WO2021232717A1 - Conjugate containing mono-phosphorylated lipid a and glycoantigen, and preparation method therefor and use thereof - Google Patents

Abstract

A conjugate containing mono-phosphorylated lipid A and glycoantigen, and a preparation method therefor and the use thereof. The conjugate of the mono-phosphorylated lipid A and the glycoantigen is a compound of general formula (I) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of general formula (I). Further disclosed is the use of the conjugate in the preparation of a drug for preventing and/or treating cancers. Mono-phosphorylated lipid A can improve the immunogenicity of a Tn glycoantigen.

Description

Conjugate containing monophosphorylated lipid A and carbohydrate antigen, and preparation method and application thereof Technical field

The invention relates to a conjugate containing monophosphorylated lipid A and a sugar antigen, and a preparation method and application thereof, and belongs to the technical field of the development of anti-tumor sugar vaccines.

Background technique

Tumor vaccines have good clinical application prospects in cancer prevention and treatment. Tumor sugar vaccines that target tumor-associated carbohydrate antigens (TACAs) that are abnormally expressed on the surface of tumor cells have the advantages of high specificity, low side effects, and better curative effects. Among them, Thomsennouveau (Tn) antigen is abnormally overexpressed on the surface of malignant tumor cells such as breast cancer, prostate cancer, lung cancer and so on. It is an excellent target for carbohydrate antigen tumor vaccine design.

Carbohydrate antigens have poor immunogenicity. They need to be covalently combined with immunologically active carrier molecules to play a role. The most mature and commonly used carrier is protein. Commonly used protein carriers include KLH, BSA, DT, CRM197 and TT. These synthetic glycoprotein vaccines have the following shortcomings: uncertain coupling sites, unstable coupling rates, complex components, and "epitopes" caused by proteins. Suppress" effect.

In order to avoid these shortcomings, the introduction of fully synthetic carbohydrate antigen vaccines with embedded adjuvants has become a new research strategy. The fully synthetic glycolipid vaccine can remove unnecessary immunogenic components and only contains those essential elements that trigger an effective immune response. Generally, lipid adjuvants (such as TLR ligands based on lipopeptides or lipoamino acids) are incorporated into vaccine constructs and are called endogenous adjuvants, such as those of various subtypes of TLR (Toll-like receptors). Agonists and KRN7000 agonists that can stimulate iNKT immune cells, etc.

Bacterial lipopolysaccharide (Lipidpolysacchrides, LPS) is the surface glycolipid of the bacterial outer membrane. Lipid A of the hydrophobic part of LPS is the ligand of Toll-like receptor 4 (TLR4). Lipid A can be used as an adjuvant to produce anti-cancer effects by initiating a strong Th1 response. However, Lipid A cannot be used clinically due to its high toxicity. Researchers found that MPLA (Monophosphoryl lipid A) obtained by removing the phosphate at position 1 in the Lipid A structure (the reaction formula is shown in the following formula) can still bind to TLR4 in a targeted manner, with significantly reduced toxicity and insignificant changes in activity.

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 analogue, has been clinically tested in patients with refractory solid tumors and has shown good tolerance.

The GUO research team has done a lot of research on MPLA as a fully synthetic tumor vaccine embedded adjuvant, mainly by combining MPLA with a variety of carbohydrate antigens to prepare antibacterial and antitumor glycoconjugate vaccines, such as GM3-MPLA, MPLA- Immunological studies of sTn, GM2-MPLA and other carbohydrate antigen vaccines have shown that carbohydrate antigen vaccines mainly induce the production of IgG antibodies. The conjugate vaccine still triggers a strong immune response without the assistance of external adjuvants, indicating its self-assistance Properties (Chemical Biology, 2012, 7: 235; Scientific reports, 2017, 7: 11403; Biomolecular Chemistry, 2014, 12: 3238). In particular, the Guo subject is combined into a conjugate of Globo H and optimized MPLA, MPLA-Globo H. Without an external adjuvant, it can produce about 2 times stronger than Globo H-KLH (the adjuvant is CFA). Times the IgG antibody. Therefore, MPLA has proven to be a newly designed and powerful embedded adjuvant for fully synthetic glycoconjugate cancer vaccines (Chemical Science, 2015, 6:7112.).

The present invention uses phosphorylated lipid A (MPLA) as a built-in adjuvant to conjugate carbohydrate antigen Tn to obtain a monophosphorylated lipid A and carbohydrate antigen conjugate. The conjugate is used as a vaccine and can effectively prevent And/or treat a variety of cancers.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide a conjugate containing monophosphorylated lipid A and carbohydrate antigen.

In order to achieve the above object, the technical solution adopted by the present invention is: a conjugate containing monophosphorylated lipid A and carbohydrate antigen, characterized in that the conjugate is a compound of general formula (I) or Isomers, pharmaceutically acceptable salts, hydrates or solvent compounds of the compound of general formula (I);

in:

n is an integer of 2-6;

R ₁ and R ₃ are -(CH ₂ )mCH ₃ , and m is an integer of 10-14;

R ₂ , R ₄ and R ₅ are -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.

In the present invention, monophosphorylated lipid A (MPLA) is used as a built-in adjuvant to conjugate carbohydrate antigen Tn to obtain a conjugate of monophosphorylated lipid A and carbohydrate antigen. MPLA can overcome the poor immunogenicity of Tn carbohydrate antigen Tn sugar antigen is presented to the corresponding immune cells, causing a specific immune response against the sugar antigen Tn, achieving the purpose of killing tumor cells; the conjugate is used as a vaccine, which can effectively prevent and/or treat multiple Kind of cancer.

As a preferred embodiment of the conjugate of the present invention, the conjugate is a compound of structural formula (II) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of a compound of structural formula (II);

in:

R ₁ and R ₃ are -(CH ₂ )mCH ₃ , and m is an integer of 10-14;

R ₂ , R ₄ and R ₅ are -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.

As a preferred embodiment of the conjugate of the present invention, the conjugate is a compound of structural formula (III) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of a compound of structural formula (III);

Wherein: n is an integer of 2-6.

As a preferred embodiment of the conjugate of the present invention, the conjugate is a compound of structural formula (IV) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of a compound of structural formula (IV);

As a preferred embodiment of the conjugate of the present invention, the monophosphorylated lipid A is a compound of general formula (V) or an isomer, pharmaceutically acceptable salt, hydrated compound of general formula (V) Substance or solvent compound;

in:

R ₅ is -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.

The present invention also provides pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula (I), (II), (III) and (IV), wherein the pharmaceutically acceptable salts include, but are not limited to, alkalis such as sodium, magnesium, A pharmaceutically acceptable salt formed by the reaction of potassium, calcium, and lithium. The compounds of formula (I), (II), (III) and (IV) provided by the present invention can be crystallized or recrystallized with hydrates or organic solvents. In this case, various solvates can be formed.

Another object of the present invention is to provide a preparation method of the conjugate, which includes the following steps:

(1) Dissolve compound 1 and compound 2 in an organic solvent, and add a catalyst to react to obtain compound 3;

(2) The compound 3 described in step (1) is catalyzed by ethylenediamine to obtain compound 4;

(3) Take the compound 4 described in step (2) and the fatty acid chain, and perform peptide-forming and ester-forming reactions under the conditions of a condensing agent to obtain compound 5;

(4) Dissolve the compound 5 described in step (3) in an organic solvent, add a catalyst, and undergo a reduction reaction to obtain compound 6;

(5) Dissolve the compound 6 described in step (4) in an organic solvent, add a catalyst, and undergo a phospholipidization reaction to obtain compound 7;

(6) Dissolve compound 8 and compound 7 described in step (5) in an organic solvent, and add a catalyst to react to obtain compound 9;

(7) The compound 9 described in step (6) is dissolved in an organic solvent, a catalyst is added, and the conjugate is obtained by debenzylation reaction;

The structural formulas of the compound 1 to the compound 9 are as follows:

in:

R ₀ is STol, SPh, Set or OC(NH)CCl ₃ ;

n is an integer of 2-6;

R ₁ and R ₃ are -(CH ₂ )mCH ₃ , and m is an integer of 10-14;

R ₂ , R ₄ and R ₅ are -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.

The reaction formula of the preparation method of the present invention is as follows:

in:

R ₀ is STol, SPh, Set or OC(NH)CCl ₃ ;

n is an integer of 2-6;

R ₁ and R ₃ are -(CH ₂ )mCH ₃ , and m is an integer of 10-14;

R ₂ , R ₄ and R ₅ are -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.

The preparation method of the present invention uses phosphorylated lipid A (MPLA) as an embedded adjuvant to conjugate carbohydrate antigen Tn to obtain MPLA-Tn tumor vaccine. The synthesis route is short, the reaction conditions are mild, the yield is high, and the operation is convenient. , Can be used for industrial production.

In step (1) of the preparation method of the present invention, compound 1 and compound 2 are reacted 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 step (1), when R ₀ is STol, SPh or Set, the catalyst is N-iodosuccinimide and selected from trifluoromethanesulfonic acid , Silver trifluoromethanesulfonate, boron trifluoride ethyl ether, trimethylsilyl trifluoromethanesulfonate, or when R ₀ is OC(NH)CCl ₃ , the catalyst is selected from trifluoromethanesulfonate Any one of methanesulfonic acid, boron trifluoride diethyl ether, and trimethylsilyl trifluoromethanesulfonate; the organic solvent is selected from any one of dichloromethane, diethyl ether, and tetrahydrofuran; the temperature of the reaction It is -40～-20℃.

More preferably, in step (1), when R ₀ is STol, the catalyst is a mixture of N-iodosuccinimide (NIS) and the catalyst trifluoromethanesulfonic acid; the organic solvent is dichloromethane .

In step (2) of the preparation method of the present invention, compound 3 is catalyzed by ethylenediamine to remove the phthaloyl group at the C-2 position, the C-2' position and the acetyl group at the C-3' position. , To obtain compound 4 with exposed amino and hydroxyl groups.

In step (3) of the preparation method of the present invention, compound 4 and fatty acid chain are reacted to form peptides and esters under the conditions of a condensing agent to obtain compound 5; as a preferred embodiment of the preparation method of the present invention, In step (3), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl iodide.

In step (4) of the preparation method of the present invention, compound 5 selectively reduces the 4-position sugar hydroxyl group under the action of a catalyst to obtain compound 6; as a preferred embodiment of the preparation method of the present invention, in 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 step (5) of the preparation method of the present invention, the compound 6 described in step (4) is dissolved in an organic solvent, a catalyst is added, and the compound 7 is obtained through phospholipidization reaction; as the preparation method of the present invention In a preferred embodiment, in step (5), the organic solvent is a mixed solution of dichloromethane and acetonitrile; the catalyst is dibenzyl diisopropyl phosphoramidite, triazole and tert-butanol peroxide mixture.

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 through a click reaction under the action of a catalyst; the organic solvent is dichloromethane , A mixed solution of methanol and water, the catalyst is a mixture of cuprous iodide, N,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 organic solvent is a mixture of methylene chloride, methanol and water Solution, the catalyst is a mixture of hydrogen, palladium on carbon and palladium hydroxide.

The application of the conjugate according to another object of the present invention in the preparation of drugs for the prevention and/or treatment of cancer.

As a preferred embodiment of the application of the present invention, the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, bowel cancer, renal cell carcinoma, cellular lymphoma, Thyroid cancer, brain cancer, stomach cancer or leukemia.

Compared with the prior art, the beneficial effects of the present invention are:

(1) A conjugate containing monophosphorylated lipid A and carbohydrate antigen provided by the present invention is obtained by conjugating carbohydrate antigen Tn with monophosphorylated lipid A (MPLA) as an embedded adjuvant. MPLA can Overcome the weak immunogenicity of Tn carbohydrate antigen, and present the Tn carbohydrate antigen to the corresponding immune cells to cause a specific immune response against the carbohydrate antigen Tn to achieve the purpose of killing tumor cells; the conjugate is used as a vaccine, Can effectively prevent and/or treat a variety of cancers, including breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, bowel cancer, renal cell carcinoma, cellular lymphoma, and thyroid gland Cancer, brain cancer, stomach cancer or leukemia.

(2) MPLA can improve the immunogenicity of Tn sugar antigen, present Tn sugar antigen to corresponding immune cells, produce a higher titer specific immune response against tumor sugar antigen Tn, and it induces the production of IgG The antibody titer and the ability to specifically recognize the antigen are significantly higher than those induced by the glycoprotein vaccine CRM197-Tn; therefore, the present invention provides a conjugate containing monophosphorylated lipid A (MPLA) and sugar antigen As a fully synthetic glycoantigen vaccine, it is expected to become a new generation of anti-tumor drugs.

(3) The present invention provides a method for preparing a conjugate of monophosphorylated lipid A and carbohydrate antigen. The preparation method has a short synthetic route, mild reaction conditions, high yield, and convenient operation. For industrial preparation.

Description of the drawings

Figure 1 is an evaluation diagram of antibody immune activity of the glycoprotein vaccine MPLA-Tn of Example 1 of the present invention and the glycoprotein vaccine CRM197-Tn of Comparative Example 1;

2 is a flow cytometric evaluation diagram of the antibody serum produced by the glycoprotein vaccine MPLA-Tn of Example 1 of the present invention and the glycoprotein vaccine CRM197-Tn of Comparative Example 1 respectively inducing mice to specifically recognize tumor cells MCF-7.

Detailed ways

In order to express the technical solution of the present invention more clearly, the following further description is combined with specific embodiments, but it cannot be used to limit the present invention. These are only some embodiments of the present invention.

Example 1

This example is a conjugate containing monophosphorylated lipid A and carbohydrate antigen provided by the present invention. The structural formula of the monophosphorylated lipid A and carbohydrate antigen conjugate is as shown in formula (IV) Show:

The preparation method of the conjugate containing monophosphorylated lipid A and carbohydrate antigen includes the following steps:

(1) Dissolve compound 1 and compound 2 in an organic solvent and add a catalyst to react to obtain compound 3. The reaction formula for obtaining compound 3 is shown in the following formula:

The specific operation of step (1) is: dissolving the vacuum-dried compound 1 (0.4g, 0.8mmol), compound 2 (0.3g, 0.5mmol) and the molecular sieve after high temperature drying in anhydrous grade dichloromethane (10.0mL) (2.0g), stirred for 4 hours under the protection of nitrogen; after cooling to -30°C, quickly add N-iodosuccinimide (360.0mg, 1.6mmol), and stir at -30°C After reacting for 1 hour, cool the reaction solution to -40°C, quickly add trifluoromethanesulfonic acid (11.9μL, 130.0μmol) and stir for 15 minutes, add saturated sodium bicarbonate solution to neutralize, and then add sodium thiosulfate The red color of the aqueous solution to the reaction solution fades. The water layer was removed and the organic layer was collected, washed twice with water and once with saturated brine. The organic layer was collected, dried with anhydrous sodium sulfate, and the organic solution was distilled off under reduced pressure to obtain a crude product, which was separated and purified by column chromatography to obtain colorless Sticky substance compound 3 (430.0 g, 82.0%). 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) The compound 3 described in step (1) is catalyzed by ethylenediamine to obtain compound 4; the reaction formula for obtaining compound 4 is shown in the following formula:

The specific operation of step (2) is: dissolving compound 3 (500.0 mg, 0.5 mmol) in methanol (30.0 mL), adding ethylenediamine (5.0 mL) dropwise, heating the reaction solution to 80° C., heating and refluxing for overnight reaction, the mixture After cooling to room temperature and removing the solvent, toluene (4.0 mL) was added to remove excess ethylenediamine to obtain a yellow oily liquid. The crude product was separated and purified by a silica gel column to obtain compound 4 (240.0 mg, 70.0%) as a white solid. ¹ H NMR (400MHz, CDCl ₃ ) δ 7.57–7.32 (m, 15H, Ar-H), 5.56 (s, 1H), 4.94 (dd, J = 34.8, 11.1 Hz, 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, 8.1 Hz, 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) Take the compound 4 described in step (2) and the fatty acid chain, and carry out peptide-forming and ester-forming reactions under the conditions of a condensing agent to obtain compound 5; the reaction formula for obtaining compound 5 is shown in the following formula:

The specific operation of step (3) is: under the protection of nitrogen, dichloromethane dissolves compound 4 (100.0mg, 150.0μmol), homemade fatty acid (300.0mg, 670.0μmol), 4-dimethylaminopyridine (1.0mg, 4.0 μmol), stir the reaction, reduce the temperature of the mixture to 0℃, add the catalyst 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl iodide (220.0mg, 740.0μmol) and stir for reaction 2 After hours, the reaction solution was diluted with dichloromethane, washed with saturated brine 3 times, the organic layer was collected, dried over anhydrous sodium sulfate, and the organic solution was removed to obtain the crude product, which was separated and purified by silica gel column to obtain the white solid compound 5 (160.0mg, 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 = 42.0 Hz, 2H), 4.87 (d, J = 7.3 Hz, 1H, H-1), 4.77 (dd, J = 16.2, 9.8 Hz, 2H), 4.66 (d, J = 11.0 Hz ,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 .89,173.73,173.49,170.31,170.18,170.07,138.17,137.84,136.87,129.04,128.42,128.35,128.14,127.88,127.79,127.73,127.66,126.11,101.45,101.38,99.75,17.97,78.78,78.77,78. ,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 ,29.50,29.47,29.36,29.33,29.29,29.26,29.19,29.10,29.07,25.55,25.23,25.00,24.95,24.92,22.65,14.04.ESI-TOF HRMS m/z:calcdfor C ₁₁₉ H ₁₉₉ N ₅ O ₁₈ ,[M+Na] ⁺ :2009.4702,found:2009.4637.

(4) Take the compound 5 described in step (3) and dissolve it in an organic solvent, add a catalyst, and undergo a reduction reaction to obtain compound 6; the reaction formula for obtaining compound 6 is shown in the following formula:

The specific operation of step (4) is: dissolving compound 5 (180.0mg, 90.1μmol) and molecular sieve (1.0g) in dichloromethane (10mL), airtight stirring for 15 minutes, cooling to -78℃, adding triethylsilane (52.0 μL, 326.4μmol) and trifluoromethanesulfonic acid (24μL, 271.8μmol), stir the reaction for 60 minutes, add 1.0mL triethylamine and methanol mixture (1:10), quench the reaction, filter to remove the gray insolubles, and remove The organic solvent yielded a gray solid crude product, which was separated and purified on a silica gel column to obtain a white solid compound 6 (110.8 mg, 61.57%). 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 = 7.6 Hz, 1H), 4.78–4.41 (m, 7H), 4.04 (m, J = 18.1, 9.8 Hz, 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 = 6.7 Hz, 18H). ¹³ C NMR (100MHz, CDCl ₃ ) δ174.26,173.70,173.40,171.42,169.96,169.54,138.32, 137.96,137.90,128.42,128.38,127.85,127.82,127.71,127.67,127.63,101.10,99.53,80.60,78.17,77.28,76.14,74.83,74.46,74.39,74.30,73.60,71.05,70.98,70.92,70.18,70.98,70.92,70.18,70.98 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, 29.38, 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) Take the compound 6 described in step (4) and dissolve it in an organic solvent, add a catalyst, and undergo a phospholipidization reaction to obtain compound 7; the reaction formula for obtaining compound 7 is shown in the following formula:

The specific operation of step (5) is: under the protection of nitrogen, dissolve compound 6 (80.0 mg, 40.2 μmol) in a mixed solution of dichloromethane and acetonitrile, and add dibenzyl diisopropyl phosphoramidite (300.0 μL, 913.0 μmol) ), triazole (1.3mL, 913.0μmol), stirred and reacted for 3 hours. After the mixture was cooled to 0°C, tert-butanol peroxide (280.0μL, 1540.0μmol) was slowly added dropwise and the reaction solution was slowly warmed to room temperature , The reaction was stirred for 1 hour; the solvent was removed to obtain the crude product, which was separated and purified by silica gel column to obtain white solid compound 7 (74.0 mg, yield of 81.8% in two steps). ¹ H NMR (400MHz, CDCl ₃ ) δ7.43-7.15 (m, 25H), 6.18 (d, J = 7.8 Hz, 1H), 5.97 (d, J = 8.1 Hz, 1H), 5.41 (t, J = 9.6Hz, 1H), 5.13 (m, J = 17.5, 6.0, 4.9 Hz, 3H), 4.89 (d, J = 7.8 Hz, 1H), 4.83 (d, J = 7.9 Hz, 4H), 4.73 (t, J = 10.2 Hz, 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, 4.1 Hz, 1H), 2.52–2.10 (m, 12H), 1.57 (t, J = 6.7 Hz, 12H), 1.24 (t, J = 4.3 Hz, 108H), 0.88 (t, J = 6.7 Hz, 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, 127.88,127.69,127.66,127.59,127.51,100.15,99.57,80.67,78.26,77.37,77.25,77.05,76.83,76.74,74.81,74.47,74.39,74.04,73.33,72.78,71.05,70.65,70.23,69.64,69.64 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. 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) Dissolve compound 8 and compound 7 described in step (5) in an organic solvent, and add a catalyst to react to obtain compound 9; the reaction formula for obtaining compound 9 is shown in the following formula:

The specific operation of step (6) is: tetrahydrofuran and methanol (1:2, 3.0mL) dissolve compound 7 (68.0mg, 30.2μmol), compound 8 (10.0mg, 20.4μmol), cuprous iodide (195.0mg, 1.0 mmol), add N,N-diisopropylethylamine (168.0μL, 1.0mmol), stir at room temperature for 12 hours; filter out the insoluble matter with Celite, and distill the filtrate under reduced pressure to remove the solvent to obtain the crude product; silica gel column separation and purification A white solid compound 9 (20.0 mg, yield 35.7%) was obtained. ¹ H NMR (600MHz, CDCl ₃ ) δ 7.98 (d, J = 26.3 Hz, 1H), 7.78-7.48 (m, 1H), 7.28 (td, J = 23.6, 22.7, 8.3 Hz, 29H), 6.81 ( s, 1H), 5.45 (d, J = 9.9 Hz, 1H), 5.25–5.06 (m, 2H), 4.93 (d, J = 9.3 Hz, 4H), 4.71 (m, J = 29.3, 19.0 Hz, 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 69.40,68.68,68.41,67.37,62.60,56.59,55.68,55.25,51.32,41.54,41.06,39.44,39.13,34.54,34.50,34.43,34.29,34.12,31.94,29.74,29.70,29.65,29.59,29.53,29.53,29. 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) The compound 9 described in step (6) is dissolved in an organic solvent, a catalyst is added, and the debenzylation reaction is carried out to obtain the conjugate containing monophosphorylated lipid A and carbohydrate antigen; to obtain the conjugate The reaction formula of compound (II) (MPLA-Tn) is shown in the following formula:

The specific operation of step (7) is: dissolve compound 9 (8.0mg) in dichloromethane/methanol/water (5:5:1, 10.0mL), add palladium hydroxide (5.0mg) and palladium on carbon (5.0mg), Hydrogen gas was passed through, sealed and stirred for 24 hours, the insoluble matter was filtered through diatomaceous earth, washed three times with 30.0 mL dichloromethane/methanol/water (5:5:1), and the filtrate was distilled under reduced pressure to remove the solvent to obtain a white solid compound. 10, which is 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)) δ 3.68 (s, 8H), 3.11 (d, J = 92.4 Hz, 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

This comparative example is the CRM197-Tn glycoprotein vaccine provided by the present invention. The structural formula is as follows:

The preparation method of the CRM197-Tn glycoprotein vaccine includes the following steps:

(1) Monosaccharide compound 10 is catalyzed by palladium on carbon and acetic acid to reduce azide to amino to obtain compound 11; compound 11 and bis(N-hydroxysuccinimide) suberate in dimethylformamide solution The reaction yields compound 12; specifically: dissolve compound 10 (100 mg, 0.244 mmol) in methanol (5 mL), add palladium on carbon (100 mg) and acetic acid (0.01 ml), seal, blow in hydrogen replacement gas 5 times, seal and stir for 20 Hours; diatomaceous earth was filtered to remove the insoluble matter, the filtrate was distilled under reduced pressure to remove the solvent to obtain a white solid. After dissolving in water, it was pre-frozen in a refrigerator at -80°C for 6 hours, then transferred to a lyophilizer and lyophilized to obtain a white solid compound 11 (86mg , 73%). Compound 11 was reacted with bis(N-hydroxysuccinimide) suberate (57.5mg, 36.6mmol) in dimethylformamide solution for 5 hours, and the organic solvent was removed under reduced pressure to obtain the crude product. Use ethyl acetate The solid was washed 8 times to obtain compound 12 (7 mg) as a white solid;

(2) Compound 12 was coupled with CRM197 protein in 0.1mol PBS (pH=7.8) buffered salt solution to obtain Tn-CRM197 glycoprotein vaccine; specifically: compound 12 was dissolved in 0.1mol PBS (pH=7.8) buffered salt solution and added CRM197 protein, stirred and reacted at room temperature for 2.5 days, transfer the reaction solution to a dialysis bag for dialysis for 2 days, change distilled water every 6 hours, and finally transfer the suspension in the dialysis bag to a sample bottle, pre-frozen at -80℃6 After hours, it was lyophilized with a lyophilizer to obtain a white solid compound Tn-CRM197 (5 mg).

The reaction formula of the above preparation method is as follows:

Experimental example 1

In this experimental example, mice were immunized with the fully synthetic sugar vaccine (MPLA-Tn) prepared in Example 1 and the glycoprotein vaccine (CRM197-Tn) prepared in Comparative Example 1, and their immune effects were preliminarily evaluated through the ELSA experiment and activated by fluorescence. Cell sorting (FACS) technology proves that antibody serum can specifically recognize tumor cells (MCF-7).

1. ELISA immunoassay

(1) Mouse immunity:

Twelve C57BL/6 mice aged 6-8 weeks were randomly divided into 2 groups. After the fully synthetic sugar vaccine MPLA-Tn and the glycoprotein vaccine CRM197-Tn were prepared into liposomes, the immunization test was carried out by subcutaneous injection of mice, using one initial immunization and three enhanced immunization programs, respectively in the 0th and 14th , 21, 28 days of injection of the prepared vaccine, each injection volume of 0.1mL (MPLA-Tn sugar vaccine contains 6μg Tn antigen); on the 38th day, each mouse from 0.1mL to 0.2mL, placed at 0 ℃ 60 Centrifuge at 4000 rpm for 15 minutes, and take the upper clear serum for ELISA detection and analysis.

(2) ELISA immunoassay:

Dissolve Tn-BSA in 0.1M carbonate buffer (pH 9.6), prepare a 2.0μg/mL solution, add 100.0μL per well to a 96-well plate, and incubate overnight at 4°C; the next day, 37°C incubator Incubate for one hour; wash the plate 3 times (300 μL/well/time) with PBST (PBS+0.05% Tween-20). After washing the plate, add PBS/1% BSA, add 250.0 μl to each well, incubate for one hour at room temperature, and wash the plate 3 times with PBST. The serum samples of 6 mice in each group were mixed in equal amounts and diluted with PBS 300, 900, 2700, 8100, 24300, 72900, 218700 and 656 100 times; the diluted serum was added to a 96-well plate at 100.0 μL per well, each The dilution gradient is made into three auxiliary wells in parallel; placed in a 37°C incubator and incubated for two hours, and the plate is washed 3 times. Add 100.0 μL of HRP (horseradish peroxidase) labeled IgG (diluted 2000 times) to each well, incubate for one hour at room temperature, and wash the plate 3 times. Add TMB solution, add 100.0μL to each well, and develop color in the dark at room temperature for 20 minutes, add 0.5M H ₂ SO ₄ solution, add 100.0μL to each well. Immediately detect the absorbance with a microplate reader, the detection wavelength is 450nm, and 570nm is used as the background wavelength.

(3) Plot the absorbance (OD) value against the dilution value of the antiserum, and obtain the best fit line. Use the equation of this line to calculate the dilution value when the OD value reaches 0.2, and calculate the antibody titer based on the reciprocal of the dilution value as shown in Figure 1.

(4) Experimental results:

It can be seen from Figure 1 that the MPLA-Tn sugar vaccine synthesized in Example 1 of the present invention, without an external adjuvant, can produce a specific immune response against the tumor sugar antigen Tn in mice, and produce more quickly High titer specific IgG antibody, and the IgG antibody titer is significantly higher than the glycoprotein vaccine CRM197-Tn.

2. Flow Cytometry (FACS)

Experimental method: Breast cancer cells MCF-7 overexpressing Tn sugar antigen and tumor cells MDA-231 not expressing Tn sugar antigen were cultured in MEM medium containing 10% fetal bovine serum (FBS) (37℃, 5 %CO2); trypsin digestion, harvest the cells, count the cells under the microscope, aliquot 2.0×105 cells into each test tube, add 1 mL of 3% FBS in PBS buffer (FACS buffer) to resuspend, and centrifuge 2 After 10 minutes, remove the supernatant and wash twice with FACS buffer; add the prepared mouse serum, incubate on ice for 1 hour, wash twice with FACS buffer, add fluorescently labeled secondary antibody, and protect from light on ice Incubate for one hour, wash twice with FACS buffer, resuspend in 0.8 mL of FACS buffer, and detect with flow cytometry. The results are shown in Figure 2.

Experimental results: As shown in Figure 2, MCF-7 is a breast cancer cell that overexpresses Tn antigen, and MDA-231 tumor cells that do not express Tn antigen are used as a negative control. In MCF-7 cells, compared with the pre-immune serum, the fluorescence peak of the antibody serum induced by the MPLA-Tn sugar vaccine synthesized in Example 1 of the present invention shifted to the right significantly. There is no obvious difference. The results showed that the antibody induced by the vaccine can specifically recognize MCF-7 cells expressing Tn antigen. The fluorescence peak of the antibody serum induced by the MPLA-Tn glycovaccine is higher than the fluorescence peak of the antibody serum induced by the glycoprotein vaccine CRM197-Tn. This indicates that the antibodies induced by the MPLA-Tn carbohydrate vaccine have a stronger ability to specifically recognize antigens.

In summary, the experimental results show that MPLA can improve the immunogenicity of Tn sugar antigens, present Tn sugar antigens to corresponding immune cells, and produce higher titer specific immune responses against tumor sugar antigen Tn, and its The titers of induced IgG antibodies and the ability to specifically recognize antigens are significantly higher than those induced by the glycoprotein vaccine CRM197-Tn; therefore, the invention provides a monophosphorylated lipid A (MPLA) and sugar antigen As a fully synthetic carbohydrate antigen vaccine, it is expected to become a new generation of anti-tumor drugs.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

1. A conjugate containing monophosphorylated lipid A and carbohydrate antigen, characterized in that the conjugate is a compound of general formula (I) or an isomer of a compound of general formula (I), Pharmaceutical salts, hydrates or solvent compounds;

   

   in:

   n is an integer of 2-6;

   R ₁ and R ₃ are -(CH ₂ )mCH ₃ , and m is an integer of 10-14;

   R ₂ , R ₄ and R ₅ are -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

   R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.
2. The conjugate according to claim 1, wherein the conjugate is a compound of structural formula (II) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of the compound of structural formula (II) ；

   

   in:

   R ₁ and R ₃ are -(CH ₂ )mCH ₃ , and m is an integer of 10-14;

   R ₂ , R ₄ and R ₅ are -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

   R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.
3. The conjugate according to claim 1, wherein the conjugate is a compound of structural formula (III) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of the compound of structural formula (III) ；

   

   Wherein: n is an integer of 2-6.
4. The conjugate according to claim 3, wherein the conjugate is a compound of structural formula (IV) or an isomer, pharmaceutically acceptable salt, hydrate or solvent compound of the compound of structural formula (IV) ；

   
5. The conjugate according to any one of claims 1-3, wherein the monophosphorylated lipid A is a compound of general formula (V) or an isomer of a compound of general formula (V), Pharmaceutical salts, hydrates or solvent compounds;

   

   in:

   R ₅ is -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

   R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.
6. The preparation method of the conjugate according to any one of claims 1 to 5, characterized by comprising the following steps:

   (1) Dissolve compound 1 and compound 2 in an organic solvent, and add a catalyst to react to obtain compound 3;

   (2) The compound 3 described in step (1) is catalyzed by ethylenediamine to obtain compound 4;

   (3) Take the compound 4 described in step (2) and the fatty acid chain, and perform peptide-forming and ester-forming reactions under the conditions of a condensing agent to obtain compound 5;

   (4) Dissolve the compound 5 described in step (3) in an organic solvent, add a catalyst, and undergo a reduction reaction to obtain compound 6;

   (5) Dissolve the compound 6 described in step (4) in an organic solvent, add a catalyst, and undergo a phospholipidization reaction to obtain compound 7;

   (6) Dissolve compound 8 and compound 7 described in step (5) in an organic solvent, and add a catalyst to react to obtain compound 9;

   (7) The compound 9 described in step (6) is dissolved in an organic solvent, a catalyst is added, and the conjugate is obtained by debenzylation reaction;

   The structural formulas of the compound 1 to the compound 9 are as follows:

   

   

   in:

   R ₀ is STol, SPh, Set or OC(NH)CCl ₃ ;

   n is an integer of 2-6;

   R ₁ and R ₃ are -(CH ₂ )mCH ₃ , and m is an integer of 10-14;

   R ₂ , R ₄ and R ₅ are -(CH ₂ )pCH ₃ , and p is an integer of 8-12;

   R ₆ is -CO(CH ₂ )rCH ₃ or -(CH ₂ )rCH ₃ , and r is an integer of 8-14.
7. The preparation method according to claim 6, characterized in that, in step (1), when R ₀ is STol, SPh or Set, the catalyst is N-iodosuccinimide and selected from trifluoromethyl Any one of sulfonic acid, silver trifluoromethanesulfonate, boron trifluoride ether, trimethylsilyl trifluoromethanesulfonate, or when R ₀ is OC(NH)CCl ₃ , the catalyst is selected from Any one of trifluoromethanesulfonic acid, boron trifluoride ethyl ether, and trimethylsilyl trifluoromethanesulfonate; the organic solvent is selected from any one of dichloromethane, diethyl ether, and tetrahydrofuran; the reaction The temperature is -40～-20℃;

   In step (3), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methyl iodide;

   In 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 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-butanol peroxide;

   In step (6), the organic solvent is a mixed solution of dichloromethane, methanol and water, and the catalyst is a mixture of cuprous iodide, N,N-diisopropylethylamine and glacial acetic acid;

   In step (7), the organic solvent is a mixed solution of methylene chloride, methanol and water, and the catalyst is a mixture of hydrogen, palladium on carbon and palladium hydroxide.
8. The preparation method according to claim 7, characterized in that, in step (1), when R ₀ is STol, the catalyst is N-iodosuccinimide (NIS) and the catalyst trifluoromethanesulfonic acid Mixture; the organic solvent is dichloromethane.
9. The use of the conjugate according to any one of claims 1 to 5 in the preparation of drugs for the prevention and/or treatment of cancer.
10. The application according to claim 8, wherein the cancer is breast cancer, uterine cancer, ovarian cancer, lung cancer, liver cancer, prostate cancer, melanoma, pancreatic cancer, bowel cancer, renal cell carcinoma, cellular lymphoma, thyroid gland Cancer, brain cancer, stomach cancer or leukemia.

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