CN111588847B - Conjugate containing monophosphorylated lipid A and saccharide antigen and preparation method and application thereof - Google Patents
Conjugate containing monophosphorylated lipid A and saccharide antigen and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a conjugate containing monophosphorylated lipid A and a saccharide antigen, and a preparation method and application thereof, belonging to the technical field of anti-tumor saccharide vaccine development. The invention provides a conjugate containing mono-phosphorylated lipid A and a glycoantigen, wherein the conjugate of the mono-phosphorylated lipid A and the glycoantigen is a compound with a general formula (I) or a pharmaceutically acceptable salt thereof. The single phosphorylated lipid A can improve the immunogenicity of the Tn saccharide antigen, presents the Tn saccharide antigen to corresponding immune cells, and generates immune response with higher titer aiming at the specificity of the tumor saccharide antigen Tn, so the conjugate is expected to become a new generation of antitumor drugs as a fully synthesized saccharide antigen vaccine.
Description
Technical Field
The invention relates to a conjugate containing monophosphorylated lipid A and a saccharide antigen, and a preparation method and application thereof, belonging to the technical field of anti-tumor saccharide vaccine development.
Background
The tumor vaccine has better clinical application prospect in preventing and treating cancers, and the tumor vaccine which takes 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 Thomsennouveau (Tn) antigen is abnormally and excessively expressed on the surfaces of malignant tumor cells such as breast cancer, prostate cancer, lung cancer and the like, and is an excellent target for designing a carbohydrate antigen tumor vaccine.
The saccharide antigens themselves are poorly immunogenic and they need to be covalently bound to immunologically active carrier molecules in order to function, the most mature and commonly used carrier being a protein. 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 suppression" effect caused by proteins.
To avoid these drawbacks, fully synthetic carbohydrate antigen vaccines incorporating embedded adjuvants have become a new strategy to study. Fully synthetic glycolipid vaccines are able to remove unnecessary immunogenic components, containing only those essential elements that elicit an effective immune response. Typically, lipid adjuvants (e.g., lipopeptides-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 TLR (Toll-like receptor) and KRN7000 agonists capable of eliciting iNKT immune cells, and the like.
Bacterial Lipopolysaccharides (LPS) are surface glycolipids of the outer membrane of bacteria. Lipid A of the hydrophobic part of LPS is a ligand of Toll-like receptor 4 (TLR 4), and Lipid A can be used as an adjuvant to generate anticancer effect by initiating strong Th1 reaction. However, lipid A cannot be used clinically due to its high toxicity. Researchers found that MPLA (Monophosphoryl Lipid A) obtained by removing the 1-position phosphoric acid in the structure of Lipid A (the reaction formula is shown as follows) can still be combined with TLR4 in a targeted manner, the toxicity is obviously reduced, the activity change is not obvious,
MPLA is used as an adjuvant in clinical trials for many different types of cancers, such as stage IV melanoma, ovarian cancer, lung cancer, thrombocytopenia, leukemia, sarcoma, merkel cell carcinoma and non-hodgkin lymphoma. OM-174, a diacylated lipid a analog, has been tested clinically in patients with refractory solid tumors and shows good tolerability.
The GUO subject group carried out a great deal of research on MPLA as an embedded adjuvant for fully synthetic tumor vaccines, mainly by combining MPLA with various saccharide antigens to prepare antibacterial and antitumor saccharide conjugate vaccines, such as GM3-MPLA, MPLA-sTn, GM2-MPLA and other saccharide antigen vaccines, the immunological research shows that the saccharide antigen vaccine mainly induces IgG antibody production, and the conjugate vaccine still induces strong immune response without the assistance of external adjuvants, indicating its self-assistance property (Chemical Biology,2012,7:235;Scientific reports,2017,7:11403;Biomolecular Chemistry,2014,12:3238). In particular, the Guo task, which is combined into a conjugate MPLA-Globo H of Globo H and optimized MPLA, can more rapidly produce IgG antibodies about 2-fold stronger than Globo H-KLH (adjuvant CFA) without an additional adjuvant. Thus, MPLA proved to be a powerful embedded adjuvant for the entirely new design of fully synthetic glycoconjugate cancer vaccines (Chemical Science,2015, 6:7112.).
The invention uses phosphorylated lipid A (MPLA) as an embedded adjuvant to conjugate with saccharide antigen Tn to obtain a conjugate of the phosphorylated lipid A and the saccharide antigen, and the conjugate can be used as a vaccine to effectively prevent and/or treat various cancers.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a conjugate containing mono-phosphorylated lipid A and a saccharide antigen.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a conjugate comprising a monophosphorylated lipid a and a carbohydrate antigen, wherein the conjugate is a compound of formula (i) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of a compound of formula (i);
wherein:
n is an integer from 2 to 6;
R 1 and R is 3 Is- (CH) 2 )mCH 3 M is an integer of 10 to 14;
R 2 、R 4 and R is 5 Is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
According to the invention, the mono-phosphorylated lipid A (MPLA) is used as an embedded adjuvant to conjugate the saccharide antigen Tn to obtain a conjugate of the mono-phosphorylated lipid A and the saccharide antigen, the MPLA can overcome the defect of poor immunogenicity of the Tn saccharide antigen, and the Tn saccharide antigen is presented to corresponding immune cells to cause specific immune reaction aiming at the saccharide antigen Tn, so that the purpose of killing tumor cells is achieved; the conjugate can be used as vaccine, and can effectively prevent and/or treat various cancers.
As a preferred embodiment of the conjugate according to the present invention, the conjugate is a compound of formula (ii) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of a compound of formula (ii);
wherein:
R 1 and R is 3 Is- (CH) 2 )mCH 3 M is an integer of 10 to 14;
R 2 、R 4 and R is 5 Is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
As a preferred embodiment of the conjugate according to the invention, the conjugate is a compound of formula (iii) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of formula (iii);
wherein: n is an integer of 2 to 6.
As a preferred embodiment of the conjugate according to the present invention, the conjugate is a compound of formula (iv) or an isomer, pharmaceutically acceptable salt, hydrate or solvate of a compound of formula (iv);
as a preferred embodiment of the conjugate according to the invention, the mono-phosphorylated lipid a is a compound of formula (v) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvent compound of formula (v);
wherein:
R 5 is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
The present invention also provides pharmaceutically acceptable salts, hydrates or solvates of the compounds of formulas (I), (II), (III) and (IV), wherein the pharmaceutically acceptable salts include, but are not limited to, pharmaceutically acceptable salts formed by reaction with bases such as sodium, magnesium, potassium, calcium, lithium and the like. The compounds of formulae (I), (II), (III) and (IV) provided herein can be crystallized or recrystallized from hydrates or organic solvents, in which case various solvates can be formed.
It is another object of the present invention to provide a method for preparing the conjugate, comprising the steps of:
(1) Dissolving a compound 1 and a compound 2 in an organic solvent, and adding a catalyst to react to obtain a compound 3;
(2) Compound 3 in the step (1) is catalyzed by ethylenediamine to obtain compound 4;
(3) Taking the compound 4 and fatty acid chain in the step (2), and carrying out peptide-forming and ester-forming reactions under the condition of condensing agents 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 performing phosphating reaction to obtain a compound 7;
(6) Dissolving the compound 8 and the compound 7 in the step (5) in an organic solvent, and adding a catalyst for reaction 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 formulas of the compounds 1 to 9 are shown as follows:
wherein:
R 0 for STol, SPh, set or OC (NH) CCl 3 ；
n is an integer from 2 to 6;
R 1 and R is 3 Is- (CH) 2 )mCH 3 M is an integer of 10 to 14;
R 2 、R 4 and R is 5 Is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
The reaction formula of the preparation method is shown as follows:
wherein:
R 0 for STol, SPh, set or OC (NH) CCl 3 ；
n is an integer from 2 to 6;
R 1 and R is 3 Is- (CH) 2 )mCH 3 M is an integer of 10 to 14;
R 2 、R 4 and R is 5 Is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
The preparation method of the invention uses phosphorylated lipid A (MPLA) as an embedded adjuvant to conjugate with saccharide antigen Tn to obtain the MPLA-Tn tumor vaccine, and has the advantages of short synthetic route, mild reaction condition, high yield and convenient operation, and can be used for industrialized 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 preparation method of the present invention, in the step (1), when R 0 In the case of STol, SPh or Set, the catalyst is N-iodosuccinimide and any one selected from trifluoromethanesulfonic acid, silver trifluoromethanesulfonate, boron trifluoride diethyl ether and trifluoromethanesulfonic acid trimethylsilicone ester, or when R 0 Is OC (NH) CCl 3 When the catalyst is selected from any one of trifluoromethanesulfonic acid, boron trifluoride diethyl ether and trifluoromethanesulfonic acid trimethylsilyl ester; the organic solvent is selected from any one of dichloromethane, diethyl ether and tetrahydrofuranThe method comprises the steps of carrying out a first treatment on the surface of the The temperature of the reaction is-40 to-20 ℃.
More preferably, in step (1), when R 0 Is STol, the catalyst is a mixture of N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid; the organic solvent is dichloromethane.
In the step (2) of the preparation method, the compound 3 is catalyzed by ethylenediamine to remove the phthaloyl at the C-2 position and the C-2 'position and the acetyl at the C-3' position to obtain the compound 4 with exposed amino and hydroxyl.
Step (3) in the preparation method, the compound 4 and the fatty acid chain are subjected to peptide-forming and ester-forming reactions under the condition of a condensing agent to obtain a compound 5; as a preferred embodiment of the preparation method of the present invention, in the step (3), the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methyl iodide.
Step (4) in the preparation method, the compound 5 selectively reduces the hydroxyl of the sugar at the 4-position under the action of a catalyst to obtain a compound 6; in the step (4), the catalyst is a mixture of triethylsilane and trifluoromethanesulfonic acid, and the organic solvent is selected from any one of dichloromethane, methanol and tetrahydrofuran.
In the step (5) of the preparation method, the compound 6 in the step (4) is taken and dissolved in an organic solvent, a catalyst is added, and the compound 7 is obtained through phosphating 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 the step (7), the compound 9 is dissolved in an organic solvent and reacted under the action of a catalyst to obtain the compound 10; the catalyst is a mixture of hydrogen, palladium carbon and palladium hydroxide.
The use of a conjugate according to another object of the invention for the preparation of a medicament for the prevention and/or treatment of cancer.
As a preferred embodiment of the use according to the invention, the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell carcinoma, cellular lymphoma, thyroid cancer, brain cancer, gastric cancer or leukemia.
Compared with the prior art, the invention has the beneficial effects that:
(1) The conjugate containing the mono-phosphorylated lipid A and the saccharide antigen is obtained by using the mono-phosphorylated lipid A (MPLA) as an embedded adjuvant to conjugate with the saccharide antigen Tn, the MPLA can overcome the defect of poor immunogenicity of the Tn saccharide antigen, and the Tn saccharide antigen is presented to corresponding immune cells to cause specific immune reaction aiming at the saccharide antigen Tn, so that the aim of killing tumor cells is fulfilled; the conjugate can be used as a vaccine to effectively prevent and/or treat various cancers, including breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, intestinal cancer, renal cell carcinoma, cell lymphoma, carcinoma of the thyroid gland, brain cancer, gastric cancer or leukemia.
(2) The MPLA can improve the immunogenicity of the Tn sugar antigen, presents the Tn sugar antigen to corresponding immune cells, generates immune response specific to the tumor sugar antigen Tn with higher titer, and the titer of IgG antibodies induced by the MPLA and the capability of specifically recognizing the antigen are obviously higher than those induced by glycoprotein vaccine CRM 197-Tn; therefore, the conjugate containing the mono-phosphorylated lipid A (MPLA) and the saccharide antigen provided by the invention is hopeful to become a new generation of antitumor drugs as a fully synthesized saccharide antigen vaccine.
(3) The preparation method of the conjugate containing the mono-phosphorylated lipid A and the saccharide antigen provided by the invention has the advantages of short synthetic route, mild reaction condition, high yield and convenience in operation, and can be used for industrial preparation.
Drawings
FIG. 1 is a graph showing the evaluation of the immunological activity of antibodies against the glycoprotein vaccine MPLA-Tn of example 1 and the glycoprotein vaccine CRM197-Tn of comparative example 1 of the present invention;
FIG. 2 is a flow cytometry evaluation chart of the antibody serum specificity recognition tumor cells MCF-7 induced by the glycoprotein vaccine MPLA-Tn of example 1 and the glycoprotein vaccine CRM197-Tn of comparative example 1, respectively, of the present invention.
Detailed Description
In order to more clearly describe the technical solution of the present invention, the following description is further given by way of specific examples, but not by way of limitation, only some examples of the present invention.
Example 1
The embodiment is a conjugate containing mono-phosphorylated lipid A and a glycoantigen, and the structural formula of the mono-phosphorylated lipid A and glycoantigen conjugate is shown as a formula (IV):
the preparation method of the conjugate containing the mono-phosphorylated lipid A and the saccharide antigen comprises the following steps:
(1) Dissolving a compound 1 and a compound 2 in an organic solvent, and adding a catalyst to react to obtain a compound 3; the reaction scheme for obtaining compound 3 is shown below:
the specific operation of the step (1) is as follows: anhydrous grade dichloromethane (10.0 mL) dissolved vacuum dried Compound 1 (0.4 g,0.8 mmol), compound 2 (0.3 g,0.5 mmol) and high temperature dried molecular sieves (2.0 g) and stirred under nitrogen for 4 hoursThe method comprises the steps of carrying out a first treatment on the surface of the After cooling to-30 ℃, N-iodosuccinimide (360.0 mg,1.6 mmol) was rapidly added, and after stirring at-30 ℃ for 1 hour, the reaction solution was cooled to-40 ℃ again, trifluoromethanesulfonic acid (11.9 μl,130.0 μmol) was rapidly added and stirred for 15 minutes, saturated sodium bicarbonate solution was added for neutralization, and then sodium thiosulfate aqueous solution was added until the red color of the reaction solution faded. The organic layer was collected by removing the water layer, washed 2 times with water, washed 1 time with saturated brine, the organic layer was collected, dried over anhydrous sodium sulfate, and then the organic solution was distilled off under reduced pressure to give a crude product, which was purified by column chromatography to give compound 3 (430.0 g, 82.0%) as a colorless viscous substance. 1 H NMR(400MHz,CDCl 3 )δ7.67–6.75(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-CH 3 ). 13 C NMR(100MHz,CDCl 3 )δ170.27,137.70,137.40,136.91,134.24,133.66,129.19,128.45,128.28,128.06,127.98,127.89,127.40,126.26,123.56,123.21,101.67,98.40(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) Compound 3 in the step (1) is catalyzed by ethylenediamine to obtain compound 4; the reaction scheme for obtaining compound 4 is shown below:
the specific operation of the step (2) is as follows: methanol (30.0 mL) was used to dissolve Compound 3 (500.0 mg,0.5 mmol), ethylenediamine (5.0 mL) was added dropwise, the reaction mixture was heated to 80℃and refluxed overnight, the mixture was cooled to room temperature, toluene (4.0 mL) was added after the solvent was removed, and excess ethylenediamine was removed to give a yellow oily liquid, and the crude product was purified by silica gel column separation to give Compound 4 (240.0 mg, 70.0%) as a white solid.
1 H NMR(400MHz,CDCl 3 )δ7.57–7.32(m,15H,Ar-H),5.56(s,1H),4.94(dd,J＝34.8,11.1Hz,2H,Ar-CH 2 -O-),4.72(dd,J＝32.9,11.1Hz,2H,Ar-CH 2 -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,-CH 2 -N 3 ). 13 C NMR(100MHz,CDCl 3 )δ138.14,137.77,137.10,129.31,128.65,128.58,128.39,128.33,128.05,128.02,127.93,127.89,127.87,126.29,104.96,103.95(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 and fatty acid chain in the step (2), and carrying out peptide-forming and ester-forming reactions under the condition of condensing agents to obtain a compound 5; the reaction scheme for obtaining compound 5 is shown below:
the specific operation of the step (3) is as follows: under the protection of nitrogen, compound 4 (100.0 mg,150.0 mu mol), self-made fatty acid (300.0 mg,670.0 mu mol) and 4-dimethylaminopyridine (1.0 mg,4.0 mu mol) are dissolved in methylene chloride, the mixture is stirred and reacted, the temperature of the mixture is reduced to 0 ℃, a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methyl iodide salt (220.0 mg,740.0 mu mol) is added, the reaction solution is stirred and reacted for 2 hours, the methylene chloride is used for diluting, saturated saline is used for flushing 3 times, an organic layer is collected, anhydrous sodium sulfate is dried, the organic solution is removed to obtain a crude product, and then the crude product is separated and purified by a silica gel column to obtain a white solid compound 5 (160.0 mg, 56.0%). 1 H NMR(400MHz,CDCl 3 )δ7.58–7.08(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). 13 C NMR(100MHz,CDCl 3 )δ173.89,173.73,173.49,170.31,170.18,170.07,138.17,137.84,136.87,129.04,128.42,128.35,128.14,127.88,127.79,127.73,127.66,126.11,101.45,101.38,99.75,80.97,78.78,78.77,78.17,78.17,74.62,74.53,74.43,71.67,71.22,70.96,69.98,68.52,68.14,67.44,66.20,55.70,54.35,50.70,41.46,41.10,39.00,34.47,34.27,33.95,33.85,31.89,29.66,29.62,29.54,29.50,29.47,29.36,29.33,29.29,29.26,29.19,29.10,29.07,25.55,25.23,25.00,24.95,24.92,22.65,14.04.ESI-TOF HRMS m/z:calcdfor C 119 H 199 N 5 O 18 ,[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 for obtaining compound 6 is shown below:
the specific operation of the step (4) is as follows: dichloromethane (10 mL) was used to dissolve compound 5 (180.0 mg, 90.1. Mu. Mol) and molecular sieve (1.0 g), the mixture was stirred in a closed condition for 15 minutes, cooled to-78 ℃, triethylsilane (52.0. Mu.L, 326.4. Mu. Mol) and trifluoromethanesulfonic acid (24. Mu.L, 271.8. Mu. Mol) were added, the mixture was stirred for 60 minutes, 1.0mL of a mixture of triethylamine and methanol (1:10) was added, the reaction was quenched, the grey insoluble matter was removed by filtration, the organic solvent was removed to obtain a crude grey solid, and the crude product was isolated and purified by silica gel column to obtain compound 6 (110.8 mg, 61.57%) as a white solid.
1 H NMR(400MHz,CDCl 3 )δ7.57–7.13(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). 13 C NMR(100MHz,CDCl 3 )δ174.26,173.70,173.40,171.42,169.96,169.54,138.32,137.96,137.90,128.42,128.38,127.85,127.82,127.71,127.67,127.63,101.10,99.53,80.60,78.17,77.28,76.14,74.83,74.46,74.39,74.30,73.60,71.05,70.98,70.92,70.18,70.05,67.92,67.46,55.94,53.73,50.73,41.71,41.50,40.08,34.70,34.52,34.49,34.13,34.09,31.94,29.74,29.70,29.67,29.65,29.61,29.57,29.54,29.52,29.41,29.38,29.31,29.25,29.16,25.32,25.28,25.15,25.05,24.99,24.97,22.70,14.13.ESI-TOF HRMS m/z:calcdfor C 119 H 201 N 5 O 18 ,[M+Na] + :2011.4859,found:2011.4877.
(5) Dissolving the compound 6 in the step (4) in an organic solvent, adding a catalyst, and performing phosphating reaction to obtain a compound 7; the reaction scheme for obtaining compound 7 is shown below:
the specific operation of the step (5) is as follows: under the protection of nitrogen, dissolving a compound 6 (80.0 mg,40.2 mu mol) in a dichloromethane-acetonitrile mixed solution, adding dibenzyl diisopropylphosphoramidite (300.0 mu L,913.0 mu mol), and triazole (1.3 mL,913.0 mu mol), stirring and 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 and 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 (74.0 mg, two-step yield 81.8%) as a white solid. 1 H NMR(400MHz,CDCl 3 )δ7.43–7.15(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). 13 C NMR(101MHz,CDCl 3 )δ173.75,173.56,173.43,170.17,170.04,169.94,138.34,138.21,137.90,135.63,135.56,135.53,128.58,128.47,128.41,128.33,128.06,127.97,127.93,127.88,127.69,127.66,127.59,127.51,100.15,99.57,80.67,78.26,77.37,77.25,77.05,76.83,76.74,74.81,74.47,74.39,74.04,73.33,72.78,71.05,70.65,70.23,69.64,69.55,69.50,68.67,68.34,67.69,56.18,55.69,50.73,41.71,41.19,39.65,34.52,34.32,34.09,31.96,29.75,29.71,29.66,29.61,29.59,29.56,29.54,29.47,29.41,29.39,29.33,29.30,29.24,29.17,25.29,25.24,25.15,25.06,25.00,22.72,14.14.ESI-TOF HRMS m/z:calcdfor C 133 H 214 N 5 O 21 P,[M+Na] + :2271.5461,found:2271.5423.
(6) Dissolving the compound 8 and the compound 7 in the step (5) in an organic solvent, and adding a catalyst for reaction to obtain a compound 9; the reaction scheme for obtaining compound 9 is shown below:
the specific operation of the step (6) is as follows: tetrahydrofuran was dissolved with methanol (1:2, 3.0 mL) to give compound 7 (68.0 mg, 30.2. Mu. Mol), compound 8 (10.0 mg, 20.4. Mu. Mol), and cuprous iodide (195.0 mg,1.0 mmol), N-diisopropylethylamine (168.0. Mu.L, 1.0 mmol) was added thereto, and the mixture was stirred at room temperature for 12 hours; filtering insoluble substances out by diatomite, and distilling the filtrate under reduced pressure to remove the solvent to obtain a crude product; the obtained white solid was purified by silica gel column chromatography to give compound 9 (20.0 mg, yield 35.7%). 1 H NMR(600MHz,CDCl 3 )δ7.98(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). 13 C NMR(151MHz,CDCl 3 )δ173.79,173.65,173.49,171.21,171.04,170.32,170.17,138.17,138.04,137.77,135.47,135.42,128.63,128.58,128.50,128.43,128.36,128.13,127.99,127.89,127.72,127.63,127.57,100.25,100.23,99.30,80.97,78.07,74.50,74.41,74.09,73.72,73.22,72.52,71.10,70.54,70.42,70.01,69.77,69.74,69.61,69.57,69.40,68.68,68.41,67.37,62.60,56.59,55.68,55.25,51.32,41.54,41.06,39.44,39.13,34.54,34.50,34.43,34.29,34.12,31.94,29.74,29.70,29.65,29.59,29.53,29.47,29.41,29.38,29.33,29.29,29.22,29.18,25.29,25.14,25.04,23.08,22.71,17.99,14.13.ESI-TOF HRMS m/z:calcdfor C 154 H 249 N 8 O 31 P,[M+H] + :2738.7964,found:2738.7970.
(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 containing the mono-phosphorylated lipid A and the saccharide 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, 10.0 mL) dissolved compound 9 (8.0 mg), palladium hydroxide (5.0 mg) and palladium on carbon (5.0 mg) were added, hydrogen gas was introduced, stirring was performed for 24 hours under sealing, insoluble matter was filtered off with celite, three times of washing with 30.0mL dichloromethane/methanol/water (5:5:1) was performed, and the filtrate was distilled off under reduced pressure to remove the solvent, to obtain compound 10 as a white solid, namely, the conjugate MPLA-Tn (5.5 mg, 83.3%). 1 H NMR(400MHz,MeOD/CDCl 3 /D 2 O(20：30：1，v/v/v，0.6mL))δ3.68(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 C 119 H 219 N 8 O 31 P,[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 to amino under the catalysis of palladium carbon and acetic acid by monosaccharide compound 10 to obtain compound 11; reacting the compound 11 with bis (N-hydroxysuccinimide) suberate in a dimethylformamide solution to obtain a compound 12; the method comprises the following steps: compound 10 (100 mg,0.244 mmol) was dissolved in methanol (5 mL), palladium on carbon (100 mg) and acetic acid (0.01 mL) were added, the mixture was sealed, and hydrogen was introduced for 5 times with stirring in a sealed manner for 20 hours; insoluble matter was filtered off with celite, and the solvent was distilled off from the filtrate under reduced pressure to give a white solid, which was dissolved in water and pre-frozen in a-80 ℃ refrigerator for 6 hours, and transferred to a freeze dryer to freeze-dry to give compound 11 (86 mg, 73%) as a white solid. Compound 11 was reacted with bis (N-hydroxysuccinimide) suberate (57.5 mg,36.6 mmol) in dimethylformamide solution for 5 hours, the organic solvent was distilled off under reduced pressure to give a crude product, and the solid was washed with ethyl acetate 8 times to give Compound 12 (7 mg) as a white solid;
(2) Coupling compound 12 with CRM197 protein in 0.1mol PBS (ph=7.8) buffer salt solution to give Tn-CRM197 glycoprotein vaccine; the method comprises the following steps: compound 12 was dissolved in 0.1mol of PBS (ph=7.8) buffer salt 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 and dialyzed for 2 days, distilled water was changed every 6 hours, finally, the suspension in the dialysis bag was transferred to a sample bottle, and after prefreezing for 6 hours at-80 ℃, the white solid compound Tn-CRM197 (5 mg) was obtained by lyophilization in a lyophilizer.
The reaction formula of the preparation method is as follows:
experimental example 1
This experimental example immunized mice were respectively subjected to the whole synthetic sugar vaccine (MPLA-Tn) prepared in example 1 and the glycoprotein vaccine (CRM 197-Tn) prepared in comparative example 1, and their immunization was primarily evaluated by ELSA experiments, and it was confirmed that antibody serum specifically recognized tumor cells (MCF-7) by Fluorescence Activated Cell Sorting (FACS) technique.
ELISA immunoassay
(1) Immunization of mice:
12C 57BL/6 mice of 6-8 weeks of age were randomly divided into 2 groups. After preparing the fully synthetic sugar vaccine MPLA-Tn and glycoprotein vaccine CRM197-Tn into liposome respectively, performing an immune test by means of subcutaneous injection of mice, and injecting the prepared vaccine on days 0, 14, 21 and 28 respectively by adopting an initial immune and three-time enhanced immune scheme, wherein the injection amount of each injection is 0.1mL (the MPLA-Tn sugar vaccine contains 6 mug Tn antigen); on day 38, 0.1 to 0.2mL of blood was collected from each mouse, left at 0℃for 60 minutes, centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected for ELISA assay.
(2) ELISA immunoassay:
0.1M carbonate buffer (pH 9.6) dissolved Tn-BSA, formulated as a 2.0. Mu.g/mL solution, added to 96-well plates at 100.0. Mu.L per well, and incubated overnight at 4 ℃; the next day the incubator incubates at 37 ℃ for one hour; plates were washed 3 times (300. Mu.L/well/time) with PBST (PBS+0.05% Tween-20). After washing the plates, 250.0. Mu.l of PBS/1% BSA was added to each well and incubated at room temperature for one hour, and the plates were washed 3 times with PBST. Equal amounts of each group of 6 mouse serum samples were mixed and diluted 300, 900, 2700, 8100, 24300, 72900, 218700 and 656100 fold with PBS; adding 100.0 mu L of diluted serum into a 96-well plate, and making three auxiliary wells in parallel for each dilution gradient; incubate at 37℃for two hours and wash the plate 3 times. HRP (horseradish peroxidase) -labeled IgG (2000-fold dilution) was added to 100.0 μl per well and incubated for one hour at room temperature, and the plates were washed 3 times. Adding TMB solution, adding 100.0 μl per well, developing at room temperature in dark for 20 min, adding 0.5M H 2 SO 4 Solution, 100.0 μl/well. The absorbance was immediately detected with a microplate reader at 450nm and 570nm as background wavelengths.
(3) Absorbance (OD) values were plotted against antisera dilution values and a best fit line was obtained. The equation for this line was used to calculate the dilution at which the OD reached 0.2, and the antibody titer was calculated from the inverse of the dilution as shown in figure 1.
(4) Experimental results:
as can be seen from fig. 1, the MPLA-Tn saccharide vaccine synthesized in example 1 of the present invention can generate immune response specific to tumor saccharide antigen Tn in mice without external adjuvant, more rapidly generate high-titer specific IgG antibodies, and the IgG antibody titer is significantly higher than that of glycoprotein vaccine CRM197-Tn.
2. Flow cytometry experiments (FACS)
The experimental method comprises the following steps: breast cancer cells MCF-7 which are used for over-expressing Tn sugar antigen and tumor cells MDA-231 which are used for not expressing Tn sugar antigen are respectively cultured in MEM culture medium containing 10% Fetal Bovine Serum (FBS) (37 ℃ C., 5% CO 2); pancreatin digestion, cell collection, microscopic cell count, split charging of 2.0X105 cells per tube, resuspension with 1mL PBS buffer containing 3% FBS (FACS buffer), centrifugation for 2 min, removal of supernatant, washing twice with FACS buffer; prepared mouse serum was added, incubated in ice for 1 hour, FACS buffer was washed twice, fluorescence-labeled secondary antibody was added, incubated in ice protected from light for one hour, FACS buffer was washed twice, resuspended in 0.8mL FACS buffer, and the results were detected with a flow cytometer as shown in fig. 2.
Experimental results: as shown in FIG. 2, MCF-7 was breast cancer cells overexpressing Tn antigen, and MDA-231 tumor cells not expressing Tn antigen were used as negative controls. In MCF-7 cells, compared with preimmune serum, the fluorescence peak of antibody serum induced by the MPLA-Tn saccharide vaccine synthesized in the embodiment 1 of the invention is obviously shifted to the right, and the serum and the antibody serum are not obviously different before MDA-231 immunization. The result shows that the antibody induced by the vaccine can specifically recognize MCF-7 cells expressing 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, thereby indicating that the antibody induced by the MPLA-Tn sugar vaccine has stronger capability of specifically recognizing antigen.
In conclusion, experimental results show that the MPLA can improve the immunogenicity of the Tn sugar antigen, present the Tn sugar antigen to corresponding immune cells, generate immune response specific to the tumor sugar antigen Tn with higher titer, and the titer of IgG antibodies induced by the MPLA and the ability of specifically recognizing the antigen are obviously higher than those induced by glycoprotein vaccine CRM 197-Tn; therefore, the conjugate containing the mono-phosphorylated lipid A (MPLA) and the saccharide antigen provided by the invention is hopeful to become a new generation of antitumor drugs as a fully synthesized saccharide antigen vaccine.
Finally, it should be noted that the foregoing examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the foregoing examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made and all equivalent substitutions are intended to be included in the scope of the present invention.
Claims (9)
1. A conjugate comprising a monophosphorylated lipid a and a saccharide antigen, wherein said conjugate is a compound of formula (i) or a pharmaceutically acceptable salt thereof;
wherein:
n is an integer from 2 to 6;
R 1 and R is 3 Is- (CH) 2 )mCH 3 M is an integer of 10 to 14;
R 2 、R 4 and R is 5 Is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
2. The conjugate of claim 1, wherein the conjugate is a compound of formula (ii) or a pharmaceutically acceptable salt thereof;
wherein:
R 1 and R is 3 Is- (CH) 2 )mCH 3 M is an integer of 10 to 14;
R 2 、R 4 and R is 5 Is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
3. The conjugate of claim 1, wherein the conjugate is a compound of formula (iii) or a pharmaceutically acceptable salt thereof;
wherein: n is an integer of 2 to 6.
4. A conjugate according to claim 3, wherein the conjugate is a compound of formula (iv) or a pharmaceutically acceptable salt thereof;
5. a conjugate according to any one of claims 1 to 3, wherein the mono-phosphorylated lipid a is a compound of formula (v) or a pharmaceutically acceptable salt thereof;
wherein:
R 5 is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8-14.
6. The method of preparing a conjugate according to any one of claims 1 to 5, comprising the steps of:
(1) Dissolving a compound 1 and a compound 2 in an organic solvent, and adding a catalyst to react to obtain a compound 3;
(2) Compound 3 in the step (1) is catalyzed by ethylenediamine to obtain compound 4;
(3) Taking the compound 4 and fatty acid chain in the step (2), and carrying out peptide-forming and ester-forming reactions under the condition of condensing agents 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 performing phosphating reaction to obtain a compound 7;
(6) Dissolving the compound 8 and the compound 7 in the step (5) in an organic solvent, and adding a catalyst for reaction 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 formulas of the compounds 1 to 9 are shown as follows:
wherein:
R 0 for STol, SPh, set or OC (NH) CCl 3 The method comprises the steps of carrying out a first treatment on the surface of the n is an integer from 2 to 6;
R 1 and R is 3 Is- (CH) 2 )mCH 3 M is an integer of 10 to 14;
R 2 、R 4 and R is 5 Is- (CH) 2 )pCH 3 P is an integer of 8 to 12;
R 6 is-CO (CH) 2 )rCH 3 Or- (CH) 2 )rCH 3 R is an integer of 8 to 14;
in step (1), when R 0 In the case of STol, SPh or Set, the catalyst is N-iodosuccinimide and is selected from the group consisting of silver triflate, boron trifluoride diethyl etherate, and trimethylsilicon triflateAny one of the esters, or when R 0 Is OC (NH) CCl 3 When the catalyst is selected from any one of trifluoromethanesulfonic acid, boron trifluoride diethyl ether and trifluoromethanesulfonic acid trimethylsilyl ester; the organic solvent is selected from 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;
in the step (4), the catalyst is a mixture of triethylsilane and trifluoromethanesulfonic acid, and the organic solvent is selected from 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.
7. The process according to claim 6, wherein in step (1), when R 0 Is STol, the catalyst is a mixture of N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid; the organic solvent is dichloromethane.
8. Use of a conjugate according to any one of claims 1-5 for the preparation of a medicament for the prevention and/or treatment of cancer.
9. The use of 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, cellular lymphoma, thyroid cancer, brain cancer, gastric cancer or leukemia.
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