffer, and the plate was centrifuged as before. The supernatant was aspirated and the remaining pellet was washed in FACs buffer (PBS + 1% FBQ. After centrifugation and the supernatant was removed, the pellet was resuspended in 150 ⁇ L of FACs buffer. Transfer the sample to a suitable one.
  • IX Pharmingen Lyse Buffer BD Pharmingen
  • 96-well plates run on the HTS 96-well system of the BD LSR II flow cytometer. Data were acquired using excitation and emission wavelengths appropriate for the fluorophore used and percent positive cells were obtained using Cell Quest Software. Results were initially analyzed using FACS analysis (Flow Jo) Analysis. IC 50 values were calculated using XLfit v3, Equation 201.
  • the preferred compound of the present application has a strong inhibitory activity against B cells in human whole blood, and its IC 50 value is less than 200 nM.
  • test compound was dissolved in acetonitrile to prepare a stock solution having a concentration of 0.5 mM.
  • phosphate buffer 100 mM, pH 7.4
  • liver microsome protein concentration of 20 mg/ml suspension
  • BD Gentest a species of liver microsomes. Human, dog, rat, and mouse were separately added; 158 ⁇ L of phosphate buffer (100 mM, pH 7.4) was added to the control group.
  • step 3 Prepare the mixed system in step 2, pre-incubated for 3 minutes in a 37 ° C water bath, then add 40 ⁇ L of NADPH production system (containing NADP +: 6.5 mM, glucose 6-phosphate: 16.5 mM, MgCl 2 : 16.5 mM, glucose 6 - Phosphate dehydrogenase: 2 U/ml) The reaction was initiated and incubated for 1 hour in a 37 ° C water bath.
  • NADPH production system containing NADP +: 6.5 mM, glucose 6-phosphate: 16.5 mM, MgCl 2 : 16.5 mM, glucose 6 - Phosphate dehydrogenase: 2 U/ml
  • Preparation method of parallel preparation 0 minute reaction sample The prepared mixed system in step 2 was taken out in a 37 ° C water bath for 3 minutes, and then taken out, 400 ⁇ L of acetonitrile was added, and then 40 ⁇ L of NADPH generation system was added. After vortexing for 3 minutes, centrifugation (13,000 rpm, 4 ° C) for 5 minutes, and the supernatant was taken to detect the drug concentration C0 by HPLC.
  • some of the preferred compounds of the present application exhibit better microsomal stability with a residual percentage >30% in liver microsomes of various genera.
  • CYP enzyme metabolism is the main pathway for drug biotransformation, and its quantity and activity directly affect the activation and metabolism of drugs in the body.
  • cytochrome CYP is an important drug phase I metabolizing enzyme that catalyzes the oxidation and reductive metabolism of various exogenous compounds.
  • the CYP enzyme plays a very important role in the elimination of the drug, and is also the main factor in the drug interaction caused by the combination.
  • METHODS This experiment used the cocktail probe drug method to simultaneously determine the inhibitory effect of compounds on five CYP450 enzymes in human liver microsomes.
  • the human microsomes were from BD Gentest.
  • the reaction was carried out in 100 mM phosphate buffer in a total volume of 200 ⁇ L.
  • the concentration of the microsomes in the reaction system was 0.25 mg/mL, and the concentration of the test compound was 20 ⁇ M, 6.67 ⁇ M, 2.22 ⁇ M, 0.74 ⁇ M, 0.25 ⁇ M.
  • the specific probe substrate and concentration were phenacetin (CYP1A2) 40 ⁇ M, respectively.
  • the incubation system was pre-incubated for 5 minutes in a 37-degree constant temperature shaker, and the reaction was started by adding a NADPH-producing system (containing 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 0.4 U/L glucose 6-phosphate dehydrogenase, 3.3 mM MgCL2). After incubation for 45 minutes, the reaction was stopped by adding an equal volume of acetonitrile, vortexed, centrifuged at 13,000 rpm, and the supernatant was subjected to LC-MS-MS injection to determine the amount of metabolite production.
  • a NADPH-producing system containing 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 0.4 U/L glucose 6-phosphate dehydrogenase, 3.3 mM MgCL2. After incubation for 45 minutes, the reaction was stopped by adding an equal volume of acetonitrile, vortexed, centrifuged at 13,000 rpm, and
  • the specific metabolites were acetaminophen (CYP1A2), dextrorphan (CYP2D6), 4-hydroxydiclofenac (CYP2C9), 4-hydroxyfenfenin (CYP2C19), and 6 ⁇ -hydroxytestosterone (CYP3A4).
  • the specific inhibitors were furaphylline (CYP1A2), quinidine (CYP2D6), sulfaphenazole (CYP2C9), tranylcypromine (CYP2C19), ketoconazole (CYP3A4).
  • some preferred compounds of the present application have only strong inhibition or no inhibition on various CYP enzymes, indicating that they have less influence on the metabolism of other drugs.
  • 2.9 SD rats were randomly divided into 3 groups, 3 in each group, one group was administered by intragastric administration, and the other group was administered by tail vein injection. Rats in the gavage-administered group were fasted overnight before administration.
  • the collected blood samples were centrifuged at 12000 rpm for 5 minutes at 4 ° C, then the upper plasma samples were collected and stored in a refrigerator at -20 ° C for testing.
  • LC-MS/MS liquid phase Waters Acquity UPLC (USA) and mass spectrometry 5500Q Trap (Applied Biosystem/MDS SCIEX) or HPLC-MS ⁇ MS: liquid phase Agilent 1200 series (USA) and mass spectrometry API 4000 (Applied Biosystem/MDS SCIEX) detects the concentration of compounds in plasma.
  • Typical test conditions are as follows:
  • the compounds which have been determined in the present application exhibit a good bioavailability (>40%).
  • IC 50 values may be determined for a compound hERG inhibition. Only weak inhibition present in preferred compounds of the application or no hERG inhibition, an IC 50 value greater than 1000nM.
  • the immunodeficiency serious defect NOD.SCID mouse was purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and was raised in the SPF animal room. After the TMD-8 cells were cultured to a sufficient amount, the cells were collected by centrifugation and washed twice with PBS. Finally, the cells were resuspended in serum-free RPMI 1640 medium and Matrigel (1:1 v/v). Using a 1 ml syringe and a 25G syringe needle, 0.2 ml of the cell suspension was injected into the right flank area of each mouse.
  • novel 5-aminopyrazolecarboxamide derivative provided by the embodiment of the present invention is an effective, safe and selective inhibitor of protein kinase BTK, and can be used as a medicine for treating BTK-mediated diseases.

Abstract

本申请公开了一种新型5-氨基吡唑甲酰胺化合物，如式（I）所示，其立体异构体，或药学上可接受的盐，或溶剂化物，或前药。此外，本申请还公开了上述化合物的制备方法、药物组合物及其用途。

Description

作为BTK抑制剂的5-氨基吡唑甲酰胺衍生物及其制备方法和药物组合物 技术领域
本发明实施方案属于药物化学技术领域，涉及一种新型高效、选择性好的、具有良好的药代动力学性质的、作为BTK抑制剂的5-氨基吡唑甲酰胺衍生物衍生物及其制备方法和药物组合物。
背景
蛋白激酶是人体内生物酶中的最大家族，包括超500种蛋白质。特别地，对于酪氨酸激酶，其酪氨酸残基上的酚官能团能被磷酸化，从而发挥重要的生物信号传导的作用。酪氨酸激酶家族拥有控制细胞生长、迁移和分化的成员。异常的激酶活性己经被阐明了与许多人体疾病密切相关，这些疾病包括癌症、自身免疫疾病和炎性疾病。
布鲁顿酪氨酸激酶(BTK)是一种细胞质非受体酪氨酸激酶，属于TEC激酶家族(共有5个成员BTK，TEC，ITK，TXK，BMX)一员。BTK基因位于X-染色体的Xq21.33-Xq22，共有19外显子，跨越37.5kb基因组DNA。
除了T细胞和浆细胞外，BTK表达在几乎所有造血细胞上，尤其在B淋巴细胞发生，分化，信号和生存中发挥必不可少的作用。B细胞是通过B细胞受体(BCR)被活化的，而BTK在BCR信号通路中起到了决定性的作用。B细胞上的BCR被活化后，会引起BTK的激活，然后导致下游的磷脂酶C(PLC)浓度增加，并激活IP3和DAG信号通路。这一信号通路可以促进细胞的增殖、粘附和存活。BTK基因突变会导致一种罕见的遗传性B细胞特异性免疫缺陷疾病，被称为X-连锁无丙种球蛋白血症(X-Iinked agammaglobulinemia，XLA)。在这种疾病中，BTK的功能被抑制，从而导致了B细胞的产生或成熟受阻。患有XLA疾病的男性，体内基本没有B细胞，循环抗体也很少，容易出现严 重甚至致命的感染。这有力证明了BTK在B细胞的生长和分化中起着极其重要的作用。
小分子BTK抑制剂能与BTK结合，抑制BTK自身磷酸化，阻止BTK的激活。这能阻断BCR通路的信号传导，抑制B淋巴瘤细胞的增殖，破坏瘤细胞的粘附，从而促进瘤细胞的凋亡。并诱导细胞凋亡。这使BTK在B细胞有关的癌症中成为引人注目的药物靶点，尤其是对于B细胞淋巴瘤和白血病，比如非霍奇金淋巴瘤(NHL)、慢性淋巴细胞白血病(CLL)、和抗复发性或难治性套细胞淋巴瘤(MCL)等。
BTK抑制剂除了可以对抗B细胞淋巴瘤和白血病，还可以抑制B细胞自身抗体和细胞因子的产生。在自身免疫性疾病中，B细胞呈递自身抗原，促进T细胞活化分泌致炎症因，既造成组织损伤，同时又激活B细胞产生大量抗体，触发自身免疫反应。T和B细胞相互作用形成反馈调节链，导致自身免疫反应失控，加重组织病理损伤。所以，BTK可以作为自身免疫性疾病，比如类风湿性关节炎、系统性红斑狼疮(SLE)、过敏性疾病(例如食道炎等疾病)的药物靶点。
此外也有报道BTK抑制剂可与化疗药或免疫检查点抑制剂联用，在临床试验中对多种实体瘤表现出较好的治疗效果。
在目前已上市的药物中，依鲁替尼是由Pharmacyclics和强生公司联合开发的一种不可逆BTK抑制剂，己分别于2013年11月和2014年2月获得FDA批准，用于治疗套细胞淋巴瘤(MCL)和慢性淋巴性白血病(CLL)。依鲁替尼被FDA定为“突破性”新药，它通过与BTK中的半胱氨酸的巯基发生反应，并形成共价键，使BTK酶失活而发挥疗效。然而，依鲁替尼在给药过程中，易被代谢(被代谢酶氧化代谢成双羟化产物或者被其他含巯基的酶、半胱氨酸、谷胱甘肽等进攻而失活)而影响药效。其临床给药剂量达到了560mg每天，而使病人负担加重。此外，依鲁替尼对除BTK外的一些激酶也有一定的抑制作用，尤其是对EGFR的抑制可导致较严重的皮疹、腹泻等不良反应。因此，本领域仍需发展新一类更为高效、选择性好，良好的药代动力学性质的BTK抑制剂用于相关疾病的治疗
发明概述
以下是对本申请详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本发明人研发了一种新型5-氨基吡唑甲酰胺类衍生物，这些新化合物是蛋白激酶BTK的有效、安全、选择性高的抑制剂。
本发明实施方案提供一种新型5-氨基吡唑甲酰胺衍生物。它是一种新的共价键抑制剂，通过改变其和半胱氨酸反应率，来改善与靶标的亲和性，以提高疗效，选择性和安全性。
本发明实施方案还提供上述衍生物的制备方法。
本发明实施方案还提供含有上述衍生物的药物组合物。
本发明实施方案还提供上述衍生物的用途。
具体地说，在本发明的实施方案中，本发明实施方案提供了一种新型5-氨基吡唑甲酰胺化合物，如式(I)所示，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
Figure PCTCN2017081906-appb-000001
其中，
n，m独立地取自于0、1或2；
L是O，-C(O)NH-，-CH2-，S，S(O)，NH或S(O)2；
A取自于取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与母核及L的连接位点可以任选；
B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点可以任选；
R1和R2各自独立地选自氢、C1-C4烷基、卤素、氰基，或者R1和R2与它们相连的碳原子一起形成三元碳环或四元碳环，或者R1和R2合并为氧代基；
Y选自氰基、
Figure PCTCN2017081906-appb-000002
R3、R4、R5和R6各自独立地选自氢、未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH2)qN(RaRb)，其中，q为1、2、3、或4，Ra和Rb各自独立地选自氢、未取代的C1-C4烷基；
并规定，当R1和R2其中一个为氢而另一个为甲基，且Y为氰基、A为苯环、L为O、m为1和n为2时，B不是取代或未取代的苯环；当R1和R2都为氢，且A为苯环、m为1和n为2时，B不是取代或未取代的苯环、和取代或未取代的吡啶；当R1和R2都为氢，且A为吡啶环、m为1和n为2时，B不是取代或未取代的苯环；当R1和R2都是氢，且A为苯环、L为O、m为1和n为1时，B不是取代或未取代的苯环。
在本发明的一种实施方案中，本发明实施方案提供的一种如式(I)所示的5-氨基吡唑甲酰胺化合物，其中，n，m独立地取自于0、1或2；L为O，-C(O)NH-，-CH2-，NH或S，更优选地为O，-C(O)NH-，NH。
在本发明的一种实施方案中，本发明实施方案提供的一种如式(I)所示的5-氨基吡唑甲酰胺化合物，其中，A取自于取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与母核及L的连接位点可以任选；B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点可以任选；其中：
所述取代的苯环是指苯基上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；优选地，所述取代的苯环为氟取代的苯基、或氯取代的苯基，更优选地为2,4-二氟苯基、或4-氯苯基；
所述未取代的杂芳环是指呋喃、吡咯、噻吩、恶唑、异恶唑、吡唑、咪唑、噻唑、异噻唑、恶二唑、三氮唑、噻二唑、四氮唑、吡啶、嘧啶、吡嗪、哒嗪、三嗪；所述取代的杂芳环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；更优选地，所述取代的吡啶为氯代吡啶，特别优选地为4-氯-吡啶-2-基；
所述未取代的脂肪环是指环丙烷、环丁烷、环戊烷、环己烷、环庚烷、环辛烷；所述取代的脂肪环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；
所述未取代的杂环是指四氢呋喃、四氢吡喃、四氢吡咯、哌啶、
Figure PCTCN2017081906-appb-000003
Figure PCTCN2017081906-appb-000004
其中w取自0、1或2；所述取代的杂环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基。
在本发明的一种实施方案中，本发明实施方案提供的一种如式(I)所示的5-氨基吡唑甲酰胺化合物，其中，优选地，R1和R2都是氢，或者其中一个为氢、而另一个为C1-C4烷基(甲基、乙基、正丙基、异丙基、正丁基、异丁基、或叔丁基)，或者R1和R2与它们相连的碳原子一起形成环丙基；更优选地，R1和R2都是氢，或者其中一个是氢而另一个为甲基，或者R1和R2与它们相连的碳原子一起形成环丙基。
在本发明的一种优选实施方案中，本发明实施方案提供了一种如式(II)所示的5-氨基吡唑甲酰胺化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
Figure PCTCN2017081906-appb-000005
其中，L、A、B和Y的定义如上述式(I)；
并规定，当A为苯环时，B不是取代或未取代的苯环、和取代或未取代的吡啶；当A为吡啶环时，B不是取代或未取代的苯环。
在本发明的一种更优选实施方案中，本发明实施方案提供的一种如式(II)所示的5-氨基吡唑甲酰胺化合物为下列化合物中的一种：
Figure PCTCN2017081906-appb-000006
其中，式(II-1)或(II-2)中L、B和Y的定义如上述式(I)；
并规定，B不是取代或未取代的苯环、和取代或未取代的吡啶。
在本发明的一种优选实施方案中，本发明实施方案提供了一种如式(III)所示的5-氨基吡唑甲酰胺化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
Figure PCTCN2017081906-appb-000007
其中，L、A、B和Y的定义如上述式(I)；
并规定，当Y为氰基、A为苯环、L为O时，B不是取代或未取代的苯环。
在本发明的一种更优选实施方案中，本发明实施方案提供的一种如式(III)所示的5-氨基吡唑甲酰胺化合物为下列化合物中的一种：
Figure PCTCN2017081906-appb-000008
其中，式(III-1)、(III-2)、(III-3)和(III-4)中L、B和Y的定义如上述式(I)；
并规定，当Y为氰基、L为O时，B不是取代或未取代的苯环。
在本发明的一种优选实施方案中，本发明实施方案提供了一种如式(IV)所示的5-氨基吡唑甲酰胺化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
Figure PCTCN2017081906-appb-000009
其中，L、A、B和Y的定义如上述式(I)。
在本发明的一种更优选实施方案中，本发明实施方案提供的一种如式(IV)所示的5-氨基吡唑甲酰胺化合物为下列化合物中的一种：
Figure PCTCN2017081906-appb-000010
其中，式(IV-1)或(IV-2)中L、B和Y的定义如上述式(I)。
在本发明的一种更优选实施方案中，本发明实施方案提供的式(I)、(II)、(II-1)、(II-2)、(III)、(III-1)、(III-2)、(III-3)、(III-4)、(IV)、(IV-1)、或(IV-2)化合物，其中，L为O；
B为
Figure PCTCN2017081906-appb-000011
Y为-CN、
Figure PCTCN2017081906-appb-000012
其中，R3、R4、R5和R6各自独立地选自氢、未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH2)qN(RaRb)，其中，q为1、2、3、或4，Ra和Rb各自独立地选自氢、未取代的C1-C4烷基。
在本发明的一种特别优选实施方案中，本发明实施方案提供的5-氨基吡唑甲酰胺类化合物，选自下列化合物中的一种：
Figure PCTCN2017081906-appb-000013
Figure PCTCN2017081906-appb-000014
Figure PCTCN2017081906-appb-000015
Figure PCTCN2017081906-appb-000016
Figure PCTCN2017081906-appb-000017
在本发明的实施方案中，所述“药学上可接受的盐”是指药学上可接受的酸加成盐和药学上可接受的碱加成盐：
所述“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其他副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、丙酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐和水杨酸盐等。这些盐可通过本领域已知的方法制备。
所述“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它副作用的盐。这些盐是通过将无机碱或有机碱添加至游离酸而制备的。衍生自无机碱的盐包括但不限于钠盐，钾盐，钙盐和镁盐等。衍生自有机碱的盐包括但不限于铵盐，三乙胺盐，赖氨酸盐，精氨酸盐等。这些盐可通过本领域已知的方法制备。
在本发明的实施方案中，所述“溶剂化物”是指本发明实施方案的化合物与溶剂形成的配合物。它们或者在溶剂中反应或者从溶剂中沉淀析出或者结晶出来。例如，一个与水形成的配合物称为“水合物”。
在本发明的实施方案中，本发明实施方案的化合物可以含有一个或多个手性中心，并以不同的光学活性形式存在。当化合物含有一个手性中心时，化合物包含对映异构体。本发明实施方案包括这两种异构体和异构体的混合物，如外消旋混合物。对映异构体可以通过本专业已知的方法拆分，例如结晶以及手性色谱等方法。当式(I)化合物含有多于一个的手性中心时，可以存在非对映异构体。本发明实施方案包括拆分过的光学纯的特定异构体以及非对映异构体的混合物。非对映异构体可由本领域已知方法拆分，比如结晶以及制备色谱。
在本发明的实施方案中，所述的前药是指已知的氨基保护基和羧基保护基，在生理条件下被水解或经由酶反应释放得到母体化合物。具体的前药制备方法可参照(Saulnier,M.G.；Frennesson,D.B.；Deshpande,M.S.；Hansel,S.B and Vysa,D.M.Bioorg.Med.Chem Lett.1994,4,1985-1990.Greenwald,R.B.；Choe,Y.H.；Conover,C.D.；Shum,K.；Wu,D.；Royzen,M.J.Med.Chem.2000,43,475.)。
第二方面，本发明实施方案提供了上述如式(I)所示的5-氨基吡唑甲酰胺化合物的制备方法，包括如下步骤：
(1)将式(V)化合物与式(VI)化合物反应，得到式(VII)化合物；
Figure PCTCN2017081906-appb-000018
(2)将式(VII)化合物发生水解反应，得到式(VIII)化合物；
Figure PCTCN2017081906-appb-000019
(3)将式(VIII)化合物经脱保护基PG得到式(IX)化合物；
Figure PCTCN2017081906-appb-000020
(4)将式(IX)化合物与式(X)化合物反应，得到式(I)化合物；
Figure PCTCN2017081906-appb-000021
在上述的式(V)、式(VI)、式(VII)、式(VIII)、式(IX)和式(X)中涉及的取代基R1、R2、L、A、B、Y和n、m定义如上面的式(I)，PG为氨基保护基(合适的氨基保护基包括酰基(例如乙酰基)、碳甲酰胺类(carbamate)(例如2',2',2'-三氯乙氧基羰基、Cbz苄氧基羰基或BOC叔丁氧基羰基)和芳基烷基(例如Bn苄基)，其可在适当时通过水解(例如使用酸，例如氯化氢的二噁烷溶液或三氟乙酸的二氯甲烷溶液)或通过还原方式(例如苄基或苄氧基羰基的氢解、或使用乙酸中的锌还原性除去2',2',2'-三氯乙氧基羰基)而除去。其它合适的氨基保护基包括三氟乙酰基(-COCF3)，其可通过碱催化 的水解除去苄氧基羰基或叔丁氧基羰基，本领域技术人员可参考T.W.Greene‘Protective Groups in Organic Synthesis’(第4版，J.Wiley and Sons，2006))，R3为C1-C4的烷基(优选地为乙基)，X为氯、溴或羟基。
在本发明的实施方案中，本发明实施方案提供的上述如式(I)所示的5-氨基吡唑甲酰胺化合物的制备方法，其中，式(VI)化合物可以通过如下的方式获得：
Figure PCTCN2017081906-appb-000022
其中，以上化合物中取代基R4为C1-C4的烷基(优选地为甲基)；X为氯、或溴，优选地为氯；R3为C1-C4的烷基(优选地为乙基)；L、A和B的定义同式(I)化合物；
更具体说，可以采用下面的合成路线：
Figure PCTCN2017081906-appb-000023
在本发明的实施方案中，本发明实施方案提供的上述5-氨基吡唑甲酰胺化合物如式(I)所示的制备方法，其中，式(V)化合物可以参考下列的方法来制备：
Figure PCTCN2017081906-appb-000024
在本发明的实施方案中，本发明实施方案还提供了用于上述5-氨基吡唑甲酰胺化合物合成的中间体化合物，包括但不限于：
Figure PCTCN2017081906-appb-000025
其中，Boc为叔丁氧羰酰基，Cbz为苄氧羰酰基。
第三方面，本发明实施方案提供了一种包含有效剂量的一种或多种本发明上述实施方案的5-氨基吡唑甲酰胺化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐的药物组合物，所述药物组合物还包括药学上可接受的辅料。
本发明实施方案的药物组合物可以被配制为固态、半固态、液态或气态制剂，如片剂、胶囊、粉剂、颗粒剂、膏剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶。
本发明实施方案的药物组合物可以通过制药领域中公知的方法制备。例如，制备药物组合物的实际方法为本领域技术人员所已知，例如可参见The Science and Practice of Pharmacy(制药科学与实践)，20th Edition(Philadelphia College of Pharmacy and Science,2000)。
本发明实施方案的药物组合物的给药途径包括但不限于口服、局部、经皮、肌肉、静脉、吸入、肠胃外、舌下、直肠、阴道及鼻内。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明实施方案的式(I)化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括但不限于水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。具体的给药方式和剂型取决于化合物本身的理化性质以及所应用疾病的严重程度等。本领域技术人员能够根据上述因素并结合其自身具有的知识来确定具体的给药途径。例如可参见：李俊，《临床药理学》，人民卫生出版社，2008.06；丁玉峰，论临床剂型因素与合理用药，医药导报，26(5)，2007；Howard C.Ansel,Loyd V.Allen,Jr.,Nicholas G.Popovich著，江志强主译，《药物剂型与给药体系》，中国医药科技出版社，2003.05。
本发明实施方案的药物组合物可以以每单位剂量含有预定量的活性成分的单位剂型存在。优选的单位剂量组合物为含有日剂量或亚剂量、或其适当分数的活性成分的那些。因此，这种单位剂量可以一天给药多于一次。优选 的单位剂量组合物为含有本申请如上所述的日剂量或亚剂量(一天给药多于一次)、或其适当分数的活性成分的那些。
本发明实施方案的药物组合物以符合医学实践规范的方式配制，定量和给药。本发明实施方案的化合物的“治疗有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。通常，经胃肠道外给药的剂量可以是1-200mg/kg，口服给药的剂量可以是1-1000mg/kg。
本申请中所提供的有效剂量的范围并非意图限制本发明实施方案的范围，而是代表优选的剂量范围。但是，最优选的剂量可针对个别个体而进行调整，这是本领域技术人员所了解且可决定的(例如参阅Berkow等人编著，Merck手册，第16版，Merck公司，Rahway,N.J.,1992)。
第四方面，本发明实施方案提供了上述5-氨基吡唑甲酰胺化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐在制备用于预防或治疗由BTK介导疾病的药物中的用途。
本发明实施方案提供了一种抑制BTK活性的方法，包含给予生物体系本发明上述实施方案的5-氨基吡唑甲酰胺化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐或包含本发明上述实施方案的5-氨基吡唑甲酰胺化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐的药物组合物。
在一些实施方式中，所述生物体系是酶、细胞或哺乳动物。
本发明实施方案还提供了一种预防或治疗由BTK介导的疾病的方法，其包括对有需要的患者联合给予治疗有效剂量的一种或多种本发明上述实施方案的5-氨基吡唑甲酰胺化合物或其立体异构体、互变异构体、溶剂化物或其药学上可接受的盐和一种或多种选自以下的药物：免疫调节剂、免疫检查点抑制剂、糖皮质激素、非甾体抗炎药、Cox-2特异性抑制剂、TNF-α结合蛋白、干扰素、白细胞介素和化疗药物。
在本发明的实施方式中，所述由BTK介导的疾病包括自身免疫性疾病、炎性疾病、异种免疫性情况或疾病、血栓栓塞疾病和癌症。在一些具体实施方式中，所述癌症包括B细胞性慢性淋巴细胞白血病、急性淋巴细胞性白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、急性髓性白血病、弥漫性大B细胞淋巴瘤、多发性骨髓瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、华氏巨球蛋白 血症、实体瘤。在一些具体实施方式中，所述自身免疫性疾病和炎性疾病选自类风湿性关节炎、骨关节炎、青少年关节炎、慢性阻塞性肺疾病、多重硬化、系统性红斑狼疮、银屑病、银屑病关节炎、克罗恩病、溃疡性结肠炎和肠道易激综合症。在一些具体实施方式中，所述异种免疫性情况或疾病包括移植物抗宿主病、移植、输血、过敏反应、变态反应、I型超敏反应、过敏性结膜炎、过敏性鼻炎或特应性皮炎。
实验数据证明，本发明实施方案提供了上述5-氨基吡唑甲酰胺化合物是蛋白激酶BTK的有效、安全的抑制剂。
附图简述
附图用来提供对本发明实施例的进一步理解，并且构成说明书的一部分。下面结合附图和详细描述对本发明实施方案做更进一步的具体说明，并不构成对本发明实施方案的限制。
图1表示的是本发明实施方案的化合物显著抑制弥漫性大B细胞淋巴瘤细胞株TMD-8体内的生长，并显示出与对照化合物依鲁替尼相同的抗肿瘤效果。
详细描述
以下对本发明实施例进行详细说明。应当理解的是，下文所描述的实验、合成方法以及所涉及的中间体等具体实施例是对本申请的阐明，并不限制本申请的范围。需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互任意组合。
本申请中实验所使用的起始原料或购买自试剂供应商或经由本领域公知的方法由已知原料制备。除非另有说明，本申请的实施例应用下述条件：
温度的单位是摄氏度(℃)；室温的定义是18-25℃；
有机溶剂使用无水硫酸镁或无水硫酸钠干燥；使用旋转蒸发仪在减压升温条件下旋干(例如：15mmHg，30℃)；
快速柱色谱分离时使用200-300目硅胶作为载体，薄层色谱表示薄层色谱法；
通常情况下，反应的进度通过薄层色谱或LC-MS监测；
最终产品的鉴定由核磁共振(Bruker AVANCE 300，300MHz)和LC-MS(Bruker esquine 6000，Agilent 1200series)完成。
实施例1
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(WS-400)的制备
Figure PCTCN2017081906-appb-000026
第一步 6-甲基哌啶-3-醇的制备
将5-羟基-2-甲基吡啶(1g,9mmol)溶解于乙酸中(20mL)，然后加入二氧化铂(0.25g)。反应混合物振摇于50psi压力的氢气气氛下过夜。溶液滤出并旋干，得到粗产物6-甲基哌啶-3-醇。
第二步 5-羟基-2-甲基哌啶-1-甲酸苄酯的制备
将6-甲基哌啶-3-醇溶解于二氯甲烷(100mL)中，然后逐滴加入三乙胺(7.5eq)，之后加入苄氧基甲酰氯(2g，11.2mmol)。反应于搅拌16小时后完成。反应液旋干后，残留物用硅胶柱进行纯化，得到所需产物5-羟基-2-甲基哌啶-1-甲酸苄酯(无色油状物,60％)。
第三步 2-甲基-5-氧代哌啶-1-甲酸苄酯的制备
将5-羟基-2-甲基哌啶-1-甲酸苄酯(1g,3.96mmol)溶解于二氯甲烷中(10mL)，冷至零度，加入Dess-Martin试剂(1eq)。反应物在零度下搅拌1小时，然后在室温下搅拌5小时。反应用硫代硫酸钠饱和水溶液小心淬灭，然后用 水和二氯甲烷进行稀释。保留有机层，将其用饱和食盐水洗涤，然后用无水硫酸钠干燥。有机层经浓缩后，所得粗产物2-甲基-5-氧代哌啶-1-甲酸苄酯可不经纯化直接用于下步反应。
第四步 (5E)-5-[(叔丁氧基羰基)亚肼基]-2-甲基哌啶-1-甲酸苄酯的制备
将2-甲基-5-氧代哌啶-1-甲酸苄酯(2.00g,8.09mmol)溶解于四氢呋喃中(10mL)，然后在室温下加入叔丁氧基羰基肼(1.2eq)。反应物回流2.5小时。之后蒸发溶剂得到粗产物(5E)-5-[(叔丁氧基羰基)亚肼基]-2-甲基哌啶-1-甲酸苄酯(白色固体)。
第五步 5-[2-(叔丁基氧基羰基)肼基]-2-甲基哌啶-1-甲酸苄酯的制备
将(5E)-5-[(叔丁氧基羰基)亚肼基]-2-甲基哌啶-1-甲酸苄酯(1.2g,4mmol)溶解于四氢呋喃(10mL)中，然后室温下加入氰基硼氢化钠(1eq)及逐滴加入对甲苯磺酸一水合物(1eq)的四氢呋喃(2mL)溶液。反应液在室温下搅拌20小时。反应液经减压浓缩后，加入乙酸乙酯(100mL)，依次用饱和碳酸氢钠水溶液、1N氢氧化钠水溶液、水和饱和食盐水洗涤，然后用无水硫酸钠干燥。溶液浓缩后得到白色固体粗产物5-[2-(叔丁基氧基羰基)肼基]-2-甲基哌啶-1-甲酸苄酯。
第六步 5-肼基-2-甲基哌啶-1-甲酸苄酯的制备
将5-[2-(叔丁基氧基羰基)肼基]-2-甲基哌啶-1-甲酸苄酯(1.78g,4.9mmol)溶解于二氯甲烷(10mL)中，然后在室温下逐滴加入三氟乙酸(5mL)。反应液在室温下搅拌5小时。反应液经浓缩后得到淡黄色粗产物5-肼基-2-甲基哌啶-1-甲酸苄酯。
第七步 4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯的制备
4-羟基苯甲酸甲酯(6.5g,49mmol)、5-氯-2-氟吡啶(5.0g,33mmol)以及碳酸铯(20g,65mmol)溶于N,N-二甲基甲酰胺(50mL)。反应液在110℃回流反应12小时，薄层色谱(石油醚：乙酸乙酯＝5:1)检测，反应完毕，旋干溶剂。粗品化合物加入乙酸乙酯(250mL)和水(250mL)分液萃取。有机相用无水硫酸钠干燥，过滤，减压蒸干。粗品化合物经柱层析分离纯化,得产品4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯(9.0g,93％)。
MS:m/z 264.2[M+1]
第八步 4-((5-氯吡啶-2-基)氧基)苯甲酸的制备
4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯(6.5g,49mmol)溶于甲醇(100mL)和水(5mL)中,然后向反应体系中加入氢氧化锂(2.3g)。反应液在45℃条件下反应12小时，薄层色谱(石油醚：乙酸乙酯＝1:1)检测，反应完毕，旋干溶剂。粗品化合物加入稀盐酸(100mL，1M)调至PH＝7，此时有大量固体生成，将固体过滤，烘干，得到产品4-((5-氯吡啶-2-基)氧基)苯甲酸(6.5g,白色固体,84％)。
MS:m/z 250.1[M+1]
第九步 4-((5-氯吡啶-2-基)氧基)苯甲酰氯的制备
将4-((5-氯吡啶-2-基)氧基)苯甲酸(6.5g，23mmol)加入到二氯亚砜(20mL)中，反应液在80℃条件下反应3小时。薄层色谱(石油醚:乙酸乙酯＝3:1)检测，反应完毕，旋干溶剂，粗品4-((5-氯吡啶-2-基)氧基)苯甲酰氯(7g,黄色固体)直接用于下一步反应。
第十步 2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈的制备
0℃条件下，将丙二睛(3.72g，56.4mmol)溶于无水四氢呋喃(50mL)，然后缓慢向其中加入氢化钠(3.6g,90mmol,60％),加入完毕后，将反应升至室温搅拌1小时，然后再降至0℃，将4-((5-氯吡啶-2-基)氧基)苯甲酰氯(6g，22.2mmol)溶于无水四氢呋喃(50mL)中，缓慢滴加入上述反应液，滴加完毕后，反应液在0℃条件下搅拌1小时。薄层色谱(石油醚：乙酸乙酯＝3:1)检测，反应完毕，新点生成。反应液加入饱和氯化铵(100mL)淬灭，乙酸乙酯(100mL*3)萃取3次，有机相分别用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，旋干，粗品化合物用石油醚:乙酸乙酯＝50:1打浆,得到2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈(8.0g，淡黄色固体，82％)。
MS:m/z298[M+1]
第十一步 2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈的制备
将2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈(5.0g，16.8mmol)加入到原甲酸三乙酯(50mL)中，反应升温至80℃搅拌12小时。薄层色谱(石油醚：乙酸乙酯＝3:1)显示有产物生成，将反应液过滤，滤液旋干，得到固体用甲醇打浆，得到2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈(1.5g，白色产物，27.7％)。
MS:m/z 326.0[M+1]
第十二步 5-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-2-甲基哌啶-1-甲酸苄酯的制备
2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈(1.0g,3.09mmol)、5-肼基-2-甲基哌啶-1-甲酸苄酯(1.28g,3.71mmol)以及三乙胺(1.56g,15.5mmol)溶于乙醇(20mL)。反应液在25℃反应12小时，薄层色谱(石油醚：乙酸乙酯＝2:1)检测，反应完毕，加水淬灭。乙酸乙酯(25mL*3)萃取。有机相用无水硫酸钠干燥，过滤，旋干。粗品化合物用石油醚:乙酸乙酯＝50:1打浆2次，得产品5-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-2-甲基哌啶-1-甲酸苄酯(1.5g,88％)。
MS:m/z 555.2[M+1]
第十三步 5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺的制备
室温下，将5-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-2-甲基哌啶-1-甲酸苄酯(750mg，1.35mmol)加入到90％的浓硫酸(10分钟)中，搅拌15分钟，然后将反应体系升温至30℃，反应24小时。薄层色谱(二氯甲烷：甲醇＝10:1)检测，反应完毕.将反应液缓慢倒入氨水(50ml)中，调至PH＝7,乙酸乙酯(30mL*3)萃取三次，合并的有机相用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，旋干，粗品化合物过柱纯化，得到5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(480mg，78％，淡黄色固体)。
MS:m/z 439.2[M+1]
第十四步 5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-((3R,6S)-1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺的制备
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(50mg,0.113mmol)溶于N,N-二甲基甲酰胺(5mL)，加入碳酸铯(110mg,0.342mmol)和溴化氰(12.5mg,0.113mmol)，反应液室温搅拌2小时，点板检测，反应完毕,反应液倒入乙酸乙酯(50mL)，水洗(20mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝50：1),得 产品5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(15mg,15％)。
MS:m/z 452.2[M+1]
实施例2
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-((3R,6S)-1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(WS-401)的制备
Figure PCTCN2017081906-appb-000027
实施例1第十四步反应的产物经手性拆分后可得实施例2化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。MS:m/z 452.2[M+1]
实施例3
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-((3S,6R)-1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(WS-402)的制备
Figure PCTCN2017081906-appb-000028
实施例1第十四步反应的产物经手性拆分后可得实施例3化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。MS:m/z 452.2[M+1]
实施例4
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-((3R,6R)-1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(WS-403)的制备
Figure PCTCN2017081906-appb-000029
实施例1第十四步反应的产物经手性拆分后可得实施例4化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。MS:m/z 452.2[M+1]
实施例5
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-((3S,6S)-1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(WS-404)的制备
Figure PCTCN2017081906-appb-000030
实施例1第十四步反应的产物经手性拆分后可得实施例5化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。MS:m/z 452.2[M+1]
实施例6
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(WS-405)的制备
Figure PCTCN2017081906-appb-000031
第一步 4-羟基丁酸甲酯的制备
将二氢呋喃-2(3H)-酮(100g，1.163mol)和三乙胺(460g,4.65mol)加入到甲醇(1L)溶液中，反应液在60℃，条件下反应24小时，薄层色谱(石油醚:乙酸乙酯＝2:1)检测反应完毕，将反应液旋干，得到4-羟基丁酸甲酯(120g，87.6％，黄色液体)，直接用于下一步反应。
第二步 4-氧代丁酸甲酯的制备
将4-羟基丁酸甲酯(120g，1.02mol)加入到二氯甲烷(1.2L)溶液中，然后将氯铬酸吡啶(330g，1.53mol)加入到上述反应液中，室温条件下反应12小时，薄层色谱(石油醚:乙酸乙酯＝3:1)检测反应完毕，将反应液通过硅藻土过滤，旋干，得到4-氧代丁酸甲酯(60g，50％，黄色液体)，直接用于下一步反应。
第三步 4-羟基-5-硝基戊酸甲酯的制备
冰水浴中，将4-氧代丁酸甲酯(60g，0.46mol)，硝基甲烷(42g，0.69mol),四氢呋喃(300mL)，叔丁醇(300mL)加入到反应瓶中，然后将叔丁醇钾(5g)缓慢加入上述反应体系中，升至室温，反应2小时，薄层色谱(石油醚:乙酸乙 酯＝3:1)检测反应完毕，加水(30mL)淬灭反应，旋干溶剂，加入水(300mL)和乙酸乙酯(300mL)分液，有机相用饱和食盐水洗涤，无水硫酸钠干燥，旋干，得到粗品4-羟基-5-硝基戊酸甲酯(45g，淡黄色油状液体)直接用于下一步。
第四步 5-羟基哌啶-2-酮的制备
将甲基4-羟基-5-硝基戊酸甲酯(45g，0.23mol)和钯碳(2.1g)加入到甲醇(500mL)溶液中，反应液在60℃，H2条件下反应24小时，薄层色谱(石油醚:乙酸乙酯＝1:1)检测反应完毕，将反应液通过硅藻土过滤，滤液旋干，得到5-羟基哌啶-2-酮(10g，黄色固体,38％)，直接用于下一步反应。
第五步 1-苄基-5-(苄氧基)哌啶-2-酮的制备
室温下，将5-羟基哌啶-2-酮(10g，0.1mol)，加入到二甲基亚砜(100mL)中，然后将氢化钠(10g，0.25mol)缓慢加入到上述反应体系中，加入完毕后，将苄溴(43.5g，0.25mol)加入到反应液中，搅拌过夜，薄层色谱(石油醚:乙酸乙酯＝1:1)检测反应完毕，向反应体系中加入饱和氯化铵(100ml)淬灭反应，乙酸乙酯(100mL*3)萃取三次，饱和食盐水洗涤，无水硫酸钠干燥，旋干，过柱纯化，得到1-苄基-5-(苄氧基)哌啶-2-酮(16g，黄色固体,54％)。
第六步 4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷的制备
-78℃氮气保护下，将1-benzy1-苄基-5-(苄氧基)哌啶-2-酮15g，50mmol)溶于无水四氢呋喃(150mL)中，然后向反应瓶中缓慢滴加乙基溴化镁(150mL)，滴加完毕后，再将钛酸四丙酯(45g，150mmol)加入到上述反应体系中，滴加完毕，将反应升至室温，搅拌2小时，薄层色谱(石油醚:乙酸乙酯＝10:1)检测反应完毕，向反应体系中加入饱和氯化铵(100ml)淬灭反应，乙酸乙酯(100mL*3)萃取三次，饱和食盐水洗涤，无水硫酸钠干燥，旋干，过柱纯化，得到4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷(5.1g，黄色固体,31％)。
第七步 4-氮杂螺[2.5]辛-6-醇的制备
将4-苄基-6-(苄氧基)-4-氮杂螺[2.5]辛烷(5.5g，18mmol)和钯碳(2g,1.8mmol)加入到甲醇(200mL)和氯化氢(2mL)溶液中，反应液在60℃，H2条件下反应48小时，薄层色谱(石油醚:乙酸乙酯＝10:1)检测反应完毕，将反应液通过硅藻土过滤，滤液旋干，得到4-氮杂螺[2.5]辛-6-醇(2.5g，黄色固体)，直接用于下一步反应。
第八步 6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将4-氮杂螺[2.5]辛-6-醇(2.5g，21mmol)和碳酸氢钠(3.8g,45mmol)加入到四氢呋喃(100mL)溶液中，然后将苄氧基甲酰氯(4.25g，25mmol)滴加到上述反应体系中，反应液在室温条件下反应48小时，薄层色谱(石油醚:乙酸乙酯＝3:1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g)。
第九步 6-氧代-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将6-羟基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g，16mmol)和2-碘酰基苯甲酸(6.7g,24mmol)加入到丙酮(100mL)溶液中，然后将反应液升至60℃反应12小时，薄层色谱(石油醚:乙酸乙酯＝2:1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-氧代-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(3.6g,85％)。
1H-NMR(400MHz,DMSO-d6):δppm 7.34-7.35(m,5H),5.15(s,2H),4.07(s,2H),2.55-2.58(m,2H),1.95-1.98(m,2H),1.09-1.11(m,2H),0.85-0.86(m,2H).
第十步 (E)-6-(2-(叔丁氧基羰基)亚肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将6-氧代-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(3.6g，13.9mmol)和叔丁氧基羰基肼(1.98g,15mmol)加入到四氢呋喃(50mL)溶液中，然后将反应液升至70℃反应2小时，薄层色谱(石油醚:乙酸乙酯＝2:1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到(E)-6-(2-(叔丁氧基羰基)亚肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(5.0g,90％)。
第十一步 6-(2-(叔丁氧基羰基)肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将(E)-6-(2-(叔丁氧基羰基)亚肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(5.0g，13.4mmol)和氰基硼氢化钠(1.4g,20mmol)加入到四氢呋喃(100mL)溶液中，然后将对甲苯磺酸(3.8g，20mmol)滴加到上述反应体系中，反应液在室温条件下反应36小时，薄层色谱(石油醚:乙酸乙酯＝1:1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-(2-(叔丁氧基羰基)肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g,80.4％)。
第十二步 6-肼基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
将6-(2-(叔丁氧基羰基)肼基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(4.2g，11mmol)加入到氯化氢/乙酸乙酯(50mL)溶液中，室温反应12小时，薄层色谱(石油醚:乙酸乙酯＝2:1)检测反应完毕，将反应液萃取，滤液旋干，过柱纯化，得到6-肼基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(3.5g)。
1H-NMR(400MHz,DMSO-d6):δppm 7.21-7.35(m,10H),4.46-4.54(m,2H),3.89-3.93(m,1H),3.78-3.81(m,1H),3.68-3.73(m,1H),2.86-2.90(m,1H),2.63-2.68(m,1H),2.08-2.12(m,1H),1.57-1.85(m,2H),1.24-1.29(m,1H),0.65(s,2H),0.41-0.44(m,2H)
第十三步 4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯的制备
4-羟基苯甲酸甲酯(6.5g,49mmol)、5-氯-2-氟吡啶(5.0g,33mmol)以及碳酸铯(20g,65mmol)溶于N,N-二甲基甲酰胺(50mL)。反应液在110℃回流反应12小时，薄层色谱(石油醚：乙酸乙酯＝5:1)检测，反应完毕，旋干溶剂。粗品化合物加入乙酸乙酯(250mL)和水(250mL)分液萃取。有机相用无水硫酸钠干燥，过滤，减压蒸干。粗品化合物经柱层析分离纯化，得产品4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯(9.0g,93％)。
MS:m/z 264.2[M+1]
第十四步 4-((5-氯吡啶-2-基)氧基)苯甲酸的制备
4-((5-氯吡啶-2-基)氧基)苯甲酸甲酯(6.5g,49mmol)溶于甲醇(100mL)和水(5mL)中,然后向反应体系中加入氢氧化锂(2.3g)。反应液在45℃条件下反应12小时，薄层色谱(石油醚：乙酸乙酯＝1:1)检测，反应完毕，旋干溶剂。粗品化合物加入稀盐酸(100mL，1M)调至PH＝7，此时有大量固体生成，将固体过滤，烘干，得到产品4-((5-氯吡啶-2-基)氧基)苯甲酸(6.5g,白色固体,84％)。
MS:m/z 250.1[M+1]
第十五步 4-((5-氯吡啶-2-基)氧基)苯甲酰氯的制备
将4-((5-氯吡啶-2-基)氧基)苯甲酸(6.5g，23mmol)加入到二氯亚砜(20mL)中，反应液在80℃条件下反应3小时。薄层色谱(石油醚:乙酸乙酯＝3:1)检测，反应完毕，旋干溶剂，粗品4-((5-氯吡啶-2-基)氧基)苯甲酰氯(7g,黄色固体)直接用于下一步反应。
第十六步 2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈的制备
0℃条件下，将丙二睛(3.72g，56.4mmol)溶于无水四氢呋喃(50mL)，然后缓慢向其中加入氢化钠(3.6g,90mmol,60％),加入完毕后，将反应升至室温搅拌1小时，然后再降至0℃，将4-((5-氯吡啶-2-基)氧基)苯甲酰氯(6g，22.2mmol)溶于无水四氢呋喃(50mL)中，缓慢滴加入上述反应液，滴加完毕后，反应液在0℃条件下搅拌1小时。薄层色谱(石油醚：乙酸乙酯＝3:1)检测，反应完毕，新点生成。反应液加入饱和氯化铵(100mL)淬灭，乙酸乙酯(100mL*3)萃取3次，有机相分别用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，旋干，粗品化合物用石油醚:乙酸乙酯＝50:1打浆,得到2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈(8.0g，淡黄色固体，82％)。
MS:m/z298[M+1]
第十七步 2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈的制备
将2-((4-((5-氯吡啶-2-基)氧基)苯基)(羟基)亚甲基)丙二腈(5.0g，16.8mmol)加入到原甲酸三乙酯(50mL)中，反应升温至80℃搅拌12小时。薄层色谱(石油醚：乙酸乙酯＝3:1)显示有产物生成，将反应液过滤，滤液旋干，得到固体用甲醇打浆，得到2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈(1.5g，白色产物，27.7％)。
MS:m/z 326.0[M+1]
第十八步 6-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯的制备
2-((4-((5-氯吡啶-2-基)氧基)苯基)(乙氧基)亚甲基)丙二腈(1.0g,3.09mmol)、6-肼基-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(1.28g,3.71mmol)以及三乙胺(1.56g,15.5mmol)溶于乙醇(20mL)。反应液在25℃反应12小时，薄层色谱(石油醚：乙酸乙酯＝2:1)检测，反应完毕，加水淬灭。乙酸乙酯(25mL*3)萃取。有机相用无水硫酸钠干燥，过滤，旋干。粗品化合物用石油醚:乙酸乙酯＝50:1打浆2次,得产品6-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(1.5g,88％)。
MS:m/z 555.2[M+1]
第十九步 5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺的制备
室温下，将6-(5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-4-氰基-1H-吡唑-1-基)-4-氮杂螺[2.5]辛烷-4-甲酸苄酯(750mg，1.35mmol)加入到90％的浓硫酸(10分钟)中，搅拌15分钟，然后将反应体系升温至30℃，反应24小时。薄层色谱(二氯甲烷：甲醇＝10:1)检测，反应完毕.将反应液缓慢倒入氨水(50ml)中，调至PH＝7,乙酸乙酯(30mL*3)萃取三次，合并的有机相用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，旋干，粗品化合物过柱纯化，得到5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(480mg，78％，淡黄色固体)。
MS:m/z 439.2[M+1]
第二十步 5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺的制备
5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(50mg,0.113mmol)溶于N,N-二甲基甲酰胺(5mL)，加入碳酸铯(110mg,0.342mmol)和溴化氰(12.5mg,0.113mmol)，反应液室温搅拌2小时，点板检测，反应完毕,反应液倒入乙酸乙酯(50mL)，水洗(20mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝50：1),得产品5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(15mg,15％)。
1H-NMR(400MHz,DMSO-d6):δppm 8.12(s,1H),7.66-7.69(dd,J1＝2.4Hz,J2＝8.4Hz 2H),7.56-7.58(d,J＝8Hz,2H),7.21-7.23(d,J＝8Hz,2H),6.92-6.94(d,J＝8Hz,1H),5.61(s,1H),5.19-5.30(m,2H),4.19-4.25(m,1H),3.52-3.66(m,2H),2.34-2.45(m,2H),2.19(s,1H),1.25-1.30(m,1H),1.15-1.20(m,1H),0.78-0.89(m,2H),0.66-0.71(m,1H).
MS:m/z 446.2[M+1]
实施例7
(E)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-(4-羟基丁-2-烯酰基)-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(WS-406)的制备
Figure PCTCN2017081906-appb-000032
以实施例6第十九步反应的产物为起始物，实施例7经过以下的步骤制备：
第一步 (E)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-(4-羟基丁-2-烯酰基)-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺的制备
将4-羟基-2-丁烯酸(100mg,0.94mmol)，1-羟基苯并三唑(280mg,0.72mmol)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(260mg,0.72mmol)和三乙胺(160mg,1.44mmol)溶于二氯甲烷(2mL)中，室温搅拌15分钟，然后将5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(100mg,0.226mmol)加入到上述反应液中，室温搅拌12小时，点板检测，反应完毕,减压蒸干。残余物溶于乙酸乙酯(20mL*3)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物制备板纯化(二氯甲烷:甲醇＝10：1),得产品(E)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-(4-羟基丁-2-烯酰基)-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(17mg,15％)。
1H-NMR(400MHz,DMSO-d6):δppm 8.17(s,1H),7.68-7.69(d,J＝4Hz,1H),7.36-7.57(m,2H),7.24(brs.,2H),6.87-6.94(m,2H),6.37-6.43(m,1H),5.75-5.79(m,1H),4.66-4.70(m,1H),3.73-4.06(m,2H),3.28-3.32(m,1H),2.55-2.58(m,1H),2.11-2.21(m,2H),1.18-1.35(m,2H),1.15-1.16(brs,1H),0.72-0.79(m,2H).
MS:m/z 523.2[M+1]
实施例8
1-(4-丙烯酰基-4-氮杂螺[2.5]辛-6-基)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺(WS-407)的制备
Figure PCTCN2017081906-appb-000033
以实施例6第十九步反应的产物为起始物，实施例8经过以下的步骤制备：
第一步 1-(4-丙烯酰基-4-氮杂螺[2.5]辛-6-基)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺的制备
将丙烯酸(8mg,0.113mmol)，加入2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(65mg,0.0.171mmol)和三乙胺(35mg,0.342mmol)溶于二氯甲烷(2mL)，搅拌15分钟，然后将5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(50mg,0.113mmol)加入上述反应液中，室温搅拌2小时，点板检测，反应完毕,减压蒸干。残余物溶于乙酸乙酯(20mL*3)，水洗(30mL*3)，无水硫酸钠干燥，过滤，减压蒸干。粗产物通过制备HPLC，得产品1-(4-丙烯酰基-4-氮杂螺[2.5]辛-6-基)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺(8mg,红色固体)。
1H-NMR(400MHz,DMSO-d6):δppm 8.17(s,1H),7.68-7.69(d,J＝4Hz,1H),7.36-7.57(m,2H),7.24(brs.,2H),6.87-6.94(m,2H),6.37-6.43(m,1H),5.75-5.79(m,1H),4.66-4.70(m,1H),3.73-4.06(m,2H),3.28-3.32(m,1H),2.55-2.58(m,1H),2.11-2.21(m,2H),1.18-1.35(m,2H),1.15-1.16(brs,1H),0.72-0.79(m,2H).
MS:m/z 493.2[M+1]
实施例9
5-氨基-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-408)的制备
Figure PCTCN2017081906-appb-000034
实施例9从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,DMSO-d6):δppm 7.47-7.49(dd,J1＝8Hz,J2＝8.4Hz2H),7.08-7.14(m,1H),6.87-7.02(m,4H),5.56(s,1H),5.13-5.16(s,2H),4.17-4.22(m,1H),3.60-3.66(m,1H),3.48-3.51(m,1H),2.34-2.41(m,2H),2.15-2.17(s,1H),1.26-1.30(m,1H),1.14-1.19(m,1H),0.78-0.88(m,2H),0.66-0.70(m,1H).
MS:m/z 465.2[M+1]
实施例10
(E)-5-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1-(4-(4-羟基丁-2-烯酰基)-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(WS-409)的制备：
Figure PCTCN2017081906-appb-000035
实施例10从对应的起始物开始，采用与实施例7类似的方法制备而得。
1H-NMR(400MHz,DMSO-d6):δppm 7.49-7.51(d,J＝8Hz,2H),7.07-7.18(m,1H),7.69-7.01(m,3H),6.87-6.94(m,2H),5.74(s,2H),5.16(s,1H),4.62(brs.,1H),4.40(s,2H),3.93(s,1H),3.22(m,1H),2.14-2.17(d,J＝12Hz,2H),1.99-2.00(m,1H),1.36-1.42(m,2H),1.16-1.21(m,1H),0.69-0.73(m,2H).
MS:m/z 524.4[M+1]
实施例11
1-(4-丙烯酰基-4-氮杂螺[2.5]辛-6-基)-5-氨基-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-410)的制备
Figure PCTCN2017081906-appb-000036
实施例11从对应的起始物开始，采用与实施例8类似的方法制备而得。
1H-NMR(400MHz,DMSO-d6):δppm 7.46-7.49(d,J＝12Hz,2H),7.08-7.18(m,1H),6.99-7.01(m,2H),6.42-6.43(d,J＝4Hz,1H),5.63-5.77(brs.,1H),5.62(s,2H),5.09(brs.,2H),4.64(m,1H),3.91(m,1H),3.24(m,1H),2.57(m,1H),2.01-2.18(m,2H),1.31-1.40(m,1H),1.22-1.24(m,1H),1.12-1.18(m,1H),0.68-0.77(m,2H).
MS:m/z 494.3[M+1]
实施例12
(R)-5-氨基-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-411)和(S)-5-氨基-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-412)的制备
Figure PCTCN2017081906-appb-000037
实施例9的产物经手性拆分后可得实施例12化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。
WS-411谱图数据：
1H-NMR(400MHz,CDCl3):δppm 7.48(d,J＝8.4Hz,2H),7.15-7.09(m,1H),7.02-6.95(m,3H),6.92-6.88(m,1H),5.75(s,2H),5.29(s,2H),4.32-4.26(m,1H),3.65-3.52(m,2H),2.42-2.30(m,2H),2.18-2.15(m,1H),1.19-1.14(m,1H),0.87-0.67(m,4H).
MS:m/z 465.4[M+H]
WS-412谱图数据：
1H-NMR(400MHz,CDCl3):δppm 7.48(d,J＝8.4Hz,2H),7.15-7.09(m,1H),7.02-6.95(m,3H),6.92-6.88(m,1H),5.75(s,2H),5.29(s,2H),4.32-4.26(m,1H),3.65-3.52(m,2H),2.42-2.30(m,2H),2.18-2.15(m,1H),1.19-1.14(m,1H),0.87-0.67(m,4H).
MS:m/z 465.4[M+H]
实施例13
(R)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(WS-413)和(S)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1-(4-氰基-4-氮杂螺[2.5]辛-6-基)-1H-吡唑-4-甲酰胺(WS-414)的制备
Figure PCTCN2017081906-appb-000038
实施例6的产物经手性拆分后可得实施例13化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。
WS-413谱图数据：
1H-NMR(400MHz,CDCl3):δppm 8.12(s,1H),7.69-7.67(m,1H),7.57(d,J＝8.4Hz,2H),7.22(d,J＝8.4Hz,2H),6.93(d,J＝8.8Hz,1H),5.60(s,2H),5.24(s,2H),4.25-4.20(m,1H),3.67-3.48(m,2H),2.42-2.35(m,2H),2.19-2.16(m,1H),1.30-1.26(m,1H),1.21-1.16(m,1H),0.90-0.79(m,2H),0.71-0.67(m,1H).
MS:m/z 464.4[M+H]
WS-414谱图数据：
1H-NMR(400MHz,CDCl3):δppm 8.12(s,1H),7.69-7.67(m,1H),7.57(d,J＝8.4Hz,2H),7.22(d,J＝8.4Hz,2H),6.93(d,J＝8.8Hz,1H),5.60(s,2H),5.24(s,2H),4.25-4.20(m,1H),3.67-3.48(m,2H),2.42-2.35(m,2H),2.19-2.16(m,1H),1.30-1.26(m,1H),1.21-1.16(m,1H),0.90-0.79(m,2H),0.71-0.67(m,1H).
MS:m/z 464.4[M+H]
实施例14
5-氨基-1-(4-(2-丁炔酰基)-4-氮杂螺[2.5]辛-6-基)-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺(WS-415)的制备
Figure PCTCN2017081906-appb-000039
实施例14从对应的起始物开始，采用与实施例8类似的方法制备而得。
1H-NMR(400MHz,MeOD):δppm 8.08(s,1H),7.85(d,J＝8.8Hz,1H),7.61-7.57(m,2H),7.23-7.20(m,2H),7.05(d,J＝8.8Hz,1H),4.40-4.37(m,1H),4.22-3.90(m,2H),3.60-3.31(m,1H),2.34-2.31(m,1H),2.16-2.09(m,2H),2.06(s,3H),1.14-0.75(m,4H).
MS:m/z 505.1[M+H]
实施例15
(R)-5-氨基-1-(4-(2-丁炔酰基)-4-氮杂螺[2.5]辛-6-基)-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺(WS-416)和(S)-5-氨基-1-(4-(2-丁炔酰基)-4-氮杂螺[2.5]辛-6-基)-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺(WS-417)的制备
Figure PCTCN2017081906-appb-000040
实施例14的产物经手性拆分后可得实施例15化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。
WS-416谱图数据：
1H-NMR(400MHz,MeOD):δppm 8.08(s,1H),7.85(d,J＝8.8Hz,1H),7.61-7.57(m,2H),7.23-7.20(m,2H),7.05(d,J＝8.8Hz,1H),4.40-4.37(m,1H),4.22-3.90(m,2H),3.60-3.31(m,1H),2.34-2.31(m,1H),2.16-2.09(m,2H),2.06(s,3H),1.14-0.75(m,4H).
MS:m/z 505.1[M+H]
WS-417谱图数据：
1H-NMR(400MHz,MeOD):δppm 8.08(s,1H),7.85(d,J＝8.8Hz,1H),7.61-7.57(m,2H),7.23-7.20(m,2H),7.05(d,J＝8.8Hz,1H),4.40-4.37(m,1H),4.22-3.90(m,2H),3.60-3.31(m,1H),2.34-2.31(m,1H),2.16-2.09(m,2H),2.06(s,3H),1.14-0.75(m,4H).
MS:m/z 505.1[M+H]
实施例16
(R)-1-(4-丙烯酰基-4-氮杂螺[2.5]辛-6-基)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺(WS-418)和(S)-1-(4-丙烯酰基-4-氮杂螺[2.5]辛-6- 基)-5-氨基-3-(4-((5-氯吡啶-2-基)氧基)苯基)-1H-吡唑-4-甲酰胺(WS-419)的制备
Figure PCTCN2017081906-appb-000041
实施例8的产物经手性拆分后可得实施例16化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。
WS-418谱图数据：
1H-NMR(400MHz,CDCl3):δppm 8.12(s,1H),7.69-7.66(m,1H),7.59(d,J＝8.4Hz,2H),7.21(d,J＝8.4Hz,2H),6.94-6.88(m,3H),6.41-6.37(m,1H),5.75(s,2H),5.43(s,2H),4.68-4.66(m,1H),4.01-3.95(m,1H),3.31-3.19(m,2H),2.63-2.56(m,1H),2.19-2.12(m,2H),1.40-1.37(m,2H),1.25-1.10(m,2H).
MS:m/z 493.1[M+H]
WS-419谱图数据：
1H-NMR(400MHz,CDCl3):δppm 8.12(s,1H),7.69-7.66(m,1H),7.59(d,J＝8.4Hz,2H),7.21(d,J＝8.4Hz,2H),6.94-6.88(m,3H),6.41-6.37(m,1H),5.75(s,2H),5.43(s,2H),4.68-4.66(m,1H),4.01-3.95(m,1H),3.31-3.19(m,2H),2.63-2.56(m,1H),2.19-2.12(m,2H),1.40-1.37(m,2H),1.25-1.10(m,2H).
MS:m/z 493.1[M+H]
实施例17
5-氨基-1-(4-(2-丁炔酰基)-4-氮杂螺[2.5]辛-6-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-420)的制备
Figure PCTCN2017081906-appb-000042
实施例17从对应的起始物开始，采用与实施例8类似的方法制备而得。
1H-NMR(400MHz,CDCl3):δppm 7.52(d,J＝8.0Hz,1H),7.13-7.10(m,1H),7.03-6.95(m,3H),6.91-6.88(m,1H),5.65(s,2H),5.18(s,2H),4.56-3.92(m,3H),3.25-3.20(m,1H),2.52-2.51(m,1H),2.21-2.14(m,2H),2.09(s,3H),1.08-0.88(m,2H),0.72-0.65(m,2H).
MS:m/z 506.4[M+H]
实施例18
(R)-5-氨基-1-(4-(2-丁炔酰基)-4-氮杂螺[2.5]辛-6-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-421)和(R)-5-氨基-1-(4-(2-丁炔酰基)-4-氮杂螺[2.5]辛-6-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-422)的制备
Figure PCTCN2017081906-appb-000043
实施例17的产物经手性拆分后可得实施例18化合物。拆分条件为：Supercritical fluid chromatography(ChiralPak AD 5μ,21x250mm col,27％甲醇,70mL/分钟)。
WS-421谱图数据：
1H-NMR(400MHz,CDCl3):δppm 7.52(d,J＝8.0Hz,1H),7.13-7.10(m,1H),7.03-6.95(m,3H),6.91-6.88(m,1H),5.65(s,2H),5.18(s,2H),4.56-3.92(m,3H),3.25-3.20(m,1H),2.52-2.51(m,1H),2.21-2.14(m,2H),2.09(s,3H),1.08-0.88(m,2H),0.72-0.65(m,2H).
MS:m/z 506.4[M+H]
WS-422谱图数据：
1H-NMR(400MHz,CDCl3):δppm 7.52(d,J＝8.0Hz,1H),7.13-7.10(m,1H),7.03-6.95(m,3H),6.91-6.88(m,1H),5.65(s,2H),5.18(s,2H),4.56-3.92(m,3H),3.25-3.20(m,1H),2.52-2.51(m,1H),2.21-2.14(m,2H),2.09(s,3H),1.08-0.88(m,2H),0.72-0.65(m,2H).
MS:m/z 506.4[M+H]
实施例19
5-氨基-1-(1-氰基-吡咯烷-3-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-423)的制备
Figure PCTCN2017081906-appb-000044
实施例19从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CDCl3):δppm 7.50(d,J＝8.4Hz,2H),7.16-7.10(m,1H),7.02-6.95(m,3H),6.92-6.88(m,1H),5.52(s,2H),5.34(s,2H),4.69-4.66(m,1H),3.87-3.76(m,3H),3.60-3.54(m,1H),2.56-2.51(m,1H),2.35-2.30(m,1H).
MS:m/z 425.3[M+H]
实施例20
5-氨基-1-(1-氰基哌啶-4-基)-3-(4-(2,4-二氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-424)的制备
Figure PCTCN2017081906-appb-000045
实施例20从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CDCl3):δppm 7.49(d,J＝8.8Hz,2H),7.15-7.09(m,1H),7.02-6.92(m,3H),6.92-6.88(m,1H),5.43(s,2H),5.21(s,2H),3.99-3.93 (m,1H),3.66-3.62(m,2H),3.23-3.17(m,2H),2.41-2.32(m,1H),2.03-2.01(m,1H).
MS:m/z 439.2[M+H]
实施例21
1-(1-丙烯酰基-6-甲基哌啶-3-基)-5-氨基-3-(4-(2-氯-4-氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-425)的制备
Figure PCTCN2017081906-appb-000046
实施例21从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CDCl3):δppm 7.52-7.49(m,2H),7.25-7.25(m,1H),7.09-6.97(m,4H),6.59-6.57(m,1H),6.32-6.12(m,1H),5.87-5.85(m,2H),5.68-5.60(m,2H),4.70-4.25(m,1H),3.88-3.85(m,1H),3.48-3.46(m,1H),2.72-2.61(m,1H),2.04-1.97(m,2H),1.82-1.77(m,2H),1.41-1.35(m,3H).
MS:m/z 498.2[M+H]
实施例22
5-氨基-3-(4-(2-氯-4-氟苯氧基)苯基)-1-(1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(WS-426)的制备
Figure PCTCN2017081906-appb-000047
实施例22从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CDCl3):δppm 7.49(d,J＝8.4Hz,2H),7.23-7.24(m,1H),7.07-7.11(m,1H),6.97-7.04(m,3H),6.55(s,1H),5.55(s,1H),5.21(s,1H),4.03-4.09(m,1H),3.60-3.71(m,2H),3.34-3.40(m,1H),2.29-2.35(m,1H),1.93-2.04(m,2H),1.82-1.86(m,1H),1.38(d,J＝6.8Hz,2H).
MS:m/z 469.3[M+H]
实施例23
5-氨基-1-(1-(2-丁炔酰基)-6-甲基哌啶-3-基)-3-(4-(2-氯-4-氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-427)的制备
Figure PCTCN2017081906-appb-000048
实施例23从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CDCl3):δppm 7.52-7.49(m,2H),7.29-7.23(m,1H),7.06-6.97(m,4H),5.63-5.61(m,2H),5.35-5.23(m,2H),4.91-4.72(m,1H), 3.91-3.81(m,1H),3.26-3.17(m,1H),2.51-2.42(m,2H),2.06(d,J＝16.8Hz,2H),1.81-1.76(m,2H),1.41-1.34(m,3H).
MS:m/z 510.2[M+H]
实施例24
5-氨基-3-(4-(4-氯-2-氟苯氧基)苯基)-1-(1-氰基-6-甲基哌啶-3-基)-1H-吡唑-4-甲酰胺(WS-428)的制备
Figure PCTCN2017081906-appb-000049
实施例24从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CD3OD):δppm 7.49(d,J＝8.8Hz,2H),7.40-7.36(m,1H),7.25-7.16(m,2H),7.07(d,J＝8.8Hz,2H),4.33(s,2H),3.70-3.63(m,1H),3.60-3.57(m,1H),3.42-3.38(m,1H),2.28-2.25(m,1H),2.19-2.19(m,3H),1.37(d,J＝6.8Hz,3H).
MS:m/z 469.1[M+H]
实施例25
1-(1-丙烯酰基-6-甲基哌啶-3-基)-5-氨基-3-(4-(4-氯-2-氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-429)的制备
Figure PCTCN2017081906-appb-000050
实施例25从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CDOD3):δppm 7.55-7.53(m,2H),7.40-7.38(m,1H),7.25-7.16(m,2H),7.09(d,J＝8.4Hz,1H),6.85-6.73(m,1H),6.24-6.15(m,1H),5.78-5.72(m,1H),4.70-4.40(m,2H),4.23-4.01(m,1H),2.38-2.32(m,1H),2.04-2.02(m,1H),1.91-1.77(m,2H),1.37(d,J＝6.8,3H).
MS:m/z 498.1[M+H]
实施例26
5-氨基-1-(1-(2-丁炔酰基)-6-甲基哌啶-3-基)-3-(4-(4-氯-2-氟苯氧基)苯基)-1H-吡唑-4-甲酰胺(WS-430)的制备
Figure PCTCN2017081906-appb-000051
实施例26从对应的起始物开始，采用与实施例6类似的方法制备而得。
1H-NMR(400MHz,CDCl3):δppm 7.54-7.50(m,2H),7.25-7.22(m,1H),7.20-7.02(m,4H),5.37(s,2H),5.92-5.91(m,1H),4.61-4.59(m,1H),4.68-4.41(m,1H),3.94-3.83(m,1H),3.69-3.63(m,1H),2.52-2.43(m,1H),2.03(d,J＝16.8Hz,3H),2.011-1.76(m,3H),1.41-1.34(m,3H).
MS:m/z 510.1[M+H]
实施例27～40
采用与本发明实施例1～26类似的方法，可制备实施例27～40所示结构的化合物。
Figure PCTCN2017081906-appb-000052
Figure PCTCN2017081906-appb-000053
Figure PCTCN2017081906-appb-000054
Figure PCTCN2017081906-appb-000055
实施例41
激酶活性抑制实验(BTK)
在基于时间分辨荧光共振能量转移方法的试验中，测试本申请公开的化合物对BTK激酶活性的抑制作用。重组的Btk与本申请公开的化合物在室温下在含有50mM Tris pH7.4、10mM MgCl2、2mM MnCl2、0.1mM EDTA、1mM DTT、20nM SEB、0.1％BSA、0.005％tween-20的试验缓冲液中预先温育1 小时。通过加入ATP(在ATP Km浓度下)和肽底物(Biotin-AVLESEEELYSSARQ-NH2)来引发反应。在室温下温育1小时后，加入等体积的含有50mM HEPES pH7.0、800mM KF、20mM EDTA、0.1％BSA、连接Eu穴合物的p-Tyr66抗体和链霉亲和素标记的XL665的终止液以终止反应。盘在室温下再温育1小时，然后在BMG PHERAstar FS仪器上读取TR-FRET信号(ex337nm，em 620nm/665nm)。基于615nm处的荧光与665nm处的荧光的比值，计算化合物浓度增加情况下残余酶活性。各化合物的IC50通过Graphpad Prism软件的四参数逻辑方程拟合数据而得到。
按照上述的实验方法，本申请中的部分化合物表现出较强的激酶抑制活性(IC50<1000nM)，其中一些优选化合物的激酶抑制活性很强(IC50<100nM)。具体结果见下表。
Figure PCTCN2017081906-appb-000056
激酶抑制活性等级分为A、B、C，具体地A(IC50<100nM)，B(100nM<IC50<1000nM)，C(IC50>1000nM)。
实施例42
体外激酶选择性实验
采用基于时间分辨荧光共振能量转移方法建立了EGFR，ITK激酶活性检测平台；采用Z’-Lyte方法建立了LCK，SRC，LYN激酶活性检测平台；采 用Lance Ultra方法建立了TEC和JAK3激酶活性检测平台，分别测试本申请公开的化合物对不同激酶活性的抑制作用。每个化合物分别在11个浓度下测定酶活性数据，用Graphpad Prism软件计算该化合物的IC50值。
按照上述的实验方法，本申请中的一些化合物表现出很强的激酶选择性，明显优于对照化合物依鲁替尼。结果见下表。
实施例编号 LCK SRC LYN EGFR ITK TEC
WS-411 B C B C C A
WS-413 C C C B C A
WS-416 C C C C C A
依鲁替尼 A A A A A A
激酶抑制活性等级分为A、B、C，具体地A(IC50<100nM)，B(100nM<IC50<1000nM)，C(IC50>1000nM)
实施例43
B细胞抑制实验
在活体外短暂暴露至BTK抑制剂足以抑制正常人类B细胞中的B细胞激活。此方案模拟活体内细胞至抑制剂的预测暴露，并且显示尽管清洗抑制剂但对B细胞的抑制仍得以保持。
B细胞是使用若赛特赛普(RosetteSep)人类B细胞富集混合剂通过阴性选择自健康供体血液纯化得到。将细胞铺板于生长培养基(10％RPMI+10％胎牛血清)中并添加指定浓度的抑制剂。于37℃下培育1小时后，将细胞洗涤三次，每次洗涤均利用于生长培养基中进行8倍稀释。随后将细胞用10μg/mL IgM F(ab')2在37℃下剌激18小时。随后用抗CD69-PE抗体对细胞进行染色并通过流式细胞术使用标准条件进行分析。
依照以上的方法进行测定，本申请中优选的化合物对B细胞具有较强的抑制活性，其IC50值小于10nM。
实施例44
T细胞抑制实验
T细胞是使用若赛特赛普(RosetteSep)人类T细胞富集混合剂通过阴性选择自健康供体血液纯化得到。将细胞铺板于生长培养基(10％RPMI+10％胎牛血清)中并添加指定浓度的抑制剂。于37℃下培育1小时后，将细胞洗涤三次，每次洗涤均利用于生长培养基中进行10倍稀释。随后将细胞用anti-CD3/CD28包被珠(珠/细胞比例为1:1)在37℃下剌激18小时。随后用抗CD69-PE抗体对细胞进行染色并通过流式细胞术使用标准条件进行分析。依照以上的方法进行测定，本申请中优选的化合物对T细胞具有很弱的抑制活性或无抑制，其IC50值大于4000nM。
实施例45
人全血B细胞抑制实验
人全血(hWB)获自健康志愿者，通过静脉穿刺将血液收集到用肝素钠抗凝化的Vacutainer管中。测试化合物在PBS中稀释至10倍所需初始药物浓度)，接着在10％的在PBS中的DMSO中三倍系列稀释，以得到9点的剂量响应曲线。将5.5μL的每种化合物稀释液一式两份添加到aiil 96孔V型底的板上；向对照和无刺激孔中添加5.5μL的10％在PBS中的DMSO。向每孔添加人全血(100μL)，在混合后将板在37C，5％CO2，100％湿度温育30分钟。在搅拌下向每孔(无刺激孔除外)添加羊F(ab')2抗人IgM(Southern Biotech)(10μL的500μg/mL溶液，50μg/mL最终浓度)，并且将板温育另外20小时。在20小时温育结束时，将样品与荧光探针标记的20μL APC小鼠抗人CD69(BD Pharmingen)在37C，5％CO2，100％湿度温育30分钟。包括用于补偿调节和初始电压设置的诱导对照、未染色的和单染色剂。然后将样品用1ml的IX Pharmingen Lyse Buffer(BD Pharmingen)裂解，并且将板在1500rpm离心5分钟。通过抽吸除去上清液，将残留的团粒用另外1ml的IX Pharmingen Lyse Buffer再次裂解，并且将板如前离心。吸出上清液，将残留的团粒在FACs缓 冲液(PBS+1％FBQ中洗涤。离心后并除去上清液后，将团粒重悬浮在150μL的FACs缓冲液中。将样品转移至适于在BD LSR II流式细胞器的HTS 96孔体系上运行的96孔板。采用适合所用荧光团的激发和发射波长，获取数据并且采用Cell Quest Software获得百分比阳性细胞值。结果最初用FACS分析软件(Flow Jo)分析。IC50值使用XLfit v3，公式201计算。
依照以上的方法进行测定，本申请中优选的化合物对人全血中B细胞具有较强的抑制活性，其IC50值小于200nM。
实施例46
化合物在肝微粒体中的稳定性研究
1.待测化合物溶解在乙腈中，制成浓度为0.5mM的储备液。
2.2μL储备液加入1.5ml离心管中，然后加入148μL磷酸缓冲液(100mM，pH 7.4)和10μL肝微粒体(蛋白浓度为20mg/ml)悬液【BD Gentest公司】，肝微粒体的种属分别为人，狗，大鼠，小鼠；对照组加入158μL磷酸缓冲液(100mM，pH 7.4)。
3.步骤2中制备好的混合体系，于37℃水浴中预孵3分钟，然后加入40μL NADPH发生体系(含有NADP+：6.5mM，葡萄糖6-磷酸：16.5mM,MgCl2：16.5mM，葡萄糖6-磷酸脱氢酶:2U/ml)启动反应，并于37℃水浴中孵育1小时。
4.反应进行1小时后，将离心管从水浴中取出，并加入400μL乙腈终止反应，然后涡旋震荡3分钟，最后离心(13000rpm，4℃)5分钟，取上清液用HPLC检测剩余药物浓度Cr。
5.平行制备0分钟反应样品的制备方法：步骤2中制备好的混合体系，于37℃水浴中预孵3分钟后取出，加入400μL乙腈，然后加入40μL NADPH发生体系。涡旋震荡3分钟后，离心(13000rpm，4℃)5分钟，取上清液用HPLC检测药物浓度C0。
6.经60分钟孵育后，药物在孵育体系中的剩余百分比按照下式计算：
药物剩余(％)＝Cr÷C0×100％
按照上述的实验方法，本申请中的一些优选化合物表现出较好的微粒体稳定性，其在各种属的肝微粒体中的剩余百分比>30％。
实施例47
评价化合物对CYP酶抑制作用
CYP酶代谢是药物生物转化的主要途径，其数量和活性大小直接影响药物在体内的活化与代谢。作为外源性化合物的主要代谢酶，细胞色素CYP是重要的药物I相代谢酶，可以催化多种外源性化合物的氧化和还原代谢。CYP酶在药物的消除过程中起着非常重要的作用，同时也是引起联合用药时药物相互作用产生的主要因素。
方法：本实验采用cocktail探针药物法同时测定化合物对人源肝微粒体中五种CYP450酶的抑制作用，人源微粒体来自BD Gentest公司。
实验步骤如下：
反应在100mM磷酸盐缓冲液中进行，总体积200μL。反应体系中微粒体浓度为0.25mg/mL，待测化合物浓度为20μM、6.67μM、2.22μM、0.74μM、0.25μM，特异性探针底物及浓度分别为非那西汀(CYP1A2)40μM、右美沙芬(CYP2D6)5μM、双氯芬酸(CYP2C9)10μM、S-美芬妥英(CYP2C19)40μM、睾酮(CYP3A4)80μM。孵育体系在37度恒温振荡器中预孵育5分钟，加入NADPH发生体系(含1.3mM NADP+、3.3mM葡萄糖6-磷酸、0.4U/L葡萄糖6-磷酸脱氢酶、3.3mM MgCL2)开始反应。孵育45分钟后加入等体积的乙腈终止反应，涡旋，13000rpm离心，取上清LC-MS-MS进样测定代谢产物生成量。特异性代谢产物分别为对乙酰氨基酚(CYP1A2)、右啡烷(CYP2D6)、4-羟基双氯芬酸(CYP2C9)、4-羟基美芬妥英(CYP2C19)、6β-羟基睾酮(CYP3A4)。特异性抑制剂分别为呋拉茶碱(CYP1A2)、奎尼丁(CYP2D6)、磺胺苯吡唑(CYP2C9)、反苯环丙胺(CYP2C19)、酮康唑(CYP3A4)。本实验最终结果为计算半数抑制浓度IC50值。IC50＝((50％-低抑制率％)/(高抑制率％-低抑制率％))×(高浓度-低浓度)+低浓度。
按照上述的实验方法，本申请的一些优选化合物对各种CYP酶均只有不强的抑制或无抑制，说明其对其它药物的代谢影响较小。
实施例48
化合物在大鼠体内的药物代谢动力学研究方法
1.雄性SD大鼠【华阜康】买入后，在本实验室适应性饲养7天。
2.9只SD大鼠随机分为3组，每组3只，一组用于灌胃给药，另一组用于尾静脉注射给药。灌胃给药组的大鼠，给药前需过夜禁食。
3.大鼠给药后，采用眼眶静脉丛采血的方法在以下时间点采集血样：I.V.:(给药前)，0.08小时，0.25小时，0.5小时，1小时，2小时，4小时，8小时，24小时。P.O.:0.08小时，0.25小时，0.5小时，1小时，2小时，4小时，8小时，24小时。每个采血时间点采血量约为300μl。
4.采集的血样在4℃以12000rpm的转速离心5分钟，然后采集上层血浆样品，并于-20℃冰箱中保存待测。
5.实验操作总结见表4：
表4、化合物在大鼠体内的药物代谢动力学试验设计
Figure PCTCN2017081906-appb-000057
6.使用LC-MS/MS(UPLC-MS/MS:液相Waters Acquity UPLC(USA)和质谱5500Q Trap(Applied Biosystem/MDS SCIEX)或者HPLC-MS\MS:液相Agilent 1200series(USA)和质谱API 4000(Applied Biosystem/MDS SCIEX))检测血浆中的化合物浓度。典型的检测条件如下：
Figure PCTCN2017081906-appb-000058
Figure PCTCN2017081906-appb-000059
Figure PCTCN2017081906-appb-000060
使用药代动力学专业软件WinNonlin 【型号：PhoenixTM
Figure PCTCN2017081906-appb-000061
6.1厂家：Pharsight Corporation】计算药代动力学参数【Phoenix 1.1User’s Guide:p251-p300】。
按照上述的实验方法，本申请中已测定的化合物表现出较好的生物利用度(>40％)。
实施例49
hERG结合实验(Dofetillide法)
依照专利US20050214870A1上描述的方法，可测定化合物对hERG抑制的IC50值。本申请中优选的化合物对hERG只有很弱的抑制作用或无抑制作用，其IC50值大于1000nM。
实施例50
药效学实验
免疫功能严重缺陷NOD.SCID小鼠购自北京维通利华实验动物技术有限公司，饲养于SPF级动物房。TMD-8细胞培养到足够数量后，离心收集细胞，PBS洗2遍。最后细胞用不含血清的RPMI1640培养基和基质胶(1:1v/v)重悬。采用1ml的注射器和25G注射器针头，将0.2ml的细胞悬液注入每只小鼠右侧翼皮下区域。植入一周左右用游标卡尺测量肿瘤大小，用以下公式计算肿瘤体积：肿瘤体积＝(长×宽2)/2。当肿瘤体积达到100-200mm3左右，将小鼠分组灌胃给药，连续给药21天。
化合物WS411，WS413，WS416，WS422能显著抑制弥漫性大B细胞淋巴瘤细胞株TMD-8体内的生长，并显示出与对照化合物依鲁替尼相同的抗肿瘤效果(实验结果请见图1)。
工业实用性
本发明实施方案所提供的新型5-氨基吡唑甲酰胺类衍生物，是蛋白激酶BTK的有效、安全、选择性高的抑制剂，可用作治疗BTK介导疾病的药品。

Claims (11)

  1. 一种5-氨基吡唑甲酰胺化合物，如式(I)所示，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
    Figure PCTCN2017081906-appb-100001
    其中，
    n，m独立地取自于0、1或2；
    L是O，-C(O)NH-，-CH2-，S，S(O)，NH或S(O)2；
    A取自于取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与母核及L的连接位点是任选的；
    B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点是任选的；
    R1和R2各自独立地选自氢、C1-C4烷基、卤素、氰基，或者R1和R2与它们相连的碳原子一起形成三元碳环或四元碳环，或者R1和R2合并为氧代基；
    Y选自氰基、
    Figure PCTCN2017081906-appb-100002
    R3、R4、R5和R6各自独立地选自氢、未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH2)qN(RaRb)，其中，q为1、2、3、或4，Ra和Rb各自独立地选自氢、未取代的C1-C4烷基；
    并规定，当R1和R2其中一个为氢而另一个为甲基，且Y为氰基、A为苯环、L为O、m为1和n为2时，B不是取代或未取代的苯环；当R1和R2都为氢，且A为苯环、m为1和n为2时，B不是取代或未取代的苯环、和取代或未取代的吡啶；当R1和R2都为氢，且A为吡啶环、m为1和n为2 时，B不是取代或未取代的苯环；当R1和R2都是氢，且A为苯环、L为O、m为1和n为1时，B不是取代或未取代的苯环。
  2. 如权利要求1所述的化合物，其中，n，m独立地取自于0、1或2；L为O，-C(O)NH-，-CH2-，NH或S，更优选地为O，-C(O)NH-，NH。
  3. 如权利要求1所述的化合物，其中，A取自于取代或未取代的杂环、取代或未取代的苯环、或着取代或未取代的杂芳环，并且与母核及L的连接位点是任选的；B独立地取自于取代或未取代的脂肪环、取代或未取代的杂环、取代或未取代的苯环、或者取代或未取代的杂芳环，并且与L的连接位点是任选的；其中：
    所述取代的苯环是指苯基上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；优选地，所述取代的苯环为氟取代的苯基、或氯取代的苯基，更优选地为2,4-二氟苯基、或4-氯苯基；
    所述未取代的杂芳环是指呋喃、吡咯、噻吩、恶唑、异恶唑、吡唑、咪唑、噻唑、异噻唑、恶二唑、三氮唑、噻二唑、四氮唑、吡啶、嘧啶、吡嗪、哒嗪、三嗪；所述取代的杂芳环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；更优选地，所述取代的吡啶为氯代吡啶，特别优选地为4-氯-吡啶-2-基；
    所述未取代的脂肪环是指环丙烷、环丁烷、环戊烷、环己烷、环庚烷、环辛烷；所述取代的脂肪环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基；
    所述未取代的杂环是指四氢呋喃、四氢吡喃、四氢吡咯、哌啶、
    Figure PCTCN2017081906-appb-100003
    Figure PCTCN2017081906-appb-100004
    其中w取自0、1或2；所述取代的杂环是指以上基团上任意位置被任选的下列取代基所取代，所述取代基选自氢、甲基、甲氧基、氟、氯、三氟甲基、三氟甲氧基或氰基。
  4. 如权利要求1所述的化合物，其中，优选地，R1和R2都是氢，或者其中一个为氢、而另一个为C1-C4烷基，或者R1和R2与它们相连的碳原子一起形成环丙基；更优选地，R1和R2都是氢，或者其中一个是氢而另一个为甲基，或者R1和R2与它们相连的碳原子一起形成环丙基。
  5. 如权利要求1所述的化合物，其中，优选地，为如式(II)所示的5-氨基吡唑甲酰胺化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
    Figure PCTCN2017081906-appb-100005
    其中，L、A、B和Y的定义如上述式(I)；
    并规定，当A为苯环时，B不是取代或未取代的苯环、和取代或未取代的吡啶；当A为吡啶环时，B不是取代或未取代的苯环；
    更优选地，式(II)所示的5-氨基吡唑甲酰胺化合物为下列化合物中的一种：
    Figure PCTCN2017081906-appb-100006
    其中，式(II-1)或(II-2)中L、B和Y的定义如上述式(I)；
    并规定，B不是取代或未取代的苯环、和取代或未取代的吡啶；或：
    优选地，为如式(III)所示的5-氨基吡唑甲酰胺化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
    Figure PCTCN2017081906-appb-100007
    其中，L、A、B和Y的定义如上述式(I)；
    并规定，当Y为氰基、A为苯环、L为O时，B不是取代或未取代的苯环；
    更优选地，式(III)所示的5-氨基吡唑甲酰胺化合物为下列化合物中的一种：
    Figure PCTCN2017081906-appb-100008
    其中，式(III-1)、(III-2)、(III-3)和(III-4)中L、B和Y的定义如上述式(I)；
    并规定，当Y为氰基、L为O时，B不是取代或未取代的苯环；或：
    优选地，为如式(IV)所示的5-氨基吡唑甲酰胺化合物，其立体异构体、互变异构体，或药学上可接受的盐，或溶剂化物，或前药：
    Figure PCTCN2017081906-appb-100009
    其中，L、A、B和Y的定义如上述式(I)；
    更优选地，式(IV)所示的5-氨基吡唑甲酰胺化合物为下列化合物中的一种：
    Figure PCTCN2017081906-appb-100010
    其中，式(IV-1)或(IV-2)中L、B和Y的定义如上述式(I)。
  6. 如权利要求5所述的化合物，其中，L为O；
    B为
    Figure PCTCN2017081906-appb-100011
    和
    Y为-CN、
    Figure PCTCN2017081906-appb-100012
    其中，R3、R4、R5和R6各自独立地选自氢、未取代的C1-C4烷基、羟基取代的C1-C4烷基、C1-C4烷氧基C1-4烷基、卤素、氰基、或-(CH2)qN(RaRb)，其中，q为1、2、3、或4，Ra和Rb各自独立地选自氢、未取代的C1-C4烷基。
  7. 选自下列化合物中的一种，或药学上可接受的盐，或溶剂化物，或前药：
    Figure PCTCN2017081906-appb-100013
    Figure PCTCN2017081906-appb-100014
    Figure PCTCN2017081906-appb-100015
    Figure PCTCN2017081906-appb-100016
    Figure PCTCN2017081906-appb-100017
  8. 权利要求1-7中任一项所述化合物的制备方法，包括如下步骤：
    (1)将式(V)化合物与式(VI)化合物反应，得到式(VII)化合物；
    Figure PCTCN2017081906-appb-100018
    (2)将式(VII)化合物发生水解反应，得到式(VIII)化合物；
    Figure PCTCN2017081906-appb-100019
    (3)将式(VIII)化合物经脱保护基PG得到式(IX)化合物；
    Figure PCTCN2017081906-appb-100020
    (4)将式(IX)化合物与式(X)化合物反应，得到式(I)化合物；
    Figure PCTCN2017081906-appb-100021
    在上述的式(V)、式(VI)、式(VII)、式(VIII)、式(IX)和式(X)中涉及的取代基R1、R2、L、A、B、Y和n、m定义如上面的式(I)，PG为氨基保护基，R3为C1-C4的烷基，优选地为乙基，X为氯、溴或羟基。
  9. 包含权利要求1-7中任一项所述化合物或其药学上可接受的盐的药物组合物。
  10. 权利要求1-7中任一项所述化合物或权利要求9所述药物组合物在制备BTK抑制剂药物中的用途。
  11. 如权利要求10所述的用途，其中，BTK抑制剂是指预防或治疗由 BTK介导的疾病，所述疾病选自自身免疫性疾病、炎性疾病、异种免疫性情况或疾病、血栓栓塞疾病和癌症。
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