CN109053682B - TDO small molecule inhibitor derivative, anti-tumor conjugate thereof and preparation method - Google Patents
TDO small molecule inhibitor derivative, anti-tumor conjugate thereof and preparation method Download PDFInfo
- Publication number
- CN109053682B CN109053682B CN201810845558.8A CN201810845558A CN109053682B CN 109053682 B CN109053682 B CN 109053682B CN 201810845558 A CN201810845558 A CN 201810845558A CN 109053682 B CN109053682 B CN 109053682B
- Authority
- CN
- China
- Prior art keywords
- tdo
- formula
- molecule inhibitor
- derivative
- small
- 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.)
- Expired - Fee Related
Links
- 239000003112 inhibitor Substances 0.000 title claims abstract description 50
- -1 TDO small molecule Chemical class 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 21
- 150000001875 compounds Chemical group 0.000 claims abstract description 102
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 44
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 29
- 229960004316 cisplatin Drugs 0.000 claims description 28
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 27
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 27
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 26
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 25
- 239000000376 reactant Substances 0.000 claims description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 229960004768 irinotecan Drugs 0.000 claims description 20
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 14
- 239000012046 mixed solvent Substances 0.000 claims description 13
- 238000010898 silica gel chromatography Methods 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 7
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 239000012317 TBTU Substances 0.000 claims description 6
- 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 claims description 6
- 230000003197 catalytic effect Effects 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N hydrogen peroxide Substances OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 4
- JRNVZBWKYDBUCA-UHFFFAOYSA-N N-chlorosuccinimide Chemical compound ClN1C(=O)CCC1=O JRNVZBWKYDBUCA-UHFFFAOYSA-N 0.000 claims description 4
- 239000003054 catalyst Substances 0.000 claims description 4
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000012265 solid product Substances 0.000 claims description 4
- 229940014800 succinic anhydride Drugs 0.000 claims description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 3
- XVCCOEWNFXXUEV-UHFFFAOYSA-N 2-pyridin-3-ylacetic acid;hydrochloride Chemical compound Cl.OC(=O)CC1=CC=CN=C1 XVCCOEWNFXXUEV-UHFFFAOYSA-N 0.000 claims description 3
- OLNJUISKUQQNIM-UHFFFAOYSA-N indole-3-carbaldehyde Chemical compound C1=CC=C2C(C=O)=CNC2=C1 OLNJUISKUQQNIM-UHFFFAOYSA-N 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000009833 condensation Methods 0.000 claims description 2
- 230000005494 condensation Effects 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 46
- 150000003384 small molecules Chemical class 0.000 abstract description 25
- 230000036039 immunity Effects 0.000 abstract description 5
- 101710136122 Tryptophan 2,3-dioxygenase Proteins 0.000 description 68
- 102000057288 Tryptophan 2,3-dioxygenases Human genes 0.000 description 68
- 210000004027 cell Anatomy 0.000 description 64
- YGPSJZOEDVAXAB-UHFFFAOYSA-N kynurenine Chemical compound OC(=O)C(N)CC(=O)C1=CC=CC=C1N YGPSJZOEDVAXAB-UHFFFAOYSA-N 0.000 description 44
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 37
- 230000000694 effects Effects 0.000 description 30
- 230000005764 inhibitory process Effects 0.000 description 27
- 239000000460 chlorine Substances 0.000 description 25
- 239000000243 solution Substances 0.000 description 24
- 238000012360 testing method Methods 0.000 description 24
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 22
- 230000002401 inhibitory effect Effects 0.000 description 18
- 238000011580 nude mouse model Methods 0.000 description 17
- 229940125904 compound 1 Drugs 0.000 description 13
- 230000012010 growth Effects 0.000 description 12
- 201000011510 cancer Diseases 0.000 description 11
- 229940079593 drug Drugs 0.000 description 11
- 239000003814 drug Substances 0.000 description 11
- 239000002207 metabolite Substances 0.000 description 11
- 230000001472 cytotoxic effect Effects 0.000 description 10
- 229910052697 platinum Inorganic materials 0.000 description 10
- 238000005160 1H NMR spectroscopy Methods 0.000 description 9
- 201000007270 liver cancer Diseases 0.000 description 9
- 208000014018 liver neoplasm Diseases 0.000 description 9
- 230000035755 proliferation Effects 0.000 description 9
- 210000004881 tumor cell Anatomy 0.000 description 9
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 8
- 239000002246 antineoplastic agent Substances 0.000 description 8
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 7
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 7
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 7
- 201000005202 lung cancer Diseases 0.000 description 7
- 208000020816 lung neoplasm Diseases 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 229960004799 tryptophan Drugs 0.000 description 7
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 6
- 206010009944 Colon cancer Diseases 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 208000029742 colonic neoplasm Diseases 0.000 description 6
- 210000002865 immune cell Anatomy 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 241000699660 Mus musculus Species 0.000 description 5
- 208000005718 Stomach Neoplasms Diseases 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 229940044683 chemotherapy drug Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 206010017758 gastric cancer Diseases 0.000 description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 201000011549 stomach cancer Diseases 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 229940125782 compound 2 Drugs 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000010998 test method Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000008076 immune mechanism Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 231100000053 low toxicity Toxicity 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical class 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
- 230000008859 change Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000005965 immune activity Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000005917 in vivo anti-tumor Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- NDBYXKQCPYUOMI-UHFFFAOYSA-N platinum(4+) Chemical compound [Pt+4] NDBYXKQCPYUOMI-UHFFFAOYSA-N 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000009790 rate-determining step (RDS) Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 210000003606 umbilical vein Anatomy 0.000 description 2
- BQUACMGZAUJSBF-RXCNPGAZSA-N 4-[[(z)-[7-[(8z)-8-[[(1,5-dimethyl-3-oxo-2-phenylpyrazol-4-yl)amino]methylidene]-1,6-dihydroxy-3-methyl-7-oxo-5-propan-2-ylnaphthalen-2-yl]-3,8-dihydroxy-6-methyl-2-oxo-4-propan-2-ylnaphthalen-1-ylidene]methyl]amino]-1,5-dimethyl-2-phenylpyrazol-3-one Chemical compound C12=C(O)C(C=3C(=C4C(=C/NC=5C(N(C=6C=CC=CC=6)N(C)C=5C)=O)/C(=O)C(O)=C(C(C)C)C4=CC=3C)O)=C(C)C=C2C(C(C)C)=C(O)C(=O)\C1=C/NC(C1=O)=C(C)N(C)N1C1=CC=CC=C1 BQUACMGZAUJSBF-RXCNPGAZSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 206010051779 Bone marrow toxicity Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- JJEDWBQZCRESJL-UHFFFAOYSA-N N-[(5-methyl-2-furanyl)methylideneamino]-2-phenoxybenzamide Chemical compound O1C(C)=CC=C1C=NNC(=O)C1=CC=CC=C1OC1=CC=CC=C1 JJEDWBQZCRESJL-UHFFFAOYSA-N 0.000 description 1
- BYHJHXPTQMMKCA-QMMMGPOBSA-N N-formyl-L-kynurenine Chemical compound [O-]C(=O)[C@@H]([NH3+])CC(=O)C1=CC=CC=C1NC=O BYHJHXPTQMMKCA-QMMMGPOBSA-N 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 231100000366 bone marrow toxicity Toxicity 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000007983 brain glioma Diseases 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000012038 nucleophile Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000006069 physical mixture Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 150000003058 platinum compounds Chemical class 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- GJAWHXHKYYXBSV-UHFFFAOYSA-N quinolinic acid Chemical compound OC(=O)C1=CC=CN=C1C(O)=O GJAWHXHKYYXBSV-UHFFFAOYSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0086—Platinum compounds
- C07F15/0093—Platinum compounds without a metal-carbon linkage
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
The invention relates to a TDO small molecule inhibitor derivative, an anti-tumor conjugate thereof and a preparation method thereof, wherein the compound structure of the TDO small molecule inhibitor derivative is shown as formulas I2 and I3,
in formula I, formula I1 is a known small molecule inhibitor of TDO; formula I2 and formula I3 are both derivatives of TDO small molecule inhibitor formula I1, abbreviated as TDO-OH. The structure of the conjugate is shown as formula II:
in the formula II-A, Y is Cl or OH; in the formulas II-A and II-B, TDO represents a derivative group of a TDO small molecule inhibitor I1. The invention is applied to the aspects of tumor resistance and immunity.
Description
Technical Field
The invention relates to an antitumor immunoconjugate containing a tryptophan 2, 3-dioxygenase (TDO) small molecule inhibitor, in particular to a compound for axially introducing a TDO small molecule inhibitor group in a tetravalent platinum octahedral structure containing a cis-platinum framework, and a compound for introducing the TDO small molecule inhibitor group on a hydroxyl group of an irinotecan active group; the invention also relates to a preparation method of the compound and application of the compound in the aspects of tumor resistance and immunity.
Background
Tryptophan is essential amino acid for human body, not only can synthesize protein, but also can be decomposed and metabolized through a canine uric acid channel, so that tryptophan is converted into a series of metabolites with biological activity, such as kynurenine, quinolinic acid and the like, and the rate-limiting step is the conversion of tryptophan into N-formyl kynurenine. Tryptophan 2, 3-dioxygenase (TDO) is one of the first step rate-limiting enzymes in the metabolic rate-limiting step of this pathway, and its expression is regulated by various signals such as glucocorticoid, L-tryptophan, kynurenine, etc. It was found that TDO is mainly expressed in the liver and regulates the levels of systemic tryptophan, and in addition to the liver, it is also expressed in brain sites. Research shows that TDO can participate in immune escape of tumors and promote growth of the tumors, and the TDO expression is detected in some tumor cells such as human brain malignant glioma, melanoma, colon cancer, liver cancer, lung cancer and the like. TDO can cause tryptophan deficiency and accumulation of biological metabolites by catalyzing the degradation of tryptophan, so that the proliferation of T lymphocytes is inhibited, even the apoptosis of the T lymphocytes can be caused, and the growth, migration and deterioration of tumor cells can be promoted. Because TDO plays a very important role in mediating tumor immune tolerance, immune escape and maintaining immune homeostasis in the body, TDO inhibitors have become one of the hot spots of recent research.
The TDO inhibitor can promote the activation and proliferation of T cells and improve the system immunity through the inhibition of the expression of the TDO protein, thereby reversing the tumor immunosuppression and playing the role of treating tumors. However, single immunotherapy has limited effectiveness, and combination therapy is therefore increasingly favored. The therapeutic method by coupling the TDO small-molecule inhibitor and the chemotherapeutic drug not only can exert the anti-cancer activity of the chemotherapeutic drug and the immune activity of immune small molecules, but also can overcome some defects of the chemotherapeutic drug.
Cisplatin, the first clinically used metal antineoplastic drug, mainly acts on guanine N7 site target of DNA, and forms an adduct by crosslinking with DNA, so that tumor cells undergo apoptosis, resulting in cell arrest and cell death. However, cisplatin drugs have several serious drawbacks: one is the corresponding toxicity, mainly renal toxicity and bone marrow toxicity; secondly, the drug resistance is generated after the drug is used, and the defects limit the application of the cisplatin bivalent platinum drugs to a certain extent. In recent years, tetravalent platinum complexes have attracted much attention due to their characteristics such as good stability and reduced reactivity with nucleophiles in vivo. Research shows that platinum (IV) ions can react with cancer cell DNA to cause apoptosis after being reduced in vivo to generate platinum (II) ions. Therefore, the functional ligand is introduced to the tetravalent platinum complex derived from the divalent platinum drug in the axial direction, so that the targeting property and the fat solubility of the divalent platinum can be improved, the toxic and side effects are overcome, and the antitumor activity of the compound is increased.
Irinotecan (Ir) is one of the antitumor drugs on the market in camptothecin derivatives, is also a semi-synthetic camptothecin derivative, exists in the form of hydrochloride, and has the characteristics of strong anticancer activity and good water solubility. Irinotecan has obvious curative effect on various cancers such as colon cancer, breast cancer, gastric cancer, leukemia and the like, and has wide application in China. Because of no obvious cross drug resistance with most common anticancer drugs, the compound is also commonly used as a combined drug with other drugs. With the progress of research, irinotecan has been recognized more and more as being effective against tumors, but it still has some drawbacks, such as poor stability, significant toxic and side effects, etc., and thus is very beneficial to the improvement of the pharmaceutical properties of irinotecan.
The invention aims to design and prepare a relative conjugate of a TDO small-molecule inhibitor and a chemotherapeutic drug cisplatin or irinotecan, and hopes to combine the advantages of chemotherapy and immunotherapy to obtain a drug which has high efficiency, low toxicity and the effect of improving immunity.
Disclosure of Invention
The technical problem is as follows: one of the purposes of the invention is to utilize the space structure of a tetravalent platinum complex and introduce a TDO small molecule inhibitor group at an axial position to obtain a tetravalent platinum compound which is coupled with a TDO small molecule inhibitor derivative and contains a cis-platinum framework; the other purpose of the invention is to utilize the spatial structure of irinotecan and introduce a TDO small-molecule inhibitor group at a hydroxyl position to obtain a compound containing an irinotecan skeleton and coupled with a TDO small-molecule inhibitor derivative; the invention also provides a preparation method of the compounds and application of the compounds in the aspects of tumor resistance and immunity.
The technical scheme is as follows: the invention provides a TDO small molecule inhibitor derivative, the compound structure of which is shown in formulas I2 and I3,
in formula I, formula I1 is a known small molecule inhibitor of TDO; formula I2 and formula I3 are both derivatives of TDO small molecule inhibitor formula I1, abbreviated as TDO-OH.
The preparation of TDO-OH is carried out according to a reaction formula shown in a formula V,
the TDO-OH is prepared as formula I2, and formula I3 adopts the following preparation method:
1) preparation of compound formula I1: adding 1.5 times of pyridine-3-acetic acid hydrochloride and 3.8 times of triethylamine into dry dioxane, stirring for 10 minutes at room temperature, adding equimolar indole-3-formaldehyde and 2.2 times of pyridine into reaction liquid, refluxing for 24 hours, cooling the reaction liquid to room temperature, concentrating the reaction liquid, and separating by silica gel column chromatography, wherein an eluent is a mixed solvent of petroleum ether and ethyl acetate (2:1), so as to obtain an orange product, namely a formula I1;
2) dissolving formula I1 in CH2Cl2Adding 1.5 times of succinic anhydride and catalytic amount of DMAP, reacting at 45 deg.C overnight, cooling to room temperature, concentrating the reaction solution, separating by silica gel column chromatography, eluting with CH2Cl2And CH3OH (20:1) mixed solvent to obtain a light yellow product, namely a formula I2;
3) dissolving formula I1 in CH2Cl2Adding 1.5 times of glutaric anhydride and catalytic amount of DMAP, reacting at 45 ℃ overnight, cooling to room temperature, concentrating the reaction solution, separating by silica gel column chromatography, wherein the eluent is CH2Cl2And CH3OH (20:1) mixed solvent to obtain a light yellow product, namely a formula I3.
The structure of the conjugate is shown as formula II:
in the formula II-A, Y is Cl or OH; in formulas II-A and II-B, TDO represents a derivative group of a TDO small molecule inhibitor I1.
The conjugate is carried out according to a reaction formula shown in III-A,
in the formula III-A, Y is Cl or OH, TBTU represents a coupling reagent O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate, TEA represents a catalyst triethylamine, and DMSO represents a solvent dimethylsulfoxide; TDO represents a derivative group of a TDO small molecule inhibitor I1, TDO-OH represents a derivative of a TDO small molecule inhibitor I1, and DMF represents a solvent N, N-dimethylformamide; pt (IV) reactant A represents cis, trans- [ Pt (NH)3)2Cl2(OH)Cl](ii) a Pt (IV) reactant B stands for cis, trans- [ Pt (NH)3)2Cl2(OH)2]。
The conjugate is carried out according to a reaction formula shown in a formula III-B,
in the formula III-B, EDCI represents a condensing agent 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, DMAP represents a catalyst 4-dimethylaminopyridine, TDO represents a derivative group of a TDO small-molecule inhibitor I1, TDO-OH represents a derivative of a TDO small-molecule inhibitor I1, and DMF represents a solvent N, N-dimethylformamide.
The preparation method specifically adopts the following steps:
III-A mixing equal mole of reactant TDO-OH and coupling reagent TBTU in anhydrous DMF or DMSO, stirring at room temperature, adding equal mole of TEA and then equal mole of Pt (IV) and reactant A (Y ═ Cl) or B (Y ═ OH), stirring at 30-60 deg.C for 12-48 hr under nitrogen protection, removing solvent under reduced pressure, separating the concentrated solution by silica gel column chromatography, elutingThe liquid is CH2Cl2And CH3OH mixed solvent to obtain yellow solid product;
the preparation method of the anti-tumor conjugate containing the TDO small-molecule inhibitor derivative specifically adopts the following steps:
III-B mixing equal molar reactant TDO-OH and condensation reagent EDCI in anhydrous DMF, stirring at room temperature, adding DMAP equal molar reactant TDO-OH, adding irinotecan equal molar reactant TDO-OH, stirring the reaction solution at room temperature for 12-48 hr, removing solvent under reduced pressure, separating the concentrated solution by silica gel column chromatography, eluting with CH2Cl2And CH3And (5) mixing the OH with the solvent to obtain a light yellow solid product.
The Pt (IV) reactants A and B have the structure shown in the formula IV,
wherein IV-A represents cis, trans- [ Pt (NH)3)2Cl2(OH)Cl]The cisplatin and the chlorosuccinimide are reacted in water to prepare the cisplatin modified cisplatin; IV-B represents cis, trans- [ Pt (NH)3)2Cl2(OH)2]The cisplatin-hydrogen peroxide is prepared by the reaction of cisplatin and hydrogen peroxide in water.
Has the advantages that:
(I) in vitro antitumor Activity
The prepared compound was evaluated for anti-tumor activity in vitro using human lung cancer cells A549 and NCI-H460 expressed by TDO, colon cancer cell HCT-116, liver cancer cell HepG-2 and gastric cancer cell SGC-7901 not expressed by TDO, and cisplatin and irinotecan were used as positive controls. The inhibition of tumor cell growth by the compounds at different concentrations was observed.
The experimental data for compounds 1-3 and T1-T4 containing TDO inhibitor groups are shown in Table 2. The compounds 1-3 have no toxicity to cancer cells, the inhibitory activities of the compounds T1 and T3 to the cancer cells A549, HepG-2, HCT-116, NCI-H460 and SGC-7901 are similar to those of cisplatin, and the inhibitory activities of the compounds T2 and T4 to the cancer cells are remarkableHigher than cisplatin, compound T2 has the highest activity and IC50IC values between 0.27-0.70. mu.M for HepG-250The value was 0.27. mu.M, which is higher than that of cisplatin (IC)50Value of 9.33 μ M) was about 35 times. The data in Table 2 show that the introduction of TDO inhibitor group leads the antitumor activity of the platinum (IV) complex to be obviously improved compared with that of cisplatin, and the activity of the compounds T2 and T4 containing hydroxyl groups in the axial direction is better than that of the compounds T1 and T3 containing chlorine atoms in the axial direction, and the activity of the compound T2 connected through succinic anhydride is higher than that of the compound T4 connected through glutaric anhydride.
The experimental data for compounds T5-T6 containing TDO inhibitor groups are shown in Table 3. The activity of the compounds T5 and T6 on cancer cells A549, HepG-2, HCT-116, NCI-H460 and SGC-7901 is greatly improved compared with that of irinotecan, wherein the IC of T550IC of T6 with a value of between 1.82 and 2.87. mu.M50Values between 1.99 and 3.86. mu.M, and less toxic to HUVEC normal cells than irinotecan. Among them, T5 and T6 showed the greatest cytotoxicity against cancer cell HepG-2, IC50The values were 1.82. mu.M and 1.99. mu.M, respectively. Experimental data show that irinotecan has a significant improvement in antitumor activity after the introduction of a TDO small-molecule inhibitor, and both target compounds have a greater improvement in the tested cancer cell inhibitory activity, with T5 being slightly better than T6.
The results show that the conjugates formed by introducing the TDO small-molecule inhibitor group to the platinum (IV) complex taking cisplatin as a parent and irinotecan show effective antitumor activity.
(II) research on related immune mechanisms
(1) Based on cytotoxicity studies, optimal compounds T2 and T5 were selected for TDO enzyme activity inhibition studies, as shown in FIG. 1, compounds 1, 2, T2 and T5 for enzyme inhibition IC50The values are 0.22. mu.M, 0.68. mu.M, 0.89. mu.M and 0.78. mu.M respectively, which shows that the derivative 2 of 1 and the coupling compounds T2 and T5 retain the biological activity of 1 and have obvious inhibitory effect on TDO.
(2) The level of TDO protein expression was determined on selected cancer cells, and as shown in FIG. 2, the expression of TDO was detected on all of the A549, HepG-2, HCT-116 and NCI-H460 cell lines, with the highest HepG-2 expression level, but not on the SGC-7901 cell line (data not shown). HepG-2 was therefore selected as a representative cell for subsequent in vitro immune mechanism studies.
(3) The inhibition of the expression level of TDO protein in HepG-2 cells by compounds T2 and T5 was evaluated, with cisplatin, irinotecan and compound 1 and its derivatives 2,3 as controls, and the results are shown in fig. 3. In FIG. 3A, cisplatin had no inhibitory effect on TDO expression, and Compound 1, a mixture of cisplatin and 1, Compound 2, and Compound T2 all showed inhibitory effects on the TDO protein, with Compound T2 being most effective; in fig. 3B, irinotecan had no inhibitory effect on TDO protein, compounds 1, 2 and 3 had more pronounced effects on TDO, and compounds T5 and T6 were more pronounced compared to compounds 1-3, with T5 being most preferred.
(4) Compounds T2 and T5 were chosen to evaluate the inhibition of the metabolite kynurenine produced by tryptophan, and compound 1 was used as a control. As shown in fig. 4(a and B), both compound 1 and T2 inhibited the expression level of kynurenine compared to the blank, wherein compound T2 was more effective than compound 1; compared with 1, the compound T5 shows that the content of kynurenine is obviously reduced, which indicates that the compound T5 has stronger inhibition effect on the kynurenine.
(5) The compounds T2 and T5 were selected to be mixed with HepG-2 and PBMCs (human peripheral blood mononuclear cells) cells, respectively, and cultured to detect the proliferation promoting effect on immune cells, and compound 1 was used as a control. The results are shown in FIG. 5(A and B), and the compounds tested were all against immune T cell CD4 compared to the blank group+And CD8+The cell has proliferation promoting effect, wherein the compounds T2 and T5 have better effect than compound 1, and the compound T5 has better effect than the mixture of compound 1 and irinotecan.
Combining the above experimental results, compounds T2 and T5 have better inhibitory effect on TDO, and also have better inhibitory effect on TDO protein overexpressed in HepG-2 cells, and the inhibition of TDO in cancer cells leads to the inhibition of the production of kynurenine, a metabolite, and as a result, the proliferation of T cells. These indicate that compounds T2 and T5 have potent immunological activity and may enhance the anti-tumor therapeutic effect of chemotherapeutic drugs.
(III) in vivo antitumor Activity
Cisplatin is used as a positive control, the inhibition effect of a compound T2(5, 10 and 20mg/kg) on the growth of human liver cancer cell HepG-2 nude mouse xenograft tumor is examined, the expression level of a metabolite kynurenine is tested, and the experimental result is shown in Table 4 (the inhibition effect of a tested sample on the growth of the human liver cancer cell HepG-2 nude mouse xenograft tumor). FIG. 6 shows the effect of the test sample on the growth volume change of human hepatoma cell HepG-2 nude mouse xenograft tumor, FIG. 7 shows the effect of the test sample on the growth of human hepatoma cell HepG-2 nude mouse xenograft tumor, FIG. 8 shows the effect of the test sample on the body weight of human hepatoma cell HepG-2 nude mouse xenograft tumor, and FIG. 9 shows the effect of the test sample on the expression level of kynurenine in human hepatoma cell HepG-2 nude mouse xenograft tumor. As can be seen from the data in Table 4, the tumor inhibition rate of compound T2 was slightly lower than that of cisplatin (5mg/kg) at the low dose of 5mg/kg and the intermediate dose of 10mg/kg, and that of compound T2 was slightly higher than that of cisplatin (5mg/kg) at the high dose of 20 mg/kg. As shown in fig. 6 and 7, the different doses of compound T2 and cisplatin significantly inhibited the growth of xenografts in nude mice, and the high dose of compound T2 was most effective, regardless of the volume or weight data of the transplants. It is noted that in fig. 8, the compound T2 with different doses has little effect on the body weight of the tested animals, while cisplatin has a greater effect on the body weight of the tested animals, which indicates that the compound T2 has less toxicity, high efficiency and low toxicity. Finally, as shown in fig. 9, compound T2 showed a dose-dependent inhibitory effect on the metabolite kynurenine, with the best high-dose inhibitory effect, indicating that compound T2 can inhibit the production of the metabolite kynurenine, indirectly indicating that compound T2 can promote the proliferation of T cells.
Drawings
FIG. 1. inhibitory Activity of test samples on TDO enzyme
FIG. 2 expression levels of TDO in selected cancer cells
FIG. 3. inhibitory Effect of test samples on TDO in HepG-2 cells
FIG. 4 inhibition of the metabolite kynurenine expression level by test samples
FIG. 5 test sample pair CD4+And CD8+Proliferation-promoting action of immune cells
FIG. 6 shows the effect of test samples on the growth volume change of human hepatoma cell HepG-2 nude mouse xenograft tumor
FIG. 7 shows the inhibitory effect of test samples on the growth of human hepatoma cell HepG-2 nude mouse xenograft tumor
FIG. 8 shows the effect of the test samples on the body weight of human hepatoma cell HepG-2 nude mouse xenograft tumor
FIG. 9 inhibitory Effect of test sample on the expression level of kynurenine, a metabolite in tumor tissues
Detailed Description
The structures of the compounds T1-T6 prepared by the invention are shown in Table 1,
TABLE 1 Structure of Compounds T1-T6
The molecular structure of the compound of the conjugate prepared by the method is determined by nuclear magnetic hydrogen spectrum and mass spectrum, and some compounds are characterized by nuclear magnetic carbon spectrum. In the mass spectrum of the compound containing platinum, multiple isotope peaks of platinum element appear in the excimer ion peak.
The conjugate shown in table 1 has a significant inhibitory effect on some human cancer cells. These cells include lung cancer, colon cancer, liver cancer, and gastric cancer cells. The in vitro immune related mechanism and T2 in vivo test of the compounds T2 and T5 show that the compound containing the TDO small molecule inhibitor group shows high-efficiency and low-toxicity antitumor immune activity and can be applied to the preparation of anticancer immune drugs.
The invention is further illustrated by the following examples, which are not intended to limit the invention. Some reactants include, unless otherwise indicated, the TDO small molecule inhibitor compound 1, cis, trans- [ Pt (NH)3)2Cl2(OH)Cl]Cis, trans- [ Pt (NH)3)2Cl2(OH)2]All prepared by literature methods.
(I) preparation of Compounds
EXAMPLE 1 preparation of Compound T1
118.4mg (0.37mmol) of Compound 2 and 118.8mg (0.37mmol) of TBTU are dissolved in 15mL of anhydrous DMF and stirred at room temperature for 10 minutes, then 37.4mg (0.37mmol) of TEA are added and stirring is continued for 5 minutes, and then 130.4mg (0.37mmol) of cis, trans- [ Pt (NH)3)2Cl3(OH)]And reacting for 48 hours at 50 ℃ under the protection of nitrogen. The reaction solution was concentrated, and the concentrated solution was separated by silica gel column chromatography using a mixed solvent of dichloromethane and methanol (10:1) as the eluent, to give 60mg of a yellow product in 30% yield.1HNMR(400MHz,DMSO-d6)8.83(d,J＝2.0Hz,1H),8.48–8.39(m,2H),8.28(s,1H),8.16– 8.08(m,2H),7.60(d,J＝16.7Hz,1H),7.46–7.36(m,4H),6.20(s,6H),3.26(t,J＝6.7Hz,2H),2.79(t,J＝6.6Hz,2H).13C NMR(100MHz,DMSO-d6)179.74,171.81,148.63,148.49,136.32, 133.71,132.80,128.65,125.72,125.54,125.47,124.27,122.32,120.70,119.49,116.70,31.84, 31.09.HRMS(ESI)m/z calculated for C19H21Cl3N4O3Pt[M–H]–:653.03537,found:653.03537.
EXAMPLE 2 preparation of Compound T2
With the reactants cis, trans- [ Pt (NH)3)2Cl2(OH)2]Instead of cis, trans- [ Pt (NH)3)2Cl2(OH)Cl]Prepared as described in example 1 to give a yellow product in 13% yield.
1H NMR(400MHz,DMSO-d6)8.83(s,1H),8.46(d,J＝4.6Hz,1H),8.42(d,J＝7.7Hz,1H), 8.28(s,1H),8.11(dd,J＝18.4,7.6Hz,2H),7.59(d,J＝16.7Hz,1H),7.44–7.37(m,4H),5.97 (dd,J＝64.1,38.7Hz,6H),3.23(t,J＝6.6Hz,2H),2.70(t,J＝6.6Hz,2H).13CNMR(100MHz, DMSO-d6)180.05,172.04,148.63,148.50,136.32,133.72,132.78,128.63,125.70,125.63, 125.41,124.27,124.24,122.35,120.70,119.41,116.70,31.94,31.14.HRMS(ESI)m/z calculated for C19H22Cl2N4O4Pt[M+H]+:637.07508,found:637.07508.
EXAMPLE 3 preparation of Compound T3
120.3mg (0.36mmol) of Compound 3Dissolved in 5mL of anhydrous DMF, 115.6mg (0.36mmol) of TBTU was added, and the mixture was stirred at room temperature for 5 minutes, then 36.4mg (0.36mmol) of TEA was added, and after stirring for another 10 minutes, 126.9mg (0.36mmol) of cis-, trans- [ Pt (NH)3)2Cl2(OH)Cl]And reacting at room temperature for 24 hours under the protection of nitrogen. The reaction mixture was concentrated, and the concentrate was subjected to silica gel column chromatography using a mixed solvent of methylene chloride and methanol (10:1) as an eluent to give 65mg of a yellow solid in 27% yield.
1H NMR(400MHz,DMSO-d6)8.84(s,1H),8.45(dd,J＝12.0,5.4Hz,2H),8.28(s,1H), 8.12(t,J＝7.0Hz,2H),7.61(d,J＝16.7Hz,1H),7.41(dt,J＝12.9,6.8Hz,4H),6.23(s,6H),3.18 (t,J＝7.3Hz,2H),2.42(t,J＝6.9Hz,2H),1.99–1.88(m,2H).13C NMR(100MHz,DMSO-d6) 180.22,172.53,162.79,148.62,148.51,136.21,133.68,132.79,128.68,125.74,125.66,125.43, 124.25,122.24,120.64,119.39,116.64,36.28,35.04,21.29.HRMS(ESI)m/z calculated for C20H23Cl3N4O3Pt[M–H]–:667.05207,found:667.05207.
EXAMPLE 4 preparation of Compound T4
With the reactants cis, trans- [ Pt (NH)3)2Cl2(OH)2]Instead of cis, trans- [ Pt (NH)3)2Cl2(OH)Cl]Prepared as described in example 3 to give a yellow product in 34.0mg as a yellow solid in 18% yield.
1H NMR(400MHz,DMSO-d6)8.84(d,J＝1.9Hz,1H),8.48–8.41(m,2H),8.30(s,1H),8.12(t,J＝8.4Hz,2H),7.61(d,J＝16.7Hz,1H),7.44–7.38(m,4H),6.15–5.90(m,6H),3.15(t, J＝7.5Hz,2H),2.34(t,J＝6.9Hz,2H),1.94–1.89(m,2H).13C NMR(100MHz,DMSO-d6)180.75,172.65,148.61,148.52,136.20,133.72,132.79,128.67,125.86,125.72,125.37,124.26, 122.32,120.67,119.35,116.64,35.98,35.18,21.52.HRMS(ESI)m/z calculatedfor C20H24Cl2N4O4Pt[M+H]+:651.24809,found:651.24809.
EXAMPLE 5 preparation of Compound T5
99.2mg (0.31mmol) of Compound 2 and 59.4mg (0.31mmol) of EDCI are dissolved in 10mL of anhydrous DMF and stirred at room temperature, 37.9mg (0.31mmol) of DMAP is added, and after stirring for 5 minutes, 180.0mg (0.31mmol) of irinotecan is added and reacted at room temperature for 24 hours. The reaction solution was concentrated, and the concentrated solution was separated by silica gel column chromatography, eluting with a mixed solvent of dichloromethane and methanol (20:1), to give 87mg of a pale yellow solid, yield: 51.7 percent.
1H NMR(600MHz,CDCl3)8.68(d,J＝1.7Hz,1H),8.46(dd,J＝4.7,1.3Hz,1H),8.32(d, J＝9.1Hz,1H),8.25(d,J＝8.1Hz,1H),7.87(d,J＝2.3Hz,1H),7.79(d,J＝7.9Hz,1H),7.72(d, J＝7.9Hz,1H),7.62(dd,J＝9.1,2.4Hz,1H),7.55(s,1H),7.50(s,1H),7.27(d,J＝6.7Hz,1H), 7.18–7.11(m,2H),7.08(d,J＝16.5Hz,1H),6.65(t,J＝7.9Hz,1H),5.68(d,J＝16.9Hz,1H), 5.38(d,J＝16.9Hz,1H),5.22–5.12(m,2H),4.45(dd,J＝59.9,12.9Hz,2H),3.37(ddd,J＝15.5, 9.3,5.2Hz,1H),3.26–3.17(m,2H),3.12(dd,J＝13.3,7.5Hz,2H),2.98–2.91(m,2H),2.73(s, 6H),2.28(dd,J＝14.1,7.4Hz,1H),2.17(dd,J＝14.2,7.4Hz,1H),2.10(d,J＝11.0Hz,1H),2.04 (d,J＝8.0Hz,1H),1.76(s,6H),1.53(s,2H),1.37(t,J＝7.6Hz,3H),1.03(t,J＝7.5Hz,3H).13C NMR(150MHz,CDCl3)171.55,169.15,167.70,157.38,153.16,151.61,150.47,148.42,148.19, 147.17,146.73,146.29,145.23,136.36,133.09,132.39,131.81,128.13,127.51,127.18,125.98, 125.82,125.47,124.03,123.59,122.70,121.61,120.43,119.64,119.31,116.80,114.62,96.76,76.55,66.99,62.34,50.22,49.27,44.43,44.09,31.59,30.60,29.70,28.35,28.21,27.49,26.05, 24.54,23.15,14.00,7.67.HRMS(ESI)m/z calculated for C52H52N6O8[M+H]+:889.37752,found: 889.37752.
EXAMPLE 6 preparation of Compound T6
Prepared as described in example 5, substituting reactant 3 for reactant 2, to give a pale yellow product, 92 mg of yellow solid, 43.4% yield.
1H NMR(600MHz,CDCl3)8.67(d,J＝1.7Hz,1H),8.45(dd,J＝4.7,1.3Hz,1H),8.32(d, J＝9.1Hz,1H),8.25(d,J＝8.0Hz,1H),7.87(d,J＝2.4Hz,1H),7.78(d,J＝8.0Hz,1H),7.72(d, J＝7.9Hz,1H),7.63(dd,J＝9.1,2.4Hz,1H),7.55(s,1H),7.50(s,1H),7.28–7.24(m,1H),7.19 –7.12(m,2H),7.08(d,J＝16.5Hz,1H),6.70–6.58(m,1H),5.67(d,J＝16.9Hz,1H),5.38(d,J ＝16.9Hz,1H),5.17(dd,J＝45.1,18.6Hz,2H),4.48(d,J＝13.0Hz,1H),4.38(d,J＝12.6Hz, 1H),3.41–3.31(m,1H),3.27–3.16(m,2H),3.12(dd,J＝13.6,6.9Hz,2H),2.98–2.89(m,2H), 2.61(s,5H),2.45(s,3H),2.29–2.24(m,1H),2.16(dd,J＝14.2,7.4Hz,1H),1.99(t,J＝14.3Hz, 2H),1.69–1.60(m,6H),1.49(s,2H),1.37(t,J＝7.7Hz,3H),1.03(t,J＝7.5Hz,3H).13C NMR (150MHz,CDCl3)171.56,169.15,167.70,157.37,153.08,151.67,150.31,148.38,148.15, 147.20,146.71,146.28,145.26,136.35,133.11,132.42,131.86,128.13,127.49,127.21,125.87, 125.80,125.45,124.03,123.62,122.73,121.62,120.41,119.65,119.33,116.79,114.69,96.77, 76.54,66.99,62.75,50.08,49.27,44.05,43.69,31.57,31.44,30.61,30.19,29.70,28.36,27.55, 26.73,24.80,23.77,23.15,14.01,7.68.HRMS(ESI)m/z calculated forC53H54N6O8[M+H]+: 903.37503,found:903.37503.
EXAMPLE 7 preparation of Compounds 1-3
(1) Preparation of Compound 1
Synthesis of Compound 1 according to the literature method (J.Med. chem.2011,54,5320-5334), 2.7g (15.5mmol) of pyridine-3-acetic acid hydrochloride and 3.9g (38.5mmol) of triethylamine are added into dried dioxane, stirred at room temperature for 10 minutes, 1.5g (10.3mmol) of indole-3-formaldehyde and 1.9g (22.3mmol) of pyridine are added into the reaction solution, reflux reaction is carried out for 24 hours, the reaction solution is cooled to room temperature, the reaction solution is concentrated, and separation by column chromatography is carried out to obtain an orange-yellow product 1, and the eluent is petroleum ether and ethyl acetate is a mixed solvent of 2: 1;
1H NMR(400MHz,DMSO-d6)11.42(s,1H),8.77(d,J＝2.1Hz,1H),8.38(dd,J＝4.7,1.5 Hz,1H),8.06(d,J＝7.8Hz,1H),8.02(dt,J＝8.0,1.9Hz,1H),7.69(d,J＝2.6Hz,1H),7.57(d,J ＝16.6Hz,1H),7.45(d,J＝8.0Hz,1H),7.36(dd,J＝7.9,4.7Hz,1H),7.21–7.17(m,1H),7.16– 7.10(m,2H).HRMS(ESI)m/z calculated for C15H12N2[M+H]+:221.10375,found:221.10375.
(2) preparation of Compound 2
1.0g (4.5mmol) of 1 are dissolved in 50mL of CH2Cl2Adding 0.9g (9.0mmol) of succinic anhydride and a catalytic amount of DMAP, reacting at 45 ℃ overnight, cooling to room temperature, concentrating the reaction solution, and performing column chromatography separation to obtain a light yellow product 2, wherein the eluent is a mixed solvent of dichloromethane and methanol in a ratio of 20: 1.
1H NMR(400MHz,DMSO-d6)12.27(s,1H),8.83(d,J＝2.1Hz,1H),8.46(dd,J＝4.7,1.5 Hz,1H),8.40(dd,J＝7.2,1.4Hz,1H),8.32(s,1H),8.13(dd,J＝6.8,1.6Hz,1H),8.10(dt,J＝7.9, 1.8Hz,1H),7.58(d,J＝16.7Hz,1H),7.44–7.38(m,4H),3.32(d,J＝6.8Hz,2H),2.72–2.69(m, 2H).HRMS(ESI)m/z calculated for C19H16N2O3[M+H]+:321.12104,found:321.12104.
(3) Preparation of Compound 3
1.0g (4.5mmol) of 1 are dissolved in 50mL of CH2Cl2Adding 1.0g (9mmol) of glutaric anhydride and a catalytic amount of DMAP, reacting at 45 ℃ overnight, cooling to room temperature, concentrating the reaction solution, and performing column chromatography separation to obtain a light yellow product 3, wherein the eluent is a mixed solvent of dichloromethane and methanol in a ratio of 20: 1.
1H NMR(400MHz,DMSO-d6)12.15(s,1H),8.82(d,J＝2.0Hz,1H),8.46(dd,J＝4.7,1.3 Hz,1H),8.43(d,J＝7.9Hz,1H),8.25(s,1H),8.11(dd,J＝16.2,7.7Hz,2H),7.57(d,J＝16.7Hz, 1H),7.43–7.36(m,4H),3.13(t,J＝7.3Hz,2H),2.41(t,J＝7.3Hz,2H),1.96(dd,J＝14.6,7.3 Hz,2H).HRMS(ESI)m/z calculated for C20H18N2O3[M+H]+:335.14297,found:335.14297.
(II) in vitro cytotoxic Activity assay of Compounds
The experimental method comprises the following steps: the compound T1-T6 prepared by the invention is tested for cytotoxic activity by MTT method. Cells in the logarithmic growth phase were counted and plated in 96-well plates at about 8000-10000 cells per well. Overnight culture, and after the cells adhere to the wall, the cells are administered, and an administration group, a positive control group and a negative control group are respectively arranged. Chemical combination to be testedThe stock solutions were prepared in saline, DMF or DMSO and diluted to a range of concentrations in cell culture medium just prior to use, with the final concentration of DMF or DMSO not exceeding 4 ‰ (the same applies to the experiments below). Each concentration was provided with 3 multiple wells. After addition of the reagent, the cells were cultured for 72 hours, 20. mu.L of MTT at a concentration of 5mg/mL was added, the cells were incubated at 37 ℃ for 4 hours, the supernatant was removed, and 150. mu.L of DMSO was added to dissolve formazan. Measuring the OD value of each hole by using a microplate reader at 490 wavelength, calculating the inhibition rate, and calculating the IC by making a concentration-inhibition rate curve50The value is obtained.
Test example 1 cytotoxic Activity of Compounds T1-T4
The cytotoxic activity of the compounds 1-3 and T1-T4 against liver cancer cell HepG-2, colon cancer cell HCT-116, lung cancer cell A549, lung cancer cell NCI-H460, stomach cancer cell SGC-7901 and human umbilical vein endothelial cell HUVEC was tested, and cisplatin was used as a positive control. Observing the inhibition of the compound on the growth of tumor cells under different concentrations, and calculating the inhibition rate and IC thereof50The cytotoxic activity of the compounds was evaluated and the results are shown in table 2.
TABLE 2 cytotoxic Activity of Compounds T1-T4
Test example 2 cytotoxic Activity of Compounds T5-T6
The cytotoxic activity of the compound T5-T6 on liver cancer cell HepG-2, colon cancer cell HCT-116, lung cancer cell A549, lung cancer cell NCI-H460, stomach cancer cell SGC-7901 and human umbilical vein endothelial cell HUVEC was tested, and irinotecan (Ir) and its physical mixture (molar ratio 1:1) with the compound 1 were used as positive controls. Observing the inhibition of the compound on the growth of tumor cells under different concentrations, and calculating the inhibition rate and IC thereof50The cytotoxic activity of the drug was evaluated and the results are shown in table 3.
TABLE 3 cytotoxic Activity of Compounds T5-T6
(III) study of the relevant immune mechanisms
Test example 1 inhibition of TDO enzyme by Compounds
The experimental method comprises the following steps: the inhibitory enzyme activity of the related compounds was evaluated by measuring the level of kynurenine, a product of TDO catalysis, by a microplate reader. A96-well plate was prepared, and after thawing the TDO reaction solution, 180. mu.L of the solution was added to the plate per well, 10. mu.L of the compound-containing DMF solution was added as a drug administration group, and 10. mu.L of the blank DMF solution was added as a positive control group and a negative control group, respectively. Adding 10 μ L TDO buffer solution into negative control group, adding 10 μ L TDO enzyme solution into administration group and positive control group, incubating at room temperature for 90 min, measuring OD value of each well at 321 wavelength with enzyme labeling instrument, calculating inhibition rate, and calculating IC by making concentration-inhibition rate curve50The value is obtained. Testing the inhibition ability of the compounds 1, 2, T2 and T5 on enzyme, observing the inhibition of the compounds on the enzyme in different compounds, and calculating the inhibition rate and IC thereof50The enzyme activity of the drug was evaluated and the results are shown in FIG. 1.
Test example 2 expression level of TDO in tumor cells
The test method comprises the following steps: the WB method was used to detect the level of TDO expression in tumor cells studied in accordance with the invention. Cells in logarithmic growth phase were taken and inoculated in 6-well culture plates. After 24 hours, the cells were harvested, the extracted protein was isolated, and the protein concentration was measured by BCA protein assay reagent. Each aliquot of protein was fractionated on a 12% SDS polyacrylamide gel electrophoresis and transferred to a PVDF Hybond-P membrane (GE Healthcare). Membranes were incubated with Tween 20(TBST) buffer and Tris buffered saline containing 5% skim milk for 1 hour, then spun slowly overnight at 4 ℃. The membrane was then incubated with primary antibody overnight at 4 ℃. Next, the membrane was incubated with peroxidase-labeled secondary antibody at RT (25 ℃) for 2 hours. Western blots were detected by chemiluminescence reagent (Thermo Fischer sciences Ltd.). Beta-actin was used as a control and the results are shown in figure 2.
Experimental example 3 inhibition of TDO expression levels in tumor cells by Compounds T2 and T5
The test method comprises the following steps: representative compounds prepared according to the present invention were evaluated for their ability to inhibit TDO in tumor cells with high expression of TDO using the WB method. The HepG-2 cells with the best activity of the compounds T2 and T5 and high expression of the TDO protein are selected for research. After incubating the compounds with HepG-2 cells for 24 hours, the cells were collected and the experimental treatment was performed according to test example 2, and the results are shown in FIG. 3.
Test example 4 Effect of Compounds T2 and T5 on the expression level of the metabolite kynurenine
The test method comprises the following steps: the representative compounds prepared by the present invention were evaluated for the expression level of kynurenine, a metabolite, by HPLC. Taking cells in logarithmic growth phase, inoculating the cells into a 6-well culture plate, culturing until the cells are attached to the wall, and then administering, adding compound T2, T5 and 1 solution respectively, and using a blank group as a control. After 48 hours, the cells were collected by centrifugation, 100. mu.L of 20% trichloroacetic acid was added to precipitate the protein, the supernatant was collected by centrifugation, and the sample was assayed by HPLC at an absorbance of 360nm to detect the expression level of kynurenine, and the results are shown in FIG. 4.
Test example 5 Compound Pair CD4+And CD8+Proliferation of immune cells
The test method comprises the following steps: the proliferation-promoting effect of the compounds T2 and T5 of the present invention on immune cells was tested by the Mixed Leukocyte Reaction (MLR) method. HepG-2 cells in the logarithmic phase of growth were seeded in 6-well plates, cultured for 24 hours, replaced with fresh medium, and then co-incubated at 37 ℃ with the addition of compounds. After two days of incubation, human Peripheral Blood Mononuclear Cells (PBMCs) were embedded, mixed and incubated for 6 days, and PBMCs were harvested by centrifugation, stained with monoclonal antibodies APC-anti CD4 and PE-anti CD8, and then assayed by flow analysis. Test CD4+And CD8+The number of immune cells after administration, compound 1 and blank were used as controls and the results are shown in fig. 5.
(IV) in vivo antitumor Activity of Compound T2
The experimental method comprises the following steps: the nude mice are adopted to evaluate the inhibition effect and the action intensity of the tested sample on the growth of human liver cancer cell HepG-2 nude mouse xenograft tumor and the inhibition effect of kynurenine. The test animals were: BALB/c nude mice, supplied by Shanghai Ling Biotech, Inc. Production license of experimental animal: SCXK (Shanghai) 2013-0018, certificate number: 2013001829927, laboratory animal use license: SYXK (Su) 2012-. The age in days: 5w, body weight: 18-20g, sex: female, number of animals: each group had 5, 25 total. Drugs and reagents: cisplatin and compound T2, cisplatin dissolved in normal saline, T2 dissolved in appropriate amount of DMF and normal saline by ultrasound.
The groups and dosing schedule were as follows:
model control group: tail vein injection of physiological saline, 0.1ml/10g, once per week for 4 times;
cisplatin (5 mg/kg): tail vein injecting 5mg/kg medicinal solution 0.1ml/10g once a week for 4 times;
t2(5 mg/kg): tail vein injecting 5mg/kg medicinal solution 0.1ml/10g once a week for 4 times;
t2(10 mg/kg): tail vein injecting 10mg/kg medicinal solution, 0.1ml/10g, once per week for 4 times;
t2(20 mg/kg): tail vein injecting 20mg/kg medicinal solution, 0.1ml/10g, once per week for 4 times;
collecting cultured human liver cancer HepG-2 cell suspension with concentration of 1 × 107one/mL, 0.1mL each, was inoculated subcutaneously into the right axilla of nude mice. The diameter of the transplanted tumor of the nude mouse is measured by a vernier caliper, and the tumor grows to 100-150mm after 18 days of inoculation3Animals were randomly grouped into groups of 5 animals each. Meanwhile, each group of nude mice starts to be dosed, the dosing scheme is shown in the group and the dosing scheme, and the antitumor effect of the tested sample is dynamically observed by using a method for measuring the tumor size. After the experiment, the nude mice were sacrificed immediately, and the tumor mass was removed by surgery and weighed.
The formula for Tumor Volume (TV) is: TV 1/2ab2Wherein a and b represent length and width, respectively.
Calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows:
RTV＝Vt/V0in which V is0When administered separately from the cage (i.e. d)0) Measurement of the resulting tumor volume, VtFor the tumor volume at each measurement. Evaluation indexes of antitumor activity: relative tumor proliferation rateT/C (%), the formula:
wherein, TRTV: treatment group RTV; cRTV: model set RTV.
Evaluation indexes of antitumor activity: the tumor growth inhibition (%) is calculated as follows:
the mean values are represented by X + -SD, the analysis between groups is statistically processed by t-test, and the results are statistically analyzed using SPSS (Standard Package for the Social science)17.0, and the results are shown in Table 4 and FIGS. 6-8.
Tumor tissues of the mice in each group are taken respectively, the tumor tissues are homogenized by perchloric acid containing 01 percent of ascorbic acid and 0.1 percent of EDTA to release the kynurenine, and after ten minutes, the supernatant is collected by centrifugation, and the content of the kynurenine in the samples is detected by HPLC. The expression level of kynurenine in each group of samples was tested, and the kynurenine level in the tumor region of mice after administration was analyzed to indirectly detect the proliferation level of immune cells, and the results are shown in fig. 9.
TABLE 4 inhibitory Effect of test samples on the growth of human hepatoma cell HepG-2 nude mouse xenograft tumors
P <0.05, P <0.01 compared to model control.
Claims (9)
1. A TDO small molecule inhibitor derivative is characterized in that the compound structure of the TDO small molecule inhibitor derivative is shown as formulas I2 and I3,
in the formula I, the formula I2 and the formula I3 are both derivatives of TDO small molecule inhibitors, and are abbreviated as TDO-OH.
2. A method for preparing TDO small molecule inhibitor derivative as claimed in claim 1, wherein the preparation of TDO-OH is performed according to the reaction formula shown in formula V,
3. the process for preparing the derivative of TDO small molecule inhibitor according to claim 2, wherein the TDO-OH is represented by formula I2, formula I3 is prepared as follows:
1) preparation of compound formula I1: adding 1.5 times of pyridine-3-acetic acid hydrochloride and 3.8 times of triethylamine into dried dioxane, stirring for 10 minutes at room temperature, adding equimolar indole-3-formaldehyde and 2.2 times of pyridine into reaction liquid, refluxing for 24 hours, cooling the reaction liquid to room temperature, concentrating the reaction liquid, separating by silica gel column chromatography, wherein eluent is petroleum ether and ethyl acetate is a mixed solvent of 2:1 to obtain an orange product, namely a formula I1;
2) dissolving formula I1 in CH2Cl2Adding 1.5 times of succinic anhydride and catalytic amount of DMAP, reacting at 45 deg.C overnight, cooling to room temperature, concentrating the reaction solution, separating by silica gel column chromatography, eluting with CH2Cl2And CH3OH is a mixed solvent of 20:1 to obtain a light yellow product, namely a formula I2;
3) dissolving formula I1 in CH2Cl2Adding 1.5 times of glutaric anhydride and catalytic amount of DMAP, reacting at 45 ℃ overnight, cooling to room temperature, concentrating the reaction solution, separating by silica gel column chromatography, wherein the eluent is CH2Cl2And CH3OH is 20:1 mixed solvent to obtain a light yellow product, namely formula I3.
4. A class of anti-tumor conjugates comprising the TDO small molecule inhibitor derivative of claim 1, wherein the structure of said conjugates is represented by formula II:
in the formula II-A, Y is Cl or OH; in the formulas II-A and II-B, TDO represents a group of a derivative of a TDO small molecule inhibitor shown as a formula I2 or a formula I3, which loses hydroxyl.
5. A method for preparing the anti-tumor conjugate containing the TDO small-molecule inhibitor derivative according to claim 4, wherein the conjugate is prepared according to the reaction formula shown in III-A,
in the formula III-A, Y is Cl or OH, TBTU represents a coupling reagent O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate, TEA represents a catalyst triethylamine, and DMSO represents a solvent dimethylsulfoxide; TDO-OH represents a derivative of the TDO small-molecule inhibitor shown in a formula I2 or a formula I3, TDO represents a group of the derivative of the TDO small-molecule inhibitor shown in a formula I2 or a formula I3, wherein hydroxyl is lost, and DMF represents N, N-dimethylformamide serving as a solvent; pt (IV) reactant A represents cis, trans- [ Pt (NH)3)2Cl2(OH)Cl](ii) a Pt (IV) reactant B stands for cis, trans- [ Pt (NH)3)2Cl2(OH)2]。
6. A method for preparing the anti-tumor conjugate containing the TDO small-molecule inhibitor derivative according to claim 4, wherein the conjugate is prepared according to the reaction formula shown in the formula III-B,
in the formula III-B, EDCI represents a condensing agent 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, DMAP represents a catalyst 4-dimethylaminopyridine, TDO-OH represents a derivative of a TDO small-molecule inhibitor shown in a formula I2 or a formula I3, TDO represents a group of a derivative of the TDO small-molecule inhibitor shown in a formula I2 or a formula I3 which loses a hydroxyl group, and DMF represents a solvent N, N-dimethylformamide.
7. The method for preparing the anti-tumor conjugate containing the derivative of the TDO small-molecule inhibitor as claimed in claim 5, wherein the method specifically comprises the following steps:
III-A, mixing equal molar reactant TDO-OH and coupling reagent TBTU in anhydrous DMF or DMSO, stirring at room temperature, adding equal molar reactant TDO-OH TEA, then equal molar reactant TDO-OH Pt (IV) reactant A or B, stirring the reaction solution at 30-60 deg.C for 12-48 hr under nitrogen protection, removing solvent under reduced pressure, separating the concentrated solution by silica gel column chromatography, eluting with CH2Cl2And CH3OH is mixed with the solvent to obtain a yellow solid product.
8. The method for preparing the anti-tumor conjugate containing the derivative of the TDO small-molecule inhibitor as claimed in claim 6, wherein the method specifically comprises the following steps:
III-B mixing equal molar reactant TDO-OH and condensation reagent EDCI in anhydrous DMF, stirring at room temperature, adding DMAP equal molar reactant TDO-OH, adding irinotecan equal molar reactant TDO-OH, stirring the reaction solution at room temperature for 12-48 hr, removing solvent under reduced pressure, separating the concentrated solution by silica gel column chromatography, eluting with CH2Cl2And CH3And (5) mixing the OH with the solvent to obtain a light yellow solid product.
9. The method for preparing the anti-tumor conjugate containing the TDO small-molecule inhibitor derivative as claimed in claim 5, wherein the Pt (IV) reactants A and B have the structure shown in formula IV,
wherein IV-A represents cis, trans- [ Pt (NH)3)2Cl2(OH)Cl]The cisplatin and the chlorosuccinimide are reacted in water to prepare the cisplatin modified cisplatin; IV-B represents cis, trans- [ Pt (NH)3)2Cl2(OH)2]The cisplatin-hydrogen peroxide is prepared by the reaction of cisplatin and hydrogen peroxide in water.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810845558.8A CN109053682B (en) | 2018-07-27 | 2018-07-27 | TDO small molecule inhibitor derivative, anti-tumor conjugate thereof and preparation method |
| PCT/CN2018/100399 WO2020019376A1 (en) | 2018-07-27 | 2018-08-14 | Tdo small molecule inhibitor derivative and antitumor conjugate thereof, and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810845558.8A CN109053682B (en) | 2018-07-27 | 2018-07-27 | TDO small molecule inhibitor derivative, anti-tumor conjugate thereof and preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109053682A CN109053682A (en) | 2018-12-21 |
| CN109053682B true CN109053682B (en) | 2020-10-27 |
Family
ID=64835983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810845558.8A Expired - Fee Related CN109053682B (en) | 2018-07-27 | 2018-07-27 | TDO small molecule inhibitor derivative, anti-tumor conjugate thereof and preparation method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN109053682B (en) |
| WO (1) | WO2020019376A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110229112B (en) * | 2019-07-03 | 2021-05-04 | 南京君若生物医药研究院有限公司 | Anti-tumor immunoregulation conjugate and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102481285A (en) * | 2009-06-23 | 2012-05-30 | 国家科学研究中心 | Use of derivatives of indoles for the treatment of cancer |
| CN105622674A (en) * | 2016-02-29 | 2016-06-01 | 东南大学 | Tetravalent platinum complex with bioactive group and preparation method of tetravalent platinum complex |
| CN108164520A (en) * | 2018-01-15 | 2018-06-15 | 东南大学 | The coupling compound and its preparation of anoxic inhibitor and antitumor drug and application |
-
2018
- 2018-07-27 CN CN201810845558.8A patent/CN109053682B/en not_active Expired - Fee Related
- 2018-08-14 WO PCT/CN2018/100399 patent/WO2020019376A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102481285A (en) * | 2009-06-23 | 2012-05-30 | 国家科学研究中心 | Use of derivatives of indoles for the treatment of cancer |
| CN105622674A (en) * | 2016-02-29 | 2016-06-01 | 东南大学 | Tetravalent platinum complex with bioactive group and preparation method of tetravalent platinum complex |
| CN108164520A (en) * | 2018-01-15 | 2018-06-15 | 东南大学 | The coupling compound and its preparation of anoxic inhibitor and antitumor drug and application |
Non-Patent Citations (3)
| Title |
|---|
| A Pt(IV) Pro-drug Preferentially Targets Indoleamine-2,3-dioxygenase, Providing Enhanced Ovarian Cancer Immuno-Chemotherapy;Awuah,Samuel G等;《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》;20151112;第137卷(第47期);全文 * |
| Phosphine-Mediated Coupling of Gramines with Aldehydes: A Remarkably Simple Synthesis of 3-Vinylindoles;Kam Hung Low等;《ORGANIC LETTERS》;20050421;第7卷(第10期);全文 * |
| Tryptophan 2,3-Dioxygenase (TDO) Inhibitors. 3-(2-(Pyridyl)ethenyl)indoles as Potential Anticancer Immunomodulators;Eduard Dolusic等;《Journal of Medicinal Chemistry》;20110704;第54卷(第15期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109053682A (en) | 2018-12-21 |
| WO2020019376A1 (en) | 2020-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105622674B (en) | A kind of tetravalence platinum complex containing bio-active group and preparation method thereof | |
| KR20200067132A (en) | Programmable polymeric drugs | |
| CN110981870B (en) | Beta-carboline-cycloenone derivative based on dual responses of pH and GSH and application thereof | |
| KR100442096B1 (en) | Antitumor derivative of double dicarboxylic acid diaminoplatin complex, process for the preparing thereof, the pharmaceutical composition containing the same and application of the derivative | |
| CN111393483B (en) | Tetravalent platinum naphthalimide complex, preparation method and application thereof | |
| CN101948500A (en) | New derivative of camptothecin 20-site coupled bile acid | |
| CN104163823B (en) | camptothecin and artesunate conjugate as well as preparation method and application thereof | |
| CN114605475B (en) | Oral Pt (IV) anticancer prodrugs containing 3-bromopyruvate ligand axially | |
| CN109053682B (en) | TDO small molecule inhibitor derivative, anti-tumor conjugate thereof and preparation method | |
| CN112940059B (en) | Glycosyl modified naphthalimide-polyamine conjugate, preparation method and application thereof | |
| Jaycox et al. | Potential DNA bis‐intercalating agents: Synthesis and antitumor activity of novel, conformationally restricted bis (9‐aminoacridines) | |
| KR101138438B1 (en) | Methods for Preparing Powder Chlorophyll a and Photosensitizer from Spirulina | |
| CN101402667A (en) | Glycosylation modified nitric oxide donor type oleaolic acid compounds, preparation and uses thereof | |
| CN110229112B (en) | Anti-tumor immunoregulation conjugate and preparation method and application thereof | |
| CN114835759A (en) | Melatonin-platinum (IV) -carbon nitrogen long-chain complex, preparation method and application thereof in tumor drugs | |
| CN114177177A (en) | Preparation method of hypoxia tumor selective activation prodrug | |
| CN114163479A (en) | Platinum compounds for treating cancer and preparation method thereof | |
| KR101106756B1 (en) | Methods for preparing powder chlorophyll a and photosensitizer from spirulina | |
| CN114933599B (en) | Double beta-carboline compound and medicinal salt, preparation method and application thereof | |
| CN113929639B (en) | Antitumor compound with GSTP1 as target spot, and preparation method and application thereof | |
| CN115894490B (en) | Hydroxy evodiamine and erlotinib split drug containing indoloquinone unit, and preparation method and application thereof | |
| CN110577501A (en) | indoleamine 2, 3-dioxygenase modulators, method for the production and use thereof | |
| KR101138437B1 (en) | Methods for Preparing Powder Chlorophyll a and Photosensitizer from Spirulina | |
| CN112608327B (en) | Furanoquinoline derivative, preparation method and application thereof | |
| EP1758904B1 (en) | Flavopereirine derivatives for cancer therapy |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20201027 |