WO2023035927A1 - Compound for targeted degradation of tyrosinase, pharmaceutical composition, and method for synthesizing compound and use thereof - Google Patents
Compound for targeted degradation of tyrosinase, pharmaceutical composition, and method for synthesizing compound and use thereof Download PDFInfo
- Publication number
- WO2023035927A1 WO2023035927A1 PCT/CN2022/114098 CN2022114098W WO2023035927A1 WO 2023035927 A1 WO2023035927 A1 WO 2023035927A1 CN 2022114098 W CN2022114098 W CN 2022114098W WO 2023035927 A1 WO2023035927 A1 WO 2023035927A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tyrosinase
- compound
- degradation
- synthesis
- nmr
- Prior art date
Links
- 102000003425 Tyrosinase Human genes 0.000 title claims abstract description 86
- 108060008724 Tyrosinase Proteins 0.000 title claims abstract description 86
- 230000015556 catabolic process Effects 0.000 title claims abstract description 63
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 63
- 150000001875 compounds Chemical class 0.000 title claims abstract description 56
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 title claims abstract description 9
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 4
- 230000008685 targeting Effects 0.000 claims abstract description 40
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 claims abstract description 17
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 claims abstract description 17
- 239000002537 cosmetic Substances 0.000 claims abstract description 12
- 238000011282 treatment Methods 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 11
- 239000002994 raw material Substances 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 10
- 208000017520 skin disease Diseases 0.000 claims abstract description 10
- 230000002087 whitening effect Effects 0.000 claims abstract description 9
- 239000000651 prodrug Substances 0.000 claims abstract description 4
- 229940002612 prodrug Drugs 0.000 claims abstract description 4
- 150000003839 salts Chemical class 0.000 claims abstract description 4
- 239000012453 solvate Substances 0.000 claims abstract description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 35
- 238000003786 synthesis reaction Methods 0.000 claims description 34
- 150000003384 small molecules Chemical class 0.000 claims description 22
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 claims description 21
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 claims description 21
- 229960004502 levodopa Drugs 0.000 claims description 14
- 208000003351 Melanosis Diseases 0.000 claims description 9
- 125000005647 linker group Chemical group 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 239000003937 drug carrier Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- DZAUWHJDUNRCTF-UHFFFAOYSA-N 3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound OC(=O)CCC1=CC=C(O)C(O)=C1 DZAUWHJDUNRCTF-UHFFFAOYSA-N 0.000 claims description 3
- OBOXTJCIIVUZEN-UHFFFAOYSA-N [C].[O] Chemical group [C].[O] OBOXTJCIIVUZEN-UHFFFAOYSA-N 0.000 claims description 3
- 208000000069 hyperpigmentation Diseases 0.000 claims description 3
- 230000003810 hyperpigmentation Effects 0.000 claims description 3
- 206010067248 Congenital naevus Diseases 0.000 claims description 2
- 206010014970 Ephelides Diseases 0.000 claims description 2
- 201000009139 Mongolian Spot Diseases 0.000 claims description 2
- 206010048218 Xeroderma Diseases 0.000 claims description 2
- 206010021198 ichthyosis Diseases 0.000 claims description 2
- 208000004942 nevus of Ota Diseases 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims 2
- 238000011865 proteolysis targeting chimera technique Methods 0.000 abstract description 25
- 108010026668 snake venom protein C activator Proteins 0.000 abstract description 25
- 230000000694 effects Effects 0.000 abstract description 24
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract description 22
- 239000003446 ligand Substances 0.000 abstract description 21
- 102000004169 proteins and genes Human genes 0.000 abstract description 21
- 108090000623 proteins and genes Proteins 0.000 abstract description 21
- 229940124823 proteolysis targeting chimeric molecule Drugs 0.000 abstract description 20
- 238000000338 in vitro Methods 0.000 abstract description 7
- 231100000053 low toxicity Toxicity 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 4
- 231100000419 toxicity Toxicity 0.000 abstract 1
- 230000001988 toxicity Effects 0.000 abstract 1
- 238000005481 NMR spectroscopy Methods 0.000 description 85
- 238000004896 high resolution mass spectrometry Methods 0.000 description 28
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 26
- 238000006243 chemical reaction Methods 0.000 description 25
- 210000003491 skin Anatomy 0.000 description 23
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 18
- 241000252212 Danio rerio Species 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 14
- 229910052799 carbon Inorganic materials 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 230000003078 antioxidant effect Effects 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 10
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 10
- 230000001939 inductive effect Effects 0.000 description 10
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-VMNATFBRSA-N methanol-d1 Chemical compound [2H]OC OKKJLVBELUTLKV-VMNATFBRSA-N 0.000 description 9
- 229960003433 thalidomide Drugs 0.000 description 9
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 8
- 239000003963 antioxidant agent Substances 0.000 description 8
- 235000006708 antioxidants Nutrition 0.000 description 8
- 150000003254 radicals Chemical class 0.000 description 8
- 208000012641 Pigmentation disease Diseases 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- -1 small molecule compounds Chemical class 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 229930003268 Vitamin C Natural products 0.000 description 6
- BEJNERDRQOWKJM-UHFFFAOYSA-N kojic acid Chemical compound OCC1=CC(=O)C(O)=CO1 BEJNERDRQOWKJM-UHFFFAOYSA-N 0.000 description 6
- 229960004705 kojic acid Drugs 0.000 description 6
- WZNJWVWKTVETCG-UHFFFAOYSA-N kojic acid Natural products OC(=O)C(N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-UHFFFAOYSA-N 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 239000011718 vitamin C Substances 0.000 description 6
- 235000019154 vitamin C Nutrition 0.000 description 6
- 101000606090 Homo sapiens Tyrosinase Proteins 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 239000006180 TBST buffer Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 230000008099 melanin synthesis Effects 0.000 description 5
- 239000012074 organic phase Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 210000002257 embryonic structure Anatomy 0.000 description 4
- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 230000019612 pigmentation Effects 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 3
- AFSDNFLWKVMVRB-UHFFFAOYSA-N Ellagic acid Chemical compound OC1=C(O)C(OC2=O)=C3C4=C2C=C(O)C(O)=C4OC(=O)C3=C1 AFSDNFLWKVMVRB-UHFFFAOYSA-N 0.000 description 3
- ATJXMQHAMYVHRX-CPCISQLKSA-N Ellagic acid Natural products OC1=C(O)[C@H]2OC(=O)c3cc(O)c(O)c4OC(=O)C(=C1)[C@H]2c34 ATJXMQHAMYVHRX-CPCISQLKSA-N 0.000 description 3
- 229920002079 Ellagic acid Polymers 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000001588 bifunctional effect Effects 0.000 description 3
- 229960002852 ellagic acid Drugs 0.000 description 3
- 235000004132 ellagic acid Nutrition 0.000 description 3
- FAARLWTXUUQFSN-UHFFFAOYSA-N methylellagic acid Natural products O1C(=O)C2=CC(O)=C(O)C3=C2C2=C1C(OC)=C(O)C=C2C(=O)O3 FAARLWTXUUQFSN-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- AHMIDUVKSGCHAU-UHFFFAOYSA-N Dopaquinone Natural products OC(=O)C(N)CC1=CC(=O)C(=O)C=C1 AHMIDUVKSGCHAU-UHFFFAOYSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 101000941994 Homo sapiens Protein cereblon Proteins 0.000 description 2
- 239000002211 L-ascorbic acid Substances 0.000 description 2
- 235000000069 L-ascorbic acid Nutrition 0.000 description 2
- AHMIDUVKSGCHAU-LURJTMIESA-N L-dopaquinone Chemical compound [O-]C(=O)[C@@H]([NH3+])CC1=CC(=O)C(=O)C=C1 AHMIDUVKSGCHAU-LURJTMIESA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 101710147108 Tyrosinase inhibitor Proteins 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 229960000271 arbutin Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 150000004677 hydrates Chemical class 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000036556 skin irritation Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 229960000401 tranexamic acid Drugs 0.000 description 2
- GYDJEQRTZSCIOI-LJGSYFOKSA-N tranexamic acid Chemical compound NC[C@H]1CC[C@H](C(O)=O)CC1 GYDJEQRTZSCIOI-LJGSYFOKSA-N 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 230000006663 ubiquitin-proteasome pathway Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- WEVYNIUIFUYDGI-UHFFFAOYSA-N 3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide Chemical compound NC(=O)C1=CC=CC(C=2N=CN=C(NC=3C=CC(OC(F)(F)F)=CC=3)C=2)=C1 WEVYNIUIFUYDGI-UHFFFAOYSA-N 0.000 description 1
- BBCQSMSCEJBIRD-UHFFFAOYSA-N 3-fluorophthalic acid Chemical compound OC(=O)C1=CC=CC(F)=C1C(O)=O BBCQSMSCEJBIRD-UHFFFAOYSA-N 0.000 description 1
- WWJAZKZLSDRAIV-UHFFFAOYSA-N 4-fluoro-2-benzofuran-1,3-dione Chemical compound FC1=CC=CC2=C1C(=O)OC2=O WWJAZKZLSDRAIV-UHFFFAOYSA-N 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 108091005625 BRD4 Proteins 0.000 description 1
- 206010051779 Bone marrow toxicity Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100029895 Bromodomain-containing protein 4 Human genes 0.000 description 1
- 102000015367 CRBN Human genes 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010008570 Chloasma Diseases 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102100039193 Cullin-2 Human genes 0.000 description 1
- 101710094489 Cullin-2 Proteins 0.000 description 1
- 102100028907 Cullin-4A Human genes 0.000 description 1
- 206010048768 Dermatosis Diseases 0.000 description 1
- 102100037813 Focal adhesion kinase 1 Human genes 0.000 description 1
- 101000916245 Homo sapiens Cullin-4A Proteins 0.000 description 1
- 101000878536 Homo sapiens Focal adhesion kinase 1 Proteins 0.000 description 1
- 101000979001 Homo sapiens Methionine aminopeptidase 2 Proteins 0.000 description 1
- 101000864342 Homo sapiens Tyrosine-protein kinase BTK Proteins 0.000 description 1
- 240000004322 Lens culinaris Species 0.000 description 1
- 235000014647 Lens culinaris subsp culinaris Nutrition 0.000 description 1
- 102100023174 Methionine aminopeptidase 2 Human genes 0.000 description 1
- 229940079156 Proteasome inhibitor Drugs 0.000 description 1
- 102100032783 Protein cereblon Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 206010039580 Scar Diseases 0.000 description 1
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 1
- 206010040880 Skin irritation Diseases 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 206010057040 Temperature intolerance Diseases 0.000 description 1
- 102100029823 Tyrosine-protein kinase BTK Human genes 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 150000001335 aliphatic alkanes Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 231100000366 bone marrow toxicity Toxicity 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000007854 depigmenting agent Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000012395 formulation development Methods 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 230000008543 heat sensitivity Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000036564 melanin content Effects 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- VMOWKUTXPNPTEN-UHFFFAOYSA-N n,n-dimethylpropan-2-amine Chemical compound CC(C)N(C)C VMOWKUTXPNPTEN-UHFFFAOYSA-N 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- MYDQAEQZZKVJSL-UHFFFAOYSA-N pentyl carbamate Chemical compound CCCCCOC(N)=O MYDQAEQZZKVJSL-UHFFFAOYSA-N 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229960000688 pomalidomide Drugs 0.000 description 1
- UVSMNLNDYGZFPF-UHFFFAOYSA-N pomalidomide Chemical compound O=C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O UVSMNLNDYGZFPF-UHFFFAOYSA-N 0.000 description 1
- 230000000270 postfertilization Effects 0.000 description 1
- 230000026938 proteasomal ubiquitin-dependent protein catabolic process Effects 0.000 description 1
- 239000003207 proteasome inhibitor Substances 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000003134 recirculating effect Effects 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000002020 sage Nutrition 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 230000036620 skin dryness Effects 0.000 description 1
- 231100000475 skin irritation Toxicity 0.000 description 1
- 208000031019 skin pigmentation disease Diseases 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- DPLOGSUBQDREOU-UHFFFAOYSA-N tert-butyl n-(5-aminopentyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCCCCN DPLOGSUBQDREOU-UHFFFAOYSA-N 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000037317 transdermal delivery Effects 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
- A61K8/4926—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having six membered rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
Definitions
- the present invention relates to a compound, a pharmaceutical composition, a synthesis method and an application for targeted degradation of tyrosinase, in particular to PROTAC (proteolysis-targeting chimeras, namely targeted proteolysis) for targeted degradation of tyrosinase (tyrosinase, referred to as TYR).
- PROTAC proteolysis-targeting chimeras, namely targeted proteolysis
- TYR targeted proteolysis
- Chimera and its synthesis method the pharmaceutical composition containing the compound and its application in skin management belong to the technical field of molecular synthesis.
- Hyperpigmentation is the process by which melanocytes in the skin and hair follicles synthesize melanin, which protects the epidermis from UV radiation, environmental pollutants, and toxic drugs and chemicals.
- melanin can lead to serious skin conditions including freckles, melasma, senile lentils and pigmented acne scars.
- epidermal skin pigmentation increases markedly with skin wound healing and UV exposure. Therefore, regulation of melanin production is an important avenue for the treatment of medical pigmentation disorders and safe cosmetic practice.
- melanin is determined by the main rate-limiting enzyme, tyrosinase (EC 1.14.18.1, TYR for short), which catalyzes the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine ( DOPA) and dopa are oxidized to dopaquinone.
- tyrosinase EC 1.14.18.1, TYR for short
- DOPA 3,4-dihydroxyphenylalanine
- Dopaquinone then forms melanin, which is transferred to basal cells and then carried throughout the epidermis as epidermal cells migrate.
- tyrosinase inhibitors used in cosmetics and dermatology are screened based on cheap and easy-to-obtain mushroom tyrosinase (mTYR), such as hydroquinone (HQ), arbutin, L-ascorbic acid, ellagic acid and tranexamic acid etc.
- mTYR mushroom tyrosinase
- hydroquinone is toxic to human cells and can cause skin irritation and bone marrow toxicity.
- the natural form of arbutin is chemically unstable and releases hydroquinone.
- L-Ascorbic acid is easily spoiled because of its heat sensitivity.
- Ellagic acid is insoluble and has poor bioavailability.
- the mechanism by which tranexamic acid inhibits melanin formation is unclear.
- tyrosinase inhibitors need to continuously occupy the active site of the target protein, but high doses can cause undesirable off-target effects and cause damage to the skin.
- depigmenting agents with low toxicity are urgently needed to meet societal needs in terms of safe cosmetic practice and medical pigmentation treatment, and should be screened directly based on human tyrosinase.
- PROTACs Targeted proteolytic chimeras
- UPS ubiquitin-proteasome system
- PROTAC molecules usually consist of three parts: target protein-binding ligand, connecting chain, and E3 ubiquitin ligase ligand. In vivo, this bifunctional small molecule brings the target protein and the E3 ubiquitin ligase into proximity so that the target protein can be labeled with ubiquitin and then degraded by the intracellular ubiquitin-proteasome pathway.
- CRBN Cereblon, a substrate recognition ligand for Cullin 4A E3 ligase
- VHL von Hippel-Lindau, a targeted recruitment subunit in cullin 2 E3 ligase
- PROTACs Compared with traditional inhibitors, PROTACs only provide binding activity and bring the target protein closer to the E3 enzyme to trigger degradation. It belongs to the "event-driven" model and does not need to directly inhibit the functional activity of the target protein. In addition, PROTAC has the ability to destroy the entire protein, can act in an enzyme-independent manner, and only requires a catalytic amount of PROTAC to remove overexpressed and disease-causing proteins, so a longer-lasting effect can be obtained. Patent documents such as CN103265635A and CN107257800A have also introduced in detail the effect of this technology on degrading the target protein.
- the key of CN103265635A is to provide a specific ligand that can bind to the active site of the target protein, and
- the targeting protein chimeric molecular compound comprising a recognition ligand capable of combining with ubiquitin ligase E3 can cause specific degradation of the target protein through the ubiquitin-proteasome pathway;
- the key of CN107257800A is to provide a covalently bonded A bifunctional compound composed of a compound capable of binding to ubiquitin ligase and a targeting ligand capable of binding to a target protein.
- the present invention is a compound targeting the degradation of tyrosinase proposed based on PROTAC technology.
- the linker group L can be used to couple the tyrosinase inhibitor to the E3 ubiquitin ligase ligand to obtain the PROTAC molecule.
- the present invention realizes through following technical scheme:
- L is a linker group, and its structural formula is as follows:
- Z 0 , Z 1 , Z 2 are Any group of -O- or -S-, m 0 , m 1 , m 2 , m 3 , m 4 , m 5 , m 6 are any integer from 0 to 15.
- a pharmaceutical composition used as raw material for skin whitening cosmetics and skin disease treatment containing a therapeutically effective amount of the above compound and at least one pharmaceutically acceptable carrier.
- the present invention has the following advantages and beneficial effects:
- the present invention couples tyrosinase inhibitors to E3 ubiquitin ligase ligands through linkers of different types and chain lengths, and successfully prepares PROTAC molecules targeting tyrosinase, which can effectively target the target protein , and reduce the content of tyrosinase in cells, and at the same time, it can regulate the pigment of zebrafish.
- the effect is better than the new raw materials of skin whitening cosmetics and skin disease treatment drugs commonly used in the market and clinical practice.
- it has low toxicity to normal cells, which conforms to the characteristics of high efficiency and low toxicity.
- the present invention also has the following characteristics:
- the combination of the ligand targeting tyrosinase and the ligand of E3 ubiquitin ligase provided by the present invention can induce the degradation of tyrosinase, but the known ligand targeting tyrosinase and E3 There are many ligands for ubiquitin ligase. Therefore, the present invention not only overcomes the technical difficulty of screening from the existing numerous ligands, but also finds two suitable ligands that can be combined to target the degradation of tyrosine. Enzymes provide new compositions.
- PROTAC molecule of the present invention is mainly used in the preparation of raw materials of skin whitening cosmetics and skin disease treatment medicine, therefore, the stability of PROTAC molecule in vivo is the premise that guarantees its quality and effectiveness, for this reason, the present invention adopts specific structure
- the linker group L, the two ends of the structure can well connect the tyrosinase inhibitor and the E3 ubiquitin ligase ligand, and obtain a relatively stable compound in vivo, which is a PROTAC molecule targeting tyrosinase Formulation development has laid the foundation.
- Fig. 1 is a screening diagram of a series of small molecule compounds degrading tyrosinase of the present invention.
- Fig. 2 is a diagram of the concentration-dependent degradation of tyrosinase of the small molecule compound L-C5.
- Figure 3 is the verification of the mechanism of degradation of tyrosinase by the small molecule compound L-C5,
- (A) is the effect of L-C5 and ligand (levodopa, thalidomide, abbreviated as THL) on the degradation of tyrosinase;
- (B) evaluated the tyrosinase induced by L-C5 Mechanisms of degradation by the proteasomal pathway.
- Figure 4 is the preliminary physical and chemical property evaluation of the small molecule compound L-C5.
- Fig. 5 is the evaluation of the inhibitory effect of different concentrations of compound L-C5, L-Dopa and Kojic acid on zebrafish melanin production after administration.
- Fig. 6 is an in vitro antioxidant activity study of different concentrations of L-C5, levodopa, thalidomide and vitamin C.
- the present invention provides a kind of PROTAC molecule targeting to degrade tyrosinase, and its synthetic structure general formula is shown in the following formula I:
- L is a linker group
- Z 0 , Z 1 , Z 2 are Any group of -O- or -S-, m 0 , m 1 , m 2 , m 3 , m 4 , m 5 , m 6 are any integer from 0-15.
- the representative structural formula of the PROTAC molecule targeting tyrosinase of the present invention is shown below, but not limited to the following structural formula:
- PROTAC molecules targeting degradation of tyrosinase in the present invention also include derivatives such as salts, prodrugs, hydrates or solvates of the compound shown in formula I.
- the compound of the invention can be used to prepare new raw materials of skin whitening cosmetics and medicines for treating skin diseases.
- the compounds of the present invention can be used alone, or together with pharmaceutically acceptable carriers or excipients in the form of pharmaceutical compositions.
- a therapeutically effective amount of the compound of the present invention and one or more pharmaceutically acceptable carriers or diluents are usually combined to make an appropriate administration form or dosage form.
- the present invention therefore also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention and at least one pharmaceutically acceptable carrier.
- the pharmaceutical composition of the compound of the present invention can be implemented in any of the following ways: oral administration, spray inhalation, rectal administration, nasal cavity administration, vaginal administration, and topical administration.
- Parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intraventricular, intrasternal or intracranial injection or infusion, or administration via an explanted reservoir, wherein spraying and skin application are preferred.
- the synthetic route of the target degradation tyrosinase carbon-oxygen chain (L-O1--L-O2) compound is as follows:
- Embodiment 5 be used for the experimental method of embodiment 1 synthetic route
- Embodiment 6 be used for the experimental method of embodiment 2 synthetic routes
- Embodiment 7 be used for the experimental method of embodiment 3 synthetic routes
- Embodiment 8 be used for the experimental method of embodiment 4 synthetic routes
- Embodiment 9 Experimental part
- A375 and HepG2 cells were maintained in RPMI-1640 medium (A10491, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and cultured at 37°C in an incubator with 5% CO2 .
- MG132 and pomalidomide were purchased from Carsmart.
- Cells were seeded into 24-well plates and cultured overnight, and treated with small molecules as shown in Figure I. Cells were then collected into 1.5mL EP tubes, washed with 1 ⁇ cold PBS, and incubated in 50L of 1 ⁇ protein loading buffer (50mM Tris-HCl pH 6.8, 100mM DTT, 0.01% bromophenol blue, 2% SDS, 10 % lysate), and then heated at 100°C for 15 minutes. Western blotting was performed following standard protocols. Briefly, 12 L (for tyrosinase and GAPDH) samples were loaded into wells of a 10% polyacrylamide gel. The voltage was set to 90V in the stacking gel and to 100V for separation.
- 1 ⁇ protein loading buffer 50mM Tris-HCl pH 6.8, 100mM DTT, 0.01% bromophenol blue, 2% SDS, 10 % lysate
- Proteins were transferred to NC membranes (Millipore, 0.2m) by electrophoresis at 100V in an ice-water bath for 1.5 hours. Membranes were incubated in 5% nonfat milk (1x TBST) for 1 hour at room temperature, then membranes were washed with 1x TBST, cleaved and incubated with primary antibodies overnight at 4°C. The next day, the primary antibody was recovered and the membrane was washed 3 times with 1x TBST for 10 minutes at room temperature. Membranes were then incubated with goat anti-rabbit (Thermo Fisher) secondary antibody for 1 hr at room temperature and then washed 3 more times in 1x TBST. Finally, membranes were treated with ECL reagent (Engreen) and photographed by the MiniChemi system (Sage Creation).
- Tyrosinase and GAPDH antibodies were purchased from Abcam and diluted 1:3000 in TBST with 5% BSA.
- Figure 1 is for the screening and evaluation of the designed compounds with different chain lengths and compositions (L-C2–L-O2, 50 ⁇ M) to induce tyrosinase degradation activity, as shown in Figure 1, although these compounds are all It can down-regulate the level of tyrosinase in cells, but compared with other compounds, L-C5 with a chain length of 5 carbon atoms has the strongest effect on inducing the degradation of tyrosinase. When the length of the carbon chain is shortened or extended, the degradation efficiency will be reduced to varying degrees. The possible reason is that the change of the length of the linking chain will affect the mutual proximity between the two ligands, so that a stable ternary spatial conformation cannot be formed.
- Figure 2 is the evaluation of the degradation effect of tyrosinase induced by different concentrations of L-C5, as shown in Figure 2, the level of tyrosinase decreased in a concentration-dependent manner.
- the DC 50 value concentration at which 50% of the protein is degraded
- the maximum activity is reached when the administration concentration is 100 ⁇ M
- the maximum level of inducing tyrosinase (Dmax) degradation is about 63%.
- the efficiency of small molecules to induce tyrosinase degradation will be affected by the length and composition of the linker, which also indicates that the composition and length of the linker chain should be considered when optimizing the degradation agent.
- L-C5 the most active degradation small molecule, was screened out, and it was verified that it could induce tyrosinase degradation in a concentration-dependent manner.
- Cytotoxicity of L-C5 on A375 and HepG2 cells was assessed using a standard cell viability protocol (CCK8 assay).
- A375 and HepG2 cells were incubated for 24 hours and then treated with various concentrations (0, 1.25, 2.5, 5, 10, 20 ⁇ M) of L-C5 for 24 hours. Then, measure the absorbance at 450nm with a microplate reader (before using the microplate reader, turn it on for 10 minutes in advance for preheating). After the measurement is completed, the data is saved and processed using Graphpad Prism 8.0.
- the partition coefficient of L-C5 was measured using the shake flask method. A weighed amount of L-C5 was added to a mixture of equal volumes of octanol and purified water, followed by shaking on a mechanical shaker at room temperature for 24 h. Then we pipetted 200 ⁇ L dissolved in octanol and 200 ⁇ L of aqueous phase into 96-well plates, and measured the UV absorption peak intensity at 270 nm with the UV analysis module of the microplate reader to obtain Abs1 and Abs2 respectively.
- PROTAC L-C5 has a molecular weight of less than 540 Da, suggesting its potential for improved delivery and bioavailability.
- the physicochemical properties of skin pigmentation treatments often affect their transdermal delivery.
- Figure 4 is the preliminary physical and chemical property evaluation of the small molecule compound L-C5, as shown in Figure 4, it has good water solubility (11.4mg/mL), so it is possible to avoid kojic acid (0.055mg/mL) and ellagic acid (0.0097mg/mL) has the disadvantage of poor solubility in clinical application.
- L-C5 long term solubility
- the good water solubility of L-C5 may make it unnecessary for harsh solvent conditions, which would otherwise cause skin dryness and irritation.
- the distribution coefficient (log Po/w) of L-C5 is-0.65
- the research on the physicochemical properties of L-C5 will help to provide guidance for the development of future pigmentation treatment drugs.
- Zebrafish were handled according to the general instructions on the care of animals used for scientific purposes and in accordance with operating procedures approved by the Sichuan Provincial Animal Protection and Use Committee.
- the wild-type AB line was maintained in a recirculating aquaculture system according to standard protocols described in the Zebrafish Book. Embryos were incubated and staged at 28.5 °C as described in Zebrafish Embryo Developmental Stages. L-C5 was then dissolved in dimethyl sulfoxide (DMSO) and diluted with embryo medium to the set concentration for all treatments. Embryos at the guard period [6 hours post fertilization (hpf)] were transferred to 24-well plates and incubated with diluted compound solutions. Embryos were assessed for phenotypic changes at the prim-25 stage (48hpf).
- DMSO dimethyl sulfoxide
- zebrafish The body of zebrafish is optically transparent, can develop rapidly in vitro within a short growth period, and zebrafish tyrosinase is highly homologous to human tyrosinase.
- melanin of zebrafish covers the entire skin surface, and the pigmentation process can be observed without complicated experimental procedures. Due to these advantages, it is often used as an animal model for pigmentation studies. Therefore, zebrafish was also used as a model organism to study the melanogenesis-inhibitory activity of L-C5. Then, at 6hpf after fertilization (hpf), which is the developmental stage of zebrafish neurons, the candidate compound L-C5 (25, 50 and 100 ⁇ M) was co-incubated with the embryos.
- L-C5 can significantly reduce the production of melanin in the zebrafish model, and has better activity than kojic acid and levodopa. Therefore, L-C5 has the potential to be developed as a drug for the treatment of skin pigmentation diseases.
- 1,1-diphenyl-2-trinitrophenylhydrazine (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl,DPPH) assay
- the assay solution consisted of 100 ⁇ L DPPH (150 ⁇ M), 20 ⁇ L of test compounds at increasing concentrations, and the volume in each well was adjusted to 200 ⁇ L with Pure water. The reaction mixture was then incubated at room temperature for 30 minutes. Ascorbic acid (vitamin C, Vc), levodopa (L-Doap) and thalidomide (Thalidomide) were used as controls. The absorption wavelength at 517 nm was measured by using a microplate reader. Each concentration was analyzed in three independent experiments in triplicate.
- Thalidomide TBL
- L-Dopa L-Dopa
- L-C5 positive control vitamin C
- Vitamin C, Vc positive control vitamin C
- the concentrations were 0, 20, 50, 100, 200, 500 ⁇ M , respectively mixed with 150 ⁇ M 1,1-diphenyl-2-trinitrophenylhydrazyl (1,1-diphenyl-2-picrylhydrazyl, DPPH), and then the antioxidant capacity of the candidate compounds was determined.
- L-C5 With the increase of concentration, L-C5, vitamin C and levodopa can obviously scavenge DPPH free radicals and play an antioxidant role, while thalidomide has a weaker effect on scavenging free radicals , indicating that our designed compound possesses antioxidant capacity due to the presence of levodopa.
- L-C5 Compared with Vc and L-Dopa, L-C5 has the strongest ability to scavenge free radicals, and it is concentration-dependent. When its concentration is 20 ⁇ M, it can scavenge about 28% of free radicals, and when the concentration increases to 500 ⁇ M, it can scavenge about 55% of free radicals.
- the stronger in vitro free radical scavenging ability of L-C5 than L-Dopa may be attributed to the better water solubility of L-C5 than L-Dopa.
- L-C5 has stronger in vitro antioxidant activity, and has the potential to protect skin from oxidative damage while regulating skin melanin production.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Cosmetics (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Disclosed in the present invention are a compound for the targeted degradation of tyrosinase, a pharmaceutical composition, and a method for synthesizing the compound and the use thereof, which belong to the technical field of new skin whitening cosmetics raw materials and skin disease treatment drugs. The technical problem to be solved by the present invention is to provide a compound for the targeted degradation of tyrosinase by means of PROTAC, and a salt, a prodrug, a hydrate or a solvate thereof. The structural formula of the PROTAC molecule is as shown in formula (I). According to the present invention, a tyrosinase ligand is coupled with an E3 ligase ligand by means of linkers of different types and different chain lengths, so that a series of PROTAC molecules targeting tyrosinase are successfully prepared. The PROTAC molecules can effectively target a target protein, reduce the content of tyrosinase in cells, have a good effect in terms of reducing melanin in vivo and in vitro, and also have a lower toxicity to normal cells, thereby meeting the characteristics of high efficiency and low toxicity.
Description
本发明是靶向降解酪氨酸酶的化合物、药物组合物及合成方法和应用，具体涉及靶向降解酪氨酸酶(tyrosinase,简称TYR)的PROTAC(proteolysis-targeting chimeras，即靶向蛋白水解嵌合体)及其合成方法，含有该化合物的药物组合物及其在皮肤管理中的应用，属于分子合成技术领域。The present invention relates to a compound, a pharmaceutical composition, a synthesis method and an application for targeted degradation of tyrosinase, in particular to PROTAC (proteolysis-targeting chimeras, namely targeted proteolysis) for targeted degradation of tyrosinase (tyrosinase, referred to as TYR). Chimera) and its synthesis method, the pharmaceutical composition containing the compound and its application in skin management belong to the technical field of molecular synthesis.
色素沉着是皮肤和毛囊中的黑色素细胞合成黑色素的过程，黑色素保护表皮免受紫外线辐射、环境污染物、有毒药物和化学物质的影响。然而，黑色素水平异常会导致严重的皮肤病，包括雀斑、黄褐斑、老年扁豆和色素性痤疮疤痕。此外，伴随皮肤伤口愈合和紫外线照射，表皮皮肤色素沉着显着增加。因此，调节黑色素的产生是医学色素沉着障碍治疗和安全美容实践的重要途径。实际上，黑色素的合成是由主要的限速酶——酪氨酸酶(EC 1.14.18.1，简称TYR)决定的，它催化酪氨酸羟基化为3,4-二羟基苯丙氨酸(DOPA)和多巴氧化为多巴醌。然后，多巴醌形成黑色素，该黑色素被转移到基底细胞，然后随着表皮细胞的迁移而携带到整个表皮。迄今为止，几乎所有用于化妆品和皮肤科的酪氨酸酶抑制剂都是基于廉价易得的蘑菇酪氨酸酶(mTYR)筛选获得的，如对苯二酚(HQ)、熊果苷、L-抗坏血酸、鞣花酸和氨甲环酸等。同时，目前文献中报道的绝大多数酪氨酸酶抑制剂活性也是直接使用蘑菇酪氨酸酶进行评价，仅少数抑制剂使用表达酪氨酸酶的细胞粗提物和同源重组体进行评价，从而导致对人源酪氨酸酶(hTYR)和蘑菇酪氨酸酶的抑制活性存在巨大差异。例如，氢醌对mTYR(IC
50＝1.1μM)的活性比对hTYR(IC
50＝4400μM)的活性高4000倍。另一方面，绝大多数使用的酪氨酸酶抑制剂显示出某些副作用。例如，对苯二酚对人体细胞有毒，可引起皮肤刺激和骨髓毒性。熊果苷的天然形式在化学上不稳定，会释放对苯二酚。L-抗坏血酸因其对热敏感而容易变质。鞣花酸不溶且生物利用度差。氨甲环酸抑制黑色素形成的机制尚不清楚。此外，为了产生抑制作用，酪氨酸酶抑制剂需要持续占据靶蛋白的活性位点，但高剂量会导致不良的脱靶效应，对皮肤造成伤害。总体而言，在安全美容实践和医学色素沉着治疗方面，迫切需要低毒的脱色剂以满足社会需求，且应直接基于人酪氨酸酶进行筛选。
Hyperpigmentation is the process by which melanocytes in the skin and hair follicles synthesize melanin, which protects the epidermis from UV radiation, environmental pollutants, and toxic drugs and chemicals. However, abnormal levels of melanin can lead to serious skin conditions including freckles, melasma, senile lentils and pigmented acne scars. In addition, epidermal skin pigmentation increases markedly with skin wound healing and UV exposure. Therefore, regulation of melanin production is an important avenue for the treatment of medical pigmentation disorders and safe cosmetic practice. In fact, the synthesis of melanin is determined by the main rate-limiting enzyme, tyrosinase (EC 1.14.18.1, TYR for short), which catalyzes the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine ( DOPA) and dopa are oxidized to dopaquinone. Dopaquinone then forms melanin, which is transferred to basal cells and then carried throughout the epidermis as epidermal cells migrate. So far, almost all tyrosinase inhibitors used in cosmetics and dermatology are screened based on cheap and easy-to-obtain mushroom tyrosinase (mTYR), such as hydroquinone (HQ), arbutin, L-ascorbic acid, ellagic acid and tranexamic acid etc. At the same time, the activity of most tyrosinase inhibitors reported in the literature is directly evaluated by using mushroom tyrosinase, and only a few inhibitors are evaluated by using crude extracts of cells expressing tyrosinase and homologous recombinants , resulting in a huge difference in the inhibitory activity against human tyrosinase (hTYR) and mushroom tyrosinase. For example, hydroquinone is 4000-fold more active on mTYR (IC 50 =1.1 μM) than on hTYR (IC 50 =4400 μM). On the other hand, the vast majority of tyrosinase inhibitors used show some side effects. For example, hydroquinone is toxic to human cells and can cause skin irritation and bone marrow toxicity. The natural form of arbutin is chemically unstable and releases hydroquinone. L-Ascorbic acid is easily spoiled because of its heat sensitivity. Ellagic acid is insoluble and has poor bioavailability. The mechanism by which tranexamic acid inhibits melanin formation is unclear. In addition, in order to produce inhibition, tyrosinase inhibitors need to continuously occupy the active site of the target protein, but high doses can cause undesirable off-target effects and cause damage to the skin. Overall, depigmenting agents with low toxicity are urgently needed to meet societal needs in terms of safe cosmetic practice and medical pigmentation treatment, and should be screened directly based on human tyrosinase.
靶向蛋白水解嵌合体(PROTAC)是新开发的化学工具，可通过诱导泛素-蛋白酶体系统(UPS)降解目标蛋白。PROTAC分子通常由三部分组成：靶蛋白结合配体、连接链和 E3泛素连接酶配体。在体内，该双功能小分子使靶蛋白和E3泛素连接酶靠近，因此靶蛋白可以被泛素标记，然后被细胞内的泛素-蛋白酶体途径降解。Crews等人于2001年首次提出PROTAC的概念(PNAS,2001；98(15):8554-8559)，并成功设计并合成了一系列双功能分子以降解甲硫氨酰氨肽酶2(MetAP-2)。受此工具的鼓励，靶向降解其他蛋白靶点的PROTAC分子也陆续被报道(Acta Pharmaceutica Sinica B 2020；10(2):207-238)，例如BCR-ABL、FAK、BRD4、STAT3、BTK等靶点，并广泛使用了CRBN(Cereblon，Cullin 4A E3连接酶的底物识别配体)和VHL(von Hippel-Lindau，在cullin 2 E3连接酶中靶向募集亚基)作为E3泛素连接酶。Targeted proteolytic chimeras (PROTACs) are newly developed chemical tools that degrade target proteins by inducing the ubiquitin-proteasome system (UPS). PROTAC molecules usually consist of three parts: target protein-binding ligand, connecting chain, and E3 ubiquitin ligase ligand. In vivo, this bifunctional small molecule brings the target protein and the E3 ubiquitin ligase into proximity so that the target protein can be labeled with ubiquitin and then degraded by the intracellular ubiquitin-proteasome pathway. Crews et al first proposed the concept of PROTAC in 2001 (PNAS, 2001; 98(15):8554-8559), and successfully designed and synthesized a series of bifunctional molecules to degrade methionyl aminopeptidase 2 (MetAP- 2). Encouraged by this tool, PROTAC molecules targeting the degradation of other protein targets have also been reported (Acta Pharmaceutica Sinica B 2020; 10(2):207-238), such as BCR-ABL, FAK, BRD4, STAT3, BTK, etc. target, and extensively used CRBN (Cereblon, a substrate recognition ligand for Cullin 4A E3 ligase) and VHL (von Hippel-Lindau, a targeted recruitment subunit in cullin 2 E3 ligase) as E3 ubiquitin ligases .
与传统抑制剂相比，PROTAC仅提供结合活性并将靶蛋白更靠近E3酶以触发降解，属于是“事件驱动”模型，不需要直接抑制靶蛋白的功能活性。另外，PROTAC具有破坏整个蛋白质的能力，能够通过不依赖酶的方式发挥作用，且仅需要催化量的PROTAC即可去除过表达和致病的蛋白质，因此能获得更持久的效果。CN103265635A、CN107257800A等专利文献也详细的介绍了这种技术在降解靶点蛋白上的作用，其中，CN103265635A的关键在于提供一种既包含能够与靶蛋白活性位点相结合的特异性配体，又包含能够与泛素连接酶E3相结合的识别配体的靶向蛋白嵌合体分子化合物，能够使靶蛋白经过泛素蛋白酶体通路发生特异性降解；CN107257800A的关键是提供一种通过共价结合使能够结合泛素连接酶的化合物、能够结合靶蛋白的靶向配体组成的双功能化合物。Compared with traditional inhibitors, PROTACs only provide binding activity and bring the target protein closer to the E3 enzyme to trigger degradation. It belongs to the "event-driven" model and does not need to directly inhibit the functional activity of the target protein. In addition, PROTAC has the ability to destroy the entire protein, can act in an enzyme-independent manner, and only requires a catalytic amount of PROTAC to remove overexpressed and disease-causing proteins, so a longer-lasting effect can be obtained. Patent documents such as CN103265635A and CN107257800A have also introduced in detail the effect of this technology on degrading the target protein. Among them, the key of CN103265635A is to provide a specific ligand that can bind to the active site of the target protein, and The targeting protein chimeric molecular compound comprising a recognition ligand capable of combining with ubiquitin ligase E3 can cause specific degradation of the target protein through the ubiquitin-proteasome pathway; the key of CN107257800A is to provide a covalently bonded A bifunctional compound composed of a compound capable of binding to ubiquitin ligase and a targeting ligand capable of binding to a target protein.
发明内容Contents of the invention
本发明是基于PROTAC技术而提出的一种靶向降解酪氨酸酶的化合物，利用连接体基团L可将酪氨酸酶抑制剂与E3泛素连接酶配体偶联以得到PROTAC分子，可用于靶向降解酪氨酸酶。为此，本发明还提出了该化合物的合成方法，含有该化合物的药物组合物以及其在制备皮肤美白化妆品新原料及皮肤疾病治疗药物中的应用。The present invention is a compound targeting the degradation of tyrosinase proposed based on PROTAC technology. The linker group L can be used to couple the tyrosinase inhibitor to the E3 ubiquitin ligase ligand to obtain the PROTAC molecule. Can be used for targeted degradation of tyrosinase. Therefore, the present invention also proposes a synthesis method of the compound, a pharmaceutical composition containing the compound and its application in the preparation of new raw materials for skin whitening cosmetics and medicines for treating skin diseases.
本发明通过下述技术方案实现：The present invention realizes through following technical scheme:
(一)靶向降解酪氨酸酶的化合物，结构式如下式I所示：(1) A compound targeting degradation of tyrosinase, the structural formula of which is shown in formula I below:
其中，L为连接体基团，其结构式如下：Wherein, L is a linker group, and its structural formula is as follows:
其中，Z
0、Z
1、Z
2为
-O-或-S-中任一基团，m
0、m
1、m
2、m
3、m
4、m
5、m
6为0-15中任一整数。
Among them, Z 0 , Z 1 , Z 2 are Any group of -O- or -S-, m 0 , m 1 , m 2 , m 3 , m 4 , m 5 , m 6 are any integer from 0 to 15.
进一步的，其化学结构包括但不限于以下结构式：Further, its chemical structure includes but not limited to the following structural formula:
(二)具有上述化学结构的化合物的盐、前药、水合物或溶剂合物。(2) Salts, prodrugs, hydrates or solvates of compounds having the above chemical structures.
(三)合成上述化合物的方法，包括但不限于以下合成路线：(3) The method for synthesizing the above-mentioned compounds, including but not limited to the following synthetic routes:
1)采用靶向E3泛素链接酶小分子的合成；1) Synthesis of small molecules targeting E3 ubiquitin ligase;
2)采用左旋多巴或3,4-二羟基苯丙酸为靶向小分子的合成；2) Using levodopa or 3,4-dihydroxyphenylpropionic acid as the synthesis of targeted small molecules;
3)靶向降解酪氨酸酶的烷烃碳链化合物的合成；3) Synthesis of alkane carbon chain compounds targeting the degradation of tyrosinase;
4)靶向降解酪氨酸酶碳氧链化合物的合成。4) Synthesis of carbon-oxygen chain compounds targeting degradation of tyrosinase.
(四)上述化合物在制备皮肤美白化妆品原料及皮肤疾病治疗药物中的应用，所述皮肤为肤质差并且色素过度沉着的肤质，包括但不限于雀斑、黄褐斑、黑素瘤、黑皮病、咖啡斑、蒙古斑、太田痣、焦油黑变病、瑞尔黑变病、种族性黑皮病、着色干皮病、肢端色素沉着。(4) The application of the above-mentioned compounds in the preparation of raw materials for skin whitening cosmetics and medicines for the treatment of skin diseases. Dermatosis, coffee spot, Mongolian spot, nevus of Ota, tar melanosis, rael's melanosis, ethnic melanosis, pigmented xeroderma, acral hyperpigmentation.
(五)用于皮肤美白化妆品原料及皮肤疾病治疗的药物组合物，含有治疗有效量的上述化合物及至少一种可药用载体。(5) A pharmaceutical composition used as raw material for skin whitening cosmetics and skin disease treatment, containing a therapeutically effective amount of the above compound and at least one pharmaceutically acceptable carrier.
本发明与现有技术相比，具有以下优点及有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
本发明通过不同种类、不同链长的linker将酪氨酸酶抑制剂与E3泛素连接酶配体偶联，成功制备得到了靶向酪氨酸酶的PROTAC分子，能有效靶向于目标蛋白，并降低细胞中酪氨酸酶的含量，同时能够对斑马鱼的色素起到调控作用。在动物模型验证上，效果好于市场及临床上常用的皮肤美白化妆品新原料及皮肤疾病治疗药物。另外，对正常细胞毒性较低，符合高效低毒的特征。The present invention couples tyrosinase inhibitors to E3 ubiquitin ligase ligands through linkers of different types and chain lengths, and successfully prepares PROTAC molecules targeting tyrosinase, which can effectively target the target protein , and reduce the content of tyrosinase in cells, and at the same time, it can regulate the pigment of zebrafish. In terms of animal model verification, the effect is better than the new raw materials of skin whitening cosmetics and skin disease treatment drugs commonly used in the market and clinical practice. In addition, it has low toxicity to normal cells, which conforms to the characteristics of high efficiency and low toxicity.
进一步的，本发明还具有以下特点：Further, the present invention also has the following characteristics:
(1)本发明所提供的靶向酪氨酸酶的配体和E3泛素连接酶的配体的组合能够诱导酪氨酸酶降解，但公知的靶向酪氨酸酶的配体和E3泛素连接酶的配体众多，为此，本发明不仅克服了从现有众多配体中进行筛选的技术难题，更找到了适宜的可组合的两种配体，为靶向降解酪氨酸酶提供了新的组合物。(1) The combination of the ligand targeting tyrosinase and the ligand of E3 ubiquitin ligase provided by the present invention can induce the degradation of tyrosinase, but the known ligand targeting tyrosinase and E3 There are many ligands for ubiquitin ligase. Therefore, the present invention not only overcomes the technical difficulty of screening from the existing numerous ligands, but also finds two suitable ligands that can be combined to target the degradation of tyrosine. Enzymes provide new compositions.
(2)本发明的PROTAC分子主要用于制备皮肤美白化妆品原料及皮肤疾病治疗药物，因此，PROTAC分子在体内的稳定性是保证其质量和有效性的前提，为此，本发明采用特定结构的连接体基团L，该结构的两端能够很好的连接酪氨酸酶抑制剂和E3泛素连接酶配体，并得到在体内比较稳定的化合物，为靶向酪氨酸酶的PROTAC分子制剂的研发奠定了基础。(2) PROTAC molecule of the present invention is mainly used in the preparation of raw materials of skin whitening cosmetics and skin disease treatment medicine, therefore, the stability of PROTAC molecule in vivo is the premise that guarantees its quality and effectiveness, for this reason, the present invention adopts specific structure The linker group L, the two ends of the structure can well connect the tyrosinase inhibitor and the E3 ubiquitin ligase ligand, and obtain a relatively stable compound in vivo, which is a PROTAC molecule targeting tyrosinase Formulation development has laid the foundation.
图1是本发明系列小分子化合物降解酪氨酸酶筛选图。Fig. 1 is a screening diagram of a series of small molecule compounds degrading tyrosinase of the present invention.
图2为小分子化合物L-C5的浓度依赖性降解酪氨酸酶图。Fig. 2 is a diagram of the concentration-dependent degradation of tyrosinase of the small molecule compound L-C5.
图3为小分子化合物L-C5降解酪氨酸酶机理的验证，Figure 3 is the verification of the mechanism of degradation of tyrosinase by the small molecule compound L-C5,
其中，(A)为L-C5和配体(左旋多巴，沙利度胺，缩写为THL)对酪氨酸酶降解的影响；(B)评估了L-C5诱导的酪氨酸酶通过蛋白酶体途径降解的机制。Among them, (A) is the effect of L-C5 and ligand (levodopa, thalidomide, abbreviated as THL) on the degradation of tyrosinase; (B) evaluated the tyrosinase induced by L-C5 Mechanisms of degradation by the proteasomal pathway.
图4为小分子化合物L-C5初步理化性质评价。Figure 4 is the preliminary physical and chemical property evaluation of the small molecule compound L-C5.
图5为不同浓度的物化合物L-C5、L-Dopa和Kojic acid给药后对斑马鱼黑色素生成的抑制效果评价。Fig. 5 is the evaluation of the inhibitory effect of different concentrations of compound L-C5, L-Dopa and Kojic acid on zebrafish melanin production after administration.
图6为不同浓度的L-C5、左旋多巴、沙利度胺及维生素C的体外抗氧化活性研究。Fig. 6 is an in vitro antioxidant activity study of different concentrations of L-C5, levodopa, thalidomide and vitamin C.
下面将本发明的发明目的、技术方案和有益效果作进一步详细的说明。The purpose, technical solution and beneficial effects of the present invention will be further described in detail below.
应该指出，以下详细说明都是示例性的，旨在对所要求的本发明提供进一步的说明，除非另有说明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通 常理解的相同含义。It should be pointed out that the following detailed description is exemplary and is intended to provide further explanation to the claimed invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used by those of ordinary skill in the art to which the invention belongs. understand the same meaning.
本发明提供了一种靶向降解酪氨酸酶的PROTAC分子，其化合物合成结构通式如下式I所示：The present invention provides a kind of PROTAC molecule targeting to degrade tyrosinase, and its synthetic structure general formula is shown in the following formula I:
其中，L为连接体基团。Wherein, L is a linker group.
进一步的，L的结构式如下：Further, the structural formula of L is as follows:
其中，Z
0、Z
1、Z
2为
-O-或-S-中任一基团，m
0、m
1、m
2、m
3、m
4、m
5、m
6为0-15中任何一整数。
Among them, Z 0 , Z 1 , Z 2 are Any group of -O- or -S-, m 0 , m 1 , m 2 , m 3 , m 4 , m 5 , m 6 are any integer from 0-15.
优选的，本发明的靶向酪氨酸酶的PROTAC分子的代表性结构式如下所示，但不限于如下结构式：Preferably, the representative structural formula of the PROTAC molecule targeting tyrosinase of the present invention is shown below, but not limited to the following structural formula:
本发明靶向降解酪氨酸酶的PROTAC分子还包括式I所示化合物的盐、前药、水合物或溶剂合物等衍生物。The PROTAC molecules targeting degradation of tyrosinase in the present invention also include derivatives such as salts, prodrugs, hydrates or solvates of the compound shown in formula I.
本发明的化合物，可用于制备皮肤美白化妆品新原料及皮肤疾病治疗药物。The compound of the invention can be used to prepare new raw materials of skin whitening cosmetics and medicines for treating skin diseases.
本发明所述化合物可以单独使用，也可以与可药用的载体或赋形剂一起以药物组合物的形式使用。当以药物组合物的形式使用时，通常将治疗有效量的本发明化合物以及一种或者多种可药用载体或稀释剂结合制成适当的施用形式或者剂量形式。因此本发明还提供了药物组合物，它包括治疗有效量的本发明所述的化合物以及至少一种可药用载体。The compounds of the present invention can be used alone, or together with pharmaceutically acceptable carriers or excipients in the form of pharmaceutical compositions. When used in the form of a pharmaceutical composition, a therapeutically effective amount of the compound of the present invention and one or more pharmaceutically acceptable carriers or diluents are usually combined to make an appropriate administration form or dosage form. The present invention therefore also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention and at least one pharmaceutically acceptable carrier.
本发明化合物的药用组合物，可以以下方面的任意方式实施：口服、喷雾吸入、直肠给药、鼻腔给药、阴道给药、局部给药。非肠道给药如皮下，静脉，肌肉、腹膜内、心室内、胸骨内或颅内注射或者输入，或者借助一种外植的储器用药，其中优选喷雾、皮肤涂抹给药方式。The pharmaceutical composition of the compound of the present invention can be implemented in any of the following ways: oral administration, spray inhalation, rectal administration, nasal cavity administration, vaginal administration, and topical administration. Parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intraventricular, intrasternal or intracranial injection or infusion, or administration via an explanted reservoir, wherein spraying and skin application are preferred.
下面结合实施例对本发明的具体实施方式作进一步描述，并不因此将本发明限制在所述的实施例范围内。The specific embodiments of the present invention will be further described below in conjunction with the examples, and the present invention is not limited to the scope of the examples.
实施例1：Example 1:
靶向E3泛素链接酶小分子的合成路线如下：The synthetic route of the small molecule targeting E3 ubiquitin ligase is as follows:
实施例2：Example 2:
以左旋多巴或3,4-二羟基苯丙酸为靶向小分子的设计及合成路线如下：The design and synthesis route of targeting small molecules with levodopa or 3,4-dihydroxyphenylpropionic acid are as follows:
实施例3：Example 3:
靶向降解酪氨酸酶的烷烃碳链化合物(L-C2—L-C10)的合成路线如下：The synthetic route of the alkane carbon chain compound (L-C2—L-C10) targeting the degradation of tyrosinase is as follows:
实施例4：Example 4:
靶向降解酪氨酸酶碳氧链(L-O1--L-O2)化合物的合成路线如下：The synthetic route of the target degradation tyrosinase carbon-oxygen chain (L-O1--L-O2) compound is as follows:
实施例5：用于实施例1合成路线的实验方法Embodiment 5: be used for the experimental method of embodiment 1 synthetic route
首先，靶向E3连接酶中间体2的合成：First, targeting the synthesis of E3 ligase intermediate 2:
合成步骤操作实验：Synthesis steps operation experiment:
在100mL的干燥圆底烧瓶中，加入2.0g(10.87mmol)3-氟邻苯二甲酸，然后再加入20mL的无水乙酸酐，混合均匀后，放置于油浴锅中，反应的温度缓慢升高至145℃，达到指定的温度后，均匀搅拌2h。在反应的过程中，原料会完全溶解，且溶剂会回流。2h后，反应完成，无需点板监测，直接高温旋干溶剂(90℃)成灰色的固体粉末，再用2x10mL石油醚洗涤粉末两次，过滤，抽干,最后在无水乙酸酐中重结晶得白色固体1.65g，产率92％。In a dry 100mL round bottom flask, add 2.0g (10.87mmol) 3-fluorophthalic acid, then add 20mL of anhydrous acetic anhydride, mix well, place in an oil bath, and the temperature of the reaction rises slowly As high as 145 ° C, after reaching the specified temperature, stir evenly for 2 hours. During the course of the reaction, the starting material was completely dissolved and the solvent was refluxed. After 2 hours, the reaction is complete, no need to spot the plate to monitor, directly spin-dry the solvent at high temperature (90°C) to form a gray solid powder, then wash the powder twice with 2x10mL petroleum ether, filter, drain, and finally recrystallize in anhydrous acetic anhydride 1.65 g of white solid was obtained with a yield of 92%.
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,CDCl
3):δ7.58(t,J＝8.0Hz,1H),7.86(d,J＝7.2Hz,1H),7.92-7.97(m,1H).
1 H NMR (400MHz, CDCl 3 ): δ7.58(t, J=8.0Hz, 1H), 7.86(d, J=7.2Hz, 1H), 7.92-7.97(m, 1H).
然后，靶向E3连接酶的化合物4合成：Then, compound 4 targeting E3 ligase was synthesized:
合成步骤操作实验：Synthesis steps operation experiment:
在干燥过的50mL圆底烧瓶中，加入上一步反应的得到的化合物4-氟异苯并呋喃-1,3-二酮90mg(0.54mmol,1.0equiv)，化合物3-氨基哌啶-2,6-二酮盐酸盐88mg(0.54mmol, 1.0equiv)以及53mg(0.65mmol,2.0equiv)无水乙酸钠，然后加入10mL的冰乙酸。缓慢搅拌并升高温度至145℃，回流反应12h，反应的进程TLC点板监测。反应完成后，冷却至室温，加入大量的水稀释，再加入3x15mL乙酸乙酯萃取三次，合并有机相，经饱和食盐水洗涤，再无水硫酸镁干燥，过滤，旋干得粗产物，最后将粗产物经柱层析纯化(DCM：MeOH＝20:1,v/v)得白色产物107mg，产率72％。In a dried 50mL round bottom flask, add 90mg (0.54mmol, 1.0equiv) of the compound 4-fluoroisobenzofuran-1,3-dione obtained in the previous step, compound 3-aminopiperidine-2, 88mg (0.54mmol, 1.0equiv) of 6-diketone hydrochloride and 53mg (0.65mmol, 2.0equiv) of anhydrous sodium acetate were then added to 10mL of glacial acetic acid. Stir slowly and increase the temperature to 145° C., reflux for 12 hours, and monitor the progress of the reaction by TLC spot plate. After the reaction was completed, cool to room temperature, add a large amount of water to dilute, then add 3x15mL ethyl acetate to extract three times, combine the organic phases, wash with saturated brine, then dry over anhydrous magnesium sulfate, filter, spin dry to obtain the crude product, and finally The crude product was purified by column chromatography (DCM:MeOH=20:1, v/v) to obtain 107 mg of white product with a yield of 72%.
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,DMSO-d6)δ11.15(s,1H),7.96(ddd,J＝8.3,7.3,4.5Hz,1H),7.82–7.71(m,2H),5.17(dd,J＝13.0,5.4Hz,1H),2.90(ddd,J＝17.1,13.9,5.4Hz,1H),2.65–2.47(m,2H),2.10–2.04(m,1H).
1 H NMR (400MHz, DMSO-d6) δ11.15 (s, 1H), 7.96 (ddd, J = 8.3, 7.3, 4.5Hz, 1H), 7.82–7.71 (m, 2H), 5.17 (dd, J = 13.0,5.4Hz,1H),2.90(ddd,J＝17.1,13.9,5.4Hz,1H),2.65–2.47(m,2H),2.10–2.04(m,1H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(100MHz,DMSO-d
6)δ173.2,170.1,166.5,164.4,157.3，138.5,133.9,123.5,120.5,117.5,49.6,31.4,22.3.
13 C NMR (100MHz, DMSO-d 6 ) δ173.2, 170.1, 166.5, 164.4, 157.3, 138.5, 133.9, 123.5, 120.5, 117.5, 49.6, 31.4, 22.3.
实施例6：用于实施例2合成路线的实验方法Embodiment 6: be used for the experimental method of embodiment 2 synthetic routes
靶向酪氨酸酶化合物6的合成：Synthesis of compound 6 targeting tyrosinase:
合成步骤操作实验：Synthesis steps operation experiment:
在100mL的圆底烧瓶中，用10mL1,4-二氧六环溶解称量好的左旋多巴(2.0g,10.0mmol,1.0 equiv)，然后加入Boc
2O(2.4g,11.0mmol,1.1equiv)。另外，配置1mol/L的NaOH溶液11mL，并用一次性吸管缓慢滴加到搅拌的反应液中，大约30min，滴加完成后，室温反应6h。反应的进度通过TLC进行监测，反应完成后，旋走反应瓶中1,4-二氧六环，然后加入1mol/L的盐酸溶液调节反应液pH＝2，再加入乙酸乙酯萃取三次(3x25mL)，饱和食盐水洗涤有机相，干燥，过滤，旋干得目标化合物2.6g，产率86.2％。
In a 100mL round bottom flask, dissolve the weighed levodopa (2.0g, 10.0mmol, 1.0 equiv) with 10mL 1,4-dioxane, then add Boc 2 O (2.4g, 11.0mmol, 1.1 equiv ). In addition, 11 mL of 1 mol/L NaOH solution was prepared, and slowly added dropwise to the stirred reaction solution with a disposable straw for about 30 min. After the dropwise addition was completed, react at room temperature for 6 h. The progress of the reaction was monitored by TLC. After the reaction was completed, the 1,4-dioxane in the reaction flask was spun away, and then 1mol/L hydrochloric acid solution was added to adjust the pH of the reaction solution to 2, and ethyl acetate was added to extract three times (3×25mL ), the organic phase was washed with saturated brine, dried, filtered, and spin-dried to obtain 2.6 g of the target compound, with a yield of 86.2%.
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,CDCl
3)δ6.61(m,3H),5.15(m,1H),4.48(m,1H),2.92(m,2H),1.38(s,9H).
1 H NMR (400MHz, CDCl 3 )δ6.61(m,3H),5.15(m,1H),4.48(m,1H),2.92(m,2H),1.38(s,9H).
实施例7：用于实施例3合成路线的实验方法Embodiment 7: be used for the experimental method of embodiment 3 synthetic routes
(1)靶向降解酪氨酸酶的中间体8d的合成：(1) Synthesis of intermediate 8d targeting degradation of tyrosinase:
合成步骤操作实验：Synthesis steps operation experiment:
将2.66g(22.9mmol,5.0equiv)1,5-戊二胺加入到500mL的反应烧瓶当中，然后加入100mL的二氯甲烷(CH
2Cl
2)，然后将1.0g(4.6mmol,1.0 equiv)Boc
2O用50mL二氯甲烷溶解，然后借助滴液漏斗缓慢滴加至反应液中，室温下搅拌反应12h，反应可由TLC监测，在监测反应的过程，需利用配置好的的碘缸显色，原料1,5-戊二胺在碘缸中呈现黑色，反应产物为黄绿色。反应完成后，过滤，旋干，经柱层析分离纯化(DCM:MeOH＝20:1)得无色油状液体0.86g，产率86％。
2.66g (22.9mmol, 5.0equiv) 1,5-pentanediamine was added to a 500mL reaction flask, then 100mL of dichloromethane (CH 2 Cl 2 ) was added, and 1.0g (4.6mmol, 1.0 equiv) Boc 2 O was dissolved in 50mL of dichloromethane, then slowly added dropwise to the reaction solution with the help of a dropping funnel, and stirred at room temperature for 12 hours. The reaction can be monitored by TLC. During the monitoring of the reaction process, a well-equipped iodine cylinder should be used for color development. , The raw material 1,5-pentanediamine is black in the iodine tank, and the reaction product is yellow-green. After the reaction was completed, it was filtered, spin-dried, and separated and purified by column chromatography (DCM:MeOH=20:1) to obtain 0.86 g of a colorless oily liquid with a yield of 86%.
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,CDCl
3,δ(ppm)4.68(s,1H)3.05(s,2H),2.63(q,J＝7.40Hz,2H),1.38(m,15H),1.28(m,2H),1.13(s,1H).
1 H NMR (400MHz, CDCl 3 , δ (ppm) 4.68 (s, 1H) 3.05 (s, 2H), 2.63 (q, J = 7.40Hz, 2H), 1.38 (m, 15H), 1.28 (m, 2H ),1.13(s,1H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(100MHz,CDCl
3):δ(ppm)24.01,28.40,29.86,32.95,40.46,41.88,79.04,155.98.
13 C NMR (100MHz, CDCl 3 ): δ (ppm) 24.01, 28.40, 29.86, 32.95, 40.46, 41.88, 79.04, 155.98.
(2)靶向降解酪氨酸酶的中间体9d的合成：(2) Synthesis of intermediate 9d targeting degradation of tyrosinase:
合成步骤操作实验：Synthesis steps operation experiment:
将上一步反应合成的5-氨基戊基氨基甲酸叔丁酯(216mg,1.0mmol,1.0equiv)加入到干燥过的100mL圆底烧瓶中，再加入2-(2,6-二氧杂哌啶-3-基-4-氟异吲哚-1,3-二酮(276mg,1.0mmol,1.0equiv)，并加入10mLN,N-二甲基甲酰胺溶解，缓慢升高温度至90℃，在升温的过程中，缓慢滴加0.25mL N,N-二甲基异丙胺，反应12h，反应混合液会变为深绿色，同点板监测反应的情况。反应完成后，冷却至室温，然后加入大量的水稀释，并用乙酸乙酯萃取三次(3x40mL)，合并有机相，经饱和食盐水洗涤，无水硫酸镁干燥，过滤，旋干为粗产物，再经柱层析(DCM：MeOH＝25:1)纯化得绿色固体212mg，产率46.3％。Add tert-butyl 5-aminopentylcarbamate (216mg, 1.0mmol, 1.0equiv) synthesized in the previous step into a dried 100mL round bottom flask, and then add 2-(2,6-dioxapiperidine -3-yl-4-fluoroisoindole-1,3-dione (276mg, 1.0mmol, 1.0equiv), and add 10mL N,N-dimethylformamide to dissolve, slowly raise the temperature to 90°C, in During the heating process, slowly add 0.25mL N,N-dimethylisopropylamine dropwise, react for 12h, the reaction mixture will turn dark green, monitor the reaction with the spot plate. After the reaction is completed, cool to room temperature, and then add a large amount of diluted with water, and extracted three times with ethyl acetate (3x40mL), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and spin-dried to obtain a crude product, which was then subjected to column chromatography (DCM: MeOH=25: 1) Purified to obtain 212mg of green solid with a yield of 46.3%.
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,CDCl
3)δ8.47(s,1H),7.50(dd,J＝8.5,7.1Hz,1H),7.09(d,J＝7.1Hz,1H),6.88(d,J＝8.5Hz,1H),6.25(t,J＝5.7Hz,1H),4.98–4.87(m,1H),4.62(t,J＝6.0Hz, 1H),3.27(td,J＝7.0,5.6Hz,2H),3.14(q,J＝6.7Hz,2H),2.94–2.68(m,3H),2.19–2.08(m,1H),1.81(s,1H),1.69(p,J＝7.2Hz,2H),1.59–1.44(m,2H),1.45(s,10H).
1 H NMR (400MHz, CDCl 3 ) δ8.47(s, 1H), 7.50(dd, J=8.5, 7.1Hz, 1H), 7.09(d, J=7.1Hz, 1H), 6.88(d, J= 8.5Hz, 1H), 6.25(t, J＝5.7Hz, 1H), 4.98–4.87(m, 1H), 4.62(t, J＝6.0Hz, 1H), 3.27(td, J＝7.0, 5.6Hz, 2H), 3.14(q, J＝6.7Hz, 2H), 2.94–2.68(m, 3H), 2.19–2.08(m, 1H), 1.81(s, 1H), 1.69(p, J＝7.2Hz, 2H ),1.59–1.44(m,2H),1.45(s,10H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,CDCl
3)δ171.24,169.51,168.50,167.63,156.01,146.92,136.13,132.48,116.63,111.44,109.89,79.19,48.88,42.54,40.38,31.42,29.85,28.92,28.43,24.15,22.80.
13 C NMR(101MHz,CDCl 3 )δ171.24,169.51,168.50,167.63,156.01,146.92,136.13,132.48,116.63,111.44,109.89,79.19,48.88,42.54,40.38,31.42,29.85,28.92,28.43,24.15,22.80 .
(3)靶向降解酪氨酸酶的中间体10d的合成：(3) Synthesis of intermediate 10d targeting degradation of tyrosinase:
合成步骤操作实验：Synthesis steps operation experiment:
将212mg(0.46mmol)5-(2-(2,6-二氧杂哌啶-3-基)-1,3-二氧异吲哚-4-基氨基)戊基氨基甲酸叔丁酯用10mL二氯甲烷溶解，然后缓慢滴加1mL的三氟乙酸(CF
3COOH)，滴加完成后，室温下缓慢搅拌30min，溶液由绿色变为橙黄色。反应的进度可点板监测，反应产物相对于原料极性变大。反应完成后，取出搅拌子，直接浓缩并旋干至油状液体，然后用油泵抽30min，再无需进一步的纯化，直接用于下一步的酰胺缩合反应。
212 mg (0.46 mmol) of tert-butyl 5-(2-(2,6-dioxaperidin-3-yl)-1,3-dioxoisoindol-4-ylamino)pentylcarbamate 10 mL of dichloromethane was dissolved, and then 1 mL of trifluoroacetic acid (CF 3 COOH) was slowly added dropwise. After the addition was completed, the solution was slowly stirred at room temperature for 30 min, and the solution changed from green to orange yellow. The progress of the reaction can be monitored by pointing the plate, and the polarity of the reaction product becomes larger relative to the raw material. After the reaction was completed, the stirring bar was taken out, directly concentrated and spin-dried to an oily liquid, and then pumped with an oil pump for 30 minutes, and was directly used in the next step of amide condensation reaction without further purification.
靶向降解酪氨酸酶的中间体11d的合成Synthesis of intermediate 11d targeting degradation of tyrosinase
合成步骤操作实验：Synthesis steps operation experiment:
称取297mg中间体6(1.0mmol,1.0equiv)Boc-L-Dopa，然后加入到装有上一步产物的圆底烧瓶中，然后加入91mg(1.0mmol,1.0equiv)1-乙基-3(3-二甲基丙胺)碳二亚胺(EDCI)和61mg(0.5mmol,0.5equiv)4-二甲氨基吡啶(DMAP)，最后加入10mLN,N-二甲基甲酰胺，放置于油浴锅中，设置温度为25℃。搅拌反应12h，TLC监测反应的情况，反应结束后，加入5mL的1mol/L的盐酸溶液，并搅拌5min，再加入50mL的饱和食盐水，最后用二氯甲烷萃取3次(3x15mL)，分离下层有机相，并合并有机相，加入无水硫酸镁干燥，过滤，旋转蒸干得粗产物，粗产物无需进一步的纯化，可直接用于下一步反应。Take by weighing 297mg intermediate 6 (1.0mmol, 1.0equiv) Boc-L-Dopa, then join in the round bottom flask that the previous step product is housed, then add 91mg (1.0mmol, 1.0equiv) 1-ethyl-3 ( 3-Dimethylpropylamine) carbodiimide (EDCI) and 61mg (0.5mmol, 0.5equiv) 4-dimethylaminopyridine (DMAP), and finally add 10mL N,N-dimethylformamide, place in an oil bath , set the temperature to 25°C. Stir the reaction for 12 hours, and monitor the reaction by TLC. After the reaction, add 5 mL of 1 mol/L hydrochloric acid solution and stir for 5 minutes, then add 50 mL of saturated saline, and finally extract 3 times with dichloromethane (3x15 mL), and separate the lower layer The organic phase was combined, dried by adding anhydrous magnesium sulfate, filtered, and evaporated to dryness to obtain a crude product, which was directly used in the next reaction without further purification.
(4)靶向降解酪氨酸酶的小分子L-C5的合成(4) Synthesis of small molecule L-C5 targeting degradation of tyrosinase
合成步骤操作实验：Synthesis steps operation experiment:
用10mL二氯甲烷溶解上一步的产物，然后加入2mL三氟乙酸，在常温下，搅拌反应30min，TLC监测，反应完成后，加入大量的水稀释反应混合液，然后用饱和NaHCO3溶液调节溶液至PH＝7，然后用二氯甲烷萃取三次(3x30mL)，合并有机相，用饱和食盐水洗涤两次，无水硫酸镁干燥，过滤，旋干，最后柱层析纯化分离(DCM:MeOH＝20:1)得淡绿色固体120mg，产率23％。Dissolve the product of the previous step with 10 mL of dichloromethane, then add 2 mL of trifluoroacetic acid, and stir the reaction at room temperature for 30 min, monitored by TLC. After the reaction is complete, add a large amount of water to dilute the reaction mixture, and then adjust the solution to PH=7, then extracted three times with dichloromethane (3x30mL), combined the organic phases, washed twice with saturated brine, dried over anhydrous magnesium sulfate, filtered, spin-dried, and finally purified and separated by column chromatography (DCM:MeOH=20 : 1) 120 mg of a light green solid was obtained, with a yield of 23%.
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ7.60–7.50(m,1H),7.04(t,J＝7.7Hz,2H),6.80–6.61(m,2H),6.58–6.46(m,1H),5.04(dd,J＝12.7,5.4Hz,1H),3.54(t,J＝7.1Hz,1H),3.33–3.15(m,2H),3.14–2.99(m,2H),2.93–2.62(m,5H),2.09(dtd,J＝13.0,5.5,2.7Hz,1H),1.64(p,J＝7.2Hz,2H),1.53–1.39(m,2H),1.38–1.28(m,2H).
1 H NMR (400MHz, Methanol-d 4 ) δ7.60–7.50(m,1H),7.04(t,J＝7.7Hz,2H),6.80–6.61(m,2H),6.58–6.46(m,1H ), 5.04(dd, J＝12.7, 5.4Hz, 1H), 3.54(t, J＝7.1Hz, 1H), 3.33–3.15(m, 2H), 3.14–2.99(m, 2H), 2.93–2.62( m,5H), 2.09(dtd,J=13.0,5.5,2.7Hz,1H),1.64(p,J=7.2Hz,2H),1.53–1.39(m,2H),1.38–1.28(m,2H) .
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(1o1MHz,MeOD)δ174.16,173.33,170.28,169.41,167.97,146.88,144.92,143.85,135.89,132.44,128.33,120.40,116.73,116.11,115.05,110.36,109.47,56.29,48.79,41.86,40.10,38.69,35.59,30.81,30.29,28.56,23.72,22.38.
13 C NMR(1o1MHz,MeOD)δ174.16,173.33,170.28,169.41,167.97,146.88,144.92,143.85,135.89,132.44,128.33,120.40,116.73,116.11,115.05,110.36,109.47,56.29,48.79,41.86,40.10, 38.69, 35.59, 30.81, 30.29, 28.56, 23.72, 22.38.
HR-MS:calculated for C
27H
31N
5O
7[M+H]
+,538.2257；found,538.2299.
HR-MS: calculated for C 27 H 31 N 5 O 7 [M+H] + ,538.2257; found, 538.2299.
(5)靶向降解酪氨酸酶的小分子L-C2的合成(5) Synthesis of small molecule L-C2 targeting degradation of tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,DMSO-d
6)δ7.60(dd,J＝8.5,7.1Hz,1H),7.22(d,J＝8.6Hz,2H),7.04(d,J＝7.1Hz,1H),6.76(t,J＝6.1Hz,2H),6.65–6.50(m,2H),6.42(dd,J＝7.8,2.0Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),3.27(d,J＝5.5Hz,3H),2.60(d,J＝3.3Hz,1H),2.55(d,J＝5.8Hz,1H),2.47–2.37(m,2H),2.07–1.96(m,2H).
1 H NMR (400MHz, DMSO-d 6 ) δ7.60(dd, J=8.5,7.1Hz,1H),7.22(d,J=8.6Hz,2H),7.04(d,J=7.1Hz,1H) ,6.76(t,J=6.1Hz,2H),6.65–6.50(m,2H),6.42(dd,J=7.8,2.0Hz,1H),5.06(dd,J=12.9,5.4Hz,1H), 3.27(d, J=5.5Hz, 3H), 2.60(d, J=3.3Hz, 1H), 2.55(d, J=5.8Hz, 1H), 2.47–2.37(m, 2H), 2.07–1.96(m ,2H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,DMSO-d
6)δ175.95,173.25,170.25,168.40,167.88,146.58,144.92,143.78,135.85,133.93,132.63,122.67,120.30,116.61,116.02,114.98,110.75,56.51,48.44,40.43,30.76,30.33,29.35,22.29.
13 C NMR(101MHz,DMSO-d 6 )δ175.95,173.25,170.25,168.40,167.88,146.58,144.92,143.78,135.85,133.93,132.63,122.67,120.30,116.61,116.02,114.98,110.75,56.51,48.44,40.43 ,30.76,30.33,29.35,22.29.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
24H
25N
5O
7[M+H]
+496.1788；found,496.1828.
HR-MS: calculated for C 24 H 25 N 5 O 7 [M+H] + 496.1788; found, 496.1828.
(6)靶向降解酪氨酸酶的小分子L-C3的合成(6) Synthesis of small molecule L-C3 targeting degradation of tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ7.61–7.52(m,1H),7.03(dd,J＝16.7,7.8Hz,2H),6.74–6.59(m,2H),6.55(dd,J＝8.0,2.1Hz,1H),5.07(dd,J＝12.4,5.4Hz,1H),3.52(t,J＝7.0Hz,1H),3.27–3.14(m,3H),3.01(s,2H),2.94–2.78(m,1H),2.81–2.71(m,3H),2.19–2.07(m,1H),1.75(p,J＝6.6Hz,2H).
1 H NMR (400MHz, Methanol-d 4 ) δ7.61–7.52(m,1H),7.03(dd,J＝16.7,7.8Hz,2H),6.74–6.59(m,2H),6.55(dd,J =8.0,2.1Hz,1H),5.07(dd,J=12.4,5.4Hz,1H),3.52(t,J=7.0Hz,1H),3.27–3.14(m,3H),3.01(s,2H) ,2.94–2.78(m,1H),2.81–2.71(m,3H),2.19–2.07(m,1H),1.75(p,J=6.6Hz,2H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δ174.70,173.25,170.25,167.93,163.45,146.67,144.97,143.89,135.88,132.50,128.38,120.34,116.69,116.06,115.01,110.46,109.78,56.44,48.79,40.15,36.37,30.25,29.35,28.40,22.39.
13 C NMR(101MHz,MeOD)δ174.70,173.25,170.25,167.93,163.45,146.67,144.97,143.89,135.88,132.50,128.38,120.34,116.69,116.06,115.01,110.46,109.78,56.44,48.79,40.15,36.37, 30.25, 29.35, 28.40, 22.39.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
25H
27N
5O
7[M+H]
+510.1944；found,510.1986.
HR-MS: calculated for C 25 H 27 N 5 O 7 [M+H] + 510.1944; found, 510.1986.
(7)靶向降解酪氨酸酶的小分子L-C4的合成(7) Synthesis of small molecule L-C4 targeting degradation of tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ7.63–7.45(m,1H),7.07–6.99(m,2H),6.76(d,J＝8.0Hz,1H),6.70(d,J＝2.2Hz,1H),6.59(dd,J＝8.1,2.2Hz,1H),5.08(dd,J＝12.9,5.4Hz,1H),3.33(dt,J＝25.0,5.2Hz,4H),3.17–3.02(m,1H),3.03–2.64(m,7H),2.11(tdd,J＝8.2,5.5,2.8Hz,1H),1.52(d,J＝6.0Hz,4H).
1 H NMR (400MHz, Methanol-d 4 ) δ7.63–7.45(m,1H),7.07–6.99(m,2H),6.76(d,J=8.0Hz,1H),6.70(d,J=2.2 Hz, 1H), 6.59(dd, J=8.1, 2.2Hz, 1H), 5.08(dd, J=12.9, 5.4Hz, 1H), 3.33(dt, J=25.0, 5.2Hz, 4H), 3.17–3.02 (m,1H),3.03–2.64(m,7H),2.11(tdd,J=8.2,5.5,2.8Hz,1H),1.52(d,J=6.0Hz,4H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δ173.32,170.34,169.40,168.19,167.91,146.76,145.31,144.64,135.88,132.45,125.45,120.44,116.67,116.10,115.33,110.44,109.57,54.70,48.77,41.58,38.75,36.85,31.69,31.67,29.38,26.19,22.39.
13 C NMR(101MHz,MeOD)δ173.32,170.34,169.40,168.19,167.91,146.76,145.31,144.64,135.88,132.45,125.45,120.44,116.67,116.10,115.33,110.44,109.57,54.70,48.77,41.58,38.75, 36.85, 31.69, 31.67, 29.38, 26.19, 22.39.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
26H
29N
5O
7[M+H]
+524.2010；found,524.2138.
HR-MS: calculated for C 26 H 29 N 5 O 7 [M+H] + 524.2010; found, 524.2138.
(8)靶向降解酪氨酸酶的小分子L-C6的合成(8) Synthesis of small molecule L-C6 targeting degradation of tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ(ppm)7.56(dd,J＝8.6,7.1Hz,1H),7.05(dd,J＝7.9,3.5Hz,2H),6.75–6.61(m,2H),6.53(dd,J＝8.0,2.1Hz,1H),5.07(dd,J＝12.5,5.4Hz,1H),3.46(t,J＝7.0Hz,1H),3.33(dt,J＝3.4,1.7Hz,4H),3.22(dt,J＝13.5,6.8Hz,1H),3.12–2.99(m,1H),2.95–2.65(m,5H),2.12(dtd,J＝12.9,4.9,2.3Hz,1H),1.65(q,J＝7.3Hz,2H),1.50–1.35(m,4H),1.33–1.20(m,2H).
1 H NMR (400MHz, Methanol-d 4 ) δ (ppm) 7.56 (dd, J = 8.6, 7.1 Hz, 1H), 7.05 (dd, J = 7.9, 3.5 Hz, 2H), 6.75–6.61 (m, 2H ),6.53(dd,J=8.0,2.1Hz,1H),5.07(dd,J=12.5,5.4Hz,1H),3.46(t,J=7.0Hz,1H),3.33(dt,J=3.4, 1.7Hz, 4H), 3.22(dt, J＝13.5, 6.8Hz, 1H), 3.12–2.99(m, 1H), 2.95–2.65(m, 5H), 2.12(dtd, J＝12.9, 4.9, 2.3Hz ,1H),1.65(q,J＝7.3Hz,2H),1.50–1.35(m,4H),1.33–1.20(m,2H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δ174.72,173.23,170.24,169.43,167.92,146.92,144.93,143.81,135.83,132.50,128.51,120.28,116.64,116.03,114.94,110.31,109.55,56.47,48.79,41.95,40.47,38.76,30.80,28.82,28.76,26.22,26.21,22.40.
13 C NMR(101MHz,MeOD)δ174.72,173.23,170.24,169.43,167.92,146.92,144.93,143.81,135.83,132.50,128.51,120.28,116.64,116.03,114.94,110.31,109.55,56.47,48.79,41.95,40.47, 38.76, 30.80, 28.82, 28.76, 26.22, 26.21, 22.40.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
28H
33N
5O
7[M+H]
+552.2414；found,552.2456.
HR-MS: calculated for C 28 H 33 N 5 O 7 [M+H] + 552.2414; found, 552.2456.
(9)靶向降解酪氨酸酶的小分子L-C7的合成(9) Synthesis of small molecule L-C7 targeting degradation of tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)
1H NMR(400MHz,Methanol-d
4)δ7.56(t,J＝7.9Hz,1H),7.09–7.01(m,2H),6.78–6.62(m,2H),6.53(d,J＝8.0Hz,1H),5.06(dd,J＝12.5,5.3Hz,1H),3.47(s,1H),3.34(d,J＝6.7Hz,2H),3.19(dq,J＝15.5,7.1Hz,2H),3.06(dt,J＝13.3,6.8 Hz,1H),2.86(d,J＝16.5Hz,2H),2.78(s,2H),2.77–2.65(m,2H),2.12(dd,J＝11.3,5.8Hz,1H),1.67(p,J＝6.9Hz,2H),1.42(h,J＝6.8,6.1Hz,4H),1.36–1.23(m,2H).
1 H NMR (400MHz, Methanol-d 4 ) 1 H NMR (400MHz, Methanol-d 4 ) δ7.56(t, J=7.9Hz, 1H), 7.09–7.01(m, 2H), 6.78–6.62(m ,2H),6.53(d,J=8.0Hz,1H),5.06(dd,J=12.5,5.3Hz,1H),3.47(s,1H),3.34(d,J=6.7Hz,2H),3.19 (dq,J=15.5,7.1Hz,2H),3.06(dt,J=13.3,6.8Hz,1H),2.86(d,J=16.5Hz,2H),2.78(s,2H),2.77–2.65( m, 2H), 2.12(dd, J=11.3, 5.8Hz, 1H), 1.67(p, J=6.9Hz, 2H), 1.42(h, J=6.8, 6.1Hz, 4H), 1.36–1.23(m ,2H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δ173.28,170.28,169.42,167.94,166.75,146.91,144.94,143.83,135.84,132.49,128.49,120.29,116.62,116.01,114.95,110.31,109.52,56.46,48.78,42.00,40.47,38.83,30.80,28.83,28.79,28.67,26.48,26.41,22.40.
13 C NMR(101MHz,MeOD)δ173.28,170.28,169.42,167.94,166.75,146.91,144.94,143.83,135.84,132.49,128.49,120.29,116.62,116.01,114.95,110.31,109.52,56.46,48.78,42.00,40.47, 38.83, 30.80, 28.83, 28.79, 28.67, 26.48, 26.41, 22.40.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
29H
35N
5O
7[M+H]
+566.2570；found,566.2619.
HR-MS: calculated for C 29 H 35 N 5 O 7 [M+H] + 566.2570; found, 566.2619.
(10)靶向降解酪氨酸酶的小分子L-C8的合成(10) Synthesis of small molecule L-C8 targeting degradation of tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ(ppm)7.57(dd,J＝8.5,7.1Hz,1H),7.09–7.02(m,2H),6.73(d,J＝8.0Hz,2H),6.66(dd,J＝14.2,2.1Hz,2H),6.54(ddd,J＝11.9,8.1,2.1Hz,2H),5.07(dd,J＝12.5,5.5Hz,1H),3.78–3.65(m,2H),3.36(d,J＝4.2Hz,1H),3.23(dt,J＝13.8,6.9Hz,1H),3.13–2.66(m,7H),1.68(q,J＝7.2Hz,2H),1.55–1.12(m,10H).
1 H NMR (400MHz, Methanol-d 4 ) δ (ppm) 7.57 (dd, J = 8.5, 7.1 Hz, 1H), 7.09–7.02 (m, 2H), 6.73 (d, J = 8.0Hz, 2H), 6.66(dd, J=14.2,2.1Hz,2H),6.54(ddd,J=11.9,8.1,2.1Hz,2H),5.07(dd,J=12.5,5.5Hz,1H),3.78–3.65(m, 2H), 3.36(d, J=4.2Hz, 1H), 3.23(dt, J=13.8, 6.9Hz, 1H), 3.13–2.66(m, 7H), 1.68(q, J=7.2Hz, 2H), 1.55–1.12(m,10H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δδ(ppm)173.28,170.30,169.41,168.09,167.92,146.90,145.34,144.67,135.86,132.47,125.40,120.39,116.62,116.06,115.26,110.33,109.50,54.67,48.77,42.01,39.19,36.85,30.81,30.75,30.28,28.85,28.69,26.49,26.40,22.40.
13 C NMR(101MHz,MeOD)δδ(ppm)173.28,170.30,169.41,168.09,167.92,146.90,145.34,144.67,135.86,132.47,125.40,120.39,116.62,116.06,115.26,110.33,109.50,54.67,48.77, 42.01, 39.19, 36.85, 30.81, 30.75, 30.28, 28.85, 28.69, 26.49, 26.40, 22.40.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
30H
37N
5O
7[M+H]
+580.2727；found,580.2727.
HR-MS: calculated for C 30 H 37 N 5 O 7 [M+H] + 580.2727; found, 580.2727.
(11)靶向降解酪氨酸酶的小分子L-C10的合成(11) Synthesis of small molecule L-C10 targeting degradation of tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ(ppm)7.55(dd,J＝8.5,7.1Hz,1H),7.03(dd,J＝7.8,4.3Hz,2H),6.87–6.72(m,1H),6.70(d,J＝2.1Hz,1H),6.58(dd,J＝8.1,2.2Hz,1H),5.06(dd, J＝12.5,5.4Hz,1H),3.92(t,J＝7.4Hz,1H),3.38–3.18(m,3H),3.16–2.98(m,1H),3.02–2.65(m,5H),2.12(dtd,J＝12.9,4.9,2.3Hz,1H),1.67(p,J＝7.0Hz,2H),1.59–1.10(m,12H).
1 H NMR (400MHz, Methanol-d 4 ) δ (ppm) 7.55 (dd, J = 8.5, 7.1 Hz, 1H), 7.03 (dd, J = 7.8, 4.3 Hz, 2H), 6.87–6.72 (m, 1H ), 6.70(d, J=2.1Hz, 1H), 6.58(dd, J=8.1, 2.2Hz, 1H), 5.06(dd, J=12.5, 5.4Hz, 1H), 3.92(t, J=7.4Hz ,1H),3.38–3.18(m,3H),3.16–2.98(m,1H),3.02–2.65(m,5H),2.12(dtd,J＝12.9,4.9,2.3Hz,1H),1.67(p ,J＝7.0Hz,2H),1.59–1.10(m,12H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δ(ppm)173.30,170.29,169.40,168.08,167.94,146.89,145.36,144.69,135.84,132.47,125.36,120.37,116.58,116.03,115.26,110.33,109.50,54.68,48.77,42.01,39.23,36.88,30.81,29.19,29.10,29.00,28.93,28.89,28.74,26.55,26.52,22.40.
13 C NMR(101MHz,MeOD)δ(ppm)173.30,170.29,169.40,168.08,167.94,146.89,145.36,144.69,135.84,132.47,125.36,120.37,116.58,116.03,115.26,110.33,109.50,54.68,48.77, 42.01, 39.23, 36.88, 30.81, 29.19, 29.10, 29.00, 28.93, 28.89, 28.74, 26.55, 26.52, 22.40.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
32H
41N
5O
7[M+H]
+608.3040；found,608.3077.
HR-MS: calculated for C 32 H 41 N 5 O 7 [M+H] + 608.3040; found, 608.3077.
合成实验步骤同实施例4Synthetic experimental procedure is the same as embodiment 4
靶向降解酪氨酸酶的小分子L-O1的合成Synthesis of Small Molecule L-O1 Targeting Degradation of Tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ7.55(dd,J＝8.5,7.1Hz,1H),7.06(dd,J＝10.8,7.8Hz,2H),6.77–6.68(m,2H),6.57(dt,J＝8.0,1.8Hz,1H),4.05–3.94(m,1H),3.67(t,J＝5.2Hz,2H),3.63–3.43(m,5H),3.42–3.26(m,2H),3.05(ddd,J＝14.0,7.0,4.3Hz,1H),2.96–2.87(m,1H),2.84–2.59(m,3H),2.05(dtd,J＝14.9,7.1,6.1,3.4Hz,1H).
1 H NMR (400MHz, Methanol-d 4 ) δ7.55 (dd, J＝8.5, 7.1Hz, 1H), 7.06 (dd, J＝10.8, 7.8Hz, 2H), 6.77–6.68 (m, 2H), 6.57(dt,J=8.0,1.8Hz,1H), 4.05–3.94(m,1H),3.67(t,J=5.2Hz,2H),3.63–3.43(m,5H),3.42–3.26(m, 2H), 3.05(ddd, J＝14.0, 7.0, 4.3Hz, 1H), 2.96–2.87(m, 1H), 2.84–2.59(m, 3H), 2.05(dtd, J＝14.9, 7.1, 6.1, 3.4 Hz,1H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δ173.22,170.37,169.45,168.40,167.84,146.75,145.33,144.66,135.91,132.37,125.50,120.50,116.86,116.12,115.35,110.72,109.80,68.90,54.60,53.41,48.78,41.63,39.15,36.79,30.76,22.32.
13 C NMR(101MHz,MeOD)δ173.22,170.37,169.45,168.40,167.84,146.75,145.33,144.66,135.91,132.37,125.50,120.50,116.86,116.12,115.35,110.72,109.80,68.90,54.60,53.41,48.78, 41.63, 39.15, 36.79, 30.76, 22.32.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
26H
29N
5O
8[M+H]
+539.2050；found,540.2091.
HR-MS: calculated for C 26 H 29 N 5 O 8 [M+H] + 539.2050; found, 540.2091.
实施例8：用于实施例4合成路线的实验方法Embodiment 8: be used for the experimental method of embodiment 4 synthetic routes
靶向降解酪氨酸酶的小分子L-O2的合成Synthesis of Small Molecule L-O2 Targeting Degradation of Tyrosinase
核磁共振氢谱(
1H NMR)确认结构：
Proton nuclear magnetic resonance ( 1 H NMR) confirmed the structure:
1H NMR(400MHz,Methanol-d
4)δ7.55(dd,J＝8.5,7.1Hz,1H),7.06(dd,J＝10.8,7.8Hz,2H),6.77–6.68(m,2H),6.57(dt,J＝8.0,1.8Hz,1H),4.05–3.94(m,1H),3.67(t,J＝5.2Hz,2H),3.63–3.43(m,5H),3.42–3.26(m,2H),3.05(ddd,J＝14.0,7.0,4.3Hz,1H),2.96–2.87(m,1H),2.84–2.59(m,3H),2.05(dtd,J＝14.9,7.1,6.1,3.4Hz,1H).
1 H NMR (400MHz, Methanol-d 4 ) δ7.55 (dd, J＝8.5, 7.1Hz, 1H), 7.06 (dd, J＝10.8, 7.8Hz, 2H), 6.77–6.68 (m, 2H), 6.57(dt,J=8.0,1.8Hz,1H), 4.05–3.94(m,1H),3.67(t,J=5.2Hz,2H),3.63–3.43(m,5H),3.42–3.26(m, 2H), 3.05(ddd, J＝14.0, 7.0, 4.3Hz, 1H), 2.96–2.87(m, 1H), 2.84–2.59(m, 3H), 2.05(dtd, J＝14.9, 7.1, 6.1, 3.4 Hz,1H).
核磁共振碳谱(
13C NMR)确认结构：
Carbon nuclear magnetic resonance ( 13 C NMR) confirmed the structure:
13C NMR(101MHz,MeOD)δ173.39,170.40,169.36,168.33,167.85,146.73,145.29,144.62,135.85,132.41,125.37,120.46,116.83,116.07,115.31,110.72,109.86,70.19,69.86,69.11,68.92,54.56,48.77,41.76,39.16,36.76,30.74,22.40.
13 C NMR(101MHz,MeOD)δ173.39,170.40,169.36,168.33,167.85,146.73,145.29,144.62,135.85,132.41,125.37,120.46,116.83,116.07,115.31,110.72,109.86,70.19,69.86,69.11,68.92, 54.56, 48.77, 41.76, 39.16, 36.76, 30.74, 22.40.
高分辨质谱(HR-MS)确认结构：High-resolution mass spectrometry (HR-MS) confirmed the structure:
HR-MS:calculated for C
28H
33N
5O
9[M+H]
+584.2312；found,584.2356.
HR-MS: calculated for C 28 H 33 N 5 O 9 [M+H] + 584.2312; found, 584.2356.
实施例9：实验部分Embodiment 9: Experimental part
(一)细胞系和材料(1) Cell lines and materials
将A375和HepG2细胞维持在补充有10％胎牛血清的RPMI-1640培养基(A10491，Invitrogen，Carlsbad，CA)中，并在37℃下于5％CO
2的培养箱中培养。MG132和pomalidomide购自Carsmart。
A375 and HepG2 cells were maintained in RPMI-1640 medium (A10491, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and cultured at 37°C in an incubator with 5% CO2 . MG132 and pomalidomide were purchased from Carsmart.
(二)蛋白质免疫印迹(2) Western blot
将细胞接种到24孔板中并培养过夜，并如图I所示用小分子处理。然后将细胞收集到1.5mL EP试管中，用1×冷PBS洗涤，并在50L 1×蛋白质上样缓冲液(50mM Tris-HCl pH 6.8，100mM DTT，0.01％溴酚蓝，2％SDS，10％裂解液)中裂解甘油)，然后在100℃下加热15分钟。按照标准方案进行蛋白质印迹。简而言之，将12L(用于酪氨酸酶和GAPDH)样品装入10％聚丙烯酰胺凝胶孔中。在堆叠凝胶中将电压设定为90V，并且将其设为100V以进行分离。通过在100V的冰水浴中电泳1.5小时，将蛋白质转移到NC膜(Millipore，0.2m)上。将膜在5％脱脂牛奶(1×TBST)中于室温孵育1小时，然后将膜用1x TBST洗涤，切割并与一抗在4℃孵育过夜。第二天，回收一抗体，将膜在室温下以1x TBST洗涤3次，每次10分钟。然后将膜与山羊抗兔(Thermo Fisher)二抗在室温下孵育1小时，然后在1x TBST中再洗涤3次。最后，用ECL试剂(Engreen)处理膜，并通过MiniChemi系统(Sage Creation)拍摄照片。Cells were seeded into 24-well plates and cultured overnight, and treated with small molecules as shown in Figure I. Cells were then collected into 1.5mL EP tubes, washed with 1× cold PBS, and incubated in 50L of 1× protein loading buffer (50mM Tris-HCl pH 6.8, 100mM DTT, 0.01% bromophenol blue, 2% SDS, 10 % lysate), and then heated at 100°C for 15 minutes. Western blotting was performed following standard protocols. Briefly, 12 L (for tyrosinase and GAPDH) samples were loaded into wells of a 10% polyacrylamide gel. The voltage was set to 90V in the stacking gel and to 100V for separation. Proteins were transferred to NC membranes (Millipore, 0.2m) by electrophoresis at 100V in an ice-water bath for 1.5 hours. Membranes were incubated in 5% nonfat milk (1x TBST) for 1 hour at room temperature, then membranes were washed with 1x TBST, cleaved and incubated with primary antibodies overnight at 4°C. The next day, the primary antibody was recovered and the membrane was washed 3 times with 1x TBST for 10 minutes at room temperature. Membranes were then incubated with goat anti-rabbit (Thermo Fisher) secondary antibody for 1 hr at room temperature and then washed 3 more times in 1x TBST. Finally, membranes were treated with ECL reagent (Engreen) and photographed by the MiniChemi system (Sage Creation).
(三)抗体信息(3) Antibody information
酪氨酸酶和GAPDH抗体购自Abcam，并用5％BSA在TBST中按1：3000稀释。Tyrosinase and GAPDH antibodies were purchased from Abcam and diluted 1:3000 in TBST with 5% BSA.
(四)化合物L-C2–L-O2的分子结构对诱导酪氨酸酶降解的效果(4) Effect of the molecular structure of compound L-C2–L-O2 on inducing tyrosinase degradation
根据上述合成路线路线设计并合成了化合物L-C2–L-O2，该系列化合物具有不同长度和组成的linkers。为了评估候选化合物L-C2–L-O2诱导酪氨酸酶降解的效果，我们将L-C2–L-O2和A375细胞也共同孵育了24h，然后分析了细胞内酪氨酸酶的水平。Compounds L-C2–L-O2 were designed and synthesized according to the above synthetic route. This series of compounds have linkers of different lengths and compositions. In order to evaluate the effect of the candidate compound L-C2–L-O2 on inducing tyrosinase degradation, we also co-incubated L-C2–L-O2 and A375 cells for 24 h, and then analyzed the level of intracellular tyrosinase.
实验结果：图1为筛选和评估所设计的具有不同链长和组成的化合物(L-C2–L-O2,50μM)诱导酪氨酸酶降解的活性，如图1所示，这些化合物虽然都能下调细胞内的酪氨酸酶水平，但是同其他化合物相比，连接链为5个碳原子长度的L-C5诱导酪氨酸酶降解的作用最强。当缩短或延长碳链的长度，会导致降解效率不同程度地降低，可能原因是连接链长度的改变会影响两个配体之间的互相靠近，从而不能形成稳定的三元空间构象。此外，将烷烃链(L-C5,L-C8)变换为相同原子数(L-O1,L-O2)的聚乙二醇(PEG)链，会大大削弱其诱导酪氨酸酶降解的活性，表明当改变连接链的组成，也会明显导致降解效率降低，可能原因是连接链组成的改变影响整个分子的亲疏水性，从而影响细胞对小分子的吸收，进而导致细胞内的药物浓度偏低。也有可能是小分子的进入细胞后，连接链的组成变化引起分子整体构象发生变化，从而不能同时有效的结合两端的配体。以上这些结果表明链的长度和组成对这些化合物的活性具有深远的影响。Experimental results: Figure 1 is for the screening and evaluation of the designed compounds with different chain lengths and compositions (L-C2–L-O2, 50μM) to induce tyrosinase degradation activity, as shown in Figure 1, although these compounds are all It can down-regulate the level of tyrosinase in cells, but compared with other compounds, L-C5 with a chain length of 5 carbon atoms has the strongest effect on inducing the degradation of tyrosinase. When the length of the carbon chain is shortened or extended, the degradation efficiency will be reduced to varying degrees. The possible reason is that the change of the length of the linking chain will affect the mutual proximity between the two ligands, so that a stable ternary spatial conformation cannot be formed. In addition, changing the alkane chain (L-C5, L-C8) to a polyethylene glycol (PEG) chain with the same number of atoms (L-O1, L-O2) will greatly weaken its activity of inducing tyrosinase degradation , indicating that when the composition of the linking chain is changed, the degradation efficiency will also be significantly reduced. The possible reason is that the change of the composition of the linking chain affects the hydrophilicity and hydrophobicity of the entire molecule, thereby affecting the absorption of small molecules by cells, which in turn leads to low drug concentration in cells . It is also possible that after the small molecule enters the cell, the change in the composition of the connecting chain causes the overall conformation of the molecule to change, so that it cannot effectively bind the ligands at both ends at the same time. These results above indicate that chain length and composition have a profound effect on the activity of these compounds.
(五)化合物L-C5的浓度对诱导酪氨酸酶降解的效果(5) Effect of the concentration of compound L-C5 on inducing tyrosinase degradation
进一步的，研究活性最好的化合物L-C5在不同给药浓度(25、50和100μM)时诱导酪氨酸酶降解的效果。Further, the effect of the most active compound L-C5 on inducing tyrosinase degradation at different concentrations (25, 50 and 100 μM) was studied.
实验结果：图2为不同浓度的L-C5诱导酪氨酸酶降解效果评价，如图2所示，酪氨酸酶水平呈浓度依赖性降低。经计算，DC
50值(蛋白质降解50％的浓度)约为50μM，而且给药浓度为100μM时达到最大活性，最多可诱导酪氨酸酶(Dmax)降解得水平大约为63％。综上，小分子诱导酪氨酸酶降解的效率会受到linker的长度和组成的影响，这也表明在优化降解剂时应考虑连接链的组成和长度。另外，筛选出活性最好的降解小分子L-C5，并验证了其能够以浓度依赖性方式诱导酪氨酸酶降解。
Experimental results: Figure 2 is the evaluation of the degradation effect of tyrosinase induced by different concentrations of L-C5, as shown in Figure 2, the level of tyrosinase decreased in a concentration-dependent manner. After calculation, the DC 50 value (concentration at which 50% of the protein is degraded) is about 50 μM, and the maximum activity is reached when the administration concentration is 100 μM, and the maximum level of inducing tyrosinase (Dmax) degradation is about 63%. In summary, the efficiency of small molecules to induce tyrosinase degradation will be affected by the length and composition of the linker, which also indicates that the composition and length of the linker chain should be considered when optimizing the degradation agent. In addition, L-C5, the most active degradation small molecule, was screened out, and it was verified that it could induce tyrosinase degradation in a concentration-dependent manner.
(六)化合物L-C5诱导酪氨酸酶降解的机理(6) Mechanism of Compound L-C5 Inducing Tyrosinase Degradation
为了探究L-C5诱导细胞内酪氨酸酶的降解是否和文献报道PROTACs靶向诱导目标蛋白降解的机理一致。首先研究了在细胞内酪氨酸酶，L-C5和募集的E3连接酶之间的三元复合物是否形成，从而才会导致随后的酪氨酸酶降解。如图3(A)所示，我们发现L-Dopa(100μM)，沙利度胺(100μM)在给药24h后完全无效诱导酪氨酸酶降解，表明两端的配体对酪氨酸酶的水平没有影响。进一步的，探讨由L-C5诱导的酪氨酸酶降解是否通过泛素-蛋白酶体系统。因此，A375细胞用蛋白酶体抑制剂MG132(1μM)和预处理30min，然后再加入100μM L-C5并孵育24h。实验结果如图3(B)所示，L-C5可以明显诱导酪氨酸 酶降解，然而，当细胞经过MG132预处理后，L-C5则无法诱导酪氨酸酶降解，表明L-C5诱导的酪氨酸酶降解取决于泛素化和蛋白酶体级联反应。In order to explore whether the degradation of intracellular tyrosinase induced by L-C5 is consistent with the mechanism reported in the literature that PROTACs target and induce the degradation of target proteins. We first investigated whether a ternary complex forms between intracellular tyrosinase, L-C5, and a recruited E3 ligase that would lead to subsequent tyrosinase degradation. As shown in Figure 3(A), we found that L-Dopa (100 μM) and thalidomide (100 μM) were completely ineffective in inducing tyrosinase degradation after 24 hours of administration, indicating that the ligands at both ends have an effect on tyrosinase. level has no effect. Further, to explore whether the degradation of tyrosinase induced by L-C5 is through the ubiquitin-proteasome system. Therefore, A375 cells were pretreated with proteasome inhibitor MG132 (1 μM) for 30 min, and then added with 100 μM L-C5 and incubated for 24 h. The experimental results are shown in Figure 3(B), L-C5 can obviously induce tyrosinase degradation, however, when cells are pretreated with MG132, L-C5 cannot induce tyrosinase degradation, indicating that L-C5 induces Tyrosinase degradation depends on ubiquitination and the proteasomal cascade.
(七)细胞毒性实验(7) Cytotoxicity test
使用标准细胞活力方案(CCK8分析)评估了L-C5对A375和HepG2细胞的细胞毒性。将A375和HepG2细胞孵育24小时，然后用各种浓度(0、1.25、2.5、5、10、20μM)的L-C5处理24小时。然后，用酶标仪测量450nm处的吸光度(在使用酶标仪之前，提前打开10分钟进行预热)。测量完成后，使用Graphpad Prism 8.0保存并处理数据。Cytotoxicity of L-C5 on A375 and HepG2 cells was assessed using a standard cell viability protocol (CCK8 assay). A375 and HepG2 cells were incubated for 24 hours and then treated with various concentrations (0, 1.25, 2.5, 5, 10, 20 μM) of L-C5 for 24 hours. Then, measure the absorbance at 450nm with a microplate reader (before using the microplate reader, turn it on for 10 minutes in advance for preheating). After the measurement is completed, the data is saved and processed using Graphpad Prism 8.0.
(八)化合物L-C5理化性质初步测定实验(8) Preliminary determination of physical and chemical properties of compound L-C5
将10mL纯净水加入25mL锥形瓶中，然后将已称重的一定量的L-C5缓慢加入烧瓶中。并且，我们在室温下摇动烧瓶30秒，直到L-C5完全溶解。然后，我们继续缓慢加入L-C5，直到仍然有明显的固体，摇晃30秒后它没有溶解。然后，我们过滤，烘干，并称重未溶解的L-C5(m1)，此时记录添加的总L-C5(m)的质量。L-C5的溶解度(S)使用以下等式测量。Add 10mL of pure water into a 25mL Erlenmeyer flask, and then slowly add a certain amount of L-C5 that has been weighed into the flask. And, we shake the flask at room temperature for 30 seconds until the L-C5 is completely dissolved. We then continued to add L-C5 slowly until there was still a noticeable solid, which did not dissolve after shaking for 30 seconds. We then filtered, dried, and weighed the undissolved L-C5(m1), at which point the mass of the total L-C5(m) added was recorded. The solubility (S) of L-C5 was measured using the following equation.
S＝(m-m1)/10S=(m-m1)/10
使用摇瓶法测量L-C5的分配系数。将称重的L-C5加入等体积的辛醇和纯净水的混合物中，然后在室温下在机械摇床上摇晃24小时。然后我们移液枪分别吸辛醇溶解的200μL和水相200μL至96孔板中，并用酶标仪的紫外分析模块测定270nm处的紫外吸收峰强度，分别得Abs1，Abs2。然后再测定不含L-C5的辛醇(200μL)及纯净水(200μL)的溶剂背景在270nm处的紫外吸收强度Abs3，Abs4，最后使用以下等式测量log P
O/W。
The partition coefficient of L-C5 was measured using the shake flask method. A weighed amount of L-C5 was added to a mixture of equal volumes of octanol and purified water, followed by shaking on a mechanical shaker at room temperature for 24 h. Then we pipetted 200 μL dissolved in octanol and 200 μL of aqueous phase into 96-well plates, and measured the UV absorption peak intensity at 270 nm with the UV analysis module of the microplate reader to obtain Abs1 and Abs2 respectively. Then measure the ultraviolet absorption intensity Abs3 and Abs4 at 270 nm of the solvent background of octanol (200 μL) and pure water (200 μL) without L-C5, and finally use the following equation to measure log P O/W .
log P
O/W＝log[(Abs1-Abs3)/(Abs2-Abs4)]
log P O/W = log[(Abs1-Abs3)/(Abs2-Abs4)]
tPSA以及LpgKp(Skon permeation)则tPSA and LpgKp (Skon permeation) are
大多数报道的PROTACs分子量较大，一般为700-1000Da，这可能使PROTACs生物利用度较差，并且是进入临床的重要障碍。但PROTAC L-C5的分子量小于540Da，表明其具有改善递送和生物利用度的潜力。此外，皮肤色素沉着治疗药物的理化性质通常会影响其透皮给药。图4为小分子化合物L-C5初步理化性质评价，如图4所示，其具有良好的水溶性(11.4mg/mL)，因此有可能避免像曲酸(0.055mg/mL)和鞣花酸(0.0097mg/mL)在临床应用中溶解度差的缺点。而且对于皮肤给药，L-C5的良好水溶性可能使其不需要苛刻的溶剂条件，否则会引起皮肤干燥和刺激。并且，通过摇瓶法，在正辛醇/纯水两相中我们确定L-C5的分配系数(log Po/w)为-0.65，表明L-C5具有很高的亲水性，可以适当地制成微乳状液的膏剂或装入微针贴剂中以提高其可药用性。总体而言，对L-C5理化性质的研究将有助于为未来色素沉着治疗药物的开发提供指导。Most of the reported PROTACs have large molecular weight, generally 700-1000Da, which may make the bioavailability of PROTACs poor and be an important obstacle to enter the clinic. However, PROTAC L-C5 has a molecular weight of less than 540 Da, suggesting its potential for improved delivery and bioavailability. In addition, the physicochemical properties of skin pigmentation treatments often affect their transdermal delivery. Figure 4 is the preliminary physical and chemical property evaluation of the small molecule compound L-C5, as shown in Figure 4, it has good water solubility (11.4mg/mL), so it is possible to avoid kojic acid (0.055mg/mL) and ellagic acid (0.0097mg/mL) has the disadvantage of poor solubility in clinical application. And for dermal administration, the good water solubility of L-C5 may make it unnecessary for harsh solvent conditions, which would otherwise cause skin dryness and irritation. And, by shake flask method, in n-octanol/pure water two-phase, we determine that the distribution coefficient (log Po/w) of L-C5 is-0.65, shows that L-C5 has very high hydrophilicity, can suitably Made into a microemulsion paste or packed into a microneedle patch to improve its drugability. Overall, the research on the physicochemical properties of L-C5 will help to provide guidance for the development of future pigmentation treatment drugs.
(九)斑马鱼实验：(9) Zebrafish experiment:
斑马鱼的处理是根据关于保护用于科学目的的动物的一般性指示并按照四川省动物保护和使用委员会批准的操作程序进行的。根据Zebrafish Book中描述的标准方案，将野生型AB品系维持在循环水产养殖系统中。根据斑马鱼胚胎发育阶段中的描述，将胚胎孵育并分阶段在28.5℃下进行。然后将L-C5溶于二甲基亚砜(DMSO)中，并用胚胎培养基稀释至所有处理的设定浓度。将保护期[受精后6小时(hpf)]的胚胎转移到24孔板中，并与稀释的化合物溶液一起孵育。在prim-25阶段(48hpf)评估胚胎的表型变化。Zebrafish were handled according to the general instructions on the care of animals used for scientific purposes and in accordance with operating procedures approved by the Sichuan Provincial Animal Protection and Use Committee. The wild-type AB line was maintained in a recirculating aquaculture system according to standard protocols described in the Zebrafish Book. Embryos were incubated and staged at 28.5 °C as described in Zebrafish Embryo Developmental Stages. L-C5 was then dissolved in dimethyl sulfoxide (DMSO) and diluted with embryo medium to the set concentration for all treatments. Embryos at the guard period [6 hours post fertilization (hpf)] were transferred to 24-well plates and incubated with diluted compound solutions. Embryos were assessed for phenotypic changes at the prim-25 stage (48hpf).
斑马鱼的身体具有光学透明性，能够在较短的生长周期内在体外快速发育，并且斑马鱼酪氨酸酶同人源的酪氨酸酶高度同源。此外，斑马鱼的黑色素覆盖于整个皮肤表面，可以观察色素沉着过程，无需复杂的操作实验程序。由于这些优点，它通常被用作色素沉着研究的动物模型。因此，也以斑马鱼作为模型生物来研究L-C5抑制黑色素生成活性。然后，在受精(hpf)后的6hpf(此时为斑马鱼神经细胞的发育阶段)，将候选化合物L-C5(25，50和100μM)和胚胎共同孵育。然后，选择左旋多巴和曲酸作为阳性对照组，以等摩尔浓度给药，并以DMSO的给药组作为空白对照。给药48hpf后，可观察到斑马鱼的状态和形态没有受到影响，这表明L-C5对斑马鱼的毒性很低(图5)。另外，L-C5可以明显地减少斑马鱼的眼睛，背部，腹部和尾巴中的黑色素产生。但是，对于左旋多巴和曲酸给药组，斑马鱼皮肤表面的黑色素则几乎没有明显的变化(图5)。斑马鱼皮肤表面黑色素经过ImageJ处理后，分析斑马鱼皮肤上的黑色素相对含量的变化，结果如图5所示，可发现当给药浓度从25μM增加到100μM时，这些化合物都能下调斑马鱼皮肤表面的黑色素含量，但是，L-C5的效果远远优于左旋多巴和曲酸。The body of zebrafish is optically transparent, can develop rapidly in vitro within a short growth period, and zebrafish tyrosinase is highly homologous to human tyrosinase. In addition, the melanin of zebrafish covers the entire skin surface, and the pigmentation process can be observed without complicated experimental procedures. Due to these advantages, it is often used as an animal model for pigmentation studies. Therefore, zebrafish was also used as a model organism to study the melanogenesis-inhibitory activity of L-C5. Then, at 6hpf after fertilization (hpf), which is the developmental stage of zebrafish neurons, the candidate compound L-C5 (25, 50 and 100 μM) was co-incubated with the embryos. Then, choose levodopa and kojic acid as the positive control group, administer with equimolar concentration, and use the administration group of DMSO as the blank control. After 48 hpf administration, it was observed that the status and morphology of the zebrafish were not affected, which indicated that L-C5 had very low toxicity to zebrafish (Fig. 5). In addition, L-C5 can significantly reduce melanin production in the eyes, back, abdomen and tail of zebrafish. However, for the levodopa and kojic acid administration groups, there was almost no significant change in the melanin on the zebrafish skin surface (Fig. 5). After the melanin on the surface of the zebrafish skin was treated with ImageJ, the changes in the relative content of the melanin on the zebrafish skin were analyzed. Surface melanin content, however, the effect of L-C5 is far superior to levodopa and kojic acid.
综上所述，L-C5可以明显减少斑马鱼模型中黑色素的产生，并且比曲酸和左旋多巴的活性更好。因此，L-C5有潜力被开发为治疗皮肤色素沉着性疾病的药物。In summary, L-C5 can significantly reduce the production of melanin in the zebrafish model, and has better activity than kojic acid and levodopa. Therefore, L-C5 has the potential to be developed as a drug for the treatment of skin pigmentation diseases.
(十)化合物L-C5体外抗氧化能力实验(10) In vitro antioxidant capacity test of compound L-C5
通过1，1-二苯基-2-三硝基苯肼(1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl,DPPH)测定法来确定L-C5的抗氧化活性。测定溶液由100μL DPPH(150μM)、20μL浓度渐增的测试化合物组成，并用纯净水(Pure water)将每个孔中的体积调整为200μL。然后将反应混合物在室温下孵育30分钟。抗坏血酸(维生素C,Vc)，左旋多巴(L-Doap)及沙利度胺(Thalidomide)作为对照。通过使用酶标仪测定517nm处的吸收波长。每个浓度在三个独立的实验中进行分析，实验一式三份。By 1,1-diphenyl-2-trinitrophenylhydrazine (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl,DPPH) assay To determine the antioxidant activity of L-C5. The assay solution consisted of 100 μL DPPH (150 μM), 20 μL of test compounds at increasing concentrations, and the volume in each well was adjusted to 200 μL with Pure water. The reaction mixture was then incubated at room temperature for 30 minutes. Ascorbic acid (vitamin C, Vc), levodopa (L-Doap) and thalidomide (Thalidomide) were used as controls. The absorption wavelength at 517 nm was measured by using a microplate reader. Each concentration was analyzed in three independent experiments in triplicate.
人体皮肤经太阳光照射后会产生不稳定的自由基，然后自由基与体内各种物质发生氧化反应，破坏人体正常的新陈代谢，导致皮肤老化、干燥、敏感、痘肌及暗沉等。而且，随着 年龄的增长，皮肤中自身的抗氧化剂含量也会逐渐降低，再加上外界压力和环境恶化(吸烟、尾气、污染、辐射、油炸食品等)，使得自由基对皮肤氧化侵蚀会更加严重，唯一的解决办法就是将抗氧化“外套”涂在皮肤上。鉴于L-C5含有邻二酚羟基，因此我们进行了体外抗氧化研究，以评价其是否能够在调控皮肤黑色素产生的同时还可以作为皮肤的抗氧化成分进行应用。Human skin will produce unstable free radicals after being irradiated by sunlight, and then the free radicals will undergo oxidation reactions with various substances in the body, destroying the normal metabolism of the human body, leading to skin aging, dryness, sensitivity, acne and dullness. Moreover, with age, the skin's own antioxidant content will gradually decrease, coupled with external pressure and environmental deterioration (smoking, exhaust, pollution, radiation, fried food, etc.), causing free radicals to oxidize and corrode the skin will get worse, and the only solution is to apply an antioxidant "coat" to the skin. Since L-C5 contains ortho-diphenolic hydroxyl groups, we conducted an in vitro antioxidant study to evaluate whether it can be used as an antioxidant component of the skin while regulating the production of melanin in the skin.
设置四组实验，包括沙利度胺(Thalidomide,THL)，L-Dopa，L-C5和阳性对照维生素C(Vitamin C,Vc)组，浓度分别为0，20，50，100，200，500μM，分别与150μM的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)混匀，再测定候选化合物的抗氧化能力。Set up four groups of experiments, including Thalidomide (THL), L-Dopa, L-C5 and positive control vitamin C (Vitamin C, Vc) groups, the concentrations were 0, 20, 50, 100, 200, 500 μM , respectively mixed with 150 μM 1,1-diphenyl-2-trinitrophenylhydrazyl (1,1-diphenyl-2-picrylhydrazyl, DPPH), and then the antioxidant capacity of the candidate compounds was determined.
实验结果如图6所示，随着浓度的增加，L-C5、维生素C和左旋多巴可以明显清除由DPPH自由基，起到抗氧化作用，而沙利度胺清除自由基的作用较弱，表明我们设计的化合物因左旋多巴的存在而具有抗氧化能力。与Vc和L-Dopa相比，L-C5清除自由基的能力最强，且具有浓度依赖性。当其浓度为20μM时，可清除约28％的自由基，当浓度增加至500μM时，则可清除约55％的自由基。而L-C5比L-Dopa具有更强的体外自由基清除能力的可能归因于L-C5比L-Dopa具有更好的水溶性。The experimental results are shown in Figure 6. With the increase of concentration, L-C5, vitamin C and levodopa can obviously scavenge DPPH free radicals and play an antioxidant role, while thalidomide has a weaker effect on scavenging free radicals , indicating that our designed compound possesses antioxidant capacity due to the presence of levodopa. Compared with Vc and L-Dopa, L-C5 has the strongest ability to scavenge free radicals, and it is concentration-dependent. When its concentration is 20 μM, it can scavenge about 28% of free radicals, and when the concentration increases to 500 μM, it can scavenge about 55% of free radicals. The stronger in vitro free radical scavenging ability of L-C5 than L-Dopa may be attributed to the better water solubility of L-C5 than L-Dopa.
综上，相比于实际生活及临床应用的维生素C，L-C5具有更强大的体外抗氧化活性，而且具有能够在调控皮肤黑色素产生的同时保护皮肤不受氧化损伤的潜力。In summary, compared with vitamin C in real life and clinical application, L-C5 has stronger in vitro antioxidant activity, and has the potential to protect skin from oxidative damage while regulating skin melanin production.
以上所述，仅是本发明的较佳实施例，并非对本发明做任何形式上的限制，凡是依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化，均落入本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Any simple modifications and equivalent changes made to the above embodiments according to the technical essence of the present invention all fall within the scope of the present invention. within the scope of protection.
Claims (6)
- 靶向降解酪氨酸酶的化合物，其特征在于：结构式如下式I所示：The compound targeting degradation of tyrosinase is characterized in that: the structural formula is as shown in formula I below:
其中，L为连接体基团，其结构式如下：Wherein, L is a linker group, and its structural formula is as follows:
其中，Z 0、Z 1、Z 2为-O-或-S-中任一基团，m 0、m 1、m 2、m 3、m 4、m 5、m 6为0-15中任一整数。 Among them, Z 0 , Z 1 , Z 2 are
Any group of -O- or -S-, m 0 , m 1 , m 2 , m 3 , m 4 , m 5 , m 6 are any integer from 0 to 15. - 根据权利要求1所述的化合物，其特征在于：其化学结构包括但不限于以下结构式：The compound according to claim 1, characterized in that: its chemical structure includes but not limited to the following structural formula:
- 根据权利要求1所述的化合物的盐、前药、水合物或溶剂合物。A salt, prodrug, hydrate or solvate of the compound according to claim 1.
- 合成权利要求1所述化合物的方法，其特征在于：包括但不限于以下合成路线：The method for synthesizing the compound described in claim 1 is characterized in that: including but not limited to the following synthetic routes:1)采用靶向E3泛素链接酶小分子的合成；1) Synthesis of small molecules targeting E3 ubiquitin ligase;2)采用左旋多巴或3,4-二羟基苯丙酸为靶向小分子的合成；2) Using levodopa or 3,4-dihydroxyphenylpropionic acid as the synthesis of targeted small molecules;3)靶向降解酪氨酸酶的烷烃碳链化合物的合成；3) Synthesis of alkane carbon chain compounds targeting the degradation of tyrosinase;4)靶向降解酪氨酸酶碳氧链化合物的合成。4) Synthesis of carbon-oxygen chain compounds targeting degradation of tyrosinase.
- 根据权利要求1所述的化合物在制备皮肤美白化妆品原料及皮肤疾病治疗药物中的应用，其特征在于：所述皮肤为肤质差并且色素过度沉着的肤质，包括但不限于雀斑、黄褐斑、黑素瘤、黑皮病、咖啡斑、蒙古斑、太田痣、焦油黑变病、瑞尔黑变病、种族性黑皮病、着色干皮病、肢端色素沉着。The application of the compound according to claim 1 in the preparation of raw materials for skin whitening cosmetics and medicines for treating skin diseases, characterized in that: the skin is poor in quality and hyperpigmented, including but not limited to freckles, tan Melanoma, melanoma, coffee spot, Mongolian spot, nevus of Ota, tar melanosis, rael's melanosis, ethnic melanosis, xeroderma pigmentosa, acral hyperpigmentation.
- 用于皮肤美白化妆品原料及皮肤疾病治疗的药物组合物，其特征在于：含有治疗有效量的权利要求1～3任一项所述的化合物及至少一种可药用载体。The pharmaceutical composition for skin whitening cosmetic raw materials and treatment of skin diseases is characterized in that it contains a therapeutically effective amount of the compound described in any one of claims 1 to 3 and at least one pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111043833.2 | 2021-09-07 | ||
| CN202111043833.2A CN113735824B (en) | 2021-09-07 | 2021-09-07 | PROTAC for targeted degradation of tyrosinase and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023035927A1 true WO2023035927A1 (en) | 2023-03-16 |
Family
ID=78736544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/114098 WO2023035927A1 (en) | 2021-09-07 | 2022-08-23 | Compound for targeted degradation of tyrosinase, pharmaceutical composition, and method for synthesizing compound and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113735824B (en) |
| WO (1) | WO2023035927A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113735824B (en) * | 2021-09-07 | 2023-06-20 | 中国科学院成都生物研究所 | PROTAC for targeted degradation of tyrosinase and application thereof |
| CN115028678A (en) * | 2022-07-08 | 2022-09-09 | 杭州医学院 | Bifunctional molecule based on BCR-ABL protein degradation induced by VHL ligand and preparation method and application thereof |
| CN115124510B (en) * | 2022-07-14 | 2023-04-28 | 广州医科大学 | PROTAC molecule, preparation method and application thereof |
| CN115089588A (en) * | 2022-08-22 | 2022-09-23 | 云南大学 | Application of Dasabovir as novel E3 ligase ligand in construction of PROTAC |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5100654A (en) * | 1989-04-07 | 1992-03-31 | Yale University | Phosphorylated derivatives of l-dopa and compositions and methods for increasing the melanin content in mammalian skin and hair |
| US20080317692A1 (en) * | 2007-06-25 | 2008-12-25 | Te-Sheng Chang | Suicide substrates of tyrosinase and use thereof |
| CN102018631A (en) * | 2009-09-11 | 2011-04-20 | 财团法人工业技术研究院 | Composition used for whitening skin, plant extraction whitening composition and composition with whitening function |
| US20120207809A1 (en) * | 2009-01-29 | 2012-08-16 | Kambiz Thomas Moazed | Method and system for effecting changes in pigmented tissue |
| CN103265635A (en) * | 2013-04-28 | 2013-08-28 | 中山大学附属第一医院 | Construction method of universal targeted protein chimeric molecular compound |
| CN104771329A (en) * | 2015-04-16 | 2015-07-15 | 欧米克斯生命科学股份有限公司 | Composition using imatinib mesylate for skin whitening and application of composition |
| CN105112470A (en) * | 2015-10-10 | 2015-12-02 | 董晓娜 | Production process of kojic acid for whitening and freckle-removing cosmetics |
| CN107257800A (en) * | 2014-12-23 | 2017-10-17 | 达纳-法伯癌症研究所股份有限公司 | The method that target protein is degraded is induced by bifunctional molecule |
| CN110684015A (en) * | 2018-07-06 | 2020-01-14 | 四川大学 | ALK-targeting PROTAC and application thereof |
| WO2020252397A1 (en) * | 2019-06-12 | 2020-12-17 | Baylor College Of Medicine | Small molecule proteolysis-targeting chimeras and methods of use thereof |
| CN113354619A (en) * | 2021-06-24 | 2021-09-07 | 皖南医学院 | Compound for targeted ubiquitination degradation of tyrosinase and preparation method and application thereof |
| CN113735824A (en) * | 2021-09-07 | 2021-12-03 | 中国科学院成都生物研究所 | PROTAC for targeted degradation of tyrosinase and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551102B (en) * | 2018-06-01 | 2021-01-12 | 四川大学 | ALK covalent inhibitors and uses thereof |
| CN112409376A (en) * | 2019-08-20 | 2021-02-26 | 华东师范大学 | Protein degradation targeting chimera based on DCAF15, and preparation method and application thereof |
| CN112979747A (en) * | 2019-12-16 | 2021-06-18 | 江苏恒瑞医药股份有限公司 | Hydroxyproline derivative for preparing protein degradation targeting chimera |
| CN112939965B (en) * | 2021-02-08 | 2023-02-24 | 沈阳药科大学 | Compound for simultaneously inducing degradation of EGFR (epidermal growth factor receptor) and PARP (para-amyloid peptide) proteins as well as preparation method and application thereof |
-
2021
- 2021-09-07 CN CN202111043833.2A patent/CN113735824B/en active Active
-
2022
- 2022-08-23 WO PCT/CN2022/114098 patent/WO2023035927A1/en unknown
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5100654A (en) * | 1989-04-07 | 1992-03-31 | Yale University | Phosphorylated derivatives of l-dopa and compositions and methods for increasing the melanin content in mammalian skin and hair |
| US20080317692A1 (en) * | 2007-06-25 | 2008-12-25 | Te-Sheng Chang | Suicide substrates of tyrosinase and use thereof |
| US20120207809A1 (en) * | 2009-01-29 | 2012-08-16 | Kambiz Thomas Moazed | Method and system for effecting changes in pigmented tissue |
| CN102018631A (en) * | 2009-09-11 | 2011-04-20 | 财团法人工业技术研究院 | Composition used for whitening skin, plant extraction whitening composition and composition with whitening function |
| CN103265635A (en) * | 2013-04-28 | 2013-08-28 | 中山大学附属第一医院 | Construction method of universal targeted protein chimeric molecular compound |
| CN107257800A (en) * | 2014-12-23 | 2017-10-17 | 达纳-法伯癌症研究所股份有限公司 | The method that target protein is degraded is induced by bifunctional molecule |
| CN104771329A (en) * | 2015-04-16 | 2015-07-15 | 欧米克斯生命科学股份有限公司 | Composition using imatinib mesylate for skin whitening and application of composition |
| CN105112470A (en) * | 2015-10-10 | 2015-12-02 | 董晓娜 | Production process of kojic acid for whitening and freckle-removing cosmetics |
| CN110684015A (en) * | 2018-07-06 | 2020-01-14 | 四川大学 | ALK-targeting PROTAC and application thereof |
| WO2020252397A1 (en) * | 2019-06-12 | 2020-12-17 | Baylor College Of Medicine | Small molecule proteolysis-targeting chimeras and methods of use thereof |
| CN113354619A (en) * | 2021-06-24 | 2021-09-07 | 皖南医学院 | Compound for targeted ubiquitination degradation of tyrosinase and preparation method and application thereof |
| CN113735824A (en) * | 2021-09-07 | 2021-12-03 | 中国科学院成都生物研究所 | PROTAC for targeted degradation of tyrosinase and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113735824A (en) | 2021-12-03 |
| CN113735824B (en) | 2023-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2023035927A1 (en) | Compound for targeted degradation of tyrosinase, pharmaceutical composition, and method for synthesizing compound and use thereof | |
| RU2635627C2 (en) | Pharmaceutical or cosmetic composition for alopecia treatment | |
| WO2008152159A2 (en) | 6 -acyloxy- 7-meth0xy- 2, 2 -dimethylchromane compounds and their use as pigmentation-regulators | |
| CN103282348B (en) | Make the method for keratin material decolouring using resorcinol derivative | |
| EP2861555B1 (en) | Process for depigmenting keratin materials using novel resorcinol-based compounds | |
| CN109069473B (en) | Compositions and methods for lightening skin and reducing hyperpigmentation | |
| CN114181170B (en) | 10H-phenothiazine iron death inhibitor and preparation method and application thereof | |
| KR100716891B1 (en) | Pyridine thiazole carboxamide derivatives, the preparation method thereof, and the composition for skin whitening containing the same | |
| US8617524B2 (en) | Depigmenting keratin materials utilizing dithiolane compounds | |
| JP5731653B2 (en) | 2,2'-Furoin derivative and its use for lightening the skin | |
| CN110121491B (en) | 3,4,5-trimethoxycinnamate derivative, method for preparing the same, and skin whitening composition containing the same | |
| KR20040102354A (en) | Hydroxamic acid and its derivatives as inhibitors of melanocyte tyrosinase for topical skin lighteners | |
| KR20130050751A (en) | Novel compound from ganoderma lucidum and use of the same | |
| JP4541699B2 (en) | Carboxylate-gated-nitroxide (CGN) compounds and compositions and methods of use thereof | |
| CN104326957B (en) | Aminoacetophenone thiosemicarbazone derivative and application thereof | |
| KR101689318B1 (en) | Compositions for prevention or treatment of skin-aging comprising Evodiamine | |
| US20190254945A1 (en) | Inhibition of melanogenesis by chemically modified curcumins | |
| AU2015346022B2 (en) | Methods and compositions for enhancing skin pigmentation | |
| KR100708613B1 (en) | Phenyl imidazol sulfonamide derivatives and preparation method thereof and whitening cosmetic composition containing it | |
| KR100851044B1 (en) | 3,5-dihydroxy benzamide derivatives having depigmenting activity and the whitening cosmetic composition containing the same | |
| KR100706475B1 (en) | Hydroxypyranone dimmers and preparation method thereof and whitening cosmetic composition containing it | |
| JP2012193146A (en) | Composition for anti-depilation against depilation induced by cancer chemotherapy | |
| KR100543954B1 (en) | 1N-benzyl-4-methylbenzene-1,2-diamine, a Method for Production of the Compound and a Cosmetic Composition Containing the Compound for Depigmentation | |
| US20120183482A1 (en) | Melanin production inhibitor | |
| KR100492809B1 (en) | 4-hydroxycinnamamide derivatives as antioxidants, process for preparing thereof and pharmaceutical compositions containing them |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22866397 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |