CN117580575A - Glutarimide substituted isoxazole fused ring compound and application thereof - Google Patents
Glutarimide substituted isoxazole fused ring compound and application thereof Download PDFInfo
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- CN117580575A CN117580575A CN202280043489.3A CN202280043489A CN117580575A CN 117580575 A CN117580575 A CN 117580575A CN 202280043489 A CN202280043489 A CN 202280043489A CN 117580575 A CN117580575 A CN 117580575A
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- compound
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- mmol
- ethyl acetate
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 405
- 150000002545 isoxazoles Chemical class 0.000 title abstract description 4
- KNCYXPMJDCCGSJ-UHFFFAOYSA-N piperidine-2,6-dione Chemical group O=C1CCCC(=O)N1 KNCYXPMJDCCGSJ-UHFFFAOYSA-N 0.000 title abstract description 4
- 150000003839 salts Chemical class 0.000 claims abstract description 26
- 238000006467 substitution reaction Methods 0.000 claims description 31
- 239000003814 drug Substances 0.000 claims description 19
- 229940079593 drug Drugs 0.000 claims description 17
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 14
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- 125000004432 carbon atom Chemical group C* 0.000 claims description 10
- 125000006570 (C5-C6) heteroaryl group Chemical group 0.000 claims description 9
- 125000000592 heterocycloalkyl group Chemical group 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 8
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 125000004076 pyridyl group Chemical group 0.000 claims description 5
- 125000001072 heteroaryl group Chemical group 0.000 claims description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 615
- 238000006243 chemical reaction Methods 0.000 description 257
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 214
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 193
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 185
- 230000015572 biosynthetic process Effects 0.000 description 164
- 238000003786 synthesis reaction Methods 0.000 description 161
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 152
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 142
- 239000012074 organic phase Substances 0.000 description 139
- 239000000243 solution Substances 0.000 description 136
- 239000011541 reaction mixture Substances 0.000 description 134
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 132
- 229910052757 nitrogen Inorganic materials 0.000 description 118
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 110
- 239000000706 filtrate Substances 0.000 description 109
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 92
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 87
- 239000000203 mixture Substances 0.000 description 87
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 78
- 210000004027 cell Anatomy 0.000 description 76
- 239000003208 petroleum Substances 0.000 description 76
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 66
- 238000004440 column chromatography Methods 0.000 description 62
- 239000012043 crude product Substances 0.000 description 60
- 239000003480 eluent Substances 0.000 description 60
- 239000002904 solvent Substances 0.000 description 58
- -1 tetrahydrothiophen-2-yl Chemical group 0.000 description 54
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 45
- 239000012065 filter cake Substances 0.000 description 40
- 239000008346 aqueous phase Substances 0.000 description 39
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 38
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 34
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 33
- 238000003756 stirring Methods 0.000 description 32
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- 239000012634 fragment Substances 0.000 description 28
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 26
- 238000012360 testing method Methods 0.000 description 26
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 25
- 239000012071 phase Substances 0.000 description 24
- 230000002378 acidificating effect Effects 0.000 description 23
- 238000001914 filtration Methods 0.000 description 23
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 22
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 21
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 21
- 238000002953 preparative HPLC Methods 0.000 description 20
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 19
- 206010028980 Neoplasm Diseases 0.000 description 18
- 238000012054 celltiter-glo Methods 0.000 description 18
- 230000000694 effects Effects 0.000 description 18
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 18
- 229910000027 potassium carbonate Inorganic materials 0.000 description 17
- 238000000605 extraction Methods 0.000 description 16
- 239000005457 ice water Substances 0.000 description 16
- LPNYRYFBWFDTMA-UHFFFAOYSA-N potassium tert-butoxide Chemical compound [K+].CC(C)(C)[O-] LPNYRYFBWFDTMA-UHFFFAOYSA-N 0.000 description 16
- 239000007787 solid Substances 0.000 description 16
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 14
- 230000005764 inhibitory process Effects 0.000 description 14
- 241000282414 Homo sapiens Species 0.000 description 13
- 206010060862 Prostate cancer Diseases 0.000 description 13
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 13
- 238000004113 cell culture Methods 0.000 description 13
- 238000000926 separation method Methods 0.000 description 12
- 125000001424 substituent group Chemical group 0.000 description 12
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 12
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 239000012046 mixed solvent Substances 0.000 description 10
- 229920006395 saturated elastomer Polymers 0.000 description 10
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 10
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 9
- 241000700159 Rattus Species 0.000 description 9
- 102000001307 androgen receptors Human genes 0.000 description 9
- 108010080146 androgen receptors Proteins 0.000 description 9
- 238000001035 drying Methods 0.000 description 9
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- 235000011056 potassium acetate Nutrition 0.000 description 9
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 9
- 239000012321 sodium triacetoxyborohydride Substances 0.000 description 9
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 8
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
- 125000004429 atom Chemical group 0.000 description 8
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- CYPYTURSJDMMMP-WVCUSYJESA-N (1e,4e)-1,5-diphenylpenta-1,4-dien-3-one;palladium Chemical compound [Pd].[Pd].C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1 CYPYTURSJDMMMP-WVCUSYJESA-N 0.000 description 7
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 7
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- IEJIGPNLZYLLBP-UHFFFAOYSA-N dimethyl carbonate Chemical compound COC(=O)OC IEJIGPNLZYLLBP-UHFFFAOYSA-N 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000012452 mother liquor Substances 0.000 description 7
- 239000012299 nitrogen atmosphere Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000001632 sodium acetate Substances 0.000 description 7
- 235000017281 sodium acetate Nutrition 0.000 description 7
- 229910002091 carbon monoxide Inorganic materials 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000001816 cooling Methods 0.000 description 6
- PQVSTLUFSYVLTO-UHFFFAOYSA-N ethyl n-ethoxycarbonylcarbamate Chemical compound CCOC(=O)NC(=O)OCC PQVSTLUFSYVLTO-UHFFFAOYSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 125000005647 linker group Chemical group 0.000 description 6
- 229940040692 lithium hydroxide monohydrate Drugs 0.000 description 6
- GLXDVVHUTZTUQK-UHFFFAOYSA-M lithium hydroxide monohydrate Substances [Li+].O.[OH-] GLXDVVHUTZTUQK-UHFFFAOYSA-M 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- XBXCNNQPRYLIDE-UHFFFAOYSA-M n-tert-butylcarbamate Chemical compound CC(C)(C)NC([O-])=O XBXCNNQPRYLIDE-UHFFFAOYSA-M 0.000 description 6
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 6
- NROKBHXJSPEDAR-UHFFFAOYSA-M potassium fluoride Chemical compound [F-].[K+] NROKBHXJSPEDAR-UHFFFAOYSA-M 0.000 description 6
- 229910000160 potassium phosphate Inorganic materials 0.000 description 6
- 235000011009 potassium phosphates Nutrition 0.000 description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 6
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 239000012224 working solution Substances 0.000 description 6
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
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- 125000000524 functional group Chemical group 0.000 description 5
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
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- 125000000217 alkyl group Chemical group 0.000 description 4
- 239000003146 anticoagulant agent Substances 0.000 description 4
- 229940127219 anticoagulant drug Drugs 0.000 description 4
- 238000003149 assay kit Methods 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- NKLCNNUWBJBICK-UHFFFAOYSA-N dess–martin periodinane Chemical compound C1=CC=C2I(OC(=O)C)(OC(C)=O)(OC(C)=O)OC(=O)C2=C1 NKLCNNUWBJBICK-UHFFFAOYSA-N 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 4
- 238000002796 luminescence method Methods 0.000 description 4
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- 230000017854 proteolysis Effects 0.000 description 4
- 229910052717 sulfur Inorganic materials 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- CWXPZXBSDSIRCS-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCNCC1 CWXPZXBSDSIRCS-UHFFFAOYSA-N 0.000 description 4
- TYXKOMAQTWRDCR-UHFFFAOYSA-N 4-isothiocyanato-2-(trifluoromethyl)benzonitrile Chemical compound FC(F)(F)C1=CC(N=C=S)=CC=C1C#N TYXKOMAQTWRDCR-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 3
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 3
- 239000012346 acetyl chloride Substances 0.000 description 3
- 235000019270 ammonium chloride Nutrition 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 229910052805 deuterium Inorganic materials 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- SACNIGZYDTUHKB-UHFFFAOYSA-N ditert-butyl-[2-[2,4,6-tri(propan-2-yl)phenyl]phenyl]phosphane Chemical group CC(C)C1=CC(C(C)C)=CC(C(C)C)=C1C1=CC=CC=C1P(C(C)(C)C)C(C)(C)C SACNIGZYDTUHKB-UHFFFAOYSA-N 0.000 description 3
- PQJJJMRNHATNKG-UHFFFAOYSA-N ethyl bromoacetate Chemical compound CCOC(=O)CBr PQJJJMRNHATNKG-UHFFFAOYSA-N 0.000 description 3
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- 125000002950 monocyclic group Chemical group 0.000 description 3
- XBXHCBLBYQEYTI-UHFFFAOYSA-N piperidin-4-ylmethanol Chemical compound OCC1CCNCC1 XBXHCBLBYQEYTI-UHFFFAOYSA-N 0.000 description 3
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- MHYGQXWCZAYSLJ-UHFFFAOYSA-N tert-butyl-chloro-diphenylsilane Chemical compound C=1C=CC=CC=1[Si](Cl)(C(C)(C)C)C1=CC=CC=C1 MHYGQXWCZAYSLJ-UHFFFAOYSA-N 0.000 description 3
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- FTVLMFQEYACZNP-UHFFFAOYSA-N trimethylsilyl trifluoromethanesulfonate Chemical compound C[Si](C)(C)OS(=O)(=O)C(F)(F)F FTVLMFQEYACZNP-UHFFFAOYSA-N 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- IBYHHJPAARCAIE-UHFFFAOYSA-N 1-bromo-2-chloroethane Chemical compound ClCCBr IBYHHJPAARCAIE-UHFFFAOYSA-N 0.000 description 2
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- 125000004105 2-pyridyl group Chemical group N1=C([*])C([H])=C([H])C([H])=C1[H] 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
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- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 2
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- 238000006646 Dess-Martin oxidation reaction Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
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- RSIJVJUOQBWMIM-UHFFFAOYSA-L sodium sulfate decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].[O-]S([O-])(=O)=O RSIJVJUOQBWMIM-UHFFFAOYSA-L 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003419 tautomerization reaction Methods 0.000 description 1
- IWSFZKCIZFXAFT-UHFFFAOYSA-N tert-butyl 4-(2-bromoethyl)piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCN(CCBr)CC1 IWSFZKCIZFXAFT-UHFFFAOYSA-N 0.000 description 1
- WOEQSXAIPTXOPY-UHFFFAOYSA-N tert-butyl 4-methylsulfonyloxypiperidine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCC(OS(C)(=O)=O)CC1 WOEQSXAIPTXOPY-UHFFFAOYSA-N 0.000 description 1
- XKXIQBVKMABYQJ-UHFFFAOYSA-M tert-butyl carbonate Chemical compound CC(C)(C)OC([O-])=O XKXIQBVKMABYQJ-UHFFFAOYSA-M 0.000 description 1
- IOGXOCVLYRDXLW-UHFFFAOYSA-N tert-butyl nitrite Chemical compound CC(C)(C)ON=O IOGXOCVLYRDXLW-UHFFFAOYSA-N 0.000 description 1
- 239000012414 tert-butyl nitrite Substances 0.000 description 1
- DEGRJODPOICGRU-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate;hydrochloride Chemical compound Cl.CC(C)(C)OC(=O)N1CCNCC1 DEGRJODPOICGRU-UHFFFAOYSA-N 0.000 description 1
- 125000004192 tetrahydrofuran-2-yl group Chemical group [H]C1([H])OC([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000003507 tetrahydrothiofenyl group Chemical group 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- 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
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/42—Oxazoles
- A61K31/423—Oxazoles condensed with carbocyclic 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/4525—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
-
- A—HUMAN NECESSITIES
- 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
- A61K31/4965—Non-condensed pyrazines
- A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
Abstract
A series of glutarimide substituted isoxazole fused ring compounds and application thereof, in particular discloses a compound shown in a formula (II) and pharmaceutically acceptable salts thereof.
Description
The present application claims priority as follows:
CN2021106741905, filing date: 2021, 6, 17;
CN2021112224376, filing date: 2021, 10, 20.
The invention relates to a series of glutarimide substituted isoxazole fused ring compounds and application thereof, in particular to a compound shown in a formula (II) and pharmaceutically acceptable salts thereof.
Protein degradation targeting chimeras (Proteolysis Targeting Chimera, PROTAC) are a technique that uses the ubiquitin-proteasome system to target specific proteins and induce their degradation in cells. Ubiquitin-proteasome systems are the main pathway of protein degradation in cells, and their normal physiological functions are mainly responsible for eliminating intracellular denatured, mutated or harmful proteins, and more than 80% of protein degradation in cells depends on ubiquitin-proteasome systems. ProTAC utilizes the protein destruction mechanism of the cell itself to eliminate specific target proteins in the cell.
The present invention provides bifunctional or proteolytically targeted chimeric (PROTAC) compounds that were found to be modulators of targeted ubiquitination and Androgen Receptor (AR) degradation.
Disclosure of Invention
The invention provides a compound shown as a formula (II) or pharmaceutically acceptable salt thereof,
wherein,
R 1 selected from H and CH 3 ；
Linker is selected fromWherein E is 1 The end is connected with the ABM;
R 2 selected from H and methyl;
E 1 selected from single bond, O, NH and-C (=o) NH-;
E 2 selected from single bonds, - (CH) 2 ) m -、 and-E 5 -(CH 2 ) m -；
E 3 Selected from- (CH) 2 ) n -、-(CH 2 ) p O(CH 2 ) q -、
E 4 Selected from single bond, O and-C (=o) NH-;
E 5 selected from 5-6 membered heterocycloalkyl;
T 1 and T 2 Each independently selected from CH and N;
m is selected from 1, 2 and 3;
n is selected from 1, 2, 3, 4, 5, 6 and 7;
p and q are each independently selected from 0, 1 and 2;
ring Q is selected from phenyl or
Ring a is selected from phenyl and 5-6 membered heteroaryl;
ABM is selected from drugs or derivatives thereof that target binding to AR proteins.
In some aspects of the invention, E is as described above 2 Selected from single bond, -CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、 The other variables are as defined herein.
In some embodiments of the invention, the structural unit-E described above 1 -E 2 -is selected from single bond, -O-, -OCH 2 CH 2 -、 The other variables are as defined herein.
In some aspects of the invention, E is as described above 3 Selected from single bond, -CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 4 -、-(CH 2 ) 5 -、 The other variables are as defined herein.
In some embodiments of the invention, the structural unit-E described above 3 -E 4 -is selected from single bond, -CH 2 -、-(CH 2 ) 2 O-、-(CH 2 ) 5 O-、 The other variables are as defined herein.
In some embodiments of the invention, the Linker is selected from The other variables are as defined herein.
In some aspects of the invention, the structural units described aboveSelected from the group consisting ofThe other variables are as defined herein.
In some embodiments of the invention, the above ABM is selected from the structures of formulas (ABM-1) and (ABM-2):
wherein,
R 3 and R is 4 Selected from methyl;
alternatively, R 3 And R is R 4 With co-linked carbon atoms to C 4-6 Cycloalkyl;
ring B is selected from phenyl and pyridyl, said phenyl and pyridylThe radicals optionally being substituted by 1, 2 or 3R a Substitution;
ring C is selected from phenyl and 6 membered heteroaryl, optionally substituted with 1, 2 or 3R b Substitution;
R a selected from F, cl, br, I, CN, CH 3 、CF 3 And NO 2 ；
R b Selected from F and Cl;
ring D is selected from
In some embodiments of the invention, the above ABM is selected from structures of formulas (ABM-1 a), (ABM-2 b) and (ABM-3 a):
wherein,
R b1 selected from H and F;
t is selected from 1, 2 and 3;
T 1 selected from CH and N;
R a as defined herein.
In some embodiments of the invention, the ABM is selected from The other variables are as defined herein.
The invention also provides a compound shown in the formula (I) or pharmaceutically acceptable salt thereof,
wherein,
R 1 Selected from H and CH 3 ；
Linker is selected fromWherein E is 1 The end is connected with the ABM;
R 2 selected from H and methyl;
E 1 selected from single bond, O, NH and-C (=o) NH-;
E 2 selected from single bonds, - (CH) 2 ) m -、 and-E 5 -(CH 2 ) m -；
E 3 Selected from- (CH) 2 ) n -、-(CH 2 ) p O(CH 2 ) q -、
E 4 Selected from single bond, O and-C (=o) NH-;
E 5 selected from 5-6 membered heterocycloalkyl;
T 1 and T 2 Each independently selected from CH and N;
m is selected from 1, 2 and 3;
n is selected from 1, 2, 3, 4, 5, 6 and 7;
p and q are each independently selected from 0, 1 and 2;
ring a is selected from the group consisting of absent, phenyl, and 5-6 membered heteroaryl;
ABM is selected from drugs or derivatives thereof that target binding to AR proteins.
In some aspects of the invention, E is as described above 2 Selected from single bond, -CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、 The other variables are as defined herein.
In some embodiments of the invention, the structural unit-E described above 1 -E 2 -is selected from single bond, -O-, -OCH 2 CH 2 -、 The other variables are as defined herein.
In some aspects of the invention, E is as described above 3 Selected from-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 4 -、-(CH 2 ) 5 -、 The other variables are as defined herein.
In some embodiments of the invention, the structural unit-E described above 3 -E 4 -is selected from-CH 2 -、-(CH 2 ) 2 O-、-(CH 2 ) 5 O-、 The other variables are as defined herein.
In some embodiments of the invention, the Linker is selected from The other variables are as defined herein.
In some aspects of the invention, the structural units described aboveSelected from the group consisting of The other variables are as defined herein.
In some embodiments of the invention, the above ABM is selected from the structures of formulas (ABM-1) and (ABM-2):
wherein,
R 3 and R is 4 Selected from methyl;
alternatively, R 3 And R is R 4 With co-linked carbon atoms to C 4-6 Cycloalkyl;
ring B is selected from phenyl and pyridinyl, optionally substituted with 1, 2 or 3R a Substitution;
ring C is selected from phenyl and 6 membered heteroaryl, optionally substituted with 1, 2 or 3R b Substitution;
R a selected from F, cl, br, I, CN, CH 3 、CF 3 And NO 2 ；
R b Selected from F and Cl;
ring D is selected from
In some embodiments of the invention, the above ABM is selected from structures of formulas (ABM-1 a), (ABM-2 a) and (ABM-2 b):
wherein,
R b1 selected from H and F;
t is selected from 1, 2 and 3;
R a as defined herein.
In some embodiments of the invention, the ABM is selected from The other variables are as defined herein.
Still other embodiments of the present invention are derived from any combination of the variables described above.
The invention also provides the following compounds or pharmaceutically acceptable salts thereof:
technical effects
The compound has excellent AR protein degradation effect, cell proliferation inhibition effect and remarkable tumor inhibition effect, and particularly has excellent treatment effect on prostate cancer; has good drug properties.
Definition and description
The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.
The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Certain specific compounds of the invention contain basic and acidic functionalities that can be converted to either base or acid addition salts.
Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
The term "targeting protein" refers to a protein or polypeptide that binds to a compound of the invention and is degraded.
The term "drug or derivative thereof" includes drugs or derivatives thereof that have been developed to bind to the targeting protein and drugs or derivatives thereof that have been developed in the future to bind to the targeting protein.
The compounds of the invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present invention.
Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of each other.
Unless otherwise indicated, the term "diastereoisomer" refers to stereoisomers of a molecule having two or more chiral centers and having a non-mirror relationship between the molecules.
Unless otherwise indicated, "(+)" means dextrorotation, "(-)" means levorotatory, "(±)" means racemization.
Unless otherwise indicated, with solid wedge bondsAnd a wedge-shaped dotted bondRepresenting the absolute configuration of a solid centre by straight solid keysAnd straight dotted line keyRepresenting the relative configuration of the three-dimensional center by wavy linesSolid key representing wedge shapeOr wedge-shaped dotted bondOr by wave linesRepresenting straight solid keysAnd straight dotted line key
Unless otherwise specified, when a group has one or more bondable sites, any one or more of the sites of the group may be bonded to other groups by chemical bonds. The chemical bond of the site and other groups can be a straight solid line bondStraight dotted line keyOr wave linesAnd (3) representing. For example-OCH 3 The straight solid line bond in (a) represents the connection to other groups through the oxygen atom in the group; Straight shape of (3)The dashed bonds represent the attachment to other groups through both ends of the nitrogen atom in the group;the wavy line in (2) represents the attachment to other groups through carbon atoms at positions 1 and 2 in the phenyl group.
The compounds of the invention may be present in particular. Unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to the fact that at room temperature, different functional group isomers are in dynamic equilibrium and are capable of rapid interconversion. If tautomers are possible (e.g., in solution), chemical equilibrium of the tautomers can be reached. For example, proton tautomers (also known as proton tautomers) (prototropic tautomer) include interconversions by proton transfer, such as keto-enol isomerisation and imine-enamine isomerisation. Valence isomer (valance tautomer) includes the interconversion by recombination of some of the bond-forming electrons. A specific example of where keto-enol tautomerization is the interconversion between two tautomers of pentane-2, 4-dione and 4-hydroxypent-3-en-2-one.
Unless otherwise indicated, the terms "enriched in one isomer", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100% and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or enantiomers. For example, where one isomer or enantiomer is present in an amount of 90% and the other isomer or enantiomer is present in an amount of 10%, the isomer or enantiomer excess (ee value) is 80%.
Optically active (R) -and (S) -isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it may be prepared by asymmetric synthesis or derivatization with chiral auxiliary wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereomeric resolution is carried out by conventional methods well known in the art, and then the pure enantiomer is recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amine).
The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, compounds can be labeled with radioisotopes, such as tritium @, for example 3 H) Iodine-125% 125 I) Or C-14% 14 C) A. The invention relates to a method for producing a fibre-reinforced plastic composite For example, deuterium can be substituted for hydrogen to form a deuterated drug, and the bond between deuterium and carbon is stronger than the bond between normal hydrogen and carbon, so that the deuterated drug has the advantages of reducing toxic and side effects, increasing the stability of the drug, enhancing the curative effect, prolonging the biological half-life of the drug and the like compared with the non-deuterated drug. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
The term "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, which may include deuterium and variants of hydrogen, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =o), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on the aromatic group. The term "optionally substituted" means that the substituents may or may not be substituted, and the types and numbers of substituents may be arbitrary on the basis that they can be chemically achieved unless otherwise specified.
When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 to 2R, the group may optionally be substituted with up to two R's, and R's in each case have independent options. Furthermore, combinations of substituents and/or variants thereof are only permissible if such combinations result in stable compounds.
When the number of one linking group is 0, such as- (CRR) 0 -it is meant that the linking group is a single bond.
When a substituent is absent, it is meant that the substituent is absent, e.g., X in A-X is absent, meaning that the structure is actually A. When the listed substituents do not indicate which atom is attached to the substituted group, such substituents may be bonded through any atom thereof, for example, a pyridyl group may be attached to the substituted group as a substituent through any carbon atom on the pyridine ring.
The term "C1-3 alkyl" is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms, unless otherwise specified. The C is 1-3 Alkyl includes C 1-2 And C 2-3 Alkyl groups, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C (C) 1- 3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
Unless otherwise specified, the number of atoms on a ring is generally defined as the number of ring elements, e.g., "5-7 membered ring" refers to a "ring" of 5-7 atoms arranged around a ring.
Unless otherwise specified, "C 4-6 Cycloalkyl "means a saturated cyclic hydrocarbon group consisting of 4 to 6 carbon atoms, which includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spiro, fused and bridged rings. The C is 4-6 Cycloalkyl includes C 4-6 、C 4-5 Or C 5-6 Cycloalkyl groups, and the like; it may be monovalent, divalent or multivalent. C (C) 4-6 Examples of cycloalkyl groups include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
Unless otherwise specified, C n-n+m Or C n -C n+m Comprising any one of the specific cases of n to n+m carbons, e.g. C 1-12 Comprises C 1 、C 2 、C 3 、C 4 、C 5 、C 6 、C 7 、C 8 、C 9 、C 10 、C 11 And C 12 Also included is any one of the ranges n to n+m, e.g. C 1-12 Comprises C 1- 3 、C 1-6 、C 1-9 、C 3-6 、C 3-9 、C 3-12 、C 6-9 、C 6-12 And C 9-12 Etc.; similarly, n-membered to n+m-membered means that the number of atoms on the ring is n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and any one of n to n+m is also included, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring, 5-7-membered ring, 6-8-membered ring, 6-10-membered ring, and the like.
Unless otherwise specified, the term "5-6 membered heterocycloalkyl" by itself or in combination with other terms, denotes a saturated cyclic group consisting of 5 to 6 ring atoms, 1,2, 3 or 4 of which are heteroatoms independently selected from O, S and N, the remainder being carbon atoms, wherein the nitrogen atoms are optionally quaternized and the nitrogen and sulfur heteroatoms may optionally be oxidized (i.e. NO and S (O) p, p being 1 or 2), respectively. It includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spiro, fused and bridged rings. In addition, in the case of the "5-6 membered heterocycloalkyl" group, the heteroatom may occupy the position of attachment of the heterocycloalkyl group to the remainder of the molecule. The 5-6 membered heterocycloalkyl group includes 5-and 6-membered heterocycloalkyl groups. Examples of 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl, and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1, 2-oxazinyl, 1, 2-thiazinyl, hexahydropyridazinyl, etc.
The terms "5-6 membered heteroaryl ring" and "5-6 membered heteroaryl" are used interchangeably herein unless otherwise specified, the term "5-6 membered heteroaryl" meaning a monocyclic group having a conjugated pi-electron system consisting of 5 to 6 ring atoms, 1,2,3 or 4 of which are heteroatoms independently selected from O, S and N, the remainder being carbon atoms. Wherein the nitrogen atom is optionally quaternized and the nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S (O) p, p being 1 or 2). The 5-6 membered heteroaryl group may be attached to the remainder of the molecule through a heteroatom or carbon atom. The 5-6 membered heteroaryl groups include 5-and 6-membered heteroaryl groups. Examples of the 5-6 membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl, 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, etc.), triazolyl (1H-1, 2, 3-triazolyl, 2H-1,2, 3-triazolyl, 1H-1,2, 4-triazolyl, 4H-1,2, 4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, etc.), furanyl (including 2-furanyl, 3-furanyl, etc.), thienyl (including 2-thienyl, 3-thienyl, etc.), pyridyl (including 2-pyridyl, 4-pyridyl, etc.), pyrimidyl (including 2-pyridyl, 4-pyrimidyl, etc.), pyrimidyl, etc.
Unless otherwise specified, when a group has one or more bondable sites, any one or more of the sites of the group may be bonded to other groups by chemical bonds. When the connection mode of the chemical bond is not positioned and the H atoms exist in the connectable site, the number of the H atoms of the site can be correspondingly reduced to be changed into the corresponding valence group along with the number of the connected chemical bond when the chemical bond is connected. The chemical bond of the site and other groups can be a straight solid line bondStraight dotted line keyOr wave linesAnd (3) representing. For example-OCH 3 The straight solid line bond in (a) represents the connection to other groups through the oxygen atom in the group;the straight dashed bonds in (a) represent the attachment to other groups through both ends of the nitrogen atom in the group;the wavy line means that the carbon atoms at positions 1 and 2 in the phenyl group are attached to other groups;representing any available ligatable site on the naphthalene ring that may be attached to other groups by 1 chemical bondConnected at least comprisesThese 6 connection modes.
The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.
The compounds of the present invention may be structured by conventional methods well known to those skilled in the art, and if the present invention relates to the absolute configuration of a compound, the absolute configuration may be confirmed by conventional means in the art. For example, single crystal X-ray diffraction (SXRD), the grown single crystal is collected from diffraction intensity data using a Bruker D8 vent diffractometer, and the light source is cukα radiation, scanning:after scanning and collecting the relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by a direct method (Shellxs 97).
The solvent used in the present invention is commercially available.
Compounds are either prepared according to the general nomenclature of the art or are usedSoftware naming, commercial compounds are referred to by vendor catalog names.
The present invention is described in detail below by way of examples, but is not meant to be limiting in any way. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the invention without departing from the spirit and scope of the invention.
Reference example 1: fragment BB-1
The synthetic route is as follows:
step 1: synthesis of intermediate BB-1-2
Compound BB-1-1 (100 g,465.02 mmol), dimethyl carbonate (167.55 g,1.86mol,156.59 mL) was dissolved in tetrahydrofuran (1L), replaced with nitrogen three times, cooled to 0℃under nitrogen protection, potassium t-butoxide (313.08 g,2.79 mol) was added, and the temperature was raised to 70℃and stirred for 12 hours. The solvent was removed by concentrating under reduced pressure, methyl tert-butyl ether (500 mL) was added, slowly poured into ice water (1000 mL), the organic phase was washed with aqueous sodium hydroxide (2N, 250 mL), the aqueous phases were combined, the pH was adjusted to 2-3 with hydrochloric acid (6N), filtration was performed, the filter cake was rinsed with water (500 mL. Times.3), and the filter cake was collected to give intermediate BB-1-2. 1 H NMR(400MHz，DMSO_d 6 )δ：7.73(d，J＝8.8Hz，1H)，7.67(d，J＝1.6Hz，1H)，7.53(dd，J＝1.8，8.2Hz，1H)，5.62(s，1H)。
Step 2: synthesis of intermediate BB-1-3
Intermediate BB-1-2 (120 g,497.85 mmol) was dissolved in ethanol (1.2L), sodium acetate (142.94 g,1.74 mol) and hydroxylamine hydrochloride (121.09 g,1.74 mol) were added, and the mixture was stirred at 80℃for 15 hours under nitrogen. The reaction solution was cooled to room temperature, filtered, the filter cake was rinsed with ethanol (200 mL), the filtrate was concentrated under reduced pressure to remove most of the ethanol, ph=8 was adjusted with aqueous sodium bicarbonate, extracted with ethyl acetate (500 ml×2), and the organic phase was discarded; the aqueous phase was adjusted to pH 2-3 with dilute hydrochloric acid (2N), extracted with ethyl acetate (1 L×2), and the organic phase was extracted with The phases were washed with saturated brine (1L), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate BB-1-3. 1 H NMR(400MHz，DMSO_d 6 )δ：8.11(d，J＝1.2Hz，1H)，7.82(d，J＝8.4Hz，1H)，7.59(dd，J＝1.2，8.4Hz，1H)，4.11(s，2H)。
Step 3: synthesis of intermediate BB-1
Concentrated sulfuric acid (9.73 g,97.25mmol,5.29mL, purity: 98%) was added to a solution of intermediate BB-1-3 (83 g,324.15 mmol) in ethanol (1L), stirred at 80℃for 15 hours, additional concentrated sulfuric acid (0.5 mL, purity: 98%) was added, and stirring was continued for 15 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure to remove most of ethanol, diluted with ice water (500 mL) and dichloromethane (1L), separated, the aqueous phase was extracted with dichloromethane (200 mL), the organic phases were combined, washed with saturated aqueous sodium bicarbonate (500 mL) and saturated brine (500 mL) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-1. 1 H NMR(400MHz，CDCl 3 )δ：7.78(d，J＝1.2Hz，1H)，7.59(d，J＝8.4Hz，1H)，7.46(dd，J＝1.6，8.4Hz，1H)，4.22(q，J＝7.2Hz，2H)，4.02(s，2H)，1.27(t，J＝7.2Hz，3H)。
Reference example 2: fragment BB-2
The synthetic route is as follows:
step 1: synthesis of intermediate BB-2-1
Intermediate BB-1 (3 g,10.56 mmol) and tert-butyl carbamate (6.18 g,52.80 mmol) were added to toluene (150 mL) and water (15 mL), and potassium phosphate (6.72 g,31.68 mmol), tris (dibenzylideneacetone) dipalladium (676.87 mg, 739.16. Mu. Mol) and 2-di-tert-butylphosphino 2',4',6' -tris (Tri-phenylpyruvate)Isopropylbiphenyl (627.76 mg,1.48 mmol) was successively added to the above reaction solution, and the mixture was replaced with nitrogen gas 3 times, and then heated to 100℃and stirred for 12 hours. After the completion of the reaction, the reaction mixture was cooled to room temperature, diluted with water (300 mL), extracted with ethyl acetate (100 mL. Times.3), and the organic phases were combined, washed with saturated brine (100 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separating and purifying by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-5/1, volume ratio) to obtain intermediate BB-2-1. 1 H NMR(400MHz，CDCl 3 )δ：7.89(s，1H)，7.56(d，J＝8.4Hz，1H)，7.14(br d，J＝8.8Hz，1H)，4.12(q，J＝7.1Hz，2H)，3.98(s，2H)，1.54(s，9H)，1.28-1.23(m，3H)。
Step 2: synthesis of intermediate BB-2-2
Intermediate BB-2-1 (3.5 g,10.93 mmol) and acrylamide (621.27 mg,8.74mmol, 603.18. Mu.L) were added to N, N-dimethylformamide (5 mL), the reaction solution was cooled to 0℃and potassium t-butoxide (1.23 g,10.93 mmol) was slowly added in portions to the reaction solution, and the mixture was stirred at 0℃for 1 hour after 3 times of displacement with nitrogen. After completion of the reaction, the reaction mixture was slowly poured into saturated ammonium chloride (1200 mL), extracted with ethyl acetate (200 mL. Times.2), and the organic phases were combined, washed with water (200 mL. Times.2) and saturated brine in this order (200 mL. Times.2), and the organic phases were collected and concentrated under reduced pressure. The crude product is separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-1/1, volume ratio) to obtain intermediate BB-2-2. 1 H NMR(400MHz，DMSO_d 6 )δ：11.06(s，1H)，9.85(s，1H)，7.92(s，1H)，7.69(d，J＝8.8Hz，1H)，7.34(dd，J＝1.6，8.8Hz，1H)，4.51(dd，J＝5.0，11.8Hz，1H)，2.84-2.68(m，1H)，2.65-2.55(m，1H)，2.48-2.38(m，1H)，2.25-2.13(m，1H)，1.50(s，9H)。
Step 3: synthesis of intermediate BB-2-3
Intermediate BB-2-2 (400 mg,1.16 mmol) was added to N, N-dimethylformamide (5 mL), methyl iodide (246.60 mg,1.74mmol, 108.16. Mu.L) and potassium carbonate (320.16 mg,2.32 mmol) were slowly added to the reaction solution, and the mixture was stirred at 25℃for 2 hours after 3 times of nitrogen substitution. After completion of the reaction, the reaction mixture was slowly poured into water (2)0 mL), extracted with ethyl acetate (20 ml×2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-3:1, volume ratio) to give intermediate BB-2-3.MS-ESI m/z:359.9[ M+H ] ] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：9.85(s，1H)，7.93(s，1H)，7.69(d，J＝8.4Hz，1H)，7.33(dd，J＝1.4，8.6Hz，1H)，4.61(dd，J＝4.8，12.0Hz，1H)，3.05(s，3H)，2.95-2.82(m，1H)，2.79-2.69(m，1H)，2.44-2.29(m，1H)，2.25-2.10(m，1H)，1.50(s，9H)。
Step 4: synthesis of hydrochloride of intermediate BB-2
Intermediate BB-2-3 (45 mg, 125.22. Mu. Mol) was added to ethyl acetate (2 mL), and a hydrochloric acid/ethyl acetate solution (4M, 2 mL) was slowly added to the reaction mixture, and the mixture was stirred at 25℃for 12 hours after 3 times of nitrogen substitution. After the reaction is finished, the reaction solution is decompressed and concentrated to obtain the hydrochloride of the intermediate BB-2.
Reference example 3: fragment BB-3
The synthetic route is as follows:
step 1: synthesis of intermediate BB-3-2
Compound BB-3-1 (15 g,69.11 mmol) was dissolved in methylene chloride (400 mL), cooled to 0-5 ℃, boron trifluoride diethyl ether (98.08 g,691.05 mmol) was added, acetic anhydride (56.95 g,557.81mmol,52.24 mL) was then added dropwise, and after the addition was completed, the reaction solution was stirred at room temperature for 2 hours. After completion of the reaction, water (200 mL) was added to the reaction mixture, the pH was adjusted to 7-8 with sodium hydroxide solution (6M), the mixture was extracted with methylene chloride (300 mL. Times.3), and the organic phases were combined and concentrated with saturated brine(1000 mL) was washed, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was stirred for 0.5 hour with petroleum ether (200 mL) and ethyl acetate (20 mL), and then filtered to collect the cake, to give intermediate BB-3-2.MS-ESI m/z:259.0[ M+H ]] + ，261.0[M+2+H] + 。
Step 2: synthesis of intermediate BB-3-3
Intermediate BB-3-2 (35 g,135.09 mmol) was dissolved in dichloromethane (300 mL), cooled to-60 to-40℃and a boron trichloride/dichloromethane solution (1M, 135.09 mL) was added dropwise. After the dripping is finished, the temperature is raised to 0-5 ℃ under the protection of nitrogen, and the mixture is stirred for 2 hours. After completion of the reaction, the reaction solution was slowly poured into ice water (300 mL), the solution was separated, the aqueous phase was extracted with methylene chloride (200 ml×2), the organic phases were combined, washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was stirred for 0.5 hour with petroleum ether (200 mL) and ethyl acetate (20 mL), and then filtered to collect the cake, to give intermediate BB-3-3.
Step 3: synthesis of intermediate BB-3-4
Intermediate BB-3-3 was dissolved in tetrahydrofuran (300 mL), dimethyl carbonate (47.05 g,522.30mmol,43.97 mL) was added, the temperature was lowered to 0℃and potassium t-butoxide (87.91 g,783.45 mmol) was added. Under the protection of nitrogen, the temperature is raised to 70 ℃ and stirred for about 16 hours. After the reaction, slowly pouring the reaction solution into ice water (200 mL), regulating the pH to 8-9 by using hydrochloric acid solution (2M), collecting a water phase after liquid separation, continuously regulating the pH of the water phase to 2-3 by using hydrochloric acid solution (6M), separating out solids, filtering, washing a filter cake by using water (100 mL), collecting the filter cake, and drying to obtain an intermediate BB-3-4. 1 H NMR(400MHz，DMSO_d 6 )δ：12.65(br s，1H)，7.90(s，1H)，7.16(s，1H)，5.49(s，1H)，3.94(s，3H)。
Step 4: synthesis of Compound BB-3-5
In a previously dried reaction flask were added intermediate BB-3-4 (12 g,44.27 mmol) and ethanol (150 mL), followed by hydroxylamine hydrochloride (10.77 g,154.95 mmol) and sodium ethoxide (10.54 g,154.95 mmol) in sequence, and the mixture was heated to 80℃under nitrogen and stirred for 4 hours. After the reaction is completed, the reaction is reversedThe reaction solution was concentrated under reduced pressure to give a residue, to which was added water (200 mL), stirred for 0.5 hours, filtered, and the cake was rinsed with water (50 mL), and the cake was collected. Ethyl acetate (50 mL) and petroleum ether (50 mL) were added to the filter cake, stirred for 0.5 hour, and then filtered to collect the filter cake, thereby obtaining intermediate BB-3-5.MS-ESI m/z:286.0[ M+H ]] + ，288.0[M+2+H] + 。
Step 5: synthesis of Compound BB-3-6
Intermediate BB-3-5 (12 g,41.95 mmol) was dissolved in ethanol (150 mL), concentrated sulfuric acid (3.68 g,36.77mmol,2.00mL, purity: 98%) was added, and the mixture was heated to 80℃under nitrogen protection and stirred for 4 hours. After the reaction, the reaction solution was concentrated under reduced pressure to 1/2 of the volume, solids were precipitated, petroleum ether (100 mL) was added, stirred for 0.5 hour, and the cake was collected by filtration to give intermediate BB-3-6. 1 H NMR(400MHz，CDCl 3 )δ：7.89(s，1H)，7.05(s，1H)，4.23(q，J＝7.2Hz，2H)，3.99(s，3H)，3.97(s，2H)，1.29(t，J＝7.2Hz，3H)。
Step 6: synthesis of intermediate BB-3-7
Intermediate BB-3-6 (5 g,15.92 mmol) was dissolved in toluene (100 mL) and water (20 mL), tert-butyl carbamate (3.73 g,31.83 mmol), potassium phosphate (10.14 g,47.75 mmol), tris (dibenzylideneacetone) dipalladium (1.17 g,1.27 mmol) and 2-di-tert-butyl phosphino-2 ',4',6' -triisopropylbiphenyl (1.08 g,2.55 mmol) were added sequentially, nitrogen was replaced three times, and the temperature was raised to 100℃and stirred for 36 hours. After the completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain a residue. To the residue were added water (50 mL) and methylene chloride (50 mL), filtered, the filtrate was separated, the aqueous phase was extracted with methylene chloride (60 ml×3), and the organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was separated by column chromatography (eluent: petroleum ether/dichloromethane/ethyl acetate=50/50/1-25/25/1, volume ratio) to give intermediate BB-3-7. 1 H NMR(400MHz，DMSO_d 6 )δ：8.35(br s，1H)，7.27(s，1H)，7.02(s，1H)，4.23(q，J＝7.2Hz，2H)，4.00-3.97(m，5H)，1.55(s，9H)，1.29(t，J＝7.2Hz，3H)。
Step 7: synthesis of intermediate BB-3-8
Intermediate BB-3-7 (0.9 g,2.57 mmol) was dissolved in dichloromethane (20 mL), nitrogen was displaced three times, cooled to 0℃and anhydrous aluminum trichloride (1.71 g,12.84mmol, 701.88. Mu.L) was added, and the reaction mixture was warmed to 40℃and stirred overnight. The reaction solution was cooled to 0℃and supplemented with anhydrous aluminum trichloride (1.37 g,10.27mmol, 561.51. Mu.L), followed by heating to 40℃for further reaction overnight. After the completion of the reaction, water (20 mL) was slowly added to the reaction mixture, and the mixture was stirred at 40℃for 0.5 hour. The mixture was separated, the aqueous phase was extracted with a mixed solvent of dichloromethane and ethanol (20 mL. Times.5, 10:1, volume ratio), and the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=10/1-1.5/1, volume ratio) to give intermediate BB-3-8. 1 H NMR(400MHz，DMSO_d 6 )δ：6.88(s，1H)，6.74(s，1H)，4.12(q，J＝7.2Hz，2H)，3.95(s，2H)，1.19(t，J＝7.2Hz，3H)。
Step 8: synthesis of intermediate BB-3
Intermediate BB-3-8 (0.3 g,1.27 mmol) was dissolved in methylene chloride (3 mL) at room temperature, chloroacetyl chloride (172.12 mg,1.52mmol, 121.21. Mu.L) and triethylamine (192.76 mg,1.90mmol, 265.15. Mu.L) were added, and after reaction for 0.5 hours, the mixture was concentrated under reduced pressure to give a residue. To the residue were added xylene (3 mL) and pyridine p-toluenesulfonate (319.15 mg,1.27 mmol), and the temperature was raised to 140℃and the reaction was continued for 4 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and concentrated under reduced pressure to obtain a residue. To the residue was added water (10 mL), extracted with ethyl acetate (10 mL. Times.3), and the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product is separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=10/1-5/1, volume ratio) to obtain intermediate BB-3. 1 H NMR(400MHz，DMSO_d 6 )δ：8.27(s，1H)，8.24(s，1H)，5.12(s，2H)，4.28(s，2H)，4.15(q，J＝7.1Hz，2H)，1.20(t，J＝7.0Hz，3H)。
Reference example 4: fragment BB-4
The synthetic route is as follows:
step 1: synthesis of intermediate BB-4-2
Compound BB-4-1 (10 g,78.67 mmol) was dissolved in dichloromethane (120 mL) and acetone (60 mL) under nitrogen protection at 0-5℃and cyano-trimethylsilane (12.45 g,125.50mmol,15.70 mL) was slowly added dropwise in this order, trimethylsilyl triflate (820.00 mg,3.69mmol, 666.67. Mu.L) and the reaction mixture was stirred at 25℃for 2 hours. After the reaction, the mixture was cooled to 0-5℃and diluted with water (200 mL) and extracted with ethyl acetate (200 mL. Times.3). The combined organic phases were washed with saturated brine (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=10/1-5/1, volume ratio) to give intermediate BB-4-2. 1 H NMR(400MHz，CDCl 3 )δ：6.83(t，J＝9.0Hz，1H)，6.74(dd，J＝2.4，12.0Hz，1H)，6.66-6.59(m，1H)，1.65(s，6H)。
Step 2: synthesis of intermediate BB-4-3
Intermediate BB-4-2 (8 g,45.40 mmol) was dissolved in N, N-dimethylacetamide (150 mL) at room temperature under nitrogen protection, 4-isothiocyanato-2- (trifluoromethyl) benzonitrile (10.36 g,45.40 mmol) was added in portions to the reaction solution, the reaction mixture was stirred at 25℃for 12 hours, methanol (60 mL), diluted hydrochloric acid (2M, 60 mL) was added, the reaction mixture was stirred at 70℃for 3 hours, after completion of the reaction, cooled to room temperature, diluted with water (500 mL) was added, and extracted with ethyl acetate (200 mL. Times.3). The organic phases were combined, washed with saturated brine (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure from the filtrate . The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=3/1-1/1, volume ratio) to give intermediate BB-4-3. 1 H NMR(400MHz，CDCl 3 )δ：8.02-7.94(m，2H)，7.85(dd，J＝1.8，8.2Hz，1H)，7.19-7.10(m，2H)，6.99-6.90(m，2H)，1.58(s，6H)。
Step 3: synthesis of intermediate BB-4-4
Intermediate BB-4-3 (2 g,4.72 mmol) was dissolved in N, N-dimethylformamide (50 mL) at room temperature under nitrogen, tert-butyl-4- (2-bromoethyl) piperazine-1-carboxylate (1.66 g,5.67 mmol), potassium carbonate (1.31 g,9.45 mmol) and potassium iodide (784.17 mg,4.72 mmol) were added sequentially, and the reaction mixture was heated to 80℃and stirred for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (100 mL. Times.3). The organic phases were combined, washed with saturated brine (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=3/1-1/1, volume ratio) to give intermediate BB-4-4.MS-ESI m/z:636.3[ M+H ]] + 。
Step 4: synthesis of hydrochloride of intermediate BB-4-5
Intermediate BB-4-4 (2.0 g,3.15 mmol) was dissolved in ethyl acetate (50 mL) at room temperature under nitrogen protection, hydrochloric acid/ethyl acetate (4M, 3.93 mL) was slowly added dropwise to the reaction mixture, the reaction mixture was stirred at 25℃for 12 hours, and after completion of the reaction, the reaction mixture was concentrated under reduced pressure to give hydrochloride of intermediate BB-4-5. 1 H NMR(400MHz，CD 3 OD)δ：8.21-8.12(m，2H)，7.98(dd，J＝2.0，8.4Hz，1H)，7.38(t，J＝9.0Hz，1H)，7.30(dd，J＝2.4，11.6Hz，1H)，7.22(br d，J＝8.8Hz，1H)，4.68-4.59(m，2H)，3.86-3.77(m，6H)，3.73-3.63(m，4H)，1.57(s，6H)。
Step 5: synthesis of intermediate BB-4-6
Intermediate BB-4-5 (1.5 g,2.62mmol, hydrochloride) was dissolved in acetonitrile (50 mL) at room temperature under nitrogen, followed by the addition of ethyl bromoacetate (875.85 mg,5.24mmol, 580.04. Mu.L) and potassium carbonate (724.86mg,5.24 mmol) and the reaction mixture was heated to 80℃and stirred for 5 hours, after the reaction was completed, cooled to room temperature, diluted with water (100 mL) and extracted with ethyl acetate (100 mL. Times.3). The organic phases were combined, washed with saturated brine (100 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=3/1-1/1, volume ratio) to give intermediate BB-4-6.MS-ESI m/z:622.2[ M+H ]] + 。
Step 6: synthesis of intermediate BB-4
Intermediate BB-4-6 (1.5 g,2.42 mmol) was dissolved in ethanol (50 mL) and lithium hydroxide (1M, 7.25 mL) was slowly added dropwise to the reaction mixture at 25℃under stirring for 12 hours under nitrogen. After the reaction, the pH of the reaction mixture was adjusted to 2-3 with hydrochloric acid solution (2M), and concentrated under reduced pressure to give intermediate BB-4.MS-ESI m/z:594.1[ M+H ]] + 。
Reference example 5: fragment BB-5
The synthetic route is as follows:
step 1: synthesis of intermediate BB-5-2
The compound N- [ ((1, 3-trans) -3-hydroxy-2, 4-tetramethylcyclobutyl)]Tert-butyl carbamate (0.8 g,3.29 mmol) was added to N, N-dimethylformamide (10 mL), the reaction system was cooled to 0℃and sodium hydride (262.98 mg,6.58mmol, purity: 60%) was slowly added thereto, after three times of nitrogen substitution, the mixture was stirred for 0.5 hour, and Compound BB-5-1 (613.68 mg,3.95 mmol) was added thereto and the mixture was heated to 70℃for 1 hour. After completion of the reaction, water (10 mL) was added to the reaction mixture, the mixture was stirred at room temperature for 0.5 hour, extracted with ethyl acetate (15 mL. Times.3), and the organic phase was usedSaturated brine (15 mL multiplied by 3), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure, and the obtained crude product is separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=8/1-4/1, volume ratio) to obtain intermediate BB-5-2.MS-ESI m/z:379.1[ M+H ]] + 。 1 H NMR(400MHz，CDCl 3 )δ：7.56(d，J＝8.8Hz，1H)，6.94(d，J＝2.4Hz，1H)，6.78(dd，J＝2.4Hz，8.8Hz，1H)，4.67(br d，J＝7.6Hz，1H)，3.68(br d，J＝8.4Hz，1H)，1.46(s，9H)，1.19(s，6H)，1.15(s，6H)。
Step 2: synthesis of hydrochloride of intermediate BB-5
Intermediate BB-5-2 (0.6 g,1.58 mmol) was added to a hydrochloric acid/ethyl acetate solution (4M, 5 mL) and reacted at room temperature for 1 hour. After the reaction, the mixture was filtered, and the cake was rinsed with ethyl acetate (40 mL) and collected to give the hydrochloride salt of intermediate BB-5. MS-ESI m/z:279.1[ M+H] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：8.52(br s，3H)，7.89(d，J＝8.8Hz，1H)，7.21(d，J＝2.4Hz，1H)，6.99(dd，J＝2.4，8.8Hz，1H)，4.36(s，1H)，3.06(s，1H)，1.31(s，6H)，1.08(s，6H)。
Reference example 6: fragment BB-6
The synthetic route is as follows:
step 1: synthesis of intermediate BB-6-2
Compound BB-6-1 (15.10 g,89.80mmol,13.25 mL) was added to dimethyl sulfoxide (200 mL), potassium carbonate (31.03 g,224.51 mmol) and 1-t-butoxycarbonyl piperazine (20 g,89.80 mmol) were added sequentially, and after three nitrogen substitutions, the temperature was raised to 120℃and the reaction was carried out overnight. After the reaction, pouring the reaction solutionInto water (250 mL), extracted with ethyl acetate (200 mL. Times.3), the organic phase was washed with saturated brine (300 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was purified by column chromatography (eluent: petroleum ether/ethyl acetate=8/1-3/1, volume ratio) to give intermediate BB-6-2.MS-ESI m/z:335.2[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：7.79(d，J＝8.8Hz，2H)，6.97(d，J＝9.2Hz，2H)，4.24(q，J＝7.2Hz，2H)，3.48-3.42(m，4H)，3.32-3.26(m，4H)，1.42(s，9H)，1.28(t，J＝7.0Hz，3H)。
Step 2: synthesis of intermediate BB-6-3
Intermediate BB-6-2 (4 g,11.96 mmol) was added to tetrahydrofuran (20 mL) and water (20 mL), sodium hydroxide (956.84 mg,23.92 mmol) was added, and after three nitrogen substitutions, the temperature was raised to 70℃and the reaction was carried out overnight. After the completion of the reaction, the reaction system was concentrated under reduced pressure to give a residue, ethyl acetate (40 mL) and water (30 mL) were added to the residue, the mixture was separated, the aqueous phase was collected, the pH was adjusted to 5-6 with hydrochloric acid solution (1M), extraction was performed with ethyl acetate (50 mL. Times.2), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-6-3.MS-ESI m/z:307.2[ M+H ] ] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：7.78(d，J＝8.8Hz，2H)，6.95(d，J＝8.8Hz，2H)，3.49-3.40(m，4H)，3.32-3.22(m，4H)，1.42(s，9H)。
Step 3: synthesis of hydrochloride of intermediate BB-6
Intermediate BB-6-3 (3.2 g,10.45 mmol) was added to a hydrochloric acid/ethyl acetate solution (4M, 30 mL) and reacted at room temperature for 2 hours. After the reaction, the mixture was filtered, and the cake was rinsed with ethyl acetate (50 mL) and collected to give the hydrochloride salt of intermediate BB-6. MS-ESI m/z:207.2[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：9.57(br s，2H)，7.80(d，J＝9.2Hz，2H)，7.02(d，J＝9.2Hz，2H)，3.60-3.50(m，4H)，3.17(br s，4H)。
Reference example 7: fragment BB-7
The synthetic route is as follows:
step 1: synthesis of intermediate BB-7-2
Compound BB-7-1 (200.00 g,930.04 mmol), dimethyl carbonate (335.10 g,3.72mol,313.18 mL) was dissolved in tetrahydrofuran (2L), cooled to 0℃and potassium tert-butoxide (626.17 g,5.58 mol) was added in portions, and the mixture was stirred for 0.5 hour at a slow temperature of 20℃and then at a temperature of 70℃for 12 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, dissolved in ice water (3L), extracted with methyl tert-butyl ether (1L. Times.2), the aqueous phase was collected, the organic phase was washed with aqueous sodium hydroxide solution (2N, 500 mL. Times.3), and the aqueous phase was collected. Combining the two aqueous phases, adjusting the pH to 2-3 with a hydrochloric acid solution (6N), precipitating solids, filtering, leaching the filter cake with water (500 mL. Times.3), and collecting the filter cake to obtain an intermediate BB-7-2. 1 H NMR(400MHz，DMSO_d 6 )δ：12.85(br s，1H)，7.89(d，J＝2.4Hz，1H)，7.79(dd，J＝2.6Hz，8.6Hz，1H)，7.35(d，J＝8.8Hz，1H)，5.65(s，1H)。
Step 2: synthesis of intermediate BB-7-3
Intermediate BB-7-2 (255 g,1.06 mol) was dissolved in ethanol (2.5L), hydroxylamine hydrochloride (257.30 g,3.70 mol) and sodium acetate (303.74 g,3.70 mol) were added at 20℃and stirred for 1 hour. Heating to 80 ℃ and stirring for 12 hours. Cooling to room temperature, filtering, rinsing the filter cake with ethyl acetate (400 ml×3), discarding the solid, concentrating the mother liquor to the remaining 1/3, adjusting the pH to 8 with aqueous sodium bicarbonate, extracting with ethyl acetate (2L), collecting the aqueous phase, washing the organic phase with aqueous saturated sodium bicarbonate (300 ml×3), and collecting the aqueous phase. The aqueous phases were combined, the pH was adjusted to 2 with hydrochloric acid solution (2N), solids were precipitated, dissolved and extracted with ethyl acetate (2L X3), the organic phases were combined, washed with saturated brine (1L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-7-3.
Step 3: synthesis of intermediate BB-7
Intermediate BB-7-3 (120 g,468.65 mmol) was dissolved in ethanol (1200 mL), sulfuric acid (9.20 g,91.92mmol,5mL, purity: 98%) was added, and the mixture was stirred at 80℃for 16 hours. After the completion of the reaction, the reaction solution was cooled to room temperature, concentrated under reduced pressure, ethyl acetate (600 mL) was added, the pH was adjusted to 8 with saturated aqueous sodium hydrogencarbonate solution, the solution was separated, the organic phase was washed with saturated aqueous sodium hydrogencarbonate solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-7. 1 H NMR(400MHz，CDCl 3 )δ：7.88(d，J＝1.6Hz，1H)，7.66(dd，J＝1.8，9.0Hz，1H)，7.48(d，J＝9.2Hz，1H)，4.24(q，J＝7.2Hz，2H)，4.02(s，2H)，1.29(t，J＝7.2Hz，3H)。
Reference example 8: fragment BB-8
The synthetic route is as follows:
step 1: synthesis of intermediate BB-8-1
Intermediate BB-1 (6 g,21.12 mmol) was added to dioxane (60 mL) followed by the sequential addition of geminal pinacol borate (6.44 g,25.34 mmol), [1, 1-bis (diphenylphosphine) ferrocene]Palladium dichloride dichloromethane (1.72 g,2.11 mmol) and potassium acetate (8.29 g,84.48 mmol) were reacted for 5 hours after nitrogen substitution three times at a temperature of 100 ℃. After completion of the reaction, the reaction mixture was poured into water (55 mL), extracted with ethyl acetate (65 mL. Times.3), the organic phases were combined, washed with saturated brine (50 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate) Ethyl acetate=10/1-6/1, volume ratio) to obtain intermediate BB-8-1.MS-ESI m/z:332.2[ M+H ]] + 。
Step 2: synthesis of intermediate BB-8-2
Intermediate BB-8-1 (8 g,24.16 mmol) was added to tetrahydrofuran (80 mL) and water (40 mL), sodium hydrogen carbonate (4.06 g,48.31 mmol) was added, the reaction system was cooled to 0℃and hydrogen peroxide (14.32 g,126.27mmol,12.13mL, purity: 30%) was slowly added dropwise, and after the dropwise addition was completed, the reaction was continued for 2 hours. After the completion of the reaction, 15% aqueous sodium sulfite solution (50 mL) was added, stirred for 10 minutes, pH was adjusted to 5-6 with hydrochloric acid solution (1N), extracted with ethyl acetate (50 ml×3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=9:1-4:1, volume ratio) to obtain intermediate BB-8-2.MS-ESI m/z:222.1[ M+H ]] + 。 1 H NMR(400MHz，CDCl 3 )δ：7.49(d，J＝8.4Hz，1H)，6.93(d，J＝1.6Hz，1H)，6.85(dd，J＝2.0，8.4Hz，1H)，4.23(q，J＝7.1Hz，2H)，4.00(s，2H)，1.28(t，J＝7.0Hz，3H)。
Step 3: synthesis of intermediate BB-8-3
Intermediate BB-8-2 (4.5 g,20.34 mmol) was added to N, N-dimethylformamide (50 mL), potassium carbonate (8.43 g,61.03mmol,3 eq), sodium iodide (304.93 mg,2.03 mmol) and 6-bromo-1-hexene (3.98 g,24.41mmol,3.26 mL) were added in this order, and after three nitrogen substitutions, the temperature was raised to 80℃and the reaction was carried out for 5 hours. After the completion of the reaction, the reaction mixture was poured into water (60 mL), extracted with ethyl acetate (50 ml×3), the organic phases were combined, washed with saturated brine (70 ml×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=9/1-4/1, volume ratio) to obtain intermediate BB-8-3.MS-ESI m/z:304.2[ M+H ] ] + 。
Step 4: synthesis of intermediate BB-8-4
Intermediate BB-8-3 (3.2 g,10.55 mmol) was added to tetrahydrofuran (30 mL) followed by the addition ofAcrylamide (899.74 mg,12.66 mmol) and potassium t-butoxide (1.54 g,13.71 mmol) were added, and after three nitrogen substitutions, the reaction was performed at room temperature for 1 hour. After the completion of the reaction, the reaction solution was poured into a hydrochloric acid solution (1 m,20 mL), water (20 mL) was added, extraction was performed with ethyl acetate (25 ml×3), the organic phases were combined, washed with saturated brine (30 ml×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=5/1-1/1, volume ratio) to obtain intermediate BB-8-4.MS-ESI m/z:329.1[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：11.08(s，1H)，7.68(d，J＝8.8Hz，1H)，7.29(d，J＝1.6Hz，1H)，6.97(dd，J＝1.6，8.8Hz，1H)，5.89-5.75(m，1H)，5.04(dd，J＝1.4，17.0Hz，1H)，4.97(dd，J＝1.0，10.2Hz，1H)，4.51(dd，J＝5.0Hz，11.8Hz，1H)，4.08(t，J＝6.4Hz，2H)，2.81-2.69(m，1H)，2.64-2.55(m，1H)，2.48-2.40(m，1H)，2.23-2.14(m，1H)，2.13-2.05(m，2H)，1.81-1.71(m，2H)，1.53-1.46(m，2H)。
Step 5: synthesis of intermediate BB-8
Intermediate BB-8-4 (0.4 g,1.22 mmol) was added to tetrahydrofuran (9 mL) and water (3 mL), cooled to 0℃and potassium osmium dihydrate (44.88 mg, 121.82. Mu. Mol) and sodium periodate (1.04 g,4.87 mmol) were slowly added and reacted at room temperature for 1 hour. After the completion of the reaction, the reaction solution was poured into a saturated sodium sulfite solution (10 mL) and water (30 mL), extracted with ethyl acetate (10 mL. Times.3), and the organic phases were combined, washed with saturated brine (15 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-8.MS-ESI m/z:331.1[ M+H ] ] + 。
Reference example 9: fragment BB-9
The synthetic route is as follows:
step 1: synthesis of intermediate BB-9-2
Compound BB-9-1 (5 g,23.25mmol,1 eq) and dimethyl carbonate (8.38 g,93.00mmol,7.83mL,4 eq) were dissolved in tetrahydrofuran (200 mL), cooled to 0-5℃in an ice bath, potassium tert-butoxide (15.65 g,139.51mmol,6 eq) was added in portions, and after the addition was completed, the temperature was raised to 70℃for 20 hours, followed by a reaction at 90℃for 15 hours. The reaction solution was cooled to room temperature, poured into hydrochloric acid solution (1N, 150 mL), extracted with ethyl acetate (100 mL. Times.3), and the organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude intermediate BB-9-2. 1 H NMR(400MHz，CDCl 3 )δ：12.50(s，1H)，7.80-7.78(m，1H)，7.67-7.65(m，1H)，6.88-6.84(m，1H)，4.05(s，2H)，3.78(s，3H)。
Step 2: synthesis of intermediate BB-9-3
Intermediate BB-9-2 (7 g) and potassium carbonate (4.70 g,34.02mmol,1.5 eq) were added to N, N-dimethylformamide (70 mL), and the mixture was warmed to 90℃and allowed to react overnight with an open mouth. Cooling to room temperature, filtering, eluting the filter cake with water (50 mL) and ethanol (50 mL) in sequence, and collecting the filter cake to obtain an intermediate BB-9-3.MS-ESI m/z:241.1[ M+H ]] + ，243.1[M+2+H] + 。
Step 3: synthesis of intermediate BB-9-4
Intermediate BB-9-3 (5.1 g,21.16mmol,1 eq), hydroxylamine hydrochloride (8.09 g,116.37mmol,5.5 eq) and sodium acetate (9.55 g,116.37mmol,5.5 eq) were dissolved in ethanol (60 mL) and warmed to 90℃for reaction overnight. The reaction solution was cooled to room temperature, concentrated under reduced pressure to remove most of the solvent, poured into water (100 mL), extracted with ethyl acetate (100 mL. Times.3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to give intermediate BB-9-4.MS-ESI m/z:256.0[ M+H ] ] + ，258.1[M+2+H] + 。
Step 4: synthesis of intermediate BB-9-5
Intermediate is preparedBB-9-4 (2.7 g,10.54mmol,1 eq) and concentrated sulfuric acid (1.88 g,18.76mmol,1.02mL, purity: 98%,1.78 eq) were added to ethanol (40 mL), and the mixture was allowed to react overnight at 90 ℃. The reaction solution was cooled to 40-50 ℃, the solvent was removed by concentration under reduced pressure, the pH of the residue was adjusted to 5-6 with saturated sodium bicarbonate solution, extraction was performed with ethyl acetate (30 mL), the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether=1/5) to give intermediate BB-9-5. 1 H NMR(400MHz，CDCl 3 )δ：7.73(d，J＝7.6Hz，1H)，7.67(d，J＝8.0Hz，1H)，7.23(t，J＝8.0Hz，1H)，4.22(q，J＝7.2Hz，2H)，4.04(s，2H)，1.27(q，J＝7.2Hz，3H)。
Step 5: synthesis of intermediate BB-9-6
Intermediate BB-9-5 (1.7 g,5.98 mmol) was added to dioxane (20 mL) and water (4 mL), followed by pinacol vinylborate (1.11 g,7.18mmol,1.22 mL), potassium carbonate (1.65 g,11.97 mmol) and [1, 1-bis (diphenylphosphine) ferrocene]Palladium dichloride dichloromethane (244.33 mg, 299.18. Mu. Mol), after three nitrogen substitutions, the temperature was raised to 90℃and the reaction was carried out for 4 hours. After the completion of the reaction, the reaction mixture was poured into water (25 mL), extracted with ethyl acetate (35 ml×3), the organic phase was washed with saturated brine (30 ml×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=10/1-5/1, volume ratio) to give intermediate BB-9-6.MS-ESI m/z:232.2[ M+H ] ] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：7.75(d，J＝7.6Hz，1H)，7.69(d，J＝7.2Hz，1H)，7.39(t，J＝7.6Hz，1H)，6.97(dd，J＝11.4，17.8Hz，1H)，6.26(d，J＝17.6Hz，1H)，5.63(d，J＝11.2Hz，1H)，4.23(s，2H)，4.14(q，J＝7.2Hz，2H)，1.19(t，J＝7.0Hz，3H)。
Step 6: synthesis of intermediate BB-9-7
Intermediate BB-9-6 (0.6 g,2.59 mmol) was added to tetrahydrofuran (13 mL), acrylamide (221.30 mg,3.11 mmol) and potassium tert-butoxide (320.26 mg, 2.85)mmol), nitrogen was replaced three times, and the reaction was carried out at room temperature for 1 hour. After the completion of the reaction, the reaction solution was poured into hydrochloric acid solution (1 m,10 mL), water (10 mL) was added, extraction was performed with ethyl acetate (15 ml×3), the organic phases were combined, washed with saturated brine (15 ml×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=5/1-1/1, volume ratio) to obtain intermediate BB-9-7.MS-ESI m/z:257.1[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：10.12(br s，1H)，7.78(d，J＝8.0Hz，1H)，7.70(d，J＝7.2Hz，1H)，7.39(d，J＝7.6Hz，1H)，6.99(dd，J＝11.2，17.6Hz，1H)，6.27(d，J＝17.6Hz，1H)，5.65(d，J＝11.6Hz，1H)，4.64(dd，J＝4.8，12.0Hz，1H)，2.84-2.72(m，1H)，2.68-2.59(m，1H)，2.59-2.51(m，1H)，2.27-2.16(m，1H)。
Step 7: synthesis of intermediate BB-9
Compound BB-9-7 (0.16 g, 624.38. Mu. Mol) was added to tetrahydrofuran (6 mL) and water (2 mL), cooled to 0℃and potassium osmium dihydrate (23.01 mg, 62.44. Mu. Mol) and sodium periodate (534.19 mg,2.50 mmol) were slowly added, and after three nitrogen substitutions, the reaction was carried out at room temperature for 1 hour. After completion of the reaction, the reaction mixture was poured into water (5 mL), extracted with ethyl acetate (10 mL. Times.3), and the organic phase was washed with saturated brine (5 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-9.MS-ESI m/z:259.1[ M+H ] ] + 。
Reference example 10: fragment BB-10
The synthetic route is as follows:
step 1: synthesis of intermediate BB-10-1
4-piperidineethanol (4.8 g,37.15 mmol) was added to N, N-dimethylformamide (60 mL), followed by potassium carbonate (15.40 g,111.46 mmol) and BB-6-1 (6.25 g,37.15mmol,5.48 mL) and after three nitrogen substitutions, the temperature was raised to 110℃and stirred for 16 hours. After the completion of the reaction, the reaction mixture was poured into water (70 mL), extracted with ethyl acetate (65 ml×3), the organic phase was washed with saturated brine (70 ml×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=4/1-1/1, volume ratio) to give intermediate BB-10-1.MS-ESI m/z:278.2[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：7.76(d，J＝8.8Hz，2H)，6.93(d，J＝9.2Hz，2H)，5.75(s，1H)，4.23(q，J＝7.2Hz，2H)，3.87(d，J＝12.8Hz，2H)，3.50-3.44(m，2H)，2.82-2.75(m，2H)，1.74-1.70(m，2H)，1，67-1，55(m，1H)，1.40-1.36(m，2H)，1.28(t，J＝7.2Hz，3H)，1.20-1.14(m，2H)。
Step 2: synthesis of intermediate BB-10-2
Intermediate BB-10-1 (4.7 g,16.95 mmol) was added to dichloroethane (60 mL), the reaction system was cooled to 0℃and N, N-diisopropylethylamine (6.57 g,50.84mmol,8.85 mL), 4-dimethylaminopyridine (207.02 mg,1.69 mmol) and methanesulfonyl chloride (3.26 g,28.46mmol,2.20 mL) were added in this order, and after three nitrogen substitutions, the reaction was continued for 1 hour. After the completion of the reaction, the reaction mixture was poured into water (50 mL), extracted with methylene chloride (45 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-10-2.MS-ESI m/z:356.2[ M+H ] ] + 。
Step 3: synthesis of intermediate BB-10-3
1-Boc piperazine (5.26 g,23.63 mmol) was added to acetonitrile (70 mL), followed by potassium carbonate (5.44 g,39.39 mmol) and intermediate BB-10-2 (7 g,19.69 mmol), nitrogen substitution three times, and then heated to 60℃for reaction overnight. After the reaction, the reaction mixture was poured into water (100 mL), extracted with ethyl acetate (75 mL. Times.3), and the organic phases were combined and saturatedAnd brine (60 mL x 3), drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to obtain crude product, and separating and purifying the crude product by column chromatography (eluent: petroleum ether/ethyl acetate=5/1-1/1, volume ratio) to obtain intermediate BB-10-3.MS-ESI m/z:446.4[ M+H ]] + 。 1 H NMR(400MHz，CD 3 OD)δ：7.84(br d，J＝8.8Hz，2H)，6.91(br d，J＝8.8Hz，2H)，4.29(q，J＝7.1Hz，2H)，3.89(br d，J＝12.8Hz，2H)，3.43(br s，4H)，2.81(br t，J＝12.4Hz，2H)，2.48-2.34(m，6H)，1.79(br d，J＝12.4Hz，2H)，1.56-1.48(m，3H)，1.45(s，9H)，1.35(t，J＝7.0 Hz，3H)，1.31-1.24(m，2H)。
Step 4: synthesis of intermediate BB-10-4
Intermediate BB-10-3 (3 g,6.73 mmol) was added to tetrahydrofuran (10 mL) and water (10 mL), sodium hydroxide (1.35 g,33.66 mmol) was added, and after three nitrogen substitutions, the temperature was raised to 75℃and the reaction was carried out for 48 hours. After the completion of the reaction, the reaction system was concentrated under reduced pressure to give a residue, ethyl acetate (40 mL) and water (30 mL) were added to the residue, the liquid was separated by extraction, the aqueous phase was collected, the pH was adjusted to 5-6 with hydrochloric acid solution (1M), and concentrated under reduced pressure to give intermediate BB-10-4.MS-ESI m/z:418.3[ M+H ] ] + 。
Step 5: synthesis of hydrochloride of intermediate BB-10
Intermediate BB-10-4 (2.4 g,5.75 mmol) was added to hydrochloric acid/ethyl acetate (4N, 20 mL) and stirred at room temperature for 2 hours. After the reaction was completed, the mixture was filtered, and the cake was rinsed with ethyl acetate (20 mL) and collected to give the hydrochloride salt of intermediate BB-10. MS-ESI m/z:318.2[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：12.10(br s，1H)，10.18(br s，2H)，7.87(br d，J＝8.4Hz，2H)，7.39(br s，2H)，3.80-3.63(m，4H)，3.53-3.25(m，6H)，3.24-3.00(m，4H)，1.83(br d，J＝12.4Hz，2H)，1.76-1.61(m，3H)，1.60-1.30(m，2H)。
Reference example 11: fragment BB-11
The synthetic route is as follows:
step 1: synthesis of hydrochloride of intermediate BB-11-2
Compound BB-11-1 (5 g,18.43 mmol) was dissolved in ethyl acetate (20 mL), hydrochloric acid/ethyl acetate solution (4M, 23.03 mL) was added at 20deg.C, the reaction mixture was stirred for 12 hours at 20deg.C, and after completion of the reaction, the reaction mixture was concentrated under reduced pressure to give hydrochloride of intermediate BB-11-2. 1 H NMR(400MHz，DMSO_d 6 )δ：9.45-8.62(m，2H)，4.06(q，J＝7.2Hz，2H)，3.18(br d，J＝12.8Hz，2H)，2.91-2.74(m，2H)，2.26(d，J＝6.8Hz，2H)，2.02-1.88(m，1H)，1.77(br d，J＝12.8Hz，2H)，1.49-1.34(m，2H)，1.18(t，J＝7.0Hz，3H)。
Step 2: synthesis of intermediate BB-11-3
Intermediate BB-11-2 (2 g,9.63mmol, hydrochloride) was dissolved in N, N-dimethylformamide (50 mL), 1-bromo-2-chloro-ethane (13.81 g,96.29mmol,7.98 mL) and N, N-diisopropylethylamine (3.73 g,28.89mmol,5.03 mL) were sequentially added to the reaction mixture, and the reaction mixture was stirred at 25℃for 12 hours. After the completion of the reaction, ice water (100 mL) and ethyl acetate (100 mL) were added, and the separated organic phase was washed with saturated brine (20 ml×2), dried over anhydrous sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and the resulting residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/1, volume ratio) to give intermediate BB-11-3. 1 H NMR(400MHz，CDCl 3 )δ：4.13(q，J＝7.1Hz，2H)，3.58(t，J＝7.2Hz，2H)，2.90(br d，J＝11.6Hz，2H)，2.71(t，J＝7.2Hz，2H)，2.23(d，J＝6.8Hz，2H)，2.15-2.04(m，2H)，1.76-1.66(m，3H)，1.40-1.28(m，2H)，1.26(t，J＝7.2Hz，3H)。
Step 3: synthesis of intermediate BB-11-4
Intermediate BB-11-3 (1.10 g,4.72mmol,1 eq), BB-4-3 (2 g,4.72 mmol), potassium iodide (784.17 mg,4.72 mmol) and potassium carbonate (1.31 g,9.45 mmol) were added sequentially to N, N-dimethylformamide (50 mL) at room temperature under nitrogen protection, the reaction mixture was stirred at 80℃for 12 hours, the reaction mixture was cooled to room temperature, ice water (100 mL) and ethyl acetate (100 mL) were added, the separated organic phase was washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and the obtained residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/1, volume ratio) to obtain intermediate BB-11-4.MS-ESI m/z:621.2[ M+H ]] + 。
Step 4: synthesis of intermediate BB-11
Intermediate BB-11-4 (1.5 g,2.42 mmol) was dissolved in ethanol (50 mL), lithium hydroxide solution (1M, 7.25 mL) was added, and the reaction mixture was stirred at 25℃for 12 hours. After the reaction, the pH of the reaction mixture was adjusted to 2-3 with dilute hydrochloric acid (2M, 10 mL), and the reaction mixture was concentrated under reduced pressure to give intermediate BB-11.MS-ESI m/z:593.3[ M+H ]] + 。
Reference example 12: fragment BB-12
The synthetic route is as follows:
step 1: synthesis of intermediate BB-12-1
Intermediate BB-7 (5 g,17.60 mmol) and tert-butyl carbamate (10.31 g,88.00 mmol) were added to toluene (25 mL) and water (2.5 mL), potassium phosphate (11.21 g,52.80mmol,3 eq), tris (dibenzylideneacetone) dipalladium (1.13 g,1.23 mmol) and 2-di-tert-butyl phosphino-2 ',4',6' -triisopropylbiphenyl (1.05 g,2.46 mmol) were slowly added in this order to the reaction, the mixture was replaced 3 times with nitrogen and then warmed to 100 Stirred at c for 12 hours. The reaction solution was cooled to room temperature, and concentrated under reduced pressure to remove most of the organic solvent. Dilute with ethyl acetate (300 mL), wash the organic phase with water (300 mL x 2), collect the organic phase, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The crude product was separated by column chromatography (eluent: petroleum ether/ethyl acetate=10/1-4/1) to give intermediate BB-12-1 (purity: 67.60%). MS-ESI m/z:320.8[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：9.86(s，1H)，7.92(s，1H)，7.66(d，J＝8.4Hz，1H)，7.35(dd，J＝1.8，8.6Hz，1H)，4.18-4.07(m，4H)，1.50(s，9H)，1.18(t，J＝7.2Hz，5H)。
Step 2: synthesis of intermediate BB-12-2
Intermediate BB-12-1 (0.5 g,1.06mmol, purity: 67.60%) and acrylamide (60.00 mg, 844.10. Mu. Mol, 58.25. Mu.L) were added to N, N-dimethylformamide (10 mL) at 0℃and potassium t-butoxide (118.40 mg,1.06 mmol) was added slowly in portions to the reaction, and the mixture was stirred at 0℃for 1 hour after 3 nitrogen substitutions. The reaction mixture was slowly added to saturated ammonium chloride (20 mL), extracted with ethyl acetate (20 mL. Times.2), and the organic phases were combined, washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated by column chromatography (eluent: petroleum ether/ethyl acetate=10/1-1/1) to give intermediate BB-12-2 (purity: 83.94%). MS-ESI m/z:346.2[ M+H ]] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：11.06(s，1H)，9.85(s，1H)，7.91(s，1H)，7.67(d，J＝8.8Hz，1H)，7.32(dd，J＝1.6，8.8Hz，1H)，4.50(dd，J＝5.0，11.8Hz，1H)，2.80-2.68(m，1H)，2.63-2.53(m，1H)，2.46-2.37(m，1H)，2.27-2.10(m，1H)，1.49(s，9H)。
Step 3: synthesis of intermediate BB-12
Intermediate BB-12-2 (50 mg, 121.53. Mu. Mol, purity: 83.94%) was added to ethyl acetate (5 mL), a hydrochloric acid/ethyl acetate solution (4M, 2 mL) was slowly added to the reaction, and the mixture was stirred at 25℃for 12 hours after 3 times of nitrogen substitution. The reaction solution was concentrated under reduced pressure to give the hydrochloride of intermediate BB-12.
Reference example 13: fragment BB-13
The synthetic route is as follows:
step 1: synthesis of intermediate BB-13-1
Intermediate BB-4-3 (0.5 g,1.18 mmol) and tert-butyl 4-methylsulfonyloxypiperidine-1-carboxylate (494.86 mg,1.77 mmol) were added to N, N-dimethylformamide (5 mL) at room temperature, followed by addition of potassium carbonate (326.43 mg,2.36 mmol) and stirring of the reaction at 80℃under nitrogen for 12 hours. After the completion of the reaction, ethyl acetate (20 mL) was added for extraction, followed by washing with water (20 ml×2) and saturated brine (20 mL), drying over anhydrous sodium sulfate, concentrating under reduced pressure, and separating the obtained residue by column chromatography (petroleum ether/ethyl acetate=1/0-5/1, volume ratio) to obtain intermediate BB-13-1.MS-ESI m/z:507.1[ M-100+H ]] + 。
Step 2: synthesis of hydrochloride of intermediate BB-13-2
Intermediate BB-13-1 (0.6 g, 849.71. Mu. Mol) was added to ethyl acetate (10 mL), hydrochloric acid/ethyl acetate (4M, 4 mL) was slowly added to the reaction, and the mixture was stirred at 25℃for 12 hours after 3 times of displacement with nitrogen. After the reaction is finished, the reaction solution is decompressed and concentrated to obtain the hydrochloride of the intermediate BB-13-2.
Step 3: synthesis of intermediate BB-13-3
Intermediate BB-13-2 (0.5 g, 987.14. Mu. Mol, hydrochloride) and ethyl bromoacetate (329.71 mg,1.97mmol, 218.35. Mu.L) were added to acetonitrile (10 mL), potassium carbonate (272.86 mg,1.97 mmol) was slowly added to the reaction, and the mixture was stirred at 80℃for 2 hours after 3 exchanges with nitrogen. The reaction solution was cooled to 25℃and concentrated under reduced pressure to remove most of the organic solvent. Ethyl acetate (20 mL) and water (20 mL) were added and separatedThe organic phase was extracted with ethyl acetate (20 ml×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the resulting residue was separated by column chromatography (petroleum ether/ethyl acetate=1/0-1/1, volume ratio) to give intermediate BB-13-3.MS-ESI m/z:593.1[ M+H ]] + 。
Step 4: synthesis of intermediate BB-13
Intermediate BB-13-3 (500 mg, 843.73. Mu. Mol) was added to ethanol (5 mL) and water (1 mL), lithium hydroxide monohydrate (70.81 mg,1.69mmol) was slowly added to the reaction, and the mixture was stirred at 25℃for 2 hours after 3 times of displacement with nitrogen. The reaction solution was concentrated under reduced pressure to remove most of the organic solvent, water (20 mL) was added, the pH of the aqueous phase was adjusted to 2 with dilute hydrochloric acid solution, and the aqueous phase was lyophilized to give intermediate BB-13.MS-ESI m/z:565.5[ M+H ] ] + 。
Reference example 14: fragment BB-14
The synthetic route is as follows:
step 1: synthesis of hydrochloride of intermediate BB-14
Intermediate BB-2-2 (50 mg, 121.53. Mu. Mol) was added to ethyl acetate (5 mL), hydrochloric acid/ethyl acetate (4M, 2 mL) was slowly added to the reaction, and the mixture was stirred at 25℃for 12 hours after 3 times of nitrogen substitution. The reaction solution was concentrated under reduced pressure to give the hydrochloride salt of intermediate BB-14.
Reference example 15: fragment BB-15
The synthetic route is as follows:
step 1: synthesis of intermediate BB-15-2
N-benzyl-bis-o-chloroethylamino hydrochloride (24.33 g,90.57 mmol), ethanol (150 mL), compound BB-15-1 (24.33 g,90.57 mmol) and N, N-diisopropylethylamine (117.05 g,905.70mmol,157.75 mL) were added to a reaction flask dried in advance, and the reaction was warmed to 90℃and stirred for 16 hours. After the completion of the reaction, the solvent was removed by concentration under reduced pressure, water (100 mL) was added to the residue, extraction was performed with methylene chloride (100 ml×3), the organic phases were combined, washed with saturated brine (300 ml×2), dried over anhydrous sodium sulfate, filtered, the filtrate was removed by concentration under reduced pressure, and the obtained residue was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=50/1 to 10/1, volume ratio) to obtain intermediate BB-15-2.MS-ESI m/z:289.3[ M+H ] ] + 。 1 H NMR(400MHz，CDCl 3 )δ：7.29-7.18(m，5H)，4.11-4.06(q，J＝6.8Hz，2H)，3.45(s，2H)，2.92(s，4H)，2.31(s，4H)，1.23-1.19(m，5H)，0.85-0.83(m，2H)。
Step 2: synthesis of hydrochloride of intermediate BB-15-3
Intermediate BB-15-2 (8 g,27.74 mmol) was dissolved in dichloromethane (80 mL), chloroethyl chloroformate (5.79 g,40.50 mmol) was added at 0deg.C, and the reaction system was warmed to 20deg.C and stirred for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was redissolved in ethanol (80 mL) and the reaction was continued for 16 hours at 85 ℃. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure to remove the solvent. Ethyl acetate (80 mL) was added to the residue, stirred for 20 minutes, and then filtered to collect a cake, to give the hydrochloride salt of intermediate BB-15-3. 1 H NMR(400MHz，CD 3 OD)δ：4.22-4.11(m，2H)，3.59-3.35(m，4H)，3.31-3.12(m，4H)，1.57-1.27(m，4H)，1.27-1.18(m，3H)。
Step 3: synthesis of Compound BB-15-4
Intermediate BB-15-3%3.5g,14.91mmol, hydrochloride) in a mixed solvent of dioxane (30 mL) and water (10 mL), the reaction system was cooled to 0deg.C, sodium bicarbonate (3.76 g,44.73mmol,1.74 mL) and tert-butyl carbonate (3.25 g,14.91mmol,3.43 mL) were added, and the mixture was returned to 20deg.C and stirred for 12 hours. After completion of the reaction, water (20 mL) and methylene chloride (20 mL) were added to the reaction mixture, the organic phase was separated, the aqueous phase was extracted with methylene chloride (20 mL. Times.3), the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=0/1-25/2, volume ratio) to give intermediate BB-15-4. 1 H NMR(400MHz，CDCl 3 )δ：4.12(q，J＝7.1Hz，2H)，3.31(br s，4H)，2.90(br s，4H)，1.47(s，9H)，1.32-1.27(m，2H)，1.25(t，J＝7.2Hz，3H)，0.94(q，J＝3.7Hz，2H)。
Step 4: synthesis of intermediate BB-15-5
Intermediate BB-15-4 (3.1 g,10.39 mmol) was dissolved in tetrahydrofuran (35 mL), nitrogen was replaced three times, cooled to-65℃and lithium aluminum hydride (1M tetrahydrofuran solution, 31.17 mL) was slowly added thereto, and stirred at 0℃for 1 hour. After the reaction was completed, sodium sulfate decahydrate (30 g) was slowly added to the reaction solution at 0 ℃, filtration was performed, the cake was rinsed with methylene chloride, and the filtrate was concentrated under reduced pressure to obtain intermediate BB-15-5. 1 H NMR(400MHz，CDCl 3 )δ：3.58(s，2H)，3.35(t，J＝4.8Hz，4H)，2.69(t，J＝4.8Hz，4H)，1.45(s，9H)，0.71-0.68(m，2H)，0.55-0.52(m，2H)。
Step 5: synthesis of intermediate BB-15-6
Intermediate BB-4-3 (2.39 g,5.66 mmol) was dissolved in tetrahydrofuran (50 mL) under nitrogen, then intermediate BB-15-5 (2.9 g,11.31 mmol) and triphenylphosphine (2.97 g,11.31 mmol) were added, the reaction system was cooled to 0deg.C, and diisopropyl azodicarboxylate (2.29 g,11.31mmol,2.20 mL) was added dropwise. Heating to 40 ℃ and stirring for 12 hours. After completion of the reaction, water (20 mL) and ethyl acetate (50 mL) were added to the reaction mixture, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (50 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL. Times.3), and anhydrous sodium sulfateDrying, filtering, concentrating the filtrate under reduced pressure to remove the solvent, and separating and purifying the obtained residue by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-4/5, volume ratio) to obtain intermediate BB-15-6. 1 H NMR(400MHz，CDCl 3 )δ：8.00-7.96(m，2H)，7.85-7.82(m，1H)，7.08-7.02(m，3H)，4.05(s，2H)，2.81(s，4H)，2.69(s，4H)，1.59(s，6H)，1.46(s，9H)，0.75-0.70(m，4H)。
Step 6: synthesis of hydrochloride of intermediate BB-15-7
Intermediate BB-15-6 (3.2 g,4.84 mmol) was dissolved in ethyl acetate (5 mL), and hydrochloric acid/ethyl acetate (4M, 30 mL) was added to react at 15℃for 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, methyl tert-butyl ether (10 mL) was added and stirred for 5 minutes, and the mixture was filtered, and the cake was rinsed with methyl tert-butyl ether (2 mL. Times.2), and the cake was collected and concentrated under reduced pressure to remove the solvent, to give the hydrochloride salt of intermediate BB-15-7.
Step 7: synthesis of Compound BB-15-8
Intermediate BB-15-7 (0.2 g, 334.42. Mu. Mol, hydrochloride) was dissolved in tetrahydrofuran (3 mL), nitrogen was purged to 0deg.C, triethylamine (203.04 mg,2.01mmol, 279.28. Mu.L) was added dropwise, and after the addition, a solution of ethyl bromoacetate (111.70 mg, 668.84. Mu. Mol, 73.97. Mu.L) in tetrahydrofuran (1 mL) was added dropwise and the mixture was stirred for 2 hours at 20deg.C. To the reaction solution was added water (10 mL), extracted with ethyl acetate (10 mL. Times.2), and the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to give intermediate BB-15-8.
Step 8: synthesis of intermediate BB-15
Intermediate BB-15-8 (210 mg, 324.23. Mu. Mol) was dissolved in a mixed solvent of tetrahydrofuran (2 mL) and water (0.5 mL), cooled to 0℃and lithium hydroxide monohydrate (68.02 mg,1.62 mmol) was added thereto, followed by stirring at 20℃for 1 hour. The pH of the reaction solution was adjusted to 3-4 with hydrochloric acid solution (1M), and concentrated under reduced pressure to give intermediate BB-15.
Reference example 16: fragment BB-16
The synthetic route is as follows:
step 1: synthesis of intermediate BB-16-1
Intermediate BB-4-5 (600 mg,1.12 mmol) and ethyl bromopropionate (405.62 mg,2.24mmol, 285.65. Mu.L) were added to acetonitrile (10 mL), followed by potassium carbonate (464.51 mg,3.36 mmol) and sodium iodide (167.93 mg,1.12 mmol), and stirred at 80℃under nitrogen for 2 hours. After the reaction, cooling to room temperature, adding ethyl acetate (50 mL) and water (50 mL), extracting, washing an organic phase by saturated saline (30 mL multiplied by 2), drying by anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and separating and purifying the obtained crude product by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-10/1, volume ratio) to obtain an intermediate BB-16-1.MS-ESI m/z:636.0[ M+H ]] + 。
Step 2: synthesis of intermediate BB-16
Intermediate BB-16-1 (0.7 g, 963.22. Mu. Mol) was added to ethanol (10 mL) and water (2 mL), stirring was turned on, and then lithium hydroxide monohydrate (80.84 mg,1.93 mmol) was added, and the reaction solution was stirred under nitrogen atmosphere at 25℃for 12 hours. After the reaction, the solvent was removed by concentrating under reduced pressure, diluted with water (10 mL), adjusted to pH 3 with hydrochloric acid solution (2N), and lyophilized from the aqueous phase to give crude intermediate BB-16. MS-ESI m/z:608.1[ M+H ] ] + 。
Reference example 17: fragment BB-17
The synthetic route is as follows:
step 1: synthesis of intermediate BB-17-2
Compound BB-17-1 (25 g,105.44mmol,62.50 mL) was dissolved in methylene chloride (1000 mL), acetyl chloride (8.28 g,105.44mmol,7.52 mL) was added, nitrogen was replaced three times, aluminum trichloride (29.53 g,221.43mmol,12.10 mL) was added in portions at 5-15℃and stirred at room temperature for 4 hours. The reaction solution was slowly poured into ice water (500 mL), extracted with methylene chloride (200 mL. Times.3), and the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-17-2.MS-ESI m/z:265.0[ M+H ]] + ，267.0[M+2+H] + 。
Step 2: synthesis of intermediate BB-17-3
Intermediate BB-17-2 (20 g,75.44 mmol) and dimethyl carbonate (27.18 g,301.77mmol,25.40 mL) were dissolved in tetrahydrofuran (200 mL), nitrogen was replaced three times, cooled to 0deg.C, potassium tert-butoxide (50.79 g,452.66 mmol) was slowly added, and the mixture was warmed to 70deg.C and stirred overnight. The reaction solution was concentrated under reduced pressure to give a yellow residue, ice water (700 mL) was added to the residue, the pH was adjusted to 2-3 with hydrochloric acid solution (6M), a large amount of solids were precipitated, filtration was performed, the cake was rinsed with water (200 mL) and methyl tert-butyl ether (200 mL) in this order, and the cake was concentrated under reduced pressure to give intermediate BB-17-3.MS-ESI m/z:291 [ M+H ] ] + ，293.0[M+2+H] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：9.91(d，J＝9.2Hz，1H)，8.21(s，1H)，8.01(d，J＝8.8Hz，1H)，7.70(d，J＝9.2Hz，1H)，7.45(d，J＝8.8Hz，1H)，5.15(s，1H)。
Step 3: synthesis of intermediate BB-17-4
Intermediate BB-17-3 (21 g,72.14 mmol) was dissolved in ethanol (300 mL), hydroxylamine hydrochloride (32.59 g,468.92 mmol) and sodium acetate (20.71 g,252.49 mmol) were added, replaced with nitrogen three times, and the mixture was stirred at 90℃for 96 hours. The reaction solution was cooled to room temperature, filtered, the filter cake was rinsed with ethyl acetate (500 mL), the filtrate was concentrated under reduced pressure to a residual 1/4, the pH was adjusted to 8-9 with sodium hydroxide solution (1M), filtered, the filter cake was rinsed with ethyl acetate (200 mL), and the filtrate was separated and the aqueous phase was collected. The aqueous and solid phases were collected, the pH was adjusted to 3-4 with hydrochloric acid solution (2M) and there was a large amount of insolubles, ethyl acetate (500 mL) was added and the organic phase was collected after separation. The aqueous phase was extracted with ethyl acetate (300 mL. Times.3), the organic phases were combined, washed successively with citric acid solution (10%, 300 mL) and saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate BB-17-4.
Step 4: synthesis of intermediate BB-17-5
Intermediate BB-17-4 (10 g,23.87 mmol) was dissolved in ethanol (100 mL), concentrated sulfuric acid (3.11 g,31.03mmol,1.69mL, purity: 98%,1.3 eq) was added, nitrogen was replaced three times, and the temperature was raised to 70℃and stirred overnight. The reaction solution was concentrated under reduced pressure to give a dark yellow residue, to which were added water (200 mL) and ethyl acetate (300 mL), and the organic phase was collected after separation. The aqueous phase was extracted with ethyl acetate (200 mL. Times.4), the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was dissolved in 2-methyltetrahydrofuran (50 mL), petroleum ether (100 mL) was added, a large amount of solids was precipitated, filtration was performed, the cake was rinsed with petroleum ether (10 mL), the cake was concentrated under reduced pressure to give a solid, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether=0/1-15/85, volume ratio) to give intermediate BB-17-5.MS-ESI m/z:334.0[ M+H ] ] + ，336.0[M+2+H] + 。 1 H NMR(400MHz，CDCl 3 )δ：8.16(d，J＝1.6Hz，1H)，7.99(d，J＝8.8Hz，1H)，7.88(d，J＝8.8Hz，1H)，7.79-7.71(m，2H)，4.32(s，2H)，4.23(q，J＝7.2Hz，2H)，1.22(t，J＝7.0Hz，3H)。
Step 5: synthesis of Compound BB-17-6
Intermediate BB-17-5 (7.9 g,23.64mmol,1 eq), tert-butyl carbamate (3.32 g,28.37mmol,1.2 eq) was dissolved in toluene (100 mL) and water (20 mL), potassium phosphate (20.07 g,94.56mmol,4 eq), 2-di-tert-butyl phosphino-2 ',4',6' -triisopropylbiphenyl (1.41 g,3.31mmol,0.14 eq), tris (dibenzylideneacetone) dipalladium (1.52 g,1.65mmol,0.07 eq) was added and nitrogen was replaced three times with litersStir at 100 ℃ overnight. The reaction solution was concentrated under reduced pressure to give a dark yellow residue, to which were added water (100 mL) and ethyl acetate (200 mL), and the organic phase was collected after separation. The aqueous phase was extracted with ethyl acetate (100 mL. Times.3), the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (eluent: ethyl acetate/petroleum ether=0/1-15/85) to give intermediate BB-17-6.MS-ESI m/z:371.2[ M+H ]] + 。 1 H NMR(400MHz，CDCl 3 )δ：8.20(s，1H)，8.03(d，J＝8.8Hz，1H)，7.90(d，J＝9.2Hz，1H)，7.68(d，J＝9.2Hz，1H)，7.53(dd，J＝2.4Hz，8.8Hz，1H)，6.71(s，1H)，4.31(s，2H)，4.22(q，J＝7.2Hz，2H)，1.57(s，9H)，1.21(t，J＝7.0Hz，3H)。
Step 6: synthesis of intermediate BB-17-7
Intermediate BB-17-6 (0.25 g, 674.95. Mu. Mol) was dissolved in tetrahydrofuran (3 mL), cooled to 0deg.C, and acrylamide (47.97 mg, 674.95. Mu. Mol, 46.58. Mu.L) and potassium t-butoxide (113.61 mg,1.01 mmol) were added. The reaction system was stirred at 20℃for 1 hour. The reaction solution was slowly poured into a saturated ammonium chloride solution (10 mL), ethyl acetate (10 mL) was added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (10 ml×3), the organic phases were combined, washed with saturated brine (10 ml×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the resulting residue was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=0/1-1/1, volume ratio) to give intermediate BB-17-7.MS-ESI m/z:396.2[ M+H ] ] + 。 1 H NMR(400MHz，DMSO_d 6 )δ：11.12(s，1H)，9.66(s，1H)，8.36(s，1H)，8.12(d，J＝9.2Hz，1H)，8.07(d，J＝9.2Hz，1H)，7.85(d，J＝8.8Hz，1H)，7.68(dd，J＝2.0，9.2Hz，1H)，5.00(dd，J＝4.8Hz，11.2Hz，1H)，2.89-2.76(m，1H)，2.68-2.53(m，2H)，2.41-2.31(m，1H)，1.52(s，9H)。
Step 7: synthesis of hydrochloride of intermediate BB-17
Intermediate BB-17-7 (120 mg, 303.48. Mu. Mol) was dissolved in a hydrochloric acid/ethyl acetate solution (5 mL, 4M) and reacted at 25℃for 30 minutes. The reaction solution was filtered, and the cake was collected by washing the cake with ethyl acetate (3 mL. Times.3), to give the hydrochloride salt of intermediate BB-17.
Reference example 18: fragment BB-18
The synthetic route is as follows:
step 1: synthesis of Compound BB-18-2
Compound BB-18-1 (10 g,53.13 mmol) was dissolved in methylene chloride (300 mL), and acetyl chloride (4.17 g,53.13mmol,3.79 mL) and aluminum trichloride (10.63 g,79.68mmol,4.35 mL) were added in this order, followed by stirring at room temperature under nitrogen for 2 hours. After the reaction, the reaction mixture was slowly poured into ice water (400 mL), and the organic phase was collected after separation. The aqueous phase was extracted with methylene chloride (3X 100 mL), and the combined organic phases were washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound BB-18-2.MS-ESI m/z:231.1[ M+H ]] + . 1 H NMR(400MHz，CDCl 3 )δ：7.78(d，J＝9.2Hz，1H)，7.70(d，J＝9.2Hz，1H)，7.26(d，J＝9.2Hz，1H)，7.17(dd，J＝2.4Hz，9.2Hz，1H)，7.10(d，J＝2.4Hz，1H)，3.96(s，3H)，3.91(s，3H)，2.65(s，3H).
Step 2: synthesis of Compound BB-18-3
Compound BB-18-2 (24.3 g,105.53 mmol) was dissolved in methylene chloride (250 mL), nitrogen was replaced three times, cooled to-60℃and a methylene chloride solution of boron trichloride (1M, 105.53 mL) was added dropwise at-60℃to-40 ℃. After the dripping is finished, the temperature is raised to 0-5 ℃ under the protection of nitrogen and the mixture is stirred for 2 hours. After the reaction was completed, the reaction solution was slowly poured into ice water (300 mL), and the organic phase was collected after separation. The aqueous phase was extracted with dichloromethane (3X 100 mL) The combined organic phases were washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give BB-18-3.MS-ESI m/z:217.1[ M+H ]] + . 1 H NMR(400MHz，CDCl 3 )δ：13.17(s，1H)，8.02(d，J＝9.6Hz，1H)，7.81(d，J＝8.8Hz，1H)，7.25(dd，J＝2.8Hz，9.2Hz，1H)，7.13(d，J＝9.2Hz，2H)，3.93(s，3H)，2.85(s，3H).
Step 3: synthesis of Compound BB-18-4
Compound BB-18-3 (10 g,46.25 mmol) was dissolved in tetrahydrofuran (150 mL), dimethyl carbonate (16.66g,184.99 mmol,15.57mL) was added, the temperature was lowered to 0℃and potassium tert-butoxide (31.14 g,277.48 mmol) was added. The reaction was warmed to 70 ℃ under nitrogen protection and stirred for 12 hours. After the completion of the reaction, the reaction mixture was slowly added to ice water (200 mL), ph=2 to 3 was adjusted with 6M hydrochloric acid, a white solid was precipitated, and the mixture was filtered, and the cake was washed with water (100 mL) and dried under reduced pressure to give compound BB-18-4.MS-ESI m/z:243.1[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：12.94(s，1H)，9.18(d，J＝9.6Hz，1H)，8.10(d，J＝9.2Hz，1H)，7.49(dd，J＝3.0Hz，5.8Hz，2H)，7.33(dd，J＝2.8Hz，9.6Hz，1H)，5.80(s，1H)，3.89(s，3H).
Step 4: synthesis of Compound BB-18-5
Compound BB-18-4 (11 g,45.41 mmol) was dissolved in dichloromethane (150 mL), replaced with nitrogen three times, cooled to-50℃and boron tribromide (39.82 g,158.94mmol,15.31 mL) was added dropwise at-50℃to-30℃and the reaction mixture was slowly brought back to 15℃and stirred for 12 hours. After the reaction was completed, the reaction mixture was poured into ice water (300 mL), filtered, and the cake was washed with water (100 mL) to collect the cake. The filtrate was extracted with 2-methyltetrahydrofuran (4X 200 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. And combining the filter cake with the filtrate, and concentrating under reduced pressure to obtain the compound BB-18-5.MS-ESI m/z:229.2[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：12.76(s，1H)，9.11(d，J＝8.8Hz，1H)，7.98(d，J＝9.2Hz，1H)，7.41(d，J＝8.8Hz，1H)，7.26-7.22(m，2H)，5.72(s，1H).
Step 5: synthesis of Compound BB-18-6
Compound BB-18-5 (11.5 g,50.39 mmol) was dissolved in ethanol (170 mL), hydroxylamine hydrochloride (12.26 g,176.38 mmol) and sodium ethoxide (12.00 g,176.38 mmol) were added in this order, and the mixture was stirred for 12 hours at 80℃under nitrogen. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to half the volume, water (200 mL) was added to the residue, the mixture was extracted with ethyl acetate (3X 200 mL), the organic phases were combined, washed with saturated brine (2X 500 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound BB-18-6.MS-ESI m/z:244.2[ M+H ]] + .
Step 6: synthesis of Compound BB-18-7
Compound BB-18-6 (11 g,39.20mmol, purity: 86.67%) was dissolved in ethanol (110 mL), sulfuric acid (4.05 g,40.45mmol,2.20mL, purity: 98%) was added, nitrogen was replaced three times, and the temperature was raised to 70℃and stirred for 12 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain a yellow residue, to which were added water (100 mL) and ethyl acetate (200 mL), and the organic phase was collected after separation. The aqueous phase was extracted with ethyl acetate (3X 100 mL), the combined organic phases were washed with saturated brine (200 mL), the organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a yellow residue. The residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-4/1) to give compound BB-18-7.MS-ESI m/z:272.1[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：9.89(s，1H)，7.99(d，J＝5.6Hz，1H)，7.97(d，J＝5.6Hz，1H)，7.80(d，J＝9.2Hz，1H)，7.40(d，J＝2.4Hz，1H)，7.27(dd，J＝2.4，9.2Hz，1H)，4.44(s，2H)，4.15(q，J＝7.2Hz，2H)，1.16(t，J＝7.0Hz，3H).
Step 7: synthesis of Compound BB-18-8
Compound BB-18-7 (2 g,7.37 mmol) was dissolved in N, N-dimethylformamide (20 mL), and potassium carbonate (2.04 g,14.75 mmol) and allyl bromide (891.93 mg,7.37 mmol) were added, nitrogen substitution was performed three times, and the temperature was raised to 50℃and stirred for 12 hours. After the reaction, water is added into the reaction solution(20 mL) and ethyl acetate (20 mL), and the organic phase was collected after separation. The aqueous phase was extracted with ethyl acetate (2X 20 mL), the organic phases were combined and washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a yellow residue. The residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-17/3) to give compound BB-18-8.MS-ESI m/z:312.1[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：8.08(d，J＝9.2Hz，1H)，8.04(d，J＝8.8Hz，1H)，7.87(d，J＝9.2Hz，1H)，7.65(d，J＝2.4Hz，1H)，7.43(dd，J＝2.6Hz，9.0Hz，1H)，6.17-6.08(m，1H)，5.47(dd，J＝1.2Hz，17.2Hz，1H)，5.31(d，J＝10.4Hz，1H)，4.72(d，J＝5.2Hz，2H)，4.47(s，2H)，4.15(q，J＝7.0Hz，2H)，1.16(t，J＝7.0Hz，3H).
Step 8: synthesis of Compound BB-18-9
Compound BB-18-8 (1 g,3.21 mmol) was dissolved in tetrahydrofuran (20 mL), and potassium tert-butoxide (540.63 mg,4.82 mmol) and acrylamide (228.30 mg,3.21 mmol) were added at 0℃and stirred at room temperature for 1 hour. After the completion of the reaction, the reaction mixture was slowly added to 0.5M hydrochloric acid to adjust ph=6 to 7, extracted with ethyl acetate (3×20 mL), and the combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a yellow residue. The residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=9/1-13/7) to give compound BB-18-9.MS-ESI m/z:337.1[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.12(s，1H)，8.15(d，J＝9.2Hz，1H)，8.09(d，J＝9.2Hz，1H)，7.89(d，J＝9.2Hz，1H)，7.66(d，J＝2.4Hz，1H)，7.39(dd，J＝2.6Hz，9.0Hz，1H)，6.18-6.08(m，1H)，5.47(dd，J＝1.6Hz，17.2Hz，1H)，5.31(dd，J＝1.6Hz，10.4Hz，1H)，5.03(dd，J＝4.8Hz，11.2Hz，1H)，4.73(d，J＝5.2Hz，2H)，2.92-2.77(m，1H)，2.68-2.54(m，2H)，2.42-2.30(m，1H).
Step 9: synthesis of Compound BB-18
Compound BB-18-9 (120 mg, 356.78. Mu. Mol) was dissolved in tetrahydrofuran (3 mL) and water (1 mL)Potassium osmium dihydrate (13.15 mg, 35.68. Mu. Mol) and sodium periodate (305.25 mg,1.43mmol, 79.08. Mu.L) were added to the mixed solvent at 0℃and the reaction system was reacted at room temperature for 3 hours with nitrogen replaced three times. After completion of the reaction, water (5 mL) was added to the reaction mixture, the mixture was extracted with ethyl acetate (10 mL. Times.3), and the organic phase was collected, washed with saturated brine (10 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give Compound BB-18.MS-ESI m/z:339.1[ M+H ]] + .
Reference example 19: fragment BB-19
The synthetic route is as follows:
step 1: synthesis of Compound BB-19-1
5-hexenoic acid (82.57 mg, 723.43. Mu. Mol, 85.92. Mu.L) was dissolved in N, N-dimethylformamide (3 mL), followed by addition of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (343.84 mg, 904.29. Mu. Mol) and N, N-diisopropylethylamine (311.66 mg,2.41mmol, 420.03. Mu.L), the reaction system was purged with nitrogen three times, and after stirring at 25℃for 20 minutes, the hydrochloride (200 mg, 602.86. Mu. Mol) of Compound BB-17 was added, and the reaction solution was stirred at 25℃for 12 hours. After the completion of the reaction, water (10 mL) and ethyl acetate (10 mL) were added to the reaction mixture, the organic phase was separated, ethyl acetate (20 mL. Times.3) was added to the aqueous phase to extract, the combined organic phases were washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product, which was separated by plate chromatography (developer: petroleum ether: ethyl acetate=0:1) to give compound BB-19-1.MS-ESI m/z:392.2[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.13(s，1H)，10.20(s，1H)，8.52(d，J＝2.0Hz，1H)，8.18(J＝9.2Hz，1H)，8.10(d，J＝8.8Hz，1H)，7.88(d，J＝9.2Hz，1H)，7.79(dd，J＝2.2Hz，9.0Hz，1H)，5.90-5.79(m，1H)，5.09-4.98(m，3H)，2.88-2.78(m，1H)，2.68-2.61(m，1H)，2.60-2.55(m，1H)，2.54-2.51(m，1H)，2.40(t，J＝7.6Hz，2H)，2.10(q，J＝7.0Hz，2H)，1.78-1.68(m，2H).
Step 2: synthesis of Compound BB-19
Compound BB-19-1 (120 mg, 306.58. Mu. Mol) was dissolved in a mixed solvent of tetrahydrofuran (3 mL) and water (1 mL), sodium periodate (262.30 mg,1.23mmol, 67.95. Mu.L) and osmium tetraoxide dihydrate (11.30 mg, 30.66. Mu. Mol) were added at 0℃and the reaction system was replaced with nitrogen three times, stirred at 25℃for 3 hours, water (5 mL) was added to the reaction solution after the completion of the reaction, then ethyl acetate (10 mL) was added for dilution, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (20 mL. Times.3), and the combined organic phase was washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound BB-19.MS-ESI m/z:394.2[ M+H ]] + .
Reference example 20: fragment BB-20
The synthetic route is as follows:
step 1: synthesis of Compound BB-20-2
Compound BB-20-1 (5 g,18.03 mmol) was dissolved in dichloromethane (50 mL) and acetone (25 mL) under nitrogen, and methyltrimethylsilane (2.68 g,27.04mmol,3.38 mL) was slowly added dropwise in this order, trimethylsilyl triflate (400.67 mg,1.80mmol, 325.74. Mu.L) and the reaction mixture was stirred at 20℃for 2 hours. After the completion of the reaction, the solvent was removed from the reaction mixture under reduced pressure. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-4/1, volume ratio) to give compound BB-20-2.
Step 2: synthesis of Compound BB-20
Compound BB-20-2 (10 g,29.03 mmol) was dissolved in N, N-dimethylacetamide (100 mL), 4-cyano-3-trifluoromethylphenyl isothiocyanate (6.62 g,29.03 mmol) was added in portions to the reaction mixture under nitrogen protection at room temperature, the reaction mixture was stirred at 20℃for 3 hours, methanol (100 mL), diluted hydrochloric acid (2M, 56.32 mL) was added, the reaction mixture was stirred at 70℃for 2 hours, after completion of the reaction, cooled to room temperature, diluted with water (100 mL) and extracted with ethyl acetate (100 mL. Times.3). The organic phases were combined, washed with saturated brine (100 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue obtained was separated by column chromatography (eluent: dichloromethane/methanol=20/1 to 10/1, volume ratio) to give compound BB-20. 1 H NMR(400MHz，CDCl 3 )δ：7.99(d，J＝8.8Hz，1H)，7.96(d，J＝1.6Hz，1H)，7.84(dd，J＝2.0，8.4Hz，1H)，7.22(d，J＝9.2Hz，2H)，7.04(d，J＝8.8Hz，2H)，3.64-3.57(m，4H)，3.45-3.38(m，4H)，1.58(s，6H).
Reference example 21: fragment BB-21
The synthetic route is as follows:
step 1: synthesis of Compound BB-21-2
Triphenyl phosphite (63.92 g,206.00mmol,54.17 mL) was dissolved in dichloromethane (400 mL), the reaction system was cooled to-70℃under nitrogen protection, liquid bromine (35.91 g,224.73mmol,11.59 mL), triethylamine (24.64 g,243.46mmol,33.89 mL) and a solution of compound BB-21-1 (33 g,187.28 mmol) in dichloromethane (100 mL) were added sequentially, and after the dropwise addition, the reaction system was slowly warmed to room temperature and stirred for reaction 15 hours. After the completion of the reaction, the reaction solution was slowly poured into a saturated aqueous sodium sulfite solution (300 mL), stirred for 10 minutes, extracted with methylene chloride (400 mL), and the organic phase was collected, washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate=20/1-7/1) to give compound BB-21-2. 1 H NMR(400MHz，DMSO_d 6 )δ：7.33(d，J＝8.4Hz，1H)，6.83-6.76(m，2H)，6.33(t，J＝4.8Hz，1H)，3.76(s，3H)，2.77(t，J＝8.2Hz，2H)，2.33-2.25(m，2H).
Step 2: synthesis of Compound BB-21-3
Compound BB-21-2 (40 g,167.29 mmol) was added to toluene (500 mL), and dichlorodicyanobenzoquinone (41.77 g,184.02 mmol) was slowly added at 0℃and stirred at 0℃for 1 hour, and then returned to room temperature and stirred for 14 hours. After the completion of the reaction, a saturated aqueous sodium sulfite solution (200 mL) was added dropwise thereto, stirring was carried out for 10 minutes, 1mol/L aqueous sodium hydroxide solution (150 mL) was added, extraction was carried out with ethyl acetate (240 mL. Times.3), and the organic phase was collected, washed with a saturated brine (320 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product is separated and purified by column chromatography (eluent: petroleum ether: ethyl acetate=5/1-1/1) to obtain compound BB-21-3. 1 H NMR(400MHz，DMSO_d 6 )δ：8.02(d，J＝9.2Hz，1H)，7.87(d，J＝8.4Hz，1H)，7.68(d，J＝7.2Hz，1H)，7.42(s，1H)，7.37(t，J＝7.8Hz，1H)，7.31(d，J＝9.2Hz，1H)，3.90(s，3H).
Step 3: synthesis of Compound BB-21-4
Acetyl chloride (32.78 g,417.56mmol,29.80 mL) was dissolved in dichloromethane (1L), the reaction system was cooled to 0℃and aluminum trichloride (106.29 g,797.16 mmol) was added thereto, the mixture was reacted at 0℃for 30 minutes under nitrogen protection, and Compound BB-21-3 (90 g,379.60 mmol) was added thereto, and after continuing the reaction for 5.5 hours, the reaction was resumed at room temperature for 10 hours. After the completion of the reaction, the reaction mixture was poured into water (1000 mL), extracted with dichloromethane (500 mL. Times.3), and the organic phase was collected, washed with saturated brine (800 mL. Times.2), dried over anhydrous sodium sulfate, and concentrated Filtering, and concentrating the filtrate under reduced pressure to obtain compound BB-21-4.MS-ESI m/z:279.0[ M+H ]] + .281.0[M+H+2] + .
Step 4: synthesis of Compound BB-21-5
Compound BB-21-4 (42.5 g,152.26 mmol) was added to methylene chloride (600 mL), the reaction system was cooled to-60℃and boron trichloride (26.76 g,228.39mmol,29.70 mL) was slowly added thereto, and the reaction was allowed to proceed to room temperature under nitrogen protection for 5 hours. After the completion of the reaction, the reaction mixture was poured into 1mol/L hydrochloric acid (100 mL), stirred at room temperature for 0.5 hour, extracted with methylene chloride (500 mL. Times.3), and the organic phase was collected, washed with saturated brine (600 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give Compound BB-21-5.MS-ESI m/z:265.0[ M+H ]] + .267.0[M+H+2] + .
Step 5: synthesis of Compound BB-21-6
Dimethyl carbonate (54.37 g,603.54mmol,50.81 mL) was added to tetrahydrofuran (1L), and compound BB-21-5 (40 g,150.89 mmol) and potassium tert-butoxide (101.58 g,905.31 mmol) were slowly added and the mixture was allowed to react at 70℃under nitrogen for 12 hours. After the completion of the reaction, the reaction mixture was cooled to room temperature, the solvent was removed by concentration under reduced pressure, ice water (250 mL) was added, extraction was performed with methyl tert-butyl ether (250 mL. Times.2), and the aqueous phase was collected. The organic phase was extracted with 2M aqueous sodium hydroxide (150 mL. Times.2) and the aqueous phase was collected. The aqueous phases were combined, ph=2-3 was adjusted with 6M hydrochloric acid solution, solids precipitated, filtered, the filter cake rinsed with water (150 ml×2) and the filter cake collected to give compound BB-21-6.MS-ESI m/z:291 [ M+H ] ] + ，292.9[M+H+2] + . 1 H NMR(400MHz，DMSO_d 6 )δ：9.38(d，J＝8.8Hz，1H)，8.42(d，J＝9.2Hz，1H)，7.96(d，J＝6.8Hz，1H)，7.67(d，J＝9.2Hz，1H)，7.58(t，J＝7.6Hz，1H)，5.89(s，1H).
Step 6: synthesis of Compound BB-21-7
Compound BB-21-6 (80 g,274.82 mmol) was added to ethanol (1L), hydroxylamine hydrochloride (66.84 g,961.88 mmol) and sodium acetate (78.90 g,961.88 mmol) were slowly added, and the temperature was raised to 80℃under nitrogen protection to react for 12 hours. ReactionAfter completion, the reaction solution was cooled to room temperature, filtered, the filter cake was rinsed with ethyl acetate (600 mL), the filter cake was discarded, the filtrate was concentrated to the remaining 1/3, ph=8 was adjusted with aqueous sodium bicarbonate, extracted with ethyl acetate (600 mL), the organic phase was washed with aqueous saturated sodium bicarbonate (200 ml×2), the aqueous phases were combined, ph=2 was adjusted with 4mol/L hydrochloric acid, and the filter cake was collected to give compound BB-21-7.MS-ESI m/z:306.0[ M+H ]] + ，308.0[M+H+2] + . 1 H NMR(400MHz，CD 3 OD)δ：8.40(d，J＝9.2Hz，1H)，8.32(d，J＝8.4Hz，1H)，7.83(d，J＝6.8Hz，1H)，7.79(d，J＝9.2Hz，1H)，7.52(t，J＝8.0Hz，1H)，4.15(s，2H).
Step 7: synthesis of Compound BB-21
Compound BB-21-7 (30 g,98.00 mmol) was added to ethanol (300 mL), and concentrated sulfuric acid (9.81 g,98.00mmol,5.33mL, purity: 98%) was slowly added thereto, and the mixture was allowed to react at 70℃for 2 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, the solvent was removed by concentration under reduced pressure, ethyl acetate (200 mL) was added, ph=8 of the solution was adjusted with saturated sodium hydrogencarbonate, extraction was performed, the organic phase was washed with saturated aqueous sodium hydrogencarbonate (150 ml×2), dried over anhydrous sodium sulfate, filtration was performed, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate=8/1-3/1) to give compound BB-21.MS-ESI m/z:334.1[ M+H ] ] + ，336.1[M+H+2] + . 1 H NMR (400MHz，DMSO_d 6 )δ：8.41(d，J＝9.6Hz，1H)，8.17(d，J＝8.4Hz，1H)，8.06(d，J＝9.2Hz，1H)，7.99(d，J＝7.6Hz，1H)，7.66(t，J＝8.0Hz，1H)，4.53(s，2H)，4.14(q，J＝7.2Hz，2H)，1.15(t，J＝7.2Hz，3H).
Reference example 22: fragment BB-22
The synthetic route is as follows:
step 1: synthesis of Compound BB-22-1
Tert-butyl carbamate (2.31 g,19.75 mmol) was added to toluene (60 mL) and water (20 mL), followed by addition of compound BB-21 (6 g,17.96 mmol), 2-di-tert-butylphosphine-2 ',4',6' -triisopropylbiphenyl (762.45 mg,1.80 mmol), tris (dibenzylideneacetone) dipalladium (822.10 mg, 897.76. Mu. Mol) and potassium phosphate (15.25 g,71.82 mmol), and the mixture was heated to 100℃under nitrogen for 12 hours. After the completion of the reaction, the reaction mixture was poured into water (100 mL), extracted with ethyl acetate (90 mL. Times.3), and the organic phase was collected, washed with saturated brine (70 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product is separated and purified by column chromatography (eluent: petroleum ether: ethyl acetate=6/1-2/1) to obtain compound BB-22-1. 1 H NMR(400MHz，DMSO_d 6 )δ：9.47(s，1H)，8.29(d，J＝9.6Hz，1H)，7.92(d，J＝7.6Hz，1H)，7.91(d，J＝9.2Hz，1H)，7.70(t，J＝8.0Hz，1H)，7.65(d，J＝6.8Hz，1H)，4.50(s，2H)，4.15(q，J＝7.0Hz，2H)，1.50(s，9H)，1.16(t，J＝7.2Hz，3H).
Step 2: synthesis of Compound BB-22-2
Compound BB-22-1 (2.3 g,6.21 mmol) was added to tetrahydrofuran (25 mL) at room temperature, and acrylamide (529.63 mg,7.45 mmol) and potassium t-butoxide (1.05 g,9.31 mmol) were added in this order to react for 1 hour under nitrogen. After the completion of the reaction, the reaction mixture was added to a 1 mol/L hydrochloric acid solution, extracted with ethyl acetate (45 mL. Times.3), and the organic phase was collected, washed with saturated brine (30 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product is separated and purified by column chromatography (eluent: petroleum ether: ethyl acetate=5/1-1/1) to obtain compound BB-22-2.MS-ESI m/z:396.2[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.14(s，1H)，9.47(s，1H)，8.28(d，J＝9.6Hz，1H)，8.04(d，J＝7.2Hz，1H)，7.93(d，J＝9.6Hz，1H)，7.68(t，J＝7.8Hz，1H)，7.63(d，J＝7.6Hz，1H)，5.07(dd，J＝4.6Hz，11.4Hz，1H)，2.89-2.81(m，1H)，2.65-2.55(m，2H)，2.41-2.33(m，1H)，1.50(s，9H).
Step 3: synthesis of Compound BB-22 hydrochloride
Compound BB-22-2 (0.7 g,1.77 mmol) was added to ethyl acetate hydrochloride (4M, 10 mL) at room temperature and stirred for 2 hours. After the reaction, the reaction mixture was filtered, the cake was rinsed with ethyl acetate (30 mL), and the cake was collected to give the hydrochloride of compound BB-22. MS-ESI m/z:296.1[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.14(s，1H)，8.34(d，J＝9.6Hz，1H)，7.98(d，J＝9.2Hz，1H)，7.90(s，1H)，7.63(t，J＝8.0Hz，1H)，7.39(d，J＝7.6Hz，1H)，5.05(dd，J＝4.4Hz，11.4Hz，1H)，2.89-2.80(m，1H)，2.68-2.61(m，1H)，2.60-2.53(m，1H)，2.41-2.35(m，1H).
Reference example 23: fragment BB-23
The synthetic route is as follows:
step 1: synthesis of Compound BB-23-2
BB-23-1 (10 g,57.95 mmol) was dissolved in N, N-dimethylformamide (100 mL), and tert-butyl piperazine-1-carboxylate (10.79 g,57.95 mmol) and N, N-diisopropylethylamine (22.47 g,173.84 mmol) were added and the reaction mixture was warmed to 100℃and reacted for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the cake was rinsed with methyl tert-butyl ether (150 mL) and collected to give Compound BB-23-2.MS-ESI m/z:323.2[ M+H ]] + .
Step 2: synthesis of Compound BB-23-3
Compound BB-23-2 (2.6 g,8.07 mmol) was dissolved in a mixed solvent of methanol (5 mL), tetrahydrofuran (30 mL) and water (7 mL), lithium hydroxide monohydrate (676.92 mg,16.13 mmol) was added, and the reaction was stirred at 20℃for 14 hours under nitrogen. After the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was adjusted to ph=2 to 3 with 1mol/L hydrochloric acid to precipitate a solid. Filtering, collecting filter cake, and vacuum drying to obtain compound BB-23-3. 1 H NMR(400MHz，DMSO_d 6 )δ：7.85(d，J＝9.6Hz，1H)，7.29(d，J＝9.6Hz，1H)，3.74(t，J＝5.4Hz，4H)，3.47(t，J＝5.0Hz，4H)，1.43(s，9H).
Step 3: synthesis of Compound BB-23-5
The hydrochloride salt of compound BB-23-4 (0.78 g,2.72 mmol) and BB-23-3 (837.44 mg,2.72 mmol) were dissolved in N, N-dimethylformamide (10 mL), followed by the sequential addition of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.55 g,4.07 mmol) and N, N-diisopropylethylamine (1.05 g,8.15mmol,1.42 mL) and stirring under nitrogen at 20℃for 12 hours. After the completion of the reaction, water (30 mL) was added to the reaction mixture, stirred for 0.5 hour, then filtered, and the cake was collected and dried in vacuo to give Compound BB-23-5. 1 H NMR(400MHz，CDCl 3 )δ：8.04(d，J＝9.6Hz，1H)，7.86(d，J＝8.0Hz，1H)，7.56(d，J＝8.8Hz，1H)，7.03-6.96(m，2H)，6.86(dd，J＝2.4Hz，8.8Hz，1H)，4.37-4.26(m，1H)，4.11-4.00(m，1H)，3.82-3.71(m，4H)，3.66-3.55(m，4H)，2.26-2.13(m，4H)，1.75-1.64(m，2H)，1.50(s，9H)，1.49-1.38(m，2H).
Step 4: synthesis of hydrochloride of Compound BB-23
Compound BB-23-5 (1.25 g,2.31 mmol) was dissolved in methylene chloride (3 mL), and ethyl acetate hydrochloride (4M, 8 mL) was added to react at 20℃for 12 hours. The reaction solution was filtered, and the cake was washed with ethyl acetate (10 mL), and the cake was collected and dried to give the hydrochloride of Compound BB-23. MS-ESI m/z:441.1[ M+H ]] + . 1 H NMR(400MHz，CD 3 OD)δ：8.36(d，J＝9.6Hz，1H)，8.00(d，J＝10.0Hz，1H)，7.69(d，J＝8.8Hz，1H)，7.20(d，J＝2.4Hz，1H)，7.05(dd，J＝2.4，8.8Hz，1H)，4.57-4.45(m，1H)，4.16(t，J＝5.4Hz，4H)，4.04-3.94(m，1H)，3.50(t，J＝5.4Hz，4H)，2.26-2.18(m，2H)，2.15-2.05(m，2H)，1.74-1.61(m，4H).
Reference example 24: fragment BB-24
The synthetic route is as follows:
step 1: synthesis of Compound BB-24-1
5-hexenoic acid (75.69 mg, 663.14. Mu. Mol, 78.76. Mu.L) was dissolved in N, N-dimethylformamide (5 mL), followed by addition of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (343.84 mg, 904.29. Mu. Mol) and N, N-diisopropylethylamine (233.75 mg,1.81mmol, 315.02. Mu.L), the reaction system was purged with nitrogen three times, and after stirring at 20℃for 30 minutes, the hydrochloride (200 mg, 602.86. Mu. Mol) of Compound BB-22 was added, and the reaction solution was stirred at 20℃for 2 hours. After the completion of the reaction, water (15 mL) was added to the reaction mixture, extraction was performed with ethyl acetate (20 mL. Times.3), and the combined organic phases were washed with saturated brine (15 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product, which was separated by column chromatography (eluent: petroleum ether/ethyl acetate=2/1-1/2, volume ratio) to give Compound BB-24-1.MS-ESI m/z:392.2[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.15(s，1H)，10.11(s，1H)，8.28(d，J＝9.6Hz，1H)，8.08(s，1H)，7.97(d，J＝9.6Hz，1H)，7.75-7.65(m，2H)，5.95-5.82(m，1H)，5.16-4.95(m，3H)，2.88-2.78(m，1H)，2.69-2.61(m，2H)，2.59-2.55(m，1H)，2.43-2.32(m，2H)，2.20-2.10(m，2H)，1.82-1.72(m，2H).
Step 2: synthesis of Compound BB-24
Compound BB-24-1 (100 mg, 255.48. Mu. Mol) was dissolved in a mixed solvent of tetrahydrofuran (12 mL) and water (4 mL), sodium periodate (218.58 mg,1.02mmol, 56.63. Mu.L) and osmium tetraoxide dihydrate (9.41 mg, 25.55. Mu. Mol) were added at 0℃to the reaction system, the nitrogen was replaced three times, the reaction was stirred at 20℃for 1 hour, the reaction mixture was poured into water (20 mL), the water phase was extracted with ethyl acetate (15 mL. Times.3), and the combined organic phases were washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound BB-24.MS-ESI m/z:394.2[ M+H ]] + .
Reference example 25
The synthetic route is as follows:
step 1: synthesis of Compound BB-25-1
Compound BB-21 (300 mg, 897.76. Mu. Mol), 1-tert-butyloxycarbonyl piperazine (299.91 mg,1.35 mmol), tris (dibenzylideneacetone) dipalladium (41.10 mg, 44.89. Mu. Mol), cesium carbonate (41.10 mg, 44.89. Mu. Mol) and 2-dicyclohexylphosphine-2, 6-diisopropyloxy-1, 1-biphenyl (41.89 mg, 89.78. Mu. Mol) were dissolved in 1, 4-dioxane (5 mL) at room temperature under nitrogen, and the reaction mixture was warmed to 100℃and stirred for 2 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, water (20 mL) was added, and the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phases were combined, washed with saturated brine (30 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the residual solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=5/1-1/1, volume ratio) to give compound BB-25-1.MS-ESI m/z:440.3[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：8.47(d，J＝9.2Hz，1H)，7.89(d，J＝9.6Hz，1H)，7.84(d，J＝8.4Hz，1H)，7.67(t，J＝8.0Hz，1H)，7.30(d，J＝7.6Hz，1H)，4.48(s，2H)，4.15(q，J＝7.2Hz，2H)，3.95-3.40(m，4H)，3.15-2.80(m，4H)，1.45(s，9H)，1.17(t，J＝7.0Hz，3H).
Step 2: synthesis of Compound BB-25-2
Compound BB-25-1 (100 mg, 227.53. Mu. Mol) and acrylamide (19.41 mg, 273.04. Mu. Mol, 18.84. Mu.L) were dissolved in tetrahydrofuran (2 mL) at room temperature under nitrogen, cooled to 0℃and potassium t-butoxide (38.30 mg, 341.29. Mu. Mol) was added, and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was poured into dilute hydrochloric acid (0.1N, 10 mL) and extracted with ethyl acetate (20 mL. Times.2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure to remove the solvent, and the resulting residue was purified by plate separation (developer: petroleum ether/ethyl acetate=1/1, volume ratio) to give compound BB-25-2.MS-ESI m/z:465.3[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.12(s，1H)，8.49(d，J＝9.6Hz，1H)，7.95(d，J＝7.2Hz，1H)，7.91(d，J＝9.2Hz，1H)，7.66(t，J＝7.8Hz，1H)，7.31(d，J＝8.0Hz，1H)，5.04(dd，J＝4.6，11.4Hz，1H)，4.04-3.50(m，4H)，3.15-2.90(m，4H)，2.90-2.79(m，1H)，2.70-2.59(m，1H)，2.40-2.31(m，2H)，1.45(s，9H).
Step 3: hydrochloride synthesis of compound BB-25
Compound BB-25-2 (40 mg, 86.11. Mu. Mol) was dissolved in ethyl acetate (2 mL) at room temperature, an ethyl acetate solution of hydrochloric acid (3 mL, 4M) was added, and the reaction mixture was stirred at room temperature for 1 hour. After the completion of the reaction, the solvent was removed by concentration under reduced pressure to give hydrochloride of Compound BB-25. MS-ESI m/z:365.0[ M+H ]] + .
Reference example 26: fragment BB-26
The synthetic route is as follows:
step 1: synthesis of Compound BB-26-2
Compound BB-26-1 (25 g,144.87 mmol) and 4-piperidinemethanol (16.69 g,144.87 mmol) were dissolved in 1, 4-dioxane (500 mL) at room temperature, potassium carbonate (60.07 g,434.61 mmol) and tetrabutylammonium iodide (5.35 g,14.49 mmol) were added in this order, and the reaction mixture was warmed to 100℃and stirred for 48 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, the cake was rinsed with methylene chloride (700 mL), water (300 mL) was added to the mother liquor, and the mixture was extracted with a methylene chloride/methanol mixed solution (10/1, volume ratio, 300 mL. Times.5), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. Adding petroleum ether/dichloromethane mixed solution (1/2, volume ratio, 250 mL) into the crude product, stirring at room temperature for 1 hour, filtering, leaching the filter cake with petroleum ether/dichloromethane mixed solution (1/2, volume ratio, 70mL multiplied by 2), collecting the filter cake, and vacuum drying to obtain the compound BB-26-2. 1 H NMR(400MHz，CDCl 3 )δ：7.85(d，J＝9.6Hz，1H)，6.87(d，J＝9.6Hz，1H)，4.61(d，J＝13.2Hz，2H)，3.99(s，3H)，3.55(d，J＝5.6Hz，2H)，3.10-3.00(m，2H)，1.97-1.84(m，3H)，1.39-1.23(m，2H).
Step 2: synthesis of Compound BB-26-3
Compound BB-26-2 (71.5 g,284.54 mmol) and imidazole (23.25 g,341.45 mmol) were dissolved in dichloromethane (700 mL) at room temperature, cooled to 0-10℃and tert-butyldiphenylchlorosilane (78.21 g,284.54mmol,73.09 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, a saturated aqueous ammonium chloride solution (500 mL) was added, and the mixture was extracted with methylene chloride (300 mL. Times.3). The organic phases were combined, washed with saturated aqueous ammonium chloride (500 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. Petroleum ether (300 mL) is added into the crude product, stirred for 0.5 hour at room temperature, filtered, the filter cake is rinsed with petroleum ether (50 mL multiplied by 3), the filter cake is collected, and vacuum dried, thus obtaining the compound BB-26-3.
Step 3: synthesis of Compound BB-26-4
Compound BB-26-3 (42 g,85.77 mmol) was dissolved in tetrahydrofuran (1.25L), sodium hydroxide (2M, 85.77 mL) was added, and the reaction mixture was stirred at room temperature for 1 hour. After the reaction, three batches are combined, the pH value is regulated to be between 6 and 7 by using 2N hydrochloric acid, and the solvent is removed by decompression concentration, so that the compound BB-26-4 is obtained.
Step 4: synthesis of Compound BB-26-6
Sodium hydrogen (27.87 g,696.74mmol, purity: 60%) was dissolved in N, N-dimethylformamide (500 mL), cooled to 0℃and a solution of compound BB-26-5 (100 g,464.49 mmol) in N, N-dimethylformamide (750 mL) was added dropwise under nitrogen atmosphere at room temperature, and the reaction mixture was stirred at 0℃for 0.5 hours. A solution of 2-chloro-4-hydroxybenzonitrile (86.71 g,557.39 mmol) in N, N-dimethylformamide (300 mL) was added dropwise, and the reaction mixture was stirred at 0deg.C for 0.5 hours. Then the temperature was slowly raised to room temperature and the reaction was stirred for 2 hours. After the completion of the reaction, the reaction mixture was poured into 0-5℃diluted hydrochloric acid (1N, 800 mL) and extracted with ethyl acetate (700 mL. Times.3). The organic phases were combined, washed with saturated brine (700 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. N-heptane (500 mL) was added to the crude, stirred at room temperature for 30 min, filtered, the filter cake rinsed with n-heptane (100 mL. Times.3), the filter cake collected and dried in vacuo to give compound BB-26-6. 1 H NMR(400MHz，CDCl 3 )δ：7.55(d，J＝8.8Hz，1H)，6.97(d，J＝2.0Hz，1H)，6.83(dd，J＝2.4，8.8Hz，1H)，4.54-4.33(m，1H)，4.30-4.20(m，1H)，3.53(br s，1H)，2.19-2.02(m，4H)，1.69-1.54(m，2H)，1.46(s，9H)，1.36-1.21(m，2H).
Step 5: hydrochloride synthesis of compound BB-26-7
Compound BB-26-6 (92 g,262.23 mmol) was dissolved in ethyl acetate (60 mL) at room temperature, an ethyl acetate solution of hydrochloric acid (4M, 750 mL) was added, and the reaction mixture was stirred at room temperature overnight. After the reaction, the two batches were combined, filtered, the filter cake was rinsed with ethyl acetate (100 mL. Times.2), and the filtrate was collectedThe cake was dried under vacuum to give the hydrochloride salt of compound BB-26-7. 1 H NMR(400MHz，D 2 O)δ：7.64(d，J＝8.8Hz，1H)，7.14(d，J＝2.4Hz，1H)，6.96(dd，J＝2.4，8.8Hz，1H)，4.50-4.39(m，1H)，3.34-3.17(m，1H)，2.28-2.07(m，4H)，1.68-1.42(m，4H).
Step 6: synthesis of Compound BB-26-8
Compound BB-26-4 (60.5 g,127.19 mmol) and N, N-diisopropylethylamine (82.19 g,635.97 mmol) were dissolved in N, N-dimethylformamide (700 mL) at room temperature, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (58.04 g,152.63 mmol) was added, and the reaction mixture was stirred at room temperature for 5 minutes, and the hydrochloride (32.88 g,114.47 mmol) of compound BB-26-7 was added, and the reaction mixture was stirred at room temperature overnight. After completion of the reaction, water (3000 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (1000 mL. Times.3). The organic phases were combined, washed with saturated brine (1000 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. Methyl tert-butyl ether (300 mL) was added to the crude product, stirred at room temperature for 0.5 hour, filtered, the filter cake rinsed with methyl tert-butyl ether (100 mL. Times.2), the filter cake collected and dried in vacuo to give compound BB-26-8. 1 H NMR(400MHz，DMSO_d 6 )δ：8.58(d，J＝8.4Hz，1H)，7.85(d，J＝8.8Hz，1H)，7.80(d，J＝9.2Hz，1H)，7.63-7.56(m，4H)，7.50-7.40(m，6H)，7.38(d，J＝2.0Hz，1H)，7.34(d，J＝9.6Hz，1H)，7.14(dd，J＝2.4，8.8Hz，1H)，4.63-4.44(m，3H)，3.94-3.77(m，1H)，3.54(d，J＝6.0Hz，2H)，3.07-2.96(m，2H)，2.17-2.06(m，2H)，1.96-1.86(m，3H)，1.84-1.74(m，2H)，1.71-1.59(m，2H)，1.58-1.45(m，2H)，1.32-1.21(m，2H)，0.99(s，9H).
Step 7: synthesis of Compound BB-26-9
Compound BB-26-8 (53 g,74.82 mmol) was dissolved in N, N-dimethylformamide (530 mL) at room temperature, potassium fluoride (30.43 g,523.74 mmol) was added, and the reaction mixture was warmed to 100℃and stirred overnight. After the reaction, cooling to room temperature, combining the two batches, and adding into the reaction solutionWater (3L) was extracted with ethyl acetate (1L. Times.3). The organic phases were combined, washed with saturated brine (1 L×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (eluent: ethyl acetate/petroleum ether=1/2-1/0, volume ratio) to give compound BB-26-9.MS-ESI m/z:470.2[ M+H ]] + . 1 H NMR(400MHz，CDCl 3 )δ：7.99(d，J＝9.6Hz，1H)，7.87(br d，J＝8.0Hz，1H)，7.57(d，J＝8.8Hz，1H)，7.04-6.97(m，2H)，6.86(dd，J＝2.4，8.4Hz，1H)，4.56(br d，J＝12.8Hz，2H)，4.38-4.28(m，1H)，4.13-3.99(m，1H)，3.57(br d，J＝5.6Hz，2H)，3.16-3.02(m，2H)，2.26-2.12(m，4H)，1.97-1.84(m，3H)，1.76-1.62(m，4H)，1.54-1.44(m，2H).
Step 8: synthesis of Compound BB-26
BB-26-9 (2 g,4.26 mmol) was added to methylene chloride (30 mL), dess-martin oxidant (2.17 g,5.11mmol,1.58 mL) was slowly added and reacted under nitrogen atmosphere for 2 hours at 20℃and after the reaction was completed, the reaction solution was filtered, saturated sodium bicarbonate solution (20 mL) was added to the filtrate and stirred for 15 minutes, extracted with methylene chloride (25 mL. Times.2), the organic phase was collected, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/1 to 1/5, volume ratio) to obtain BB-26.MS-ESI m/z:468.2[ M+H ] ] + .
Reference example 27: fragment BB-27
The synthetic route is as follows:
step 1: synthesis of Compound BB-27-2
Compound BB-27-1 (0.5 g,2.90 mmol) and endo-3-azabicyclo [3.1.0]Hexane-6-methanol (328.16 mg,2.90 mmol) was dissolved in 1, 4-dioxane (10 mL), potassium carbonate (1.20 g,8.70 mmol) and tetrabutylammonium iodide (107.02 mg, 290.00. Mu. Mol) were added sequentially, and the reaction mixture was warmed to 100℃and stirred overnight. After the reaction was completed, cooled to room temperature, filtered, the cake was rinsed with dichloromethane (70 mL), water (30 mL) was added to the mother liquor, extracted with a dichloromethane/methanol mixed solution (10/1, volume ratio, 30ml×5), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. And adding petroleum ether/dichloromethane mixed solution (2/1, volume ratio, 10 mL) into the obtained crude product, stirring at room temperature for 1 hour, filtering, leaching a filter cake with the petroleum ether/dichloromethane mixed solution (2/1, volume ratio, 5mL multiplied by 2), collecting the filter cake, and drying in vacuum to obtain the compound BB-27-2. 1 H NMR(400MHz，CDCl 3 )δ：7.87(d，J＝9.6Hz，1H)，6.58(d，J＝9.6Hz，1H)，4.20-3.75(m，5H)，3.73-3.52(m，4H)，1.74(br s，2H)，1.12-0.97(m，1H).
Step 2: synthesis of Compound BB-27-3
Compound BB-27-2 (0.541 g,2.17 mmol) and imidazole (192.08 mg,2.82 mmol) were dissolved in dichloromethane (10 mL) at room temperature under nitrogen, cooled to 0deg.C, tert-butyldiphenylchlorosilane (596.55 mg,2.17 mmol) was added dropwise, the reaction mixture was slowly warmed to room temperature, and stirred for 1 hour. After completion of the reaction, a saturated aqueous ammonium chloride solution (10 mL) was added, and the mixture was extracted with methylene chloride (5 mL. Times.3). The organic phases were combined, washed with saturated brine (10 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. N-heptane (5 mL) was added to the crude, stirred at room temperature for 10 min, filtered, the filter cake rinsed with n-heptane (2 mL. Times.3), the filter cake collected and dried in vacuo to give compound BB-27-3. 1 H NMR(400MHz，CDCl 3 )δ：7.86(d，J＝9.6Hz，1H)，7.71-7.65(m，4H)，7.47-7.36(m，6H)，6.55(d，J＝9.6Hz，1H)，4.20-3.72(m，5H)，3.67(d，J＝6.0Hz，2H)，3.63-3.51(m，2H)，1.62(br s，2H)，1.06(s，9H)，0.97-0.90(m，1H).
Step 3: synthesis of Compound BB-27-4
Compound BB-27-3 (902 mg,1.85 mmol) was dissolved in tetrahydrofuran (10 mL) at room temperature, and aqueous sodium hydroxide solution (2M, 1.85 mL) was added to stir the reaction mixture at room temperature for 2 hours. After the reaction, hydrochloric acid (1N) was added to the reaction mixture to adjust ph=6-7, and the solvent was removed by concentration under reduced pressure to obtain compound BB-27-4.
Step 4: synthesis of Compound BB-27-5
Compound BB-27-4 (1.02 g,2.15 mmol) and N, N-diisopropylethylamine (1.39 g,10.77 mmol) were dissolved in N, N-dimethylformamide (10 mL) at room temperature, and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (781.60 mg,2.06 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 0.5 hours. The hydrochloride salt of compound BB-26-7 (491.92 mg,1.71 mmol) was further added, and the reaction mixture was stirred overnight. After completion of the reaction, water (50 mL) was added thereto, and the mixture was extracted with ethyl acetate (20 mL. Times.3). The organic phases were combined, washed with saturated brine (20 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The crude product obtained was purified by column chromatography (eluent: ethyl acetate/petroleum ether=1/3-1/2, volume ratio) to give compound BB-27-5.
Step 5: synthesis of Compound BB-27-6
Compound BB-27-5 (1 g,1.42 mmol) was dissolved in N, N-dimethylformamide (20 mL) at room temperature, potassium fluoride (575.75 mg,9.91 mmol) was added, and the reaction mixture was warmed to 110℃and stirred overnight. After the completion of the reaction, the reaction mixture was cooled to room temperature, water (50 mL) was added, and the mixture was extracted with ethyl acetate (20 mL. Times.3). The organic phases were combined, washed with saturated brine (20 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Methyl tert-butyl ether (3 mL) was added to the crude product, stirred at room temperature for 20 minutes, filtered, the filter cake rinsed with methyl tert-butyl ether (2 mL. Times.2), the filter cake collected and dried in vacuo to give compound BB-27-6. 1 H NMR(400MHz，CDCl 3 )δ：7.98(d，J＝9.6Hz，1H)，7.89(br d，J＝8.0Hz，1H)，7.57(d，J＝8.8Hz，1H)，7.01(d，J＝2.4Hz，1H)，6.86(dd，J＝2.4，8.8Hz，1H)，6.68(d，J＝9.2Hz，1H)，4.38-4.28(m，1H)，4.11-4.00(m，1H)，3.49(br s，2H)，3.70-3.57(m，4H)，2.25-2.13(m，4H)，1.77(br s，2H)，1.72-1.63(m，2H)，1.52-1.40(m，2H)，1.11-1.03(m，1H).
Step 6: synthesis of Compound BB-27
Compound BB-27-6 (100 mg, 213.70. Mu. Mol) was dissolved in methylene chloride (2 mL) at room temperature under nitrogen, and dess-Martin reagent (135.96 mg, 320.55. Mu. Mol) was added to stir the reaction mixture at room temperature for 3 hours. After completion of the reaction, filtration was performed, a saturated sodium hydrogencarbonate solution (10 mL) was added to the filtrate, stirred at room temperature for 5 minutes, extracted with methylene chloride (20 ml×2), and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, whereby compound BB-27 was obtained. MS-ESI m/z:466.1[ M+H ] ] + .
Reference example 28: fragment BB-28
The synthetic route is as follows:
step 1: synthesis of Compound BB-28-2
Compound BB-28-1 (9.5 g,54.29 mmol) and triethylamine (10.99 g,108.58mmol,15.11 mL) were dissolved in ethanol (100 mL) at room temperature, palladium acetate (609.43 mg,2.71 mmol) and 4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene (2.51 g,4.34 mmol) were added, and the reaction mixture was warmed to 85℃under an atmosphere of carbon monoxide (15 psi) at room temperature and stirred for 14 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, ethyl acetate (100 mL) was added, the mixture was filtered, the cake was rinsed with ethyl acetate (100 mL. Times.4), and the filtrate was concentrated under reduced pressure to remove the solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=3/1-1/1, volume ratio) to give compound BB-28-2.MS-ESI m/z:169.2[ M+H ]] + . 1 H NMR(400MHz，CDCl 3 )δ：8.80(s，1H)，6.54-5.89(m，2H)，4.51(q，J＝7.2Hz，2H)，1.45(t，J＝7.2Hz，3H).
Step 2: synthesis of Compound BB-28-3
Compound BB-28-2 (1.8 g,10.70 mmol) and cupric chloride dihydrate (2.19 g,12.85 mmol) were dissolved in acetonitrile (30 mL) at room temperature under nitrogen, tert-butyl nitrite (1.66 g,16.06mmol,1.91 mL) was added, and the reaction mixture was warmed to 60℃and stirred for 1 hour. After completion of the reaction, the reaction mixture was cooled to room temperature, hydrochloric acid (1N, 30 mL) was added thereto, and the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phases were combined, washed with saturated brine (80 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=15/1-5/1, volume ratio) to give compound BB-28-3.MS-ESI m/z:188.0[ M+H ] ] + . 1 H NMR(400MHz，CDCl 3 )δ：9.12(s，1H)，4.59(q，J＝7.2Hz，2H)，1.50(t，J＝7.2Hz，3H).
Step 3: synthesis of Compound BB-28-4
Intermediate BB-28-3 (0.65 g,3.47 mmol) and 4-piperidinemethanol (598.63 mg,5.20 mmol) were dissolved in 1, 4-dioxane (10 mL) at room temperature under nitrogen, N-diisopropylethylamine (1.34 g,10.40mmol,1.81 mL) was added, and the reaction mixture was warmed to 100℃and stirred for 14 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solvent was removed by vacuum concentration. To the residue was added water (20 mL), and the mixture was extracted with ethyl acetate (20 mL. Times.4). The organic phases were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Petroleum ether (20 mL) and ethyl acetate (4 mL) were added to the residue, stirred at room temperature for 0.5 h, filtered, the filter cake was washed with petroleum ether (20 mL), the filter cake was collected, and dried in vacuo to give compound BB-28-4.MS-ESI m/z:267.1[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：8.68(s，1H)，5.22-4.56(m，2H)，4.53(t，J＝5.2Hz，1H)，4.35(q，J＝7.2Hz，2H)，3.28(t，J＝5.6Hz，2H)，3.17-3.00(m，2H)，1.87-1.68(m，3H)，1.32(t，J＝7.2Hz，3H)，1.22-1.06(m，2H).
Step 4: synthesis of Compound BB-28-5
Compound BB-28-4 (0.54 g,2.03 mmol) and imidazole (186.37 mg,2.74 mmol) were dissolved in dichloromethane (10 mL) at room temperature under nitrogen, and tert-butyldiphenylchlorosilane (613.11 mg,2.23mmol, 573.00. Mu.L) was added. The reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, a saturated ammonium chloride solution (20 mL) was added, and the mixture was extracted with methylene chloride (20 mL. Times.2). The organic phases were combined, washed with a saturated ammonium chloride solution (40 mL. Times.3) and a saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, to give Compound BB-28-5.MS-ESI m/z:505.3[ M+H ] ] + 。
Step 5: synthesis of Compound BB-28-6
Compound BB-28-5 (1 g,1.98 mmol) was dissolved in ethanol (10 mL), tetrahydrofuran (10 mL) and water (3 mL) under nitrogen at room temperature, and lithium hydroxide monohydrate (249.44 mg,5.94 mmol) was added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure. To the residue was added water (10 mL), ph=2-3 was adjusted with hydrochloric acid (1N), and extraction was performed with ethyl acetate (20 ml×4). The organic phases were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to give Compound BB-28-6.MS-ESI m/z:477.2[ M+H ]] + .
Step 6: synthesis of Compound BB-28-7
Compound BB-28-6 (1.1 g,2.31 mmol) and N, N-diisopropylethylamine (1.19 g,9.23mmol,1.61 mL) were dissolved in N, N-dimethylformamide (10 mL) under nitrogen, 2- (7-azobenzotriazole) -N, N, N, N-tetramethylurea hexafluorophosphate (1.14 g,3.00 mmol) was further added, and the reaction mixture was stirred at room temperature for 5 minutes, and the hydrochloride (662.77 mg,2.31 mmol) of intermediate BB-26-7 was added, and the reaction mixture was further stirred at room temperature for 55 minutes. After completion of the reaction, water (20 mL) was added thereto, and the mixture was extracted with ethyl acetate (20 mL. Times.3). The organic phases were combined, washed with saturated brine (50 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was reduced in pressure Concentrating to remove the solvent. The residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=3/1-1/1, volume ratio) to give compound BB-28-7.MS-ESI m/z:709.3[ M+H ]] + . 1 H NMR(400MHz，CDCl 3 )δ：8.82(s，1H)，7.68-7.64(m，4H)，7.57(d，J＝8.8Hz，2H)，7.46-7.38(m，6H)，7.01(d，J＝2.4Hz，1H)，6.86(dd，J＝2.4，8.8Hz，1H)，5.17-4.70(m，2H)，4.35-4.28(m，1H)，4.09-4.03(m，1H)，3.55(d，J＝5.6Hz，2H)，3.12-3.00(m，2H)，2.24-2.13(m，4H)，1.99-1.86(m，3H)，1.76-1.64(m，2H)，1.52-1.43(m，2H)，1.36-1.29(m，2H)，1.06(s，9H).
Step 7: synthesis of Compound BB-28-8
Compound BB-28-7 (0.5 g, 704.87. Mu. Mol) was dissolved in N, N-dimethylformamide (6 mL) at room temperature, potassium fluoride (409.51 mg,7.05 mmol) was added, and the reaction mixture was warmed to 100℃and stirred overnight. After the reaction was completed, the reaction mixture was cooled to room temperature. Water (30 mL) was added and extracted with ethyl acetate (20 mL. Times.3). The organic phases were combined, washed with saturated brine (60 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=3/1-1/2, volume ratio) to give compound BB-28-8.MS-ESI m/z:471.2[ M+H ]] + . 1 H NMR(400MHz，CDCl 3 )δ：8.82(s，1H)，7.56(d，J＝8.4Hz，2H)，7.00(d，J＝2.0Hz，1H)，6.86(dd，J＝2.4，8.8Hz，1H)，4.98(br s，2H)，4.37-4.25(m，1H)，4.09-4.02(m，1H)，3.57(d，J＝5.6Hz，2H)，3.07(brt，J＝12.4Hz，2H)，2.22-2.14(m，4H)，1.99-1.82(m，3H)，1.75-1.67(m，2H)，1.52-1.41(m，2H)，1.37-1.28(m，2H).
Step 8: synthesis of Compound BB-28
Compound BB-28-8 (0.257 g, 545.70. Mu. Mol) was dissolved in methylene chloride (5 mL) at room temperature under nitrogen, cooled to 0℃and a dess-Martin oxidant (347.18 mg, 818.56. Mu. Mol) was added thereto, and the reaction mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the mixture was filtered, and the cake was washed with ethyl acetate (30 mL). Directional filtering To the solution was added saturated sodium bicarbonate (20 mL), and the mixture was stirred for 5 minutes, followed by extraction with ethyl acetate (20 mL. Times.3). The organic phases were combined, washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to give Compound BB-28.MS-ESI m/z:469.2[ M+H ]] + 。
Reference example 29: fragment BB-29
The synthetic route is as follows:
step 1: synthesis of Compound BB-29-2
Compound BB-29-1 (5 g,28.97 mmol) was dissolved in dimethyl sulfoxide (80 mL), 4-piperidinemethanol (3.34 g,28.97 mmol) and N, N-diisopropylethylamine (7.49 g,57.95mmol,10.09 mL) were sequentially added, the reaction mixture was stirred at 120℃for 12 hours under nitrogen protection, the reaction mixture was cooled to room temperature, ethyl acetate (200 mL) and deionized water (200 mL) were added, the organic phase was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and the resulting crude product was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/1, volume ratio) to give compound BB-29-2.MS-ESI m/z:273.9[ M+Na] + . 1 H NMR(400MHz，DMSO_d 6 )δ：8.62(d，J＝1.2Hz，1H)，8.35(d，J＝1.2Hz，1H)，4.55-4.46(m，3H)，3.80(s，3H)，3.27(t，J＝5.8Hz，2H)，3.03-2.92(m，2H)，1.80-1.64(m，3H)，1.20-1.05(m，2H).
Step 2: synthesis of Compound BB-29-3
Compound BB-29-2 (5 g,19.90 mmol) was dissolved in a mixed solvent of methanol (100 mL) and deionized water (25 mL), lithium hydroxide (2.50 g,59.69 mmol) was added, and the reaction mixture was under nitrogen Stirring at 25 ℃ for 12 hours, concentrating the reaction mixture under reduced pressure to remove methanol after the reaction is finished, cooling at 0-5 ℃, adjusting the pH value of the solution to be 3-4 by using dilute hydrochloric acid (1M), precipitating white precipitate in the reaction mixture, filtering under reduced pressure, washing a filter cake by using deionized water, collecting the filter cake, and drying in vacuum at 40-45 ℃ to obtain the compound BB-29-3.MS-ESI m/z:237.9[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：12.63(br s，1H)，8.61(s，1H)，8.33(s，1H)，4.63-4.39(m，3H)，3.27(br d，J＝6.0Hz，2H)，3.02-2.89(m，2H)，1.80-1.63(m，3H)，1.19-1.04(m，2H).
Step 3: synthesis of Compound BB-29-4
Compound BB-26-7 (4 g,13.93 mmol) and compound BB-29-3 (3.30 g,13.93 mmol) were dissolved in N, N-dimethylformamide (100 mL) under nitrogen atmosphere at room temperature, triethylamine (7.05 g,69.64mmol,9.69 mL), 1-hydroxybenzotriazole (2.82 g,20.89 mmol) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (4.01 g,20.89 mmol) were added in this order, and the reaction mixture was stirred at 25℃for 12 hours. After completion of the reaction, ice water (300 mL) and ethyl acetate (200 mL) were added, the organic phase was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and the crude product obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/1, volume ratio) to give compound BB-29-4.MS-ESI m/z:470.1[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：8.58(d，J＝0.8Hz，1H)，8.23(d，J＝1.2Hz，1H)，8.07(d，J＝8.4Hz，1H)，7.85(d，J＝8.8Hz，1H)，7.35(d，J＝2.0Hz，1H)，7.11(dd，J＝2.4，8.8Hz，1H)，4.56-4.41(m，4H)，3.90-3.73(m，1H)，3.27(t，J＝5.4Hz，2H)，3.00-2.88(m，2H)，2.14-2.02(m，2H)，1.92-1.82(m，2H)，1.80-1.66(m，3H)，1.65-1.43(m，4H)，1.19-1.05(m，2H).
Step 4: synthesis of Compound BB-29
Compound BB-29-4 (2 g,4.26 mmol) was dissolved in dichloromethane (100 mL) at room temperature under nitrogen, and dess-Martin reagent (2.71 g,6.38mmol,1.98 mL) was added and the reaction mixture was cooled to room temperatureStirring for 2 hours, quenching by adding saturated sodium sulfite solution (50 mL), separating organic phase, washing with saturated saline (100 mL), drying with anhydrous sodium sulfate, filtering, decompressing filtrate to remove solvent, separating the crude product by column chromatography (eluent: petroleum ether/ethyl acetate=1/1, volume ratio) to obtain compound BB-29.MS-ESI m/z:468.2[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：9.62(s，1H)，8.59(d，J＝0.8Hz，1H)，8.27(d，J＝0.8Hz，1H)，8.10(d，J＝8.4Hz，1H)，7.85(d，J＝8.8Hz，1H)，7.36(d，J＝2.0Hz，1H)，7.12(dd，J＝2.2，9.0Hz，1H)，4.57-4.45(m，1H)，4.35-4.20(m，2H)，3.90-3.75(m，1H)，3.29-3.18(m，2H)，2.75-2.60(m，1H)，2.15-2.02(m，2H)，2.00-1.83(m，4H)，1.69-1.44(m，6H).
Example 1
The synthetic route is as follows:
step 1: synthesis of Compound WX001-1
Compound BB-3 (0.14 g, 475.08. Mu. Mol) and the hydrochloride salt of compound BB-4-5 (285.34 mg, 498.83. Mu. Mol) were dissolved in acetonitrile (8 mL), followed by addition of N, N-diisopropylethylamine (184.20 mg,1.43mmol, 248.25. Mu.L) and stirring at 50℃under nitrogen. After the completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (eluent: dichloromethane/methanol=100/1-30/1, volume ratio) to give compound WX001-1. 1 H NMR(400MHz，DMSO_d 6 )δ：8.39(d，J＝8.4Hz，1H)，8.27(d，J＝1.6Hz，1H)，8.17(dd，J＝0.8Hz，4.8Hz，2H)，8.06(dd，J＝1.6Hz，8.4Hz，1H)，7.35(t，J＝9.2Hz，1H)，7.30(dd，J＝2.4Hz，12.0Hz，1H)，7.15(d，J＝8.8Hz，1H)，4.27(s，2H)，4.22(t，J＝5.4Hz，2H)，4.15(q，J＝7.2Hz，2H)，3.90(s，2H)，2.81-2.72(m，2H)，2.65-2.52(m，8H)，1.50(s，6H)，1.20(t，J＝7.2Hz，3H).
Step 2: synthesis of Compound WX001
Compound WX001-1 (0.012 g, 15.12. Mu. Mol) was dissolved in anhydrous tetrahydrofuran (0.8 mL), cooled to-60℃and acrylamide (2.15 mg, 30.23. Mu. Mol) and potassium tert-butoxide (5.09 mg, 45.35. Mu. Mol) were added, and stirred at-60℃for 1 hour with nitrogen substitution three times, and slowly returned to 15℃for 12 hours. After the completion of the reaction, the reaction solution was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX001.MS-ESI m/z:819.3[ M+H ] ] + . 1 H NMR(400MHz，CD 3 OD)δ：8.15(d，J＝8.0Hz，2H)，8.11(s，1H)，7.97(dd，J＝1.6Hz，8.4Hz，1H)，7.91(s，1H)，7.35(t，J＝9.2Hz，1H)，7.31(dd，J＝1.6Hz，11.6Hz，1H)，7.24-7.20(m，1H)，4.62(dd，J＝4.8Hz，10.8Hz，1H)，4.56(t，J＝4.4Hz，2H)，4.13(s，2H)，3.72(t，J＝4.4Hz，2H)，3.47-3.35(m，4H)，3.28-3.19(m，4H)，2.86-2.80(m，2H)，2.70-2.58(m，1H)，2.45-2.37(m，1H)，1.56(s，6H).
Example 2
The synthetic route is as follows:
step 1: synthesis of Compound WX002-1
Compound BB-8 (0.4 g,1.21 mmol) was added to 1, 2-dichloroethane (10 mL), followed by addition of hydrochloride of compound BB-6 (293.89 mg,1.21 mmol) and acetic acidPotassium (118.84 mg,1.21 mmol) was stirred at room temperature for 0.5 hours, and sodium triacetoxyborohydride (384.96 mg,1.82 mmol) was slowly added thereto and reacted at room temperature for 11.5 hours. After the completion of the reaction, 1mol/L hydrochloric acid (10 mL) was added to the reaction mixture, stirred at 20℃for 0.5 hour, filtered, and the cake was collected to give Compound WX002-1.MS-ESI m/z:519.3[ M-H ]] + .
Step 2: synthesis of Compound WX002
The compound WX002-1 (0.12 g, 230.51. Mu. Mol) was added to N, N-dimethylformamide (3 mL), and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (113.94 mg, 299.67. Mu. Mol), N, N-diisopropylethylamine (89.38 mg, 691.54. Mu. Mol, 120.45. Mu. L) was added in this order, followed by three nitrogen substitutions, and then the temperature was raised to 50℃for reaction for 1 hour. Cooling to 50℃and adding hydrochloride of Compound BB-5 (72.67 mg, 230.51. Mu. Mol), and reacting at 20℃for 2 hours. After the completion of the reaction, the reaction mixture was poured into water (5 mL), extracted with ethyl acetate (5 mL. Times.3), the organic phase was washed with saturated brine (10 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was separated and purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX002.MS-ESI m/z:781.4[ M+H ] ] + . 1 H NMR(400MHz，CD 3 OD)δ：7.80(d，J＝8.8Hz，2H)，7.72(d，J＝8.8Hz，1H)，7.64(d，J＝8.8Hz，1H)，7.16(d，J＝1.6Hz，1H)，7.13(d，J＝2.4Hz，1H)，7.09(d，J＝8.8Hz，2H)，6.99(t，J＝2.8Hz，1H)，6.97(t，J＝2.8Hz，1H)，4.44(d，J＝5.0Hz，10.6Hz，1H)，4.29(s，1H)，4.15(t，J＝6.0Hz，3H)，4.05(d，J＝13.6Hz，2H)，3.71(d，J＝12.0Hz，2H)，3.29-3.21(m，4H)，3.15(t，J＝12.2Hz，2H)，2.82-2.76(m，2H)，2.58-2.47(m，1H)，2.39-2.31(m，1H)，1.98-1.85(m，4H)，1.69-1.61(m，2H)，1.28(s，6H)，1.23(s，6H).
Example 3
The synthetic route is as follows:
step 1: synthesis of Compound WX003-1
Compound BB-9 (0.12 g, 464.70. Mu. Mol) was added to 1, 2-dichloroethane (8 mL), the hydrochloride of compound BB-10 (164.45 mg, 464.70. Mu. Mol) and potassium acetate (45.61 mg, 464.70. Mu. Mol) were sequentially added, and after three times of nitrogen substitution, the reaction was continued for 0.5 hour at 20℃and sodium triacetoxyborohydride (147.73 mg, 697.06. Mu. Mol) was added, and stirring was continued for 11.5 hours at 20 ℃. After the completion of the reaction, 1mol/L hydrochloric acid (5 mL) was added to the reaction mixture, stirred at 20℃for 0.5 hour, filtered, and the cake was collected to give Compound WX003-1.MS-ESI m/z:560.2[ M+H ]] + .
Step 2: synthesis of Compound WX003
Compound WX003-1 (0.13 g, 232.29. Mu. Mol) was added to N, N-dimethylformamide (3 mL), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (114.82 mg, 301.97. Mu. Mol) and N, N-diisopropylethylamine (90.06 mg, 696.86. Mu. Mol, 121.38. Mu. L) were added in this order, and after nitrogen substitution, the mixture was heated to 50℃for reaction for 1 hour. The temperature was lowered to 20℃and the hydrochloride salt of Compound BB-5 (73.23 mg, 232.29. Mu. Mol) was added thereto, and the reaction was continued at 20℃for 2 hours. After the completion of the reaction, the reaction mixture was poured into water (5 mL), extracted with ethyl acetate (5 mL. Times.3), the organic phase was washed with saturated brine (10 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was separated and purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX003.MS-ESI m/z:820.4[ M+H ] ] + . 1 H NMR(400MHz，CD 3 OD)δ：8.06-7.98(m，3H)，7.95-7.88(m，1H)，7.84(d，J＝8.4Hz，2H)，7.73(d，J＝8.8Hz，1H)，7.57-7.50(m，1H)，7.13(d，J＝2.0Hz，1H)，6.99(dd，J＝2.4Hz，8.8Hz，1H)，4.82(s，2H)，4.61(dd，J＝5.0Hz，11.0Hz，1H)，4.30(s，1H)，4.18(s，1H)，3.95-3.80(m，3H)，3.79-3.65(m，9H)，3.41(t，J＝7.0Hz，2H)，2.86-2.81(m，1H)，2.64-2.58(m，1H)，2.51-2.43(m，1H)，2.42-2.33(m，1H)，2.17(d，J=14.4Hz，2H)，2.04- 1.98(m，1H)，1.94-1.84(m，4H)，1.30(s，6H)，1.23(s，6H).
Example 4
The synthetic route is as follows:
step 1: synthesis of Compound WX004 hydrochloride
The compound BB-11 (0.2 g, 337.49. Mu. Mol) and BB-12 (82.76 mg, 337.49. Mu. Mol) were added sequentially to N, N-dimethylformamide (10 mL), followed by N, N-diisopropylethylamine (218.09 mg,1.69mmol, 293.93. Mu. L) and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (192.49 mg, 506.24. Mu. Mol), and the reaction mixture was stirred at 25℃for 3 hours. After completion of the reaction, ice water (50 mL), ethyl acetate (50 mL) and the organic phase was added, washed with saturated brine (50 mL. Times.2), dried over anhydrous sodium sulfate, filtered, the solvent was removed from the filtrate under reduced pressure, and the resulting residue was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.05% hydrochloric acid) to give hydrochloride of compound WX 004. MS-ESI m/z:820.3[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.15(s，1H)，10.72(s，1H)，10.39(s，1H)，8.40(d，J＝8.4Hz，1H)，8.28(d，J＝0.4Hz，1H)，8.21-8.15(m，1H)，8.07(d，J＝8.0Hz，1H)，7.76-7.69(m，2H)，7.45-7.36(m，2H)，7.24(t，J＝8.0Hz，1H)，4.65-4.54(m，3H)，3.64-3.50(m，4H)，3.21-3.06(m，2H)，2.87-2.74(m，1H)，2.71-2.60(m，1H)，2.46-2.37(m，1H)，2.34(d，J＝6.8Hz，2H)，2.27-2.18(m，1H)，2.13-2.01(m，1H)，1.90(d，J＝13.6Hz，2H)，1.74-1.60(m，2H)，1.52(s，6H).
Example 5
The synthetic route is as follows:
step 1: synthesis of Compound WX005
Compound BB-13 (120 mg, 212.56. Mu. Mol) and the hydrochloride salt of compound BB-12 (87.33 mg, 277.57. Mu. Mol) were added to N, N-dimethylformamide (3 mL), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (121.23 mg, 318.84. Mu. Mol) and triethylamine (64.53 mg, 637.67. Mu. Mol) were slowly added to the reaction, and the mixture was stirred at 25℃for 2 hours after being purged 3 times with nitrogen. The reaction solution was poured into 20mL of water and 30mL of ethyl acetate, and the organic phase was separated, washed with water (20 mL. Times.2), and the organic phase was collected and dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure. The crude product was purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.05% hydrochloric acid) to give compound WX005.MS-ESI m/z:792.1[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.15(s，1H)，10.38(br s，1H)，8.39(d，J＝8.4Hz，1H)，8.27(d，J＝1.6Hz，1H)，8.18(d，J＝6.0Hz，1H)，8.06(dd，J＝1.6，8.0Hz，1H)，7.82-7.72(m，2H)，7.51-7.41(m，1H)，7.37(d，J＝12.8Hz，1H)，7.25-7.15(m，1H)，4.92-4.68(m，1H)，4.63(dd，J＝5.0，11.8Hz，1H)，4.28(d，J＝13.2Hz，2H)，3.71-3.50(m，1H)，3.48-3.35(m，2H)，2.90-2.75(m，1H)，2.72-2.59(m，1H)，2.45-2.36(m，1H)，2.35-2.20(m，4H)，2.19-1.98(m，2H)，1.52(s，6H).
Example 6
The synthetic route is as follows:
step 1: synthesis of Compound WX006
Compound BB-4 (100 mg, 168.47. Mu. Mol) and the hydrochloride salt of compound BB-2 (54.80 mg, 185.32. Mu. Mol) were added to N, N-dimethylformamide (20 mL), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (128.11 mg, 336.94. Mu. Mol) and N, N-diisopropylethylamine (54.43 mg, 421.18. Mu. Mol, 73.36. Mu. L) were slowly added to the reaction, and the mixture was stirred at 25℃for 12 hours after 3 times of nitrogen substitution. The reaction solution was diluted with ethyl acetate (30 mL), the organic phase was washed with water (20 mL. Times.3), the organic phase was collected and dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying under reduced pressure was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.05% hydrochloric acid) to give compound WX006.MS-ESI m/z:835.2[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.20(s，1H)，8.39(d，J＝8.0Hz，1H)，8.28(s，1H)，8.22(s，1H)，8.06(d，J＝8.4Hz，1H)，7.82(d，J＝8.8Hz，1H)，7.52(d，J＝8.0Hz，1H)，7.43(t，J＝9.0Hz，1H)，7.38(dd，J＝1.8，11.8Hz，1H)，7.22(d，J＝8.4Hz，1H)，4.66(dd，J＝5.0，11.8Hz，1H)，4.64-4.58(m，2H)，4.20-3.91(m，2H)，3.90-3.62(m，10H)，3.05(s，3H)，2.97-2.83(m，1H)，2.81-2.70(m，1H)，2.47-2.27(m，1H)，2.25-2.13(m，1H)，1.52(s，6H).
Example 7
The synthetic route is as follows:
step 1: synthesis of hydrochloride salt of Compound WX007
The hydrochloride salt of Compound BB-20 (77.78 mg, 152.52. Mu. Mol) was dissolved in 1, 2-dichloroethane (2 mL), followed by addition of Compound BB-19 (60 mg, 152.52. Mu. Mol) and potassium acetate (25.15 mg, 256.23. Mu. Mol), displacement of nitrogen gas by the reaction system was three times, and after stirring at 25℃for 20 minutes, sodium borohydride acetate (48.49 mg, 228.78. Mu. Mol) was added and stirring at 25℃for 12 hours. After completion of the reaction, water (5 mL) and methylene chloride (10 mL) were added to the reaction mixture, the organic phase was separated, the aqueous phase was extracted with methylene chloride (20 mL. Times.3) and the combined organic phases were washed with saturated brine (20 mL. Times.2), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product which was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give hydrochloride of Compound WX 007. MS-ESI m/z:851.4[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.14(s，1H)，10.61(s，1H)，10.43(s，1H)，8.55(s，1H)，8.38(d，J＝8.0Hz，1H)，8.29(s，1H)，8.19(d，J＝8.0Hz， 1H)，8.09(t，J＝9.2Hz，2H)，7.87(t，J＝10.8Hz，2H)，7.24(d，J＝8.0Hz，2H)，7.14(d，J＝8.0Hz，2H)，5.02(dd，J＝3.8，11.0Hz，1H)，3.93(d，J＝11.6Hz，2H)，3.61(d，J＝9.6Hz，2H)，3.23-3.12(m，6H)，2.90-2.75(m，1H)，2.70-2.55(m，3H)，2.45-2.30(m，2H)，1.88-1.77(m，2H)，1.76-1.64(m，2H)，1.48(s，6H).
Example 8
The synthetic route is as follows:
step 1: synthesis of Compound WX008
Compound BB-4 (60 mg, 101.08. Mu. Mol) and the hydrochloride salt of compound BB-14 (34.17 mg, 121.30. Mu. Mol) were added to N, N-dimethylformamide (10 mL), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (76.87 mg, 202.16. Mu. Mol) and N, N-diisopropylethylamine (39.19 mg, 303.24. Mu. Mol, 52.82. Mu. L) were slowly added to the reaction, and the mixture was stirred at 25℃for 30 minutes after 3 times of nitrogen substitution. The reaction solution was diluted with ethyl acetate (30 mL), and the organic phase was washed with water (20 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure. The crude product obtained was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.05% hydrochloric acid) to give compound WX008.MS-ESI m/z:821.4[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.09(s，1H)，10.97(s，1H)，8.40(d，J＝8.4Hz，1H)，8.28(s，1H)，8.22(s，1H)，8.06(d，J＝8.0Hz，1H)，7.81(d，J＝8.4Hz，1H)，7.50(d，J＝8.4Hz，1H)，7.43(d，J＝8.8Hz，1H)，7.38(dd，J＝2.4，11.6Hz，1H)，7.22(d，J＝8.8Hz，1H)，4.63-4.52(m，3H)，4.18-3.60(m，12H)，2.85-2.72(m，1H)，2.69-2.57(m，1H)，2.48-2.32(m，1H)，2.26-2.16(m，1H)，1.52(s，6H).
Example 9
The synthetic route is as follows:
step 1: synthesis of Compound WX009
Compound BB-15 (0.08 g, 129.11. Mu. Mol) was placed in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (50.06 mg, 387.33. Mu. Mol, 67) was added46. Mu.L) and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (98.18 mg, 258.22. Mu. Mol) were added, followed by addition of the hydrochloride salt of Compound BB-14 (44.33 mg, 180.75. Mu. Mol), and the reaction solution was stirred at 20℃for 12 hours, followed by heating to 45℃and stirring for 6 hours. After the reaction, the reaction solution was directly concentrated to obtain a crude product, which was purified by preparative HPLC separation (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to obtain compound WX009.MS-ESI m/z:847.4[ M+H ] ] + . 1 H NMR(400MHz，CD 3 OD)δ：8.22(d，J＝1.2Hz，1H)，8.16(d，J＝2.8Hz，1H)，8.14(d，J＝3.6Hz，1H)，7.98(dd，J＝1.8，8.2Hz，1H)，7.75(d，J＝8.4Hz，1H)，7.34(dd，J＝1.6，8.8Hz，1H)，7.29(d，J＝2.4Hz，1H)，7.28-7.23(m，1H)，7.20-7.17(m，1H)，4.49(dd，J＝5.0，11.0Hz，1H)，4.27(s，2H)，4.14(s，2H)，3.48-3.35(m，8H)，2.82-2.77(m，2H)，2.60-2.50(m，1H)，2.40-2.30(m，1H)，1.57(s，6H)，1.02-0.88(m，4H).
Example 10
The synthetic route is as follows:
step 1: synthesis of Compound WX010
Compound BB-16 (100 mg, 164.58. Mu. Mol) and the hydrochloride of compound BB-14 (86.95 mg, 197.50. Mu. Mol, purity: 63.98%) were added to N, N-dimethylformamide (10 mL), followed by addition of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (125.15 mg, 329.16. Mu. Mol) and N, N-diisopropylethylamine (63.81 mg, 493.74. Mu. Mol, 86.00. Mu. L), and the reaction mixture was stirred under nitrogen at 25℃for 2 hours. After the completion of the reaction, ethyl acetate (30 mL) was added for dilution, and the organic phase was washed with water (20 mL. Times.3),the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the crude product obtained by spin-drying under reduced pressure was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.05% hydrochloric acid) to give compound WX010.MS-ESI m/z:835.2[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.08(s，1H)，10.81(s，1H)，8.40(d，J＝8.4Hz，1H)，8.27(d，J＝1.2Hz，1H)，8.22(s，1H)，8.06(dd，J＝1.6，8.0Hz，1H)，7.78(d，J＝8.8Hz，1H)，7.45-7.33(m，3H)，7.24-7.16(m，1H)，4.54(d，J＝4.8，11.6Hz，1H)，4.52-4.35(m，2H)，3.77-3.45(m，10H)，3.13-2.85(m，4H)，2.82-2.73(m，1H)，2.69-2.64(m，1H)，2.40-2.30(m，1H)，2.25-2.15(m，1H)，1.52(s，6H).
Example 11
The synthetic route is as follows:
step 1: synthesis of Compound WX011
Compound BB-4 (107.36 mg, 180.86. Mu. Mol) was dissolved in N, N-dimethylformamide (2 mL), followed by addition of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (137.53 mg, 361.71. Mu. Mol) and N, N-diisopropylethylamine (116.87 mg, 904.29. Mu. Mol, 157.51. Mu. L), stirring at 25℃for 30 minutes, addition of hydrochloride (60 mg, 180.86. Mu. Mol) of compound BB-17, and stirring at 25℃for 3 hours. To the reaction solution were added water (5 mL) and ethyl acetate (10 mL), the organic phase was separated, the aqueous phase was extracted with ethyl acetate (20 mL. Times.3), the combined organic phases were washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX011.MS-ESI m/z:871.3 [M+H] + . 1 H NMR(400MHz，CD 3 OD)δ：8.42(d，J＝2.0Hz，1H)，8.22-8.13(m，3H)，8.03(d，J＝9.2Hz，1H)，7.98(dd，J＝1.8，8.2Hz，1H)，7.89(dd，J＝2.0，8.8Hz，1H)，7.78(d，J＝9.2Hz，1H)，7.36(t，J＝9.2Hz，1H)，7.31(dd，J＝2.0，11.2Hz，1H)，7.22(d，J＝8.8Hz，1H)，4.95(dd，J＝5.0，10.2Hz，1H)，4.54(t，J＝4.6Hz，2H)，3.71-3.62(m，4H)，3.60-3.50(m，3H)，3.28-3.05(m，3H)，2.92-2.75(m，2H)，2.74-2.62(m，1H)，2.55-2.47(m，1H)，2.03(s，2H)，1.57(s，6H).
Example 12
The synthetic route is as follows:
step 1: synthesis of Compound WX012
Compound BB-4-5 hydrochloride (185.99 mg, 325.14. Mu. Mol) was dissolved in 1, 2-dichloroethane (5 mL), and Compound BB-18 (110 mg, 325.14. Mu. Mol) and sodium acetate (53.35 mg, 650.28. Mu. Mol) were added, followed by stirring for 30 minutes, and sodium triacetoxyborohydride (137.82 mg, 650.28. Mu. Mol) was added to react at 25℃for 12 hours. After completion of the reaction, water (5 mL) and methylene chloride (10 mL) were added, the organic phase was separated, the aqueous phase was extracted with methylene chloride (20 mL. Times.3), the organic phases were combined, washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX012.MS-ESI m/z:858.3[ M+H ]] + . 1 H NMR(400MHz，CD 3 OD)δ：8.20-8.14(m，3H)，8.05(d，J＝9.6Hz，1H)，7.98(dd，J＝1.8Hz，8.2Hz，1H)，7.78(d，J＝9.2Hz，1H)，7.65(d，J＝2.8Hz，1H)，7.49(dd，J＝2.6，9.0Hz，1H)，7.37(t，J＝8.8Hz，1H)，7.31(dd，J＝2.4，11.6Hz，1H)，7.25-7.21(m，1H)，4.93(dd，J＝5.0，10.2Hz，2H)，4.62-4.55 (m，4H)，3.82-3.65(m，11H)，2.90-2.75(m，2H)，2.73-2.60(m，1H)，2.55-2.45(m，1H)，1.56(s，6H).
Example 13
The synthetic route is as follows:
step 1: synthesis of Compound WX013-1
Compound BB-20 (0.2 g, 422.37. Mu. Mol) and ethyl 2-chloroethoxyacetate (140.74 mg, 844.75. Mu. Mol) were dissolved in N, N-dimethylformamide (3 mL), and potassium carbonate (233.50 mg,1.69 mmol) and potassium iodide (14.02 mg, 84.47. Mu. Mol) were added thereto, and the reaction was stirred under nitrogen for 15 hours at 90 ℃. Water (10 mL) was added to the reaction mixture, the mixture was extracted with ethyl acetate (3X 10 mL), and the organic phases were combined, washed with saturated brine (3X 30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/1-0/1, volume ratio) to give compound WX013-1.MS-ESI m/z:604.3[ M+H ] ] + . 1 H NMR(400MHz，CDCl 3 )δ：8.01-7.95(m，2H)，7.85(dd，J＝1.6，8.0Hz，1H)，7.17(d，J＝8.4Hz，2H)，7.01(d，J＝8.8Hz，2H)，4.25(q，J＝7.2Hz，2H)，4.14(s，2H)，3.95-3.60(m，4H)，3.50-3.20(m，4H)，2.85-2.60(m，4H)，1.58(s，6H)，1.31(t，J＝7.2Hz，3H).
Step 2: synthesis of Compound WX013-2
Compound WX013-1 (0.135 g, 223.64. Mu. Mol) was dissolved in a mixed solvent of ethanol (0.5 mL), tetrahydrofuran (2 mL) and water (0.5 mL)Lithium hydroxide monohydrate (28.15 mg, 670.91. Mu. Mol) was then added, and the reaction mixture was stirred under nitrogen at 20℃for 14 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure, and 1M aqueous hydrochloric acid (10 mL) was added to the obtained residue, which was extracted with a mixed solvent of methylene chloride/ethanol (10/1, volume ratio) (5X 10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give Compound WX013-2. 1 H NMR(400MHz，CDCl 3 )δ：12.81(s，1H)，8.01-7.95(m，2H)，7.84(dd，J＝1.8Hz，8.2Hz，1H)，7.30-7.28(m，2H)，7.21(d，J＝8.4Hz，2H)，4.20(s，2H)，4.14-4.08(m，2H)，4.00-3.86(m，4H)，3.83-3.77(m，2H)，3.43-3.38(m， 2H)，3.37-3.28(m，2H)，1.59(s，6H).
Step 3: synthesis of Compound WX013
The hydrochloride salt of compound BB-17 (28.82 mg, 86.87. Mu. Mol) and compound WX013-2 (0.05 g, 86.87. Mu. Mol) were dissolved in N, N-dimethylformamide (2 mL), followed by the addition of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (49.54 mg, 130.30. Mu. Mol) and N, N-diisopropylethylamine (33.68 mg, 260.60. Mu. Mol, 45.39. Mu.L) and stirring under nitrogen for 14 hours at 20 ℃. After the reaction was completed, the filtrate was filtered and purified by preparative HPLC separation (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX013.MS-ESim/z:853.3[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.13(s，1H)，10.30(s，1H)，8.54(d，J＝1.6Hz，1H)，8.39(d，J＝8.4Hz，1H)，8.29(d，J＝1.6Hz，1H)，8.23(d，J＝8.8Hz，1H)，8.13(d，J＝8.8Hz，1H)，8.08(dd，J＝1.6，8.4Hz，1H)，7.91(d，J＝9.2Hz，2H)，7.25(d，J＝8.8Hz，2H)，7.17(d，J＝9.2Hz，2H)，5.03(dd，J＝4.8Hz，11.6Hz，1H)，4.31(s，2H)，4.03-3.95(m，4H)，3.83-3.73(m，2H)，3.57-3.47(m，2H)，3.31-3.18(m，4H)，2.88-2.77(m，1H)，2.69-2.59(m，3H)，1.49(s，6H).
Example 14
The synthetic route is as follows:
step 1: synthesis of Compound WX014-2
Compound WX014-1 (10 g,78.67 mmol) was dissolved in methylene chloride (100 mL) and acetone (50 mL) at room temperature, and trimethylcyanosilane (11.71 g,118.00mmol,14.76 mL) and trimethylsilyl triflate (1.75 g,7.87mmol,1.42 mL) were added thereto at 0℃and the reaction mixture was stirred under nitrogen for 2 hours at 20 ℃. After the reaction was completed, the filtrate was concentrated under reduced pressure to remove the solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-4/1, volume ratio) to give compound WX014-2.
Step 2: synthesis of Compound WX014-3
Compound WX014-2 (9.83 g,50.62 mmol) was dissolved in N, N-dimethylacetamide (90 mL) at room temperature, 4-cyano-3-trifluoromethylphenyl isothiocyanate (11.55 g,50.62 mmol) was added thereto, the reaction mixture was stirred at room temperature under nitrogen for 3 hours, then hydrochloric acid (2M, 98.20 mL) and methanol (90 mL) were added respectively, and the reaction mixture was heated to 70℃and stirred for 2 hours. After the completion of the reaction, the mixture was cooled to room temperature, water (150 mL) was added thereto, and the mixture was extracted with ethyl acetate (150 mL. Times.3). The organic phases were combined, washed successively with saturated brine (150 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-4/1, volume ratio) to give compound WX014-3. 1 H NMR(400MHz，CDCl 3 )δ：7.98(d，J＝8.4Hz，1H)，7.96(d，J＝1.6Hz，1H)，7.84(dd，J＝2.0，8.4Hz，1H)，7.16(t，J＝8.8Hz，1H)，7.06(dd，J＝1.2，10.8Hz，1H)，7.01-6.95(m，1H)，1.59(s，6H).
Step 3: synthesis of Compound WX014-4
Compound WX014-3 (1 g,2.22mmol, purity: 93.85%) was dissolved in methylene chloride (10 mL) at room temperature, and trifluoromethanesulfonic anhydride (938.11 mg,3.32mmol, 548.60. Mu.L) and triethylamine (672.91 mg,6.65mmol, 925.59. Mu.L) were added thereto, and the reaction mixture was stirred under nitrogen at room temperature for 2 hours. After completion of the reaction, water (15 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (15 mL. Times.3). The organic phases were combined, washed successively with saturated brine (15 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-9/1, volume ratio) to give compound WX014-4.
Step 4: synthesis of Compound WX014-5
Compound WX014-4 (756 mg,1.36 mmol) and piperazine-1-carboxylic acid tert-butyl ester hydrochloride (1.52 g,6.81 mmol) were dissolved in toluene (10 mL) and water (1 mL) under nitrogen, potassium phosphate (866.74 mg,4.08 mmol), tris (dibenzylideneacetone) dipalladium (87.25 mg, 95.27. Mu. Mol) and 2-di-tert-butylphosphine-2 ',4',6' -triisopropylbiphenyl (80.92 mg, 190.55. Mu. Mol) were added respectively, and the reaction mixture was heated to 100℃and stirred for 12 hours. After completion of the reaction, the mixture was cooled to room temperature, water (10 mL) was added thereto, and the mixture was extracted with ethyl acetate (10 mL. Times.3). The organic phases were combined, washed successively with saturated brine (10 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue obtained was separated by column chromatography (eluent: petroleum ether/ethyl acetate=1/0-5/1, volume ratio) to give compound WX014-5.
Step 5: synthesis of Compound WX014-6
Compound WX014-5 (333 mg, 562.86. Mu. Mol) was dissolved in ethyl hydrochloride (4M, 2.83 mL) at room temperature, and the reaction mixture was stirred under nitrogen at 20℃for 12 hours. After the reaction was completed, the filtrate was concentrated under reduced pressure to remove the solvent. Compound WX014-6 was obtained. 1 H NMR(400MHz，CDCl 3 )δ：10.0(br s，1H)，7.99(d，J＝8.0Hz，1H)，7.95(s，1H)，7.83(d，J＝8.0Hz，1H)，7.18-6.97(m，3H)，3.75-3.25(m，8H)，1.60(s，6H).
Step 6: synthesis of Compound WX014
Compound WX014-6 hydrochloride (80.52 mg, 152.52. Mu. Mol) was dissolved in 1, 2-dichloroethane (5 mL), and Compound BB-24 (50 mg, 127.10. Mu. Mol) and potassium acetate (18.71 mg, 190.65. Mu. Mol) were added, followed by stirring at 20℃for 1 hour, and sodium triacetoxyborohydride (61.96 mg, 292.33. Mu. Mol) was added to react at 20℃for 11 hours under nitrogen atmosphere. After completion of the reaction, the reaction mixture was poured into dilute hydrochloric acid (10 mL), 5mL of water was added, extraction was performed with ethyl acetate (15 mL. Times.3), and the organic phase was washed with saturated brine (10 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX014.MS-ESI m/z:869.3[ M+H ]] + . 1 H NMR(400MHz，CD 3 OD)δ：8.26(d，J＝9.6Hz，1H)，8.19-8.14(m，3H)，7.98(dd，J＝1.6，8.4Hz，1H)，7.84(d，J＝9.2Hz，1H)，7.74(t，J＝7.8Hz，1H)，7.68(d，J＝7.2Hz，1H)，7.27-7.16(m，3H)，5.00(dd，J＝5.0Hz，10.2Hz，1H)，3.72(d，J＝11.6Hz，4H)，3.42-3.33(m，4H)，3.22(t，J＝11.6Hz，2H)，2.93-2.78(m，2H)，2.75-2.65(m，3H)，2.56-2.47(m，1H)，2.03-1.86(m，4H)，1.56(s，6H).
Example 15
The synthetic route is as follows:
step 1: synthesis of Compound WX015
The hydrochloride salt of Compound BB-23 (75.24 mg, 157.60. Mu. Mol) was dissolved in 1, 2-dichloroethane (2 mL), and Compound BB-19 (62 mg, 157.60. Mu. Mol), sodium triacetoxyborohydride (66.81 mg, 315.21. Mu. Mol) and acetic acid (0.1 mL) were added under nitrogen atmosphere 2 Potassium acetate (30.94 mg, 315.21. Mu. Mol) was added thereto with stirring at 0℃for 20 minutes, and stirring was continued at 20℃for 12 hours. After completion of the reaction, water (5 mL) and methylene chloride (10 mL) were added, the organic phase was separated, the aqueous phase was extracted with methylene chloride (20 mL. Times.3), the combined organic phases were washed with saturated brine (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue which was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) and purified to give compound WX015.MS-ESI m/z:818.4[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.13(s，1H)，10.80(s，1H)，10.40(s，1H)，8.68(d，J＝8.4Hz，1H)，8.53(d，J＝1.2Hz，1H)，8.19(d，J＝8.4Hz，1H)，8.09(d，J＝8.8Hz，1H)，7.97-7.80(m，4H)，7.48(d，J＝9.6Hz，1H)，7.38(d，J＝2.4Hz，1H)，7.13(dd，J＝2.4Hz，8.8Hz，1H)，5.03(dd，J＝4.8Hz，11.2Hz，1H)，4.59(d，J＝14.4Hz，2H)，4.54-4.47(m，1H)，3.92-3.83(m，1H)，3.61(d，J＝10.8Hz，2H)，3.56-3.48(m，2H)，3.23-3.05(m，4H)，2.88-2.77(m，1H)，2.70-2.58(m，2H)，2.45-2.32(m，2H)，2.15-2.05(m，2H)，1.95-1.77(m，4H)，1.75-1.58(m，4H)，1.57-1.45(m，2H).
Example 16
The synthetic route is as follows:
step 1: synthesis of Compound WX016
Compound BB-23 (50 mg, 127.10. Mu. Mol) was added to 1, 2-dichloroethane (10 mL) at room temperature, the hydrochloride of compound BB-24 (72.81 mg, 152.52. Mu. Mol) and potassium acetate (18.71 mg, 190.65. Mu. Mol) were slowly added, and after stirring for 1 hour, sodium triacetoxyborohydride (61.96 mg, 292.33. Mu. Mol) was added to react under nitrogen atmosphereOvernight. The reaction solution was poured into dilute hydrochloric acid (10 mL), water (5 mL) was added, extraction was performed with ethyl acetate (15 mL. Times.3), and the organic phase was collected, washed with saturated brine (10 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give compound WX016.MS-ESI m/z:818.4[ M+H ] ] + . 1 H NMR(400MHz，CD 3 OD)δ：8.25(d，J＝9.6Hz，1H)，8.18-8.11(m，2H)，7.83(d，J＝9.6Hz，1H)，7.73(t，J＝8.0Hz，1H)，7.71-7.65(m，2H)，7.62(d，J＝9.6Hz，1H)，7.20(d，J＝2.4Hz，1H)，7.04(dd，J＝2.4，8.8Hz，1H)，5.05-4.95(m，5H)，4.75-4.65(m，2H)，4.55-4.46(m，1H)，4.04-3.95(m，1H)，3.84-3.73(m，2H)，3.61-3.52(m，2H)，2.90-2.79(m，2H)，2.76-2.66(m，3H)，2.57-2.47(m，1H)，2.25-2.17(m，2H)，2.13-2.06(m，2H)，2.02-1.95(m，2H)，1.94-1.84(m，2H)，1.71-1.58(m，4H).
Example 17
The synthetic route is as follows:
step 1: synthesis of Compound WX017
Compound BB-4 (85.88 mg, 144.69. Mu. Mol) was added to N, N-dimethylformamide (8 mL), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (68.77 mg, 180.86. Mu. Mol) and N, N-diisopropylethylamine (77.92 mg, 602.86. Mu. Mol, 105.01. Mu. L) were added in this order, and after stirring for 0.5 hour, the hydrochloride (40 mg, 120.57. Mu. Mol) of compound BB-22 was added and reacted under nitrogen atmosphere for 2.5 hours. The reaction mixture was poured into water (15 mL), extracted with ethyl acetate (10 mL. Times.3), and the organic phase was collected and saturated withAnd brine (15 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to give crude product. The crude product was purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% HCl) to give compound WX017.MS-ESI m/z:871.3[ M+H ]] + . 1 H NMR(400MHz，CD 3 OD)δ：8.27(d，J＝9.6Hz，1H)，8.22-8.13(m，3H)，7.98(d，J＝8.4Hz，1H)，7.84(d，J＝9.2Hz，1H)，7.77-7.73(m，2H)，7.36(t，J＝9.6Hz，1H)，7.31(dd，J＝1.6，11.2Hz，1H)，7.23(d，J＝8.4Hz，1H)，4.99(dd，J＝5.2，10.4Hz，1H)，4.58(t，J＝4.0Hz，2H)，4.18-4.12(m，2H)，3.80-3.73(m，4H)，3.63-3.55(m，3H)，2.92-2.78(m，2H)，2.77-2.60(m，2H)，2.56-2.47(m，1H)，1.57(s，6H)，1.45-1.35(m，1H)，1.34-1.23(m，1H).
Example 18
The synthetic route is as follows:
the hydrochloride salt of compound BB-25 (33 mg, 82.32. Mu. Mol) was dissolved in N, N-dimethylformamide (1 mL) at room temperature under nitrogen, and compound BB-26 (38.52 mg, 82.32. Mu. Mol), potassium acetate (24.24 mg, 246.97. Mu. Mol) and glacial acetic acid (0.1 mL) were added, and the reaction mixture was stirred at room temperature for 0.5 hour, and sodium triacetoxyborohydride (52.34 mg, 246.97. Mu. Mol) was further added, and the reaction mixture was stirred for 1 hour. After completion of the reaction, the solvent was removed by concentration under reduced pressure, and N, N-dimethylformamide (1 mL) and hydrochloric acid (1N, 1 mL) were added to the residue, followed by filtration. The filtrate was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% HCl) to give the hydrochloride salt of compound WX 018. MS-ESI m/z:816.3[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.15(s，1H)，10.06(br s，1H)，8.60(br d，J＝8.4Hz，1H)，8.47(br d，J＝9.6Hz，1H)，8.07-7.92(m，2H)，7.85(br d，J＝8.0Hz，2H)，7.71(br t，J＝6.8Hz，1H)，7.47-7.32(m，3H)，7.13(br d，J＝8.0Hz，1H)，5.11-5.01(m，1H)，4.66-4.41(m，4H)，3.94-3.82(m，4H)，3.74-3.71(m，2H)，3.14-3.03(m，4H)，2.92-2.78(m，3H)，2.75-2.60(m，4H)，2.16-2.06(m，2H)，2.02-1.83(m，4H)，1.73-1.43(m，4H)，1.37-1.24(m，2H).
Example 19
The synthetic route is as follows:
compound BB-27 (99 mg, 169.55. Mu. Mol) was dissolved in N, N-dimethylformamide (3 mL) at room temperature under nitrogen, and the hydrochloride (67.97 mg, 169.55. Mu. Mol) of compound BB-25, potassium acetate (49.92 mg, 508.66. Mu. Mol) and glacial acetic acid (0.1 mL) were added to stir the reaction mixture at room temperature for 0.5 hours. Sodium triacetoxyborohydride (107.81 mg, 508.66. Mu. Mol) was further added thereto, and the reaction was stirred for 1 hour. After completion of the reaction, the solvent was removed by concentration under reduced pressure, and N, N-dimethylformamide (1 mL) and hydrochloric acid (1N, 1 mL) were added to the residue, followed by filtration. The filtrate was separated by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% hydrochloric acid) to give hydrochloride salt of compound WX 019. MS-ESI m/z:814.4[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.14(s，1H)，10.95(s，1H)，8.57(d，J＝8.4Hz，1H)，8.47(d，J＝9.6Hz，1H)，8.01(d，J＝7.6Hz，1H)，7.95(d，J＝9.2Hz，1H)，7.90-7.83(m，2H)，7.70(t，J＝8.0Hz，1H)，7.38(d，J＝2.4Hz，1H)，7.35(d，J＝7.6Hz，1H)，7.13(dd，J＝2.4，8.8Hz，1H)，7.06(d，J＝9.6Hz，1H)，5.06(dd，J＝4.6，11.4Hz，1H)，4.59-4.48(m，1H)，4.00-3.90(m，2H)，3.90-3.79(m，1H)，3.76-3.66(m，2H)，3.64-3.57(m，2H)，3.36-3.20(m，6H)，2.93-2.79(m，1H)，2.70-2.56(m，3H)，2.41-2.30(m，1H)，2.16-2.01(m，5H)，1.95-1.85(m，2H)，1.71-1.57(m，2H)，1.57-1.44(m，2H)，1.14(s，1H).
Example 20
The synthetic route is as follows:
BB-28 (0.07 g, 149.27. Mu. Mol) was dissolved in N, N-dimethylformamide (3 mL) at room temperature under nitrogen, followed by addition of the hydrochloride salt of intermediate BB-25 (59.84 mg, 149.27. Mu. Mol), potassium acetate (43.95 mg, 447.82. Mu. Mol) and acetic acid (105.00 mg,1.75mmol,0.1 mL), and the reaction mixture was stirred at room temperature for 0.5 hours, and sodium triacetoxyborohydride (94.91 mg, 447.82. Mu. Mol) was added, and the reaction mixture was stirred for further 12 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure. N, N-dimethylformamide (1 mL) and hydrochloric acid (1N, 1 mL) were added to the residue, and the mixture was filtered, and the filtrate was separated and purified by preparative HPLC (mobile phase: acetonitrile/water; acidic system: 0.04% HCl) to give hydrochloride of compound WX 020. MS-ESI m/z:817.3[ M+H ] ] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.14(s，1H)，10.31(br s，1H)，8.73(s，1H)，8.69(d，J＝8.0Hz，1H)，8.48(d，J＝9.2Hz，1H)，8.05-7.99(m，1H)，7.96(d，J＝9.2Hz，1H)，7.86(d，J＝8.8Hz，1H)，7.71(t，J＝8.0Hz，1H)，7.39(d，J＝2.4Hz，1H)，7.36(d，J＝7.6Hz，1H)，7.14(dd，J＝2.4，8.8Hz，1H)，5.06(br dd，J＝4.6，11.4Hz，1H)，4.79(br s，2H)，4.60-4.46(m，1H)，3.95-3.81(m，1H)，3.77-3.65(m，2H)，3.55-3.44(m，4H)，3.23-3.13(m，4H)，2.93-2.78(m，1H)，2.70-2.52(m，4H)，2.44-2.34(m，2H)，2.17-2.07(m，2H)，2.06-1.97(m，2H)，1.94-1.83(m，2H)，1.75-1.59(m，2H)，1.58-1.47(m，2H)，1.39-1.25(m，2H).
Example 21
The synthetic route is as follows:
compound BB-29 (119.75 mg, 255.90. Mu. Mol) and the hydrochloride of BB-25 (102.58 mg, 255.90. Mu. Mol) were added to methylene chloride (5 mL), and sodium triacetylborohydride (81.35 mg, 383.85. Mu. Mol) and sodium acetate (41.98 mg, 511.80. Mu. Mol) were slowly added to the reaction, and the reaction solution was stirred at 25℃for 2 hours after being replaced with nitrogen gas 3 times. The reaction mixture was slowly added to water (50 mL), extracted with dichloromethane (50 mL. Times.2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by plate chromatography (developer: ethyl acetate) to give compound WX021.MS-ESI m/z:816.6[ M+H ]] + . 1 H NMR(400MHz，DMSO_d 6 )δ：11.14(s，1H)，8.59(d，J＝0.8Hz，1H)，8.44(d，J＝9.6Hz，1H)，8.25(s，1H)，8.07(d，J＝8.4Hz，1H)，7.96-7.88(m，2H)，7.85(d，J＝8.8Hz，1H)，7.65(t，J＝7.8Hz，1H)，7.37(d，J＝2.4Hz，1H)，7.30(d，J＝7.6Hz，1H)，7.12(dd，J＝2.4，8.8Hz，1H)，5.03(br dd，J＝5.0，11.4Hz，1H)，4.65-4.35(m，3H)，3.91-3.73(m，1H)，3.16-2.94(m，6H)，2.90-2.76(m，2H)，2.75-2.52(m，5H)，2.42-2.21(m，3H)，2.15-2.02(m，2H)，1.98-1.79(m，5H)，1.66-1.43(m，4H)，1.20-1.10(m，2H).
Biological testing
Test example 1: evaluation of anti-proliferation effects in human prostate cancer LNCaP cells for experimental purposes
The assay detects the inhibition of cell proliferation by a test compound in human prostate cancer LNCaP cells.
Experimental materials:
1. cell lines and methods of culture (see table 1):
TABLE 1
| Cell lines | Tumor type | Growth characteristics | Culture method |
| LNCaP | Human prostate cancer | Wall-attaching | Phenol red-free 1640+10% FBS |
2. Culture medium and reagents (see table 2):
TABLE 2
| Culture medium and reagent | Manufacturer (S) | Goods number |
| Phenol red-free RPMI 1640 | GIBCO | 11835030 |
| Dulbecco′s PBS | CORNING | 21-031-CVC |
| FBS | ExCell Bio | FSP500 |
| Penicillin-Streptomycin Solution | HyClone | SV30010 |
| 0.25％Trypsin | GIBCO | 25200072 |
3. Perforated plate
Greiner 96-well plate, flat bottom blackboard (with cover and transparent bottom), #655090.
4. Reagent and instrument for cell activity experiment
(1) Promega CellTiter-Glo luminescence method cell activity assay kit (Promega-G7573).
(2)2104 Plate reader, perkinElmer.
Experimental protocol
1. Cell culture
Tumor cell lines were cultured at 37℃under the above conditions with 5% CO 2 Is cultured in an incubator of (a). Cells in the logarithmic growth phase were taken for plating at regular passages.
2. Cell plating
(1) Staining cells with trypan blue and counting living cells;
(2) Adjusting the cell concentration to the appropriate concentration (see table 3);
TABLE 3 Table 3
| Cell line | Density(per 96-well) |
| LNCaP | 4500 |
(3) Adding 90 mu L of cell suspension into each hole of the culture plate according to the table, and adding cell-free culture solution into blank control air;
(4) The culture plate is incubated at 37 ℃ with 5% CO 2 And 100% relative humidity overnight.
3. Preparation of compound storage plate
Preparation of a mother liquor storage plate at 400 times the initial concentration of the compound: compounds were diluted with DMSO from highest concentration gradient to lowest concentration. Is prepared at any time.
Preparation of working solution with 10 times of initial concentration of compound and treatment of cells with compound
(1) 78 μl of cell culture broth was added to a 96-well plate with a V-shaped bottom, and 2 μl of the compound was pipetted from a mother liquor storage plate with a 400-fold concentration of the initial concentration of the compound and added to the cell culture broth of the 96-well plate. 2. Mu.L DMSO was added to the vehicle control and the blank. After adding the compound or DMSO, the mixture is blown and evenly mixed by a gun.
(2) Adding the medicine: mu.L of working solution 10 times the initial concentration of the compound was added to the cell culture plate. To the vehicle control and the blank control, 10. Mu.L of DMSO-cell culture medium mixture was added.
(3) The 96-well cell plate was returned to the incubator for 6 days.
Cell Activity detection by CellTiter-Glo luminescence method
The following steps were performed according to the instructions of the Promega CellTiter-Glo luminescence cell activity assay kit (Promega-G7573).
(1) Thawing CellTiter-Glo buffer solution and standing to room temperature;
(2) Placing CellTiter-Glo substrate to room temperature;
(3) Adding 100mL of CellTiter-Glo buffer solution to a bottle of CellTiter-Glo substrate to dissolve the substrate, thereby preparing CellTiter-Glo working solution;
(4) Slowly vortex oscillating to make the materials fully dissolved;
(5) Taking out the cell culture plate, standing for 30 min to allow the cell culture plate to equilibrate to room temperature;
(6) Add 50. Mu.L of CellTiter-Glo working fluid per well (equal to half the volume of cell culture fluid per well). Wrapping the cell plate with aluminum foil paper to avoid light;
(7) Shaking the culture plate on an orbital shaker for 2 minutes to induce cell lysis;
(8) The plates were left at room temperature for 10 minutes to stabilize the luminescence signal;
(9) The luminescence signal is detected at 2104 EnVision reader.
5. Data analysis
The Inhibition Rate (IR) of the test compound was calculated using the following formula: IR (%) = (RLU vehicle control-RLU compound)/(RLU vehicle control-RLU blank) ×100% inhibition rate of compounds at different concentrations was calculated in Excel, and then GraphPad Prism software was used for inhibition graph and relevant parameters including minimum inhibition were calculatedRate of production, maximum inhibition rate and IC 50 。
Experimental results:
the test results are shown in Table 4.
TABLE 4 inhibition of cell proliferation of compounds of the invention in LNCaP cell lines
| Compounds of formula (I) | LNCaP IC 50 (nM) |
| WX008 | 117 |
| WX009 | 125 |
| WX020 hydrochloride | 89 |
| WX021 | 130 |
Conclusion: the compounds of the present invention exhibit excellent inhibition of cell proliferation in human prostate cancer cells LNCaP.
Test example 2: evaluation of antiproliferative effects in human prostate cancer VCaP cells
Purpose of experiment
The present assay detects the inhibition of cell proliferation by a test compound in human prostate cancer VCaP cells.
Experimental materials:
1. the cell lines and the culture methods are shown in Table 5.
TABLE 5
| Cell lines | Tumor type | Growth characteristics | Culture conditions |
| VCaP | Human prostate cancer | Wall-attaching | DMEM+10％FBS |
2. The culture medium and the reagents are shown in Table 6.
TABLE 6
| Culture medium and reagent | Manufacturer (S) | Goods number |
| DMEM | GIBCO | 11995-065 |
| Dulbecco′s PBS | Thermo | SH30028.02B |
| FBS | ExCell Bio | FSP500 |
| Penicillin-Streptomycin Solution | HyClone | SV30010 |
| 0.25％Trypsin | GIBCO | 25200072 |
3. Perforated plate
Greiner CELLSTAR 96-well plate, flat bottom blackboard (with cover and transparent bottom), #655090.
4. Reagent and instrument for cell activity experiment
(1) Promega CellTiter-Glo luminescence method cell activity assay kit (Promega-G7573).
(2)2104 EnVision Plate reader, perkinElmer.
Experimental protocol
1. Cell culture
Tumor cell lines were cultured at 37℃under the above conditions with 5% CO 2 Is cultured in an incubator of (a). Cells in the logarithmic growth phase were taken for plating at regular passages.
2. Cell plating
(1) Staining cells with trypan blue and counting living cells;
(2) Adjusting the cell concentration to a suitable concentration;
(3) Adding 90 mu L of cell suspension into each hole of the culture plate, and adding cell-free culture solution into blank control air;
(4) The culture plate is incubated at 37 ℃ with 5% CO 2 And 100% relative humidity overnight.
3. Preparation of compound storage plate
Preparation of a mother liquor storage plate at 400 times the initial concentration of the compound: compounds were diluted with DMSO from highest concentration gradient to lowest concentration. Is prepared at any time.
Preparation of working solution with 10 times of initial concentration of compound and treatment of cells with compound
(1) 78 μl of cell culture broth was added to a 96-well plate with a V-shaped bottom, and 2 μl of the compound was pipetted from a mother liquor storage plate with a 400-fold concentration of the initial concentration of the compound and added to the cell culture broth of the 96-well plate. 2. Mu.L DMSO was added to the vehicle control and the blank. After adding the compound or DMSO, the mixture is blown and evenly mixed by a gun.
(2) Adding the medicine: mu.L of working solution 10 times the initial concentration of the compound was added to the cell culture plate. To the vehicle control and the blank control, 10. Mu.L of DMSO-cell culture medium mixture was added.
(3) The 96-well cell plate was returned to the incubator for 6 days.
Cell Activity detection by CellTiter-Glo luminescence method
The following steps were performed according to the instructions of the Promega CellTiter-Glo luminescence cell activity assay kit (Promega-G7573).
(1) Thawing CellTiter-Glo buffer solution and standing to room temperature;
(2) Placing CellTiter-Glo substrate to room temperature;
(3) Adding 100mL of CellTiter-Glo buffer solution to a bottle of CellTiter-Glo substrate to dissolve the substrate, thereby preparing CellTiter-Glo working solution;
(4) Slowly vortex oscillating to make the materials fully dissolved;
(5) Taking out the cell culture plate, standing for 30 min to allow the cell culture plate to equilibrate to room temperature;
(6) Add 50. Mu.L of CellTiter-Glo working fluid per well (equal to half the volume of cell culture fluid per well). Wrapping the cell plate with aluminum foil paper to avoid light;
(7) Shaking the culture plate on an orbital shaker for 2 minutes to induce cell lysis;
(8) The plates were left at room temperature for 10 minutes to stabilize the luminescence signal;
(9) The luminescence signal is detected at 2104 EnVision reader.
5. Data analysis
The Inhibition Rate (IR) of the test compound was calculated using the following formula: IR (%) = (RLU vehicle control-RLU compound)/(RLU vehicle control-RLU blank) ×100% inhibition rate of compounds at different concentrations was calculated in Excel, and then GraphPad Prism software was used as inhibition graph and relevant parameters including minimum inhibition rate, maximum inhibition rate and IC were calculated 50 。
Experimental results:
TABLE 7 inhibition of cell proliferation of the compounds of the invention in VCaP cell lines
| Compounds of formula (I) | VCaP IC 50 (nM) |
| WX008 | 81 |
Conclusion: the compounds of the present invention exhibit excellent inhibition of cell proliferation in human prostate cancer cells VCaP.
Test example 3: in vivo pharmacodynamics study of human prostate cancer LNCaP cell subcutaneous xenograft tumor CB-17 SCID model
Cell culture
In vitro monolayer culture of human prostate cancer LNcap cells (ECACC-89110211) under the conditions of adding 10% fetal bovine serum, 100U/mL penicillin and 100 μg/mL streptomycin in RPMI-1640 medium, and 37 ℃ 5% CO 2 Incubator culture. Passaging was performed twice a week with conventional digestion treatments with pancreatin-EDTA. When the saturation of the cells is 80% -90% and the number reaches the requirement, the cells are collected, counted and inoculated.
Experimental animal
CB-17 SCID mice, males 6-8 weeks old, weighing 18-22 grams.
Experimental protocol
0.2mL (1X 10) 7 (v) LNcap cells (matrigel added, volume ratio 1:1) were inoculated subcutaneously on the right back of each mouse with an average tumor volume of up to about 80mm 3 The mice were then castrated at 166mm 3 The administration of the packets was started at that time.
Table 8 experimental animal grouping and dosing regimen
Note that:
n: number of mice per group
2. Dosing volume: based on the weight of the mice, 10. Mu.L/g. If body weight decreases by more than 15%, the dosing regimen should be adjusted accordingly.
BID time interval of 8hr
Tumor measurement and experimental index
Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b 2 A and b represent the major and minor diameters of the tumor, respectively.
The tumor-inhibiting effect of the compound was evaluated by TGI (%). TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [ (1- (mean tumor volume at the end of the treatment group administration-mean tumor volume at the beginning of the treatment group administration))/(mean tumor volume at the end of the treatment with solvent control group-mean tumor volume at the beginning of the treatment with solvent control group) ]x100%.
Experimental results
The test results are shown in Table 9.
Tumor inhibiting effect of compounds of table 9 on LNCaP xenograft tumor model
Conclusion: the compound of the invention shows remarkable tumor inhibition effect on a human prostate cancer LNCaP xenograft tumor model.
Test example 4: in vitro test of the modulation of AR protein levels in human prostate cancer LNCaP cells
The purpose of the experiment is as follows: testing Compounds for degradation of androgen receptor AR in LNCap cells by ICW method
The experimental method comprises the following steps:
first day
1. Cell culture medium, 0.025% trypsin and phosphate buffer were pre-heated in a 37 ℃ water bath.
2. Trypsinized cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded and the cells resuspended in fresh medium.
3. Cells were seeded at a density of 4 k/36. Mu.L/well in COL-Coated CellCarrier-384 plates.
4. The 384-cell plate inoculated with the cells was left to stand in an ultra clean bench for 10 minutes.
5. The cell plates were placed in a carbon dioxide cell incubator for overnight incubation.
The next day
1. The compound was diluted to a concentration of 10mM to 3mM (3. Mu.L+7. Mu.L dimethyl sulfoxide) before being subjected to the double dilution.
2. Compounds were serially diluted 3-fold in Echo plates (4. Mu.L+8. Mu.L) using a Bravo instrument.
3. ZPE and HPE control wells were set, and the ZPE control wells were dimethyl sulfoxide.
4. After centrifugation of the Echo plate for 1 min at 1000 rpm, diluted compounds were transferred from the Echo plate to the intermediate plate with Echo, 200 nL/well.
5. Cell culture medium was added to the intermediate plate with a multitropcombi, 20 μl/well.
6. The intermediate plate was placed on a horizontal shaker and shaken for 1 minute to allow the compound to mix well with the medium.
7. The compounds in the intermediate dilution plates were transferred to the cell plates with Bravo, 4 μl/well.
8. The cell plates were placed on a horizontal shaker with shaking for 1 minute and centrifuged at 800 rpm for 30 seconds.
9. The cell plates were returned to the carbon dioxide incubator for continued incubation for 24 hours.
Third day
1. 4% paraformaldehyde is taken out of the refrigerator and left to stand at room temperature.
2. The cell plates were removed from the incubator.
3. 4% paraformaldehyde, 40. Mu.L/well was added directly to the cell plates.
4. Incubation at room temperature for 20 min
5. The culture medium in the cell plate is manually thrown away, the cell plate is inverted in a centrifuge, and the cell plate is centrifuged for 7 seconds at 800 revolutions, so that the residual culture medium is fully removed.
6. Cell plates were added 4% paraformaldehyde, 25 μl/well.
7. Incubate for 30 minutes at room temperature.
8. Manually throwing away the paraformaldehyde solution in the cell plate, inverting the solution in a centrifugal machine, and centrifuging for 7 seconds at 800 revolutions.
9. 0.1% station, 25. Mu.L/well was added.
10. Incubate at room temperature for 10 min.
11. The cell plates were manually spun off with 0.1% of the Triton solution, inverted into a centrifuge, and centrifuged at 800 rpm for 7 seconds.
12. Adding Intercept(PBS) Blocking Buffer, 25. Mu.L/well.
13. Incubate for 1 hour at room temperature.
14. Intercept in manual cell-free plate(PBS) Blocking Buffer solution, inverted in centrifuge, and centrifuged at 800 rpm for 7 seconds.
15. Add primary antibody solution, 20. Mu.L/well. The cell plates were placed in a centrifuge and centrifuged at 800 rpm for 7 seconds.
16. Cell plates were incubated overnight at 4 ℃ after membrane sealing.
Fourth day
1. The primary antibody solution in the cell plate was manually thrown off, inverted in a centrifuge, and centrifuged at 800 rpm for 7 seconds.
2.110. Mu.L PBS was washed 3 times.
3. The cell plates were inverted in a centrifuge and centrifuged at 800 rpm for 7 seconds.
4. Secondary antibody and DAPI solution were added at 20. Mu.L/well. The cell plates were placed in a centrifuge and centrifuged at 800 rpm for 7 seconds.
5. Incubate for 1 hour at room temperature.
6. The secondary antibody and DAPI solution in the cell plates were manually spun off and inverted into a centrifuge for 7 seconds at 800 rpm.
7. After washing 3 times with 110. Mu.L of PBS, 30. Mu.L of PBS remained in the cell plate
8. The cell plates were placed in a centrifuge and centrifuged at 800 rpm for 7 seconds.
Cell plate scanning by operatta instrument
Experimental results
The test results are shown in Table 10.
TABLE 10 degradation of AR proteins by the compounds of the invention in LNCaP cell lines
| # | Numbering of compounds | DC 50 (nM) | D max |
| 1 | Hydrochloride salt of WX018 | 42.0 | 106.15 |
| 2 | Hydrochloride of WX019 | 9.5 | 80.77 |
| 3 | Hydrochloride of WX020 | 7.94 | 110.84 |
| 4 | WX021 | 13.4 | 107.74 |
Conclusion: the compounds of the present invention exhibit good degradation activity for AR proteins.
Test example 5: mouse pharmacokinetic evaluation of Compounds
The purpose of the experiment is as follows:
the test compounds were selected from CB-17 SCID male mice and the pharmacokinetic profile of the test drugs in the mice was evaluated by measuring the drug concentration in plasma at various time points of intravenous or oral administration of the test compound and the reference compound using LCMS/MS method.
Experimental materials:
CB-17 SCID mice (Male, 20-30g,7-10 weeks old, beijing Vitolihua or Shanghai Srilk).
Experimental operation:
the clarified solution or suspension of the test compound is injected into mice via the tail vein (non-fasting), or administered to mice parenterally (non-fasting). Intravenous administration was performed for 0h (pre-administration) and 0.083,0.25,0.5,1,2,4,8, 24h post-administration, blood was collected from jugular vein puncture, placed in an anticoagulant tube with EDTA-K2 added, and the mixture was thoroughly vortex mixed at 4 ℃ and centrifuged at 13000rpm for 10 minutes; oral gavage administration was performed for 0h (pre-administration) and 0.25,0.5,1,2,4,8, 24h post-administration blood was collected from jugular vein puncture, placed in an anticoagulant tube with EDTA-K2 added, and the mixture was thoroughly vortexed and centrifuged at 13000rpm for 10 minutes. The blood concentration was determined by LC-MS/MS method and the relevant pharmacokinetic parameters were calculated by non-compartmental linear logarithmic trapezoidal method using winnonlin tmversion 6.3 (Pharsight, mountainView, CA) pharmacokinetic software.
Experimental results: the test results are shown in Table 11.
TABLE 11 pharmacokinetic parameters of the compounds of the invention in mice
Conclusion: the compounds of the invention exhibit good drug formation in mice.
Test example 6: rat pharmacokinetic evaluation of Compounds
The purpose of the experiment is as follows:
SD male rats are selected as test compounds for this study, and the pharmacokinetic profile of the test compounds in rats is evaluated by measuring the drug concentration in plasma of rats at different time points when the test compounds and the reference compounds are administered intravenously or orally by LCMS/MS method.
Experimental materials:
SD rats (male, beijing velutinib).
Experimental operation:
the clarified solution or suspension of the test compound is injected into rats via the tail vein (non-fasted) or administered to rats parenterally (non-fasted). Intravenous administration was performed at 0h (pre-administration) and 0.083h,0.25h,0.5h,1h,2h,4h,6h,8h,24h from jugular vein puncture, placed in an anticoagulant tube with heparin sodium added, and after blood sample collection placed on ice and plasma was centrifuged within 1 hour (centrifugation conditions: 6000g,3 min, 2-8deg.C); oral gavage administration blood was collected from jugular vein punctures at 0h (pre-administration) and 0.25h,0.5h,1h,2h,4h,6h,8h,24h post-administration, placed in an anticoagulant tube with heparin sodium added, and after blood sample collection placed on ice and plasma was centrifuged within 1 hour (centrifugation conditions: 6000g,3 min, 2-8 ℃). The blood concentration was determined by LC-MS/MS, and the relevant pharmacokinetic parameters were calculated by non-compartmental linear logarithmic trapezoidal method using winnonlin tmversion 8.2.0 (Pharsight, mountain View, CA) pharmacokinetic software.
Experimental results: the test results are shown in Table 12.
TABLE 12 pharmacokinetic parameters of the compounds of the invention in rats
Conclusion: the compounds of the invention exhibit good drug formation in rats.
Claims (11)
- A compound represented by the formula (II) or a pharmaceutically acceptable salt thereof,wherein,R 1 selected from H and CH 3 ；Linker is selected fromWherein E is 1 The end is connected with the ABM;R 2 selected from H and methyl;E 1 selected from single bond, O, NH and-C (=o) NH-;E 2 selected from single bonds, - (CH) 2 ) m -、 and-E 5 -(CH 2 ) m -；E 3 Selected from- (CH) 2 ) n -、-(CH 2 ) p O(CH 2 ) q -、E 4 Selected from single bond, O and-C (=o) NH-;E 5 selected from 5-6 membered heterocycloalkyl;T 1 and T 2 Each independently selected from CH and N;m is selected from 1, 2 and 3;n is selected from 0, 1, 2, 3, 4, 5, 6 and 7;p and q are each independently selected from 0, 1 and 2;ring Q is selected from phenyl orRing a is selected from phenyl and 5-6 membered heteroaryl;ABM is selected from drugs or derivatives thereof that target binding to AR proteins.
- The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein E 2 Selected from single bond, -CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、
- The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein structural unit-E 1 -E 2 -is selected from single bond, -O-, -OCH 2 CH 2 -、
- The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein E 3 Selected from single bond, -CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 4 -、-(CH 2 ) 5 -、
- The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein structural unit-E 3 -E 4 -is selected from single bond, -CH 2 -、-(CH 2 ) 2 O-、-(CH 2 ) 5 O-、
- The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Linker is selected from the group consisting of
- The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the structural unitSelected from the group consisting of
- The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein ABM is selected from the structures of formulae (ABM-1) and (ABM-2):wherein,R 3 and R is 4 Selected from methyl;alternatively, R 3 And R is R 4 With co-linked carbon atoms to C 4-6 Cycloalkyl;ring B is selected from phenyl and pyridinyl, optionally substituted with 1, 2 or 3R a Substitution;ring C is selected from phenyl and 6 membered heteroaryl, optionally substituted with 1, 2 or 3R b Substitution;R a selected from F, cl, br, I, CN, CH 3 、CF 3 And NO 2 ；R b Selected from F and Cl;ring D is selected from
- The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein ABM is selected from the structures represented by formulas (ABM-1 a), (ABM-2 b), and (ABM-3 a):wherein,R b1 selected from H and F;t is selected from 1, 2 and 3;T 1 selected from CH and N;R a as defined in claim 8.
- The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein ABM is selected from the group consisting of
- A compound of the formula or a pharmaceutically acceptable salt thereof, selected from:
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| PCT/CN2022/099015 WO2022262782A1 (en) | 2021-06-17 | 2022-06-15 | Glutarimide-substituted isoxazole fused ring compound, and application thereof |
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| WO2017197046A1 (en) * | 2016-05-10 | 2017-11-16 | C4 Therapeutics, Inc. | C3-carbon linked glutarimide degronimers for target protein degradation |
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