CN110075291B - Monophosphoryl ester A conjugated Tn anti-tumor vaccine and application thereof - Google Patents

Monophosphoryl ester A conjugated Tn anti-tumor vaccine and application thereof Download PDF

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CN110075291B
CN110075291B CN201910105176.6A CN201910105176A CN110075291B CN 110075291 B CN110075291 B CN 110075291B CN 201910105176 A CN201910105176 A CN 201910105176A CN 110075291 B CN110075291 B CN 110075291B
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刘中秋
廖国超
高玲强
曾莉茗
马磊磊
李文伟
杨德盈
胡丽琴
吴鹏
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Guangzhou University of Traditional Chinese Medicine
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Abstract

The invention provides a monophosphoryl ester A conjugated Tn anti-tumor vaccine which is a compound with a general formula (I): Y-L-X (I). The invention uses the second generation of the new structure of TLR4 ligand type I compound to replace MPLA and the compound (Tn) with clinical development potential to couple and obtain the two-component vaccine with definite structure and stronger anti-tumor effect, and has better anti-tumor application prospect.

Description

Monophosphoryl ester A conjugated Tn anti-tumor vaccine and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a monophosphoryl ester A conjugated Tn anti-tumor vaccine and an application thereof.
Background
The prevention and treatment of cancer become an important public health problem in China, the morbidity and mortality of cancer are rapidly increased along with the aggravation of environmental pollution and the change of life style of people in China, the life safety of people in China is seriously threatened, and meanwhile, the social and personal economic burden is increased. Although the progress of targeted antitumor drugs such as small molecule targeted drugs, monoclonal antibody drugs and Antibody Drug Conjugates (ADCs) and tumor immunotherapy aiming at programmed cell death molecules and agonists thereof (PD-1, PD-L1) has been great in recent years, the progress has certain limitations, and the defects of large toxic and side effects, easiness in drug resistance generation, high recurrence risk and the like of tumor drugs cannot be overcome at present.
At present, the Tumor vaccine mainly comprises protein, nucleic acid, carbohydrate vaccine and the like, wherein the glycoprotein vaccine has become one of hot spots of domestic and foreign research, and the reason is that the Tumor-associated carbohydrate antigens (TACAs) are usually expressed in abnormal excess on the surface of Tumor cells and play a key role in the aspects of Tumor cell metastasis, signal transduction and the like. Therefore, the glycotumor vaccine has the advantages of high specificity, small side effect, potential of improving or curing tumors and the like, and is an ideal target spot of a therapeutic anti-tumor vaccine.
TACAs belong to T-independent antigens, are poorly immunogenic, do not induce T-cell responses, can only activate B-cells, can only produce a low affinity IgM antibody, and cannot produce high affinity IgG antibodies. To solve this difficulty, the traditional strategy is to covalently link the glycoprotein antigen with a carrier protein containing T cell epitopes to prepare glycoprotein vaccines, and when such vaccines enter the body, antigen Presenting Cells (APCs) phagocytose and hydrolyze the glycoprotein and present the protein to T cells, activate the T cells, induce proliferation of B cells and produce IgG antibodies, and at the same time produce B cells with immunological memory.
Among them, tn antigen (alpha-D-GalNAc-Ser/Thr) is a common carbohydrate antigen related to cancer, and is over-expressed in cluster form on the surface of malignant cells such as breast cancer, prostate cancer, lung cancer, pancreatic cancer and the like.
As the acetylgalactosamine derivative, the Tn antigen is capable of further elongating sugar chains in normal cells to form complex oligosaccharides, so that it is recessive to normal cells; in most cancer cells, the Tn antigen is exposed on the cell surface due to the inhibition of sugar chain extension. Therefore, the over-expression of Tn antigen has high specificity to some cancer cells, and is an important reference in early diagnosis of cancer. Tn antigens are also very attractive targets for immunotherapy in the study of designing synthetic anti-cancer vaccines. Tumor vaccines containing Tn antigens as single tumor antigens or as multivalent vaccine components have been advanced to phase I and phase II clinical trials. Researchers have done a lot of work on Tn-related tumor vaccine studies, and a brief summary and overview of the relevant studies is provided below.
Singhal, A. Et al found that antigen vaccines coupled with asialo goat submaxillary gland mucin (A-OSM) and Tn antigen can significantly express the Tn antigen; antigen vaccines coupled with protein vectors (KLH or sheep serum protein) and dimeric Tn can also effectively prevent metastasis of TA3HA mouse breast cancer, showing strong expression of TN antigen. And a dimer Tn antigen and tripalmitoyl-s-glycerocysteinyl serine (P3 CS) coupled vaccine is synthesized, is a completely synthesized immunogen with low molecular weight and no carrier, and triggers immune response aiming at glycoprotein expressing Tn. This is a precedent that the first synthetic small carbohydrate antigen can generate an immune response against tumor-associated carbohydrate antigens without the use of macromolecular carriers or adjuvants.
By combining the analysis, a plurality of two-component tumor protein vaccines combined with Tumor Associated Carbohydrate Antigens (TACAs) are synthesized by taking TLR4 (Toll-like receptor 4) agonist MPLA (Monophosphoryl lipid A) as a carrier, and have certain feasibility.
Disclosure of Invention
In order to solve the technical problems, the invention provides a monophosphoryl ester A conjugated Tn anti-tumor vaccine which has a good anti-tumor application prospect.
The invention adopts the following technical scheme to realize the purpose of the invention: a monophosphoryl ester A conjugated Tn anti-tumor vaccine is a compound with a general formula (I):
Y-L-X(I)
or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate of the compound of formula (I);
wherein:
y is selected from the group having the following structure:
Figure BDA0001966552110000031
R 1 is selected from-CH 2 -CH(-OR 4 )(CH 2 ) m CH 3 ，R 4 is-C (O) - (CH) 2 ) n CH 3 M and n are integers of 6 to 18;
R 2 is selected from-CH 2 -CH(-OR 5 )(CH 2 ) P CH 3 ，R 5 is-C (O) - (CH) 2 ) q CH 3 P and q are integers of 6 to 18;
R 3 is selected from-CH 2 -CH(-OR 6 )(CH 2 ) r CH 3 ，R 6 Is- (CH) 2 ) s CH 3 or-C (O) - (CH) 2 ) t CH 3 R, s, t are integers from 6 to 18;
l is selected from the formula- (CH) 2 ) 2 -NHC(O)-(CH 2 ) a -C(O)NH-(CH 2 ) b -or structure
Figure BDA0001966552110000032
The connecting arm of (1); wherein a, b, c are integers selected from 2 to 6;
x is selected from the group having the following structure:
Figure BDA0001966552110000033
preferably, said Y is selected from the group having the following structure:
Figure BDA0001966552110000034
R 1 is-CH 2 -CH(-OR 4 )(CH 2 ) 10 CH 3 ，R 4 is-C (O) - (CH) 2 ) 12 CH 3 ；
R 2 is-CH 2 -CH(-OR 5 )(CH 2 ) 10 CH 3 ，R 5 is-C (O) - (CH) 2 ) 12 CH 3 ；
R 3 is-CH 2 -CH(-OR 6 )(CH 2 ) 10 CH 3 or-CH 2 -CH(-OR 6 )(CH 2 ) 12 CH 3 R6 is- (CH) 2 ) 11 CH 3 、 -C(O)-(CH 2 ) 10 CH 3 or-C (O) - (CH) 2 ) 12 CH 3 。
Preferably, L is of the structure
Figure BDA0001966552110000035
The connecting arm of (a).
Preferably, the general formula (I) is:
Figure BDA0001966552110000041
or a pharmaceutically acceptable salt of a compound of formula (I).
The invention also provides application of the compound in preparing a medicament for treating cancer.
Preferably, the medicament further comprises medically acceptable auxiliary materials.
Preferably, the cancer is breast cancer, prostate cancer, intestinal cancer, melanoma, liver cancer, lung cancer, renal cell carcinoma, uterine cancer, ovarian cancer, cellular lymphoma, brain cancer, stomach cancer, pancreatic cancer, a cancer of the nail head, or leukemia.
The invention uses the second generation of TLR4 agonist compound of formula I with brand new structure as vaccine carrier, and couples with the compound of formula II (Tn) with clinical development potential to obtain two-component vaccine with definite structure and anti-tumor effect, and has better anti-tumor application prospect.
Drawings
FIG. 1 is a graph showing the results of ELISA IgG antibody titer for the compounds 1,2,3 and 4 of the present invention.
FIG. 2 is a graph showing the results of ELISA IgM antibody titers of the compounds 1,2,3 and 4 of the present invention.
FIG. 3 is a graph showing the cell lysis rate of the compound 1,2,3 antibody-mediated complement-dependent cytotoxicity assay of the present invention.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
The vaccines of the present invention (compounds of formula I) can be prepared using synthetic methods known in the art and described below (schemes 1-3):
scheme 1: synthesis of Compound 10
Figure BDA0001966552110000051
The synthesis of compound 10 is shown in scheme 1: a monosaccharide molecule 5 is obtained by protecting a two-position amino group with phthaloyl, acetylating, one-position connecting p-toluenesulfonyl, deacetylating and four-position and six-position protecting glucosamine which is sold in the market. Tert-butyldimethylsilyl chloride is used to protect the three-position hydroxyl under the action of imidazole. The six position was then selectively cleaved with borane tetrahydrofuran and silver triflate. Then, the hydroxyl at the six-position is reacted with bromopropyne under the catalysis of sodium hydride and tetrabutylammonium bromide to obtain 8. Then, N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) were used as catalysts and docked with homemade azidoethanol to give compound 9. Removal of the phthaloyl group with ethylenediamine in refluxing methanol gives compound 10.
Scheme 2: synthesis of Compound 15
Figure BDA0001966552110000061
Synthesis of Compound 15 is shown in scheme 2: triethylamine trihydrofluoride was used to remove the three-tert-butyldimethylsilyl group. The free amino and hydroxyl groups are acylated with fatty acid 12 in the presence of 4-dimethylaminopyridine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methiodide, and in the one-pot process. The four p-methoxybenzyl groups were then removed with 5% trifluoroacetic acid. The compound 14 is phosphorylated by using tetrazole, dibenzyl N, N-diisopropylphosphoramidite and t-butyl peroxy-alcohol in a two-step one-pot method to obtain a synthetic compound 15.
Scheme 3: synthesis of target product compounds 1,2,3 and 4 of the invention
Figure BDA0001966552110000071
Lipid A:
Figure BDA0001966552110000072
The synthesis of compounds 1,2,3, 4 is shown in scheme 3: under acidic conditions, the azide group of 15 is reduced with Zn. Condensed with four fatty acids (Lipid A-1, 2,3, 4) respectively in the presence of N, N-diisopropylethylamine. Then, the antigen Tn is connected with the antigen Tn through covalent bond triazole under the action of N, N-diisopropylethylamine and catalytic amount of cuprous iodide. Finally, debenzylation was performed with hydrogen and palladium on carbon to give compounds 1,2,3, 4.
The synthesis of compounds 1,2,3, 4 is achieved by the following specific examples:
example 1: preparation of Compound 1
Synthesis of [3-R- (dodecanoic acid carboxylate) dodecanoylamino ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoic acid carboxylate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoic acid carboxylate) tetradecanoyl ] -beta-D-glucoside (Compound 1)
Step 1: preparation of 2-deoxy-2- (2-carboxybenzoyl) -D-glucose (Compound 25)
Figure BDA0001966552110000081
Adding D-glucosamine hydrochloride (80.00g, 0.37mol) into a mixed solution of water (350.0 mL) and methanol (150.0 mL), cooling the mixed solution to 0 ℃, adding sodium hydroxide (15.60g, 0.39mol) under stirring, stirring in an ice bath for 1 hour, then adding phthalic anhydride (63.20g, 0.43mol), slowly returning the mixed solution to the room temperature, continuing stirring overnight, gradually precipitating white solids in the reaction solution, detecting the reaction process by using silica gel Thin Layer Chromatography (TLC), stopping stirring and cooling in the ice bath for 1 hour after detecting that raw materials completely disappear, layering, carrying out vacuum filtration under reduced pressure to obtain a yellowish solid, washing with a small amount of water for 3 times, and carrying out vacuum drying to obtain a compound 25 (67.35g, 55%).
Step 2: preparation of 1,3,4, 6-tetra-O-acetyl-2-deoxy-2-phthalamido-beta-D-glucose (Compound 26)
Figure BDA0001966552110000082
Compound 25 (20.00g, 0.06mol) and anhydrous sodium acetate (13.50g, 2.69mol) were added to 270.0mL of acetic anhydride, and the mixture was refluxed with stirring at 198 ℃ for 10 hours, and then subjected to thin layer chromatography to detect that the reaction (ethyl acetate: petroleum ether = 1.
And step 3: preparation of p-tolylthio 3,4, 6-tri-O-acetyl-2-deoxy-2-phthalamido-1-thio-beta-D-glucoside (Compound 27)
Figure BDA0001966552110000091
Compound 26 (13.35g, 0.03mol) and p-methylphenylthiol (4.50g, 0.04mol) were dissolved in dry anhydrous dichloromethane (45.0 mL), the mixture was cooled to 0 ℃, then under the protection of nitrogen, boron trifluoride ether complex catalyst (5.30ml, 0.04mol) was slowly added dropwise, the mixture was slowly warmed to room temperature, the color of the reaction solution gradually changed from yellow to black, the reaction was continued for 48 hours, the reaction was monitored by Thin Layer Chromatography (TLC) (ethyl acetate: toluene =1 3), after detecting complete disappearance of the raw materials, the reaction solution was diluted with dichloromethane under ice bath conditions, quenched with saturated sodium bicarbonate solution, repeatedly washed 3 times until no bubble was generated, the organic layer was collected, after drying with anhydrous sodium sulfate, the organic solvent was distilled off under reduced pressure to give a crude yellow solid, and compound 27 (12.8785% was obtained after separation and purification by column chromatography (ethyl acetate: petroleum ether = 1.
And 4, step 4: preparation of p-toluenesulfonyl 2-deoxy-2-phthalamido-1-thio-beta-D-glucoside (compound 28)
Figure BDA0001966552110000092
Dissolving compound 27 (226.00g, 0.42mol) with anhydrous dichloromethane, preparing a sodium methoxide/methanol solution (0.4M), weighing 1.47g of metallic sodium, adding the metallic sodium into 160.0mL of methanol solution in batches, cooling to room temperature, adding the solution into the dichloromethane solution containing 27 under the condition of an ice bath, stirring for 90 minutes under the protection of nitrogen, slowly precipitating a solid in the reaction solution to obtain a yellow suspension, monitoring the reaction process by TLC (methanol: dichloromethane = 1), detecting that the raw materials completely disappear, placing a reaction bottle into the ice bath for two hours to ensure that the product is precipitated as much as possible, filtering under reduced pressure to obtain a white solid, collecting filtrate, neutralizing the filtrate with glacial acetic acid, removing the organic solvent to obtain a crude product, recrystallizing the crude product with methanol and dichloromethane, and performing suction filtration under reduced pressure to obtain a white solid compound 28 (130.00g, 74%).
And 5: preparation of p-tolylthio 4, 6-oxy-p-methoxybenzylidene-2-deoxy-2-phthalamido-1-thio-beta-D-glucoside (Compound 5)
Figure BDA0001966552110000101
Compound 28 (1.00g, 2.40mmol) and p-toluenesulfonic acid monohydrate (0.02g, 0.12mmol) were dissolved in 4.6mL of anhydrous N, N-dimethylformamide, p-methoxybenzaldehyde dimethyl acetal (0.7ml, 3.60 mmol) was added dropwise under nitrogen protection, the reaction was stirred, occasionally evacuated, the produced methanol was removed, the reaction was allowed to proceed in the forward direction, after 3 hours of reaction, the reaction process was monitored by TLC (ethyl acetate: petroleum ether = 1), after detecting the complete disappearance of the raw materials, the reaction solution was diluted with 30.0mL of ethyl acetate, quenched with saturated sodium bicarbonate solution, washed with water 2 times (30.0 mL × 2), the organic layer was collected, after drying with anhydrous sodium sulfate, the whole solvent was distilled off under reduced pressure to give a white solid crude product, which was isolated and purified by column chromatography (ethyl acetate: petroleum ether = 1) to give compound 5 (1.00g, 80%) as a white solid.
Step 6: preparation of p-tolylthio 4, 6-oxy-p-methoxybenzylidene-2-deoxy-2-phthalamido-3-oxy-tert-butyldimethylsilyl-1-thio-. Beta. -D-glucoside (Compound 6)
Figure BDA0001966552110000102
Dissolving compound 5 (1.00g, 1.87mmol) and imidazole (0.77g, 11.25mmol) in anhydrous N, N-dimethylformamide, cooling the mixed solution to 0 ℃, adding tert-butyldimethylchlorosilane (0.56g, 3.75mmol), reacting and stirring under the protection of nitrogen overnight,the reaction process was monitored by TLC (ethyl acetate: petroleum ether =1 = 4), and after complete disappearance of the starting material (reaction solution was a white suspension), the reaction solution was diluted and dissolved with 130.0mL of ethyl acetate, followed by addition of 100.0mL of saturated sodium bicarbonate solution and washing 2 times, and finally washing 1 time with 100.0mL of saturated brine, and the organic layer was collected, dried over anhydrous sodium sulfate, and then the organic solvent was removed by reduced pressure distillation to obtain a crude product. After the crude product was isolated and purified by column chromatography (ethyl acetate: petroleum ether =1:20,1:15,1) compound 6 was obtained as a white solid (0.80g, 66%). 1 HNMR(400MHz，CDCl 3 )，δ(ppm)：7.87(dd， J＝21.1，5.6Hz，2H，Ar-H)，7.80–7.67(m，2H，Ar-H)，7.39(d，J＝8.7Hz，2H，Ar-H)，7.26(d， J＝8.0Hz，2H，Ar-H)，7.05(d，J＝8.0Hz，2H，Ar-H)，6.89(d，J＝8.9Hz，2H，Ar-H)，5.58(d， J＝10.7Hz，1H，H-1)，5.48(s，1H，CH)，4.62(t，J＝9.2Hz，1H)，4.33(m，2H)，3.80(s，3H， Ac-CH 3 )，3.79(t，J＝10.03，1H)，3.67(td，J＝9.6，4.8Hz，1H)，3.54(t，J＝9.0Hz，1H)，2.30(s， 3H，Ar-CH 3 )，0.57(s，9H，Si-(CH3)3-0.14(s，3H，Si-CH 3 )，-0.31(s，3H，Si-CH 3 )。 13 CNMR(100MHz， CDCl 3 )，δ(ppm)：168.39，167.44，160.06，138.25，134.29，134.21，133.04，131.73，129.70，129.63，128.22，127.68，123.67，123.25，113.50，101.90，84.63，82.43，70.70，70.59，68.66， 56.77，55.27，25.54，25.41，21.18，17.74，-4.10，-5.34。 ESI-TOFHRMSm/z:calcdforC 35 H 41 NO 7 SSiNa[M+Na] + 670.2265，found 670.2257。
And 7: preparation of p-tolylthio 4-oxy-p-methoxybenzyl-2-deoxy-2-phthalamido-3-oxy-tert-butyldimethylsilyl-1-thio-beta-D-glucoside (compound 7)
Figure BDA0001966552110000111
Compound 6 (1.00g, 1.54mmol), borane tetrahydrofuran complex (7.70mL, 7.70mmol) was added to anhydrous grade bisMethyl chloride (30.0 mL), stirred for 15 minutes under closed conditions, then the catalyst silver trifluoromethanesulfonate (0.06 g, 0.23 mmol) was added to the reaction solution, stirred for about 30 minutes, the reaction process was monitored by TLC (ethyl acetate: petroleum ether = 1. 1 H NMR(400MHz，CDCl 3 )，δ(ppm)：7.96–7.63(m，4H，Ar-H)，7.30–7.15(m， 4H，Ar-H)，7.05(d，J＝7.8Hz，2H，Ar-H)，6.88(d，J＝8.4Hz，2H，Ar-H)，5.57(d，J＝10.6Hz， 1H，H-1)，4.77(d，J＝11.1Hz，1H，CH 2 -H)，4.58(d，J＝11.2Hz，1H，CH 2 -H)，4.49(t，J＝8.9Hz， 1H，H-3)，4.23(t，J＝10.2Hz，1H，H-2)，3.88(d，J＝11.9Hz，1H，H-6)，3.80(s，3H，-O-CH 3 )， 3.68(dd，J＝12.0，4.0Hz，1H，H-6)，3.62–3.43(m，2H，H-4，H-5)，2.30(s，3H，Ar-CH 3 )， 0.74(s，9H，Si-(CH3)3)，-0.00(s，3H，Si-CH 3 )，-0.41(s，3H，Si-CH 3 )。 13 CNMR(100MHz， CDCl 3 )，δ(ppm)：168.67，167.44，159.15，138.23，134.28，133.00，132.02，131.74，130.14， 129.73，128.97，128.24，123.63，123.25，113.80，83.64，79.59，79.41，74.53，73.36，62.08， 56.90，55.27，25.70，21.16，17.66，-4.05，-4.60。ESI-TOF HRMS m/z:calcd for C 35 H 43 NO 7 SSiNa[M+Na] + 672.2422，found 672.2424。
And step 8: preparation of p-tolylthio 4-oxy-p-methoxybenzyl-6-oxy-propargyl-2-deoxy-2-phthalamido-3-oxy-tert-butyldimethylsilyl-1-thio-beta-D-glucoside (compound 8)
Figure BDA0001966552110000121
After adding compound 7 (1.00g, 1.16mmol) after vacuum drying and a vacuum high-temperature dried molecular sieve to 16.0mL of anhydrous N, N-dimethylformamide, stirring for 2 hours under the protection of nitrogen, reducing the temperature of the mixture to 0 ℃, rapidly adding sodium hydride (0.23g, 5.78mmol), continuing to stir for 15 minutes under the condition of 0 ℃, then slowly adding bromopropyne (1.0 mL, 11.56mmol) and a catalytic amount of tetrabutylammonium bromide to the reaction solution, slowly raising the mixture to room temperature, continuing to stir for 3 hours, monitoring the reaction process by TLC (ethyl acetate: petroleum ether = 1), after detecting that the raw materials completely disappear, adding 2.0mL of isopropanol to quench the reaction and stirring for 30 minutes, diluting the reaction solution with 30.0mL of dichloromethane, adding a saturated sodium bicarbonate solution to neutralize the reaction, washing the organic phase with water for 3 times, collecting the organic layer, drying with anhydrous sodium sulfate, removing the crude organic solvent under reduced pressure to obtain a crude product (black distilled product, and purifying the crude product by a yellow solid column chromatography (1.1: 798, 798% of acetic acid). 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.82(t，J＝21.9Hz，2H，Ar-H)，7.77–7.61(m，2H，Ar-H)， 7.26(t，J＝8.7Hz，4H，Ar-H)，7.03(d，J＝8.0Hz，2H，Ar-H)，6.86(d，J＝8.6Hz，2H，Ar-H)， 5.50(d，J＝10.5Hz，1H，H-1)，4.74(d，J＝11.1Hz，1H，-CH2-H)，4.65(d，J＝11.1Hz，1H，-CH2-)， 4.46(dd，J＝9.6，8.5Hz，1H，H-3)，4.33–4.04(m，3H，H-2，-O-CH2-)，3.86–3.70(m，5H， -O-CH3，H-6)，3.70–3.58(m，1H，H-4)，3.58–3.45(m，1H，H-5)，2.39–2.31(m，1H，CH)， 2.28(s，3H，Ar-CH3)，0.73(s，9H，Si-(CH3)3)，-0.04(s，3H，Si-CH3)，-0.44(s，3H，Si-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：168.69，167.40，159.00，138.02，134.22，133.17， 132.08，131.77，130.46，129.59，128.78，128.47，123.59，123.20，113.67，83.77，79.76， 79.37，79.27，74.77，74.28，73.51，68.38，58.75，56.83，55.26，25.72，21.18，17.66，-4.06， -4.59。ESI-TOFHRMSm/z:calcd for C 38 H 46 NO 7 SSi[M+H] + 407.1925，found 670.1915。
And step 9: preparation of azidoethyl 4-oxo-p-methoxybenzyl-6-oxo-propargyl-2-deoxy-2-phthalamido-3-oxo-tert-butyldimethylsilyl-beta-D-glucoside (compound 9)
Figure BDA0001966552110000122
The vacuum dried compound 8 (0.20g, 0.29mmol), azidoethanol (0.05g, 0.58mmol) and the molecular sieve after drying at high temperature (0.65 g) were added to 5.0mL of anhydrous dichloromethane, and the mixture was hermetically stirred for 4 hours under the protection of nitrogen. After the mixed solution is cooled to-30 ℃ from room temperature, N-iodosuccinimide (0.19g, 0.87mmol) is rapidly added, after the mixed solution is stirred and reacts for 1 hour at-30 ℃, the reaction solution is cooled to-40 ℃, trifluoromethanesulfonic acid (6.4uL, 0.87 mmol) is rapidly added and stirred and reacts for 15 minutes, the color of the reaction solution becomes wine red, the TLC monitors the reaction process (ethyl acetate: petroleum ether =1: 3), after the raw materials are detected to be completely disappeared, the reaction solution is diluted by dichloromethane and then is neutralized by adding saturated sodium bicarbonate solution, and sodium thiosulfate is added until the red of the reaction solution fades. The organic layer was washed with water 2 times and saturated brine 1 time, and the organic layer was collected, dried over anhydrous sodium sulfate, and then the organic solution was distilled off under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (ethyl acetate: petroleum ether =1, 18, 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.82(dd，J＝18.8，15.9Hz，2H，Ar-H)，7.78-7.64(m， 2H，Ar-H)，7.27(d，J＝6.9Hz，2H，Ar-H)，6.87(d，J＝8.5Hz，2H，Ar-H)，5.25(d，J＝8.5Hz， 1H，H-1)，4.75(t，J＝11.8Hz，1H-CH2-)，4.66(d，J＝11.1Hz，1H，-CH2-)，4.43(dd，J＝10.1， 8.3Hz，1H，H-3)，4.34–4.08(m，3H，H-2，-O-CH2-)，4.02–3.89(m，1H，-CH2-)，3.88–3.70(m， 5H，Ar-CH3，H-6)，3.68–3.48(m，3H，H-4，H-5，-CH2-)，3.34(ddd，J＝12.1，8.2，3.5Hz， 1H，-CH2-)，3.15(dt，J＝13.2，4.1Hz，1H，-CH2-)2.33(dd，J＝14.7，12.7Hz，1H，≡CH)， 0.74(s，9HSi-(CH3)3)，-0.02(d，J＝9.0Hz，4H，Si-CH3)，-0.42(s，3H，Si-CH3)。 13 CNMR(100MHz， CDCl3)，δ(ppm)：159.08，134.06，132.05，130.46，128.82，123.45，113.67，98.14，79.56， 74.33，72.34，68.25，68.16，58.70，57.36，55.26，50.49，25.69，17.64，-4.11，-4.60。
Step 10: preparation of azidoethyl 4-oxo-p-methoxybenzyl-6-oxo-propargyl-2-deoxy-2-amino-3-oxo-tert-butyldimethylsilyl-beta-D-glucoside (Compound 10)
Figure BDA0001966552110000131
Compound 9 (0.50g, 0.76mmol), ethylenediamine (5.0 mL) was added to 30.0mL of methanol, stirred to complete dissolution, then the reaction solution was heated to 80 ℃ again, refluxed overnight, TLC monitored the reaction process (ethyl acetate: petroleum ether =1: 2), after detecting complete disappearance of the raw materials, the reaction solution was cooled to room temperature, after removing most of the solvent under reduced pressure, toluene (4.0 mL) was added, and excess ethylenediamine was removed by distillation under reduced pressure 3 times to give a crude product (yellow oily liquid), which was isolated and purified by column chromatography (ethyl acetate: petroleum ether =1, 12,1, 8, 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.33–7.12(m，2H，Ar-H)，6.78(d，J＝8.6Hz，2H，Ar-H)，4.65(d，J＝11.1Hz，1H，Ar-H)，4.51(d，J＝11.1Hz，1HAr-H)，4.20–4.06(m，3H，-O-CH2-， H-1)，4.03–3.94(m，1H，-CH2-)，3.71(s，3H，-O-CH3，)，3.66–3.56(m，3H，H-6，-CH2-)， 3.51–3.40(m，2H，-CH2-，H-3)，3.40–3.32(m，2H，H-4，H-5)，3.21(ddd，J＝13.2，4.8， 3.5Hz，1H，-CH2-)，2.72(dd，J＝9.0，8.1Hz，1H，H-2)，2.25(t，J＝2.3Hz，1H，≡CH)，0.86(s， 9H，Si-(CH3)3)，0.07(s，3H，Si-CH3)，-0.01(d，J＝5.8Hz，3H，Si-CH3)。 13 CNMR(100MHz， CDCl3)，δ(ppm)：159.04，130.43，128.97，113.67，103.71，79.56，78.17，77.50，75.03， 74.88，74.26，68.70，68.48，58.64，58.14，55.26，50.80，29.71，26.04，18.17，-3.74，-3.91.ESI-TOF， HRMS m/z:calcd for C 25 H 41 N 4 O 6 Si[M+H] + 521.2793，found 521.2790。
Step 11: preparation of azidoethyl 4-oxo-p-methoxybenzyl-6-oxo-propargyl-2-deoxy-2-amino-beta-D-glucoside (Compound 11)
Figure BDA0001966552110000141
Compound 10 (0.20g, 0.38mmol) was dissolved in 1.5mL of tetrahydrofuran solution, triethylamine trihydrofluoride (1.50ml, 9.20mmol) was added dropwise, the reaction was stirred at room temperature for 40 hours, TLC monitored for the reaction process (ethyl acetate 100%), after detecting complete disappearance of the raw material, the reaction solution was diluted with dichloromethane, a saturated sodium bicarbonate solution was added to quench the reaction until no bubble was produced, and then extracted with ethyl acetate 3 times by 30.0mL each time, the organic layer was collected, after drying anhydrous sodium sulfate, the organic solvent was distilled off under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (methanol: dichloromethane =1, 100,1, 50, 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.31–7.13(m，2H， Ar-H)，6.81(d，J＝8.5Hz，2H，Ar-H)，4.71–4.55(m，2H，Ar-CH2-)，4.28–4.07(m，3H， -CH2-，H-1)，4.00(dt，J＝10.4，4.2Hz，1H，-CH2-)，3.82–3.65(m，4H，-O-CH3，H-6)， 3.60(tt，J＝15.4，7.7Hz，1H，-CH2-)，3.45(ddd，J＝14.5，9.0，5.0Hz，1H，-CH2-)，3.41–3.31(m， 3H，H-3，H-4，H-5)，3.27–3.17(m，1H，-CH2-)，2.63(t，J＝8.7Hz，1H，H-2)，2.36(t，J＝2.3Hz， 1H，≡CH)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：159.45，130.42，129.76，113.99，104.13， 79.58，77.53，74.90，74.84，74.29，68.65，68.46，58.69，57.30，55.29，50.77。
Step 12: preparation of azidoethyl 4-oxo-p-methoxybenzyl-6-oxo-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 13)
Figure BDA0001966552110000151
Compound 11 (0.20g, 0.49mmol), compound 12 (0.62g, 1.37mmol) and 4-dimethylaminopyridine (1.8 mg, 0.014 mmol) were all dissolved in a dichloromethane solution treated without water, and the reaction was stirred under nitrogen protection, then the reaction solution was cooled to 0 ℃, a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methyliodide salt (0.73g, 2.46mmol) was added, and after stirring for 30 minutes, the reaction solution was slowly heated to 10 ℃, and the reaction process was monitored by TLC (ethyl acetate: petroleum ether =1, 2.5), and after detecting complete disappearance of the raw material, the reaction solution was diluted with 20.0mL of dichloromethane and then washed 3 times with 30.0mL of saturated brine, an organic layer was collected, dried with anhydrous sodium sulfate, and the organic solution was removed by distillation under reduced pressure to obtain a crude compound 13 (0.41g, 65%) which was purified by column chromatography (ethyl acetate: petroleum ether =1, 8, 1) to obtain a white solid. 1 H NMR(400MHz， CDCl3)，δ(ppm)：7.20(d，J＝8.3Hz，2H，Ar-2H)，6.85(d，J＝8.4Hz，2H，Ar-2H)，6.05(d， J＝8.7Hz，1H，-NH-)，5.24–5.02(m，3H，H-3，-OCOCH-，-OCOCH-)，4.63(t，J＝10.6Hz， 1H，H-1)，4.56(s，2H，PMPCH2-)，4.35–4.09(m，2H，-OCH2C-)，4.03–3.91(m，1H， -OCH2CH2-)，3.87–3.60(m，8H，-OCH3，H-2，H-6，H-4，-OCH2CH2-)，3.59–3.38(m， 2H，H-5，-OCH2CH2-)，3.29(dt，J＝13.2，4.2Hz，1H，-CH2N3)，2.53–2.26(m，7H，-COCH2-6H， ≡CH)，2.21(t，J＝7.5Hz，2H，-NHCOCH2-)，1.58(d，J＝17.9Hz，8H)，1.25(s，76H，-CH2-)，， 0.95–0.75(m，12H，-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：73.83，173.26，170.31， 169.92，159.38，129.94，129.58，113.84，100.75，79.49，75.09，74.92，74.81，74.06，70.86， 69.83，68.02，67.64，58.73，55.23，54.38，50.64，41.84，39.09，34.53，34.42，34.20，31.95， 29.73，29.71，29.69，29.64，29.58，29.47，29.39，29.25，29.20，25.27，25.15，25.02，22.71， 14.13。ESI-TOFLRMSm/z:calcd for C 75 H 131 N 4 O 12 [M+H] + 1278.9，found 1279.9。
Step 13: preparation of azidoethyl 6-oxy-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxy- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 14)
Figure BDA0001966552110000152
After 0.3mL of trifluoroacetic acid was diluted with 2.5mL of anhydrous dichloromethane, a few drops of 1, 8-diazabicycloundecen-7-ene were added dropwise and the reaction was stirred for 30 minutes. Then, compound 13 (0.20g, 0.16mmol) was dissolved in 2.6mL of anhydrous grade dichloromethane, the temperature was decreased to 0 ℃, a diluted trifluoroacetic acid solution was slowly added dropwise to the reaction solution, the temperature was slowly raised to room temperature, the reaction process was monitored by TLC (ethyl acetate: petroleum ether = 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：6.04(d， J＝8.5Hz，1H-NH-)5.20–5.00(m，3H，H-3，-OCOCH-)，4.67(d，J＝8.3Hz，1H，H-1)，4.22(s， 2H，-OCH2CCH)，4.06–3.93(m，1H，-OCH2CH2-)，3.92–3.73(m，3H，H-2，H-6)，3.75–3.51(m， 3H，H-4，H-5，-OCH2CH2-)，3.52–3.38(m，1H，-CH2N3)，3.28(d，J＝13.2Hz，1H，-CH2N3)， 2.62–2.20(m，9H，≡CH，-COCH2-)，1.58(s，8H，-CH2-)，1.25(s，76H，-CH2-)，0.87(t， J＝6.6Hz，12H，-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：174.48，173.69，171.56， 169.98，100.71，79.43，76.06，74.87，74.80，71.13，70.99，69.50，69.04，67.74，58.87， 53.91，50.64，41.83，40.27，34.81，34.52，34.50，34.26，31.94，29.73，29.70， 29.68，29.63，29.60，29.56，29.48，29.38，29.35，29.32，29.24，29.18，25.30， 25.17，25.02，24.96，22.70，14.13。ESI-TOF LRMS m/z:calcd for C 67 H 123 N 4 O 11 [M+H] + 1158.9，found 1160.1。
Step 14: preparation of azidoethyl 4-oxy- (di-oxy-benzylphosphoryl) -6-oxy-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxy- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 15)
Figure BDA0001966552110000161
Compound 14 (0.20g, 0.17mmol) was dissolved in redistilled anhydrous dichloromethane, dibenzyl N, N-diisopropylphosphoramidite (0.2ml, 0.52mmol) and triazole (0.45m, 1.9ml, 0.86mmol) were added, the reaction was stirred for 3 hours under the protection of nitrogen, the reaction process was monitored by TLC (ethyl acetate: petroleum ether = 1. 1 H NMR(400MHz，CDCl3)，δ (ppm)：7.30(dd，J＝19.4，8.8Hz，10H，Ar-H)，6.22(d，J＝7.6Hz，1H，-NH-)，5.55–5.42(m， 1H，H-3)，5.16(d，J＝5.2Hz，2H，-OCOCH-)，5.07(d，J＝8.2Hz，1H，H-1)，5.04–4.85(m， 4H，ArCH2-)，4.46–4.32(m，1H，H-4)，4.10(q，J＝15.8Hz，2H，-OCH2CCH)，3.98(dd， J＝18.2，13.0Hz，1H，-OCH2CH2-)，3.89(t，J＝13.0Hz，1H，H-6)，3.65(ddd，J＝15.8，14.5， 6.3Hz，3H，H-6，H-5，-OCH2CH2-)，3.56–3.39(m，2H，H-2，-OCH2CH2-)，3.29(d，J＝13.3Hz， 1H，-CH2N3)，2.57–2.16(m，9H，≡CH，-CH2-)，1.71–1.39(m，8H)，1.25(s，76H，-CH2-)， 0.88(t，J＝6.3Hz，12H，-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：173.63，173.56， 170.42，170.05，135.60，135.54，135.47，128.63，128.59，128.11，128.03，99.77，79.43， 74.76，74.12，74.06，73.80，73.74，72.43，70.64，70.29，69.74，69.68，69.62，68.24，68.08， 58.72，55.88，50.60，41.45，39.79，34.59，34.50，34.33，31.94，29.69，29.63，29.59，29.50， 29.39，29.28，29.22，25.26，25.15，25.05，25.01，22.7114.13。31P NMR(162MHz，CDCl3)， δ(ppm)：-2.28。ESI-TOF LRMS m/z:calcd for C 81 H 137 N 4 O 14 P[M+H] + 1419.9，found 1420.3。
Step 15 preparation of aminoethyl 4-oxy- (di-oxy-benzylphosphoryl) -6-oxy-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxy- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 16)
Figure BDA0001966552110000171
Compound 15 (0.10g, 0.07mmol) was dissolved in 6.0mL of dichloromethane, zinc powder (0.23 g, 3.50mmol) activated with hydrochloric acid was added, 50uL of glacial acetic acid was further added, the reaction was stirred at normal temperature for 1 hour, the progress of the reaction was monitored by TLC (ethyl acetate: petroleum ether = 1), the complete disappearance of the raw material was detected, the reaction solution was diluted with dichloromethane, the reaction solution was neutralized with a saturated sodium bicarbonate solution, the reaction solution was washed once with water and a saturated saline solution, the organic layer was collected, dried over anhydrous sodium sulfate overnight, and the solvent was distilled off under reduced pressure to give compound 16, which was directly subjected to the next step without being subjected to column chromatography purification.
Step 16: preparation of [3-R- (dodecanoic acid ester group) dodecanoylamino ] ethyl 4-oxo- (di-oxo-benzylphosphoryl) -6-oxo-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoic acid ester group) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoic acid ester group) tetradecanoyl ] -beta-D-glucoside (Compound 17)
Figure BDA0001966552110000181
Compound 16 (0.10g, 0.07mmol) and Lipid A-3 (0.05g, 0.10mmol) were added to 10.0mL of anhydrous treated methylene chloride, and N, N-diisopropylethylamine (15.3uL, 0.09mmol) was added dropwise to the mixture to adjust the pH to about 8, and the mixture was stirred under nitrogen atmosphere for 3 hours. The reaction process was monitored with a thin layer silica gel plate (methanol: dichloromethane =1, ethyl acetate: petroleum ether = 1), the starting material was detected to be completely disappeared, the solvent was distilled off at normal temperature under reduced pressure, and the crude product was isolated and purified by a silica gel column to obtain compound 17 (ethyl acetate: petroleum ether =1: 1 H NMR(400MHz， CDCl3)，δ(ppm)：7.44–7.21(m，10H，Ar-H)，6.63(d，J＝30.1Hz，1H，-NH-)，6.34 (s，1H，-NH-)，5.40–5.27(m，1H，H-3)，5.27–5.05(m，3H-OCOCH-)，5.04–4.89(m，4 H，Ar-CH2-)，4.83(s，1H，H-1)，4.44–4.30(m，1H，H-4)，4.10(q，J＝15.5Hz，2H，-OCH2CCH)， 3.72(ddd，J＝30.8，24.7，9.0Hz 6H，H-6-AH-6-B，H-2，H-5，-OCH2CH2-)，3.39(d，J＝27.9 Hz，2H-CH2N3)，2.56–2.14(m，13H，≡CH，-OCH2，-COCH2-)，1.52(d，J＝60.7Hz，12 H)，1.25(s，102H)，0.87(d，J＝6.6Hz，18H，-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)： 173.58，170.53，170.12，169.95，135.48，128.71，128.65，128.62，128.15，128.04，100.57， 79.19，75.15，75.07，74.99，74.38，74.01，71.94，71.36，71.23，70.25，69.75，69.43，68.36， 58.68，55.34，52.82，41.40，39.66，34.62，34.49，34.43，31.94，29.69，29.60，29.39，29.34， 29.28，29.22，25.33，25.15，25.10，25.05，22.70，14.13。 31 P NMR(162MHz，CDCl3)， δ(ppm)：-1.91。ESI-TOF LRMS m/z:calcd for C 105 H 182 N 2 O 17 P[M+H] + 1774.3，found 1774.7。
and step 17: preparation of [3-R- (dodecanoic acid carboxylate) dodecanoylamino ] ethyl 4-oxo- (di-oxo-benzylphosphoryl) -6-oxo-6- [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 21)
Figure BDA0001966552110000182
Compound 17 (0.06g, 0.03mmol), tn (11.7mg, 0.04mmol) and cuprous iodide (0.06g, 0.44mmol) were dissolved in a mixed solution of tetrahydrofuran (1.2 mL) and methanol (2.4 mL), and N, N-diisopropylethylamine (56.0 uL, 0.44mmol) was added to the mixture and the mixture was stirred at room temperature overnight. The progress of the reaction was monitored with a thin layer silica gel plate (ethyl acetate: petroleum ether =1, methanol: dichloromethane = 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.52–7.10(m， 11H，Ar-H，＝CH)，5.40–5.24(m，1H)，5.10(m，3H，-OCOCH-)，5.06–3.12(m，H’s of sugar and linker)，2.57–1.94(m，12H of lipid，3H of-NHAc)，1.70-1.07(m，-CH2-of lipid)， 0.89(dt，J＝18.2，10.3Hz，18H，-CH3of lipid)。 31 P NMR(162MHz，CDCl3)，δ(ppm)： -2.02。ESI-TOF LRMS m/z:calcd for C 115 H 199 N 6 O 22 PNa[M+Na] + 2086.4，found 2086.8。
Step 18: preparation of [3-R- (dodecanoic acid carboxylate) dodecanoylamino ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl- α -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoyl) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoyl) tetradecanoyl ] - β -D-glucoside (Compound 1)
Figure BDA0001966552110000191
The compound is21 (9.0 mg) to 20mL of dichloromethane/methanol/water (6. 1H NMR (400MHz, CDCl3/MeOD =1: 1). 31 P NMR(162MHz，CDCl3/MeOD＝1:1)，δ(ppm)：-1.15。ESI-TOF HRMS m/z:calcd for C 100 H 186 N 6 O 24 P[M-H]-1882.3312，found 1882.3235。
Example 2: preparation of Compound 2
Synthesis of [3-R- (dodecanoic acid carboxylate) tetradecanamido ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanamido ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanamido ] -beta-D-glucoside (Compound 2)
Step 1: preparation of [3-R- (dodecanoic acid ester group) tetradecanoylamino ] ethyl 4-oxo- (di-oxo-benzylphosphoryl) -6-oxo-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoate group) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate group) tetradecanoyl ] -beta-D-glucoside (Compound 18)
Figure BDA0001966552110000201
Following the synthetic procedure of step 16 in example 1, compound 16 (0.10g, 0.07mmol) was reacted with Lipid a-2 (0.05 g,0.10 mmol), N-diisopropylethylamine (15.3ul, 0.09mmol) to give a crude product, which was isolated and purified by column chromatography (ethyl acetate: petroleum ether = 1. 1 H NMR(400MHz， CDCl3)，δ(ppm)：7.42–7.27(m，10H，Ar-H)，6.67(s，1H，-NH-)，6.35(d，J＝7.8Hz， 1H，-NH-)，5.34(t，J＝9.4Hz，1H，H-3)，5.14(dt，J＝55.0，17.5Hz，3H，-OCOCH-)，5.06–4.87(m， 4H，Ar-CH2-)，4.83(d，J＝8.1Hz，1H，H-1)，4.35(dd，J＝17.9，8.6Hz，1H，H-4)，4.10(q， J＝15.8Hz，2H，-OCH2CCH)，3.88(d，J＝10.6Hz，1H，H-6-A)，3.83–3.53(m，5H，H-6-B， H-2，H-5，-OCH2CH2-)，3.39(d，J＝26.8Hz，2H，-CH2N3)，2.53–2.14(m，13H，≡CH，-OCH2， -COCH2-)，1.73–1.38(m，12H)，1.25(s，102H)，0.88(t，J＝6.3Hz，18H，-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：172.57，172.57，172.41，169.51，169.32，168.93， 134.46，134.39，127.69，127.63，127.60，127.13，127.02，99.68，78.17，74.04，72.99，72.63， 71.55，70.33，69.94，69.22，68.78，68.73，68.67，68.28，67.32，57.63，54.32，52.40，40.66， 40.36，38.69，33.59，33.48，33.41，30.92，28.69，28.67，28.65，28.58，28.51，28.48，28.36， 28.26，28.20，24.32，24.13，24.07，24.02，21.68，21.60，13.10。 31 P NMR(162MHz，CDCl3)， δ(ppm)：-2.00。ESI-TOF LRMS m/z:calcd for C 107 H 187 N 2 O 17 P[M+H] + 1802.3,found 1802.7。
Step 2: preparation of [3-R- (dodecanoic acid carboxylate) tetradecanoylamino ] ethyl 4-oxo- (di-oxo-benzylphosphoryl) -6-oxo-6- [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 22)
Figure BDA0001966552110000211
Following the synthetic procedure of step 17 in example 1, compound 18 (0.07g, 0.04mmol), tn (0.01g, 0.05mmol), cuprous iodide (0.07g, 0.37mmol) and N, N-diisopropylethylamine (60.0ul, 0.37mmol) were reacted to obtain a crude product, which was separated and purified by column chromatography (methanol: dichloromethane = 60, 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.31(m，11H，Ar-H，＝CH)，5.31(m，1H)，5.12(m， 3H，-OCOCH-)，5.03–3.12(m，H’s of sugar and linker)，2.66–1.91(m，15H，12H of lipid， 3H of-NHAc)，1.83-1.04(m，-CH2-of lipid)，0.88(m，18H，-CH3of lipid)。 31 P NMR(162MHz， CDCl3)，δ(ppm)：-2.02。ESI-TOF LRMS m/z:calcd forC 117 H 205 N 6 O 22 P[M+2H]2 + 1046.8，found 1047.2。
And step 3: preparation of [3-R- (dodecanoic acid carboxylate) tetradecanamido ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl-alpha-D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanamido ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanamido ] -beta-D-glucoside (Compound 2)
Figure BDA0001966552110000212
Compound 22 (10.0 mg), palladium on carbon (45.0 mg) and hydrogen were reacted by the synthetic method of step 18 in example 1 to obtain compound 2 (8.2mg, 95%) as a white solid. 1 H NMR(400MHz,CDCl3/MeOD＝1：1)，31P NMR (162MHz,CDCl3/MeOD＝1:1),δ(ppm)：-1.13。MALDI-TOF MS m/z:calcd for C 103 H 190 N 6 O 23 P[M-H]-1910.369,found 1910.307。
Example 3: preparation of Compound 3
Synthesis of [3-R- (tetradecanoate) tetradecanoylamino ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 3)
Step 1: preparation of [3-R- (tetradecanoate) tetradecanoylamino ] ethyl 4-oxo- (di-oxo-benzylphosphoryl) -6-oxo-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 19)
Figure BDA0001966552110000221
According to the synthetic method of step 16 in example 1, compound 16 (0.10 mg, 0.07mmol), lipid a-1 (0.05 mg, 0.09 mmol) and N, N-diisopropylethylamine (15.0ul, 0.09mmol) were reacted to completion to obtain a crude product, which was separated and purified by column chromatography (ethyl acetate: petroleum ether = 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)： 7.42–7.25(m,10H,Ar-H),6.63(s,1H,-NH-),,6.31(d,J＝7.3Hz,1H,-NH-),5.34(t,J＝9.4Hz,1H,H-3),5.26 –5.05(m,3H,-OCOCH-),5.07–4.88(m,4H,Bn-),4.84(d,J＝7.9Hz,1H,H-1),4.35(q,J＝9.1Hz,1H,H-4), 4.10(q,J＝15.9Hz,2H,-OCH2CCH),3.88(d,J＝10.4Hz,1H,H-6),3.83–3.54(m,5H,H-6,H-2,H-5,-OCH 2CH2-),3.39(d,J＝21.2Hz,2H,-CH2N3),2.54–2.15(m，13H，≡CH)，1.74–1.37(m，12H)，1.25(s， 106H)，0.88(t，J＝6.1Hz，18H，-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：172.57，172.43， 169.51，169.33，168.94，134.50，134.45，134.38，129.89，127.82，127.69，127.63，127.60， 127.13，127.03，99.69，78.16，74.05，73.04，72.62，71.55，70.33，69.94，69.21，68.79， 68.73，68.67，68.31，67.3，64.55，57.63，54.29，40.67，40.38，38.71，38.62，33.59，33.48， 33.46，33.41，30.92，29.55，28.70，28.67，28.57，28.51，28.48，28.37，28.26，28.21，24.32， 24.12，24.07，24.02，21.68，18.17，13.11，12.72。31P NMR(162MHz，CDCl3)，δ(ppm)： -1.90。ESI-TOF LRMS m/z:calcd for C 109 H 191 N 2 O 17 P[M+H] + 1830.3，found 1830.7。
Step 2: preparation of [3-R- (tetradecanoate) tetradecanoylamino ] ethyl 4-oxo- (di-oxo-benzylphosphoryl) -6-oxo-6- [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 23)
Figure BDA0001966552110000231
According to the synthesis method of step 17 in example 1, compound 19 (0.09mg, 0.0499mmol), tn (0.02 g, 0.0599 mmol), cuprous iodide (0.10 mg, 0.50mmol) and N, N-diisopropylethylamine (82.6 ul, 0.488mmol) were reacted to obtain a crude product, which was separated and purified by column chromatography (methanol: dichloromethane =1, 60, 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.49–7.14(m，11H，Ar-H，＝CH)，5.35(m， 1H，H'-3)，5.10(m，3H，-OCOCH-)，5.08–3.14(m，H’sof sugarandlinker)，2.58-1.88(m， 15H，12H of lipid，3H of-NHAc)，1.70-1.07(m，-CH2-of lipid)，0.87(m，18H，-CH3 of lipid)。 31 P NMR(162MHz，CDCl3)，δ(ppm)：-1.98。ESI-TOF LRMS m/z:calcd for C119H211N6O23P[M+2H]2+1060.8，found 1061.5。
And step 3: preparation of [3-R- (tetradecanoate) tetradecanamido ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanamido ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanamido ] -beta-D-glucoside (Compound 3)
Figure BDA0001966552110000232
The synthesis of step 18 in example 1 was carried out in the same manner as in the above-mentioned step (3-step), except that the reaction of compound 23 (10.0 mg), palladium on carbon (45.0 mg) and hydrogen was completed to give compound 3 (8.5mg, 93%) as a white solid. 1 H NMR(400MHz，CDCl3/MeOD＝1：1)， 31 P NMR(162MHz，CDCl3/MeOD＝1:1)，δ(ppm)：-1.18。MALDI-TOF MS m/z:calcd forC 105 H 194 N 6 O 23 PH]-1938.401，found1938.401。
Example 4: preparation of Compound 4
Synthesis of [3-R- (tetradecyloxy) tetradecanoylamino ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 4)
Step 1: preparation of [3-R- (tetradecyloxy) tetradecanoyl ] ethyl 4-oxy- (di-oxy-benzylphosphoryl) -6-oxy-propargyl-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxy- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 20)
Figure BDA0001966552110000241
Following the synthetic procedure of step 16 in example 1, compound 16 (0.10g, 0.07mmol), lipid a-4 (0.05 g,0.10 mmol) and N, N-diisopropylethylamine (15.0ul, 0.09mmol) were reacted to obtain a crude product, which was separated and purified by column chromatography (ethyl acetate: petroleum ether =1: 1 H NMR (400MHz，CDCl3)，δ(ppm)：7.49–7.18(m，10H，Ar-H)，6.76(s，1H，-NH-)，6.30(d， J＝7.4Hz，1H，-NH-)，5.33(t，J＝9.5Hz，1H，H-3)，5.14(s，2H，-OCOCH-)，5.07–4.88(m， 4H，Ar-CH2-)，4.84(d，J＝8.1Hz，1H，H-1)，4.35(dd，J＝17.8，8.9Hz，1H，H-4)，4.11(dd， J＝33.8，15.8Hz，2H，-OCH2CCH)，3.86(t，J＝15.7Hz，1H，H-6-A)，3.83–3.54(m，5H， H-2，H-5，H-6-B，-OCH2CH2-)，3.43(d，J＝5.9Hz，3H，-CH2NH-，-OCH-)，2.52–2.14(m， 9H，≡CH，-OCH2，-COCH2-)1.69–1.14(m，124H)，0.87(d，J＝6.6Hz，18H，-CH3)。 13 CNMR(100MHz，CDCl3)，δ(ppm)：173.59，173.53，171.73，170.46，170.34，135.56， 135.49，130.92，128.85，28.69，28.64，128.62，128.14，128.04，100.46，79.29，74.97，74.09， 72.70，70.89，70.24，69.73，69.67，69.62，69.27，68.26，65.58，58.64，55.35，41.91，41.60， 39.66，39.39，34.62，34.57，34.50，34.41，31.95，30.58，30.20，29.72，29.70，29.60，29.55， 29.43，29.39，29.29，29.22，26.22，25.42，25.31，25.15，25.03，22.71，19.20，14.13，13.74。 31 P NMR(162MHz，CDCl3)，δ(ppm)：-1.98。ESI-TOF LRMS m/z:calcd for C 109 H 192 N 2 O 17 P[M+H] + 1816.4，found 1816.8。
and 2, step: preparation of [3-R- (tetradecyloxy) tetradecanoylamino ] ethyl 4-oxy- (di-oxy-benzylphosphoryl) -6-oxy-6- [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene-2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxy- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 24)
Figure BDA0001966552110000251
Following the synthetic procedure of step 17 in example 1, compound 20 (0.08g, 0.04mmol), tn (15.3 mg, 0.05 mmol), cuprous iodide (0.06g, 0.44mmol) and N, N-diisopropylethylamine (56.0 ul, 0.44mmol) were reacted to give a crude product, which was separated and purified by column chromatography (methanol: dichloromethane = 60,1, 40, 1. 1 H NMR(400MHz，CDCl3)，δ(ppm)：7.44–7.14(m，1H，Ar-H，＝CH)， 5.335.33(m，1H，H-3)，5.12(m，2H-OCOCH-)，5.06–3.22(m，H’s of sugar and linker)， 2.53–1.96(m，15H，12H of lipid，3H of-NHAc)，1.71–1.07(m，-CH2-of lipid)，0.87(t， J＝7.0Hz，18H，-CH3of lipid)。 31 P NMR(162MHz，CDCl3)，δ(ppm)：-2.04。ESI-TOF LRMS m/z:calcd for C 119 H 211 N 6 O 22 P[M+2H] 2+ 1053.8，found 1054.4。
And 3, step 3: preparation of [3-R- (tetradecyloxy) tetradecanoylamino ] ethyl 4-oxo-phosphoryl-6-oxo-6- { [ (ethyl 2-deoxy-acetyl-. Alpha. -D-galactosyl) -1-hydro-1, 2, 3-triazolyl-4 ] methylene } -2-deoxy-2- [ (R) -3- (tetradecanoate) tetradecanoyl ] -3-oxo- [ (R) -3- (tetradecanoate) tetradecanoyl ] -beta-D-glucoside (Compound 4)
Figure BDA0001966552110000252
Compound 24 (10.0 mg), palladium on carbon (45.0 mg) and hydrogen were reacted by the synthesis method of step 18 in example 1 to obtain compound 4 (8.0 mg, 9%) as a white solid. 1 H NMR(400MHz，CDCl3/MeOD＝1：1)， 31 P NMR(162MHz，CDCl3/MeOD＝1:1)，δ(ppm)：-1.17。MALDI-TOF MS m/z:calcd for C 105 H 196 N 6 O 22 P[M-H] - 1924.421，found1924.712。
The NMR spectra of the compounds (1, 2,3, 4) of examples 1 to 4 were determined on a Bruker 400MHz, 600MHz NMR spectrometer. The HRMS spectrogram is measured on an Agilent 6540 high-resolution Q-TOF mass spectrometer, thin Layer Chromatography (TLC) analysis is carried out on an HF254 silica gel plate, and a 20% concentrated sulfuric acid ethanol solution is used as a color developing agent baking plate. Silica gel column chromatography uses mixed solvent of methanol, ethyl acetate, toluene, petroleum ether (bp 60-90 deg.C), n-hexane, etc. as eluent. 200-300 mesh silica gel is used as a silica gel column, and the molecular sieve is dried in vacuum at high temperature before use and then is restored to room temperature under the protection of nitrogen. The dichloromethane used for the glycosidation reaction is anhydrous.
EXAMPLE 5 anticancer Activity of Compounds 1,2,3, 4
The experimental method comprises the following steps:
24 female C57BL6/J mice 6-8 weeks old were divided into 4 groups on average, and on days 0, 14, 21, and 28, compounds 1,2,3, and 4 were injected with 100. Mu.l of the drug (containing 10. Mu.g of Tn antigen) subcutaneously via the abdomen, respectively; collecting blood through tail vein on days 0, 27 and 38 respectively, centrifuging to obtain serum, and storing at-80 deg.C before use.
Enzyme-linked immunosorbent assay (ELISA): tn-BSA was first prepared as a 2. Mu.g/ml solution in 0.1M carbonate buffer (pH 9.6), added to a 96-well plate in an amount of 100. Mu.l per well, and incubated overnight at 4 ℃; placing the mixture into an incubator at 37 ℃ for incubation for one hour on the next day; the plates were washed 3 times with PBST (PBS +0.05% Tween-20) and 300. Mu.l of wash solution was added to each well. Then adding blocking buffer (PBST/1% BSA); add 250. Mu.l per well; incubate at room temperature for one hour, wash the plate 3 times with PBST. Serum samples from compound 1,2,3, 4 on day 38 were then taken, and individual mouse sera from each group were pooled and diluted in PBS from 1:300 times diluted to 1:656100 times; the diluted serum was added to a 96-well plate at 100. Mu.l per well, 3 replicates per sample were placed in a 37 ℃ incubator for two hours, and the plates were washed three times. Then, the goat anti-mouse IgG secondary antibody marked by HRP (horse radish peroxidase) is diluted by 1000 times, 100 mu l of the secondary antibody is added into each hole, and the secondary antibody is shaken for one hour at the room temperature and the rpm of 400; the plate was washed 3 times. Then, 100. Mu.l of TMB (3, 3', 5' -tetramethylbenzidine) solution was added to each well, and the mixture was shaken at 400rpm at room temperature for 0.5 hour. The absorbance was measured at a wavelength of 450nm with a microplate reader. Finally, absorbance (OD) values were plotted against antiserum dilution values and a line of best fit was obtained. The equation of the line was used to calculate a dilution value to an OD of 0.2, and the antibody titer was calculated at the reciprocal of the dilution value.
The experimental results are as follows:
the ELISA experiment result shows that the target compounds 1,2,3 and 4 can cause specific immune response under the condition of no any additional adjuvant, as shown in figures 1 and 2; as is evident from the figure, compound 3 caused relatively low IgG and IgM antibody titers; the IgG and IgM antibody titers caused by the compounds 2 and 4 are similar and are higher than that of the compound 3, while the IgG and IgM antibody titers caused by the compound 1 are the highest among the four compounds, and the antibody titer is about twice of that of the compound 3, which indicates that the compound 1 can stimulate a mouse to generate a strong immune response and can be applied to an anti-tumor vaccine.
EXAMPLE 6 Compounds 1,2,3 Glyco-antibody mediated Complement Dependent Cytotoxicity (CDC) assay generated by immunization of mice
The experimental method comprises the following steps:
MCF-7 cells (human breast cancer cells) in logarithmic growth phase were trypsinized at 1X 10 per well 4 The individual cells were seeded in 96-well plates, cultured overnight at 37 ℃ and the plates were washed twice with serum-free MEM medium. Serum samples of 6 mice were collected from the group of Compound 1, which were bled at 38 days, and 7. Mu.l of each sample was collected, and then the serum samples of 6 mice were mixed and diluted 50-fold with serum-free MEM to obtain mouse serum dilutions. Dilutions of mouse sera from compound 2, compound 3, and placebo were prepared as described above. The compound 1, the compound 2, the compound 3 and the blank control group are respectively provided with a sample maximum enzyme activity control group, a sample control group and a sample treatment group, each group is parallelly provided with 6 multiple holes, 100 mu l of diluted mouse serum solution is added into each hole, and a 96-hole plate is placed at 37 ℃ for culturing for 2h. After washing the plate twice with serum-free MEM medium, 100. Mu.l of rabbit complement serum solution was added to each well of the sample-treated group, 100. Mu.l of LDH release solution was added to each well of the maximum enzyme activity control group, and 100. Mu.l of serum-free MEM medium was added to each well of the sample control group, and the mixture was incubated at 37 ℃ for 1 hour.
And adding 100 mu l of PBS solution into each new 96-well plate, carefully sucking 40 mu l of cell supernatant of the 96-well plate, adding 60 mu l of LDH detection solution into each new 96-well plate, and incubating for 30min in a dark place. Detection was carried out with a microplate reader at 490 nm.
Cell lysis rate (%) = (sample treatment absorbance-sample control absorbance)/(maximum enzyme activity control absorbance-sample control absorbance) × 100%
Results of the experiment
The results of antibody-mediated Complement Dependent Cytotoxicity (CDC) experiments are shown in fig. 3, and antibodies generated by compounds 1,2 and 3 have the ability of killing tumor cells, the cell lysis rates are higher than those of the blank control group, and the significant difference (P < 0.001) is obtained between the blank control group and the antibodies.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims (7)

1. A monophosphoryl ester A conjugated Tn anti-tumor vaccine, which is characterized by being a compound with a general formula (I):
Y-L-X (I)
or a pharmaceutically acceptable salt of a compound of formula (I);
wherein:
y is selected from the group having the following structure:
Figure FDA0003845433740000011
R 1 is selected from-CH 2 -CH(-OR 4 )(CH 2 ) m CH 3 ，R 4 is-C (O) - (CH) 2 ) n CH 3 M and n are integers of 6 to 18;
R 2 is selected from-CH 2 -CH(-OR 5 )(CH 2 ) P CH 3 ，R 5 is-C (O) - (CH) 2 ) q CH 3 P and q are integers of 6 to 18;
R 3 is selected from-CH 2 -CH(-OR 6 )(CH 2 ) r CH 3 ，R 6 Is- (CH) 2 ) s CH 3 or-C (O) - (CH) 2 ) t CH 3 R, s, t are integers from 6 to 18;
l is selected from the structure
Figure FDA0003845433740000012
The connecting arm of (1); wherein c is an integer selected from 2 to 6;
x is selected from the group having the following structure:
Figure FDA0003845433740000013
2. a monophosphoryl ester a conjugated Tn anti-tumor vaccine according to claim 1 wherein Y is selected from the group having the structure:
Figure FDA0003845433740000014
R 1 is-CH 2 -CH(-OR 4 )(CH 2 ) 10 CH 3 ，R 4 is-C (O) - (CH) 2 ) 12 CH 3 ；
R 2 is-CH 2 -CH(-OR 5 )(CH 2 ) 10 CH 3 ，R 5 is-C (O) - (CH) 2 ) 12 CH 3 ；
R 3 is-CH 2 -CH(-OR 6 )(CH 2 ) 10 CH 3 or-CH 2 -CH(-OR 6 )(CH 2 ) 12 CH 3 R6 is- (CH) 2 ) 11 CH 3 、-C(O)-(CH 2 ) 10 CH 3 or-C (O) - (CH) 2 ) 12 CH 3 。
3. A monophosphoryl ester A conjugated Tn anti-tumor vaccine according to claim 1 wherein L is of the structure
Figure FDA0003845433740000021
The connecting arm of (1).
4. A monophosphoryl ester a conjugated Tn anti-tumor vaccine according to claim 1, wherein the general formula (I) is:
Figure FDA0003845433740000022
or a pharmaceutically acceptable salt of the above compound.
5. Monophosphoryl ester A with general formula (I) conjugated Tn antitumor vaccine and its medicinal salt in preparing medicine for treating cancer.
6. The use of claim 5, wherein the medicament further comprises a medically acceptable excipient.
7. The use of claim 5, wherein the cancer is breast cancer, prostate cancer, intestinal cancer, melanoma, liver cancer, lung cancer, renal cell carcinoma, uterine cancer, ovarian cancer, cellular lymphoma, brain cancer, gastric cancer, pancreatic cancer, cancer of the nail, or leukemia.
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CN111588847B (en) * 2020-05-18 2023-05-26 广州中医药大学(广州中医药研究院) Conjugate containing monophosphorylated lipid A and saccharide antigen and preparation method and application thereof
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