CN117624284A - Liquid phase synthesis method of linker and synthesis product thereof - Google Patents
Liquid phase synthesis method of linker and synthesis product thereof Download PDFInfo
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- CN117624284A CN117624284A CN202311624284.7A CN202311624284A CN117624284A CN 117624284 A CN117624284 A CN 117624284A CN 202311624284 A CN202311624284 A CN 202311624284A CN 117624284 A CN117624284 A CN 117624284A
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- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 28
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 28
- 239000007791 liquid phase Substances 0.000 title claims abstract description 22
- 238000001308 synthesis method Methods 0.000 title claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 22
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 claims abstract description 18
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 66
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 66
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 52
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 36
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 34
- 239000002904 solvent Substances 0.000 claims description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 239000003795 chemical substances by application Substances 0.000 claims description 26
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 24
- 238000006482 condensation reaction Methods 0.000 claims description 22
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 21
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 21
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 17
- GQHTUMJGOHRCHB-UHFFFAOYSA-N DBU Substances C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 claims description 13
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 12
- 239000002585 base Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 150000007530 organic bases Chemical class 0.000 claims description 12
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 10
- 239000003513 alkali Substances 0.000 claims description 10
- 238000002425 crystallisation Methods 0.000 claims description 10
- 230000008025 crystallization Effects 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
- 230000035484 reaction time Effects 0.000 claims description 10
- AXKGIPZJYUNAIW-UHFFFAOYSA-N (4-aminophenyl)methanol Chemical compound NC1=CC=C(CO)C=C1 AXKGIPZJYUNAIW-UHFFFAOYSA-N 0.000 claims description 9
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 9
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 9
- XBNGYFFABRKICK-UHFFFAOYSA-N 2,3,4,5,6-pentafluorophenol Chemical compound OC1=C(F)C(F)=C(F)C(F)=C1F XBNGYFFABRKICK-UHFFFAOYSA-N 0.000 claims description 8
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 8
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims description 8
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 claims description 8
- IZUPBVBPLAPZRR-UHFFFAOYSA-N pentachloro-phenol Natural products OC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl IZUPBVBPLAPZRR-UHFFFAOYSA-N 0.000 claims description 8
- 238000005809 transesterification reaction Methods 0.000 claims description 8
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 6
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- GKQLYSROISKDLL-UHFFFAOYSA-N EEDQ Chemical compound C1=CC=C2N(C(=O)OCC)C(OCC)C=CC2=C1 GKQLYSROISKDLL-UHFFFAOYSA-N 0.000 claims description 6
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 6
- ACBQROXDOHKANW-UHFFFAOYSA-N bis(4-nitrophenyl) carbonate Chemical compound C1=CC([N+](=O)[O-])=CC=C1OC(=O)OC1=CC=C([N+]([O-])=O)C=C1 ACBQROXDOHKANW-UHFFFAOYSA-N 0.000 claims description 6
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical compound NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 claims description 6
- 238000004537 pulping Methods 0.000 claims description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 6
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 5
- FPDWHGAQJGAILQ-UHFFFAOYSA-N C(O)(O)=O.[N+](=O)([O-])C1=CC=CC=C1.[N+](=O)([O-])C1=CC=CC=C1 Chemical compound C(O)(O)=O.[N+](=O)([O-])C1=CC=CC=C1.[N+](=O)([O-])C1=CC=CC=C1 FPDWHGAQJGAILQ-UHFFFAOYSA-N 0.000 claims description 5
- 239000008346 aqueous phase Substances 0.000 claims description 4
- -1 diazabicyclo Chemical compound 0.000 claims description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- JBTWLSYIZRCDFO-UHFFFAOYSA-N ethyl methyl carbonate Chemical compound CCOC(=O)OC JBTWLSYIZRCDFO-UHFFFAOYSA-N 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 abstract description 11
- DALMAZHDNFCDRP-VMPREFPWSA-N 9h-fluoren-9-ylmethyl n-[(2s)-1-[[(2s)-5-(carbamoylamino)-1-[4-(hydroxymethyl)anilino]-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamate Chemical compound O=C([C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(C)C)NC1=CC=C(CO)C=C1 DALMAZHDNFCDRP-VMPREFPWSA-N 0.000 abstract description 5
- ZJLNQOIFTYLCHC-VXKWHMMOSA-N (2s)-5-(carbamoylamino)-2-[[(2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=O)C(O)=O)C3=CC=CC=C3C2=C1 ZJLNQOIFTYLCHC-VXKWHMMOSA-N 0.000 abstract description 4
- 150000001413 amino acids Chemical class 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 239000013078 crystal Substances 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 229920001184 polypeptide Polymers 0.000 abstract description 2
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 20
- 239000000047 product Substances 0.000 description 14
- 239000007787 solid Substances 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 7
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
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- 239000000706 filtrate Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000003756 stirring Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 101710112752 Cytotoxin Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 231100000599 cytotoxic agent Toxicity 0.000 description 2
- 239000002619 cytotoxin Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000010626 work up procedure Methods 0.000 description 2
- JPJMNCROLRPFHI-QFIPXVFZSA-N (2,5-dioxopyrrolidin-1-yl) (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoate Chemical compound O=C([C@@H](NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(C)C)ON1C(=O)CCC1=O JPJMNCROLRPFHI-QFIPXVFZSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- USMYACISHVPTHK-PXLJZGITSA-N [4-[[(2s)-5-(carbamoylamino)-2-[[(2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate Chemical compound O=C([C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(C)C)NC(C=C1)=CC=C1COC(=O)OC1=CC=C([N+]([O-])=O)C=C1 USMYACISHVPTHK-PXLJZGITSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- FJPRYKYQVBQRTD-UHFFFAOYSA-N aniline methanol Chemical compound CO.Nc1ccccc1 FJPRYKYQVBQRTD-UHFFFAOYSA-N 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 230000002349 favourable effect Effects 0.000 description 1
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- 239000012634 fragment Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
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- 238000011403 purification operation Methods 0.000 description 1
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Classifications
-
- 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
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of polypeptide conjugate synthesis, in particular to a liquid phase synthesis method of a linker and a synthesis product thereof. The method comprises the steps of synthesizing the compound by referring to the following synthesis paths:. The new synthetic route can solve the problems of poor crystal form, difficult suction filtration and difficult purification caused by Fmoc-Val-Cit formed by sequentially connecting amino acids in the prior art, greatly improves the purity and yield of the product and reduces the time cost. Meanwhile, the Fmoc-Val-OH and Cit-PAB have high conversion rate, and the yield of the intermediate C is improved.
Description
Technical Field
The invention relates to the technical field of polypeptide conjugate synthesis, in particular to a liquid phase synthesis method of a linker and a synthesis product thereof.
Background
The ADC medicine consists of three parts, namely an antibody, a Linker and cytotoxin, wherein the antibody can specifically target a specific antigen, is over-expressed in tumor cells, and is under-expressed and not expressed in normal cells. Linker is used as bridge of ADC medicine, and is connected with antibody through cleavable and non-cleavable Linker, and the Linker has the characteristics of stability under physiological state and release at specific position. The cytotoxin has the characteristics of high toxicity activity and low immunity, and has active groups connected with a Linker and a definite action mechanism.
The compound represented by the following formula (1) is a Linker (Linker)
Fmoc-Val-OH, H-Cit-OH, 4-aminobenzyl alcohol and p-nitrobenzoic acid ester are adopted to react in the existing synthesis process, and then the linker is formed by a scheme of sequentially connecting amino acids from front to back, but the product in Fmoc-Val-Cit synthesis has poor crystal form, extremely large purification operation difficulty and difficult operation, so that the intermediate has low purity; the reaction rate is slow in the synthesis of Fmoc-Val-Cit-PAB, the reaction time is as long as 36 hours, the time cost is high, and the purity is not high; the liquid phase is adopted in the synthesis of Fmoc-Val-Cit-PAB-PNP to obtain the compound with higher purity, the purification cost is high, and the yield is lower, so that the method is not beneficial to industrial production.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a liquid phase synthesis method of a linker and a synthesis product thereof. The embodiment of the invention provides a novel liquid phase synthesis method, which improves the conversion rate of raw materials and can improve the yield and purity of products.
The invention is realized in the following way:
in a first aspect, the present invention provides a liquid phase synthesis method of a linker, which synthesizes with reference to the following synthesis route:
in an alternative embodiment, the step of forming intermediate a comprises: carrying out condensation reaction on Fmoc-L-Cit and p-aminobenzyl alcohol by a condensing agent;
preferably, the step of forming intermediate a comprises: mixing the Fmoc-L-Cit, the para-aminobenzyl alcohol, the condensing agent and a solvent for condensation reaction;
or the step of forming intermediate a comprises: mixing the Fmoc-L-Cit, the para-aminobenzyl alcohol, the condensing agent, alkali and a solvent for condensation reaction;
preferably, the method further comprises: and (3) after the condensation reaction is finished, carrying out post-treatment, wherein the post-treatment comprises filtering, pulping methyl tertiary butyl ether and methylene dichloride, and then filtering and drying.
In alternative embodiments, the condensing agent is selected from any one or two or more of N, N "-diisopropylcarbodiimide, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline, N" -dicyclohexylcarbodiimide, N "-carbonyldiimidazole, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; preferably N, N "-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole;
the organic solvent is any one or mixture of several of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol and chloroform, preferably DMF;
the adopted alkali is any one or a mixture of a plurality of triethylamine, N-diisopropylethylamine, sodium bicarbonate, sodium carbonate or DBU with any proportion, preferably N, N-diisopropylethylamine;
preferably, the molar ratio of Fmoc-L-Cit, p-aminobenzyl alcohol and condensing agent is 1:1-1.3:2-2.8;
preferably, the molar ratio of Fmoc-L-Cit, said para-aminobenzyl alcohol, said N, N "-dicyclohexylcarbodiimide and said 1-hydroxybenzotriazole is 1:1-1.3:1-1.5:1-1.3, preferably 1:1.05:1.2:1.3;
preferably, the temperature of the condensation reaction is from-10 to 0 ℃, for a time of 10-30 hours, preferably at-10 ℃; the time was 18 hours.
In an alternative embodiment, the step of forming intermediate B comprises: removing Fmoc from the intermediate A by using an organic base;
preferably, the step of forming intermediate B comprises: mixing the intermediate A, the organic base and a solvent for reaction;
preferably, the method further comprises: and after the reaction is finished, carrying out post-treatment, wherein the post-treatment comprises the following steps: the solvent was removed by concentration, then the pH was adjusted and extraction was performed with ethyl acetate, followed by collection of the aqueous phase.
In an alternative embodiment, the organic base comprises any one of piperidine, diazabicyclo, diethylamine, ethanolamine, cyclohexylamine, and morpholine, preferably diethylamine;
the solvent comprises any one or a mixture of a plurality of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol and chloroform in any proportion, and DMF is preferred;
preferably, the mass ratio of the intermediate A to the organic base is 1:0.5-3, preferably 1:2.5;
the reaction temperature is 0 to 40 ℃, preferably 25 ℃, for 1 to 8 hours, preferably 6 hours.
In an alternative embodiment, the step of forming intermediate C comprises: condensing Fmoc-L-Val-OH and an intermediate B through a condensing agent to perform condensation reaction;
preferably, the step of forming intermediate C comprises: mixing the Fmoc-L-Val-OH, the intermediate B, the condensing agent, a solvent and alkali for condensation reaction;
preferably, the method further comprises: after the condensation reaction is finished, post-treatment is carried out, wherein the post-treatment comprises: crystallizing the reaction liquid, filtering and drying.
In an alternative embodiment, the condensing agent comprises: any one or two or more of N, N '-diisopropylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with 1-hydroxybenzotriazole;
the solvent comprises any one or a mixture of a plurality of ethyl acetate, dichloromethane, tetrahydrofuran, water, acetonitrile, DMF, dichloromethane and chloroform in any proportion, and DMF is preferred;
the base comprises any one of triethylamine, N-diisopropylethylamine, sodium bicarbonate, sodium carbonate and DBU, preferably triethylamine;
preferably, the molar ratio of Fmoc-L-Val-OH, intermediate B and condensing agent is 1:1-1.3:2-2.8;
preferably, the molar ratio of Fmoc-L-Val-OH, intermediate B, 1-hydroxybenzotriazole and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1-1.3:1-1.3:1 to 1.5; the most preferred is 1:1.05:1.1:1.2;
preferably, the reaction temperature is from-10 ℃ to 0 ℃, preferably from-5 ℃; the reaction time is 10 to 20 hours, preferably 15 hours.
In an alternative embodiment, the method comprises: performing transesterification reaction on the intermediate C and di (p-nitrobenzene) carbonate under alkaline conditions;
preferably, it comprises: mixing the intermediate C, the di (p-nitrobenzene) carbonate, alkali and a solvent for transesterification;
preferably, it comprises: after the transesterification reaction, the reaction solution is added into an ester solvent for crystallization, and then filtered and dried.
In alternative embodiments, the base includes any one of triethylamine, N-diisopropylethylamine, pyridine, N-methylmorpholine, and DBU; preferably N-methylmorpholine;
the solvent comprises any one or a mixture of a plurality of 1, 4-dioxane, dimethyl sulfoxide, DMF, tetrahydrofuran, dichloromethane and chloroform in any proportion, and is preferably 1, 4-dioxane;
preferably, the molar ratio of intermediate C, di (p-nitrophenyl) carbonate and base is 1:1-4:1-4, most preferably 1:1.5:2.5;
preferably, the temperature is from-10 to 30 ℃, most preferably 25 ℃, and the reaction time is from 2 to 8 hours, most preferably 7 hours.
In a second aspect, the present invention provides a liquid phase synthesis product prepared by the liquid phase synthesis method of the linker of any one of the previous embodiments.
The invention has the following beneficial effects: the embodiment of the invention provides a new synthesis route, which is characterized in that an intermediate fragment is synthesized firstly, the front-section amino acid is butted, and finally the rear-end activated ester is connected, so that the problems of poor crystal form, difficult suction filtration and difficult purification caused by Fmoc-Val-Cit formed by sequentially connecting the amino acids in the prior art can be solved, the purity and the yield of the product are greatly improved, and the time cost is reduced. Meanwhile, the Fmoc-Val-OH and Cit-PAB have high conversion rate, and the yield of the intermediate C is improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a plot of a comparison of the dot panels provided in comparative example 3 of the present invention;
FIG. 2 is a graph of HPLC of the reaction product provided in comparative example 4 of the present invention;
FIG. 3 is a graph of HPLC of the reaction product provided in comparative example 5 of the present invention;
FIG. 4 is a plot of a comparison of the dot panels provided in comparative example 6 of the present invention;
FIG. 5 is a HPLC profile of the reaction product provided in comparative example 7 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The embodiment of the invention provides a liquid phase synthesis method of a connector, which comprises the following steps:
it is synthesized with reference to the following synthesis route:
specifically, the operation is as follows:
(1) Intermediate a is formed:
carrying out condensation reaction on Fmoc-L-Cit and p-aminobenzyl alcohol by a condensing agent; specifically, mixing Fmoc-L-Cit, p-aminobenzyl alcohol, condensing agent and solvent for condensation reaction; alkali may be added during the reaction. And after the reaction is finished, carrying out post-treatment, wherein the post-treatment comprises filtering, pulping methyl tertiary butyl ether and methylene dichloride, and then filtering and drying.
The pulping with a specific solvent is beneficial to improving the purity of the product, and if the pulping solvent is changed, the purity of the product may be reduced.
Wherein the condensing agent is one of N, N ' -Diisopropylcarbodiimide (DIC), 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ), N ' -Dicyclohexylcarbodiimide (DCC), N ' -Carbonyldiimidazole (CDI) or is combined with two of N-hydroxysuccinimide (HOSu), 1-Hydroxybenzotriazole (HOBT), p-nitrophenol (PN) and pentafluorophenol (PFP); DCC and 1-Hydroxybenzotriazole (HOBT) are preferred.
The organic solvent is one or more of Ethyl Acetate (EA), dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile, DMF, methanol and chloroform, preferably DMF.
The base used is triethylamine (Et 3N), N-Diisopropylethylamine (DIEA), sodium bicarbonate, sodium carbonate, DBU or no base added, preferably N, N-Diisopropylethylamine (DIEA).
The reaction conditions are as follows: the molar ratio of Fmoc-L-Cit, p-aminobenzene methanol and condensing agent is 1:1-1.3:2-2.8; specifically, the mole ratio of Fmoc-L-Cit, para-aminobenzyl alcohol (PAB), DCC and HOBT is 1:1-1.3:1-1.5:1-1.3, preferably 1:1.05:1.2:1.3. the condensation reaction temperature is-10 to 0 ℃, preferably-10 ℃, preferably for 10-30 hours, preferably 18 hours.
The adoption of the synthesis conditions is favorable for obtaining a product with higher purity, and the yield of the intermediate A is also improved.
(2) Intermediate B is formed:
removing Fmoc from the intermediate A by using an organic base; the specific operation is as follows: mixing the intermediate A, the organic base and a solvent for reaction; after the reaction, the solvent was removed by concentration, the pH was adjusted, ethyl acetate was used for extraction, and then the aqueous phase was collected.
According to the embodiment of the invention, through a specific method, the collected water phase can be directly used as a reaction raw material to carry out the next reaction without further post-treatment, and the water in the water phase can not influence the subsequent reaction.
Wherein the organic base is one of piperidine, diazabicyclo (DBU), diethylamine, ethanolamine, cyclohexylamine and morpholine, preferably diethylamine.
The solvent is one or more of Ethyl Acetate (EA), dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile, DMF, methanol and chloroform, preferably DMF.
The reaction conditions include: the mass ratio of the intermediate A to the organic base is 1:0.5-3, optimally 1:2.5. the reaction temperature is 0-40 ℃, optimally 25 ℃, and the reaction time is 1-8h, optimally 6h.
(3) Forming intermediate C;
condensing Fmoc-L-Val-OH and an intermediate B through a condensing agent to perform condensation reaction; specifically, the Fmoc-L-Val-OH, the intermediate B, the condensing agent, a solvent and alkali are mixed for condensation reaction; and cooling the reaction liquid for crystallization after the reaction is finished, filtering and drying.
According to the embodiment of the invention, cooling crystallization is adopted, and compared with concentration to remove the solvent, the intermediate C with higher purity can be obtained.
Further, the condensing agent is a combination of two of N, N '-Diisopropylcarbodiimide (DIC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (edc.hcl), N' -Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (HOSu), 1-Hydroxybenzotriazole (HOBT), p-nitrophenol (PN) and pentafluorophenol (PFP); edc, hcl and HOBT are preferred.
The solvent is one or more of Ethyl Acetate (EA), dichloromethane (DCM), tetrahydrofuran (THF), water, acetonitrile, DMF, dichloromethane and chloroform, preferably DMF.
The base used was triethylamine (Et 3 N), N-Diisopropylethylamine (DIEA), sodium bicarbonate, sodium carbonate, DBU, preferably triethylamine (Et) 3 N)。
The reaction conditions include: the molar ratio of Fmoc-L-Val-OH, the intermediate B and the condensing agent is 1:1-1.3:2-2.8; preferably, the molar ratio of Fmoc-L-Val-OH, intermediate B, HOBT to EDC.HCL is 1:1-1.3:1-1.3:1-1.5, optimally 1:1.05:1.1:1.2; preferably, the temperature of the condensation reaction is-10-0deg.C, optimally-5deg.C, preferably the reaction time is 10-20h, optimally 15h.
(4) Synthesizing a linker;
performing transesterification reaction on the intermediate C and di (p-nitrobenzene) carbonate under alkaline conditions; specifically, the intermediate C, the di (p-nitrophenyl) carbonate, a base, and a solvent are mixed to perform a transesterification reaction. After the reaction, the reaction solution is added into an ester solvent for crystallization, and then filtered and dried.
Wherein the base comprises any one of triethylamine, N-diisopropylethylamine, pyridine, N-methylmorpholine and DBU; preferably N-methylmorpholine;
the solvent comprises any one or a mixture of a plurality of 1, 4-dioxane, dimethyl sulfoxide, DMF, tetrahydrofuran, dichloromethane and chloroform in any proportion, and is preferably 1, 4-dioxane;
preferably, the molar ratio of intermediate C, di (p-nitrophenyl) carbonate and base is 1:1-4:1-4, most preferably 1:1.5:2.5;
preferably, the temperature is from-10 to 30 ℃, most preferably 25 ℃, and the reaction time is from 2 to 8 hours, most preferably 7 hours.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a method for synthesizing a connector, which comprises the following steps:
intermediate a was formed with reference to the following synthetic pathway:
10.00g Fmoc-L-Cit and 3.25g p-aminobenzyl alcohol were dissolved in 50ml DMF. Cooled to-10 ℃ and EEDQ 9.33g is added. After reacting for 18 hours, carrying out suction filtration, pouring the filtrate into 500mL of methyl tertiary butyl ether, stirring for 30 minutes, filtering, pulping a filter cake with 100mL of dichloromethane solution for 2 times, and carrying out suction filtration to obtain a white solid, wherein 9.8g of white solid is obtained after drying at 50 ℃, and the yield is 77.1% (12.7 g) and the purity is 99.3%; MHz, (C) 2 D 6 OS):δ＝1.34-1.50(m，2H)，1.56-1.71(m，2H)，2.91-3.09(m，2H)，4.07-4.26(m，4H)，4.41-4.43(d，2H)，，5.09-5.12(t，3H)，5.45-5.49(d，2H)，5.99-6.05(m，1H)，7.22-7.85(m，13H)，9.99(s，1H)。
Intermediate B was synthesized with reference to the following synthetic pathway:
2. 5.00g Fmoc-L-Cit-PAB was dissolved in 15mL DMF, 7.5mL diethylamine was added for reaction at 25℃for 6 hours, after the reaction was completed, diethylamine was first concentrated at 45℃and 45mL water was then added, the pH of the solution was adjusted to 4 with 1mol/L aqueous hydrochloric acid solution, and the impurities were extracted 3 times with 15mL ethyl acetate, and the aqueous phase was collected and used directly for the next reaction.
Synthesizing intermediate C with reference to the synthesis pathway described below;
3.18g Fmoc-L-Val-OH and 1.39g HOBT were dissolved in the 2 filtrate. 2.15g EDCI and 1.82g DIEA were added to the reaction mixture at-5℃and reacted for 15 hours. After the reaction, the reaction mixture was added to water for crystallization, suction filtration and drying at 50℃to give 3.82g of a white solid, the purity of which was 98% by HPLC detection and the yield of which was 67.8% (5.63 g). 1 HNMR(400MHz，C 2 D 6 OS):δ＝0.82-0.86(m，6H)，1.30-1.71(m，4H)，1.92-2.01(m，1H)，2.84-3.04(m，2H)，3.88-3.92(m，1H)，4.17-4.23(m，2H)，4.37-4.42(m，3H)，5.06-5.09(t，1H)，5.39(s，2H)，5.94-5.97(t，1H)，7.19-8.09(m，14H)，9.95(s，1H)。
Synthesizing the linker with reference to the synthetic pathway;
5.00g Fmoc-Val-Cit-PAB was added to 50mL DMF and dissolved with stirring, 3.79g di (p-nitrophenyl) carbonate was added, 1.68-g N-methylmorpholine was added when the ice salt bath was cooled to 0deg.C, and the reaction was resumed at room temperature for 7 hours. After the reaction, the reaction mixture was added to ethyl acetate for crystallization, suction filtration and drying at 40℃to give 5.10g of a white solid, with an HPLC purity of 98% and a yield of 80.0% (6.37 g). 1 HNMR(400MHz，C 2 D 6 OS):δ＝0.79-0.87(m，6H)，1.32-1.71(m，4H)，1.96-2.03(m，1H)，2.88-3.03(m，2H)，3.89-4.43(m，5H)，5.21(s，2H)，5.40(s，2H)，5.95-5.98(t，1H)，7.27-8.28(m，18H)，10.12(s，1H)。
Comparative example 1
Intermediate C was synthesized with reference to the following synthetic pathway:
(1) 10.00g of Fmoc-L-Val-OH and 3.73g of NHS are dissolved in 50ml of THF, the temperature is reduced to below 10 ℃, 7.29g of DCC is added, the reaction is carried out for 10 hours after natural temperature return, the by-product DCU is filtered off by suction after the reaction is finished, filtrate is collected, after the filtrate is concentrated to be oily, 4 times of MTBE crystallization is added, white solid is obtained by suction filtration, and the yield is 62% (7.90 g).
(2) 5g of Fmoc-L-Val-OSU and 2.21g L-Cit-OH were dissolved in THF and water. 2.22g DIEA was added to the reaction mixture at-5℃and the reaction mixture was allowed to react at room temperature for 12 hours. After the reaction was completed, the product was extracted with ethyl acetate, and the ethyl acetate phase was washed with acid water, clear water, dried over anhydrous sodium sulfate, suction filtered, and concentrated at 50℃to give 5.01g of a white solid in 87.8% (5.01 g).
(3) 5.00g Fmoc-Val-Cit-OH and 2.48g p-aminobenzyl alcohol were dissolved in 50ml DMF. Cooling to-10deg.C. EEDQ 4.98g was added. After 36 hours of reaction at room temperature in the absence of light, suction filtration is carried out, the filtrate is poured into 500mL of methyl tertiary butyl ether, stirring is carried out for 30min, white solid is obtained through suction filtration, 3.15 yellow solid is obtained through drying at 50 ℃, the purity is 94% through HPLC detection, and the yield is 51.9% (3.15 g).
It can be seen that when intermediate C is synthesized from Fmoc-L-Val-OH, the yield of intermediate C in this comparative example is only 28.2%, while the yield of intermediate C in this example is 67.8%, and the yield of intermediate C formed by the method of the present invention is significantly improved.
Comparative example 2
Synthesizing the linker with reference to the synthetic pathway;
intermediate C3.00g Fmoc-Val-Cit-PAB formed in comparative example 1 was added to 50mL DMF and dissolved with stirring, 2.27g di (p-nitrophenyl) carbonate was added, 1.32g DIEA was added when the ice salt bath was cooled to 0℃and the reaction was resumed for 7 hours at room temperature. After the reaction, the reaction mixture was added to MTBE for crystallization, suction filtration and oven drying at 40℃to give 2.71g of yellow solid with a purity of 91% by HPLC and a yield of 71.0% (2.71 g).
Comparative example 3
Intermediate a was prepared with reference to the preparation method provided in example 1, except that: the reaction temperature was room temperature (25 ℃ C.) and after the same reaction time of 18 hours, the reaction system for forming intermediate A of example 1 and the reaction system of this comparative example were spotted and then observed.
Referring to fig. 1, it can be seen from fig. 1 that modification of the reaction temperature of intermediate a results in an increase in impurity content.
Comparative example 4
Intermediate a was prepared with reference to the preparation method provided in example 1, except that: after the reaction is finished, ethyl acetate is adopted; that is, 500mL of methyl tertiary butyl ether of example 1 was stirred for 30min, filtered, the cake was slurried 2 times with 100mL of methylene chloride solution, changed to 500mL of ethyl acetate, stirred for 30min, filtered, and the cake was slurried 2 times with 100mL of ethyl acetate solution. The product of this comparative example was then tested for purity of 96.9% with reference to fig. 2 and hplc with reference to fig. 2.
The purity of intermediate a of comparative example 1 was 99.3%, and it was found that modification of the work-up procedure resulted in a significant decrease in the purity of intermediate a.
Comparative example 5
Intermediate C was prepared with reference to the preparation method provided in example 1, except that: after the reaction was completed, the reaction solvent was removed by rotary evaporation, and then dried at 50℃to obtain a solid having a purity of 97.3% and HPLC as shown in FIG. 3.
The purity of intermediate C in comparative example 1 was 98%, and it was found that modification of the work-up procedure resulted in a significant decrease in the purity of intermediate C.
Comparative example 6
The linker was prepared with reference to the preparation method provided in example 1, except that: the reaction temperature was room temperature, and after the same reaction time of 7 hours, the linker-forming reaction system of example 1 and the reaction system of this comparative example were spotted and then observed.
Referring to FIG. 4, it can be seen from FIG. 4 that the impurity content increases by changing the reaction temperature of the linker.
Comparative example 7
The linker was prepared with reference to the preparation method provided in example 1, except that: after the reaction, the reaction solution was added to methyl tert-butyl ether for crystallization, followed by suction filtration and drying at 40℃to give a product with a purity of 91.9% and HPLC as shown in FIG. 5.
The purity of the linker of comparative example 1 was 98%, and it was found that the modification of the post-treatment method resulted in a significant decrease in the purity of the linker.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A liquid phase synthesis method of a linker, characterized in that it comprises the steps of synthesizing by referring to the following synthesis routes:
2. the method of liquid phase synthesis of a linker according to claim 1, wherein the step of forming intermediate a comprises: carrying out condensation reaction on Fmoc-L-Cit and p-aminobenzyl alcohol by a condensing agent;
preferably, the step of forming intermediate a comprises: mixing the Fmoc-L-Cit, the para-aminobenzyl alcohol, the condensing agent and a solvent for condensation reaction;
or the step of forming intermediate a comprises: mixing the Fmoc-L-Cit, the para-aminobenzyl alcohol, the condensing agent, alkali and a solvent for condensation reaction;
preferably, the method further comprises: and (3) after the condensation reaction is finished, carrying out post-treatment, wherein the post-treatment comprises filtering, pulping methyl tertiary butyl ether and methylene dichloride, and then filtering and drying.
3. The method for liquid-phase synthesis of a linker according to claim 2, wherein the condensing agent is selected from any one or two or more of N, N ' -diisopropylcarbodiimide, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline, N ' -dicyclohexylcarbodiimide, N ' -carbonyldiimidazole, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; preferably N, N' -dicyclohexylcarbodiimide and 1-hydroxybenzotriazole;
the organic solvent is any one or mixture of several of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol and chloroform, preferably DMF;
the adopted alkali is any one or a mixture of a plurality of triethylamine, N-diisopropylethylamine, sodium bicarbonate, sodium carbonate or DBU with any proportion, preferably N, N-diisopropylethylamine;
preferably, the molar ratio of Fmoc-L-Cit, p-aminobenzyl alcohol and condensing agent is 1:1-1.3:2-2.8;
preferably, the molar ratio of Fmoc-L-Cit, said para-aminobenzyl alcohol, said N, N' -dicyclohexylcarbodiimide and said 1-hydroxybenzotriazole is 1:1-1.3:1-1.5:1-1.3, preferably 1:1.05:1.2:1.3;
preferably, the temperature of the condensation reaction is from-10 to 0 ℃, for a time of 10-30 hours, preferably at-10 ℃; the time was 18 hours.
4. The method of liquid phase synthesis of a linker according to claim 1, wherein the step of forming intermediate B comprises: removing Fmoc from the intermediate A by using an organic base;
preferably, the step of forming intermediate B comprises: mixing the intermediate A, the organic base and a solvent for reaction;
preferably, the method further comprises: and after the reaction is finished, carrying out post-treatment, wherein the post-treatment comprises the following steps: the solvent was removed by concentration, then the pH was adjusted and extraction was performed with ethyl acetate, followed by collection of the aqueous phase.
5. The method for liquid phase synthesis of a linker according to claim 4, wherein the organic base comprises any one of piperidine, diazabicyclo, diethylamine, ethanolamine, cyclohexylamine and morpholine, preferably diethylamine;
the solvent comprises any one or a mixture of a plurality of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, DMF, methanol and chloroform in any proportion, and DMF is preferred;
preferably, the mass ratio of the intermediate A to the organic base is 1:0.5-3, preferably 1:2.5;
the reaction temperature is 0 to 40 ℃, preferably 25 ℃, for 1 to 8 hours, preferably 6 hours.
6. The method of liquid phase synthesis of a linker according to claim 1, wherein the step of forming intermediate C comprises: condensing Fmoc-L-Val-OH and an intermediate B through a condensing agent to perform condensation reaction;
preferably, the step of forming intermediate C comprises: mixing the Fmoc-L-Val-OH, the intermediate B, the condensing agent, a solvent and alkali for condensation reaction;
preferably, the method further comprises: after the condensation reaction is finished, post-treatment is carried out, wherein the post-treatment comprises: crystallizing the reaction liquid, filtering and drying.
7. The method for liquid-phase synthesis of a linker according to claim 6, wherein the condensing agent comprises: any one or two or more of N, N '-diisopropylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N' -dicyclohexylcarbodiimide, N-hydroxysuccinimide, 1-hydroxybenzotriazole, p-nitrophenol and pentafluorophenol; preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with 1-hydroxybenzotriazole;
the solvent comprises any one or a mixture of a plurality of ethyl acetate, dichloromethane, tetrahydrofuran, water, acetonitrile, DMF, dichloromethane and chloroform in any proportion, and DMF is preferred;
the base comprises any one of triethylamine, N-diisopropylethylamine, sodium bicarbonate, sodium carbonate and DBU, preferably triethylamine;
preferably, the molar ratio of Fmoc-L-Val-OH, intermediate B and condensing agent is 1:1-1.3:2-2.8;
preferably, the molar ratio of Fmoc-L-Val-OH, intermediate B, 1-hydroxybenzotriazole and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1-1.3:1-1.3:1 to 1.5; the most preferred is 1:1.05:1.1:1.2;
preferably, the reaction temperature is from-10 ℃ to 0 ℃, preferably from-5 ℃; the reaction time is 10 to 20 hours, preferably 15 hours.
8. The method for liquid-phase synthesis of a linker according to claim 1, comprising: performing transesterification reaction on the intermediate C and di (p-nitrobenzene) carbonate under alkaline conditions;
preferably, it comprises: mixing the intermediate C, the di (p-nitrobenzene) carbonate, alkali and a solvent for transesterification;
preferably, it comprises: after the transesterification reaction, the reaction solution is added into an ester solvent for crystallization, and then filtered and dried.
9. The method for liquid phase synthesis of a linker according to claim 8, wherein the base comprises any one of triethylamine, N-diisopropylethylamine, pyridine, N-methylmorpholine and DBU; preferably N-methylmorpholine;
the solvent comprises any one or a mixture of a plurality of 1, 4-dioxane, dimethyl sulfoxide, DMF, tetrahydrofuran, dichloromethane and chloroform in any proportion, and is preferably 1, 4-dioxane;
preferably, the molar ratio of intermediate C, di (p-nitrophenyl) carbonate and base is 1:1-4:1-4, most preferably 1:1.5:2.5;
preferably, the temperature is from-10 to 30 ℃, most preferably 25 ℃, and the reaction time is from 2 to 8 hours, most preferably 7 hours.
10. A liquid phase synthesis product, characterized in that it is prepared by the liquid phase synthesis method of the linker according to any one of claims 1 to 9.
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