High-activity human Toll-like Receptor2 agonist

Abstract

The invention relates to the technical field of medicinal chemistry and pharmacology, in particular to a synthetic method of a high-activity human Toll-like Receptor 2 agonist. In the technical route, an acetal aliphatic chain in the agonist is introduced by using a method for activating dithiol acetal under an oxidation condition. By adjusting the length of an acetal aliphatic chain, a high-activity immune agonist aiming at human-derived TLR2 is created, and the EC50 aiming at TNF-alpha is equal to 116pM. The technical route is as follows: D-galactose which is low in price and easy to obtain is taken as an initial raw material, and the high-activity human TLR2 agonist is obtained in a gram-level scale through six-step chemical conversion, so that a technical route and a material basis are provided for subsequent application in vaccine adjuvants and tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry and pharmacology, and in particular to the synthesis and activity evaluation of human Toll-like Receptor 2 agonist CaLGL-1 analogs. Background Art

[0002] The innate immune response is the body's first line of defense, helping the host effectively resist invasion by foreign pathogens. Pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), initiating downstream responses to type I interferons (IFNs) and proinflammatory cytokines. Different PAMPs are specific for specific microorganisms. Lipopolysaccharides, peptidoglycans and lipocholic acid, flagellin and lipoproteins, yeast, and DNA and RNA are all well-known PAMPs. With the exception of some NLRs, PRRs detect PAMP signals and induce the transcription of inflammatory genes, encoding proinflammatory cytokines, type I interferons (IFNs), chemokines, and antimicrobial proteins.

[0003] Of all the PRR families, Toll-like receptors (TLRs) are the most characterized and play a key role in innate immunity; they can mediate both innate and acquired immunity. Among the various TLRs, TLR2 is relatively special and requires heterodimerization with TLR1 or TLR6 to sense the widest range of PAMPs, thereby expanding its ligand diversity against pathogens. In general, over the past 20 years, various skeletons have been reported to activate TLR2, including naturally occurring lipoproteins, synthetic lipopeptides, and several heterocyclic small molecules, demonstrating the importance of this receptor in various diseases and in the development of vaccine adjuvants. Therefore, the development of novel, highly active TLR2 agonists is particularly important.

[0004] Naturally occurring and synthetic TLR agonists can enhance and regulate the antigen immune effects of vaccines in these endogenous immune signaling pathways, becoming new adjuvants for many vaccines. Such agonists are not only important for the development of vaccines against infectious diseases, but also for immunotherapy of cancer, allergies, Alzheimer's disease and other diseases. Each TLR has its own specific tissue localization and downstream gene signaling pathways. TLR agonists can be combined with other TLRs or alternative adjuvants to produce combination adjuvants with synergistic or regulatory effects, which provides researchers with the opportunity to precisely customize adjuvants with specific immune effects.

[0005] Some members of the human gut microbiota profoundly affect the host's physiology, health, and therapeutic responses, but the responsible molecules and mechanisms are largely unknown. As part of a project to identify immunomodulators produced by gut microbes. In 2023, Clardy's group isolated CaLGL-1 from the gut bacteria Collinsella aerofaciens of healthy humans (JACS2023,145,7071), and used dendritic cells (mBMDCs) and cytokine data for phenotypic analysis to guide screening and preliminary mechanistic studies, and discovered TLR-2-dependent signaling (EC 50 =3.2uM).

[0006] However, CaLGL-1 has a weak immune response to human TLR2. Based on the compound skeleton, the present invention adjusts the length of the lipid chain and conducts structure-activity relationship research to create a highly active human TLR2 immune agonist.

[0007] The structural formula of CaLGL-1 and its derivatives

[0008] The purpose of this invention is to develop a highly active human TLR2 agonist to provide a technical route and material basis for subsequent research on TLR-2 agonists in vaccine adjuvants, cancer immunotherapy and other related drugs. Summary of the Invention

[0009] The present invention is based on a natural TLR2 agonist, CaLGL-1, and has developed a highly active immune agonist targeting human TLR2 through structure-activity relationship research. Its structural formula is as follows (IR and IS):

[0010] The present invention aims to provide a highly active human TLR2 agonist, the technical route of which is as follows: using cheap and readily available D-galactose as a starting material, II is obtained after TBS protection; glycosylation reaction with propylene glycol under the action of TMSI produces III; then, the propylene glycol is selectively removed under acidic conditions to obtain diol IV; esterification reaction under EDC conditions produces monolipid V; then, the TBS protecting group is removed to obtain VI; and finally, acetalization reaction promoted by an oxidant produces IR and IS.

[0011] The method for synthesizing a human high-activity TLR2 agonist is characterized by comprising the following steps:

[0012] Commercially obtained D-galactose was treated with anhydrous N,N-dimethylformamide as solvent and imidazole as base at room temperature and protected with TBS to obtain product (II).

[0013] Compound (II) obtained in

[0012] was reacted in anhydrous dichloromethane as solvent, N-ethyldiisopropylamine as base, (S)-(2,2-dimethyl-1,3-dioxolane-4-yl)methanol and activated 4A molecular sieves as additives, under an argon atmosphere at -60°C, and reacted at room temperature to obtain product (III).

[0014] The compound (III) obtained in

[0013] was reacted in trifluoroacetic acid: water: dichloromethane = 2.5:2.5:95 at a temperature from zero to room temperature for 30 minutes to obtain the large-scale product (IV).

[0015] The compound (IV) obtained in

[0014] was reacted in toluene as solvent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a condensing agent, the corresponding acid as a reaction raw material, and a catalytic amount of 4-dimethylaminopyridine as an additive, and the reaction was carried out at room temperature for 12 hours to obtain a large-scale product (V).

[0016] The obtained compound (V) in

[0015] was dissolved in anhydrous tetrahydrofuran as a solvent, and hydrogen fluoride pyridine was slowly added dropwise thereto at zero degrees, followed by reaction at room temperature for 2 hours. After the reaction was complete as detected by thin layer chromatography, the compound was placed in an ice bath, and a saturated sodium bicarbonate solution was slowly added dropwise to quench the reaction. The compound was then extracted three times with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and finally subjected to column chromatography to obtain a large-scale product (VI).

[0017] Compound (VI) obtained in

[0016] was reacted in anhydrous dichloromethane as a solvent, p-toluenethiophenol-activated acetals of different chain lengths as reaction materials, and N-iodosuccinimide as an oxidant under an argon atmosphere at -30°C for 3 hours. After completion of the reaction as determined by thin-layer chromatography, the product was placed in an ice bath and quenched by the slow dropwise addition of 10% sodium thiosulfate. The product was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Finally, large-scale products IR and IS were obtained by column chromatography.

[0018] Compound (VI) obtained in

[0016] was reacted in anhydrous dichloromethane as a solvent, acetals activated by p-toluenethiophenol of different chain lengths as reaction materials, and N-bromosuccinimide as an oxidant under an argon atmosphere at -20°C to room temperature for 3 hours. After completion of the reaction as determined by thin-layer chromatography, the product was placed in an ice bath and quenched by the slow dropwise addition of 10% sodium thiosulfate. The product was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Finally, large-scale products IR and IS were obtained by column chromatography.

[0019] Compound (VI) obtained in

[0016] was reacted in anhydrous dichloromethane as solvent, acetals activated by p-toluene thiophenol of different chain lengths as reaction materials, and dibromohydroxyin as oxidant under argon atmosphere at -20°C to room temperature for 3 hours. After completion of the reaction by thin-layer chromatography, the mixture was transferred to an ice bath and quenched by slowly adding 10% sodium thiosulfate. The mixture was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Finally, large-scale products IR and IS were obtained by column chromatography.

[0020] A highly active human TLR2 agonist, tested on human immune cells THP-1.

[0021] A highly active human TLR2 agonist, IR-4, activates TNF-α in ECs in a cell model 50 =116pM.

[0022] The present invention has the advantages of safe operation, environmental friendliness, mild reaction conditions and high yield. The raw materials and additives used in the present invention are cheap and easily available, and the environmental pressure is small.

[0023] The invention meets the requirements of green synthesis and has great practical application value.

[0024] The technical solution of the present invention is further described in detail below through examples. DETAILED DESCRIPTION

[0025] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following specific embodiments are provided to further illustrate the above-mentioned contents of the present invention. However, this should not be construed as limiting the scope of the present invention. All technologies implemented based on the above-mentioned contents of the present invention fall within the scope of the present invention.

[0026] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0027] In the experimental methods described in the following examples, all solvents and chemicals used were of analytical or chemical grade. Anhydrous solvents were processed according to standard methods. Column chromatography and silica gel plates were of conventional type. Anhydrous sodium sulfate was used as the drying agent for the organic phase obtained from extraction, unless otherwise specified. LC-MS was performed using an Agilent 1260 HPLC-ion trap mass spectrometer (ESI source) with a diode array detector (DAD) at 210 nm and 254 nm. Unless otherwise specified, all system specifications are conventional. Reagents and materials described were commercially available.

[0028] The synthesis method of the highly active Toll-like Receptor 2 agonist is specifically described as follows:

[0029] Step 1: Commercially available D-galactose (30 mmol) was added to a 250 mL round-bottom flask equipped with a magnetic rod. Anhydrous N,N-dimethylformamide (DMF, 150 mL) was added to the system and the mixture was placed at 0°C. Imidazole (6.6 equivalents) and tert-butyldimethylsilyl chloride (TBSCl, 7.25 equivalents) were then slowly added to the system in portions. The reaction was then allowed to react at room temperature for 48 hours. After completion of the reaction as determined by thin-layer chromatography, the reaction system was slowly poured into ice water in portions. The aqueous phase was then extracted with dichloromethane (100 mL × 3) and the organic phases were combined. The organic phases were then washed sequentially with 5% hydrochloric acid, saturated sodium bicarbonate, and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Anhydrous methanol (20 mL) was then added to the system to precipitate a white solid, which was filtered to obtain product (II) in an 80% yield. 1 H NMR(400MHz,Chloroform-d)δ5.15(d,J=2.4Hz,1H),4.09(t,J=3.8Hz,1H),4.00(t,J=4.1Hz,1H),3.92(t,J=2.8Hz,1H) ,3.78–3.70(m,1H),3.67(dd,J=9.9,6.3Hz,1H),3.55(dd,J=9.8,5.6Hz,1H),0.88(m,45H),0.14–0.02(m,30H).[M+Na] + =773.7.

[0030] Step 2: The obtained product (II) (10 mmol) and activated 4A molecular sieves were added to a 250 mL round-bottom flask with a magnetic rod and protected with argon, and then anhydrous dichloromethane (DCM, 100 mL) was added thereto, stirred at room temperature for 30 minutes, and then placed at -40 ° C., trimethylsilyl iodide (TMSI, 1.2 equivalents) was added thereto, and after reacting for 30 minutes, N-ethyldiisopropylamine (DIPEA, 2.0 equivalents) was slowly added thereto. ), (S)-(2,2-dimethyl-1,3-dioxolane-4-yl)methanol ((S)-(+)-1,2-Isopropylideneglycerol, 1.2 equivalents), followed by reaction at room temperature for 2 hours. After the reaction was complete as detected by thin layer chromatography, the liquid obtained by molecular sieve was filtered off, and the organic phase was washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product (III) was obtained by separation and purification by column chromatography with a yield of 74%. 1H NMR(400MHz,Chloroform-d)δ4.81(d,J=2.2Hz,1H),4.28(m,1H)4.11(dd,J=5.8,3.3Hz,1H),4 .05(m,1H),4.01(m,1H),3.93(dd,J=5.8,3.0Hz,1H),3.81(dd,J=8.3,6.2Hz,1H),3.77(dd,J= 10.0Hz,4.7Hz,1H),3.75(m,1H),3.65(dd,J=9.9,6.7Hz,1H),3.57(dd,J=9.8,5.8Hz,1H),3.4 1(dd,J=10.2,6.7Hz,1H),1.39(s,3H),1.34(s,3H),0.87(m,36H),0.10–0.03(m,24H).[M+Na] + =773.6.

[0031] Step 3: The product (III) (8.5 mmol) obtained by

[0030] was added to a 500 mL round-bottom flask with a magnetic rod, 400 mL of dichloromethane (DCM) was added thereto, and then the mixture was placed in an ice bath. Trifluoroacetic acid (TFA, 10.6 mL) and water (H2O, 10.6 mL) were added thereto, and the mixture was placed at room temperature to react for 30 minutes. After the reaction was detected to be complete by thin layer chromatography, the mixture was placed in an ice bath, and a saturated sodium bicarbonate solution was slowly added dropwise to quench the reaction. Then, the mixture was extracted three times with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The product (IV) was obtained by column chromatography with a yield of 83%. 1 H NMR(600MHz,Chloroform-d)δ4.83(d,J=2.3Hz,1H),4.12(dd,J=4.7,3.0Hz,1H),4.02–3.96(m,2H),3.82(q,J=4.6Hz,1H),3.74(td,J=6.1,3.9Hz,1H),3.6 7(d,J=4.8Hz,2H),3.63(dd,J=10.2,6.6Hz,1H),3.58(m,2H),3.33(d,J=4.3H z,1H),2.41(t,J=6.1Hz,1H),0.90–0.85(m,36H),0.12–0.03(m,24H).[M+Na] + =733.6.

[0032] Step 4: The obtained product (IV) (5 mmol) was added to a 100 mL round-bottom flask with a magnetic rod, and anhydrous toluene (60 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 2.0 equivalents), 4-dimethylaminopyridine (DMAP, 0.1 equivalents) and lauric acid (2.0 equivalents) were added thereto. The mixture was reacted at room temperature for 12 hours. After the reaction was complete by thin layer chromatography, the mixture was returned to room temperature, ethyl acetate (100 mL) was added thereto, and the organic phase was washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product (V) was obtained by column chromatography separation and purification with a yield of 92%. 1 H NMR(600MHz,Chloroform-d)δ4.84(d,J=2.5Hz,1H),4.13(dd,J=5.0,3.2Hz,1H),4.12–4.09(m,2H),4.02–3.94(m,3H),3.74(m,1H),3.66–3.6 0(m,2H),3.60–3.54(m,2H),3.22(d,J=3.3Hz,1H),2.32(t,J=7.6Hz,2H ),1.61(m,2H),1.26(m,16H),0.88(m,39H),0.11–0.03(m,24H).[M+Na] + =915.8.

[0033] Step 5: The obtained product (V) (5 mmol) was added to a 100 mL plastic reaction bottle with a magnetic rod, anhydrous tetrahydrofuran (THF) was added thereto, and hydrogen fluoride pyridine (6.0 equivalents) was slowly added dropwise at 0°C, followed by reaction at room temperature for 2 hours. After the reaction was complete as detected by thin layer chromatography, it was placed in an ice bath, and a saturated sodium bicarbonate solution was slowly added dropwise to quench the reaction. Then, the mixture was extracted three times with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The product (VI) was obtained by column chromatography with a yield of 84%. 1H NMR(600MHz,Chloroform-d)δ5.22(s,2H),4.96(s,1H),4.86(d,J=2.6Hz,1H),4.81–4.70(m,1H),4.58(s,1H),4.08(d,J=10.8Hz,3H),3.99(d,J= 15.1Hz,2H),3.76(m,5H),3.59–3.43(m,1H),2.32(t,J=7.6Hz,2H),1.58 (p,J=7.2Hz,2H),1.26(d,J=15.8Hz,16H),0.86(t,J=7.0Hz,3H).[M+Na] + =459.3.

[0034] Step 6: (VI) (3.6 mmol) obtained in

[0033] and N-iodosuccinimide (NIS, 2.4 equivalents) were added to a 100 mL round-bottom flask equipped with a magnetic rod. Anhydrous dichloromethane (40 mL) was added thereto under argon and the mixture was placed at -30°C. Then, VII (1.2 equivalents) was slowly added thereto. The mixture was reacted at -30°C for 3 hours. After the reaction was complete as determined by thin-layer chromatography, the mixture was placed in an ice bath and 10% sodium thiosulfate was slowly added dropwise to quench the reaction. The mixture was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure and purified by column chromatography to obtain IR and IS in yields of 72-86%. (IR-1,n=8), 1 H NMR (600MHz, CD3OD) δ4.90(t,J=4.8Hz,1H),4.88(d,J=1.5Hz,1H),4.21–4.06(m,3H),3.98(m,1H),3.96–3.94(m,1H),3.92–3.85(m,2H),3.84–3. 80(m,1H),3.79–3.71(m,1H),3.49(m,1H),2.36(t,J=7.2Hz,2H),1.69–1 .58(m,4H),1.47–1.38(m,2H),1.35–1.25(m,38H),0.90(t,J=7.2Hz,6H). 13 C NMR (151MHz, CD3OD) δ175.4,109.7,106.4,84.5,83.4,79.2,76.8,69.9,69.9,69.5,69.4,67.4,66.4 ,35.1,35.0,33.1,33.1,30.8,30.7,30.7,30.7,30.6,30.5,30.5,30.4,30.2,26.0,25.0,23.7,14.5. (I-S-1,n=8), 1 H NMR(600 MHz,CD3OD)δ4.98(t,J=4.8 Hz,1H),4.88(d,J=1.7Hz,1H),4.21(m,1H),4.18–4.14(m,1H),4.12–4.07(m,2H),3.99–3.94(m,2H),3.90–3.85(m,2H),3.82(m,1H),3.75(m,1H),3.49(m,1H),2.35(t,J=7.2 Hz,2H),1.65–1.56(m,4H),1.40(m,2H),1.34–1.27(m,28H),0.90(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CD3OD)δ175.4,109.7,106.4,84.5,83.5,79.2,76.9,69.9,69.5,69.4,67.4,66.4,35.1,35.0,33.1,33.1,30.8,30.728,30.7,30.7,30.6,30.5,30.5,30.4,30.2,26.0,25.0,23.7,14.5. (I-R-2,n=9), 1 H NMR(400 MHz,CD3OD)δ4.96(t,J=4.8 Hz,1H),4.88(d,J=1.5Hz,1H),4.21–4.15(m,1H),4.15–4.07(m,2H),3.98(m,1H),3.94(m,1H),3.88(m,2H),3.85–3.80(m,1H),3.80–3.70(m,1H),3.49(m,1H),2.36(t,J=7.5 Hz,1H),2.28(t,J=7.4 Hz,1H),1.62(m,4H),1.42(m,2H),1.29(m,30H),0.90(t,J=7.2 Hz,6H). 13 C NMR(101 MHz,CD3OD)δ175.3,109.7,106.4,84.5,83.5,79.2,76.8,69.9,69.5,69.4,67.3,66.3,35.1,34.9,33.1,30.8,30.7,30.7,30.7,30.637,30.6,30.5,30.5,30.4,30.2,26.0,25.0,23.7,14.5. (I-S-2,n=9), 1 H NMR(600 MHz,CD3OD)δ4.98(t,J=4.8 Hz,1H),4.21(m,1H),4.18–4.12(m,2H),4.11–4.07(m,1H),3.98(m,1H),3.96–3.93(m,1H),3.89(m,1H),3.86(m,1H),3.83–3.80(m,1H),3.75(m,1H),3.49(m,1H),2.69(s,1H),2.36(t,J=7.2 Hz,2H),1.67–1.56(m,4H),1.46–1.38(m,2H),1.30(m,30H),0.90(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CD3OD)δ175.4,109.7,106.4,84.5,83.5,79.2,76.9,69.9,69.9,69.5,69.4,67.4,66.4,35.1,35.0,33.1,30.8,30.7,30.7,30.7,30.6,30.6,30.5,30.5,30.5,30.2,26.0,25.0,23.8,14.5. (I-R-3,n=10), 1 H NMR(600 MHz,CDCl3)δ5.08(d,J=2.8 Hz,1H),4.90(t,J=5.1Hz,1H),4.33(m,1H),4.21(m,1H),4.18–4.13(m,2H),4.12–4.05(m,2H),4.05–4.02(m,1H),4.01(s,1H),3.97(m,1H),3.82(m,2H),3.73(m,1H),3.60–3.54(m,1H),3.01(s,1H),2.71(s,1H),2.34(t,J=7.2 Hz,2H),1.70–1.61(m,4H),1.43–1.36(m,2H),1.29–1.22(m,32H),0.88(t,J=7.2Hz,6H). 13C NMR(151 MHz,CDCl3)δ174.2,108.9,108.6,105.8,86.6,86.6,75.6,69.0,69.0,68.4,68.3,66.5,65.3,65.2,34.3,33.5,32.1,29.8,29.7,29.6,29.6,29.6,29.6,29.5,29.4,29.3,25.1,24.3,22.8,14.3. (I-S-3,n=10), 1 H NMR(600 MHz,CDCl3)δ5.05(d,J=3.0 Hz,1H),4.95(t,J=4.9Hz,1H),4.27(m,1H),4.19(m,1H),4.16–4.14(m,1H),4.12(m,1H),4.10(m,1H),4.06(m,1H),4.02(m,1H),3.97(m,1H),3.90(m,1H),3.83–3.69(m,1H),3.57(m,1H),2.34(t,J=7.2Hz,2H),1.62(m,4H),1.39–1.32(m,2H),1.26(m,32H),0.87(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CDCl3)δ174.1,109.0,108.7,106.4,86.4,86.3,79.1,78.8,75.4,69.0,68.5,68.4,67.1,65.2,65.2,34.3,34.3,33.9,32.0,29.8,29.7,29.7,29.6,29.5,29.4,29.3,25.1,23.9,22.8,14.3. (I-R-4,n=11), 1H NMR(600 MHz,CDCl3)δ5.08(s,1H),4.90(t,J=5.1 Hz,1H),4.33(m,1H),4.21(m,1H),4.16(m,1H),4.13–4.09(m,1H),4.08–4.04(m,2H),4.02(m,2H),3.97(m,1H),3.84(m,1H),3.75–3.71(m,1H),3.59(m,1H),3.03(s,1H),2.72(s,1H),2.34(t,J=7.2 Hz,2H),1.69–1.61(m,4H),1.41–1.35(m,2H),1.31–1.21(m,34H),0.88(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CDCl3)δ174.2,108.6,105.8,86.6,78.8,78.6,75.5,69.0,68.2,66.5,65.2,34.3,33.5,32.1,29.9,29.8,29.8,29.8,29.8,29.7,29.6,29.6,29.6,29.5,29.5,29.4,29.3,25.1,24.4,22.8,14.3. (I-S-4,n=11) 1 H NMR(600 MHz,CDCl3)δ5.06(s,1H),4.95(t,J=4.9 Hz,1H),4.27(m,1H),4.23–4.18(m,2H),4.14(m,1H),4.11(m,2H),4.08–4.02(m,2H),4.02(s,1H),3.90(m,1H),3.71(m,1H),3.59(m,1H),3.09(s,1H),2.90–2.71(m,1H),2.34(t,J=7.2 Hz,2H),1.62(m,4H),1.35(m,2H),1.25(m,34H),0.88(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CDCl3)δ174.2,108.7,106.4,86.4,79.1,78.6,75.3,69.0,68.4,67.1,65.1,34.3,33.9,32.1,29.8,29.8,29.8,29.7,29.7,29.6,29.5,29.5,29.4,29.3,25.0,23.9,22.8,14.3. (CaLGL-1,I-R-5,n=12), 1 H NMR(600 MHz,CD3OD)δ4.90(t,J=4.8 Hz,1H),4.88(d,J=1.5Hz,1H),4.19–4.07(m,3H),4.02–3.92(m,3H),3.91–3.85(m,2H),3.82(m,1H),3.75(m,1H),3.49(m,1H),2.36(t,J=7.2 Hz,2H),1.69–1.59(m,4H),1.43(m,2H),1.35–1.26(m,36H),0.90(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CD3OD)δ175.5,109.9,106.6,85.5,83.4,79.4,77.8,70.0,69.7,67.1,66.5,35.1,35.0,33.2,30.9,30.9,30.9,30.8,30.8,30.8,30.7,30.6,30.5,30.3,26.1,25.1,23.8,14.5.MS(ESI)m / z 653.6[M+Na] + (I-S-5,n=12), 1 H NMR(600 MHz,CD3OD)δ4.98(t,J=4.8 Hz,1H),4.88(d,J=1.7Hz,1H),4.23–4.14(m,2H),4.13–4.10(m,2H),3.99–3.97(m,2H),3.94(m,1H),3.91–3.85(m,2H),3.82(m,1H),3.75(m,1H),3.53–3.45(m,1H),2.35(t,J=7.2 Hz,2H),1.66–1.57(m,4H),1.41(m,2H),1.35–1.27(m,36H),0.90(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CD3OD)δ175.3,109.7,106.4,84.5,83.4,79.2,76.9,70.0,69.5,69.5,67.4,66.4,35.1,35.0,33.1,30.8,30.8,30.8,30.7,30.7,30.7,30.7,30.6,30.5,30.4,30.2,26.0,25.0,23.7,14.5. MS(ESI)m / z 653.6[M+Na] + (I-R-6,n=14), 1 H NMR(600 MHz,CDCl3)δ5.07(d,J=3.0 Hz,1H),4.90(t,J=5.1Hz,1H),4.32(m,1H),4.22–4.11(m,3H),4.09–4.00(m,4H),3.96(m,1H),3.83(m,1H),3.73(m,1H),3.61–3.52(m,1H),2.34(t,J=7.2 Hz,2H),1.64(m,4H),1.38(m,2H),1.30–1.21(m,40H),0.88(t,J=7.2 Hz,6H). 13 C NMR(151 MHz,CDCl3)δ174.2,108.9,108.659,105.8,86.5,79.0,78.6,78.6,75.6,69.0,68.9,68.4,68.3,66.4,65.3,65.2,34.3,33.5,32.1,32.0,29.8,29.8,29.8,29.8,29.7,29.7,29.6,29.6,29.6,29.5,29.5,29.4,29.3,25.1,24.4,22.8,14.3. (I-S-6,n=14), 1 H NMR(600 MHz,CDCl3)δ5.05(d,J=2.9 Hz,1H),4.97–4.93(t,J=5.1 Hz,1H),4.27(m,1H),4.22–4.17(m,1H),4.16–4.09(m,4H),4.06(m,1H),4.04–3.94(m,2H),3.90(m,1H),3.76(m,1H),3.57(m,1H),3.16(s,1H),2.91(s,1H),2.34(t,J=7.2Hz,2H),1.66–1.59(m,4H),1.37–1.34(m,2H),1.30–1.22(m,40H),0.87(t,J=7.2Hz,6H). 13C NMR(151MHz,CDCl3)δ174.1,109.0,108.7,106.4,86.4,79.1,78.8,75.4,69.0,69.0,68.5,68.4,67.1,65.2,65.2,34.3,34.3,33.9,32.1,32.1,29.8,29.8,29.8,29.7,29.7,29.6,29.5,29.5,29.4,29.3,25.1,24.0,22.8,14.3. (I-R-7,n=16), 1 H NMR(600MHz,CD3OD)δ4.90(t,J=4.8Hz,1H),4.88(d,J=1.4Hz,1H),4.20–4.08(m,3H),4.01–3.93(m,3H),3.91–3.85(m,2H),3.82(m,1H),3.79–3.71(m,1H),3.49(m,1H),2.36(t,J=7.2Hz,2H),1.70–1.59(m,4H),1.43(m,2H),1.36–1.27(m,44H),0.90(t,J=7.2Hz,6H). 13 C NMR(151MHz,CD3OD)δ175.3,109.7,106.4,85.4,83.3,79.3,77.7,69.9,69.5,67.0,66.3,35.0,34.9,33.1,30.8,30.8,30.8,30.7,30.5,30.5,30.3,26.0,25.0,23.8,14.5. (I-S-7,n=16), 1 H NMR(600MHz,CD3OD)δ4.98(t,J=4.8Hz,1H),4.88(d,J=1.8Hz,1H),4.23–4.07(m,4H),3.99–3.93(m,2H),3.90–3.85(m,2H),3.82(m,1H),3.75(m,1H),3.50(m,1H),2.35(t,J=7.5Hz,2H),1.66–1.57(m,4H),1.41(m,2H),1.30(m,44H),0.90(t,J=7.2Hz,6H). 13C NMR(151MHz,CD3OD)δ175.3,109.7,106.4,84.5,83.4,79.2,76.9,69.9,69.5,69.4,67.4,66.3,35.1,35.0,33.1,30.8,30.8,30.8,30.7,30.7,30.5,30.5,30.3,26.0,25.0,23.8,14.5. (I-R-8,n=18), 1 H NMR(600MHz,CD3OD)δ4.90(t,J=4.8Hz,1H),4.88(d,J=1.5Hz,1H),4.19–4.07(m,3H),3.97(m,3H),3.91–3.85(m,2H),3.82(m,1H),3.75(m,1H),3.53–3.45(m,1H),2.36(t,J=7.2Hz,2H),1.65(m,4H),1.43(m,2H),1.29(m,48H),0.90(t,J=7.2Hz,6H). 13 C NMR(151MHz,CD3OD)δ175.3,109.7,106.4,85.4,83.4,79.3,77.7,69.9,69.5,69.5,66.9,66.3,35.0,34.9,33.1,30.8,30.8,30.8,30.7,30.6,30.5,30.5,30.5,30.2,26.0,25.0,23.7,14.5. (I-S-8,n=18), 1 H NMR(600MHz,CD3OD)δ4.98(t,J=4.8Hz,1H),4.88(d,J=1.7Hz,1H),4.20(m,1H),4.17–4.06(m,3H),4.00–3.93(m,2H),3.91–3.85(m,2H),3.82(m,1H),3.75(m,1H),3.50(m,1H),2.35(t,J=7.2Hz,2H),1.61(m,4H),1.40(m,2H),1.30(m,48H),0.90(t,J=7.2Hz,6H). 13C NMR (151MHz, CD3OD) δ175.3,109.7,106.4,84.5,83.5,79.2,76.9,69.9,69.5,67.4,66.3 ,35.1,35.0,33.1,30.8,30.8,30.7,30.7,30.7,30.5,30.5,30.3,26.0,25.0,23.8,14.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 PMA induces TNF-α production in THP-1 macrophages, and IR-4 promotes TNF-α production

[0036] Figure 2 EC50 of PMA-induced TNF-α and IR-4 production in THP-1 macrophages

[0037] Figure 3 PMA induced the mRNA expression of TNF-α in THP-1 macrophages in a dose-dependent manner.

Claims

1. A highly active human Toll-like receptor 2 agonist, the structure of which is shown in IR and IS:

2. Its synthesis technology route comprises the following steps:

3. According to claim 2, in step 6, compound VI reacts with dithiol acetal under oxidizing conditions at low temperature to obtain a series of TLR2 agonists.

4. according to claim 3, its dithiol acetal general formula is VII Wherein R1 and R2 can be alkyl, cycloalkyl, phenyl, substituted phenyl, aryl and substituted aryl.

5. According to claim 3, the dithiol acetal has the general formula VII, wherein n is any number from 6 to 20.

6. According to claim 3, the oxidant is a commonly used oxidizing thioether reagent, including iodide, bromide, perchlorate, iodine element, bromine element, etc.

7. According to claim 3, the solvent is a common anhydrous solvent that is compatible with the oxidant. The optimal solvents in this patent are dichloromethane and 1,2-dichloroethane. The reaction temperature can be adjusted appropriately depending on the strength of the oxidant. When using N-iodosuccinimide as the oxidant, the optimal temperature is -35 to -15 degrees Celsius.

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