CN116375644B - Aporphine alkaloid compound and preparation method and application thereof - Google Patents
Aporphine alkaloid compound and preparation method and application thereof Download PDFInfo
- Publication number
- CN116375644B CN116375644B CN202310237740.6A CN202310237740A CN116375644B CN 116375644 B CN116375644 B CN 116375644B CN 202310237740 A CN202310237740 A CN 202310237740A CN 116375644 B CN116375644 B CN 116375644B
- Authority
- CN
- China
- Prior art keywords
- column
- alkaloid compound
- aporphine alkaloid
- fractions
- aporphine
- 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.)
- Active
Links
- -1 Aporphine alkaloid compound Chemical class 0.000 title claims abstract description 121
- 229930014594 aporphine alkaloid Natural products 0.000 title claims abstract description 116
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 102100040061 Indoleamine 2,3-dioxygenase 1 Human genes 0.000 claims abstract description 42
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 12
- 101710136122 Tryptophan 2,3-dioxygenase Proteins 0.000 claims abstract description 10
- 102000057288 Tryptophan 2,3-dioxygenases Human genes 0.000 claims abstract description 10
- 201000010099 disease Diseases 0.000 claims abstract description 8
- 230000001404 mediated effect Effects 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 117
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 69
- 238000013375 chromatographic separation Methods 0.000 claims description 54
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 39
- 238000010828 elution Methods 0.000 claims description 27
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 26
- 239000000741 silica gel Substances 0.000 claims description 26
- 229910002027 silica gel Inorganic materials 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 26
- 238000011068 loading method Methods 0.000 claims description 25
- 238000000605 extraction Methods 0.000 claims description 24
- 150000003839 salts Chemical class 0.000 claims description 22
- 229930013930 alkaloid Natural products 0.000 claims description 21
- 239000000499 gel Substances 0.000 claims description 21
- 238000007254 oxidation reaction Methods 0.000 claims description 21
- 238000002386 leaching Methods 0.000 claims description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- QELDJEKNFOQJOY-ZDUSSCGKSA-N Isocorydine Chemical compound CN1CCC2=CC(OC)=C(OC)C3=C2[C@@H]1CC1=CC=C(OC)C(O)=C13 QELDJEKNFOQJOY-ZDUSSCGKSA-N 0.000 claims description 18
- DRVVGOFZCWCCSA-UHFFFAOYSA-N isocorydine Natural products COc1cc2CCN(C)C3Cc4cccc(C)c4c(c1O)c23 DRVVGOFZCWCCSA-UHFFFAOYSA-N 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 18
- 238000001953 recrystallisation Methods 0.000 claims description 17
- 239000012071 phase Substances 0.000 claims description 16
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 14
- 239000012452 mother liquor Substances 0.000 claims description 13
- 238000010829 isocratic elution Methods 0.000 claims description 12
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 claims description 12
- 230000003647 oxidation Effects 0.000 claims description 12
- 239000008194 pharmaceutical composition Substances 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 12
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 8
- 150000003797 alkaloid derivatives Chemical class 0.000 claims description 8
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002798 polar solvent Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 6
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 5
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims description 5
- 101710120843 Indoleamine 2,3-dioxygenase 1 Proteins 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 5
- 239000003929 acidic solution Substances 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 5
- 239000010452 phosphate Substances 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 229940095064 tartrate Drugs 0.000 claims description 5
- 239000003513 alkali Substances 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 4
- 239000012295 chemical reaction liquid Substances 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 claims description 3
- 229910002651 NO3 Inorganic materials 0.000 claims description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 241001083527 Dicentra Species 0.000 claims 5
- 239000004480 active ingredient Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 101001037256 Homo sapiens Indoleamine 2,3-dioxygenase 1 Proteins 0.000 abstract description 39
- 206010028980 Neoplasm Diseases 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 9
- 230000005764 inhibitory process Effects 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 4
- 239000003112 inhibitor Substances 0.000 abstract description 4
- 208000030159 metabolic disease Diseases 0.000 abstract description 4
- 206010006187 Breast cancer Diseases 0.000 abstract description 3
- 208000026310 Breast neoplasm Diseases 0.000 abstract description 3
- 206010027406 Mesothelioma Diseases 0.000 abstract description 3
- 208000008589 Obesity Diseases 0.000 abstract description 3
- 208000005017 glioblastoma Diseases 0.000 abstract description 3
- 201000010536 head and neck cancer Diseases 0.000 abstract description 3
- 208000014829 head and neck neoplasm Diseases 0.000 abstract description 3
- 102000006639 indoleamine 2,3-dioxygenase Human genes 0.000 abstract description 3
- 108020004201 indoleamine 2,3-dioxygenase Proteins 0.000 abstract description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 abstract description 3
- 235000020824 obesity Nutrition 0.000 abstract description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 abstract description 3
- 206010005003 Bladder cancer Diseases 0.000 abstract description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 abstract description 2
- 201000005112 urinary bladder cancer Diseases 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 19
- 241001361205 Dactylicapnos Species 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 150000001875 compounds Chemical class 0.000 description 16
- 238000005481 NMR spectroscopy Methods 0.000 description 15
- 238000004809 thin layer chromatography Methods 0.000 description 12
- 239000000546 pharmaceutical excipient Substances 0.000 description 10
- 150000008441 aporphines Chemical class 0.000 description 9
- 238000001228 spectrum Methods 0.000 description 9
- 239000002253 acid Substances 0.000 description 8
- 238000004440 column chromatography Methods 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- YGPSJZOEDVAXAB-UHFFFAOYSA-N kynurenine Chemical compound OC(=O)C(N)CC(=O)C1=CC=CC=C1N YGPSJZOEDVAXAB-UHFFFAOYSA-N 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- OKKJLVBELUTLKV-VMNATFBRSA-N methanol-d1 Chemical compound [2H]OC OKKJLVBELUTLKV-VMNATFBRSA-N 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 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 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000002425 crystallisation Methods 0.000 description 5
- 230000008025 crystallization Effects 0.000 description 5
- 239000003480 eluent Substances 0.000 description 5
- 238000001704 evaporation Methods 0.000 description 5
- 239000008187 granular material Substances 0.000 description 5
- 238000009169 immunotherapy Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 229960004799 tryptophan Drugs 0.000 description 5
- 241000218180 Papaveraceae Species 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- BZKUYNBAFQJRDM-UHFFFAOYSA-N aporphine Chemical compound C12=CC=CC=C2CC2N(C)CCC3=CC=CC1=C32 BZKUYNBAFQJRDM-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 150000004677 hydrates Chemical class 0.000 description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- YRYCIFUZSUMAAY-UHFFFAOYSA-N (RS)-1-benzyl-1,2,3,4-tetrahydroisoquinoline Chemical class N1CCC2=CC=CC=C2C1CC1=CC=CC=C1 YRYCIFUZSUMAAY-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 244000117040 Dactylicapnos scandens Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 229940042040 innovative drug Drugs 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 238000002211 ultraviolet spectrum Methods 0.000 description 3
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 2
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 description 2
- WSVLPVUVIUVCRA-KPKNDVKVSA-N Alpha-lactose monohydrate Chemical compound O.O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O WSVLPVUVIUVCRA-KPKNDVKVSA-N 0.000 description 2
- 241001081440 Annonaceae Species 0.000 description 2
- 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
- 241000133570 Berberidaceae Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000218164 Menispermaceae Species 0.000 description 2
- 208000002193 Pain Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- QCMQEZNBBPGFKQ-UHFFFAOYSA-N Thalisopynine Natural products CN1CCC2=C(OC)C(OC)=C(OC)C3=C2C1CC1=C3C=C(OC)C(O)=C1 QCMQEZNBBPGFKQ-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 229960002749 aminolevulinic acid Drugs 0.000 description 2
- 239000012752 auxiliary agent Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 229930013397 isoquinoline alkaloid Natural products 0.000 description 2
- 125000002183 isoquinolinyl group Chemical class C1(=NC=CC2=CC=CC=C12)* 0.000 description 2
- FVRABHGHBLRNNR-UHFFFAOYSA-N liriodenine Natural products O=C1C=CC=c2c1cc3nccc4cc5OCOc5c2c34 FVRABHGHBLRNNR-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000531362 Dactylicapnos lichiangensis Species 0.000 description 1
- 102000016680 Dioxygenases Human genes 0.000 description 1
- 108010028143 Dioxygenases Proteins 0.000 description 1
- 241000249877 Doronicum kamaonense Species 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000612122 Myrsine Species 0.000 description 1
- 241000218201 Ranunculaceae Species 0.000 description 1
- 229940124639 Selective inhibitor Drugs 0.000 description 1
- 208000033749 Small cell carcinoma of the bladder Diseases 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- XQAXGZLFSSPBMK-UHFFFAOYSA-M [7-(dimethylamino)phenothiazin-3-ylidene]-dimethylazanium;chloride;trihydrate Chemical compound O.O.O.[Cl-].C1=CC(=[N+](C)C)C=C2SC3=CC(N(C)C)=CC=C3N=C21 XQAXGZLFSSPBMK-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 150000008622 benzophenanthridines Chemical class 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000010485 coping Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- NFDFQCUYFHCNBW-SCGPFSFSSA-N dienestrol Chemical compound C=1C=C(O)C=CC=1\C(=C/C)\C(=C\C)\C1=CC=C(O)C=C1 NFDFQCUYFHCNBW-SCGPFSFSSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 102000012427 human indoleamine 2,3-dioxygenase 1 Human genes 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 230000007108 local immune response Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 238000005691 oxidative coupling reaction Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 101150020000 tdo gene Proteins 0.000 description 1
- 208000004371 toothache Diseases 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 201000007710 urinary bladder small cell neuroendocrine carcinoma Diseases 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/18—Ring systems of four or more rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Child & Adolescent Psychology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of medicine development, and particularly relates to an aporphine alkaloid compound, and a preparation method and application thereof. The aporphine alkaloid compound provided by the invention has a double-selective and high-efficiency inhibition effect on indoleamine 2, 3-dioxygenase (IDO 1) and tryptophan 2, 3-dioxygenase (TDO), can be used as a TDO/IDO1 double-target inhibitor, and can be used for TDO/IDO1 mediated diseases, such as glioblastoma, head and neck cancer, mesothelioma, non-small cell lung cancer, bladder cancer, breast cancer and other tumors and obesity and other metabolic disorder diseases singly or in combination with other medicines.
Description
Technical Field
The invention belongs to the technical field of medicine development, and particularly relates to an aporphine alkaloid compound, and a preparation method and application thereof.
Background
Indoleamine 2, 3-dioxygenase 1 (IDO 1) is the rate-limiting enzyme for the degradation of tryptophan to kynurenine in humans, and plays an important role in the metabolic pathway for the breakdown of tryptophan, which is ubiquitous in all tissues in humans. In the tumor microenvironment, IDO1 over-expressed by tumor cells causes excessive consumption of tryptophan and aggregation of the metabolite kynurenine, inhibiting local immune responses and forming immune evasions. In addition to IDO1, tryptophan Dioxygenase (TDO) of the same family is also able to catalyze the same biochemical reaction in humans, but with different tissue distribution and substrate selectivity. Thus, inhibition of IDO1 activity is one of the strategies for tumor immunotherapy, and drug design for this target has been the focus of research for over twenty years.
TDO/IDO1 is expressed in a variety of tumor cells and surrounding microenvironment cells. TDO and IDO1 are expressed in different tumor cells, at different sites in the same tumor, and some tumor cells express two proteins. Thus targeting tumor cells with 50% greater efficiency for bi-directional selective inhibition of both enzymes than 31% and 34% response rates of tumor cells expressing only TDO or IDO 1. The effectiveness of tumor immunotherapy can be improved complementarily, and the T cell can attack tumors better by coping with epidemic prevention systems of organisms. IDO1 and TDO become important targets for tumor immunotherapy, and simultaneously, the effects of inhibiting the activities of the TDO and the IDO1 on improving the tumor immunotherapy are complementary, so that the response rate to stem cells can be greatly improved, IDO1/TDO dual inhibitors are developed, and the novel drug development important strategy in the field of immunotherapy is realized. The use of inhibitors in combination with other immune checkpoint inhibitors or with conventional methods of chemotherapy, radiation therapy, etc. is also desirable.
The aporphine alkaloid is one of the isoquinoline alkaloids, and is mainly distributed in dozens of plant groups such as Papaveraceae (Papaveraceae), annonaceae (Annonaceae), menispermaceae (Menispermaceae), ranunculaceae (Ranunc mu Laceae), berberidaceae (Berberidaceae) and the like, and is mainly formed by synthesizing benzyl tetrahydroisoquinoline alkaloids through tyrosine biosynthesis, generating dienone or dienol through oxidative coupling of benzyl tetrahydroisoquinoline alkaloids, and then carrying out various rearrangements; or directly forming other types of alkaloids by secondary cyclization, C-C bond and C-N bond cleavage and other reactions of benzyl tetrahydroisoquinoline alkaloids, and forming aporphine by the alkaloids. In addition, the aporphine alkaloid can also be formed by migration and rearrangement of a C-C bond. The aporphine alkaloid has wide physiological activities, including functions of regulating immunity, resisting tumor, resisting bacteria, easing pain, etc. The aporphine alkaloid has abundant compounds with antitumor activity, and the compounds have special and strong anticancer effects and relatively special action mechanisms, so that the aporphine alkaloid is worthy of further research as candidate anticancer drugs and lead compounds. The development of innovative drugs based on natural products is an effective and classical way of drug development worldwide, china has abundant medicinal plant resources, and long-term natural product research accumulation provides a solid foundation and a rich compound library for the development of the innovative drugs. These provide a rich plant resource and compound resource library for the research of innovative drugs based on aporphine alkaloids.
Papaveraceae (Papaveraceae) Dacticapnos plants are rich in apofeine alkaloids, 7 species worldwide, and are distributed Yu Xima Laya regions to the western region of our country. There are 4 kinds of Chinese, including Dactylicapnos (D. Scandens), myrsine (D. Tor. Mu.losa), lijiang Dactylicapnos (D. Lichiangensis) and Dactylicapnos (D. Roylei), in southwest, respectively. The plants are rich in aporphine alkaloids, in particular to dactylicapnos (Dactylicapnos scandens) which is a traditional and conventional medicinal material in white nationality of Yunnan province, and are also called as 'hard-turning' in white nationality, such as radix kadsurae, radix Pisi Sativi, radix Hedychii and the like. The root is collected in summer and autumn, dried in the sun, and then used as a medicine, has bitter taste and cool nature, has the effects of easing pain, stopping bleeding, diminishing inflammation and reducing blood pressure, and is widely used for toothache, nervous headache, stomachache, cancer resistance and the like by the white nationalities. To date, 70 or more isoquinoline alkaloids isolated from dactylicapnos have been involved in the framework types including aporphines, pra Luo Tuo, morphine, benzophenanthridines, etc., especially aporphines.
However, no study and report on dual inhibition of TDO and IDO1 by the above alkaloids have been made in the prior art.
Disclosure of Invention
The invention aims to provide an aporphine alkaloid compound, a preparation method and application thereof, and the aporphine alkaloid compound provided by the invention has a double-selective and high-efficiency inhibition effect on indoleamine 2, 3-dioxygenase (IDO 1) and tryptophan 2, 3-dioxygenase (TDO), can be used as a TDO/IDO1 double-target inhibitor, and can be used for TDO/IDO1 mediated diseases, such as glioblastoma, head and neck cancer, mesothelioma, non-small cell lung cancer, bladder cancer, breast cancer and other tumors and obesity and other metabolic disorder diseases singly or in combination with other medicines.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an aporphine alkaloid compound, which comprises a structure shown in a formula 1, a formula 2 or a formula 3:
the invention provides pharmaceutically acceptable salts, hydrates or solvent compounds of the aporphine alkaloid compounds in the technical scheme, wherein the pharmaceutically acceptable salts of the aporphine alkaloid compounds comprise inorganic acid salts and/or organic acid salts;
the inorganic acid salt comprises one or more of hydrochloride, hydrobromide, nitrate, sulfate and phosphate;
the organic acid salt comprises one or more of tartrate, citrate, formate, acetate and oxalate.
The invention provides a preparation method of an aporphine alkaloid compound, which comprises the following steps:
(1) Leaching dactylicapnos with polar solvent, concentrating the obtained leaching solution to obtain extract;
(2) Dispersing the extract in a dilute acid aqueous solution, and performing first extraction on the obtained acidic solution by using first ethyl acetate to obtain a first ethyl acetate phase and a first water phase; the pH value of the first aqueous phase is regulated to 7-8, and then second extraction is carried out by using second ethyl acetate, so as to obtain a second ethyl acetate phase; concentrating the second ethyl acetate phase to obtain total alkaloids of dactylicapnos root;
(3) Mixing the dactylicapnos alkaloid total alkali and silica gel, loading the obtained mixture to a silica gel column for first column chromatographic separation, performing gradient elution on the first column chromatographic separation according to the volume ratio from large to small by adopting a chloroform-methanol system with the volume ratio of chloroform to methanol of 1:0-1:1, and merging the same fractions to obtain six sections of fractions which are named as fractions Fr.I-Fr.VI; recrystallizing the fraction Fr.II to obtain a recrystallization mother liquor and isocorydine;
(4) Loading the recrystallization mother liquor to a first chromatographic column for second column chromatographic separation, wherein the second column chromatographic separation adopts a methanol-water system with the volume percentage of 20-90% for gradient elution according to the volume percentage of methanol from small to large, and the same fractions are combined to obtain four sections of fractions which are named as fractions Fr.II-1-Fr.II-4 respectively; loading the fraction Fr.II-3 to a first gel chromatographic column for third column chromatographic separation, wherein the third column chromatographic separation adopts methanol for isocratic elution, and the same fractions are combined to obtain three sections of fractions which are named as fractions Fr.II-3-1 to Fr.II-3-3 respectively; loading the fraction Fr.II-3-1 to a second chromatographic column, and performing fourth column chromatographic separation, wherein the fourth column chromatographic separation is performed by adopting an acetonitrile-water system with the volume percentage of 20-35% to perform continuous gradient elution according to the volume percentage of acetonitrile to obtain an aporphine alkaloid compound with the structure shown in the formula 1;
(5) Mixing the isocorydine, manganese dioxide and an organic solvent for oxidation reaction, and removing the solvent from the reaction liquid obtained after solid-liquid separation to obtain an oxidation product; loading the oxidation product to a second gel chromatographic column for fifth column chromatographic separation, wherein methanol is adopted for isocratic elution in the fifth column chromatographic separation, and the same fractions are combined to respectively obtain a fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and a fraction containing the aporphine alkaloid compound with the structure shown in the formula 3; respectively loading the fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and the fraction containing the aporphine alkaloid compound with the structure shown in the formula 3 into third chromatographic columns, respectively carrying out sixth column chromatographic separation, and carrying out continuous gradient elution by adopting an acetonitrile-water system with the volume percentage of acetonitrile from small to large to obtain the aporphine alkaloid compound with the structure shown in the formula 2 and the aporphine alkaloid compound with the structure shown in the formula 3;
there is no chronological limitation between said step (4) and said step (5).
Preferably, the polar solvent is methanol, the leaching times are 4 times, the leaching temperature is normal temperature, the time of each leaching is 2 days, and the ratio of the mass of the dactylicapnos root to the volume of the polar solvent in each leaching is 1kg:10L.
Preferably, the dilute acid aqueous solution is hydrochloric acid solution, the mass percentage of the hydrochloric acid solution is 0.5%, the pH value of the acid solution is 2-3, the first extraction and the second extraction are both equal-volume extraction, and the times of the first extraction and the second extraction are all 3 times.
Preferably, in the step (3), the silica gel used in the mixing is 100-200 mesh silica gel, and the mass ratio of the total alkaloids of the golden dragon to the silica gel is 1:2 during the mixing;
the packing of the silica gel column is 200-300 meshes of silica gel.
Preferably, the first chromatographic column is an RP-18 medium pressure chromatographic column, and the second chromatographic column and the third chromatographic column are both C18 columns;
the first gel chromatographic column and the second gel chromatographic column are gel Sephadex LH-20 chromatographic columns.
The invention provides an aporphine alkaloid compound prepared by the technical scheme or the preparation method of the technical scheme, or application of the aporphine alkaloid compound in preparing medicines of indoleamine 2, 3-dioxygenase 1 and/or tryptophan 2, 3-dioxygenase mediated diseases, wherein the aporphine alkaloid compound is pharmaceutically acceptable salt, hydrate or solvent compound.
The invention provides a pharmaceutical composition, which comprises a pharmaceutical active component and a pharmaceutically acceptable carrier;
the pharmaceutical active component comprises the aporphine alkaloid compound prepared by the technical scheme or the preparation method of the technical scheme, or pharmaceutically acceptable salts, hydrates and solvent compounds of the aporphine alkaloid compound.
Preferably, the mass percentage of the active components of the medicine is more than or equal to 10 percent.
The invention provides an aporphine alkaloid compound, which comprises a structure shown in a formula 1, a formula 2 or a formula 3. The interaction between the aporphine alkaloid compound provided by the invention and TDO and IDO1 is detected by a nuclear magnetic resonance saturated transfer difference spectrum (NMR STD) method. If the aporphine alkaloid compound provided by the invention is combined with TDO and IDO1, saturation is carried out through intermolecular 1 H- 1 H cross-relaxation transferred to the aporphine alkaloid compound and exhibited a complete decrease in intensity. Thus, NMR STD spectra may show only with eggsWhite matter (TDO and IDO 1) in intimate contact. As can be seen from the results of the examples, the amplified STD spectra show H-3, H-4, H-5, H-8, H-9, 1-OCH 3 、10-OCH 3 And 11-OCH 3 Protons have a pronounced STD signal, indicating that these hydrogen atoms may bind to IDO1 and TDO. H-8, 10-OCH 3 And 11-OCH 3 The strongest proton intensity in the NMR STD spectrum indicates tighter binding to the protein. It was inferred that the 8-position hydrogen atom and the methoxy hydrogen atoms at the C-10 and C-11 positions were most closely bonded to the protein. The NMR STD results further confirmed that the compound of the structure shown in formula 1 was directly bound to IDO1 (A) and TDO (B), respectively. Meanwhile, experiments of half inhibition concentration of the compound also show that the aporphine alkaloid compound with the structure shown in the formula 1, the formula 2 or the formula 3 has double selective inhibition on IDO1/TDO, and the aporphine alkaloid compound with the structure shown in the formula 1 has the strongest inhibition effect.
Drawings
FIG. 1 is a flow chart showing the oxidation reaction of isocorydine with manganese dioxide in example 1 of the present invention;
FIG. 2 is a graph showing the binding of the aporphine alkaloid compound prepared in example 1 of the present invention to IDO1 and TDO.
Detailed Description
The invention provides an aporphine alkaloid compound, which comprises a structure shown in a formula 1, a formula 2 or a formula 3:
in the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
The invention provides pharmaceutically acceptable salts, hydrates or solvates of the aporphine alkaloid compounds in the technical scheme, wherein the pharmaceutically acceptable salts of the aporphine alkaloid compounds comprise inorganic acid salts and/or organic acid salts.
In the present invention, the inorganic acid salt preferably includes one or more of hydrochloride, hydrobromide, nitrate, sulfate and phosphate.
In the present invention, the organic acid salt includes one or more of tartrate, citrate, formate, acetate and oxalate.
The invention provides a preparation method of an aporphine alkaloid compound, which comprises the following steps:
(1) Leaching dactylicapnos with polar solvent, concentrating the obtained leaching solution to obtain extract;
(2) Dispersing the extract in a dilute acid aqueous solution, and performing first extraction on the obtained acidic solution by using first ethyl acetate to obtain a first ethyl acetate phase and a first water phase; the pH value of the first aqueous phase is regulated to 7-8, and then second extraction is carried out by using second ethyl acetate, so as to obtain a second ethyl acetate phase; concentrating the second ethyl acetate phase to obtain total alkaloids of dactylicapnos root;
(3) Mixing the dactylicapnos alkaloid total alkali and silica gel, loading the obtained mixture to a silica gel column for first column chromatographic separation, performing gradient elution on the first column chromatographic separation according to the volume ratio from large to small by adopting a chloroform-methanol system with the volume ratio of chloroform to methanol of 1:0-1:1, and merging the same fractions to obtain six sections of fractions which are respectively named as fractions Fr.I-VI; recrystallizing the fraction Fr.II to obtain a recrystallization mother liquor and isocorydine;
(4) Loading the recrystallization mother liquor to a first chromatographic column for second column chromatographic separation, wherein the second column chromatographic separation comprises gradient elution according to the volume percentage of methanol from small to large by adopting a methanol-water system of 20-90%, and merging the same fractions to obtain four sections of fractions which are named as fractions Fr.II-1-II-4 respectively; loading the fraction Fr.II-3 to a first gel chromatographic column for third column chromatographic separation, performing isocratic elution on the third column chromatographic separation by adopting methanol, and combining the same fractions to obtain three sections of fractions which are named as fractions Fr.II-3-1-II-3-3 respectively; loading the fraction Fr.II-3-1 to a second chromatographic column, and performing fourth column chromatographic separation, wherein the fourth column chromatographic separation is performed by adopting an acetonitrile-water system with the volume percentage of 20-35% to perform continuous gradient elution according to the volume percentage of acetonitrile to obtain an aporphine alkaloid compound with the structure shown in the formula 1;
(5) Mixing the isocorydine, manganese dioxide and an organic solvent for oxidation reaction, and removing the solvent from the reaction liquid obtained after solid-liquid separation to obtain an oxidation product; loading the oxidation product to a second gel chromatographic column for fifth column chromatographic separation, performing isocratic elution by adopting methanol, and combining the same fractions to obtain a fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and a fraction containing the aporphine alkaloid compound with the structure shown in the formula 3; and respectively loading the fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and the fraction containing the aporphine alkaloid compound with the structure shown in the formula 3 into third chromatographic columns, and respectively carrying out sixth chromatographic separation, wherein the sixth chromatographic separation adopts an acetonitrile-water system with the volume percentage of acetonitrile from small to large to carry out continuous gradient elution to respectively obtain the aporphine alkaloid compound with the structure shown in the formula 2 and the aporphine alkaloid compound with the structure shown in the formula 3.
The invention uses polar solvent to extract dactylicapnos root, and the obtained extract liquor is concentrated to obtain extract.
In the invention, the polar solvent is preferably methanol, the leaching times are preferably 4 times, the leaching temperature is preferably normal temperature, the time of each leaching is preferably 2 days, and the ratio of the mass of the dactylicapnos root to the volume of the polar solvent in each leaching is preferably 1 kg/10L. The specific embodiment of the concentration is preferably concentration under reduced pressure.
After an extract is obtained, the extract is dispersed in a dilute acid aqueous solution, and the obtained acid solution is subjected to first extraction by using first ethyl acetate to obtain a first ethyl acetate phase and a first water phase; the pH value of the first aqueous phase is regulated to 7-8, and then second extraction is carried out by using second ethyl acetate, so as to obtain a second ethyl acetate phase; concentrating the second ethyl acetate phase to obtain the total alkaloids of the dactylicapnos root.
In the present invention, the dilute acid aqueous solution is preferably a hydrochloric acid solution, the mass percentage of the hydrochloric acid solution is preferably 0.5%, the pH value of the acidic solution is preferably 2 to 3, the first extraction is preferably an equal volume extraction, and the number of times of the first extraction is preferably 3. The second extraction is preferably an equal volume extraction and the number of second extractions is preferably 3.
After obtaining the total alkaloids of the dactylicapnos root, the invention mixes the total alkaloids of the dactylicapnos root with silica gel, the obtained mixture is loaded on a silica gel column to carry out first column chromatography separation, the first column chromatography separation is carried out by gradient elution according to the volume ratio from large to small by adopting a chloroform-methanol system with the volume ratio of chloroform to methanol of 1:0-1:1, and the same fractions are combined to obtain six sections of fractions which are respectively named as fractions Fr.I-VI; recrystallizing the fraction Fr.II to obtain a recrystallization mother liquor and isocorydine.
In the invention, the silica gel used in the mixing is preferably 100-200 mesh silica gel, and the mass ratio of the total alkaloids of the golden dragon alkaloid to the silica gel is preferably 1:2 during the mixing. In the present invention, the packing of the silica gel column is preferably 200 to 300 mesh silica gel.
In the present invention, the first column chromatography separation is preferably performed under room temperature conditions. The first column chromatographic separation is preferably: the volume ratio is 1:0, 40: 1. 20: 1. 10: 1. 5: 1. gradient elution was performed with a 1:1 chloroform-methanol system. The 10 column volumes were eluted separately for each ratio, and the eluents were then combined for each ratio. The obtained eluate was subjected to Thin Layer Chromatography (TLC), and based on the result of the thin layer chromatography, the same fractions were combined to obtain six fractions, which were named fraction fr.i, fraction fr.ii, fraction fr.iii, fraction fr.iv, fraction fr.v and fraction fr.vi, respectively. In the present invention, the concentration is preferably concentration under reduced pressure.
The fraction Fr.II is recrystallized to obtain recrystallization mother liquor and isocorydine. In the present invention, the solvent for recrystallization is preferably methanol, the recrystallization is preferably evaporative crystallization, and the number of times of recrystallization is preferably 2. In the present invention, the specific embodiment of the recrystallization is preferably: dissolving the fraction Fr.II in methanol to obtain a fraction Fr.II solution; evaporating the fraction Fr.II solution at room temperature to remove the solvent, and evaporating the methanol to remove one third of the solvent to obtain a primary crystallization product; dissolving the crystallization product in methanol to obtain a primary crystallization product solution; and evaporating the primary crystallization product solution at room temperature to remove the solvent, and evaporating the methanol to remove one third of the solvent to obtain a crude product of isocorydine and a recrystallization mother liquor. In the invention, a methanol-water system with the volume percentage of 50% is preferably adopted to flush the crude product of the isocorydine, so as to obtain the pure product of the isocorydine.
After obtaining a recrystallization mother liquor, loading the recrystallization mother liquor to a first chromatographic column for second column chromatographic separation, wherein the second column chromatographic separation adopts a methanol-water system with the volume percent of 20-90% for gradient elution according to the volume percent of methanol from small to large, and the same fractions are combined to obtain four sections of fractions which are named as fractions Fr.II-1-II-4 respectively; loading the fraction Fr.II-3 to a first gel chromatographic column for third column chromatographic separation, performing isocratic elution on the third column chromatographic separation by adopting methanol, and combining the same fractions to obtain three sections of fractions which are named as fractions Fr.II-3-1-II-3-3 respectively; loading the fraction Fr.II-3-1 to a second chromatographic column for fourth column chromatographic separation, wherein the fourth column chromatographic separation adopts an acetonitrile-water system with the volume percentage of 20-35% to perform continuous gradient elution according to the volume percentage of acetonitrile to obtain the aporphine alkaloid compound with the structure shown in the formula 1.
In the present invention, the first chromatographic column is preferably an RP-18 medium pressure chromatographic column. The second column chromatographic separation is preferably carried out at room temperature. The second column chromatographic separation is as follows: sequentially adopting methanol-water systems with the volume percentage of 20%, 40%, 60%, 80% and 90% of methanol to carry out gradient elution. In the case of the gradient elution, the flow rate of the methanol-water system is preferably 30mL/min. The 8 column volumes were eluted separately for each ratio, and the eluents for each ratio were then combined. And carrying out thin-layer chromatography on the obtained eluent, merging the same fractions according to the thin-layer chromatography result, and concentrating to dryness to obtain four sections of fractions which are named as fraction Fr.II-1, fraction Fr.II-2, fraction Fr.II-3 and fraction Fr.II-4 respectively. In the present invention, the concentration is preferably concentration under reduced pressure.
The method comprises the steps of loading the fraction Fr.II-3 to a first gel chromatographic column for third column chromatography separation, performing isocratic elution on the third column chromatography by adopting methanol, and merging the same fractions to obtain three sections of fractions which are named as fractions Fr.II-3-1-II-3-3 respectively. In the present invention, the first gel column is preferably a gel Sephadex LH-20 column. The third column chromatography separation is preferably performed at room temperature. In the case of the isocratic elution, the flow rate of the methanol is preferably 0.5mL/min. And eluting 5 column volumes by using methanol, performing color band observation and thin layer chromatography analysis on the obtained eluent, merging the same fractions, and concentrating to dryness to obtain three sections of fractions which are named as fraction Fr.II-3-1, fraction Fr.II-3-2 and fraction Fr.II-3-3 respectively. In the present invention, the concentration is preferably concentration under reduced pressure.
The fraction Fr.II-3-1 is loaded to a second chromatographic column for fourth column chromatographic separation, and the fourth column chromatographic separation is carried out by adopting an acetonitrile-water system with the volume percentage of 20-35% to carry out continuous gradient elution according to the volume percentage of acetonitrile to obtain the aporphine alkaloid compound with the structure shown in the formula 1. In the present invention, the second chromatography column is preferably a C18 column, and the fourth column chromatography separation is preferably performed at room temperature. The fourth column chromatographic separation is as follows: sequentially adopting an acetonitrile-water system with the acetonitrile volume percentage of 20% -35% to carry out continuous gradient elution for 45min. In the case of the gradient elution, the flow rate of the acetonitrile-water system is preferably 8mL/min. The invention collects the fraction with the retention time of 28min and then concentrates the fraction to be dried to obtain the aporphine alkaloid compound with the structure shown in the formula 1. In the present invention, the concentration is preferably concentration under reduced pressure.
After isocorydine is obtained, the isocorydine, manganese dioxide and an organic solvent are mixed for oxidation reaction, and a reaction liquid obtained after solid-liquid separation is used for removing the solvent to obtain an oxidation product; loading the oxidation product to a second gel chromatographic column for fifth column chromatographic separation, performing isocratic elution by adopting methanol, and combining the same fractions to obtain a fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and a fraction containing the aporphine alkaloid compound with the structure shown in the formula 3; and respectively loading the fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and the fraction containing the aporphine alkaloid compound with the structure shown in the formula 3 into third chromatographic columns, and respectively carrying out sixth chromatographic separation, wherein the sixth chromatographic separation adopts an acetonitrile-water system with the volume percentage of acetonitrile from small to large to carry out continuous gradient elution to respectively obtain the aporphine alkaloid compound with the structure shown in the formula 2 and the aporphine alkaloid compound with the structure shown in the formula 3.
In the present invention, the organic solvent is preferably methylene chloride. The mass ratio of the isocorydine to the manganese dioxide is preferably 2:5. The oxidation reaction is carried out at room temperature, the time of the oxidation reaction is preferably 2 hours, and the oxidation reaction is carried out under stirring. The present invention preferably employs TLC to detect completion of the oxidation reaction. In the present invention, the solid-liquid separation is preferably filtration. The invention preferably adopts a reduced pressure evaporation mode to remove the solvent to obtain the oxidation product.
After an oxidation product is obtained, the oxidation product is loaded to a second gel chromatographic column for fifth column chromatographic separation, methanol is adopted for isocratic elution in the fifth column chromatographic separation, and the same fractions are combined to obtain a fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and a fraction containing the aporphine alkaloid compound with the structure shown in the formula 3. In the present invention, the second gel chromatographic column is preferably a gel Sephadex LH-20 chromatographic column. The fifth column chromatography separation is preferably performed at room temperature. In the case of the isocratic elution, the flow rate of the methanol is preferably 0.5mL/min. Eluting 5 column volumes with methanol, performing color band observation and thin layer chromatography detection on the obtained eluent, combining the same fractions, and concentrating to dryness to obtain a fraction containing the aporphine alkaloid compound with the structure shown in formula 2 and a fraction containing the aporphine alkaloid compound with the structure shown in formula 3. In the present invention, the concentration is preferably concentration under reduced pressure.
The method comprises the steps of respectively loading a fraction containing an aporphine alkaloid compound with a structure shown in a formula 2 and a fraction containing an aporphine alkaloid compound with a structure shown in a formula 3 into a third chromatographic column, respectively carrying out sixth column chromatographic separation, and carrying out gradient elution on the sixth column chromatographic separation according to the acetonitrile volume percentage from small to large by adopting an acetonitrile-water system with a volume percentage of 50-65% to respectively obtain the aporphine alkaloid compound with the structure shown in the formula 2 and the aporphine alkaloid compound with the structure shown in the formula 3. In the present invention, the third column is preferably a C18 column, and the sixth column chromatographic separation is preferably performed at room temperature. The sixth column chromatographic separation is as follows: sequentially adopting an acetonitrile-water system with the acetonitrile volume percentage of 50% -65% to carry out continuous gradient elution for 45min. The flow rate of the acetonitrile-water system is preferably 8mL/min at the time of the continuous gradient elution. The aporphine alkaloid compound with the structure shown in the formula 2 is obtained by collecting fractions with retention time of 26min and concentrating the fractions to dryness. The invention collects the fraction with the retention time of 41min and then concentrates the fraction to be dried to obtain the aporphine alkaloid compound with the structure shown in the formula 3. In the present invention, the concentration is preferably concentration under reduced pressure.
The invention provides an aporphine alkaloid compound prepared by the technical scheme or the preparation method of the technical scheme or application of the aporphine alkaloid compound in preparing medicines of indoleamine 2, 3-dioxygenase and/or tryptophan 2, 3-dioxygenase mediated diseases in pharmaceutically acceptable salts, hydrates or solvent compounds.
In the present invention, the indoleamine 2, 3-dioxygenase 1 and/or tryptophan 2, 3-dioxygenase mediated diseases are diseases in which TDO and/or IDO1 expression or activity is significantly enhanced.
In the present invention, the indoleamine 2, 3-dioxygenase 1 and/or tryptophan 2, 3-dioxygenase mediated diseases include cancer or metabolic disorder diseases.
The cancer includes glioblastoma, head and neck cancer, mesothelioma, non-small cell lung cancer, bladder cancer, or breast cancer.
The metabolic disorder disease includes obesity.
The invention provides a pharmaceutical composition, which comprises a pharmaceutical active component and a pharmaceutically acceptable carrier;
the pharmaceutical active component comprises the aporphine alkaloid compound prepared by the technical scheme or the aporphine alkaloid compound prepared by the preparation method of the technical scheme or pharmaceutically acceptable salt of the aporphine alkaloid compound.
The kind of the pharmaceutically acceptable carrier is not particularly limited in the present invention, and may be any kind known to those skilled in the art.
In the invention, the mass percentage of the aporphine alkaloid compound or the pharmaceutically acceptable salt thereof in the pharmaceutical composition is more than or equal to 10%, more preferably more than or equal to 20%, and most preferably more than or equal to 50%. .
The present invention is not limited in any particular manner to the form of administration of the pharmaceutical composition, and forms of administration known to those skilled in the art may be employed.
In the present invention, the route of administration of the pharmaceutical composition is preferably oral or non-oral. The daily dosage is preferably 1 to 1000mg. When the route of administration is oral, the pharmaceutical composition also preferably comprises pharmaceutically acceptable adjuvants; the pharmaceutical auxiliary agent preferably comprises one or more of excipient, disintegrating agent, binder, lubricant, antioxidant, coating agent, colorant, flavoring agent and surfactant; the mass percentage of the medicinal auxiliary agent in the pharmaceutical composition is preferably more than or equal to 10%, more preferably more than or equal to 20%, and most preferably more than or equal to 50%; the pharmaceutical composition is preferably administered in the form of granules, capsules or tablets. When the route of administration is parenteral, the pharmaceutical composition is preferably administered in the form of an injection, infusion or suppository. As one or more embodiments of the present invention, the pharmaceutically acceptable carrier preferably includes one or more of a-lactose monohydrate, starch, and cellulose.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Drying radix Dactylicapni (D.scandens) sample 10kg, pulverizing, leaching with methanol at room temperature for 4 times, each time with solvent amount (volume) 10 times of sample mass, each time for 2 days, and recovering the extractive solution under reduced pressure to obtain total extract. Dissolving the total extract completely with 0.5% HCl solution, adjusting pH to 2-3, extracting with equal volume of ethyl acetate for 3 times, mixing with rotary evaporator, concentrating under reduced pressure to obtain non-alkaloid component, and removing non-alkaloid component; adjusting pH of the acid water part to 7-8 with 10% ammonia water, extracting with equal volume of ethyl acetate for 3 times, combining ethyl acetate layers, and concentrating under reduced pressure with a rotary evaporator to obtain radix Dactylicapni total alkaloids. Mixing with silica gel with the total alkaloid mass of twice 100-200 meshes, performing silica gel column chromatography with 200-300 meshes, performing gradient elution with chloroform-methanol (from 1:0, 40:1, 20:1, 10:1,5:1 to 1:1), detecting by TLC, and combining the same fractions to obtain six sections of fractions named fractions Fr.I-VI. Dissolving fraction Fr.II (80.0 g) with methanol, standing at room temperature, volatilizing the solvent for one third, crystallizing, dissolving the precipitated crystal with methanol again, standing at room temperature, volatilizing the solvent for one third, and precipitating pure isocorydine crystal again to obtain recrystallization mother liquor and isocorydine.
The recrystallization mother liquor is firstly eluted by 20 percent, 40 percent, 60 percent and 80 percent of methanol water sequentially through an RP-18 medium-pressure chromatographic column, detected by TLC, four sections of fractions are obtained by combining the same fractions, which are named as fractions Fr.II-1 to II-4, the fraction Fr.II-3 is eluted by methanol through a gel Sephadex LH-20 chromatographic column, observed by a color band and detected by thin layer chromatography, and is combined into three sections of fractions, which are named as fractions Fr.II-3-1 to II-3-3, the fraction Fr.II-3-1 is purified by HPLC (C18 preparation column, gradient elution is carried out for 45min by 20 to 35 percent acetonitrile), and the fraction with the retention time of 28min peak is collected, thus obtaining the pure aporphine alkaloid compound (4.0 mg) with the structure shown in the formula 1.
An aporphine alkaloid compound of the structure shown in formula 1: a dark green powder; ultraviolet spectrum (MeOH) lambda (max): 241,312,405,588nm; high resolution mass spectrum HRESIMS m/z 374.0999[ M+Na ]] + Molecular formula C 20 H 17 NO 5 。 1 HNMR(500MHz,methanol-d 4 )δppm 8.21(d,J＝6.2Hz,1H,H-5),8.13(d,J＝8.8Hz,1H,H-8),7.90(d,J＝6.2Hz,1H,H-4),7.24(d,J＝8.8Hz,1H,H-9),7.19(s,1H,H-3),4.66(s,3H,N-CH 3 ),4.09(s,3H,1-OCH 3 ),4.01(s,3H,10-OCH 3 ),3.76(s,3H,11-OCH 3 ). 13 C NMR(125MHz,methanol-d 4 )δ178.2(s,C-7),170.0(s,C-2),164.5(s,C-1),158.5(s,C-10),146.6(s,C-11),140.5(s,C-3a),138.1(d,C-5),131.5(s,C-11a),129.7(s,C-6a),128.9(s,C-7a),127.0(s,C-1b),124.9(d,C-8),122.6(d,C-4),112.2(d,C-9),105.9(s,C-1a),104.1(d,C-3),60.4(q,11-OCH 3 ),57.0(q,1-OCH 3 ),56.7(q,10-OCH 3 ),49.5(q,N-CH 3 ).HRESIMS m/z 374.0999[M+Na] + (calc.for C 20 H 17 NO 5 Na,374.0999)。
Example 2
Isocorydine (20 mg,29.32 mmol) prepared in example 1 was dissolved in 2mL of methylene chloride, and MnO was added thereto 2 (50 mg,290.69 mmol) was stirred at room temperature for 2h (oxidation scheme shown in FIG. 1), TLC was monitored to complete the reaction, and the mixture was filtered to remove unreacted MnO 2 The solvent was evaporated under reduced pressure, and after eluting with methanol, the fractions were combined to obtain a fraction containing the aporphine alkaloid compound of the structure shown in formula 2 and a fraction containing the aporphine alkaloid compound of the structure shown in formula 3, which were purified by HPLC (C18 column 50-65% acetonitrile water was eluted for 45 min), and the fraction having a retention time of 26min was collected to obtain an aporphine alkaloid compound pure product (2.5 mg) of the structure shown in formula 2, and the fraction having a retention time of 41min was collected to obtain an aporphine alkaloid compound pure product (2.0 mg) of the structure shown in formula 3.
An aporphine alkaloid compound of the structure shown in formula 2: blue-green powder; ultraviolet spectrum (MeOH)
(max) 232,270,344,398,594nm; mass spectrum ESIMS m/z 685.2526[ M+Na ]] + Molecular formula C 39 H 38 N 2 O 8 . Nuclear magnetic data: 1 H NMR(500MHz,methanol-d 4 )δ H 7.71(s,1H),7.36(d,J＝8.3Hz,1H),7.15(d,J＝8.3Hz,1H),7.07(s,1H),6.72(s,1H),4.76(s,1H),3.93(s,3H),3.86(s,3H),3.73(s,3H),3.70(s,3H),3.62(m,2H),3.53(s,3H),3.47(s,3H),3.21(dd,J＝13.2,3.4Hz,1H),3.14(m,2H),3.02(m,1H),3.02(m,1H),2.99(m,1H),2.88(dd,J＝12.5,3.4Hz,1H),2.64(d,J＝15.8,1H),2.48(m,1H),2.39(t,J＝13.2Hz,1H),2.54(s,3H). 13 C NMR(125MHz,methanol-d 4 )δ C 186.6,178.9,170.7,154.5,153.6,153.3,152.0,146.8,144.4,139.6,138.7,131.9,131.2,130.0,129.9,129.5,127.1,127.0,124.6，118.5,118.1,113.6,113.0,112.9,103.6,101.9,64.5,61.4,60.7,56.8,56.6,56.3,53.9,51.3,44.0,40.5,35.8,29.5,29.2.
an aporphine alkaloid compound of the structure shown in formula 3: blue-green powder; ultraviolet spectrum (MeOH)
(max) 232,311,343,398,588nm; mass spectrum ESIMS m/z 1000.4042[ M-H ]] - Molecular formula C 59 H 59 N 3 O 12 The method comprises the steps of carrying out a first treatment on the surface of the Nuclear magnetic data: 1 H NMR(600MHz,methanol-d 4 )δ H :7.77(s,1H),7.25(s,1H),7.16(s,1H),7.15(s,1H),6.91(s,1H),6.78(s,1H),4.96(s,1H),4.02(s,3H),4.00(s,3H),3.98(s,3H),3.93(s,3H),3.78(s,3H),3.77(s,3H),3.69(m,2H),3.62(s,3H),3.56(s,3H),3.24(m,1H),3.18(m,1H),3.03(m,1H),2.96(m,2H),2.95(m,2H),2.84(overlap,2H),2.78(m,1H),2.69(m,2H),2.49(m,2H),2.27(s,6H),2.13(m,2H). 13 C NMR(125MHz,methanol-d 4 )δ C :186.6,179.4,166.3,154.6,153.7,153.3,153.2,153.0,151.6,150.2,144.6,144.5,144.3,139.8,139.0,131.6,131.3,130.9,130.7,130.0,129.7,129.6,129.5,128.8,127.2,126.6,124.7,123.8,121.5,118.6,118.1,114.6,114.0,113.7,112.9,112.8,103.6,101.9,64.2,64.0,62.3,61.5,60.7,56.9,56.9,56.8,56.5,56.3,53.7,53.5,51.4,43.7,43.5,40.6,33.2,33.1,29.6,29.4,29.0.
test example 1
Cloning and inducible expression of the genes IDO1 (uniprot ID: P14202), TDO (uniprot ID: P48775): the optimized DNA fragment of the human IDO1 is amplified by in vitro PCR and is constructed into a vector pET28 a. The correctness of the insert and the constructed vector is determined by genetic sequencing. After the sequencing results were correct, the plasmid was transformed into BL21 (DE 3) E.coli for protein expression and purification. The expression and purification process of the protein is as follows: the transformed E.coli was selected and cultured overnight in LB medium in a shaker at 37 ℃. The next day the bacterial liquid is amplified into 1L LB culture medium, and when the bacterial liquid grows to OD600 in logarithmic growth phase: 0.6-0.8, 5-aminolevulinic acid (ALA) (final concentration 0.5 mM) was added to adjust the rotation speed to 120rpm, IPTG (final concentration 0.5 mM) was added after half an hour, and cells were collected after induction at 30℃for 6 hours. After the thalli are mixed and resuspended, the thalli are crushed under ice bath condition until the cell suspension is nearly clear, the protein solution is separated from the sediment by high-speed centrifugation at 4 ℃, and the protein expression quantity in the supernatant is detected by SDS-PAGE electrophoresis. TDO gene cloning and induced expression are identical to IDO1.
Purification and concentration measurement of protein: the protein solution contains fusion protein IDO1, and as the N end of the vector pET28a is provided with a His-tag fusion tag, the fusion protein IDO1 is provided with the His-tag and can be combined with metal ion Ni 2+ The special interaction occurs, and the fusion protein IDO1 is adsorbed on a nickel column by utilizing the characteristics of the protein surface, so that the protein is separated and purified. To increase the protein purity, molecular sieve Superdex G75 was further used for purification. Finally, the purity of the protein reaches more than 95 percent through SDS-PAGE identification. TDO protein was purified as IDO1.
IDO1, TDO protein concentration determination: the purified protein concentration was measured by BCA ProteinAssay Kit using a bovine serum protein standard curve as a reference. In each detection process, different dilution concentrations are used for simultaneous detection, so that errors in the concentration detection process are ensured.
Enzymatic activity reaction system: the reaction system is 80 mu L, and the final concentration of each component is respectively as follows: 50mM potassium phosphate buffer (Ph 6.5), 40mM ascorbic acid, 200. Mu.g/mL catalase, 20uM methylene blue, 300uM substrate L-tryptophan. The final concentration of DMSO was controlled to within 2%. Enzyme was added to the system, preheated in an oven at 37℃for five minutes, and then the compound was added to react in a buffer for 30 minutes. The compound concentrations were: 0. Mu.M, 0.064. Mu.M, 0.320. Mu.M, 1.600. Mu.M, 8.000. Mu.M, 40.000. Mu.M. After the subsequent addition of 30% (w/v) trichloroacetic acid (TCA), the reaction was terminated by heating at 65℃for 15 minutes. And the reaction mixture was centrifuged at 12000rpm for 10min. The supernatant was transferred to 96-well microplates and added to acetic acid 2% (w/v) of p-Dimethylaminobenzaldehyde (DMAB) was added. Kynurenine concentration was determined by measuring absorbance at 490nm using a Bio-Rad microplate reader. Determination of enzyme IC by nonlinear regression analysis using GraphPad Prism 8.0.1 50 Values.
Experimental results: half inhibition concentration of compound experimental steps such as enzyme activity experiments, the compound concentration is set as follows: 0. 0.064, 0.320, 1.600, 8.000, 40.000 μm. As shown in Table 1, the results of the enzymatic IDO1/TDO inhibitory activity of the compounds are shown in Table 1, and it is clear from Table 1 that the aporphine alkaloid compounds of the structures shown in formula 1, formula 2 or formula 3 prepared in example 1 are IDO1/TDO dual-selective inhibitors, and the aporphine alkaloid compounds of the structures shown in formula 1 are the most effective.
TABLE 1 inhibitory Activity of aporphine alkaloid compounds prepared in example 1
Test example 2: characterization in combination with IDO1 and TDO
Nuclear magnetic resonance saturation transfer differential spectroscopy (NMR STD) techniques are commonly used to detect protein-ligand interactions. If the ligand binds to the protein, saturation occurs through intermolecular 1 H- 1 H cross-relaxation is transferred to the ligand and appears as a complete decrease in intensity. Thus, NMR STD spectra may only show ligand signals in intimate contact with the protein. The reference spectrum of the aporphine alkaloid compound of the structure shown in formula 1 alone (C in fig. 2) and its STD spectrum in the presence of IDO1 (a in fig. 2) and TDO (B in fig. 2) are shown in fig. 2. The amplified STD spectra showed H-3, H-4, H-5, H-8, H-9, 1-OCH 3 、10-OCH 3 And 11-OCH 3 Protons have a pronounced STD signal, indicating that these hydrogen atoms may bind to IDO1 and TDO. H-8, 10-OCH 3 And 11-OCH 3 The strongest proton intensity in the NMR STD spectrum indicates tighter binding to the protein. It was inferred from this that the hydrogen proton at the 8-position was most closely bound to the methoxy hydrogen atom at the C-10 and C-11 positions and the protein. The NMR STD results further confirm that the structure of aporphine shown in FIG. 1The alkaloid-like compounds are directly bound to IDO1 (a) and TDO (B), respectively. Important groups are also derived that bind and interact intimately with the protein.
Example 3
Preparation of sulfate:
the 3 aporphine alkaloid compounds with chemical structures prepared in the example 1 are respectively added with sulfuric acid ethanol solution with the mass concentration of 4% to pH=4, filtered, and the filtrate is dried to obtain the sulfate of the aporphine alkaloid compound.
Example 4
Preparation of hydrochloride:
the 3 aporphine alkaloid compounds with chemical structures prepared in example 1 are respectively added with hydrochloric acid ethanol solution with mass concentration of 4% to pH=4, filtered, and the filtrate is dried to obtain the hydrochloride of the aporphine alkaloid compound.
Example 5
Preparation of phosphate:
The 3 aporphine alkaloid compounds with chemical structures prepared in example 1 are respectively added with phosphoric acid solution with mass concentration of 4% to pH=4, filtered, and the filtrate is dried to obtain the phosphate of the aporphine alkaloid compound.
Example 6
Preparation of tartrate:
the 3 aporphine alkaloid compounds with chemical structures prepared in example 1 are respectively added with tartaric acid solution with mass concentration of 4% to pH=4, filtered, and the filtrate is dried to obtain tartrate of the aporphine alkaloid compound.
Example 7
Preparation of citrate:
the 3 aporphine alkaloid compounds with chemical structures prepared in example 1 are respectively added with citric acid solution with mass concentration of 4% to pH=4, filtered, and the filtrate is dried to obtain the citrate of the aporphine alkaloid compound.
Example 8
Preparation of formate:
the 3 aporphine alkaloid compounds with chemical structures prepared in example 1 are respectively added with formic acid solution with mass concentration of 4% to pH=4, filtered, and the filtrate is dried to obtain formate of the aporphine alkaloid compound.
Example 9
Preparation of oxalate:
the 3 aporphine alkaloid compounds with chemical structures prepared in the example 1 are respectively added with oxalic acid solution with the mass concentration of 4% to the pH value of 4, filtered, and the filtrate is dried to obtain oxalic acid salt of the aporphine alkaloid compound.
Example 10
Preparation of injection:
the salt prepared in examples 2 to 8 was added with water for injection, finely filtered, filled and sterilized to obtain an injection.
Example 11
Preparation of powder injection:
dissolving the salt prepared in the examples 2-8 in sterile water for injection, stirring until the salt is dissolved, filtering by using a sterile suction filter funnel, performing sterile fine filtration, packaging in an ampoule, performing low-temperature freeze drying, and performing sterile sealing to obtain the powder injection.
Example 12
Preparation of the powder:
the salt prepared in example 2 was mixed with excipients in a mass ratio of 9:1 to give a powder.
Wherein the excipient is a-lactose monohydrate.
Example 13
Preparation of tablets:
the salt prepared in examples 2 to 8 was mixed with an excipient at a mass ratio of 1:10, and tabletted to obtain tablets.
Wherein the excipient is cellulose.
Example 14
The salt prepared in example 3 and the excipient are mixed according to the mass ratio of 5:1, and the salt prepared in examples 2-8 and the excipient are prepared into capsules, granules or medicinal granules.
Wherein the excipient is starch.
Example 15
The salt prepared in example 4 and the excipient are mixed according to the mass ratio of 3:1, and the salt prepared in examples 2-8 and the excipient are prepared into capsules, granules or medicinal granules.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.
Claims (6)
1. An aporphine alkaloid compound is characterized by comprising the following structure:
2. the pharmaceutically acceptable salt of the aporphine alkaloid compound of claim 1, wherein the pharmaceutically acceptable salt of the aporphine alkaloid compound comprises an inorganic acid salt and/or an organic acid salt;
the inorganic acid salt comprises one or more of hydrochloride, hydrobromide, nitrate, sulfate and phosphate;
the organic acid salt comprises one or more of tartrate, citrate, formate, acetate and oxalate.
3. The process for preparing an aporphine alkaloid compound of claim 1, comprising the steps of:
(1) Leaching dactylicapnos with methanol, and concentrating the obtained leaching solution to obtain an extract; the leaching times are 4 times, the leaching temperature is normal temperature, the time of each leaching is 2 days, and the ratio of the mass of the dactylicapnos root to the volume of the polar solvent in each leaching is 1kg:10L;
(2) Dispersing the extract in a hydrochloric acid solution, wherein the mass percentage of the hydrochloric acid solution is 0.5%, performing first extraction on the obtained acidic solution by using first ethyl acetate, and obtaining a first ethyl acetate phase and a first water phase, wherein the pH value of the acidic solution is 2-3; the pH value of the first aqueous phase is regulated to 7-8, and then second extraction is carried out by using second ethyl acetate, so as to obtain a second ethyl acetate phase; concentrating the second ethyl acetate phase to obtain total alkaloids of dactylicapnos root; the first extraction and the second extraction are both equal-volume extraction, and the times of the first extraction and the second extraction are both 3 times;
(3) Mixing the dactylicapnos root alkaloid total alkali and silica gel, wherein the silica gel used in the mixing is 100-200 meshes silica gel, and the mass ratio of the dactylicapnos root alkaloid total alkali to the silica gel is 1:2 during the mixing; loading the obtained mixture to a silica gel column for first column chromatographic separation, wherein the packing of the silica gel column is 200-300 meshes of silica gel, the first column chromatographic separation adopts chloroform-methanol system with the volume ratio of chloroform to methanol of 1:0-1:1 for gradient elution according to the volume ratio of the silica gel column to the silica gel column, and the same fractions are combined to obtain six sections of fractions which are respectively named as fractions Fr.I-Fr.VI; recrystallizing the fraction Fr.II to obtain a recrystallization mother liquor and isocorydine;
(4) Loading the recrystallization mother liquor to a first chromatographic column for second column chromatographic separation, wherein the first chromatographic column is RP-18 medium-pressure chromatographic column, the second column chromatographic separation adopts a methanol-water system with the volume percent of 20-90% for gradient elution according to the volume percent of methanol from small to large, and the same fractions are combined to obtain four sections of fractions which are named as fractions Fr.II-1-Fr.II-4 respectively; loading the fraction Fr.II-3 to a first gel chromatographic column for third column chromatographic separation, wherein the third column chromatographic separation adopts methanol for isocratic elution, and the same fractions are combined to obtain three sections of fractions which are named as fractions Fr.II-3-1 to Fr.II-3-3 respectively; loading the fraction Fr.II-3-1 to a second chromatographic column, and performing fourth column chromatographic separation, wherein the fourth column chromatographic separation is performed by adopting an acetonitrile-water system with the volume percentage of 20-35% to perform continuous gradient elution according to the volume percentage of acetonitrile to obtain an aporphine alkaloid compound with the structure shown in the formula 1;
(5) Mixing the isocorydine, manganese dioxide and an organic solvent for oxidation reaction, and removing the solvent from the reaction liquid obtained after solid-liquid separation to obtain an oxidation product; loading the oxidation product to a second gel chromatographic column for fifth column chromatographic separation, wherein methanol is adopted for isocratic elution in the fifth column chromatographic separation, and the same fractions are combined to respectively obtain a fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and a fraction containing the aporphine alkaloid compound with the structure shown in the formula 3; respectively loading the fraction containing the aporphine alkaloid compound with the structure shown in the formula 2 and the fraction containing the aporphine alkaloid compound with the structure shown in the formula 3 into third chromatographic columns, respectively carrying out sixth column chromatographic separation, and carrying out continuous gradient elution by adopting an acetonitrile-water system with the volume percentage of acetonitrile from small to large to obtain the aporphine alkaloid compound with the structure shown in the formula 2 and the aporphine alkaloid compound with the structure shown in the formula 3;
The second chromatographic column and the third chromatographic column are both C18 columns; the first gel chromatographic column and the second gel chromatographic column are gel Sephadex LH-20 chromatographic columns;
there is no chronological limitation between said step (4) and said step (5).
4. Use of an aporphine alkaloid compound according to claim 1 or an aporphine alkaloid compound prepared by the preparation method according to claim 3 or an aporphine alkaloid compound according to claim 2 in the preparation of a pharmaceutically acceptable salt for the treatment of indoleamine 2, 3-dioxygenase 1 and/or tryptophan 2, 3-dioxygenase mediated diseases.
5. A pharmaceutical composition comprising a pharmaceutically active ingredient and a pharmaceutically acceptable carrier;
the pharmaceutically active component comprises an aporphine alkaloid compound prepared by the preparation method of claim 1 or an aporphine alkaloid compound prepared by the preparation method of claim 3 or a pharmaceutically acceptable salt of the aporphine alkaloid compound of claim 2.
6. The pharmaceutical composition according to claim 5, wherein the mass percentage of the pharmaceutically active component in the pharmaceutical composition is not less than 10%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310237740.6A CN116375644B (en) | 2023-03-14 | 2023-03-14 | Aporphine alkaloid compound and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310237740.6A CN116375644B (en) | 2023-03-14 | 2023-03-14 | Aporphine alkaloid compound and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116375644A CN116375644A (en) | 2023-07-04 |
| CN116375644B true CN116375644B (en) | 2024-01-16 |
Family
ID=86964795
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310237740.6A Active CN116375644B (en) | 2023-03-14 | 2023-03-14 | Aporphine alkaloid compound and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116375644B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1923813A (en) * | 2005-08-31 | 2007-03-07 | 中国科学院上海药物研究所 | Aporphine and use of oxidized aporphine alkaloid |
| CN109806259A (en) * | 2019-02-28 | 2019-05-28 | 中国科学院昆明植物研究所 | Application of the proto-berberine compounds with TDO selective inhibitory activity in pharmacy |
| CN109824753A (en) * | 2018-11-20 | 2019-05-31 | 中国科学院昆明植物研究所 | Tanshinone IIA derivative with IDO/TDO double selectivity inhibitory activity |
| CN115490637A (en) * | 2022-06-21 | 2022-12-20 | 海南医学院 | Preparation method and application of aporphine alkaloid compound |
-
2023
- 2023-03-14 CN CN202310237740.6A patent/CN116375644B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1923813A (en) * | 2005-08-31 | 2007-03-07 | 中国科学院上海药物研究所 | Aporphine and use of oxidized aporphine alkaloid |
| CN109824753A (en) * | 2018-11-20 | 2019-05-31 | 中国科学院昆明植物研究所 | Tanshinone IIA derivative with IDO/TDO double selectivity inhibitory activity |
| CN109806259A (en) * | 2019-02-28 | 2019-05-28 | 中国科学院昆明植物研究所 | Application of the proto-berberine compounds with TDO selective inhibitory activity in pharmacy |
| CN115490637A (en) * | 2022-06-21 | 2022-12-20 | 海南医学院 | Preparation method and application of aporphine alkaloid compound |
Non-Patent Citations (1)
| Title |
|---|
| M.-F. Bao et al..Discovery and biological evaluation of a new type of dual inhibitors of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase from ethnomedicinal plant Dactylicapnos scandens.Phytochemistry.2023,第214卷113794. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116375644A (en) | 2023-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107986951B (en) | Novel topoisomerase I inhibitor, pharmaceutical composition thereof, preparation method and application thereof | |
| CN108484699B (en) | Bipyridine alkaloid, preparation method and application thereof | |
| WO2017220042A2 (en) | Amrinone pharmaceutical composition and application thereof in hypertension treatment | |
| Luo et al. | Isolation, synthesis and bioactivity evaluation of isoquinoline alkaloids from Corydalis hendersonii Hemsl. against gastric cancer in vitro and in vivo | |
| CN113563409B (en) | Natural triterpene-iridoid glycoside dimer heterocomplex, preparation method thereof and application thereof in preparation of ACL inhibitor | |
| CN101993370A (en) | Glaucocalyxin A acid ester derivative as well as preparation method and application of Glaucocalyxin A acid ester derivative | |
| CN107445952B (en) | Method for extracting gladiolide and coptisine and application | |
| Men et al. | Chemical constituents and ACE inhibitory activity of desert plant Suaeda physophora Pall. | |
| CN116375644B (en) | Aporphine alkaloid compound and preparation method and application thereof | |
| CN101612183B (en) | Glaucescent fissistigma root saponin extract, pharmaceutical composition and preparation method and application thereof | |
| CN102050832B (en) | Melonines bisindole compound, medicine composition, preparation method and application thereof | |
| CN115154453B (en) | Application of aspulvinone compound in preparation of antidiabetic drugs | |
| CN109810113A (en) | A kind of alkaloid compound and the preparation method and application thereof | |
| CN113679727B (en) | Use of natural triterpene-iridoid glycoside dimer hybrid in preparation of acetyl coenzyme A carboxylase1 inhibitor | |
| CN113214214B (en) | Preparation method and application of terpenoid in Atractylodes lancea | |
| CN101993359A (en) | Fatty acid derivative of glaucocalyxin A, as well as preparation method and applications thereof | |
| CN112979640B (en) | Alkaloid dimer compound and application thereof in preparation of PD-1/PD-L1 pathway inhibitor | |
| CN110256468B (en) | Bisindole alkaloid compound or pharmaceutically acceptable salt thereof, and preparation method and application thereof | |
| CN110498805B (en) | Bis-furoxanthone and bis-furoanthraquinone compounds, and preparation method and application thereof | |
| EP2999480B1 (en) | Extract of hippeastrum papilio rich in galanthamine | |
| CN101851271A (en) | Glaucocalyxin D derivative, preparation method and application thereof | |
| CN115073481B (en) | Furana quebracho base dimer or pharmaceutically acceptable salt thereof, and preparation method and application thereof, and pharmaceutical composition | |
| WO2017220051A2 (en) | Benserazide hydrochloride pharmaceutical composition and medical use thereof for lowering blood sugar | |
| CN109678873B (en) | Coumarin compound and preparation method and application thereof | |
| CN115991687B (en) | Diisopentenyl substituted aspulvinone compound and preparation method and application thereof |
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 |