CN111588847A - Conjugate containing monophosphorylated lipid A and saccharide antigen and its prepn and application - Google Patents
Conjugate containing monophosphorylated lipid A and saccharide antigen and its prepn and application Download PDFInfo
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Abstract
The invention relates to a conjugate containing monophosphorylated lipid A and saccharide antigen, and a preparation method and application thereof, and belongs to the technical field of development of anti-tumor saccharide vaccines. The conjugate of the monophosphorylated lipid A and the carbohydrate antigen is a compound with a general formula (I) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound with the general formula (I). The monophosphorylated lipid A can improve the immunogenicity of Tn saccharide antigen, and the Tn saccharide antigen is presented to corresponding immunocyte to generate higher-titer immunological reaction specific to tumor saccharide antigen Tn, so that the conjugate is used as a fully synthetic oneThe saccharide antigen vaccine is expected to become a new generation of antitumor drugs;
wherein: n is an integer of 2 to 6; r1And R3Is- (CH)2)mCH3M is an integer of 10 to 14; r2、R4And R5Is- (CH)2)pCH3P is an integer of 8 to 12; r6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
Description
Technical Field
The invention relates to a conjugate containing monophosphorylated lipid A and saccharide antigen, and a preparation method and application thereof, and belongs to the technical field of development of anti-tumor saccharide vaccines.
Background
The tumor vaccine has better clinical application prospect in preventing and treating cancer, and the tumor carbohydrate vaccine taking Tumor Associated Carbohydrate Antigens (TACAs) abnormally expressed on the surface of tumor cells as targets has the advantages of high specificity, small side effect, better curative effect and the like. The Thomsenouveau (Tn) antigen is abnormally over-expressed on the surfaces of malignant tumor cells of breast cancer, prostatic cancer, lung cancer and the like, and is an excellent target point for designing a carbohydrate antigen tumor vaccine.
Carbohydrate antigens are inherently poorly immunogenic and require covalent attachment to an immunologically active carrier molecule to function, the most mature and commonly used carrier being a protein. The commonly used protein carriers are KLH, BSA, DT, CRM197 and TT, and these synthetic glycoprotein vaccines suffer from the following disadvantages: uncertain coupling sites, unstable coupling rate, complex composition and epitope inhibition effect caused by protein.
To avoid these disadvantages, fully synthetic saccharide antigen vaccines incorporating built-in adjuvants have become a new strategy. Fully synthetic glycolipid vaccines can remove unwanted immunogenic components and contain only those elements necessary to elicit an effective immune response. Typically, lipid adjuvants (e.g., lipopeptide-based or lipoamino acid-based TLR ligands) are incorporated into vaccine constructs and are referred to as endogenous adjuvants, such as agonists of various subtypes of TLRs (Toll-like receptors) and KRN7000 agonists that can stimulate iNKT immune cells, among others.
Bacterial Lipopolysaccharides (LPS) are surface glycolipids of the bacterial outer membrane. Lipid a, the hydrophobic part of LPS, is a ligand for Toll-like receptor 4(TLR4) and can act as an adjuvant to produce anti-cancer effects by initiating a strong Th1 response. However, due to its high toxicity, Lipid A cannot be clinically used. Researchers find that MPLA (monophosphoryl Lipid A) obtained by removing phosphate at position 1 in the Lipid A structure (the reaction formula is shown as the following formula) can still be combined with TLR4 in a targeted way, the toxicity is obviously reduced, the activity is not obviously changed,
MPLA is used as an adjuvant in clinical trials for many different types of cancer, such as stage IV melanoma, ovarian cancer, lung cancer, thrombocytopenia, leukemia, sarcoma, merkel cell carcinoma and non-hodgkin's lymphoma. OM-174, a diacylated lipid a analog, has been clinically tested in patients with refractory solid tumors and has been shown to be well tolerated.
GUO topic group made a lot of studies on MPLA as a fully synthetic tumor vaccine inline adjuvant, mainly by combining MPLA with various saccharide antigens to prepare antibacterial and antitumor glycoconjugate vaccines, such as GM3-MPLA, MPLA-sTn, GM2-MPLA and other saccharide antigen vaccines, immunological studies showed that the saccharide antigen vaccine mainly induces the production of IgG antibodies, and the conjugate vaccine still elicited strong immune response without the assistance of external adjuvant, indicating its self-adjuvanting property (Chemical Biology,2012,7: 235; Scientific reports,2017, 11403; biomolecular chemistry,2014,12: 3238). In particular, the Guo subject combined MPLA-Globo H, a conjugate of Globo H and optimized MPLA, produced IgG antibodies about 2-fold stronger than Globo H-KLH (CFA as adjuvant) more rapidly without the addition of adjuvant. Thus, MPLA proved to be a completely new and powerful inline adjuvant designed for use in a fully synthetic glycoconjugate cancer vaccine (Chemical Science,2015,6: 7112.).
The invention uses phosphorylated lipid A (MPLA) as an embedded adjuvant to conjugate sugar antigen Tn to obtain a conjugate of monophosphorylated lipid A and sugar antigen, and the conjugate can be used as a vaccine and can effectively prevent and/or treat various cancers.
Disclosure of Invention
The present invention aims to overcome the disadvantages of the prior art and to provide a conjugate comprising monophosphorylated lipid a and a saccharide antigen.
In order to achieve the purpose, the invention adopts the technical scheme that: a conjugate comprising a monophosphorylated lipid a and a saccharide antigen, wherein the conjugate is a compound of formula (i) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of formula (i);
wherein:
n is an integer of 2 to 6;
R1and R3Is- (CH)2)mCH3M is an integer of 10 to 14;
R2、R4and R5Is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
The invention uses monophosphoryl lipid A (MPLA) as an embedded adjuvant to conjugate with a carbohydrate antigen Tn to obtain a conjugate of monophosphoryl lipid A and the carbohydrate antigen, wherein the MPLA can overcome the defect of poor immunogenicity of the Tn carbohydrate antigen, and the Tn carbohydrate antigen is presented to corresponding immune cells to cause specific immune reaction aiming at the carbohydrate antigen Tn, thereby achieving the purpose of killing tumor cells; the conjugate can be used as vaccine, and can effectively prevent and/or treat various cancers.
As a preferred embodiment of the conjugate of the present invention, the conjugate is a compound of structural formula (II) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of structural formula (II);
wherein:
R1and R3Is- (CH)2)mCH3M is an integer of 10 to 14;
R2、R4and R5Is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
As a preferred embodiment of the conjugate of the present invention, the conjugate is a compound of structural formula (III) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of structural formula (III);
wherein: n is an integer of 2 to 6.
As a preferred embodiment of the conjugate of the present invention, the conjugate is a compound of structural formula (IV) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of structural formula (IV);
as a preferred embodiment of the conjugate according to the invention, the monophosphorylated lipid a is a compound of general formula (v) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of a compound of general formula (v);
wherein:
R5is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
The present invention also provides pharmaceutically acceptable salts, hydrates or solvates of the compounds of formulae (I), (II), (III) and (IV), including but not limited to those formed by reaction with a base such as sodium, magnesium, potassium, calcium, lithium and the like. The compounds of formulae (I), (II), (III) and (IV) provided by the present invention may be crystallized or recrystallized from hydrates or organic solvents, in which case various solvates may be formed.
It is another object of the present invention to provide a method for preparing the conjugate, comprising the steps of:
(1) dissolving the compound 1 and the compound 2 in an organic solvent, and adding a catalyst to react to obtain a compound 3;
(2) obtaining a compound 4 from the compound 3 in the step (1) under the catalytic action of ethylenediamine;
(3) taking the compound 4 in the step (2) and fatty acid chains, and carrying out peptide-forming and ester-forming reactions under the condition of a condensing agent to obtain a compound 5;
(4) dissolving the compound 5 in the step (3) in an organic solvent, adding a catalyst, and carrying out reduction reaction to obtain a compound 6;
(5) dissolving the compound 6 in the step (4) in an organic solvent, adding a catalyst, and carrying out a phosphitylation reaction to obtain a compound 7;
(6) dissolving a compound 8 and the compound 7 in the step (5) in an organic solvent, and adding a catalyst to react to obtain a compound 9;
(7) dissolving the compound 9 in the step (6) in an organic solvent, adding a catalyst, and carrying out debenzylation reaction to obtain the conjugate;
the structural formulae of the compound 1 to the compound 9 are shown below:
wherein:
R0is STol, SPh, Set or OC (NH) CCl3；
n is an integer of 2 to 6;
R1and R3Is- (CH)2)mCH3M is an integer of 10 to 14;
R2、R4and R5Is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
The reaction formula of the preparation method is as follows:
wherein:
R0is STol, SPh, Set or OC (NH) CCl3；
n is an integer of 2 to 6;
R1and R3Is- (CH)2)mCH3M is an integer of 10 to 14;
R2、R4and R5Is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
The preparation method of the invention uses phosphorylated lipid A (MPLA) as an embedded adjuvant to conjugate with a saccharide antigen Tn to obtain the MPLA-Tn tumor vaccine, and the preparation method has the advantages of short synthetic route, mild reaction condition, high yield and convenient operation, and can be used for industrial preparation.
In the step (1) of the preparation method, the compound 1 and the compound 2 react under the conditions of a catalyst and an organic solvent to obtain a coupling product compound 3.
As a preferred embodiment of the production process of the present invention, in step (1), when R is0In the case of STol, SPh or Set, the catalyst is N-iodosuccinimide and any one selected from trifluoromethanesulfonic acid, silver trifluoromethanesulfonate, boron trifluoride diethyl etherate, trimethylsilyl trifluoromethanesulfonate, or when R is0Is OC (NH) CCl3When the catalyst is selected from one of trifluoromethanesulfonic acid, boron trifluoride diethyl etherate and trimethylsilyl trifluoromethanesulfonate; the organic solvent is any one of dichloromethane, diethyl ether and tetrahydrofuran; the temperature of the reaction is-40 to-20 ℃.
More preferably, in step (1), when R is0Is STol, and the catalyst is a mixture of N-iodosuccinimide (NIS) and a catalyst of trifluoromethanesulfonic acid; the organic solvent is dichloromethane.
In the step (2) of the preparation method, the compound 3 is catalyzed by ethylenediamine to remove phthaloyl at C-2 position, phthaloyl at C-2 'position and acetyl at C-3' position, and a compound 4 with naked amino and hydroxyl is obtained.
In the step (3) of the preparation method of the present invention, the compound 4 and the fatty acid chain undergo peptide-forming and ester-forming reactions under the condition of a condensing agent to obtain a compound 5; in a preferred embodiment of the production method of the present invention, in the step (3), the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methiodide.
In the step (4) of the preparation method, the compound 5 is selectively reduced to the sugar hydroxyl at the 4-position under the action of a catalyst to obtain a compound 6; in a preferred embodiment of the preparation method of the present invention, in the step (4), the catalyst is a mixture of triethylsilane and trifluoromethanesulfonic acid, and the organic solvent is any one selected from dichloromethane, methanol, and tetrahydrofuran.
In the step (5) of the preparation method, the compound 6 in the step (4) is dissolved in an organic solvent, a catalyst is added, and a compound 7 is obtained through a phosphitylation reaction; as a preferred embodiment of the preparation method of the present invention, in the step (5), the organic solvent is a mixed solution of dichloromethane and acetonitrile; the catalyst is a mixture of dibenzyl diisopropyl phosphoramidite, triazole and tert-butyl peroxide.
As a preferred embodiment of the preparation method of the present invention, in step (6), compound 7 and compound 8 are dissolved in an organic solvent, and compound 9 is obtained by a click reaction under the action of a catalyst; the organic solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of cuprous iodide, N-diisopropylethylamine and glacial acetic acid.
As a preferred embodiment of the preparation method of the present invention, in step (7), compound 9 is dissolved in an organic solvent and reacted under the action of a catalyst to obtain compound 10; the catalyst is a mixture of hydrogen, palladium carbon and palladium hydroxide.
The conjugate of another object of the present invention is used for preparing a medicament for preventing and/or treating cancer.
In a preferred embodiment of the use of the present invention, the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell carcinoma, cell lymphoma, a cancer of the nail, brain cancer, stomach cancer or leukemia.
Compared with the prior art, the invention has the beneficial effects that:
(1) the conjugate containing monophosphorylated lipid A and carbohydrate antigen is obtained by taking monophosphorylated lipid A (MPLA) as an embedded adjuvant to conjugate carbohydrate antigen Tn, wherein MPLA can overcome the defect of poor immunogenicity of the Tn carbohydrate antigen, and the Tn carbohydrate antigen is presented to corresponding immune cells to cause specific immune reaction aiming at the carbohydrate antigen Tn, so that the aim of killing tumor cells is fulfilled; the conjugate can be used as a vaccine and can effectively prevent and/or treat various cancers, including breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostatic cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell carcinoma, cell lymphoma, cancer of the nail gland, brain cancer, gastric cancer or leukemia.
(2) MPLA can improve the immunogenicity of Tn sugar antigen, presents the Tn sugar antigen to corresponding immune cells, generates an immune response with higher titer specific to the tumor sugar antigen Tn, and the titer of IgG antibody generated by the MPLA induction and the capacity of specific antigen recognition are both obviously higher than those generated by glycoprotein vaccine CRM197-Tn induction; therefore, the conjugate containing the monophosphorylated lipid A (MPLA) and the saccharide antigen provided by the invention is expected to become a new generation of antitumor drugs as a fully synthetic saccharide antigen vaccine.
(3) The preparation method of the conjugate containing the monophosphorylated lipid A and the carbohydrate antigen provided by the invention has the advantages of short synthetic route, mild reaction conditions, high yield and convenience in operation, and can be used for industrial preparation.
Drawings
FIG. 1 is a graph showing the evaluation of the antibody immunity of MPLA-Tn of a glycoprotein vaccine of example 1 of the present invention and CRM197-Tn of a glycoprotein vaccine of comparative example 1;
FIG. 2 is a flow cytometry experiment evaluation chart of tumor cell MCF-7 specifically recognized by antibody serum generated by mice induced by the carbohydrate vaccine MPLA-Tn of example 1 and the glycoprotein vaccine CRM197-Tn of comparative example 1.
Detailed Description
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.
Example 1
This example is a conjugate containing monophosphorylated lipid a and a saccharide antigen provided by the present invention, where the structural formula of the conjugate is shown in formula (iv):
the preparation method of the conjugate containing the monophosphorylated lipid A and the carbohydrate antigen comprises the following steps:
(1) dissolving the compound 1 and the compound 2 in an organic solvent, and adding a catalyst to react to obtain a compound 3; the reaction scheme to give compound 3 is shown below:
the specific operation of the step (1) is as follows: anhydrous dichloromethane (10.0mL) dissolved the vacuum dried compound 1(0.4g, 0.8mmol), compound 2(0.3g, 0.5mmol) and the high temperature dried molecular sieve (2.0g), and stirred under nitrogen for 4 hours; cooling to-30 ℃, quickly adding N-iodosuccinimide (360.0mg, 1.6mmol), stirring at-30 ℃ for reaction for 1 hour, cooling the reaction liquid to-40 ℃, quickly adding trifluoromethanesulfonic acid (11.9 mu L, 130.0 mu mol), stirring for reaction for 15 minutes, adding saturated sodium bicarbonate solution for neutralization, and adding sodium thiosulfate aqueous solution until the reaction liquid is red and faded. The aqueous layer was removed and the organic layer was collected, washed with water 2 times, washed with saturated brine 1 time, and the organic layer was collected, dried over anhydrous sodium sulfate, and the organic solution was distilled off under reduced pressure to obtain a crude product, which was purified by column chromatography to give compound 3(430.0g, 82.0%) as a colorless viscous substance.1H NMR(400MHz,CDCl3)7.67–6.75(m,23H,Ar-H),5.86(t,1H),5.59–5.54(d,J＝8.8Hz,1H,H-1),5.55(s,1H,),5.10(d,J＝8.7Hz,1H,H-1),4.64(dd,J＝37.0,11.6Hz,2H),4.51–4.29(m,4H),4.20(t,1H),4.07(d,J＝6.7Hz,2H),3.96–3.65(m,5H),3.63–3.40(m,3H),3.30–3.15(m,3H),3.06-2.98(d,1H),1.89(s,3H,-CO-CH3).13CNMR(100MHz,CDCl3)170.27,137.70,137.40,136.91,134.24,133.66,129.19,128.45,128.28,128.06,127.98,127.89,127.40,126.26,123.56,123.21,101.67,98.40(C-1),98.08(C-1’),79.48,79.22,79.07,74.93,74.83,74.64,69.83,68.69,68.19,68.05,66.31,55.50,55.27,50.36,20.62.
(2) Obtaining a compound 4 from the compound 3 in the step (1) under the catalytic action of ethylenediamine; the reaction scheme to give compound 4 is shown below:
the specific operation of the step (2) is as follows: methanol (30.0mL) dissolved compound 3(500.0mg, 0.5mmol), ethylenediamine (5.0mL) was added dropwise, the reaction was heated to 80 ℃ and refluxed overnight, the mixture was cooled to room temperature, after the solvent was removed, toluene (4.0mL) was added to remove excess ethylenediamine to give a yellow oily liquid, and the crude product was purified by silica gel column separation to give compound 4(240.0mg, 70.0%) as a white solid.
1H NMR(400MHz,CDCl3)7.57–7.32(m,15H,Ar-H),5.56(s,1H),4.94(dd,J＝34.8,11.1Hz,2H,Ar-CH2-O-),4.72(dd,J＝32.9,11.1Hz,2H,Ar-CH2-O-),4.39–4.21(m,3H),4.21–4.02(m,2H),3.91–3.20(m,11H),2.89(m,J＝16.9,8.1Hz,2H,-CH2-N3).13C NMR(100MHz,CDCl3)138.14,137.77,137.10,129.31,128.65,128.58,128.39,128.33,128.05,128.02,127.93,127.89,127.87,126.29,104.96,103.95(C-1),101.94(C-1’),84.93,81.34,78.98,77.38,77.26,77.06,76.74,75.55,74.99,74.93,73.28,68.93,68.80,68.74,66.51,57.74,56.94,50.76.
(3) Taking the compound 4 in the step (2) and fatty acid chains, and carrying out peptide-forming and ester-forming reactions under the condition of a condensing agent to obtain a compound 5; the reaction scheme to give compound 5 is shown below:
the specific operation of the step (3) is as follows: under the protection of nitrogen, the compound 4(100.0mg, 150.0 mu mol), the self-made fatty acid (300.0mg, 670.0 mu mol) and the 4-dimethylaminopyridine (1.0mg, 4.0 mu mol) are dissolved in dichloromethane, the mixture is stirred for reaction, the temperature of the mixed solution is reduced to 0 ℃, and the catalyst 1-(3-dimethylaminopropyl) -3-ethylcarbodiimide iodonium salt (220.0mg, 740.0. mu. mol) was reacted with stirring for 2 hours, the reaction mixture was diluted with dichloromethane, washed with saturated brine for 3 times, the organic layer was collected, dried over anhydrous sodium sulfate, and the organic solution was removed to obtain a crude product, which was then separated and purified by a silica gel column to obtain Compound 5(160.0mg, 56.0%) as a white solid.1H NMR(400MHz,CDCl3)7.58–7.08(m,15H),6.12(t,J＝8.4Hz,2H),5.48(s,1H),5.44–5.24(m,2H),5.20-5.09(d,J＝42.0Hz,2H),4.87(d,J＝7.3Hz,1H,H-1),4.77(dd,J＝16.2,9.8Hz,2H),4.66(d,J＝11.0Hz,1H),4.58(d,J＝10.6Hz,1H),4.28(s,1H),4.15–3.82(m,4H),3.81–3.39(m,8H),3.33(d,J＝11.8Hz,1H),2.60–2.00(m,12H),1.80–1.47(m,12H),1.25(s,108H),0.87(d,J＝6.3Hz,18H).13C NMR(100MHz,CDCl3)173.89,173.73,173.49,170.31,170.18,170.07,138.17,137.84,136.87,129.04,128.42,128.35,128.14,127.88,127.79,127.73,127.66,126.11,101.45,101.38,99.75,80.97,78.78,78.77,78.17,78.17,74.62,74.53,74.43,71.67,71.22,70.96,69.98,68.52,68.14,67.44,66.20,55.70,54.35,50.70,41.46,41.10,39.00,34.47,34.27,33.95,33.85,31.89,29.66,29.62,29.54,29.50,29.47,29.36,29.33,29.29,29.26,29.19,29.10,29.07,25.55,25.23,25.00,24.95,24.92,22.65,14.04.ESI-TOF HRMS m/z:calcdfor C119H199N5O18,[M+Na]+:2009.4702,found:2009.4637.
(4) Dissolving the compound 5 in the step (3) in an organic solvent, adding a catalyst, and carrying out reduction reaction to obtain a compound 6; the reaction scheme to give compound 6 is shown below:
the specific operation of the step (4) is as follows: dichloromethane (10mL) dissolved compound 5(180.0mg, 90.1. mu. mol) and molecular sieve (1.0g), closed stirring for 15 minutes, cooling to-78 ℃, adding triethylsilane (52.0. mu.L, 326.4. mu. mol) and trifluoromethanesulfonic acid (24. mu.L, 271.8. mu. mol), stirring to react for 60 minutes, adding 1.0mL of a mixed solution of triethylamine and methanol (1: 10), quenching, filtering to remove gray insoluble substances, removing organic solvent to obtain a crude gray solid, and separating and purifying with silica gel column to obtain compound 6(110.8mg, 61.57%) as a white solid.
1H NMR(400MHz,CDCl3)7.57–7.13(m,15H),6.07(d,J＝8.0Hz,1H),6.00(d,J＝8.7Hz,1H),5.19–5.08(m,2H),5.08–4.93(m,2H),4.83(d,J＝7.6Hz,1H),4.78–4.41(m,7H),4.04(m,J＝18.1,9.8Hz,2H),3.98–3.84(m,2H),3.79–3.58(m,6H),3.56–3.48(m,2H),3.47–3.39(m,2H),3.32–3.24(m,2H),2.62–2.14(m,12H),1.56(s,12H),1.25(s,108H),0.88(t,J＝6.7Hz,18H).13C NMR(100MHz,CDCl3)174.26,173.70,173.40,171.42,169.96,169.54,138.32,137.96,137.90,128.42,128.38,127.85,127.82,127.71,127.67,127.63,101.10,99.53,80.60,78.17,77.28,76.14,74.83,74.46,74.39,74.30,73.60,71.05,70.98,70.92,70.18,70.05,67.92,67.46,55.94,53.73,50.73,41.71,41.50,40.08,34.70,34.52,34.49,34.13,34.09,31.94,29.74,29.70,29.67,29.65,29.61,29.57,29.54,29.52,29.41,29.38,29.31,29.25,29.16,25.32,25.28,25.15,25.05,24.99,24.97,22.70,14.13.ESI-TOF HRMS m/z:calcdfor C119H201N5O18,[M+Na]+:2011.4859,found:2011.4877.
(5) Dissolving the compound 6 in the step (4) in an organic solvent, adding a catalyst, and carrying out a phosphitylation reaction to obtain a compound 7; the reaction scheme to give compound 7 is shown below:
the specific operation of the step (5) is as follows: under the protection of nitrogen, dissolving a compound 6(80.0mg, 40.2 mu mol) in a dichloromethane acetonitrile mixed solution, adding dibenzyl diisopropyl phosphoramidite (300.0 mu L, 913.0 mu mol) and triazole (1.3mL, 913.0 mu mol), stirring for reacting for 3 hours, cooling the mixed solution to 0 ℃, slowly dropwise adding tert-butyl peroxide (280.0 mu L, 1540.0 mu mol), slowly heating the reaction solution to room temperature, and stirring for reacting for 1 hour; the solvent was removed to give a crude product, which was purified by silica gel column separation to give compound 7(74.0mg, two-step yield 81.8%) as a white solid.1H NMR(400MHz,CDCl3)7.43–7.15(m,25H),6.18(d,J＝7.8Hz,1H),5.97(d,J＝8.1Hz,1H),5.41(t,J＝9.6Hz,1H),5.13(m,J＝17.5,6.0,4.9Hz,3H),4.89(d,J＝7.8Hz,1H),4.83(d,J＝7.9Hz,4H),4.73(t,J＝10.2Hz,1H),4.64–4.37(m,7H),4.12–3.91(m,3H),3.81–3.71(m,2H),3.71–3.41(m,8H),3.27(m,J＝13.5,4.1Hz,1H),2.52–2.10(m,12H),1.57(t,J＝6.7Hz,12H),1.24(t,J＝4.3Hz,108H),0.88(t,J＝6.7Hz,18H).13C NMR(101MHz,CDCl3)173.75,173.56,173.43,170.17,170.04,169.94,138.34,138.21,137.90,135.63,135.56,135.53,128.58,128.47,128.41,128.33,128.06,127.97,127.93,127.88,127.69,127.66,127.59,127.51,100.15,99.57,80.67,78.26,77.37,77.25,77.05,76.83,76.74,74.81,74.47,74.39,74.04,73.33,72.78,71.05,70.65,70.23,69.64,69.55,69.50,68.67,68.34,67.69,56.18,55.69,50.73,41.71,41.19,39.65,34.52,34.32,34.09,31.96,29.75,29.71,29.66,29.61,29.59,29.56,29.54,29.47,29.41,29.39,29.33,29.30,29.24,29.17,25.29,25.24,25.15,25.06,25.00,22.72,14.14.ESI-TOF HRMS m/z:calcdfor C133H214N5O21P,[M+Na]+:2271.5461,found:2271.5423.
(6) Dissolving a compound 8 and the compound 7 in the step (5) in an organic solvent, and adding a catalyst to react to obtain a compound 9; the reaction scheme to give compound 9 is shown below:
the specific operation of the step (6) is as follows: tetrahydrofuran and methanol (1: 2, 3.0mL) dissolved compound 7(68.0mg, 30.2. mu. mol), compound 8(10.0mg, 20.4. mu. mol), cuprous iodide (195.0mg, 1.0mmol), added N, N-diisopropylethylamine (168.0. mu.L, 1.0mmol), and stirred at room temperature for 12 hours; filtering out insoluble substances with diatomite, and distilling the filtrate under reduced pressure to remove the solvent to obtain a crude product; purification on silica gel column gave compound 9(20.0mg, 35.7% yield) as a white solid.1H NMR(600MHz,CDCl3)7.98(d,J＝26.3Hz,1H),7.78–7.48(m,1H),7.28(td,J＝23.6,22.7,8.3Hz,29H),6.81(s,1H),5.45(d,J＝9.9Hz,1H),5.25–5.06(m,2H),4.93(d,J＝9.3Hz,4H),4.71(m,J＝29.3,19.0Hz,5H),4.60–4.26(m,4H),4.26–3.34(m,22H),2.68–1.91(m,18H),1.49(s,12H),1.26(s,110H),0.89(q,J＝7.4,6.8Hz,18H).13C NMR(151MHz,CDCl3)173.79,173.65,173.49,171.21,171.04,170.32,170.17,138.17,138.04,137.77,135.47,135.42,128.63,128.58,128.50,128.43,128.36,128.13,127.99,127.89,127.72,127.63,127.57,100.25,100.23,99.30,80.97,78.07,74.50,74.41,74.09,73.72,73.22,72.52,71.10,70.54,70.42,70.01,69.77,69.74,69.61,69.57,69.40,68.68,68.41,67.37,62.60,56.59,55.68,55.25,51.32,41.54,41.06,39.44,39.13,34.54,34.50,34.43,34.29,34.12,31.94,29.74,29.70,29.65,29.59,29.53,29.47,29.41,29.38,29.33,29.29,29.22,29.18,25.29,25.14,25.04,23.08,22.71,17.99,14.13.ESI-TOF HRMS m/z:calcdfor C154H249N8O31P,[M+H]+:2738.7964,found:2738.7970.
(7) Dissolving the compound 9 in the step (6) in an organic solvent, adding a catalyst, and performing debenzylation reaction to obtain the conjugate containing the monophosphorylated lipid A and the carbohydrate antigen; the reaction scheme for obtaining conjugate (II) (MPLA-Tn) is shown below:
the specific operation of the step (7) is as follows: dichloromethane/methanol/water (5: 5: 1, 10.0mL) dissolved compound 9(8.0mg), palladium hydroxide (5.0mg) and palladium on carbon (5.0mg) were added, hydrogen gas was introduced, the mixture was stirred under sealed conditions for 24 hours, the insoluble matter was filtered off with celite, the mixture was washed three times with 30.0mL dichloromethane/methanol/water (5: 5: 1), and the filtrate was distilled under reduced pressure to remove the solvent, whereby compound 10 was obtained as a white solid, i.e., MPLA-Tn (5.5mg, 83.3%) as the conjugate.1H NMR(400MHz,MeOD/CDCl3/D2O(20：30：1，v/v/v，0.6mL))3.68(s,8H),3.11(d,J＝92.4Hz,28H),2.14(s,18H),1.81–1.50(m,12H),1.43–1.09(m,110H),0.90(m,J＝14.1,12.5Hz,18H).ESI-TOF HRMS m/z:calcdfor C119H219N8O31P,[M+K++Na+]2+:1174.7534,found:1174.7434.
Comparative example 1
The comparative example is the CRM197-Tn glycoprotein vaccine provided by the invention, and the structural formula is shown as follows:
the preparation method of the CRM197-Tn glycoprotein vaccine comprises the following steps:
(1) reducing azide into amino to obtain a compound 11 by using the monosaccharide compound 10 under the catalysis of palladium carbon and acetic acid; reacting the compound 11 with bis (N-hydroxysuccinimide) suberate in a dimethylformamide solution to obtain a compound 12; the method specifically comprises the following steps: dissolving compound 10(100mg, 0.244mmol) with methanol (5mL), adding palladium carbon (100mg) and acetic acid (0.01mL), sealing, introducing hydrogen to replace gas for 5 times, sealing, and stirring for 20 hr; insoluble matter was filtered off with celite, and the filtrate was distilled off under reduced pressure to remove the solvent to give a white solid, which was dissolved with water and then pre-frozen at-80 ℃ for 6 hours in a refrigerator, and transferred to a lyophilizer to be lyophilized to give compound 11(86mg, 73%) as a white solid. Reacting the compound 11 with bis (N-hydroxysuccinimide) suberate (57.5mg, 36.6mmol) in a dimethylformamide solution for 5 hours, distilling under reduced pressure to remove the organic solvent to obtain a crude product, washing the solid with ethyl acetate for 8 times to obtain a white solid compound 12(7 mg);
(2) compound 12 was coupled to CRM197 protein in 0.1mol PBS (pH 7.8) buffered saline to give a Tn-CRM197 glycoprotein vaccine; the method specifically comprises the following steps: compound 12 was dissolved in 0.1mol PBS (pH 7.8) buffer solution, CRM197 protein was added, the reaction was stirred at room temperature for 2.5 days, the reaction solution was transferred to a dialysis bag for dialysis for 2 days, distilled water was changed every 6 hours, and finally the suspension inside the dialysis bag was transferred to a sample bottle, pre-frozen at-80 ℃ for 6 hours, and lyophilized with a lyophilizer to obtain a white solid compound Tn-CRM197(5 mg).
The reaction formula of the preparation method is as follows:
experimental example 1
In this experimental example, mice were immunized with the total synthetic saccharide vaccine (MPLA-Tn) prepared in example 1 and the glycoprotein vaccine (CRM197-Tn) prepared in comparative example 1, and the immunization was preliminarily evaluated by ELSA, and it was confirmed that the antibody serum specifically recognized tumor cells (MCF-7) by fluorescence-activated cell sorting (FACS) technique.
ELISA immunoassay
(1) Mouse immunization:
12C 57BL/6 mice, 6-8 weeks old, were randomly divided into 2 groups. After the total synthetic saccharide vaccine MPLA-Tn and the glycoprotein vaccine CRM197-Tn are respectively prepared into liposome, an immune test is carried out by a mouse subcutaneous injection mode, the prepared vaccine is respectively injected on 0 th, 14 th, 21 th and 28 th days by adopting a scheme of primary immunity and three times of enhanced immunity, and the injection amount of each injection is 0.1mL (the MPLA-Tn saccharide vaccine contains 6 mug Tn antigen); on day 38, each mouse was bled from 0.1mL to 0.2mL, placed at 0 ℃ for 60 minutes, centrifuged at 4000 rpm for 15 minutes, and the supernatant clear serum was taken for ELISA assay.
(2) ELISA immunoassay:
Tn-BSA was dissolved in 0.1M carbonate buffer (pH 9.6) to prepare a solution of 2.0. mu.g/mL, and 100.0. mu.L/well was added to a 96-well plate, and the plate was incubated overnight at 4 ℃; incubating the incubator at 37 ℃ for one hour the next day; the plates were washed 3 times (300. mu.L/well/time) with PBST (PBS + 0.05% Tween-20). After washing, PBS/1% BSA was added, 250.0. mu.l/well, incubated at room temperature for one hour, and washed 3 times with PBST. Serum samples from 6 mice per group were mixed in equal amounts and diluted 300, 900, 2700, 8100, 24300, 72900, 218700 and 656100 fold with PBS; adding the diluted serum into a 96-well plate at a volume of 100.0 mu L per well, and making three auxiliary wells in parallel for each dilution gradient; incubate at 37 ℃ for two hours in an incubator and wash the plate 3 times. HRP (horseradish peroxidase) -labeled IgG (diluted 2000-fold) was added to each well at 100.0. mu.L, incubated for one hour at room temperature, and the plate was washed 3 times. Adding TMB solution, adding 100.0 μ L per well, developing at room temperature in dark for 20 min, adding 0.5M H2SO4Solution, 100.0. mu.L per well. Immediately, the absorbance was measured by a microplate reader, and the measurement wavelength was 450nm, 570nm was used as the background wavelength.
(3) Absorbance (OD) values were plotted against antiserum dilution values and a best fit line was obtained. The equation of the line was used to calculate a dilution value at which the OD value reached 0.2, and the antibody titer was calculated from the reciprocal of the dilution value as shown in fig. 1.
(4) The experimental results are as follows:
as can be seen from FIG. 1, the MPLA-Tn saccharide vaccine synthesized in example 1 of the present invention, without any additional adjuvant, can generate an immune response specific to tumor saccharide antigen Tn in mice, and more rapidly generate a high-titer specific IgG antibody, and the IgG antibody titer is significantly higher than that of glycoprotein vaccine CRM 197-Tn.
2. Flow cytometry (FACS)
The experimental method comprises the following steps: culturing the breast cancer cell MCF-7 over-expressing the Tn sugar antigen and the tumor cell MDA-231 not expressing the Tn sugar antigen in MEM medium containing 10% Fetal Bovine Serum (FBS) (37 ℃, 5% CO 2); trypsinizing, collecting cells, counting the number of cells under a microscope, subpackaging 2.0 × 105 cells per tube, adding 1mL of 3% FBS-containing PBS buffer (FACS buffer) for resuspension, centrifuging for 2 minutes, removing supernatant, and washing twice with the FACS buffer; the prepared mouse serum was added, incubated for 1 hour in ice, washed twice with FACS buffer, a fluorescently labeled secondary antibody was added, incubated for one hour in ice away from light, washed twice with FACS buffer, resuspended in 0.8mL FACS buffer, and detected with flow cytometry, with the results shown in fig. 2.
The experimental results are as follows: as shown in FIG. 2, MCF-7 is a Tn antigen overexpressing breast cancer cell, and MDA-231 tumor cells that do not express the Tn antigen are negative controls. In MCF-7 cells, the antibody serum induced by the MPLA-Tn saccharide vaccine synthesized in example 1 of the present invention has a significant rightward shift of the fluorescence peak compared with the preimmune serum, and there is no significant difference between the antibody serum and the preimmune serum in MDA-231. The result shows that the antibody induced by the vaccine can specifically recognize MCF-7 cells expressing the Tn antigen, and the fluorescence peak of the antibody serum induced by the MPLA-Tn sugar vaccine is shifted to the right compared with the fluorescence peak of the antibody serum induced by the glycoprotein vaccine CRM197-Tn, so that the antibody specifically recognized by the MPLA-Tn sugar vaccine has stronger antigen capability.
In conclusion, experimental results show that the MPLA can improve the immunogenicity of Tn sugar antigen, the Tn sugar antigen is presented to corresponding immune cells, an immune response with higher titer specific to the tumor sugar antigen Tn is generated, and the titer of IgG antibody generated by the MPLA and the capacity of specific antigen recognition are obviously higher than those generated by glycoprotein vaccine CRM 197-Tn; therefore, the conjugate containing the monophosphorylated lipid A (MPLA) and the saccharide antigen provided by the invention is expected to become a new generation of antitumor drugs as a fully synthetic saccharide antigen vaccine.
Finally, it should be noted that the above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be regarded as equivalent substitutions and are included in the protection scope of the present invention.
Claims (10)
1. A conjugate comprising a monophosphorylated lipid a and a saccharide antigen, wherein the conjugate is a compound of formula (i) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of formula (i);
wherein:
n is an integer of 2 to 6;
R1and R3Is- (CH)2)mCH3M is an integer of 10 to 14;
R2、R4and R5Is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
2. The conjugate of claim 1, wherein the conjugate is a compound of structural formula (ii) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of a compound of structural formula (ii);
wherein:
R1and R3Is- (CH)2)mCH3M is an integer of 10 to 14;
R2、R4and R5Is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
3. The conjugate of claim 1, wherein the conjugate is a compound of structural formula (iii) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of a compound of structural formula (iii);
wherein: n is an integer of 2 to 6.
4. The conjugate of claim 3, wherein the conjugate is a compound of structural formula (IV) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of a compound of structural formula (IV);
5. the conjugate of any of claims 1-3, wherein the monophosphorylated lipid A is a compound of formula (V) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of a compound of formula (V);
wherein:
R5is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
6. A process for the preparation of a conjugate as claimed in any one of claims 1 to 5, comprising the steps of:
(1) dissolving the compound 1 and the compound 2 in an organic solvent, and adding a catalyst to react to obtain a compound 3;
(2) obtaining a compound 4 from the compound 3 in the step (1) under the catalytic action of ethylenediamine;
(3) taking the compound 4 in the step (2) and fatty acid chains, and carrying out peptide-forming and ester-forming reactions under the condition of a condensing agent to obtain a compound 5;
(4) dissolving the compound 5 in the step (3) in an organic solvent, adding a catalyst, and carrying out reduction reaction to obtain a compound 6;
(5) dissolving the compound 6 in the step (4) in an organic solvent, adding a catalyst, and carrying out a phosphitylation reaction to obtain a compound 7;
(6) dissolving a compound 8 and the compound 7 in the step (5) in an organic solvent, and adding a catalyst to react to obtain a compound 9;
(7) dissolving the compound 9 in the step (6) in an organic solvent, adding a catalyst, and carrying out debenzylation reaction to obtain the conjugate;
the structural formulae of the compound 1 to the compound 9 are shown below:
wherein:
R0is STol, SPh, Set or OC (NH) CCl3；
n is an integer of 2 to 6;
R1and R3Is- (CH)2)mCH3M is an integer of 10 to 14;
R2、R4and R5Is- (CH)2)pCH3P is an integer of 8 to 12;
R6is-CO (CH)2)rCH3Or- (CH)2)rCH3And r is an integer of 8 to 14.
7. The method according to claim 6, wherein in the step (1), when R is0In the case of STol, SPh or Set, the catalyst is N-iodosuccinimide and any one selected from trifluoromethanesulfonic acid, silver trifluoromethanesulfonate, boron trifluoride diethyl etherate, trimethylsilyl trifluoromethanesulfonate, or when R is0Is OC (NH) CCl3When the catalyst is selected from one of trifluoromethanesulfonic acid, boron trifluoride diethyl etherate and trimethylsilyl trifluoromethanesulfonate; the organic solvent is any one of dichloromethane, diethyl ether and tetrahydrofuran; the temperature of the reaction is-40 to-20 ℃;
in the step (3), the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methyl iodide salt;
in the step (4), the catalyst is a mixture of triethylsilane and trifluoromethanesulfonic acid, and the organic solvent is any one of dichloromethane, methanol and tetrahydrofuran;
in the step (5), the organic solvent is a mixed solution of dichloromethane and acetonitrile; the catalyst is a mixture of dibenzyl diisopropyl phosphoramidite, triazole and tert-butyl peroxide;
in the step (6), the organic solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of cuprous iodide, N-diisopropylethylamine and glacial acetic acid;
in the step (7), the organic solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of hydrogen, palladium carbon and palladium hydroxide.
8. The method according to claim 7, wherein in the step (1), when R is0Is STol, and the catalyst is a mixture of N-iodosuccinimide (NIS) and a catalyst of trifluoromethanesulfonic acid; the organic solvent is dichloromethane.
9. Use of a conjugate according to any one of claims 1 to 5 for the preparation of a medicament for the prophylaxis and/or treatment of cancer.
10. The use according to claim 8, wherein the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell carcinoma, cell lymphoma, a cancer of the nail, brain, stomach or leukemia.
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| WO2021232717A1 (en) * | 2020-05-18 | 2021-11-25 | 广州中医药大学(广州中医药研究院) | Conjugate containing mono-phosphorylated lipid a and glycoantigen, and preparation method therefor and use thereof |
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