WO2016112846A1 - 3-乙炔基吡唑并嘧啶衍生物及其制备方法和用途 - Google Patents
3-乙炔基吡唑并嘧啶衍生物及其制备方法和用途 Download PDFInfo
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- WO2016112846A1 WO2016112846A1 PCT/CN2016/070725 CN2016070725W WO2016112846A1 WO 2016112846 A1 WO2016112846 A1 WO 2016112846A1 CN 2016070725 W CN2016070725 W CN 2016070725W WO 2016112846 A1 WO2016112846 A1 WO 2016112846A1
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- 0 C*(C)c1c(*)c(*)c(*)c(*)c1* Chemical compound C*(C)c1c(*)c(*)c(*)c(*)c1* 0.000 description 29
- AJGLFRJMVRQXSZ-UHFFFAOYSA-N CC(C)(C)CN1CCN(C)CC1 Chemical compound CC(C)(C)CN1CCN(C)CC1 AJGLFRJMVRQXSZ-UHFFFAOYSA-N 0.000 description 1
- CSLXWXSYBCBLKZ-UHFFFAOYSA-N CC(C)(C)OC(N(CC1)CCC1[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1)=O Chemical compound CC(C)(C)OC(N(CC1)CCC1[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1)=O CSLXWXSYBCBLKZ-UHFFFAOYSA-N 0.000 description 1
- ODIQTOYGORNLPE-UHFFFAOYSA-N CC(C)N1CCN(C)CC1 Chemical compound CC(C)N1CCN(C)CC1 ODIQTOYGORNLPE-UHFFFAOYSA-N 0.000 description 1
- ZUHZYTLWIPYSSW-UHFFFAOYSA-N CC(C)[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1 Chemical compound CC(C)[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1 ZUHZYTLWIPYSSW-UHFFFAOYSA-N 0.000 description 1
- TVJIDCCWASCDEI-UHFFFAOYSA-N CC(C)[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(NC(c2ccc(CN3CCN(C)CC3)cc2)=O)c1 Chemical compound CC(C)[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(NC(c2ccc(CN3CCN(C)CC3)cc2)=O)c1 TVJIDCCWASCDEI-UHFFFAOYSA-N 0.000 description 1
- LJPCNSSTRWGCMZ-UHFFFAOYSA-N CC1COCC1 Chemical compound CC1COCC1 LJPCNSSTRWGCMZ-UHFFFAOYSA-N 0.000 description 1
- DLEWDCPFCNLJEY-UHFFFAOYSA-N CCC(N1CCOCC1)=O Chemical compound CCC(N1CCOCC1)=O DLEWDCPFCNLJEY-UHFFFAOYSA-N 0.000 description 1
- UTCKUKOWEDNSQA-UHFFFAOYSA-N CCCC(CCN)[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1 Chemical compound CCCC(CCN)[n](c1ncnc(N)c11)nc1C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1 UTCKUKOWEDNSQA-UHFFFAOYSA-N 0.000 description 1
- TXIOGJHPPVXTOY-UHFFFAOYSA-N CCN1CCN(C)CC1 Chemical compound CCN1CCN(C)CC1 TXIOGJHPPVXTOY-UHFFFAOYSA-N 0.000 description 1
- DNKDQXQJMZPTJU-UHFFFAOYSA-N CCNc1c(c(C)n[n]2CC)c2ncn1 Chemical compound CCNc1c(c(C)n[n]2CC)c2ncn1 DNKDQXQJMZPTJU-UHFFFAOYSA-N 0.000 description 1
- DDEFQBOZTZUKCK-UHFFFAOYSA-O CC[NH2+]c1ncnc(N)c1C(C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1)=N Chemical compound CC[NH2+]c1ncnc(N)c1C(C#Cc1c(C)ccc(C(Nc2cc(C(F)(F)F)c(CN3CCN(C)CC3)cc2)=O)c1)=N DDEFQBOZTZUKCK-UHFFFAOYSA-O 0.000 description 1
- CXOQHMNHYCMCQK-UHFFFAOYSA-N CC[N](C)(C)(C)(C)=CC Chemical compound CC[N](C)(C)(C)(C)=CC CXOQHMNHYCMCQK-UHFFFAOYSA-N 0.000 description 1
- MYOSJPRSRNFNSQ-UHFFFAOYSA-N CCc1n[nH]c2ncnc(N)c12 Chemical compound CCc1n[nH]c2ncnc(N)c12 MYOSJPRSRNFNSQ-UHFFFAOYSA-N 0.000 description 1
- FXLGXUBFIWFPJA-UHFFFAOYSA-N Cc(c(C#C)c1)ccc1C(O)=O Chemical compound Cc(c(C#C)c1)ccc1C(O)=O FXLGXUBFIWFPJA-UHFFFAOYSA-N 0.000 description 1
- QXMYBKYVOMIHGI-UHFFFAOYSA-N Cc(c(C#C[Si](C)(C)C)c1)ccc1C(OC)=O Chemical compound Cc(c(C#C[Si](C)(C)C)c1)ccc1C(OC)=O QXMYBKYVOMIHGI-UHFFFAOYSA-N 0.000 description 1
- NKMHAOTZPFVSPC-UHFFFAOYSA-N Cc(c(I)c1)ccc1C(OC)=O Chemical compound Cc(c(I)c1)ccc1C(OC)=O NKMHAOTZPFVSPC-UHFFFAOYSA-N 0.000 description 1
- VRCBGBBYFHLJRE-UHFFFAOYSA-N Cc(ccc(C(Nc1cc(C(F)(F)F)c(CN2CCN(C)CC2)cc1)=O)c1)c1C#Cc1n[n](C(CC2)CCN2C(C=C)=O)c2ncnc(N)c12 Chemical compound Cc(ccc(C(Nc1cc(C(F)(F)F)c(CN2CCN(C)CC2)cc1)=O)c1)c1C#Cc1n[n](C(CC2)CCN2C(C=C)=O)c2ncnc(N)c12 VRCBGBBYFHLJRE-UHFFFAOYSA-N 0.000 description 1
- CWEOEBZJBKISJK-UHFFFAOYSA-N Cc(ccc(C(Nc1cc(C(F)(F)F)c(CN2CCN(C)CC2)cc1)=O)c1)c1C#Cc1n[n](C2CCC2)c2ncnc(N)c12 Chemical compound Cc(ccc(C(Nc1cc(C(F)(F)F)c(CN2CCN(C)CC2)cc1)=O)c1)c1C#Cc1n[n](C2CCC2)c2ncnc(N)c12 CWEOEBZJBKISJK-UHFFFAOYSA-N 0.000 description 1
- LNLJMGHNVYZKPU-UHFFFAOYSA-N Cc(ccc(C(Nc1cc(C(F)(F)F)c(CN2CCN(C)CC2)cc1)=O)c1)c1C#Cc1n[n](C2CCCC2)c2ncnc(N)c12 Chemical compound Cc(ccc(C(Nc1cc(C(F)(F)F)c(CN2CCN(C)CC2)cc1)=O)c1)c1C#Cc1n[n](C2CCCC2)c2ncnc(N)c12 LNLJMGHNVYZKPU-UHFFFAOYSA-N 0.000 description 1
- BYPNAYSWCCERGN-UHFFFAOYSA-N Cc(ccc(C(Nc1ccc(CN2CCN(C)CC2)c(C(F)(F)F)c1)=O)c1)c1C#C Chemical compound Cc(ccc(C(Nc1ccc(CN2CCN(C)CC2)c(C(F)(F)F)c1)=O)c1)c1C#C BYPNAYSWCCERGN-UHFFFAOYSA-N 0.000 description 1
- LDDHMKANNXWUAK-UHFFFAOYSA-N Cc(ccc(C(O)=O)c1)c1I Chemical compound Cc(ccc(C(O)=O)c1)c1I LDDHMKANNXWUAK-UHFFFAOYSA-N 0.000 description 1
- IJWDCLWLOGCXOQ-UHFFFAOYSA-N Cc(ccc(C(OC)=O)c1)c1C#C Chemical compound Cc(ccc(C(OC)=O)c1)c1C#C IJWDCLWLOGCXOQ-UHFFFAOYSA-N 0.000 description 1
- BLHTXORQJNCSII-UHFFFAOYSA-N Cc1c[n](C)cn1 Chemical compound Cc1c[n](C)cn1 BLHTXORQJNCSII-UHFFFAOYSA-N 0.000 description 1
- MHDIEAWIDWABPN-UHFFFAOYSA-N Ic1n[nH]c2ncnc(NI)c12 Chemical compound Ic1n[nH]c2ncnc(NI)c12 MHDIEAWIDWABPN-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—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
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the invention belongs to the field of chemical medicine, and particularly relates to a 3-ethynylpyrazolopyrimidine derivative, a preparation method thereof and use thereof.
- Protein kinase is an important phosphotransferase whose function is to catalyze the transfer of the ⁇ -phosphate group at the end of adenosine triphosphate (ATP) to the amino acid residues of specific substrate proteins, so that the protein is phosphorylated and thus exerts its physiology. Biochemical function. Protein kinases are a key component of cellular signaling pathways. Protein kinases are involved in the regulation of multiple cellular processes, including cell growth, energy metabolism, cell cycle, transcription, apoptosis, and differentiation. In addition, protein kinases play a crucial role in maintaining intercellular connections, homeostasis, and immune system function. Abnormal regulation of protein kinases is associated with the development of a variety of diseases, particularly tumors. Protein kinases have become important therapeutic targets for diseases and have attracted widespread attention.
- ATP adenosine triphosphate
- protein kinase drugs have become the fastest growing unit in the global pharmaceutical market. As of 2014, more than 20 protein kinase drugs have been approved worldwide. Listed, including more than ten kinds of cancer treatment drugs, such as Imatinib, Gefitinib, Erlotinib, Sorafenib, Sunitinib ), Dasatinib, Nilotinib, Lapinib, Pazopanib and Regorafenib.
- cancer treatment drugs such as Imatinib, Gefitinib, Erlotinib, Sorafenib, Sunitinib ), Dasatinib, Nilotinib, Lapinib, Pazopanib and Regorafenib.
- the high development success rate of protein kinase drugs especially the successful development of various protein kinase targeted anti-tumor drugs, has made it a hot research direction in the scientific and pharmaceutical industry.
- the present invention provides a 3-ethynylpyrazolopyrimidine derivative having the structure shown in Formula I:
- R 1 is -H, C 1 -C 4 alkyl
- R 2 is -H, C 1 -C 8 alkyl, R 8 substituted C 3 -C 8 cycloalkyl, C 3 - C 8 alkylene oxide,
- R 3 to R 7 are independently -H, C 1 -C 8 alkyl, -OH, C 1 -C 8 alkoxy, halogen,
- R 8 to R 11 are independently -H, C 1 -C 8 alkyl, halogen, -OH,
- R 12 to R 14 are independently
- R 15 to R 19 are independently -H, C 1 -C 8 alkyl, -OH, C 1 -C 8 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 20 to R 38 are independently -H, halogen, C 1 -C 8 alkyl, C 1 -C 8 cycloalkyl, -OCF 3 or -CF 3 ;
- R 39 to R 42 are independently a C 1 -C 8 alkyl group, a C 3 -C 8 cycloalkyl group or a C 1 -C 8 hydroxyalkyl group;
- n 0 to 6.
- R 1 is -H or R 2 is -H, C 1 -C 4 alkyl, R 8 substituted C 3 -C 8 cycloalkyl, C 3 - C 8 alkylene oxide, R 3 to R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, R 8 to R 11 are independently -H, C 1 -C 4 alkyl, halogen, -OH, R 12 to R 14 are independently R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 , R 20 to R 38 are independently -H, halogen, C 1 -C 4 alkyl, C 1 -C 4 cycloalkyl, -OCF 3 or -CF 3 ; R 39 to R 42 are independently C 1 -C 4- alkyl, R 8 substituted C 3 -
- R 1 is -H or R 2 is -H, C 1 -C 4 alkyl, R 8 substituted C 3 -C 8 cycloalkyl, C 3 - C 8 alkylene oxide, R 3 to R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH, R 12 to R 14 are independently R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 , R 20 to R 38 are independently -H, halogen, C 1 -C 4 alkyl, C 1 -C 4 cycloalkyl, -OCF 3 or -CF 3 ; R 39 to R 42 are independently C 1 -C 4- alkyl, C 3 -C 8 cyclo
- R 1 is -H or R 2 is -H, C 1 -C 4 alkyl, R 8 substituted C 3 -C 8 cycloalkyl, C 3 - C 8 alkylene oxide, R 3 to R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH, R 12 to R 14 are independently R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 , R 20 to R 38 are independently -H, halogen, C 1 -C 4 alkyl, C 1 -C 4 cycloalkyl, -OCF 3 or -CF 3 ; R 39 to R 42 are independently C 1 -C 4- alkyl, C 3 -C 8 cyclo
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl or halogen;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 substituted C 3 -C 8 cycloalkyl, C 3 - C 8 alkylene oxide, n 0 to 4;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -F or -Cl;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, methyl or -Cl;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, methyl or -Cl;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 12 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, methoxy, -F, -Cl, -CF 3 , -OCF 3 or
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 2 is a C 1 -C 4 alkyl group
- R 8 and R 9 are independently -H, C 1 -C 4 alkyl, -OH
- R 1 is -H or
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen
- R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 2 is a C 1 -C 4 alkyl group, an R 8 -substituted C 3 -C 8 cycloalkyl group, C 3 - C 8 alkylene oxide, R 8 and R 9 are independently -H, C 1 -C 4 alkyl, -OH, R 1 is -H or R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 , R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 2 is a C 1 -C 4 alkyl group, a R 8 -substituted C 3 -C 8 cycloalkyl group, C 3 - C 8 alkylene oxide, R 8 and R 9 are independently -H, C 1 -C 4 alkyl, R 1 is -H or R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 , R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH or halogen;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH or -Cl;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH, R 13 is R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH, R 13 is R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 20 to R 38 are independently -H or C 1 -C 4 alkyl;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 1 is -H or R 2 is a C 1 -C 4 alkyl group, a R 8 -substituted C 3 -C 8 cycloalkyl group, C 3 - C 8 alkylene oxide, R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH or -Cl; R 8 and R 9 are independently -H, C 1 -C 4 alkyl, R 13 is R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -CF 3 or R 20 to R 38 are independently -H or C 1 -C 4 alkyl.
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 2 is a C 1 -C 4 alkyl group
- R 8 and R 9 are independently -H, C 1 -C 4 alkyl, -OH
- R 1 is -H or
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen
- R 15 to R 19 are independently -H, C 1 - C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 2 is a C 1 -C 4 alkyl group, an R 8 -substituted C 3 -C 8 cycloalkyl group, C 3 - C 8 alkylene oxide, R 8 and R 9 are independently -H, C 1 -C 4 alkyl, -OH, R 1 is -H or R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; and R 15 to R 19 are independently -H, C 1 - C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 2 is a C 1 -C 4 alkyl group, a R 8 -substituted C 3 -C 8 cycloalkyl group, C 3 - C 8 alkylene oxide, R 8 and R 9 are independently -H, C 1 -C 4 alkyl, R 1 is -H or R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; and R 15 to R 19 are independently -H, C 1 - C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH or halogen;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH or -Cl;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3 ,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; and
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -CF 3 or
- R 1 is -H or R 2 is a C 1 -C 4 alkyl group, a R 8 -substituted C 3 -C 8 cycloalkyl group, C 3 - C 8 alkylene oxide, R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH or -Cl; R 8 and R 9 are independently -H, C 1 -C 4 alkyl, R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -CF 3 or
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 9 is C 1 -C 4 alkyl, -OH,
- R 1 is -H or
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 15 to R 19 are independently -H, C 1 - C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 2 is a C 1 -C 4 alkyl group
- R 9 is C 1 -C 4 alkyl, -OH or R 1 is -H or
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 15 to R 19 are independently -H, C 1 - C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 2 is a C 1 -C 4 alkyl group or R 9 is C 1 -C 4 alkyl;
- R 1 is -H or R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen;
- R 15 to R 19 are independently -H, C 1 - C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH or halogen;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl or halogen;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 substituted C 3 -C 8 cycloalkyl, C 3 - C 8 alkylene oxide, n 0 to 4;
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl or -Cl;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, C 1 -C 4 alkyl or -CF 3 .
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy, halogen or -CF 3 ;
- R 1 is -H or
- R 2 is -H, C 1 -C 4 alkyl,
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; and
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H, C 1 -C 4 alkyl, halogen or -CF 3 ;
- R 1 is -H or
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; and
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 15 to R 19 are independently -H or -CF 3 ;
- R 1 is -H or
- R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl, -OH, C 1 -C 4 alkoxy or halogen; and
- R 8 to R 11 are independently -H, C 1 -C 4 alkyl, -OH,
- R 1 is -H
- R 2 is C 1 -C 4 alkyl or R 3 to R 5 and R 7 are independently -H, C 1 -C 4 alkyl or -Cl
- R 9 is C 1 -C 4 alkyl
- R 15 to R 19 are independently -H or -CF 3 .
- the invention also provides a preparation method of the above 3-ethynylpyrazolopyrimidine derivative:
- reaction conditions in the above reaction formula are:
- Compound 1 is halogenated in a usual halogenated reagent (such as NIS (N-iodosuccinimide), NBS (N-bromosuccinimide), Br 2 , I 2 , ICl, IBr)
- a usual halogenated reagent such as NIS (N-iodosuccinimide), NBS (N-bromosuccinimide), Br 2 , I 2 , ICl, IBr
- R 2 haloalkane bromine or iodo
- the corresponding sulfonate methanesulfonate, p-toluenesulfonate, p-nitrobenzenesulfonate, etc.
- alkaline conditions such as KOH, NaOH
- compound 3 was prepared by substituting compound 2 with K 2 CO 3 , Na 2 CO 3 , Cs 2 CO 3 , NaH).
- the corresponding carboxyl group-containing compound is condensed with a corresponding amino group-containing compound under usual condensation conditions (e.g., a condensing agent method, a mixed acid anhydride method, an activated hair, etc.) to obtain a corresponding amide bond-bonded compound 7 to 10.
- condensation conditions e.g., a condensing agent method, a mixed acid anhydride method, an activated hair, etc.
- the corresponding intermediate is catalyzed by a transition metal to a coupling reaction with an alkyne reagent having a protecting group at one end to obtain a corresponding intermediate, and then deprotected to obtain a corresponding alkynyl-containing compound 4, compound 6, and compound 7. , Compound 10 and Compound 13.
- the corresponding halogen-containing intermediate and the alkynyl-containing intermediate are subjected to a coupling reaction under transition metal catalysis to obtain a compound of the formula II, III, IV and V, and the coupling reaction is carried out using a palladium catalyst (such as Pd 4 ( PPh 3 ) 4 , PdAc 2 , Pd 2 (dba) 3 , PdPPh 3 Cl 2 , etc.), copper salts (such as cuprous chloride, bromide, cuprous iodide, etc.), and appropriate organic bases or inorganic a base (such as triethylamine, DIPEA (diisopropylethylamine), potassium carbonate, sodium carbonate, sodium hydrogencarbonate, etc.) in a suitable solvent (such as THF (tetrahydrofuran), toluene, DMF (N, N-dimethyl A) It is obtained by reacting at 20 to 150 ° C in an amide), 1,4-dio
- compound 14 is reacted with triphosgene in a suitable base (such as triethylamine, DIPEA, etc.) and a solvent (such as EA (ethyl acetate), THF, dichloromethane, etc.) and temperature (0-50 degrees) to obtain a compound.
- a suitable base such as triethylamine, DIPEA, etc.
- a solvent such as EA (ethyl acetate), THF, dichloromethane, etc.
- compound 15 and compound 6 are reacted in a suitable solvent (such as EA (ethyl acetate), THF, dichloromethane, toluene, DMF, etc.) at 20 to 120 ° C to obtain compound 16.
- a suitable solvent such as EA (ethyl acetate), THF, dichloromethane, toluene, DMF, etc.
- R 1 is -H, C 1 -C 4 alkyl
- R 2 is -H, C 1 -C 8 alkyl, R 8 substituted C 3 -C 8 cycloalkyl, C 3 - C 8 alkylene oxide
- R 3 to R 7 are independently -H, C 1 -C 8 alkyl, -OH, C 1 -C 8 alkoxy, halogen
- R 8 to R 11 are independently -H, C 1 -C 8 alkyl, halogen, -OH
- R 12 to R 14 are independently R 15 to R 19 are independently -H, C 1 -C 8 alkyl, -OH, C 1 -C 8 alkoxy, halogen, -CF 3 , -OCF 3
- R 20 to R 38 are independently -H, halogen, C 1 -C 8 alkyl, C 1 -C 8 cycloalkyl, -OCF 3 or -CF 3
- R 39 to R 42 are independently C 1 -
- the above 3-ethynylpyrazolopyrimidine derivatives of the present invention include their isotopic compounds, racemates, optically active isomers, polymorphic forms or mixtures thereof.
- the present invention also provides a pharmaceutically acceptable salt of the above 3-ethynylpyrazolopyrimidine derivative.
- prodrugs of the compounds of the invention are derivatives of the above compounds which may themselves have weak or even no activity, but after administration, under physiological conditions (for example It is converted to the corresponding biologically active form by metabolism, solvolysis or otherwise.
- the present invention also provides a pharmaceutically acceptable hydrate of the above 3-ethynylpyrazolopyrimidine derivative.
- the present invention also provides a pharmaceutical composition prepared by adding the above-mentioned 3-ethynylpyrazolopyrimidine derivative provided by the present invention to a pharmaceutically acceptable auxiliary component.
- the structure of the 3-ethynylpyrazolopyrimidine derivative provided by the present invention is as shown in Formulas I to V.
- the present invention also provides the use of the above 3-ethynylpyrazolopyrimidine derivative, a salt or hydrate thereof for the preparation of a kinase inhibitor.
- the above kinase inhibitor is an inhibitor of the SRC family tyrosine protein kinase (Blk tyrosine protein kinase, Fgr tyrosine protein kinase, Frk tyrosine protein kinase, Fyn tyrosine protein kinase, Hck tyrosine protein kinase, Lck tyrosine protein kinase, Lyn tyrosine protein kinase, c-SRC tyrosine protein kinase , YES tyrosine protein kinase), FLT3 (human FMS-like tyrosine kinase 3), Abl (Abl tyrosine protein kinase), VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2 (vascular endothelial growth factor receptor) 2), VEGFR3 (vascular endothelial growth factor receptor 3), RET (RET
- the present invention also provides the use of the above 3-ethynylpyrazolopyrimidine derivative, a salt or hydrate thereof for the preparation of an antitumor drug.
- the above tumor is leukemia or solid tumor.
- the solid tumor is at least one of lung cancer, breast cancer, pancreatic cancer, melanoma, glioma, liver cancer, thyroid tumor, cervical cancer, stomach cancer or colorectal cancer.
- the above leukemia is acute myeloid leukemia or mixed leukemia.
- the 3-ethynylpyrazolopyrimidine derivative provided by the invention exhibits a good inhibitory effect on tumors such as human breast cancer, human lung cancer, human pancreatic cancer, human malignant melanoma, and human leukemia.
- Methyl 4-methyl-3-(trifluoromethyl)benzoate was dissolved in 1,2-dichloroethane (DCE) and NBS (N-bromosuccinimide) (1.81 g) was added with stirring. , 10.18 mmol, 1.2 eq), AIBN (azobisisobutyronitrile) (0.139 g, 0.848 mmol, 0.1 eq) was replaced with nitrogen three times, and after reacting at 80 ° C for 30 h, the reaction liquid was saturated with saturated aqueous sodium hydrogen carbonate solution. The aqueous solution of sodium chloride and water were washed, and the DCE layer was dried over magnesium sulfate and evaporated to dryness.
- DCE 1,2-dichloroethane
- NBS N-bromosuccinimide
- AIBN azobisisobutyronitrile
- N-(3-iodo-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzamide is prepared:
- N-(3-ethynyl-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzamide was prepared.
- 6-Trifluoromethyl-pyridine-2-carboxylic acid (229.33 mg, 1.2 mmol, 1.2 eq) was dissolved in thionyl chloride, refluxed at 80 ° C for 3 h, and dichlorosulfoxide was evaporated to dryness to give 6-trifluorobenzene.
- N-(4-chloro-3-ethynylphenyl)-3-(trifluoromethyl)benzamide is prepared:
- the upper oil was dissolved in THF, and cuprous iodide (1.1 g, 5.74 mmol, 0.1 eq) and tetratriphenylphosphine palladium (3.3 g, 2.86 mmol, 0.05 eq) were added, three times of nitrogen, and triethylamine was added. (11.57 g, 114.28 mmol, 2.0 eq) and trimethylsilylacetylene (8.4 g, 85.71 mmol, 1.5 eq), stirred at room temperature, and the reaction was completed after 24 h. The solvent was evaporated to dryness under reduced pressure.
- the upper step oil was dissolved in methanol, potassium carbonate (3.95 g, 28.57 mmol, 0.5 eq) was added, and the mixture was stirred at room temperature for 10 min, and then the reaction was completed.
- the solvent was evaporated to dryness in vacuo, and then dissolved in DCM.
- the sodium aqueous solution and water were extracted once, and the DCM layer was dried over anhydrous sodium sulfate.
- the product obtained in the above step was dissolved in methanol, and 10 mL of a saturated aqueous sodium hydroxide solution was added thereto, and the mixture was stirred at room temperature overnight. After completion of the reaction, the solvent was evaporated to dryness under reduced pressure, and water (20 mL) was added, and the pH was adjusted to about 3 with hydrochloric acid, and a large amount of white solid was precipitated, and extracted with DCM three times. The DCM layer was combined, and the DCM layer was washed once with water.
- N-(3-(trifluoromethyl)phenyl)-3-((trimethylsilyl)ethynyl)benzamide is prepared:
- Triphosgene (1.79 g, 6.0 mmol, 1.5 eq) was dissolved in EA and stirred at room temperature.
- m-Trifluoromethylaniline (967 mg, 6.0 mmol, 1.5 eq) was dissolved in EA, placed in a dropping funnel, and slowly added dropwise to the phosgene EA solution. After 30 min, the addition was completed. Then, triethylamine (1.2 g, 12 mmol, 2.0 eq) was slowly added to precipitate a large amount of white solid, which was reacted at room temperature. After 2 h, the solvent was evaporated to dryness under reduced pressure, EA was added, and the insoluble material was filtered, and filtrate was collected.
- a compound 2-119 was obtained by a similar method as described in Example 28, using aryl acetylene and a halogen compound containing different substituents as starting materials.
- the purpose of this experiment was to test the inhibitory activity of the compounds of the present invention against protein kinases in vitro by the isotope labeling method (labeling the gamma phosphate group on ATP).
- This experiment is for Abl(T315I)(h), ALK(h), ARK5(h), Axl(h), Blk(h), Bmx(h), BTK(h), B-Raf(h), cKit (h), cSRC (h), CDK7, CHK1 (h), c-RAF (h), DDR2 (h), EGFR (h), EphA1 (h), EphA2 (h), EphA8 (h), EphB2 ( h), EphB4 (h), ErbB2 (h), FAK (h), Fer (h), FGFR1 (h), Flt3 (h), Fms (h), Fyn (h), Hck (h), GSK3 ⁇ ( h), IKK ⁇ (h), IKK
- the kinase inhibitory activity of the test compound is expressed by the IC 50 (half inhibitory concentration) or the inhibition rate of the test compound to the kinase activity at a concentration of 10 ⁇ M.
- IC 50 values may be obtained for kinase activity inhibition rate calculated by a series of different concentrations of the test compound.
- the experimental method is as follows: in a reaction tube, a buffer (8 mM MOPS, pH 7.0, 0.2 mM EDTA, 10 mM MnCl 2 ), a kinase to be tested (5-10 mU), a substrate for the kinase to be tested, and 10 mM magnesium acetate are sequentially added. And ⁇ 33P-ATP solution, as well as different concentrations of test compounds.
- Table 1 gives the IC50 values of partial test compounds for partial kinase inhibitory activity. ("--" in the following tables The test was not tested. )
- test compounds are related to kinases such as Abl, Abl (T315I), c-Src (1-530), c-Src (T341M), B-Raf (V600E), B-Raf, c-RAF.
- Table 2 gives the IC 50 values of the test compound for c-Src (1-530) kinase inhibitory activity.
- Compound IC 50 (nM) Compound IC 50 (nM) 1 4 17 4 5 2 18 4 6 9 19 8 7 19 20 7 8 7 twenty one 7 9 15 twenty four 59 10 83 25 3191 11 10 26 11 12 139 29 5 13 11 30 20 14 3 120 12 16 7 121 15
- Table 3 shows compounds 3, 31, 120 and 121 at a concentration of 10 ⁇ M for Abl(T315I)(h), ALK(h), ARK5(h), Axl(h), Blk(h), Bmx, respectively.
- h BTK(h), B-Raf(h), cKit(h), cSRC(h), CDK7, CHK1(h), c-RAF(h), DDR2(h), EGFR(h), EphA1 (h), EphA2 (h), EphA8 (h), EphB2 (h), EphB4 (h), ErbB2 (h), FAK (h), Fer (h), FGFR1 (h), Flt3 (h), Fms (h), Fyn(h), Hck(h), GSK3 ⁇ (h), IKK ⁇ (h), IKK ⁇ (h), Itk(h), JAK3(h), JNK1 ⁇ 1(h), KDR(h), Lyn (h), MAPK1 (h
- test compounds are Abl(T315I), Axl, Blk, Bmx, BTK, B-Raf, cSRC, c-RAF, DDR2, EphA1, EphA2, EphA8, EphB2, EphB4, ErbB2, FGFR1, Flt3, Fms, Fyn, Hck, IKK ⁇ , IKK ⁇ , Itk, JAK3, KDR, Lyn, PDGFR ⁇ , Ret, RIPK2, Src(1- 530), TAK1, Tec, Tie2, TrkA, ULK3, Yes have better inhibitory activity.
- Some of the test compounds have moderate inhibitory activity against ALK, cKit, EGFR, FAK, Fer, JNK1 ⁇ 1, PKA, and PKB ⁇ .
- Table 4 shows the inhibition rates of kinase activity of Abl, ACK1, ALK, etc., respectively, of Compound 93 and Compound 100 at a concentration of 10 ⁇ M (values indicate the percentage of remaining active protein).
- test compounds were Abl, Abl (T315I), Arg, Blk, Bmx, BRK, cKit (D816H), cKit (V560G), CSK, cSRC, DDR1, EGFR (T790M), EGFR (T790M).
- the purpose of this experiment was to examine the inhibitory activity of the compounds of the present invention against human tumor cell proliferation in vitro, using the MTT (tetramethylazozolium salt) colorimetric method.
- RPMI-1640, fetal calf serum, trypsin, etc. were purchased from Gibco BRL (Invitrogen Corporation, USA), and DMEM medium was purchased from ATCC (American Type Culture Collection). Tetramethylazozolium salt (MTT) and dimethyl sulfoxide (DMSO) are products of Sigma (USA).
- MMTT Tetramethylazozolium salt
- DMSO dimethyl sulfoxide
- Compound 3 Compound 31, Compound 93, and Compound 100 were synthesized by the inventors. In vitro, 100% DMSO was used to prepare a 10 mM stock solution, stored in a refrigerator at -20 ° C in the dark, and diluted to the desired concentration with a complete medium. .
- Cell line and culture Human breast cancer cell lines MDA-MB-231, MCF-7, SKBR-3, BT474, MDA-MB-468, MDA-MB-453, MDA-MB-435, large B used in this experiment
- Cell lymphoma cell line LY-10, HBL-1, human pancreatic cancer cell line Panc-1, Miapaca-2, human lung cancer cell line A549, H358, H1975, human leukemia cell line THP-1, human hepatoma cell line Hepg2, Human melanoma cell line A2058 and the like were purchased from American ATCC (American type culture collection) and kept by the laboratory.
- All of the above lymphoma cell lines, large B cell lymphoma cell lines, and T cell lymphoma cell lines were treated with RPMI-1640 complete medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin. Incubate at %CO2 and 37 °C. The remaining cell lines were cultured in DMEM complete medium containing 10% fetal bovine serum (20% of MV4-11 cells), 100 U/ml penicillin, and 100 ⁇ g/mL streptomycin at 5% CO 2 at 37 °C.
- a cell suspension having a cell concentration of 1 to 2 ⁇ 10 4 /mL was adjusted with a complete cell culture solution, and seeded in a 96-well plate, and 200 ⁇ L of the cell suspension per well was cultured overnight. On the next day, the supernatant was aspirated (the supernatant was aspirated after centrifugation), and then the cells were treated with a gradient concentration of the test compound. At the same time, a drug-free negative control group and an equal volume of solvent control group were used, and the DMSO concentration was 0.1%. Three doses of each well were set in each dose group, and cultured at 37 ° C, 5% CO 2 .
- the proliferation inhibition activity test was carried out, and the results are shown in Table 5.
- Test Compound 3 and Compound 31 have strong inhibitory activities against MV4-11, Miapaca-2, MDA-MB-231, MDA-MB-435, and A375 cells; Test Compound 3 and Compound 31 It also has moderate inhibitory activity against other tumor cell lines including CFPAC, U87, MM.1S, HCT116, HT29, A549 and the like.
- test compound 93 and the compound 100 have strong inhibitory activities against MV4-11, Miapaca-2, MDA-MB-231, MDA-MB-435, and A375 cells; the test compound 93 and the compound 100 It also has moderate inhibitory activity against other tumor cell lines including U251, H4, HCT116, Hela and the like.
- IC 50 ⁇ 100 nM is represented by the symbol ++++
- 100 nM ⁇ IC 50 ⁇ 500 nM is represented by the symbol +++
- 500 nM ⁇ IC 50 ⁇ 1000 nM is represented by the symbol ++
- IC 50 >1000 nM is represented by the symbol +.
- the purpose of this experiment was to test the in vivo antitumor effect of the compounds of the invention.
- This experiment tested the in vivo antitumor activity of inventive compound 3, compound 31, compound 93 and compound 100 using a SCID mouse subcutaneous tumor model.
- the cell line used was a breast cancer cell line MDA-MB-231.
- Dastinib a marketed drug for clinical use of the anti-breast cancer drug paclitaxel and breast cancer, was used as a positive control.
- Fetal bovine serum, trypsin and the like were purchased from Gibco BRL (Invitrogen Corporation, USA), DMEM medium was purchased from ATCC (American Type Culture Collection), and human breast cancer cell line MDA-MB-231 was purchased from ATCC, USA, SCID was small.
- the mouse was purchased from Beijing Huakang Biotechnology Co., Ltd., China.
- Paclitaxel was purchased from Shanghai Muxiang Biotechnology Co., Ltd., China.
- Dasatinib was purchased from Nanjing Kangmanlin Chemical Industry Co., Ltd., China.
- the drug solvent control group was orally administered with a blank solvent of 200 uL per day;
- Compound 3 was orally administered orally at a dose of 30 mg/kg per day;
- Compound 3 was orally administered orally at a dose of 15 mg/kg per day.
- Compound 3 was orally administered orally at a dose of 7.5 mg/kg per day;
- the positive control paclitaxel was administered by tail vein injection at a dose of 10 mg/kg per week; the positive control dasatinib was orally administered orally at a dose of 40 mg/kg per day.
- the drug solvent control group was orally administered with a blank solvent of 200 uL per day;
- Compound 31 was orally administered orally at a dose of 40 mg/kg per day;
- Compound 31 was orally administered orally at a dose of 20 mg/kg per day;
- Compound 31 was orally administered orally at a dose of 10 mg/kg per day;
- the positive control paclitaxel was administered by tail vein injection at a dose of 10 mg/kg per week; the positive control dasatinib was orally administered orally at a dose of 40 mg/kg per day.
- the drug solvent control group was orally administered with a blank solvent of 200 uL per day;
- Compound 93 was orally administered orally at a dose of 40 mg/kg per day;
- Compound 93 was orally administered orally at a dose of 20 mg/kg per day.
- Compound 93 was orally administered orally at a dose of 10 mg/kg per day;
- the positive control paclitaxel was administered by tail vein injection at a dose of 10 mg/kg per week; the positive control dasatinib was orally administered orally at a dose of 40 mg/kg per day.
- the drug solvent control group was orally administered with a blank solvent of 200 uL per day;
- Compound 100 was orally administered orally at a dose of 40 mg/kg per day;
- Compound 100 was orally administered orally at a dose of 20 mg/kg per day;
- Compound 100 was orally administered orally at a dose of 10 mg/kg per day;
- the positive control paclitaxel was administered by tail vein injection at a dose of 10 mg/kg per week; the positive control dasatinib was orally administered orally at a dose of 40 mg/kg per day.
- mice in each group were observed daily for diarrhea, convulsions, rash, and significant weight loss.
- the experimental growth of the experimental group 1 tumor growth curve is shown in Figure 1, the experimentally measured experimental group 2 tumor growth curve As shown in Fig. 2, the experimental growth of the experimental group 1 tumor growth curve is shown in Fig. 3, and the experimentally measured experimental group 2 tumor growth curve is shown in Fig. 4.
- test compound 3 had significant growth inhibition effect on the breast cancer cell line MDA-MB-231, and the tumor growth was significantly inhibited at a dose of 30 mg/kg per day, and showed superiority to the positive control (paclitaxel).
- Compound 31 has significant growth inhibitory effect on breast cancer cell line MDA-MB-231. At a dose of 20 mg/kg per day, it can significantly inhibit tumor growth and show superiority to positive controls (paclitaxel and dasatinib).
- Compound 93 has a significant growth inhibitory effect on breast cancer cell line MDA-MB-231, and can significantly inhibit tumor growth at a dose of 20 mg/kg per day, and exhibits an inhibitory effect superior to the positive control (paclitaxel).
- Compound 100 breast cancer cell line MDA-MB-231 has significant growth inhibition in vivo, and can significantly inhibit tumor growth at a dose of 40 mg/kg per day, and exhibits superiority to positive controls (paclitaxel and dasatinib). Inhibitory effect. No adverse effects such as weight loss, rash, diarrhea, etc. were observed in the mice during the administration, indicating that the test compound 3, the compound 31, the compound 93 and the compound 100 were less toxic in the dose range of the test at the test dose.
- the purpose of this experiment was to examine the inhibitory activity of the inventive compounds on neovascularization in vivo by examining the inhibition of the intercellular vascularity of the transgenic fluorescent zebrafish FLK1-GFP at various concentrations.
- the neovascularization inhibitory activity of the test compound was expressed by the degree of inhibition of the zebrafish internode blood vessels by the test compound at a concentration of 10 ug/mL, 5 ug/mL, and 2.5 ug/mL.
- the marketed drug dasatinib which is clinically studied in breast cancer, was used as a positive control.
- the zebrafish used in the laboratory is a vascular fluorescent transgenic zebrafish (FLK1: GFP).
- FLK1 vascular fluorescent transgenic zebrafish
- the cultivation and cultivation of zebrafish is based on the method of Westerfield. On the day before the eggs were taken, the male and female zebrafish were paired in a 1:1 ratio. The next day, at a temperature of about 28 ° C and sufficient light, they naturally mate to lay eggs. Sufficient zebrafish embryos were collected, washed, placed in embryo culture medium, and placed in a 28 ° C incubator. Survival is identified at any time using morphological and developmental criteria. Dead embryos are white and should be removed in time to prevent deterioration of water quality.
- Drug treatment 10 hours after zebrafish embryo fertilization (microscopic observation of zebrafish embryo development to bud stage), healthy embryos were randomly selected and added into 24-well plates, 10 zebrafish embryos per well, and the culture medium was absorbed. Add different concentrations of compound solution.
- the compound 31 concentration settings were 10 ug/mL, 5 ug/mL, and 2.5 ug/mL, respectively.
- the dasatinib concentration was set at 10 ug/mL.
- a blank control was set, and no compound was added. It was then placed in a 28 ° C incubator for cultivation.
- Figure 5 reflects the vascular inhibition of compound 31 to FLK1 transgenic zebrafish at various concentrations. The results showed that Compound 31 was able to inhibit angiogenesis in zebrafish well compared with the control group. The results of the present experiment indicate that the compound 31 produced in the examples of the present invention has a good inhibitory activity against the angiogenesis of the FLK1 transgenic zebrafish, and this result reflects that the compound 31 has a good inhibitory activity against VEGFR2.
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Abstract
本发明属于化学医药领域，具体涉及3-乙炔基吡唑并嘧啶衍生物及其制备方法和用途。本发明提供了一种3-乙炔基吡唑并嘧啶衍生物，其结构如式Ⅰ所示。本发明还提供了3-乙炔基吡唑并嘧啶衍生物的制备方法和用途，包含该衍生物的药物组合物，以及该化合物和药物组合物用于治疗和预防肿瘤的用途。
Description
本发明属于化学医药领域，具体涉及3-乙炔基吡唑并嘧啶衍生物及其制备方法和用途。
蛋白激酶是一类重要的磷酸转移酶，其功能是催化腺苷三磷酸(ATP)末端的γ-磷酸基团转移到特定底物蛋白质的氨基酸残基上，使蛋白质磷酸化，从而发挥其生理生化功能。蛋白激酶是细胞信号转导通路的关键组件。蛋白激酶参与调控多个细胞过程，包括细胞的生长、能量代谢、细胞周期、转录、凋亡以及分化等。此外，蛋白激酶在维持细胞间联系、体内平衡、免疫系统功能等方面也起到至关重要的作用。蛋白激酶的异常调节与多种疾病特别是肿瘤的发生发展相关。蛋白激酶已成为重要的疾病治疗靶标，引起了广泛的关注。
至2001年首个酪氨酸蛋白激酶药物伊马替尼(Imatinib)上市以来，蛋白激酶药物已成为全球药物市场增长最快的单元，截止2014年已有20多个蛋白激酶药物在全球范围批准上市，其中包括十余种肿瘤治疗药物，例如伊马替尼(Imatinib)，吉非替尼(Gefitinib)，厄洛替尼(Erlotinib)，索拉非尼(Sorafenib)，舒尼替尼(Sunitinib)，达沙替尼(Dasatinib)，尼洛替尼(Nilotinib)，拉帕替尼(Laptinib)，帕唑帕尼(Pazopanib)和瑞格非尼(Regorafenib)等。蛋白激酶类药物的高研发成功率，特别是多种蛋白激酶靶向抗肿瘤药物的研发成功，促使其成为科学及制药工业界的热点研究方向。
当前蛋白激酶药物的研发虽然取得了巨大的成功，但仍然存在很大的发展空间和潜力。特别是研发新型的蛋白激酶抑制剂用于难治性肿瘤、耐药性肿瘤、高转移性肿瘤的治疗，例如三阴性乳腺癌、耐药性肺癌、肝癌、胰腺癌、黑色素瘤、多种白血病等，仍然是当前的研究热点。
发明内容
本发明提供了一种3-乙炔基吡唑并嘧啶衍生物，其结构如式Ⅰ所示：
其中，R1为-H、C1～C4烷基、
R2为-H、C1～C8烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R7独立地为-H、C1～C8烷基、-OH、C1～C8烷氧基、卤素、
R8～R11独立地为-H、C1～C8烷基、卤素、-OH、
R12～R14独立地为
R15～R19独立地为-H、C1～C8烷基、-OH、C1～C8烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、卤素、C1～C8烷基、C1～C8环烷基、-OCF3或-CF3；
R39～R42独立地为C1～C8烷基、C3～C8环烷基或C1～C8羟基烷基；
n＝0～6。
作为本发明优选的技术方案，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、
R8～R11独立地为-H、C1～C4烷基、卤素、-OH、
R12～R14独立地为
R15～R19独立地为-H、
C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、卤素、C1～C4烷基、C1～C4环烷基、-OCF3或-CF3；R39～R42独立地为C1～C4烷基、C3～C8环烷基或C1～C4羟基烷基；n＝0～4。
优选的，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、
R8～R11独立地为-H、C1～C4烷基、-OH、
R12～R14独立地为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、卤素、C1～C4烷基、C1～C4环烷基、-OCF3或-CF3；R39～R42独立地为C1～C4烷基、C3～C8环烷基或C1～C4羟基烷基；n＝0～4。
进一步优选的，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、
R8～R11独立地为-H、C1～C4烷基、-OH、
R12～R14独立地为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、卤素、C1～C4烷基、C1～C4环烷基、-OCF3或-CF3；R39～R42独立地为C1～C4烷基、C3～C8环烷基或C1～C4羟基烷基；n＝0～4。
更进一步优选的，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、
R8～R11独立地为-H、C1～C4烷基、-OH、
R12～R14独立地为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、卤素、C1～C4烷基、C1～C4环烷基、-OCF3或-CF3；n＝0～2。
最优的，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、-F、-Cl、
R8～R11独立地为-H、C1～C4烷基、-OH、
R12～R14独立地为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、-F、-Cl、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3；n＝0或1。
上述3-乙炔基吡唑并嘧啶衍生物，当R6为
时，其结构如式Ⅱ所示：
其中，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R1为-H或
R2为C1～C4烷基、R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R1为-H或
R2为C1～C4烷基、R8取代的C3～C8环烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
再进一步优选的，R1为-H或
R2为C1～C4烷基、R8取代的C3～C8环烷基、
n＝0～2；R8～R11独立地为-H、C1～C4烷基、-OH、
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
更进一步优选的，R1为-H或
R2为C1～C4烷基、R8取代的C3～C8环烷基、
n＝0或1；R8～R11独立地为-H、C1～C4烷基、-OH、
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R3～R5、R7独立地为-H、C1～C4烷基或卤素；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4
烷基、-OH、
R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R3～R5、R7独立地为-H、C1～C4烷基、-F或-Cl；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
更进一步优选的，R3～R5、R7独立地为-H、甲基或-Cl；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R12为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～
C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R15～R19独立地为-H、C1～C4烷基、C1～C4烷氧基、卤素、-CF3、-OCF3或
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R12为
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R15～R19独立地为-H、C1～C4烷基、甲氧基、-F、-Cl、-CF3、-OCF3或
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R12为
R20～R38独立地为-H、C1～C4烷基或-CF3。
最优的，R1为-H或
R2为C1～C4烷基、R8取代的C3～C8环烷基、
n＝0或1；R3～R5、R7独立地为-H、甲基或-Cl；R8～R11独立地为-H、C1～C4烷基、-OH、
R12为
R15～R19独立地为-H、C1～C4烷基、甲氧基、-F、-Cl、-CF3、-OCF3或
R20～R38独立地为-H、C1～C4烷基或-CF3。
上述3-乙炔基吡唑并嘧啶衍生物，当R6为
时，其结构如式Ⅲ所示：
其中，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R2为C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8、R9独立地为-H、C1～C4烷基、-OH、
R1为-H或
R3～R5、R7独立地为
-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R2为C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R8、R9独立地为-H、C1～C4烷基、-OH、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
再进一步优选的，R2为C1～C4烷基、R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R8、R9独立地为-H、C1～C4烷基、-OH、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
更进一步优选的，R2为C1～C4烷基、R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R8、R9独立地为-H、C1～C4烷基、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R3～R5、R7独立地为-H、C1～C4烷基、-OH或卤素；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R3～R5、R7独立地为-H、C1～C4烷基、-OH或-Cl；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地
为-H、C1～C4烷基、-OH、
R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R13为
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R15～R19独立地为-H、C1～C4烷基、C1～C4烷氧基、卤素、-CF3或
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R13为
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R15～R19独立地为-H、C1～C4烷基、卤素、-CF3或
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R13为
R20～R38独立地为-H、C1～C4烷基或-CF3。
更进一步优选的，R15～R19独立地为-H、C1～C4烷基、-CF3或
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R13为
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R20～R38独立地为-H或C1～C4烷基；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R13为
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
最优的，R1为-H或
R2为C1～C4烷基、R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R5、R7独立地为-H、C1～C4烷基、-OH或-Cl；R8、R9独立地为-H、C1～C4烷基、
R13为
R15～R19独立地为-H、C1～C4烷基、-CF3或
R20～R38独立地为-H或C1～C4烷基。
上述3-乙炔基吡唑并嘧啶衍生物，当R6为
时，其结构如式Ⅳ所示：
其中，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
优选的，R2为C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧
烷基、
n＝0～4；R8、R9独立地为-H、C1～C4烷基、-OH、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
进一步优选的，R2为C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R8、R9独立地为-H、C1～C4烷基、-OH、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
再进一步优选的，R2为C1～C4烷基、R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R8、R9独立地为-H、C1～C4烷基、-OH、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
更进一步优选的，R2为C1～C4烷基、R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R8、R9独立地为-H、C1～C4烷基、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
优选的，R3～R5、R7独立地为-H、C1～C4烷基、-OH或卤素；R1为-H或
R2为-H、
C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
进一步优选的，R3～R5、R7独立地为-H、C1～C4烷基、-OH或-Cl；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
优选的，R15～R19独立地为-H、C1～C4烷基、C1～C4烷氧基、卤素、-CF3或
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
进一步优选的，R15～R19独立地为-H、C1～C4烷基、卤素、-CF3或
R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
更进一步优选的，R15～R19独立地
为-H、C1～C4烷基、-CF3或
最优的，R1为-H或
R2为C1～C4烷基、R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R5、R7独立地为-H、C1～C4烷基、-OH或-Cl；R8、R9独立地为-H、C1～C4烷基、
R15～R19独立地为-H、C1～C4烷基、-CF3或
上述3-乙炔基吡唑并嘧啶衍生物，当R6为
R14为
时，其结构如式Ⅴ所示：
其中，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立
地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R2为C1～C4烷基、
C3～C8环氧烷基、
n＝0或1；R9为C1～C4烷基、-OH、
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R2为C1～C4烷基、
R9为C1～C4烷基、-OH或
R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
再进一步优选的，R2为C1～C4烷基或
R9为C1～C4烷基；R1为-H或
R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R3～R5、R7独立地为-H、C1～C4烷基、-OH或卤素；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4
烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
进一步优选的，R3～R5、R7独立地为-H、C1～C4烷基或卤素；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
更进一步优选的，R3～R5、R7独立地为-H、C1～C4烷基或-Cl；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R8～R11独立地为-H、C1～C4烷基、-OH、
R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、C1～C4烷基或-CF3。
优选的，R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素或-CF3；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
进一步优选的，R15～R19独立地为-H、C1～C4烷基、卤素或-CF3；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
更进一步优选的，R15～R19独立地为-H或-CF3；R1为-H或
R2为-H、C1～C4烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、
最优的，R1为-H；R2为C1～C4烷基或
R3～R5、R7独立地为-H、C1～C4烷基或-Cl；R9为C1～C4烷基；R15～R19独立地为-H或-CF3。
上述3-乙炔基吡唑并嘧啶衍生物，其结构式为：
本发明还提供了上述3-乙炔基吡唑并嘧啶衍生物的制备方法：
当R6为
时，式Ⅱ所示化合物的合成路线为：
当R6为
R13为
时，式Ⅲ所示化合物的合成路线为：
当R6为
R13为
时，式Ⅳ所示化合物的合成路线为：
当R6为
R14为
时，式Ⅴ所示化合物的合成路线为：
上述反应式中的反应条件为：
a、化合物1在通常的卤代试剂(如NIS(N-碘代丁二酰亚胺)、NBS(N-溴代丁二酰亚胺)、Br2、I2、ICl、IBr)进行卤代反应制备得到化合物2。
b、R2的卤代烷(溴带或者碘代)或相应的磺酸酯(甲磺酸酯、对甲苯磺酸酯、对硝基苯磺酸酯等)在碱性条件下(如KOH、NaOH、K2CO3、Na2CO3，Cs2CO3、NaH)与化合物2取代制得化合物3。
c、相应的含羧基化合物在通常的缩合条件下(如缩合剂法、混合酸酐法、活化发等)与相应的含氨基化合物缩合得到对应的以酰胺键连接的化合物7～10。
d、使相应的中间体在过渡金属催化下与一端带保护基团的炔试剂进行偶联反应得到相应的中间体，然后再脱保护得到相应的含有炔基的化合物4、化合物6、化合物7、化合物10及化合物13。
e、相应的含卤素的中间体与含炔基的中间体在过渡金属催化下进行偶联反应得到通式Ⅱ、Ⅲ、Ⅳ、Ⅴ所示化合物，偶联反应采用钯催化剂(如Pd4(PPh3)4、PdAc2、Pd2(dba)3、PdPPh3Cl2等)、铜盐(如氯化亚铜、溴化亚酮、碘化亚铜等)，和适当的有机碱或者无机碱(如三乙胺、DIPEA(二异丙基乙胺)、碳酸钾、碳酸钠、碳酸氢钠等)在适当溶剂(如THF(四氢呋喃)、甲苯、DMF(N,N-二甲基甲酰胺)、1,4-二氧六环等)中于20～150℃反应得到。
f、化合物12在相应的醇溶液(如甲醇、乙醇等)、浓硫酸催化下得到相应的酯化中间体。
g、化合物14在适当的碱(如三乙胺、DIPEA等)和溶剂(如EA(乙酸乙酯)、THF、二氯甲烷等)及温度下(0-50度)与三光气反应得到化合物15。
h、化合物15与化合物6在适当的溶剂(如EA(乙酸乙酯)、THF、二氯甲烷、甲苯、DMF等)中于20～120℃反应得到得到化合物16。
其中，R1为-H、C1～C4烷基、
R2为-H、C1～C8烷基、
R8取代的C3～C8环烷基、
C3～C8环氧烷基、
R3～R7独立地为-H、C1～C8烷基、-OH、C1～C8烷氧基、卤素、
R8～R11独立地为-H、C1～C8烷基、卤素、-OH、
R12～R14独立地为
R15～R19独立地为-H、C1～C8烷基、-OH、C1～C8烷氧基、卤素、-CF3、-OCF3、
R20～R38独立地为-H、卤素、C1～C8烷基、C1～C8环烷基、-OCF3或-CF3；R39～R42独立地为C1～C8烷基、C3～C8环烷基或C1～C8羟基烷基；n＝0～6。
本发明上述的3-乙炔基吡唑并嘧啶衍生物，包括了它们的同位素化合物、外消旋体、旋光活性异构体、多晶型形式或其混合物。
本发明还提供了上述3-乙炔基吡唑并嘧啶衍生物药学上可接受的盐。
本发明还提供了本发明化合物的前药，依据本发明，前药是上述化合物的衍生物，它们自身可能具有较弱的活性或甚至没有活性，但是在给药后，在生理条件下(例如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。
本发明还提供了上述3-乙炔基吡唑并嘧啶衍生物药学上可接受的水合物。
本发明还提供一种药物组合物，是由本发明提供的上述3-乙炔基吡唑并嘧啶衍生物添加药学上可以接受的辅助性成分制备而成的。本发明提供的3-乙炔基吡唑并嘧啶衍生物结构如式Ⅰ～Ⅴ所示。
本发明还提供了上述3-乙炔基吡唑并嘧啶衍生物、它的盐或水合物在制备激酶抑制剂中的用途。
进一步的，上述激酶抑制剂为抑制SRC家族络氨酸蛋白激酶(Blk络氨酸蛋白激酶、
Fgr络氨酸蛋白激酶、Frk络氨酸蛋白激酶、Fyn络氨酸蛋白激酶、Hck络氨酸蛋白激酶、Lck络氨酸蛋白激酶、Lyn络氨酸蛋白激酶、c-SRC络氨酸蛋白激酶、YES酪氨酸蛋白激酶)、FLT3(人FMS样酪氨酸激酶3)、Abl(Abl络氨酸蛋白激酶)、VEGFR1(血管内皮生长因子受体1)、VEGFR2(血管内皮生长因子受体2)、VEGFR3(血管内皮生长因子受体3)、RET(RET受体酪氨酸激酶)、c-RAF(c-RAF丝氨酸/苏氨酸蛋白激酶)、B-RAF(B-RAF丝氨酸/苏氨酸蛋白激酶)、c-KIT(酪氨酸蛋白激酶KIT)、PDGFα(血小板衍生生长因子受体α)、PDGFβ(血小板衍生生长因子受体β)、FGFR1(成纤维细胞生长因子受体1)、FGFR2(成纤维细胞生长因子受体2)、FGFR3(成纤维细胞生长因子受体3)、EphA2(EphA2酪氨酸激酶)、EphB2(EphB2酪氨酸激酶)、EphB4(EphB4酪氨酸激酶)、ALK(间变性淋巴瘤激酶)、Met(Met酪氨酸激酶)DDR1(DDR1络氨酸激酶)、DDR2(DDR2络氨酸激酶)、Btk(Btk络氨酸激酶)、BMX(BMX络氨酸激酶)、TAK1(转化生长因子激酶1)、Arg(Arg络氨酸蛋白激酶)、BRK(BRK络氨酸激酶)、CSK(CSK络氨酸激酶)、EGFR(T790M)(T790M突变型表皮生长因子受体络氨酸激酶)、EGFR(T790M,L858R)(T790M、L958R双突变型表皮生长因子受体络氨酸激酶)、Flt1(FLT1络氨酸激酶)、Flt4(Flt4络氨酸激酶)、LIMK1(人单丝氨酸蛋白激酶1)、Mer(Mer络氨酸激酶)、PTK5(PTK5络氨酸激酶)、Pyk2(Pyk2络氨酸激酶)、Ret(Ret络氨酸激酶)、SAPK2b(应激活化蛋白激酶2b)、Tie2(Tie2络氨酸激酶)、Txk(Txk络氨酸激酶)中至少一种激酶的药物。
本发明还提供了上述3-乙炔基吡唑并嘧啶衍生物、它的盐或水合物在制备抗肿瘤药物中的用途。
进一步的，上述肿瘤为白血病或实体瘤。
进一步的，上述实体肿瘤为肺癌、乳腺癌、胰腺癌、黑色素瘤、神经胶质瘤、肝癌、甲状腺瘤、宫颈癌、胃癌或结直肠癌中的至少一种。其中，上述的白血病为急性髓性白血病或混合型白血病。
本发明提供的3-乙炔基吡唑并嘧啶衍生物对人乳腺癌、人肺癌、人胰腺癌、人恶性黑色素瘤、人白血病等肿瘤均表现出了良好的抑制效果。
图1 化合物3对裸小鼠的体内药效学实验。
图2 化合物31对裸小鼠的体内药效学实验。
图3 化合物93对裸小鼠的体内药效学实验。
图4 化合物100对裸小鼠的体内药效学实验。
图5 化合物31在不同浓度下对FLK1转基因斑马鱼的血管抑制情况。
实施例1 3-乙炔基-1-异丙基-1H-吡唑并[3,4-d]嘧啶-4-胺(中间体1)的制备
第一步，制备3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺：
1H-吡唑并[3,4-d]嘧啶-4-胺(20g,148.0mmol,1.0eq)于三口瓶中，加入150mL DMF(N,N-二甲基甲酰胺)，搅拌并氮气置换3次，加入NIS(N-碘代丁二酰亚胺)(50g,222.0mmol,1.5eq)后于80℃反应，TLC监控，22h后反应完毕。停止反应，减压浓缩DMF至剩一半，加入饱和Na2O3S2水溶液150mL搅拌，减压过滤，滤饼依次用饱和Na2O3S2水溶液及水洗涤至滤液无色，真空干燥得到目标产物，淡黄色粉末(33.9g，收率87.8％)。1H NMR(400MHz,DMSO-d6)δ8.17(s,1H)。MS m/z(ESI):262.1[M+H]。
第二步，制备3-碘-1-异丙基-1H-吡唑并[3,4-d]嘧啶-4-胺：
3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺(5g,19.2mmol,1.0eq)于三口瓶中，加入40mL DMF及碳酸钾(5.3g,38.4mmol,2.0eq)后，用氮气置换3次，加入2-溴丙烷(1.9mL,20.1mmol,1.05eq)后于80℃反应，TLC监控，4h后反应完毕。停止反应，减压蒸去DMF，用DCM(二氯甲烷)及水萃取3次，合并DCM层后蒸干，用体积比为EA(乙酸乙酯)︰PE(石油醚)＝1︰3的混合溶剂重结晶得目标产物，重结晶母液中目标产物可经柱层析分离得到，淡黄色粉末(5.4g,收率92.8％)。1H NMR(400MHz,DMSO-d6)δ8.19(s,1H),4.99-4.93(m,1H),1.42(d,J＝6.7Hz,6H)。MS m/z(ESI):304.0[M+H]。
第三步，制备1-异丙基-3-((三甲基甲硅烷基)乙炔基)-1H-吡唑并[3,4-d]嘧啶-4-胺：
3-碘-1-异丙基-1H-吡唑并[3,4-d]嘧啶-4-胺(5.4g,17.8mmol,1.0eq)于三口瓶中，加入DMF 40mL,CuI(339mg,1.78mmol,0.1eq),Pd(PPh3)4(1g,0.89mmol,0.05eq)，氮气置换3次后加入三乙胺(5mL,35.6mmol,2.0eq)，三甲基硅乙炔(2.65mL,18.7mmol,1.05eq)，于80℃反应2.5h，TLC监控反应完毕后减压蒸去DMF，余下残余物经柱层析分离得目标产物，直接用于下步反应。MS m/z(ESI):274.2[M+H]。
第四步，制备3-乙炔基-1-异丙基-1H-吡唑并[3,4-d]嘧啶-4-胺：
1-异丙基-3-((三甲基甲硅烷基)乙炔基)-1H-吡唑并[3,4-d]嘧啶-4-胺于20mL MeOH(甲醇)中，加入碳酸钾(4.9g,35.6mmol,2.0eq)室温搅拌10min，TLC监控反应完毕，减压蒸去MeOH后用水分散，DCM洗涤水层3次，合并DCM层，蒸干后经柱层析分离得中间体1，淡灰色粉末(1.6g，收率44.7％)。1H NMR(400MHz,DMSO-d6)δ8.22(s,1H),5.02–4.98(m,1H),4.62(s,1H),1.44(d,J＝6.7Hz,6H)。MS m/z(ESI):202.1[M+H]。
实施例2 中间体2～10的制备
采用不同卤代烷烃与1H-吡唑并[3,4-d]嘧啶-4-胺反应，添加碳酸铯、碳酸钾、DIPEA(二异丙基乙胺)或者其他无机或有机碱，PdCl2(PPh3)2或Pd(PPh3)4等原料，通过与制备中间体1所述类似的方法得到以下中间体2～10。
实施例3 3-碘-1-(1-(甲磺酰)哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺(中间体11)的制备
第三步，制备3-碘-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺：
叔丁基-3-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-羧酸酯(2.9g)于甲醇35mL中溶解，加入4MHCl二氧六环溶液35mL，室温搅拌8h后析出固体，然后将反应液降温至0℃后减压过滤，滤饼分散于水中，调PH至8后析出固体，减压过滤，水洗固体，固体于乙醇中，减压蒸去乙醇后真空干燥得目标产物(2.2g，收率93.1％)。MS m/z(ESI):345.1[M+H]。
第四步，制备3-碘-1-(1-(甲磺酰)哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺：
3-碘-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺(172.1mg,0.5mmol,1.0eq)于4mL DCM中，加入DIPEA(129.24mg,1.0mmol,2.0eq)后于0℃搅拌，缓慢滴加甲磺酰氯(57.3mg,0.5mmol,1.0eq)，加毕后TLC，确认反应完毕后反应液依次用饱和碳酸氢钠水溶液，饱和氯化钠水溶液以及水洗涤，DCM层经无水硫酸镁干燥，过滤后蒸干得中间体11，白色粉末(197.8mg，收率93.7％)。1H NMR(400MHz,DMSO-d6)δ8.23(s,1H),4.83-4.65(m,1H),3.69(d,J＝7.2Hz,1H),3.59(d,J＝11.1Hz,1H),3.07(t,J＝10.9Hz,1H),2.91(s,3H),2.80(t,J＝10.9Hz,1H),2.45-2.00(m,2H),1.95(d,J＝13.6Hz,1H),1.76-1.59(m,1H)。MS m/z(ESI):423.0[M+H]。
实施例4 中间体12～19的制备
以不同烷基醇或烷基磺酸酯与1H-吡唑并[3,4-d]嘧啶-4-胺反应，添加碳酸铯、碳酸钾、
其他无机或有机碱，甲磺酰氯或者对硝基甲磺酰氯，HCl乙醇溶液，HCl乙醚溶液，三氟乙酸等原料，通过与制备中间体11所述类似的方法得到以下中间体12～19。
实施例5 (R)-3-碘-1-(1-甲基哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺(中间体20)的制备
(R)-3-碘-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺(750mg,2mmol,1.0eq)溶于18mL 1,2-二氯乙烷︰甲醇＝8︰1(体积比)的混合溶剂中，缓慢滴加甲醛水溶液(37％,0.82mL,10mmol,5.0eq)，加毕后室温搅拌10min，分两批加入NaBH3CN(502.7mg,8mmol,4.0eq)，10min后TLC监控反应完毕，向反应液中滴加水3mL，加毕后减压蒸干溶剂，残余物分散于水中，调pH至碱性，过滤，水洗固体，然后固体于乙醇中减压蒸干，残余物经重结晶得中间体20，白色粉末(500mg，收率70.0％)。1H NMR(400MHz,DMSO-d6)δ8.20(s,1H),4.73–4.66(m,1H),4.41(t,J＝4.8Hz,1H),3.48-3.41(m,1H),2.88(dd,J＝11.1,3.7Hz,1H),2.77(d,J＝11.1Hz,1H),2.21(s,3H),1.97-1.72(m,3H),1.71-1.61(m,1H)。MS m/z(ESI):359.1[M+H]。
实施例6 中间体21～24的制备
以不同带芳基的仲胺与1H-吡唑并[3,4-d]嘧啶-4-胺反应，添加烷基醛或者芳基醛等原料，通过与制备中间体20所述类似的方法得到以下中间体21～24。
实施例7 N,1-二乙基-3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺(中间体25)的制备
3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺(2.6g，10mmol，1.0eq)于三口瓶中，加入35mL DMF及碳酸铯(9.8g，30mmol，3.0eq)后，用氮气置换3次，加碘乙烷(2.0mL，25mmol，2.5eq)后于100℃反应，10h后TLC监控。反应完毕后停止反应，减压蒸去DMF，用DCM(二氯甲烷)及水萃取3次，合并DCM层后蒸干，经柱层析分离得到中间体25，米白色粉末(1.75g，收率55.1％)。MS m/z(ESI):318.0[M+H]。
实施例8 (R)-1-((R)-3-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-基)-2-羟基丙烷-1-酮(中间体26)的制备
将D-2-羟基丙酸(157mg,1.74mmol,1.2eq)，HOBT(1-羟基苯并三唑)(235.1mg,1.74mmol,1.2eq)，EDCI(1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐)(417mg,2.2mmol,1.5eq)溶于DMF中，加入三乙胺(0.61mL,4.35mmol,3.0eq)，室温搅拌0.5h后加入(R)-3-碘-1-(哌啶-3-基)-1H-吡唑并[3,4-d]嘧啶-4-胺(500mg,1.45mmol,1.0eq)室温反应约5h，完毕后蒸干DMF，用饱和碳酸氢钠水溶液同二氯甲烷萃取2次，合并有机层后蒸干，残余物经柱层析分离得中间体26，白色固体(442mg，收率73.3％)。
1H NMR(400MHz,DMSO-d6)δ8.22(s,1H),5.04–4.94(m,1H),4.77-4.51(m,1H),4.50–4.44(m,1H),4.40-4.26(m,1H),4.19-4.03(m,1H),3.13–2.98(m,1H),2.76(t,J＝12.1Hz,1H),2.25-2.11(m,1H),2.06–2.04(m,1H),1.86(t,J＝13.8Hz,1H),1.62–1.49(m,1H),1.24–1.13(m,3H)。MS m/z(ESI):417.1[M+H]。
实施例9 中间体27～32的制备
以不同带芳基的仲胺，不同构型的乳酸等原料，采用与制备中间体25所述类似的方法得到以下中间体27～32。
实施例10 顺/反-4-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)环己烷-1-醇(中间体33及34)的制备
第一步：参照实施例3第一步所述的方法，以4-羟基环己酮乙二醇缩醛及甲基磺酰氯为原料合成得目标产物。MS m/z(ESI):236.1[M+H]。
第二步，制备3-碘-1-(1,4-二氧杂螺[4.5]癸烷-8-基)-1H-吡唑并[3,4-d]嘧啶-4-胺：
参照实施例3第二步所述方法以1,4-二氧杂螺[4.5]癸烷-8-基甲磺酸酯及3-碘-1H-吡唑并[3,4-d]嘧啶-4-胺为原料合成得目标产物。1H NMR(400MHz,DMSO-d6)δ8.19(s,1H),4.79-4.63(m,1H),3.98-3.82(m,4H),2.19-2.11(m,2H),1.91-1.64(m,6H)。MS m/z(ESI):402.2[M+H]。
第三步，制备4-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)环己烷-1-酮：
3-碘-1-(1,4-二氧杂螺[4.5]癸烷-8-基)-1H-吡唑并[3,4-d]嘧啶-4-胺(1.94g,4.84mmol)于40mL丙酮中，加入1MHCl 20mL，于70℃3h后降至室温反应过夜，完毕后减压蒸去丙酮，余下水溶液调pH至10，析出大量固体，过滤，干燥得目标产物，白色固体(1.68g，收率97.1％)。1H NMR(400MHz,DMSO-d6)δ8.27(s,1H),5.21–5.15(m,1H),2.75-2.61(m,2H),2.37–2.26(m,4H),2.20–2.17(m,2H)。MS m/z(ESI):358.1[M+H]。
第四步，制备4-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)环己烷-1-醇：
4-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)环己烷-1-酮(1.68g,4.7mmol,1.0eq)于甲醇中，降温至0℃后分批加入硼氢化钠(183mg，4.7mmol,1.0eq)，加毕后自然升至室温反应，1h后反应完毕，加入5mL水搅拌5min后蒸干溶剂，残余物经二氯甲烷与饱和碳酸氢钠水溶液萃取后柱层析得中间体，前方点(Rf＝0.63,DCM/MeOH＝8)为中间体33，顺-4-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)环己烷-1-醇，白色固体(375mg，收率22.2％)。1H NMR(400MHz,DMSO-d6)δ8.19(s,1H),4.64–4.60(m,1H),4.49(d,J＝2.4Hz,1H),3.88(s,1H),2.41–2.20(m,2H),1.87–1.69(m,2H),1.66–1.57(m,4H)。MS m/z(ESI):360.2[M+H]。后方点(Rf＝0.55,DCM/MeOH＝8)为中间体34，反-4-(4-氨基-3-碘-1H-吡唑并[3,4-d]嘧啶-1-基)环己烷-1-醇，白色固体(844mg，收率50.1％)。1H NMR(400MHz,DMSO-d6)δ8.19(s,1H),4.68(d,J＝4.3Hz,1H),4.62-4.50(m,1H),3.56-3.49(m,1H),2.03-1.79(m,5H),1.46-1.29(m,3H)。MS m/z(ESI):360.2[M+H]。
实施例11 N-(3-碘-4-甲基苯基)-4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯酰胺(中间体35)的制备
第一步，制备4-甲基-3-三氟甲基-苯甲酸甲酯：
4-甲基-3-(三氟甲基)苯甲酸(2.04g,10mmol,1.0eq)于25mL MeOH中，搅拌下加入2mL
浓硫酸，回流反应，TLC监控，24h小时后反应完毕，蒸干MeOH，DCM溶解，依次用饱和碳酸氢钠水溶液，饱和氯化钠水溶液以及水洗涤，DCM层经无水硫酸镁干燥，过滤后蒸干得产品(1.85g，收率84.8％)用于下步。
第二步，制备4-溴甲基-3-三氟甲基-苯甲酸甲酯：
4-甲基-3-(三氟甲基)苯甲酸甲酯溶于1,2-二氯乙烷(DCE)中，搅拌下加入NBS(N-溴代丁二酰亚胺)(1.81g,10.18mmol,1.2eq)，AIBN(偶氮二异丁腈)(0.139g,0.848mmol,0.1eq)后氮气置换3次，80℃反应30h后，反应液依次用饱和碳酸氢钠水溶液，饱和氯化钠水溶液以及水洗涤，DCE层经硫酸镁干燥后，蒸干得产物直接用于下步，按收率80％计。
第三步，制备4-(4-甲基哌嗪-1-基甲基)-3-三氟甲基-苯甲酸甲酯：
4-溴甲基-3-三氟甲基苯甲酸甲酯(6.78mmol,1.0eq)，三乙胺(1.03g,10.2mmol,1.5eq)，氮甲基哌嗪(0.681g,6.8mmol,1.0eq)于氯仿中，80℃反应，1h后反应完毕。反应液依次用饱和碳酸氢钠水溶液，饱和氯化钠水溶液及水洗涤，氯仿层蒸干后残余物经柱层析分离得目标产物，淡黄色油状物(1.72g,收率79.9％)。MS m/z(ESI):317.2[M+H]。
第四步，制备4-(4-甲基哌嗪-1-基甲基)-3-三氟甲基-苯甲酸：
4-(4-甲基哌嗪-1-基甲基)-3-三氟甲基-苯甲酸甲酯(1.72g，5.4mmol)于20ml乙醇中，加入5M NaOH水溶液3ml，室温搅拌过夜后pH至6，减压浓缩，浓缩液加入四氢呋喃后减压过滤，滤液蒸干得目标产物，淡黄色粉末(1.47g，收率89.9％)。
第五步，制备N-(3-碘-4-甲基苯基)-4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯酰胺：
4-(4-甲基哌嗪-1-基甲基)-3-三氟甲基-苯甲酸(0.755g，2.5mmol，1.0eq)，HATU(O-(7-偶氮苯并三氮唑-1-氧)-N,N,N’,N’-四甲基脲鎓六氟磷酸盐)(1.14g，3mmol，1.2eq)，DIPEA(1.29g，10mmol，4.0eq)溶于二氯甲烷中室温搅拌0.5h，加入3-碘-4-甲基苯胺(582.6mg，2.5mmol，1.0eq)后于45℃反应过夜，反应完毕后蒸干反应液，饱和碳酸氢钠水溶液与二氯甲烷萃取三次，合并有机层后减压浓缩，残余物经柱层析分离得目标产物，淡黄色固体(892mg，收率69.0％)。1H NMR(400MHz,CDCl3)8.10(s,1H),8.08(d,J＝2Hz,1H),8.08-8.05(m,1H),8.0(s,1H),7.95(s,1H),7.55(dd,J＝4,2Hz,1H),7.20(d,J＝8Hz,1H),3.75(s,2H)2.60-2.41(m,8H),2.40(s,3H),2.30(s,3H)。MS m/z(ESI):518.1[M+H]。
实施例12 N-(3-碘-4-甲基苯基)-4-((4-甲基哌嗪-1-基)甲基)苯酰胺(中间体36)
按照中间体35所述第五步类似的缩合方法得到中间体36，淡黄色固体(532mg，盐酸盐，收率76.7％)。1H NMR(400MHz,DMSO-d6)δ10.35(br.s,1H),10.26(s,1H),8.34(s,1H),7.95(d,J＝7.6Hz,2H),7.72(d,J＝8.3Hz,1H),7.47(d,J＝7.6Hz,2H),7.30(d,J＝8.3Hz,1H),3.64(s,2H),3.39(br.s,2H),3.01(br.s,2H),2.87(br.s,2H),2.73(s,3H),2.42(br.s,2H),2.34(s,3H)。MS m/z(ESI):450.1[M+H]。
实施例13 N-(3-乙炔基-4-甲基苯基)-4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯酰胺(中间体37)的制备
第一步，制备3-三甲基硅乙炔基-4-甲基苯胺：
将3-碘-4-甲基苯胺(11.65g，50mmol，1.0eq)、碘化亚铜(0.9g，4.7mmol，0.1eq)、双三苯基膦二氯化钯(1.75g，2.5mmol，0.05eq)溶于二氧六环中，氮气置换三次，加入DIPEA(1.29g，100mmol，2.0eq)、三甲基硅乙炔(6.4g，65mmol，1.3eq)。升温至75℃反应，17h后反应完毕，减压蒸干溶剂，残留物经柱层析分离得到目标产品，直接用于下步反应。
第二步，制备3-乙炔基-4-甲基苯胺：
3-三甲基硅乙炔基-4-甲基苯胺用甲醇溶解，加入碳酸钾(3.45g，25mmol，0.5eq)，常温搅拌10min后反应完毕。减压蒸干溶剂，残留物用DCM、水萃取3次，DCM层干燥后蒸干，得到油状物，经柱层析分离得到产品3-乙炔基-4-甲基苯胺，黄棕色油状物(4.0g，收率61.0％)。1H NMR(400MHz,CDCl3)δ6.99(d,J＝8.1Hz,1H),6.81(s,1H),6.61(d,J＝8.1Hz,1H),3.44(s,2H),3.23(s,1H),2.34(s,3H)。MS m/z(ESI):132.2[M+H]。
第三步，制备N-(3-乙炔基-4-甲基苯基)-4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯酰胺：
按照中间体35第五步所述类似的缩合方法得到中间体37，淡黄色固体(1.2g，盐酸盐，收率71.7％)。1H NMR(400MHz,DMSO-d6)δ11.24(br.s,1H),10.58(s,1H),8.33(d,J＝6.4Hz,2H),8.14(s,1H),7.92(s,1H),7.72(d,J＝8.3Hz,1H),7.28(d,J＝8.3Hz,1H),4.39(s,1H),4.06(s,2H),3.47(d,J＝10.9Hz,2H),3.22(br.s,2H),3.13(br.s,2H),2.89(br.s,2H),2.77(s,3H),2.36(s,3H)。MS m/z(ESI):416.3[M+H]。
实施例14 N-(3-乙炔基-4-甲基苯基)-6-(三氟甲基)甲基吡啶酰胺(中间体38)的制备
将6-三氟甲基-吡啶-2-甲酸(229.33mg，1.2mmol，1.2eq)用二氯亚砜溶解，80℃回流3h，减压蒸干二氯亚砜，得到6-(三氟甲基)吡啶-2-甲酰氯，无色油状物，用DCM溶解。将3-乙炔基-4-甲基苯胺(131.17mg，1.0mmol，1.0eq)和三乙胺(202.4mg，2.0mmol，2.0eq)用DCM溶解，冷却至-5℃，将6-三氟甲基-吡啶-2-甲酰氯DCM溶液缓慢滴加到其中。滴加完毕后，自然升温至常温，15min后反应完毕。往反应液中加入氢氧化钠水溶液，DCM萃取2次。合并DCM层，再依次用稀盐酸、水萃取，DCM层用无水硫酸镁干燥，蒸干得中间体38，白色固体(225mg，收率73.9％)。1H NMR(400MHz,DMSO-d6)δ10.47(s,1H),8.08-8.05(m,2H),7.93(dd,J＝7.3,1.4Hz,1H),7.76(d,J＝2.2Hz,1H),7.54(dd,J＝8.3,2.2Hz,1H),7.06(d,J＝8.3Hz,1H),4.16(s,1H),2.13(s,3H)。MS m/z(ESI):305.1[M+H]。
实施例15 中间体39～64的制备
以含不同取代基的芳基甲酸、芳基酰氯、五元杂环酰氯及含不同取代基的苯胺为原料，采用与制备中间体38所述类似的方法得到以下中间体39～64。
实施例16 2-叔丁基-N-(3-乙炔基-4-甲基苯基)噻唑-5-甲酰胺(中间体65)的制备
第一步，制备2-叔丁基-噻唑-5-羧酸乙酯：
将3-溴丙酮酸乙酯(1.95g，10mmol，1.0eq)和2,2,2-三甲基硫代乙酰胺(1.17g，10mmol，1.0eq)加入到20mL乙醇中，常温搅拌48h。反应完毕后，减压蒸干溶剂，残留物用碳酸氢钠水溶液与DCM萃取，DCM层用无水硫酸镁干燥后蒸干，残留物经柱层析分离得到2-叔丁基-噻唑-5-羧酸乙酯，无色油状物(1.15g，收率55.0％)。1H NMR(400MHz,CDCl3)δ8.01(s,1H),4.38(q,J＝7.1Hz,2H),1.46(s,9H),1.37(t,J＝7.1Hz,3H)。MS m/z(ESI):214.0[M+H]。
第二步，制备2-叔丁基-噻唑-5-羧酸：
将2-叔丁基-噻唑-5-羧酸乙酯溶于10mL THF，加入氢氧化锂(0.45g，10.9mmol，1.1eq)水溶液5mL，常温搅拌约16h。反应完毕后，减压蒸干溶剂，残留物用5mL水溶解，用2M HCl调节pH至约为3，析出大量白色絮状固体，抽滤，用水(2×5mL)洗涤滤饼，得到2-叔丁基-噻唑-5-羧酸，白色固体(0.55g，收率55.0％)。MS m/z(ESI):184.0[M-H]。
第三步，制备2-叔丁基-噻唑-5-羰基氯化：
将2-叔丁基-噻唑-5-羧酸(203.7mg，1.1mmol)溶于3ml二氯亚砜中，80℃回流2h后减压蒸干溶剂，直接用于下一步。
第四步，制备2-叔丁基-N-(3-乙炔基-4-甲基苯基)噻唑-5-甲酰胺：
将3-乙炔基4-甲基-苯胺(131.2mg，1.0mmol，1.0eq)溶于4mL DCM，加入三乙胺(151.8mg，1.5mmol，1.5eq)，降温至0℃后缓慢滴加上步2-叔丁基-噻唑-5-羰基氯化的二氯甲烷溶液(4mL)，加毕后自然升至室温反应10min，然后依次用饱和氯化铵水溶液，饱和碳酸氢钠水溶液，饱和氯化钠水溶液洗涤反应液，有机层经无水硫酸镁干燥后过滤，减
压蒸干溶剂，3mL石油醚洗涤残余物后得目标产物，淡黄色粉末(253.6mg，收率85.1％)。1H NMR(400MHz,DMSO-d6)δ9.95(s,1H),8.30(s,1H),7.95(d,J＝2.0Hz,1H),7.76(dd,J＝8.3,2.0Hz,1H),7.28(d,J＝8.3Hz,1H),4.39(s,1H),2.37(s,3H),1.47(s,9H)。MS m/z(ESI):299.1[M+H]。
实施例17 N-(4-氯-3-乙炔基苯基)-3-(三氟甲基)苯酰胺(中间体66)的制备
第一、二步，制备3-乙炔基-4-氯苯胺：
以3-碘-4-氯苯胺为原料，按照中间体37第一、二步所述的方法，得目标产物，淡黄色油状物(2.1g，收率77.8％)。1H NMR(400MHz,DMSO-d6)δ7.12(d,J＝8.7Hz,1H),6.75(d,J＝2.8Hz,1H),6.60(dd,J＝8.7,2.8Hz,1H),5.39(s,2H),4.34(s,1H)。MSm/z(ESI):518.3[M+H]。
第三步，制备N-(4-氯-3-乙炔基苯基)-3-(三氟甲基)苯酰胺：
采用中间体38所述的类似方法得到中间体66，淡黄色粉末(291mg，收率90.1％)。1HNMR(400MHz,DMSO-d6)δ10.64(s,1H),8.30(s,1H),8.26(d,J＝8.0Hz,1H),8.07(d,J＝2.5Hz,1H),8.00(d,J＝8.0Hz,1H),7.89-7.82(m,1H),7.82-7.75(m,1H),7.57(d,J＝8.8Hz,1H),4.61(s,1H)。MS m/z(ESI):324.1[M+H]。
实施例18 3-碘-4-甲基-N-(4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)苯酰胺(中间体67)的制备
第一步，制备1-溴甲基-4-硝基-2-三氟甲基-苯：
将1-甲基-4-硝基-2-三氟甲基-苯(5.62g，27.4mmol，1.0eq)溶于50ml 1,2-二氯乙烷，然后依次加入N-溴代丁二酰亚胺(5.85g，32.8mmol，1.2eq)和AIBN(450mg，2.7mmol，0.1eq)，加热回流过夜。然后将反应液冷却至室温，依次用饱和碳酸氢钠溶液，饱和氯化钠溶液及水洗涤，无水硫酸镁干燥，过滤后减压浓缩得目标产物，直接用于下步反应。
第二步，制备1-甲基-4-(4-硝基-2-(三氟甲基)苯甲基)哌嗪：
将上步1-溴甲基-4-硝基-2-三氟甲基-苯(20.55mmol)溶于50mL DCM中，加入三乙胺(3.1g，30.8mmol，1.5eq)和N-甲基哌嗪(4.12g，41.1mmol，2.0eq)，室温反应，反应完毕后减压浓缩，DCM溶解，依次用饱和碳酸氢钠溶液，饱和氯化钠溶液及水洗涤，无水硫酸镁干燥，过滤后减压浓缩，残余物经柱层析分离得目标产物，黄色固体。MS m/z(ESI):304.2[M+H]
第三步，制备4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯胺：
1-甲基-4-(4-硝基-2-(三氟甲基)苯甲基)哌嗪(5g)溶于65mL 75％乙醇中，加入0.5g 10％钯碳，氮气置换后，反应液在氢气下室温搅拌5h，反应完毕后减压过滤，滤液蒸干后得目标产物，淡黄色固体。MS m/z(ESI):274.2[M+H]。
第四步，制备3-碘-4-甲基-N-(4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)苯酰胺：
按照中间体35第五步所述类似的缩合方法得到中间体67，淡黄色固体(2.2g，收率74.3％)。1H NMR(400MHz,DMSO-d6)δ10.49(s,1H),8.43(s,1H),8.18(d,J＝1.5Hz,1H),8.04(d,J＝8.5Hz,1H),7.92(d,J＝8.0Hz,1H),7.70(d,J＝8.5Hz,1H),7.50(d,J＝8.0Hz,1H),3.56(s,2H),2.45(s,3H),2.36(d,J＝20.7Hz,8H),2.16(s,3H)。MS m/z(ESI):518.3[M+H]。
实施例19 中间体68～73的制备
以含不同取代基的苯甲酸与含不同取代基的苯胺为原料，采用中间体67所述类似方法得到以下中间体68～73。
实施例20 3-乙炔基-4-甲基-N-(4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)苯酰胺(中间体74)的制备
第一步，制备3-碘-4-甲基苯甲酸甲酯：
将3-碘-4-甲基苯甲酸(15g，57.14mmol)用甲醇溶解，将浓硫酸缓慢滴入其中，反应液放热，滴加完毕后升温至70℃反应。TLC监测，48h后反应完全，停止反应，减压蒸干甲醇，得到棕色油状物，将油状物缓慢倒入200mL水中，呈白色乳浊状，且放热，水用DCM萃取2次，DCM层再依次用碳酸氢钠水溶液、饱和氯化钠水溶液、水萃取一次，DCM层用无水硫酸钠干燥，减压蒸干DCM后得目标产物，黄棕色油状物。
第二步，制备3-三甲基硅乙炔基-4-甲基苯甲酸甲酯：
将上步油状物用THF溶解，加入碘化亚铜(1.1g，5.74mmol，0.1eq)和四三苯基膦钯(3.3g，2.86mmol，0.05eq)，氮气置换三次，加入三乙胺(11.57g，114.28mmol，2.0eq)和三甲基硅乙炔(8.4g，85.71mmol，1.5eq)，常温搅拌，24h后反应完毕。减压蒸干溶剂，柱层析分离得目标产品，黄色油状物。
第三步，制备3-乙炔基-4-甲基苯甲酸甲酯：
将上步油状物用甲醇溶解，加入碳酸钾(3.95g，28.57mmol，0.5eq)，常温搅拌10min后反应完毕，减压蒸干溶剂，DCM溶解，再依次用碳酸氢钠水溶液、饱和氯化钠水溶液、水萃取一次，DCM层用无水硫酸钠干燥，经柱层析分离得到目标产品，淡黄色油状物。
第四步，制备3-乙炔基-4-甲基苯甲酸：
将上步得到的产品用甲醇溶解，加入饱和氢氧化钠水溶液10mL，常温搅拌过夜。反应完毕后，减压蒸干溶剂，加入水约20mL，用盐酸将pH调成约为3，析出大量白色固体，用DCM萃取3次，合并DCM层，用水洗涤DCM层一次，无水硫酸钠干燥后，减压蒸干溶剂得到产品3-乙炔基-4-甲基苯甲酸，淡黄色固体粉末(8.0g，总收率87.2％)。1H NMR(400MHz,DMSO-d6)δ13.04(br.s,1H),7.93(s,1H),7.85(d,J＝8.0Hz,1H),7.43(d,J＝8.0Hz,1H),4.48(s,1H),2.45(s,3H)。MS m/z(ESI):159.0[M-H]。
第五步，制备3-乙炔基-4-甲基-N-(4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)苯酰胺：
将3-乙炔基-4-甲基苯甲酸(500mg，3.12mmol，1.05eq)用DCM溶解，加入HATU(1.42g，
3.74mmol，1.2eq)、DIPEA(806mg，6.24mmol，2.0eq)，常温搅拌约30min后，加入3-三氟甲基-4-[(4-甲基哌嗪-1-基)甲基]苯胺(812mg，2.97mmol，1.0eq)，升温至45℃回流，20h后反应完毕。减压蒸干溶剂，用DCM和饱和碳酸氢钠水溶液萃取2次，DCM层蒸干后得到黄色油状物，经柱层析分离得到中间体74，黄棕色固体(807mg，收率约65.4％)。1H NMR(400MHz,CDCl3)δ8.33(s,1H),7.91(s,1H),7.86(d,J＝13.7Hz,2H),7.73(t,J＝7.1Hz,2H),7.28(s,1H),3.60(s,2H),3.32(s,1H),2.48(br.s,11H),2.30(s,3H)。MS m/z(ESI):416.3[M+H]。
实施例21 中间体75～79的制备
以含不同取代基的苯胺或者芳基胺为原料，采用中间体74所述类似的方法得到以下中间体75～79。
实施例22 3-乙炔基-4-甲基-N-(3-(4-甲基-1H-咪唑-1-基)-5-(三氟甲基)苯基)苯酰胺(中间体80)的制备
第一步，制备3-(4-甲基-咪唑-1-基)-5-三氟甲基-苯胺：
将3-溴-5-三氟甲基-苯胺(500mg，2.1mmol，1.0eq)，4-甲基-1H-咪唑(20.5mg，2.5mmol，1.2eq)，碘化亚铜(60mg，0.3mmol，0.14eq)和8-羟基喹啉(44mg，0.3mmol，0.14eq)溶解在3mL二甲亚砜中，氮气置换3次后升温至120℃反应过夜，反应完毕后加水稀释，DCM萃取，有机层依次用稀氨水溶液，饱和氯化钠水溶液洗涤，无水硫酸镁干燥，过滤，减压浓缩后残余物经柱层析分离得目标产物，黄色固体(392mg，收率77.3％)。1HNMR(400MHz,CDCl3)δ7.73(s,1H),6.98(s,1H),6.92(s,1H),6.83(s,1H),6.77(s,1H),4.14(s,2H),2.27(s,3H)。MS m/z(ESI):242.1[M+H]。
第二步，制备3-乙炔基-4-甲基-N-(3-(4-甲基-1H-咪唑-1-基)-5-(三氟甲基)苯基)苯酰胺：
采用中间体35第五步所述类似的缩合方法得到中间体80，淡黄色固体(350mg，收率65.7％)。1H NMR(400MHz,DMSO-d6)δ10.68(s,1H),8.29(s,1H),8.22(s,1H),8.16(s,1H),8.12(s,1H),7.93(d,J＝8.0Hz,1H),7.74(s,1H),7.51(d,J＝9.0Hz,2H),4.56(s,1H),2.48(s,3H),2.19(s,3H)。MS m/z(ESI):384.1[M+H]。
实施例23 3-乙炔基-4-甲基-N-(3-((4-甲基哌嗪-1-基)甲基)-5-(三氟甲基)苯基)苯酰胺(中间体81)的制备
第一步，制备3-硝基-5-三氟甲基苯甲酰氯：
3-硝基-5-三氟甲基苯甲酸(3.52g，14.97mmol，1.0eq)溶于20mL二氯亚砜中，80℃回流2h，反应完毕后减压蒸干溶剂，余下残余物为目标产物，直接用于下步反应。
第二步，制备(4-甲基哌嗪-1-基)(3-硝基-5-(三氟甲基)苯基)甲酮：
N-甲基哌嗪(1.5g，15mmol，1.05eq)，三乙胺(2.2g，22.4mmol，1.5mmol)溶于20ml DCM中，上步产物3-硝基-5-三氟甲基苯甲酰氯溶于6mL DCM中，缓慢滴加至反应液中，加毕后室温反应0.5h，反应完毕后依次用饱和碳酸氢钠溶液，饱和氯化钠溶液及水洗涤，无水硫酸镁干燥，过滤，减压蒸干后直接用于下步反应。MS m/z(ESI):318.1[M+H]
第三步，制备1-甲基-4-(3-硝基-5-(三氟甲基)苯甲基)哌嗪：
将(4-甲基哌嗪-1-基)(3-硝基-5-(三氟甲基)苯基)甲酮(14.97mmol)于无水四氢呋喃中，氮气保护，降温至0℃。用无水四氢呋喃将2mol/L的硼烷二甲硫醚(22.5mL，45mmol，3.0eq)稀释至50mL，然后缓慢滴加至反应液中，加毕后升温至65℃反应过夜，反应完毕后降温
至0℃，缓慢滴加6M HCl 22.5mL，加毕后升温至65℃反应1h，然后降温至0℃，缓慢滴加4MNaOH水溶液调pH至9，反应液用乙酸乙酯萃取3次，合并有机层，无水硫酸镁干燥，减压过滤，滤液蒸干后残余物经柱层析分离得目标产物，淡黄色油状物(2.4g，收率53.2％)。MS m/z(ESI):304.1[M+H]
第四步，制备3-((4-甲基哌嗪-1-基)甲基)-5-(三氟甲基)苯胺：
1-甲基-4-(3-硝基-5-(三氟甲基)苯甲基)哌嗪(2.4g，7.96mmol)溶于75％乙醇中，加入10％钯碳(240mg，10％eq)，氮气置换后通氢气于50℃下反应，反应完毕后，减压过滤，滤液蒸干得目标产物，白色固体(2.1g，收率96.1％)。MS m/z(ESI):274.1[M+H]
第五步，制备3-乙炔基-4-甲基-N-(3-((4-甲基哌嗪-1-基)甲基)-5-(三氟甲基)苯基)苯酰胺：
采用中间体35第五步所述类似的缩合方法得到中间体81，淡黄色固体(260mg，盐酸盐，收率61.7％)。1H NMR(400MHz,DMSO-d6)δ10.55(s,1H),9.28(br.s,1H),8.10(s,3H),7.91(d,J＝8.1Hz,1H),7.49(d,J＝8.1Hz,1H),7.41(s,1H),4.56(s,1H),3.66(s,2H),3.39(d,J＝11.1Hz,2H),3.14-2.99(m,2H),2.94(d,J＝12.5Hz,2H),2.80(s,3H),2.47(s,3H),2.33(t,J＝11.9Hz,2H)。MS m/z(ESI):416.3[M+H]。
实施例24 3-乙炔基-N-(3-(三氟甲基)苯基)苯酰胺(中间体82)的制备
第一步，制备3-碘-N-(3-(三氟甲基)苯基)苯酰胺：
将间碘苯甲酸(1.29g，5.2mmol，1.05eq)、TBTU(O-(苯并三唑-1-氧)-N,N,N’,N’-四甲基脲鎓六氟硼酸盐)(1.93g，6.0mmol，1.2eq)、三乙胺(1.01g，10mmol，2.0eq)加入DCM中，常温搅拌0.5h，加入间三氟甲基苯胺(805mg，5.0mmol，1.0eq)，升温至45℃回流。12h后反应完毕。减压蒸干溶剂，用饱和碳酸氢钠水溶液、DCM萃取一次，DCM层蒸干，经柱层析分离得到目标产物。1H NMR(400MHz,DMSO-d6)δ10.60(s,1H),8.33(s,1H),8.22(s,1H),8.05(d,J＝8.0Hz,1H),7.99(dd,J＝7.8,1.2Hz,2H),7.61(t,J＝8.0Hz,1H),7.47(d,J＝7.8Hz,1H),7.37(t,J＝8.0Hz,1H)。MS m/z(ESI):392.0[M+H]。
第二步，制备N-(3-(三氟甲基)苯基)-3-((三甲基甲硅烷基)乙炔基)苯酰胺：
将上步产物溶于THF(四氢呋喃)，加入碘化亚铜(95.23mg，0.5mmol，0.1eq)、双三苯基膦二氯化钯(175.5mg，0.25mmol，0.05eq)，氮气置换三次，再加入DIPEA(1.29g，10mmol，2.0eq)、三甲基硅乙炔(737mg，7.5mmol，1.5eq)，常温搅拌，4h后反应完毕。停止反应，经柱层析分离得到目标产物，直接用于下步反应。MS m/z(ESI):362.2[M+H]。
第三步，制备3-乙炔基-N-(3-(三氟甲基)苯基)苯酰胺：
将上步产物用甲醇溶解，加入碳酸钾(345.5mg，2.5mmol，0.5eq)，常温搅拌，10min后反应完毕。蒸干溶剂，残留物经柱层析分离得到中间体82，淡黄色固体(875.0mg，收率约60.5％)。1H NMR(400MHz,DMSO-d6)δ10.61(s,1H),8.25(s,1H),8.10(s,1H),8.06(d,J＝8.3Hz,1H),8.00(d,J＝7.9Hz,1H),7.72(d,J＝7.7Hz,1H),7.63–7.56(m,2H),7.47(d,J＝7.9Hz,1H),4.35(s,1H)。MS m/z(ESI)：290.1[M+H]。
实施例25 4-氯-3-乙炔基-N-(3-(三氟甲基)苯基)苯酰胺(中间体83)的制备
以4-氯-3-碘苯甲酸同3-三氟甲基苯胺为原料，采用中间体82所述类似的方法得中间体83。1H NMR(400MHz,CDCl3)δ7.98(s,1H),7.91(s,1H),7.85(d,J＝7.6Hz,1H),7.77(d,J＝7.2Hz,1H),7.46(d,J＝8.4Hz,2H),7.44-7.39(m,2H),3.43(s,1H)。MS m/z(ESI)：324.1[M+H]。
实施例26 1-(3-乙炔基苯基)-3-(3-(三氟甲基)苯基)脲(中间体84)的制备
将三光气(1.79g，6.0mmol，1.5eq)溶于EA，常温搅拌。将间三氟甲基苯胺(967mg，6.0mmol，1.5eq)溶于EA，置于滴液漏斗中，缓慢滴加到三光气的EA溶液中，30min后滴加完毕。然后缓慢加入三乙胺(1.2g，12mmol，2.0eq)，析出大量白色固体，常温反应。2h后，减压蒸干溶剂，加入EA，滤去不溶物，收集滤液。向滤液中加入间乙炔基苯胺(469mg，4.0mmol,1.0eq)，有固体产生。TLC监测反应完毕后，减压蒸干溶剂，用体积比乙醚：石油醚＝1：1重结晶得到中间体84，白色固体(543mg，收率约44.6％)。1H NMR(400MHz,DMSO-d6)δ9.35(s,1H),9.15(s,1H),8.01(s,1H),7.68(s,1H),7.59(d,J＝8.4Hz,1H),7.52(t,J＝7.9Hz,1H),7.44(d,J＝8.2Hz,1H),7.31(d,J＝7.8Hz,2H),7.10(d,J＝7.6Hz,1H),4.17(s,1H)。MS m/z(ESI):304.2[M+H]。
实施例27 中间体85～86的制备
以含不同取代基的苯胺为原料，采用中间体84所述类似的方法得到以下中间体85～86。
实施例28 N-(3-((4-氨基-1-异丙基-1H-吡唑并[3,4-d]嘧啶-3-基)乙炔基)-4-甲基苯基)-4-((4-甲基哌嗪-1-基)甲基)苯酰胺(化合物1)的制备
N-(3-碘-4-甲基苯基)-4-((4-甲基哌嗪-1-基)甲基)苯酰胺(中间体36)(118mg,0.263mmol,1.0eq)，3-乙炔基-1-异丙基-1H-吡唑并[3,4-d]嘧啶-4-胺(中间体1)(55.57mg,0.276mmol,1.05eq)，CuI(5.7mg,0.03mmol,0.1eq)，PdCl2(PPh3)2(21.06mg,0.03mmol,0.1eq)于三口瓶中，加入2mL DMF溶解后氮气置换三次，加入三乙胺(53.23mg,0.526mmol,2.0eq)于80℃反应过夜，TLC监控反应完毕后，减压蒸去DMF，残余物经柱层析分离得化合物1，淡黄色固体(62mg，收率45.1％)。1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),8.27(s,1H),8.10(s,1H),7.94(d,J＝7.6Hz,2H),7.75(d,J＝8.2Hz,1H),7.47(d,J＝7.6Hz,2H),7.34(d,J＝8.2Hz,1H),5.20-4.93(m,1H),3.56(s,2H),2.58(br.s,8H),2.47(s,3H),2.34(s,3H),1.49(d,J＝6.4Hz,6H).MS m/z(ESI):523.2930[M+H]。
实施例29 化合物2-119的制备
以含不同取代基的芳基乙炔与卤代物为原料，采用实施例28所述类似的方法得到化合物2-119。
实施例30 3-((4-氨基-1-(哌啶-4-基)-1H-吡唑并[3,4-d]嘧啶-3-基)乙炔基)-4-甲基-N-(4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)苯酰胺(化合物120)的制备
叔-丁基-4-(4-氨基-3-((2-甲基-5-((4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)氨基甲酰)苯基)乙炔基)-1H-吡唑并[3,4-d]嘧啶-1-基)哌啶-1-羧酸酯(1.46g，2mmol)溶于20mL DCM中，降温至0℃后加入10ml三氟乙酸，自然升至室温反应，0.5h后反应完毕，减压蒸干溶剂，残余物用水溶解后调PH至8，析出大量固体，减压过滤，水洗滤饼，固体经真空干燥后得化合物120，淡黄色粉末(1.2g，收率94.9％)。
1H NMR(400MHz,DMSO-d6)δ10.56(s,1H),8.35(s,1H),8.28(s,1H),8.22(s,1H),8.08(d,J＝7.9Hz,1H),7.98(d,J＝7.9Hz,1H),7.72(d,J＝8.2Hz,1H),7.55(d,J＝8.2Hz,1H),5.09-4.98(m,1H),4.36-4.32(m,1H),3.58(s,2H),3.21-3.08(m,2H),2.99(d,J＝7.0Hz,2H),2.58(s,3H),2.42(br.s,8H),2.31(d,J＝11.7Hz,2H),2.22(s,3H),2.12(d,J＝11.6Hz,2H)。MS m/z(ESI):632.3071[M+H]。
实施例31 3-((1-(1-丙烯酰哌啶-4-基)-4-氨基-1H-吡唑并[3,4-d]嘧啶-3-基)乙炔基)-4-甲基-N-(4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)苯酰胺(化合物121)的制备
3-((4-氨基-1-(哌啶-4-基)-1H-吡唑并[3,4-d]嘧啶-3-基)乙炔基)-4-甲基-N-(4-((4-甲基哌嗪-1-基)甲基)-3-(三氟甲基)苯基)苯酰胺(510mg，0.807mmol，1.0eq)溶于10mL DCM中，加入三乙胺(163mg，1.6mmol，2.0eq)，降温至0℃，缓慢滴加丙烯酰氯(72μL，0.888mmol，1.1eq)的二氯甲烷液(4mL)，加毕后反应完全。然后依次用饱和氯化铵水溶液，饱和碳酸氢钠水溶液，饱和氯化钠水溶液洗涤反应液，余下有机层用无水硫酸镁干燥，过滤，滤液减压蒸干，残余物经丙酮：乙醚(1:1)洗涤得化合物121，淡黄色粉末(440.1mg，收率72.3％)。
1H NMR(400MHz,DMSO-d6)δ10.54(s,1H),8.33(s,1H),8.27(s,1H),8.21(s,1H),8.06(d,J＝7.7Hz,1H),7.96(d,J＝7.7Hz,1H),7.71(d,J＝7.8Hz,1H),7.54(d,J＝7.8Hz,1H),6.88(dd,J＝17.7,10.2Hz,1H),6.14(d,J＝17.7Hz,1H),5.71(d,J＝10.2Hz,1H),5.08-4.94(m,1H),4.56(d,J＝10.5Hz,1H),4.21(d,J＝10.9Hz,1H),3.57(s,2H),2.97-2.84(m,1H),2.73-2.62(m,1H),2.57(s,3H),2.39(br.s,8H),2.19(s,3H),2.01(br.s,4H)。MS m/z(ESI):686.3181[M+H]。
实施例32 激酶抑制实验
本实验的目的是检测本发明化合物对体外蛋白激酶的抑制活性，采用的方法为同位素标记法(标记ATP上的γ磷酸基团)。本实验分别对Abl(T315I)(h)、ALK(h)、ARK5(h)、Axl(h)、Blk(h)、Bmx(h)、BTK(h)、B-Raf(h)、cKit(h)、cSRC(h)、CDK7、CHK1(h)、c-RAF(h)、DDR2(h)、EGFR(h)、EphA1(h)、EphA2(h)、EphA8(h)、EphB2(h)、EphB4(h)、ErbB2(h)、FAK(h)、Fer(h)、FGFR1(h)、Flt3(h)、Fms(h)、Fyn(h)、Hck(h)、GSK3β(h)、IKKα(h)、IKKβ(h)、Itk(h)、JAK3(h)、JNK1α1(h)、KDR(h)、Lyn(h)、MAPK1(h)、MEK1(h)、Met(h)、mTOR(h)、PAK1(h)、PDGFRα(h)、Pim-1(h)、PKA(h)、PKBα(h)、PKBβ(h)、PKCα(h)、Ret(h)、RIPK2(h)、Src(1-530)(h)、TAK1(h)、TBK1(h)、Tec(h)activated、Tie2(h)、TrkA(h)、ULK3(h)、Yes(h)、PI3Kinase a(h)等激酶进行体外活性抑制测试。受试化合物的激酶抑制活性用IC50(半数抑制浓度)或受试化合物在10μM浓度下对激酶活性的抑制率来表示。IC50值可通过受试化合物在一系列不同浓度下对激酶活性的抑制率计算而获得。实验方法如下：在一个反应管中，依次加入缓冲液(8mM MOPS,pH 7.0,0.2mM EDTA,10mM MnCl2)，待测激酶(5-10mU)、待测激酶的底物，10mM的醋酸镁和γ33P-ATP溶液，以及不同浓度的受试化合物。然后向反应中加入MgATP以启动酶反应过程，并在室温下孵育40分钟。最终用5μl的3％磷酸盐缓冲液终止反应，并把10μL的反应液滴定到Filtermat A膜上，用75mM的磷酸盐溶液洗三次，每次5分钟，再用甲醇洗一次。最后干燥Filtermat A膜并对其进行闪烁计数，闪烁计数值的大小反映了底物被磷酸化的程度，从而可以表征激酶活性被抑制情况。其中，剩余活性蛋白百分比＝实验组活性蛋白量÷对照组活性蛋白量×100％
表1给出了部分受试化合物对部分激酶抑制活性的IC50值。(以下各表中的“--”均表
示未进行测试。)
表1 化合物3、31、93、120和121对各种激酶的抑制活性(IC50:nM)
表1的结果表明，部分受试化合物对激酶如Abl、Abl(T315I)、c-Src(1-530)、c-Src(T341M)、B-Raf(V600E)、B-Raf、c-RAF、Yes、Fyn、Blk、Bcr-Abl、KDR、FGFR1、FGFR2、EphA2、EphB2、EphB4、ErbB2、DDR1、DDR2、TAK1、TrkA、Btk、Bmx、Arg、BRK、CSK、EGFR(T790M)、EGFR(T790M,L858R)、Flt1、Flt4、Hck、Lck、LIMK1、Lyn、Mer、PTK5、Pyk2、Ret、SAPK2b、Tie2、Txk有较好的抑制活性，对部分激酶如IKKα、IKKβ、Axl、PDGFRα有中等的抑制活性。
表2给出了部分受试化合物对c-Src(1-530)激酶抑制活性的IC50值。
表2 部分受试化合物对c-Src激酶的抑制活性(IC50:nM)
| 化合物 | IC50(nM) | 化合物 | IC50(nM) |
| 1 | 4 | 17 | 4 |
| 5 | 2 | 18 | 4 |
| 6 | 9 | 19 | 8 |
| 7 | 19 | 20 | 7 |
| 8 | 7 | 21 | 7 |
| 9 | 15 | 24 | 59 |
| 10 | 83 | 25 | 3191 |
| 11 | 10 | 26 | 11 |
| 12 | 139 | 29 | 5 |
| 13 | 11 | 30 | 20 |
| 14 | 3 | 120 | 12 |
| 16 | 7 | 121 | 15 |
表2的结果表明，部分受试化合物对Src激酶有较好的抑制活性。
表3给出了化合物3、31、120和121在10μM的浓度下分别对Abl(T315I)(h)、ALK(h)、ARK5(h)、Axl(h)、Blk(h)、Bmx(h)、BTK(h)、B-Raf(h)、cKit(h)、cSRC(h)、CDK7、CHK1(h)、c-RAF(h)、DDR2(h)、EGFR(h)、EphA1(h)、EphA2(h)、EphA8(h)、EphB2(h)、EphB4(h)、ErbB2(h)、FAK(h)、Fer(h)、FGFR1(h)、Flt3(h)、Fms(h)、Fyn(h)、Hck(h)、GSK3β(h)、IKKα(h)、IKKβ(h)、Itk(h)、JAK3(h)、JNK1α1(h)、KDR(h)、Lyn(h)、MAPK1(h)、MEK1(h)、Met(h)、mTOR(h)、PAK1(h)、PDGFRα(h)、Pim-1(h)、PKA(h)、PKBα(h)、PKBβ(h)、PKCα(h)、Ret(h)、RIPK2(h)、Src(1-530)(h)、TAK1(h)、TBK1(h)、Tec(h)activated、Tie2(h)、TrkA(h)、ULK3(h)、Yes(h)、PI3Kinase a(h)激酶活性的抑制情况(数值表示剩余活性蛋白百分比)。
表3 化合物3、31、120和121在10μM的浓度下对部分激酶的抑制率
表3的结果表明，部分受试化合物对Abl(T315I)、Axl、Blk、Bmx、BTK、B-Raf、cSRC、
c-RAF、DDR2、EphA1、EphA2、EphA8、EphB2、EphB4、ErbB2、FGFR1、Flt3、Fms、Fyn、Hck、IKKα、IKKβ、Itk、JAK3、KDR、Lyn、PDGFRα、Ret、RIPK2、Src(1-530)、TAK1、Tec、Tie2、TrkA、ULK3、Yes有较好的抑制活性。部分受试化合物对ALK、cKit、EGFR、FAK、Fer、JNK1α1、PKA、PKBβ有中等的抑制活性。
表4给出了化合物93和化合物100在10μM的浓度下分别对Abl、ACK1、ALK等激酶活性的抑制率(数值表示剩余活性蛋白百分比)。
表4 化合物93和化合物100在10μM的浓度下对部分激酶的抑制率
表4到结果表明，部分受试化合物对Abl、Abl(T315I)、Arg、Blk、Bmx、BRK、cKit(D816H)、cKit(V560G)、CSK、cSRC、DDR1、EGFR(T790M)、EGFR(T790M,L858R)、EphA1、EphA2、EphA3、EphA4、EphA5、EphA7、EphA8、EphB2、EphB1、EphB3、EphB4、ErbB2、ErbB4、FGFR1、FGFR2、FGFR3、Fgr、Flt1、Flt4、Fms、Fyn、Hck、JAK1、KDR、Lck、LIMK1、Lyn、Mer、PDGFRα(D842V)、PDGFRα(V561D)、PTK5、Pyk2、Ret、Rse、SAPK2a、SAPK2b、Src、TAK1、TAO2、TAO3、Tie2、TrkB、Txk、Yes等有较好的抑制活性。
实施例33 细胞增殖抑制实验
本实验的目的是检测本发明化合物对体外人肿瘤细胞增殖抑制活性，采用的方法为MTT(四甲基偶氮唑盐)比色法。
1)实验材料：
主要试剂：RPMI-1640、胎牛血清、胰酶等购自Gibco BRL公司(Invitrogen Corporation，USA)，DMEM培养基购自ATCC(American Type Culture Collection)。四甲基偶氮唑盐(MTT)、二甲基亚砜(DMSO)为Sigma公司(USA)产品。化合物3、化合物31、化合物93、化合物100由发明人合成，体外实验时用100％DMSO配制成10mM储存液，置-20℃冰箱避光保存备用，临用时用完全培养液稀释至所需浓度。
细胞系及培养：本实验所用的人乳腺癌细胞株MDA-MB-231、MCF-7、SKBR-3、BT474、MDA-MB-468、MDA-MB-453、MDA-MB-435，大B细胞淋巴瘤细胞株LY-10、HBL-1，人胰腺癌细胞株Panc-1、Miapaca-2，人肺癌细胞株A549、H358、H1975，人白血病细胞株THP-1，人肝癌细胞株Hepg2，人黑色素瘤细胞株A2058等均购于美国ATCC(American type culture collection)，由本实验室保存。以上所有人淋巴瘤细胞株、大B细胞淋巴瘤细胞株、T细胞淋巴瘤细胞株用含10％胎牛血清、100U/mL青霉素、100μg/mL链霉素的RPMI-1640完全培养基在5％CO2、37℃条件下培养。其余细胞株使用含10％胎牛血清(MV4-11细胞为20％)、100U/ml青霉素、100μg/mL链霉素的DMEM完全培养基在5％CO2、37℃条件下培养。
2)实验方法：
用完全细胞培养液调整细胞浓度为1～2×104个/mL的细胞悬液，接种于96孔板，每孔200μL细胞悬液，培养过夜。次日，吸弃上清(悬浮细胞离心后吸取上清)，然后分别用梯度浓度的受试化合物处理细胞。同时设不含药物的阴性对照组和等体积的溶剂对照组，DMSO浓度为0.1％，每个剂量组设3个复孔，在37℃，5％CO2条件下培养。72小时后，每孔加入浓度为5mg/mL的MTT试剂20μL，再培养2-4h后，弃上清，每孔再加入DMSO 150μL，振荡混匀15min，用酶标仪(λ＝570nm)测定吸光度(A)值(A值与活细胞数成正比)，取其平均值。相对细胞增殖抑制率＝(对照组A570-实验组A570)×100％/对照
组A570。实验至少重复3次。实验数据用均数表示，数据统计资料采用t检验，P<0.05为差异有统计学意义。以下各化合物对细胞增殖抑制作用均用IC50或抑制率表示。
3)实验结果：
采用以上方法，对人乳腺癌细胞株MDA-MB-231、MCF-7、SKBR-3、BT474、MDA-MB-468、MDA-MB-453、MDA-MB-435，大B细胞淋巴瘤细胞株LY-10、HBL-1，人胰腺癌细胞株Panc-1、Miapaca-2，人肺癌细胞株A549、H358，人白血病细胞株THP-1，人肝癌细胞株Hepg2，人黑色素瘤细胞株A2058等进行了增殖抑制活性测试，结果见表5。
表5 化合物3和化合物31对各种细胞株的增殖抑制活性(IC50:μM)
| 细胞株 | 化合物3 | 化合物31 | 细胞株 | 化合物3 | 化合物31 |
| HBL-1 | ～10 | -- | Hela | 5.41 | 7.23 |
| OCI-LY10 | 2.168 | -- | PC-3 | -- | 3.974 |
| RAMOS | ～10 | -- | MDA-MB-231 | 0.0113 | 0.01533 |
| MV4-11 | 0.020 | -- | MDA-MB-435 | 0.008654 | 0.0147 |
| THP-1 | 5.11 | 7.378 | MDA-MB-453 | 3.369 | -- |
| PANC-1 | 6.556 | -- | MDA-MB-468 | 4.478 | 3.216 |
| Miapaca-2 | 0.01277 | 0.04036 | BT474 | >10 | >10 |
| CFPAC | 0.607 | 0.4721 | MCF-7 | -- | 1.987 |
| H4 | -- | 1.807 | SKBR-3 | 4.523 | 1.265 |
| U87 | 0.742 | 5.420 | MM.1S | 0.872 | 2.383 |
| A2058 | 1.908 | -- | HCT116 | 0.178 | 0.2303 |
| H358 | 1.665 | -- | HT29 | 0.1663 | 0.3496 |
| A549 | 0.1988 | 1.423 | SW1990 | 0.3559 | 0.2061 |
| HepG2 | 8.672 | 6.167 | A375 | 0.02088 | 0.02657 |
| plc/prf/5 | -- | 3.974 |
表5的结果表明，受试化合物3和化合物31对MV4-11、Miapaca-2、MDA-MB-231、MDA-MB-435、A375细胞具有较强的抑制活性；受试化合物3和化合物31对其它肿瘤细胞株包括CFPAC、U87、MM.1S、HCT116、HT29、A549等也具有中等的抑制活性。
表6 化合物93和化合物100对各种细胞株的增殖抑制活性(IC50:μM)
表6的结果表明，受试化合物93和化合物100对MV4-11、Miapaca-2、MDA-MB-231、MDA-MB-435、A375细胞具有较强的抑制活性；受试化合物93和化合物100对其它肿瘤细胞株包括U251、H4、HCT116、Hela等也具有中等的抑制活性。
部分受试化合物对MDA-MB-231和MDA-MB-435细胞的增殖抑制活性见表7。其中，IC50<100nM用符号++++表示，100nM<IC50<500nM用符号+++表示，500nM<IC50<1000nM用符号++表示，IC50>1000nM用符号+表示。
表7 化合物对MDA-MB-231和MDA-MB-435细胞的增殖抑制活性
表7的结果表明，部分受试化合物对MDA-MB-231和MDA-MB-435细胞较强的增殖抑制活性。
实施例34 化合物3、化合物31、化合物93和化合物100对SCID裸鼠的体内药效学实验
本实验的目的是检测本发明化合物的体内抗肿瘤效果。本实验使用SCID小鼠皮下肿瘤模型，测试发明化合物3、化合物31、化合物93和化合物100的体内抗肿瘤活性。所用细胞株为乳腺癌细胞株MDA-MB-231。以正在使用的抗乳腺癌药物紫杉醇和乳腺癌临床在研的上市药物达沙替尼为阳性对照。
1)实验材料：
胎牛血清、胰酶等购自Gibco BRL公司(Invitrogen Corporation，USA)，DMEM培养基购自ATCC(American Type Culture Collection)，人乳腺癌细胞株MDA-MB-231购于美国ATCC公司，SCID小鼠购于中国北京华阜康生物科技股份有限公司。紫杉醇购自中国上海瀚香生物技术有限公司。达沙替尼购自中国南京康满林化工实业有限公司。
2)实验方法：
使用6～8周龄SCID小鼠，按照约5×106个/0.1mL/只MDA-MB-231细胞浓度接种于
小鼠皮下后肋部，待肿瘤长到200mm3后(约15天)，小鼠分组(n＝6)并开始给药。各组药物溶解于5％DMSO+25％PEG-400+70％水。
实验分组一：
药物溶剂对照组，每天口服灌胃空白溶剂200uL；
化合物3按剂量30mg/kg每天口服灌胃给药；
化合物3按剂量15mg/kg每天口服灌胃给药.；
化合物3按剂量7.5mg/kg每天口服灌胃给药；
阳性对照紫杉醇按剂量10mg/kg每周尾静脉注射给药；阳性对照达沙替尼按剂量40mg/kg每天口服灌胃给药。
实验分组二：
药物溶剂对照组，每天口服灌胃给药空白溶剂200uL；
化合物31按剂量40mg/kg每天口服灌胃给药；
化合物31按剂量20mg/kg每天口服灌胃给药；
化合物31按剂量10mg/kg每天口服灌胃给药；
阳性对照紫杉醇按剂量10mg/kg每周尾静脉注射给药；阳性对照达沙替尼按剂量40mg/kg每天口服灌胃给药。
实验分组三：
药物溶剂对照组，每天口服灌胃空白溶剂200uL；
化合物93按剂量40mg/kg每天口服灌胃给药；
化合物93按剂量20mg/kg每天口服灌胃给药.；
化合物93按剂量10mg/kg每天口服灌胃给药；
阳性对照紫杉醇按剂量10mg/kg每周尾静脉注射给药；阳性对照达沙替尼按剂量40mg/kg每天口服灌胃给药。
实验分组四：
药物溶剂对照组，每天口服灌胃给药空白溶剂200uL；
化合物100按剂量40mg/kg每天口服灌胃给药；
化合物100按剂量20mg/kg每天口服灌胃给药；
化合物100按剂量10mg/kg每天口服灌胃给药；
阳性对照紫杉醇按剂量10mg/kg每周尾静脉注射给药；阳性对照达沙替尼按剂量40mg/kg每天口服灌胃给药。
观察指标：每三天测量一次小鼠体重及肿瘤长径、短径并计算肿瘤体积(length×width2×0.5)。每天观察各组小鼠有无腹泻，抽搐，皮疹，体重明显降低等反应。
3)实验结果：
实验测得的实验分组一的肿瘤生长曲线见图1，实验测得的实验分组二的肿瘤生长曲线
见图2，实验测得的实验分组一的肿瘤生长曲线见图3，实验测得的实验分组二的肿瘤生长曲线见图4。
实验结果表明，受试化合物3对乳腺癌细胞株MDA-MB-231具有明显的体内生长抑制作用，在每天30mg/kg的剂量下，可以明显抑制肿瘤生长，并表现出优于阳性对照(紫杉醇)的抑制效果。化合物31对乳腺癌细胞株MDA-MB-231具有明显的体内生长抑制作用，在每天20mg/kg的剂量下，可以明显抑制肿瘤生长，并表现出优于阳性对照(紫杉醇及达沙替尼)的抑制效果。化合物93对乳腺癌细胞株MDA-MB-231具有明显的体内生长抑制作用，在每天20mg/kg的剂量下，可以明显抑制肿瘤生长，并表现出优于阳性对照(紫杉醇)的抑制效果。化合物100乳腺癌细胞株MDA-MB-231具有明显的体内生长抑制作用，在每天40mg/kg的剂量下，可以明显抑制肿瘤生长，并表现出优于阳性对照(紫杉醇及达沙替尼)的抑制效果。给药过程中未发现小鼠出现体重降低、皮疹、腹泻等不良反应，表明在测试剂量下，受试化合物3、化合物31、化合物93和化合物100在给药剂量范围内毒性较低。
实施例35 化合物31的转基因斑马鱼血管生成的抑制活性测试
本实验的目的是检测发明化合物对体内新生血管的抑制活性，采用的方法是考察多个浓度下本发明化合物对转基因荧光斑马鱼FLK1-GFP体节间血管的抑制情况。受试化合物的新生血管抑制活性用受试化合物在10ug/mL、5ug/mL、2.5ug/mL浓度下对斑马鱼体节间血管的抑制程度来表示。以乳腺癌临床在研的上市药物达沙替尼为阳性对照。
1)实验材料：
转基因斑马鱼(FLK1-GFP)：本实验室养殖
实验试剂：二甲基亚砜(DMSO)；受试化合物；达沙替尼
主要实验仪器：荧光显微镜；体视显微镜；CCD相机等。
2)实验方法：
斑马鱼胚胎的获得：实验室所用的斑马鱼为血管荧光转基因斑马鱼(FLK1：GFP)。斑马鱼的养殖和培育参照Westerfield的方法。在取卵的前一天，将雌雄斑马鱼按1:1比例配对。第二天在28℃左右的温度和充足的光照下使其自然交配产卵。采集足够的斑马鱼胚胎，清洗后放于胚胎培养液中，并放入28℃培养箱培养。利用形态和发育学标准来随时鉴定存活情况，已死的胚胎呈白色，应及时取出以防止水质变坏。
药物处理：在斑马鱼胚胎受精后10h(显微镜下观察斑马鱼胚胎发育至bud期)后，随机选取健康的胚胎分组，加入24孔板，每孔10个斑马鱼胚胎，吸净培养液，再加入不同浓度化合物溶液。化合物31浓度设置分别为10ug/mL、5ug/mL、2.5ug/mL。达沙替尼浓度设置分别为10ug/mL。同时设空白对照，不加入任何化合物。然后将其放入28℃培养箱培养。
结果观察：在斑马鱼胚胎受精31h后，取出斑马鱼并剥卵。然后将其放于载玻片上，加入1‰三卡因溶液麻醉鱼体并用1.5％甲基纤维素固定鱼体，接着在荧光显微镜下对体节间血管(Inter segmental vessel，ISV)进行观察计数拍照。
3)实验结果：
图5反映的是化合物31在不同浓度下对FLK1转基因斑马鱼的血管抑制情况。结果表明，和对照组相比，化合物31能很好地抑制斑马鱼的血管生成。本实验结果表明，本发明实施例中制得的化合物31对FLK1转基因斑马鱼的新生血管很好的抑制活性，这一结果反映了化合物31对VEGFR2具有很好的抑制活性。
Claims (18)
- 3-乙炔基吡唑并嘧啶衍生物，其结构如式Ⅰ所示：其中，R1为-H、C1～C4烷基、R2为-H、C1～C8烷基、R8取代的C3～C8环烷基、C8环氧烷基、或R3～R7独立地为-H、C1～C8烷基、-OH、C1～C8烷氧基、卤素、R8～R11独立地为-H、C1～C8烷基、卤素、-OH、R12～R14独立地为R15～R19独立地为-H、C1～C8烷基、-OH、C1～C8烷氧基、卤素、-CF3、-OCF3、R20～R38独立地为-H、卤素、C1～C8烷基、C1～C8环烷基、-OCF3或-CF3；R39～R42独立地为C1～C8烷基、C3～C8环烷基或C1～C8羟基烷基；n＝0～6。
- 根据权利要求1所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：R1为-H或R2为-H、C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、R8～R11独立地为-H、C1～C4烷基、-OH、R12～R14独立地为R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、R20～R38独立地为-H、卤素、C1～C4烷基、C1～C4环烷基、-OCF3或-CF3；R39～R42独立地为C1～C4烷基、C3～C8环烷基或C1～C4羟基烷基；n＝0～4。
- 根据权利要求2所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：R1为-H或R2为-H、C1～C4烷基、R8取代的C3～C8环烷基、C3～ C8环氧烷基、R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、R8～R11独立地为-H、C1～C4烷基、-OH、R12～R14独立地为R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、R20～R38独立地为-H、卤素、C1～C4烷基、C1～C4环烷基、-OCF3或-CF3；n＝0～2。
- 根据权利要求3所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：R1为-H或R2为-H、C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、R3～R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、-F、-Cl、R8～R11独立地为-H、C1～C4烷基、-OH、R12～R14独立地为R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、-F、-Cl、-CF3、-OCF3、R20～R38独立地为-H、C1～C4烷基或-CF3；n＝0或1。
- 根据权利要求2所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：当R6为时，其结构如式Ⅱ所示：其中，R1为-H或R2为-H、C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、R12为R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、R20～R38独立地为-H、C1～C4烷基或-CF3。
- 根据权利要求5所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：R1为-H或R2为C1～C4烷基、R8取代的C3～C8环烷基、n＝0或1；R3～R5、R7独立地为-H、甲基或-Cl；R8～R11独立地为-H、C1～C4烷基、-OH、R12为R15～R19独立地为-H、C1～C4烷基、甲氧基、-F、-Cl、-CF3、-OCF3或R20～R38独立地为-H、C1～C4烷基或-CF3。
- 根据权利要求2所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：当R6为时，其结构如式Ⅲ所示：其中，R1为-H或R2为-H、C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、R13为R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、R20～R38独立地为-H、C1～C4烷基或-CF3。
- 根据权利要求7所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：R1为-H或C1～C4烷基、R2为C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、R3～R5、R7独立地为-H、C1～C4烷基、-OH或-Cl；R8、R9独立地为-H、C1～C4烷基、R13为R15～R19独立地为-H、C1～C4烷基、-CF3或R20～R38独立地为-H或C1～C4烷基。
- 根据权利要求7所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：当R13为时，其结构如式Ⅳ所示：其中，R1为-H或R2为-H、C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、
- 根据权利要求9所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：R1为-H或R2为C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、R3～R5、R7独立地为-H、C1～C4烷基、-OH或-Cl；R8、R9独立地为-H、C1～C4烷基、R15～R19独立地为-H、C1～C4烷基、-CF3或
- 根据权利要求2所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：当R6为R14为时，其结构如式Ⅴ所示：其中，R1为-H或R2为-H、C1～C4烷基、R8取代的C3～C8环烷基、C3～C8环氧烷基、n＝0～4；R3～R5、R7独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基或卤素；R8～R11独立地为-H、C1～C4烷基、-OH、R15～R19独立地为-H、C1～C4烷基、-OH、C1～C4烷氧基、卤素、-CF3、-OCF3、R20～R38独立地为-H、C1～C4烷基或-CF3。
- 根据权利要求11所述的3-乙炔基吡唑并嘧啶衍生物，其特征在于：R1为-H；R2为C1～C4烷基或R3～R5、R7独立地为-H、C1～C4烷基或-Cl；R9为C1～C4烷基；R15～R19独立地为-H或-CF3。
- 3-乙炔基吡唑并嘧啶衍生物，其特征在于：其结构式为
- 权利要求1～13任一项所述的3-乙炔基吡唑并嘧啶衍生物药学上可接受的盐。
- 权利要求1～13任一项所述的3-乙炔基吡唑并嘧啶衍生物药学上可接受的水合物。
- 一种药物组合物，是由1～13任一项所述的3-乙炔基吡唑并嘧啶衍生物、权利要求14所述的盐或权利要求15所述的水合物添加药学上可以接受的辅助性成分制备而成的。
- 权利要求1～13任一项所述的3-乙炔基吡唑并嘧啶衍生物、权利要求14所述的盐或权利要求15所述的水合物在制备激酶抑制剂中的用途。
- 权利要求1～13任一项所述的3-乙炔基吡唑并嘧啶衍生物、权利要求14所述的盐或权利要求15所述的水合物在制备治疗肿瘤药物中的用途。
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| US10266537B2 (en) | 2015-01-13 | 2019-04-23 | St. Chuan University | 3-acetylenyl-pyrazole-pyrimidine derivative, and preparation method therefor and uses thereof |
| US11174250B2 (en) | 2016-03-01 | 2021-11-16 | Propellon Therapeutics Inc. | Substituted carboxamides as inhibitors of WDR5 protein-protein binding |
| US11319299B2 (en) | 2016-03-01 | 2022-05-03 | Propellon Therapeutics Inc. | Substituted carboxamides as inhibitors of WDR5 protein-protein binding |
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