CN113831346A - 多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用 - Google Patents
多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用 Download PDFInfo
- Publication number
- CN113831346A CN113831346A CN202111184936.0A CN202111184936A CN113831346A CN 113831346 A CN113831346 A CN 113831346A CN 202111184936 A CN202111184936 A CN 202111184936A CN 113831346 A CN113831346 A CN 113831346A
- Authority
- CN
- China
- Prior art keywords
- small molecule
- derivative
- compound
- tumor
- dmso
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000003384 small molecules Chemical class 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 37
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 24
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 9
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims abstract description 17
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 claims abstract description 16
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 9
- 230000037361 pathway Effects 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims description 94
- 238000000034 method Methods 0.000 claims description 23
- 239000003814 drug Substances 0.000 claims description 10
- 239000003276 histone deacetylase inhibitor Substances 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 7
- -1 methyl ester compound Chemical class 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical group [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000556 agonist Substances 0.000 claims description 3
- 239000003534 dna topoisomerase inhibitor Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 229940121372 histone deacetylase inhibitor Drugs 0.000 claims description 3
- 239000001257 hydrogen Chemical group 0.000 claims description 3
- 229940044693 topoisomerase inhibitor Drugs 0.000 claims description 3
- JXASPPWQHFOWPL-UHFFFAOYSA-N Tamarixin Natural products C1=C(O)C(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(CO)O2)O)C(=O)C2=C(O)C=C(O)C=C2O1 JXASPPWQHFOWPL-UHFFFAOYSA-N 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 229910052731 fluorine Inorganic materials 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 150000007522 mineralic acids Chemical class 0.000 claims description 2
- 150000007524 organic acids Chemical class 0.000 claims description 2
- 229940034982 antineoplastic agent Drugs 0.000 claims 1
- 125000005843 halogen group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 28
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 7
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 4
- 231100000053 low toxicity Toxicity 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 3
- 101710183280 Topoisomerase Proteins 0.000 abstract description 2
- 231100001084 no genetic toxicology Toxicity 0.000 abstract 1
- 230000001875 tumorinhibitory effect Effects 0.000 abstract 1
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 84
- 239000000543 intermediate Substances 0.000 description 54
- 206010028980 Neoplasm Diseases 0.000 description 34
- 230000005764 inhibitory process Effects 0.000 description 30
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 27
- 239000007787 solid Substances 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 26
- 238000005160 1H NMR spectroscopy Methods 0.000 description 24
- 102000003964 Histone deacetylase Human genes 0.000 description 23
- 108090000353 Histone deacetylase Proteins 0.000 description 23
- 108020004414 DNA Proteins 0.000 description 22
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- WAEXFXRVDQXREF-UHFFFAOYSA-N vorinostat Chemical compound ONC(=O)CCCCCCC(=O)NC1=CC=CC=C1 WAEXFXRVDQXREF-UHFFFAOYSA-N 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 9
- 229940079593 drug Drugs 0.000 description 8
- 229960000237 vorinostat Drugs 0.000 description 8
- 229910001868 water Inorganic materials 0.000 description 8
- 239000013641 positive control Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 239000003292 glue Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- 102100039996 Histone deacetylase 1 Human genes 0.000 description 4
- 101001035024 Homo sapiens Histone deacetylase 1 Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000001472 cytotoxic effect Effects 0.000 description 4
- 239000012065 filter cake Substances 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 208000032839 leukemia Diseases 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 238000004809 thin layer chromatography Methods 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108090000323 DNA Topoisomerases Proteins 0.000 description 3
- 102000003915 DNA Topoisomerases Human genes 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 102100022537 Histone deacetylase 6 Human genes 0.000 description 3
- 101000899330 Homo sapiens Histone deacetylase 6 Proteins 0.000 description 3
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 3
- WWGBHDIHIVGYLZ-UHFFFAOYSA-N N-[4-[3-[[[7-(hydroxyamino)-7-oxoheptyl]amino]-oxomethyl]-5-isoxazolyl]phenyl]carbamic acid tert-butyl ester Chemical compound C1=CC(NC(=O)OC(C)(C)C)=CC=C1C1=CC(C(=O)NCCCCCCC(=O)NO)=NO1 WWGBHDIHIVGYLZ-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 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
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000001738 genotoxic effect Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- AVRPFRMDMNDIDH-UHFFFAOYSA-N 1h-quinazolin-2-one Chemical compound C1=CC=CC2=NC(O)=NC=C21 AVRPFRMDMNDIDH-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 238000011729 BALB/c nude mouse Methods 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 102000003893 Histone acetyltransferases Human genes 0.000 description 2
- 108090000246 Histone acetyltransferases Proteins 0.000 description 2
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000001028 anti-proliverative effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 229940127093 camptothecin Drugs 0.000 description 2
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 231100000024 genotoxic Toxicity 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- LZMKLGSLUKISQE-UHFFFAOYSA-N hydroxylamine;methanol Chemical compound OC.ON LZMKLGSLUKISQE-UHFFFAOYSA-N 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000005917 in vivo anti-tumor Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000003475 lamination Methods 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- FBDOJYYTMIHHDH-OZBJMMHXSA-N (19S)-19-ethyl-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-2,4,6,8,10,14,20-heptaen-18-one Chemical compound CC[C@@]1(O)C(=O)OCC2=CN3Cc4cc5ccccc5nc4C3C=C12 FBDOJYYTMIHHDH-OZBJMMHXSA-N 0.000 description 1
- GLGNXYJARSMNGJ-VKTIVEEGSA-N (1s,2s,3r,4r)-3-[[5-chloro-2-[(1-ethyl-6-methoxy-2-oxo-4,5-dihydro-3h-1-benzazepin-7-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide Chemical compound CCN1C(=O)CCCC2=C(OC)C(NC=3N=C(C(=CN=3)Cl)N[C@H]3[C@H]([C@@]4([H])C[C@@]3(C=C4)[H])C(N)=O)=CC=C21 GLGNXYJARSMNGJ-VKTIVEEGSA-N 0.000 description 1
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 description 1
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 1
- CHMAEAFFKRYPQT-UHFFFAOYSA-N (7-methoxy-7-oxoheptyl)azanium;chloride Chemical compound Cl.COC(=O)CCCCCCN CHMAEAFFKRYPQT-UHFFFAOYSA-N 0.000 description 1
- VQFVRLVJEKZQMM-UHFFFAOYSA-N 1,3-dimethyl-7-(naphthalen-1-ylmethyl)-8-[(4-phenylpiperazin-1-yl)methyl]purine-2,6-dione Chemical compound C=1C=CC2=CC=CC=C2C=1CN1C=2C(=O)N(C)C(=O)N(C)C=2N=C1CN(CC1)CCN1C1=CC=CC=C1 VQFVRLVJEKZQMM-UHFFFAOYSA-N 0.000 description 1
- WLJVXDMOQOGPHL-PPJXEINESA-N 2-phenylacetic acid Chemical compound O[14C](=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-PPJXEINESA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 239000012623 DNA damaging agent Substances 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 108091005772 HDAC11 Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 102100039385 Histone deacetylase 11 Human genes 0.000 description 1
- 102100021454 Histone deacetylase 4 Human genes 0.000 description 1
- 101000899259 Homo sapiens Histone deacetylase 4 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 239000012819 MDM2-Inhibitor Substances 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 1
- LGRFSURHDFAFJT-UHFFFAOYSA-N Phthalic anhydride Natural products C1=CC=C2C(=O)OC(=O)C2=C1 LGRFSURHDFAFJT-UHFFFAOYSA-N 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 239000012317 TBTU Substances 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- NCNRHFGMJRPRSK-MDZDMXLPSA-N belinostat Chemical compound ONC(=O)\C=C\C1=CC=CC(S(=O)(=O)NC=2C=CC=CC=2)=C1 NCNRHFGMJRPRSK-MDZDMXLPSA-N 0.000 description 1
- 229960003094 belinostat Drugs 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- JHIWVOJDXOSYLW-UHFFFAOYSA-N butyl 2,2-difluorocyclopropane-1-carboxylate Chemical compound CCCCOC(=O)C1CC1(F)F JHIWVOJDXOSYLW-UHFFFAOYSA-N 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 229940125758 compound 15 Drugs 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000004049 epigenetic modification Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 150000002367 halogens Chemical group 0.000 description 1
- 230000006195 histone acetylation Effects 0.000 description 1
- 230000006197 histone deacetylation Effects 0.000 description 1
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000002350 laparotomy Methods 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 229960005184 panobinostat Drugs 0.000 description 1
- FWZRWHZDXBDTFK-ZHACJKMWSA-N panobinostat Chemical compound CC1=NC2=CC=C[CH]C2=C1CCNCC1=CC=C(\C=C\C(=O)NO)C=C1 FWZRWHZDXBDTFK-ZHACJKMWSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000006343 physiological stress response Effects 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028617 response to DNA damage stimulus Effects 0.000 description 1
- OHRURASPPZQGQM-GCCNXGTGSA-N romidepsin Chemical compound O1C(=O)[C@H](C(C)C)NC(=O)C(=C/C)/NC(=O)[C@H]2CSSCC\C=C\[C@@H]1CC(=O)N[C@H](C(C)C)C(=O)N2 OHRURASPPZQGQM-GCCNXGTGSA-N 0.000 description 1
- 229960003452 romidepsin Drugs 0.000 description 1
- OHRURASPPZQGQM-UHFFFAOYSA-N romidepsin Natural products O1C(=O)C(C(C)C)NC(=O)C(=CC)NC(=O)C2CSSCCC=CC1CC(=O)NC(C(C)C)C(=O)N2 OHRURASPPZQGQM-UHFFFAOYSA-N 0.000 description 1
- 108010091666 romidepsin Proteins 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000003744 tubulin modulator Substances 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一类多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用。该类小分子的化学结构如式I所示，其衍生物涉及所述小分子的药学上可接受的盐。该类小分子及其衍生物在体内外具有高效的肿瘤抑制作用，对细胞和生物体毒性低，不产生基因毒性；通过多靶点作用实现抗肿瘤活性，具有泛HDAC抑制活性，同时还可以有效激活p53通路，抑制拓扑异构酶活性，可用于制备抗肿瘤药物；制备方法简便，易操作。
Description
技术领域
本发明涉及一种多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用，尤其涉及一种具有组蛋白去乙酰化酶抑制剂、拓扑异构酶抑制剂、p53通路激动剂中至少一种机制的多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用。
背景技术
组蛋白乙酰化和去乙酰化分别由组蛋白乙酰转移酶(HATs)和组蛋白去乙酰化酶(HDAC)完成，这是调节染色质结构和基因转录的关键表观遗传修饰。根据其与酵母蛋白的同源性和辅因子依赖性，HDAC被分为不同的种类。HDAC I、II类(再分为IIa和IIb亚型)和IV类是锌依赖性酶，而HDACIII类是烟酰胺腺嘌呤二核苷酸(NAD+)依赖性酶。I类同工酶(HDAC1、2、3、8)主要位于细胞核内，IIa类(HDAC4、5、7、9)和IV类(HDAC11)穿梭于细胞核和细胞质之间。相反，IIb类(HDAC6、10)主要存在于细胞质中。I类HDACs，特别是HDAC1、2、3和IIb类HDAC6被认为是癌症治疗的关键靶点。HDACs在多种癌症中均有过表达，抑制HDACs可通过降低癌细胞活力、阻滞迁移和侵袭、抑制血管生成、诱导细胞凋亡与克服DNA修复等多种机制产生抗癌作用。因此，HDAC已成为开发新型抗癌药物的重要靶点。迄今为止，四种HDAC抑制剂(vorinostat、romidepsin、belinostat和panobinostat)已获得FDA批准用于癌症治疗。还有一些HDAC抑制剂正处于临床研究阶段。
不过目前上市的HDAC抑制剂主要用于治疗白血病，它们对实体瘤的治疗效果一般。最近的研究表明HDAC抑制剂可与多种抗肿瘤药物发挥协同作用，如DNA损伤试剂、微管蛋白调节剂等，特别是HDAC抑制剂能够与MDM2抑制剂协同抑制肿瘤细胞增殖。尽管联合用药带来了较好的疗效，但是也造成了副作用的增强、药物与药物之间的作用和不可预见的药代性质等缺陷。
p53是控制细胞生死的细胞级联反应的关键激活因子。它在生理和非生理应激反应中被激活，如氧化应激、病毒应激、致癌应激和基因毒性应激以及缺氧应激。在肿瘤进化过程中，虽然p53基因TP53经常发生突变，但是超过50％的人类肿瘤中含有野生型基因，因此p53通路是治疗癌症的一个重要靶点。有研究认为p53通路的激活可以增强对肿瘤细胞的杀伤，是细胞周期阻滞和凋亡通路的关键激活因子。
发明内容
发明目的：本发明的第一目的是提供一种多靶点抗肿瘤小分子及其衍生物，第二目的是提供所述多靶点抗肿瘤小分子及其衍生物的制备方法，第三目的是提供一种包含所述多靶点抗肿瘤小分子和/或其衍生物的药物组合物，第四目的是提供所述多靶点抗肿瘤小分子及其衍生物在制备抗肿瘤药物中的应用。
技术方案：作为本发明涉及的第一方面，本发明的多靶点抗肿瘤小分子及其衍生物具有式I的结构，所述衍生物为所述小分子的药学上可接受的盐：
其中：
M为
或羟基；
R为卤素、氢或C1-C4烷基；
n为1-5的整数。
本发明以邻苯二氮基[1,2-b]喹唑啉酮结构单元作为表面识别区，以2-氨基苯胺或羟肟酸结构单元作为锌离子结合基团，通过烷基碳链作为连接子将二者结合，得到一类以I/II类HDAC为靶点的具有抗肿瘤作用的化合物。
针对R取代基，所述小分子及其衍生物结构中：R优选为Br、Cl、F、CH3或H。
针对M取代基，所述小分子及其衍生物结构中：M优选为羟基；当M优选为羟基时，R更优选为CH3或H，n更优选为4-5的整数。
具体地，所述小分子为以下任一化合物：
针对上述小分子的药学上可接受的盐，其具体为上述小分子与酸形成的盐，所述酸为无机酸(例如：盐酸、氢溴酸、硫酸、磷酸等)或有机酸(例如：甲磺酸、苯磺酸、对甲苯磺酸、萘磺酸、柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、马来酸、琥珀酸、富马酸、水杨酸、苯基乙酸或杏仁酸等)。
作为本发明涉及的第二方面，本发明的小分子及其衍生物的制备方法为：
甲酯类化合物6与邻苯二胺类化合物或羟胺类化合物反应得到化合物I；
其中，R、n的定义同上所述；
将相应的酸与以上方法制备的化合物I成盐，即得所述小分子的药学上可接受的盐。
作为本发明涉及的第三方面，本发明的药物组合物包含所述小分子和/或其衍生物以及药学上可接受的载体。
所述小分子及其衍生物可以添加药学上可接受的载体制成常见的药用制剂，如片剂、胶囊、糖浆、悬浮剂或注射剂，制剂可以加入香料、甜味剂、液体/固体填料、稀释剂等常用药用辅料。
作为本发明涉及的第四方面，本发明的小分子及其衍生物可制备为抗肿瘤药物，所述药物是作为组蛋白去乙酰化酶抑制剂、拓扑异构酶抑制剂、p53通路激动剂中的至少一种来发挥药效的。
有益效果：与现有技术相比，本发明具有如下显著优点：
(1)该类小分子及其衍生物和药物组合物具有高效的抗肿瘤作用，体外癌细胞的抑制IC50值最优达到纳摩尔浓度级别，体内抑瘤率最高可达80％以上；
(2)通过多靶点作用实现抗肿瘤活性，其对I/II类HDAC亚型的抑制作用均可达到纳摩尔浓度级别，具有泛HDAC抑制活性，同时还可以有效激活p53通路，抑制拓扑异构酶活性，可用于制备抗肿瘤药物；
(3)安全性高、毒性低，对细胞的抑制具有选择性，对正常细胞抑制作用低，对DNA无明显抑制作用，未对正常组织产生基因毒性，并且对动物体重影响甚小；
(4)制备方法简便、易操作。
附图说明
图1为化合物I22在不同浓度下在HepG2细胞中对p-p53和p53蛋白表达的影响；
图2为化合物I22在不同浓度下对DNA的作用；其中，泳道1：DNA，泳道2：EB(5μM)，泳道3-泳道8：化合物I22(10,50,100,300,500,1000μM)+DNA；
图3为不同浓度的化合物I22在拓扑异构酶I存在下对DNA的作用；其中，泳道1：DNA，泳道2：Topo I+DNA，泳道3：CPT(200μM)+Topo I+DNA，泳道4-9：化合物I22(10,50,100,300,500,1000μM)+Topo I+DNA；
图4为化合物I22对人肝癌细胞HepG2裸鼠异种移植瘤生长体积变化的影响；
图5为化合物I22对人肝癌细胞HepG2裸鼠异种移植瘤裸鼠体重的影响。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明。
除本发明制备的中间体、目标化合物之外，其余均为市售试剂。按本发明方法制备的化合物经核磁氢谱、碳谱及高分辨质谱确定了化合物的分子结构。
实施例1：中间体的制备
(1)中间体2的制备(以中间体2e为例：R＝H)
将5.1g(37.2mmol)反应物1e(邻氨基苯甲酸)溶解于35mL THF中，0℃条件下边搅拌边加入6.6g(22.3mmol)三光气(BTC)，混合液35℃反应12h，反应结束后过滤，滤饼用Et2O洗涤(20mL×3)，得到褐色固体(中间体2e)。
1H NMR(600MHz,DMSO-d6)δ11.73(s,1H),7.91–7.89(m,1H),7.74–7.71(m,1H),7.25–7.22(m,1H),7.15(d,J＝8.0Hz,1H)ppm.
(2)中间体3的制备(以中间体3e为例：R＝H)
将3.8g(23.3mmol)中间体2e和水合肼(85％)依次加入50mL圆底烧瓶中，加入回流3h，用薄层色谱法检测反应过程。反应完成后冷却至0℃过滤，滤饼用EtOH洗涤(20mL×3)，得到白色固体(中间体3e)。
1H NMR(600MHz,DMSO-d6)δ9.47(s,1H),7.42–7.41(m,1H),7.13–7.10(m,1H),6.69–6.68(m,1H),6.49–6.46(m,1H),6.33(s,2H),4.41(s,2H)ppm.
(3)中间体4的制备(以中间体4e为例：R＝H)
将5.0g(33.1mmol)中间体3e溶于乙二醇(40mL)中，加入5.4g(36.41mmol)邻苯二甲酸酐，加热回流3h，用薄层色谱法检测反应过程。反应完成后冷却至0℃过滤，滤饼用水洗涤(20mL×3)，干燥后和三氯氧磷(25mL)依次加入50mL圆底烧瓶中，加热回流6h，用薄层色谱法检测反应过程。反应完成后减压除去大部分溶剂，然后加入20g冰块融化后过滤，滤饼用硅胶柱层析提纯，洗脱液为CH2Cl2:PE(1:3)混合溶剂。得到浅绿色固体(中间体4e)。
1H NMR(600MHz,CDCl3)δ8.99–8.98(m,1H),8.52(d,J＝7.9Hz,1H),8.16–8.14(m,1H),7.97–7.90(m,2H),7.88–7.85(m,2H),7.56–7.54(m,1H)ppm.
(4)中间体6的制备(以中间体6e3为例：n＝5，R＝H)
将1.0g(3.8mmol)中间体4e与1.5g(7.6mmol)反应物5a(7-氨基庚酸甲酯盐酸盐)依次加入100mL圆底烧瓶中，甲苯(50mL)作溶剂，滴加1.2g(11.4mmol)三乙胺后加热回流6h，用薄层色谱法检测反应过程。反应完成后减压除去溶剂，用硅胶柱层析提纯，洗脱液为CH2Cl2:MeOH(100:1)混合溶剂。得到淡黄色固体(中间体6e3)。
1H NMR(600MHz,CDCl3)δ9.03(d,J＝7.5Hz,1H),8.46–8.45(m,1H),7.86–7.81(m,2H),7.80–7.74(m,3H),7.47–7.45(m,1H),5.41(s,1H),3.62(s,3H),3.62–3.59(m,2H),2.24(t,J＝7.5Hz,2H),1.73–1.68(m,2H),1.59–1.53(m,2H),1.40–1.35(m,2H),1.32–1.28(m,2H)ppm.
实施例2：化合物I7的制备
将中间体6a1(400mg,0.88mmol)加入15mL THF中溶解，滴加氢氧化锂(92.4mg,2.2mmol)水溶液(5mL)，室温搅拌过夜，减压除去溶剂得到乳白色固体，用30mL水将其溶解，用1M盐酸溶液调节pH至5，过滤得到淡黄色固体，干燥后与TBTU(340.4mg,1.06mmol)在DMF(10mL)溶液中室温搅拌5min，加入115.4mg(1.14mmol)三乙胺继续搅拌2min，再加入285.4mg(2.64mmol)邻苯二胺，在35℃下反应2h。浓缩反应液，浓缩液经硅胶柱层析分离，洗脱液为CH2Cl2和MeOH混合溶剂(30:1)，得到深红色固体135.3mg，产率：30％。
1H NMR(600MHz,DMSO-d6)δ9.58(s,1H),8.82(d,J＝7.6Hz,1H),8.30(d,J＝7.6Hz,1H),8.24(s,1H),7.99–7.85(m,4H),7.73–7.40(m,3H),7.23(d,J＝7.6Hz,1H),7.06(t,J＝7.1Hz,1H),6.97(d,J＝7.4Hz,1H),6.85–6.82(m,1H),3.58(d,J＝4.5Hz,2H),2.56(t,J＝6.7Hz,2H),2.15–2.07(m,2H)ppm；13C NMR(150MHz,DMSO–d6):δ172.1,156.7,149.3,145.2,144.1,136.9,133.6,132.8,129.2,128.9,128.8,126.8,126.5,126.1,123.5,122.0,121.6,119.3,118.2,41.4,34.1,24.7ppm.HRMS(ESI)m/z calcd for C25H21BrN6O2[M+Na]+:539.0802,found:539.0827.
实施例3：化合物I8的制备
用中间体6a2代替中间体6a1参照实施例1所述方法制备，得到深红色固体，产率35％。
1H NMR(600MHz,DMSO-d6)δ9.12(s,1H),8.80–8.77(m,1H),8.27(d,J＝7.8Hz,1H),8.24(d,J＝2.3Hz,1H),7.94–7.87(m,3H),7.65(d,J＝8.7Hz,1H),7.56(t,J＝5.1Hz,1H),7.15(d,J＝7.8Hz,1H),6.89–6.87(m,1H),6.72–6.70(m,1H),6.53–6.50(m,1H),4.82(s,2H),3.51–3.48(m,2H),2.39(t,J＝7.4Hz,2H),1.82–1.77(m,2H),1.73–1.68(m,2H),1.51–1.46(m,2H)ppm；13C NMR(150MHz,DMSO–d6):δ171.7,156.7,149.2,145.1,144.1,142.4,136.9,133.5,132.7,129.8,128.9,128.7,126.7,126.2,125.8,124.1,123.4,122.0,121.6,118.1,116.6,116.4,41.7,36.3,28.2,26.9,25.7ppm.HRMS(ESI)m/z calcd forC27H25BrN6O2[M+H]+:545.1295,found:545.1309.
实施例4：化合物I9的制备
用中间体6a3代替中间体6a1参照实施例1所述方法制备，得到深红色固体，产率36％。
1H NMR(600MHz,DMSO-d6)δ9.12(s,1H),8.69(d,J＝7.8Hz,1H),8.22(d,J＝7.9Hz,1H),8.17(d,J＝2.1Hz,1H),7.88(t,J＝7.4Hz,1H),7.84–7.79(m,2H),7.56(d,J＝8.7Hz,1H),7.44(t,J＝4.8Hz,1H),7.17(d,J＝7.7Hz,1H),6.90(t,J＝7.5Hz,1H),6.73(d,J＝7.9Hz,1H),6.54(t,J＝7.5Hz,1H),4.84(s,2H),3.46–3.43(m,2H),2.37(t,J＝7.4Hz,2H),1.77–1.74(m,2H),1.68–1.64(m,2H),1.48–1.41(m,2H)ppm；13C NMR(150MHz,DMSO–d6):δ171.7,156.5,149.1,145.0,143.9,142.4,136.7,133.3,132.5,129.7,128.8,128.6,126.6,126.2,125.8,124.1,123.4,121.9,121.5,118.1,116.7,116.4,41.8,36.3,29.1,28.4,27.2,25.8ppm.HRMS(ESI)m/z calcd for C28H27BrN6O2[M+H]+:559.1452,found:559.1486.
实施例5：化合物I10的制备
用中间体6a4代替中间体6a1参照实施例1所述方法制备，得到深红色固体，产率38％。
1H NMR(600MHz,DMSO-d6)δ9.11(s,1H),8.81(dd,J＝7.9,1.1Hz,1H),8.29(d,J＝7.8Hz,1H),8.25(d,J＝2.3Hz,1H),7.95–7.92(m,1H),7.91–7.89(m,2H),7.67(d,J＝8.7Hz,1H),7.53(t,J＝5.1Hz,1H),7.15(dd,J＝7.8,1.2Hz,1H),6.89–6.87(m,1H),6.71(dd,J＝7.9,1.2Hz,1H),6.54–6.51(m,1H),4.88(s,2H),3.49–3.46(m,2H),2.33(t,J＝7.4Hz,2H),1.78–1.73(m,2H),1.64–1.60(m,2H),1.43–1.36(m,6H)ppm；13C NMR(150MHz,DMSO–d6):δ171.7,156.7,149.2,145.2,144.1,142.3,136.9,133.6,132.7,129.9,128.9,128.8,126.8,126.2,125.8,124.1,123.5,122.0,121.7,118.2,116.7,116.4,41.8,36.3,29.3,29.2,28.5,27.3,25.8ppm.HRMS(ESI)m/z calcd for C29H29BrN6O2[M+H]+:573.1608,found:573.1646.
实施例6：化合物I11的制备
用中间体6b3代替中间体6a1参照实施例1所述方法制备，得到深红色固体，产率35％。
1H NMR(600MHz,DMSO-d6)δ9.12(s,1H),8.72–8.70(m,1H),8.23(d,J＝7.9Hz,1H),8.03(d,J＝2.4Hz,1H),7.89–7.83(m,2H),7.72–8.70(m,1H),7.65(d,J＝8.7Hz,1H),7.46(t,J＝4.9Hz,1H),7.16(d,J＝7.1Hz,1H),6.90–6.87(m,1H),6.72–6.71(m,1H),6.54–6.51(m,1H),4.83(s,2H),3.47–3.44(m,2H),2.36(t,J＝7.4Hz,2H),1.79–1.74(m,2H),1.68–1.63(m,2H),1.49–1.38(m,4H)ppm；13C NMR(150MHz,DMSO–d6):δ171.7,156.7,149.1,144.8,143.8,142.4,134.1,133.4,132.5,129.9,129.6,128.6,126.6,126.2,125.8,125.7,124.1,123.4,121.9,121.1,116.7,116.4,41.8,36.3,29.1,28.4,27.1,25.8ppm.HRMS(ESI)m/z calcd for C28H27ClN6O2[M+H]+:515.1957,found:515.1987.
实施例7：化合物I12的制备
用中间体6c3代替中间体6a1参照实施例1所述方法制备，得到深红色固体，产率32％。
1H NMR(600MHz,DMSO-d6)δ9.11(s,1H),8.76(dd,J＝7.7,1.4Hz,1H),8.25(d,J＝7.5Hz,1H),7.90–7.85(m,2H),7.81(dd,J＝8.8,3.0Hz,1H),7.77–7.74(m,2H),7.66–7.63(m,2H),7.47(t,J＝5.0Hz,1H),7.16–7.15(m,1H),6.90–6.87(m,1H),6.73–6.71(m,1H),6.54–6.51(m,1H),4.85(s,2H),3.49–3.45(m,2H),2.36(t,J＝7.4Hz,2H),1.79–1.74(m,2H),1.68–1.63(m,2H),1.48–1.40(m,4H)ppm.13C NMR(150MHz,DMSO-d6)δ171.7,160.6,158.9,157.0(d,J＝4.5Hz),149.1,143.2(d,J＝19.5Hz),142.4,133.2,132.6,130.3(d,J＝9.0Hz),128.8,126.5,126.2,125.8,124.1,123.4,123.0(d,J＝24.0Hz),121.8,121.2(d,J＝9.0Hz),116.7,116.4,111.1(d,J＝22.5Hz),41.8,36.3,29.0,28.4,27.1,25.8ppm.HRMS(ESI)m/z calcd for C28H27FN6O2[M+H]+:499.2252,found:499.2237.
实施例8：化合物I13的制备
用中间体6d3代替中间体6a1参照实施例1所述方法制备，得到深红色固体，产率40％。
1H NMR(600MHz,DMSO-d6)δ9.12(s,1H),8.81(d,J＝7.9Hz,1H),8.27(d,J＝7.2Hz,1H),7.98(s,1H),7.90–7.86(m,2H),7.64(d,J＝8.3Hz,1H),7.59(d,J＝8.3Hz,1H),7.40(s,1H),7.17(d,J＝7.7Hz,1H),6.89(t,J＝7.5Hz,1H),6.73(d,J＝7.9Hz,1H),6.53(t,J＝7.5Hz,1H),4.89(s,2H),3.50–3.47(m,2H),2.44(s,3H),2.35(t,J＝7.3Hz,2H),1.80–1.75(m,2H),1.68–1.63(m,2H),1.49–1.40(m,4H)ppm；13C NMR(150MHz,DMSO-d6)δ171.7,157.6,148.9,144.3,142.9,142.4,135.6,133.0,132.5,129.0,127.3,126.5,126.2,126.1,125.8,124.1,123.4,121.8,120.1,116.7,116.4,41.8,36.3,29.0,28.5,27.1,25.8,21.4ppm.HRMS(ESI)m/z calcd for C29H30N6O2[M+H]+:495.2503,found:495.2536.
实施例9：化合物I14的制备
用中间体6e3代替中间体6a1参照实施例1所述方法制备，得到深红色固体，产率41％。
1H NMR(600MHz,DMSO-d6)δ9.12(s,1H),8.85–8.83(m,1H),8.28(d,J＝7.3Hz,1H),8.24(d,J＝7.9Hz,1H),7.91–7.87(m,2H),7.82–7.79(m,1H),7.76(d,J＝8.1Hz,1H),7.49(t,J＝7.6Hz,1H),7.45(t,J＝5.0Hz,1H),7.17(d,J＝7.6Hz,1H),6.90(t,J＝7.6Hz,1H),6.73(d,J＝7.8Hz,1H),6.55(t,J＝7.5Hz,1H),5.04(s,2H),3.51–3.47(m,2H),2.36(t,J＝7.4Hz,2H),1.79–1.75(m,2H),1.68–1.63(m,2H),1.48–1.42(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ171.7,157.7,148.9,146.3,143.6,142.4,134.1,133.3,132.6,128.9,127.4,127.0,126.7,126.2,125.9,125.8,124.1,123.4,122.0,120.3,116.7,116.4,41.8,36.3,29.1,28.5,27.1,25.8ppm.HRMS(ESI)m/z calcd for C28H28N6O2[M+H]+:481.2347,found:481.2374.
实施例10：化合物I15的制备
将盐酸羟胺(4.67g,67mmol)溶解在MeOH(24mL)中，在0℃滴加氢氧化钾(5.61g,100mmol)和MeOH(12mL)形成的溶液，然后在0℃下搅拌30min，过滤，滤液为羟胺甲醇溶液。将化合物6a1(300mg,0.66mmol)溶于5mL新制备的羟胺甲醇中，室温搅拌1h，然后用2M盐酸将pH调至7，浓缩后用水洗涤得粗品，粗品经重结晶得到化合物15，乳白色固体，产率50％。
1H NMR(600MHz,DMSO-d6)δ10.46(s,1H),8.70(s,2H),8.22(s,1H),8.17(s,1H),7.90–7.82(m,3H),7.63–7.54(m,2H),3.46(s,2H),2.22–2.12(m,2H),2.04–1.95(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.5,156.6,149.1,145.0,143.9,136.8,133.4,132.6,129.7,128.8,128.6,126.6,123.4,121.9,121.5,118.1,41.5,30.8,24.7ppm.HRMS(ESI)m/z calcd for C19H16BrN5O3[M+H]+:442.0509,found:442.0535.
实施例11：化合物I16的制备
用中间体6a2代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率52％。
1H NMR(600MHz,DMSO-d6)δ10.41(s,1H),8.78(d,J＝6.6Hz,1H),8.70(s,1H),8.31(s,1H),8.23(s,1H),7.93(s,1H),7.89(s,2H),7.65(d,J＝7.5Hz,1H),7.61(s,1H),3.45(s,2H),2.02(t,J＝6.7Hz,2H),1.78–1.73(m,2H),1.62–1.56(m,2H),1.42–1.36(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.6,156.7,149.2,145.2,144.1,136.9,133.6,132.7,129.9,128.9,128.7,126.7,123.6,122.0,121.6,118.1,41.7,32.8,28.2,26.9,25.5ppm.HRMS(ESI)m/z calcd for C21H20BrN5O3[M+H]+:470.0822,found:470.0837.
实施例12：化合物I17的制备
用中间体6a3代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率55％。
1H NMR(600MHz,DMSO-d6)δ10.38(s,1H),8.75(d,J＝7.8Hz,1H),8.69(s,1H),8.27(d,J＝7.5Hz,1H),8.20(d,J＝2.2Hz,1H),7.92(t,J＝7.2Hz,1H),7.88–7.84(m,2H),7.62(d,J＝8.7Hz,1H),7.51(s,1H),3.45–3.42(m,2H),1.99(t,J＝7.3Hz,2H),1.75–1.71(m,2H),1.56–1.51(m,2H),1.43–1.38(m,2H),1.35–1.31(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.7,156.6,149.1,145.1,144.0,136.8,133.5,132.6,129.8,128.9,128.7,126.7,123.5,121.9,121.6,118.1,41.8,32.8,29.0,28.4,27.1,25.7ppm.HRMS(ESI)m/z calcdfor C22H22BrN5O3[M+Na]+:506.0798,found:506.0846.
实施例13：化合物I18的制备
用中间体6a4代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率52％。
1H NMR(600MHz,DMSO-d6)δ10.37(s,1H),8.69(dd,J＝7.9,0.9Hz,2H),8.21(d,J＝8.0Hz,1H),8.15(d,J＝2.3Hz,1H),7.89–7.86(m,1H),7.84(d,J＝7.4Hz,1H),7.82–7.80(m,1H),7.56(d,J＝8.7Hz,1H),7.45(t,J＝5.0Hz,1H),3.42–3.39(m,2H),1.96(t,J＝7.4Hz,2H),1.73–1.69(m,2H),1.53–1.48(m,2H),1.41–1.37(m,2H),1.35–1.32(m,2H),1.29–1.26(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.7,156.6,149.1,145.1,144.0,136.7,133.4,132.6,129.8,128.9,128.6,126.7,123.4,121.9,121.6,118.1,41.9,32.8,29.3,29.2,28.5,27.3,25.7ppm.HRMS(ESI)m/z calcd for C23H24BrN5O3[M+H]+:498.1135,found:498.1133.
实施例14：化合物I19的制备
用中间体6b3代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率58％。
1H NMR(600MHz,DMSO-d6)δ10.35(s,1H),8.94–8.37(m,2H),8.25(d,J＝7.8Hz,1H),8.06(d,J＝2.4Hz,1H),7.93–7.84(m,2H),7.76–7.74(m,1H),7.69(d,J＝8.8Hz,1H),7.48(t,J＝5.0Hz,1H),3.46–3.43(m,2H),1.98(t,J＝7.4Hz,2H),1.76–1.71(m,2H),1.57–1.52(m,2H),1.43–1.38(m,2H),1.36–1.31(m,2H)ppm.13C NMR(150MHz,DMSO-d6)δ169.7,156.6,149.1,144.8,143.8,134.0,133.3,132.5,129.9,129.6,128.6,126.6,125.7,123.4,121.9,121.1,41.9,32.8,29.0,28.4,27.1,25.7ppm.HRMS(ESI)m/z calcd forC22H22ClN5O3[M+Na]+:462.1303,found:462.1324.
实施例15：化合物I20的制备
用中间体6c3代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率58％。
1H NMR(600MHz,DMSO-d6)δ10.36(s,1H),8.70(d,J＝7.8Hz,2H),8.21(d,J＝7.8Hz,1H),7.87–7.81(m,2H),7.76(dd,J＝8.8,2.8Hz,1H),7.71–7.69(m,1H),7.62–7.59(m,1H),7.42(t,J＝4.8Hz,1H),3.44–3.41(m,2H),1.99(t,J＝7.3Hz,2H),1.75–1.70(m,2H),1.57–1.52(m,2H),1.43–1.38(m,2H),1.36–1.31(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.7,160.5,158.9,157.0(d,J＝3.0Hz),149.0,143.1(d,J＝19.5Hz),133.2,132.5,130.3(d,J＝7.5Hz),128.7,126.5,123.3,122.9(d,J＝24.0Hz),121.8,121.1(d,J＝9.0Hz),111.0(d,J＝22.5Hz),41.8,32.8,29.0,28.4,27.1,25.7ppm.HRMS(ESI)m/z calcdfor C22H22FN5O3[M+H]+:424.1779,found:424.1796.
实施例16：化合物I21的制备
用中间体6d3代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率60％。
1H NMR(600MHz,DMSO-d6)δ10.36(s,1H),8.82–8.80(m,1H),8.68(s,1H),8.28(d,J＝7.3Hz,1H),7.99(s,1H),7.92–7.87(m,2H),7.65(d,J＝8.3Hz,1H),7.61(d,J＝7.3Hz,1H),7.40–7.43(m,1H),3.48–3.45(m,2H),2.46(s,3H),1.97(t,J＝7.3Hz,2H),1.76–1.71(m,2H),1.56–1.51(m,2H),1.43–1.38(m,2H),1.35–1.31(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.6,157.6,148.9,144.3,142.9,135.6,133.1,132.6,129.1,127.4,126.5,126.1,123.4,121.8,120.1,41.8,32.8,29.0,28.5,27.1,25.6,21.5ppm.HRMS(ESI)m/z calcdfor C23H25N5O3[M+H]+:420.2030,found:420.2060.
实施例17：化合物I22的制备
用中间体6e3代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率60％。
1H NMR(600MHz,DMSO-d6)δ10.37(s,1H),8.83(d,J＝7.9Hz,1H),8.69(s,1H),8.27(d,J＝7.4Hz,1H),8.22(d,J＝7.9Hz,1H),7.91–7.86(m,2H),7.81(t,J＝7.4Hz,1H),7.74(d,J＝8.1Hz,1H),7.49(t,J＝7.4Hz,1H),7.44–7.40(m,1H),3.47–3.44(m,2H),1.99(t,J＝7.3Hz,2H),1.75–1.71(m,2H),1.56–1.51(m,2H),1.42–1.38(m,2H),1.35–1.30(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.7,157.7,148.9,146.3,143.6,134.1,133.3,132.6,128.9,127.4,127.0,126.6,125.9,123.4,121.9,120.3,41.8,32.8,29.0,28.4,27.1,25.7ppm.HRMS(ESI)m/z calcd for C22H23N5O3[M+H]+:406.1874,found:406.1898.
实施例18：化合物I23的制备
用中间体6b4代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率59％。
1H NMR(600MHz,DMSO-d6)δ10.36(s,1H),8.68(d,J＝7.8Hz,2H),8.21(d,J＝7.9Hz,1H),7.99(d,J＝2.3Hz,1H),7.86(t,J＝7.2Hz,1H),7.82(t,J＝7.5Hz,1H),7.69–7.67(m,1H),7.62(d,J＝8.7Hz,1H),7.42(t,J＝4.8Hz,1H),3.43–3.39(m,2H),1.96(t,J＝7.3Hz,2H),1.74–1.69(m,2H),1.54–1.49(m,2H),1.42–1.36(m,2H),1.35–1.31(m,2H),1.30–1.25(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.7,156.6,149.0,144.8,143.8,134.0,133.3,132.5,129.9,129.6,128.6,126.6,125.7,123.4,121.9,121.1,41.8,32.8,29.2,29.2,28.5,27.3,25.7ppm.HRMS(ESI)m/z calcd for C23H24ClN5O3[M+H]+:454.1640,found:454.1678.
实施例19：化合物I24的制备
用中间体6c4代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率61％。
1H NMR(600MHz,DMSO-d6)δ10.36(s,1H),8.72–8.69(m,2H),8.23(d,J＝7.8Hz,1H),7.87(t,J＝7.4Hz,1H),7.84(t,J＝7.4Hz,1H),7.77–7.75(m,1H),7.73–7.71(m,1H),7.64–7.60(m,1H),7.44(t,J＝4.6Hz,1H),3.44–3.41(m,2H),1.95(t,J＝7.3Hz,2H),1.74–1.69(m,2H),1.53–1.48(m,2H),1.41–1.36(m,2H),1.35–1.31(m,2H),1.30–1.24(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ170.9,162.1,160.5,158.4(d,J＝3.0Hz),150.5,144.7(d,J＝26.3Hz),134.4,133.6,131.6(d,J＝8.4Hz),130.4,127.8,124.3,124.0(d,J＝24Hz),123.3,122.7(d,J＝8.7Hz),112.1(d,J＝22.5Hz),43.1,36.7,34.0,31.6,29.8,28.5,26.9ppm.HRMS(ESI)m/z calcd for C23H24FN5O3[M+H]+:438.1936,found:438.1960.
实施例20：化合物I25的制备
用中间体6d4代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率64％。
1H NMR(600MHz,DMSO-d6)δ10.35(s,1H),8.79–8.65(m,2H),8.26(d,J＝7.5Hz,1H),7.97(s,1H),7.90–7.85(m,2H),7.62–7.61(m,1H),7.58(d,J＝8.3Hz,1H),7.41–7.37(m,1H),3.47–3.44(m,2H),2.44(s,3H),1.95(t,J＝7.3Hz,2H),1.74–1.70(m,2H),1.53–1.48(m,2H),1.41–1.36(m,2H),1.35–1.31(m,2H),1.29–1.24(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.7,157.6,148.9,144.3,142.9,135.6,133.0,132.5,129.0,127.3,126.5,126.1,123.4,121.8,120.1,41.8,32.8,29.2,29.1,28.6,27.3,25.6,21.5ppm.HRMS(ESI)m/z calcd for C24H27N5O3[M+Na]+:456.2006,found:456.2045.
实施例21：化合物I26的制备
用中间体6e4代替中间体6a1参照实施例9所述方法制备，得到乳白色固体，产率61％。
1H NMR(600MHz,DMSO-d6)δ10.36(s,1H),8.83–8.81(m,1H),8.69(s,1H),8.27(d,J＝7.6Hz,1H),8.22(d,J＝7.8Hz,1H),7.91–7.86(m,2H),7.81–7.78(m,1H),7.74(d,J＝8.1Hz,1H),7.47(t,J＝7.4Hz,1H),7.43(t,J＝4.8Hz,1H),3.47–3.44(m,2H),1.95(t,J＝7.3Hz,2H),1.74–1.69(m,2H),1.53–1.48(m,2H),1.41–1.35(m,2H),1.35–1.30(m,2H),1.29–1.23(m,2H)ppm；13C NMR(150MHz,DMSO-d6)δ169.7,157.7,148.9,146.3,143.6,134.1,133.3,132.6,128.9,127.4,127.0,126.7,125.9,123.4,122.0,120.3,41.8,32.8,29.2,28.5,27.3,25.6ppm.HRMS(ESI)m/z calcd for C23H25N5O3[M+H]+:420.2030,found:420.2081.
实施例22：化合物体外细胞毒活性测试
实验方法：采用MTT方法对本发明的代表性化合物进行了细胞毒活性测试。取对数生长期的细胞计数，接种于96孔培养板内，每孔约8000-10000个细胞。培养过夜，待细胞贴壁后进行给药，分别设给药组和对照组。待测的化合物用DMSO溶液配制成贮液，临用前用蒸馏水稀释成一系列浓度，其中DMSO的终浓度不超过4‰(下面实验类同)。每个浓度设3个复孔。加药后培养72h，加20μL浓度为5mg/mL的MTT，37℃孵育4h，弃去上清，加入150μL的DMSO溶解。用酶标仪在490纳米波长下测定每孔的OD值，并计算抑制率，做浓度-抑制率曲线计算IC50值，结果见表1。
测试了代表性化合物对人前列腺癌细胞PC-3、人乳腺癌细胞MCF-7、人结肠癌细胞HCT-116和人肝癌细胞HepG2及人肝细胞LO2和人脐静脉内皮细胞HUVEC的体外抗增殖活性，上市药物伏立诺他(SAHA)为阳性对照。观察化合物在不同浓度下对肿瘤细胞生长的抑制情况，计算抑制率及其IC50值来评价药物的细胞毒活性，结果见表1。此外，还测试了两个代表性化合物I22和I26对三种白血病细胞K562、RS；11和HL60的IC50值，结果见表2。
表1代表性化合物的细胞毒活性
表2化合物I22和I26对三种白血病细胞的抗增殖活性
由表1数据可见，化合物对四种癌细胞PC-3(人前列腺癌)、MCF-7(人乳腺癌)、HCT-116(人结直肠癌)和HepG2(人肝癌)均具有较强的抗肿瘤活性，其中大部分化合物对MCF-7、HCT-116和HepG2三种癌细胞(HDAC高表达，p53正常表达)的抑制活性明显优于PC-3(HDAC高表达，p53缺失)细胞，显示出多靶点的作用效果。化合物I15-I26对癌细胞的抑制作用优于化合物I7-I14。活性最优的化合物对相关癌细胞的IC50值达到纳摩尔级别，是阳性对照药物伏立诺他活性的十几倍。此外，还选取LO2和HUVEC两株正常细胞作为对照，结果表明多数目标化合物对肿瘤细胞的抑制作用高于对正常细胞的毒性，具有选择性。这表明本发明所述的多靶点化合物具有高效低毒的抗肿瘤活性。
由表2数据可见，两个代表性化合物I22和I26对白血病细胞K562、RS4；11和HL60也具有良好的抑制作用，优于阳性药物伏立诺他。
实施例23：化合物对人组蛋白去乙酰化酶的抑制检测
实验方法：通过人组蛋白脱乙酰基酶(HDAC)酶联免疫分析试剂盒检测了代表性化合物对HDAC1、2、3、8、4和6的抑制作用。待测的化合物用DMSO溶液配制成贮液，临用前用蒸馏水稀释成一系列浓度，其中DMSO的终浓度不超过4‰(下面实验类同)。分别设空白孔(空白对照孔不加样品及酶标试剂)和待测样品孔。在酶标包被板上标准品(800ng/L)准确加样50μL，空白孔加50μL标准品稀释液，待测样品孔中先加样品稀释液40μL，然后再加待测样品10μL。将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。用封板膜封板后置于37℃温育30min。然后弃去液体用1倍浓缩洗涤液洗涤3次。样品孔加入酶标试剂50μL，置于37℃温育30min。然后弃去液体再用1倍浓缩洗涤液洗涤3次。每孔先加入显色剂A 50μL，再加入显色剂B 50μL，轻轻震荡混匀，37℃避光显色10min。每孔加终止液50μL，终止反应。用酶标仪在450nm处检测各组吸光度(OD值)。计算抑制率，做浓度-抑制率曲线计算IC50值。
观察化合物在不同浓度下对人组蛋白去乙酰化酶(HDAC1、2、3、8、4和6)抑制情况，上市药物伏立诺他(SAHA)为阳性对照，相关化合物的IC50值见表3。
表3化合物对人组蛋白去乙酰化酶的抑制
*.-代表未测。
由表3数据可知，化合物I15-I26表现为一种广泛的HDAC抑制剂，对I/II类HDAC亚型的抑制作用能够达到纳摩尔级。当表面识别区邻苯二氮基[1,2-b]喹唑啉酮结构单元中的芳环无取代基或有供电子基团时，化合物的活性较高。连接子的碳链长度是6或7时，对HDAC亚型的抑制作用影响不大，其中化合物I21、I22、I25和I26的活性较高，均优于阳性对照药物伏立诺他。由于化合物I能够同时抑制HDACI类亚型(HDAC1、2、3、8)和HDAC6，这可能是其具有较高抗癌活性的原因。
实施例24：化合物对p53信号通路作用的检测
实验方法：采用Western Blot实验对本发明的化合物I22进行了检测。将生长在对数期的细胞计数接种于6孔板内，每孔约105-106个细胞。在37℃含CO25％的孵箱内培养24h。代表性化合物溶解在DMSO中，用培养基稀释至不同浓度后加入给药组，对照组更换培养基。24h后收集并裂解细胞，取上清液备用。用BCA蛋白试剂盒(Thermo，Waltham，MA)对蛋白含量进行测定。
将干净的玻璃板安装在凝胶架上并检漏。先加入10％的分离胶至1/2界面处，用去离子水封胶，分离胶凝固后倒掉去离子水。接着加入15％的积层胶，插入梳子等待积层胶凝固。将玻璃板转移至电泳槽中，倒满电泳缓冲液。缓慢拔除梳子，依次加入受试样品到各个孔内，用60V电压进行电泳。结束后将胶板取出计算条带的剪切区域。将条带放入半干转膜缓冲液中，室温下电压25V转膜50min。转膜完成后，将条带置于脱脂牛奶中，37℃振荡1h，然后用PBST洗涤3次(每次8min)。置于一抗中4℃振荡过夜。弃去一抗，将膜转移至PBST溶液中清洗3次(每次8min)。加入二抗室温孵育1h。弃去二抗，PBST溶液振荡清洗3次(每次8min)，再用PBS洗涤5min。最后将条带取出置于干净的玻璃板上待用。滴加AB发光液(A:B＝1:1)作用约10s左右，去除多余的发光液后盖上薄膜，并将胶片覆于薄膜上，置于预热好的曝光机中曝光。将胶片洗出，观察条带。该操作过程需要避光。实验结果见图1。
通过Western Blot实验检测化合物I22(0.5、1和2μM)对p-p53和p53蛋白表达情况的影响。随着化合物I22浓度的增大，p-p53蛋白表达含量逐渐增加，存在浓度剂量依赖性，而对p53蛋白的表达含量影响不大。图1结果表明，化合物I22能够有效激活p53通路，发挥抗肿瘤活性。
实施例25：化合物对DNA作用的检测
实验方法：采用琼脂糖凝胶电泳法对本发明的化合物I22进行了DNA作用检测。将琼脂糖溶解在TAE(1X)缓冲溶液中，使其浓度为1％。在微波炉内加热到澄清透明，倒入制胶槽内冷却凝固。代表性化合物和EB溶解在DMSO中，用去离子水稀释至不同浓度。分别加入体积为0.5μL的pBR322 DNA(5μg/μL)，加去离子水使最终体积为10μL。混匀后37℃恒温水浴3h。加入2μL(6X)溴酚蓝溶液终止反应。将其转移至琼脂糖胶槽中，电压设置为80V/cm，2h后结束，用EB溶液染色30min。用凝胶成像仪成像拍照。实验结果见图2。
通过琼脂糖凝胶电泳实验，检测化合物I22对pBR322质粒的裂解，可确定DNA超螺旋闭环结构转化为开环结构的比例，实验选用DNA拓扑异构酶I(Topo I)抑制剂喜树碱(CPT)为阳性对照。
采用琼脂糖凝胶电泳的方式研究了化合物I22与DNA的相互作用，化合物I22(10-1000μM)与pBR322质粒DNA预处理后，在DNA构象变化或DNA的迁移率方面与阴性对照组相比几乎没有区别，但是在阳性对照组中，DNA的迁移率显著受到抑制。图2的结果表明，化合物I22对DNA没有明显抑制作用。
实施例26：化合物对拓扑异构酶I抑制作用的检测
实验方法：采用琼脂糖凝胶电泳法对本发明的化合物I22进行了Topo I抑制作用检测。将琼脂糖溶解在TAE(1X)缓冲溶液中，使其浓度为1％。在微波炉内加热到澄清透明，倒入制胶槽内冷却凝固。代表性化合物和喜树碱(CPT)溶解在DMSO中，用去离子水稀释至不同浓度。分别加入0.5μL的pBR322 DNA、1μL的DNA Topoisomerase I Buffer、0.5μL的TopoI(1U/μL)和1μL的BSA。加去离子水使最终体积为10μL。混匀后37℃恒温水浴0.5h。加入2μL(6X)溴酚蓝溶液终止反应。将其转移至琼脂糖胶槽中，电压设置为80V/cm，2h后结束，用EB溶液染色30min。用凝胶成像仪成像拍照。实验结果见图3。
图3显示，化合物I22和CPT一样能够明显抑制Topo I的活性，导致Topo I无法有效解旋超螺旋型DNA，并且这种活性抑制作用随着化合物浓度的增加而增强。结果表明，化合物I22在激活p53的同时，并未引发细胞DNA损伤反应(即不会对正常组织施加基因毒性效应)，对Topo I有抑制作用。
实施例27：化合物体内抗肿瘤活性的测试
实验方法：采用BALB/c裸鼠，评价本发明的化合物I22对HepG2裸鼠异种移植瘤模型的抑制效果。
动物模型构建：取对数生长期的HepG2细胞，制备细胞悬液浓度为5×107cell/mL，在无菌条件下，接种于BALB/c裸鼠右腋下。体重16～18g，雄性，由南京大学动物模式中心提供(许可证号：SCXK(苏)2015-0001)。饲养于SPF级饲养环境中，室内温度控制在23±2℃，自由饮食和摄水。动物总数20只。接种前适应性饲养7天。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生长至100mm3后将动物随机分成4组。
药物及试剂：伏立诺他(SAHA)、化合物I22，用生理盐水溶解，然后用DMSO助溶；如难溶，可加助剂助溶。
组别及给药方案如下：
空白对照组：腹腔注射，每2天1次，连续21天。
阳性对照组：腹腔注射，(SAHA剂量为12mg/kg)，每2天1次，连续21天。
化合物I22低浓度组：腹腔注射，(剂量为12mg/kg)，每2天1次，连续21天。
化合物I22高浓度组：腹腔注射，(剂量为24mg/kg)，每2天1次，连续21天。
肿瘤直径的测量次数为每3天测1次，同时每3天检查一次小鼠体重。4周后，注射水合氯醛处死所有小鼠，剖腹观察，摘除肿瘤，剔除无关组织，用D-Hanks液洗涤2～3次，洗去血液，沥干水分保存，称取重量和测量体积。肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b2，其中a、b分别表示长宽。
据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0，其中V0为分笼给药时(即d0)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标：相对肿瘤增殖率T/C(％)，计算公式如下：
其中，TRTV：治疗组RTV；CRTV：模型组RTV。
抗肿瘤活性的评价指标：肿瘤生长抑制率(％)，计算公式如下：
实验结果见图4-图5及表4。
表4化合物I22在HepG2移植瘤模型中的体内活性
如图4所示，在建立的HepG2异种移植瘤模型中，阳性对照药物伏立诺他(12mg/kg)最终抑瘤率为49.28％。当化合物I22与伏立诺他等剂量给药剂量时，其抑瘤率达到63.10％，显著优于伏立诺他；当化合物I22的给药剂量进一步提高到24mg/kg时，抑瘤率达到了80.19％。有关化合物I22的相对肿瘤增殖率和肿瘤抑制率数据见表4。与伏立诺他相比，化合物I22在HepG2异种移植瘤模型中具有更高的体内抗肿瘤活性。图5中小鼠体重变化显示化合物I22对受试动物体重几乎没有影响，表明化合物I22的毒性较小，具有高效低毒的特点。
通过对I类与II类人组蛋白去乙酰化酶和DNA拓扑异构酶I的抑制作用及体内外抗肿瘤活性试验表明，本发明所述的多靶点小分子抗肿瘤化合物对所测试的癌细胞具有显著的抑制作用，可用于制备抗肿瘤药物。
Claims (10)
1.一种多靶点抗肿瘤小分子及其衍生物，其特征在于，所述多靶点抗肿瘤小分子及其衍生物具有式I的结构，所述衍生物为所述小分子的药学上可接受的盐：
其中：M为
或羟基；R为卤素、氢或C1-C4烷基；n为1-5的整数。
2.根据权利要求1所述的小分子及其衍生物，其特征在于，所述小分子及其衍生物结构中：R为Br、Cl、F、CH3或H。
3.根据权利要求1所述的小分子及其衍生物，其特征在于，所述小分子及其衍生物结构中：M为羟基。
4.根据权利要求3所述的小分子及其衍生物，其特征在于，所述小分子及其衍生物结构中：R为CH3或H；n为4-5的整数。
5.根据权利要求1-4任一所述的小分子及其衍生物，其特征在于，所述小分子为以下任一化合物：
6.根据权利要求1所述的小分子及其衍生物，其特征在于，所述药学上可接受的盐为所述小分子与酸形成的盐，所述酸为有机酸或无机酸。
7.一种权利要求1～6任一所述的小分子及其衍生物的制备方法，其特征在于，所述制备方法为：
甲酯类化合物6与邻苯二胺类化合物或羟胺类化合物反应得到化合物I；
其中，R、n的定义如权利要求1-5任一所述；
将相应的酸与以上方法制备所得的化合物I成盐，即得所述小分子的药学上可接受的盐。
8.一种药物组合物，其特征在于，所述药物组合物包含权利要求1～7任一所述小分子和/或其衍生物以及药学上可接受的载体。
9.一种权利要求1～7任一所述的小分子及其衍生物在制备抗肿瘤药物中的应用。
10.根据权利要9所述的应用，其特征在于，所述抗肿瘤药物为组蛋白去乙酰化酶抑制剂、拓扑异构酶抑制剂、p53通路激动剂中的至少一种。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111184936.0A CN113831346B (zh) | 2021-10-12 | 2021-10-12 | 多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111184936.0A CN113831346B (zh) | 2021-10-12 | 2021-10-12 | 多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113831346A true CN113831346A (zh) | 2021-12-24 |
| CN113831346B CN113831346B (zh) | 2022-06-14 |
Family
ID=78968641
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111184936.0A Active CN113831346B (zh) | 2021-10-12 | 2021-10-12 | 多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113831346B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102241678A (zh) * | 2011-04-26 | 2011-11-16 | 辽宁利锋科技开发有限公司 | 含有脂环结构化合物的抗肿瘤作用与应用 |
| CN105461723A (zh) * | 2015-12-22 | 2016-04-06 | 广西师范大学 | 酞嗪并[1,2,b]喹唑啉-8-酮化合物及其制备方法和在抗肿瘤药物中的应用 |
-
2021
- 2021-10-12 CN CN202111184936.0A patent/CN113831346B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102241678A (zh) * | 2011-04-26 | 2011-11-16 | 辽宁利锋科技开发有限公司 | 含有脂环结构化合物的抗肿瘤作用与应用 |
| CN105461723A (zh) * | 2015-12-22 | 2016-04-06 | 广西师范大学 | 酞嗪并[1,2,b]喹唑啉-8-酮化合物及其制备方法和在抗肿瘤药物中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| LIU JU ET AL.,: "Synthesis of new substituted 5-amino-8H-phthalazino[1,2-b]quinazolin-8-one derivatives", 《JOURNAL OF CHEMICAL RESEARCH》 * |
| ZHANG GUO-HAI ET AL.,: "Phthalazino[1,2‑b]quinazolinones as p53 Activators: Cell Cycle Arrest, Apoptotic Response and Bak−Bcl-xl Complex Reorganization in Bladder Cancer Cells", 《J. MED. CHEM.》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113831346B (zh) | 2022-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4405602B2 (ja) | ヒストン脱アセチル化酵素阻害剤 | |
| JP6940039B2 (ja) | Ｐａｒｐ阻害剤の製造方法、結晶型、及びその使用 | |
| JP2018513876A (ja) | Ｄｎａアルキル化剤 | |
| CA3012078A1 (en) | Selective estrogen receptor degraders and uses thereof | |
| KR20030001551A (ko) | ＩＬ-1β 및 ＴＮＦ-α 억제제로서의 벤조페논 | |
| KR20160103986A (ko) | N-벤질 트립탄트린 유도체, 및 이의 제조 방법 및 적용 | |
| CN107176933B (zh) | 一种含氮烷基化和芳基化亚砜亚胺的吲哚胺-2,3-双加氧酶抑制剂 | |
| CN106220641B (zh) | 含愈创兰烃薁结构的吲哚螺环类化合物及其制备方法与应用 | |
| WO2022174525A1 (zh) | 一类化合物及其制备方法和用途 | |
| CN107879975A (zh) | 组蛋白去乙酰化酶抑制剂及其应用 | |
| CN103319402A (zh) | 二苯基硫脲类衍生物及其制备方法和应用 | |
| CN105384738A (zh) | 作为蛋白激酶抑制剂的杂环类化合物及其制备方法和用途 | |
| CN113831346A (zh) | 多靶点抗肿瘤小分子及其衍生物、制法、药物组合物和应用 | |
| US11370766B2 (en) | Sulfonyl amidine as indoleamine-2,3-dioxygenase inhibitor, and preparation method therefor and use thereof | |
| TW201922690A (zh) | 環－amp反應元素結合蛋白的抑制劑 | |
| CN105601618B (zh) | 芳香族酰亚胺类化合物及其制备方法与应用 | |
| CN115490689A (zh) | 不可逆krasg12c抑制剂的制备及其应用 | |
| CN103288803B (zh) | 苯并咪唑酰胺类化合物及其制备方法和应用 | |
| TWI609013B (zh) | 用於治療癌症之新穎化合物 | |
| KR20210151849A (ko) | 퀴놀린 유도체 및 암의 치료를 위한 그의 용도 | |
| CN110028439B (zh) | 邻苯二甲酰亚胺类衍生物及其制备方法和用途 | |
| CN117756781B (zh) | 一种具有抗肿瘤作用的吲哚类组蛋白去乙酰化酶家族抑制剂 | |
| CN115340526B (zh) | 邻二甲酰亚胺类化合物及其药物组合物、制备方法和用途 | |
| ES2555802B1 (es) | Derivados de isoindolino-1,3-diona | |
| CN108467365B (zh) | 一种ido酶抑制剂化合物及其制备方法与应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |