WO2018161876A1 - 二氢喹喔啉类溴结构域识别蛋白抑制剂及制备方法和用途 - Google Patents

二氢喹喔啉类溴结构域识别蛋白抑制剂及制备方法和用途 Download PDF

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WO2018161876A1
WO2018161876A1 PCT/CN2018/078051 CN2018078051W WO2018161876A1 WO 2018161876 A1 WO2018161876 A1 WO 2018161876A1 CN 2018078051 W CN2018078051 W CN 2018078051W WO 2018161876 A1 WO2018161876 A1 WO 2018161876A1
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alkyl
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熊兵
沈竞康
缪泽鸿
胡剑萍
王迎庆
宋姗姗
孟韬
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中国科学院上海药物研究所
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Priority to US16/491,202 priority Critical patent/US11078188B2/en
Publication of WO2018161876A1 publication Critical patent/WO2018161876A1/zh

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    • 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
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    • 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
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
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    • 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
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
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    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • 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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    • C07D403/10—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 linked by a carbon chain containing aromatic rings
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    • C07D405/10—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 two hetero rings linked by a carbon chain containing aromatic rings
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    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D417/02—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 two hetero rings
    • C07D417/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D417/10—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 two hetero rings linked by a carbon chain containing aromatic rings
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    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
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    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/027—Organoboranes and organoborohydrides

Definitions

  • the present invention relates to a novel class of bromodomain recognition protein inhibitors and methods for their preparation, and to the use of such compounds in the manufacture of a medicament for the treatment of a disease mediated by a bromodomain recognition protein.
  • bromodomains a region was found in Drosophila and female sterile homologous proteins and was named bromodomains (BRDs).
  • the bromodomain is a conserved domain in many chromatin and transcription related proteins that recognize acetylated lysine. So far, 61 bromodomains have been found on 46 different proteins in the human genome, which can be divided into eight families, of which the BET (Bromo-and Extra-terminal) family is more studied.
  • the BET family includes BRD2, BRD3, BRD4, and BRDT specifically expressed in testis tissue, which are widely expressed in tissues.
  • bromodomain protein abnormalities are closely related to many diseases, such as cancer, inflammation, immune diseases and cardiovascular diseases.
  • the relationship between bromodomain protein abnormalities and tumors is complex, and there is no specific pathway that causes tumors.
  • One of the mechanisms may be that a bromodomain protein can form an oncogenic fusion protein, such as an oncogenic fusion protein found in line cancer (NMC) in the NUT (a gene located on chromosome 15).
  • NMC line cancer
  • the NUT coding region was inserted into chromosome 19 to form the BRD4-NUT fusion protein at the 3' end of the BRD4 gene, and in a few cases, it was found to be a BRD3-NUT fusion protein.
  • bromodomain protein recognizes acetylated lysine and can recruit transcription factors to induce downstream gene expression, such as the oncogene C-MYC, etc., and it is difficult to find small molecules to directly inhibit these genes, so it can be indirectly inhibited by inhibiting bromodomain proteins. Inhibit gene expression. Therefore, it is meaningful to study the synthesis of selective inhibitors of bromodomain proteins.
  • kinase inhibitors can also inhibit bromodomain proteins, especially BRD4, and it has been found that BRD4 exhibits the properties of atypical kinases, which can phosphorylate the RNAC IIC-terminal serine at position 2.
  • BRD4 exhibits the properties of atypical kinases, which can phosphorylate the RNAC IIC-terminal serine at position 2.
  • the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 have good activity against BRD4 with IC 50 of 25 nM and 130 nM, respectively.
  • the wavy line indicates that the configuration is an R-type or an S-type or a racemate
  • Ring A is a 5-10 membered heteroaryl group, a 5-8 membered heterocyclic group, or a C6-C10 aryl group;
  • X is C or N
  • R 4 is a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted benzyl group, 5-10 membered heterocyclyl or 5-10 membered heteroaryl;
  • R 5 is absent, hydrogen, a substituted or unsubstituted C1-C6 alkyl
  • R 6 and R 7 are each independently hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted a substituted benzyl group, a substituted or unsubstituted 5-10 membered heterocyclic group, or a substituted or unsubstituted 5-10 membered heteroaryl; or R 6 and R 7 together with the nitrogen atom to which they are attached form a substitution or not Substituted 5-15 membered heterocyclic group;
  • substitution means having one or more substituents selected from the group consisting of halogen, hydroxy, nitro, cyano, tert-butoxycarbonyl, C1-C6 alkyl, C1-C6 alkoxy, C3-C10 ring Alkyl, NR 8 R 9 ; each R 8 , each R 9 is independently hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), armor Oxycarbonyl (Fmoc), methoxycarbonyl, ethoxycarbonyl, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), pivaloyl, benzoyl, triphenyl (Trt), 2,4-dimethoxybenzyl (Dmb), p-methoxybenzyl (PMB), or
  • the A ring is a C6-C10 aryl group, a 5-6 membered heterocyclic group or a 5-6 membered heteroaryl group.
  • R 1 is hydrogen, a substituted or unsubstituted C1-C4 alkyl group, or a substituted or unsubstituted C1-C4 alkoxy group, said substituent being a halogen, a hydroxyl group, an amino group, a nitro group or Cyano group.
  • R 2 is a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted C1-C4 alkoxy group, and the substituent is a halogen, a hydroxyl group, an amino group, a nitro group or a cyano group.
  • R 4 is a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted hetero atom having 1-3 selected from N, O and S. a group of 5-8 membered heterocyclic or 5-8 membered heteroaryl, said substituent having 1-3 substituents, each substituent being independently: halogen, hydroxy, amino, nitro, cyano , C1-C4 alkyl, or C1-C4 alkoxy.
  • R 5 is a hydrogen-free, substituted or unsubstituted C1-C4 alkyl group
  • R 6 and R 7 are each independently selected from hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C6-C14 aryl, substituted or unsubstituted Substituted benzyl, substituted or unsubstituted group having 1-3 heteroatoms selected from N, O and S: a 5-8 membered heterocyclic group and a 5-8 membered heteroaryl group, said substitution being Refers to having 1-3 substituents, each substituent being independently: halogen, hydroxy, amino, methylamino, cyano, N(C1-C4 alkyl)(C1-C4 alkyl), C1-C4 alkyl or C1 -C4 alkoxy;
  • R 6 and R 7 together with the nitrogen atom to which they are attached form a substituted or unsubstituted 5-10 membered heterocyclic group having 1 to 3 N, O, S heteroatoms, said substitution being 1-3 a substituent, each substituent being independently: halogen, NR 8 R 9 , hydroxy, nitro, cyano, tert-butyloxycarbyl, C1-C4 alkyl, C3-C8 cycloalkyl, or C1-C4 alkane
  • Each of R 8 and each R 9 is independently hydrogen, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), methoxycarbonyl, ethoxycarbonyl, p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), pivaloyl, benzoyl, 2,4-dimethoxybenzyl (Dmb), p-methoxybenzyl (PMB
  • the A ring is selected from the group consisting of phenyl, pyridyl, pyrimidinyl, triazolyl, tetrazolyl, thiazolidinyl, pyrazolyl, oxazolyl, isoxazolyl and imidazole base.
  • R 1 is methyl, ethyl, propyl or isopropyl.
  • R 2 is methyl, ethyl, propyl or isopropyl.
  • R 4 is a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted benzyl group, a substituted or unsubstituted one or three hetero atoms selected from N, O and S. a group having 5-6 membered heterocyclic group or 5-6 membered heteroaryl group, said substituent having 1-3 substituents, each substituent being independently: fluorine, chlorine, bromine, hydroxyl group, amino group, nitrate Base, cyano, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy or isopropoxy.
  • R 5 is a hydrogen atom, a substituted or unsubstituted methyl group, a substituted or unsubstituted ethyl group, a substituted or unsubstituted propyl group, a substituted or unsubstituted isopropyl group
  • R 6 and R 7 are each independently selected from a hydrogen atom, a substituted or unsubstituted methyl group, a substituted or unsubstituted ethyl group, a substituted or unsubstituted propyl group, a substituted or unsubstituted isopropyl group, a substituted or unsubstituted group.
  • Substituted piperidinyl said substituent means having 1-3 substituents, each substituent being independently: fluorine, chlorine, bromine, hydroxy, amino, methylamino, cyano, -N(CH 3 ) 2 ,- N(CH 2 CH 3 ) 2 , -N(CH 3 )(CH 2 CH 3 ), methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy base;
  • R 6 and R 7 together with the nitrogen atom to which they are attached form a substituted or unsubstituted group: piperazinyl, homopiperazinyl, piperidinyl,
  • the substitution means having 1-3 substituents, and each substituent is independently: fluorine, chlorine, bromine, hydroxyl, nitro, cyano, methyl, ethyl, propyl, isopropyl, cyclopropyl, Cyclobutyl, cyclopentyl, cyclohexyl, tert-butoxycarbonyl, NR 8 R 9 ; each R 8 , each R 9 is independently hydrogen, tert-butoxycarbonyl (Boc), methoxycarbonyl, ethoxycarbonyl, Pivaloyl, benzoyl, benzyl (Bn).
  • the A ring is selected from the group consisting of phenyl, triazolyl, tetrazolyl, thiazolidinyl, pyrazolyl and isoxazolyl; and X is C or N.
  • R 1 is a methyl group.
  • R 2 is a methyl group.
  • R 4 is a substituted or unsubstituted group: phenyl, oxazolyl, 1,3-dioxolanyl, and said substituent means having 1-3 substituents, each The substituents are independently: fluorine, chlorine and methyl;
  • R 5 is none, hydrogen atom, methyl group
  • R 6 and R 7 are independently selected from a hydrogen atom, a substituted or unsubstituted methyl group, a substituted or unsubstituted ethyl group; and the substituent means having 1-3 substituents, each substituent being independently:- N(CH 3 ) 2 , methyl (preferably the substitution means substitution by -N(CH 3 ) 2 ); or R 6 and R 7 together with the nitrogen atom to which they are attached form a substituted or unsubstituted Group: piperazinyl, homopiperazinyl, The substitution means having 1-3 substituents, and each substituent is independently methyl, ethyl, isopropyl, cyclopropyl, NH 2 , N(CH 3 ) 2 , NHBoc.
  • the R 1 , R 2 , R 3 , R 4 , R 5 , X, A ring and the wavy line are each independently the corresponding group of each specific compound in the examples.
  • the compound is:
  • a process for the preparation of a compound of the first aspect which comprises the step of preparing a compound of formula I by a compound of formula V,
  • the preparation method comprises the step of preparing a compound of formula I by a compound of formula V,
  • the preparation of the compound is as shown in the following scheme.
  • a pharmaceutical composition comprising the compound of the formula (I) according to the first aspect, or a stereoisomer, a prodrug thereof, a protein targeted degradation conjugate, a solvent
  • a pharmaceutically acceptable carrier One or more of a compound, a hydrate, a crystalline form, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the related disease mediated by the bromodomain recognition protein is selected from the group consisting of a malignant tumor, an immune disease, a cardiovascular disease, or inflammation.
  • the malignant tumor is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, B cell chronic lymphocytic leukemia, chronic myelomonocytic leukemia, midline cancer, lung cancer, B cell lymphoma, prostate cancer , gastric cancer, colorectal cancer, kidney cancer, liver cancer, breast cancer, pancreatic cancer.
  • the protein targeted degradation conjugate is synthesized using a PROTAC (proteolysis targeting chimeric molecule) technique.
  • C1-C6 means having 1 to 6 carbon atoms
  • C3-C10 means having 3 to 10 carbon atoms, and so on.
  • 5-8 yuan means that there are 5-8 atoms on the ring, 5-10 yuan means 5-10 atoms on the ring, and so on.
  • Alkyl means a saturated aliphatic hydrocarbon group which is a branched alkyl group or a linear alkyl group.
  • Cycloalkyl means an all-carbon monocyclic ring, spiro ring, bridged ring or fused ring such as cyclopropyl, cyclopentyl or cyclohexyl.
  • Alkoxy means -O-(alkyl), such as methoxy, ethoxy;
  • Aryl means an all-carbon monocyclic ring or a fused ring polycyclic group having a complete conjugated ⁇ -electron system, and examples of aryl groups are, but not limited to, phenyl, naphthyl, and anthracenyl;
  • Heterocyclyl means a monocyclic, spiro, bridge or fused ring containing one, two, three, four or five ring heteroatoms selected from N, O, S and P, and the remaining ring atoms, if The existence of C, such a ring may also have one or more double bonds, but such a ring does not have a complete conjugated ⁇ -electron system;
  • Heteroaryl means a monocyclic or fused ring containing one, two, three or four ring heteroatoms selected from N, O, S and P, the remainder if the ring atom is C, and furthermore, Has a complete conjugated ⁇ -electron system.
  • the compound represented by the formula (I), the compound of the formula I, and the compound of the formula I all refer to a bromodomain recognition protein inhibitor having the following structure:
  • the compound represented by the formula (I) may contain an asymmetric or chiral center and thus may exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, and mixtures thereof (eg, racemic mixtures) are included in the present invention. In the range.
  • the compounds of the formula (I) may also exist in different tautomeric forms, all of which are included in the scope of the invention.
  • tautomer or “tautomeric form” refers to structural isomers of different energies that are converted to each other via a low energy barrier.
  • the compound of formula (I) may exist in unsolvated as well as solvated forms containing pharmaceutically acceptable solvents such as water, ethanol, and the like, and the compounds of the present invention include both solvated and unsolvated forms.
  • the compound represented by the formula (I) has a basic group and thus can form a pharmaceutically acceptable salt (ie, a pharmaceutically acceptable salt) with an inorganic or organic acid, including a pharmaceutically acceptable acid addition salt, by using an inorganic
  • a pharmaceutically acceptable salt can be obtained by treating the free base of the compound of the formula (I) with an acid or an organic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid, and the organic acid such as ascorbic acid.
  • niacin citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, oxalic acid, malic acid, glycolic acid, succinic acid, propionic acid, acetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzene Sulfonic acid, p-toluenesulfonic acid, etc.
  • the invention also encompasses isotopically-labeled compounds of the invention, in addition to the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number common in nature.
  • isotopes that may be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as: 2 hydrogen, 3 hydrogen, 11 carbon, 13 carbon, 14 carbon, respectively. , 13 nitrogen, 15 nitrogen, 15 oxygen, 17 oxygen, 18 oxygen, 31 phosphorus, 32 phosphorus, 35 sulfur, 18 fluorine, 123 iodine, 125 iodine and 36 chlorine.
  • isotopically-labeled compounds of the invention are used in compound and/or substrate tissue distribution assays.
  • Deuterated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred because they are easy to prepare and detect.
  • substitution of heavier isotopes such as deuterium (ie, 2 H) may provide certain therapeutic advantages resulting from greater metabolic stability (eg, increased in vivo half-life or reduced dosage requirements) and, in some cases, may be Preferred.
  • Positron emission isotopes such as 15 O, 13 N, 11 C, and 18 F are used in positron emission tomography (PET) studies to examine substrate receptor occupancy.
  • Isotopically labeled compounds of the invention can generally be prepared by replacing the non-isotopically labeled reagent with an isotopically labeled reagent, following procedures similar to those disclosed in the Schemes and/or the Examples below.
  • reaction schemes shown below provide possible pathways for the synthesis of the compounds of the invention as well as key intermediates.
  • the compounds of the formula (I) of the present invention can be synthesized by methods including those well known in the chemical arts, especially in accordance with the description of the present invention.
  • the starting materials are generally available from commercial sources such as Sigma Aldrich or are readily prepared using methods well known to those skilled in the art.
  • the compound in the reaction scheme includes a salt thereof, for example, a salt as defined by the compound of the formula (I), etc., i.e., a free base of the compound treated with an organic acid or a mineral acid, to give a salt of the corresponding compound.
  • a salt thereof for example, a salt as defined by the compound of the formula (I), etc., i.e., a free base of the compound treated with an organic acid or a mineral acid, to give a salt of the corresponding compound.
  • the preparation method of the above compound represented by the structural formula (I) includes
  • Step a Compound 1A is reacted with thionyl chloride and methanol to obtain compound 1B;
  • Step b Compound 1B is reacted with a different primary amine R 3 NH 2 by nucleophilic substitution to give compound 1C;
  • Step c compound 1C is reduced under iron powder and ammonium chloride to obtain compound 1D;
  • Step d 1) Compound 1D with a different 2-bromoalkanoyl bromide The reaction gives an intermediate, 2) the intermediate is obtained by intramolecular nucleophilic reaction under N,N-diisopropylethylamine to obtain compound 1E;
  • Step e Compound 1E is reacted with R 1 I or R 1 Br under sodium hydride to obtain compound 1F;
  • Step f Compound 1F is reacted with hydrazine hydrate to obtain compound 1G;
  • Step g Compound 1G with a different sulfamide Guanhuan gives compound 1H,
  • Step a compound 2A and a different amine R 4 NH 2 by condensation reaction to obtain compound 2B;
  • Step b compound 2B and a different primary amine R 3 NH 2 by nucleophilic substitution reaction to obtain compound 2C;
  • Step c compound 2C is reacted with Lawesson reagent to obtain compound 2D;
  • Step d compound 2D and trimethylsilyl azide are reacted under mercury acetate to obtain compound 2E;
  • Step e compound 2E is reduced under tin dichloride dihydrate to obtain compound 2F;
  • Step f 1) Compound 2F with a different 2-bromoalkanoyl bromide The reaction gives an intermediate, 2) the intermediate is obtained by intramolecular nucleophilic reaction under N,N-diisopropylethylamine to obtain compound 2G;
  • Step g Compound 2G is reacted with R 1 I or R 1 Br under sodium hydride to obtain compound 2H.
  • Step a compound 3A with thioglycolic acid and a different amine R 4 NH 2 under dicyclohexylcarbodiimide conditions to obtain compound 3B;
  • Step b compound 3B is reacted with a different primary amine R 3 NH 2 by nucleophilic substitution to give compound 3C;
  • Step c compound 3C is reduced under tin dichloride dihydrate to obtain compound 3D;
  • Step d 1) Compound 3D with a different 2-bromoalkanoyl bromide The reaction gives an intermediate, 2) the intermediate is obtained by intramolecular nucleophilic reaction under N,N-diisopropylethylamine to obtain compound 3E;
  • Step e Compound 3E is reacted with R 1 I or R 1 Br under sodium hydride to obtain compound 3F.
  • Step a Compound 4A is reacted with amino acid NH 2 R 2 COOH under potassium carbonate to obtain compound 4B;
  • Step b Compound 4B is reacted under the conditions of sodium dithionite and potassium carbonate to obtain compound 4C;
  • Step c Compound 4C with a different ketone
  • the compound 4D is obtained by a reductive amination reaction or a condensation reaction with a different acid chloride under the conditions of phenylsilane and dibutyltin dichloride;
  • Step d compound 4D is reacted with R 1 I or R 1 Br under sodium hydride to obtain compound 4E;
  • Step e compound 4E and boronic acid pinacol ester in potassium acetate and [1,1 '-bis (diphenylphosphino) ferrocene] palladium chloride dichloromethane complex conditions to obtain compound 4F;
  • Step f Compound 4F with a different ring Coupling reaction under the conditions of sodium hydrogencarbonate and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride methylene chloride complex to obtain compound 4G,
  • Step a Compound 5A is reacted with thionyl chloride and methanol to obtain compound 5B;
  • Step b Compound 5B with an amino acid NH 2 R 2 COOH to give compound 5C under conditions of potassium carbonate;
  • Step c compound 5C is reacted under the conditions of sodium dithionite and potassium carbonate to obtain compound 5D;
  • Step d Compound 5D with different ketones Reaction under phenylsilane and dibutyltin dichloride to obtain compound 5E;
  • Step e Compound 5E is reacted with R 1 I or R 1 Br under sodium hydride to obtain compound 5F;
  • Step f compound 5F is reacted with hydrazine hydrate to obtain compound 5G;
  • Step g Compound 5G with a different sulfamide Guanhuan gave compound 5H.
  • Scheme 5 provides another method for synthesizing the A ring to be a triazole, similar to Scheme 1, but Scheme 5 provides a synthetic method for the parent core R 2 to be an R form, an S form, or a racemate. Only a synthetic method in which the parent core R 2 is a racemate can be provided.
  • R 1 -R 5 and the X and A rings are as defined above, and the dotted line indicates the absence or a single bond.
  • a “pharmaceutical composition” comprises one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, and a pharmaceutically acceptable carrier, for example, Other chemical components such as a mixture of physiologically/pharmaceutically acceptable carriers and excipients; the purpose of the pharmaceutical composition is to aid in the administration of the compound to an organism.
  • physiologically/pharmaceutically acceptable carrier means that the carrier, excipient or diluent does not cause significant irritation to the organism and does not abrogate the biological activity and properties of the administered compound.
  • “Pharmaceutically acceptable excipient” means an inert property that is added to a pharmaceutical composition to further aid in the administration of the compound.
  • excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starch types. , cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • the pharmaceutical composition of the present invention comprises a therapeutically effective amount of a compound represented by the formula (I), and stereoisomers thereof, pharmaceutically acceptable salts, prodrugs, solvates, hydrates and crystals thereof One or more of the types, and at least one excipient, diluent or carrier.
  • “Therapeutically effective amount” means an amount of a compound administered which will alleviate one or more conditions of the condition being treated to some extent, and for the treatment of cancer, a therapeutically effective amount means that the amount has at least one of the following effects :
  • the compound represented by the general formula (I) of the present invention, and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates or crystal forms thereof can be used in monotherapy or combination therapy.
  • the compounds of the formula (I) of the present invention, and stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates and crystal forms thereof are generally associated with small molecule-based compounds, radiation Antibody therapy (eg, Herceptin and Rituxima) is used in combination with anti-cancer vaccination, gene therapy, cell therapy, hormone therapy, or cytokine therapy.
  • a typical formulation is prepared by mixing the compound of the formula (I) of the present invention with a carrier, diluent or excipient.
  • Suitable carriers, diluents or excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oil, A substance such as a solvent or water.
  • the particular carrier, diluent or excipient used will depend on the mode and purpose for which the compound of the invention is applied.
  • the solvent is generally selected based on a solvent which is considered safely (GRAS) to a mammal in the art.
  • safe solvents are non-toxic aqueous solvents such as water, as well as other non-toxic solvents that are soluble in water or miscible with water.
  • Suitable aqueous solvents include mixtures of one or more of water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG300), and the like.
  • the formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, a coloring agent, sweetener, flavoring agent, flavoring agent or other known additive to provide a beautiful presentation of the drug (ie, a compound of the invention or a pharmaceutical composition thereof), or to assist the pharmaceutical product (also That is, the manufacture of drugs.
  • This formulation can be prepared using conventional dissolution mixing procedures.
  • a block-like drug substance i.e., a compound represented by the formula (I) of the present invention or a stabilized form of the compound (e.g., with a ring)
  • the dextrin derivative or a complex of other known complexing agents is dissolved in a suitable solvent.
  • the compound represented by the general formula (I) of the present invention is typically formulated into a pharmaceutical dosage form to provide easy control of the drug. The dose, and provides the patient with an easy to handle product.
  • a compound of the invention or a combination of a compound of the invention and at least one other agent is preferably administered in the form of a pharmaceutical composition.
  • the compounds or combinations of the invention can be administered orally, rectally, transdermally, parenterally (e.g., intravenously, intramuscularly, or subcutaneously) intracerebrocerally, intravaginally, intraperitoneally, intravesically, locally (e.g., Powder, ointment or droplets, buccal or nasal dosage forms, administered separately or together to a patient.
  • compositions suitable for parenteral injection generally comprising a pharmaceutically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and for reconstitution into a sterile injectable solution or dispersion Bacteria powder.
  • Suitable aqueous or non-aqueous vehicles or diluents including solvents and carriers, including mixtures of one or more of water, ethanol, polyol (propylene glycol, polyethylene glycol, glycerol, etc.); vegetable oils (such as olives) Oil); and injectable organic esters such as ethyl oleate.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, in the case of dispersions, by the maintenance of the required particle size, or by the use of surfactants.
  • compositions may also contain excipients such as preservatives, wetting agents, emulsifying agents and dispersing agents. Microbial contamination of the composition can be avoided by various bactericides and fungicides, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. These compositions may also include isotonic agents such as sugars, sodium chloride, and the like. The absorption of the injectable pharmaceutical compositions can also be extended by the use of agents which delay absorption, such as aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration can include capsules, tablets, powders, and granules.
  • the compound or combination of the invention is admixed with at least one inert excipient, diluent or carrier.
  • Suitable excipients, diluents or carriers include those such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders (such as starch, lactose, sucrose, mannitol, silicic acid, etc.); b) binders (such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic, etc.); (c) wetting agents (such as glycerin, etc.); (d) disintegrants (such as Agar, calcium carbonate, potato or tapioca starch, alginic acid, specific complex silicate, sodium carbonate, etc.); (e) solution blockers (such as paraffin, etc.); (f) accelerated absorbers (such as sodium
  • the dosage form may also include a buffer.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose as well as high molecular weight polyethylene glycols and the like as excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents; solubilizers and emulsifiers such as ethanol, isopropyl alcohol, ethyl carbonate, acetic acid Ethyl ester, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide; oils (such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, etc.) Glycerin; tetrahydrofurfuryl alcohol; fatty acid ester of polyethylene glycol and sorbitan; or a mixture of several of these.
  • an inert diluent commonly used in the art, such as water or other solvents
  • compositions may also include excipients such as one or more of wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, and flavoring agents.
  • excipients such as one or more of wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, and flavoring agents.
  • a carrier such as a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan ester, microcrystals may be further included.
  • a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan ester, microcrystals.
  • compositions for rectal or vaginal administration preferably include suppositories, which may be prepared by admixing a compound or combination of the present invention with a suitable non-irritating excipient or carrier, such as a cocoa butter, Ethylene glycol or suppository wax, which is solid at room temperature and liquid at body temperature, and thus can be melted in the rectum or vagina to release the active compound.
  • a suitable non-irritating excipient or carrier such as a cocoa butter, Ethylene glycol or suppository wax, which is solid at room temperature and liquid at body temperature, and thus can be melted in the rectum or vagina to release the active compound.
  • the dosage form of the compound of the present invention and the compound of the present invention in combination with a blood cancer or an inflammatory drug for topical administration may include ointments, powders, sprays, and inhalants.
  • the medicament can be mixed under sterile conditions with a pharmaceutically acceptable excipient, diluent or carrier, and any preservative, buffer or propellant required.
  • Ophthalmic formulations, ophthalmic ointments, powders and solutions are also intended to be encompassed within the scope of the invention.
  • the compounds (or compositions) of the invention can be placed in drinking water whereby a therapeutic dose of the compound is ingested along with the daily water supply.
  • the compound can be metered directly into the drinking water, preferably in the form of a liquid water-soluble concentrate such as an aqueous solution of a water-soluble salt.
  • a paste formulation can be prepared by dispersing the drug in a pharmaceutically acceptable oil such as peanut oil, sesame oil, corn oil or the like.
  • a pill containing an effective amount of a compound, pharmaceutical composition or combination of the present invention may be prepared by mixing a compound or composition of the present invention with a diluent such as a carbow wax, palm wax or the like; A lubricant such as magnesium stearate or calcium stearate to enhance the pelleting process.
  • a diluent such as a carbow wax, palm wax or the like
  • a lubricant such as magnesium stearate or calcium stearate to enhance the pelleting process.
  • the compound represented by the formula (I) of the present invention and a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form thereof, are selective inhibitors of a protein recognized by a bromine domain, It inhibits the recognition of lysine acetylation by the bromodomain recognition protein.
  • the present invention also provides a compound represented by the formula (I), and a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form thereof as a selective inhibitor of a bromodomain recognition protein.
  • a compound represented by the formula (I) and a stereoisomer, a pharmaceutically acceptable salt, a prodrug, a solvate, a hydrate or a crystal form thereof as a selective inhibitor of a bromodomain recognition protein.
  • bromodomain recognition protein inhibitors acute lymphoblastic leukemia, acute myeloid leukemia, B-cell chronic lymphocytic leukemia, chronic myelomonocytic leukemia, etc.
  • Malignant tumors midline cancer, lung cancer, B cell lymphoma, prostate cancer, stomach cancer, colorectal cancer, kidney cancer, liver cancer, breast cancer, pancreatic cancer, immune diseases, cardiovascular diseases, and inflammation.
  • related diseases mediated by bromodomain recognition proteins include, but are not limited to, hematological malignancies, midline cancer, and diseases such as inflammation.
  • the compound represented by the formula (I) of the present invention can be used for the preparation of therapeutic hematological malignancy.
  • a drug for diseases such as tumors, midline cancer, and inflammation, preferably for preparing blood malignant tumors including, but not limited to, acute lymphocytic leukemia, acute myeloid leukemia, B cell chronic lymphocytic leukemia, chronic myelomonocytic leukemia, and the like
  • Drugs for diseases such as midline cancer, lung cancer, B cell lymphoma, prostate cancer, stomach cancer, colorectal cancer, kidney cancer, liver cancer, breast cancer, pancreatic cancer, immune diseases, cardiovascular diseases, and inflammation.
  • the present invention also provides a method of treating a disease, condition and/or dysfunction mediated by a bromodomain recognition protein inhibitor, the method comprising administering to a patient an effective amount of a compound of formula (I) And its stereoisomers, pharmaceutically acceptable salts, prodrugs, solvates, hydrates and crystal forms.
  • the structure of the compound is determined by nuclear magnetic resonance ( 1 H-NMR) and/or mass spectrometry (MS).
  • the NMR measurement was carried out by a Varian company's Mercury-400 NMR spectrometer, and the solvent was deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), deuterated dimethyl sulfoxide (DMSO-d 6 ) or deuterated.
  • Acetonitrile (CD 3 CN), TMS is an internal standard.
  • the measurement of MS was carried out using a Thermo Finnigan LCQ-Deca XP type (ESI) liquid chromatography-mass spectrometer. Separation and purification of the product by column chromatography using ISCO Rf 75 rapid preparation chromatograph, the carrier uses 200-300 mesh silica gel from Qingdao Ocean Chemical Plant.
  • the synthesis yield is a molar yield.
  • Reagents and conditions a) thionyl chloride (SOCl 2 ), methanol, reflux at 12 ° C for 12 hours; b) cyclopropylamine, 1,2-dichloroethane, reflux at 80 ° C for 12 hours; c) iron powder, chlorination Ammonium solution, ethanol, reacted at 80 ° C for 1 hour; d) 1.2-bromopropionyl bromide, N,N-diisopropylethylamine (DIPEA), dichloromethane, reacted at room temperature for 2 hours; 2.
  • DIPEA 1.2-bromopropionyl bromide, N,N-diisopropylethylamine
  • Reagents and conditions a) p-methylaniline, 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), DIPEA, DMF, Reactive overnight at room temperature; b) cyclopropylamine, 1,2-dichloroethane, refluxed at 80 ° C for 12 hours; c) Lawson's reagent, toluene, refluxed at 110 ° C overnight; d) 1. Hydrazine hydrate, reaction at room temperature for 4 hours; .
  • Reagents and conditions a) p-methylaniline, 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), DIPEA, DMF, Reactive overnight at room temperature; b) cyclopropylamine, 1,2-dichloroethane, refluxed at 80 ° C for 12 hours; c) Lawson's reagent, toluene, refluxed at 110 ° C overnight; d) tetrahydrofuran, mercuric acetate, trimethylsilyl azide , 0 ° C reaction for 4 hours; e) tin dichloride dihydrate, concentrated hydrochloric acid, room temperature reaction for 3 hours; f) 1.2-bromopropionyl bromide, DIPEA, dichloromethane, room temperature reaction for 2 hours; 2. acetonitrile, DIPEA, The reaction was carried out at 80 °
  • Compound 4 was prepared in the same manner as in Example 3 except that cyclopentylamine was used instead of cyclopropylamine in the step b.
  • Reagents and conditions a) p-toluidine, thioglycolic acid, dicyclohexylcarbodiimide, tetrahydrofuran, 0 ° C - room temperature, 12 hours; b) cyclopropylamine, 1,2-dichloroethane, reflux at 80 ° C for 12 hours ; c) tin dichloride dihydrate, concentrated hydrochloric acid, 0 ° C - room temperature, 3 hours; d) 1.2-bromopropionyl bromide, N, N-diisopropylethylamine (DIPEA), dichloromethane, room temperature reaction 2 hours; 2. Acetonitrile, DIPEA, reaction at 80 ° C overnight; e) sodium hydride, N,N-dimethylformamide (DMF), methyl iodide, reaction at room temperature for 1 hour.
  • DIPEA 1.2-bromopropionyl bromide, N, N-
  • Reagents and conditions a) D-aminopropionic acid, potassium carbonate, ethanol, water, 80 ° C, 8 hours; b) potassium carbonate, sodium dithionite, water, 60 ° C reaction overnight; c) benzene silane, cyclopentanone, Dibutyltin dichloride, THF, room temperature, 10 hours; d) NaH, DMF, methyl iodide, 0 ° C - room temperature, 4 hours; e) boranoic acid pinacol ester, potassium acetate, dioxane, [ 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex, refluxed at 120 ° C overnight; f) 5-bromo-1-(p-tolyl)-1H-1, Ethyl 2,4-triazole-3-carboxylate, sodium hydrogencarbonate, THF, [1,1'-bis(diphenylphosphino)
  • Compound 10 was prepared in the same manner as in Example 6 except that 1-ethylpiperazine was used instead of N-methylpiperazine in the step h.
  • Compound 11 was prepared in the same manner as in Example 6 except that 1-cyclopropylpiperazine was used instead of N-methylpiperazine in the step h.
  • Compound 12 was prepared in the same manner as in Example 6 except that (2S,6R)-2,6-dimethylpiperazine was used instead of N-methylpiperazine in the step h.
  • Compound 13 was prepared in the same manner as in Example 6 except that N 1 , N 1 , N 2 -trimethylethane-1,2-diamine was used instead of N-methylpiperazine in the step h.
  • Compound 14 was prepared in the same manner as in Example 6 except that 8-methyl-3,8-diazabicyclo[3.2.1]octane was used instead of N-methylpiperazine in the step h.
  • Compound 15 was prepared in the same manner as in Example 6 except that 1-isopropylpiperazine was used instead of N-methylpiperazine in the step h.
  • Compound 18 was prepared in the same manner as in Example 6 except that 3-methyl-3,8-diazabicyclo[3.2.1]octane was used instead of N-methylpiperazine in the step h.
  • Compound 19 was prepared in the same manner as in Example 6 except that 1-methyl-1,4-diazepane was used instead of N-methylpiperazine in the step h.
  • Compound 20 was prepared in the same manner as in Example 6 except that 8-azaspiro[4.5]decane was used instead of N-methylpiperazine in the step h.
  • Compound 21 was prepared in the same manner as in Example 6 except that 2-oxo-8-azaspiro[4.5]decane was used instead of N-methylpiperazine in the step h.
  • Compound 22 was prepared in the same manner as in Example 6 except that 1,4-diazabicyclo[4.3.0]decane was used instead of N-methylpiperazine in the step h.
  • the operation method of the step a in the embodiment 32 is the same as the step a in the first embodiment, and the operation method of the step be is the same as the step ad in the sixth embodiment.
  • the operation method of the step fg is the same as the step fg in the first embodiment.
  • the operation method of the step ad in the embodiment 33 is the same as that in the first embodiment, and the operation method of the step eh is the same as the eh in the sixth embodiment.
  • the compound was prepared in the same manner as in Example 12 except that 3,3-difluorocyclobutanone was used instead of cyclopentanone in the step c.
  • the compound was prepared in the same manner as in Example 12 except that the cyclopentanone was replaced with 2-oxo-7-azaspiro[3.5]decane-7-carboxylic acid tert-butyl ester in the step c.
  • the compound was prepared in the same manner as in Example 12 except that 2-oxo-spiro[3.3]heptane-6-one was used instead of cyclopentanone in the step c.
  • Reagents and conditions a); b); c) dinacol borate, potassium acetate, dioxane, [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride Methane complex, refluxed at 120 ° C overnight; d) lithium hydroxide, THF, H 2 O, room temperature, 12 hours; e) HATU, DMF, cis-2,6-dimethylpiperazine, DIPEA, room temperature reaction Overnight; f) sodium hydrogencarbonate, THF, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex, refluxed at 80 ° C overnight.
  • the compound was prepared in the same manner as in Example 44 except that propionyl chloride was used instead of cyclopropylcarbonyl chloride in the step a.
  • the compound was prepared in the same manner as in Example 44 except that isobutyl chloride was used instead of cyclopropylcarbonyl chloride in the step a.
  • Reagents and conditions a) sodium borohydride, THF, tetrahydrofuran, room temperature for 2 hours; b) Example 6 Intermediate F, sodium bicarbonate, THF, [1,1'-bis(diphenylphosphino)ferrocene] Palladium dichloride methylene chloride complex, refluxed at 80 ° C overnight; c) methanesulfonyl chloride, triethylamine, dichloromethane, room temperature for 1 hour; d) cis-2,6-dimethylpiperazine, N , N-diisopropylethylamine, DMF, overnight at 60 ° C
  • the binding activity of the compound to BRD4 (I) was tested using a fluorescence anisotropy test (Fluorescence Anisotropy, FA).
  • the principle of FA test is to calculate the fluorescence polarization values in the horizontal and vertical directions by detecting the change of molecular weight before and after the interaction of fluorescein-labeled small molecules with other molecules. If the equilibrium of the binding between the fluorescently labeled small molecule and the macromolecule is established, it will move slowly when excited, and the measured fluorescence polarization value will increase.
  • the binding between the fluorescently labeled small molecule and the macromolecule is replaced by another ligand, its rotation or flipping speed in the free state will be faster, and the emitted light will be depolarized relative to the plane of the excitation light, and the measured polarized light value Decrease to calculate the fluorescence anisotropy of the sample.
  • BRD4(I) recognition domain colonies of newly transformed plasmid DNA from E. coli BL21(DE3)-condon plus-RIL cells containing 50 ⁇ g/mL kanamycin and 34 ⁇ g/mL The cells were grown overnight at 37 ° C in 50 mL of Terrific Broth medium (starting culture). The starter culture was then diluted 100-fold in 1 L of fresh TB medium and the cells were grown at 37 °C to an optical density of about 0.8 at OD600 and then the temperature was lowered to 16 °C.
  • lysis buffer [50 mmol 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), pH 7.5 at 25 ° C, 500 mmol NaCl, 10 mmol imidazole, 5% glycerol and fresh 0.5 mmol of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 1 mmol of phenylmethanesulfonyl fluoride (PMSF) were added and lysed at 4 ° C with a JN 3000PLUS high pressure homogenizer (JNBIO-Guangzhou, China) .
  • HEP 4-hydroxyethylpiperazineethanesulfonic acid
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • PMSF phenylmethanesulfonyl fluoride
  • the lysate was clarified by centrifugation (12,000 x g for 1 hour at 4 ° C) and applied to a nickel-nitriloacetate agarose column.
  • the column was washed once with 50 mL of wash buffer containing 30 mmol of imidazole.
  • the protein was eluted using imidazole in a stepwise elution in an elution buffer (100-250 mmol imidazole in 50 mmol HEPES at 25 ° C, pH 7.5, 500 mmol NaCl, 5% glycerol).
  • Samples were monitored by SDS-polyacrylamide gel electrophoresis and concentrated to 8-10 mg/mL with gel filtration buffer, 10 mmol Hepes pH 7.5, 500 mM NaCl, 1 mmol DTT, and used for protein binding assays and crystallization.
  • the fluorogenic substrate is (+)-JQ1 linked to a fluorescent molecule and has a working concentration of 5 nM.
  • BRD4(I) protein working concentration is 10nM
  • total reaction system is 40 ⁇ L
  • buffer is 50mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) pH 7.4, 150mM NaCl, 0.5mM 3-[3-(cholamide Dimethylamino]propanesulfonic acid inner salt (CHAPS).
  • the initial screening concentration of the compound was 1 ⁇ M, and the IC 50 of the compound having an inhibition rate greater than 60% under this condition was determined.
  • the final concentration of DMSO was chosen to be 0.2%, taking into account the solubility of the compound and the effect of DMSO on the assay.
  • the test used Corning's all-black, low-side, NBS surface 384-well microplate (Cat. No. CLS3575).
  • the test instrument was a BioTek synergy 2 detector with an excitation of 485 nM and an emission of 530 nM. Use buffer as the blank value for the system reading.
  • the S curve is taken as the concentration of the compound and the corresponding inhibition rate. To give the corresponding compound of IC 50.
  • Fluorescent substrate refers to the product of (+)-JQ1 linked to a fluorescent molecule through a linker
  • Pharmacological data The pharmacological test results of some of the compounds of the present invention are disclosed in Table 1 below, and the control used in the test is a bromodomain egg recognition white BRD4 inhibitor (+)-JQ1.
  • the compounds of the present invention have a good inhibitory activity against the BRD4(I) protein, and the compounds listed in the table have better molecular activities than the positive (+)-JQ1 activities.
  • the MM.1S cell line was tested for cell viability.
  • the test methods were as follows: human myeloma cells MM.1S were added and treated with compound for 72h, and the proliferation inhibition effect and degree of the compound were detected by CCK-8 method.
  • Pharmacological data The pharmacological test results of some of the compounds of the present invention are disclosed in Table 2 below, and the control used in the test is a bromodomain egg recognition white BRD4 inhibitor (+)-JQ1.
  • the compounds of the present invention have a good inhibitory activity against the MM.1S cell line, especially the compounds of Examples 1, 6, 12, 14, 18, 33, 34, 35, 36 and 37.
  • the .1S cell line has better inhibitory activity against the MM.1S cell line than the positive (+)-JQ1.
  • the system was 150 ⁇ l of human liver microsomes (final concentration 0.5 mg/ml) for metabolic stability incubation.
  • the system contained reduced coenzyme II (NADPH) (final concentration 1 mM) and 1 ⁇ M compound, positive control or negative control, respectively.
  • NADPH reduced coenzyme II
  • the reaction was terminated with acetonitrile containing (imiprozine, batch number: 3221; tinidazole, given) at 0 min, 5 min, 10 min, and 30 min, vortexed for 10 min, centrifuged at 15000 rpm for 10 min, and 50 ⁇ l of the supernatant was injected into a 96-well plate.
  • the metabolic stability of the compound was calculated by measuring the relative reduction in the original drug.
  • HLM Clint refers to the clearance of compounds in human liver microsomes in ul/min/mg
  • mLM Clint (ul/min/mg) refers to compounds in mouse liver microsomes. Metabolic clearance in ul/min/mg
  • HLM t 1/2 (min) refers to the half-life of the compound in human liver microsomes, in minutes
  • mLM t 1/2 (min) refers to the compound The half-life of metabolism in mouse human liver microsomes, in minutes
  • TDI refers to the mechanism inhibition test of compounds on CYP enzyme, evaluating whether the compound has time-dependent inhibition of the enzyme, if the calculated k obs is greater than 200, indicating the compound There is a mechanism of inhibition of the enzyme.
  • the compounds of the present invention are highly stable in human and mouse liver microsomes, especially that compounds 12 and 27 are very stable in liver microsome metabolism in mice, with half-lives of 193 minutes and 248 minutes. . Moreover, this class of compounds has no direct inhibition or mechanism inhibition on CYP450 enzyme.
  • Balb/C nude mice (6 weeks, female, Beijing Huakang Biotechnology Co., Ltd.) were purchased, and the animals were conditioned for about one week before the test.
  • MM.1S cells were cultured in vitro, and the cells in logarithmic growth phase were resuspended in serum-free RPMI1640 medium to adjust the cell concentration.
  • the cell suspension was injected into the Balb/C nude mice with a syringe before the right armpits, each Animals were injected with 150 ⁇ L (8.0 ⁇ 10 6 /piece).
  • the animals were divided into 4 groups by the randomized block method, the solvent-containing control group, the 50 mg/kg OTX-015 group, and the 20 and 50 mg/kg compound 6 groups, each group of 6 Animals were divided into groups and administered for 19 days.
  • the tumor diameter was measured twice a week, the body weight of the animals was weighed, the living state of the animals was observed, and abnormal conditions were recorded.
  • TV 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width, respectively.
  • RTV relative tumor volume
  • T/C (%) (T RTV / C RTV ) ⁇ 100%, T RTV : treatment group RTV; C RTV : negative Control group RTV.
  • OTX-015 is a novel BRD2/3/4 inhibitor with antiproliferative activity against some B cell tumor cell lines with an IC 50 of 192 nM for DLBCL cells.
  • Compound 6 had a significant inhibitory effect on the growth of MM.1S nude mice xenografts by oral administration of 50 mg/kg per day.

Abstract

本发明涉及一种二氢喹喔啉类溴结构域识别蛋白抑制剂及制备方法和用途。本发明的抑制剂为通式(I)所示的化合物，或其立体异构体、可药用盐、前药、溶剂化物、水合物和晶型，各取代基的定义如说明书和权利要求书所述。本发明通式(I)所示的化合物能够抑制溴结构域识别蛋白，可以用于制备调控细胞的表观状态和治疗由溴结构域识别蛋白介导的一系列疾病和症状的药物。

Description

二氢喹喔啉类溴结构域识别蛋白抑制剂及制备方法和用途 技术领域
本发明涉及一类新型结构的溴结构域识别蛋白抑制剂及其制备方法，还涉及此类化合物在制备用于治疗由溴结构域识别蛋白介导的疾病的药物中的应用。
背景技术
1992年，在果蝇和雌性不育同源蛋白中共同发现了一个区域并且被命名为溴结构域(bromodomains，BRDs)。溴结构域在许多与染色质和转录相关蛋白中是一个保守的结构域可以去识别乙酰化赖氨酸。到目前为止，在人类基因组的46种不同的蛋白上发现61种溴结构域，可以分为八大家族，其中对BET(Bromo-and Extra-terminal)家族研究较多。BET家族包括在组织中广泛表达的BRD2，BRD3，BRD4和在睾丸组织中特定表达的BRDT。
研究发现溴结构域蛋白异常与很多疾病密切相关，比如癌症，炎症，免疫性疾病以及心血管疾病等。溴结构域蛋白异常与肿瘤的关系很复杂，没有一个引起肿瘤的特定通路。其中一个机制可能是溴结构域蛋白可以形成致癌融合蛋白，比如在NUT(位于染色体15上的一个基因)中线癌(NMC)中发现致癌融合蛋白。在大多数NMC中，NUT编码区域插入到染色体19上BRD4基因3’端形成BRD4-NUT融合蛋白，在少数情况下，发现是BRD3-NUT融合蛋白。敲除BRD4-NUT和BRD3-NUT融合蛋白导致细胞鳞状分化和细胞复制停止。而且溴结构域蛋白识别乙酰化赖氨酸之后可以招募转录因子引起下游基因表达，比如癌基因C-MYC等，而很难找到小分子直接抑制这些基因，所以可以通过抑制溴结构域蛋白来间接抑制基因表达。因此研究合成溴结构域蛋白选择性抑制剂很有意义。
在2010年，人们发现了两个BET家族选择性抑制剂(+)-JQ1和I-BET762，从而引起人们对溴结构域抑制剂的广泛关注。随着研究人员的不断努力，越来越多BET家族选择性抑制剂被发现而且近几年对非BET家族的溴结构域蛋白的抑制剂研究越来越多，这些溴结构域蛋白抑制剂可以帮助更好地了解蛋白的功能以及它相关的疾病。
近几年研究发现很多激酶抑制剂也可以抑制溴结构域蛋白尤其是BRD4，并且研究发现BRD4显示出非典型激酶的性质，即可以使RNA聚合酶ⅡC端丝氨酸2位磷酸化。在这些激酶抑制剂中PLK1抑制剂BI2536以及JAK2抑制剂TG101209对BRD4有很好的活性，IC 50分别是25nM和130nM。
本领域仍需开发选择性溴结构域识别蛋白抑制剂，希望可以减少脱靶的副作用或者为以后药物联合作用提供更多的可能。
发明内容
本发明的目的在于提供一种选择性的溴结构域识别蛋白抑制剂。
本发明的第一方面，提供一种如通式(I)所示的化合物，或其立体异构体、前药、 溶剂化物、水合物、晶型、或其药学上可接受的盐：
Figure PCTCN2018078051-appb-000001
式中：
R 1、R 2、R 3各自独立地为氢、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C3-C10环烷基、取代或未取代的3-8元杂环基、取代或未取代的-C(＝O)R X或取代或未取代的苄基；其中，R X为C1-C6烷基、C1-C6烷氧基或C3-C10环烷基；
波浪线表示构型是R型或者S型或者消旋体；
A环为5-10元杂芳基、5-8元杂环基、或C6-C10芳基；
X为C或者N；
R 4为取代或未取代的C6-C20芳基、取代或未取代的苄基、5-10元杂环基或5-10元杂芳基；
R 5为无、氢、取代或未取代的C1-C6烷基、
Figure PCTCN2018078051-appb-000002
其中，R 6、R 7各自独立地为氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C10环烷基、取代或未取代的C6-C20芳基、取代或未取代的苄基、取代或未取代的5-10元杂环基、或取代或未取代的5-10元杂芳基；或者，R 6和R 7和它们连接的氮原子一起形成取代或未取代的5-15元杂环基；
所述取代是指具有选自下组的一个或多个取代基：卤素、羟基、硝基、氰基、叔丁氧羰基、C1-C6烷基、C1-C6烷氧基、C3-C10环烷基、NR 8R 9；各R 8、各R 9独立地为氢、C1-C6烷基、C3-C10环烷基、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、芴甲氧羰基(Fmoc)、甲氧羰基、乙氧羰基、邻苯二甲酰基(Pht)、对甲苯磺酰基(Tos)、三氟乙酰基(Tfa)、特戊酰基、苯甲酰基、三苯甲基(Trt)、2,4-二甲氧基苄基(Dmb)、对甲氧基苄基(PMB)、或苄基(Bn)。
在另一优选例中，A环为C6-C10芳基、5-6元杂环基或5-6元杂芳基。
在另一优选例中，R 1为氢、取代或未取代的C1-C4烷基、或取代或未取代的C1-C4烷氧基，所述取代基为卤素、羟基、氨基、硝基或氰基。
在另一优选例中，R 2为取代或未取代的C1-C4烷基、取代或未取代的C1-C4烷氧基，所述取代基为卤素、羟基、氨基、硝基或氰基。
在另一优选例中，R 3为取代或未取代的C1-C4烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C3-C6环烷基、取代或未取代的3-8元杂环基、取代或未取代的-C(＝O)R X或取代或未取代的苄基，其中，R X为C1-C6烷基、C1-C6烷氧基或C3-C6环烷基，所述取代基选自下组：卤素、羟基、氨基、硝基、氰基、C1-C3烷基、或者C1-C3烷氧基。
在另一优选例中，R 4为取代或未取代的C6-C14芳基、取代或未取代的苄基、取代或未取代的含有1-3个选自N、O和S的杂原子的如下基团：5-8元杂环基或5-8元杂芳基，所述取代是指具有1-3个取代基，各取代基独立为：卤素、羟基、氨基、硝基、氰基、C1-C4烷基、或者C1-C4烷氧基。
在另一优选例中，R 5为无、氢、取代或未取代的C1-C4烷基、
Figure PCTCN2018078051-appb-000003
Figure PCTCN2018078051-appb-000004
其中R 6、R 7各自独立地选自氢、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C14芳基、取代或未取代的苄基、取代或未取代的含有1-3个选自N、O和S的杂原子的如下基团：5-8元杂环基和5-8元杂芳基，所述取代是指具有1-3个取代基，各取代基独立为：卤素、羟基、氨基、甲氨基、氰基、N(C1-C4烷基)(C1-C4烷基)、C1-C4烷基或者C1-C4烷氧基；
或者，R 6和R 7和它们连接的氮原子一起形成取代或未取代的含有1-3个N、O、S杂原子的5-10元杂环基，所述取代是指具有1-3个取代基，各取代基独立为：卤素、NR 8R 9、羟基、硝基、氰基、叔丁基氧碳基、C1-C4烷基、C3-C8环烷基、或者C1-C4烷氧基；各R 8、各R 9独立地为氢、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、甲氧羰基、乙氧羰基、对甲苯磺酰基(Tos)、三氟乙酰基(Tfa)、特戊酰基、苯甲酰基、2,4-二甲氧基苄基(Dmb)、对甲氧基苄基(PMB)、苄基(Bn)。
在另一优选例中，A环选自如下基团：苯基、吡啶基、嘧啶基、三唑基、四唑基、噻唑烷基、吡唑基、噁唑基、异噁唑基和咪唑基。
在另一优选例中，R 1为甲基、乙基、丙基或异丙基。
在另一优选例中，R 2为甲基、乙基、丙基或异丙基。
在另一优选例中，R 3为环丙基、环丁基、环戊基、环己基、
Figure PCTCN2018078051-appb-000005
Figure PCTCN2018078051-appb-000006
或-C(＝O)R X；其中，R X为C1-C4烷基或C3-C6环烷基。
在另一优选例中，R 4为取代或未取代的C6-C10芳基、取代或未取代的苄基、取代或未取代的含有1-3个选自N、O和S的杂原子的如下基团：5-6元杂环基或5-6元杂芳基，所述取代是指具有1-3个取代基，各取代基独立为：氟、氯、溴、羟基、氨基、硝基、氰基、甲基、乙基、丙基、异丙基、甲氧基、乙氧基、丙氧基或者异丙氧基。
在另一优选例中，R 5为氢原子、取代或未取代的甲基、取代或未取代的乙基、取代或未取代的丙基、取代或未取代的异丙基、
Figure PCTCN2018078051-appb-000007
其中R 6、R 7各自独立地选自氢原子、取代或未取代的甲基、取代或未取代的乙基、取代或未取代的丙基、取代或未取代的异丙基、取代或未取代的哌啶基；所述取代是指具有1-3个取代基，各取代基独立为：氟、氯、溴、羟基、氨基、甲氨基、氰基、-N(CH 3) 2、-N(CH 2CH 3) 2、-N(CH 3)(CH 2CH 3)、甲基、乙基、丙基、异丙基、甲氧基、乙氧基、丙氧基、异丙氧基；
或者，R 6和R 7和它们连接的氮原子一起形成如下取代或未取代的基团：哌嗪基、高哌嗪基、哌啶基、
Figure PCTCN2018078051-appb-000008
所述取代是指具有1-3个取代基，各取代基独立为：氟、氯、溴、羟基、硝基、氰基、甲基、乙基、丙基、异丙基、环丙基、环丁基、环戊基、环己基、叔丁氧碳基、NR 8R 9；各R 8、各R 9独立地为氢、叔丁氧羰基(Boc)、甲氧羰基、乙氧羰基、特戊酰基、苯甲酰基、苄基(Bn)。
在另一优选例中，A环选自如下基团：苯基、三唑基、四唑基、噻唑烷基、吡唑基和异噁唑基；X为C或者N。
在另一优选例中，R 1为甲基。
在另一优选例中，R 2为甲基。
在另一优选例中，R 3为环丙基、环戊基、
Figure PCTCN2018078051-appb-000009
或-C(＝O)R X；其中，R X为C1-C4烷基或C3-C6环烷基。
在另一优选例中，R 4为取代或未取代的如下基团：苯基、噁唑基、1,3-二氧戊烷基，所述取代是指具有1-3个取代基，各取代基独立为：氟、氯和甲基；
在另一优选例中，R 5为无、氢原子、甲基、
Figure PCTCN2018078051-appb-000010
其中R 6、R 7独立地选自为氢原子、取代或未取代的甲基、取代或未取代的乙基；所述取代是指具有1-3个取代基，各取代基独立为：-N(CH 3) 2、甲基(较佳地所述取代是指被-N(CH 3) 2取代)；或者，R 6和R 7和它们连接的氮原子一起形成如下取代或未取代的基团：哌嗪基、高哌嗪基、
Figure PCTCN2018078051-appb-000011
所述取代是指具有1-3个取代基，各取代基独立为：甲基、乙基、异丙基、环丙基、NH 2、N(CH 3) 2、NHBoc。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、X、A环和波浪线各自独立为实施例中各具体化合物中相应的基团。
在另一优选例中，所述化合物为：
Figure PCTCN2018078051-appb-000012
Figure PCTCN2018078051-appb-000013
Figure PCTCN2018078051-appb-000014
本发明的第二方面，提供一种第一方面所述的化合物的制备方法，所述制备方法包括式V化合物制备式I化合物的步骤，
Figure PCTCN2018078051-appb-000015
所述制备方法包括式V化合物制备式I化合物的步骤，
各式中，各取代基和波浪线的定义如前所述。
在另一优选例中，所述化合物的制备方法如以下路线所示。
Figure PCTCN2018078051-appb-000016
各式中，各取代基和波浪线的定义如前所述。
本发明的第三方面，提供一种药物组合物，包括第一方面所述的通式(I)所示的化合物，或其立体异构体、前药、蛋白靶向降解偶联物、溶剂化物、水合物、晶型、或其药学上可接受的盐中的一种或多种，以及药学上可接受的载体。
本发明的第四方面，提供第一方面所述的通式(I)所示的化合物，或其立体异构体、前药、蛋白靶向降解偶联物、溶剂化物、水合物、晶型、或其药学上可接受的盐或第三方面所述的药物组合物的用途，(i)用于制备溴结构域识别蛋白的选择性抑制剂；或(ii)用于制备预防和/或治疗由溴结构域识别蛋白介导的相关疾病的药物。
在另一优选例中，所述由溴结构域识别蛋白介导的相关疾病选自：恶性肿瘤、免疫性疾病、心血管疾病或炎症。
在另一优选例中，所述恶性肿瘤选自：急性淋巴细胞白血病、急性骨髓性白血病、B细胞慢性淋巴细胞白血病、慢性骨髓单核细胞白血病、中线癌、肺癌、B细胞淋巴瘤、前列腺癌、胃癌、结肠直肠癌、肾癌、肝癌、乳腺癌、胰腺癌。
本发明的一优选实施方式中，所述蛋白靶向降解偶联物采用PROTAC(proteolysis targeting chimeric molecule)技术合成。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本申请的发明人经过广泛而深入地研究，首次研发出一种结构新颖的二氢喹喔啉类溴结构域识别蛋白抑制剂，结构如式I所示。在此基础上，完成了本发明。
定义：
除非另有述及，否则于本专利说明书和权利要求书使用的下列术语具有下文所讨论的意义。在本部分中定义的变量，例如，R 1-R 5以及X等仅供此部分内参考，并非意味具有如可使用于此定义部分外的相同意义。再者，此处定义的许多基团可选择性地被取代。在此定义部分中典型取代基的清单是作为举例，并非意欲限制本专利说明书和权利要求书中别处所定义的取代基。
C1-C6是指具有1-6个碳原子，C3-C10是指具有3-10个碳原子，依此类推。
5-8元是指环上具有5-8个原子，5-10元是指环上具有5-10个原子，依此类推。
“烷基”是指饱和脂肪烃基团，为支链烷基或直链烷基。
“环烷基”是指全碳单环状环、螺环、桥环或稠环，如环丙基、环戊基、环己基。
“烷氧基”是指-O-(烷基)，如甲氧基、乙氧基；
“芳基”是指全碳单环状环或稠合环多环基团，具有完整共轭π电子系统，芳基的实例是但不限于苯基、萘基以及蒽基；
“杂环基”是指单环状、螺环、桥环或稠环，含有一、二、三、四或五个选自N、O、S以及P的环杂原子，其余环原子，如果存在为C，这样的环亦可具有一或多个双键，但是这样的环并未具有完整共轭π电子系统；
“杂芳基”是指单环状或稠合环，含有一、二、三或四个选自N、O、S以及P的环杂原子，其余，如果存在环原子为C，且此外，具有完整共轭π电子系统。
式I化合物
本发明中，通式(I)所示的化合物，式I化合物，式I所示的化合物，均是指具有如下结构的溴结构域识别蛋白抑制剂：
Figure PCTCN2018078051-appb-000017
各取代基和波浪线的定义如前。
通式(I)所示的化合物可以含有不对称或手性中心，因此可以以不同立体异构体形式存在。本发明化合物的所有立体异构体形式，包括但不限于非对映异构体、对映异构体和阻转异构体以及它们的混合物(如外消旋混合物)，均包括在本发明的范围内。
通式(I)所示的化合物还可以以不同互变异构形式存在，所有这些形式均包括在本发明范围内。术语“互变异构体”或“互变异构形式”是指经由低能垒相互转化的不同能量的结构异构体。
通式(I)所示的化合物可以以非溶剂化形式和含有药学上可接受的溶剂(如水、乙醇等)的溶剂化形式存在，本发明的化合物包括溶剂化和非溶剂化形式。
通式(I)所示的化合物具有碱性基团，因此可与无机酸或有机酸形成药学上可接受的盐(即可药用盐)，包括可药用酸加成盐，通过用无机酸或有机酸处理通式(I)所示的化合物的游离碱，可以得到药学上可接受的盐，所述的无机酸如盐酸、氢溴酸、磷酸和硫酸，所述的有机酸如抗坏血酸、烟酸、柠檬酸、酒石酸、乳酸、马来酸、丙二酸、富马酸、草酸、苹果酸、乙醇酸、琥珀酸、丙酸、乙酸、甲磺酸、三氟甲磺酸、苯磺酸、对甲苯磺酸等。
本发明也涵盖经同位素标记的本发明化合物，除了一个或多个原子是被原子质量或质量数不同于自然中常见的原子质量或质量数之一原子所置换的事实之外，其是与此述者相同。可纳入本发明的化合物中的同位素实例，包括氢、碳、氮、氧、磷、硫、氟、碘及氯之同位素，其分别诸如： 2氢、 3氢、 11碳、 13碳、 14碳、 13氮、 15氮、 15氧、 17氧、 18氧、 31磷、 32磷、 35硫、 18氟、 123碘、 125碘及 36氯。
某些同位素标记的本发明的化合物(例如用 3H和 14C标记的那些)用于化合物和/或底物组织分布试验。特别优选氚化(即 3H)和碳-14(即 14C)同位素，因为它们容易制备和检测。而且，较重的同位素如氘(即 2H)进行取代可以提供由较大的代谢稳定性导致的某些治疗优点(例如体内半衰期增加或剂量需求减小)，因而在某些情况下可能是优选的。正电子发射同位素，例如 15O、 13N、 11C和 18F用于正电子发射体层摄影术(PET)研究，以检查底物受体占用率。同位素标记的本发明的化合物一般可以遵循类似于在方案和/或下文实施例中所公开的方法，通过用同位素标记的试剂替代非同位素标记的试剂来制备。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
制备方法
为了说明之用，下列所示的反应流程图提供用于合成本发明的化合物以及关键中间产物的可能途径。有关个别反应步骤的更详细的说明，请见后述的实施例部分。本发明通式(I)所示的化合物可以通过包括化学领域众所周知的那些方法来合成，尤其根据本发明的说明来合成。原料一般可以从商业来源如西格玛奥德里奇公司获得，或者使用本领域技术人员熟知的方法容易地制备。
在反应路线中的化合物包括其盐，例如，如具有通式(I)的化合物定义的盐类等，即用有机酸或无机酸处理化合物的游离碱的形式，得到相应化合物的盐。
上述具有结构通式(I)表示的化合物的制备方法包括，
反应路线一：
Figure PCTCN2018078051-appb-000018
步骤a：化合物1A与二氯亚砜和甲醇反应得到化合物1B；
步骤b：化合物1B与不同的伯胺R 3NH 2通过亲核取代反应得到化合物1C；
步骤c：化合物1C在铁粉和氯化铵条件下还原得到化合物1D；
步骤d：1)化合物1D与不同的2-溴烷酰基溴
Figure PCTCN2018078051-appb-000019
反应得到中间体，2)中间体在N,N-二异丙基乙胺条件下通过分子内亲核反应得到化合物1E；
步骤e：化合物1E与R 1I或R 1Br在氢化钠条件下反应得到化合物1F；
步骤f：化合物1F与水合肼反应得到化合物1G；
步骤g：化合物1G与不同的硫酰胺
Figure PCTCN2018078051-appb-000020
关环得到化合物1H，
或者反应路线二：
Figure PCTCN2018078051-appb-000021
步骤a：化合物2A与不同的胺R 4NH 2通过缩合反应的得到化合物2B；
步骤b：化合物2B与不同的伯胺R 3NH 2通过亲核取代反应得到化合物2C；
步骤c：化合物2C与劳森试剂反应得到化合物2D；
步骤d：化合物2D与三甲基硅叠氮在醋酸汞条件下反应得到化合物2E；
步骤e：化合物2E在二水合二氯化锡条件下还原得到化合物2F；
步骤f：1)化合物2F与不同的2-溴烷酰基溴
Figure PCTCN2018078051-appb-000022
反应得到中间体，2)中间体在N,N-二异丙基乙胺条件下通过分子内亲核反应得到化合物2G；
步骤g：化合物2G与R 1I或R 1Br在氢化钠条件下反应得到化合物2H，
或者反应路线三：
Figure PCTCN2018078051-appb-000023
步骤a：化合物3A与巯基乙酸和不同的胺R 4NH 2在二环己基碳二亚胺条件下关环得到化合物3B；
步骤b：化合物3B与不同的伯胺R 3NH 2通过亲核取代反应得到化合物3C；
步骤c：化合物3C在二水合二氯化锡条件下还原得到化合物3D；
步骤d：1)化合物3D与不同的2-溴烷酰基溴
Figure PCTCN2018078051-appb-000024
反应得到中间体，2)中间体在N,N-二异丙基乙胺条件下通过分子内亲核反应得到化合物3E；
步骤e：化合物3E与R 1I或R 1Br在氢化钠条件下反应得到化合物3F，
或者反应路线四：
Figure PCTCN2018078051-appb-000025
步骤a：化合物4A与氨基酸NH 2R 2COOH在碳酸钾条件下反应得到化合物4B；
步骤b：化合物4B在连二亚硫酸钠和碳酸钾的条件下反应得到化合物4C；
步骤c：化合物4C与不同的酮
Figure PCTCN2018078051-appb-000026
在苯硅烷和二丁基二氯化锡条件下经过还原氨化反应或者与不同的酰氯发生缩合反应得到化合物4D；
步骤d：化合物4D与R 1I或R 1Br在氢化钠条件下反应得到化合物4E；
步骤e：化合物4E与联硼酸频那醇酯在醋酸钾和[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物条件下反应得到化合物4F；
步骤f：化合物4F与不同的环
Figure PCTCN2018078051-appb-000027
在碳酸氢钠和[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物条件下偶联反应得到化合物4G，
或者反应路线五：
Figure PCTCN2018078051-appb-000028
步骤a：化合物5A与二氯亚砜和甲醇反应得到化合物5B；
步骤b：化合物5B与氨基酸NH 2R 2COOH在碳酸钾条件下反应得到化合物5C；
步骤c：化合物5C在连二亚硫酸钠和碳酸钾的条件下反应得到化合物5D；
步骤d：化合物5D与不同的酮
Figure PCTCN2018078051-appb-000029
在苯硅烷和二丁基二氯化锡条件下反应得到化合物5E；
步骤e：化合物5E与R 1I或R 1Br在氢化钠条件下反应得到化合物5F；
步骤f：化合物5F与水合肼反应得到化合物5G；
步骤g：化合物5G与不同的硫酰胺
Figure PCTCN2018078051-appb-000030
关环得到化合物5H。
反应路线五提供了合成A环是三唑的另一种方法，类似于反应路线一，但是反应路线五可提供母核R 2是R型、S型或者消旋体的合成方法，反应路线一只能提供母核R 2是消旋体的合成方法。
在以上反应路线中，R 1-R 5以及X和A环的定义如前，虚线表示不存在或为单键。
药物组合物
“药物组合物”包含一种或多种本文中所述的化合物，或其生理学上/药学上可接受的盐、溶剂化物、水合物或前体药物，与药学上可接受的载体，例如与其它化学 成分例如生理学上/药学上可接受的载体与赋形剂的混合物；药物组合物的目的是帮助化合物对生物体的给药。
于本文中使用的“生理学上/药学上可接受的载体”是指载体、赋形剂或稀释剂不会对生物体造成显著刺激，且不会消除所施用化合物的生物学活性与性质。
“药学上可接受的赋形剂”是指被加入药物组合物中以进一步帮助化合物给药的惰性性质，赋形剂的实例包括但不限于碳酸钙、磷酸钙、各种糖类与淀粉类型、纤维素衍生物、明胶、植物油以及聚乙二醇。
在另一优选例中，本发明的药物组合物包括治疗有效量的通式(I)表示的化合物，及其立体异构体、其可药用盐、前药、溶剂化物、水合物和晶型中的一种或多种，以及至少一种赋形剂、稀释剂或载剂。
“治疗有效量”是指所施用化合物的量，其将使被治疗病症的一种或多种病症缓解达某种程度，关于癌症的治疗，治疗有效量是指该量具有至少一种下列作用：
(1)降低肿瘤大小；
(2)抑制(意即，减慢至某种程度，优选为停止)肿瘤转移；
(3)抑制肿瘤生长达某种程度(意即，减慢至某种程度，优选为停止)
(4)使与癌症有关联的一种或多种病症，缓解达某种程度(或优选为消除)。
进一步地，本发明通式(I)表示的化合物，及其立体异构体、可药用盐、前药、溶剂化物、水合物或晶型可用于单一治疗或联合治疗中。当用于联合治疗中时，本发明通式(I)表示的化合物，及其立体异构体、可药用盐、前药、溶剂化物、水合物和晶型通常与基于小分子化合物、辐射、抗体的疗法(例如赫塞汀和利妥希玛)抗癌接种、基因疗法、细胞疗法、激素疗法或细胞因子疗法一起使用。
典型的配方是通过混合本发明的通式(I)表示的化合物及载剂、稀释剂或赋形剂而制备之。适宜的载剂、稀释剂或赋形剂是本领域技术人员所熟知的，包括诸如碳水化合物、蜡、水溶性及/或可膨胀性聚合物、亲水性或疏水性物质、明胶、油、溶剂、水等的物质。所用的特定载剂、稀释剂或赋形剂，将依施用本发明的化合物的方式与目的而定。一般以本领域技术人员认为可安全(GRAS)地投药至一哺乳类动物的溶剂为基础，而选择溶剂。一般而言，安全的溶剂是无毒性含水溶剂诸如水，以及其他可溶于水或与水混溶的无毒性溶剂。适宜的含水溶剂包括水、乙醇、丙二醇、聚乙二醇(如PEG400、PEG300)等中的一种或多种的混合物。该配方也可包括一种或多种缓冲剂、安定剂、表面活性剂、润湿剂、润滑剂、乳化剂、悬浮剂、防腐剂、抗氧化剂、遮光剂、助流剂、加工助剂、着色剂、增甜剂、香料剂、调味剂或其他已知的添加剂，以提供该药物之一优美的呈现形式(亦即本发明的化合物或其药学组合物)，或协助该药学产物(亦即药物)之制造。
该配方可使用常规的溶解混合程序而制备。例如，在上述的一种或多种赋形剂的之存在下，将块状的药物物质(亦即本发明的通式(I)表示的化合物或该化合物的稳 定化形式(如与一环糊精衍生物或其他已知的复合剂的络合物)溶于一适宜溶剂中。典型地将本发明的通式(I)表示的化合物配制成药学剂型，以提供该药物的容易控制的剂量，及提供患者一种容易处理的产物。
依据本发明的方法，本发明的一种化合物或本发明的一种化合物与至少一种其他药剂的组合(在此称作“组合”)，优选是以药学组合物的形式投药。因此，本发明的化合物或组合能以任一已知的口服、直肠、透皮、胃肠外(例如静脉内、肌内或皮下)脑池内、阴道内、腹膜内、膀胱内、局部(例如粉末、油膏或液滴)、颊或鼻剂型，而分开或一起投药至一病患。
适用于非经肠注射的组合物，一般包括药学上可接受的无菌含水或非水溶液、分散液、悬浮液或乳化液，及用于重组成为无菌的可注射性溶液或分散液的无菌粉末。适宜的含水或非水载剂或稀释剂(包括溶剂与载体)，包括水、乙醇、多元醇(丙二醇、聚乙二醇、甘油等)中的一种或多种的混合物；植物油(诸如橄榄油)；及可注射性有机酯诸如油酸乙酯。例如可通过使用一涂层诸如卵磷脂，在分散液的情况下，维持所需的颗粒尺寸，或通过使用表面活性剂，维持适宜的流动性。
这些组成物亦可含有赋形剂，诸如防腐剂、润湿剂、乳化剂及分散剂。可通过各种的杀细菌剂与杀真菌剂，例如对羟苯甲酸酯、氯丁醇、苯酚、山梨酸等，而避免微生物污染该组合物。这些组成物亦可包括等渗压剂诸如糖类、氯化钠等。还可通过使用能延迟吸收的药剂，诸如单硬脂酸铝与明胶，而延长可注射式药学组合物之吸收。
用于口服投药的固态剂型可包括胶囊、片剂、粉末及颗粒。在固态剂型中，本发明的化合物或组合是与至少一种惰性赋形剂、稀释剂或载剂混合。适宜的赋形剂、稀释剂或载剂包括诸如柠檬酸钠或磷酸二钙的物质，或(a)填料或增量剂(如淀粉、乳糖、蔗糖、甘露糖醇、硅酸等)；(b)粘合剂(如羧甲基纤维素、褐藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖、阿拉伯胶等)；(c)湿润剂(如甘油等)；(d)崩解剂(如琼脂、碳酸钙、马铃薯或木薯淀粉、褐藻酸、特定的络合硅酸盐、碳酸钠等)；(e)溶液阻滞剂(如石蜡等)；(f)加速吸收剂(如季铵化合物等)；(g)润湿剂(如乙酰基醇、单硬脂酸甘油酯等)；(h)吸附剂(如高岭土、膨润土等)；及/或i)润滑剂(如滑石、硬脂酸钙、硬脂酸镁、固态聚乙二醇、月桂基硫酸钠等)。在胶囊与片剂的情况下，该剂型亦可包括缓冲剂。类似类型的固态组合物亦可作为软式与硬式填充明胶胶囊中的填料，其使用乳糖以及高分子量聚乙二醇等作为赋形剂。
用于口服投药的液态剂型包括药学上可接受的乳化液、溶液、悬浮液、糖浆液与酏剂。除了本发明的化合物或其组合物之外，该液态剂型可含有本领域中常用的惰性稀释剂，诸如水或其他溶剂；增溶剂及乳化剂诸如乙醇、异丙基醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄基酯、丙二醇、1,3-丁二醇、二甲基甲酰胺；油类(如棉籽 油、落花生油、玉米胚芽油、橄榄油、蓖麻油、芝麻油等)；甘油；四氢糠基醇；聚乙二醇与脱水山梨糖醇的脂肪酸酯；或这些物质中的几种的混合物等。
除了这些惰性稀释剂之外，该组合物也可包括赋形剂，诸如润湿剂、乳化剂、悬浮剂、增甜剂、调味剂与香料剂中的一种或多种。
就悬浮液而言，除了本发明的化合物或组合之外，可进一步含有载剂诸如悬浮剂，如乙氧基化异硬脂醇、聚氧乙烯山梨醣醇、脱水山梨醣醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂及黄耆胶，或这些物质中几种的混合物等。
用于直肠或阴道投药之组合物优选包括栓剂，可通过将本发明的化合物或组合与适宜的非刺激性赋形剂或载剂混合而制备，赋形剂或载剂诸如可可豆脂、聚乙二醇或栓剂蜡，其在一般室温为固态而在体温为液态，及因此可在直肠或阴道中熔化而释出活性化合物。
本发明化合物和本发明化合物与血液性癌症或者炎症药物的组合用于局部投药之剂型，可包括油膏、粉末、喷剂及吸入剂。该药物可在无菌条件下与药学上可接受的赋形剂、稀释剂或载剂以及所需要的任一防腐剂、缓冲剂或推进剂混合。眼用配方、眼用油膏、粉末与溶液，亦意欲涵盖于本发明的范围内。
已知地，本发明的化合物(或组合物)可置入饮水中，借此随同每日的饮水供应而摄入治疗剂量的该化合物。该化合物可直接计量置入饮水中，优选以液态水溶性浓缩物(诸如水溶性盐的水溶液)的形式。
可通过将药物分散于一种药学上可接受的油诸如花生油、芝麻油、玉米油等中，而制备糊状配方。
可通过将本发明的一种化合物或组合物与一种稀释剂诸如碳蜡、棕榈蜡等混合，而制备含有有效量的本发明的一种化合物、药学组合物或组合的丸剂；亦可添加一种润滑剂诸如硬脂酸镁或硬脂酸钙，以增进制丸制程。
用途
本发明的通式(I)表示的化合物，及其立体异构体、可药用盐、前药、溶剂化物、水合物或晶型，为作用于溴结构域识别蛋白的选择性抑制剂，其能对溴结构域识别蛋白识别赖氨酸乙酰化的作用进行抑制。
因此，本发明还提供通式(I)表示的化合物，及其立体异构体、可药用盐、前药、溶剂化物、水合物或晶型作为溴结构域识别蛋白的选择性抑制剂的用途，以及在制备治疗由溴结构域识别蛋白介导的相关疾病、病况及/或机能失调的药物中的用途。
初步研究表明，以下的疾病、病症和/或障碍由溴结构域识别蛋白抑制剂所介导：急性淋巴细胞白血病、急性骨髓性白血病、B细胞慢性淋巴细胞白血病、慢性骨髓单核细胞白血病等血液性恶性肿瘤、中线癌、肺癌、B细胞淋巴瘤、前列腺癌、胃癌、结肠直肠癌、肾癌、肝癌、乳腺癌、胰腺癌、免疫性疾病、心血管疾病以及炎症等。 但由溴结构域识别蛋白介导的相关疾病包括但不限于血液学恶性肿瘤、中线癌以及炎症等疾病。
在另一优选例中，本发明的通式(I)表示的化合物，及其立体异构体、可药用盐、前药、溶剂化物、水合物或晶型可以用于制备治疗血液学恶性肿瘤、中线癌以及炎症等疾病的药物，较佳地，用于制备包括但不限于急性淋巴细胞白血病、急性骨髓性白血病、B细胞慢性淋巴细胞白血病、慢性骨髓单核细胞白血病等血液性恶性肿瘤、中线癌、肺癌、B细胞淋巴瘤、前列腺癌、胃癌、结肠直肠癌、肾癌、肝癌、乳腺癌、胰腺癌、免疫性疾病、心血管疾病以及炎症等疾病的药物。
治疗方法
因此，本发明还提供了一种治疗由溴结构域识别蛋白抑制剂所介导的疾病、病况及/或机能失调的方法，该方法包括向患者施用有效剂量的通式(I)表示的化合物，及其立体异构体、可药用盐、前药、溶剂化物、水合物和晶型。
本发明提到的上述特征，或实施例提到的特征可以任意组合。本案说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
原料可以从商业途径获得，或者通过本领域已知的方法制备，或根据本文所述方法制备。
化合物的结构通过核磁共振( 1H-NMR)和/或质谱(MS)来确定。NMR测定是用Varian公司的Mercury-400型核磁共振仪，测定溶剂为氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)、氘代二甲亚砜(DMSO-d 6)或氘代乙腈(CD 3CN)，TMS为内标。MS的测定用Thermo Finnigan LCQ-Deca XP型(ESI)液相色谱-质谱联用仪。柱层析分离纯化产物使用的是ISCO
Figure PCTCN2018078051-appb-000031
Rf 75快速制备色谱仪，载体采用青岛海洋化工厂的200-300目硅胶。
实施例中，合成收率为摩尔收率。
实施例1：
Figure PCTCN2018078051-appb-000032
试剂与条件：a)二氯亚砜(SOCl 2),甲醇，60℃回流12小时；b)环丙胺，1,2-二氯乙烷，80℃回流12小时；c)铁粉，氯化铵溶液，乙醇，80℃反应1小时；d)1.2-溴丙酰溴，N,N-二异丙基乙胺(DIPEA)，二氯甲烷，室温反应2小时；2.乙腈，DIPEA，80℃反应过夜；e)氢化钠，N,N-二甲基甲酰胺(DMF)，碘甲烷，室温反应1小时；f)水合肼，乙醇，80℃反应24小时；g)N-(对甲苯基)乙硫代酰胺，醋酸汞，醋酸，四氢呋喃(THF)，0℃反应3小时，室温24小时。
a)化合物A(10g,54.02mmol)溶于26mL甲醇中，冷却至0℃，缓慢加入SOCl 2(8.24mL,113.45mmol)，60℃回流12小时，用TLC板监测，反应完后蒸干溶剂，冷却至0℃，用饱和NaHCO 3调pH至7～8，然后用二氯甲烷(40mL*2)和40mL水萃取，合并有机层，用80mL饱和食盐水洗一次，有机相无水硫酸钠干燥，过滤蒸干得10.2g浅黄色固体B，收率95％。MS(EI)[M] +：199； 1H NMR(400MHz,CDCl 3)δ8.11(t,J＝7.8Hz,1H),7.97(s,1H),7.94(d,J＝3.0Hz,1H),3.98(s,3H)。
b)化合物B(10g,50.25mmol)溶于50mL 1,2-二氯乙烷中，加入环丙胺(6.95mL,100.50mmol)，然后80℃回流12小时，用TLC板监测，反应完后蒸干溶剂，用二氯甲烷(50mL*2)和50mL水萃取，合并有机层，用100mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相蒸干得10.2g红色固体C，收率86％。MS(EI)[M] +：236； 1H NMR(400MHz,DMSO-d 6)δ8.15(d,J＝8.8Hz,1H),8.03(s,1H),7.92(d,J＝1.7Hz,1H),7.22(dd,J＝8.8,1.8Hz,1H),3.89(s,3H),2.72–2.65(m,1H),0.94–0.84(m,2H),0.70–0.60(m,2H)。
c)化合物C(10g,42.33mmol)溶于30mL乙醇中，加入氯化铵溶液(11.33g,10mL H 2O)，再加入铁粉(11.86g,211.67mmol)，80℃反应1小时，用TLC板监测，反应完后用硅藻土滤去铁粉，用乙酸乙酯(40mL*2)和40mL水萃取，合并有机层，用80mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相蒸干得5.2g黄色固体D，产率60％。MS(ESI)[M+H] +：207.67。
d)化合物D(5.2g,25.21mmol)溶于干燥的20mL二氯甲烷中，冷却至0℃，加入DIPEA(8.78mL,50.42mmol)和2-溴丙酰溴(3.96mL,37.82mmol)，然后室温反应2小时，用TLC板监测，反应完后用二氯甲烷(40mL*2)和40mL水萃取，合并有机层，用80mL饱和食盐水洗一次，有机相无水硫酸钠干燥，过滤然后蒸干得中间 体，该中间体溶于20mL乙腈中，加入9mL DIPEA，然后80℃反应过夜，用TLC板监测，反应完后蒸干溶剂，用二氯甲烷(40mL*2)和40mL水萃取，合并有机层，用80mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～40％梯度洗脱，得到3g白色固体E，产率46％。MS(ESI)[M+H] +：261.20； 1H NMR(400MHz,CDCl 3)δ8.61(s,1H),7.75(d,J＝1.6Hz,1H),7.53(dd,J＝8.1,1.8Hz,1H),6.78(d,J＝8.1Hz,1H),4.09(q,J＝6.9Hz,1H),3.90(s,3H),2.54–2.45(m,1H),1.24(d,J＝6.9Hz,3H),1.09–1.02(m,1H),0.87–0.78(m,1H),0.69–0.60(m,1H),0.60–0.52(m,1H)。
e)化合物E(3g,11.53mmol)溶于8mL DMF中，冷却至0℃，加入氢化钠(0.83g,34.58mmol)，0℃反应半小时后加入碘甲烷(1.08mL,17.30mmol)，然后室温反应1小时，用TLC板监测，反应完后冷却至0℃，加入稀盐酸调pH至7～8，然后用二氯甲烷(20mL*2)和40mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～25％梯度洗脱，得到2.8g无色透明液体F，产率89％。MS(ESI)[M+H] +：275.18； 1H NMR(400MHz,CDCl 3)δ7.75(d,J＝1.9Hz,1H),7.59(dd,J＝8.3,1.9Hz,1H),6.93(d,J＝8.4Hz,1H),4.12(q,J＝6.9Hz,1H),3.89(s,3H),3.35(s,3H),2.48–2.42(m,1H),1.15(d,J＝6.9Hz,3H),1.07–0.99(m,1H),0.84–0.76(m,1H),0.66–0.58(m,1H),0.56–0.48(m,1H)。
f)化合物F(2.8g,10.21mmol)溶于20mL乙醇中，加入水合肼(2.48mL,51.05mmol)，然后80℃反应24小时，用TLC板监测，反应完后蒸干溶剂，用甲苯带几次水，然后干燥得到2.5g白色泡沫状固体G，产率89％。
g)化合物G(0.6g,2.19mmol)溶于3mL THF和4.2mL醋酸中，加入N-(对甲苯基)乙硫代酰胺(0.36g,2.19mmol)，冷却至0℃，加入醋酸汞(1.05g,3.29mmol)，0℃反应3小时，然后室温反应24小时，用TLC板监测，反应完后用饱和碳酸氢钠中和pH至7～8，然后用乙酸乙酯(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用甲醇/二氯甲烷＝0～10％梯度洗脱，得到300mg白色粉末化合物1，产率35％。MS(ESI)[M+H] +：388.41； 1H NMR(400MHz,CDCl 3)δ7.30(d,J＝8.0Hz,2H),7.15(d,J＝1.9Hz,1H),7.09(d,J＝8.3Hz,2H),6.98(dd,J＝8.3,1.9Hz,1H),6.81(d,J＝8.4Hz,1H),4.04(q,J＝6.8Hz,1H),3.29(s,3H),2.43(s,3H),2.34(s,3H),2.20–2.15(m,1H),1.09(d,J＝6.8Hz,3H),0.73–0.65(m,1H),0.64–0.56(m,1H),0.55–0.48(m,1H),0.23–0.14(m,1H)。
实施例2
Figure PCTCN2018078051-appb-000033
Figure PCTCN2018078051-appb-000034
试剂与条件：a)对甲基苯胺，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，DIPEA，DMF，室温反应过夜；b)环丙胺，1,2-二氯乙烷，80℃回流12小时；c)劳森试剂，甲苯，110℃回流过夜；d)1.水合肼，室温反应4小时；2.原甲酸三甲酯，DMF，醋酸，室温反应过夜；e)二水合二氯化锡，浓盐酸，室温反应3小时；f)1.2-溴丙酰溴，DIPEA，二氯甲烷，室温反应2小时；2.乙腈，DIPEA，80℃反应过夜；g)氢化钠，N,N-二甲基甲酰胺(DMF)，碘甲烷，室温反应1小时。
a)化合物A(2g,10.80mmol)溶于10mL DMF中，加入HATU(4.11g,10.80mmol)，室温反应半小时后加入对甲基苯胺(1.16g,10.80mmol)和DIPEA(1.88mL,10.80mmol)，室温反应过夜，用TLC板监测，反应完后用乙酸乙酯(20mL*2)和60mL饱和碳酸氢钠萃取，合并有机层，分别用40mL稀盐酸和40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，得到2.2g黄色固体B，收率74％。 1H NMR(400MHz,DMSO-d 6)δ10.49(s,1H),8.31(t,J＝8.1Hz,1H),8.10(d,J＝12.0Hz,1H),7.96(d,J＝8.5Hz,1H),7.65(d,J＝8.4Hz,2H),7.19(d,J＝8.3Hz,2H),2.29(s,3H)。
b)化合物B(2g,7.29mmol)溶于10mL1,2-二氯乙烷中，加入环丙胺(1.01mL,14.58mmol)，然后80℃回流12小时，用TLC板监测，反应完后蒸干溶剂，用二氯甲烷(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相蒸干得2.1g红色固体C，收率93％。 1H NMR(400MHz,CDCl 3)δ8.23(d,J＝8.8Hz,1H),8.12(s,1H),7.86(s,1H),7.80(s,1H),7.53(d,J＝8.1Hz,2H),7.19(d,J＝8.3Hz,2H),7.01(d,J＝8.7Hz,1H),2.69–2.62(m,1H),2.35(s,3H),1.00–0.94(m,2H),0.71–0.66(m,2H)。
c)化合物C(2g,6.42mmol)溶于10mL甲苯中，加入劳森试剂(1.36g,3.37mmol)，然后110℃回流过夜，用TLC板监测，反应完后蒸干溶剂，用50mL二氯甲烷溶解后硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～15％梯度洗脱，得1.8g红色固体D，收率86％。MS(ESI)[M+H] +：328.11； 1H NMR(400MHz,DMSO-d 6)δ11.97(s,1H),8.12(d,J＝8.8Hz,2H),7.73(d,J＝8.2Hz,2H),7.65(s,1H),7.26(d,J＝8.2Hz,2H),7.07(d,J＝8.8Hz,1H),2.71(s,1H),2.33(s,3H),0.89(d,J＝5.0Hz,2H),0.68(s,2H)。
d)化合物D(1.8g,5.50mmol)溶于5mL甲醇和20mL THF的混合溶剂中，加入水合肼(2.67mL,55.05mmol)，然后室温反应4小时，用TLC板监测，反应完后蒸干溶剂，干燥得中间体，中间体溶于5mL DMF中，加入4mL醋酸和原甲酸三甲酯(1.62mL,14.85mmol)，然后室温反应过夜，用TLC板监测，反应完后用饱和NaHCO 3 调pH至7～8，然后用乙酸乙酯(40mL*2)和80mL饱和碳酸氢钠萃取，合并有机层，用80mL饱和食盐水洗一次，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～30％梯度洗脱，得1.5g红色固体E，收率81％。 1H NMR(400MHz,CDCl 3)δ8.34(s,1H),8.10(d,J＝8.8Hz,1H),8.03(s,1H),7.45(s,1H),7.31(d,J＝7.9Hz,2H),7.17(d,J＝8.2Hz,2H),6.81(d,J＝8.8Hz,1H),2.44(s,3H),2.36–2.29(m,1H),0.74–0.68(m,2H),0.51–0.46(m,2H)。
e)化合物E(1.5g,4.47mmol)溶于4mL浓盐酸中，加入二水合二氯化锡(5.04g,22.35mmol)的浓盐酸(4mL)溶液，然后室温反应3小时，用TLC板监测，反应完后蒸干溶剂，再用饱和碳酸氢钠中和pH至7～8，然后用乙酸乙酯(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相蒸干得1.2g黄色泡沫状粉末F，收率88％。
f)化合物F(1.2g,3.93mmol)溶于干燥的8mL二氯甲烷中，冷却至0℃，加入DIPEA(1.37mL,7.86mmol)和2-溴丙酰溴(0.62mL,5.90mmol)，然后室温反应2小时，用TLC板监测，反应完后用二氯甲烷(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，过滤蒸干得到0.7g红色油状中间体，该中间体溶于5mL乙腈中，加入2mL DIPEA，然后80℃反应过夜，用TLC板监测，反应完后蒸干溶剂，用二氯甲烷(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～20％梯度洗脱，得到0.8g浅黄色粉末G，收率57％。
g)化合物G(0.8g,2.23mmol)溶于2mL DMF中，冷却至0℃，加入氢化钠(0.16g,6.69mmol)，0℃反应半小时，在0℃条件下加入碘甲烷(0.21mL,3.35mmol)，然后室温反应1小时，用TLC板监测，反应完后用饱和碳酸氢钠中和pH至7～8，然后用乙酸乙酯(20mL*2)和40mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用甲醇/二氯甲烷＝0～10％梯度洗脱，得到0.22g浅黄色粉末化合物2，产率26％。MS(ESI)[M+H] +：374.31； 1H NMR(400MHz,CDCl 3)δ8.28(s,1H),7.29–7.24(m,2H),7.17–7.14(m,2H),7.14–7.12(m,1H),7.04(dd,J＝8.3,1.9Hz,1H),6.85(d,J＝8.3Hz,1H),4.05(q,J＝6.9Hz,1H),3.31(s,3H),2.42(s,3H),2.22–2.14(m,1H),1.10(d,J＝6.8Hz,3H),0.72–0.64(m,1H),0.58–0.47(m,2H),0.21–0.13(m,1H)。
实施例3
Figure PCTCN2018078051-appb-000035
Figure PCTCN2018078051-appb-000036
试剂与条件：a)对甲基苯胺，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，DIPEA，DMF，室温反应过夜；b)环丙胺，1,2-二氯乙烷，80℃回流12小时；c)劳森试剂，甲苯，110℃回流过夜；d)四氢呋喃，醋酸汞，三甲基硅叠氮，0℃反应4小时；e)二水合二氯化锡，浓盐酸，室温反应3小时；f)1.2-溴丙酰溴，DIPEA，二氯甲烷，室温反应2小时；2.乙腈，DIPEA，80℃反应过夜；g)氢化钠，N,N-二甲基甲酰胺(DMF)，碘甲烷，室温反应1小时。
化合物D的制备方法参见化合物2制备方法中步骤a-c。
d)化合物D(2g,6.12mmol)溶于10mL四氢呋喃中，冷却至0℃，加入醋酸汞(3.88g,12.24mmol)和三甲基硅叠氮(8.05mL,61.2mmol)，然后0℃反应4小时，用TLC板监测，反应完后蒸干溶剂，用乙酸乙酯(30mL*2)和30mL水萃取，合并有机层，用60mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～10％梯度洗脱，得到1.8g红色固体E，收率87％。MS(ESI)[M+H] +：337.09； 1H NMR(400MHz,CDCl 3)δ8.14(d,J＝8.9Hz,1H),8.03(s,1H),7.53(d,J＝1.8Hz,1H),7.35(d,J＝8.2Hz,2H),7.32–7.27(m,2H),6.86(dd,J＝8.9,1.9Hz,1H),2.46(s,3H),2.39–2.31(m,1H),0.76–0.68(m,2H),0.55–0.44(m,2H)。
e)化合物E(1.8g,5.35mmol)溶于4mL浓盐酸中，加入二水合二氯化锡(6.04g,26.75mmol)的浓盐酸(4mL)溶液，然后室温反应3小时，用TLC板监测，反应完后用饱和碳酸氢钠中和pH至7～8，然后用乙酸乙酯(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相蒸干得1.6g黄色泡沫状粉末F，收率93％。
f)化合物F(1.5g,4.90mmol)溶于干燥的8mL二氯甲烷中，冷却至0℃，加入DIPEA(1.71mL,9.80mmol)和2-溴丙酰溴(0.77mL,7.35mmol)，然后室温反应2小时，用TLC板监测，反应完后用二氯甲烷(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，过滤蒸干得到0.8g红色油状中间体，该中间体溶于5mL乙腈中，加入2mL DIPEA，然后80℃反应过夜，用TLC板监测，反应完后蒸干溶剂，用二氯甲烷(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～20％梯度洗脱，得到1.0g浅黄色粉末G，收率57％。MS(ESI)[M+H] +：361.11； 1H NMR(400MHz,CDCl 3)δ9.62(s,1H),7.33–7.28(m,4H),7.23(d,J＝1.8Hz,1H),7.02(dd,J＝8.1,1.8Hz,1H),6.82(d,J＝8.1Hz,1H), 4.02(q,J＝6.8Hz,1H),2.44(s,3H),2.27–2.22(m,1H),1.20(d,J＝6.9Hz,3H),0.84–0.80(m,1H),0.76–0.68(m,1H),0.56–0.53(m,1H),0.26–0.20(m,1H)。
g)化合物G(0.6g,1.67mmol)溶于2mL DMF中，冷却至0℃，加入氢化钠(0.12g,5.01mmol)，0℃反应半小时，在0℃条件下加入碘甲烷(0.19mL,3.01mmol)，然后室温反应1小时，用TLC板监测，反应完后用饱和碳酸氢钠中和pH至7～8，然后用乙酸乙酯(20mL*2)和40mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用甲醇/二氯甲烷＝0～10％梯度洗脱，得到180mg浅黄色粉末化合物3，产率29％。MS(ESI)[M+H] +：375.20； 1H NMR(400MHz,CDCl 3)δ7.36–7.28(m,4H),7.23(d,J＝1.7Hz,1H),7.15(dd,J＝8.3,1.8Hz,1H),6.91(d,J＝8.3Hz,1H),4.08(q,J＝6.9Hz,1H),3.33(s,3H),2.45(s,3H),2.25–2.15(m,1H),1.13(d,J＝6.8Hz,3H),0.76–0.65(m,1H),0.58–0.48(m,2H),0.26–0.15(m,1H)。
实施例4
除了在步骤b中用环戊胺代替环丙胺之外，其它采用与实施例3中相同的方法制备化合物4。
Figure PCTCN2018078051-appb-000037
MS(ESI)[M+H] +：403.13。 1H NMR(400MHz,CDCl 3)δ7.30(q,J＝8.4Hz,4H),7.13(d,J＝8.3Hz,1H),6.93(d,J＝8.1Hz,2H),4.15(q,J＝6.7Hz,1H),3.48(dd,J＝14.9,7.4Hz,1H),3.34(s,3H),2.43(s,3H),1.83(dd,J＝14.2,8.8Hz,2H),1.74–1.66(m,1H),1.64–1.48(m,5H),0.99(d,J＝6.8Hz,3H)。
实施例5
Figure PCTCN2018078051-appb-000038
Figure PCTCN2018078051-appb-000039
试剂与条件：a)对甲苯胺，巯基乙酸，二环己基碳二亚胺，四氢呋喃，0℃-室温，12小时；b)环丙胺，1,2-二氯乙烷，80℃回流12小时；c)二水合二氯化锡，浓盐酸，0℃-室温，3小时；d)1.2-溴丙酰溴，N,N-二异丙基乙胺(DIPEA)，二氯甲烷，室温反应2小时；2.乙腈，DIPEA，80℃反应过夜；e)氢化钠，N,N-二甲基甲酰胺(DMF)，碘甲烷，室温反应1小时。
a)对甲苯胺(2g,18.69mmol)和化合物A(6.3g,37.38mmol)在0℃条件下溶于干燥的四氢呋喃中，0℃搅拌10分钟，然后加入巯基乙酸(3.9mL,56.07mmol)继续在0℃条件下搅拌10分钟，然后加入二环己基碳二亚胺(4.62g,22.43mmol)，然后室温搅拌过夜，用TLC板监测，反应完后过滤，滤液硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～10％梯度洗脱，得到3.5g浅黄色固体B，收率56％。 1H NMR(400MHz,DMSO-d 6)δ8.06(t,J＝8.1Hz,1H),7.70(d,J＝12.0Hz,1H),7.47(d,J＝8.5Hz,1H),7.24(d,J＝8.2Hz,2H),7.12(d,J＝8.2Hz,2H),6.61(s,1H),4.11(d,J＝15.6Hz,1H),3.89(d,J＝15.6Hz,1H),2.21(s,3H)。
b)化合物B(2g,6.02mmol)溶于10mL1,2-二氯乙烷中，加入环丙胺(0.83mL,12.04mmol)，然后80℃反应过夜，用TLC板监测，反应完后蒸干溶剂，然后用二氯甲烷(30mL*2)和30mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～15％梯度洗脱，得到1.8g红色固体C，收率81％。 1H NMR(400MHz,CDCl 3)δ8.11(d,J＝8.9Hz,1H),8.07(s,1H),7.13–7.11(m,4H),6.66(dd,J＝8.9,1.9Hz,1H),6.05(d,J＝1.0Hz,1H),3.99(dd,J＝15.8,1.6Hz,1H),3.87(d,J＝15.8Hz,1H),2.54–2.47(m,1H),2.28(s,3H),0.92–0.84(m,2H),0.60–0.54(m,2H)。
c)化合物C(1.5g,4.06mmol)溶于4mL浓盐酸中，冷却至0℃，加入二水合二氯化锡(4.35g,19.29mmol)的浓盐酸(4mL)溶液，然后室温反应4小时，用TLC板监测，反应完后用6N氢氧化钠中和pH至7～8，然后用乙酸乙酯(40mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相蒸干得到1.1g浅黄色粉末D，收率80％。
d)化合物D(1.1g,3.24mmol)溶于干燥的8mL二氯甲烷中，冷却至0℃，加入DIPEA(1.13mL,6.48mmol)和2-溴丙酰溴(0.41mL,3.89mmol)，然后室温反应2小时，用TLC板监测，反应完后用二氯甲烷(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱 柱纯化，使用乙酸乙酯/石油醚＝0～30％梯度洗脱，得到200mg黄色固体中间体， 1H NMR(400MHz,CDCl 3)δ7.78(s,1H),7.18(d,J＝8.0Hz,1H),7.06(s,4H),7.03(d,J＝1.9Hz,1H),6.70(dd,J＝8.1,2.0Hz,1H),6.04(s,1H),4.56–4.47(m,1H),3.96(dd,J＝15.8,1.6Hz,1H),3.84(d,J＝15.8Hz,1H),2.42–2.34(m,1H),2.24(s,3H),1.88(d,J＝7.0Hz,3H),0.80–0.70(m,2H),0.47–0.38(m,2H)；该中间体溶于5mL乙腈中，加入1mL DIPEA，然后80℃反应过夜，用TLC板监测，反应完后蒸干溶剂，用二氯甲烷(20mL*2)和20mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相未纯化直接蒸干得到0.1g白色固体E，产率8％。
e)化合物E(0.1g,0.25mmol)溶于2mL DMF中，冷却至0℃，加入氢化钠(0.02g,0.76mmol)，0℃反应半小时，在0℃条件下加入碘甲烷(0.03mL,0.51mmol)，然后室温反应1小时，用TLC板监测，反应完后用饱和碳酸氢钠中和pH至7～8，然后用乙酸乙酯(20mL*2)和40mL水萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～30％梯度洗脱，得到15mg白色粉末化合物5，收率15％。MS(ESI)[M+H] +：408.21； 1H NMR(400MHz,CDCl 3)δ7.11–7.04(m,3H),7.02(s,1H),7.01–6.97(m,1H),6.86–6.79(m,1H),6.78–6.75(m,1H),6.04(d,J＝34.1Hz,1H),4.06(q,J＝6.9Hz,1H),3.96(dd,J＝15.8,9.5Hz,1H),3.87(d,J＝15.8Hz,1H),3.27(d,J＝4.1Hz,3H),2.38–2.30(m,1H),2.25(d,J＝3.0Hz,3H),1.11(dd,J＝8.7,7.1Hz,3H),0.94–0.85(m,1H),0.82–0.71(m,1H),0.63–0.54(m,1H),0.41–0.29(m,1H)。
实施例6
Figure PCTCN2018078051-appb-000040
试剂与条件：a)D-氨基丙酸，碳酸钾，乙醇，水，80℃，8小时；b)碳酸钾，连二亚硫酸钠，水，60℃反应过夜；c)苯硅烷，环戊酮，二丁基二氯化锡，THF， 室温，10小时；d)NaH，DMF，碘甲烷，0℃-室温，4小时；e)联硼酸频那醇酯，醋酸钾，二氧六环，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，120℃回流过夜；f)5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯，碳酸氢钠，THF，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，80℃回流过夜；g)氢氧化锂，THF，H 2O，室温，12小时；h)HATU，DMF，N-甲基哌嗪，DIPEA，室温反应过夜。
a)将4-溴-2氟硝基苯(25g,113.64mmol)，D-氨基丙酸(11.12g,125.0mmol)和碳酸钾(17.25g,125.0mmol)溶于500mL乙醇：水＝3：1的混合溶剂中，加热回流8小时，用TLC板监测，反应完后冷却至室温，用1N HCl调节pH至2-3，将固体过滤，用200mL石油醚洗涤固体，抽干并且用旋转蒸发干燥得28.7g黄色固体B，收率88％。 1H NMR(400MHz,CDCl 3)δ8.35(d,J＝6.9Hz,1H),8.06(d,J＝9.1Hz,1H),6.90(s,1H),6.85(d,J＝9.2Hz,1H),4.33(p,J＝7.0Hz,1H),1.67(d,J＝7.0Hz,3H)。
b)将B(28.7g,99.31mmol)和K 2CO 3(27.41g,198.62mmol)溶于500mL水中，分批加入连二亚硫酸钠(86.45g,496.55mmol)，60℃反应过夜。产生大量沉淀，用TLC板监测，反应完后将固体过滤，用300mL水洗涤，干燥，得9g黄色固体C，收率38％。 1H NMR(400MHz,CDCl 3)δ9.42(s,1H),6.89–6.83(dd,J＝8.2,2.0Hz,1H),6.81(s,1H),6.68–6.59(dd,J＝13.1,7.6Hz,1H),4.02(q,J＝6.7Hz,1H),3.92(s,1H),1.45(d,J＝6.7Hz,3H)。
c)将C(9g,37.34mmol)，苯硅烷(11.90g,113.14mmol)，环戊酮(10.0mL,113.14mmol)以及二丁基二氯化锡(17.02g,56.01mmol)溶于100mL THF中，常温搅拌10小时，用TLC板监测，反应完后将溶剂蒸干，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～30％梯度洗脱，得10.4g棕色油状物D，收率90％。MS(ESI)[M+H] +：309.03； 1H NMR(400MHz,CDCl 3)δ9.68(s,1H),6.92(d,J＝1.9Hz,1H),6.88(dd,J＝8.2,2.0Hz,1H),6.69(d,J＝8.2Hz,1H),4.10(q,J＝6.8Hz,1H),3.88–3.75(m,1H),2.08–1.94(m,2H),1.78–1.55(m,6H),1.14(d,J＝6.8Hz,3H)。
d)将NaH(1.61g,67.27mmol)悬浮于100mL干燥的DMF中，在冰浴下搅拌5分钟，加入80mL化合物D(10.4g,33.63mmol)的DMF溶液，搅拌20分钟，缓慢滴入碘甲烷(3.14mL,50.45mmol)，将体系移至室温反应4小时，用TLC板监测，反应完后在冰浴下加水淬灭，加入300mL水，用乙酸乙酯萃取两次，合并有机相，用600mL饱和食盐水洗一次，无水硫酸钠干燥，蒸干溶剂，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～25％梯度洗脱，得10g棕色油状物E，收率92％。 1H NMR(400MHz,CDCl 3)δ6.97(dd,J＝8.4,2.1Hz,1H),6.92(d,J＝2.1Hz,1H),6.78(d,J＝8.5Hz,1H),4.17(q,J＝6.8Hz,1H),3.81–3.72(m,1H),3.33(s,3H),2.08–1.96(m,2H),1.84–1.74(m,1H),1.72–1.58(m,5H),1.05(d,J＝6.8Hz,3H)。
e)将E(10g,30.96mmol)，联硼酸频那醇酯(8.65g,34.06mmol)以及醋酸钾(6.08g,61.92mmol)溶于400mL无水二氧六环中，通氩气10分钟，加入[1,1'-双(二 苯基膦)二茂铁]二氯化钯二氯甲烷络合物(1.26g,1.55mmol)，继续通气2分钟，氩气保护下加热至120℃回流过夜，用TLC板监测，反应完后用二氯甲烷(400mL*2)和40mL水萃取，合并有机层，用800mL饱和食盐水洗一次，无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～10％梯度洗脱，得9.5g棕色油状物F，收率83％。MS(ESI)[M+H] +：371.14； 1H NMR(400MHz,CDCl 3)δ7.36(d,J＝8.3Hz,1H),7.29(s,1H),6.95(d,J＝7.9Hz,1H),4.17(q,J＝6.8Hz,1H),3.94–3.85(m,1H),3.37(s,3H),2.09–1.98(m,2H),1.83–1.74(m,1H),1.73–1.58(m,5H),1.35(s,12H),1.03(d,J＝6.9Hz,3H)。
f)将F(1g,2.70mmol)，5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯(1.17g,3.78mmol)以及饱和碳酸氢钠(0.45g,5.40mmol)溶液溶于10mL THF中，通氩气10分钟，加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(0.22g,0.27mmol)，继续通气2分钟，氩气保护下加热至80℃回流过夜蒸干溶剂，用TLC板监测，反应完后用40mL二氯甲烷萃取，饱和食盐水洗，无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚＝0～25％梯度洗脱，得0.8g棕色油状物G，收率63％。MS(ESI)[M+H] +：474.35； 1H NMR(400MHz,CDCl 3)δ7.29(d,J＝8.4Hz,2H),7.24(d,J＝8.3Hz,2H),7.20–7.16(m,1H),6.90(d,J＝8.4Hz,1H),6.85(s,1H),4.53(q,J＝7.3Hz,2H),3.52–3.42(m,1H),3.35(s,3H),2.40(s,3H),1.84–1.77(m,1H),1.74–1.64(m,3H),1.61–1.48(m,5H),1.45(t,J＝7.1Hz,3H),0.97(d,J＝6.8Hz,3H)。
g)将G(0.8g,1.69mmol)以及氢氧化锂(0.28g,6.76mmol)溶于10mL THF：H 2O＝4：1的混合溶剂中，然后室温反应12小时，用TLC板监测，反应完后用1N HCl调节pH至5-6，用乙酸乙酯(20mL*2)和20mL水萃取，合并有机层，用20mL饱和食盐水洗一次，有机相无水硫酸钠干燥，过滤，直接蒸干得到0.75g白色粉末H，收率99％。MS(ESI)[M+H] +：446.30； 1H NMR(400MHz,CDCl 3)δ8.81(s,1H),7.30(d,J＝8.2Hz,2H),7.26–7.19(m,3H),6.93(d,J＝8.2Hz,1H),6.84(s,1H),4.19(q,J＝6.7Hz,1H),3.50–3.40(m,1H),3.35(s,3H),2.39(s,3H),1.84–1.74(m,1H),1.71–1.62(m,1H),1.61–1.42(m,5H),1.36–1.24(m,1H),0.98(d,J＝6.8Hz,3H)。
h)化合物H(0.75g,1.69mmol)溶于5mL DMF中，加入HATU(0.64g,1.69mmol),室温反应半小时后加入N-甲基哌嗪(0.19mL,1.69mmol)和DIPEA(0.29mL,1.69mmol)，室温反应过夜，用TLC板监测，反应完后用乙酸乙酯(20mL*2)和60mL饱和碳酸氢钠萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，机相硅胶拌样通过色谱柱纯化，使用甲醇/二氯甲烷＝0～10％梯度洗脱，得到0.5g白色固体化合物6，收率56％。MS(ESI)[M+H] +：528.41； 1H NMR(400MHz,CDCl 3)δ7.29–7.26(m,2H),7.25–7.22(m,2H),7.14(dd,J＝8.3,1.9Hz,1H),6.90(d,J＝8.4Hz,1H),6.84(d,J＝1.9Hz,1H),4.12(q,J＝6.8Hz,1H),4.01–3.92(m,2H),3.90–3.83(m,2H),3.48–3.40(m,1H),3.34(s,3H),2.54–2.49(m,2H),2.49–2.45 (m,2H),2.39(s,3H),2.32(s,3H),1.84–1.74(m,1H),1.72–1.63(m,1H),1.61–1.43(m,5H),1.35–1.27(m,1H),0.97(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.08,160.70,156.86,154.45,139.61,135.72,135.52,132.26,130.15(2×C),125.67(2×C),122.18,120.21,116.16,114.56,59.05,55.48,54.71,54.56,47.10,46.10,42.43,30.78,30.35,29.24,24.01,23.55,21.30,14.14。
实施例7
除了在步骤f中用5-溴-1-苯基-1H-1,2,4-三唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以外，其它采用与实施例6中相同的方法制备化合物7。
Figure PCTCN2018078051-appb-000041
MS(ESI)[M+H] +：514.44； 1H NMR(400MHz,CDCl 3)δ7.47–7.36(m,5H),7.11(dd,J＝8.3,1.5Hz,1H),6.89(d,J＝8.3Hz,1H),6.84(s,1H),4.11(q,J＝6.7Hz,1H),3.99–3.92(m,2H),3.89–3.82(m,2H),3.47–3.37(m,1H),3.33(s,3H),2.54–2.44(m,4H),2.31(s,3H),1.82–1.72(m,1H),1.70–1.61(m,1H),1.60–1.42(m,5H),1.34–1.25(m,1H),0.96(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.01,160.59,156.96,154.52,138.14,135.53,132.29,129.57(2×C),129.29,125.78(2×C),122.03,120.14,116.13,114.54,59.02,55.43,54.65,54.49,47.06,46.05,42.40,30.72,30.32,29.20,23.96,23.50,14.16。
实施例8
除了在步骤f中用5-溴-1-苯基-1H-1,2,4-三唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以及在步骤h中用1-甲基-1,4-二氮杂环庚烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物8。
Figure PCTCN2018078051-appb-000042
MS(ESI)[M+H] +：528.43； 1H NMR(400MHz,CDCl 3)δ7.47–7.42(m,3H),7.41–7.37(m,2H),7.14–7.08(m,1H),6.89(d,J＝8.3Hz,1H),6.86(dd,J＝4.2,1.8Hz,1H),4.16–4.09(m,1H),3.99–3.94(m,1H),3.93–3.85(m,2H),3.83(t,J＝6.3Hz,1H),3.49–3.39(m,1H),3.34(s,3H),2.82–2.77(m,2H),2.71–2.63(m,2H),2.40(d,J＝8.1Hz,3H),2.10–1.99(m,2H),183–1.74(m,1H),1.71–1.62(m,1H),1.61–1.42(m,6H),0.98(dd,J＝6.8,1.3Hz,3H)。
实施例9
除了在步骤f中用5-溴-1-(2,4-二甲基苯基)-1H-1,2,4-三唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以外，其它采用与实施例6中相同的方法制备化合物9。
Figure PCTCN2018078051-appb-000043
MS(ESI)[M+H] +：542.45； 1H NMR(400MHz,CDCl 3)δ7.20–7.15(m,2H),7.15–7.13(m,1H),7.13–7.09(m,1H),6.89(d,J＝1.9Hz,1H),6.86(d,J＝8.5Hz,1H),4.11(q,J＝6.8Hz,1H),4.00–3.94(m,2H),3.89–3.82(m,2H),3.40–3.33(m,1H),3.31(s,3H),2.54–2.50(m,2H),2.50–2.46(m,2H),2.38(s,3H),2.32(s,3H),1.98(s,3H),1.83–1.75(m,1H),1.74–1.66(m,1H),1.66–1.55(m,2H),1.55–1.44(m,3H),1.36–1.27(m,1H),0.95(d,J＝6.8Hz,3H)； 13C NMR(151MHz,CDCl 3)δ169.04,160.67,156.77,155.01,140.48,135.57,135.13,134.96,132.22,132.15,127.98,127.45,122.09,119.66,115.09,114.58,58.95,55.44,54.69,54.23,47.08,46.06,42.43,30.79,30.35,29.19,23.94,23.43,21.33,17.64,14.17。
实施例10
除了在步骤h中用1-乙基哌嗪代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物10。
Figure PCTCN2018078051-appb-000044
MS(ESI)[M+H] +：542.42； 1H NMR(400MHz,CDCl 3)δ7.30–7.26(m,2H),7.25–7.22(m,2H),7.15(dd,J＝8.3,1.8Hz,1H),6.90(d,J＝8.4Hz,1H),6.86(d,J＝1.8Hz,1H),4.13(q,J＝6.8Hz,1H),4.05–3.95(m,2H),3.94–3.84(m,2H),3.50–3.41(m,1H),3.35(s,3H),2.59–2.51(m,4H),2.47(q,J＝7.2Hz,2H),2.40(s,3H),1.84–1.75(m,1H),1.73–1.64(m,1H),1.62–1.44(m,5H),1.36–1.28(m,1H),1.11(t,J＝7.2Hz,3H),0.98(d,J＝6.8Hz,3H)。
实施例11
除了在步骤h中用1-环丙基哌嗪代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物11。
Figure PCTCN2018078051-appb-000045
MS(ESI)[M+H] +：554.44； 1H NMR(400MHz,CDCl 3)δ7.30–7.21(m,4H),7.15(d,J＝7.2Hz,1H),6.90(d,J＝8.4Hz,1H),6.85(s,1H),4.21–4.04(m,1H),3.93–3.86(m,2H),3.84–3.76(m,2H),3.50–3.39(m,1H),3.34(s,3H),2.75–2.62(m,4H),2.39(s,3H),2.28–2.15(m,1H),1.82–1.74(m,1H),1.69–1.63(m,2H),1.58–1.46(m,5H),0.97(d,J＝6.7Hz,3H),0.87–0.82(m,2H),0.49–0.43(m,2H)； 13C NMR(126MHz,CDCl 3)δ169.05,160.68,156.92,154.41,139.56,135.72,135.50,132.24,130.13(2×C),125.66(2×C),122.19,120.20,116.16,114.53,59.03,54.54,53.77,52.97,47.21,42.53,38.41,30.77,30.34,29.77,29.21,24.00,23.53,21.28,14.12,5.99。
实施例12
除了在步骤h中用(2S，6R)-2,6-二甲基哌嗪代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物12。
Figure PCTCN2018078051-appb-000046
MS(ESI)[M+H] +：542.43； 1H NMR(400MHz,CDCl 3)δ7.30–7.21(m,4H),7.16–7.11(m,1H),6.90(dd,J＝8.3,2.7Hz,1H),6.85(d,J＝7.3Hz,1H),4.68(d,J＝12.9Hz,1H),4.44(d,J＝11.9Hz,1H),4.12(q,J＝6.8Hz,1H),3.49–3.39(m,1H),3.33(s,3H),3.04–2.89(m,2H),2.89–2.78(m,1H),2.48–2.40(m,1H),2.38(s,3H),2.34(s,1H),1.82–1.74(m,1H),1.72–1.63(m,1H),1.62–1.42(m,6H),1.14(d,J＝6.2Hz,3H),1.07(d,J＝5.8Hz,3H),0.97(d,J＝6.6Hz,3H)； 13C NMR(151MHz,CDCl 3)δ169.04,160.50(d,J＝5.1Hz),156.88,154.42,139.60,135.63,135.50,132.22,130.13(2×C),125.63(2×C),122.10,120.15,116.07,114.55(d,J＝4.2Hz),58.95,54.52,53.49(d,J＝4.0Hz),51.62,50.79,48.70,30.77,30.32(d,J＝4.3Hz),29.21,23.98,23.51,21.28,19.38,19.19,14.07(d,J＝5.5Hz)。
实施例13
除了在步骤h中用N 1,N 1,N 2-三甲基乙烷-1,2-二胺代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物13。
Figure PCTCN2018078051-appb-000047
MS(ESI)[M+H] +：530.32。 1H NMR(400MHz,CDCl 3)δ7.26–7.19(m,4H),7.13(dt,J＝8.3,2.1Hz,1H),6.90(dd,J＝8.4,4.1Hz,1H),6.77(dd,J＝19.2,1.6Hz,1H),4.10(q,J＝6.7Hz,1H),3.85–3.79(m,1H),3.78–3.69(m,2H),3.45–3.35(m,1H),3.34(s,2H),3.32(s,3H),2.84(t,J＝6.5Hz,1H),2.74–2.66(m,1H),2.49(s,3H),2.37(s,3H),2.24(s,3H),1.81–1.70(m,1H),1.69–1.59(m,1H),1.60–1.40(m,5H),1.31–1.24(m,1H),0.95(dd,J＝6.8,2.7Hz,3H)。
实施例14
除了在步骤h中用8-甲基-3,8-二氮杂双环[3.2.1]辛烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物14。
Figure PCTCN2018078051-appb-000048
MS(ESI)[M+H] +：554.44； 1H NMR(400MHz,CDCl 3)δ7.30–7.20(m,4H),7.14(d,J＝8.2Hz,1H),6.89(d,J＝8.4Hz,1H),6.85(s,1H),5.15(s,1H),4.87(d,J＝5.9Hz,1H),4.12(q,J＝6.7Hz,1H),3.51–3.39(m,1H),3.33(s,3H),2.74(d,J＝10.1Hz,2H),2.50–2.42(m,2H),2.38(s,3H),2.25(s,3H),2.03–1.91(m,4H),1.83–1.74(m,1H),1.71–1.63(m,1H),1.61–1.42(m,6H),0.97(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.03,157.24,157.01,154.41,139.52,135.80,135.44,132.18,130.08(2×C),125.67(2×C),122.27,120.20,116.18,114.49,61.95,60.28,59.03,56.44,54.57,52.85,45.18,30.74,30.32,29.20,28.66,26.78,23.97,23.52,21.28,14.11。
实施例15
除了在步骤h中用1-异丙基哌嗪代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物15。
Figure PCTCN2018078051-appb-000049
MS(ESI)[M+H] +：556.44； 1H NMR(400MHz,CDCl 3)δ7.30–7.26(m,2H),7.26 –7.22(m,2H),7.15(dd,J＝8.3,1.9Hz,1H),6.91(d,J＝8.4Hz,1H),6.84(d,J＝1.8Hz,1H),4.13(q,J＝6.8Hz,1H),4.01–3.94(m,2H),3.91–3.83(m,2H),3.48–3.40(m,1H),3.34(s,3H),2.83–2.70(m,1H),2.69–2.57(m,4H),2.39(s,3H),1.84–1.75(m,1H),1.72–1.64(m,1H),1.63–1.44(m,5H),1.34–1.27(m,1H),1.06(d,J＝6.6Hz,6H),0.98(d,J＝6.8Hz,3H)； 13C NMR(151MHz,CDCl 3)δ169.10,160.58,156.87,154.47,139.62,135.70,135.53,132.26,130.17(2×C),125.69(2×C),122.18,120.22,116.16,114.58,59.01,54.84,54.58,49.31,48.32,47.45,42.75,30.80,30.35,29.25,24.03,23.56,21.32,18.45(2×C),14.11。
实施例16
除了在步骤f中用5-溴-1-(3-氟-4-甲基苯基)-1H-1,2,4-三唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以外，其它采用与实施例6中相同的方法制备化合物16。
Figure PCTCN2018078051-appb-000050
MS(ESI)[M+H] +：546.40； 1H NMR(400MHz,CDCl 3)δ7.22(t,J＝8.0Hz,1H),7.11–7.03(m,3H),6.89(d,J＝8.4Hz,1H),6.83(d,J＝1.7Hz,1H),4.11(q,J＝6.8Hz,1H),3.94–3.88(m,2H),3.87–3.79(m,2H),3.53–3.43(m,1H),3.32(s,3H),2.52–2.42(m,4H),2.29(s,3H),2.28(d,J＝1.5Hz,3H),1.84–1.75(m,1H),1.72–1.62(m,1H),1.60–1.43(m,5H),1.37–1.24(m,1H),0.96(d,J＝6.8Hz,3H)。
实施例17
除了在步骤f中用5-溴-1-(4-氯苯基)-1H-1,2,4-三唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以外，其它采用与实施例6中相同的方法制备化合物17。
Figure PCTCN2018078051-appb-000051
MS(ESI)[M+H] +：548.38； 1H NMR(400MHz,CDCl 3)δ7.44–7.39(m,2H),7.37–7.32(m,2H),7.08(dd,J＝8.3,1.7Hz,1H),6.91(d,J＝8.3Hz,1H),6.82(d,J＝1.7Hz,1H),4.14(q,J＝6.8Hz,1H),4.00–3.90(m,2H),3.90–3.79(m,2H),3.55–3.43(m,1H),3.34(s,3H),2.55–2.50(m,2H),2.50–2.45(m,2H),2.32(s,3H),1.87–1.78(m,1H),1.74–1.66(m,1H),1.63–1.46(m,5H),1.37–1.28(m,1H),0.99(d,J＝6.8 Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.00,160.42,157.21,154.67,136.56,135.71,135.35,132.52,129.80(2×C),127.00(2×C),121.74,120.14,116.06,114.67,59.11,55.46,54.68,54.55,47.07,46.08,42.45,30.81,30.43,29.25,24.05,23.57,14.22。
实施例18
除了在步骤h中用3-甲基-3,8-二氮杂双环[3.2.1]辛烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物18。
Figure PCTCN2018078051-appb-000052
MS(ESI)[M+H] +：554.42； 1H NMR(400MHz,CDCl 3)δ7.29–7.21(m,4H),7.15(d,J＝8.3Hz,1H),6.90(d,J＝8.4Hz,1H),6.83(t,J＝2.0Hz,1H),5.21(s,1H),4.88(d,J＝6.2Hz,1H),4.12(q,J＝6.8Hz,1H),3.47–3.38(m,1H),3.33(s,3H),2.85–2.77(m,2H),2.50(dd,J＝15.8,11.1Hz,2H),2.38(s,3H),2.29(d,J＝2.2Hz,3H),2.06–1.86(m,4H),1.82–1.73(m,1H),1.73–1.63(m,1H),1.60–1.42(m,6H),0.96(d,J＝6.8Hz,3H)； 13C NMR(151MHz,CDCl 3)δ169.03,157.06,156.97,154.50,139.59,135.69,135.43,132.20,130.11(2×C),125.65(2×C),122.14,120.19,116.12,114.54,61.83,60.20,58.96,56.33,54.56,52.81,45.12,30.73,30.28,29.19,28.52,26.59,23.96,23.50,21.27,14.06(d,J＝2.9Hz)。
实施例19
除了在步骤h中用1-甲基-1,4-二氮杂环庚烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物19。
Figure PCTCN2018078051-appb-000053
MS(ESI)[M+H] +：542.39。 1H NMR(400MHz,CDCl 3)δ7.30–7.26(m,2H),7.25–7.22(m,2H),7.14(ddd,J＝8.1,6.2,1.8Hz,1H),6.91(d,J＝8.4Hz,1H),6.86(dd,J＝3.9,1.8Hz,1H),4.18–4.10(m,1H),4.00–3.95(m,1H),3.91(t,J＝6.4Hz,1H),3.89–3.86(m,1H),3.83(t,J＝6.4Hz,1H),3.50–3.41(m,1H),3.35(s,3H),2.85–2.78(m,2H),2.73–2.64(m,2H),2.45–2.38(m,6H),2.09–1.98(m,2H),1.84–1.76(m,1H),1.73–1.65(m,1H),1.63–1.45(m,5H),1.37–1.27(m,1H),0.99(dd,J＝6.8,1.3Hz,3H)。
实施例20
除了在步骤h中用8-氮杂螺[4.5]癸烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物20。
Figure PCTCN2018078051-appb-000054
1H NMR(400MHz,CDCl 3)δ7.28–7.24(m,2H),7.21(d,J＝8.3Hz,2H),7.13(dd,J＝8.3,1.9Hz,1H),6.88(d,J＝8.4Hz,1H),6.84(d,J＝1.8Hz,1H),4.11(q,J＝6.8Hz,1H),3.80–3.70(m,4H),3.48–3.38(m,1H),3.32(s,3H),2.37(s,3H),1.83–1.73(m,1H),1.69–1.58(m,5H),1.56–1.43(m,13H),1.33–1.24(m,1H),0.96(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ168.99,160.82,157.27,154.26,139.39,135.73,135.42,132.12,130.04(2×C),125.60(2×C),122.27,120.15,116.13,114.46,58.98,54.49,45.36,41.48,40.54,38.21,37.78,37.73,37.01,30.72,30.29,29.16,24.34(2×C),23.95,23.48,21.23,14.07。
实施例21
除了在步骤h中用2-氧杂-8-氮杂螺[4.5]癸烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物21。
Figure PCTCN2018078051-appb-000055
MS(ESI)[M+H] +：569.49。 1H NMR(400MHz,CDCl 3)δ7.31–7.26(m,2H),7.23(d,J＝8.3Hz,2H),7.14(dd,J＝8.3,1.5Hz,1H),6.90(d,J＝8.4Hz,1H),6.85(s,1H),4.13(q,J＝6.8Hz,1H),3.89(t,J＝7.1Hz,3H),3.80–3.69(m,2H),3.60(d,J＝1.0Hz,2H),3.47–3.39(m,1H),3.34(s,3H),2.39(s,3H),1.86–1.76(m,4H),1.72–1.65(m,5H),1.60–1.45(m,6H),0.98(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.06,160.90,157.08,154.43,139.56,135.74,135.52,132.26,130.14(2×C),125.66(2×C),122.23,120.20,116.16,114.54,67.42,59.05,54.55,45.37,42.38,40.73,37.33,37.29,35.78,34.60,30.79,30.36,29.23,24.02,23.55,21.30,14.15。
实施例22
除了在步骤h中用1,4-二氮杂双环[4.3.0]壬烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物22。
Figure PCTCN2018078051-appb-000056
MS(ESI)[M+H] +：554.46。 1H NMR(400MHz,CDCl 3)δ7.31–7.20(m,4H),7.14(d,J＝8.1Hz,1H),6.90(d,J＝8.4Hz,1H),6.88–6.83(m,1H),4.84(dd,J＝54.8,12.9Hz,1H),4.56(dd,J＝26.1,13.0Hz,1H),4.12(q,J＝6.9Hz,1H),3.49–3.39(m,1H),3.34(s,3H),3.11(t,J＝8.5Hz,2H),3.07–2.98(m,1H),2.70–2.61(m,1H),2.39(s,3H),2.30(t,J＝11.1Hz,1H),2.23–2.14(m,1H),2.12–2.01(m,1H),1.95–1.63(m,6H),1.62–1.41(m,6H),0.97(d,J＝6.7Hz,3H)。
实施例23
除了在步骤h中用(7S)-5-氮杂螺[2.4]庚烷-7-基氨基甲酸叔丁酯代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物23。
Figure PCTCN2018078051-appb-000057
MS(ESI)[M+H] +：640.31。 1H NMR(400MHz,CDCl 3)δ7.27–7.19(m,5H),7.16–7.09(m,1H),6.87(dd,J＝8.4,3.2Hz,1H),6.82(d,J＝3.5Hz,1H),4.89–4.79(m,1H),4.29(dd,J＝12.1,5.3Hz,1H),4.18–4.07(m,2H),4.00(dd,J＝12.9,5.7Hz,1H),3.92–3.85(m,1H),3.83–3.69(m,2H),3.53–3.38(m,2H),3.31(d,J＝2.7Hz,3H),2.37(s,3H),1.81–1.72(m,1H),1.69–1.61(m,1H),1.59–1.44(m,6H),1.42–1.36(m,10H),0.95(dd,J＝6.8,1.4Hz,3H)。
实施例24
Figure PCTCN2018078051-appb-000058
a)化合物23(0.1g,0.16mmol)溶于1mL二氯甲烷中，加入0.5mL三氟醋酸，然后室温反应3小时，用TLC板监测，反应完后用饱和NaHCO3调pH至7～8，然后用乙酸乙酯(10mL*2)和10mL水萃取，合并有机相，用20mL饱和食盐水洗一次，有机相无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用甲醇/二氯甲烷＝0～5％梯度洗脱，得50mg白色固体化合物24，收率58％。 1H NMR(400MHz,CDCl 3)δ7.30–7.22(m,4H),7.20–7.11(m,1H),6.93–6.88(m,1H),6.87–6.84(m,1H),4.36–4.19 (m,1H),4.13(q,J＝6.8Hz,1H),4.04–3.94(m,1H),3.87(dd,J＝37.4,12.0Hz,1H),3.66(dd,J＝56.3,13.2Hz,1H),3.50–3.40(m,1H),3.34(d,J＝2.3Hz,3H),3.18–3.11(m,1H),2.40(s,3H),2.00(s,2H),1.85–1.74(m,1H),1.73–1.64(m,1H),1.61–1.43(m,6H),0.98(d,J＝6.8Hz,3H),0.83–0.76(m,1H),0.70–0.54(m,3H)。
实施例25
除了在步骤f中用4-溴-5-甲基-3-苯基异噁唑代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯和没有步骤g-h以外，其它采用与实施例6中相同的方法制备化合物25。
Figure PCTCN2018078051-appb-000059
MS(ESI)[M+H] +：402.19； 1H NMR(400MHz,CDCl 3)δ7.58–7.55(m,2H),7.36–7.30(m,3H),7.00(d,J＝8.2Hz,1H),6.82(dd,J＝8.1,1.8Hz,1H),6.66(d,J＝1.8Hz,1H),4.20(q,J＝6.8Hz,1H),3.66–3.56(m,1H),3.41(s,3H),2.29(s,3H),1.90–1.82(m,1H),1.81–1.67(m,2H),1.62–1.46(m,5H),1.08(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.21,164.42,160.19,136.06,130.61,129.71,128.70(2×C),128.10,127.08(2×C),125.59,120.31,117.29,116.33,114.97,58.99,55.01,30.87,30.79,29.19,24.21,23.74,13.91,10.87。
实施例26
除了在步骤f中用5-溴-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以外，其它采用与实施例6中相同的方法制备化合物26。
Figure PCTCN2018078051-appb-000060
MS(ESI)[M+H] +：527.32； 1H NMR(400MHz,CDCl 3)δ7.19–7.10(m,4H),6.89–6.83(m,3H),6.47(d,J＝1.1Hz,1H),4.17–4.05(m,3H),3.87–3.78(m,2H),3.39–3.29(m,4H),2.55–2.43(m,4H),2.31(s,6H),1.79–1.71(m,1H),1.67–1.60(m,1H),1.58–1.38(m,5H),1.20–1.17(m,1H),0.95(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.02,162.73,147.23,143.83,138.03,137.45,135.34,130.80,129.62(2×C),125.40(2×C),124.80,119.47,116.22,114.50,109.54,58.79,55.52,54.77,46.89,45.96,42.33,30.68,30.19,29.69,29.08,23.97,23.52,21.08,13.76。
实施例27
除了在步骤f中用5-溴-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以及步骤h中用(2S，6R)-2,6-二甲基哌嗪代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物27。
Figure PCTCN2018078051-appb-000061
MS(ESI)[M+H] +：541.36； 1H NMR(400MHz,CDCl 3)δ7.18–7.12(m,4H),6.93–6.87(m,2H),6.85(s,1H),6.47(d,J＝3.0Hz,1H),4.92(d,J＝12.9Hz,1H),4.69(d,J＝13.2Hz,1H),4.11(q,J＝6.8Hz,1H),3.50–3.39(m,2H),3.34(s,3H),3.15–3.03(m,2H),3.01–2.92(m,1H),2.63–2.54(m,1H),2.34(s,3H),1.80–1.72(m,1H),1.70–1.61(m,1H),1.60–1.37(m,6H),1.26–1.18(m,6H),0.97(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.16,162.79,146.78,144.23,138.32,137.38,135.47,130.97,129.79(2×C),125.40(2×C),124.66,119.62,116.29,114.68,109.47,58.85,54.83,52.48(d,J＝12.8Hz),51.75,49.55,47.81,30.76,30.26,29.21,27.07,24.05,23.60,21.16,18.30(d,J＝23.3Hz),13.86。
实施例28
除了在步骤f中用5-溴-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以及步骤h中用3-甲基-3,8-二氮杂双环[3.2.1]辛烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物28。
Figure PCTCN2018078051-appb-000062
MS(ESI)[M+H] +：553.40； 1H NMR(400MHz,CDCl 3)δ7.21–7.14(m,4H),6.97(d,J＝0.5Hz,1H),6.92–6.85(m,2H),6.51–6.48(m,1H),5.39(s,1H),4.86(s,1H),4.12(q,J＝6.8Hz,1H),3.44–3.29(m,4H),2.84(t,J＝8.7Hz,2H),2.58–2.45(m,2H),2.35(s,3H),2.32(d,J＝3.0Hz,3H),2.07–1.90(m,4H),1.80–1.73(m,1H),1.70–1.62(m,1H),1.57–1.39(m,6H),0.98(d,J＝6.8Hz,3H)； 13C NMR(126MHz,CDCl 3)δ169.12,159.47,147.90,143.87,138.07,137.65,135.45,130.89,129.69(2×C),125.40(2×C),124.99,119.58,116.34,114.58,109.72,61.85,60.39,58.89,55.78,54.88,52.63(d,J＝4.9Hz),45.28,30.79,30.31,29.18,28.64(d,J＝3.8Hz),26.46(d,J＝5.7Hz),24.07,23.62,21.17,13.86。
实施例29
除了在步骤f中用5-溴-1-(对甲苯基)-1H-吡唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以及步骤h中用8-甲基-3,8-二氮杂双环[3.2.1]辛烷代替N-甲基哌嗪以外，其它采用与实施例6中相同的方法制备化合物29。
Figure PCTCN2018078051-appb-000063
MS(ESI)[M+H] +：553.40； 1H NMR(400MHz,CDCl 3)δ7.22–7.14(m,4H),7.00(s,1H),6.93–6.83(m,2H),6.51(d,J＝5.8Hz,1H),5.44(s,1H),4.89(s,1H),4.13(q,J＝6.6Hz,1H),3.44–3.33(m,4H),2.99–2.81(m,2H),2.65–2.49(m,2H),2.41–2.29(m,6H),2.11–1.92(m,4H),1.83–1.74(m,1H),1.71–1.64(m,1H),1.59–1.42(m,6H),0.99(d,J＝6.8Hz,3H)。
实施例30
除了在步骤f中用5-(2-溴苯基)噁唑代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以及没有步骤g和h之外，其它采用与实施例6中相同的方法制备化合物30。
Figure PCTCN2018078051-appb-000064
MS(ESI)[M+H] +：388.11； 1H NMR(400MHz,CDCl 3)δ7.79(s,1H),7.78–7.74(m,1H),7.43(ddd,J＝14.7,7.2,1.7Hz,2H),7.39–7.35(m,1H),6.98(d,J＝8.1Hz,1H),6.80(dd,J＝8.1,1.8Hz,1H),6.69(d,J＝1.6Hz,1H),6.34(s,1H),4.19(q,J＝6.8Hz,1H),3.72–3.60(m,1H),3.41(s,3H),1.91–1.84(m,1H),1.74–1.68(m,1H),1.66–1.47(m,6H),1.08(d,J＝6.8Hz,3H)。
实施例31
除了在步骤f中用2-(2-溴苯基)-1,3-二氧戊环代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以及没有步骤g和h之外，其它采用与实施例6中相同的方法制备化合物31。
Figure PCTCN2018078051-appb-000065
MS(ESI)[M+H] +：393.16； 1H NMR(400MHz,CDCl 3)δ7.72(s,1H),7.40(s,2H),7.35–7.27(m,1H),7.01–6.90(m,3H),4.30–4.11(m,3H),3.93(s,2H),3.86–3.74(m,1H),3.40(s,3H),2.14–1.92(m,2H),1.85–1.51(m,7H),1.08(d,J＝6.5Hz,3H)。
实施例32
Figure PCTCN2018078051-appb-000066
实施例32中步骤a的操作方法同实施例1中步骤a，步骤b-e的操作方法同实施例6中的步骤a-d，步骤f-g的操作方法同实施例1中的步骤f-g。MS(ESI)[M+H] +：416.37； 1H NMR(400MHz,CDCl 3)δ7.30(d,J＝8.1Hz,2H),7.10(d,J＝8.1Hz,2H),7.00(dd,J＝8.4,1.5Hz,1H),6.83(d,J＝8.1Hz,2H),4.10(q,J＝6.8Hz,1H),3.43(dt,J＝14.6,7.4Hz,1H),3.30(s,3H),2.42(s,3H),2.33(s,3H),1.87–1.76(m,1H),1.75–1.46(m,7H),0.96(d,J＝6.8Hz,3H)。
实施例33
Figure PCTCN2018078051-appb-000067
Figure PCTCN2018078051-appb-000068
实施例33中步骤a-d的操作方法同实施例1中b-e，步骤e-h的操作方法同实施例6中e-h。MS(ESI)[M+H] +：500.41； 1H NMR(400MHz,CDCl 3)δ7.30–7.27(m,2H),7.25–7.22(m,2H),7.17(dd,J＝8.2,1.9Hz,1H),7.14(d,J＝1.8Hz,1H),6.89(d,J＝8.2Hz,1H),4.06(q,J＝6.8Hz,1H),4.01–3.96(m,2H),3.90–3.85(m,2H),3.33(s,3H),2.52(dt,J＝14.2,5.0Hz,4H),2.41(s,3H),2.34(s,3H),2.20–2.12(m,1H),1.11(d,J＝6.8Hz,3H),0.73–0.63(m,1H),0.57–0.42(m,2H),0.21–0.11(m,1H)。
实施例34
除了在步骤f中用5-溴-1-(2,4-二甲基苯基)-1H-1,2,4-三唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以外，其它采用与实施例33中相同的方法制备化合物34。
Figure PCTCN2018078051-appb-000069
MS(ESI)[M+H] +：514.39； 1H NMR(400MHz,CDCl 3)δ7.18–7.14(m,3H),7.13(s,1H),7.09(d,J＝8.0Hz,1H),6.84(d,J＝8.5Hz,1H),4.02(q,J＝6.8Hz,1H),3.99–3.94(m,2H),3.89–3.82(m,2H),3.28(s,3H),2.55–2.45(m,4H),2.37(s,3H),2.31(s,3H),2.12–2.05(m,1H),1.98(s,3H),1.07(d,J＝6.8Hz,3H),0.70–0.62(m,1H),0.55–0.45(m,2H),0.19–0.11(m,1H)； 13C NMR(126MHz,CDCl 3)δ168.76,160.66,156.77,155.07,140.46,135.75,135.08,134.91,132.11,131.22,127.92,127.44,122.28,120.10,114.33,113.64,58.07,55.43,54.68,47.05,46.03,42.41,29.04,27.79,21.28,17.63,12.17,9.13,6.63。
实施例35
除了在步骤f中用5-溴-1-(2,4-二甲基苯基)-1H-1,2,4-三唑-3-羧酸乙酯代替5-溴-1-(对甲苯基)-1H-1,2,4-三唑-3-羧酸乙酯以及步骤h中用1-乙基哌嗪代替N-甲基哌嗪以外，其它采用与实施例33中相同的方法制备化合物35。
Figure PCTCN2018078051-appb-000070
MS(ESI)[M+H] +：528.43； 1H NMR(400MHz,CDCl 3)δ7.19–7.12(m,4H),7.10(d,J＝8.1Hz,1H),6.84(d,J＝8.2Hz,1H),4.06–3.97(m,3H),3.90–3.85(m,2H),3.29(s,3H),2.58–2.52(m,4H),2.47(q,J＝7.2Hz,2H),2.37(s,3H),2.13–2.05(m,1H),1.98(s,3H),1.12–1.04(m,6H),0.71–0.62(m,1H),0.56–0.45(m,2H),0.20–0.10(m,1H)； 13C NMR(151MHz,CDCl 3)δ168.76,160.58,156.76,155.08,140.46,135.74,135.06,134.90,132.11,131.21,127.93,127.44,122.27,120.10,114.34,113.63,58.08,53.23,52.38,52.31,47.04,42.39,29.04,27.79,21.29,17.65,12.17,11.85,9.12,6.63。
实施例36
除了在步骤h中用顺式-2,6-二甲基哌嗪代替N-甲基哌嗪以外，采用实施例33的方法制备以下化合物。
Figure PCTCN2018078051-appb-000071
MS(ESI)[M+H] +：514.29； 1H NMR(400MHz,DMSO-d 6)δ7.38(d,J＝1.0Hz,4H),7.19–7.04(m,3H),4.51(d,J＝13.0Hz,1H),4.29(s,1H),4.00–3.94(m,1H),3.25(d,J＝1.0Hz,3H),2.98(s,3H),2.40(s,3H),2.18(dt,J＝6.7,3.5Hz,1H),1.24(s,1H),1.14(d,J＝6.3Hz,3H),1.05(s,3H),1.02(dd,J＝6.9,1.6Hz,4H),0.69–0.64(m,1H),0.55–0.50(m,1H),0.45–0.34(m,1H).
实施例37
除了在步骤c中用丙酮代替环戊酮，其他采用与实施例12中相同的方法制备以下化合物。
Figure PCTCN2018078051-appb-000072
MS(ESI)[M+H] +：516.23； 1H NMR(400MHz,Chloroform-d)δ7.32–7.29(m,4H),7.05(d,J＝8.3Hz,1H),6.96(s,1H),6.91(d,J＝8.4Hz,1H),4.95–4.86(m,2H),4.18(q,J＝6.8Hz,1H),3.58(p,J＝6.6Hz,1H),3.37(s,3H),3.32–3.21(m,2H),3.13–2.97(m,2H),2.45(s,3H),1.59–1.54(m,3H),1.52–1.44(m,3H),1.16(d,J＝6.5 Hz,3H),1.13(d,J＝6.6Hz,3H),1.07(d,J＝6.8Hz,3H).
实施例38
除了在步骤c中用3-氧杂环丁酮代替环戊酮，其他采用与实施例12中相同的方法制备以下化合物。
Figure PCTCN2018078051-appb-000073
MS(ESI)[M+H] +：530.3； 1H NMR(400MHz,Chloroform-d)δ7.30(m,4H),7.25–7.15(m,1H),7.00(d,J＝8.4Hz,1H),6.22(d,J＝7.3Hz,1H),4.84(d,J＝13.6Hz,1H),4.72(d,J＝6.3Hz,2H),4.36–4.25(m,2H),4.23–4.13(m,1H),3.92(q,J＝6.9Hz,1H),3.41(s,3H),3.27(m,3H),2.88(s,1H),2.45(s,3H),1.44(d,J＝6.0Hz,3H),1.37(s,3H),1.30–1.23(m,1H),0.97(d,J＝6.8,1.6Hz,3H).
实施例39
除了在步骤c中用四氢吡喃酮代替环戊酮，其他采用与实施例12中相同的方法制备以下化合物。
Figure PCTCN2018078051-appb-000074
MS(ESI)[M+H] +：558.3； 1H NMR(400MHz,Chloroform-d)δ7.36–7.26(m,4H),7.22(d,J＝8.5Hz,1H),6.99(d,J＝8.3Hz,1H),6.92–6.85(m,1H),4.85–4.77(m,2H),4.13(q,J＝6.8Hz,1H),3.97(d,J＝11.5Hz,2H),3.38(s,3H),3.32–3.21(m,6H),2.78(s,1H),2.45(s,3H),1.80–1.72(m,1H),1.66–1.61(m,2H),1.36(s,3H),1.31–1.24(m,5H),1.05(d,J＝6.8Hz,3H).
实施例40
除了在步骤c中用4,4-二氟环已酮代替环戊酮，其他采用与实施例12中相同的方法制备以下化合物。
Figure PCTCN2018078051-appb-000075
MS(ESI)[M+H] +：592.3； 1H NMR(400MHz,Chloroform-d)δ7.33(d,J＝1.0Hz,4H),7.23(d,J＝8.4Hz,1H),7.03–6.94(m,1H),6.87(s,1H),4.92–4.83(m,2H),4.11–4.06(m,1H),3.39(s,3H),3.35–3.30(m,3H),3.17–3.12(m,1H),2.46(s,3H),2.18–2.11(m,3H),1.88–1.71(m,2H),1.64(m,5H),1.53–1.47(m,3H),1.46–1.39(m,3H),1.06(d,J＝6.8Hz,3H).
实施例41
除了在步骤c中用3,3-二氟环丁酮代替环戊酮，其他采用与实施例12中相同的方法制备化合物。
Figure PCTCN2018078051-appb-000076
MS(ESI)[M+H] +：563.3； 1H NMR(400MHz,Chloroform-d)δ7.36–7.26(m,4H),7.22(d,J＝8.4Hz,1H),7.02–6.93(m,1H),6.86(s,1H),4.92–4.83(m,2H),4.11–4.05(m,1H),3.38(s,3H),3.34–3.30(m,3H),3.17–3.12(m,1H),2.46(s,3H),2.18–2.11(m,3H),1.88–1.71(m,2H),1.64(m,2H),1.53–1.47(m,2H),1.45–1.37(m,3H),1.05(d,J＝6.8Hz,3H).
实施例42
除了在步骤c中用2-氧代-7-氮杂螺[3.5]壬烷-7-甲酸叔丁酯代替环戊酮，其他采用与实施例12中相同的方法制备化合物。
Figure PCTCN2018078051-appb-000077
MS(ESI)[M+H] +：568.3； 1H NMR(400MHz,Chloroform-d)δ7.38–7.30(m,4H),7.26–7.15(m,1H),7.01(d,J＝8.4Hz,1H),6.24(d,J＝7.2Hz,1H),4.85(d,J＝13.5Hz,1H),4.82(m,2H),4.36–4.28(m,2H),4.32–4.18(m,1H),3.90(q,J＝6.9Hz,1H),3.58–3.48(m,4H),3.41(s,3H),3.27(m,2H),2.88(s,1H),2.45(s,3H),1.85–1.72(m,4H),1.44(d,J＝6.0Hz,3H),1.37(s,3H),0.97(d,J＝6.8,1.6Hz,3H).
实施例43
除了在步骤c中用2-氧杂-螺[3.3]庚烷-6-酮代替环戊酮，其他采用与实施例12中相同的方法制备化合物。
Figure PCTCN2018078051-appb-000078
MS(ESI)[M+H] +：569.3； 1H NMR(400MHz,Chloroform-d)δ7.36–7.30(m,4H),7.25–7.15(m,1H),7.00(d,J＝8.5Hz,1H),6.22(d,J＝7.3Hz,1H),4.80(d,J＝13.6Hz,1H),4.45(d,J＝6.3Hz,2H),4.23–4.13(m,1H),4.12–4.01(m,2H),3.88(q,J＝6.9Hz,1H),3.41(s,3H),3.27(m,3H),2.88(s,1H),2.45(s,3H),1.88–1.76(m,4H),1.44(d,J＝6.0Hz,3H),1.37(s,3H),1.30–1.23(m,2H),0.97(d,J＝6.8,1.6Hz,3H).
实施例44
试剂与条件：a)；b)；c)联硼酸频那醇酯，醋酸钾，二氧六环，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，120℃回流过夜；d)氢氧化锂，THF，H 2O，室温，12小时；e)HATU，DMF，顺式-2,6-二甲基哌嗪，DIPEA，室温反应过夜；f)碳酸氢钠，THF，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，80℃回流过 夜。
Figure PCTCN2018078051-appb-000079
a)将A(300mg，1.18mmol)溶在20mL无水二氯甲烷中，室温搅拌5分钟后加入4-二甲氨基吡啶(216mg，1.17mmol)，继续搅拌5分钟后加入环丙基甲酰氯(214μL，2.36mmol)，然后室温搅拌2小时。蒸干有机相，柱层析纯化得到产物309mg,产率84％。
MS(ESI)[M+H] +：309.1； 1H NMR(400MHz,DMSO-d 6)δ10.87(s,1H),7.67(d,J＝2.1Hz,1H),7.40(dd,J＝8.3,2.1Hz,1H),7.00(d,J＝8.5Hz,1H),5.06(q,J＝7.2Hz,1H),1.99(d,J＝5.7Hz,1H),1.10(d,J＝7.2Hz,3H),1.06(m,1H),1.02–0.95(m,1H),0.88(m,1H),0.80(m,J＝7.9Hz,1H).
b)将B(300mg，0.97mmol)溶在15mL无水二氯甲烷中，室温搅拌5分钟后加入氢化钠(28mg，1.16mmol)，继续搅拌20分钟后加入碘甲烷(66μL，1.07mmol)，然后室温搅拌1小时。蒸干有机相，柱层析纯化得到产物237mg,产率76％。
MS(ESI)[M+H] +：323.2； 1H NMR(400MHz,Chloroform-d)δ7.60(s,1H),7.34(dt,J＝8.7,2.3Hz,1H),6.95(dd,J＝8.7,1.5Hz,1H),5.37(d,J＝7.4Hz,1H),3.33(s,3H),1.93–1.85(m,1H),1.32–1.25(m,1H),1.15(d,J＝7.4Hz,3H),1.01(td,J＝8.2,7.7,3.5Hz,2H),0.81–0.73(m,1H).
c)将C(80mg,0.25mmol)，联硼酸频那醇酯(70mg,0.28mmol)以及醋酸钾(49mg,0.5mmol)溶于2mL无水二氧六环中，通氩气10分钟，加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(9mg,0.012mmol)，继续通气2分钟，氩气保护下加热至120℃回流过夜，用TLC板监测，反应完后用乙酸乙酯萃取，饱和盐水洗，无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用二氯甲烷/甲醇＝0～2％ 梯度洗脱，得67mg米色固体D，收率73％。
d)将E(0.31g,1mmol)以及氢氧化锂(0.168g,4mmol)溶于6mL THF：H2O＝4：1的混合溶剂中，然后室温反应12小时，用TLC板监测，反应完后用1M HCl调节pH至5-6，用乙酸乙酯(20mL*2)和20mL水萃取，合并有机层，用饱和食盐水20mL反萃取一次，有机相无水硫酸钠干燥，过滤，直接蒸干得到0.28g白色粉末F，收率99％。
e)化合物F(0.28g,1mmol)溶于5mL DMF中，加入HATU(0.38g,1mmol),室温反应半小时后加入顺式-2,6-二甲基哌嗪(0.144mL,1mmol)和DIPEA(0.165mL,1mmol)，室温反应过夜，用TLC板监测，反应完后用乙酸乙酯(20mL*2)和60mL饱和碳酸氢钠萃取，合并有机层，用40mL饱和食盐水洗一次，有机相无水硫酸钠干燥，机相硅胶拌样通过色谱柱纯化，使用甲醇/二氯甲烷＝0～5％梯度洗脱，得到0.25g粉色固体G，收率66％。
f)将D(70mg,0.19mmol)，G(78mg,0.2mmol)以及饱和碳酸氢钠(32mg,0.38mmol)溶液溶于1mL THF中，通氩气10分钟，加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(14mg,0.019mmol)，继续通气2分钟，氩气保护下加热至80℃回流过夜蒸干溶剂，用TLC板监测，反应完后用乙酸乙酯萃取，饱和盐水洗，无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用二氯甲烷/甲醇0～5％梯度洗脱，得40mg浅棕色固体补9，收率39％。
MS(ESI)[M+H] +：542.3； 1H NMR(400MHz,Chloroform-d)δ7.64(s,1H),7.48(s,2H),7.33–7.21(m,3H),7.14(s,1H),5.47(s,1H),4.86(s,2H),3.41(s,3H),3.38–3.19(m,2H),3.01(s,1H),2.43(s,3H),1.49(d,J＝27.8Hz,6H),1.27(s,3H),1.15(s,3H),0.84(s,2H),0.70–0.41(m,2H).
实施例45
除了在步骤a中用丙酰氯代替环丙基甲酰氯，其他采用与实施例44中相同的方法制备化合物。
Figure PCTCN2018078051-appb-000080
MS(ESI)[M+H] +：530.4；1H NMR(400MHz,Chloroform-d)δ7.49–7.56(m,2H),7.32(s,4H),7.14–7.09(m,1H),4.87(d,J＝14.2Hz,2H),4.13(q,J＝7.2Hz,1H), 3.38(s,3H),3.36–2.85(m,2H),2.46(s,3H),1.62–1.32(m,7H),1.31–1.24(m,1H),1.24–0.87(m,8H).
实施例46
除了在步骤a中用异丁酰氯代替环丙基甲酰氯，其他采用与实施例44中相同的方法制备化合物。
Figure PCTCN2018078051-appb-000081
MS(ESI)[M+H] +：544.3； 1H NMR(400MHz,Chloroform-d)δ7.52–7.47(m,4H),7.35–7.24(m,2H),7.11(d,J＝9.5Hz,1H),4.87(d,J＝13.3Hz,2H),4.14(q,J＝7.2Hz,1H),3.52–3.18(m,7H),3.11–2.86(m,1H),2.48–2.42(m,4H),1.51(d,J＝7.8Hz,3H),1.44(s,3H),1.35–1.21(m,1H),1.23–0.77(m,8H).
实施例47
Figure PCTCN2018078051-appb-000082
试剂与条件：a)硼氢化钠，THF，四氢呋喃，室温2小时；b)实施例6中间体F，碳酸氢钠，THF，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物，80℃回流过夜；c)甲磺酰氯，三乙胺，二氯甲烷，室温1小时；d)顺式-2,6-二甲基哌嗪，N，N-二异丙基乙胺，DMF，60℃过夜
a)将A(500mg，1.61mmol)溶于20mL四氢呋喃/甲醇(1：1)的溶剂中，加入硼氢化钠(306mg，8.05mmol)，室温搅拌2小时。加水，用1N NaOH调pH6-7， 乙酸乙酯萃取，饱和盐水洗，无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用二氯甲烷/甲醇＝0～2％梯度洗脱，得340mg无色粘稠物B，收率78％。
b)将B(200mg，0.75mmol)和实施例6中间体F(276mg，0.75mmol)以及饱和碳酸氢钠(125mg,1.49mmol)溶液溶于20mL THF中，通氩气10分钟，加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(55mg,0.075mmol)，继续通气2分钟，氩气保护下加热至80℃回流过夜蒸干溶剂，用TLC板监测，反应完后用乙酸乙酯萃取，饱和盐水洗，无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用乙酸乙酯/石油醚50～80％梯度洗脱，得80mg浅棕色固体C，收率19％。
c)将C(60mg，0.14mmol)溶于2mL二氯甲烷中，加入三乙胺(38uL，0.28mmol)，加入甲磺酰氯(16uL，0.21mmol)，室温搅拌1小时。加入饱和碳酸氢钠溶液，用乙酸乙酯萃取，饱和盐水洗，无水硫酸钠干燥，蒸干溶剂得75mg褐色粘稠物D，不纯化直接用于下一步反应。
d)将D(75mg，0.147mmol)溶于2mL DMF中，加入顺式-2,6-二甲基哌嗪(28mg，0.24mmol)和N，N-二异丙基乙胺(47uL，0.285mmol)，60℃搅拌过夜。反应完后用乙酸乙酯萃取，饱和盐水洗，无水硫酸钠干燥，有机相硅胶拌样通过色谱柱纯化，使用二氯甲烷/甲醇8％洗脱，得36mg浅棕色固体补12，收率47％。
MS(ESI)[M+H] +：528.3； 1H NMR(400MHz,Chloroform-d)δ7.32–7.22(m,4H),7.22–7.17(m,1H),6.94(d,J＝8.4Hz,1H),6.79(d,J＝1.8Hz,1H),4.15(q,J＝6.7Hz,1H),3.84(s,2H),3.52–3.40(m,4H),3.38(s,3H),3.15(d,J＝12.5Hz,2H),2.60(dt,J＝15.9,12.0Hz,2H),2.41(s,3H),1.87–1.78(m,1H),1.77–1.65(m,3H),1.59–1.49(m,8H),1.34–1.24(m,2H),1.00(d,J＝6.8Hz,3H).
实施例48
一.溴结构域识别蛋白BRD4抑制剂酶活性测试方法
采用荧光各异向性测试方法(Fluorescence Anisotropy，FA)测试化合物与BRD4(I)的结合活性。FA测试的原理是通过检测荧光素标记的小分子与其它分子相互作用前后分子量的变化，计算水平方向及垂直方向的荧光偏振值作相关分析。如果被荧光标记小分子与大分子之间的结合平衡建立后，它受激发时运动慢，测得的荧光偏振光值会增高。如果荧光标记小分子与大分子之间的结合被其它配基取代，它在游离状态下的旋转或翻转速度会变快，发射光相对于激发光平面将去偏振化，测得的偏振光值降低，从而计算出样品的荧光各异向性。
BRD4(I)识别结构域的表达和纯化：将来自大肠杆菌BL21(DE3)-condon plus-RIL细胞中的新转化的质粒DNA的菌落在含有50μg/mL卡那霉素和34μg/mL氯霉素的50mL的Terrific Broth培养基中在37℃下生长过夜(启动培养物)。然后将 启动培养物在1L新鲜TB培养基中稀释100倍，并将细胞在37℃下生长至在OD600处约0.8的光密度，然后将温度降至16℃。当系统在16℃平衡时，OD600处的光密度约为1.2，并且在16℃下过夜用0.2mmol异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导蛋白表达。通过离心(4000×g，20分钟，4℃)收获细菌，并在-80℃下作为沉淀物储存。将表达His 6标记的蛋白质的细胞重悬浮于裂解缓冲液[50mmol 4-羟乙基哌嗪乙磺酸(HEPES)，在25℃条件下pH 7.5，500mmol NaCl，10mmol咪唑，5％甘油与新鲜加入的0.5mmol三(2-羧乙基)膦盐酸盐(TCEP)和1mmol苯基甲磺酰氟(PMSF)]并在4℃下用JN 3000PLUS高压匀浆器(JNBIO-中国广州)裂解。通过离心(在4℃下12,000×g离心1小时)澄清裂解物，并应用于镍-次氮基乙酸琼脂糖柱。将柱用50mL含有30mmol咪唑的洗涤缓冲液洗涤一次。使用咪唑在洗脱缓冲液(100-250mmol咪唑在50mmol HEPES，在25℃条件下pH 7.5，500mmol NaCl，5％甘油)中的逐步洗脱来洗脱蛋白质。收集所有级分并通过SDS-聚丙烯酰胺凝胶电泳(Bio-Rad Criterion TM Precast Gels，4-12％Bis-Tris，1.0mm，来自Bio-Rad，CA)监测。加入1mmol二硫苏糖醇(DTT)后，洗脱的蛋白质在4℃下用烟草蚀斑病毒(TEV)蛋白酶处理过夜以去除His6标签。将蛋白质浓缩，并在Superdex 75 16/60HiLoad凝胶过滤柱上用尺寸排阻色谱法进一步纯化。通过SDS-聚丙烯酰胺凝胶电泳监测样品，并用凝胶过滤缓冲液，10mmol Hepes pH 7.5,500mM NaCl，1mmol DTT中浓缩至8-10mg/mL，并用于蛋白质结合测定和结晶。
荧光底物为连接荧光分子的(+)-JQ1，工作浓度为5nM。BRD4(I)蛋白工作浓度为10nM，总反应体系为40μL，缓冲液为50mM 4-羟乙基哌嗪乙磺酸(HEPES)pH 7.4，150mM NaCl，0.5mM 3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐(CHAPS)。化合物初筛浓度为1μM，对该条件下抑制率大于60％的化合物测定其IC 50。考虑到化合物的溶解性及DMSO对测定的影响，选定DMSO终浓度为0.2％。所有测定均在该条件下进行。所有成分混合后室温下避光反应4h或4℃过夜反应后测定各向异性值。测试采用康宁(Corning)公司全黑、低边、NBS表面的384孔微孔板(货号为CLS3575)，测试仪器为BioTek synergy2检测仪，激发(excitation)为485nM，发射(emission)为530nM。以缓冲液为系统读数空白值。
数值处理：抑制率＝(C-F)/(C-B)×100％           (公式1)
其中：C：荧光底物与蛋白完全结合的各向异性值
B：荧光底物各向异性本底值
F：化合物相应浓度下的各向异性值
以化合物浓度和相应的抑制率作S曲线。得到相应化合物的IC 50。
BRD4(I)酶活性检测方法FA中使用的荧光底物结构如下：
Figure PCTCN2018078051-appb-000083
(备注：荧光底物是指(+)-JQ1通过连接链与荧光分子相连的产物)
药理学数据：以下表1中公布了部分本发明化合物的药理学试验结果，测试中采用的对照为溴结构域蛋识别白BRD4抑制剂(+)-JQ1。
表1 溴结构域蛋白BRD4抑制剂酶活性测试结果
Figure PCTCN2018078051-appb-000084
由表1可以看出，本发明的化合物对BRD4(I)蛋白有很好的抑制活性，表中所列的化合物的分子活性均比阳性(+)-JQ1活性更好。
二.溴结构域识别蛋白BRD4抑制剂细胞活性测试方法
细胞活性测试了MM.1S细胞株，测试方法分别为：采用人骨髓瘤细胞MM.1S，分别加入化合物处理72h，用CCK-8法检测化合物的增殖生长抑制作用及其程度。
药理学数据：以下表2中公布了部分本发明化合物的药理学试验结果，测试中采用的对照为溴结构域蛋识别白BRD4抑制剂(+)-JQ1。
表2 化合物对MM.1S细胞株的测试结果
Figure PCTCN2018078051-appb-000085
Figure PCTCN2018078051-appb-000086
由表2可以看出，本发明的化合物对MM.1S细胞株具有很好的抑制活性，尤其是实施例1，6，12，14，18，33，34，35，36和37化合物对MM.1S细胞株具有比阳性(+)-JQ1对MM.1S细胞株更好的抑制活性。
三.化合物在肝微粒体的代谢稳定性及酶抑制性质测试方法
3.1代谢稳定性试验测试方法
用体系为150μl的人肝微粒体(终浓度0.5mg/ml)进行代谢稳定性温孵，体系含还原型辅酶Ⅱ(NADPH)(终浓度1mM)和1μM化合物、阳性对照或阴性对照，分别在0min、5min、10min和30min用含(丙米嗪，批号：3221；替硝唑，赠送)的乙腈终止反应，涡旋10min，15000rmp离心10min，取50μl上清于96孔板中进样。通过测定原药的相对减少量计算化合物代谢稳定性。
3.2直接抑制试验(DI试验)测试方法
用体系为100μl的人肝微粒体(终浓度0.2mg/ml)进行直接抑制温孵，体系含NADPH(终浓度1mM)、10μM化合物、阳性抑制剂混合剂cocktail(酮康唑Ketoconazole 10μM，奎尼丁Quinidine 10μM，磺胺苯吡唑Sulfaphenazole 100μM，萘黄酮Naphthoflavone 10μM，反苯环丙胺Tranylcypromine 1000μM)、阴性对照10μM DMSO和混合探针底物(咪达唑仑Midazolam 10μM、睾酮Testosterone 100μM、右美沙芬Dextromethophan 10μM、二氯酚酸Diclofenac 20μM、非那西丁Phenacetin 100μM，美芬妥英Mephenytoin 100μM)，温孵20min后终止反应。通过测定代谢物的相对生成量计算酶相对活性。
3.3机制性抑制试验(TDI试验)测试方法
用体系为200μl的人肝微粒体(终浓度0.2mg/ml)进行机制性抑制温孵，10μM化合物、混合阳性抑制剂(醋竹桃霉素Troleandomycin 10μM、帕罗西汀Paroxetine 10μM、替尼酸Tienilic Acid 10μM、呋拉茶碱Furafylline 10μM)或10μM阴性对照PRO，在加入NADPH(终浓度1mM)或PBS后预温孵0min、5min、10min和30min后加入NADPH(终浓度1mM)和混合探针底物(咪达唑仑Midazolam 5μM、睾酮Testosterone 50μM、右美沙芬Dextromethophan 5μM、二氯酚酸Diclofenac 10μM、非那西丁Phenacetin 5 0μM、S-(+)-美芬妥英S-(+)-mephenytoin 50μM)，温孵10min后终止反应。阳性抑制剂CYP2C19单独做，抑制剂S-(+)-氟西汀S-(+)-fluoxetine 100μM。通过测定代谢物的相对生成量计算酶活性。计算k obs。
药理学数据：以下表3中公布了部分本发明化合物的药理学试验结果。
表3 肝微粒体的代谢稳定性及酶抑制性质测试结果
Figure PCTCN2018078051-appb-000087
HLM Clint(ul/min/mg)是指化合物在人肝微粒体中代谢的清除率，单位是ul/min/mg；mLM Clint(ul/min/mg)是指化合物在小鼠肝微粒体中代谢的清除率，单位是ul/min/mg；HLM t 1/2(min)是指化合物在人肝微粒体中代谢的半衰期，单位是分钟；mLM t 1/2(min)是指化合物在小鼠人肝微粒体中代谢的半衰期，单位是分钟；TDI是指化合物对CYP酶的机制性抑制试验，评价化合物对酶有没有时间依赖性抑制，如果计算出的k obs大于200，表明化合物对该酶有机制性抑制。
从表3中可以看出本发明的化合物在人和小鼠的肝微粒体中代谢都很稳定，尤其是化合物12和27在小鼠的肝微粒体代谢非常稳定，半衰期达到193分钟和248分钟。而且该类化合物对CYP450酶没有直接抑制和机制性抑制作用。
四.化合物对MM.1S裸鼠移植瘤体内生长的抑制作用的测试方法
购买Balb/C裸小鼠(6周，雌性，北京华阜康生物科技股份有限公司)，试验前动物适应性饲养约一周。MM.1S细胞体外培养扩增，收取对数生长期细胞重悬于无血清RPMI1640培养液中，调整细胞浓度；用注射器将细胞悬液注入Balb/C裸小鼠前右肢腋窝皮下，每只动物注射150μL(8.0×106/只)。待平均肿瘤体积生长至约～150mm3左右时，采用随机区组法将动物分为4组，含溶剂对照组、50mg/kg OTX-015组以及20和50mg/kg化合物6的组，每组6只动物；分组后开始给药，给药19天；给药期间，每周测量2次瘤径、称量动物体重，观察动物生活状态，对异常状况进行记录。
肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b 2，其中a、b分别表示长、宽。
根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝V t/V 0。其中V 0为分笼给药时(即d 0)测量所得肿瘤体积，V t为每一次测量时的肿瘤体积。
根据测量的结果计算出相对肿瘤增殖率T/C(％)，计算公式如下：T/C(％)＝(T RTV/C RTV)×100％，T RTV：治疗组RTV；C RTV：阴性对照组RTV。OTX-015是新型BRD2/3/4抑制剂，对一些B细胞肿瘤细胞株有抗增殖活性，对DLBCL细胞的IC 50为192nM。
药理学数据：以下表4中公布了化合物6的体内药效结果。
表4 体内药效结果
Figure PCTCN2018078051-appb-000088
从表4可以看出，化合物6在每天口服给药50mg/kg对MM.1S裸鼠移植瘤体内生长的有显著的抑制作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种如通式(I)所示的化合物，或其立体异构体、前药、溶剂化物、水合物、晶型、或其药学上可接受的盐：
    Figure PCTCN2018078051-appb-100001
    式中：
    R 1、R 2、R 3各自独立地为氢、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C3-C10环烷基、取代或未取代的3-8元杂环基、取代或未取代的-C(＝O)R X或取代或未取代的苄基；其中，R X为C1-C6烷基、C1-C6烷氧基或C3-C10环烷基；
    波浪线表示构型是R型或者S型或者消旋体；
    A环为5-10元杂芳基、5-8元杂环基、或C6-C10芳基；
    X为C或者N；
    R 4为取代或未取代的C6-C20芳基、取代或未取代的苄基、5-10元杂环基或5-10元杂芳基；
    R 5为无、氢、取代或未取代的C1-C6烷基、
    Figure PCTCN2018078051-appb-100002
    Figure PCTCN2018078051-appb-100003
    其中，R 6、R 7各自独立地为氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C10环烷基、取代或未取代的C6-C20芳基、取代或未取代的苄基、取代或未取代的5-10元杂环基、或取代或未取代的5-10元杂芳基；或者，R 6和R 7和它们连接的氮原子一起形成取代或未取代的5-15元杂环基；
    所述取代是指具有选自下组的一个或多个取代基：卤素、羟基、硝基、氰基、叔丁氧羰基、C1-C6烷基、C1-C6烷氧基、C3-C10环烷基、NR 8R 9；各R 8、各R 9独立地为氢、C1-C6烷基、C3-C10环烷基、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、芴甲氧羰基(Fmoc)、甲氧羰基、乙氧羰基、邻苯二甲酰基(Pht)、 对甲苯磺酰基(Tos)、三氟乙酰基(Tfa)、特戊酰基、苯甲酰基、三苯甲基(Trt)、2,4-二甲氧基苄基(Dmb)、对甲氧基苄基(PMB)、或苄基(Bn)。
  2. 一种如通式(I)所示的化合物，或其立体异构体、前药、溶剂化物、水合物、晶型、或其药学上可接受的盐：
    Figure PCTCN2018078051-appb-100004
    式中：
    R 1、R 2、R 3各自独立地为氢、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C3-C10环烷基或取代或未取代的苄基；
    波浪线表示构型是R型或者S型或者消旋体；
    A环为5-10元杂芳基、5-8元杂环基、或C6-C10芳基；
    X为C或者N；
    R 4为取代或未取代的C6-C20芳基、取代或未取代的苄基、5-10元杂环基或5-10元杂芳基；
    R 5为无、氢、取代或未取代的C1-C6烷基、
    Figure PCTCN2018078051-appb-100005
    Figure PCTCN2018078051-appb-100006
    其中，R 6、R 7各自独立地为氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C10环烷基、取代或未取代的C6-C20芳基、取代或未取代的苄基、取代或未取代的5-10元杂环基、或取代或未取代的5-10元杂芳基；或者，R 6和R 7和它们连接的氮原子一起形成取代或未取代的5-15元杂环基；
    所述取代是指具有选自下组的一个或多个取代基：卤素、羟基、硝基、氰基、叔丁氧羰基、C1-C6烷基、C1-C6烷氧基、C3-C10环烷基、NR 8R 9；各R 8、各R 9独立地为氢、C1-C6烷基、C3-C10环烷基、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、芴甲氧羰基(Fmoc)、甲氧羰基、乙氧羰基、邻苯二甲酰基(Pht)、对甲苯磺酰基(Tos)、三氟乙酰基(Tfa)、特戊酰基、苯甲酰基、三苯甲基(Trt)、2,4-二甲氧基苄基(Dmb)、对甲氧基苄基(PMB)、或苄基(Bn)
  3. 根据权利要求1或2所述的化合物，其特征在于，A环为C6-C10芳基、5-6元杂环基或5-6元杂芳基；
    R 1为氢、取代或未取代的C1-C4烷基、或取代或未取代的C1-C4烷氧基，所述取代基为卤素、羟基、氨基、硝基或氰基；
    R 2为取代或未取代的C1-C4烷基、取代或未取代的C1-C4烷氧基，所述取代基为卤素、羟基、氨基、硝基或氰基；
    R 3为取代或未取代的C1-C4烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C3-C6环烷基、取代或未取代的3-8元杂环基、取代或未取代的-C(＝O)R X或取代或未取代的苄基，其中，R X为C1-C6烷基、C1-C6烷氧基或C3-C6环烷基，所述取代基选自下组：卤素、羟基、氨基、硝基、氰基、C1-C3烷基、或者C1-C3烷氧基；
    R 4为取代或未取代的C6-C14芳基、取代或未取代的苄基、取代或未取代的含有1-3个选自N、O和S的杂原子的如下基团：5-8元杂环基或5-8元杂芳基，所述取代是指具有1-3个取代基，各取代基独立为：卤素、羟基、氨基、硝基、氰基、C1-C4烷基、或者C1-C4烷氧基；
    R 5为无、氢、取代或未取代的C1-C4烷基、
    Figure PCTCN2018078051-appb-100007
    Figure PCTCN2018078051-appb-100008
    其中R 6、R 7各自独立地选自氢、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C14芳基、取代或未取代的苄基、取代或未取代的含有1-3个选自N、O和S的杂原子的如下基团：5-8元杂环基和5-8元杂芳基，所述取代是指具有1-3个取代基，各取代基独立为：卤素、羟基、氨基、甲氨基、氰基、N(C1-C4烷基)(C1-C4烷基)、C1-C4烷基或者C1-C4烷氧基；
    或者，R 6和R 7和它们连接的氮原子一起形成取代或未取代的含有1-3个N、O、S杂原子的5-10元杂环基，所述取代是指具有1-3个取代基，各取代基独立为：卤素、NR 8R 9、羟基、硝基、氰基、叔丁基氧碳基、C1-C4烷基、C3-C8环烷基、或者C1-C4烷氧基；各R 8、各R 9独立地为氢、苄氧羰基(Cbz)、叔丁氧羰基(Boc)、甲氧羰基、乙氧羰基、对甲苯磺酰基(Tos)、三氟乙酰基(Tfa)、 特戊酰基、苯甲酰基、2,4-二甲氧基苄基(Dmb)、对甲氧基苄基(PMB)、苄基(Bn)。
  4. 根据权利要求1或2所述的化合物，其特征在于，A环选自如下基团：苯基、吡啶基、嘧啶基、三唑基、四唑基、噻唑烷基、吡唑基、噁唑基、异噁唑基和咪唑基；
    R 1为甲基、乙基、丙基或异丙基；
    R 2为甲基、乙基、丙基或异丙基；
    R 3为环丙基、环丁基、环戊基、环己基、
    Figure PCTCN2018078051-appb-100009
    或-C(＝O)R X；其中，R X为C1-C4烷基或C3-C6环烷基；
    R 4为取代或未取代的C6-C10芳基、取代或未取代的苄基、取代或未取代的含有1-3个选自N、O和S的杂原子的如下基团：5-6元杂环基或5-6元杂芳基，所述取代是指具有1-3个取代基，各取代基独立为：氟、氯、溴、羟基、氨基、硝基、氰基、甲基、乙基、丙基、异丙基、甲氧基、乙氧基、丙氧基或者异丙氧基；
    R 5为氢原子、取代或未取代的甲基、取代或未取代的乙基、取代或未取代的丙基、取代或未取代的异丙基、
    Figure PCTCN2018078051-appb-100010
    其中
    R 6、R 7各自独立地选自氢原子、取代或未取代的甲基、取代或未取代的乙基、取代或未取代的丙基、取代或未取代的异丙基、取代或未取代的哌啶基；所述取代是指具有1-3个取代基，各取代基独立为：氟、氯、溴、羟基、氨基、甲氨基、氰基、-N(CH 3) 2、-N(CH 2CH 3) 2、-N(CH 3)(CH 2CH 3)、甲基、乙基、丙基、异丙基、甲氧基、乙氧基、丙氧基、异丙氧基；
    或者，R 6和R 7和它们连接的氮原子一起形成如下取代或未取代的基团：哌嗪基、高哌嗪基、哌啶基、
    Figure PCTCN2018078051-appb-100011
    Figure PCTCN2018078051-appb-100012
    所述取代是指具有1-3个取代基，各取代基独立为：氟、氯、溴、羟基、硝基、氰基、甲基、乙基、丙基、异丙基、环丙基、环丁基、环戊基、环己基、叔丁氧碳基、NR 8R 9；各R 8、各R 9独立地为氢、叔丁氧羰基(Boc)、甲氧羰基、乙氧羰基、特戊酰基、苯甲酰基、苄基(Bn)。
  5. 根据权利要求1或2所述的化合物，其特征在于，
    A环选自如下基团：苯基、三唑基、四唑基、噻唑烷基、吡唑基和异噁唑基；X为C或者N；
    R 1为甲基；
    R 2为甲基；
    R 3为环丙基、环戊基、
    Figure PCTCN2018078051-appb-100013
    或-C(＝O)R X；其中，R X为C1-C4烷基或C3-C6环烷基；
    R 4为取代或未取代的如下基团：苯基、噁唑基、1,3-二氧戊烷基，所述取代是指具有1-3个取代基，各取代基独立为：氟、氯和甲基；
    R 5为无、氢原子、甲基、
    Figure PCTCN2018078051-appb-100014
    其中R 6、R 7独立地选自为氢原子、取代或未取代的甲基、取代或未取代的乙基，取代基为：-N(CH 3) 2；或者，R 6和R 7和它们连接的氮原子一起形成如下取代或未取代的基团：哌嗪基、高哌嗪基、
    Figure PCTCN2018078051-appb-100015
    所述取代是指具有1-3个取代基，各取代基独立为：甲基、乙基、异丙基、环丙基、NH 2、N(CH 3) 2、NHBoc。
  6. 根据权利要求1或2所述的化合物，其特征在于，所述化合物为：
    Figure PCTCN2018078051-appb-100016
    Figure PCTCN2018078051-appb-100017
    Figure PCTCN2018078051-appb-100018
  7. 一种权利要求1所述的化合物的制备方法，其特征在于，
    Figure PCTCN2018078051-appb-100019
    所述制备方法包括式V化合物制备式I化合物的步骤，
    各式中，各取代基和波浪线的定义如权利要求1所述。
  8. 一种药物组合物，其特征在于，包括权利要求1所述的通式(I)所示的化合物，或其立体异构体、前药、蛋白靶向降解偶联物、溶剂化物、水合物、晶型、或其药学上可接受的盐中的一种或多种，以及药学上可接受的载体。
  9. 一种根据权利要求1所述的通式(I)所示的化合物，或其立体异构体、前药、蛋白靶向降解偶联物、溶剂化物、水合物、晶型、或其药学上可接受的盐，或权利要求7所述的药物组合物的用途，其特征在于，(i)用于制备溴结构域识别蛋白的选择性抑制剂；或(ii)用于制备预防和/或治疗由溴结构域识别蛋白介导的相关疾病的药物。
  10. 根据权利要求9所述的用途，其特征在于，所述由溴结构域识别蛋白介导的相关疾病选自：恶性肿瘤、免疫性疾病、心血管疾病或炎症。
  11. 根据权利要求10所述的用途，其特征在于，所述恶性肿瘤选自：急性淋巴细胞白血病、急性骨髓性白血病、B细胞慢性淋巴细胞白血病、慢性骨髓单核细胞白血病、中线癌、肺癌、B细胞淋巴瘤、前列腺癌、胃癌、结肠直肠癌、肾癌、肝癌、乳腺癌、胰腺癌。
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