WO2020216343A1 - 嘧啶并[5,4-b]吡呤化合物、其光学异构体、制备方法及用途 - Google Patents

嘧啶并[5,4-b]吡呤化合物、其光学异构体、制备方法及用途 Download PDF

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WO2020216343A1
WO2020216343A1 PCT/CN2020/086787 CN2020086787W WO2020216343A1 WO 2020216343 A1 WO2020216343 A1 WO 2020216343A1 CN 2020086787 W CN2020086787 W CN 2020086787W WO 2020216343 A1 WO2020216343 A1 WO 2020216343A1
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cancer
compound
pyrimido
pharmaceutically acceptable
pyrine
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PCT/CN2020/086787
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English (en)
French (fr)
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张翱
丁健
谢华
宋子兰
薛宇
邢莉
童林江
耿美玉
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中国科学院上海药物研究所
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Priority to EP20794381.2A priority Critical patent/EP3960743A4/en
Priority to US17/606,121 priority patent/US20220204518A1/en
Publication of WO2020216343A1 publication Critical patent/WO2020216343A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—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 three hetero rings
    • C07D487/14—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to a pyrimido[5,4-b]pyrrolizin (pyrimido[5,4-b]pyrrolizin) compound, its optical isomers, preparation methods, pharmaceutical compositions and uses, and in particular to a A pyrimido[5,4-b]pyrine compound used as a small molecule inhibitor targeting BTK kinase, its preparation method, pharmaceutical composition and its preparation for treating diseases related to BTK kinase signal transduction pathway Use in medicine.
  • Bruton's tyrosine kinase is a non-receptor tyrosine kinase belonging to the TEC tyrosine kinase family.
  • the members of the TEC family include Tec, Bmx, BTK, Itk and Txk.
  • BTK is the most widely studied member of the TEC family and a key regulator in the B cell receptor (BCR) signaling pathway. It is widely expressed in many types of hematological malignancies and participates in the proliferation, differentiation and apoptosis of B cells. Therefore, BTK has become an important molecular target for the treatment of hematological malignancies.
  • Ibrutinib a small molecule inhibitor targeting BTK kinase developed by the international pharmaceutical company Pharmacyclics, was approved by the FDA in November 2013 as a breakthrough therapy for relapsed or refractory mantle cell lymphoma (Mantle Cell lymphoma, MCL) clinical treatment.
  • MCL Mantle Cell lymphoma
  • Ibrutinib also has a good inhibitory effect on Tec and EGFR kinases, the drug has relatively large side effects.
  • the second-generation BTK inhibitor Acalabrutinib (ACP-96) which was launched in 2017, has high selectivity for BTK and lacks irreversible activity on off-target kinases.
  • the overall response rate is relatively high.
  • Zanubrutinib (BGB-3111), another highly selective irreversible BTK inhibitor of BeiGene in China, has also recently released positive clinical data, especially in the indication of mantle cell lymphoma (MCL), which will be released in January 2019 Obtained Breakthrough Therapy Certification (BTD) from FDA on the 14th.
  • MCL mantle cell lymphoma
  • BTD Obtained Breakthrough Therapy Certification
  • the first-generation inhibitors have high inhibitory activity against BTK, but the target selection and bioavailability are poor; the second-generation inhibitors have good selectivity, but the inhibition rate for BTK is lower than that of the first-generation inhibitors.
  • the core skeletons of compounds currently on the market or under research are all bicyclic systems.
  • BTK kinase that has excellent BTK inhibitory activity compared with known compounds, and maintains better BTK inhibitory selectivity, better oral administration performance, and better metabolic stability Inhibitor compound;
  • a compound that has better anti-tumor activity in vivo than known compounds as a small molecule inhibitor targeting BTK kinase.
  • a pyrimido[5,4-b]pyrine compound especially the compound S17016, which not only has excellent in vivo and in vitro activities, but also has good oral administration properties. And metabolic stability, significantly better than the marketed drug ibrutinib (ibrutinib) and other control compounds, and has the potential to treat diseases related to the BTK kinase signal transduction pathway. And completed the present invention on this basis.
  • An object of the present invention is to provide a pyrimido[5,4-b]pyrine compound.
  • Another object of the present invention is to provide pharmaceutically acceptable salts, enantiomers, diastereomers, optical isomers, racemates, deuterated derivatives, and solvates of the compounds Or hydrate.
  • An object of the present invention is to provide a method for preparing the compound.
  • Another object of the present invention is to provide a compound comprising the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical isomer, racemate, deuterated derivative , Solvate or hydrated pharmaceutical composition.
  • Another object of the present invention is to provide the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical isomer, racemate, deuterated derivative, solvate Use of the compound or hydrate in the preparation of a medicine for treating diseases related to the BTK kinase signal transduction pathway.
  • Another object of the present invention is to provide the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical isomer, racemate, deuterated derivative, solvate Or hydrates in the preparation of drugs for the treatment, prevention or regulation of cancer.
  • pyrimido[5,4-b]pyrine compound a pyrimido[5,4-b]pyrine compound, pharmaceutically acceptable salts, enantiomers, and diastereomers of the general formula (I) Enantiomers, optical isomers, racemates, deuterated derivatives, solvates or hydrates:
  • R 1 and R 2 are each independently H or C 1 -C 10 alkyl, preferably H or C 1 -C 6 alkyl, more preferably H or C 1 -C 3 alkyl, more preferably H ;
  • R 3 is H or C 1 -C 10 alkyl, preferably H or C 1 -C 6 alkyl, more preferably H or C 1 -C 3 alkyl, more preferably methyl.
  • the compound of general formula I is a compound represented by formula I-1:
  • R 1 , R 2 and R 3 are as defined above.
  • the compound of general formula I is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the compound of general formula I-1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the compounds of the present invention may have asymmetric centers, chiral axes and chiral planes, and may exist in the form of enantiomers, diastereomers, optical isomers, racemates and mixtures thereof.
  • each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.
  • the pharmaceutically acceptable salt of the compound may be a conventional non-toxic salt formed by the reaction of the compound with an inorganic acid or an organic acid.
  • a conventional non-toxic salt can be prepared by reacting a compound with an inorganic acid or organic acid.
  • the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, etc.
  • the organic acid includes lysine.
  • Acid arginine, ornithine, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, Malay Acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, hexanoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid , Ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, aspartic acid and isethionic acid.
  • the deuterated derivative of the compound of the present invention may contain any one or several hydrogen in the structure of the compound of the general formula I or I-1 A derivative in which an atom is replaced by its isotope deuterium atom.
  • the method includes the following steps:
  • R 1 , R 2 and R 3 are as defined above;
  • R 1 , R 2 and R 3 are as defined above;
  • the protective group T includes tert-butoxycarbonyl, benzyloxycarbonyl, methoxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, methyl Oxycarbonyl, ethoxycarbonyl, phthalimide, p-toluenesulfonyl, trifluoroacetyl, trityl, 2,4-dimethoxybenzyl, p-methoxybenzyl, benzyl Group, etc.; preferably tert-butoxycarbonyl, benzyloxycarbonyl, methoxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, methoxycarbonyl, ethoxycarbonyl; more preferably tert-butoxycarbonyl.
  • the method further includes the following steps:
  • the deprotection reagent includes acidic reagents and alkaline reagents, such as hydrobromic acid, hydrochloric acid, trifluoroacetic acid, sodium hydroxide, sodium carbonate, triethylamine, etc.; preferably trifluoroacetic acid;
  • the reaction solvent includes dichloromethane, ethyl acetate, DMF, etc., preferably dichloromethane; the extraction organic solvent is preferably dichloromethane;
  • the inert atmosphere is preferably a nitrogen atmosphere, an argon atmosphere, and preferably a nitrogen atmosphere.
  • the above preparation method further includes the following steps:
  • R 1 , R 2 and R 3 are as defined above;
  • Intermediate A6 is obtained by reacting intermediate A4 with substituted phenylboronic acid or boronic acid ester (A5); where -BP 1 P 2 is -B(OH) 2 or Preferably
  • the compound prepared by the above method is its optical isomer, and in this method:
  • R 1 , R 2 , R 3 , T, P 1 and P 2 are as defined above.
  • the above method includes the following steps:
  • reaction solvent includes DMF, DMSO, THF, preferably DMF
  • extraction organic solvent includes dichloromethane , Ethyl acetate, preferably ethyl acetate
  • reaction solvent includes 1,4-dioxane, THF, Preferably it is 1,4-dioxane;
  • extraction organic solvent includes dichloromethane and ethyl acetate, preferably ethyl acetate;
  • the palladium catalyst includes Pd(PPh 3 ) 4 , PdCl 2 , PdCl 2 (dppf ), Pd(OAc) 2 and Pd(PPh 3 ) 2 Cl 2 , preferably Pd(PPh 3 ) 4 ;
  • the reaction solvent includes 1,4-dioxane, THF, ethanol, toluene, methanol, DMF and One or more mixed solvents such as water, preferably 1,4-dioxane;
  • the extraction organic solvent includes dichloromethane and ethyl acetate, preferably ethyl acetate;
  • reaction solvent includes DMF, THF, acetonitrile, etc., preferably DMF
  • extraction organic solvent includes Dichloromethane, ethyl acetate, preferably ethyl acetate
  • the palladium catalyst includes Pd(PPh 3 ) 4 , PdCl 2 , PdCl 2 (dppf), Pd (OAc) 2 and Pd(PPh 3 ) 2 Cl 2 , preferably PdCl 2 (dppf);
  • the reaction solvent includes 1,4-dioxane, THF, ethanol, toluene, methanol, DMF and water, etc. Or multiple mixed solvents, preferably THF;
  • the extraction organic solvents include dichloromethane and ethyl acetate, preferably ethyl acetate;
  • the deprotection reagents include acidic reagents and alkaline reagents, such as hydrobromic acid, hydrochloric acid, trichloride Fluoroacetic acid, sodium hydroxide, sodium carbonate, triethylamine, etc.; preferably trifluoroacetic acid;
  • the reaction solvent includes dichloromethane, ethyl acetate, DMF, etc., preferably dichloromethane;
  • the extraction organic solvent includes two Methylene chloride, ethyl acetate, preferably dichloromethane;
  • the amount of the reaction solvent and the liquid reactant whose specific amount is not specified is a conventional amount. For example, based on 1 molar equivalent of the reactant in each reaction, the volume of the reaction solvent and the liquid reactant is 1 molar equivalent of 100-100000 mL/mol Reactant.
  • the inert atmosphere is preferably a nitrogen atmosphere, an argon atmosphere, and preferably a nitrogen atmosphere.
  • the compound prepared by the method is its optical isomer, and in the method:
  • step a) N-protected (S)-methanesulfonic acid 2-amino-but-3-en-1-yl ester (B1) is used in step a) instead of methanesulfonic acid 2-((tert-butoxycarbonyl)amino )-But-3-en-1-yl ester (A1), the steps of this method are the same as the above steps a) to f), and finally the compound of general formula I-1 can be obtained.
  • DIAD Diisopropyl azodicarboxylate
  • DIPEA Diisopropylethylamine
  • HATU 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • NBS N-bromosuccinimide
  • Pd(PPh 3 ) 4 Tetra(triphenylphosphine) palladium
  • Pd(PPh 3 ) 2 Cl 2 Bistriphenylphosphorus palladium dichloride
  • PE petroleum ether
  • the conditions of the above-mentioned method for preparing the compound of general formula I or the compound of general formula I-1, such as reactants, solvents, acids or bases, catalysts, condensing agents, amount of compounds used, reaction temperature, reaction time, etc. are all different Limited to the above explanation.
  • the compounds of the present invention can also be conveniently prepared by combining various synthetic methods described in this specification or known in the art. Such combinations can be easily performed by those skilled in the art to which the present invention belongs.
  • a pharmaceutical composition which contains a therapeutically effective amount of the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical Isomers, racemates, deuterated derivatives, solvates or hydrates, and contain one or more pharmaceutically acceptable carriers.
  • Carriers can include solid carriers as well as liquid carriers.
  • the pharmaceutical composition preferably contains active ingredients in a weight ratio of 1 to 99%, and the preferred ratio is the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical Isomers, racemates, deuterated derivatives, solvates or hydrates as active ingredients account for 65% to 99% of the total weight, and the remaining part is, for example, a pharmaceutically acceptable carrier.
  • the compounds and pharmaceutical compositions of the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in suitable solid or liquid carriers or diluents. Neutralization is suitable for sterilization equipment for injection or drip.
  • the unit dose of the formulation formulation contains 0.05-1000 mg of the compound, preferably, the unit dose of the formulation formulation contains 0.1 mg-500 mg of the compound.
  • the compound and pharmaceutical composition of the present invention can be clinically used in mammals, including humans and animals, and can be administered through oral, nasal, skin, lung, or gastrointestinal administration routes. Most preferably it is oral.
  • the daily dose is 0.01-1000 mg/kg body weight for one-time administration, or 0.01-500 mg/kg body weight for divided doses.
  • the individual's optimal dose should be determined based on the specific treatment. Usually start with a small dose and gradually increase the dose until the most suitable dose is found.
  • the present invention provides the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical isomer, racemate, deuterated derivative
  • a compound, a solvate or a hydrate in the preparation of a medicine for treating diseases related to the BTK kinase signal transduction pathway.
  • the present invention provides the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical isomer, racemate, deuterated derivative
  • the compound, solvate or hydrate is used to treat diseases related to BTK kinase signal transduction pathway or to prepare drugs for treating diseases related to BTK kinase signal transduction pathway.
  • the present invention provides a method for treating diseases related to BTK kinase signal transduction pathway, wherein a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof is administered to the subject , Enantiomers, diastereomers, optical isomers, racemates, deuterated derivatives, solvates or hydrates.
  • the diseases related to the BTK kinase signal transduction pathway include cancer, hyperplasia, restenosis, immune disorders and inflammation.
  • the present invention provides the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical isomer, racemate, deuterated derivative
  • the present invention provides the compound, its pharmaceutically acceptable salt, enantiomer, diastereomer, optical isomer, racemate, deuterated derivative Compounds, solvates or hydrates, used for the treatment, prevention or regulation of cancer or for the preparation of drugs for the treatment, prevention or regulation of cancer.
  • the present invention provides a method of treating, preventing or regulating cancer, wherein a therapeutically effective amount of the compound, its pharmaceutically acceptable salt, enantiomer is administered to the subject Isomers, diastereomers, optical isomers, racemates, deuterated derivatives, solvates or hydrates.
  • the cancer of the present invention includes hematological tumors and solid tumors; wherein, the hematological tumors include lymphoma, myeloma, and leukemia; preferably, the cancer includes, but is not limited to, histocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, ovarian cancer, squamous cell carcinoma of the head and neck, stomach cancer, breast cancer, childhood hepatocellular carcinoma, colorectal cancer, cervical cancer, lung cancer, sarcoma, nasopharyngeal cancer, pancreatic cancer, gelatinization Plasma cell carcinoma, prostate cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, thyroid cancer, testicular cancer, cervical cancer, endometrial cancer, esophageal cancer, renal cell carcinoma, bladder cancer, lung adenocarcinoma, liver cancer And astrocytoma; preferably mantle cell lympho
  • the compound of the present invention has an inhibitory activity on BTK at the molecular level and at the cellular level that is equivalent to or better than that of the marketed drug ibrutinib or other control compounds.
  • the compound of the present invention has high activity on BTK-sensitive human diffuse large B lymphoma Ramos cells and TMD8 cells, especially TMD8 cells, which can be taken orally, and the activity in mice is significantly better than that of ibrutinib and has good metabolic stability.
  • Is a selective inhibitor of BTK with development potential and can be used as a candidate drug for preclinical and clinical research.
  • Figure 1 Schematic diagram of experimental results of REC-1 xenograft tumor model.
  • Figure 2 Schematic diagram of the experimental results of the TMD-8 xenograft tumor model.
  • the terms “include”, “include”, “have”, “include” or any other similar terms are all open-ended transitional phrases, which are intended to cover non-exclusive inclusions.
  • a composition or article containing plural elements is not limited to the elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent in the composition or article.
  • the term “or” refers to the inclusive “or” rather than the exclusive “or”. For example, any one of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), Both A and B are true (or exist).
  • a value should be understood as having the accuracy of the number of significant digits.
  • the number 40.0 should be understood to cover the range from 39.50 to 40.49.
  • the raw materials, reaction reagents, catalysts or solvents involved in the following specific embodiments can all be purchased through commercial channels or prepared by conventional methods in the prior art.
  • reaction buffer 50mM HEPES pH 7.4, 50mM MgCl 2 , 0.5mM MnCl 2 , 0.2mM Na 3 VO 4 , 1mM DTT
  • the final concentration is 5 ⁇ M.
  • 50 ⁇ L of BTK tyrosine kinase protein diluted in reaction buffer to start the reaction.
  • Compounds S1, S10, Ibrutinib and Acalabrutinib were used as positive control compounds.
  • Compounds S1 and S10 were prepared using methods disclosed in the prior art (for example, CN108101905A) or similar methods, and Ibrutinib and Acalabrutinib were purchased from Selleck.
  • the inhibition rate of each compound is calculated by the following formula:
  • the IC 50 value was obtained by the four-parameter regression using the software that came with the microplate reader. The results are listed in Table 1 below.
  • the cell suspension (Ramos: 10000 cells/well; TMD8: 12000 cells/well) was seeded in a 96-well plate, and then placed in a 37°C incubator for 2 hours. After the cell status was stabilized, different concentrations of test compound were added to each well (Set up 3 multiple wells for each concentration), and set a blank control (wells containing only culture medium and no cells), negative control (wells with cells only, no compound) and positive compound controls. After 72 hours of dosing treatment, add 20 ⁇ L MTT (5mg/mL) to each well and incubate at 37°C for 4h, add 100 ⁇ L triple solution (10% SDS, 5% isobutanol, 0.01M HCl), and place at 37°C overnight. Measure the OD value with tunable wavelength microplate microplate reader SPECTRAMAX Plus384 at 570nm wavelength.
  • the inhibition rate of the compound is calculated by the following formula:
  • the IC 50 value was obtained by the four-parameter regression using the software that came with the microplate reader. The experiment was repeated 3 times independently, and the results are listed in Table 2 below.
  • compound S17016 is superior to the previous compounds S1, S10, S18s, S19s and S20s in inhibiting BTK-dependent cell proliferation, and is also superior to the first-generation BTK inhibitor Ibrutinib and the second-generation currently marketed.
  • the BTK inhibitor Acalabrutinib.
  • the compound S17016 of the present invention has higher proliferation inhibitory activity on Ramos cells, and higher proliferation inhibitory activity on TMD8 cells.
  • Cell culture human lymphoma TMD8 cells are cultured in suspension in vitro, and the culture conditions are RPMI 1640 medium (supplier: gibco; article number: 22400-089; production lot number: 4868546) with 10% fetal bovine serum, 100U/mL penicillin and 100 ⁇ g /mL streptomycin, 37 °C 5% CO 2 culture. Routine processing and passage are carried out twice a week. When the cell saturation is 80%-90%, the cells are collected, counted, and seeded.
  • Human mantle cell lymphoma REC-1 cells were cultured in suspension in vitro with RPMI 1640 medium (supplier: gibco; article number: 22400-089; production lot number: 1868795) with 10% fetal bovine serum, 100U/mL penicillin and 100 ⁇ g/mL streptomycin, 37°C 5% CO2 culture. Routine processing and passage are carried out twice a week. When the cell saturation is 80%-90%, the cells are collected, counted, and seeded.
  • group administration was started.
  • the group was divided into groups by a random grouping software based on Excel, with 6 mice in each group.
  • the drug needs to be mixed gently before administering to animals.
  • Tumor measurement and experimental indicators are to investigate whether tumor growth is inhibited, delayed or cured. The tumor diameter was measured with a vernier caliper three times a week.
  • the calculation formula of tumor volume is:
  • V 0.5a ⁇ b 2 .
  • a and b represent the long diameter and short diameter of the tumor, respectively.
  • TGI % or relative tumor growth rate T/C (%).
  • TGI (%) reflects the tumor growth inhibition rate.
  • TGI (%) [1-(Average tumor volume at the end of a certain treatment group-average tumor volume at the beginning of the treatment group) / (Average tumor volume at the end of the solvent control group treatment-average tumor volume at the beginning of the solvent control group Tumor volume)] ⁇ 100%.
  • T/C% T RTV /C RTV ⁇ 100% ( TRTV : relative tumor volume in the treatment group; C RTV : relative tumor volume in the negative control group).
  • RTV relative tumor volume
  • the average tumor volume at time, T RTV and C RTV take the same day data.
  • Statistical analysis including the mean and standard error (SEM) of the tumor volume at each time point in each group.
  • the treatment group showed the best therapeutic effect on the 15th day (REC-1 xenograft tumor model) and 17th day (TMD8 xenograft tumor model) after administration, so statistical analysis was performed based on this data to evaluate the group difference.
  • the comparison between three or more groups is analyzed by one-way ANOVA. If the F value is significantly different, the Games-Howell method is used to test. Use SPSS 17.0 for all data analysis. p ⁇ 0.05 considered a significant difference.
  • the in vivo efficacy of compound S17016 in human mantle cell lymphoma REC-1 xenograft tumor model is shown in Table 5 and Figure 1.
  • the tumor volume of the tumor-bearing mice in the solvent control group reached 3501 mm 3
  • the tumor volume is 1323mm 3 .
  • the tumor volume of compound S17016 15mg/kg and 30mg/kg groups were 1034 and 680mm 3 , respectively.
  • c.p value is calculated based on tumor volume.
  • c.p value is calculated based on tumor volume.
  • the compound S18s was used to repeat the above experiment in the human lymphoma TMD8 xenograft tumor model, and the T/C (%) results are listed in the table below. In the table below, the T/C (%) results of compound S17016 are also listed for comparison.
  • the intragastric administration is formulated with 0.5% sodium carboxymethylcellulose (CMC-Na) containing 1% Tween 80, and the intravenous administration is formulated with 5% DMSO/5% Tween 80/90% normal saline.
  • CMC-Na sodium carboxymethylcellulose
  • Gavage 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 hours after administration;
  • Intravenous administration 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 hours after administration;
  • venous blood was taken from the rat's posterior venous plexus, placed in a heparinized test tube, centrifuged at 11000 rpm for 5 minutes, and the plasma was separated and frozen in a refrigerator at -20°C.
  • the concentration of S17016 in rat plasma was determined by LC/MS/MS method.
  • the peak concentration C max and peak time T max are measured values
  • AUC 0-t value of area under the drug-time curve calculated by trapezoidal method
  • AUC 0- ⁇ AUC 0-t +C t /k e ,
  • C t is the blood drug concentration at the last measurable time point
  • Average residence time MRT AUMC/AUC.
  • Absolute bioavailability F (AUC intragastric gavage ⁇ D intravenous )/(AUC intravenous ⁇ D intragastric gavage ) ⁇ 100%
  • Liver microsomes were incubated in 96-well plates. The volume of each incubation system was 450 ⁇ L. The medium was 0.1M Tris buffer (pH 7.4), including liver microsomes with a final concentration of 0.33mg/mL and 0.1 ⁇ M test drug. , 5.0mM MgCl 2 , 0.01% DMSO, 0.005% BSA and 1.0mM NADPH. Incubate at 37°C. After pre-incubation for 10 minutes, NADPH was added to initiate the reaction. After 0, 7, 17, 30, and 60 minutes, 50 ⁇ L of the incubation system was added to the same volume of methanol to terminate the reaction.
  • Tris buffer pH 7.4
  • the S17016 in the incubated samples was determined by LC/MS/MS, the ion source was an electrospray ionization source (ESI source), and the scanning method was multiple reaction monitoring (MRM).
  • ESI source electrospray ionization source
  • MRM multiple reaction monitoring
  • HMM human liver microsomes
  • RLM rat liver microsomes
  • MLM mouse liver microsomes
  • S17016 is basically a prototype drug in human, mouse, and rat liver microsomes, and basically has no major metabolites. It effectively overcomes the diphenyl ether structure in compounds such as S1, S10, S18s and S19s.
  • the terminal benzene ring is hydroxylated with good metabolic stability.
  • compound S17016 is a novel structure, oral, highly selective, and highly active BTK inhibitor. Its activity in vivo and in vitro is significantly better than that of BTK inhibitors that have been marketed abroad. At the same dose, it can inhibit tumor growth. The activity is significantly better than the positive control drug Ibrutinib, which is of great development value.

Abstract

本发明涉及一种如通式I所示的嘧啶并[5,4-b]吡呤化合物、其光学异构体、制备方法、含有其的药物组合物及用途。本发明的化合物在分子水平及细胞水平对于激酶BTK的抑制活性均较好，同时，也具有良好的体内抗肿瘤活性和药代动力学性质。

Description

嘧啶并[5,4-b]吡呤化合物、其光学异构体、制备方法及用途 技术领域
本发明涉及一种嘧啶并[5,4-b]吡呤(pyrimido[5,4-b]pyrrolizin)化合物、其光学异构体、制备方法、药物组合物及用途，具体而言，涉及一种用作靶向BTK激酶的小分子抑制剂的嘧啶并[5,4-b]吡呤化合物、其制备方法、药物组合物及其在制备用于治疗BTK激酶信号转导通路相关的疾病的药物中的用途。
背景技术
布鲁顿酪氨酸激酶(Bruton’s tyrosine kinase，BTK)是隶属于TEC酪氨酸激酶家族的一种非受体酪氨酸激酶。TEC家族成员包括Tec，Bmx，BTK，Itk和Txk。BTK是TEC家族中研究最广泛的成员，是B细胞受体(BCR)信号通路中的关键调节因子，在多种类型恶性血液肿瘤中广泛表达，参与B细胞的增殖、分化与凋亡。因此，BTK已成为恶性血液肿瘤药物治疗的重要分子靶点。
国际制药公司Pharmacyclics开发的靶向BTK激酶小分子抑制剂依布鲁替尼(Ibrutinib)于2013年11月由FDA批准上市，作为突破性治疗药物用于复发或难治性套细胞淋巴瘤(Mantle cell lymphoma，MCL)的临床治疗。然而，由于依布鲁替尼对Tec及EGFR等激酶也有较好的抑制作用，导致该药物毒副作用较大。2017年上市的第二代BTK抑制剂Acalabrutinib(ACP-96)对BTK有较高的选择性，缺乏对脱靶激酶的不可逆活性，在复发/难治性慢性淋巴细胞性白血病(Chronic Lymphocytic Leukemia，CLL)患者的I/II期临床试验中，总反应率较高。国内百济神州的另一个高选择性的BTK不可逆抑制剂Zanubrutinib(BGB-3111)最近也发布积极的临床数据，特别是在套细胞淋巴瘤(MCL)适应症上表现优异，在2019年1月14日获得FDA的突破疗法认证(BTD)。总的来讲，第一代抑制剂对BTK的抑制活性高，但靶点选择和生物利用度差；第二代抑制剂选择性好，但对BTK的抑制率低于第一代抑制剂。结构上，目前上市或在研的化合物的核心骨架均是双环体系。
为了获得兼具第一代抑制剂的高活性和第二代抑制剂的良好选择性的新一代BTK抑制剂，中国科学院上海药物研究所在CN108101905A专利中公开了一类结构独特的三环化合物。其中，嘧啶并[5,4-b]吲嗪类化合物S1和S10及嘧啶并[5,4-b]吡呤类化合物S18、S19和S20显示了较高的BTK抑制活性，在进一步的工作中，还合成了S18、S19和S20的S构型的化合物(即S18s、S19s和S20s)。但进一步研究发现S1、S10、S18s和S19s这些化合物代谢过程中不稳定，末端苯基的4-位易于氧化；而化合物S20s的口服生物利用度不理想。
Figure PCTCN2020086787-appb-000001
技术问题
本发明所要解决以下技术问题之一：
提供一种相比于已知化合物具有优良的BTK抑制活性，且保持较好的BTK抑制选择性；
提供一种相比于已知化合物具有优良的BTK抑制活性，且保持较好的BTK抑制选择性、和具有更好的口服给药性能的作为靶向BTK激酶的小分子抑制剂的化合物；
提供一种相比于已知化合物具有优良的BTK抑制活性，且保持较好的BTK抑制选择性、和更好的代谢稳定性的作为靶向BTK激酶的小分子抑制剂的化合物；
提供一种相比于已知化合物具有更好的口服给药性能和更好的代谢稳定性的作为靶向BTK激酶的小分子抑制剂的化合物；
提供一种相比于已知化合物具有优良的BTK抑制活性，且保持较好的BTK抑制选择性、更好的口服给药性能、和更好的代谢稳定性的作为靶向BTK激酶的小分子抑制剂的化合物；
提供一种相比于已知化合物具有更好的体内抗肿瘤活性的作为靶向BTK激酶的小分子抑制剂的化合物。
发明内容
针对上述问题，申请人经进一步研究，发现一种嘧啶并[5,4-b]吡呤化合物，特别是其中的化合物S17016，不仅具有优异的体内外活性，同时还具有良好的口服给药性能和代谢稳定性，明显优于上市药物ibrutinib(依布鲁替尼)及其它对照化合物，具有治疗BTK激酶信号转导通路相关的疾病的潜力。并在此基础上完成了本发明。
本发明的一个目的是提供一种嘧啶并[5,4-b]吡呤化合物。
本发明的另一个目的是提供所述化合物的药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物。
本发明的一个目的是提供所述化合物的制备方法。
本发明的另一个目的是提供一种包含所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物的药物组合物。
本发明的另一个目的是提供所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物在制备用于治疗BTK激酶信号转导通路相关的疾病的药物中的用途。
本发明的另一个目的是提供所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物在制备用于治疗、预防或调控癌症的药物中的用途。
根据本发明的一个方面，其提供了一种如通式(I)所述的嘧啶并[5,4-b]吡呤化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物：
Figure PCTCN2020086787-appb-000002
其中，R 1和R 2各自独立地为H或C 1-C 10烷基，优选为H或C 1-C 6烷基，更优选为H或C 1-C 3烷基，更优选为H；
R 3为H或C 1-C 10烷基，优选为H或C 1-C 6烷基，更优选为H或C 1-C 3烷基，更优选为甲基。
优选地，所述通式I化合物为式I-1所示化合物：
Figure PCTCN2020086787-appb-000003
其中R 1，R 2，R 3如上定义。
优选地，所述通式I化合物为
Figure PCTCN2020086787-appb-000004
优选地，所述通式I-1化合物为
Figure PCTCN2020086787-appb-000005
本发明的化合物可能具有不对称中心、手性轴和手性平面并且可以以对映异构体、非对映异构体、光学异构体、外消旋体及其混合物的形式存在。例如，当化合物结构中存在手性碳原子时，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。
化合物的药学上可接受的盐可以为所述化合物与无机酸或有机酸反应形成的常规的无毒盐。例如，常规的无毒盐可通过化合物与无机酸或有机酸反应制得，所述 无机酸包括盐酸、氢溴酸、硫酸、硝酸、氨基磺酸和磷酸等，以及所述有机酸包括赖氨酸、精氨酸、鸟氨酸、柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙酸、草酸、三氟乙酸、硬脂酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对氨基苯磺酸、2-乙酰氧基苯甲酸、天冬氨酸和羟乙磺酸等。
本发明所述的化合物的氘代衍生物，即所述通式I化合物或I-1化合物的氘代形式，可以包含所述通式I化合物或I-1化合物结构中任意一个或几个氢原子被其同位素氘原子替代的衍生物。
根据本发明的一个方面，其提供了一种制备上述嘧啶并[5,4-b]吡呤化合物的方法，
该方法包括以下步骤：
Figure PCTCN2020086787-appb-000006
其中R 1，R 2，R 3如上定义；
优选为
Figure PCTCN2020086787-appb-000007
其中R 1，R 2，R 3如上定义；
将反应物A8或B8脱除保护基T，然后再与
Figure PCTCN2020086787-appb-000008
反应得到通式I化合物或通式I-1化合物，其中，所述保护基T包括叔丁氧羰基、苄氧羰基、笏甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基、邻苯二甲酰亚胺基、对甲苯磺酰基、三氟乙酰基、三苯甲基、2，4-二甲氧基苄基、对甲氧基苄基、苄基等；优选为叔丁氧羰基、苄氧羰基、笏甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基；进一步优选为叔丁氧羰基。
优选地，该方法进一步包括如下步骤：
在反应溶剂中加入A8或B8(1摩尔当量)，在搅拌下加入或滴入脱保护试剂， 在室温下搅拌2-4小时，反应完成后，将反应液旋干得到粗品，
然后再加入三乙胺(1-3摩尔当量，例如1摩尔当量)，搅拌，再加入
Figure PCTCN2020086787-appb-000009
(1-2摩尔当量，例如1.1摩尔当量)和HATU(1-2摩尔当量，例如1.1摩尔当量)，再加入反应溶剂，在冰水浴下，逐滴加入三乙胺，逐渐升温至室温，搅拌1-2小时，
反应完成后，加入水和有机溶剂萃取，合并有机层，洗涤、干燥、柱层析分离得到通式I化合物或通式I-1化合物；
所述脱保护试剂包括酸性试剂和碱性试剂，例如氢溴酸、盐酸、三氟乙酸、氢氧化钠、碳酸钠、三乙胺等；优选为三氟乙酸；
所述反应溶剂包括二氯甲烷、乙酸乙酯、DMF等，优选为二氯甲烷；所述萃取有机溶剂优选为二氯甲烷；
所述惰性气氛优选为氮气气氛、氩气气氛，优选为氮气气氛。
另一方面，在实施方式中，上述制备方法进一步包括以下步骤：
Figure PCTCN2020086787-appb-000010
其中R 1，R 2，R 3如上定义；
a)由4-氯-5-碘-7H-吡咯并[2,3-d]嘧啶(原料2)和N-保护的甲磺酸2-氨基-丁-3-烯-1-基酯(A1)反应得到中间体A3；其中，T为常用的氨基保护基，所述氨基保护基包括叔丁氧羰基、苄氧羰基、笏甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基、邻苯二甲酰亚胺基、对甲苯磺酰基、三氟乙酰基、三苯甲基、2，4-二甲氧基苄基、对甲氧基苄基、苄基等；优选为叔丁氧羰基、苄氧羰基、笏甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基；进一步优选为叔丁氧羰基；
b)由中间体A3与NR 1R 2·H 2O或NR 1R 2反应得到中间体A4；
c)将中间体A4和取代苯硼酸或硼酸酯(A5)反应得到中间体A6；其中，-BP 1P 2为-B(OH) 2或
Figure PCTCN2020086787-appb-000011
优选为
Figure PCTCN2020086787-appb-000012
d)将中间体A6和N-溴代丁二酰亚胺(NBS)反应得到中间体A7；
e)将中间体A7进行闭环反应得到中间体A8；
f)将中间体A8脱除保护基T，然后再与
Figure PCTCN2020086787-appb-000013
反应得到通式I化合物。
优选地，上述方法制备的化合物为其光学异构体，且在该方法中：
除了在步骤a)中用N-保护的(S)-甲磺酸2-氨基-丁-3-烯-1-基酯(B1)代替N-保护的甲磺酸2-氨基-丁-3-烯-1-基酯(A1)以外，该方法的步骤与上述的步骤a)至f)相同：
Figure PCTCN2020086787-appb-000014
其中R 1，R 2，R 3，T，P 1，P 2如上定义。
进一步优选地，在实施方式中，上述方法包括以下步骤：
a)在反应溶剂中加入干燥的4-氯-5-碘-7H-吡咯并[2,3-d]嘧啶(原料2)(1摩尔当量)和无水碳酸钾(1.5-2.5摩尔当量，例如2摩尔当量)，然后加入粉碎的N-保护的甲磺酸2-氨基-丁-3-烯-1-基酯(A1)(1.1-2摩尔当量，例如1.5摩尔当量)，在惰性气氛下、在50-60℃下加热搅拌9-15小时，
反应完成后，加入水和有机溶剂萃取，合并有机层，干燥、柱层析分离得到中间体A3；所述反应溶剂包括DMF、DMSO、THF，优选为DMF；所述萃取有机溶剂包括二氯甲烷、乙酸乙酯，优选为乙酸乙酯；
b)向中间体A3(1摩尔当量)中加入反应溶剂，再加入NR 1R 2·H 2O或NR 1R 2，在110-130℃下密封反应2-3小时，
反应完成后，冷却至室温，加入水和有机溶剂萃取，合并有机层，洗涤、干燥、柱层析分离得到中间体A4；其中，所述反应溶剂包括1，4-二氧六环、THF，优选为1，4-二氧六环；所述萃取有机溶剂包括二氯甲烷、乙酸乙酯，优选为乙酸乙酯；
c)将中间体A4(1摩尔当量)和取代苯硼酸或硼酸酯(A5)(1.2-2摩尔当量，例如1.4摩尔当量)混合，并加入钯催化剂(0.1-1摩尔当量，例如0.2摩尔当量)，加入反应溶剂，在惰性气氛下、在搅拌下加入碳酸钠溶液，并在80-100℃下回流搅拌4-6小时，
反应完成后，加入水和有机溶剂萃取，合并有机层，洗涤、干燥、柱层析分离、重结晶得到中间体A6；所述钯催化剂包括Pd(PPh 3) 4、PdCl 2、PdCl 2(dppf)、Pd(OAc) 2和Pd(PPh 3) 2Cl 2，优选为Pd(PPh 3) 4；所述反应溶剂包括1，4-二氧六环、THF、乙醇、甲苯、甲醇、DMF和水等一种或多种的混合溶剂，优选为1，4-二氧六环；所述萃取有机溶剂包括二氯甲烷、乙酸乙酯，优选为乙酸乙酯；
d)在反应溶剂中加入中间体A6(1摩尔当量)、N-溴代丁二酰亚胺(NBS)(1.0-1.5摩尔当量，例如1.1摩尔当量)，在室温下搅拌过夜，
反应完成后，加入水和有机溶剂萃取，合并有机层，洗涤、干燥、柱层析分离得到中间体A7；所述反应溶剂包括DMF、THF、乙腈等，优选为DMF；所述萃取有机溶剂包括二氯甲烷、乙酸乙酯，优选为乙酸乙酯；
e)将中间体A7(1摩尔当量)和钯催化剂(0.1-0.5摩尔当量，例如0.11摩尔当量)混合，加入反应溶剂，在惰性气氛下、在搅拌下加入氢氧化钠溶液，在80-90℃下回流搅拌12-18小时，
反应完成后，加入水和有机溶剂萃取，合并有机层，洗涤、干燥、柱层析分离得到中间体A8；所述钯催化剂包括Pd(PPh 3) 4、PdCl 2、PdCl 2(dppf)、Pd(OAc) 2和Pd(PPh 3) 2Cl 2，优选为PdCl 2(dppf)；所述反应溶剂包括1，4-二氧六环、THF、乙醇、甲苯、甲醇、DMF和水等一种或多种的混合溶剂，优选为THF；所述萃取有机溶剂包括二氯甲烷、乙酸乙酯，优选为乙酸乙酯；
f)在反应溶剂中加入中间体A8(1摩尔当量)，在搅拌下加入或滴入脱保护试剂，在室温下搅拌2-4小时，反应完成后，将反应液旋干得到粗品，
然后再加入三乙胺(1-3摩尔当量，例如1摩尔当量)，搅拌，再加入
Figure PCTCN2020086787-appb-000015
(1-2摩尔当量，例如1.1摩尔当量)和HATU(1-2摩尔当量，例如1.1摩尔当量)，再加入溶剂，在冰水浴下，逐滴加入三乙胺，逐渐升温至室温，搅拌1-2小时，
反应完成后，加入水和有机溶剂萃取，合并有机层，洗涤、干燥、柱层析分离得到通式I化合物；所述脱保护试剂包括酸性试剂和碱性试剂，例如氢溴酸、盐酸、三氟乙酸、氢氧化钠、碳酸钠、三乙胺等；优选为三氟乙酸；所述反应溶剂包括二氯甲烷、乙酸乙酯、DMF等，优选为二氯甲烷；所述萃取有机溶剂包括二氯甲烷、乙酸乙酯，优选为二氯甲烷；
所述反应溶剂和未指明具体用量的液体反应物用量为常规用量，例如，以每个 反应中1摩尔当量反应物计，反应溶剂和液体反应物的体积为100-100000mL/mol的1摩尔当量反应物。
所述惰性气氛优选为氮气气氛、氩气气氛，优选为氮气气氛。
进一步优选地，该方法制备的化合物为其光学异构体，且在该方法中：
除了在步骤a)中用N-保护的(S)-甲磺酸2-氨基-丁-3-烯-1-基酯(B1)代替甲磺酸2-((叔丁氧基羰基)氨基)-丁-3-烯-1-基酯(A1)以外，该方法的步骤与上述的步骤a)至f)相同，最终可得到通式I-1化合物。
在该制备方法以及本发明中，使用的术语如下所述：
9-BBN：9-硼双环[3.3.1]壬烷
DCM：二氯甲烷
DIAD：偶氮二甲酸二异丙酯
DIPEA：二异丙基乙胺
DMF：N,N-二甲基甲酰胺
EA：乙酸乙酯
HATU：2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯
NBS：N-溴代丁二酰亚胺
NIS：N-碘代丁二酰亚胺
PdCl 2(dppf)：[1,1'-双(二苯基膦基)二茂铁]二氯化钯
Pd(PPh 3) 4：四(三苯基膦)钯
PdCl 2：二氯化钯
Pd(OAc) 2：醋酸钯
Pd(PPh 3) 2Cl 2：双三苯基磷二氯化钯
PE：石油醚
THF：四氢呋喃
DMSO：二甲基亚砜
以上所述制备通式I化合物或通式I-1化合物的方法的条件，例如反应物、溶剂、酸或碱、催化剂、缩合剂、所用化合物的用量、反应温度、反应所需时间等均不限于上面的解释。本发明化合物还可以任选地将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便的制得，这样的组合可由本发明所属领域的技术人员容易地进行。
根据本发明的又一个实施方式，其提供了一种药物组合物，其含有治疗有效量的所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物，以及含有一种或多种可药用的载体。载体可以包括固体载体以及液体载体。
所述药物组合物优选含有重量比为1～99％的活性成分，其优选的比例是，所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物作为活性成分占总重量比65％～99％，其余部分例如为可药用的载体。
本发明所述的化合物和药物组合物可以是多种形式，如片剂、胶囊、粉剂、糖浆、溶液状、悬浮液和气雾剂等，并可以存在于适宜的固体或液体的载体或稀释液 中和适宜的用于注射或滴注的消毒器具中。
所述药物组合物的各种剂型可按照药学领域的常规制备方法制备。其制剂配方的单位剂量中包含0.05～1000mg所述化合物，优选地，制剂配方的单位剂量中包含0.1mg～500mg所述化合物。
本发明的化合物和药物组合物可对哺乳动物临床使用，包括人和动物，可以通过口、鼻、皮肤、肺、或者胃肠道等给药途径进行给药。最优选为口服。优选日剂量为0.01～1000mg/kg体重，一次性服用，或0.01～500mg/kg体重分次服用。不管用何种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。
根据本发明的又一个实施方式，其提供了所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物在制备用于治疗BTK激酶信号转导通路相关的疾病的药物中的用途。
根据本发明的又一个实施方式，其提供了所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物，用于治疗与BTK激酶信号转导通路相关的疾病或者用于制备治疗BTK激酶信号转导通路相关的疾病的药物。
根据本发明的又一个实施方式，其提供了一种治疗与BTK激酶信号转导通路相关的疾病的方法，其中，向受试者施用治疗有效量的所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物。
其中，所述BTK激酶信号转导通路相关的疾病包括癌症、超常增生、再狭窄、免疫病症和炎症。
根据本发明的又一个实施方式，其提供了所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物在制备用于治疗、预防或调控癌症的药物中的用途。
根据本发明的又一个实施方式，其提供了所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物，用于治疗、预防或调控癌症或者用于制备治疗、预防或调控癌症的药物。
根据本发明的又一个实施方式，其提供了一种治疗、预防或调控癌症的方法，其中，向受试者施用治疗有效量的所述化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物。
本发明所述癌症包括血液肿瘤和实体瘤；其中，所述血液肿瘤包括淋巴瘤、骨髓瘤和白血病；优选地，所述癌症包括但不限于组织细胞性淋巴瘤、套细胞淋巴瘤、弥漫大B细胞淋巴瘤、慢性淋巴细胞白血病、卵巢癌、头颈磷状上皮细胞癌、胃癌、乳腺癌、儿童肝细胞癌、结肠直肠癌、***、肺癌、肉瘤、鼻咽癌、胰腺癌、成胶质细胞癌、***癌、小细胞肺癌、非小细胞肺癌、多发性骨髓瘤、甲状腺癌、睾丸癌、***、子宫内膜癌、食道癌、肾细胞癌、膀胱癌、肺腺癌、肝癌和星形细胞瘤；优选为套细胞淋巴瘤、弥漫大B细胞淋巴瘤、慢性淋巴细胞白血病、头颈磷状上皮细胞癌、组织细胞性淋巴瘤、肺腺癌、小细胞肺癌、非小细胞肺癌、胰腺癌、***状肾细胞癌、肝癌、胃癌、结肠癌、多发性骨髓瘤和成胶质细胞瘤。
根据本发明的实验可知，本发明的化合物在分子水平及细胞水平对于BTK的抑制活性均较上市药物ibrutinib(依布鲁替尼)或其他对照化合物相当或更好。重要的是，本发明的化合物对BTK敏感的人弥漫大B淋巴瘤Ramos细胞和TMD8细胞具有较高活性，特别是TMD8细胞，可口服，且小鼠体内活性显著优于ibrutinib，代谢稳定性好，是一种具有开发潜力的BTK选择性抑制剂，可作为候选药物进行临床前和临床研究。
附图说明
图1：REC-1异种移植瘤模型的实验结果示意图。
图2：TMD-8异种移植瘤模型的实验结果示意图。
具体实施方式
为使本领域具有普通知识的人员可了解本发明的特点及效果，以下谨就说明书及申请专利范围中提及的术语及用语进行一般性的说明及定义。除非另有指明，否则文中使用的所有技术及科学上的字词，皆具有本领域技术人员对于本发明所了解的通常意义，当有冲突情形时，应以本说明书的定义为准。
在本文中，用语“包含”、“包括”、“具有”、“含有”或其他任何类似用语均属于开放性连接词(open-ended transitional phrase)，其意欲涵盖非排他性的包括物。举例而言，含有复数要素的一组合物或制品并不仅限于本文所列出的这些要素而已，而是还可包括未明确列出但却是该组合物或制品通常固有的其他要素。除此之外，除非有相反的明确说明，否则用语“或”是指涵盖性的“或”，而不是指排他性的“或”。例如，以下任何一种情况均满足条件“A或B”：A为真(或存在)且B为伪(或不存在)、A为伪(或不存在)且B为真(或存在)、A和B均为真(或存在)。此外，在本文中，用语“包含”、“包括”、“具有”、“含有”的解读应视为已具体公开并同时涵盖“由…所组成”及“实质上由…所组成”等封闭式或半封闭式连接词。
在本文中，所有以数值范围或百分比范围形式界定的特征或条件仅是为了简洁及方便。据此，数值范围或百分比范围的描述应视为已涵盖且具体公开所有可能的次级范围及范围内的个别数值，特别是整数数值。举例而言，“1至8”的范围描述应视为已经具体公开如1至7、2至8、2至6、3至6、4至8、3至8等所有次级范围，特别是由所有整数数值所界定的次级范围，且应视为已经具体公开范围内如1、2、3、4、5、6、7、8等个别数值。除非另有指明，否则前述解释方法适用于本发明全文的所有内容，不论范围广泛与否。
若数量或其他数值或参数是以范围、较佳范围或一系列上限与下限表示，则其应理解成是本文已特定公开了由任一对该范围的上限或较佳值与该范围的下限或较佳值构成的所有范围，不论这些范围是否有分别公开。此外，本文中若提到数值的范围时，除非另有说明，否则该范围应包括其端点以及范围内的所有整数与分数。
在本文中，在可实现发明目的的前提下，数值应理解成具有该数值有效位数的精确度。举例来说，数字40.0则应理解成涵盖从39.50至40.49的范围。
在本文中，对于使用马库什群组(Markush group)或选项式用语以描述本发明特征或实例的情形，本领域技术人员应了解马库什群组或选项列表内所有要素的次级群组或任何个别要素亦可用于描述本发明。举例而言，若X描述成“选自于由X 1、X 2及X 3所组成的群组”，亦表示已经完全描述出X为X1的主张与X为X 1及/或X 2 的主张。再者，对于使用马库什群组或选项式用语以描述本发明的特征或实例的情况，本领域技术人员应了解马库什群组或选项列表内所有要素的次级群组或个别要素的任何组合亦可用于描述本发明。据此，举例而言，若X描述成“选自于由X 1、X 2及X 3所组成的群组”，且Y描述成“选自于由Y 1、Y 2及Y 3所组成的群组”，则表示已经完全描述出X为X 1或X 2或X 3而Y为Y 1或Y 2或Y 3的主张。
以下具体实施方式中涉及的原料、反应试剂、催化剂或溶剂，均可通过商业途径购买得到或现有技术常规方法制备得到。
以下具体实施方式本质上仅是例示性，且并不欲限制本发明及其用途。此外，本文并不受前述现有技术或发明内容或以下具体实施方式或实施例中所描述的任何理论的限制。
制备实施例1 S17016的合成
1、中间体3的合成
Figure PCTCN2020086787-appb-000016
在250mL圆底烧瓶中加入4-氯-5-碘-7H-吡咯并[2,3-d]嘧啶(原料2,17.28g,1eq)和无水碳酸钾(2eq)，真空干燥除水。加入干燥的DMF作为溶剂，粉碎的(S)-甲磺酸2-((叔丁氧基羰基)氨基)-丁-3-烯-1-基酯(原料1,24.6g,1.5eq)，置换氮气。于55℃下加热搅拌12小时，时间可适当延长以确保反应完全。
反应完成后，加入水和乙酸乙酯萃取三次，合并酯层，用水反萃一次，用饱和食盐水洗。无水硫酸钠干燥。干法过柱(洗脱液：CHCl 3:MeOH＝100:1)得产品(S)-(1-(4-氯-5-碘-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体3,19.28g)，产率为69.5％。
1H NMR(300MHz,CDCl 3)δ8.60(s,1H),7.39(s,1H),5.82(ddd,J＝17.1,10.5,5.5Hz,1H),5.33-5.14(m,2H),4.80(s,1H),4.63-4.51(m,1H),4.51-4.42(m,1H),4.35(s,1H),1.33(s,9H)。ee>99.5％。
2、中间体4的合成
Figure PCTCN2020086787-appb-000017
在350mL耐压管中加入(S)-(1-(4-氯-5-碘-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体3,9.2g)，加入1,4-二氧六环(40mL)作为溶剂，加入氨水(40mL)。于120℃下密封反应2.5小时。
反应完成后，冷却至室温，加入水和乙酸乙酯萃取，合并酯层，用饱和食盐水洗。无水硫酸钠干燥。干法过柱(洗脱液：CHCl 3:MeOH＝30:1)得产品(S)-(1-(4- 氨基-5-碘-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体4,6.86g)，产率为78.0％。
1H NMR(300MHz,CDCl 3)δ8.25(s,1H),7.05(s,1H),5.87-5.74(m,1H),5.72(s,2H),5.34-5.13(m,3H),4.56-4.43(m,1H),4.34(dd,J＝14.8,4.9Hz,1H),4.30-4.15(m,1H),1.35(s,9H)。ee>99.5％。
3、中间体6的合成
Figure PCTCN2020086787-appb-000018
在1L圆底烧瓶中加入(S)-(1-(4-氨基-5-碘-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体4,32.9g,1eq)，N-(吡啶-2-基)-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)苯甲酰胺(原料5,34.8g,1.4eq)和Pd(PPh 3) 4(17.7g,0.2eq)。加入1,4-二氧六环(383mL)作为溶剂，并置换N 2。在搅拌下加入2M碳酸钠溶液(76.6mL)。于90℃下回流搅拌5小时。
加入水与乙酸乙酯萃取，合并酯层，用饱和食盐水洗。无水硫酸钠干燥。干法过柱，先用EA作为洗脱液除去大部分杂质，然后用CHCl 3:MeOH＝30:1的混合物作为洗脱液。产品可能含有少量杂质，可用PE重结晶析出纯品。得产品(S)-(1-(4-氨基-5-(4-(吡啶-2-基氨基甲酰基)苯基)-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体6，28.4g)，产率为74.3％。ee>99.5％。
4、中间体7的合成
Figure PCTCN2020086787-appb-000019
在1L圆底烧瓶中加入(S)-(1-(4-氨基-5-(4-(吡啶-2-基氨基甲酰基)苯基)-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体6,32.7g,1eq)，加入600mL DMF作为溶剂。在搅拌下缓慢加入NBS(12.8g,1.1eq)，于室温下搅拌过夜。
反应完成后，加入水与乙酸乙酯萃取，合并酯层，用水反萃一次，饱和食盐水洗。无水硫酸钠干燥。干法过柱，首先用CHCl 3:MeOH＝50:1的混合物作为洗脱液，然后换CHCl 3:MeOH＝30:1的混合物作为洗脱液。得产品(S)-(1-(4-氨基-6-溴-5-(4-(吡啶-2-基氨基甲酰基)苯基)-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体7，25.8g)，产率为68.2％。ee>99.5％。
5、中间体8的合成
Figure PCTCN2020086787-appb-000020
在250mL圆底烧瓶中加入(S)-(1-(4-氨基-6-溴-5-(4-(吡啶-2-基氨基甲酰基)苯基)-7H-吡咯并[2,3-d]嘧啶-7-基)丁-3-烯-2-基)氨基甲酸叔丁酯(中间体7,11.9g,1eq)和PdCl 2(dppf)(1.66g,0.11eq)，加入51mL THF作为溶剂，并置换氮气，多置换几次以确保完全。在搅拌下加入4M氢氧化钠溶液(8.2mL)。于85℃下回流搅拌15小时。
反应完全后，加入水与乙酸乙酯萃取，合并酯层，用饱和食盐水洗。无水硫酸钠干燥。干法过柱(洗脱液：CHCl 3:MeOH＝30:1)得产品(S)-(4-氨基-6-亚甲基-5-(4-(吡啶-2-基氨基甲酰基)苯基)-7,8-二氢-6H-嘧啶并[5,4-b]吡呤-7-基)氨基甲酸叔丁酯(中间体8，8.79g)，产率为85.9％。ee>99.5％。
6、S17016的合成
Figure PCTCN2020086787-appb-000021
在250mL圆底烧瓶中加入(S)-(4-氨基-6-亚甲基-5-(4-(吡啶-2-基氨基甲酰基)苯基)-7,8-二氢-6H-嘧啶并[5,4-b]吡呤-7-基)氨基甲酸叔丁酯(中间体8,2.75g,1eq)，加入110mL DCM作为溶剂。于搅拌下逐滴加入三氟乙酸(10.5mL)。于室温下搅拌3小时。反应完成后，直接将反应液旋干，多用甲醇带几次将三氟乙酸带出，旋干后得到氨基脱Boc保护粗品，直接投下一步。
将上一步产物移至50mL圆底烧瓶中，再加入三乙胺(1eq)，搅拌五分钟后再加入2-丁炔酸(0.511g,1.1eq)和HATU(2.31g,1.1eq)，加入100mL DCM作为溶剂。冰水浴降温至0℃，逐滴加入三乙胺(1.54mL+0.77mL)。逐渐升温至室温，于室温下搅拌1.5小时。反应液呈微黄色。加入水和DCM萃取，合并有机相，用饱和食盐水洗。无水硫酸钠干燥，柱层析(CHCl 3:MeOH＝30:1)后得终产物S17016(1.88g)，产率为73.3％。ee>99.5％。
1H NMR(400MHz,CDCl 3)δ8.98(s,1H),8.43(dt,J＝8.3,1.0Hz,1H),8.34(ddd,J＝5.0,1.9,0.9Hz,1H),8.22(s,1H),8.09-8.03(m,2H),7.81(ddd,J＝8.4,7.4,1.9Hz,1H),7.69-7.63(m,2H),7.13(ddd,J＝7.4,4.9,1.0Hz,1H),6.55(d,J＝8.2Hz,1H),5.67(m,J＝8.1,5.7,2.6Hz,1H),5.56(d,J＝2.3Hz,1H),5.40(s,2H),5.27(d,J＝2.3 Hz,1H),4.70(dd,J＝11.7,8.1Hz,1H),4.09-3.99(m,1H),1.99(s,3H)。
实验实施例1：布鲁顿激酶(BTK)分子水平酶活抑制活性评价
将酶反应底物Poly(Glu,Tyr) 4:1用无钾离子的PBS(10mM磷酸钠缓冲液，150mM NaCl，pH 7.2-7.4)稀释成20μg/mL包被酶标板，在37℃下反应12-16小时后，用200μL/孔的T-PBS(含0.1％Tween-20的PBS)洗板三次，于37℃烘箱中干燥酶标板1-2小时。在以上包被底物的酶标板中，首先加入用反应缓冲液(50mM HEPES pH 7.4，50mM MgCl 2，0.5mM MnCl 2，0.2mM Na 3VO 4，1mM DTT)稀释的ATP溶液49μL/孔(终浓度为5μM)。每孔中加入1μL待测试化合物(化合物孔)或含相应浓度的DMSO(阴性对照孔)，每次实验需设无酶对照孔。再加入50μL以反应缓冲液稀释的BTK酪氨酸激酶蛋白启动反应。
将上述反应体系置于37℃摇床(100rpm)中1小时，然后T-PBS洗板三次，加入一抗PY99 100μL/孔(Santa Cruz)，37℃摇床反应0.5小时。T-PBS洗板后，加入辣根过氧化物酶标记的羊抗鼠二抗稀释液100μL/孔，37℃摇床反应0.5小时。T-PBS洗板后，加入2mg/mL的OPD显色液100μL/孔，25℃避光反应1-10分钟。然后加入2M H 2SO 4 50μL/孔中止反应，用可调波长式微孔板酶标仪SPECTRA MAX Plus384读数，波长为490nm。
以化合物S1、S10、Ibrutinib和Acalabrutinib作为阳性对照化合物，其中，化合物S1和S10采用现有技术(例如CN108101905A)中公开的方法或类似方法制备，Ibrutinib和Acalabrutinib购自Selleck公司。
各化合物的抑制率通过下列公式求得：
Figure PCTCN2020086787-appb-000022
IC 50值采用酶标仪随机附带软件以四参数法回归求得。结果列于下表1中。
表1
化合物 IC 50(nM)
S1 ～1
S10 <10
Ibrutinib ～1
Acalabrutinib ～10
S17016 0.5
以上结果表明，化合物S17016对BTK的抑制活性优于前期化合物S1和S10，也优于目前已上市的第一代BTK抑制剂Ibrutinib和第二代BTK抑制剂Acalabrutinib。
实验实施例2：化合物对人B淋巴瘤细胞Ramos(Burkitt淋巴瘤)和人弥漫大B淋巴瘤细胞TMD8的体外增殖抑制活性检测
将细胞悬液(Ramos：10000细胞/孔；TMD8：12000细胞/孔)接种于96孔板中，于37℃培养箱静置2小时待细胞状态稳定后，每孔加入不同浓度的受试化合物(每个浓度设3个复孔)，并同时设置空白对照(仅包含培养液，不含细胞的孔)、阴性对照(仅加细胞，不加化合物的孔)及阳性化合物对照。加药处理72h后，每孔加入20μL MTT(5mg/mL)于37℃孵育4h，加入100μL三联液(10％SDS， 5％异丁醇，0.01M HCl)，37℃放置过夜。用可调波长式微孔板酶标仪SPECTRAMAX Plus384在570nm波长条件下测定OD值。
化合物的抑制率通过下列公式求得：
Figure PCTCN2020086787-appb-000023
IC 50值采用酶标仪随机附带软件以四参数法回归求得。实验独立重复3次，其结果列于下表2中。
同样以上文所述化合物S1、S10、Ibrutinib、Acalabrutinib、S18s、S19s和S20s作为阳性对照化合物。
表2
化合物 Ramos细胞IC 50 TMD8细胞IC 50
S1 94.73μM 0.006μM
S10 8.72μM 0.030μM
Ibrutinib 12.91μM 0.005μM
Acalabrutinib 38.16μM 0.023μM
S17016 3.15μM 0.003μM
S18s 5.04μM 0.016μM
S19s — 0.017μM
S20s 14.3μM 0.004μM
以上结果表明，在细胞水平，化合物S17016对BTK依赖的细胞增殖抑制能力优于前期化合物S1、S10、S18s、S19s和S20s，也优于目前已上市的第一代BTK抑制剂Ibrutinib和第二代BTK抑制剂Acalabrutinib。进一步需要说明的是，与其他化合物相比，本发明的化合物S17016对于Ramos细胞具有较高的增殖抑制活性，且对于TMD8细胞具有更高的增殖抑制活性。
实验实施例3：体内抗肿瘤活性评价
实验动物：
TMD8异种移植瘤模型
1)种属：小鼠
2)品系：CB-17 SCID
3)周龄及体重：6-8周；18-22g
4)性别：雌性
5)供应商：北京维通利华实验动物技术有限公司
REC-1异种移植瘤模型
1)种属：小鼠
2)品系：BALB/c裸小鼠
3)周龄及体重：6-8周；17-20g
4)性别：雌性
5)供应商：上海灵畅生物科技有限公司
细胞培养:人淋巴癌TMD8细胞体外悬浮培养，培养条件为RPMI 1640培养基 (供应商：gibco；货号：22400-089；生产批号：4868546)中加10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，37℃ 5％CO 2培养。一周两次进行常规处理传代。当细胞饱和度为80％-90％时，收取细胞，计数，接种。
人套细胞淋巴癌REC-1细胞体外悬浮培养，培养条件为RPMI 1640培养基(供应商：gibco；货号：22400-089；生产批号：1868795)中加10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，37℃ 5％CO2培养。一周两次进行常规处理传代。当细胞饱和度为80％-90％时，收取细胞，计数，接种。
肿瘤细胞接种:将0.2mL 10×10 6个人淋巴癌TMD8细胞皮下接种于每只裸小鼠的右后背(PBS:Matrigel＝1:1)。肿瘤平均体积达到104mm 3时开始分组给药。根据动物肿瘤体积通过一个基于Excel随机分组软件进行分组，每组6只小鼠。
将0.2mL 5×10 6个REC-1细胞皮下接种于每只裸小鼠的右后背(PBS:Matrigel＝1:1)。肿瘤平均体积达到100mm 3时开始分组给药。根据动物肿瘤体积通过一个基于Excel随机分组软件进行分组，每组6只小鼠。
受试物的配制:
受试物配制方法参见下表3和表4：
表3 TMD8异种移植瘤模型受试物配制方法
Figure PCTCN2020086787-appb-000024
注：在给动物给药前需要轻轻将药物充分混匀。
表4 REC-1异种移植瘤模型受试物配制方法
Figure PCTCN2020086787-appb-000025
实验动物日常观察:本实验方案的拟定及任何修改均通过了苏州药明康德新药开发股份有限公司实验动物管理与使用委员会(IACUC)的评估核准。实验动物的使用及福利遵照国际实验动物评估和认可委员会(AAALAC)的规定执行。每天监 测动物的健康状况及死亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量(仅目测)，体重变化(每周测量三次体重)，外观体征或其它不正常情况。基于各组动物数量记录了组内动物死亡数和副作用。
肿瘤测量和实验指标：实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周三次用游标卡尺测量肿瘤直径。
肿瘤体积的计算公式为：
V＝0.5a×b 2，
a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。TGI(％)，反映肿瘤生长抑制率。
TGI(％)的计算：
TGI(％)＝【1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)】×100％。
相对肿瘤增殖率T/C(％)：计算公式如下：
T/C％＝T RTV/C RTV×100％(T RTV：治疗组相对肿瘤体积；C RTV：阴性对照组相对肿瘤体积)。根据肿瘤测量的结果计算出相对肿瘤体积(RTV)，计算公式为RTV＝V t/V 0，其中V 0是分组给药时(即d 0)测量所得平均肿瘤体积，V t为某一次测量时的平均肿瘤体积，T RTV与C RTV取同一天数据。
统计分析：统计分析，包括每个组的每个时间点的肿瘤体积的平均值和标准误(SEM)。治疗组在给药后分别在第15天(REC-1异种移植瘤模型)和第17天(TMD8异种移植瘤模型)表现出最好的治疗效果，因此基于此数据进行统计学分析评估组间差异。三组或多组间比较用one-way ANOVA进行分析，如果F值有显著性差异，应用Games-Howell法进行检验。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。
化合物S17016在人套细胞淋巴癌REC-1异种移植瘤模型中的体内药效如表5和图1所示。开始给药后15天，溶剂对照组荷瘤鼠的瘤体积达到3501mm 3，Ibrutinib 25mg/kg组与溶剂对照组相比具有显著的抑瘤作用(T/C＝38％,TGI＝64％,p＝0.008)，瘤体积为1323mm 3。化合物S17016 15mg/kg和30mg/kg组的瘤体积分别为1034和680mm 3，与溶剂对照组相比具有显著的抑瘤作用(T/C值分别为30％和19％，TGI值分别为73％和83％，p＝0.004和0.003)。
表5.S17016对REC-1异种移植瘤模型的抑瘤药效评价(基于给药后第15天肿瘤体积计算得出)
Figure PCTCN2020086787-appb-000026
注：
a.平均值±SEM；
b.肿瘤生长抑制由T/C和TGI(TGI(％)＝[1-(T 15-T 0)/(V 15-V 0)]×100)计算；
c.p值根据肿瘤体积计算。
化合物S17016在人淋巴癌TMD8异种移植瘤模型中的体内药效如表6和图2所示。开始给药后17天，溶剂对照组荷瘤鼠的瘤体积达到1852mm 3，Ibrutinib 25mg/kg组与溶剂对照组相比具有显著的抑瘤作用(T/C＝35.68％,TGI＝68.18％,p<0.001)，瘤体积为661mm 3。化合物S17016 5mg/kg和10mg/kg组的瘤体积分别为912和553mm 3，与溶剂对照组相比具有显著的抑瘤作用(T/C值分别为49.27％和29.85％，TGI值分别为53.78％和74.35％，p＝0.003和<0.001)。
表6.S17016对TMD8异种移植瘤模型的抑瘤药效评价(基于给药后第17天肿瘤体积计算得出)
Figure PCTCN2020086787-appb-000027
注：
a.平均值±SEM；
b.肿瘤生长抑制由T/C和TGI(TGI(％)＝[1-(T 17-T 0)/(V 17-V 0)]×100)计算；
c.p值根据肿瘤体积计算。
结果表明，在两种BTK敏感的小鼠移植瘤模型中，化合物S17016具有显著的肿瘤生长抑制活性，明显优于目前已上市的第一代BTK抑制剂Ibrutinib。
另外，使用化合物S18s，重复上述在人淋巴癌TMD8异种移植瘤模型中的实验，T/C(％)结果列于下表中。在下表中，还列出了化合物S17016的T/C(％)结果作为对比。
Figure PCTCN2020086787-appb-000028
由上述数据可以看出，在更低剂量下(10mg/kg)，化合物S17016表现出较化合物S18s(15mg/kg)更好的肿瘤生长抑制效果。
实验实施例4：大鼠代谢性质评价
SD大鼠14只，雄性，体重200-220g，随机分成4组，每组4/3只，分别灌胃和静脉给予受试化合物，具体安排见下表7：
表7
组别 动物数 化合物 给药途径 给药剂量(mg/kg)
1 4 S17016 灌胃(po) 3
2 3 S17016 静脉(iv) 1
灌胃给药以含1％吐温80的0.5％羧甲基纤维素钠(CMC-Na)配制，静脉给药以5％DMSO/5％吐温80/90％生理盐水配制成溶液。
试验前禁食12小时，自由饮水。给药后2小时统一进食。
采血时间点及样品处理：
灌胃给药：给药后0.25，0.5，1.0，2.0，4.0，6.0，8.0和24小时；
静脉给药：给药后5分钟，0.25，0.5，1.0，2.0，4.0，6.0，8.0和24小时；
在以上设定时间点经大鼠眼球后静脉丛取静脉血0.3mL，置肝素化试管中，11000rpm离心5分钟，分离血浆，于-20℃冰箱中冷冻。
样品测试和数据分析
采用LC/MS/MS法测定大鼠血浆中S17016的浓度。
采用WinNonlin 5.3软件(美国Pharsight公司)的非房室模型计算给药后的药代动力学参数。
达峰浓度C max和达峰时间T max为实测值；
药时曲线下面积AUC 0-t值：采用梯形法计算；
AUC 0-∞＝AUC 0-t+C t/k e，
C t为最后一个可测得时间点的血药浓度，
k e为消除速率常数；
消除半衰期t 1/2＝0.693/k e；
平均滞留时间MRT＝AUMC/AUC。
清除率CL＝D/AUC 0-∞；稳态分布容积Vss＝CL×MRT
绝对生物利用度F＝(AUC 灌胃×D 静脉)/(AUC 静脉×D 灌胃)×100％
试验结果见下表8
表8
Figure PCTCN2020086787-appb-000029
以上结果表明，化合物S17016在大鼠体内清除率显著低于Ibrutinib(20倍)，血浆中药物暴露量也比Ibrutinib高达70倍，即同等剂量下S17016的口服吸收更好，且S17016具有更好的口服生物利用度。
实验实施例5：代谢稳定性试验
肝微粒体孵育于96孔板中进行，每个孵育体系体积为450μL，介质为0.1M Tris缓冲液(pH 7.4)，包括终浓度为0.33mg/mL的肝微粒体、0.1μM的受试药物，5.0mM MgCl 2，0.01％DMSO、0.005％BSA及1.0mM NADPH。采用37℃孵育，预孵10min后加入NADPH起始反应，分别在0、7、17、30、60min后从孵育体系取50μL加入同体积的甲醇终止反应。
样品测试和数据分析
LC/MS/MS测定孵化样品中的S17016，离子源为电喷雾电离源(ESI源)，扫描方式为多反应监测(MRM)。
S17016在不同种属肝微粒体(HLM：人肝微粒体；RLM：大鼠肝微粒体；MLM：小鼠肝微粒体)体外代谢稳定性研究实验结果见下表9和图3。
表9
Figure PCTCN2020086787-appb-000030
结果表明，S17016在人、小鼠、大鼠肝微粒体中基本以原型药物为主，基本无较大占比代谢产物，有效克服了S1、S10、S18s和S19s等化合物中二苯醚结构的末端苯环羟基化，代谢稳定性较好。
因此，化合物S17016是一个结构新颖的、可口服的、高选择性的、高活性的BTK抑制剂，体内外活性明显优于目前国外已上市的BTK抑制剂，在同等剂量下，对肿瘤生长抑制活性显著优于阳性对照药Ibrutinib，极具开发价值。
以上实施方式本质上仅为辅助说明，且并不欲用以限制申请目标的实施例或这些实施例的应用或用途。在本文中，用语“例示性”代表“作为一个实例、范例或说明”。本文中任一种例示性的实施形态并不必然可解读为相对于其他实施形态而言为优选或较有利者。
此外，尽管已于前述实施方式中提出至少一例示性实施例或比较例，但应了解本发明仍可存在大量的变化。同样应了解的是，本文所述的实施例并不欲用以通过任何方式限制所请求的申请目标的范围、用途或组态。相反的，前述实施方式将可提供本领域具有普通知识人员一种简便的指引以实施所述的一种或多种实施例。再者，可对要素的功能与排列进行各种变化而不脱离申请专利范围所界定的范围，且申请专利范围包含已知的均等物及在本专利申请案提出申请时的所有可预见均等物。

Claims (9)

  1. 一种如通式I所示的嘧啶并[5,4-b]吡呤化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物：
    Figure PCTCN2020086787-appb-100001
    优选为
    Figure PCTCN2020086787-appb-100002
    其中，R 1和R 2各自独立地为H或C 1-C 10烷基，优选为H或C 1-C 6烷基，更优选为H或C 1-C 3烷基；
    R 3为H或C 1-C 10烷基，优选为H或C 1-C 6烷基，更优选为H或C 1-C 3烷基。
  2. 根据权利要求1所述的嘧啶并[5,4-b]吡呤化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物，其中，所述通式I化合物为
    Figure PCTCN2020086787-appb-100003
    优选为
    Figure PCTCN2020086787-appb-100004
  3. 一种制备如权利要求1所述的嘧啶并[5,4-b]吡呤化合物的方法，该方法包括如下步骤：
    Figure PCTCN2020086787-appb-100005
    或
    Figure PCTCN2020086787-appb-100006
    其中R 1，R 2，R 3如权利要求1中定义，
    将反应物A8或B8脱除保护基T，然后再与
    Figure PCTCN2020086787-appb-100007
    反应得到通式I化合物或通式I-1化合物，其中，所述保护基T包括叔丁氧羰基、苄氧羰基、笏甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基、邻苯二甲酰亚胺基、对甲苯磺酰基、三氟乙酰基、三苯甲基、2，4-二甲氧基苄基、对甲氧基苄基、苄基等；优选为叔丁氧羰基、苄氧羰基、笏甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基；进一步优选为叔丁氧羰基。
  4. 一种药物组合物，其含有治疗有效量的根据权利要求1或2所述的嘧啶并[5,4-b]吡呤化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物和一种或多种可药用的载体。
  5. 根据权利要求4所述的药物组合物，其中，所述药物组合物包含总重量比65％～99％的所述嘧啶并[5,4-b]吡呤化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物作为活性成分。
  6. 根据权利要求1所述的嘧啶并[5,4-b]吡呤化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物或者权利要求4或5所述的药物组合物在制备用于治疗BTK激酶信号转导通路相关的疾病的药物中的用途。
  7. 根据权利要求6所述的用途，其中，所述BTK激酶信号转导通路相关的疾病包括癌症、超常增生、再狭窄、免疫病症和炎症。
  8. 根据权利要求1所述的嘧啶并[5,4-b]吡呤化合物、其药学上可接受的盐、对映异构体、非对映异构体、光学异构体、外消旋体、氘代衍生物、溶剂合物或水合物或者权利要求4或5所述的药物组合物在制备用于治疗、预防或调控癌症的药物中的用途。
  9. 根据权利要求8所述的用途，其中，所述癌症包括血液肿瘤和实体瘤，其中所述血液肿瘤包括淋巴瘤、白血病和骨髓瘤；优选地，所述癌症包括组织细胞性淋巴瘤、套细胞淋巴瘤、弥漫大B细胞淋巴瘤、慢性淋巴细胞白血病、卵巢癌、头颈磷状上皮细胞癌、胃癌、乳腺癌、儿童肝细胞癌、结肠直肠癌、***、肺癌、肉瘤、鼻咽癌、胰腺癌、成胶质细胞癌、***癌、小细胞肺癌、非小细胞肺癌、多发性骨髓瘤、甲状腺癌、睾丸癌、***、子宫内膜癌、食道癌、肾细胞癌、膀胱癌、肝癌和星形细胞瘤。
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