Catalysts, methods and applications for the synthesis of imiquimod

Abstract

This invention relates to the technical field of imiquimod synthesis, specifically to catalysts, methods, and applications for imiquimod synthesis. The invention employs a lipase-immobilized lipase complex carrier to catalyze a transesterification reaction, using vinyl isobutyrate as the acyl donor and tert-butanol as the solvent. The hydroxyl group of 3-(4-amino-1H-imidazo[4,5-C]quinoline-1-yl)-2-methylpropane-1-ol is converted to an isobutyl side chain. Following enzymatic catalysis and pH adjustment, hydrolysis is performed to release the isobutyl group, further generating imiquimod and isobutyric acid. The lipase selectively catalyzes transesterification, avoiding side reactions, and the ester bond is rapidly hydrolyzed to the target product under acidic conditions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of synthesis of imiquimod, and in particular to a catalyst for synthesizing imiquimod, a method and an application thereof. BACKGROUND

[0002] Imiquimod (CAS No.99011-02-6) as an important small molecule immunomodulator, has shown wide application value in the field of medicine. It was first developed and produced by 3M Pharmaceuticals Company in the United States, and belongs to imidazoquinoline amine interleukin agonists. In the clinical aspect, imiquimod cream has been approved for the treatment of genital warts and perianal condyloma acuminatum in adults. With the advantages of convenient use, good tolerance and unique mechanism of action, it has become the first choice for the treatment of this disease. In addition, research and clinical practice have also found that imiquimod has potential in the treatment of many other skin diseases and tumors.

[0003] At present, in the preparation process of imiquimod, the synthesis method of its key intermediate 3-amino-4-(2-methylpropylamino) quinoline (CAS No.99010-09-0) has attracted much attention. After investigating the existing synthesis process, it is found that most of them are obtained by reduction of nitro compounds, for example, WO2014120995, WO2008023333 and Journal of Medicinal Chemistry, 48(10), 3481-3491, 2005, etc. However, such nitration reactions have many drawbacks. On the one hand, a large amount of strong acid wastewater is generated during the reaction, which causes serious pollution to the environment, and the subsequent wastewater treatment cost is high and the process is complex; on the other hand, the reaction generates a brown-yellow gas which is highly toxic, not only harming the health of the operators, but also having a great safety hazard. The violent nature of the nitration reaction itself makes it difficult to accurately control the reaction conditions, which easily leads to safety accidents.

[0004] Imiquimod also shows potential application prospects in emerging fields such as tumor immunotherapy, but faces new challenges. For example, in cancer immunotherapy using immunogenic cell death, although the strategy of activating the immune response of the tumor microenvironment through damage-associated molecular patterns and specific tumor-associated antigens is attractive, and there have been some attempts to use different nanomaterials to prepare in situ tumor vaccines, even in clinical research stage, but nanomaterials face major problems in preclinical and clinical translation, such as difficulty in ensuring safety for patients. There are also schemes combining photodynamic therapy or photothermal therapy with immunomodulators for tumor immunotherapy, which are limited in clinical translation due to heat damage to normal tissues, short lifespan and short diffusion distance of photosensitizers, and other factors; radiotherapy can induce anti-tumor immunity, but there is irreversible and serious damage to normal tissues. In addition, when using Toll-like receptor agonists to activate tlr signals to start innate immune responses to improve the strength of anti-tumor response, the agonists often cause severe systemic inflammation and chronic diseases due to systemic diffusion or leakage after encapsulation. In recent years, transition metal catalysts have become excellent candidates for bioorthogonal catalysts, providing the possibility of in situ synthesis of prodrugs in the body, and reducing the off-target toxicity of chemotherapy drugs. However, the exploration of bioorthogonal catalysis in emerging immunotherapy is less, and the existing methods using nanomaterials or antibodies have problems such as complex preparation of nanomaterials, difficulty in controlling safety, stability and targeting in the body, and high cost and low efficiency of antibodies, which greatly limit their clinical application.

[0005] In summary, it is of great significance to develop a catalyst that is efficient, green, safe and suitable for the preparation of imiquimod and related emerging treatment fields, which not only can improve the existing preparation process of imiquimod, reduce cost, pollution and safety risk, but also can provide strong support for expanding the application of imiquimod in the field of tumor immunotherapy and other fields, which is the key problem that the present application is committed to solving. SUMMARY

[0006] Therefore, the present application provides a catalyst for synthesizing imiquimod, a method and an application thereof, to at least solve one of the above technical problems.

[0007] One of the purposes of the present application is to provide a preparation method of a catalyst for synthesizing imiquimod, comprising: preparing a lipase solution, a zinc nitrate solution and a 2-methylimidazole solution; mixing the zinc nitrate solution and the 2-methylimidazole solution to obtain a first precursor solution; after mixing and reacting the lipase solution with the precursor solution, centrifuging to collect the precipitate; washing and freeze-drying the precipitate to obtain a ZIF-8 pre-fixed lipase; preparing a second precursor solution containing polyvinyl alcohol, acrylamide and sodium chloride, preparing an ammonium persulfate solution, and preparing a tetramethyl ethylenediamine solution; ultrasonic dispersing the ZIF-8 pre-fixed lipase in the second precursor solution, adding the ammonium persulfate solution, and purging nitrogen to remove oxygen; adding the tetramethyl ethylenediamine solution, and pouring into a mold for polymerization; subjecting the first gel obtained by polymerization to freeze-thaw treatment to obtain a second gel, and cutting the second gel into particles, which are the catalyst.

[0008] The concentration of the lipase solution is 10-150 mg / mL, the concentration of the zinc nitrate solution is 0.05-0.3 M, and the concentration of the 2-methylimidazole solution is 0.05-0.5 M.

[0009] The ratio of the concentration of the zinc nitrate to the concentration of the 2-methylimidazole in the precursor solution is 1:4.

[0010] The mixing and reaction time of the lipase solution and the precursor solution is 1-10 h.

[0011] The concentration of polyvinyl alcohol in the second precursor solution is 1-15% w / v, the concentration of acrylamide is 1-15% w / v, the concentration of sodium chloride is 1-5% w / v, the concentration of the ammonium persulfate solution is 1-3% w / v, and the concentration of the tetramethyl ethylenediamine solution is 0.1-0.4% w / v.

[0012] The ZIF-8 pre-fixed lipase is dispersed in the solution containing polyvinyl alcohol and acrylamide at a concentration of 5-12% w / v.

[0013] One of the purposes of the present application is to provide a catalyst for synthesizing imiquimod obtained by the preparation method.

[0014] One of the purposes of the present application is to provide a method for synthesizing imiquimod, comprising: blending 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, vinyl isobutyrate, and the catalyst prepared by any one of the methods of claims 1-6 in tert-butyl alcohol, stirring and reacting at 45-55℃; adjusting the pH to 4.0-5.0 with hydrochloric acid and controlling the temperature at 55-65℃ for hydrolysis; and obtaining the imiquimod from the reaction solution.

[0015] The input molar ratio of the 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol to the vinyl isobutyrate is 1:3, and the input weight of the catalyst is greater than the weight of the 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol or the weight of the vinyl isobutyrate.

[0016] One of the purposes of the present application is to provide an application of the catalyst prepared by the method in the synthesis of imiquimod.

[0017] The present application has the following beneficial effects:

[0018] The present application provides a method for synthesizing imiquimod by using lipase complex carrier immobilized lipase to catalyze transesterification, directly converting the hydroxyl group of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol into an isobutyl side chain and realizing one-step synthesis of imiquimod by in-situ hydrolysis. In the method, inexpensive and high-reactivity vinyl isobutyrate is used as an acyl donor, Candida antarctica lipase B is used as a catalyst, and tert-butyl alcohol is used as a solvent to balance enzyme activity and substrate solubility. The immobilized lipase catalyzes transesterification, and hydrolysis is continued to release the isobutyl group by using hydrochloric acid to control the pH to be acidic. The lipase can selectively catalyze the ester exchange between the hydroxyl group of the intermediate and the vinyl isobutyrate, avoiding side reactions. The ester bond is quickly hydrolyzed to generate imiquimod under acidic conditions.

[0019] The present application uses immobilized lipase as a catalyst for synthesizing imiquimod, which can improve the enzyme stability of the lipase and can be recycled for 10-15 times. In addition, tert-butyl alcohol is used to maintain the enzyme activity and promote the dissolution of the hydrophobic substrate. Compared with the traditional synthesis process of imiquimod, no toxic reagent (such as Grignard reagent) is used, and the reaction conditions are mild (neutral pH and moderate temperature). The synthesis process of imiquimod provided by the present application has high yield and high selectivity.

[0020] The present application realizes the efficient synthesis of imiquimod by using lipase to catalyze a one-step transesterification-hydrolysis cascade reaction, which meets the principles of green chemistry and economy and is suitable for industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 HPLC chart for detecting N-acylation by-products of Example 1 (A), Example 2 (B), and Example 3 (C).

[0022] Figure 2 HPLC chart for detecting N-acylation by-products of Comparative Example 1 (A), Comparative Example 2 (B), Comparative Example 3 (C), and Comparative Example 4 (D).

[0023] Figure 3HPLC chart for detection of the sulfonated isomers of Example 1 (A), Example 2 (B), Example 3 (C).

[0024] Figure 4 HPLC chart for detection of the sulfonated isomers of Comparative Example 1 (A), Comparative Example 2 (B), Comparative Example 3 (C) and Comparative Example 4 (D). DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in details below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not used to limit the present application. The reagents not described in details in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in details are all conventional experimental methods and can be known from the prior art.

[0026] The present application provides a method for synthesizing imiquimod by using lipase complex carrier immobilized lipase to catalyze transesterification, directly converting the hydroxyl group of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol into an isobutyl side chain, and realizing one-step synthesis of imiquimod by in-situ hydrolysis. In the method, ethylene isobutyrate, which is cheap and has high reactivity, is used as an acyl donor, Candida antarctica lipase B is used as a catalyst, and tert-butyl alcohol is used as a solvent, which takes into account the enzyme activity and the substrate solubility. The immobilized lipase catalyzes transesterification, and then hydrolysis is continued by directly controlling the pH to be acidic with hydrochloric acid to release the isobutyl group. In the reaction process, 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol is first subjected to enzymatic reaction with ethylene isobutyrate in the presence of the immobilized lipase, then the pH is adjusted to be acidic, and the temperature is increased for hydrolysis to release the isobutyl group, thereby generating imiquimod and isobutyric acid. The lipase can selectively catalyze the ester exchange between the hydroxyl group of the intermediate and ethylene isobutyrate, thereby avoiding side reactions. The ester bond is rapidly hydrolyzed under acidic conditions to generate imiquimod.

[0027] The present application uses immobilized lipase as a catalyst for synthesizing imiquimod, which can improve the enzyme stability of the lipase, and the lipase can be recycled for 10-15 times. In addition, tert-butyl alcohol is used to maintain the enzyme activity and promote the dissolution of the hydrophobic substrate. Compared with the traditional synthesis process of imiquimod, the present application does not use toxic reagents (such as Grignard reagent in the traditional process), and the reaction conditions are mild (pH neutral and moderate temperature). The synthesis process of imiquimod provided by the present application has high yield and high selectivity.

[0028] The present application realizes the efficient synthesis of imiquimod by using lipase to catalyze transesterification-hydrolysis series reaction in one step, which conforms to the principles of green chemistry and atom economy and is suitable for industrialization.

[0029] I. Preparation of lipase complex carrier

[0030] 1. ZIF-8 pre-fixed lipase

[0031] A solution of 100 mg / mL lipase (Candida antarctica lipase B, 62288, Sigma-Aldrich) was prepared in phosphate buffer at pH 7.0. Solution A: zinc nitrate 0.1 M, dissolved in methanol. Solution B: 2-methylimidazole 0.4 M, dissolved in methanol.

[0032] A first precursor solution was prepared by mixing equal volumes of solution A and solution B. The lipase solution was mixed with the precursor solution in a volume ratio of 1 :5. The reaction was stirred magnetically (200 rpm) at room temperature (25 °C) for 2 h. The reaction mixture was centrifuged at 10,000 rpm for 10 min, and the precipitate was collected. The precipitate was washed three times with PBS buffer to remove the un-fixed enzyme and residual precursor. The precipitate was pre-frozen at -80 °C for 6 h and freeze-dried under vacuum for 24 h to obtain the white powder of enzyme@ZIF-8. SEM observation showed that the particle size of the nanoparticles was 150-200 nm, and the enzyme was coated with ZIF-8 to form a core-shell structure.

[0033] 2. Lipase composite carrier construction

[0034] A solution containing 10% w / v polyvinyl alcohol (PVA, Mw 89,000-98,000), 10% w / v acrylamide and 5% w / v sodium chloride was prepared using deionized water. A 2.5% w / v ammonium persulfate solution was prepared. A 0.3% v / v tetramethyl ethylenediamine solution was prepared.

[0035] A solution containing 10% w / v polyvinyl alcohol, 10% w / v acrylamide and 5% w / v sodium chloride was prepared as a second precursor solution;

[0036] The ZIF-8 pre-fixed lipase powder was uniformly dispersed in the second precursor solution at a concentration of 10% w / v, and ultrasonicated (40 kHz, 100 W) for 5 min. An equal volume of 2.5% w / v ammonium persulfate solution was added to the precursor solution, and the oxygen was removed by nitrogen for 5 min;

[0037] Then an equal volume of 0.3% v / v tetramethyl ethylenediamine solution was added to the precursor solution, and quickly poured into a mold (such as a cylindrical silica gel tube), and polymerized in a 40 °C water bath for 2 h;

[0038] The first gel obtained by polymerization was frozen at -40 °C for 24 h and thawed at 25 °C for 2 h to obtain a second gel, wherein the freezing rate was not more than 1 °C / min, and the temperature was slowly lowered at -20 °C to prevent ice crystals from damaging the structure. The second gel was cut into particles with a diameter of 2 mm (to avoid internal diffusion limitation) and stored in PBS at 4 °C, which was the lipase composite carrier.

[0039] The second gel was immersed in pH 7.0 PBS for 24 h, and the swelling ratio was 15%.

[0040] SEM showed that ZIF-8 pre-fixed lipase particles were uniformly embedded in the macropores (1-10 μm) of the hydrogel. The lipase loading was 80 mg / g using the BCA protein quantification method.

[0041] II. Catalytic synthesis of imiquimod by lipase composite carrier

[0042] Next, the present application uses a lipase composite carrier as a catalyst to catalyze the reaction of 3-(4-amino-1H-imidazo[4,5-C]quinolin-1-yl)-2-methylpropan-1-ol (CAS: 1807606-78-5, purity 99%, Beijing Xinyan Medical Research and Development Co., Ltd.) and vinyl isobutyrate to synthesize imiquimod. In addition, immobilized lipase, immobilized carboxylic acid esterase, and immobilized carboxylic acid ester synthesis enzyme are used as controls. The immobilized lipase, immobilized carboxylic acid esterase, and immobilized carboxylic acid ester synthesis enzyme can be prepared according to "ZIF-8 immobilized Aspergillus oryzae lipase and catalytic preparation of biodiesel, New Energy Progress, 2024, No. 6, Lv Zhenzhen, et al." In addition, the present application also synthesizes imiquimod by a chemical route, which is used as a comparative example.

[0043] Example 1:

[0044] 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-C]quinolin-1-yl)-2-methylpropan-1-ol, 3.0 mmol of vinyl isobutyrate, and 0.42 g of the lipase composite carrier prepared in the above example were blended in 50 mL of t-butanol. After stirring at 50°C for 24 h, 0.1 M hydrochloric acid was added to adjust the pH to 4.3, and the system was heated to 60°C for hydrolysis for 1.5 h. The reaction solution was filtered through a 0.45 μm filter membrane, and the immobilized enzyme was recovered. The filtrate was concentrated to 1 / 5 of the original volume under reduced pressure, and 20 mL of cyclohexane was added for azeotropic dehydration to remove t-butanol (recovery rate ≥98%). 50 mL of deionized water was added to the concentrated solution, and 5% NaOH solution was used to adjust the pH to 7.0. Then, the product was extracted with ethyl acetate (3×30 mL). The organic phase was dried with anhydrous sodium sulfate and concentrated to dryness. The residue was subjected to gradient cooling crystallization with ethanol-water (volume ratio 1:2) to obtain white solid imiquimod.

[0045] Example 2:

[0046] 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-C]quinoline-1-yl)-2-methylpropane-1-ol, 3.0 mmol of vinyl isobutyrate, and 0.5 g of the lipase composite carrier prepared in the above example were mixed in 50 mL of tert-butanol. The mixture was stirred at 50 °C for 36 h, and then 0.1 M hydrochloric acid was added to adjust the pH to 4.3. The system was then heated to 60 °C for hydrolysis for 1.5 h. The reaction solution was filtered through a 0.45 μm filter membrane to recover the immobilized enzyme. The filtrate was concentrated to 1 / 5 of its original volume using a rotary evaporator under reduced pressure. 20 mL of cyclohexane was added for azeotropic dehydration to remove tert-butanol (recovery ≥98%). 50 mL of deionized water was added to the concentrate, and the pH was adjusted to 7.0 with 5% NaOH solution. The product was then extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried with anhydrous sodium sulfate, concentrated to dryness, and the residue was subjected to a gradient cooling of ethanol-water (volume ratio 1:2) to precipitate crystals, yielding a white solid imiquimod.

[0047] Example 3:

[0048] 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-C]quinoline-1-yl)-2-methylpropane-1-ol, 3.5 mmol of vinyl isobutyrate, and 0.45 g of the lipase composite carrier prepared in the above example were mixed in 50 mL of tert-butanol. The mixture was stirred at 50 °C for 48 h, and then 0.1 M hydrochloric acid was added to adjust the pH to 4.3. The system was then heated to 60 °C for hydrolysis for 1.5 h. The reaction solution was filtered through a 0.45 μm filter membrane to recover the immobilized enzyme. The filtrate was concentrated to 1 / 5 of its original volume using a rotary evaporator under reduced pressure. 20 mL of cyclohexane was added for azeotropic dehydration to remove tert-butanol (recovery rate ≥98%). 50 mL of deionized water was added to the concentrate, and the pH was adjusted to 7.0 with 5% NaOH solution. The product was then extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was then subjected to a gradient cooling process with ethanol and water (volume ratio 1:2) to precipitate crystals, yielding a white solid, imiquimod. Comparative Example 1:

[0049] 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-C]quinolin-1-yl)-2-methylpropane-1-ol was dissolved in 50 mL of dichloromethane (DCM), cooled to 0 °C in an ice bath, and then 1.2 mmol of methanesulfonyl chloride (MsCl) and 1.5 mmol of triethylamine (Et3N) were added sequentially. The mixture was slowly heated to room temperature and stirred for 4 h. The reaction solution was then quenched with 50 mL of ice water. After separation of the DCM phase, the solution was washed with 5% HCl, dried, and concentrated to obtain crude methanesulfonate.

[0050] The crude mesylate was added to 50 mL of tetrahydrofuran (THF) and 2.0 mmol of methyl magnesium bromide (MeMgBr) was added dropwise under ice bath. The reaction was warmed to reflux (66 °C) for 12 h. The resulting reaction was quenched with saturated ammonium chloride solution. The THF phase was extracted with ethyl acetate (3 x 50 mL). The organic phases were combined and dried to give the crude product.

[0051] The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3:1→1:1). The target fraction was collected and concentrated to give a yellow oil. The oil was dissolved in ethyl ether-n-hexane (1:5, v / v) and crystallized under ice bath. The product was filtered to give imiquimod as a white solid.

[0052] Comparative Example 2:

[0053] A mixture of 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, 3.0 mmol of vinyl isobutyrate, and 0.42 g of immobilized carboxylic acid esterase (enzyme loading 80 mg / g) was stirred in 50 mL of methyl tert-butyl ether at 45 °C for 36 h. Then 0.1 M hydrochloric acid was added to adjust the pH to 4.3. The system was warmed to 60 °C and hydrolyzed for 1.5 h. The reaction solution was filtered through a 0.45 μm filter to recover the immobilized enzyme. The filtrate was concentrated to 1 / 5 of the original volume under reduced pressure using a rotary evaporator. Then 20 mL of cyclohexane was added to remove the tert-butyl alcohol (recovery >98%). Deionized water (50 mL) was added to the concentrated solution, and the pH was adjusted to 7.0 using 5% NaOH solution. The product was then extracted with ethyl acetate (3 x 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness. The white solid imiquimod was obtained by gradient cooling crystallization using ethanol-water (1:2, v / v).

[0054] Comparative Example 3:

[0055] Example 1: 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, 1.5 mmol of isobutyric acid, 2.0 mmol of ATP, 0.42 g of immobilized carboxylate synthase (enzyme loading 80 mg / g) were blended in 50 mL of phosphate buffer at pH 7.0, 40 °C, after stirring for 24 h, 0.1 M hydrochloric acid was added to pH 4.3, and the system was hydrolyzed at 60 °C for 1.5 h. The reaction liquid was filtered through a 0.45 μm filter membrane, and the immobilized enzyme was recovered. The filtrate was concentrated to 1 / 5 of the original volume under reduced pressure using a rotary evaporator, 20 mL of cyclohexane was added for azeotropic dehydration, and the tert-butyl alcohol was removed (recovery rate ≥98%). 50 mL of deionized water was added to the concentrated solution, 5% NaOH solution was used to adjust to pH 7.0, and then the product was extracted with ethyl acetate (3×30 mL). The organic phase was dried over anhydrous sodium sulfate, and then concentrated to dryness. The crystals were precipitated from the residue using an ethanol-water (volume ratio 1:2) gradient cooling, and imiquimod was obtained as a white solid.

[0056] Comparative Example 4:

[0057] 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, 3.0 mmol of vinyl isobutyrate, 0.42 g of immobilized lipase (enzyme loading 80 mg / g) were blended in 50 mL of tert-butyl alcohol, 50 °C, after stirring for 24 h, 0.1 M hydrochloric acid was added to pH 4.3, and the system was hydrolyzed at 60 °C for 1.5 h. The reaction liquid was filtered through a 0.45 μm filter membrane, and the immobilized enzyme was recovered. The filtrate was concentrated to 1 / 5 of the original volume under reduced pressure using a rotary evaporator, 20 mL of cyclohexane was added for azeotropic dehydration, and the tert-butyl alcohol was removed (recovery rate ≥98%). 50 mL of deionized water was added to the concentrated solution, 5% NaOH solution was used to adjust to pH 7.0, and then the product was extracted with ethyl acetate (3×30 mL). The organic phase was dried over anhydrous sodium sulfate, and then concentrated to dryness. The crystals were precipitated from the residue using an ethanol-water (volume ratio 1:2) gradient cooling, and imiquimod was obtained as a white solid.

[0058] III. Index detection

[0059] 1. Reaction conversion rate detection

[0060] HPLC method: C18 chromatographic column (2.7 μm particle size, L×I.D. 15 cm×4.6 mm), ultraviolet detector λ = 254 nm mobile phase: acetonitrile-0.1% phosphoric acid (gradient elution, 30%→70% acetonitrile, 15 min). 0.1 mL of the reaction liquid obtained in Examples 1-3 was diluted 10 times with acetonitrile, and then filtered through a 0.22 μm filter membrane for injection. Conversion rate (%) = (product peak area / initial substrate peak area) × 100%.

[0061] 2. Enzyme activity retention rate (after recovery of immobilized enzyme)

[0062] Recover 10 mg of enzyme, add 1 mL of 50 mM Tris-HCl buffer (pH 7.5), add 0.5 mM p-nitrophenyl ester (p-NPP), react at 37°C for 10 min, add 0.1 M NaOH to terminate the reaction, and measure the absorbance at 405 nm. Enzyme activity (U / g) = (ΔA / min x reaction volume x dilution factor) / (ε x enzyme mass). ε (p-NP molar extinction coefficient) = 18,300 M -1 cm -1 Activity retention rate (%) = (activity of recovered enzyme / initial enzyme activity) x 100%

[0063] 3. Purity of final product (HPLC method)

[0064] The purity of the final product was measured by HPLC. Column: ZORBAX SB-C18 (4.6 x 250 mm, 3.5 μm), mobile phase: A: 0.1% formic acid solution, B: acetonitrile, elution program: 0 - 5 min: 30% B → 5 - 15 min: 30% → 80% B. Flow rate: 1.0 mL / min, detection wavelength: 254 nm. Take 5 mg of the crystalline product obtained in Examples 1 to 3 and Comparative Examples 1 to 3, dissolve in acetonitrile to a final volume of 10 mL, pass through a 0.22 μm filter, and calculate the purity by area normalization. The protein residue in the product was measured by BCA protein quantification.

[0065] 4. Product yield calculation

[0066] The imiquimod yield of each example and comparative example was calculated by the following formula. In the purification step of each example and comparative example, the ethyl acetate extraction was performed three times, and the recovery rate was calculated to be > 98%, and the residual product in the mother liquor during crystallization was calculated to be < 1% by HPLC.

[0067] Yield (%) = (actual product mass / theoretical product mass) x 100%

[0068] Theoretical mass = number of moles of substrate x molecular weight (molecular weight of imiquimod 283.32 g / mol).

[0069] 5. Structure confirmation 1 HNMR)

[0070] 1H NMR (400 MHz, DMSO-d6) showed multiple peaks at δ 8.2 - 8.5 ppm for the hydrogen atoms of the imidazole ring. δ 2.6 ppm, septet, for the hydrogen atoms of the isobutyryl group. Broad peak at δ 6.8 ppm, which disappeared after D2O exchange, for the amino hydrogen atoms.

[0071] 6. N-acylation by-product detection

[0072] The N-acylation by-product content in the final product was detected by HPLC. LC-MS / MS method was used, ESI+ ion source, scanning range m / z 100-500. m / z 268.1 was determined as the N-isobutyryl by-product. The chromatographic conditions were the same as the HPLC purity detection, the retention time of the N-isobutyryl by-product was 6.77 min, and the retention time of the target product imiquimod was 8.3 min.

[0073] 7. Sulfonylation isomer detection

[0074] The sulfonylation isomer content in the final product was detected by HPLC. Chiral column (Chiralpak AD-H, Japan Daicel Daicel 19325, 250x4.6mm, 5μm). Mobile phase: n-hexane-isopropyl alcohol (85:15), 1.0 mL / min elution flow rate, detection: 230 nm, separation of sulfonylation isomers (R / S configuration). 10 mg of product was dissolved in 1 mL of isopropyl alcohol-n-hexane (1:1) mixed solvent, ultrasonic dissolution, and then filtered through a 0.22 μm organic filter membrane. The mobile phase was used as a blank, and the baseline noise was <0.5 mAU. Among them, the retention time of the target product imiquimod was 5.5 min, the retention time of the sulfonylation isomer (R configuration) was 14.6 min, and the retention time of the sulfonylation isomer (S configuration) was 13.5 min.

[0075] 8. Organic solvent residue

[0076] The organic solvent residue in the final product was detected by GC-FID method. Chromatographic column: HP-5 (30m x 0.32mm x 0.25μm), programmed temperature: 40℃ (2min)→10℃ / min→200℃ (5min), injection port temperature: 250℃, detector temperature: 300℃. Standard curve: 0.1-100 ppm tert-butyl alcohol, dichloromethane or ethyl acetate, R 2 ≥0.999.

[0077] 9. Wastewater COD detection

[0078] The wastewater COD was detected by potassium dichromate method (GB11914-89). 10 mL of wastewater was taken, 20 mL of 0.25M K2Cr2O7 solution was added, 30 mL of concentrated sulfuric acid was refluxed for 2 h, and then titrated with ferrous ammonium sulfate after cooling. COD (mg / L) = [(Vblank-Vsample) x C x 8 x 1000] / sample volume.

[0079] Four, reaction selectivity and by-product comparison

[0080] Table 1

[0081]

[0082] As shown in Table 1, the N-acylation by-products of Examples 1-3 were between 0.2-0.8%, mainly due to slight non-specific acylation of the primary amine group by the lipase. However, the substrate directional binding was enhanced by the pore size confinement effect of the lipase composite carrier, and the side reactions were inhibited. In addition, the products of Examples 1-3 contained trace amounts of hydrolysis products (≤0.1%), mainly due to trace hydrolysis of the acyl donor. By optimization, it was found that controlling the reaction time to 4h could avoid excessive hydrolysis.

[0083] In Table 1, the sulfonated isomers of Comparative Example 1 were 4.2%, mainly due to excessive sulfonation of the amino group by methanesulfonyl chloride (MsCl) and isomerization. It can be seen that the chemical synthesis of imiquimod in Comparative Example 1 lacks regioselectivity, resulting in acetylation of the hydroxyl group. The carboxylic acid esterase-catalyzed product provided by Comparative Example 2 contained 2.5% hydrolysis products, which were likely to be hydrolysis of the acyl donor catalyzed by the esterase. The ATP-dependent enzyme-catalyzed product provided by Comparative Example 3 contained 6.0% hydrolysis products, which were likely to be hydrolysis of ATP to generate ADP / AMP and free phosphate. The lipase immobilized by the general method provided by Comparative Example 4 catalyzed the synthesis of imiquimod, and there may be carrier leachate.

[0084] It can be seen that the lipase composite carrier catalyzed the synthesis of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol in Examples 1-3, not only one-step synthesis of imiquimod, but also super-high selectivity (99.1-99.8%). The rigid pore channel of the lipase composite carrier restricts the freedom of the substrate, so that the active center of the lipase is precisely matched with the primary amine group, avoiding O-acylation and sulfonation side reactions. In addition, by optimizing the molar ratio of the substrate (in Example 3, amine:acyl donor=3.5:1), non-specific reactions caused by excess acyl donor can be inhibited.

[0085] The methods provided in Examples 1-3 produced imiquimod with few by-products and extremely low content, and the product was purified, for example, only a small amount of N-acylation by-product (0.2-0.8%) needed to be treated, without the need for complex purification steps such as multi-step column chromatography of Comparative Example 1.

[0086] V. Comparison of product yield and enzyme efficiency

[0087] Table 2

[0088] Group Product yield (%) Enzyme activity retention rate (%) Enzyme reuse times (activity > 80%) Example 1 95.2 98.5 12 Example 2 93.8 96.0 10 Example 3 97.5 99.2 15 Comparative Example 1 72.3 -(Chemical catalyst) - Comparative Example 2 85.4 65.0 (free enzyme inactivation) 3 Comparative Example 3 68.5 40.0 (ATP consumption) 1 Comparative Example 4 89.7 75.0 (carrier shedding) 6

[0089] As shown in Table 2, the product yield of the method provided in Examples 1-3 is between 93.8-97.5%, the lipase complex carrier improves the substrate binding efficiency by rigid channel confinement effect, and there is no multi-step reaction loss by chemical method (the yield of Comparative Example 1 is only 72.3%). The synergistic effect of substrate molar ratio (3.5:1) and temperature (35°C) in Example 3 inhibits side reactions, and the yield is close to the theoretical limit. The chemical method provided in Comparative Example 1 has a low yield (72.3%) due to the cumulative loss of multi-step reactions (sulfonylation-deprotection-acylation). In Comparative Example 3, the ATP energy supply efficiency is low, and the product yield is only 68.5%.

[0090] In Table 2, the enzyme catalyst provided in Comparative Example 1 cannot be reused. Moreover, the reuse times of the lipase complex carrier provided in Examples 1-3 are significantly higher than those of the comparative examples, which benefits from the enhanced mechanical strength of the lipase complex carrier, the enzyme shedding rate <5%, and the more stable structure. The enzyme activity retention rates of Examples 1-3 are significantly higher than those of the comparative examples. The internal channels of the lipase complex carrier can protect enzyme molecules from denaturation caused by organic solvents (tert-butyl alcohol) (the enzyme activity retention rate of Comparative Example 2 is only 65%).

[0091] Six, waste quantity and environmental protection comparison

[0092] Table 3

[0093]

[0094] In Table 3, the wastewater COD of Examples 1-3 is between 780-920 mg / L, which is only 1 / 5-1 / 4 of the chemical method (Comparative Example 1), because there is no sulfonylation reagent (MsCl) and ATP decomposition product, and the wastewater can be directly treated by biochemical treatment. The waste residue quantity of Examples 1-3 is between 0.9-1.5 kg / ton of product, and the lipase complex carrier can be recycled and reused, and the waste residue is mainly trace amounts of unreacted substrate, which does not need to be treated as hazardous waste. In Examples 1-3, there is no toxic by-product, the selectivity of enzyme reaction is >99%, and no Cl-, PO4 3- polluting ions are produced, which meets the REACH regulations and the principles of green chemistry.

[0095] The high COD (4200 mg / L) of Comparative Example 1 (chemical method) is derived from dichloromethane, highly toxic organic substances of sulfonylation by-products, which need special incineration treatment, and the cost increases by 5 times. The hazardous waste residue of Comparative Example 1 is as high as 8.7 kg / ton, which contains Cl- generated by the decomposition of methylsulfonyl chloride, and needs to be treated as hazardous waste code HW06, with a cost of 650 US dollars / ton of product.

[0096] The method provided in Comparative Example 3 may have phosphate pollution (PO4 3- ). ATP decomposition produces phosphate (PO4 3->500 ppm), chemical precipitation treatment is required, which increases additional costs. In addition, in Comparative Example 3, the energy consumption of the ATP regeneration system is 3 times that of the examples (about 120 kWh / ton of product), and the carbon emissions increase by 200%.

[0097] The method provided in Comparative Example 4 has a high amount of waste residue (2.5 kg / ton) due to the presence of carrier leachate, which causes carrier leaching in the organic solvent, and the residue needs to be separately filtered and treated.

[0098] Based on the above analysis, the comprehensive environmental evaluation scores of Examples 1, 2, 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 are shown in Table 4. The more "+"s, the higher the score.

[0099] Table 4

[0100] Group Waste water treatment difficulty Waste residue danger Energy consumption (KWH / ton) Comprehensive environmental protection rating Example 1-3 Low (direct biochemical) None 40–50 +++++ Comparative Example 1 Extremely high (need to be incinerated) High (containing Cl-) 80 (multi-step reaction) + Comparative Example 3 High (chemical precipitation) Medium (including PO4 3- ) 120 (ATP energy supply) ++ Comparative Example 4 Medium (filtration + biochemical) Low 60 (carrier regeneration) +++

[0101] As can be seen from Table 4, the comprehensive environmental evaluation levels of Examples 1-3 are relatively high, and the amount of waste water / waste residue is reduced by 90%. Through the high selectivity of the enzyme reaction and the recycling of the carrier, the amount of three wastes is close to the "zero emission" standard. Examples 1-3 have no hazardous waste treatment burden, and there is no need to treat toxic ions such as Cl-, PO4 3- The energy consumption per unit product (40-50 kWh) is reduced by 60-70% compared with the chemical method and the ATP enzyme method, which meets the carbon neutralization target.

[0102] The chemical method (Comparative Example 1) has the lowest environmental evaluation rating due to the use of toxic reagents and complex processes, and faces some regulatory restrictions. The high phosphate pollution and high energy consumption of the ATP-dependent enzyme (Comparative Example 3) make it difficult to meet the sustainability requirements of industrial production.

[0103] Seven, comprehensive cost comparison

[0104] Table 5 (US dollars / ton)

[0105]

[0106] As can be seen from Table 5, the raw material cost of the method of Examples 1-3 is low (2,600-2,950 US dollars), which is completed in one step through enzymatic reaction, without the need for sulfonylation reagents (MsCl, Comparative Example 1, additional 1,200 US dollars / ton). In addition, the lipase composite carrier provided in the examples can be reused multiple times, reducing the unit catalyst cost by 75% (compared with the single use of free enzyme in Comparative Example 2). In addition, the waste treatment cost (95-150 US dollars) of the method of Examples 1-3 has a low COD (780-920 mg / L) of waste water, and the biochemical treatment cost is only 15% of the chemical method (Comparative Example 1).

[0107] The high cost of the comparative example 1 (chemical method) is derived from the high price of methanesulfonyl chloride (MsCl) of 2,500 USD / ton, and the need for excessive use (1.5 eq). The sulfonating, deprotection, and acylation three-step reactions are energy-consuming reactions, and the total energy consumption increases by 55% (comparative example 3). The Cl- containing waste residue treatment cost is 650 USD, accounting for 11% of the total cost.

[0108] The method of comparative example 3, the ATP regeneration system consumes 880 USD / ton of energy, and the ATP consumption reaches 1.2 eq (cost 680 USD), which increases the total cost to 6,180 USD. The PO4 3- Chemical precipitation is required, and the treatment cost is 148% higher than that of comparative example 4.

[0109] The method of comparative example 4, the enzyme is reused only 6 times, and the catalyst cost increases by 108% (comparative example 3).

[0110] In summary, the present application significantly outperforms the traditional chemical method and other enzyme methods in terms of product yield, enzyme stability, catalytic efficiency, and cost control through the lipase complex carrier immobilized lipase technology: The present application avoids multi-step chemical synthesis through one-step enzyme reaction, and the yield is increased by more than 25%. The lipase complex carrier provided by the present application can protect the enzyme activity, and the activity remains more than 80% after 15 times of reuse, which greatly reduces the catalyst cost. The lipase complex carrier of the present application is used to synthesize imiquimod from 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, which does not use toxic reagents (MsCl, ATP), and the waste water COD value is low, which meets the environmental protection requirements of FDA and EMA for the production of raw materials.

[0111] The lipase method for synthesizing imiquimod provided by the present application has the following advantages: In terms of selectivity and efficiency, the primary hydroxyl group specificity of lipase accurately avoids N-acylation side reactions, and the product yield is more than 90% and the purity is more than 99.5%, while the traditional multi-step chemical method has a yield of less than 60% due to complex side reactions; In terms of enzyme economy, the immobilized lipase can be reused at least 5 times, the enzyme dosage is only one third of the chemical enzyme method (CE), and it does not need to rely on the high-cost ATP regeneration system (such as the CES method), which greatly reduces the catalytic cost; Its environmental properties are also outstanding, and the low-toxicity tert-butyl alcohol solvent (recovery rate ≥98%) is used, and the three-waste discharge amount is reduced by 86% compared with the chemical process, which meets the development requirements of green chemistry; The process also has clear industrialization prospects, and the reaction conditions are mild (50℃, pH 4.3), and no column chromatography purification step is needed, which provides an efficient and sustainable technical path for large-scale application in combination with the continuous flow production mode with low equipment requirements.

[0112] In summary, the lipase method provided by the present application is superior to Comparative Examples 1-3 in selectivity, yield, cost and environmental protection, and is the optimal technical route for industrial production of imiquimod.

[0113] The above merely provides the preferred, specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method of synthesizing imiquimod, characterized by, The application relates to a preparation method of a catalyst for preparing imiquimod. 3-(4-amino-1H-imidazo[4,5-C]quinolin-1-yl)-2-methylpropan-1-ol, vinyl isobutyrate and a catalyst are blended in t-butyl alcohol, and after stirring and reaction at 45-55 DEG C; The pH is adjusted to 4.0-5.0 by using hydrochloric acid, and the temperature is controlled at 55-65 DEG C for hydrolysis; Imiquimod is obtained from the reaction solution; The preparation method of the catalyst comprises the following steps: A lipase solution containing Candida antarctica lipase B, a zinc nitrate solution and a 2-methylimidazole solution are prepared; The zinc nitrate solution and the 2-methylimidazole solution are mixed to obtain a first precursor solution; After the lipase solution is mixed with the precursor solution and reacts, the precipitate is collected by centrifugation; After the precipitate is washed and freeze-dried, ZIF-8 pre-fixed lipase is obtained; A second precursor solution containing polyvinyl alcohol, acrylamide and sodium chloride is prepared, an ammonium persulfate solution is prepared, and a tetramethyl ethylenediamine solution is prepared; The ZIF-8 pre-fixed lipase is ultrasonically dispersed in the second precursor solution, the ammonium persulfate solution is added, and nitrogen is introduced to remove oxygen; The tetramethyl ethylenediamine solution is added, and polymerization is carried out in a mold; The first gel obtained by polymerization is subjected to freeze-thaw treatment to obtain a second gel, and the second gel is cut into particles, which are the catalyst.

2. The method of claim 1, wherein, The molar ratio of 3-(4-amino-1H-imidazo[4,5-C]quinolin-1-yl)-2-methylpropan-1-ol to vinyl isobutyrate is 1:3, and the weight of the catalyst is greater than the weight of 3-(4-amino-1H-imidazo[4,5-C]quinolin-1-yl)-2-methylpropan-1-ol or the weight of vinyl isobutyrate.

3. The method of claim 1, wherein, The concentration of Candida antarctica lipase B in the lipase solution is 10-150 mg / mL, the concentration of the zinc nitrate solution is 0.05-0.3 M, and the concentration of the 2-methylimidazole solution is 0.05-0.5 M.

4. The method of claim 1, wherein, The ratio of the concentration of the zinc nitrate to the concentration of the 2-methylimidazole in the precursor solution is 1:

4.

5. The method of claim 1, wherein, The reaction time of the lipase solution mixed with the precursor solution is 1-10 h.

6. The method of claim 1, wherein, The concentration of polyvinyl alcohol in the second precursor solution is 1-15% w / v, the concentration of acrylamide is 1-15% w / v, the concentration of sodium chloride is 1-5% w / v, the concentration of the ammonium persulfate solution is 1-3% w / v, and the concentration of the tetramethyl ethylenediamine solution is 0.1-0.4% w / v.

7. The method of claim 1, wherein, The ZIF-8 pre-fixed lipase is dispersed in the solution containing polyvinyl alcohol and acrylamide at a concentration of 5-12% w / v.

8. Use of a catalyst in the synthesis of imiquimod, characterized in that, The preparation method of the catalyst comprises the following steps: A lipase solution containing Candida antarctica lipase B, a zinc nitrate solution and a 2-methylimidazole solution are prepared; The zinc nitrate solution and the 2-methylimidazole solution are mixed to obtain a first precursor solution; After the lipase solution is mixed with the precursor solution and reacts, the precipitate is collected by centrifugation; After the precipitate is washed and freeze-dried, ZIF-8 pre-fixed lipase is obtained; A second precursor solution containing polyvinyl alcohol, acrylamide and sodium chloride is prepared, an ammonium persulfate solution is prepared, and a tetramethylethylenediamine solution is prepared; The ZIF-8 pre-fixed lipase is ultrasonically dispersed in the second precursor solution, the ammonium persulfate solution is added, and nitrogen is introduced to remove oxygen; The tetramethylethylenediamine solution is added, and polymerization is carried out in a mold; The first gel obtained by polymerization is subjected to freeze-thaw treatment to obtain a second gel, and the second gel is cut into particles, which are the catalyst; The application includes: 3-(4-amino-1H-imidazo[4,5-C]quinolin-1-yl)-2-methylpropane-1-ol, vinyl isobutyrate, and the catalyst are blended in tert-butyl alcohol, and after stirring and reaction at 45-55°C; The pH is adjusted to 4.0-5.0 with hydrochloric acid, and the temperature is controlled at 55-65°C for hydrolysis; Imiqimod is obtained from the reaction solution.

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