WO2022016755A1 - Trehalose derivative and carbohydrate antigen conjugate, and preparation method therefor and application thereof - Google Patents
Trehalose derivative and carbohydrate antigen conjugate, and preparation method therefor and application thereof Download PDFInfo
- Publication number
- WO2022016755A1 WO2022016755A1 PCT/CN2020/130230 CN2020130230W WO2022016755A1 WO 2022016755 A1 WO2022016755 A1 WO 2022016755A1 CN 2020130230 W CN2020130230 W CN 2020130230W WO 2022016755 A1 WO2022016755 A1 WO 2022016755A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- conjugate
- trehalose
- preparation
- nhc
- Prior art date
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 46
- 102000036639 antigens Human genes 0.000 title claims abstract description 46
- 108091007433 antigens Proteins 0.000 title claims abstract description 46
- 150000001720 carbohydrates Chemical class 0.000 title claims abstract description 37
- 150000003625 trehaloses Chemical class 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 229960005486 vaccine Drugs 0.000 claims abstract description 22
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 16
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims description 51
- 238000006243 chemical reaction Methods 0.000 claims description 37
- 235000014633 carbohydrates Nutrition 0.000 claims description 26
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- 239000001257 hydrogen Substances 0.000 claims description 12
- 229910052739 hydrogen Inorganic materials 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 10
- 239000012453 solvate Substances 0.000 claims description 10
- OISVCGZHLKNMSJ-UHFFFAOYSA-N 2,6-dimethylpyridine Chemical compound CC1=CC=CC(C)=N1 OISVCGZHLKNMSJ-UHFFFAOYSA-N 0.000 claims description 9
- 125000005313 fatty acid group Chemical group 0.000 claims description 9
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 claims description 8
- 229940126543 compound 14 Drugs 0.000 claims description 8
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 claims description 7
- WWTBZEKOSBFBEM-SPWPXUSOSA-N (2s)-2-[[2-benzyl-3-[hydroxy-[(1r)-2-phenyl-1-(phenylmethoxycarbonylamino)ethyl]phosphoryl]propanoyl]amino]-3-(1h-indol-3-yl)propanoic acid Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)O)C(=O)C(CP(O)(=O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1C=CC=CC=1)CC1=CC=CC=C1 WWTBZEKOSBFBEM-SPWPXUSOSA-N 0.000 claims description 7
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 claims description 7
- ONBQEOIKXPHGMB-VBSBHUPXSA-N 1-[2-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 ONBQEOIKXPHGMB-VBSBHUPXSA-N 0.000 claims description 7
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 claims description 7
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 7
- KGNDCEVUMONOKF-UGPLYTSKSA-N benzyl n-[(2r)-1-[(2s,4r)-2-[[(2s)-6-amino-1-(1,3-benzoxazol-2-yl)-1,1-dihydroxyhexan-2-yl]carbamoyl]-4-[(4-methylphenyl)methoxy]pyrrolidin-1-yl]-1-oxo-4-phenylbutan-2-yl]carbamate Chemical compound C1=CC(C)=CC=C1CO[C@H]1CN(C(=O)[C@@H](CCC=2C=CC=CC=2)NC(=O)OCC=2C=CC=CC=2)[C@H](C(=O)N[C@@H](CCCCN)C(O)(O)C=2OC3=CC=CC=C3N=2)C1 KGNDCEVUMONOKF-UGPLYTSKSA-N 0.000 claims description 7
- 239000003054 catalyst Substances 0.000 claims description 7
- 229940125773 compound 10 Drugs 0.000 claims description 7
- 229940125797 compound 12 Drugs 0.000 claims description 7
- 229940126142 compound 16 Drugs 0.000 claims description 7
- 229940126208 compound 22 Drugs 0.000 claims description 7
- 229940125833 compound 23 Drugs 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 claims description 7
- 229910000104 sodium hydride Inorganic materials 0.000 claims description 7
- 239000012312 sodium hydride Substances 0.000 claims description 7
- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 claims description 6
- GLGNXYJARSMNGJ-VKTIVEEGSA-N (1s,2s,3r,4r)-3-[[5-chloro-2-[(1-ethyl-6-methoxy-2-oxo-4,5-dihydro-3h-1-benzazepin-7-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide Chemical compound CCN1C(=O)CCCC2=C(OC)C(NC=3N=C(C(=CN=3)Cl)N[C@H]3[C@H]([C@@]4([H])C[C@@]3(C=C4)[H])C(N)=O)=CC=C21 GLGNXYJARSMNGJ-VKTIVEEGSA-N 0.000 claims description 6
- 229940126657 Compound 17 Drugs 0.000 claims description 6
- LNUFLCYMSVYYNW-ZPJMAFJPSA-N [(2r,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6r)-6-[(2r,3r,4s,5r,6r)-6-[(2r,3r,4s,5r,6r)-6-[[(3s,5s,8r,9s,10s,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-3-yl]oxy]-4,5-disulfo Chemical compound O([C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1[C@@H](COS(O)(=O)=O)O[C@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1C[C@@H]2CC[C@H]3[C@@H]4CC[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@H](C)CCCC(C)C)[C@H]1O[C@H](COS(O)(=O)=O)[C@@H](OS(O)(=O)=O)[C@H](OS(O)(=O)=O)[C@H]1OS(O)(=O)=O LNUFLCYMSVYYNW-ZPJMAFJPSA-N 0.000 claims description 6
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- 229940125758 compound 15 Drugs 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 claims description 6
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 claims description 5
- OPFJDXRVMFKJJO-ZHHKINOHSA-N N-{[3-(2-benzamido-4-methyl-1,3-thiazol-5-yl)-pyrazol-5-yl]carbonyl}-G-dR-G-dD-dD-dD-NH2 Chemical compound S1C(C=2NN=C(C=2)C(=O)NCC(=O)N[C@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(N)=O)=C(C)N=C1NC(=O)C1=CC=CC=C1 OPFJDXRVMFKJJO-ZHHKINOHSA-N 0.000 claims description 5
- 239000002246 antineoplastic agent Substances 0.000 claims description 5
- 229940041181 antineoplastic drug Drugs 0.000 claims description 5
- 229940126086 compound 21 Drugs 0.000 claims description 5
- 238000006264 debenzylation reaction Methods 0.000 claims description 5
- JAXFJECJQZDFJS-XHEPKHHKSA-N gtpl8555 Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)N[C@H](B1O[C@@]2(C)[C@H]3C[C@H](C3(C)C)C[C@H]2O1)CCC1=CC=C(F)C=C1 JAXFJECJQZDFJS-XHEPKHHKSA-N 0.000 claims description 5
- XXFUZSHTIOFGNV-UHFFFAOYSA-N 1-bromoprop-1-yne Chemical compound CC#CBr XXFUZSHTIOFGNV-UHFFFAOYSA-N 0.000 claims description 4
- 229940125898 compound 5 Drugs 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 4
- NNHYAHOTXLASEA-UHFFFAOYSA-N 1-(dimethoxymethyl)-4-methoxybenzene Chemical compound COC(OC)C1=CC=C(OC)C=C1 NNHYAHOTXLASEA-UHFFFAOYSA-N 0.000 claims description 3
- XWKFPIODWVPXLX-UHFFFAOYSA-N 2-methyl-5-methylpyridine Natural products CC1=CC=C(C)N=C1 XWKFPIODWVPXLX-UHFFFAOYSA-N 0.000 claims description 3
- 229910015900 BF3 Inorganic materials 0.000 claims description 3
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 claims description 3
- LNTIKAHDNWYAGL-UHFFFAOYSA-N Br.CC#C Chemical compound Br.CC#C LNTIKAHDNWYAGL-UHFFFAOYSA-N 0.000 claims description 2
- 230000009471 action Effects 0.000 claims description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 125000006239 protecting group Chemical group 0.000 claims description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 2
- PQDJYEQOELDLCP-UHFFFAOYSA-N trimethylsilane Chemical compound C[SiH](C)C PQDJYEQOELDLCP-UHFFFAOYSA-N 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 9
- 230000005847 immunogenicity Effects 0.000 abstract description 7
- 239000002671 adjuvant Substances 0.000 abstract description 6
- 230000008878 coupling Effects 0.000 abstract description 3
- 238000010168 coupling process Methods 0.000 abstract description 3
- 238000005859 coupling reaction Methods 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 230000000259 anti-tumor effect Effects 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 238000001308 synthesis method Methods 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 92
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 64
- 239000000243 solution Substances 0.000 description 33
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 24
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 17
- 230000015572 biosynthetic process Effects 0.000 description 15
- 239000012043 crude product Substances 0.000 description 15
- 239000002904 solvent Substances 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- 210000002966 serum Anatomy 0.000 description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 12
- 239000011734 sodium Substances 0.000 description 12
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 10
- 239000000706 filtrate Substances 0.000 description 10
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 9
- 239000003921 oil Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 229910021595 Copper(I) iodide Inorganic materials 0.000 description 8
- LSXDOTMGLUJQCM-UHFFFAOYSA-M copper(i) iodide Chemical compound I[Cu] LSXDOTMGLUJQCM-UHFFFAOYSA-M 0.000 description 8
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 8
- 239000012074 organic phase Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 238000004440 column chromatography Methods 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 239000000741 silica gel Substances 0.000 description 7
- 229910002027 silica gel Inorganic materials 0.000 description 7
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 239000005909 Kieselgur Substances 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- UKMSUNONTOPOIO-UHFFFAOYSA-N docosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCC(O)=O UKMSUNONTOPOIO-UHFFFAOYSA-N 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108010060123 Conjugate Vaccines Proteins 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- UQMXIEVSCRFPEI-PKTVYVHXSA-N [(2r,3s,4s,5r,6r)-3,4,5-trihydroxy-6-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(3-nonyldodecanoyloxymethyl)oxan-2-yl]oxyoxan-2-yl]methyl 3-nonyldodecanoate Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](COC(=O)CC(CCCCCCCCC)CCCCCCCCC)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](COC(=O)CC(CCCCCCCCC)CCCCCCCCC)O1 UQMXIEVSCRFPEI-PKTVYVHXSA-N 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 229940125810 compound 20 Drugs 0.000 description 3
- 229940031670 conjugate vaccine Drugs 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 230000004727 humoral immunity Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000012044 organic layer Substances 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 3
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 235000021357 Behenic acid Nutrition 0.000 description 2
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 2
- 0 CC1(*(C*(C2)[n]3nnc(C)c3)[n]3nnc(C)c3)C22C1C2 Chemical compound CC1(*(C*(C2)[n]3nnc(C)c3)[n]3nnc(C)c3)C22C1C2 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 229940116226 behenic acid Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000012230 colorless oil Substances 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000005965 immune activity Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- KJIFKLIQANRMOU-UHFFFAOYSA-N oxidanium;4-methylbenzenesulfonate Chemical compound O.CC1=CC=C(S(O)(=O)=O)C=C1 KJIFKLIQANRMOU-UHFFFAOYSA-N 0.000 description 2
- NXJCBFBQEVOTOW-UHFFFAOYSA-L palladium(2+);dihydroxide Chemical compound O[Pd]O NXJCBFBQEVOTOW-UHFFFAOYSA-L 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- AQRLNPVMDITEJU-UHFFFAOYSA-N triethylsilane Chemical compound CC[SiH](CC)CC AQRLNPVMDITEJU-UHFFFAOYSA-N 0.000 description 2
- UVNPEUJXKZFWSJ-LMTQTHQJSA-N (R)-N-[(4S)-8-[6-amino-5-[(3,3-difluoro-2-oxo-1H-pyrrolo[2,3-b]pyridin-4-yl)sulfanyl]pyrazin-2-yl]-2-oxa-8-azaspiro[4.5]decan-4-yl]-2-methylpropane-2-sulfinamide Chemical compound CC(C)(C)[S@@](=O)N[C@@H]1COCC11CCN(CC1)c1cnc(Sc2ccnc3NC(=O)C(F)(F)c23)c(N)n1 UVNPEUJXKZFWSJ-LMTQTHQJSA-N 0.000 description 1
- -1 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl Iodonium salt Chemical class 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- WDBQJSCPCGTAFG-QHCPKHFHSA-N 4,4-difluoro-N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclohexane-1-carboxamide Chemical compound FC1(CCC(CC1)C(=O)N[C@@H](CCN1CCC(CC1)N1C(=NN=C1C)C(C)C)C=1C=NC=CC=1)F WDBQJSCPCGTAFG-QHCPKHFHSA-N 0.000 description 1
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- ASVYXWZOBCRSFB-UHFFFAOYSA-N C(C)S(=O)(O)=S.OCCN1CCNCC1 Chemical compound C(C)S(=O)(O)=S.OCCN1CCNCC1 ASVYXWZOBCRSFB-UHFFFAOYSA-N 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- RXSPASZXGHGMRQ-UHFFFAOYSA-N Cc(nn1)c[n]1I Chemical compound Cc(nn1)c[n]1I RXSPASZXGHGMRQ-UHFFFAOYSA-N 0.000 description 1
- OJAWOLWHEQUTDE-UHFFFAOYSA-N Cc1c[n](C)nn1 Chemical compound Cc1c[n](C)nn1 OJAWOLWHEQUTDE-UHFFFAOYSA-N 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- NUGPIZCTELGDOS-QHCPKHFHSA-N N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclopentanecarboxamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CC[C@@H](C=1C=NC=CC=1)NC(=O)C1CCCC1)C NUGPIZCTELGDOS-QHCPKHFHSA-N 0.000 description 1
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 description 1
- CZOGCRVBCLRHQJ-WHWAGLCYSA-N N-acetyl-alpha-neuraminyl-(2->6)-N-acetyl-alpha-D-galactosamine Chemical compound O[C@@H]1[C@H](O)[C@@H](NC(=O)C)[C@@H](O)O[C@@H]1CO[C@@]1(C(O)=O)O[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(C)=O)[C@@H](O)C1 CZOGCRVBCLRHQJ-WHWAGLCYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical class [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- ULKGULQGPBMIJU-UHFFFAOYSA-N benzene;hydron;bromide Chemical compound Br.C1=CC=CC=C1 ULKGULQGPBMIJU-UHFFFAOYSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- HRYZWHHZPQKTII-UHFFFAOYSA-N chloroethane Chemical compound CCCl HRYZWHHZPQKTII-UHFFFAOYSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- STZCRXQWRGQSJD-GEEYTBSJSA-M methyl orange Chemical compound [Na+].C1=CC(N(C)C)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 STZCRXQWRGQSJD-GEEYTBSJSA-M 0.000 description 1
- 229940012189 methyl orange Drugs 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 208000015347 renal cell adenocarcinoma Diseases 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- DPKBAXPHAYBPRL-UHFFFAOYSA-M tetrabutylazanium;iodide Chemical compound [I-].CCCC[N+](CCCC)(CCCC)CCCC DPKBAXPHAYBPRL-UHFFFAOYSA-M 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- XETCRXVKJHBPMK-MJSODCSWSA-N trehalose 6,6'-dimycolate Chemical compound C([C@@H]1[C@H]([C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](COC(=O)C(CCCCCCCCCCC3C(C3)CCCCCCCCCCCCCCCCCC)C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)O2)O)O1)O)OC(=O)C(C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)CCCCCCCCCCC1CC1CCCCCCCCCCCCCCCCCC XETCRXVKJHBPMK-MJSODCSWSA-N 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- the present invention relates to the technical field of chemistry and medicine, in particular to a conjugate of a trehalose derivative and a saccharide antigen and a preparation method and application thereof.
- TACAs Tumor-associated carbohydrate antigens
- TACAs tumor-associated carbohydrate antigens
- TACAs tumor-associated carbohydrate antigens
- the classic strategy is to conjugate carbohydrate antigens with carrier proteins to enhance their immunogenicity, but glycoprotein vaccines have disadvantages such as uncertain coupling sites, unstable coupling rates, and complex components.
- the total synthesis strategy of tumor-related carbohydrate antigen vaccines has become one of the new research hotspots; the fully synthetic carbohydrate antigen vaccines have the advantages of clear structure, stable and controllable quality, etc., which are convenient for various immunological and clinical studies.
- sugar molecules on the surface of various pathogenic bacteria can be used as embedded adjuvants for fully synthesized sugar antigen tumor vaccines, which can not only overcome the shortcomings of the weak immunogenicity of sugar antigens, but also avoid the "epitope" caused by proteins. suppress” effect.
- Trehalose-6,6-dimycolate (TDM, cord factor) is an important glycolipid component in the cell wall of Mycobacterium tuberculosis, consisting of trehalose and two mycolate chains; this glycolipid can cause inflammatory responses , and has certain cytotoxicity to tumor cells. Studies have shown that the derivatives of trehalose-6,6-dimycolate, Vizantin and TDE, have high immune activity and low toxicity, and can be used as effective adjuvants for vaccine research.
- the object of the present invention is to provide a conjugate of a trehalose derivative and a carbohydrate antigen.
- the present invention uses potent immunostimulators trehalose derivatives Vizantin and TDE as built-in adjuvants to conjugate sugar antigens (STn, Tn) respectively to obtain the conjugates, wherein trehalose derivatives can improve the immunogenicity of sugar antigens , the conjugate can induce T cell-mediated humoral immunity, produce high-concentration high-affinity IgG antibody, and achieve the purpose of specifically killing tumor cells.
- Another object of the present invention is to provide a method for preparing the conjugate of the trehalose derivative and carbohydrate antigen.
- Another object of the present invention is to provide the application of the conjugate of the trehalose derivative and the saccharide antigen in the preparation of tumor vaccine.
- Another object of the present invention is to provide the application of the conjugate of the trehalose derivative and the carbohydrate antigen in the preparation of antitumor drugs.
- X represents a carbohydrate antigen, selected from any one of the carbohydrate antigens in the following formula, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
- L represents a linker, selected from the following (CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-, (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-, -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 -(OCH 2 CH 2 ) a -CH 2 CH 2 -C(O)NH-, (CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) a -C(O)NH-, -NHC(O)-(CH 2 ) a Any of , a is any integer from 0 to 20;
- Y represents a trehalose derivative, selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
- R 1 and R 2 are each independently selected from hydrogen, -CH 2 -CH(OR 3 )-(CH 2 ) m -CH 3 , -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , R 3 is -(CH 2 ) m -CH 3 or -C(O)-(CH 2 ) m -CH 3 , m is an integer selected from 8-26.
- the L is selected from (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 (CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) a Any of , where a is any integer from 0 to 20.
- the L is selected from (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 ) a Any of , where a is any integer from 0 to 20.
- the Y is selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
- R 1 and R 2 are each independently selected from hydrogen, -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , and R 3 is -(CH 2 ) m -CH 3 or -C(O )-(CH 2 ) m -CH 3 , m is an integer selected from 8-26.
- the Y is selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
- R 1 and R 2 are each independently selected from hydrogen or -(CH 2 ) m CH 3 , and m is an integer selected from 8-26.
- the conjugate is selected from any of the following structures or a pharmaceutically acceptable salt, hydrate or solvate thereof:
- the preparation method of the conjugate of trehalose derivative and saccharide antigen when Y is , the preparation process includes:
- compound 10 reacts with compound Tn or STn, compound 11 and compound STn respectively under the action of a catalyst to obtain compound 19 or 20, compound 21;
- the preparation process includes;
- Compound 22 or 23 are respectively subjected to debenzylation protection reaction to obtain the target product;
- the catalyst of the debenzylation protection reaction is hydrogen/palladium carbon/palladium hydroxide, hydrogen/palladium carbon or hydrogen/palladium hydroxide, etc., preferably hydrogen/palladium carbon;
- the solvent used in the reaction is Dichloromethane/methanol/water, dichloromethane, methanol or dichloromethane/methanol, etc.; preferably dichloromethane/methanol/water.
- the catalyst for the reaction is cuprous iodide/N,N-diisopropylethylamine or cuprous iodide/N,N-diisopropylethylamine/glacial acetic acid, etc.; Cuprous iodide/N,N-diisopropylethylamine is preferred; the reaction solvent is dichloromethane, dichloromethane/methanol or tetrahydrofuran/methanol, etc.; tetrahydrofuran/methanol is preferred.
- the catalyst for the reaction is an aqueous solution of boron trifluoride ethyl ether or trifluoroacetic acid, etc., preferably boron trifluoride ethyl ether;
- the solvent for the reaction is acetonitrile, dichloromethane or tetrahydrofuran, etc., preferably acetonitrile.
- the condensation reagent in the reaction process can be selected from 4-dimethylaminopyridine (DMAP)/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl Iodonium salt (EDC MEI), N,N'-dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP) or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC. HCl) / 4- dimethylaminopyridine (DMAP) was the like, preferably DMAP / EDC. HCl.
- DMAP 4-dimethylaminopyridine
- EDC MEI 4-dimethylaminopyridine
- EDC MEI 4-dimethylaminopyridine
- DCC N,N'-dicyclohexylcarbodiimide
- DMAP 1-(3-dimethylaminopropyl)-3-e
- the preparation process of compound 7 is as follows:
- the catalyst for the reaction is triethylsilane/trifluoromethanesulfonic acid or sodium cyanoborohydride/diethyl ether hydrochloride; the reaction is performed in a solvent such as dichloromethane, methanol or tetrahydrofuran.
- the catalyst for the reaction is sodium cyanoborohydride/diethyl ether hydrochloride; the reaction is carried out in a solvent dichloromethane.
- the above-mentioned preparation route of the present invention is short, the reaction conditions are mild, the yield is high, and the operation is convenient, and can be used for industrial preparation.
- the tumor vaccine or anti-tumor drug is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell cancer, cellular lymphoma, thyroid cancer , brain cancer, stomach cancer or leukemia vaccines or drugs.
- the present invention has the following beneficial effects:
- the present invention uses potent immunostimulators trehalose derivatives Vizantin and TDE as built-in adjuvants to conjugate sugar antigens (STn, Tn) respectively to obtain the conjugates, wherein trehalose derivatives can improve the immunogenicity of sugar antigens , so that the conjugate can induce T cell-mediated humoral immunity, generate high concentration and high affinity IgG antibody, achieve the purpose of specifically killing tumor cells, and can be prepared as a tumor vaccine or an anti-tumor drug for application.
- FIG. 1 is a graph showing the evaluation of the immune activity of antibodies produced in mice induced by vaccines prepared with conjugates 1, 2, 3 and 4.
- FIG. 1 is a graph showing the evaluation of the immune activity of antibodies produced in mice induced by vaccines prepared with conjugates 1, 2, 3 and 4.
- Figure 2 is a graph showing the evaluation of complement-dependent cytotoxicity of antibody serum produced by vaccine-induced mice produced by conjugates 1, 2, 3 and 4 to specifically kill tumor cell MCF-7.
- test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
- reaction was carried out at room temperature for 18 h, the reaction solution was cooled to 0 °C, a small amount of methanol solution was dropped to quench the reaction, diluted with ethyl acetate, the mixture was washed with saturated brine, the organic layer was collected, concentrated to obtain the crude product, and purified by silica gel column chromatography to obtain compound 6 as a white solid (14.90 g, 78.4%).
- Tetrabutylammonium bromide (6eq); reacted at room temperature for 4h, the reaction solution was cooled to 0°C, slowly added dropwise with a small amount of methanol to quench the reaction, diluted with dichloromethane, rinsed with saturated sodium bicarbonate solution and saturated brine successively, and collected organic The phase was concentrated under reduced pressure, and the purified compound 13 (380.01 mg, 97%) was separated and purified by silica gel column.
- HEPES hydroxyethylpiperazine ethanethiosulfonic acid
- mice C57BL/6 mice aged 6-8 weeks were taken and divided into four groups with 6 mice in each group.
- the immunization test was carried out by subcutaneous injection of mice, and the liposomes 1, 2, and 3 prepared in step (1) were injected on the 1st, 14th, 21st, and 28th days respectively with the scheme of one initial immunization and three boosting immunizations. 4.
- the injection volume of each mouse is 0.1 mL. Blood is collected on the 0, 27, 35, and 49 days, and 0.1 mL to 0.2 mL of blood is collected from each mouse, placed at 4°C for half an hour, centrifuged at 5000 rpm, and separated. Top layer clearing serum.
- the blank control group pre-immune serum
- the vaccine titer was measured using the blood collected on the 35th day.
- ELISA immunoassay Use 0.1M carbonate buffer (pH 9.6) to prepare Tn-HSA or STn-HSA into a 2.0 ⁇ g/mL solution, add 100 ⁇ L per well to a 96-well plate, and put 4 Incubate overnight at °C; incubate at 37 °C for one hour the next day; wash the plate three times with PBST (PBS+0.05% Tween-20), and add 300 ⁇ L of washing solution to each well. After washing the plate, add blocking buffer (PBST/1% BSA); add 250 ⁇ L to each well; incubate at room temperature for one hour, and wash the plate three times with PBST.
- PBST blocking buffer
- the sample serum was diluted with PBS from 1:300 to 1:656100 times according to the half-dilution method; the diluted serum was added to 96-well plate at 100 ⁇ L per well, placed in a 37°C incubator for two hours, and washed. plate three times.
- 100 ⁇ L of HRP (horseradish peroxidase)-labeled Kappa, IgG, and IgM were added to each well, and incubated at room temperature for one hour; the plate was washed 3 times.
- Add TMB (3,3',5,5'-tetramethylbenzidine) solution add 100 ⁇ L to each well, and develop color at room temperature for 20 min in the dark.
- 0.5M H 2 SO 4 solution was added to each well 100 ⁇ L. Absorbance was detected with a microplate reader at dual wavelengths of 450-570 nm.
- the conjugates 1, 2, 3 and 4 of trehalose derivatives and carbohydrate antigens synthesized in Example 1 of the present invention can all induce high titers without adjuvant.
- the specific IgG antibody titer produced is more than 2 to 3 times that of IgM, indicating that the covalently conjugated carbohydrate antigen vaccine can induce T cell-mediated humoral immunity; among them, the conjugate vaccine 1 double-linked STn antigen and
- the titers of IgG antibodies elicited by 2 were slightly higher than those of conjugates 3 and 4, which were single-attached to the antigen, indicating that the more complex the space of the antigen, the more epitopes, had a certain promoting effect on the immune response;
- the fatty acid chain of trehalose derivatives was Conjugate vaccines 1 and 3 with branched CH(C 9 H 19 ) 2 induced slightly higher antibodies than conjugate vaccines 2 and 4 with fatty acid chain (CH 2 ) 20 CH 2
- CT-26 cells (mouse colon cancer cells) in logarithmic growth phase were trypsinized, seeded in 96-well plates at 1 ⁇ 10 4 cells per well, cultured overnight at 37°C, and cultured with serum-free 1640 The substrate was washed twice. Take the serum samples of 6 mice in the compound 1 group on 38 days, take 7 ⁇ l of each sample, then mix the serum samples of the 6 mice, and dilute 50 times with serum-free 1640 medium to obtain mouse serum Diluent. The mouse serum dilutions of compound group 2, group 3, group 4 and blank control group were prepared according to the above method.
- Group 1, Group 2, Group 3, Group 4 and blank control group were set as the maximum enzyme activity control group, sample control group and sample treatment group, each group was made 6 replicate wells in parallel, each well 100 ⁇ l of diluted mouse serum solution was added, and the 96-well plate was incubated at 37° C. for 2 h. After washing the plate twice with serum-free 1640 medium, 100 ⁇ l of rabbit complement serum solution was added to each well of the sample treatment group, 100 ⁇ l of LDH release solution was added to each well of the maximum enzyme activity control group, and 100 ⁇ l of LDH release solution was added to each well of the sample control group. Serum in MEM medium and cultured at 37°C for 1 h.
- Cell lysis rate (%) (absorbance of sample treatment group-absorbance of sample control group)/(absorbance of maximum enzyme activity control group-absorbance of sample control group) ⁇ 100%.
Abstract
The present invention relates to the technical field of chemistry and medicines. Disclosed are a trehalose derivative and carbohydrate antigen conjugate, and a preparation method therefor and an application thereof. According to the present invention, the conjugate is obtained by taking trehalose as an embedded adjuvant and coupling the trehalose with a tumor-associated carbohydrate antigen (STn or Tn) abnormally expressed on the surface of a tumor cell. The conjugate has the advantages of being clear in structure, simple and convenient in synthesis method, stable and controllable in product quality, and the like, and particularly, a vaccine can overcome the defect of weak immunogenicity of the carbohydrate antigen and can induce generation of a high-affinity specific IgG antibody, so that the anti-tumor effect of killing tumor cells in a targeted manner is achieved.
Description
本发明涉及化学与医药技术领域，尤其涉及一种海藻糖衍生物与糖抗原的缀合物及其制备方法与应用。The present invention relates to the technical field of chemistry and medicine, in particular to a conjugate of a trehalose derivative and a saccharide antigen and a preparation method and application thereof.
如今癌症已成为人类的第一杀手并成为全球最大的公共卫生问题，恶性肿瘤死亡率呈明显增长趋势，严重的威胁人类的生活。现治疗癌症的方法有手术、化疗、放疗、免疫疗法，其中肿瘤免疫疗法是研究肿瘤治疗的热门之一。在肿瘤细胞表明异常过量表达的肿瘤相关糖抗原(TACAs)，具有重要的生物学功能，是糖抗原肿瘤疫苗设计的优秀靶点。Nowadays, cancer has become the number one killer of human beings and the biggest public health problem in the world. The mortality rate of malignant tumors is increasing obviously, which is a serious threat to human life. The current methods of cancer treatment include surgery, chemotherapy, radiotherapy, and immunotherapy, among which tumor immunotherapy is one of the hottest topics in the study of tumor treatment. Tumor-associated carbohydrate antigens (TACAs), which are abnormally overexpressed in tumor cells, have important biological functions and are excellent targets for the design of carbohydrate antigen tumor vaccines.
但是肿瘤相关糖抗原(TACAs)是T细胞非依赖性抗原，免疫原性较弱，需要在免疫原性载体分子的协助下才能刺激T细胞并诱导持久的抗体反应。如何提高糖抗原的免疫原性，使其更高效地诱导产生高滴度、高亲和力的IgG抗体并特异性地杀死肿瘤细胞，是肿瘤疫苗的研究热点之一。经典的策略是将糖抗原与载体蛋白缀合来增强其免疫原性，但糖蛋白疫苗有偶联位点不确定、偶联率不稳定、组成成分复杂等缺点。因此，肿瘤相关糖抗原疫苗的全合成策略成了新的研究热点之一；全合成糖抗原疫苗具有明确的结构，质量稳定可控等优点，便于各种免疫学和临床研究。近年来，科研工作者发现多种病原菌表面的糖分子可作为全合成糖抗原肿瘤疫苗的内嵌佐剂，其既能克服糖抗原免疫原性弱的缺点，又可以避免蛋白质引起的“表位抑制”效应。However, tumor-associated carbohydrate antigens (TACAs) are T cell-independent antigens with weak immunogenicity and require the assistance of immunogenic carrier molecules to stimulate T cells and induce durable antibody responses. How to improve the immunogenicity of carbohydrate antigens, so as to induce the production of high-titer, high-affinity IgG antibodies more efficiently and kill tumor cells specifically, is one of the research hotspots of tumor vaccines. The classic strategy is to conjugate carbohydrate antigens with carrier proteins to enhance their immunogenicity, but glycoprotein vaccines have disadvantages such as uncertain coupling sites, unstable coupling rates, and complex components. Therefore, the total synthesis strategy of tumor-related carbohydrate antigen vaccines has become one of the new research hotspots; the fully synthetic carbohydrate antigen vaccines have the advantages of clear structure, stable and controllable quality, etc., which are convenient for various immunological and clinical studies. In recent years, researchers have found that sugar molecules on the surface of various pathogenic bacteria can be used as embedded adjuvants for fully synthesized sugar antigen tumor vaccines, which can not only overcome the shortcomings of the weak immunogenicity of sugar antigens, but also avoid the "epitope" caused by proteins. suppress" effect.
海藻糖-6,6-二霉菌酸酯(TDM，索状因子)是结核分枝杆菌细胞壁中重要的糖脂成分，由海藻糖及2条霉菌酸酯链构成；该糖脂能引起炎症反应，并且对肿瘤细胞有一定的细胞毒性。研究表明，海藻糖-6,6-二霉菌酸酯的衍生物Vizantin和TDE免疫活性高且毒性低，可作为有效佐剂用于疫苗研究。Trehalose-6,6-dimycolate (TDM, cord factor) is an important glycolipid component in the cell wall of Mycobacterium tuberculosis, consisting of trehalose and two mycolate chains; this glycolipid can cause inflammatory responses , and has certain cytotoxicity to tumor cells. Studies have shown that the derivatives of trehalose-6,6-dimycolate, Vizantin and TDE, have high immune activity and low toxicity, and can be used as effective adjuvants for vaccine research.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种海藻糖衍生物与糖抗原的缀合物。本发明以强效免疫刺激剂海藻糖衍生物Vizantin和TDE作为内嵌佐剂分别缀合糖抗原(STn、Tn)得到所述缀合物，其中海藻糖衍生物能提高糖抗原的免疫原性，所述缀合物能够诱导T细 胞介导体液免疫，产生高浓度的高亲和力的IgG抗体，达到特异性杀死肿瘤细胞的目的。The object of the present invention is to provide a conjugate of a trehalose derivative and a carbohydrate antigen. The present invention uses potent immunostimulators trehalose derivatives Vizantin and TDE as built-in adjuvants to conjugate sugar antigens (STn, Tn) respectively to obtain the conjugates, wherein trehalose derivatives can improve the immunogenicity of sugar antigens , the conjugate can induce T cell-mediated humoral immunity, produce high-concentration high-affinity IgG antibody, and achieve the purpose of specifically killing tumor cells.
本发明的另一目的在于提供所述海藻糖衍生物与糖抗原的缀合物的制备方法。Another object of the present invention is to provide a method for preparing the conjugate of the trehalose derivative and carbohydrate antigen.
本发明的再一目的在于提供所述海藻糖衍生物与糖抗原的缀合物在制备肿瘤疫苗中的应用。Another object of the present invention is to provide the application of the conjugate of the trehalose derivative and the saccharide antigen in the preparation of tumor vaccine.
本发明的还一目的在于提供所述海藻糖衍生物与糖抗原的缀合物在制备抗肿瘤药物中的应用。Another object of the present invention is to provide the application of the conjugate of the trehalose derivative and the carbohydrate antigen in the preparation of antitumor drugs.
本发明的上述目的是通过以下方案予以实现的：Above-mentioned purpose of the present invention is achieved through the following scheme:
一种海藻糖衍生物与糖抗原的缀合物，所述缀合物结构通式如下式I所示：A conjugate of a trehalose derivative and a carbohydrate antigen, the general structural formula of the conjugate is shown in the following formula I:
[X—L—Y][X—L—Y]
式(I)Formula (I)
其中，X表示糖抗原，选自下式中的任意一种糖抗原，或其可要药用盐、水合物或溶剂化物：Wherein, X represents a carbohydrate antigen, selected from any one of the carbohydrate antigens in the following formula, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
L表示连接体，选自以下
(CH
2CH
2O)
a-CH
2CH
2-C(O)NH-、
(CH
2CH
2O)
a-、-(OCH
2CH
2)
a
-NHC(O)-(CH
2CH
2O)
a-CH
2CH
2-C(O)NH-、-NHC(O)-(CH
2CH
2O)
a-CH
2CH
2
-(OCH
2CH
2)
a-CH
2CH
2-C(O)NH-、
(CH2)
a-C(O)NH-、-NHC(O)-(CH
2)
a-C(O)NH-、-NHC(O)-(CH
2)
a
中的任意一种，a是0到20中的任意一个整数；
L represents a linker, selected from the following (CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-, (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-, -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 -(OCH 2 CH 2 ) a -CH 2 CH 2 -C(O)NH-, (CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) a -C(O)NH-, -NHC(O)-(CH 2 ) a Any of , a is any integer from 0 to 20;
Y表示海藻糖衍生物，选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：Y represents a trehalose derivative, selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
其中，R
1和R
2各自独立地选自氢、-CH
2-CH(OR
3)-(CH
2)
m-CH
3，-(CH
2)
mCH
3或-(CH
2)
mCHR
3
2，R
3为-(CH
2)
m-CH
3或-C(O)-(CH
2)
m-CH
3，m是选自8-26的整数。
wherein R 1 and R 2 are each independently selected from hydrogen, -CH 2 -CH(OR 3 )-(CH 2 ) m -CH 3 , -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , R 3 is -(CH 2 ) m -CH 3 or -C(O)-(CH 2 ) m -CH 3 , m is an integer selected from 8-26.
优选地，所述L选自
(CH
2CH
2O)
a-、-(OCH
2CH
2)
a
-NHC(O)-(CH
2CH
2O)
a-CH
2CH
2
(CH2)
a-C(O)NH-、-NHC(O)-(CH
2)
a
中的任意一种，a是0到20中的任意一个整数。
Preferably, the L is selected from (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 (CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) a Any of , where a is any integer from 0 to 20.
优选地，所述L选自
(CH
2CH
2O)
a-、-(OCH
2CH
2)
a
-NHC(O)-(CH
2)
a
中的任意一种，a是0到20中的任意一个整数。
Preferably, the L is selected from (CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a -NHC(O)-(CH 2 ) a Any of , where a is any integer from 0 to 20.
优选地，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：Preferably, the Y is selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
其中，R
1和R
2各自独立地选自氢、-(CH
2)
mCH
3或-(CH
2)
mCHR
3
2，R
3为-(CH
2)
m-CH
3或-C(O)-(CH
2)
m-CH
3，m是选自8-26的整数。
wherein, R 1 and R 2 are each independently selected from hydrogen, -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , and R 3 is -(CH 2 ) m -CH 3 or -C(O )-(CH 2 ) m -CH 3 , m is an integer selected from 8-26.
优选地，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：Preferably, the Y is selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
其中，R
1和R
2各自独立地选自氢或-(CH
2)
mCH
3，m是选自8-26的整数。
wherein, R 1 and R 2 are each independently selected from hydrogen or -(CH 2 ) m CH 3 , and m is an integer selected from 8-26.
优选地，所述缀合物选自如下任一结构或其可要药用盐、水合物或溶剂化物：Preferably, the conjugate is selected from any of the following structures or a pharmaceutically acceptable salt, hydrate or solvate thereof:
本发明同时所述海藻糖衍生物与糖抗原的缀合物的制备方法，当Y为
时，制备过程包括：
At the same time of the present invention, the preparation method of the conjugate of trehalose derivative and saccharide antigen, when Y is , the preparation process includes:
S1.化合物7在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物8；S1. compound 7 reacts with propyne bromide under the catalysis of sodium hydride and tetrabutylammonium bromide to obtain compound 8;
S2.化合物8在三氟乙酸的条件下，脱去PMB基团得到化合物9；S2. Compound 8, under the condition of trifluoroacetic acid, removes the PMB group to obtain compound 9;
S3.化合物9与相应的脂肪酸链进行酯化缩合得到对应的化合物10或11；S3. Compound 9 is esterified and condensed with the corresponding fatty acid chain to obtain the corresponding compound 10 or 11;
S4.化合物10与化合物Tn或STn、化合物11与化合物STn分别在催化剂的作用 下反应得到化合物19或20、化合物21；S4. compound 10 reacts with compound Tn or STn, compound 11 and compound STn respectively under the action of a catalyst to obtain compound 19 or 20, compound 21;
S5.化合物19、20和21分别经脱苄基保护反应，即可得到目标产物；S5. Compounds 19, 20 and 21 are respectively subjected to debenzylation protection reaction to obtain the target product;
当Y为
制备过程包括；
when Y is The preparation process includes;
S11.化合物7在2,6-二甲基吡啶的催化下与叔丁基二甲基三氟甲磺酸酯反应得到化合物12；S11. Compound 7 is reacted with tert-butyldimethyl triflate under the catalysis of 2,6-lutidine to obtain compound 12;
S21.化合物12在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物化合物13；S21. Compound 12 is reacted with bromopropyne under the catalysis of sodium hydride and tetrabutylammonium bromide to obtain compound compound 13;
S31.化合物13在三氟乙酸的条件下，脱去PMB基团得到化合物14；S31. Compound 13, under the condition of trifluoroacetic acid, removes the PMB group to obtain compound 14;
S41.化合物14分别与相应的脂肪酸链进行酯化缩合得到对应的化合物15或16；S41. Compound 14 is esterified and condensed with the corresponding fatty acid chain to obtain the corresponding compound 15 or 16;
S51.化合物15或16分别脱去保护基团三甲基硅烷得到相应的化合物17或18；S51. Compound 15 or 16 respectively removes the protecting group trimethylsilane to obtain the corresponding compound 17 or 18;
S61.化合物17或18与三氟化硼乙醚反应得到相应的化合物22或23；S61. Compound 17 or 18 is reacted with boron trifluoride ether to obtain corresponding compound 22 or 23;
S71.化合物22或23分别经脱苄基保护反应，即可得到目标产物；S71. Compound 22 or 23 are respectively subjected to debenzylation protection reaction to obtain the target product;
其中，化合物7至23的结构如下所示：Among them, the structures of compounds 7 to 23 are shown below:
优选地，步骤S5和步骤S71中，脱苄基保护反应的催化剂为氢气/钯碳/氢氧化钯、氢气/钯碳或氢气/氢氧化钯等，优选氢气/钯碳；反应采用的溶剂为二氯甲烷/甲醇/水、二氯甲烷、甲醇或二氯甲烷/甲醇等；优选二氯甲烷/甲醇/水。Preferably, in step S5 and step S71, the catalyst of the debenzylation protection reaction is hydrogen/palladium carbon/palladium hydroxide, hydrogen/palladium carbon or hydrogen/palladium hydroxide, etc., preferably hydrogen/palladium carbon; the solvent used in the reaction is Dichloromethane/methanol/water, dichloromethane, methanol or dichloromethane/methanol, etc.; preferably dichloromethane/methanol/water.
优选地，步骤S4和步骤S61中，反应的催化剂为碘化亚铜/N,N-二异丙基乙胺或碘化亚铜/N,N-二异丙基乙胺/冰乙酸等；优选碘化亚铜/N,N-二异丙基乙胺；反应的溶剂为二氯甲烷、二氯甲烷/甲醇或四氢呋喃/甲醇等；优选四氢呋喃/甲醇。Preferably, in step S4 and step S61, the catalyst for the reaction is cuprous iodide/N,N-diisopropylethylamine or cuprous iodide/N,N-diisopropylethylamine/glacial acetic acid, etc.; Cuprous iodide/N,N-diisopropylethylamine is preferred; the reaction solvent is dichloromethane, dichloromethane/methanol or tetrahydrofuran/methanol, etc.; tetrahydrofuran/methanol is preferred.
优选地，步骤S51中，反应的催化剂为三氟化硼乙醚或三氟乙酸的水溶液等，优选三氟化硼乙醚；反应的溶剂为乙腈、二氯甲烷或四氢呋喃等，优选为乙腈。Preferably, in step S51, the catalyst for the reaction is an aqueous solution of boron trifluoride ethyl ether or trifluoroacetic acid, etc., preferably boron trifluoride ethyl ether; the solvent for the reaction is acetonitrile, dichloromethane or tetrahydrofuran, etc., preferably acetonitrile.
优选地，步骤S3和步骤S41中，反应过程中的缩合试剂可以选择4-二甲氨基吡啶(DMAP)/1-(3-二甲基氨丙基)-3-乙基碳二亚胺甲碘盐(EDC·MEI)，N,N’-二环己基碳二亚胺(DCC)/4-二甲氨基吡啶(DMAP)或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC
.HCl)/4-二甲氨基吡啶(DMAP)等，优选DMAP/EDC
.HCl。
Preferably, in step S3 and step S41, the condensation reagent in the reaction process can be selected from 4-dimethylaminopyridine (DMAP)/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl Iodonium salt (EDC MEI), N,N'-dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine (DMAP) or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC. HCl) / 4- dimethylaminopyridine (DMAP) was the like, preferably DMAP / EDC. HCl.
优选地，化合物7的制备过程如下：Preferably, the preparation process of compound 7 is as follows:
S31.以海藻糖为起始原料，在对甲苯磺酸的作用下，4-甲氧基苯甲醛二甲缩醛对原料进行4,6位羟基选择性保护，得到化合物5，用溴化卞对裸露的羟基进行全苄基化得化合物6；S31. take trehalose as starting raw material, under the effect of p-toluenesulfonic acid, 4-methoxybenzaldehyde dimethylacetal carries out 4,6-position hydroxyl selective protection to raw material, obtains compound 5, uses brominated Bian The exposed hydroxyl group is fully benzylated to give compound 6;
S32.化合物6选择性还原4,4’位糖羟基得到化合物7；S32. Compound 6 selectively reduces 4,4' sugar hydroxyl groups to obtain compound 7;
其中化合物5和6的结构如下：The structures of compounds 5 and 6 are as follows:
优选地，步骤S32中，反应的催化剂为三乙基硅烷/三氟甲磺酸或氰基硼氢化钠/盐酸乙醚；反应在溶剂为二氯甲烷、甲醇或四氢呋喃中进行。Preferably, in step S32, the catalyst for the reaction is triethylsilane/trifluoromethanesulfonic acid or sodium cyanoborohydride/diethyl ether hydrochloride; the reaction is performed in a solvent such as dichloromethane, methanol or tetrahydrofuran.
更优选地，步骤S32中，反应的催化剂为氰基硼氢化钠/盐酸乙醚；反应在溶剂二氯甲烷中进行。More preferably, in step S32, the catalyst for the reaction is sodium cyanoborohydride/diethyl ether hydrochloride; the reaction is carried out in a solvent dichloromethane.
本发明上述制备路线简短、反应条件温和、产率高、操作方便，能够用于工业化制备。The above-mentioned preparation route of the present invention is short, the reaction conditions are mild, the yield is high, and the operation is convenient, and can be used for industrial preparation.
所述海藻糖衍生物与糖抗原的缀合物在制备肿瘤疫苗中的应用也在本发明的保护范围之内。The application of the conjugate of the trehalose derivative and the saccharide antigen in the preparation of tumor vaccines also falls within the protection scope of the present invention.
所述海藻糖衍生物与糖抗原的缀合物在制备抗肿瘤药物中的应用也在本发明的保护范围之内。The application of the conjugate of the trehalose derivative and the saccharide antigen in the preparation of antitumor drugs also falls within the protection scope of the present invention.
优选地，所述肿瘤疫苗或抗肿瘤药物为乳腺癌、子宫癌、卵巢癌、肺癌、肝癌、前列腺癌、黑素瘤、胰腺癌、肠癌、肾细胞癌、细胞性淋巴癌、甲腺癌、脑癌、胃癌或白血病的疫苗或药物。Preferably, the tumor vaccine or anti-tumor drug is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell cancer, cellular lymphoma, thyroid cancer , brain cancer, stomach cancer or leukemia vaccines or drugs.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明以强效免疫刺激剂海藻糖衍生物Vizantin和TDE作为内嵌佐剂分别缀合糖抗原(STn、Tn)得到所述缀合物，其中海藻糖衍生物能提高糖抗原的免疫原性，使得所述缀合物能够诱导T细胞介导体液免疫，产生高浓度的高亲和力的IgG抗体，达到特异性杀死肿瘤细胞的目的，可制备成为肿瘤疫苗或抗肿瘤药物进行应用。The present invention uses potent immunostimulators trehalose derivatives Vizantin and TDE as built-in adjuvants to conjugate sugar antigens (STn, Tn) respectively to obtain the conjugates, wherein trehalose derivatives can improve the immunogenicity of sugar antigens , so that the conjugate can induce T cell-mediated humoral immunity, generate high concentration and high affinity IgG antibody, achieve the purpose of specifically killing tumor cells, and can be prepared as a tumor vaccine or an anti-tumor drug for application.
图1为缀合物1，2，3与4制备的疫苗诱导小鼠产生的抗体免疫活性评价图。FIG. 1 is a graph showing the evaluation of the immune activity of antibodies produced in mice induced by vaccines prepared with conjugates 1, 2, 3 and 4. FIG.
图2为缀合物1，2，3与4制备的疫苗诱导小鼠产生的抗体血清特异性杀死肿瘤细胞MCF-7的补体依赖性细胞毒性评价图。Figure 2 is a graph showing the evaluation of complement-dependent cytotoxicity of antibody serum produced by vaccine-induced mice produced by conjugates 1, 2, 3 and 4 to specifically kill tumor cell MCF-7.
下面结合具体实施例对本发明做出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。The present invention will be further elaborated below with reference to specific embodiments, which are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
实施例1海藻糖衍生物与糖抗原缀合物1、2、3、4与L2-3的制备Example 1 Preparation of trehalose derivatives and carbohydrate antigen conjugates 1, 2, 3, 4 and L2-3
1)化合物5的合成1) Synthesis of compound 5
将海藻糖(10.00g,0.03mol)和对甲苯磺酸一水化合物(1.10g,5.84mol)溶于二甲基甲酰胺中，在氮气保护下滴加茴香醛二甲缩醛(20mL,0.117mol)，室温搅拌反应，反应24小时后，乙酸乙酯稀释，饱和碳酸氢钠溶液，静置，有白色固体析出，抽滤，得化合物5(11.90g,70％)。
1H NMR(400MHz，CD
3OD)δ7.41-7.39(d,J＝8.8Hz,4H),6.88-6.68(d,J＝8.8Hz,4H),5.51(s,2H,PhCH),5.11(d,J＝3.6Hz,2H,H-1&H-1′),4.19(dt,J＝10.0,4.8Hz,2H,H5&H5′),4.12-4.06(m,2H,H3&H3′),4.03-3.98(t,J＝9.6,2H,H4&H4′),3.77(s，6H),3.72-3.67(t,J＝10.4Hz,2H),3.61(dd,J
1＝9.2Hz,J
2＝4.0Hz,2H),3.45(t,J＝9.6,2H),3.31-3.29(m,2H)；HR-ESI-MS(m/z):calcd for C
28H
34O
13[M+H]
+579.2072 found,579.2067.
Trehalose (10.00g, 0.03mol) and p-toluenesulfonic acid monohydrate (1.10g, 5.84mol) were dissolved in dimethylformamide, and anisaldehyde dimethylacetal (20mL, 0.117 g) was added dropwise under nitrogen protection mol), the reaction was stirred at room temperature, after 24 hours of reaction, diluted with ethyl acetate, saturated sodium bicarbonate solution, allowed to stand, a white solid was precipitated, and suction filtered to obtain compound 5 (11.90 g, 70%). 1 H NMR (400 MHz, CD 3 OD) δ 7.41-7.39 (d, J=8.8 Hz, 4H), 6.88-6.68 (d, J=8.8 Hz, 4H), 5.51 (s, 2H, PhCH), 5.11 (d,J=3.6Hz,2H,H-1&H-1′),4.19(dt,J=10.0,4.8Hz,2H,H5&H5′),4.12-4.06(m,2H,H3&H3′),4.03-3.98 (t, J = 9.6,2H, H4 & H4 '), 3.77 (s, 6H), 3.72-3.67 (t, J = 10.4Hz, 2H), 3.61 (dd, J 1 = 9.2Hz, J 2 = 4.0Hz, 2H), 3.45 (t, J=9.6, 2H), 3.31-3.29 (m, 2H); HR-ESI-MS (m/z): calcd for C 28 H 34 O 13 [M+H] + 579.2072 found , 579.2067.
2)化合物6的制备2) Preparation of compound 6
化合物5(2.98g，5.75mmol)溶于N-N-二甲基甲酰胺中，在氮气的保护下，加入 氢化钠(60％in oil；2.30g，57.5mmol)，反应液降温到0℃，依次加入溴化卞(5.5mL，45.98mmol)、四丁基碘化铵(425.01mg，1.15mmol)。室温反应18h，反应液降温至0℃，滴少量甲醇溶液进行反应的淬灭，乙酸乙酯稀释，饱和食盐水冲洗混合液，收集有机层，浓缩得粗品，硅胶柱色谱纯化得白色固体化合物6(14.90g,78.4％)。
1H NMR(400MHz，CDCl
3)δ7.34-7.24(m,24H),6.92-6.90(d,J＝8.8Hz，4H)5.51(s,2H,PhCH)，5.11(d,J＝3.8Hz，2H,H-1&H-1′),4.96(d,J＝10.8Hz,2H,PhCH
2O),4.85-4.81(dd,J＝10.8Hz,4H),4.72(d,J＝10.8Hz,2H,PhCH
2O),4.27(dt,J＝10.0,4.8Hz,2H,H5&H5′),4.14(t,J＝9.4Hz,2H,H3&H3′),4.12-4.10(m,2H,H4&H4′),3.83(S,6H),3.69-3.59(m,6H)；HR-ESI-MS(m/z):calcd for C
54H
58O
11[M+H]
+939.395 found，939.3933.
Compound 5 (2.98g, 5.75mmol) was dissolved in NN-dimethylformamide, under the protection of nitrogen, sodium hydride (60% in oil; 2.30g, 57.5mmol) was added, the reaction solution was cooled to 0°C, followed by Benzene bromide (5.5 mL, 45.98 mmol), tetrabutylammonium iodide (425.01 mg, 1.15 mmol) were added. The reaction was carried out at room temperature for 18 h, the reaction solution was cooled to 0 °C, a small amount of methanol solution was dropped to quench the reaction, diluted with ethyl acetate, the mixture was washed with saturated brine, the organic layer was collected, concentrated to obtain the crude product, and purified by silica gel column chromatography to obtain compound 6 as a white solid (14.90 g, 78.4%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.34-7.24 (m, 24H), 6.92-6.90 (d, J=8.8 Hz, 4H) 5.51 (s, 2H, PhCH), 5.11 (d, J=3.8 Hz) , 2H,H-1&H-1′),4.96(d,J=10.8Hz,2H,PhCH 2 O),4.85-4.81(dd,J=10.8Hz,4H),4.72(d,J=10.8Hz, 2H, PhCH 2 O), 4.27 (dt, J=10.0, 4.8Hz, 2H, H5&H5'), 4.14 (t, J=9.4Hz, 2H, H3&H3'), 4.12-4.10 (m, 2H, H4&H4') , 3.83(S, 6H), 3.69-3.59(m, 6H); HR-ESI-MS(m/z): calcd for C 54 H 58 O 11 [M+H] + 939.395 found, 939.3933.
3)化合物7的制备3) Preparation of compound 7
将6(2.00g，2.11mol)溶于四氢呋喃中，依次加入氰基硼氢化钠、甲基橙指示剂，冰浴下，缓慢滴加盐酸乙醚溶液，滴加至反应液的颜色由橙黄色渐变为粉红色，且半分钟内不变化，然后，缓慢升至室温，继续反应8小时，加乙酸乙酯溶液稀释，饱和食盐水洗3次，有机层减压浓缩，得黄色油状液体粗品。硅胶柱色谱纯化得呈白色油状液体化合物7(1.31g,68.2％)。
1H NMR(400MHz，CDCl
3)δ7.37-7.23(m,20H),7.19-7.16(d,J＝6.4Hz,4H),6.83-6.80(d,J＝6.4Hz,4H),5.20(d,J＝3.6Hz,2H),5.00(d,J＝11.2Hz,2H),4.80(d,J＝11.2Hz,2H),4.70-4.60(q,J＝12.0Hz,4H),4.40(q,J＝12.0Hz,4H),4.73-4.63(m,6H),4.12-4.08(dt,J
1＝13.6Hz,J
2＝7.6Hz,2H),3.89-3.87(t,J＝9.2Hz,2H),3.77(s,6H),3.67-3.65(t,J＝9.2Hz,2H)，3.53(dd,J＝9.6,3.6Hz,2H),3.51-3.43(qd,J＝10.4,4.0Hz,4H)；HR-ESI-MS(m/z):calcd for C
56H
62O
13[M+COOH]
-987.4172 found,987.4177
Dissolve 6 (2.00 g, 2.11 mol) in tetrahydrofuran, add sodium cyanoborohydride and methyl orange indicator in turn, slowly add ether hydrochloric acid solution dropwise under ice bath, and dropwise add to the reaction solution until the color of the reaction solution changes from orange to yellow It was pink and did not change within half a minute, then, slowly warmed to room temperature, continued the reaction for 8 hours, diluted with ethyl acetate solution, washed three times with saturated brine, and concentrated the organic layer under reduced pressure to obtain a crude yellow oily liquid. Purification by silica gel column chromatography gave Compound 7 (1.31 g, 68.2%) as a white oily liquid. 1 H NMR (400MHz, CDCl 3 ) δ 7.37-7.23 (m, 20H), 7.19-7.16 (d, J=6.4Hz, 4H), 6.83-6.80 (d, J=6.4Hz, 4H), 5.20 ( d,J＝3.6Hz,2H),5.00(d,J＝11.2Hz,2H),4.80(d,J＝11.2Hz,2H),4.70-4.60(q,J＝12.0Hz,4H),4.40( q, J = 12.0Hz, 4H) , 4.73-4.63 (m, 6H), 4.12-4.08 (dt, J 1 = 13.6Hz, J 2 = 7.6Hz, 2H), 3.89-3.87 (t, J = 9.2Hz ,2H),3.77(s,6H),3.67-3.65(t,J=9.2Hz,2H),3.53(dd,J=9.6,3.6Hz,2H),3.51-3.43(qd,J=10.4,4.0 Hz, 4H); HR-ESI-MS(m/z): calcd for C 56 H 62 O 13 [M+COOH] - 987.4172 found,987.4177
4)化合物8的合成4) Synthesis of compound 8
N-N'-二甲基甲酰胺溶解化合物7(1.01g,1.06mmol)，加入氢化钠(60％in oil；2.31g,57.5mmol)，反应液降温至0℃，依次加入溴丙炔(5.5mL，45.98mmol)、四丁基溴化铵(425.10mg,1.15mmol)。室温搅拌反应4h，反应液的颜色由乳白变为浊黄色，滴少量甲醇溶液进行反应的淬灭，乙酸乙酯稀释反应液，饱和食盐水冲洗混合液，有机层浓缩的粗品，硅胶柱色谱纯化得白色油状化合物8(0.45g,42.3％)。
1H NMR(400MHz,CDCl
3)δ7.40-7.27(m,20H),7.21-7.18(d,J＝8.4Hz,4H),6.84-6.81(d,J＝8.4Hz,4H),5.15-5.14(d,J＝3.6Hz,2H),4.98-4.83(q,J＝10.4Hz,4H),4.64(s,4H),4.48-4.45(d,J＝11.6Hz,4H),4.39-4.33(m,4H),4.16-4.11(m,2H),3.98-3.96(t,J＝9.2Hz,2H),3.79(s,6H)，3.53-3.48(m,6H),3.40-3.37(m,4H),2.41(t,J＝2.4Hz,2H)；HR-ESI-MS(m/z):calcd for C
62H
66O
13[M+Na]
+1041.4396 found,1041.4395.
Compound 7 (1.01 g, 1.06 mmol) was dissolved in N-N'-dimethylformamide, sodium hydride (60% in oil; 2.31 g, 57.5 mmol) was added, the reaction solution was cooled to 0 °C, and bromopropyne ( 5.5 mL, 45.98 mmol), tetrabutylammonium bromide (425.10 mg, 1.15 mmol). The reaction was stirred at room temperature for 4 hours, the color of the reaction solution changed from milky white to turbid yellow, a small amount of methanol solution was dropped to quench the reaction, the reaction solution was diluted with ethyl acetate, the mixed solution was washed with saturated brine, and the crude product was concentrated in the organic layer and purified by silica gel column chromatography Compound 8 (0.45 g, 42.3%) was obtained as a white oil. 1 H NMR (400MHz, CDCl 3 ) δ 7.40-7.27 (m, 20H), 7.21-7.18 (d, J=8.4Hz, 4H), 6.84-6.81 (d, J=8.4Hz, 4H), 5.15- 5.14(d,J＝3.6Hz,2H),4.98-4.83(q,J＝10.4Hz,4H),4.64(s,4H),4.48-4.45(d,J＝11.6Hz,4H),4.39-4.33 (m,4H),4.16-4.11(m,2H),3.98-3.96(t,J=9.2Hz,2H),3.79(s,6H),3.53-3.48(m,6H),3.40-3.37(m , 4H), 2.41 (t, J=2.4Hz, 2H); HR-ESI-MS (m/z): calcd for C 62 H 66 O 13 [M+Na] + 1041.4396 found, 1041.4395.
5)化合物9的合成5) Synthesis of compound 9
二氯甲烷溶解化合物8(50.20mg,0.0491mmol)，缓慢滴加5％TFA(0.15mL)，反应2h，二氯甲烷稀释，饱和碳酸氢钠冲洗，有机相减压浓缩得粗品，柱层析分离纯化得透明无色油状化合物9(33.20mg,85.7％)。
1H NMR(400MHz,CDCl
3)δ7.39-7.28(m,20H),5.12-5.11(d,J＝3.6Hz,2H),5.01-4.98(d,J＝10.8Hz,2H),4.86-4.83(d,J＝10.8Hz,2H),4.72-4.65(m,4H),4.46-4.37(m,4H),4.04-4.00(m,4H),3.72-3.60(m,4H),3.54-3.49(m,4H),2.49-2.48(t,J＝2.0Hz,2H)；HR-ESI-MS(m/z):calcd for C
46H
50O
11[M+COOH]
-823.3335 found,823.3311.
Compound 8 (50.20 mg, 0.0491 mmol) was dissolved in dichloromethane, 5% TFA (0.15 mL) was slowly added dropwise, reacted for 2 h, diluted with dichloromethane, washed with saturated sodium bicarbonate, and the organic phase was concentrated under reduced pressure to obtain the crude product, which was subjected to column chromatography The compound 9 (33.20 mg, 85.7%) was obtained by separation and purification as a transparent colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ 7.39-7.28 (m, 20H), 5.12-5.11 (d, J=3.6Hz, 2H), 5.01-4.98 (d, J=10.8Hz, 2H), 4.86- 4.83(d, J＝10.8Hz, 2H), 4.72-4.65(m, 4H), 4.46-4.37(m, 4H), 4.04-4.00(m, 4H), 3.72-3.60(m, 4H), 3.54- 3.49 (m, 4H), 2.49-2.48 (t, J=2.0Hz, 2H); HR-ESI-MS (m/z): calcd for C 46 H 50 O 11 [M+COOH] - 823.3335 found, 823.3311 .
6)化合物10的合成6) Synthesis of compound 10
二氯甲烷溶解化合物9(21.03mg)，自制脂肪酸链(22.00mg)，EDC/HCl(4eq)和DMAP(1eq)，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物10(20.36mg,54％)。
1H NMR(400MHz in CDCl
3)δ7.40-7.27(20H,m),5.15-5.14(2H,d,J＝3.2Hz),5.00-4.98(2H,d,J＝10.8Hz),4.86-4.84(2H,d,J＝10.8Hz),4.72-4.66(2H,q,J＝12.4Hz),4.46-4.42(2H,dd,J＝12.4Hz),4.32-4.28(2H,dd,J
1＝15.2Hz,J
2＝2.4Hz),4.19(2H,m),4.14(4H,m),4.01(2H,t,J＝9.2Hz),3.52(2H,dd,J
1＝9.6Hz,J
2＝3.2Hz),3.45(2H,t,J＝9.2Hz),2.44(2H,s),2.19(4H,d,J＝6.8Hz),1.80(2H,m),1.24(64H,m),0.87(12H,t,J＝6.4Hz).
13C NMR(100MHz in CDCl
3)δ14.15,22.71,26.56,26.51,29.37,29.64,29.67,29.95,31.93,33.71,33.78,34.86,39.08,60.14,62.59,68.89,72.95,74.62,75.75,77.25,79.26,79.70,81.33,94.02,127.44,127.58,127.77,127.92,128.11,128.48,128.55,137.78,137.71,138.45,173.24.HR-ESI-MS(m/z):calcd for C
88H
130O
13[M+Na]
+1417.9404 found,1417.9404.
Dissolve compound 9 (21.03mg) in dichloromethane, prepare fatty acid chain (22.00mg), EDC/HCl (4eq) and DMAP (1eq), heat to 53°C, reflux for 12h, dilute with dichloromethane, saturated sodium bicarbonate solution After washing, the organic phase was collected and concentrated under reduced pressure to obtain the crude product, which was separated and purified by column chromatography to obtain compound 10 (20.36 mg, 54%) as a transparent pale yellow oil. 1 H NMR (400 MHz in CDCl 3 ) δ 7.40-7.27 (20H, m), 5.15-5.14 (2H, d, J=3.2 Hz), 5.00-4.98 (2H, d, J=10.8 Hz), 4.86- 4.84(2H,d,J＝10.8Hz),4.72-4.66(2H,q,J＝12.4Hz),4.46-4.42(2H,dd,J＝12.4Hz),4.32-4.28(2H,dd,J 1 =15.2Hz,J 2 =2.4Hz),4.19(2H,m),4.14(4H,m),4.01(2H,t,J=9.2Hz),3.52(2H,dd,J 1 =9.6Hz,J 2 = 3.2Hz), 3.45 (2H, t, J = 9.2Hz), 2.44 (2H, s), 2.19 (4H, d, J = 6.8Hz), 1.80 (2H, m), 1.24 (64H, m) ,0.87(12H,t,J=6.4Hz). 13 C NMR (100MHz in CDCl 3 )δ14.15,22.71,26.56,26.51,29.37,29.64,29.67,29.95,31.93,33.71,33.78,34.86,39.08, 60.14,62.59,68.89,72.95,74.62,75.75,77.25,79.26,79.70,81.33,94.02,127.44,127.58,127.77,127.92,128.11,128.48,128.55,137.78,137.71,138.45,173.24.HR-ESI-MS ( m/z):calcd for C 88 H 130 O 13 [M+Na] + 1417.9404 found,1417.9404.
7)化合物11的制备7) Preparation of compound 11
二氯甲烷溶解化合物9(40.20mg)，Behenic Acid(52.53mg)，EDC/HCl和DMAP，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物11(46.80mg,63％)。
1H NMR(400MHz in CDCl
3)δ7.37-7.25(20H,m),5.15-5.14(2H,d,J＝3.6Hz),5.00-4.98(2H,d,J＝10.8Hz),4.86-4.84(2H,d,J＝10.8Hz),4.69(4H,m),4.37-4.33(2H,dd,J
1＝15.6Hz,J
2＝2.4Hz),4.32-4.28(2H,dd,J
1＝15.6Hz,J
2＝2.4Hz),4.15-4.03(6H,m),3.96 (2H,t,J＝9.2Hz),3.49-3.44(2H,dd,J
1＝10.0Hz,J
2＝3.6Hz),3.41-3.39(4H,t,J＝9.2Hz,2H),2.37(2H,t,J＝2.4Hz),2.25(2H,t,J＝7.6Hz),1.48(4H,m),1.20(76H,m),0.82(6H,t,J＝6.8Hz).
13C NMR(100MHz in CDCl
3)δ14.17,22.73,24.88,29.19,29.30,29.40,29.52,29.65,29.70,29.74,31.96,34.16,60.11,62.73,68.90,72.95,74.63,75.76,77.27,79.18,79.72,81.36,94.04,127.44,127.59,127.81,127.95,128.13,128.51,128.57,137.78,137.71,138.41,173.51.HR-ESI-MS(m/z):calcd for C
90H
134O
13，[M+Na]
+1445.9710 found,1445.9730.
Compound 9 (40.20 mg), Behenic Acid (52.53 mg), EDC/HCl and DMAP were dissolved in dichloromethane, heated to 53 °C, refluxed for 12 h, diluted with dichloromethane, washed with saturated sodium bicarbonate solution, collected the organic phase, reduced The crude product was obtained by pressure concentration, which was separated and purified by column chromatography to obtain compound 11 (46.80 mg, 63%) as a transparent pale yellow oil. 1 H NMR (400 MHz in CDCl 3 ) δ 7.37-7.25 (20H, m), 5.15-5.14 (2H, d, J=3.6 Hz), 5.00-4.98 (2H, d, J=10.8 Hz), 4.86- 4.84 (2H, d, J = 10.8Hz), 4.69 (4H, m), 4.37-4.33 (2H, dd, J 1 = 15.6Hz, J 2 = 2.4Hz), 4.32-4.28 (2H, dd, J 1 =15.6Hz,J 2 =2.4Hz),4.15-4.03(6H,m),3.96(2H,t,J=9.2Hz),3.49-3.44(2H,dd,J 1 =10.0Hz,J 2 =3.6 Hz),3.41-3.39(4H,t,J=9.2Hz,2H),2.37(2H,t,J=2.4Hz),2.25(2H,t,J=7.6Hz),1.48(4H,m), 1.20(76H,m),0.82(6H,t,J=6.8Hz). 13 C NMR(100MHz in CDCl 3 )δ14.17,22.73,24.88,29.19,29.30,29.40,29.52,29.65,29.70,29.74, 31.96,34.16,60.11,62.73,68.90,72.95,74.63,75.76,77.27,79.18,79.72,81.36,94.04,127.44,127.59,127.81,127.95,128.13,128.51,128.57,137.78,137.71,138.41,173.51.HR- ESI-MS(m/z): calcd for C 90 H 134 O 13, [M+Na] + 1445.9710 found, 1445.9730.
8)化合物12的合成8) Synthesis of compound 12
二氯甲烷溶解化合物7(270.21mg,0.29mmol)和2,6-二甲基吡啶(0.15mL)，0℃下缓慢加入叔丁基二甲基三氟甲磺酸酯(0.14mL)，室温下搅拌反应30min，加水淬灭，二氯甲烷萃取，有机相减压浓缩，粗品过硅胶柱，得淡黄色油状化合物12(171.14mg,56.5％)。
1H NMR(400MHz,CDCl
3)δ7.56-7.09(m,26H),6.84-6.82(d,J＝6.0Hz,4H),5.32-5.26(d,J＝3.6Hz,2H),5.10-5.01(dd,J
1＝28.4Hz,J
2＝11.6Hz,2H),4.85(d,J＝11.2Hz,1H),4.76-4.75(m,2H),4.63(d,J＝12.0Hz,2H),4.55-4.52(d,J＝12.0Hz,1H),4.45-4.32(m,4H),3.92-3.81(m,2H),3.80(s,6H),3.67-3.58(m,4H),3.51-3.43(m,4H)；HR-ESI-MS(m/z):calcd for C
62H
76O
13Si，[M+Na]
+1079.4947 found,1079.4951.
Compound 7 (270.21 mg, 0.29 mmol) and 2,6-lutidine (0.15 mL) were dissolved in dichloromethane, and tert-butyl dimethyl trifluoromethanesulfonate (0.14 mL) was slowly added at 0 °C, room temperature The reaction was stirred at low temperature for 30 min, quenched by adding water, extracted with dichloromethane, the organic phase was concentrated under reduced pressure, and the crude product was passed through a silica gel column to obtain compound 12 (171.14 mg, 56.5%) as a light yellow oil. 1 H NMR (400MHz, CDCl 3 ) δ 7.56-7.09 (m, 26H), 6.84-6.82 (d, J=6.0Hz, 4H), 5.32-5.26 (d, J=3.6Hz, 2H), 5.10- 5.01 (dd, J 1 = 28.4Hz , J 2 = 11.6Hz, 2H), 4.85 (d, J = 11.2Hz, 1H), 4.76-4.75 (m, 2H), 4.63 (d, J = 12.0Hz, 2H ),4.55-4.52(d,J=12.0Hz,1H),4.45-4.32(m,4H),3.92-3.81(m,2H),3.80(s,6H),3.67-3.58(m,4H), 3.51-3.43 (m, 4H); HR-ESI-MS (m/z): calcd for C 62 H 76 O 13 Si, [M+Na] + 1079.4947 found, 1079.4951.
9)化合物13的合成9) Synthesis of compound 13
-N'-二甲基甲酰胺溶解化合物12(366.1mg,1eq)，在氮气的保护下，加入氢化钠(60％in oil；6eq)，0℃条件下依次加入溴丙炔(6eq)与四丁基溴化铵(6eq)；室温反应4h，反应液降温至0℃，缓慢滴加少量甲醇淬灭反应，二氯甲烷稀释，依次用 饱和碳酸氢钠溶液、饱和食盐水冲洗，收集有机相，减压浓缩，过硅胶柱分离纯化的化合物13(380.01mg,97％).
1H NMR(400MHz,CDCl
3)δ7.40-7.27(m,20H),7.21-7.18(m,4H),6.84-6.81(m,4H),5.26(dd,J
1＝22.4Hz,J
2＝2.8Hz,2H),5.12(d,J＝11.6Hz,1H),4.87-4.84(d,J＝11.6Hz,2H),4.64(s,4H),4.39-4.33(m,4H),4.16-4.11(m,2H),3.79(m,6H),3.53-3.48(m,6H),3.40-3.37(m,4H),2.41(m,2H),2.43(s,1H),0.85(s,9H),0.01(dd,J＝3.2Hz,6H)；HR-ESI-MS(m/z):calcd for C
65H
78O
13Si，[M+Na]
+1117.5104found,1117.5107.
Compound 12 (366.1 mg, 1 eq) was dissolved in -N'-dimethylformamide, and under the protection of nitrogen, sodium hydride (60% in oil; 6 eq) was added, and bromopropyne (6 eq) was added successively at 0°C. Tetrabutylammonium bromide (6eq); reacted at room temperature for 4h, the reaction solution was cooled to 0°C, slowly added dropwise with a small amount of methanol to quench the reaction, diluted with dichloromethane, rinsed with saturated sodium bicarbonate solution and saturated brine successively, and collected organic The phase was concentrated under reduced pressure, and the purified compound 13 (380.01 mg, 97%) was separated and purified by silica gel column. 1 H NMR (400 MHz, CDCl 3 ) δ 7.40-7.27 (m, 20H), 7.21-7.18 (m, 4H) , 6.84-6.81 (m, 4H), 5.26 (dd, J 1 = 22.4Hz, J 2 = 2.8Hz, 2H), 5.12 (d, J = 11.6Hz, 1H), 4.87-4.84 (d, J = 11.6 Hz, 2H), 4.64(s, 4H), 4.39-4.33(m, 4H), 4.16-4.11(m, 2H), 3.79(m, 6H), 3.53-3.48(m, 6H), 3.40-3.37( m, 4H), 2.41 (m, 2H), 2.43 (s, 1H), 0.85 (s, 9H), 0.01 (dd, J=3.2Hz, 6H); HR-ESI-MS (m/z): calcd for C 65 H 78 O 13 Si, [M+Na] + 1117.5104found,1117.5107.
10)化合物14的合成10) Synthesis of compound 14
二氯甲烷溶解化合物13(50.20mg,0.049mmol)，缓慢滴加5％TFA(0.15mL)，反应2h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩，所得粗品经柱层析分离纯化，得透明无色油状化合物14(33.10mg，87.5％)；
1H NMR(400MHz,CDCl
3)δ7.32-7.12(m,20H),5.13-5.08(dd,J
1＝21.6Hz,J
2＝2.8Hz,2H),5.01-4.98(d,J＝11.6Hz,1H),5.01-4.98(d,J＝11.6Hz,1H),4.94-4.91(d,J＝11.6Hz,1H),4.82-4.80(d,J＝11.6Hz,1H),4.73-4.70(d,J＝11.6Hz,1H),4.66-4.59(m,2H),4.56-4.50(m,2H),4.38-4.30(m,2H),4.03-4.01(t,J＝9.2Hz,2H),3.93-3.83(m,2H),3.76-3.70(m,2H),3.62-3.52(m,4H),3.49-3.43(m,5H),2.47(s,1H),0.87(s,9H),0.01(d,J＝24.0Hz,6H)；
13C NMR(100MHz,CDCl
3)δ139.09,138.53,137.97,137.86,128.52,128.48,128.46,128.17,128.15,127.79,127.76,127.63,127.43,127.08,93.54,81.16,81.08,80.32,79.96,79.85,77.43,77.32,76.80,75.52,74.91,74.54,73.13,72.93,72.85,71.13,70.38,61.38,59.97,26.08,18.17,-3.66,-4.88.HR-ESI-MS(m/z):calcd for C
49H
62O
11Si,[M+COOH]
-899.4043 found,899.4034.
Compound 13 (50.20 mg, 0.049 mmol) was dissolved in dichloromethane, 5% TFA (0.15 mL) was slowly added dropwise, reacted for 2 h, diluted with dichloromethane, washed with saturated sodium bicarbonate solution, the organic phase was collected, and concentrated under reduced pressure to obtain the crude product Separation and purification by column chromatography gave compound 14 (33.10 mg, 87.5%) as a transparent colorless oil; 1 H NMR (400 MHz, CDCl 3 ) δ 7.32-7.12 (m, 20H), 5.13-5.08 (dd, J 1 =21.6Hz,J 2 =2.8Hz,2H),5.01-4.98(d,J=11.6Hz,1H),5.01-4.98(d,J=11.6Hz,1H),4.94-4.91(d,J=11.6 Hz,1H),4.82-4.80(d,J＝11.6Hz,1H),4.73-4.70(d,J＝11.6Hz,1H),4.66-4.59(m,2H),4.56-4.50(m,2H) ,4.38-4.30(m,2H),4.03-4.01(t,J=9.2Hz,2H),3.93-3.83(m,2H),3.76-3.70(m,2H),3.62-3.52(m,4H) , 3.49-3.43(m, 5H), 2.47(s, 1H), 0.87(s, 9H), 0.01(d, J=24.0Hz, 6H); 13 C NMR (100MHz, CDCl 3 ) δ 139.09, 138.53, 137.97 , 137.86,128.52,128.48,128.46,128.17,128.15,127.79,127.76,127.63,127.43,127.08,93.54,81.16,81.08,80.32,79.96,79.85,77.43,77.32,76.80,75.52,74.91,74.54,73.13,72.93 ,72.85,71.13,70.38,61.38,59.97,26.08,18.17,-3.66,-4.88.HR-ESI-MS(m/z):calcd for C 49 H 62 O 11 Si,[M+COOH] - 899.4043 found , 899.4034.
10)化合物15的合成10) Synthesis of compound 15
二氯甲烷溶解化合物14(140.01mg)，自制脂肪酸链(213.77mg)，EDC/HCl和DMAP，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物15(169.50mg,70.3％)；
1H NMR(400MHz,CDCl
3)δ7.37-7.18(m,20H),5.22-5.18(dd,J
1＝16.8Hz,J
2＝3.2Hz,2H),5.11-5.08(d,J＝11.6Hz,1H),5.00-4.97(d,J＝11.6Hz,1H),4.88-4.86(d,J＝10.4Hz 1H),4.77-4.63(m,4H),4.57-4.54(d,J＝11.6Hz,1H),4.44-4.27(dq,J
1＝15.2Hz,J
2＝2.0Hz,2H),4.18-4.04(m,6H),3.89(dd,J
1＝12.0Hz,J
2＝3.6Hz,1H),3.79(t,J＝8.8Hz,1H),3.65-3.63(t,J＝9.6Hz,1H),3.57-3.53(td,J
1＝9.2Hz,J
2＝3.2Hz,2H),3.46(t,J＝9.2Hz,1H),2.44(s,1H),2.18(m,4H),1.78(s,2H),1.26(m,64H),0.89-0.85(m,21H),0.01-0.00(d,J＝2.4Hz,6H).
13C NMR(100MHz,CDCl
3)δ14.15,22.71,26.56,26.51,29.37,29.64,29.67,29.95,31.93,33.71,33.78,34.86,39.08,60.14,62.59,68.89,72.95,74.62,75.75,77.25,79.26,79.70,81.33,94.02,127.44,127.58,127.77,127.92,128.11,128.48,128.55,137.78,137.71,138.45,173.24.HR-ESI-MS(m/z):calcd for C
91H
142O
13Si[M+Na]
+1494.0112 found,1494.0111.
Compound 14 (140.01 mg) was dissolved in dichloromethane, and the fatty acid chain (213.77 mg) was prepared with EDC/HCl and DMAP, heated to 53 °C, refluxed for 12 h, diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected, Concentrate under reduced pressure to obtain the crude product, which was separated and purified by column chromatography to obtain compound 15 (169.50 mg, 70.3%) as a transparent pale yellow oil; 1 H NMR (400 MHz, CDCl 3 ) δ 7.37-7.18 (m, 20H), 5.22-5.18 ( dd,J 1 =16.8Hz,J 2 =3.2Hz,2H),5.11-5.08(d,J=11.6Hz,1H),5.00-4.97(d,J=11.6Hz,1H),4.88-4.86(d , J=10.4Hz 1H), 4.77-4.63 (m, 4H), 4.57-4.54 (d, J=11.6Hz, 1H), 4.44-4.27 (dq, J 1 =15.2Hz, J 2 =2.0Hz, 2H ), 4.18-4.04(m, 6H), 3.89(dd, J 1 =12.0Hz, J 2 =3.6Hz, 1H), 3.79(t, J=8.8Hz, 1H), 3.65-3.63(t, J= 9.6Hz, 1H), 3.57-3.53 (td , J 1 = 9.2Hz, J 2 = 3.2Hz, 2H), 3.46 (t, J = 9.2Hz, 1H), 2.44 (s, 1H), 2.18 (m, 4H), 1.78(s, 2H), 1.26(m, 64H), 0.89-0.85(m, 21H), 0.01-0.00(d, J=2.4Hz, 6H). 13 C NMR (100MHz, CDCl 3 )δ14 . " ,127.58,127.77,127.92,128.11,128.48,128.55,137.78,137.71,138.45,173.24.HR-ESI-MS(m/z):calcd for C 91 H 142 O 13 Si[M+Na] + 1494.0112 found, 1494.0111.
11)化合物16的合成11) Synthesis of compound 16
二氯甲烷溶解化合物14(250.00mg)，Behenic Acid(398.80mg)，EDC/HCl(223.47mg)和DMAP(35.75mg)，加热至53℃，回流反应12h，二氯甲烷稀释，饱和碳酸氢钠溶液冲洗，收集有机相，减压浓缩得粗品，柱层析分离纯化得透明淡黄色油状化合物16(334.51mg,76.2％)；
1H NMR(400MHz,CDCl
3)δ7.36-7.21(m,20H),5.22-5.18(dd,J
1＝16.8Hz,J
2＝3.2Hz,2H),5.11-5.08(d,J＝11.6Hz,1H),5.00-4.97(d,J＝11.6Hz, 1H),4.88-4.86(d,J＝10.4Hz 1H),4.75-4.56(m,5H),4.44-4.20(m,2H),4.09(m,6H),3.93-3.76(m,2H),3.66-3.45(m,4H),2.43(d,J＝4Hz,1H),2.2(m,4H),1.62(s,2H),1.25(m,76H),0.88-0.85(m,15H),0.01-0.00(s,6H).
13C NMR(100MHz,CDCl
3)δ-5.02,-3.62,1.03,14.14,18.13,22.71,24.79,24.87,26.01,29.18,29.29,29.38,29.50,29.65,29.72,31.94,34.11,34.15,60.06,62.79,68.82,70.57,70.60,72.74,73.09,74.59,74.80,75.58,79.73,79.89,80.89,81.10,93.38,127.03,127.32,127.58,127.81,127.82,127.87,128.11,128.20,128.47,128.57,137.61,137.75,138.37,138.99,173.47.HR-ESI-MS(m/z)calcd for C
96H
138O
13Na,(M+Na)
+1522.0036,found 1522.0020.
Compound 14 (250.00mg), Behenic Acid (398.80mg), EDC/HCl (223.47mg) and DMAP (35.75mg) were dissolved in dichloromethane, heated to 53°C, refluxed for 12h, diluted with dichloromethane, saturated with sodium bicarbonate The solution was rinsed, the organic phase was collected, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by column chromatography to obtain compound 16 (334.51 mg, 76.2%) as a transparent pale yellow oil; 1 H NMR (400 MHz, CDCl 3 ) δ 7.36-7.21 (m, 20H), 5.22-5.18 (dd, J 1 = 16.8Hz, J 2 = 3.2Hz, 2H), 5.11-5.08 (d, J = 11.6Hz, 1H), 5.00-4.97 (d, J = 11.6Hz, 1H ), 4.88-4.86(d, J＝10.4Hz 1H), 4.75-4.56(m, 5H), 4.44-4.20(m, 2H), 4.09(m, 6H), 3.93-3.76(m, 2H), 3.66 -3.45(m, 4H), 2.43(d, J=4Hz, 1H), 2.2(m, 4H), 1.62(s, 2H), 1.25(m, 76H), 0.88-0.85(m, 15H), 0.01 -0.00(s,6H). 13 C NMR(100MHz, CDCl 3 )δ-5.02,-3.62,1.03,14.14,18.13,22.71,24.79,24.87,26.01,29.18,29.29,29.38,29.50,29.65,29.72, 31.94,34.11,34.15,60.06,62.79,68.82,70.57,70.60,72.74,73.09,74.59,74.80,75.58,79.73,79.89,80.89,81.10,93.38,127.03,127.32,127.58,127.81,127.82,127.87,128.11, 128.20,128.47,128.57,137.61,137.75,138.37,138.99,173.47.HR-ESI-MS(m/z)calcd for C 96 H 138 O 13 Na,(M+Na) + 1522.0036,found 1522.0020.
12)化合物17与18的合成12) Synthesis of compounds 17 and 18
乙腈溶解化合物16(220.01mg)，0℃下缓慢滴加三氟化硼乙醚(40μL)，反应液逐渐变黄色，反应2小时后，用饱和碳酸氢钠溶液终止中和反应，二氯甲烷溶液稀释萃取，收集有机相，减压浓缩，所得粗品经柱层析分离纯化，得透明淡黄色油状液体化合物18(140.00mg,68.9％)。
1H NMR(400MHz,CDCl
3)δ7.36-7.21(m,20H),5.17-5.13(d,J＝3.2Hz,2H),5.01-4.83(dq,J
1＝10.8Hz,J
2＝6.0Hz,4H),4.73-4.66(m,4H),4.44-4.40(dd,J
1＝15.2Hz,J
2＝2.4Hz,1H),4.34-4.28(m,2H),4.23-4.11(m,4H),4.04-4.00(t,J＝9.6Hz,2H),3.90-3.85(m,2H),3.56-3.40(m,4H),2.43(t,J＝2.4Hz,1H),2.29-2.24(m,4H),1.56(m,4H),1.25(m,76H),0.88-0.85(t,J＝2.4Hz,6H).HR-ESI-MS(m/z)calcd for C
87H
132O
13,(M+Na)
+1407.9578,found 1407.9567.
Compound 16 (220.01 mg) was dissolved in acetonitrile, and boron trifluoride ether (40 μL) was slowly added dropwise at 0°C. The reaction solution gradually turned yellow. After 2 hours of reaction, the neutralization reaction was terminated with saturated sodium bicarbonate solution, and dichloromethane solution The organic phase was collected and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography to obtain compound 18 (140.00 mg, 68.9%) as a transparent pale yellow oily liquid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.36-7.21 (m, 20H), 5.17-5.13 (d, J=3.2 Hz, 2H), 5.01-4.83 (dq, J 1 =10.8 Hz, J 2 =6.0 Hz, 4H), 4.73-4.66(m, 4H), 4.44-4.40(dd, J 1 =15.2Hz, J 2 =2.4Hz, 1H), 4.34-4.28(m, 2H), 4.23-4.11(m, 4H), 4.04-4.00(t, J＝9.6Hz, 2H), 3.90-3.85(m, 2H), 3.56-3.40(m, 4H), 2.43(t, J＝2.4Hz, 1H), 2.29-2.24 (m,4H),1.56(m,4H),1.25(m,76H),0.88-0.85(t,J=2.4Hz,6H).HR-ESI-MS(m/z)calcd for C 87 H 132 O 13, (M+Na) + 1407.9578, found 1407.9567.
同方法制得白色固体化合物17(41.76mg，60.4％)。
1H NMR(400MHz,CDCl
3)δ7.36-7.30(m,20H),5.18(d,J＝3.2Hz,2H),5.01-4.83(dq,J
1＝10.8Hz,J
2＝6.0Hz,4H), 4.75-4.56(m,4H),4.45-4.41(dd,J
1＝15.2Hz,J
2＝2.4Hz,1H),4.32-4.28(m,2H),4.23-4.11(m,4H),4.04-3.99(t,J＝9.6Hz,2H),3.89-3.85(m,2H),3.54-3.40(m,4H),2.45(s,1H),2.21-2.18(m,4H),1.80(m,2H),1.25(s,64H),0.88-0.85(t,J＝6.4Hz,12H),
13C NMR(100MHz,CDCl
3)δ14.15,22.71,26.52,26.57,29.38,29.65,29.67,29.94,31.93,33.71,33.75,33.77,33.79,34.88,34.94,39.04,39.10,60.14,62.55,68.93,69.92,70.16,72.87,73.05,74.66,75.53,75.76,78.91,79.40,79.68,80.61,81.40,94.04,94.37,127.45,127.80,127.83,127.93,128.05,128.10,128.50,128.55,137.73,137.75,138.39,138.66,173.27,174.23.HR-ESI-MS(m/z)calcd for C
85H
128O
13Na,(M+Na)
+1379.9247,found 1379.9206.
In the same way, white solid compound 17 (41.76 mg, 60.4%) was obtained. 1 H NMR (400 MHz, CDCl 3 ) δ 7.36-7.30 (m, 20H), 5.18 (d, J=3.2 Hz, 2H), 5.01-4.83 (dq, J 1 =10.8 Hz, J 2 =6.0 Hz, 4H), 4.75-4.56(m, 4H), 4.45-4.41(dd, J 1 =15.2Hz, J 2 =2.4Hz, 1H), 4.32-4.28(m, 2H), 4.23-4.11(m, 4H) ,4.04-3.99(t,J=9.6Hz,2H),3.89-3.85(m,2H),3.54-3.40(m,4H),2.45(s,1H),2.21-2.18(m,4H),1.80 (m, 2H), 1.25 (s, 64H), 0.88-0.85 (t, J=6.4Hz, 12H), 13 C NMR (100 MHz, CDCl 3 ) δ 14.15, 22.71, 26.52, 26.57, 29.38, 29.65, 29.67,29.94,31.93,33.71,33.75,33.77,33.79,34.88,34.94,39.04,39.10,60.14,62.55,68.93,69.92,70.16,72.87,73.05,74.66,75.53,75.76,78.91,79.40,79.68,80.61, 81.40,94.04,94.37,127.45,127.80,127.83,127.93,128.05,128.10,128.50,128.55,137.73,137.75,138.39,138.66,173.27,174.23.HR-ESI-MS (m / z) calcd for C 85 H 128 O 13 Na, (M+Na) + 1379.9247, found 1379.9206.
13)化合物19的合成13) Synthesis of compound 19
四氢呋喃与甲醇(1：1)溶解化合物10(40.00mg,0.028mmol)、Tn(20.00mg,0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物19(24.35mg,43％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ7.83(s,2H),7.21-7.16(m,20H),5.11(s,2H),4.86(m,4H),4.62(m,20H),4.03(m,14H),3.70(s,4H),3.49(m,13H),2.1(s,4H),1.83(s,6H),1.68(s,64H),0.78(m,12H)；HR-ESI-MS(m/z):calcd for C
108H
166N
8O
25,[M+H]
+1976.2037 found,1976.2007
Compound 10 (40.00 mg, 0.028 mmol), Tn (20.00 mg, 0.068 mmol), cuprous iodide (106.41 mg, 0.56 mmol) were dissolved in tetrahydrofuran and methanol (1:1), and N,N-diisopropylethyl acetate was added. Amine (90 μL) was stirred at room temperature for 24 hours; the insolubles were filtered off with diatomaceous earth, the filtrate was evaporated under reduced pressure to remove the solvent to obtain the crude product; the compound 19 (24.35 mg, 43%) was obtained by separation and purification on a silica gel column. 1 H NMR (400MHz, CD 3 OD/CDCl 3 ) δ 7.83(s, 2H), 7.21-7.16(m, 20H), 5.11(s, 2H), 4.86(m, 4H), 4.62(m, 20H) ),4.03(m,14H),3.70(s,4H),3.49(m,13H),2.1(s,4H),1.83(s,6H),1.68(s,64H),0.78(m,12H) ;HR-ESI-MS(m/z):calcd for C 108 H 166 N 8 O 25, [M+H] + 1976.2037 found, 1976.2007
14)化合物20的合成14) Synthesis of compound 20
四氢呋喃与甲醇(1：1)溶解化合物10(40.01mg，0.028mmol)、STn(20mg，0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物20(38mg,51.9％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ7.63(s,1H),7.38-7.24(m,20H),5.19(s,2H),5.00-4.96(d,J＝12.8Hz,4H),4.86-4.82(d,J＝8.0Hz,2H),4.76-4.69(m,8H),3.56(m,5H),4.51(s,4H),4.13(m,4H),3.97-3.89(m,5H),3.90-3.63(m,24H),3.58-3.47(m,10H),2.75-2.69(dd,J
1＝13.2Hz,J
2＝3.6Hz,4H),2.24-2.21(d,J＝6.8Hz,5H),2.03-20.1(d,J＝9.2Hz,12H),1.84(m,4H),1.26(s,64H),0.88(t,J＝6.4Hz,12H)；HR-ESI-MS(m/z):calcd for C
130H
200N
10O
41,[M+2H]
+21279.7009 found,1279.7037.
Compound 10 (40.01 mg, 0.028 mmol), STn (20 mg, 0.068 mmol), cuprous iodide (106.41 mg, 0.56 mmol) were dissolved in tetrahydrofuran and methanol (1:1), and N,N-diisopropylethylamine was added. (90 μL), the reaction was stirred at room temperature for 24 hours; the insolubles were filtered off with diatomaceous earth, the filtrate was distilled under reduced pressure to remove the solvent to obtain the crude product; the compound 20 (38 mg, 51.9%) was obtained by separation and purification on a silica gel column. 1 H NMR (400 MHz, CD 3 OD/CDCl 3 ) δ 7.63 (s, 1H), 7.38-7.24 (m, 20H), 5.19 (s, 2H), 5.00-4.96 (d, J=12.8 Hz, 4H ),4.86-4.82(d,J=8.0Hz,2H),4.76-4.69(m,8H),3.56(m,5H),4.51(s,4H),4.13(m,4H),3.97-3.89( m, 5H), 3.90-3.63 (m, 24H), 3.58-3.47 (m, 10H), 2.75-2.69 (dd, J 1 =13.2Hz, J 2 =3.6Hz, 4H), 2.24-2.21 (d, J=6.8Hz, 5H), 2.03-20.1(d, J=9.2Hz, 12H), 1.84(m, 4H), 1.26(s, 64H), 0.88(t, J=6.4Hz, 12H); HR- ESI-MS(m/z): calcd for C 130 H 200 N 10 O 41, [M+2H] +2 1279.7009 found,1279.7037.
15)化合物21的合成15) Synthesis of compound 21
四氢呋喃与甲醇(1：1)溶解化合物11(30.01mg,0.021mmol)、STn(29.01mg，0.050mmol)、碘化亚铜(79.80mg,0.42mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶 柱分离纯化得到化合物21(22.31mg,43％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ7.74-7.67(d,J＝26.4Hz,1H),7.36-7.07(m,20H),5.19(d,J＝5.2Hz,2H),5.07-4.93(d,1H),4.90-4.82(d,J＝5.2Hz,1H),4.76-4.63(m,4H),4.60-4.54(m,2H),4.41-4.09(m,20H),3.92-3.65(m,14H),3.58-3.48(m,8H),3.26-3.25(d,J＝4.8Hz,2H),2.56-2.52(m,2H),2.34-2.27(m,4H),2.08-1.98(m,12H),1.80-1.62(m,2H),1.60-1.50(m,4H),1.26(s,76H),0.88(m,6H).HR-ESI-MS(m/z):calcd for C
132H
204N
10O
41,[M+2H]
+2 1293.7165 found,1293.7167
Compound 11 (30.01 mg, 0.021 mmol), STn (29.01 mg, 0.050 mmol), cuprous iodide (79.80 mg, 0.42 mmol) were dissolved in tetrahydrofuran and methanol (1:1), and N,N-diisopropylethyl acetate was added. Amine (90 μL) was stirred at room temperature for 24 hours; the insolubles were filtered off with diatomaceous earth, the filtrate was distilled under reduced pressure to remove the solvent to obtain the crude product; the compound 21 (22.31 mg, 43%) was obtained by separation and purification on a silica gel column. 1 H NMR (400 MHz, CD 3 OD/CDCl 3 ) δ 7.74-7.67 (d, J=26.4 Hz, 1H), 7.36-7.07 (m, 20H), 5.19 (d, J=5.2 Hz, 2H), 5.07-4.93(d, 1H), 4.90-4.82(d, J=5.2Hz, 1H), 4.76-4.63(m, 4H), 4.60-4.54(m, 2H), 4.41-4.09(m, 20H), 3.92-3.65(m, 14H), 3.58-3.48(m, 8H), 3.26-3.25(d, J=4.8Hz, 2H), 2.56-2.52(m, 2H), 2.34-2.27(m, 4H), 2.08-1.98(m,12H),1.80-1.62(m,2H),1.60-1.50(m,4H),1.26(s,76H),0.88(m,6H).HR-ESI-MS(m/z ):calcd for C 132 H 204 N 10 O 41, [M+2H] +2 1293.7165 found,1293.7167
16)化合物22的合成16) Synthesis of compound 22
四氢呋喃与甲醇(1：1)溶解化合物17(35.01mg，0.026mmol)、STn(20mg，0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物22(14.5mg，31.1％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ7.77-7.71(s,1H),7.40-7.24(m,20H),5.21-5.20(dd,J
1＝6.8Hz,J
2＝3.2Hz,2H),5.01-4.87(m,5H),4.78-4.75(d,J＝11.6Hz,2H),4.71-4.47(m,8H),4.23-4.16(m,5H),4.16-4.01(m,7H),4.13(m,4H),3.97-3.44(m,21H),2.75-2.71(m,1H),2.24-2.20(m,4H),2.05(s,6H),1.84-1.77(m,4H),1.62(m,1H),1.46(m,4H),1.26(s,64H),0.88(t,J＝5.6Hz,12H)；HR-ESI-MS(m/z):calcd for C
106H
163N
5O
27,[M+H]
+1939.1608 found,1939.1629
Compound 17 (35.01 mg, 0.026 mmol), STn (20 mg, 0.068 mmol), cuprous iodide (106.41 mg, 0.56 mmol) were dissolved in tetrahydrofuran and methanol (1:1), and N,N-diisopropylethylamine was added. (90 μL), the reaction was stirred at room temperature for 24 hours; the insolubles were filtered off with diatomaceous earth, the filtrate was distilled under reduced pressure to remove the solvent to obtain the crude product; the compound 22 (14.5 mg, 31.1%) was obtained by separation and purification on a silica gel column. 1 H NMR (400 MHz, CD 3 OD/CDCl 3 ) δ 7.77-7.71 (s, 1H), 7.40-7.24 (m, 20H), 5.21-5.20 (dd, J 1 =6.8 Hz, J 2 =3.2 Hz ,2H),5.01-4.87(m,5H),4.78-4.75(d,J＝11.6Hz,2H),4.71-4.47(m,8H),4.23-4.16(m,5H),4.16-4.01(m ,7H),4.13(m,4H),3.97-3.44(m,21H),2.75-2.71(m,1H),2.24-2.20(m,4H),2.05(s,6H),1.84-1.77(m ,4H),1.62(m,1H),1.46(m,4H),1.26(s,64H),0.88(t,J=5.6Hz,12H); HR-ESI-MS(m/z):calcd for C 106 H 163 N 5 O 27, [M+H] + 1939.1608 found, 1939.1629
17)化合物23的合成17) Synthesis of compound 23
四氢呋喃与甲醇(1：1)溶解化合物18(36.01mg,0.026mmol)、STn(20mg，0.068mmol)、碘化亚铜(106.41mg，0.56mmol)，加入N,N-二异丙基乙胺(90μL)，室温下搅拌反应24小时；硅藻土过滤掉不溶物，滤液减压蒸馏除去溶剂得粗品；硅胶柱分离纯化得到化合物23(22.80mg,44.5％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ7.65(s,1H),7.38-7.28(m,20H),5.22-5.20(d,J＝7.6Hz,2H),5.01-4.90(m,5H),4.78-4.71(m,6H),4.51-4.48(m,3H),4.27-3.99(m,11H),3.60-3.43(m,7H),2.74-2.70(m,1H),2.27(s,4H),2.04-2.00(d,J＝12.0Hz,6H),1.80-1.75(m,1H),1.57s,5H),1.26(s,64H),0.88(t,J＝5.6Hz,12H)；HR-ESI-MS(m/z):calcd for C
108H
167N
5O
27,[M+H]
+1967.1921 found,1967.1908
Compound 18 (36.01 mg, 0.026 mmol), STn (20 mg, 0.068 mmol), cuprous iodide (106.41 mg, 0.56 mmol) were dissolved in tetrahydrofuran and methanol (1:1), and N,N-diisopropylethylamine was added. (90 μL), the reaction was stirred at room temperature for 24 hours; the insolubles were filtered off with diatomaceous earth, the filtrate was distilled under reduced pressure to remove the solvent to obtain the crude product; the compound 23 (22.80 mg, 44.5%) was obtained by separation and purification on a silica gel column. 1 H NMR (400MHz, CD 3 OD/CDCl 3 ) δ 7.65 (s, 1H), 7.38-7.28 (m, 20H), 5.22-5.20 (d, J=7.6Hz, 2H), 5.01-4.90 (m ,5H),4.78-4.71(m,6H),4.51-4.48(m,3H),4.27-3.99(m,11H),3.60-3.43(m,7H),2.74-2.70(m,1H),2.27 (s,4H),2.04-2.00(d,J=12.0Hz,6H),1.80-1.75(m,1H),1.57s,5H),1.26(s,64H),0.88(t,J=5.6Hz ,12H); HR-ESI-MS(m/z): calcd for C 108 H 167 N 5 O 27, [M+H] + 1967.1921 found, 1967.1908
18)化合物L2-3的合成18) Synthesis of compound L2-3
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物19(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物L2-3(4.81mg，56.7％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ8.01(s,2H),4.92-4.90(m,2H),4.88(s,2H),4.78-4.50(m,14H),4.21(m,2H),4.23-4.17(m,4H),4.01-3.92(m,4H),3.86-3.82(t,J＝9.2Hz,2H),3.72-3.68(m,4H),3.64-3.48(m, 7H),3.41-3.38(dd,J
1＝9.2Hz,J
2＝2.8Hz,2H),3.39-3.23(t,J＝9.6Hz,4H),2.97-2.92(q,J＝7.6Hz,1H),2.61(s,1H),2.20-2.18(d,J＝6.8Hz,3H),1.88(s,6H),1.75(s,2H),1.19(s,64H),0.88(t,J＝5.6Hz,12H).HR-ESI-MS(m/z):calcd for C
80H
142N
8O
25[M+H]
+1616.0159 found,1616.0162.
Compound 19 (10.05 mg) was dissolved in dichloromethane/methanol/water (3:3:1, 10.0 mL), palladium on carbon (15.64 mg) was added, hydrogen gas was passed through it, sealed and stirred for 40 hours, and the insoluble insoluble was filtered off with celite. The filtrate was evaporated under reduced pressure to remove the solvent to obtain a white solid compound L2-3 (4.81 mg, 56.7%). 1 H NMR (400MHz, CD 3 OD/CDCl 3 ) δ 8.01 (s, 2H), 4.92-4.90 (m, 2H), 4.88 (s, 2H), 4.78-4.50 (m, 14H), 4.21 (m ,2H),4.23-4.17(m,4H),4.01-3.92(m,4H),3.86-3.82(t,J＝9.2Hz,2H),3.72-3.68(m,4H),3.64-3.48(m , 7H), 3.41-3.38 (dd, J 1 = 9.2Hz, J 2 = 2.8Hz, 2H), 3.39-3.23 (t, J = 9.6Hz, 4H), 2.97-2.92 (q, J = 7.6Hz, 1H), 2.61(s, 1H), 2.20-2.18(d, J=6.8Hz, 3H), 1.88(s, 6H), 1.75(s, 2H), 1.19(s, 64H), 0.88(t, J =5.6Hz,12H).HR-ESI-MS(m/z):calcd for C 80 H 142 N 8 O 25 [M+H] + 1616.0159 found,1616.0162.
19)化合物1的合成19) Synthesis of compound 1
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物20(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物1(4.72mg，53.8％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ8.0(s,2H),4.96(s,2H),4.90-4.87(m,3H),4.6-4.53(m,5H),4.22-4.11(m,7H),3.98-3.86(m,6H),3.80-3.69(m,10H),3.65-3.47(m,10H),3.43-3.31(m,8H),2.94-2.91(dd,J
1＝11.2Hz,J
2＝4.8Hz,2H),2.60(m,2H),2.21-2.19(d,J＝6.4Hz,4H),1.93-1.75(m,12H),1.69-1.62(m,2H),1.55-1.48(m,2H),1.21(s,64H),0.88(t,J＝6.0Hz,12H).HR-ESI-MS(m/z):calcd for C
102H
176N
10O
41[M+Na]
+2220.1887 found,2220.1807.
Compound 20 (10.05 mg) was dissolved in dichloromethane/methanol/water (3:3:1, 10.0 mL), palladium on carbon (15.64 mg) was added, hydrogen was passed through, sealed and stirred for 40 hours, celite was filtered to remove the insoluble The filtrate was evaporated under reduced pressure to remove the solvent to obtain compound 1 (4.72 mg, 53.8%) as a white solid. 1 H NMR (400MHz, CD 3 OD/CDCl 3 ) δ 8.0 (s, 2H), 4.96 (s, 2H), 4.90-4.87 (m, 3H), 4.6-4.53 (m, 5H), 4.22-4.11 (m,7H),3.98-3.86(m,6H),3.80-3.69(m,10H),3.65-3.47(m,10H),3.43-3.31(m,8H),2.94-2.91(dd,J 1 =11.2Hz, J 2 =4.8Hz, 2H), 2.60(m, 2H), 2.21-2.19(d, J=6.4Hz, 4H), 1.93-1.75(m, 12H), 1.69-1.62(m, 2H) ),1.55-1.48(m,2H),1.21(s,64H),0.88(t,J=6.0Hz,12H).HR-ESI-MS(m/z):calcd for C 102 H 176 N 10 O 41 [M+Na] + 2220.1887 found, 2220.1807.
20)化合物2的合成20) Synthesis of compound 2
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物21(10.05mg)，加入钯碳(15.64 mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物2(6.10mg,54％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ8.09(s,2H),5.1(s,2H),4.97-4.96(d,J＝1.2Hz,2H),4.85-4.74(m,5H),4.43-4.13(m,10H),4.10-3.96(m,9H),3.91-3.68(m,25H),3.62-3.41(m,11H),3.05-3.00(dd,J
1＝14.4Hz,J
2＝7.2Hz,2H),2.75-2.70(m,2H),2.41-2.37(t,J＝7.2Hz,4H),2.05(s,12H),1.81(m,2H),1.66-1.62(m,4H),1.27(s,76H),0.88(t,J＝6.0Hz,6H).HR-ESI-MS(m/z):calcd for C
104H
180N
10O
41,[M+2H]
+2 1113.6226 found,1113.6251.
Compound 21 (10.05 mg) was dissolved in dichloromethane/methanol/water (3:3:1, 10.0 mL), palladium on carbon (15.64 mg) was added, hydrogen gas was passed through it, sealed and stirred for 40 hours, and the insoluble insoluble was filtered off with celite. The filtrate was evaporated under reduced pressure to remove the solvent to obtain compound 2 (6.10 mg, 54%) as a white solid. 1 H NMR (400 MHz, CD 3 OD/CDCl 3 ) δ 8.09 (s, 2H), 5.1 (s, 2H), 4.97-4.96 (d, J=1.2 Hz, 2H), 4.85-4.74 (m, 5H) ), 4.43-4.13(m, 10H), 4.10-3.96(m, 9H), 3.91-3.68(m, 25H), 3.62-3.41(m, 11H), 3.05-3.00(dd, J 1 =14.4Hz, J 2 =7.2Hz, 2H), 2.75-2.70(m, 2H), 2.41-2.37(t, J=7.2Hz, 4H), 2.05(s, 12H), 1.81(m, 2H), 1.66-1.62( m, 4H), 1.27(s, 76H), 0.88(t, J=6.0Hz, 6H). HR-ESI-MS(m/z): calcd for C 104 H 180 N 10 O 41 , [M+2H ] +2 1113.6226 found, 1113.6251.
21)化合物3的合成21) Synthesis of compound 3
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物22(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物3(6.01mg,74.1％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ8.12(s,1H),5.1(s,2H),5.00-4.96(m,2H),4.66-4.61(m,4H),4.36-4.30(m,2H),4.29-4.16(m,4H),4.09-3.97(m,5H),3.9-3.42(m,22H),3.23-3.1(m,2H),3.05-3.00(dd,J
1＝14.8Hz,J
2＝7.2Hz,2H),2.78-2.67(m,4H),2.32-2.28(m,4H),2.3(m,4H),2.05(s,6H),1.85(s,2H),1.66-1.62(m,1H),1.28(s,64H),0.88(t,J＝5.6Hz,6H).HR-ESI-MS(m/z):calcd for C
78H
139N
5O
27,[M+H]
+1578.9730 found,1578.9726
Compound 22 (10.05 mg) was dissolved in dichloromethane/methanol/water (3:3:1, 10.0 mL), palladium on carbon (15.64 mg) was added, hydrogen gas was passed through it, sealed and stirred for 40 hours, and the insoluble insoluble was filtered off with celite. The filtrate was evaporated under reduced pressure to remove the solvent to obtain compound 3 (6.01 mg, 74.1%) as a white solid. 1 H NMR (400MHz, CD 3 OD/CDCl 3 ) δ 8.12 (s, 1H), 5.1 (s, 2H), 5.00-4.96 (m, 2H), 4.66-4.61 (m, 4H), 4.36-4.30 (m,2H),4.29-4.16(m,4H),4.09-3.97(m,5H),3.9-3.42(m,22H),3.23-3.1(m,2H),3.05-3.00(dd,J 1 =14.8Hz, J 2 =7.2Hz, 2H), 2.78-2.67(m, 4H), 2.32-2.28(m, 4H), 2.3(m, 4H), 2.05(s, 6H), 1.85(s, 2H) ),1.66-1.62(m,1H),1.28(s,64H),0.88(t,J=5.6Hz,6H).HR-ESI-MS(m/z):calcd for C 78 H 139 N 5 O 27 ,[M+H] + 1578.9730 found,1578.9726
22)化合物4的合成22) Synthesis of compound 4
二氯甲烷/甲醇/水(3：3：1，10.0mL)溶解化合物23(10.05mg)，加入钯碳(15.64mg)，通入氢气，密封搅拌40个小时，硅藻土滤过掉不溶物，滤液减压蒸馏除去溶剂，得到白色固体化合物4(5.9mg，66.5％)。
1H NMR(400MHz,CD
3OD/CDCl
3)δ8.12(s,1H),5.11-5.10(d,J＝5.4Hz,2H),5.00-4.95(m,2H),4.38-4.21(m,5H),4.01-4.67(m,16H),3.63-3.45(m,6H),3.1-3.0(m,2H),2.72-2.70(d,J＝21.6Hz,1H),2.41-2.38(td,J
1＝17.2Hz,J
2＝7.6Hz,4H),2.07(s,6H),1.81-1.78(m,1H),1.66-1.62(m,4H),1.26(s,76H),0.89(t,J＝5.6Hz,6H).HR-ESI-MS(m/z):calcd for C
80H
143N
5O
27,[M+H]
+1607.0043 found,1607.0044.
Compound 23 (10.05 mg) was dissolved in dichloromethane/methanol/water (3:3:1, 10.0 mL), palladium on carbon (15.64 mg) was added, hydrogen was passed through, sealed and stirred for 40 hours, celite was filtered to remove the insoluble The filtrate was evaporated under reduced pressure to remove the solvent to obtain compound 4 (5.9 mg, 66.5%) as a white solid. 1 H NMR (400 MHz, CD 3 OD/CDCl 3 ) δ 8.12 (s, 1H), 5.11-5.10 (d, J=5.4 Hz, 2H), 5.00-4.95 (m, 2H), 4.38-4.21 (m ,5H),4.01-4.67(m,16H),3.63-3.45(m,6H),3.1-3.0(m,2H),2.72-2.70(d,J＝21.6Hz,1H),2.41-2.38(td , J 1 =17.2Hz, J 2 =7.6Hz, 4H), 2.07(s, 6H), 1.81-1.78(m, 1H), 1.66-1.62(m, 4H), 1.26(s, 76H), 0.89( t,J=5.6Hz,6H).HR-ESI-MS(m/z):calcd for C 80 H 143 N 5 O 27 ,[M+H] + 1607.0043 found,1607.0044.
实施例2：ELISA免疫分析Example 2: ELISA immunoassay
(1)将制备好的化合物1至4分别作为疫苗分子1，2，3与4，按照疫苗分子：二硬脂酰基磷脂酰胆碱把(DSPC)：胆固醇＝1:6.5:5溶解于DCM-MeOH(1:1，v/v，2mL)混合液中，旋干溶剂，使瓶壁上形成一层薄的脂质膜。加入2.0mL羟乙基哌嗪乙硫磺酸(HEPES)缓冲液(20Mm,pH＝7.5)，超声10～20min得脂质体1，2，3与4。(1) Compounds 1 to 4 were prepared as vaccine molecules 1, 2, 3 and 4, respectively, and dissolved in DCM according to vaccine molecule: distearoyl phosphatidyl choline (DSPC): cholesterol=1:6.5:5 -MeOH (1:1, v/v, 2mL) mixture, spin dry the solvent to form a thin lipid film on the bottle wall. 2.0 mL of hydroxyethylpiperazine ethanethiosulfonic acid (HEPES) buffer solution (20Mm, pH=7.5) was added, and sonicated for 10-20min to obtain liposomes 1, 2, 3 and 4.
(2)小鼠的免疫：取6-8周龄的C57BL/6小鼠，分成四组，每组6只。通过小鼠皮下注射的方式进行免疫试验，采用一次初始免疫和三次增强免疫的方案，分别在第1,14,21,28天注射步骤(1)中制备的脂质体1，2，3与4，每只每次注射量0.1mL，在第0,27,35,49天采血，每只小鼠取血0.1mL～0.2mL，4℃放置半个小时，离心，5000转/分钟，分离上层清亮血清。在本实验中，空白对照组(为免疫前血清)用到的是第一次取血分离出的血清，测定疫苗滴度用到的是第35天采血分离出的血清。(2) Immunization of mice: C57BL/6 mice aged 6-8 weeks were taken and divided into four groups with 6 mice in each group. The immunization test was carried out by subcutaneous injection of mice, and the liposomes 1, 2, and 3 prepared in step (1) were injected on the 1st, 14th, 21st, and 28th days respectively with the scheme of one initial immunization and three boosting immunizations. 4. The injection volume of each mouse is 0.1 mL. Blood is collected on the 0, 27, 35, and 49 days, and 0.1 mL to 0.2 mL of blood is collected from each mouse, placed at 4°C for half an hour, centrifuged at 5000 rpm, and separated. Top layer clearing serum. In this experiment, the blank control group (pre-immune serum) used the serum separated from the first blood collection, and the vaccine titer was measured using the blood collected on the 35th day.
(3)ELISA免疫分析：用0.1M碳酸盐缓冲液(pH 9.6)，将Tn-HSA或STn-HSA配置成2.0μg/mL溶液，以每孔100μL的量加入96孔板，放入4℃孵育过夜；第二 天放入37℃培养箱孵育一小时；用PBST(PBS+0.05％吐温-20)洗板3次，每孔加入300μL洗液。洗板后，加入blocking buffer(PBST/1％BSA)；每孔加入250μL；常温孵育一小时，用PBST洗板3次。样品血清稀释，用PBS按照对半数稀释法从1：300倍稀释至1：656100倍；将稀释好的血清以每孔100μL加入96孔板中，放置在37℃培养箱孵育两个小时，洗板三次。分别在每孔加入100μL的HRP(辣根过氧化物酶)标记的Kappa,IgG,IgM，室温孵育一个小时；洗板3次。加入TMB(3,3',5,5'-四甲基联苯胺)溶液，每孔加入100μL，室温避光显色20min。加入0.5M H
2SO
4溶液，每孔加100μL。在450-570nm双波长处，用酶标仪检测吸光度。
(3) ELISA immunoassay: Use 0.1M carbonate buffer (pH 9.6) to prepare Tn-HSA or STn-HSA into a 2.0μg/mL solution, add 100μL per well to a 96-well plate, and put 4 Incubate overnight at °C; incubate at 37 °C for one hour the next day; wash the plate three times with PBST (PBS+0.05% Tween-20), and add 300 μL of washing solution to each well. After washing the plate, add blocking buffer (PBST/1% BSA); add 250 μL to each well; incubate at room temperature for one hour, and wash the plate three times with PBST. The sample serum was diluted with PBS from 1:300 to 1:656100 times according to the half-dilution method; the diluted serum was added to 96-well plate at 100 μL per well, placed in a 37°C incubator for two hours, and washed. plate three times. 100 μL of HRP (horseradish peroxidase)-labeled Kappa, IgG, and IgM were added to each well, and incubated at room temperature for one hour; the plate was washed 3 times. Add TMB (3,3',5,5'-tetramethylbenzidine) solution, add 100 μL to each well, and develop color at room temperature for 20 min in the dark. Was added 0.5M H 2 SO 4 solution was added to each well 100μL. Absorbance was detected with a microplate reader at dual wavelengths of 450-570 nm.
4)将吸光度(OD)值相对于抗血清稀释值作图，并获得最佳拟合线。使用该线的方程式来计算OD值达到0.2时的稀释度值，并且根据稀释值的倒数计算抗体滴度如图6所示。4) Plot the absorbance (OD) value against the antiserum dilution value and obtain a line of best fit. The equation of this line was used to calculate the dilution value at which the OD value reached 0.2, and the antibody titer was calculated from the inverse of the dilution value as shown in Figure 6.
从图1可看出，本发明实施例1合成的海藻糖衍生物与糖抗原的缀合物1，2，3与4，在无外加佐剂的情况下，均能诱导产生高滴度的抗体，产生的特异性IgG抗体滴度是IgM的2～3倍多，表明共价偶联的糖抗原疫苗能诱导T细胞介导的体液免疫；其中双接STn抗原的缀合物疫苗1与2引发的IgG抗体滴度均略高于单接抗原的缀合物3与4，说明抗原的空间越复杂，表位数多，对免疫反应具有一定的促进作用；海藻糖衍生物脂肪酸链为具有支链的CH(C
9H
19)
2的缀合物疫苗1与3诱导产生的抗体分别略高于脂肪酸链为酯链(CH
2)
20CH
2的缀合物疫苗2与4，说明海藻糖衍生物脂肪酸链的个数与长短对活性有一定影响。
It can be seen from Figure 1 that the conjugates 1, 2, 3 and 4 of trehalose derivatives and carbohydrate antigens synthesized in Example 1 of the present invention can all induce high titers without adjuvant. Antibodies, the specific IgG antibody titer produced is more than 2 to 3 times that of IgM, indicating that the covalently conjugated carbohydrate antigen vaccine can induce T cell-mediated humoral immunity; among them, the conjugate vaccine 1 double-linked STn antigen and The titers of IgG antibodies elicited by 2 were slightly higher than those of conjugates 3 and 4, which were single-attached to the antigen, indicating that the more complex the space of the antigen, the more epitopes, had a certain promoting effect on the immune response; the fatty acid chain of trehalose derivatives was Conjugate vaccines 1 and 3 with branched CH(C 9 H 19 ) 2 induced slightly higher antibodies than conjugate vaccines 2 and 4 with fatty acid chain (CH 2 ) 20 CH 2 , respectively, indicating that The number and length of fatty acid chains of trehalose derivatives have certain influence on the activity.
实施例3：抗体介导的互补依赖细胞毒性(CDC)Example 3: Antibody-mediated complementation-dependent cytotoxicity (CDC)
用胰酶消化处于对数生长期的CT-26细胞(小鼠结肠癌细胞)，以每孔1×10
4个细胞种于96孔板，在37℃培养过夜，用不含血清的1640培养基洗板两次。取化合物1组在38天取血的6只小鼠血清样品，每个样品取7μl，然后将6只小鼠的血清样品混合，用不含血清的1640培养基稀释50倍，得小鼠血清稀释液。化合物2组、3组、4组以及空白对照组的小鼠血清稀释液按上述方法制备。1组、2组、3组、4组以及空白对照组(为免疫前血清)均设置样品最大酶活性对照组，样品对照组以及样品处理组，每个组平行做6个复孔，每孔加入100μl稀释后的小鼠血清溶液，将96孔板置于37℃培养2h。用不含血清的1640培养基洗板两次后，在样品处理组每孔加入100μl兔补体血清溶液，最大酶活性对照组每孔加入100μl的LDH释放液，样品对照组每孔 加入100μl不含血清的MEM培养基，置于37℃培养1h。
CT-26 cells (mouse colon cancer cells) in logarithmic growth phase were trypsinized, seeded in 96-well plates at 1×10 4 cells per well, cultured overnight at 37°C, and cultured with serum-free 1640 The substrate was washed twice. Take the serum samples of 6 mice in the compound 1 group on 38 days, take 7 μl of each sample, then mix the serum samples of the 6 mice, and dilute 50 times with serum-free 1640 medium to obtain mouse serum Diluent. The mouse serum dilutions of compound group 2, group 3, group 4 and blank control group were prepared according to the above method. Group 1, Group 2, Group 3, Group 4 and blank control group (pre-immune serum) were set as the maximum enzyme activity control group, sample control group and sample treatment group, each group was made 6 replicate wells in parallel, each well 100 μl of diluted mouse serum solution was added, and the 96-well plate was incubated at 37° C. for 2 h. After washing the plate twice with serum-free 1640 medium, 100 μl of rabbit complement serum solution was added to each well of the sample treatment group, 100 μl of LDH release solution was added to each well of the maximum enzyme activity control group, and 100 μl of LDH release solution was added to each well of the sample control group. Serum in MEM medium and cultured at 37°C for 1 h.
在新的96孔板每孔加入100μl的PBS溶液，小心吸取40μl上述96孔板的细胞上清液加至此新的96孔板中，每孔加入60μl的LDH检测液，避光孵育30min。用酶标仪在490nm处检测。Add 100 μl of PBS solution to each well of a new 96-well plate, carefully pipette 40 μl of the cell supernatant from the above 96-well plate into this new 96-well plate, add 60 μl of LDH detection solution to each well, and incubate in the dark for 30 min. Detected at 490 nm with a microplate reader.
细胞裂解率(％)＝(样品处理组吸光度-样品对照组吸光度)/(最大酶活性对照组吸光度-样品对照组吸光度)×100％。Cell lysis rate (%)=(absorbance of sample treatment group-absorbance of sample control group)/(absorbance of maximum enzyme activity control group-absorbance of sample control group)×100%.
结果如图2所示，本发明实施例1合成的1，2，3与4糖疫苗诱导产生的抗血清均能介导CT-26细胞裂解，细胞裂解率均显著高于空白对照组(P<0.001)，说明糖疫苗1、2、3与4所诱导产生的抗体都有杀死肿瘤细胞能力。The results are shown in Figure 2, the antisera induced by the 1, 2, 3 and 4 sugar vaccines synthesized in Example 1 of the present invention can all mediate the lysis of CT-26 cells, and the cell lysis rates were significantly higher than those in the blank control group (P <0.001), indicating that the antibodies induced by sugar vaccines 1, 2, 3 and 4 have the ability to kill tumor cells.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Variations or changes in other different forms are not required and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.
Claims (10)
- 一种海藻糖衍生物与糖抗原的缀合物，其特征在于，所述缀合物结构通式如下式I所示：A conjugate of trehalose derivative and carbohydrate antigen, characterized in that the general structural formula of the conjugate is shown in the following formula I:[X—L—Y][X—L—Y]式(I)Formula (I)其中，X表示糖抗原，选自下式中的任意一种糖抗原，或其可要药用盐、水合物或溶剂化物：Wherein, X represents a carbohydrate antigen, selected from any one of the carbohydrate antigens in the following formula, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
L表示连接体，选自以下(CH 2CH 2O) a-CH 2CH 2-C(O)NH-、
(CH 2CH 2O) a-、-(OCH 2CH 2) a
-NHC(O)-(CH 2CH 2O) a-CH 2CH 2-C(O)NH-、-NHC(O)-(CH 2CH 2O) a-CH 2CH 2
-(OCH 2CH 2) a-CH 2CH 2-C(O)NH-、
(CH2) a-C(O)NH-、-NHC(O)-(CH 2) a-C(O)NH-、-NHC(O)-(CH 2) a
中的任意一种，a是0到20中的任意一个整数； L represents a linker, selected from the following
(CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-,(CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a-NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2 -C(O)NH-, -NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2-(OCH 2 CH 2 ) a -CH 2 CH 2 -C(O)NH-,(CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) a -C(O)NH-, -NHC(O)-(CH 2 ) aAny of , a is any integer from 0 to 20;Y表示海藻糖衍生物，选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：Y represents a trehalose derivative, selected from any one of the following formulae, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
其中，R 1和R 2各自独立地选自氢、-CH 2-CH(OR 3)-(CH 2) m-CH 3，-(CH 2) mCH 3或-(CH 2) mCHR 3 2，R 3为-(CH 2) m-CH 3或-C(O)-(CH 2) m-CH 3，m是选自8-26的整数。 wherein R 1 and R 2 are each independently selected from hydrogen, -CH 2 -CH(OR 3 )-(CH 2 ) m -CH 3 , -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , R 3 is -(CH 2 ) m -CH 3 or -C(O)-(CH 2 ) m -CH 3 , m is an integer selected from 8-26. - 根据权利要求1所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述L选自(CH 2CH 2O) a-、-(OCH 2CH 2) a
-NHC(O)-(CH 2CH 2O) a-CH 2CH 2
(CH2) a-C(O)NH-、-NHC(O)-(CH 2) a
中的任意一种，a是0到20 中的任意一个整数。 The conjugate of trehalose derivative and carbohydrate antigen according to claim 1, wherein the L is selected from
(CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a-NHC(O)-(CH 2 CH 2 O) a -CH 2 CH 2(CH2) a -C(O)NH-, -NHC(O)-(CH 2 ) aAny of , where a is any integer from 0 to 20. - 根据权利要求2所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述L选自(CH 2CH 2O) a-、-(OCH 2CH 2) a
-NHC(O)-(CH 2) a
中的任意一种，a是0到20中的任意一个整数。 The conjugate of trehalose derivative and carbohydrate antigen according to claim 2, wherein the L is selected from
(CH 2 CH 2 O) a -, -(OCH 2 CH 2 ) a-NHC(O)-(CH 2 ) aAny of , where a is any integer from 0 to 20. - 根据权利要求1所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：The conjugate of trehalose derivative and saccharide antigen according to claim 1, wherein said Y is selected from any one of the following formulas, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
其中，R 1和R 2各自独立地选自氢、-(CH 2) mCH 3或-(CH 2) mCHR 3 2，R 3为-(CH 2) m-CH 3或-C(O)-(CH 2) m-CH 3，m是选自8-26的整数。 wherein, R 1 and R 2 are each independently selected from hydrogen, -(CH 2 ) m CH 3 or -(CH 2 ) m CHR 3 2 , and R 3 is -(CH 2 ) m -CH 3 or -C(O )-(CH 2 ) m -CH 3 , m is an integer selected from 8-26. - 根据权利要求4所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述Y选自下式中的任意一种，或其可要药用盐、水合物或溶剂化物：The conjugate of trehalose derivative and saccharide antigen according to claim 4, wherein said Y is selected from any one of the following formulas, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
其中，R 1和R 2各自独立地选自氢或-(CH 2) mCH 3，m是选自8-26的整数。 wherein, R 1 and R 2 are each independently selected from hydrogen or -(CH 2 ) m CH 3 , and m is an integer selected from 8-26. - 根据权利要求1所述海藻糖衍生物与糖抗原的缀合物，其特征在于，所述缀合物选自如下任一结构或其可要药用盐、水合物或溶剂化物：The conjugate of a trehalose derivative and a carbohydrate antigen according to claim 1, wherein the conjugate is selected from any of the following structures or a pharmaceutically acceptable salt, hydrate or solvate thereof:
- 权利要求1至6任一所述海藻糖衍生物与糖抗原的缀合物的制备方法，其特征在于，当Y为时，制备过程包括： The preparation method of the conjugate of any one of the trehalose derivatives and carbohydrate antigens according to any one of claims 1 to 6, characterized in that, when Y is
, the preparation process includes:S1.化合物7在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物8；S1. compound 7 reacts with propyne bromide under the catalysis of sodium hydride and tetrabutylammonium bromide to obtain compound 8;S2.化合物8在三氟乙酸的条件下，脱去PMB基团得到化合物9；S2. Compound 8, under the condition of trifluoroacetic acid, removes the PMB group to obtain compound 9;S3.化合物9与相应的脂肪酸链进行酯化缩合得到对应的化合物10或11；S3. Compound 9 is esterified and condensed with the corresponding fatty acid chain to obtain the corresponding compound 10 or 11;S4.化合物10与化合物Tn或STn、化合物11与化合物STn分别在催化剂的作用下反应得到化合物19或20、化合物21；S4. Compound 10 reacts with compound Tn or STn, compound 11 and compound STn respectively under the action of a catalyst to obtain compound 19 or 20, compound 21;S5.化合物19、20和21分别经脱苄基保护反应，即可得到目标产物；S5. Compounds 19, 20 and 21 are respectively subjected to debenzylation protection reaction to obtain the target product;当Y为制备过程包括； when Y is
The preparation process includes;S11.化合物7在2,6-二甲基吡啶的催化下与叔丁基二甲基三氟甲磺酸酯反应得到 化合物12；S11. compound 7 is reacted with tert-butyl dimethyl trifluoromethanesulfonate under the catalysis of 2,6-lutidine to obtain compound 12;S21.化合物12在氢化钠、四丁基溴化铵催化作用下与溴丙炔反应得到化合物化合物13；S21. Compound 12 is reacted with bromopropyne under the catalysis of sodium hydride and tetrabutylammonium bromide to obtain compound compound 13;S31.化合物13在三氟乙酸的条件下，脱去PMB基团得到化合物14；S31. Compound 13, under the condition of trifluoroacetic acid, removes the PMB group to obtain compound 14;S41.化合物14分别与相应的脂肪酸链进行酯化缩合得到对应的化合物15或16；S41. Compound 14 is esterified and condensed with the corresponding fatty acid chain to obtain the corresponding compound 15 or 16;S51.化合物15或16分别脱去保护基团三甲基硅烷得到相应的化合物17或18；S51. Compound 15 or 16 respectively removes the protecting group trimethylsilane to obtain the corresponding compound 17 or 18;S61.化合物17或18与三氟化硼乙醚反应得到相应的化合物22或23；S61. Compound 17 or 18 is reacted with boron trifluoride ether to obtain corresponding compound 22 or 23;S71.化合物22或23分别经脱苄基保护反应，即可得到目标产物；S71. Compound 22 or 23 are respectively subjected to debenzylation protection reaction to obtain the target product;其中，化合物7至23的结构如下所示：Among them, the structures of compounds 7 to 23 are shown below:
- 根据权利要求7所述海藻糖衍生物与糖抗原的缀合物的制备方法，其特征在于，化合物7的制备过程如下：The preparation method of the conjugate of trehalose derivative and carbohydrate antigen according to claim 7, is characterized in that, the preparation process of compound 7 is as follows:S31.以海藻糖为起始原料，在对甲苯磺酸的作用下，4-甲氧基苯甲醛二甲缩醛对原料进行4,6位羟基选择性保护，得到化合物5，用溴化卞对裸露的羟基进行全苄基 化得化合物6；S31. take trehalose as starting raw material, under the effect of p-toluenesulfonic acid, 4-methoxybenzaldehyde dimethylacetal carries out 4,6-position hydroxyl selective protection to raw material, obtains compound 5, uses brominated Bian The exposed hydroxyl group is fully benzylated to give compound 6;S32.化合物6选择性还原4,4’位糖羟基得到化合物7；S32. Compound 6 selectively reduces 4,4' sugar hydroxyl groups to obtain compound 7;其中化合物5和6的结构如下：The structures of compounds 5 and 6 are as follows:
- 权利要求1至6任一所述海藻糖衍生物与糖抗原的缀合物在制备肿瘤疫苗中的应用。Application of the conjugate of any one of the trehalose derivatives and carbohydrate antigens described in claims 1 to 6 in the preparation of tumor vaccines.
- 权利要求1至6任一所述海藻糖衍生物与糖抗原的缀合物在制备抗肿瘤药物中的应用。Application of the conjugate of any one of the trehalose derivatives and carbohydrate antigens according to any one of claims 1 to 6 in the preparation of antitumor drugs.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010698972.8 | 2020-07-20 | ||
| CN202010698972.8A CN111875649B (en) | 2020-07-20 | 2020-07-20 | Conjugate of trehalose derivative and saccharide antigen and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022016755A1 true WO2022016755A1 (en) | 2022-01-27 |
Family
ID=73155142
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2020/130230 WO2022016755A1 (en) | 2020-07-20 | 2020-11-19 | Trehalose derivative and carbohydrate antigen conjugate, and preparation method therefor and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN111875649B (en) |
| WO (1) | WO2022016755A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111875649B (en) * | 2020-07-20 | 2021-12-17 | 广州中医药大学(广州中医药研究院) | Conjugate of trehalose derivative and saccharide antigen and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011014771A1 (en) * | 2009-07-31 | 2011-02-03 | Wayne State University | Monophosphorylated lipid a derivatives |
| CN110075291A (en) * | 2019-02-01 | 2019-08-02 | 广州中医药大学(广州中医药研究院) | A kind of monophosphate class ester A conjugation Tn anti-tumor vaccine and its application |
| CN111875649A (en) * | 2020-07-20 | 2020-11-03 | 广州中医药大学(广州中医药研究院) | Conjugate of trehalose derivative and saccharide antigen and preparation method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102276662B (en) * | 2010-06-09 | 2014-05-14 | 北京大学 | Sialic acid (alpha-(2-6))-D-pyranose derivative and its synthetic method and use |
| CN106620682A (en) * | 2017-01-19 | 2017-05-10 | 华中师范大学 | Liposomal vaccine preparation and use thereof, as well as method for producing IgG antibody |
| CN109432415A (en) * | 2018-07-27 | 2019-03-08 | 广州粤美医药科技有限公司 | The conjugate and its preparation method and application of monophosphate class ester A (MPLA) and sugar antigens Globo H |
| CN110064050B (en) * | 2019-04-29 | 2020-10-02 | 北京大学 | Glycoconjugate containing STn or F-STn, preparation method thereof and application thereof in anti-tumor vaccine |
-
2020
- 2020-07-20 CN CN202010698972.8A patent/CN111875649B/en active Active
- 2020-11-19 WO PCT/CN2020/130230 patent/WO2022016755A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011014771A1 (en) * | 2009-07-31 | 2011-02-03 | Wayne State University | Monophosphorylated lipid a derivatives |
| CN110075291A (en) * | 2019-02-01 | 2019-08-02 | 广州中医药大学(广州中医药研究院) | A kind of monophosphate class ester A conjugation Tn anti-tumor vaccine and its application |
| CN111875649A (en) * | 2020-07-20 | 2020-11-03 | 广州中医药大学(广州中医药研究院) | Conjugate of trehalose derivative and saccharide antigen and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| WANG QIANLI, ZHOU ZHIFANG, TANG SHOUCHU, GUO ZHONGWU: "Carbohydrate-Monophosphoryl Lipid A Conjugates Are Fully Synthetic Self-Adjuvanting Cancer Vaccines Eliciting Robust Immune Responses in the Mouse", ACS CHEMICAL BIOLOGY, vol. 7, no. 1, 20 January 2012 (2012-01-20), pages 235 - 240, XP055889059, ISSN: 1554-8929, DOI: 10.1021/cb200358r * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111875649B (en) | 2021-12-17 |
| CN111875649A (en) | 2020-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DK166827B1 (en) | SACCHAROSE ALKYL-4,6 ORTHOESTERS AND A PROCEDURE FOR THE PREPARATION OF SACCHAROSE-6 ESTERS | |
| JP5923373B2 (en) | Compounds for the preparation of immune adjuvants | |
| AU2017206277A1 (en) | Methods for preparation of glycosphingolipds and uses thereof | |
| SU724087A3 (en) | Method of obtaining hydrochloride of optically active down-saminyl-derivatives of anthracyclinons | |
| CN111760021B (en) | Conjugate containing alpha-galactosylceramide analogue and saccharide antigen, and preparation method and application thereof | |
| JP2015134793A (en) | Saponin as triterpene, and method of synthesis and use thereof | |
| US20110245488A1 (en) | Process for the synthesis of l-fucosyl di- or oligo-saccharides and novel 2,3,4 tribenzyl-fucosyl derivatives intermediates thereof | |
| CN111760020B (en) | Conjugate, preparation method and application thereof | |
| CN109432415A (en) | The conjugate and its preparation method and application of monophosphate class ester A (MPLA) and sugar antigens Globo H | |
| WO2022016755A1 (en) | Trehalose derivative and carbohydrate antigen conjugate, and preparation method therefor and application thereof | |
| SU897111A3 (en) | Method of preparing anthracycline derivatives | |
| Yi et al. | Synthesis of 4, 5-disubstituted-3-deoxy-d-manno-octulosonic acid (Kdo) derivatives | |
| Lockhoff et al. | Syntheses of glycosylamides as glycolipid analogs | |
| WO2023216732A1 (en) | Chemical synthesis method for helicobacter pylori core lipopolysaccharide oligosaccharide antigen carbohydrate chain | |
| JPH01275581A (en) | Antitumor substance sf2582 derivative | |
| CA1311751C (en) | Ganglioside related compounds and method of producing the same | |
| RU2320653C2 (en) | 6r-(3,6-dideoxy-l-arabinohexoparanosyloxy)heptanoic acid for treatment of diseases associated with aging and stress and method for its preparing | |
| CN108794556B (en) | Compound and application thereof in treating cataract | |
| US4849512A (en) | 3-acylamino-3-deoxyallose derivatives | |
| Kirschning et al. | Polymer-bound haloate (I) anions by iodine (III)-mediated oxidation of polymer-bound iodide: synthetic utility in natural product transformations | |
| CN114259559B (en) | Synthetic tumor vaccine containing alpha-GalCer endogenous adjuvant | |
| NO169590B (en) | ANALOGY PROCEDURE FOR THE PREPARATION OF NEW THERAPEUTIC-ACTIVE SIALOSYLYL GYCERIDES | |
| CN117903223A (en) | Disaccharide structure compound and preparation method and application thereof | |
| CN117024494A (en) | Conjugate, preparation method and application thereof | |
| CN116120381A (en) | Monophosphate A (MPLA) conjugated saccharide antigen Tn anti-tumor vaccine and application thereof |
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: 20946303 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20946303 Country of ref document: EP Kind code of ref document: A1 |
|
| 32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 07/07/2023) |