CN110003304B - Water-soluble triptolide derivative and preparation method and application thereof - Google Patents
Water-soluble triptolide derivative and preparation method and application thereof Download PDFInfo
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- CN110003304B CN110003304B CN201910325154.0A CN201910325154A CN110003304B CN 110003304 B CN110003304 B CN 110003304B CN 201910325154 A CN201910325154 A CN 201910325154A CN 110003304 B CN110003304 B CN 110003304B
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- DFBIRQPKNDILPW-CIVMWXNOSA-N Triptolide Chemical class O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](O)[C@]21[C@H]3O1 DFBIRQPKNDILPW-CIVMWXNOSA-N 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 150000001875 compounds Chemical class 0.000 claims abstract description 76
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000000126 substance Substances 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 111
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 238000001035 drying Methods 0.000 claims description 36
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 27
- 230000005526 G1 to G0 transition Effects 0.000 claims description 25
- 239000003463 adsorbent Substances 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Natural products CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 22
- 238000001704 evaporation Methods 0.000 claims description 19
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 18
- 239000003480 eluent Substances 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 14
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 12
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 claims description 12
- 230000015572 biosynthetic process Effects 0.000 claims description 12
- 238000003786 synthesis reaction Methods 0.000 claims description 10
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- -1 hydroquinone compound Chemical class 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 8
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 claims description 6
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N 1,4-Benzenediol Natural products OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000002246 antineoplastic agent Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 229920006395 saturated elastomer Polymers 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 230000000259 anti-tumor effect Effects 0.000 claims description 4
- OQNGCCWBHLEQFN-UHFFFAOYSA-N chloroform;hexane Chemical compound ClC(Cl)Cl.CCCCCC OQNGCCWBHLEQFN-UHFFFAOYSA-N 0.000 claims description 4
- 229940125773 compound 10 Drugs 0.000 claims description 4
- 229940126214 compound 3 Drugs 0.000 claims description 4
- 229940125898 compound 5 Drugs 0.000 claims description 4
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Inorganic materials Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 claims description 4
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 claims description 4
- 229940098779 methanesulfonic acid Drugs 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 229940014800 succinic anhydride Drugs 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- 150000001252 acrylic acid derivatives Chemical class 0.000 claims description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims description 3
- 229940125904 compound 1 Drugs 0.000 claims description 3
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 claims description 3
- JPSKCQCQZUGWNM-UHFFFAOYSA-N 2,7-Oxepanedione Chemical compound O=C1CCCCC(=O)O1 JPSKCQCQZUGWNM-UHFFFAOYSA-N 0.000 claims description 2
- WDBQJSCPCGTAFG-QHCPKHFHSA-N 4,4-difluoro-N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclohexane-1-carboxamide Chemical compound FC1(CCC(CC1)C(=O)N[C@@H](CCN1CCC(CC1)N1C(=NN=C1C)C(C)C)C=1C=NC=CC=1)F WDBQJSCPCGTAFG-QHCPKHFHSA-N 0.000 claims description 2
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 claims description 2
- NUGPIZCTELGDOS-QHCPKHFHSA-N N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclopentanecarboxamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CC[C@@H](C=1C=NC=CC=1)NC(=O)C1CCCC1)C NUGPIZCTELGDOS-QHCPKHFHSA-N 0.000 claims description 2
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 claims description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims description 2
- 239000007810 chemical reaction solvent Substances 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- XHFLOLLMZOTPSM-UHFFFAOYSA-M sodium;hydrogen carbonate;hydrate Chemical class [OH-].[Na+].OC(O)=O XHFLOLLMZOTPSM-UHFFFAOYSA-M 0.000 claims description 2
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 42
- 239000007787 solid Substances 0.000 description 36
- 238000005160 1H NMR spectroscopy Methods 0.000 description 21
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 13
- 239000000203 mixture Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 7
- 210000002381 plasma Anatomy 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 231100000419 toxicity Toxicity 0.000 description 6
- 230000001988 toxicity Effects 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 5
- YYPNJNDODFVZLE-UHFFFAOYSA-N 3-methylbut-2-enoic acid Chemical compound CC(C)=CC(O)=O YYPNJNDODFVZLE-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
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- 238000000746 purification Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 102100022365 NAD(P)H dehydrogenase [quinone] 1 Human genes 0.000 description 3
- 150000001335 aliphatic alkanes Chemical class 0.000 description 3
- 239000012267 brine Substances 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- 101710157142 2-methylene-furan-3-one reductase Proteins 0.000 description 2
- CFNMUZCFSDMZPQ-GHXNOFRVSA-N 7-[(z)-3-methyl-4-(4-methyl-5-oxo-2h-furan-2-yl)but-2-enoxy]chromen-2-one Chemical compound C=1C=C2C=CC(=O)OC2=CC=1OC/C=C(/C)CC1OC(=O)C(C)=C1 CFNMUZCFSDMZPQ-GHXNOFRVSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 101000973778 Homo sapiens NAD(P)H dehydrogenase [quinone] 1 Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 101710189291 Quinone oxidoreductase Proteins 0.000 description 2
- 102100034576 Quinone oxidoreductase Human genes 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate 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](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- AZQWKYJCGOJGHM-UHFFFAOYSA-N para-benzoquinone Natural products O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- 229940002612 prodrug Drugs 0.000 description 2
- 239000000651 prodrug Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 150000005208 1,4-dihydroxybenzenes Chemical class 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000208365 Celastraceae Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 101710095135 NAD(P)H dehydrogenase [quinone] 1 Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000830536 Tripterygium wilfordii Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000003509 anti-fertility effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- LKFRZUAJFOSMJA-UHFFFAOYSA-N cyclohexa-2,5-diene-1,4-dione;propanoic acid Chemical group CCC(O)=O.O=C1C=CC(=O)C=C1 LKFRZUAJFOSMJA-UHFFFAOYSA-N 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 239000012091 fetal bovine serum Substances 0.000 description 1
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
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- 238000001990 intravenous administration Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
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- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- KKHUSADXXDNRPW-UHFFFAOYSA-N malonic anhydride Chemical compound O=C1CC(=O)O1 KKHUSADXXDNRPW-UHFFFAOYSA-N 0.000 description 1
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- ZJHUBLNWMCWUOV-UHFFFAOYSA-N oxocane-2,8-dione Chemical compound O=C1CCCCCC(=O)O1 ZJHUBLNWMCWUOV-UHFFFAOYSA-N 0.000 description 1
- RMIBXGXWMDCYEK-UHFFFAOYSA-N oxonane-2,9-dione Chemical compound O=C1CCCCCCC(=O)O1 RMIBXGXWMDCYEK-UHFFFAOYSA-N 0.000 description 1
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- 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
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a water-soluble triptolide derivative, a preparation method and application thereof, wherein the chemical structural formula of the water-soluble triptolide derivative is as follows:
wherein: n is 2,3,4 … …, n is a positive integer greater than 1. On the premise of maintaining the bioactivity of triptolide, the invention improves the water solubility of the compound, reduces the toxic and side effects of triptolide, and enables the triptolide to be safely and effectively applied to clinic.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a water-soluble triptolide derivative and a preparation method and application thereof.
Background
Triptolide, also known as triptolide, is an epoxy diterpene lactone compound separated from Tripterygium wilfordii of Celastraceae, is the main active ingredient of traditional Chinese medicine, and has antiinflammatory, immunosuppressive, antifertility and antitumor effects. Triptolide has attracted extensive attention for its anti-tumor effects. Under the condition of low concentration (nM), triptolide can not only inhibit tumor growth, but also enhance the antitumor effect of various chemotherapeutic drugs. Triptolide has poor water solubility and narrow treatment window, has large toxic and side effects on digestive systems, urogenital systems, blood systems and the like, and limits the clinical research and development of triptolide.
Disclosure of Invention
The invention introduces quinone propionyl group on 19-position free hydroxyl of triptolide through a prodrug principle to obtain the derivative. Carboxyl is introduced into the compound structure, so that the solubility of the compound in water is improved. In addition, quinone oxidoreductase (NAD (P) H: quinone oxido-reductase-1, NQO1) is abundantly expressed in tumor tissues compared to normal tissues. Therefore, after being absorbed and distributed, the medicine can be reduced by NOQ1 in tumor tissues and spontaneously hydrolyzed into triptolide, so that the selectivity of the medicine is improved, and the toxicity of the triptolide to normal tissues is reduced. Has good clinical development prospect.
(as shown below):
the technical scheme adopted by the invention is as follows:
the chemical structural formula of the water-soluble triptolide derivative is shown as the following formula (I):
wherein: n is 2,3,4 … …, n is a positive integer greater than 1.
The synthetic route of the preparation method of the water-soluble triptolide derivative is as follows:
the method comprises the following specific steps:
(1) synthesis of compound 1 (coumarin derivative): hydroquinone compounds are used as reaction raw materials, acrylic acid derivatives with the mole number of 1-2 times are added, the reaction is carried out at the temperature of 50-100 ℃, the reaction time is 2-24 hours, and the solvent used in the reaction is methanesulfonic acid. After the reaction, the mixture is extracted by ethyl acetate, purified water, saturated sodium bicarbonate water solution and saturated saline are washed in sequence, dried by anhydrous sodium sulfate, decompressed and evaporated to dryness, and recrystallized by chloroform-normal hexane to obtain the target compound.
(2) Synthesis of Compound 2: the coumarin derivative obtained in the first step is used as a raw material, 1-3 times of bromine water in mole number is added, and the reaction is carried out for 24 hours at the temperature of 25 ℃, wherein the reaction solvent is acetic acid. And (3) after the reaction is finished, decompressing and evaporating to dryness, extracting the residue by dichloromethane, washing with water, drying by anhydrous sodium sulfate, decompressing and evaporating to dryness to obtain the target compound.
(3) Synthesis of Compound 3: taking the second step product and methanol as raw materials, adding 1.5-3 times of thionyl chloride by mol at 0 ℃, and reacting for 1-3 hours at 25 ℃. Separating and purifying with adsorbent as stationary phase, and recovering and drying the eluate to obtain compound 3.
(4) Synthesis of Compound 4: adding 2.5-5 times of the molar amount of sodium azide aqueous solution into the methanol solution of the compound 3 for multiple times, reacting at room temperature for 24 hours, then decompressing and evaporating to dryness, extracting the residue by dichloromethane, washing with water, drying by anhydrous sodium sulfate, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying the eluent to obtain the compound 4.
(5) Synthesis of Compound 5: dissolving compound 4 in dichloromethane, adding equimolar triphenylphosphine for reaction for 1 hr, removing solvent under reduced pressure, adding acetic acid-containing tetrahydrofuran aqueous solution, heating under reflux for 4 hr, evaporating under reduced pressure, extracting the residue with ethyl NaHCO, and collecting the saturated NaHCO solution3And washing with brine, drying with anhydrous sodium sulfate, separating and purifying with adsorbent as stationary phase, and recovering and drying the eluate to obtain compound 5.
(6) Synthesis of Compound 6: adding 2-4 times mole of lithium hydroxide into methanol aqueous solution of compound 5, reacting at room temperature for 24 hr, removing methanol under reduced pressure, adjusting pH of the aqueous solution to 2.0, and filtering to obtain compound 6.
(7) Synthesis of compound 7: adding Boc anhydride with 2 times of mol into dichloromethane solution of compound 6, reacting at room temperature for 4 hr, removing dichloromethane under reduced pressure, separating and purifying with adsorbent as stationary phase, recovering eluate, and drying to obtain compound 7.
(8) Synthesis of compound 8: triptolide is used as a raw material, a compound 7 with the mole number of 1.5-2 times, Dicyclohexylcarbodiimide (DCC) with the mole ratio of 1-4 times and catalytic equivalent 4-Dimethylaminopyridine (DMAP) are added to react at the temperature of 20-60 ℃, the reaction is carried out for 48 hours, the reduced pressure evaporation is carried out, the residue is extracted by ethyl acetate, washed by water, dried by anhydrous sodium sulfate, separated and purified by using an adsorbent as a stationary phase, and the compound 8 is obtained by recovering and drying the eluent.
(9) Synthesis of compound 9: trifluoroacetic acid (TFA) is added into dichloromethane solution of the compound 8 to react for 4 to 8 hours at room temperature, then separation and purification are carried out by taking an adsorbent as a stationary phase, and the compound 9 is obtained by recovering and drying eluent.
(10) Biosynthesis of compound 10: alkane diacid is slowly dropped into dichloromethane solution of the compound 9 to react for 2 to 4 hours at 65 ℃, then dichloromethane is removed under reduced pressure, the pH value of the water solution is adjusted to 2.0, and the mixture is filtered to obtain a compound 10.
Further, in the technical scheme, the hydroquinone compound in the step (1) is a compound with 2,3 and 5-positions being-CH3，NH2，OCH3X, etc.
Further, in the above technical solution, the acrylic acid derivative in step (1) is an olefinic acid compound in which the hydrogen atom at the β -position is substituted by 1-2 methyl groups or amino groups, preferably 3-methyl-2-butenoic acid or acrylic acid.
Further, in the above technical solution, the alkane diacid in step (10) is any one of malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, pimelic anhydride, or octanedioic anhydride. In the step, an amido bond is formed between the amino on the hydroquinone compound and the alkane diacid.
The water-soluble triptolide derivative can be used for preparing antitumor drugs.
An anti-tumor drug comprises the water-soluble triptolide derivative.
The invention has the following positive effects:
the invention introduces a quinone propionic acid structure on the free hydroxyl of triptolide. Because the compound structurally contains a free carboxyl, the compound is convenient to form salt, and the water solubility of the compound is improved. Meanwhile, quinone oxidoreductase (NAD (P) H: quinone oxidase reductase-1, NQO1) is abundantly expressed in tumor tissues compared with normal tissues. Therefore, after being absorbed and distributed, the medicine can be reduced in tumor tissues and spontaneously hydrolyzed into triptolide, so that the selectivity of the medicine is improved, and the toxicity of the triptolide to normal tissues is reduced. The invention reduces the toxic and side effects of the triptolide on the premise of keeping the bioactivity of the triptolide, so that the triptolide can be safely and effectively applied to clinic.
Drawings
FIG. 1, (A) pharmacological activity of TP on HepG2 cells and toxicity to normal hepatocytes, (B) pharmacological activity of CX-94 on HepG2 cells and toxicity to normal hepatocytes. (n is 6)
FIG. 2 chromatogram after incubation of homogenate of CX-94 and HepG2 cells for 0h (A) and 1h (B). (C) CX-94 is metabolically converted to XC-01 and TP.
Figure 3 metabolic stability of CX-94 in human liver microsomes (a) and human plasma (B) (n-3).
Fig. 4 is a graph of mean plasma concentration of triptolide and CX-94 versus time (n ═ 6) following tail vein injection of 1.5mg/kg of CX-94 in ICR mice.
Detailed Description
The following examples are further detailed descriptions of the present invention.
Example 1
The preparation method of the water-soluble triptolide derivative comprises the following steps:
(1)1.0g (6.58mmol) of hydroquinoneMixing with 0.54g (7.50mmol) of acrylic acid, adding 10mL of methanesulfonic acid at one time, heating and stirring at 70 ℃ for 18h, detecting complete reaction by TLC, stopping stirring, adding 125mL of water, extracting with ethyl acetate, pure water, saturated sodium bicarbonate aqueous solution and saturated brine in turn, drying with anhydrous sodium sulfate, and evaporating to dryness under reduced pressure. Recrystallizing with chloroform-n-hexane to obtain yellow solid, namely compound XC-01, with the yield of about 67%. [ M + H ]]+:221.0；1H NMR,600MHz(CDCl3)δ6.45(q,1H)，3.04(s,2H),1.96(m,3H),1.92(m,3H),1.41(s,6H)。
(2)1.1g (5mmol) of the compound of step (1) dissolved in 40mL of acetic acid solution; 0.57mL (11mmol) of bromine water is added into the solution dropwise, the mixture is stirred at room temperature for reaction for 24 hours, then the mixture is decompressed and evaporated to dryness, the residue is extracted by dichloromethane, washed by water, dried by anhydrous sodium sulfate and evaporated to dryness under reduced pressure to obtain a yellow solid compound. The yield thereof was found to be about 70%. [ M-H ]]-:313.0；1H NMR(600MHz,CDCl3)δ3.04(s,2H),2.19(s,3H),2.16(s,3H),1.44(s,6H)。
(3) Dissolving 1.2g (3.9mmol) of the yellow solid compound obtained in the step (2) in 12mL of methanol, slowly dropwise adding 0.56mL (8mmol) of thionyl chloride in an ice bath, continuously stirring at room temperature for 3 hours after dropwise adding, then evaporating under reduced pressure, separating and purifying the residue by using an adsorbent as a stationary phase, and recovering and drying the eluent to obtain the yellow solid compound. The yield thereof was found to be about 64%. [ M + H ]]+:329；1H NMR(600MHz,CDCl3)δ3.56(s,3H),2.98(s,2H),2.14(s,3H),2.14(s,3H),1.43(s,6H)。
(4) Every 1.44g (4.36mmol) of the stepsDissolving the yellow solid compound obtained in the step (3) in 15mL of methanol solution; dissolving 0.85g (13.1mmol) of sodium azide compound in 5mL of water, adding the solution for multiple times, reacting at room temperature for 24 hours, then decompressing and evaporating to dryness, extracting the residue by dichloromethane, washing with water, drying by anhydrous sodium sulfate, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying the eluent to obtain a yellow solid compound. The yield thereof was found to be about 85%. [ M + H ]]+:292.0；1H NMR(500MHz,CDCl3)δ3.63(s,3H),2.99(s,2H),2.16(s,3H),1.89(s,3H),1.41(s,6H)。
(5) Dissolving the compound obtained in the step (4) in 25mL of dichloromethane every 1.22g (4.17 mmol); after 1.1g (4.17mmol) of triphenylphosphine was added in portions and reacted at room temperature for 1 hour, the solvent was removed under reduced pressure. Adding aqueous solution containing 15mL of acetic acid and 15mL of tetrahydrofuran, heating and refluxing for 4 hours, then evaporating to dryness under reduced pressure, extracting the residue with ethyl acetate, and obtaining saturated NaHCO3And washing with brine, drying with anhydrous sodium sulfate, separating and purifying with adsorbent as stationary phase, and recovering and drying the eluate to obtain red solid compound. The yield thereof was found to be about 60%. [ M + H ]]+:266.0；1H NMR(600MHz,CDCl3)δ4.61(s,2H),3.56(s,3H),2.94(s,2H),2.19(s,3H),1.84(s,3H),1.43(s,6H)。
(6) Dissolving 1.3g (4.9mmol) of the red solid compound obtained in the step (5) in a mixed solution of 16mL of methanol and 4mL of water; after reacting 360mg (15mmol) of lithium hydroxide at room temperature for 24 hours, methanol was removed under reduced pressure, the pH of the aqueous solution was adjusted to 2.0, and the mixture was filtered to obtain a purple solid compound. The yield thereof was found to be about 90%. [ M + H ]]+:252.0；1H NMR,600MHz,(CDCl3)δ11.98(s,1H),6.32(s,2H),2.80(s,2H),2.07(s,3H),1.72(s,3H),1.35(s,6H)。
(7) Every 1.26g (5.0mmol) of the purple solid compound was dissolved in 15mL of dichloromethane; by dropwise addition of 2.18g (10.0mmol) of (Boc)2And reacting the O anhydride at room temperature for 4 hours, removing dichloromethane under reduced pressure, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying eluent to obtain a purple solid compound. The yield thereof was found to be about 82%. [ M + H ]]+:352.0；1H NMR,600MHz,(CDCl3)δ11.98(s,1H),6.31(s,1H),2.82(s,2H),2.07(s,3H),1.72(s,3H),1.44(s,9H)，1.37(s,6H)。
(8) And (3) dissolving 527mg (1.5mmol) of the purple solid corresponding matter obtained in the step (7) and 360mg (1mmol) of triptolide in 15mL of dichloromethane, adding 824mg (4mmol) of DCC and 127mg (1mmol) of DMAP, stirring for 48 hours at the temperature of 60 ℃, filtering after the reaction is finished, collecting filtrate, volatilizing the filtrate, redissolving ethyl acetate, filtering, and volatilizing the filtrate to obtain a purple solid compound. The yield thereof was found to be about 30%. [ M + H ]]+:695.0；1H NMR,600MHz(CDCl3)δ6.39(s,1H),5.30(s,1H),4.97(s,1H),4.19(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),3.02(s,2H),2.65(m,1H),2.24(s,3H),2.12(m,2H),1.99(s,3H),1.97(s,3H),1.86(m,2H),1.55(m,1H),1.49(s,3H)，1.46(s,9H)，1.22(m,1H),1.01(s,3H),0.90(d,3H),0.80(d,3H)。
(9) 694mg (1mmol) of the purple solid counterpart obtained in step (8) was dissolved in 15mL of dichloromethane, and stirred at room temperature for 4-8 hours by adding 5mL of TFA, the dichloromethane was removed under reduced pressure, separation and purification were carried out using an adsorbent as a stationary phase, and the eluate was recovered and dried to obtain the compound as a purple solid. The yield thereof was found to be about 67%. [ M + H ]]+:594.0；1H NMR,600MHz(CDCl3)δ6.34(s,2H),5.30(s,1H),4.97(s,1H),4.19(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),3.02(s,2H),2.65(m,1H),2.16(s,3H),2.12(m,2H),1.96(s,3H),1.94(s,3H),1.86(m,2H),1.55(m,1H),1.49(s,3H)，1.22(m,1H),1.01(s,3H),0.90(d,3H),0.80(d,3H)。
(10) Dissolving every 594mg (1mmol) of the purple solid corresponding matter obtained in the step (9) in 15mL of dichloromethane, slowly dropwise adding 150mg (1.5mmol) of succinic anhydride by adding 5mL, reacting at 65 ℃ for 2-4 hours, removing dichloromethane under reduced pressure, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying eluent to obtain a purple solid compound. The yield thereof was found to be about 67%. [ M-H ]]-:692.0；1H NMR,600MHz(CDCl3)δ6.38(s,1H),5.31(s,1H),4.99(s,1H),4.19(d,2H),3.84(d,1H),3.57(d,1H),3.49(d,1H),3.12(s,2H),2.64(m,1H),2.42(s,4H)，2.19(s,3H),2.14(m,2H),1.96(s,3H),1.91(s,3H),1.86(m,2H),1.54(m,1H),1.49(s,3H)，1.22(m,1H),1.15(s,3H),0.90(d,3H),0.81(d,3H)。
(11) Dissolving every 594mg (1mmol) of the purple solid corresponding matter obtained in the step (9) in 15mL of dichloromethane, slowly adding 171mg (1.5mmol) of glutaric anhydride by adding 5mL, reacting at 65 ℃ for 2-4 hours, removing dichloromethane under reduced pressure, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying eluent to obtain a purple solid compound. The yield thereof was found to be about 67%. [ M-H ]]-:706.0；1H NMR,600MHz(CDCl3)δ6.39(s,1H),5.34(s,1H),4.97(s,1H),4.29(d,2H),3.86(d,1H),3.59(d,1H),3.49(d,1H),3.12(s,2H),2.65(m,1H),2.46(s,4H)，2.18(s,3H),2.12(m,2H),2.06(s,3H),1.97(s,3H),1.92(s,2H),1.86(m,2H),1.55(m,1H),1.49(s,3H)，1.24(m,1H),1.21(s,3H),0.92(d,3H),0.84(d,3H)。
Example 2
The preparation method of the water-soluble triptolide derivative comprises the following steps:
(1) every 1.0g (6.58mmol) of hydroquinone is correspondingly mixed with 0.85g (7.50mmol) of 3-methyl-2-butenoic acid, then the mixture is added into 10mL of methanesulfonic acid at one time, the mixture is heated and stirred for 4h at 70 ℃, the TLC detection reaction is complete, the stirring is stopped, 125mL of water is added, ethyl acetate is used for extraction, water, saturated sodium bicarbonate aqueous solution and saturated saline are used for washing in sequence, anhydrous sodium sulfate is used for drying, reduced pressure evaporation is carried out, and chloroform-n-hexane is used for recrystallization, so as to obtain yellow solid. [ M + H ]]+:221.0；1H NMR,600MHz(CDCl3)δ6.45(q,1H)，3.04(s,2H),1.96(m,3H),1.92(m,3H),1.41(s,6H)。
(2)6.6g (30mmol) of the compound of step (1) dissolved in 200mL of acetic acid solution; 3.4mL (66mmol) of bromine water is added into the solution dropwise, stirred at room temperature for reaction for 24 hours, then decompressed and evaporated to dryness, the residue is extracted by dichloromethane, washed by water, dried by anhydrous sodium sulfate and decompressed and evaporated to dryness to obtain a yellow solid compound. [ M-H ]]-:313.0；1H NMR(600MHz,CDCl3)δ3.04(s,2H),2.19(s,3H),2.16(s,3H),1.44(s,6H)。
(3) Dissolving 4.88g (15.5mmol) of the yellow solid compound obtained in the step (2) in 50mL of methanol, slowly dropwise adding 2.25mL (31mmol) of thionyl chloride in an ice bath, continuously stirring at room temperature for 3 hours after the dropwise adding is finished, then evaporating under reduced pressure to dryness, separating and purifying the residue by using an adsorbent as a stationary phase, and recovering and drying the eluent to obtain the yellow solid compound. [ M + H ]]+:329；1H NMR(600MHz,CDCl3)δ3.56(s,3H),2.98(s,2H),2.14(s,3H),2.14(s,3H),1.43(s,6H)。
(4) Every 2.87g (8.72mmol) of the yellow solid compound obtained in step (3) was dissolved in 30mL of a methanol solution; dissolving 1.7g (26.2mmol) of sodium azide compound in 10mL of water, adding the solution for multiple times, reacting at room temperature for 24 hours, then decompressing and evaporating to dryness, extracting the residue by dichloromethane, washing with water, drying by anhydrous sodium sulfate, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying the eluent to obtain a yellow solid compound. [ M + H ]]+:292.0；1H NMR(500MHz,CDCl3)δ3.63(s,3H),2.99(s,2H),2.16(s,3H),1.89(s,3H),1.41(s,6H)。
(5) Each 2.43g (8.35mmol) of the compound obtained in step (4) was dissolved in 50mL of dichloro chlorideIn methane; after 2.19g (8.35mmol) of triphenylphosphine was added in portions and reacted at room temperature for 1 hour, the solvent was removed under reduced pressure. Adding aqueous solution containing 30mL acetic acid and 30mL tetrahydrofuran, heating under reflux for 4 hr, evaporating under reduced pressure, extracting the residue with ethyl acetate, and collecting saturated NaHCO3And washing with brine, drying with anhydrous sodium sulfate, separating and purifying with adsorbent as stationary phase, and recovering and drying the eluate to obtain red solid compound. [ M + H ]]+:266.0；1H NMR(600MHz,CDCl3)δ4.61(s,2H),3.56(s,3H),2.94(s,2H),2.19(s,3H),1.84(s,3H),1.43(s,6H)。
(6) Dissolving 1.3g (4.9mmol) of the red solid compound obtained in the step (5) in a mixed solution of 16mL of methanol and 4mL of water; after reacting 360mg (15mmol) of lithium hydroxide at room temperature for 24 hours, methanol was removed under reduced pressure, the pH of the aqueous solution was adjusted to 2.0, and the mixture was filtered to obtain a purple solid compound. [ M + H ]]+:252.0；1H NMR,600MHz,(CDCl3)δ11.98(s,1H),6.32(s,2H),2.80(s,2H),2.07(s,3H),1.72(s,3H),1.35(s,6H)。
(7) Every 1.26g (5.0mmol) of the purple solid compound was dissolved in 15mL of dichloromethane; 2.18g (10.0mmol) of Boc anhydride is dropwise added to react at room temperature for 4 hours, dichloromethane is removed under reduced pressure, separation and purification are carried out by taking an adsorbent as a stationary phase, and the eluent is recovered and dried to obtain a purple solid compound. [ M + H ]]+:352.0；1H NMR,600MHz,(CDCl3)δ11.98(s,1H),6.31(s,1H),2.82(s,2H),2.07(s,3H),1.72(s,3H),1.44(s,9H)，1.37(s,6H)。
(8) And (3) dissolving 527mg (1.5mmol) of the purple solid corresponding matter obtained in the step (7) and 360mg (1mmol) of triptolide in 15mL of dichloromethane, adding 824mg (4mmol) of DCC and 127mg (1mmol) of DMAP, stirring for 48 hours at the temperature of 60 ℃, filtering after the reaction is finished, collecting filtrate, volatilizing the filtrate, redissolving ethyl acetate, filtering, and volatilizing the filtrate to obtain a purple solid compound. [ M + H ]]+:695.0；1H NMR,600MHz(CDCl3)δ6.39(s,1H),5.30(s,1H),4.97(s,1H),4.19(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),3.02(s,2H),2.65(m,1H),2.24(s,3H),2.12(m,2H),1.99(s,3H),1.97(s,3H),1.86(m,2H),1.55(m,1H),1.49(s,3H)，1.46(s,9H)，1.22(m,1H),1.01(s,3H),0.90(d,3H),0.80(d,3H)。
(9) 694mg (1mmol) of the purple solid counterpart obtained in step (8) was dissolved in 15mL of dichloromethane, and stirred at room temperature for 4 hours by adding 5mL of TFA, the dichloromethane was removed under reduced pressure, separation and purification were carried out using an adsorbent as a stationary phase, and the eluate was recovered and dried to obtain a purple solid compound. [ M + H ]]+:594.0；1H NMR,600MHz(CDCl3)δ6.34(s,2H),5.30(s,1H),4.97(s,1H),4.19(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),3.02(s,2H),2.65(m,1H),2.16(s,3H),2.12(m,2H),1.96(s,3H),1.94(s,3H),1.86(m,2H),1.55(m,1H),1.49(s,3H)，1.22(m,1H),1.01(s,3H),0.90(d,3H),0.80(d,3H)。
(10) Dissolving every 594mg (1mmol) of the purple solid corresponding matter obtained in the step (9) in 15mL of dichloromethane, slowly dropwise adding 150mg (1.5mmol) of succinic anhydride by adding 5mL, reacting at 65 ℃ for 2-4 hours, removing dichloromethane under reduced pressure, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying eluent to obtain a purple solid compound. [ M-H ]]-:692.0；1H NMR,600MHz(CDCl3)δ6.38(s,1H),5.31(s,1H),4.99(s,1H),4.19(d,2H),3.84(d,1H),3.57(d,1H),3.49(d,1H),3.12(s,2H),2.64(m,1H),2.42(s,4H)，2.19(s,3H),2.14(m,2H),1.96(s,3H),1.91(s,3H),1.86(m,2H),1.54(m,1H),1.49(s,3H)，1.22(m,1H),1.15(s,3H),0.90(d,3H),0.81(d,3H)。
The product obtained in example 1 was tested for various properties as follows:
CX-94 Water-soluble assay
Weighing a certain amount of CX-94, dissolving and diluting the CX-94 solution into a series of CX-94 solutions with concentration gradients by using acetonitrile, detecting the content of CX-94 by LC-MS/MS and preparing a standard curve; preparing a certain amount of CX-94 into a CX-94 supersaturated solution with water, filtering the saturated solution with a 0.22 mu m filter membrane, collecting the filtrate, and detecting the concentration of CX-94 by adopting LC-MS/MS. The results show that CX-94 has a solubility in water of 4.52mg/mL and the solubility of comparative TP in water is only 17. mu.g/mL. The water solubility of the target compound is improved by 265 times.
2. In vitro pharmacological Activity and toxicity assays
Respectively taking frozen HepG2 cells and normal hepatocytes, recovering, and adjusting cell density to 1x10 per well5Separate inoculation of cellsAnd (5) culturing in a 96-well plate for 24h by using a serum-free culture solution when the cell fusion reaches 60-70%. Subsequently, the cells were randomized to negative control, positive control (0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5. mu.M triptolide-treated cells), CX-94(0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5. mu.M) administration group. Adding 5mg/ml MTT 20 mu 1 into each well after 48h, culturing for 4h, discarding the supernatant, adding 150 mu l of dimethyl sulfoxide into each well, shaking at room temperature for 15min, and measuring the absorbance value (OD) at 570nm by using an enzyme-linked immunosorbent assay after yellow crystals are dissolved.
The results after administration of normal hepatocytes are shown in fig. 1: toxicity to cells after incubation of triptolide (figure 1A) and CX-94 (figure 1B) at different concentrations with normal liver cells and liver cancer cells, respectively. The results show that compared to triptolide (IC 50-38.9 nM), CX-94(IC 50-1250 nM) exhibited a 32-fold decrease in normal liver cytotoxicity. The structural modification of the triptolide by the method is shown to reduce the toxicity of the triptolide on normal liver cells. IC50 of the same concentrations of CX-94 and triptolide on HepG2 are respectively 38.4nM and 36.9nM, which shows that the compound CX-94 obtained by modifying the triptolide structure by the method can retain the pharmacological activity of the triptolide on HepG2 hepatoma cells.
In vitro metabolic transformation of HepG2 cell lysates
Human hepatoma cell HepG2 cells were cultured in DMEM containing 10% fetal bovine serum at 37 ℃ and 5% CO2And culturing in a carbon dioxide incubator with saturated humidity. Collecting cells in logarithmic phase, washing with PBS, homogenizing, centrifuging at 8000 Xg and 4 deg.C for 20min, collecting supernatant, measuring protein concentration of cell lysate by BCA method, and storing in refrigerator at-80 deg.C.
The reaction system contained 10. mu.g/mL of compound CX-94, 1mg/mL of HepG2 cell lysate, 1mM NADPH, and 50mM PBS buffer in a total volume of 100. mu.L. After incubation of the samples for 1h in vitro, the reaction was stopped by addition of 2 fold acetonitrile. The sample is passed through 12000 Xg, centrifuged at 4 deg.C for 20min, and the supernatant is injected, and the production of triptolide is detected by UPLC. The results are shown in FIG. 2.CX-94 is metabolically transformed in HepG2 lysate to TP and XC-01.
4. In vitro metabolic stability study
Incubating human liver microsome with CX-94 at a final concentration of 10 μ M to obtain 100 μ L of incubation system, which comprises phosphate buffer (50mM, pH 7.4), liver microsome (1mg/mL), and MgCl2(5 mM). The reaction was initiated by the addition of 10. mu.L NADPH (final concentration 1mM) at a concentration of 20mM, and quenched by the addition of 200. mu.L acetonitrile at 37 ℃ after incubation for 0, 5, 10, 20, 30, 40 and 60 min. The sample is evenly mixed by vortex oscillation at 4 ℃, centrifuged at 12000rpm for 10min, and the supernatant is taken and analyzed by LC-MS/MS injection.
CX-94 (final concentration is 10 μ M) and human plasma are incubated at 37 ℃ for 0, 5, 10, 20, 30, 40 and 60min respectively, acetonitrile with twice volume is added to stop the reaction, the sample is vortexed and uniformly mixed, centrifuged at 12000rpm for 10min at 4 ℃, the supernatant is taken, and LC-MS/MS sample injection analysis is carried out. The results are shown in FIG. 3.CX-94 is metabolically stable in plasma and in liver microsomes with a metabolic half-life of greater than 1 hour.
5. In vivo pharmacokinetic study in mice
After intravenous administration (1.5mg/kg CX-94) of mice, blood was collected from the heart at 2, 5, 10, 20, 40min, 1, 2,3,4, 8, 18 hours. Another group of mice was orally administered with 1.5mg/kg CX-94 by gastric gavage, and then blood was collected from the heart at 10, 30min, 1, 2,3,4, 5, 6, 8, and 24 hours. The collected plasma is placed in a heparinized 1.5mLEP tube, centrifuged at 1000g and 4 ℃ for 10min, and the supernatant is taken to obtain the plasma. Adding 2 times of acetonitrile according to the volume ratio, carrying out vortex mixing and oscillation, centrifuging at 12000g for 10min at 4 ℃, taking supernatant for injection, and detecting the CX-94 content in blood plasma by an LC-MS/MS method. The concentration of the compound in plasma is plotted on the ordinate and the blood collection time on the abscissa. And calculating pharmacokinetic parameters. The results of the study showed that CX-94, as a prodrug, is rapidly metabolically converted to TP in mice.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A water-soluble triptolide derivative is characterized in that: the chemical structural formula of the water-soluble triptolide derivative is shown as the following formula (I):
wherein: n is 2,3, 4.
2. The water-soluble triptolide derivative of claim 1, wherein: the chemical structural formula of the water-soluble triptolide derivative is as follows:
3. a method of preparing the water-soluble triptolide derivative of claim 1, comprising: the method comprises the following specific steps:
(1) synthesis of Compound 1: hydroquinone compound is used as reaction raw material, 1-2 times of acrylic acid derivative is added, reaction is carried out at 50-100 ℃, the reaction time is 2-24 hours, and the solvent used in the reaction is methanesulfonic acid; after the reaction is finished, sequentially extracting with ethyl acetate, washing with pure water, saturated sodium bicarbonate water solution and saturated saline, drying with anhydrous sodium sulfate, evaporating to dryness under reduced pressure, and recrystallizing with chloroform-n-hexane to obtain the target compound;
(2) synthesis of Compound 2: taking the compound 1 obtained in the first step as a raw material, adding 1-3 times of bromine water in mole number, and reacting for 24 hours at 25 ℃, wherein the reaction solvent is acetic acid; after the reaction is finished, decompressing and evaporating to dryness, extracting the residue by dichloromethane, washing with water, drying by anhydrous sodium sulfate, decompressing and evaporating to dryness to obtain a target compound;
(3) synthesis of Compound 3: taking the second step product and methanol as raw materials, adding thionyl chloride with the mol of 1.5-3 times of that of the second step product at the temperature of 0 ℃, and reacting for 1-3 hours at the temperature of 25 ℃; separating and purifying by using an adsorbent as a stationary phase, and recovering and drying eluent to obtain a compound 3;
(4) synthesis of Compound 4: adding 2.5-5 times of the molar amount of sodium azide aqueous solution into the methanol solution of the compound 3 for multiple times, reacting at room temperature for 24 hours, then decompressing and evaporating to dryness, extracting the residue by dichloromethane, washing with water, drying by anhydrous sodium sulfate, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying the eluent to obtain a compound 4;
(5) synthesis of Compound 5: dissolving compound 4 in dichloromethane, adding equimolar triphenylphosphine for reaction for 1 hr, removing solvent under reduced pressure, adding acetic acid-containing tetrahydrofuran aqueous solution, heating under reflux for 4 hr, evaporating under reduced pressure, extracting the residue with ethyl NaHCO, and collecting the saturated NaHCO solution3Washing with saline water, drying with anhydrous sodium sulfate, separating and purifying with adsorbent as stationary phase, and recovering and drying eluate to obtain compound 5;
(6) synthesis of Compound 6: adding 2-4 times of lithium hydroxide in mol into methanol aqueous solution of the compound 5, reacting at room temperature for 24 hours, removing methanol under reduced pressure, adjusting pH of the aqueous solution to 2.0, and filtering to obtain a compound 6;
(7) synthesis of compound 7: adding Boc anhydride with 2 times of mol into dichloromethane solution of compound 6, reacting at room temperature for 4 hours, removing dichloromethane under reduced pressure, separating and purifying with adsorbent as stationary phase, recovering eluate, and drying to obtain compound 7;
(8) synthesis of compound 8: taking triptolide as a raw material, adding a compound 7 with the mole number of 1.5-2 times, Dicyclohexylcarbodiimide (DCC) with the mole ratio of 1-4 times and catalytic equivalent 4-Dimethylaminopyridine (DMAP), reacting at 20-60 ℃, decompressing and evaporating to dryness after reacting for 48 hours, extracting residues through ethyl acetate, washing with water, drying through anhydrous sodium sulfate, separating and purifying by taking an adsorbent as a stationary phase, and recovering and drying eluent to obtain a compound 8;
(9) synthesis of compound 9: adding trifluoroacetic acid (TFA) into a dichloromethane solution of the compound 8, reacting at room temperature for 4-8 hours, separating and purifying by using an adsorbent as a stationary phase, and recovering and drying eluent to obtain a compound 9;
(10) biosynthesis of compound 10: slowly dropwise adding alkane dianhydride into a dichloromethane solution of the compound 9, reacting at 65 ℃ for 2-4 hours, removing dichloromethane under reduced pressure, adjusting the pH value of the aqueous solution to 2.0, and filtering to obtain a compound 10;
the synthetic route of the water-soluble triptolide derivative is as follows:
4. the method of claim 3, wherein the step of preparing the water-soluble triptolide derivative comprises: the alkane dianhydride in the step (10) is any one of succinic anhydride, glutaric anhydride and adipic anhydride.
5. The use of the water-soluble triptolide derivative of claim 1 or 2 in preparing an anti-tumor medicament.
6. An antitumor agent characterized by: the antitumor drug comprises the water-soluble triptolide derivative as claimed in claim 1 or 2.
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