WO2019157959A1 - 一种嘧啶类化合物、其制备方法及其医药用途 - Google Patents

一种嘧啶类化合物、其制备方法及其医药用途 Download PDF

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WO2019157959A1
WO2019157959A1 PCT/CN2019/073874 CN2019073874W WO2019157959A1 WO 2019157959 A1 WO2019157959 A1 WO 2019157959A1 CN 2019073874 W CN2019073874 W CN 2019073874W WO 2019157959 A1 WO2019157959 A1 WO 2019157959A1
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group
alkyl
cycloalkyl
alkoxy
cyano
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PCT/CN2019/073874
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English (en)
French (fr)
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司聚同
姜美锋
李加艳
张丽云
樊平平
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恩瑞生物医药科技（上海）有限公司
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Priority to CN201980005206.4A priority Critical patent/CN111247137A/zh
Publication of WO2019157959A1 publication Critical patent/WO2019157959A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention belongs to the field of medicine, and relates to a novel pyrimidine compound, a preparation method thereof and a pharmaceutical composition containing the same, and as a selective cyclin-dependent kinase 7 (CDK7) inhibitor in the prevention and/or treatment of human Diseases include use in cancer.
  • CDK7 selective cyclin-dependent kinase 7
  • Tumors including leukemia, are one of the major causes of clinical death in humans, with millions of patients worldwide dying of cancer each year.
  • Malignant tumors such as lung cancer, stomach cancer, breast cancer, pancreatic cancer, liver cancer, colon cancer, ovarian cancer, cervical cancer, esophageal cancer, nasopharyngeal carcinoma, leukemia and malignant lymphoma have extremely high mortality rates.
  • genetic screening, molecular diagnosis, and precision medicine for cancer have improved clinical early detection, correct diagnosis, and treatment for cancer patients, most cancers, especially advanced, refractory, relapsed, and drug-resistant malignancies have not been effective so far.
  • the method and medicine can be completely eradicated or cured. There is an urgent need for high-quality anticancer drugs with good specificity, high activity, low toxicity, and no drug resistance.
  • the mammalian cell cycle is a highly organized, orderly and precisely regulated cell mitosis process in which the genetic material of the cell replicates and is equally distributed among the two proliferating daughter cells.
  • Cell growth factors and cell cycle regulators play an important role in the cell cycle.
  • Cell cycle regulators are a class of self-synthesized proteins in cells. Abnormal activities of various cell cycle regulators (proteins) often cause abnormalities in the normal cell cycle, leading to different types of diseases, such as when cell proliferation is not controlled. Cell transformation forms cancer cells.
  • Cyclin Dependent Kinase is a group of serine/threonine protein kinases that act synergistically with cyclin (Cyclin) and are key regulators of cell cycle progression and transcription. CDK can bind to cyclins to form heterodimers, in which CDK is a catalytic subunit, cyclin is a regulatory subunit, and different Cyclin-CDK complexes are phosphorylated to different substrates in cells. Advance and transformation of different phases of the cell cycle.
  • CDK1 to CDK20 in which CDK11 has two genes CDK11A and CDK11B
  • CDKL1 to CDKL5 CDK-type genes
  • Amino acid sequences are highly evolutionarily conserved.
  • CDK direct cell cycle regulation of CDK
  • CDK such as CDK1, CDK2, CDK3, CDK4 and CDK6
  • transcriptional function CDK such as CDK7, CDK 8, CDK 9, CDK 11, CDK 12 and CDK13.
  • Direct cell cycle regulation of CDK directly regulates the progression of the cell cycle, and its phosphorylation substrate is a cell cycle-associated protein.
  • Transcriptional Function CDK regulates gene transcription by phosphorylating the RNA polymerase II complex.
  • Clinical data found that in different types of malignant tumors and leukemia patient samples such as skin cancer, melanoma, lung cancer, stomach cancer, breast cancer, pancreatic cancer, liver cancer or colon cancer and acute myeloid leukemia, different CDK frequent gene mutations, expansion Increased overexpression, which is closely related to the occurrence, development, and/or maintenance of malignant cell phenotypes, as well as patient survival and drug resistance.
  • CDK Crohn's disease .
  • CDK4/6 selective inhibitors Palbociclib, Ribociclib and Abemaciclib Otto T et al. (2017) Nat Rev Cancer 17(2): 93-115; Kwapisz D (2017) Breast Cancer Res Treat. 166(1): 41-54; Vijayaraghavan S et al. (2017) Target Oncol.2017 Dec 7; Ingham M et al. (2017) J Clin Oncol. 35 (25): 2949-2959; Abou Zahr A et al.
  • CDK4/6 and CDK5 have tumor immune regulation function, and selective inhibition of CDK4/6 or CDK5 can enhance the effect of tumor immunotherapy, further demonstrating that CDK is an important target protein for tumor therapy (Dorand RD et al. (2016) Science .353(6297): 399-403; Goel S et al. (2017) Nature. 548 (7668): 471-475; Deng J et al. (2017) Cancer Discov. 8(2); 216–33; Zhang J et al. Person (2016) Nature. 553 (7686): 91-95).
  • CDK inhibitors Over the years, many different types of CDK inhibitors have undergone extensive preclinical and clinical studies, but to date only the CDK4/6 highly selective inhibitors Pabsini, Ribociclib and Abemaciclib have been successfully applied to hormonal receptor-positive, Clinical treatment of HER2-negative advanced or recurrent breast cancer, Pabsini and Ribociclib need to be combined with letrozole, Abemaciclib alone or in combination with Fulvestrant.
  • Pan-CDK inhibitors such as Alvocidib and Seliciclib are flavonoids, and Alvocidib and ATP competitively inhibit CDK1, CDK2, CDK4 and CDK6 with IC 50 values of approximately 40 nM; Seliciclib inhibits CDK5, Cdc2 and CDK2, IC 50 were 0.2 ⁇ M, 0.65 ⁇ M and 0.7 [mu; but did not show antitumor activity in clinical research and promising pre clinic.
  • Second-generation pan-CDK inhibitors such as Dinaciclib, AT7519, Milciclib, TG02, CYC065, and RGB-286638 are highly active and inhibit multiple CDKs, although they enter different phases of clinical trials, respectively, but these inhibitors alone do not show Good clinical effects and high clinical side effects.
  • CDK7 is unique in mammalian CDK and regulates cell cycle and gene transcription.
  • CDK7 is present as a CDK activating enzyme (CAK) heterotrimeric complex that is capable of fully activating CDK1/2 by phosphorylating the CDK1/2 activation domain conserved residue (T-loop), a process that is cell cycle Required for the process; in the nucleus, CDK7 forms a complex with RNA polymerase (RNAP) II, which is responsible for phosphorylating the C-terminal domain (CTD) of RNAP II, which is a necessary step in the initiation of gene transcription.
  • RNAP RNA polymerase
  • CTD C-terminal domain
  • CDK7 selective inhibitors are expected to be effective new targeted drugs for the treatment of cancer.
  • CDK7 high-selective inhibitor SY-1365 (THZ1) has entered human clinical trials.
  • the preclinical test of this compound shows good anti-tumor activity and is a potential new type of CDK7 anti-tumor inhibitor (Lücking U et al. (2017) Chem Med Chem. 12 (21): 1776-1793; Kwiatkowski N et al. (2014) Nature. 511 (7511): 616-20), but the stability of this compound in vivo (T1/2 in mouse plasma) It is limited to 45 minutes). Therefore, there is an urgent need for a more efficient and toxic side effect CDK7 inhibitor.
  • the present inventors discovered a novel pyrimidine compound in the development of a long-term selective inhibitor of CDK7, which can effectively inhibit the growth of CDK7-positive leukemia cell MOLM-13 in vitro, and its IC 50 value can reach Yana Moore. Concentration is expected to be developed as a new and effective anti-tumor drug.
  • the object of the present invention is to provide a novel orally administrable small molecule compound which is selective, high in activity and low in toxicity, and can be used as a cyclin-dependent kinase 7 (CDK7) inhibitor for prevention and/or prevention. Treatment of human diseases including cancer.
  • the present invention relates to a novel pyrimidine compound which is capable of covalently binding to CDK7 and inhibits the growth of CDK7-positive tumor cells in vitro, and has an IC 50 value of subnanomolar concentration.
  • the present invention provides a compound of the formula (I),
  • Each R 1 is independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, nitro, -N(R y )(R z ), alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl Base, heteroaryl, -NHC(O)R x , -C(O)N(R y )(R z ), -OR u OR x , -OR x or -OR u N(R y )(R z
  • said alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is further optionally selected from the group consisting of halogen, alkyl, alkoxy, haloalkoxy, cyano, Substituted by one or more substituents of amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl,
  • R 2 and R 3 are each independently selected from hydrogen, halogen, hydroxy, cyano, nitro, alkyl, alkoxy, cycloalkyl, heterocyclyl, -NHC(O)R x or -C(O) N(R y )(R z ), wherein the alkyl, alkoxy, cycloalkyl and heterocyclic groups are optionally further selected from the group consisting of halogen, alkyl, alkoxy, haloalkoxy, cyano, amino Substituting one or more substituents of a nitro group, a hydroxyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group;
  • R 4 and R 5 are each independently selected from hydrogen, halogen, hydroxy, cyano, nitro, alkyl, alkoxy, cycloalkyl, heterocyclyl, -NHC(O)R x or -C(O) N(R y )(R z ), wherein the alkyl, alkoxy, cycloalkyl and heterocyclic groups are optionally further selected from the group consisting of halogen, alkyl, alkoxy, haloalkoxy, cyano, amino Substituting one or more substituents of a nitro group, a hydroxyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group;
  • Each R 6 is each independently selected from a Q group
  • Each R 7 is independently selected from the group consisting of -NHC(O)R and Q groups;
  • R 8 and R 9 are the same or different and are each independently selected from the group consisting of -NHC(O)R and Q groups;
  • Q is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, nitro, -N(R y )(R z ), alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or a heteroaryl group, wherein the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group is further optionally selected from the group consisting of halogen, alkyl, haloalkyl, alkoxy Substituted by one or more substituents in the group, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl;
  • R is selected from alkenyl, alkynyl, cyano, optionally further selected from halo, alkyl, haloalkyl, alkoxy, haloalkoxy or -R u N(R y ) ( Substituting one or more groups of R z );
  • R u is selected from an alkylene group, an alkenylene group, or an alkynylene group
  • R x is selected from hydrogen, alkyl, cycloalkyl, aryl or heteroaryl, and the alkyl, cycloalkyl, aryl or heteroaryl group is optionally further selected from the group consisting of halogen, alkyl, haloalkyl, alkane Substituted with one or more substituents of oxy, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl;
  • R y and R z are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; the alkyl, alkoxy, alkene
  • the base, alkynyl, cycloalkyl, aryl or heteroaryl group is optionally further selected from the group consisting of halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl Substituting one or more substituents of a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group; or
  • R y and R z together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl group, which is optionally further selected from the group consisting of halogen, alkyl, haloalkyl, alkoxy, haloalkyl Substituted by one or more substituents of oxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl;
  • R 8 is selected from -NHC(O)R, and R 9 is selected from a Q group; or,
  • R 9 is selected from -NHC(O)R, and R 8 is selected from the group consisting of Q;
  • R is selected from alkenyl, alkynyl, cyano, and the alkenyl and alkynyl are optionally further substituted with one or more groups selected from the group consisting of hydrogen, alkyl or R u N(R y )(R z ) ;
  • R u , R y , R z , Q groups are as defined in the general formula (I).
  • R 8 is selected from -NHC(O)R, and R 9 is selected from the Q group; or,
  • R 9 is selected from -NHC(O)R, and R 8 is selected from the group consisting of Q;
  • R is selected from alkenyl, alkynyl, cyano, and the alkenyl and alkynyl are optionally further substituted with one or more groups selected from the group consisting of hydrogen, alkyl or R u N(R y )(R z ) ;
  • R u is selected from a C 1 -C 6 alkylene group
  • R y and R z are each independently selected from hydrogen, C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl; or
  • R y and R z together with the nitrogen atom to which they are attached form a 5- to 7-membered nitrogen-containing heterocyclic group, preferably morpholinyl, piperidinyl, piperazinyl, azepanyl, tetrahydropyrrolyl,
  • the 5- to 7-membered nitrogen-containing heterocyclic group is optionally further selected from the group consisting of halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy Substituted by one or more groups of a C 3 -C 7 cycloalkyl group;
  • the Q group is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 7 cycloalkyl.
  • Each R 7 is each independently selected from -NHC(O)R;
  • R is selected from alkenyl, alkynyl, cyano, and the alkenyl and alkynyl are optionally further substituted with one or more groups selected from the group consisting of hydrogen, alkyl or R u N(R y )(R z ) ;
  • R u is selected from a C 1 -C 6 alkylene group
  • R y and R z are each independently selected from hydrogen, C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl; or
  • R y and R z together with the nitrogen atom to which they are attached form a 5- to 7-membered nitrogen-containing heterocyclic group, preferably morpholinyl, piperidinyl, piperazinyl, azepanyl, tetrahydropyrrolyl,
  • the 5- to 7-membered nitrogen-containing heterocyclic group is optionally further selected from the group consisting of halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy Substituted by one or more groups of a C 3 -C 7 cycloalkyl group.
  • Each R 1 is independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, nitro, C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, -NHC(O)R x , -C(O N(R y )(R z ), -OR u OR x , -OR x , wherein the C 1 -C 6 alkyl group, C 3 -C 7 cycloalkyl group is optionally further selected from halogen, cyano group Substituting one or more substituents of an amino group, a nitro group, a hydroxyl group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group;
  • R u is selected from a C 1 -C 6 alkylene group
  • R x is selected from hydrogen, C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl or 5- to 7-membered heteroaryl, said C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl or The 5- to 7-membered heteroaryl group is optionally further substituted with one or more groups of a halogen, a hydroxyl group, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group.
  • R 2 and R 3 are each independently selected from hydrogen, halogen, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, -NHC(O)R x or -C(O)N(R y ) (R z ), wherein the alkyl group, alkoxy group, cycloalkyl group and heterocyclic group are optionally further selected from the group consisting of halogen, alkyl, alkoxy, haloalkoxy, cyano, amino, nitro, hydroxy Substituted by one or more substituents of a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group;
  • R x , R y , R z are as defined in the general formula (I).
  • R 2 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, cyano, -NHC(O)R x or -C(O)N(R y )(R z );
  • R 3 is selected from hydrogen
  • R x is selected from the group consisting of hydrogen, alkyl, cycloalkyl, and the alkyl, cycloalkyl is optionally further selected from the group consisting of halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, cyano, amino, nitro Substituting one or more substituents of a hydroxyl group or a hydroxyalkyl group;
  • R y and R z are each independently selected from hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; the alkyl, cycloalkyl, aryl or heteroaryl group is optionally further selected Substituted by one or more substituents of halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, cyano, amino, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl; or
  • R y and R z together with the nitrogen atom to which they are attached form a nitrogen-containing heterocyclic group or heteroaryl group, which is optionally further selected from the group consisting of halogen, alkyl, haloalkyl, alkoxy Substituting one or more substituents of a haloalkoxy group, a cyano group, an amino group, a nitro group, a hydroxyl group, or a hydroxyalkyl group.
  • R 4 and R 5 are each independently selected from the group consisting of hydrogen, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy .
  • Each R 6 is each independently selected from a Q group
  • Q is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 7 cycloalkyl.
  • Exemplary compounds of the invention include, but are not limited to, the following compounds:
  • a tautomer a meso form, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof.
  • the intermediate compound M3 and the aniline intermediate compound M4 are reacted under an acid catalysis at a suitable temperature and a suitable solvent to obtain a compound of the formula (I);
  • the solvent is preferably isopropanol, isoamyl alcohol, secondary pentanol or dioxane;
  • the acid is preferably hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid or benzenesulfonic acid;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , n, m, p are as defined in the formula (I).
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) according to the invention, together with a pharmaceutically acceptable carrier.
  • the invention further relates to the use of a compound of the formula (I) according to the invention or a pharmaceutical composition comprising the same, for the preparation of a medicament for the prevention and/or treatment of a cancer of a mammal, including a human, said cancer Including, but not limited to, non-solid tumors such as leukemia, solid tumors such as skin cancer, melanoma, lung cancer, stomach cancer, breast cancer, pancreatic cancer, liver cancer, colon cancer.
  • non-solid tumors such as leukemia, solid tumors such as skin cancer, melanoma, lung cancer, stomach cancer, breast cancer, pancreatic cancer, liver cancer, colon cancer.
  • the invention further relates to a method of preventing and/or treating cancer in a mammal, including a human, comprising administering to a patient in need thereof a prophylactically or therapeutically effective amount of a compound of the formula (I) according to the invention or A pharmaceutical composition comprising the same, including, but not limited to, non-solid tumors such as leukemia, solid tumors such as skin cancer, melanoma, lung cancer, gastric cancer, breast cancer, pancreatic cancer, liver cancer, colon cancer.
  • non-solid tumors such as leukemia, solid tumors such as skin cancer, melanoma, lung cancer, gastric cancer, breast cancer, pancreatic cancer, liver cancer, colon cancer.
  • the invention further relates to a compound of the formula (I) according to the invention or a pharmaceutical composition comprising the same, which is used as a medicament.
  • the invention further relates to a compound of the formula (I) according to the invention or a pharmaceutical composition comprising the same, which is useful as a medicament for the prevention and/or treatment of cancer, including but not limited to, non-solid tumors Such as leukemia, solid tumors such as skin cancer, melanoma, lung cancer, stomach cancer, breast cancer, pancreatic cancer, liver cancer, colon cancer.
  • non-solid tumors Such as leukemia, solid tumors such as skin cancer, melanoma, lung cancer, stomach cancer, breast cancer, pancreatic cancer, liver cancer, colon cancer.
  • alkyl refers to a saturated aliphatic hydrocarbon group, including straight chain and branched chain groups of 1 to 20 carbon atoms. It includes a linear or branched alkyl group having from 1 to 18 carbon atoms, preferably from 1 to 10 carbon atoms, more preferably from 1 to 6 carbon atoms, even more preferably from 1 to 4 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, Isopyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-decyl, n-decyl and the like.
  • the "alkyl group” further includes a cyclic alkyl group having 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, more preferably 4 to 6 carbon atoms, such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group or a ring. Hexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclodecyl, decahydronaphthyl, norbornane, adamantyl.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkyl, alkenyl, Alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycle Alkoxy, cycloalkylthio, heterocycloalkylthio, oxo, amino, haloalkyl, hydroxyalkyl, carboxy or carboxylate.
  • alkylene refers to a saturated straight or branched aliphatic hydrocarbon radical having two residues derived from the removal of two hydrogen atoms from the same carbon atom of the parent alkane or two different carbon atoms.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 ) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-pentylene (-CH 2 CH 2 CH 2 CH 2 CH 2 - )Wait.
  • alkenyl refers to a straight or branched hydrocarbon chain radical containing at least one double bond consisting of carbon and hydrogen atoms and attached to the remainder of the molecule by a single or double bond. It preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms. Non-limiting examples include ethenyl, propenyl, butenyl, pentenyl, pentadienyl, hexenyl.
  • the alkenyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, hetero A cycloalkylthio group, an oxo group, an amino group, a halogenated alkyl group, a hydroxyalkyl group, a carboxyl group or a carboxylate group.
  • alkenylene refers to a straight or branched hydrocarbon chain radical containing at least one double bond consisting of carbon and hydrogen atoms, having two substituents removed from the same carbon atom or two different carbon atoms of the parent. A residue derived from two hydrogen atoms which is attached to the remainder of the molecule by a single bond or a double bond. It preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms. Non-limiting examples include ethenylene, propenylene, butenylene, pentenylene, hexylene, and the like.
  • alkynyl refers to a straight or branched hydrocarbon chain radical containing at least one triple bond consisting of a carbon atom and a hydrogen atom and attached to the remainder of the molecule by a single or triple bond. It preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms. Non-limiting examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl.
  • the alkynyl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, hetero A cycloalkylthio group, an oxo group, an amino group, a halogenated alkyl group, a hydroxyalkyl group, a carboxyl group or a carboxylate group.
  • alkynylene refers to a straight or branched hydrocarbon chain radical containing at least one triple bond consisting of a carbon atom and a hydrogen atom, having two identical carbon atoms or two different carbon atoms from the parent. Residues derived from two hydrogen atoms are removed which are attached to the remainder of the molecule by a single or triple bond. It preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms. Non-limiting examples include ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably the cycloalkyl ring comprises from 3 to The 10 carbon atoms, most preferably the cycloalkyl ring contains 3 to 7 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptene
  • the alkenyl group, the cyclooctyl group and the like are preferably a cyclopropyl group or a cyclohexenyl group.
  • Polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups.
  • the cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, oxo, amino, haloalkyl, hydroxyalkyl, carboxy or carboxylate groups.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising from 3 to 20 ring atoms wherein one or more ring atoms are selected from nitrogen, oxygen or S(O)m A hetero atom (where m is an integer of 0 to 2), but does not include a ring moiety of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably comprises from 3 to 12 ring atoms, wherein from 1 to 4 are heteroatoms, more preferably the heterocyclyl ring contains from 3 to 10 ring atoms, more preferably the heterocyclyl ring contains from 5 to 7 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, tetrahydrofuranyl, azepan Alkyl and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • the heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, oxo, amino, haloalkyl, hydroxyalkyl, carboxy or carboxylate groups.
  • the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cyclo
  • aryl refers to an all-carbon monocyclic or fused polycyclic ring (i.e., a ring that shares a pair of adjacent carbon atoms) having a conjugated ⁇ -electron system, preferably from 5 to 10 members, more preferably from 5 to 7 members. Even more preferred are phenyl and naphthyl, most preferably phenyl.
  • the aryl group may be a completely aromatic group such as a phenyl group, a naphthyl group, an anthracenyl group, a phenanthryl group or the like.
  • the aryl group may also contain a combination of an aromatic ring and a non-aromatic ring, for example, ruthenium, osmium, and iridium.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring wherein the ring to which the parent structure is attached is an aryl ring, non-limiting examples comprising:
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkane.
  • Base amino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkane A thio group, an amino group, a halogenated alkyl group, a hydroxyalkyl group, a carboxyl group or a carboxylate group.
  • heteroaryl refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 5 to 10 members, more preferably 5 to 7 members, even more preferably 5 or 6 members, such as thiadiazolyl, pyrazolyl, oxazolyl, oxadiazolyl, imidazolyl, or the like.
  • heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples of which include:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxy or carboxylate groups.
  • Alkoxy means -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl, cycloalkyl are as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy and the like.
  • the alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, and an alkane group.
  • haloalkyl refers to an alkyl group wherein one or more hydrogen atoms are replaced by a halogen, wherein alkyl is as defined above.
  • Non-limiting examples include chloromethyl, trifluoromethyl, 1-chloro-2-fluoroethyl, 2,2-difluoroethyl, 2-fluoropropyl, 2-fluoropropan-2-yl, 2, 2,2-Trifluoroethyl, 1,1-difluoroethyl, 1,3-difluoro-2-methylpropyl, 2,2-difluorocyclopropyl, (trifluoromethyl)cyclopropane Base, 4,4-difluorocyclohexyl and 2,2,2-trifluoro-1,1-dimethyl-ethyl.
  • haloalkoxy refers to an alkoxy group wherein one or more hydrogen atoms are replaced by a halogen, wherein the alkoxy group is as defined above.
  • halogen includes fluoro, chloro, bromo and iodo.
  • amino means -NH 2.
  • nitro refers to -NO 2 .
  • cyano refers to -CN.
  • hydroxy refers to an -OH group.
  • hydroxyalkyl refers to an alkyl group substituted by a hydroxy group, wherein the alkyl group is as defined above.
  • hydroxyalkoxy refers to an alkoxy group substituted by a hydroxy group, wherein the alkoxy group is as defined above.
  • acyl refers to -C(O)R, wherein R refers to alkyl, cycloalkyl, alkenyl, alkynyl, wherein alkyl, cycloalkyl, alkenyl, alkynyl are as defined above.
  • R refers to alkyl, cycloalkyl, alkenyl, alkynyl, wherein alkyl, cycloalkyl, alkenyl, alkynyl are as defined above.
  • Non-limiting examples include acetyl, propionyl, butyryl, valeryl, hexanoyl, vinyl, acryloyl groups.
  • acylamino refers to -NHC (O) R, or -C (O) NH 2, wherein R denotes an alkyl group, an alkenyl group, an alkynyl group, where the definition of an alkyl group, an alkenyl group, an alkynyl group as described above.
  • R denotes an alkyl group, an alkenyl group, an alkynyl group, where the definition of an alkyl group, an alkenyl group, an alkynyl group as described above.
  • Non-limiting examples include formylamino, acetylamino, propionylamino, butyrylamino, pentanoylamino, hexanoylamino, vinylamido, acrylamido.
  • ester group refers to -C(O)OR, wherein R refers to alkyl or cycloalkyl, wherein alkyl, cycloalkyl are as defined above.
  • R refers to alkyl or cycloalkyl, wherein alkyl, cycloalkyl are as defined above.
  • Non-limiting examples include ethyl ester groups, propyl ester groups, butyl ester groups, amyl ester groups, cyclopropyl ester groups, cyclobutyl ester groups, cyclopentyl ester groups, cyclohexyl ester groups.
  • substituents are selected from the group consisting of a halogen atom, an alkyl group, an alkenyl group, an alkynyl group, a halogenated alkyl group, an alkoxy group, an aryl group, a halogenated aryl group, an aryloxy group, an aralkyl group, an aralkyloxy group, and a hetero group.
  • substituents can also be further substituted.
  • the alkyl group as a substituent is also optionally selected from one or more groups selected from a halogen atom, a hydroxyl group, an alkoxy group, an alkylamino group, a pyrrolidinyl group, a phenyl group, a pyridyl group, or a halogenated phenyl group.
  • the heterocyclic group as a substituent is also optionally substituted with one or more groups selected from a halogen atom, an alkyl group, and an alkoxy group.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • Drug Combination refers to the simultaneous or sequential application of two or more drugs for therapeutic purposes.
  • the present invention mainly employs the following synthetic routes and technical solutions.
  • the intermediate compound M3 is obtained by a substitution reaction of a hydrazine intermediate M1 and a pyrimidine intermediate M2 at a suitable temperature and a base under a catalyst catalyzed in a suitable solvent;
  • the base may be, for example, hydrogenated Sodium, potassium carbonate, cesium carbonate, etc.
  • the solvent may be, for example, DMF, NMP or the like, and the catalyst may be, for example, 1-hydroxybenzotriazole (HOBT).
  • Substituted pyrimidine intermediates M2 are generally commercially available.
  • Step 1 An aniline starting material M5 and a carboxylic acid starting material M6 are subjected to a condensation reaction to obtain an intermediate M7 under a suitable temperature and basic conditions in a suitable solvent;
  • the base may be, for example, triethyl An amine, N-methylmorpholine or the like
  • the solvent may be, for example, DCM, THF, etc.
  • the condensing agent may be, for example, HATU, HBTU, TBTU, or the like;
  • Step 2 reduction of the nitro group of intermediate M7 to an amino group to obtain intermediate M8; reduction of the nitro group can be achieved, for example, in an iron powder ammonium chloride system or a H 2 /palladium carbon system;
  • Step 3 The carboxylic acid starting material M9 is reacted in a suitable solvent under the action of a suitable chlorinating reagent and a catalyst to form an acid chloride
  • the solvent may be, for example, tetrahydrofuran, dichloromethane or the like
  • the chlorinating reagent may be
  • the catalyst may be, for example, DMF or the like;
  • the resulting acid chloride is then reacted with the intermediate M8 by nucleophilic substitution in a suitable solvent under appropriate conditions and under basic conditions to give the intermediate M10, which may be, for example, triethylamine, pyridine or N-methyl.
  • a suitable solvent for example, triethylamine, pyridine or N-methyl.
  • Morpholine or the like the solvent may be, for example, DCM, THF or the like;
  • Step 4 Intermediate M10 is deprotected under acidic conditions in a suitable solvent to give intermediate M4.
  • the acidic conditions may be, for example, trifluoroacetic acid, ethyl acetate, and the like, and the solvent may be, for example, DCM. , ethyl acetate, and the like.
  • Step 1 The intermediate carboxylic acid M11 is reacted in a suitable solvent under the action of a suitable chlorinating reagent and a catalyst to form an acid chloride, which may be, for example, tetrahydrofuran, dichloromethane or the like.
  • a suitable chlorinating reagent and a catalyst for example, oxalyl chloride, thionyl chloride, phosphorus oxychloride, etc.
  • the catalyst may be, for example, DMF or the like
  • the obtained acid chloride and intermediate M8 are passed through a suitable solvent under appropriate temperature and basic conditions.
  • the nucleophilic substitution reaction produces the intermediate M12, which may be, for example, triethylamine, pyridine, N-methylmorpholine or the like, and the solvent may be, for example, DCM, THF, DMF, or the like;
  • Step 2 Intermediate M12 is obtained by a substitution reaction in an appropriate solvent under appropriate temperature and basic conditions to obtain an intermediate product M10;
  • the base may be, for example, potassium carbonate, cesium carbonate or the like, and the solvent may be, for example, DMF, NMP, etc.
  • Step 3 Intermediate M10 is deprotected under acidic conditions in a suitable solvent to form intermediate M4.
  • the acidic conditions may be, for example, trifluoroacetic acid, ethyl acetate, and the like, and the solvent may be, for example, DCM. , ethyl acetate, and the like.
  • the intermediate M3 and the aniline intermediate M4 are reacted under acid catalysis at a suitable temperature and a suitable solvent to obtain a compound of the formula (I);
  • the solvent may be, for example, isopropanol, isoamyl alcohol, secondary pentanol, dioxane or the like
  • the acid may be, for example, hydrochloric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid or the like.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , n, m, p, R u , R z , R y are as in the formula (I) Defined.
  • the pharmaceutically acceptable salt of the compound of the formula (I) of the present invention may be an acid addition salt or a base addition salt.
  • the acid may be a mineral acid including, but not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid; or may be an organic acid including, but not limited to, citric acid, maleic acid, oxalic acid, formic acid, acetic acid, propionic acid, valeric acid.
  • glycolic acid glycolic acid, benzoic acid, fumaric acid, trifluoroacetic acid, succinic acid, tartaric acid, lactic acid, glutamic acid, aspartic acid, salicylic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid, p-benzenesulfonic acid .
  • the base may be an inorganic base including, but not limited to, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide; or may be an organic base including, but not limited to, ammonium hydroxide, triethylamine, N, N- Dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzyl phenyl An amine, arginine or lysine; or may be an alkali metal salt, including but not limited to: lithium, potassium and sodium salts; or may be an alkaline earth metal salt, including but not limited to: barium, calcium and magnesium salts;
  • the transition metal salt includes, but is not limited to, a zinc salt; or other metal salts including, but not limited to, sodium hydrogen phosphate and disodium hydrogen phosphate.
  • the compound of the formula (I) or a pharmaceutically acceptable salt is prepared into a clinically usable pharmaceutical composition.
  • the pharmaceutical preparations thereof include, but are not limited to, oral preparations such as tablets, gels, soft/hard capsules, emulsions, dispersible powders, granules, water/oil suspoemulsions; injections Including intravenous injection, intramuscular injection, intraperitoneal injection, rectal suppository, intracranial injection, these dosage forms may be aqueous solutions or oily solutions; topical preparations include creams, ointments, gels, water/oil solutions and packs Formulations; inhalation dosage forms include fine powders, liquid aerosols, and various dosage forms suitable for in vivo implantation.
  • the pharmaceutical composition of the present invention may be added with a pharmaceutically acceptable carrier, diluent or excipient as needed.
  • a pharmaceutically acceptable carrier diluent or excipient as needed.
  • Carriers for solid oral formulations include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, cyclodextrin, and the intestinal absorption molecular carrier vitamin E-PEG 1000.
  • Oral formulations may incorporate suitable colorants, sweeteners, flavoring agents, and preservatives.
  • the compound of the formula (I) or a pharmaceutically acceptable salt of the present invention is administered to a warm-blooded animal at a unit dose of 0.01 to 100 mg/kg.
  • the compound represented by the formula (I) or a pharmaceutically acceptable salt of the present invention can be used alone or in combination with radiotherapy, chemotherapy, immunotherapy, tumor vaccine, and tumor which are conventionally used in clinical practice.
  • Combination therapy with one or more methods of viral, RNAi, cancer adjuvant therapy, and bone marrow transplantation and stem cell transplantation including but not limited to the following anti-tumor drugs and treatments:
  • alkylating agents such as cisplatin, cisplatin, oxaliplatin, chlorambucil, carbophosphoramide, nitrogen mustard, melphalan, temozolomide, busulfan, nitrosourea.
  • anti-tumor antibiotics such as doxorubicin, bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin C, actinomycin, genus Anti-mitotic drugs such as vincristine, vinblastine, vindesine, vinorelbine, paclitaxel, taxotere, Polo kinase inhibitors.
  • Antimetabolites and antifolates such as fluoropyrimidine, rametamine, cytarabine, azacitidine, decitabine, trebuta, hydroxyurea, IDH1/IDH2 mutant inhibitors.
  • Topoisomerase inhibitors such as epipodophyllotoxin, camptothecin, and irinotecan.
  • Cell growth inhibitors such as antiestrogens/antiandrogens, such as tamoxifen, fulvestrant, toremifene, raloxifene, ranoxifene, oxyxid, bicalutamide , flutamide, nilutamide, cyproterone acetate;
  • LHRH antagonists or LHRH agonists such as goserelin, leuprolide, and buserelin, progestogens such as megestrol acetate;
  • Aromatase inhibitors such as anastrozole, letrozole, vorozole, exemestane, 5a-reductase inhibitors such as finasteride.
  • Anti-invasive agents such as c-Src kinase family inhibitors, metalloproteinase inhibitors, inhibitors of urokinase plasminogen activator receptor function or heparanase-like antibodies.
  • inhibitors of growth function such as growth factor antibodies and growth factor receptor antibodies such as anti-HER2 antibody trastuzumab, anti-EGFR antibody panitumumab, anti-EGFR antibody cetuximab, etc.; Including other tyrosine kinase inhibitors and inhibitors of serine/threonine kinases such as Ras/Raf signaling inhibitors, cell signaling inhibitors of MEK and/or AKT kinase, c-kit inhibitors, abl kinase inhibitors , PI3 kinase inhibitors, JAKs and STAT3 inhibitors, FLT3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor kinase inhibitors, Aurora kinase inhibitors, NTRKA/B/C kinase inhibitors.
  • growth factor antibodies and growth factor receptor antibodies such as anti-HER2 antibody trastuzumab, anti-EGFR antibody panitumumab, anti-EGFR antibody
  • An anti-angiogenic agent such as bevacizumab which inhibits the action of vascular endothelial growth factor and a VEGF receptor tyrosine kinase inhibitor.
  • HDACi histone deacetylase inhibitors
  • DNMTi DNA methyltransferase inhibitors
  • histone acetyltransferase inhibitors histone demethylases Inhibitors
  • histone methyltransferase inhibitors and the like.
  • PARPi Poly ADP-ribose polymerase inhibitors
  • Tumor immunotherapy includes any in vitro and in vivo methods that increase the immunogenicity of a patient's tumor cells.
  • cytokine IL-2, IL-4 or GM-CSF for transfection; methods for reducing T cell ineffectiveness such as anti-PD-1/PD-L mAb; transfected immune cells such as cytokine transfected trees Method of squamous cell; method of cytokine transfected tumor cell line; reduction of immunosuppressive cells such as regulatory T cells, myeloid suppressor cells, or dendrites expressing guanamine 2,3-deoxygenase Functional methods of cells; methods of agonists that increase the activity of immune cells, such as STING, and cancer vaccines composed of tumor-associated antigenic proteins or peptides.
  • Tumor gene therapy such as CRISPR-Cas 9, RNAi, gene transduction.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • the NMR was measured by a (Bruker AVANCE-400) nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard was Tetramethylsilane (TMS).
  • DMSO-d6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Tetramethylsilane
  • the MS was measured by a liquid chromatography mass spectrometer (Thermo, Ultimate 3000/MSQ).
  • the HPLC was measured using a high pressure liquid chromatograph (Agilent 1260 Infinity, Gemini C18 250 x 4.6 mm, 5u column).
  • the silica gel plate HSGF245 used for thin layer chromatography has a specification of 0.15 mm to 0.2 mm, and the specification for separation and purification of thin layer chromatography is 0.9 mm to 1.0 mm (Yantai Yellow Sea).
  • the known starting materials of the present invention can be synthesized by or according to methods known in the art, or purchased from Shanghai Darui Fine Chemicals Co., Ltd., Shanghai Titan Technology Co., Ltd., Shanghai Runjie Chemical Reagent Co., Ltd., TCI, Aldrich Chemical Company.
  • the experimental methods in the examples which do not specify the specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific source are routine reagents purchased from the market.
  • the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.
  • An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon having a volume of about 1 L.
  • the solution means an aqueous solution.
  • reaction temperature is room temperature and is 20 ° C to 30 ° C.
  • 2,4-Dichloropyrimidine (7.15 g, 0.048 mol) was dissolved in DMF (80 ml), HOBT (1.0 g, 8 mmol) and potassium carbonate (11 g, 0.08 mol) were added at room temperature, and the mixture was stirred at room temperature for 15 minutes.
  • the hydrazine (4.68 g, 0.04 mol) (dissolved in DMF) was slowly added dropwise to the reaction system, and the reaction was carried out by heating at 85 ° C for 6 hours, and the reaction was confirmed by TLC.
  • the mixture was poured into water (200 ml), EtOAc (EtOAc m. Purification by column chromatography (eluent: petroleum ether / ethyl acetate) afforded 5.3 g of 1-(2-chloro-pyrimidin-4-yl)-1H-indole as a yellow solid.
  • Step 2 Preparation of (3-nitro-phenyl)-carbamic acid tert-butyl ester
  • Step 3 Preparation of (3-amino-phenyl)-carbamic acid tert-butyl ester
  • Step 4 Preparation of [3-(3-nitro-benzoylamino)-phenyl]-carbamic acid tert-butyl ester
  • Step 5 Preparation of [3-(3-Amino-benzoylamino)-phenyl]-carbamic acid tert-butyl ester
  • Step 6 Preparation of (E)- ⁇ 3-[3-(4-dimethylamino-but-2-enoylamino)-benzoylamino]-phenyl ⁇ -carbamic acid tert-butyl ester
  • Trans-4-dimethylamino croton hydrochloride (6.1 g, 0.0367 mol) was added to a 250 ml three-necked flask under a nitrogen atmosphere, and the nitrogen balloon was ventilated three times.
  • Ultra dry (anhydrous) THF 150 ml
  • 6 drops of DMF were added by syringe and cooled to 0-5 ° C in an ice water bath.
  • Oxalyl chloride (3.9 g, 0.0306 mol) was slowly added dropwise to the system, and the mixture was added dropwise, and the ice water bath was removed, and the mixture was warmed to room temperature for 2 hours.
  • EtOAc EtOAc
  • EtOAc ⁇ 3-[3-(4-Dimethylamino-but-2-enoylamino)-benzoylamino]-phenyl ⁇ -carbamic acid tert-butyl ester.
  • Step 7 Preparation of (E)-N-(3-amino-phenyl)-3-(4-dimethylamino-but-2-enoylamino)-benzamide
  • Step 8 (E)-3-(4-Dimethylamino-but-2-enoylamino)-N-[3-(4-indol-1-yl-pyrimidin-2-ylamino)-benzene Preparation of bis-benzamide
  • Example 2 Same as the preparation method in Example 1, except that 4-methoxy hydrazine (TCI) was used instead of the hydrazine in the first step of Example 1, to obtain (E)-3-(4-dimethylamino-butyl- 2-enoylamino)-N- ⁇ 3-[4-(4-methoxy-indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -benzamide.
  • TCI 4-methoxy hydrazine
  • Methyl (E)-4-bromo-2-butenoate (3 g, 0.0168 mol) was dissolved in 30 ml of THF under nitrogen atmosphere, cooled to 0-5 ° C in an ice water bath, and 10 ml of aqueous lithium hydroxide solution was slowly added dropwise. (960 mg of lithium hydroxide), after completion of the dropwise addition, the temperature was maintained at 0 to 5 ° C for 3 hours. The reaction was completely detected by TLC, 50 ml of ice water was added, and petroleum ether was extracted (100 ml ⁇ 2), and the organic phase was discarded.
  • the aqueous phase was cooled to about 0 ° C, the pH was adjusted to about 1 with concentrated hydrochloric acid, extracted with dichloromethane (70 ml ⁇ 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 1.35 g of 4-bromo croton as a solid. acid.
  • Step 3 Preparation of (E)- ⁇ 3-[3-(4-bromo-but-2-enoylamino)-benzoylamino]-phenyl ⁇ -carbamic acid tert-butyl ester
  • Step 4 Preparation of (E)- ⁇ 3-[3-(4-morpholin-4-yl-but-2-enoylamino)-benzoylamino]-phenyl ⁇ -carbamic acid tert-butyl ester
  • Step 6 (E)-N- ⁇ 3-[4-(4-Methoxy-indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -3-(4-morpholine- Preparation of 4-yl-but-2-enoylamino)-benzamide
  • Step 3 (E)-4-(4-Dimethylamino-but-2-enoylamino)-N-[3-(4- ⁇ 4-[2-(6-methyl-pyridine-3- Preparation of methoxy)-ethoxy]-indol-1-yl ⁇ -pyrimidin-2-ylamino)-phenyl]-benzamide
  • Step 2 (E)-3-(4-Dimethylamino-but-2-enoylamino)-N- ⁇ 3-[4-(4-isopropoxy-indol-1-yl)- Preparation of pyrimidin-2-ylamino]-phenyl ⁇ -benzamide
  • Step 4 (E)-N- ⁇ 3-[4-(4-Difluoromethoxy-indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -3-(4-di Preparation of methylamino-but-2-enoylamino)-benzamide
  • Example 1 Same as the preparation method of Example 1, except that 4-difluoromethoxy-1H-indole was used instead of the hydrazine in the first step of Example 1, and p-nitrobenzoic acid (TCI) was used instead of the first step in Example 1.
  • TCI p-nitrobenzoic acid
  • M-nitrobenzoic acid to give (E)-N- ⁇ 3-[4-(4-difluoromethoxy-indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -3 -(4-Dimethylamino-but-2-enoylamino)-benzamide.
  • Example 2 The same procedure as in Example 1, except that 4-methoxyindole (Dari) was used instead of the hydrazine in Step 1 of Example 1, and 4-methoxy-3-nitroaniline was used instead of Example 1.
  • m-Nitroaniline in 2 gives (E)-3-(4-dimethylamino-but-2-enoylamino)-N- ⁇ 2-methoxy-5-[4-(4-A Oxy-indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -benzamide.
  • Example 1 Same as the preparation method of Example 1, except that 4-methoxy hydrazine (Dare) was used instead of the hydrazine in the first step of Example 1, and 4-chloro-3-nitrobenzoic acid was used instead of Example 1 Step 4.
  • m-Nitrobenzoic acid to give (E)-4-chloro-3-(4-dimethylamino-but-2-enoylamino)-N- ⁇ 3-[4-(4-methoxy -Indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -benzamide.
  • 3-indolecarboxylic acid (30 g, 0.186 mol) was stirred at room temperature in 500 ml of dichloromethane and was not completely dissolved. 0.5 ml of DMF was added, and then oxalyl chloride (71.0 g, 0.56 mol) was added dropwise thereto at room temperature. After 30 minutes, the addition was completed, and the reaction was continued at room temperature for 2 hours. The reaction was completed by EtOAc (EtOAc:EtOAc) The product was used in the next step without purification.
  • 1H-indole-3-carbonyl chloride (0.186 mol, theoretical yield) obtained in the step 1 was added to 500 ml of DCM, stirred at room temperature for 30 minutes, and was not completely dissolved, and was a cloudy dispersion system.
  • 350 ml of ammonia water and 200 ml of DCM were added to a 2 L three-necked flask and stirred vigorously.
  • the dichloromethane turbid dispersion of 1H-indole-3-carbonyl chloride was slowly added dropwise to a 2 L three-necked flask at room temperature, and after 20 minutes, the reaction was continued at room temperature for 1 hour.
  • Step 3 (E)-1-(2- ⁇ 3-[3-(4-Dimethylamino-but-2-enoylamino)-benzoylamino]-phenylamino ⁇ -pyrimidine-4- Preparation of -1H-indole-3-carboxamide
  • Step 2 (E)-3-(4-Dimethylamino-but-2-enoylamino)-N- ⁇ 3-[5-fluoro-4-(4-methoxy-oxime-1- Of p-pyrimidin-2-ylamino]-phenyl ⁇ -benzamide
  • the reaction mixture was cooled to room temperature, 20 ml of methyl tert-butyl ether was added, and the solid was washed with a small amount of methyl t-butyl ether.
  • the obtained solid was dissolved in dichloromethane/methanol (50 ml/5 ml), adjusted to pH 9-10 by adding 20 ml of 0.2 mol/L dilute aqueous sodium hydroxide solution, extracted with dichloromethane (30 ml ⁇ 2), and the organic phase was saturated with NaCl solution. It was washed twice, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated.
  • Example 22 The preparation method of Example 22 was carried out except that 2,4-dichloro-5-fluoropyrimidine in Step 1 of Example 22 was replaced with 2,4,5-trichloropyrimidine (Dari) to obtain (E)-N. - ⁇ 3-[5-Chloro-4-(4-methoxy-indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -3-(4-dimethylamino-butyl- 2-enoylamino)-benzamide.
  • Example 22 The same procedure as in Example 22 was carried out except that 2,4-dichloro-5-methylpyrimidine (Dari) was used instead of 2,4-dichloro-5-fluoropyrimidine in Step 1 of Example 22 to give (E )-3-(4-Dimethylamino-but-2-enoylamino)-N- ⁇ 3-[4-(4-methoxy-indol-1-yl)-5-methyl-pyrimidine 2-ylamino]-phenyl ⁇ -benzamide.
  • Dari 2,4-dichloro-5-methylpyrimidine
  • Step 1 of Example 22 to give (E )-3-(4-Dimethylamino-but-2-enoylamino)-N- ⁇ 3-[4-(4-methoxy-indol-1-yl)-5-methyl-pyrimidine 2-ylamino]-phenyl ⁇ -benzamide.
  • Example 22 Same as the preparation method of Example 22 except that 4-isopropoxy hydrazine (prepared in Example 7) was used instead of 4-methoxyindole in Step 1 of Example 22, and 2,4,5-three was used. Chloropyrimidine (Dari) is substituted for the 2,4-dichloro-5-fluoropyrimidine in Step 1 of Example 22 to give (E)-N- ⁇ 3-[5-chloro-4-(4-isopropoxy) -Indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -3-(4-dimethylamino-but-2-enoylamino)-benzamide.
  • 4-isopropoxy hydrazine prepared in Example 7
  • 2,4,5-three was used.
  • Chloropyrimidine (Dari) is substituted for the 2,4-dichloro-5-fluoropyrimidine in Step 1 of Example 22 to give (E)-N- ⁇ 3-[5-chloro-4-(4
  • Example 22 The same procedure as in Example 22 was carried out except that 1H-indole-3-carboxamide (prepared in Step 2 of Example 21) was used instead of 4-methoxyindole in Step 1 of Example 22 to give (E)-1. -(2- ⁇ 3-[3-(4-Dimethylamino-but-2-enoylamino)-benzoylamino]-phenylamino ⁇ -5-fluoro-pyrimidin-4-yl)-1H- Indole-3-carboxamide.
  • the acid salt replaces the trans-4-dimethylamino crotonate salt of the first step of Example 1, to give 3-(4-dimethylamino-butyrylamino)-N- ⁇ 3-[4-( 4-methoxy-indol-1-yl)-pyrimidin-2-ylamino]-phenyl ⁇ -benzamide.
  • MOLM13 is a human acute myeloid leukemia cell line with positive expression of CDK7 and cell line derived from DMSZ. The cells were cultured in suspension with RPMI 1640 (Gibco) plus 10% fetal bovine serum (Gibco), 1% double antibody, and 2 mM glutamine medium.
  • the MOLM-13 suspension cells in logarithmic growth phase were collected by centrifugation (1700 rpm, 3 minutes), the supernatant was discarded, and the cells were counted.
  • the cell concentration was 2 x 10 5 cells per ml in RPMI1640 medium, inoculated into 96-well plates (Corning), 100 ⁇ l per well, and cultured overnight at 37 ° C, 5% CO 2 .
  • the final concentration of the organic solvent is not more than one thousandth, and the cells are further cultured for 3 to 6 days, and the MTT is measured.
  • the compound of the present invention and the control compounds THZ1 and THZ2 were each dissolved in DMSO (Sigma), and the purity of the compound was 98% or more.
  • the compound was stored at a concentration of 10 mM, stored at -20 ° C, and serially diluted 10 or 10 times before use.
  • the MTT detection reagent is the Dojindo CCK8 kit, and the enzyme labeling instrument is THERMO MULTISKAN FC instrument.
  • the CCK8 reagent was directly added to the drug-treated and solvent-controlled suspension cells MOLM-13, the final concentration of CCK8 was 10%, and the culture was continued for 1 to 4 hours.
  • the solvent control wells were dark yellow, the OD450nm light absorption value was measured (THERMO MULTISKAN) FC instrument), calculate the cell growth rate according to the following formula:
  • T drug treatment cell hole optical density value - blank control hole optical density value
  • T 0 cell hole optical density value before drug treatment - blank control hole optical density value
  • C solvent control cell hole optical density - blank control hole light Density value.
  • the drug concentration, i.e., IC50, at which 50 % inhibition of cell growth was calculated by GraphPad Prism7 software. The experiment was repeated 1-3 times and the data was subjected to biological statistical analysis.
  • Table 2 summarizes the results of the determination of the IC 50 concentration of the compound of the present invention for inhibiting the growth-inducing apoptosis of tumor cells MOLM-13 in vitro. The smaller the IC 50 value, the stronger the activity of the compound.
  • "*****” represents an IC 50 value of ⁇ 1 nM
  • "****” represents an IC 50 value in the range of 1 nM to ⁇ 10 nM
  • "***” represents an IC 50 value in the range of 10 nM to ⁇ 100 nM
  • **" represents an IC 50 value in the range of 100 nM to ⁇ 1000 nM
  • "*” represents an IC 50 value > 1000 nM
  • "-" represents no measurement.

Abstract

一种嘧啶类化合物、其制备方法及其医药用途。具体而言，涉及一种通式（I）所示的嘧啶类化合物，及其制备方法，以及其作为细胞周期蛋白依赖性激酶7（CDK7）抑制剂的用途，特别是在预防和/或治疗人类疾病包括癌症中的用途。其中，通式（I）中各基团的定义与说明书中的定义相同。

Description

一种嘧啶类化合物、其制备方法及其医药用途 技术领域
本发明属于医药领域，涉及一种新型的嘧啶类化合物、其制备方法及含有其的药物组合物，以及其作为选择性细胞周期蛋白依赖性激酶7(CDK7)抑制剂在预防和/或治疗人类疾病包括癌症中的用途。
背景技术
肿瘤包括白血病是导致人类临床死亡的重大疾病之一，每年全球有数百万病人死于癌症。恶性肿瘤如肺癌、胃癌、乳腺癌、胰腺癌、肝癌、肠癌、卵巢癌、***、食管癌、鼻咽癌、白血病和恶性淋巴瘤等死亡率极高。尽管癌症的遗传筛查、分子诊断和精准医疗提高了对癌症病人的临床早期发现、正确诊断和治疗效果，但至今为止多数癌症特别是晚期、难治、复发和耐药性恶性肿瘤仍没有有效的方法与药物可完全根除或治愈。临床上急需特异性好、活性高、毒性小、无耐药性产生的优质抗癌药物。
癌症的发生、发展、转移、恶化、复发及耐药性产生与许多因素有关。哺乳动物细胞周期是一个高度有组织有次序和精确调控的细胞有丝***过程，在这个过程中，细胞的遗传物质复制并均等地分配给两个增殖的子细胞中。细胞生长因子和细胞周期调控因子在细胞周期中起着重要作用。细胞周期调控因子是一类细胞内自身合成的蛋白质，各种细胞周期调控因子(蛋白)的异常活性往往引起正常细胞周期的异常，导致不同类型的疾病，例如当细胞增殖不受控时，引起细胞转化即形成癌细胞。
细胞周期蛋白依赖性激酶(Cyclin Dependent Kinase，CDK)是一组丝氨酸/苏氨酸蛋白激酶，和细胞周期蛋白(Cyclin)协同作用，是细胞周期进程和转录的关键调控因子。CDK可以和细胞周期蛋白结合形成异二聚体，其中CDK为催化亚基，细胞周期蛋白为调节亚基，不同的Cyclin-CDK复合物，通过CDK活性磷酸化细胞中的不同底物，而实现对细胞周期不同时相的推进和转化作用。至今为止已发现和鉴定出21个CDK基因(CDK1～CDK20，其中CDK11具有CDK11A和CDK11B两个基因)和五个CDK类基因CDKL(CDKL1～CDKL5)，其中在这些CDK蛋白激酶功能结构域中，氨基酸序列具有高度的进化保守性。根据CDK作用机理和功能可分为直接细胞周期调控CDK(如CDK1、CDK2、CDK3、CDK4和CDK6)和转录功能CDK(如CDK7、CDK 8、CDK 9、CDK 11、CDK 12和CDK13)。直接细胞周期调控CDK直接调节进展细胞周期阶段，其磷酸化作用底物为细胞周期相关蛋白。转录功能CDK通过磷酸化RNA聚合酶II复合物来调节基因转录。临床数据发现在不同类型恶性肿瘤和白血病病人样本如皮肤癌、黑色素瘤、肺癌、胃癌、乳腺癌、胰腺癌、肝癌或结肠癌和急性髓细胞白血病中，不同的CDK频繁的发生基因突变、扩增和过表达，这些变异与癌症的发生、发展和/或维持恶性细胞表型以 及病人存活期和耐药性具有密切的关联。同样基础研究发现CDK的异常能够驱动肿瘤的发生，抑制CDK能够有效的抑制/消除肿瘤细胞的体内外生长。CDK已广泛的被作为测试和应用癌症治疗的良好靶标，特别是CDK4/6选择性抑制剂帕博西尼(Palbociclib)、Ribociclib和Abemaciclib在临床上的成功应用(Otto T等人(2017)Nat Rev Cancer 17(2):93-115；Kwapisz D(2017)Breast Cancer Res Treat.166(1):41-54；Vijayaraghavan S等人(2017)Target Oncol.2017 Dec 7；Ingham M等人(2017)J Clin Oncol.35(25):2949-2959；Abou Zahr A等人(2017)Expert Opin Emerg Drugs.22(2):137-148；O'Leary B等人(2016)Nat Rev Clin Oncol.13(7):417-30；Coin F等人(2015)Mol Cell.59(4):513-4；Pozo K等人(2016)Trends Cancer.2(10):606-618)。近来研究发现CDK4/6和CDK5具有肿瘤免疫调节功能，选择性抑制CDK4/6或CDK5可增强肿瘤免疫治疗的效果，进一步的证明CDK是肿瘤治疗的重要靶蛋白(Dorand RD等人(2016)Science.353(6297):399-403；Goel S等人(2017)Nature.548(7668):471-475；Deng J等人(2017)Cancer Discov.8(2)；216–33；Zhang J等人(2018)Nature.553(7686):91-95)。
多年来，许多不同类型的CDK抑制剂已进行了广泛的临床前和临床研究，但至今为止只有CDK4/6高选择性抑制剂帕博西尼、Ribociclib和Abemaciclib成功地应用于荷尔蒙受体阳性、HER2阴性的晚期或复发乳腺癌的临床治疗，帕博西尼和Ribociclib需要与来曲唑(Letrozole)联合用药，Abemaciclib可单独或与氟维司琼(Fulvestrant)联合用药。泛CDK抑制剂(第一代CDK抑制剂)如Alvocidib和Seliciclib为黄酮类化合物，Alvocidib与ATP竞争性地抑制CDK1、CDK2、CDK4和CDK6，IC 50值约为40nM；Seliciclib可抑制CDK5、Cdc2和CDK2，IC 50分别为0.2μM、0.65μM和0.7μM；但没有显示出有希望的临床前和临床研究中的抗肿瘤活性。第二代泛CDK抑制剂如Dinaciclib、AT7519、Milciclib、TG02、CYC065和RGB-286638能够高活性的同时抑制多种CDK，尽管分别进入临床试验的不同期，但这些抑制剂单独应用并没有表现出良好的治疗效果而呈现出高的临床副作用。
CDK7在哺乳动物CDK中具有独特性，能够调节细胞周期和基因转录。在细胞质中，CDK7作为CDK激活酶(CAK)异三聚体复合物存在，能够通过磷酸化CDK1/2激活域保守残基(T环)使CDK1/2完全被激活，这一过程是细胞周期进程所需的；在细胞核中，CDK7与RNA聚合酶(RNAP)II形成复合物，负责磷酸化RNAP II的C-末端结构域(CTD)，这是基因转录起始的必要步骤。CDK7的双功能即CAK和CTD磷酸化能够在细胞增殖、细胞周期和基因转录的关键方面发挥重要作用。越来越多的研究数据发现抑制人类CDK7激酶活性能够抑制肿瘤细胞的体内外增殖。CDK7选择性抑制剂有望作为新型潜在治疗癌症的有效靶向药物。近来CDK7高选择性抑制剂SY-1365(THZ1)已进入人体临床试验，该化合物临床前试验呈现出良好的抗肿瘤活性，是一类潜在的新型CDK7抗肿瘤抑制剂 (Lücking U等人(2017)Chem Med Chem.12(21):1776-1793；Kwiatkowski N等人(2014)Nature.511(7511):616-20)，但此化合物动物体内的稳定性(在小鼠血浆中T1/2为45分钟)限制了其体内药效。因此临床急需更高效和毒副作用低CDK7抑制剂。
本发明人在长期CDK7新型选择性抑制剂研发过程中，发现一种新型的嘧啶类化合物，其能够有效的抑制CDK7表达阳性白血病细胞MOLM-13的体外生长，其IC 50值可达亚纳摩尔浓度，有望开发为新型有效的抗肿瘤药物。
发明内容
本发明的目的是提供一种选择性好、活性高、毒性低的新型可口服给药的小分子化合物，其可以作为细胞周期蛋白依赖性激酶7(CDK7)抑制剂，用于预防和/或治疗人类疾病包括癌症。
本发明涉及一种新型的嘧啶类化合物，其能够有效的与CDK7共价结合，抑制CDK7表达阳性肿瘤细胞的体外生长，其IC 50值可达亚纳摩尔浓度。
因此，本发明提供一种通式(I)所示的化合物，
Figure PCTCN2019073874-appb-000001
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐；
其中：
每一个R 1各自独立地选自氢、卤素、羟基、氰基、硝基、-N(R y)(R z)、烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基、-NHC(O)R x、-C(O)N(R y)(R z)、-OR uOR x、-OR x或-OR uN(R y)(R z)，其中所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
R 2和R 3各自独立地选自氢、卤素、羟基、氰基、硝基、烷基、烷氧基、环烷基、杂环基、-NHC(O)R x或-C(O)N(R y)(R z)，其中所述烷基、烷氧基、环烷基和杂环基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
R 4和R 5各自独立地选自氢、卤素、羟基、氰基、硝基、烷基、烷氧基、环烷基、杂环基、-NHC(O)R x或-C(O)N(R y)(R z)，其中所述烷基、烷氧基、环烷基和杂 环基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
每一个R 6各自独立地选自Q基团；
每一个R 7各自独立地选自-NHC(O)R和Q基团；
R 8和R 9相同或不同且各自独立地选自-NHC(O)R和Q基团；
Q选自氢、卤素、羟基、氰基、硝基、-N(R y)(R z)、烷基、烷氧基、烯基、炔基、环烷基、杂环基、芳基或杂芳基，其中所述烷基、烷氧基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基或-R uN(R y)(R z)的一个或多个基团取代；
R u选自亚烷基、亚烯基、或亚炔基；
R x选自氢、烷基、环烷基、芳基或者杂芳基，所述烷基、环烷基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
R y和R z各自独立地选自氢、烷基、烷氧基、烯基、炔基、环烷基、杂环基、芳基或杂芳基；所述烷基、烷氧基、烯基、炔基、环烷基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；或者，
R y和R z与它们所连接的氮原子一起形成杂环基或杂芳基，所述杂环基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
n为1至4的整数；m为1至4的整数；p为1至3的整数。
在本发明一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
R 8选自-NHC(O)R，R 9选自Q基团；或者，
R 9选自-NHC(O)R，R 8选自Q基团；
其中R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自氢、烷基或R uN(R y)(R z)的一个或多个基团取代；
R u、R y、R z、Q基团如通式(I)中所定义。
在本发明另一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
R 8选自-NHC(O)R，且R 9选自Q基团；或者，
R 9选自-NHC(O)R，且R 8选自Q基团；
其中R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自氢、烷基或R uN(R y)(R z)的一个或多个基团取代；
R u选自C 1～C 6亚烷基；
R y和R z各自独立地选自氢、C 1～C 6烷基、C 3～C 7环烷基；或者，
R y和R z与它们所连接的氮原子一起形成5～7元含氮杂环基，优选吗啉基、哌啶基、哌嗪基、氮杂环庚烷基、四氢吡咯基，所述5～7元含氮杂环基任选进一步被选自卤素、C 1～C 6烷基、C 1～C 6卤代烷基、C 1～C 6烷氧基、C 1～C 6卤代烷氧基、C 3～C 7环烷基的一个或多个基团所取代；
Q基团选自氢、卤素、羟基、氰基、硝基、氨基、C 1～C 6烷基、C 1～C 6烷氧基、C 3～C 7环烷基。
在本发明另一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
每一个R 7各自独立地选自-NHC(O)R；
其中R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自氢、烷基或R uN(R y)(R z)的一个或多个基团取代；
R u选自C 1～C 6亚烷基；
R y和R z各自独立地选自氢、C 1～C 6烷基、C 3～C 7环烷基；或者，
R y和R z与它们所连接的氮原子一起形成5～7元含氮杂环基，优选吗啉基、哌啶基、哌嗪基、氮杂环庚烷基、四氢吡咯基，所述5～7元含氮杂环基任选进一步被选自卤素、C 1～C 6烷基、C 1～C 6卤代烷基、C 1～C 6烷氧基、C 1～C 6卤代烷氧基、C 3～C 7环烷基的一个或多个基团所取代。
在本发明另一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
每一个R 1各自独立地选自氢、卤素、羟基、氰基、硝基、C 1～C 6烷基、C 3～C 7环烷基、-NHC(O)R x、-C(O)N(R y)(R z)、-OR uOR x、-OR x，其中所述C 1～C 6烷基、C 3～C 7环烷基任选进一步被选自卤素、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
R u选自C 1～C 6亚烷基；
R x选自氢、C 1～C 6烷基、C 3～C 7环烷基或者5～7元杂芳基，所述C 1～C 6烷基、C 3～C 7环烷基或者5～7元杂芳基任选进一步被卤素、羟基、烷基、环烷基、杂环基、芳基或杂芳基的一个或多个基团所取代。
在本发明另一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
R 2和R 3各自独立地选自氢、卤素、氰基、烷基、烷氧基、环烷基、杂环基、-NHC(O)R x或-C(O)N(R y)(R z)，其中所述烷基、烷氧基、环烷基和杂环基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
R x、R y、R z如通式(I)所定义。
在本发明另一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
R 2选自氢、烷基、环烷基、氰基、-NHC(O)R x或-C(O)N(R y)(R z)；
R 3选自氢；
R x选自氢、烷基、环烷基，所述烷基、环烷基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基的一个或多个取代基所取代；
R y和R z各自独立地选自氢、烷基、环烷基、杂环基、芳基或杂芳基；所述烷基、环烷基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基的一个或多个取代基所取代；或者，
R y和R z与它们所连接的氮原子一起形成含氮杂环基或杂芳基，所述杂环基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基中的一个或多个取代基所取代。
在本发明另一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
R 4和R 5各自独立地选自氢、卤素、氰基、C 1～C 6烷基、C 1～C 6卤代烷基、C 1～C 6烷氧基、C 1～C 6卤代烷氧基。
在本发明另一个优选的实施方案中，根据本发明所述的通式(I)所示的化合物，
其中：
每一个R 6各自独立地选自Q基团；
Q选自氢、卤素、羟基、氰基、硝基、氨基、C 1～C 6烷基、C 1～C 6烷氧基、C 3～C 7环烷基。
本发明典型的化合物包括但不限于以下化合物：
Figure PCTCN2019073874-appb-000002
Figure PCTCN2019073874-appb-000003
Figure PCTCN2019073874-appb-000004
Figure PCTCN2019073874-appb-000005
Figure PCTCN2019073874-appb-000006
Figure PCTCN2019073874-appb-000007
Figure PCTCN2019073874-appb-000008
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐。
本发明另一方面提供根据本发明所述的通式(I)所示的化合物或其药学上可接受的盐的制备方法，其包括以下步骤：
Figure PCTCN2019073874-appb-000009
将中间体化合物M3和苯胺类中间体化合物M4在适宜的温度和适当的溶剂中，在酸催化下反应，得到通式(I)化合物；
所述溶剂优选异丙醇、异戊醇、仲戊醇、二氧六环；
所述酸优选盐酸、硫酸、甲磺酸、对甲苯磺酸、苯磺酸；
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、n、m、p如通式(I)中所定义。
本发明进一步涉及一种药物组合物，其含有治疗有效量的根据本发明所述的通式(I)所示的化合物，以及药学上可接受的载体。
本发明进一步涉及根据本发明所述的通式(I)所示的化合物或者包含其的药物组合物，在制备用于预防和/或治疗哺乳动物包括人类癌症的药物中的用途，所述癌症包括但不限于，非实体瘤如白血病，实体瘤如皮肤癌、黑色素瘤、肺癌、胃癌、乳腺癌、胰腺癌、肝癌、结肠癌。
本发明进一步涉及一种预防和/或治疗哺乳动物包括人类中癌症的方法，其包括向需要其的患者施用预防或治疗有效量的根据本发明所述的通式(I)所示的化合物或者包含其的药物组合物，所述癌症包括但不限于，非实体瘤如白血病，实体瘤如皮肤癌、黑色素瘤、肺癌、胃癌、乳腺癌、胰腺癌、肝癌、结肠癌。
本发明进一步涉及根据本发明所述的通式(I)所示的化合物或者包含其的药物组合物，其用作药物。
本发明进一步涉及根据本发明所述的通式(I)所示的化合物或者包含其的药物组合物，其用作预防和/或治疗癌症的药物，所述癌症包括但不限于，非实体瘤如白血病，实体瘤如皮肤癌、黑色素瘤、肺癌、胃癌、乳腺癌、胰腺癌、肝癌、结肠癌。
具体实施方式
除非另有规定，本文使用的所有技术和科学术语具有与本领域技术人员的通常理解相同含义。所有专利、申请、公开的申请和其他出版物均以全部内容并入作为参考。倘若对于本文使用的术语有多个定义，除非另有说明，以本节中的为准。如果任何给定取代基的数量没有规定，则可以存在一个或多个取代基。例如“卤代烷基”可以含有一个或多个相同或不同的卤素。在本文的描述中，如果化学结构和化学名称彼此矛盾时，则是以其化学结构为准。当在本文使用时，对于任何保护基团、氨基酸和其他化合物的缩写，除非另有说明，以其常用的公认缩写表示，或根据IUPAC-IUB Commission on Biochemical Nomenclature表示(参见Biochem.1972,77:942-944)。
除非有相反陈述，否则下列用在说明书和权利要求书中的术语具有下述含义。
术语“烷基”指饱和的脂族烃基团，包括1至20个碳原子的直链和支链基团。其包括碳数1～18、优选碳数1～10、更优选碳数1～6、甚至更优选碳数1～4的直链或支链烷基。非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、新戊基、己基、异己基、正庚基、异庚基、正辛基、异辛基、正壬基、正癸基等。本说明书中，“烷基”还包括碳数3～10、优选碳数3～8、更优选碳数4～6的环状烷基，例如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基、环癸基、十氢萘基、降冰片烷、金刚烷基。烷基可以是取代的或未取代的，当被取代时，取代基可以在任何可使用 的连接点上被取代，优选为一个或多个以下基团，独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚戊基(-CH 2CH 2CH 2CH 2CH 2-)等。
术语“烯基”指由碳和氢原子组成的含有至少一个双键的直链或支链的烃链基团，并通过单键或双键与分子的其余部分连接。优选具有2～10个碳原子，更优选具有2～6个碳原子，甚至更优选具有2～4个碳原子。非限制性实施例包括乙烯基、丙烯基、丁烯基、戊烯基、戊二烯基、己烯基。烯基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
术语“亚烯基”指由碳和氢原子组成的含有至少一个双键的直链或支链的烃链基团，其具有2个从母体的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其通过单键或双键与分子的其余部分连接。优选具有2～10个碳原子，更优选具有2～6个碳原子，甚至更优选具有2～4个碳原子。非限制性实施例包括亚乙烯基、亚丙烯基、亚丁烯基、亚戊烯基、亚己烯基等。
术语“炔基”指由碳原子和氢原子组成的含有至少一个三键的直链或支链的烃链基团，并通过单键或三键与分子的其余部分连接。优选具有2～10个碳原子，更优选具有2～6个碳原子，甚至更优选具有2～4个碳原子。非限制性实施例包括乙炔基、丙炔基、丁炔基、戊炔基、己炔基。炔基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
术语“亚炔基”指由碳原子和氢原子组成的含有至少一个三键的直链或支链的烃链基团，其具有2个从母体的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其通过单键或三键与分子的其余部分连接。优选具有2～10 个碳原子，更优选具有2～6个碳原子，甚至更优选具有2～4个碳原子。非限制性实施例包括亚乙炔基、亚丙炔基、亚丁炔基、亚戊炔基、亚己炔基等。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，其包括3至20个碳原子，优选包括3至12个碳原子，更优选环烷基环包含3至10个碳原子，最优选环烷基环包含3至7个碳原子。单环环烷基的非限制性实施例包含环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等，优选环丙基、环己烯基。多环环烷基包括螺环、稠环和桥环的环烷基。环烷基可以是任选取代的或未取代的，当被取代时，取代基优选为一个或多个以下基团，独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包括3至20个环原子，其中一个或多个环原子选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包括3至12个环原子，其中1～4个是杂原子，更优选杂环基环包含3至10个环原子，更优选杂环基环包含5至7个环原子。单环杂环基的非限制性实施例包含吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基、吡喃基、四氢呋喃基、氮杂环庚烷基等。多环杂环基包括螺环、稠环和桥环的杂环基。杂环基可以是任选取代的或未取代的，当被取代时，取代基优选为一个或多个以下基团，独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
术语“芳基”指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为5至10元，更优选5至7元，甚至更优选苯基和萘基，最优选苯基。芳基可以是完全芳香族的基团，例如苯基、萘基、蒽基、菲基等。芳基也可以含有芳香环与非芳香环的组合，例如，茚、芴和苊等。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，非限制性实施例包含：
芳基可以是取代的或未取代的，当被取代时，取代基优选为一个或多个以下基团，独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、 羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选5至7元，甚至更优选为5元或6元，例如噻二唑基、吡唑基、噁唑基、噁二唑基、咪唑基、***基、噻唑基、呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2019073874-appb-000011
杂芳基可以是任选取代的或未取代的，当被取代时，取代基优选为一个或多个以下基团，独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
“烷氧基”指-O-(烷基)和-O-(未取代的环烷基)，其中烷基、环烷基的定义如上所述。非限定性实例包括甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基等。烷氧基可以是任选取代的或未取代的，当被取代时，取代基优选为一个或多个以下基团，独立地选自为烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、硫醇、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基或羧酸酯基。
术语“卤代烷基”指其中一个或多个氢原子被卤素取代的烷基，其中烷基的定义如上所述。非限定性实例包括氯甲基、三氟甲基、1-氯-2-氟乙基、2,2-二氟乙基、2-氟丙基、2-氟丙-2-基、2,2,2-三氟乙基、1,1-二氟乙基、1,3-二氟-2-甲基丙基、2,2-二氟环丙基、(三氟甲基)环丙基、4,4-二氟环己基和2,2,2-三氟-1,1-二甲基-乙基。
术语“卤代烷氧基”指其中一个或多个氢原子被卤素取代的烷氧基，其中烷氧基的定义如上所述。
术语“卤素”包括氟、氯、溴和碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
术语“氰基”指-CN。
术语“羟基”指-OH基团。
术语“羟烷基”指被羟基取代的烷基，其中烷基的定义如上所述。
术语“羟烷氧基”指被羟基取代的烷氧基，其中烷氧基的定义如上所述。
术语“酰基”指-C(O)R，其中R指烷基、环烷基、烯基、炔基，其中烷基、环烷基、烯基、炔基的定义如上所述。非限定性实例包括乙酰基、丙酰基、丁酰基、戊酰基、己酰基、乙烯酰基、丙烯酰基。
术语“酰氨基”指-NHC(O)R或-C(O)NH 2，其中R指烷基、烯基、炔基，其中烷基、烯基、炔基的定义如上所述。非限定性实例包括甲酰氨基、乙酰氨基、丙酰氨基、丁酰氨基、戊酰氨基、己酰氨基、乙烯酰氨基、丙烯酰氨基。
术语“酯基”指-C(O)OR，其中R指烷基或环烷基，其中烷基、环烷基的定义如上所述。非限定性实例包括乙酯基、丙酯基、丁酯基、戊酯基、环丙酯基、环丁酯基、环戊酯基、环己酯基。
本说明书中的“任选取代”是指未取代或被一个或多个(例如2、3、4个)取代基取代。其中取代基选自下组：卤素原子、烷基、烯基、炔基、卤代烷基、烷氧基、芳基、卤代芳基、芳氧基、芳烷基、芳烷基氧基、杂环基烷氧基、卤代芳基烷基氧基、烷基氨基、烷基酰基、氰基、或杂环基等。这些取代基还可以进一步被取代。例如，作为取代基的烷基还任选被选自卤素原子、羟基、烷氧基、烷基氨基、吡咯烷基、苯基、吡啶基、或卤代苯基中的一个或多个基团取代。作为取代基的杂环基还任选被选自卤素原子、烷基、烷氧基中的一个或多个基团取代。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“联合用药”(Drug Combination)是指为了达到治疗目的而采用的两种或两种以上药物同时或先后应用。
本发明通式(I)所示的化合物的制备方法。
为了完成本发明化合物的目的，本发明主要采用如下合成路线与技术方案。
Figure PCTCN2019073874-appb-000012
本发明化合物的合成主要分为三部分：
第一部分：中间体M3的合成
Figure PCTCN2019073874-appb-000013
将吲哚类中间体M1和嘧啶类中间体M2，在适当的温度和碱的存在下，在催化剂催化下，在适当的溶剂中通过取代反应得到中间体化合物M3；所述碱可以为例如氢化钠、碳酸钾、碳酸铯等，所述溶剂可以为例如DMF、NMP等，所述催化剂可以为例如1-羟基苯并***(HOBT)。
取代的嘧啶类中间体M2通常为商购可得。
第二部分：苯胺类中间体M4的合成
可以通过以下方案2或方案3进行合成。
Figure PCTCN2019073874-appb-000014
步骤1：苯胺类原料M5和羧酸类原料M6在适当的温度和碱性条件下，在缩合剂催化下，在适当的溶剂中通过缩合反应得到中间体M7；所述碱可以为例如三乙胺、N-甲基吗啉等，所述溶剂可以为例如DCM、THF等，所述缩合剂可以为例如HATU、HBTU、TBTU等；
步骤2：将中间体M7的硝基还原为氨基，得到中间体M8；硝基的还原可以在例如铁粉氯化铵体系或H 2/钯碳体系下实现；
步骤3：羧酸类原料M9在适当的溶剂中，在适合的氯化试剂和催化剂的作用下，反应生成酰氯，所述溶剂可以为例如四氢呋喃、二氯甲烷等，所述氯化试剂可以为例如草酰氯、氯化亚砜、三氯氧磷等，所述催化剂可以为例如DMF等；
然后，将得到的酰氯与中间体M8在适当的温度和碱性条件下在适当的溶剂中通过亲核取代反应生成中间体M10，所述碱可以为例如三乙胺、吡啶、N-甲基吗啉等，所述溶剂可以为例如DCM、THF等；
步骤4：中间体M10在酸性条件下，在适当的溶剂中脱掉保护基，得到中间体M4，所述酸性条件可以为例如三氟乙酸、盐酸乙酸乙酯等，所述溶剂可以为例如DCM、乙酸乙酯等。
Figure PCTCN2019073874-appb-000015
步骤1：将中间体羧酸M11在适当的溶剂中，在适合的氯化试剂和催化剂的作用下，反应生成酰氯，所述溶剂可以为例如四氢呋喃、二氯甲烷等，所述氯化试剂可以为例如草酰氯、氯化亚砜、三氯氧磷等，所述催化剂可以为例如DMF等；将得到的酰氯与中间体M8在适当的温度和碱性条件下，在适当的溶剂中，通过亲核取代反应生成中间体M12，所述碱可以为例如三乙胺、吡啶、N-甲基吗啉等，所述溶剂可以为例如DCM、THF、DMF等；
步骤2：中间体M12在适当的温度和碱性条件下，在适当的溶剂中通过取代反应得到中间产物M10；所述碱可以为例如碳酸钾、碳酸铯等，所述溶剂可以为例如DMF、NMP等；
步骤3：中间体M10在酸性条件下，在适当的溶剂中脱掉保护基，生成中间体M4，所述酸性条件可以为例如三氟乙酸、盐酸乙酸乙酯等，所述溶剂可以为例如DCM、乙酸乙酯等。
第三部分：通过吲哚类中间体M3和苯胺类中间体M4合成通式(I)的化合物
Figure PCTCN2019073874-appb-000016
将中间体M3和苯胺类中间体M4在适宜的温度和适当的溶剂中，在酸催化下反应得通式(I)的化合物；
所述溶剂可以为例如异丙醇、异戊醇、仲戊醇、二氧六环等，所述酸可以为例如盐酸、硫酸、甲磺酸、对甲苯磺酸、苯磺酸等。
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、n、m、p、R u、R z、R y如通式(I)中所定义。
本发明通式(I)所示的化合物在药学上可接受的盐，可以为酸加成盐或碱加成盐。酸可以为无机酸，包括但不限于：盐酸、硫酸、磷酸、氢溴酸；或可以为有机酸，包括但不限于：柠檬酸、马来酸、草酸，甲酸、乙酸、丙酸、戊酸、乙醇酸、苯甲酸、富马酸、三氟乙酸、琥珀酸、酒石酸、乳酸、谷氨酸、天门冬氨酸、水杨酸、丙酮酸、甲磺酸、苯磺酸、对苯磺酸。碱可以为无机碱，包括但不限于：氢氧化钠、氢氧化钾、氢氧化镁、氢氧化钙；或可以为有机碱，包括但不限于：氢氧化铵、三乙胺、N,N-二苄基乙二胺、氯普鲁卡因、胆碱、氨、二乙醇胺和其他羟基烷基胺、乙二胺、N-甲基葡糖胺、普鲁卡因、N-苄基苯乙胺、精氨酸或赖氨酸；或可以为碱金属盐，包括但不限于：锂、钾和钠盐；或可以为碱土金属盐，包括但不限于：钡、钙和镁盐；或可以为过渡金属盐，包括但不限于锌盐；或其他金属盐，包括但不限于：磷酸氢钠和磷酸氢二钠。
本发明另一方面将通式(I)所示的化合物或药学上可接受的盐制备成临床上可使用的药用组合物。根据临床适应症，给药途径与方式，其药用制剂包括但不限于口服制剂如片剂、凝胶剂、软/硬胶囊、乳剂、分散性粉剂、颗粒剂、水/油悬乳剂；注射剂包括静脉注射剂、肌肉注射剂、腹腔注射剂、直肠给药栓剂、颅内注射剂，这些剂型可为水溶液也可为油类溶液；局部制剂包括霜剂、软膏剂、凝胶剂、水/油溶液以及包合物制剂；吸入剂型包括细粉、液体气溶胶以及适合于体内植入的各种剂型。
本发明的药物组合物可以根据需要加入药学上可接受的载体、稀释剂或赋形剂。这些载体、稀释剂或赋形剂应符合药物制剂制备工艺规则，与活性成分相兼容。固体口服制剂的载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖、环糊精以及促进肠吸收分子载体维生素E-PEG1000。口服制剂可加入适当的着色剂、甜味剂、矫味剂及防腐剂。
本发明通式(I)所示的化合物或药学上可接受的盐，按0.01-100mg/kg单位剂量给予温血动物。
本发明通式(I)所示的化合物或药学上可接受的盐，在上述癌症治疗中，可单独使用，或与临床上常规使用的放射疗法、化学疗法、免疫疗法、肿瘤疫苗、融瘤病毒、RNAi、癌症辅助治疗以及骨髓移植和干细胞移植的一个或多个方法联合治疗，其中包括但不限于以下抗肿瘤类药物和治疗方法：
1)烷化剂如顺铂、顺铂、奥沙利铂、苯丁酸氮芥、卡环磷酰胺，氮芥、美法仑、替莫唑胺、白消安、亚硝基脲类。
2)抗肿瘤抗生素类如阿霉素、博来霉素、多柔比星、道诺霉素、表柔比星、伊达比星、丝裂霉素C、放线菌素、光神霉素；抗有丝***药如长春新碱、长春碱、长春地辛、长春瑞滨、紫杉醇、泰索帝、Polo激酶抑制剂。
3)抗代谢和抗叶酸剂如氟嘧啶、雷甲氨蝶呤、阿糖胞苷、阿扎胞苷、地西他滨、替曲塞、羟基脲、IDH1/IDH2突变株抑制剂。
4)拓扑异构酶抑制剂如表鬼臼毒素、喜树碱、伊立替康。
5)细胞生长抑制剂如抗***/抗雄激素类药物，如他莫昔芬、氟维司群、托瑞米芬、雷诺昔芬、屈诺昔芬、碘昔芬、比卡鲁胺、氟他胺、尼鲁米特、醋酸环丙孕酮；
LHRH拮抗剂或LHRH激动剂如戈舍瑞林、亮丙瑞林、和布舍瑞林、孕激素类如醋酸甲地孕酮；
芳香酶抑制剂如阿那曲唑、来曲唑、伏罗唑、伊西美坦、5a-还原酶抑制剂如非那雄胺。
6)抗侵袭剂如c-Src激酶家族抑制剂、金属蛋白酶抑制剂、尿激酶纤溶酶原激活物受体功能的抑制剂或者类肝素酶的抗体。
7)生长功能的抑制剂如生长因子抗体和生长因子受体抗体如抗HER2抗体曲妥珠单抗、抗EGFR抗体帕尼单抗、抗EGFR抗体西妥昔单抗等；这种抑制剂还包括其它酪氨酸激酶抑制剂以及丝氨酸/苏氨酸激酶的抑制剂如Ras/Raf信号传导抑制剂，MEK和/或AKT激酶的细胞信号传导抑制剂、c-kit抑制剂、abl激酶抑制剂、PI3激酶抑制剂、JAKs和STAT3抑制剂、FLT3激酶抑制剂、CSF-1R激酶抑制剂、IGF受体激酶抑制剂，极光激酶抑制剂，NTRKA/B/C激酶抑制剂。
8)抗血管生成剂如抑制血管内皮生长因子作用的药剂贝伐珠单抗以及VEGF受体酪氨酸激酶抑制剂。
9)表观遗传学(epigenetics)抑制剂如组蛋白去乙酰化酶抑制剂(HDACi)、DNA甲基转移酶抑制剂(DNMTi)、组蛋白乙酰转移酶抑制剂、组蛋白去甲基化酶抑制剂、组蛋白甲基转移酶抑制剂等。
10)聚腺苷二磷酸核糖聚合酶抑制剂(PARPi)如奥拉帕利(Olaparib)、瑞卡帕尼(Rucaparib)和尼拉帕尼(Niraparib)。
11)肿瘤免疫治疗法包括任何提高患者肿瘤细胞的免疫原性的体外和体内方法。如细胞因子IL-2、IL-4或者GM-CSF进行转染；降低T细胞无效能的方法如抗PD-1/PD-L单抗；使用转染的免疫细胞如细胞因子转染的树突状细胞的方法；使用细胞因子转染的肿瘤细胞系的方法；降低免疫抑制性细胞如调节性T细胞、髓源性抑制细胞、或表达吲哚胺2,3-脱氧酶的树突状细胞的功能方法；提高免疫细胞活性的激动剂如STING以及肿瘤相关抗原蛋白类或肽类组成的癌症疫苗的方法。
12)嵌合抗原受体T细胞免疫疗(CART)。
13)肿瘤基因治疗如CRISPR-Cas 9、RNAi、基因转导。
实施例
以下结合实施例进一步描述本发明，但这些实施例并非限制本发明的范围。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的单位给出。NMR的测定是用(Bruker AVANCE-400)核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)。
MS的测定用液相色谱质谱联用仪(Thermo，Ultimate3000/MSQ)。
HPLC的测定使用高压液相色谱仪(安捷伦1260 Infinity，Gemini C18 250×4.6mm，5u色谱柱)。
薄层色谱法(TLC)使用的硅胶板HSGF245采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.9mm～1.0mm(烟台黄海)。
柱层析色谱法一般使用200～300目硅胶为载体(烟台黄海硅胶)。
本发明的已知起始原料可以采用或按照本领域已知的方法来合成，或购买自上海达瑞精细化学品有限公司、上海泰坦科技股份有限公司、上海润捷化学试剂有限公司、TCI、Aldrich Chemical Company。实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例1
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-[3-(4-吲哚-1-基-嘧啶-2-基氨基)-苯基]-苯甲酰胺(化合物1)的制备
Figure PCTCN2019073874-appb-000017
步骤1：1-(2-氯-嘧啶-4-基)-1H-吲哚的制备
将2,4-二氯嘧啶(7.15g，0.048mol)溶解于DMF(80ml)中，于室温加入HOBT(1.0g,8mmol)和碳酸钾(11g，0.08mol)，继续室温搅拌15分钟。将吲哚(4.68g，0.04mol)(溶解于DMF中)慢慢滴加到反应体系中，滴毕升温85℃反应6小时，TLC检测反应完全。降温到室温，将反应液倒入水(200ml)中，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：石油醚/乙酸乙酯)纯化，得到5.3g黄色固体状的1-(2-氯-嘧啶-4-基)-1H-吲哚。
步骤2：(3-硝基-苯基)-氨基甲酸叔丁酯的制备
将间硝基苯胺(9.66g，0.07mol)和二碳酸二叔丁酯(46g，0.21mol)溶解于150mlTHF中，于室温依次加入碳酸钾(19.5g，0.14mol)和DMAP(4.27g，0.035mol)，加毕升温60℃反应12小时。TLC检测反应完全。降温到室温，将反应液倒入水(600ml)中，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，得到16.6g粗品(3-硝基-苯基)-氨基甲酸叔丁酯，无需纯化，直接用于下一步反应。
步骤3：(3-氨基-苯基)-氨基甲酸叔丁酯的制备
将步骤2中所得产物(3-硝基-苯基)-氨基甲酸叔丁酯(16.6g，0.07mol)、还原铁粉(15.68g，0.28mol)、氯化铵(26.2g，0.49mol)加入乙醇(120ml)/水(40ml)中，并将得到的混合物加热至90℃，反应2h。将反应液冷却至室温后，缓慢倒入饱和碳酸氢钠水溶液中(300ml)，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：石油醚/乙酸乙酯)纯化，得到13.5g黄色固体状的(3-氨基-苯基)-氨基甲酸叔丁酯。
步骤4：[3-(3-硝基-苯甲酰氨基)-苯基]-氨基甲酸叔丁酯的制备
步骤3得到的(3-氨基-苯基)-氨基甲酸叔丁酯(9g，0.0433mol)、间硝基苯甲酸(7.23g，0.0433mmol)、HATU(24.66g，0.0649mol)和N-甲基吗啉(13.11g，0.13mol)溶解200ml二氯甲烷中，30℃反应6小时。TLC检测反应完全，将反应液冷却至室温后，缓慢倒入水中(300ml)，用二氯甲烷萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：石油醚/乙酸乙酯)纯化，得到15.2g粗品[3-(3-硝基-苯甲酰氨基)-苯基]-氨基甲酸叔丁酯。
步骤5：[3-(3-氨基-苯甲酰氨基)-苯基]-氨基甲酸叔丁酯的制备
将步骤4中所得产物[3-(3-硝基-苯甲酰氨基)-苯基]-氨基甲酸叔丁酯(15.2g，0.043mol)、还原铁粉(9.7g，0.17mol)、氯化铵(16.2g，0.303mol)加入乙醇(100ml)/水(30ml)中，并将得到的混合物加热至90℃，反应2h。将反应液冷却至室温后，缓慢倒入饱和碳酸氢钠水溶液中(300ml)，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，得到15.5g黄色固体状的[3-(3-氨基-苯甲酰氨基)-苯基]-氨基甲酸叔丁酯，无需纯化，直接用于下一步反应。
步骤6：(E)-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯的制备
在氮气氛下，将反式-4-二甲基氨基巴豆酸盐酸盐(6.1g，0.0367mol)加入到250ml三口瓶中，氮气球换气三次。用注射器加入超干(无水)THF(150ml)和6滴DMF，冰水浴降温到0-5℃。向体系中慢慢滴加草酰氯(3.9g，0.0306mol)，滴毕，移走冰水浴，升温至室温反应2小时。在氮气氛下，于室温，将步骤5中得到的[3-(3-氨基-苯甲酰氨基)-苯基]-氨基甲酸叔丁酯(10g，0.0306mol)(溶解于80ml超干THF中)慢慢滴加到反应体系中，滴毕，继续室温反应1小时。TLC检测仍有原料剩余，将反应液缓慢倒入饱和碳酸氢钠水溶液中(300ml)，用1mol/L稀氢氧化钠水溶液调节PH在10左右，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：二氯甲烷/甲醇)纯化，得到5.8g粗品(E)-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯。
步骤7：(E)-N-(3-氨基-苯基)-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺的制备
将步骤6中所得产物(E)-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯(5.8g，0.0132mol)溶解于二氯甲烷(60ml)中，于室温加入三氟乙酸(20ml)，加毕继续室温反应12小时。TLC检测反应完全，将反应液减压浓缩，残余物缓慢加入150ml水溶解，用乙酸乙酯萃取(50ml×2)，有机相弃去，水相用1mol/L稀氢氧化钠水溶液调节PH至9-10，用乙酸乙酯萃取(60ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，得到3g黄色固体状的(E)-N-(3-氨基-苯基)-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺，无需纯化，直接用于下一步反应。
步骤8：(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-[3-(4-吲哚-1-基-嘧啶-2-基氨基)-苯基]-苯甲酰胺的制备
将步骤1中所得产物1-(2-氯-嘧啶-4-基)-1H-吲哚(200mg，0.873mmol)、步骤7中所得产物(E)-N-(3-氨基-苯基)-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(246mg，0.728mmol)和甲磺酸(209mg，2.18mmol)分散于异丙醇(20ml)中，将反应体系加热至85℃反应12小时。TLC检测原料仍有剩余，将反应液冷却到室温，过滤，固体用少量乙酸乙酯洗涤。所得固体加入二氯甲烷/甲醇(50ml/5ml)溶解，加入0.2mol/L稀氢氧化钠水溶液20ml调节PH至9-10，用二氯甲烷萃取(30ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：二氯甲烷/甲醇)纯化，得到58mg白色固体状的(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-[3-(4-吲哚-1-基-嘧啶-2-基氨基)-苯基]-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.38(s,1H),10.28(s,1H),9.78(s,1H),8.79-8.81(d,1H),8.49-8.50(d,1H),8.25(s,1H),8.17-8.18(m,2H),7.91-7.93(d,1H),7.62-7.66(m,2H),7.45-7.53(m,2H),7.38-7.40(m,1H),7.17-7.33(m,4H),6.76-6.83(m,2H),6.35-6.39(d,1H),3.25(m,2H),2.37(s,6H)。
LC-MS(ESI):532.2(M+H) +。
实施例2
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物2)的制备
Figure PCTCN2019073874-appb-000018
与实施例1中的制备方法相同，除了用4-甲氧基吲哚(TCI)代替实施例1步骤1中的吲哚，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.30(s,1H),10.29(s,1H),9.80(s,1H),8.49-8.50(d,1H),8.36-8.38(d,1H),8.25(s,1H),8.18(s,1H),8.06-8.07(d,1H),7.90-7.92(d,1H),7.63-7.65(d,1H),7.45-7.52(m,2H),7.37-7.39(m,1H),7.31-7.33(m,1H),7.16-7.24(m,2H),6.73-6.81(m,3H),6.29-6.33(d,1H),3.89(s,3H),3.07-3.08(d,2H),2.19(s,6H)。
LC-MS(ESI):562.2(M+H) +。
实施例3
(E)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-吗啉-4-基-丁-2-烯酰氨基)苯甲酰胺(化合物3)的制备
Figure PCTCN2019073874-appb-000019
步骤1：4-溴代巴豆酸的制备
在氮气氛下，将(E)-4-溴-2-丁烯酸甲酯(3g，0.0168mol)溶解于30mlTHF中，冰水浴冷却至0-5℃，慢慢滴加氢氧化锂水溶液10ml(960mg氢氧化锂)，滴毕，保持温度在0-5℃反应3小时。TLC检测反应完全，加入50ml冰水，石油醚萃取(100ml×2)，有机相弃去。水相降温到0℃左右，用浓盐酸调节PH至1左右，二氯甲烷萃取(70ml×3)，无水硫酸钠干燥，过滤，减压浓缩，得1.35g固体状的4-溴代巴豆酸。
步骤2：4-溴代巴豆酰氯的制备
在氮气氛下，将4-溴代巴豆酸(1.35g，8.23mmol)溶解于20ml二氯甲烷中，于室温加入3滴DMF，冰水浴冷却至0-5℃，慢慢滴加草酰氯(2g，15.7mmol)，滴毕，升温至室温反应1小时。将反应液减压浓缩，得1.5g黑色油状的4-溴代巴豆酰氯。
步骤3：(E)-{3-[3-(4-溴-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯的制备
在氮气氛下，将-[3-(3-氨基-苯甲酰氨基)-苯基]-氨基甲酸叔丁酯(570mg，1.743mmol)(实施例1步骤5中制备)溶解于20m二氯甲烷中，冰水浴冷却至0-5℃，慢慢滴加4-溴代巴豆酰氯(381mg，2.1mmol，溶解于10mlDCM中)，滴毕，升温至室温反应0.5小时。TLC检测反应完全，加入50ml冰水，二氯甲烷萃取(50ml×3)，无水硫酸钠干燥，过滤，减压浓缩，得900mg固体状的(E)-{3-[3-(4-溴-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯。无需纯化，直接用于下一步反应。
步骤4：(E)-{3-[3-(4-吗啉-4-基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯的制备
将步骤3中得到的(E)-{3-[3-(4-溴-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯(900mg，1.743mmol，理论收率)和吗啉(303mg，3.49mmol)溶解于20mDMF中，于室温加入碳酸钾(721mg，5.23mmol)，将反应体系加热至60℃反应1小时。TLC检测反应完全，降温至室温，将反应液倒入水(100ml)中，用乙酸乙酯萃取(50ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：二氯甲烷/甲醇)纯化，得到490mg黄色固体状的(E)-{3-[3-(4-吗啉-4-基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯。
步骤5：(E)-N-(3-氨基-苯基)-3-(4-吗啉-4-基-丁-2-烯酰氨基)-苯甲酰胺的制备
将步骤4中得到的(E)-{3-[3-(4-吗啉-4-基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基}-氨基甲酸叔丁酯(490mg，1.02mmol)溶解于20m乙酸乙酯中，于室温加入盐酸乙酸乙酯(20ml，2mol/L)，加毕，继续室温反应2小时，TLC检测反应完全，将反应液缓慢倒入饱和碳酸氢钠水溶液中(50ml)，用1mol/L稀氢氧化钠水溶液调节PH至10左右，用乙酸乙酯萃取(50ml×3)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：二氯甲烷/甲醇)纯化，得到120mg黄色固体状的(E)-N-(3-氨基-苯基)-3-(4-吗啉-4-基-丁-2-烯酰氨基)-苯甲酰胺。
步骤6：(E)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-吗啉-4-基-丁-2-烯酰氨基)-苯甲酰胺的制备
将步骤5中所得产物(E)-N-(3-氨基-苯基)-3-(4-吗啉-4-基-丁-2-烯酰氨基)-苯甲酰胺(120mg，0.316mmol)、1-(2-氯-嘧啶-4-基)-4-甲氧基-1H-吲哚(123mg，0.474mmol)(实施例2中制备)和对甲苯磺酸(65mg，0.379mmol)溶解于异戊醇(10ml)中，将反应体系加热至120℃反应12小时。TLC检测原料仍有剩余，将反应液冷却至室温，加入20ml甲基叔丁基醚，过滤，固体用少量甲基叔丁基醚洗涤。所得固体加入二氯甲烷/甲醇(50ml/5ml)溶解，加入0.2mol/L稀氢氧化钠水溶液20ml调节PH至9-10，用二氯甲烷萃取(30ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：二氯甲烷/甲醇)纯化，得到15mg白色固体状的(E)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-吗啉-4-基-丁-2-烯酰氨基)苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.38(br,1H),10.30(s,1H),9.80(s,1H),8.49-8.50(d,1H),8.35-8.39(m,1H),8.25(s,1H),8.18(s,1H),8.06-8.07(d,1H),7.92-7.94(m,1H),7.64-7.66(d,1H),7.51-7.53(d,1H),7.45-7.49(t,1H),7.38-7.40(d,1H),7.28-7.32(t,1H),7.23-7.24(d,1H),7.16-7.20(t,1H),6.73-6.81(m,3H),6.34-6.38(d,1H),3.89(s,3H),3.63(m,4H),3.16(m,2H),2.42(m,4H)。
LC-MS(ESI):604.2(M+H) +。
实施例4
(E)-4-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物4)的制备
Figure PCTCN2019073874-appb-000020
与实施例2中的制备方法相同，除了用对硝基苯甲酸(TCI)代替实施例1步骤4中的间硝基苯甲酸，得到(E)-4-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.35(s,1H),10.13(s,1H),9.77(s,1H),8.49-8.51(d,1H),8.36-8.38(m,1H),8.24(s,1H),8.06-8.07(d,1H),7.94-7.96(d,2H),7.79-7.81(d,2H),7.49-7.51(m,1H),7.39-7.41(m,1H),7.28-7.32(t,1H),7.22-7.23(d,1H),7.15-7.20(t,1H),6.73-6.83(m,3H),6.30-6.34(d,1H),3.90(s,3H),3.10-3.11(d,2H),2.21(s,6H)。
LC-MS(ESI):562.2(M+H) +。
实施例5
(E)-4-(4-二甲基氨基-丁-2-烯酰氨基)-N-[3-(4-{4-[2-(6-甲基-吡啶-3-基氧基)-乙氧基]-吲哚-1-基}-嘧啶-2-基氨基)-苯基]-苯甲酰胺(化合物5)的制备
Figure PCTCN2019073874-appb-000021
步骤1：4-(2-氯-乙氧基)-1H-吲哚的制备
将4-羟基吲哚(4g，0.03mol)和1-溴-2-氯乙烷(6.45g，0.045mol)溶解于2-丁酮(60ml)中，于室温加入碳酸铯(20g，0.0615mol)，并将反应体系加热至90℃反应16小时。TLC检测仍有原料剩余，降温至室温，将反应液缓慢倒入200ml水中，用乙酸乙酯萃取(100ml×3)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：二氯甲烷/甲醇)纯化，得到4-(2-氯-乙氧基)-1H-吲哚2.1g。
步骤2：4-[2-(6-甲基-吡啶-3-基氧基)-乙氧基]-1H-吲哚的制备
将步骤1得到的产物4-(2-氯-乙氧基)-1H-吲哚(2.1g，0.01mol)和3-羟基-6-甲基吡啶(1.4g，0.013mol)溶解于DMF(30ml)中，于室温加入碳酸铯(6.5g，0.02mol)，并将反应体系加热至90℃反应2小时。TLC检测反应完全，降温至室温，将反应液缓慢倒入100ml水，用乙酸乙酯萃取(60ml×3)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，得到1.5g黄色固体状的4-[2-(6-甲基-吡啶-3-基氧基)-乙氧基]-1H-吲哚。
步骤3：(E)-4-(4-二甲基氨基-丁-2-烯酰氨基)-N-[3-(4-{4-[2-(6-甲基-吡啶-3-基氧基)-乙氧基]-吲哚-1-基}-嘧啶-2-基氨基)-苯基]-苯甲酰胺的制备
与实施例1中的制备方法相同，除了用4-[2-(6-甲基-吡啶-3-基氧基)-乙氧基]-1H-吲哚代替实施例1步骤1中的吲哚，并用对硝基苯甲酸代替实施例1步骤4中的间硝基苯甲酸，得到(E)-4-(4-二甲基氨基-丁-2-烯酰氨基)-N-[3-(4-{4-[2-(6-甲基-吡啶-3-基氧基)-乙氧基]-吲哚-1-基}-嘧啶-2-基氨基)-苯基]-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.41(s,1H),10.16(s,1H),9.79(s,1H),8.49-8.50(d,1H),8.40-8.42(d,1H),8.24(m,2H),8.06-8.07(d,1H),7.94-7.96(d,2H),7.79-7.81(d,2H),7.49- 7.51(d,1H),7.38-7.40(m,2H),7.28-7.32(m,1H),7.16-7.23(m,3H),6.73-6.81(m,3H),6.31-6.35(d,1H),4.45(s,4H),3.14-3.15(d,2H),2.41(s,3H),2.23(s,6H)。
LC-MS(ESI):683.2(M+H) +。
实施例6
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-乙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物6)的制备
Figure PCTCN2019073874-appb-000022
与实施例1的制备方法相同，除了用4-乙氧基吲哚(TCI)代替实施例1步骤1中的吲哚，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-乙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.26(s,1H),10.26(s,1H),9.76(s,1H),8.48-8.49(d,1H),8.33-8.35(d,1H),8.237(s,1H),8.16(s,1H),8.04-8.05(d,1H),7.89-7.91(d,1H),7.62-7.63(d,1H),7.44-7.51(m,2H),7.36-7.38(m,1H),7.27-7.31(t,1H),7.21-7.22(d,1H),7.13-7.17(t,1H),6.71-6.81(m,3H),6.28-6.31(d,1H),4.13-4.18(q,2H),3.06-3.08(d,2H),2.18(s,6H),1.38-1.42(t,3H)。
LC-MS(ESI):576.3(M+H) +。
实施例7
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-异丙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物7)的制备
Figure PCTCN2019073874-appb-000023
步骤1：4-异丙氧基吲哚的制备
将4-羟基吲哚(5g，0.0376mol)、2-溴异丙烷(5.5g，0.045mol)和碳酸钾(10.4g，0.075mol)混合于30mL DMF中，加入催化量的KI，80℃油浴反应1小时，TLC检测反应完全。降温至室温，将反应液倒入水(100ml)中，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，得黑色油状物4.9g，无需纯化，直接用于下一步反应。
步骤2：(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-异丙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺的制备
与实施例1中的制备方法相同，除了用4-异丙氧基吲哚代替实施例1步骤1中的吲哚，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-异丙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.41(s,1H),10.28(s,1H),9.79(s,1H),8.49-8.50(d,1H),8.32-8.35(d,1H),8.25(s,1H),8.20(s,1H),8.04-8.05(d,1H),7.92-7.94(d,1H),7.64-7.66(d,1H),7.45-7.52(m,2H),7.38-7.40(m,1H),7.29-7.33(t,1H),7.21-7.22(d,1H),7.13-7.17(t,1H),7.73-7.83(m,3H),6.36-6.39(d,1H),4.68-4.74(m,1H),3.28-3.29(d,2H),2.33(s,6H),1.32-1.34(d,6H)。
LC-MS(ESI):590.2(M+H) +。
实施例8
(E)-N-{3-[4-(5,6-二甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物8)的制备
Figure PCTCN2019073874-appb-000024
与实施例1的制备方法相同，除了用5,6-二甲氧基吲哚(TCI)代替实施例1步骤1中的吲哚，得到(E)-N-{3-[4-(5,6-二甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.46(s,1H),10.30(s,1H),9.76(s,1H),8.47-8.48(d,1H),8.24(s,1H),8.17(s,1H),8.15(s,1H),7.96-7.97(d,1H),7.92-7.94(m,1H),7.62-7.64(d,1H),7.52-7.54(d,1H),7.42-7.48(m,2H),7.27-7.31(t,1H),7.17-7.19(d,1H),7.14(s,1H),6.76-6.83(m,1H),6.69-6.70(d,1H),6.37-6.41(d,1H),3.79(s,3H),3.69(s,3H),3.36-3.37(m,2H),2.38s,6H)。
LC-MS(ESI):592.3(M+H) +。
实施例9
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-(3-{4-[4-(3-甲氧基-丙氧基)-吲哚-1-基]-嘧啶-2-基氨基}-苯基)-苯甲酰胺(化合物9)的制备
Figure PCTCN2019073874-appb-000025
与实施例7的制备方法相同，除了用1-溴-3-甲氧基丙烷(达瑞)代替实施例7步骤1中的2-溴异丙烷，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-(3-{4-[4-(3-甲氧基-丙氧基)-吲哚-1-基]-嘧啶-2-基氨基}-苯基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.30(s,1H),10.28(s,1H),9.79(s,1H),8.49-8.50(d,1H),8.35-8.37(d,1H),8.25(s,1H),8.18(s,1H),8.06-8.07(d,1H),7.91-7.93(d,1H),7.63-7.65(d,1H),7.51-7.53(d,1H),7.45-7.49(t,1H),7.38-7.40(d,1H),7.28-7.32(t,1H),7.22-7.23(d,1H),7.14-7.18(t,1H),6.72-6.82(m,3H),6.30-6.33(d,1H),4.14-4.17(t,2H),3.52-3.55(t,2H),3.27(s,3H),3.10-3.11(d,2H),2.21(s,6H),2.01-2.04(m,2H)。
LC-MS(ESI):620.3(M+H) +。
实施例10
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-(3-{4-[4-(噻唑-2-基甲氧基)-吲哚-1-基]-嘧啶-2-基氨基}-苯基)-苯甲酰胺(化合物10)的制备
Figure PCTCN2019073874-appb-000026
与实施例7的制备方法相同，除了用2-氯甲基噻唑(达瑞)代替实施例1步骤1中的2-溴异丙烷，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-(3-{4-[4-(噻唑-2-基甲氧基)-吲哚-1-基]-嘧啶-2-基氨基}-苯基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.29(s,1H),10.28(s,1H),9.81(s,1H),8.50-8.52(d,1H),8.43-8.45(d,1H),8.25(s,1H),8.18(s,1H),8.11-8.12(d,1H),7.91-7.93(m,1H),7.87-7.88(d,1H),7.79-7.80(d,1H),7.63-7.65(d,1H),7.51-7.53(d,1H),7.44-7.48(t,1H),7.38-7.40(d,1H),7.29-7.33(t,1H),7.24-7.25(d,1H),7.16-7.20(t,1H),6.89-6.91(d,1H),6.85-6.86(d,1H),6.75-6.82(m,1H)),6.29-6.33(d,1H),3.09-3.10(d,2H),2.20(s,6H)。
LC-MS(ESI):645.3(M+H) +。
实施例11
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-氟-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物11)的制备
Figure PCTCN2019073874-appb-000027
与实施例1的制备方法相同，除了用4-氟吲哚(达瑞)代替实施例1步骤1中的吲哚，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-氟-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.27(s,1H),10.26(s,1H),9.83(s,1H),8.63-8.65(d,1H),8.53-8.54(d,1H),8.23-8.25(m,2H),8.18(s,1H),7.89-7.91(d,1H),7.62-7.64(d,1H),7.45-7.52(m,2H),7.38-7.40(m,1H),7.22-7.33(m,3H),7.02-7.04(m,1H),6.91-6.92(d,1H),6.75-6.82(m,1H),6.29-6.32(d,1H),3.08-3.09(d,2H),2.20(s,6H)。
LC-MS(ESI):550.2(M+H) +。
实施例12
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(6-氟吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物12)的制备
Figure PCTCN2019073874-appb-000028
与实施例1的制备方法相同，除了用6-氟吲哚(达瑞)代替实施例1步骤1中的吲哚，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(6-氟吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.30(s,1H),10.29(s,1H),9.87(s,1H),8.71-8.73(d,1H),8.50-8.541(d,1H),8.25(s,1H),8.19-8.20(d,1H),8.15(s,1H),7.91-7.93(d,1H),7.61-7.65(m,2H),7.52-7.54(d,1H),7.45-7.49(t,1H),7.40-7.42(m,1H),7.29--7.33(t,1H),7.25-7.26(d,1H),7.05-7.11(m,1H),6.84-6.85(d,1H),6.74-6.81(m,1H),6.29-6.33(d,1H),3.11-3.13(d,2H),2.22(s,6H)。
LC-MS(ESI):550.2(M+H) +。
实施例13
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(7-氟-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物13)的制备
Figure PCTCN2019073874-appb-000029
与实施例1的制备方法相同，除了用7-氟吲哚(达瑞)代替实施例1步骤1中的吲哚，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(7-氟-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.27(s,1H),10.25(s,1H),9.86(s,1H),8.58-8.60(d,1H),8.34(s,1H),8.19(s,1H),8.10-8.11(d,1H),7.90-7.92(d,1H),7.64-7.66(d,1H),7.46-7.53(m,3H),7.11-7.34(m,4H),6.99-7.02(t,1H),6.90-6.91(m,1H),6.76--6.82(m,1H),6.29-6.33(d,1H),3.09-3.10(d,2H),2.20(s,6H)。
LC-MS(ESI):550.2(M+H) +。
实施例14
(E)-N-{3-[4-(4-二氟甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物14)的制备
Figure PCTCN2019073874-appb-000030
步骤1：1-二氟甲氧基-2-甲基-3-硝基苯的制备
将二氟乙酸钠(7.5g，0.049mol)和碳酸钾(6.76g，0.049mol)混合于10mLDMF中，加入2mL水，室温搅拌。将2-甲基-3-硝基苯酚(3g，0.02mol)溶于7mLDMF中，慢慢滴加到反应体系中，滴毕升温至110℃反应4h，降温至室温，将反应液倒入1N NaOH水溶液(100ml)中，用乙酸乙酯萃取(100ml×1)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱 法(洗脱剂：石油醚/乙酸乙酯)纯化，得到2.2g黑色油状的1-二氟甲氧基-2-甲基-3-硝基苯。
步骤2：[2-(2-二氟甲氧基-6-硝基-苯基)-乙烯基]-二甲基-胺的制备
将步骤1中所得产物1-二氟甲氧基-2-甲基-3-硝基苯(2.2g,0.0108mol)溶于12ml DMF中，加入7ml DMF-DMA，加毕升温至110℃反应20小时。降温至室温，将反应液倒入1N NaOH水溶液(100ml)中，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过柱层析色谱法(洗脱剂：石油醚/乙酸乙酯)纯化，得到1g红色油状的[2-(2-二氟甲氧基-6-硝基-苯基)-乙烯基]-二甲基-胺。
步骤3：4-二氟甲氧基-1H-吲哚的制备
将步骤2中所得产物[2-(2-二氟甲氧基-6-硝基-苯基)-乙烯基]-二甲基-胺(500mg，1.94mmol)与铁粉(500mg，8.93mmol)混合，于10ml冰乙酸中，120℃油浴反应2小时，TLC检测反应完全，降温至室温，抽滤，滤液加入1N NaOH水溶液(100ml)中，用乙酸乙酯萃取(100ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，得到270mg粗品4-二氟甲氧基-1H-吲哚，无需纯化，直接用于下一步反应。
步骤4：(E)-N-{3-[4-(4-二氟甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺的制备
与实施例1的制备方法相同，除了用4-二氟甲氧基-1H-吲哚代替实施例1步骤1中的吲哚，并用对硝基苯甲酸(TCI)代替实施例1步骤4中的间硝基苯甲酸，得到(E)-N-{3-[4-(4-二氟甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.28-10.29(m,2H),9.85(s,1H),8.70-8.72(d,1H),8.52-8.54(d,1H),8.23-8.25(m,2H),8.18(s,1H),7.89-7.91(d,1H),7.62-7.64(d,1H),7.45-7.53(m,2H),7.38-7.40(m,1H),7.25-7.43(m,3H),7.01-7.03(d,1H),7.84-7.85(d,1H),6.75-6.81(m,1H),6.28-6.32(d,1H),3.07-3.08(d,2H),2.19(s,6H)。
LC-MS(ESI):598.2(M+H) +。
实施例15
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{2-甲氧基-5-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物15)的制备
Figure PCTCN2019073874-appb-000031
与实施例1的制备方法相同，除了用4-甲氧基吲哚(达瑞)代替实施例1步骤1中的吲哚，并用4-甲氧基-3-硝基苯胺代替实施例1步骤2中的间硝基苯胺，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{2-甲氧基-5-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.29(s,1H),9.65(s,1H),9.42(s,1H),8.45-8.47(d,1H),8.33(br,1H),8.23-8.24(d,1H),8.19(s,1H),8.13-8.04(d,1H),7.93-7.95(m,1H),7.66-7.68(d,1H),7.56-7.59(m,1H),7.46-7.50(t,1H),7.16-7.22(m,2H),7.08-7.10(d,1H),6.72-6.82(m,3H),6.28-6.32(d,1H),3.89(s,3H),3.86(s,3H),3.08-3.09(d,2H),2.19(s,6H)。
LC-MS(ESI):592.2(M+H) +。
实施例16
(E)-N-{4-氯-3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物16)的制备
Figure PCTCN2019073874-appb-000032
与实施例1的制备方法相同，除了用4-甲氧基吲哚(达瑞)代替实施例1步骤1中的吲哚，并用2-氯-5-硝基苯胺代替实施例1步骤2中的间硝基苯胺，得到(E)-N-{4-氯-3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO 3-d 6,400MHz)δ:10.73(s,1H),10.51(s,1H),9.30(s,1H),8.43-8.44(d,1H),8.23(s,1H),8.17(d,1H),7.94-8.01(m,2H),7.76-7.78(dd,1H),7.66-7.68(d,1H),7.54-7.56(d,1H),7.46-7.50(t,1H),7.19-7.21(d,1H),6.98-7.02(t,1H),6.81-6.88(m,1H),6.76-6.77(d,1H),6.68-6.70(d,1H),6.50-6.54(d,1H),3.86(s,3H),3.76-3.77(d,2H),2.64(s,6H)。
LC-MS(ESI):596.2(M+H) +。
实施例17
(E)-N-{2-氯-5-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物17)的制备
Figure PCTCN2019073874-appb-000033
与实施例1的制备方法相同，除了用4-甲氧基吲哚(达瑞)代替实施例1步骤1中的吲哚，并用4-氯-3-硝基苯胺代替实施例1步骤2中的间硝基苯胺，得到(E)-N-{2-氯-5-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.30(s,1H),10.06(s,1H),9.98(s,1H),8.52-8.54(d,1H),8.34-8.36(m,1H),8.23(s,1H),8.17-8.18(d,1H),8.03-8.04(d,1H),7.93-7.95(d,1H),7.69-7.75(m,2H),7.47-7.51(m,2H),7.22-7.28(m,2H),6.73-6.82(m,3H),6.29-6.33(d,1H),3.89(s,3H),3.07-3.08(d,2H),2.19(s,6H)。
LC-MS(ESI):596.2(M+H) +。
实施例18
(E)-4-氯-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物18)的制备
Figure PCTCN2019073874-appb-000034
与实施例1的制备方法相同，除了用4-甲氧基吲哚(达瑞)代替实施例1步骤1中的吲哚，并用4-氯-3-硝基苯甲酸代替实施例1步骤4中的间硝基苯甲酸，得到(E)-4-氯-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.35(s,1H),9.91(s,1H),9.80(s,1H),8.50-8.51(d,1H),8.36(m,2H),8.25(s,1H),8.06-8.07(d,1H),7.80-7.82(d,1H),7.68-7.70(d,1H),7.51-7.53(d,1H),7.16-7.40(m,5H),6.69-6.85(m,3H),6.50-6.54(d,1H),3.90(s,3H),3.18-3.21(m,2H),2.28(s,6H)。
LC-MS(ESI):596.2(M+H) +。
实施例19
(E)-2-氯-5-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物19)的制备
Figure PCTCN2019073874-appb-000035
与实施例1的制备方法相同，除了用4-甲氧基吲哚(达瑞)代替实施例1步骤1中的吲哚，并用2-氯-5-硝基苯甲酸代替实施例1步骤4中的间硝基苯甲酸，得到(E)-2-氯-5-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.54(s,1H),10.43(m,1H),9.81(s,1H),8.48-8.50(d,1H),8.37-8.39(d,1H),8.19(s,1H),8.06-8.07(d,1H),7.91(s,1H),7.76-7.78(m,1H),7.55-7.57(d,1H),7.50(s,1H),7.53(s,1H),7.28-7.36(m,2H),7.19-7.24(m,2H),6.76-6.83(m,3H),6.30-6.33(d,1H),3.90(s,3H),3.19-3.20(m,2H),2.27(s,6H)。
LC-MS(ESI):596.2(M+H) +。
实施例20
(E)-N-{3-[4-(3-氰基吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物20)的制备
Figure PCTCN2019073874-appb-000036
与实施例1的制备方法相同，除了用3-氰基吲哚(达瑞)代替实施例1步骤1中的吲哚，得到(E)-N-{3-[4-(3-氰基吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.41(s,1H),10.33(s,1H),10.00(s,1H),9.16(s,1H),8.85(br,1H),8.63-8.64(d,1H),8.29(s,1H),8.19(s,1H),7.91-7.93(d,1H),7.73-7.75(d,1H),7.65-7.67(d,1H),7.30-7.51(m,7H),6.76-6.83(m,1H),6.35-6.39(d,1H),3.31-3.32(m,2H),2.35(s,6H)。
LC-MS(ESI):557.2(M+H) +。
实施例21
(E)-1-(2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-嘧啶-4-基)-1H-吲哚-3-甲酰胺(化合物21)的制备
Figure PCTCN2019073874-appb-000037
Figure PCTCN2019073874-appb-000038
步骤1：1H-吲哚-3-碳酰氯的制备
3-吲哚甲酸(30g，0.186mol)于500ml二氯甲烷中室温搅拌，未能完全溶解。加入0.5mlDMF，然后于室温慢慢将草酰氯(71.0g，0.56mol)滴加其中。30分钟后滴加完毕，继续室温反应2小时。TLC检测反应完全，减压浓缩，得粗品黄色固体状的1H-吲哚-3-碳酰氯。产品无需纯化，直接用于下一步反应。
步骤2：1H-吲哚-3-甲酰胺的制备
将步骤1中得到的1H-吲哚-3-碳酰氯(0.186mol，理论收率)加入至500mlDCM中，室温搅拌30分钟，不能完全溶解，为浑浊分散体系。在2L三口瓶中加入350ml氨水和200mlDCM，并强烈搅拌。于室温将1H-吲哚-3-碳酰氯的二氯甲烷浑浊分散体系慢慢滴加到2L三口瓶中，20分钟后滴毕，继续室温反应1小时。TLC检测反应完全，过滤，固体用少量乙醇洗涤，鼓风干燥(60℃)8小时，得粗品20g黄色固体状的1H-吲哚-3-甲酰胺。产品无需纯化，直接用于下一步反应。
步骤3：(E)-1-(2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-嘧啶-4-基)-1H-吲哚-3-甲酰胺的制备
与实施例1的制备方法相同，除了用1H-吲哚-3-甲酰胺代替实施例1步骤1中的吲哚，得到(E)-1-(2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-嘧啶-4-基)-1H-吲哚-3-甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.34(s,1H),10.33(s,1H),9.92(s,1H),8.85(s,1H),8.75(br,1H),8.60-8.61(d,1H),8.31(s,1H),8.25-8.26(d,1H),8.17(s,1H),7.91-7.93(d,1H),7.76(br,1H),7.63-7.64(d,1H),7.46-7.52(m,2H),7.15-7.40(m,6H),6.77-6.81(m,1H),6.32-6.35(d,1H),3.17(m,2H),3.07-3.08(d,2H),2.25(s,6H)。
LC-MS(ESI):575.2(M+H) +。
实施例22
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[5-氟-4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物22)的制备
Figure PCTCN2019073874-appb-000039
Figure PCTCN2019073874-appb-000040
步骤1：1-(2-氯-5-氟-嘧啶-4-基)-4-甲氧基-1H-吲哚的制备
将2,4-二氯-5-氟嘧啶(1.25g，7.5mmol)溶解于DMF(30ml)中，于室温加入HOBT(135mg,1mmol)和碳酸钾(2g，15mmol)，继续室温搅拌15分钟。将4-甲氧基吲哚(735m，5mol)慢慢滴加到反应体系中，加毕继续室温反应30分钟，然后升温至85℃反应4小时，TLC检测反应完全。降温至室温，将反应液倒入水(150ml)中，室温搅拌30分钟，过滤，固体用50ml水洗涤，鼓风干燥(60℃)8小时，得到860mg黄色固体状的1-(2-氯-5-氟-嘧啶-4-基)-4-甲氧基-1H-吲哚。
步骤2：(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[5-氟-4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺的制备
将步骤1中所得产物1-(2-氯-5-氟-嘧啶-4-基)-4-甲氧基-1H-吲哚(180mg，0.65mmol)与(E)-N-(3-氨基-苯基)-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(183mg，0.54mmol)(实施例1步骤7中制备)和对甲苯磺酸(134mg，0.78mmol)溶解于异戊醇(10ml)中，将反应体系加热至120℃反应12小时。TLC检测原料仍有剩余，将反应液冷却至室温，加入20ml甲基叔丁基醚，过滤，固体用少量甲基叔丁基醚洗涤。所得固体加入二氯甲烷/甲醇(50ml/5ml)溶解，加入0.2mol/L稀氢氧化钠水溶液20ml调节PH至9-10，用二氯甲烷萃取(30ml×2)，有机相用饱和NaCl溶液洗涤两次，无水硫酸钠干燥，过滤，减压浓缩，残余物通过制备板分离(展开剂：二氯甲烷/甲醇)纯化，得到10mg白色固体状的(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[5-氟-4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.57(s,1H),10.26(s,1H),9.88(s,1H),8.67-8.68(d,1H),8.18(m,2H),7.94-7.96(m,2H),7.78(s,1H),7.65-7.67(d,1H),7.47-7.50(m,2H),7.35-7.36(m,1H),7.25-7.29(t,1H),7.16-7.20(t,1H),6.78-6.85(m,2H),6.73-6.75(d,1H),6.46-6.50(d,1H),3.90(s,3H),3.79(m,2H),2.67(s,6H)。
LC-MS(ESI):580.2(M+H) +。
实施例23
(E)-N-{3-[5-氯-4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物23)的制备
Figure PCTCN2019073874-appb-000041
与实施例22的制备方法相同，除了用2,4,5-三氯嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-N-{3-[5-氯-4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.41(s,1H),10.25(s,1H),10.11(s,1H),8.74(s,1H),8.17(s,1H),8.13(s,1H),7.92-7.94(m,1H),7.76-7.77(d,1H),7.62-7.64(d,1H),7.46-7.51(m,2H),7.41-7.43(d,1H),7.34-7.36(m,1H),7.23-7.27(t,1H),7.12-7.16(t,1H),6.79-6.83(m,1H),6.76-6.77(d,1H),6.67-6.69(d,1H),6.36-6.39(d,1H),3.90(s,3H),3.35(m,2H),2.39(s,6H)。
LC-MS(ESI):596.2(M+H) +。
实施例24
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-5-甲基-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物24)的制备
Figure PCTCN2019073874-appb-000042
与实施例22的制备方法相同，除了用2,4-二氯-5-甲基嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-5-甲基-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.30(s,1H),10.21(s,1H),9.78(s,1H),8.56(s,1H),8.17(s,1H),8.14(s,1H),7.92-7.93(d,1H),7.62-7.66(m,2H),7.54-7.56(m,1H),7.46-7.49(m,1H),7.30-7.33(m,2H),7.22(m,1H),7.12(m,1H),6.73-6.81(m,2H),6.64-6.66(d,1H),6.30-6.33(d,1H),3.90(s,3H),3.09-3.10(d,2H),2.20(s,6H),2.18(s,3H)。
LC-MS(ESI):576.2(M+H) +。
实施例25
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[5-甲氧基-4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物25)的制备
Figure PCTCN2019073874-appb-000043
与实施例22的制备方法相同，除了用2,4-二氯-5-甲氧基嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[5-甲氧基-4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.74(s,1H),10.23(s,1H),9.61(s,1H),8.53(s,1H),8.22(s,1H),8.17(s,1H),7.95-7.97(d,1H),7.87-7.88(d,1H),7.80-7.82(d,1H),7.65-7.67(m,1H),7.47-7.54(m,2H),7.28-7.30(m,1H),7.21-7.25(m,1H),7.12-7.16(t,1H),6.82-6.89(m,1H),6.73-6.74(m,1H),6.68-6.70(d,1H),6.53-6.57(d,1H),3.89(m,8H),2.72(s,6H)。
LC-MS(ESI):592.2(M+H) +。
实施例26
(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-6-甲基-嘧啶-2-基氨基]-苯基}-苯甲酰胺(化合物26)的制备
Figure PCTCN2019073874-appb-000044
与实施例22的制备方法相同，除了用2,4-二氯-6-甲基嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-3-(4-二甲基氨基-丁-2-烯酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-6-甲基-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.61(s,1H),10.28(s,1H),9.73(s,1H),8.28(m,2H),8.21(s,1H),8.05(s,1H),7.95-7.97(d,1H),7.67-7.69(d,1H),7.50-7.53(m,2H),7.15-7.35(m,4H),7.71-7.83(m,3H),6.50-6.54(d,1H),3.89(m,5H),2.77(s,6H),2.45(s,3H)。
LC-MS(ESI):576.2(M+H) +。
实施例27
(E)-N-{3-[5-氯-4-(4-乙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物27)的制备
Figure PCTCN2019073874-appb-000045
Figure PCTCN2019073874-appb-000046
与实施例22的制备方法相同，除了用4-乙氧基吲哚(达瑞)代替实施例22步骤1中的4-甲氧基吲哚，并用2,4,5-三氯嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-N-{3-[5-氯-4-(4-乙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.43(s,1H),10.25(s,1H),10.11(s,1H),8.73(s,1H),8.18(br,1H),8.13(br,1H),7.92-7.94(m,1H),7.75-7.76(d,1H),7.63-7.65(d,1H),7.46-7.51(m,2H),7.40-7.42(d,1H),7.34-7.36(m,1H),7.23-7.27(t,1H),7.10-7.14(m,1H),6.75-6.83(m,2H),6.65-6.67(d,1H),6.37-6.41(d,1H),4.13-4.20(q,2H),3.37-3.39(m,2H),2.40(s,6H),1.39-1.43(t,3H)。
LC-MS(ESI):610.2(M+H) +。
实施例28
(E)-N-{3-[5-氯-4-(4-异丙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺(化合物28)的制备
Figure PCTCN2019073874-appb-000047
与实施例22的制备方法相同，除了用4-异丙氧基吲哚(实施例7中制备)代替实施例22步骤1中的4-甲氧基吲哚，并用2,4,5-三氯嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-N-{3-[5-氯-4-(4-异丙氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.48(s,1H),10.26(s,1H),10.12(s,1H),8.73(s,1H),8.19(s,1H),8.13(s,1H),7.93-7.95(m,1H),7.74-7.75(d,1H),7.63-7.65(d,1H),7.46-7.50(m,2H),7.39-7.41(d,1H),7.34-7.36(m,1H),7.23-7.27(t,1H),7.09-7.13(m,1H),6.77-6.84(m,1H),6.71-6.72(d,1H),6.66-6.68(d,1H),6.39-6.43(d,1H),4.70-4.73(m,1H),3.50-3.51(m,2H),2.48(s,6H),1.33-1.34(d,6H)。
LC-MS(ESI):624.2(M+H) +。
实施例29
(E)-1-(5-氯-2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-嘧啶-4-基)-1H-吲哚-3-甲酰胺(化合物29)的制备
Figure PCTCN2019073874-appb-000048
与实施例22的制备方法相同，除了用1H-吲哚-3-甲酰胺(实施例21步骤2中制备)代替实施例22步骤1中的4-甲氧基吲哚，并用2,4,5-三氯嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-1-(5-氯-2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-嘧啶-4-基)-1H-吲哚-3-甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.69(s,1H),10.30(s,1H),10.21(s,1H),8.82(s,1H),8.56(s,1H),8.25-8.26(m,1H),8.17(m,2H),7.94-7.96(m,1H),7.75-7.80(m,2H),7.64-7.66(d,1H),7.47-7.52(m,2H),7.12-7.38(m,5H),6.80-6.86(m,1H),6.51-6.55(d,1H),3.90-3.92(m,2H),2.74(s,6H)。
LC-MS(ESI):609.1(M+H) +。
实施例30
(E)-1-(2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-5-甲基-嘧啶-4-基)-1H-吲哚-3-甲酰胺(化合物30)的制备
Figure PCTCN2019073874-appb-000049
与实施例22的制备方法相同，除了用1H-吲哚-3-甲酰胺(实施例21步骤2中制备)代替实施例22步骤1中的4-甲氧基吲哚，并用2,4-二氯-5-甲基嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-1-(2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-5-甲基-嘧啶-4-基)-1H-吲哚-3-甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.71(s,1H),10.27(s,1H),9.86(s,1H),8.63(s,1H),8.50(s,1H),8.27-8.28(d,1H),8.17-8.19(m,2H),7.95-7.97(m,1H),7.78(br,1H),7.65-7.72(m,2H),7.23-7.51(m,6H),7.09(br,1H),6.83-6.87(m,1H),6.51-6.55(d,1H),3.79(m,2H),2.67(s,6H),2.21(s,3H)。
LC-MS(ESI):589.2(M+H) +。
实施例31
(E)-1-(2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-5-甲氧基-嘧啶-4-基)-1H-吲哚-3-甲酰胺(化合物31)的制备
Figure PCTCN2019073874-appb-000050
与实施例22的制备方法相同，除了用1H-吲哚-3-甲酰胺(实施例21步骤2中制备)代替实施例22步骤1中的4-甲氧基吲哚，并用2,4-二氯-5-甲氧基嘧啶(达瑞)代替实施例22步骤1中的2,4-二氯-5-氟嘧啶，得到(E)-1-(2-{3-[3-(4-二甲基氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-5-甲氧基-嘧啶-4-基)-1H-吲哚-3-甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.73(s,1H),10.27(s,1H),9.71(s,1H),8.65(s,1H),8.62(s,1H),8.20-8.26(m,3H),8.07-8.09(d,1H),7.95-7.97(d,1H),7.83(br,1H),7.65-7.67(d,1H),7.47-7.51(m,2H),7.21-7.31(m,4H),7.10(br,1H),6.81-6.89(m,1H),6.52-6.56(d,1H),3.93(s,3H),3.80-3.82(d,2H),2.67(s,6H)。
LC-MS(ESI):605.3(M+H) +。
实施例32
(E)-1-(2-{3-[3-(4-二甲氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-5-氟-嘧啶-4-基)-1H-吲哚-3-甲酰胺(化合物32)的制备
Figure PCTCN2019073874-appb-000051
与实施例22的制备方法相同，除了用1H-吲哚-3-甲酰胺(实施例21步骤2中制备)代替实施例22步骤1中的4-甲氧基吲哚得到(E)-1-(2-{3-[3-(4-二甲氨基-丁-2-烯酰氨基)-苯甲酰氨基]-苯基氨基}-5-氟-嘧啶-4-基)-1H-吲哚-3-甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.28(s,1H),9.97(s,1H),8.76-8.77(d,1H),8.58-8.59(d,1H),8.25-8.28(m,2H),8.22(s,1H),8.16(s,1H),7.86-7.93(m,2H),7.63-7.65(d,1H),7.47-7.50(m,2H),7.25-7.39(m,4H),7.12(br,1H),6.77-6.85(m,1H),6.44-6.48(d,1H),2.63(s,6H)。
LC-MS(ESI):593.3(M+H) +。
参照实施例1
参照化合物(3-(4-二甲基氨基-丁酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺)(ACA1180)的制备
Figure PCTCN2019073874-appb-000052
(3-(4-二甲基氨基-丁酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺)的制备与实施例1中的制备方法相同，除了用4-甲氧基吲哚(TCI)代替实施例1步骤1中的吲哚，并用4-二甲基氨基丁酸盐酸盐代替实施例1步骤6中的反式-4-二甲基氨基巴豆酸盐酸盐，得到3-(4-二甲基氨基-丁酰氨基)-N-{3-[4-(4-甲氧基-吲哚-1-基)-嘧啶-2-基氨基]-苯基}-苯甲酰胺。
1HNMR(DMSO-d6,400MHz)δ:10.26(s,1H),10.21(s,1H),9.77(s,1H),8.49-8.50(d,1H),8.35-8.37(d,1H),8.23(s,1H),8.12(s,1H),8.05-8.06(d,1H),7.83-7.85(d,1H),7.62-7.64(d,1H),7.50-7.52(d,1H),7.43-7.47(t,1H),7.37-7.39(m,1H),7.28-7.32(m,1H),7.16-7.23(m,2H),6.79-6.80(d,1H),6.73-6.75(d,1H),3.90(s,3H),2.73(m,2H),2.51(s,6H),2.42(t,2H),1.87(m,2H)。
LC-MS(ESI)：564.2(M+H)+。
生物学评价
本发明化合物对MOLM13白血病细胞系体外生长抑制活性的测定
实验材料与方法
1.MOLM13细胞系及细胞培养
MOLM13为人急性髓细胞样白血病细胞系，CDK7表达阳性，细胞系来源于DMSZ。用RPMI1640(Gibco)加10％胎牛血清(Gibco)，1％双抗，2mM谷氨酰胺培养基悬浮培养。
2.药物处理
离心收集(1700rpm，3分钟)对数生长期的MOLM-13悬浮细胞，弃上清，计数细胞。用RPMI1640培养基配制细胞浓度为每毫升2×10 5细胞，接种到96孔板(Corning)，每孔100微升，37℃，5％CO 2培养过夜。第二天，加入待测化合物到培养细胞中，平行2孔。有机溶剂终浓度不超过千分之一，细胞继续培养3～6天，MTT测定。
本发明化合物与对照化合物THZ1和THZ2(表1)分别用DMSO(Sigma)溶解，化合物纯度达98％以上。化合物贮存浓度为10mM，-20℃保存，使用前对倍或者10倍系列稀释。
表1对照化合物
Figure PCTCN2019073874-appb-000053
Figure PCTCN2019073874-appb-000054
3.MTT检测及IC 50计算
MTT检测试剂为Dojindo CCK8试剂盒，酶标测定仪为THERMO MULTISKAN FC仪。
将CCK8试剂直接加入到药物处理和溶剂对照的悬浮细胞MOLM-13中，CCK8终浓度为10％，继续培养1～4小时，当溶剂对照孔呈现暗黄色时，测OD450nm光吸收值(THERMO MULTISKAN FC仪)，按下列公式计算细胞生长率：
细胞生长率％＝100*(T-T 0)/(C-T 0)
T＝药物处理细胞孔光密度值-空白对照孔光密度值；T 0＝药物处理前细胞孔光密度值-空白对照孔光密度值；C＝溶剂对照组细胞孔光密度-空白对照孔光密度值。通过GraphPad Prism7软件计算细胞生长50％抑制的药物浓度即IC 50。试验重复进行1-3次，并对数据进行生物学统计分析。
4. 实验结果
表2总结本发明化合物抑制肿瘤细胞MOLM-13体外生长诱导细胞凋亡的IC 50浓度的测定结果。IC 50值越小，化合物活性越强。表中“*****”代表IC 50值<1nM；“****”代表IC 50值在1nM～<10nM范围；“***”代表IC 50值在10nM～<100nM范围；“**”代表IC 50值在100nM～<1000nM范围；“*”代表IC 50值>1000nM；“-”代表无测定。
表2本发明化合物对MOLM-13细胞的体外生长抑制IC 50值
化合物 IC50
1 ***
2 ***
3 ***
4 **
5 **
6 ***
7 **
8 ***
9 ***
10 **
11 ***
12 ***
13 ***
14 **
15 **
16 *
17 **
18 ***
19 ***
20 ***
21 ***
22 ****
23 ****
24 ****
25 ***
26 *
27 ****
28 ***
29 *****
30 ****
31 ****
32 *****
ACA1180 *
THZ1 ****
THZ2 ****
结论：结果显示本发明化合物对MOLM-13具有高的生长抑制活性，IC 50值可达亚纳摩尔。

Claims (13)

  1. 一种通式(I)所示的化合物，
    Figure PCTCN2019073874-appb-100001
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐；
    其中：
    每一个R 1各自独立地选自氢、卤素、羟基、氰基、硝基、-N(R y)(R z)、烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基、-NHC(O)R x、-C(O)N(R y)(R z)、-OR uOR x、-OR x或-OR uN(R y)(R z)，其中所述烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
    R 2和R 3各自独立地选自氢、卤素、羟基、氰基、硝基、烷基、烷氧基、环烷基、杂环基、-NHC(O)R x或-C(O)N(R y)(R z)，其中所述烷基、烷氧基、环烷基和杂环基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
    R 4和R 5各自独立地选自氢、卤素、羟基、氰基、硝基、烷基、烷氧基、环烷基、杂环基、-NHC(O)R x或-C(O)N(R y)(R z)，其中所述烷基、烷氧基、环烷基和杂环基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
    每一个R 6各自独立地选自Q基团；
    每一个R 7为各自独立地选自-NHC(O)R和Q基团；
    R 8和R 9相同或不同且各自独立地选自-NHC(O)R和Q基团；
    Q选自氢、卤素、羟基、氰基、硝基、-N(R y)(R z)、烷基、烷氧基、烯基、炔基、环烷基、杂环基、芳基或杂芳基，其中所述烷基、烷氧基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
    R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基或-R uN(R y)(R z)的一个或多个基团取代；
    R u选自亚烷基、亚烯基、或亚炔基；
    R x选自氢、烷基、环烷基、芳基或者杂芳基，所述烷基、环烷基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基的一个或多个取代基所取代；
    R y和R z各自独立地选自氢、烷基、烷氧基、烯基、炔基、环烷基、杂环基、芳基或杂芳基；所述烷基、烷氧基、烯基、炔基、环烷基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；或者，
    R y和R z与它们所连接的氮原子一起形成含氮杂环基或杂芳基，所述杂环基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
    n为1至4的整数；
    m为1至4的整数；
    p为1至3的整数。
  2. 根据权利要求1所述的通式(I)所示的化合物，
    其中：
    R 8选自-NHC(O)R，且R 9选自Q基团；或者，
    R 9选自-NHC(O)-R，且R 8选自Q基团；
    其中R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自氢、烷基或-R uN(R y)(R z)的一个或多个基团取代；
    R u、R y、R z、Q基团如权利要求1中所定义。
  3. 根据权利要求1或2所述的通式(I)所示的化合物，
    其中：
    R 8选自-NHC(O)R，且R 9选自Q基团；或者，
    R 9选自-NHC(O)-R，且R 8选自Q基团；
    其中R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自氢、烷基或R uN(R y)(R z)的一个或多个基团取代；
    R u选自C 1～C 6亚烷基；
    R y和R z各自独立地选自氢、C 1～C 6烷基、C 3～C 7环烷基；或者，
    R y和R z与它们所连接的氮原子一起形成5～7元含氮杂环基，优选吗啉基、哌啶基、哌嗪基、氮杂环庚烷基、四氢吡咯基，所述5～7元含氮杂环基任选进一 步被选自卤素、C 1～C 6烷基、C 1～C 6卤代烷基、C 1～C 6烷氧基、C 1～C 6卤代烷氧基、C 3～C 7环烷基的一个或多个基团所取代；
    Q基团选自氢、卤素、羟基、氰基、硝基、氨基、C 1～C 6烷基、C 1～C 6烷氧基、C 3～C 7环烷基。
  4. 根据权利要求1至3中任一项所述的通式(I)所示的化合物，
    其中：
    每一个R 7各自独立地选自-NHC(O)R；
    其中R选自烯基、炔基、氰基，所述烯基和炔基任选进一步被选自氢、烷基或R uN(R y)(R z)的一个或多个基团取代；
    R u选自C 1～C 6亚烷基；
    R y和R z各自独立地选自氢、C 1～C 6烷基、C 3～C 7环烷基；或者，
    R y和R z与它们所连接的氮原子一起形成5～7元含氮杂环基，优选吗啉基、哌啶基、哌嗪基、氮杂环庚烷基、四氢吡咯基，所述5～7元含氮杂环基任选进一步被选自卤素、C 1～C 6烷基、C 1～C 6卤代烷基、C 1～C 6烷氧基、C 1～C 6卤代烷氧基、C 3～C 7环烷基的一个或多个基团所取代。
  5. 根据权利要求1至4中任一项所述的通式(I)所示的化合物，
    其中：
    每一个R 1各自独立地选自氢、卤素、羟基、氰基、硝基、C 1～C 6烷基、C 3～C 7环烷基、-NHC(O)R x、-C(O)N(R y)(R z)、-OR uOR x、-OR x，其中所述C 1～C 6烷基、C 3～C 7环烷基任选进一步被选自卤素、氰基、氨基、硝基、羟基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
    R u选自C 1～C 6亚烷基；
    R x选自氢、C 1～C 6烷基、C 3～C 7环烷基或者5～7元杂芳基，所述C 1～C 6烷基、C 3～C 7环烷基或者5～7元杂芳基任选进一步被卤素、羟基、烷基、环烷基、杂环基、芳基或杂芳基的一个或多个基团所取代。
  6. 根据权利要求1至5中任一项所述的通式(I)所示的化合物，
    其中：
    R 2和R 3各自独立地选自氢、卤素、氰基、烷基、烷氧基、环烷基、杂环基、-NHC(O)R x或-C(O)N(R y)(R z)，其中所述烷基、烷氧基、环烷基和杂环基任选进一步被选自卤素、烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基、芳基、杂芳基中的一个或多个取代基所取代；
    R x、R y、R z如权利要求1所定义。
  7. 根据权利要求1至6中任一项所述的通式(I)所示的化合物，
    其中：
    R 2选自氢、烷基、环烷基、氰基、-NHC(O)R x或-C(O)N(R y)(R z)；
    R 3选自氢；
    R x选自氢、烷基、环烷基，所述烷基、环烷基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基的一个或多个取代基所取代；
    R y和R z各自独立地选自氢、烷基、环烷基、杂环基、芳基或杂芳基；所述烷基、环烷基、芳基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基、环烷基、杂环基的一个或多个取代基所取代；或者，
    R y和R z与它们所连接的氮原子一起形成含氮杂环基或杂芳基，所述杂环基或杂芳基任选进一步被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、氰基、氨基、硝基、羟基、羟烷基中的一个或多个取代基所取代。
  8. 根据权利要求1至7中任一项所述的通式(I)所示的化合物，
    其中：
    R 4和R 5各自独立地选自氢、卤素、氰基、C 1～C 6烷基、C 1～C 6卤代烷基、C 1～C 6烷氧基、C 1～C 6卤代烷氧基。
  9. 根据权利要求1至8中任一项所述的通式(I)所示的化合物，
    其中：
    每一个R 6各自独立地选自Q基团；
    Q选自氢、卤素、羟基、氰基、硝基、氨基、C 1～C 6烷基、C 1～C 6烷氧基、C 3～C 7环烷基。
  10. 根据权利要求1至9中任一项所述的通式(I)所示的化合物，其选自：
    Figure PCTCN2019073874-appb-100002
    Figure PCTCN2019073874-appb-100003
    Figure PCTCN2019073874-appb-100004
  11. 根据权利要求1至10中任一项所述的通式(I)所示的化合物的制备方法，其包括以下步骤：
    Figure PCTCN2019073874-appb-100005
    将中间体化合物M3和苯胺类中间体化合物M4在适宜的温度和适当的溶剂中，在酸催化下反应，得到通式(I)化合物；
    所述溶剂优选异丙醇、异戊醇、仲戊醇、二氧六环；
    所述酸优选盐酸、硫酸、甲磺酸、对甲苯磺酸、苯磺酸；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、n、m、p如权利要求1中所定义。
  12. 一种药物组合物，其含有治疗有效量的根据权利要求1至10中任一项所述的通式(I)所示的化合物，以及药学上可接受的载体。
  13. 根据权利要求1至10中任一项所述的通式(I)所示的化合物或者根据权利要求12所述的药物组合物，在制备用于预防和/或治疗哺乳动物包括人类中癌症的药物中的用途，所述癌症选自非实体瘤如白血病和实体瘤如皮肤癌、黑色素瘤、肺癌、胃癌、乳腺癌、胰腺癌、肝癌或结肠癌。
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