Catalyst, method and application for synthesizing imiquimod

Abstract

The invention relates to the technical field of imiquimod synthesis, in particular to a catalyst for synthesizing imiquimod, a method and application. According to the method, lipase composite carrier immobilized lipase is used for catalyzing transesterification reaction, vinyl isobutyrate is used as an acyl donor, tert-butyl alcohol is used as a solvent, 3-(4-amino-1H-imidazo [4, 5-C] quinoline-1-yl)-2-methylpropane-1-alcohol hydroxyl is converted into an isobutyl side chain, after enzymatic reaction, pH is adjusted to be acidic, hydrolysis is carried out to release isobutyl, and the isobutyl side chain is separated from the isobutyric acid side chain to obtain the 3-(4-amino-1H-imidazo [4, 5-C] quinoline-1-yl)-2-methylpropane-1-alcohol. And imiquimod and isobutyric acid are further generated. Lipase selectively catalyzes ester exchange, side reaction is avoided, and ester bonds are rapidly hydrolyzed into a target product under the acidic condition.

Description

Technical Field

[0001] The present invention relates to the technical field of imiquimod synthesis, and particularly relates to a catalyst, a method and an application for synthesizing imiquimod. Background Art

[0002] Imiquimod (CAS No. 99011-02-6), as an important small molecule immunomodulator, has shown extensive application value in the medical field. It was first developed and produced by 3M Pharmaceuticals in the United States and belongs to imidazoquinolineamine interleukin agonists. Clinically, imiquimod cream has been approved for the treatment of external genital and perianal condyloma acuminata in adults. Due to its convenient use, good tolerance and unique mechanism of action, it has become the preferred treatment drug for this disease. In addition, research and clinical practice have also found that imiquimod has potential in the treatment of a variety of other skin diseases and tumors.

[0003] Currently, 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. Through investigation of existing synthesis processes, it is found that most of them are obtained by reducing nitro compounds. For example, it is recorded in documents such as WO2014120995, WO2008023333, and "Journal of Medicinal Chemistry, 48(10), 3481-3491, 2005". However, such nitrification reactions have many drawbacks. On the one hand, a large amount of strong acid wastewater is generated during the reaction, causing serious environmental pollution. The subsequent wastewater treatment cost is high and the process is complex. On the other hand, highly toxic brownish-yellow gases are generated during the reaction, which not only endanger the health of operators but also pose great safety hazards. The severity of the nitrification reaction itself makes it difficult to precisely control the reaction conditions, easily leading to safety accidents.

[0004] In emerging fields such as cancer immunotherapy, imiquimod also shows potential application prospects, but faces new challenges. For example, in cancer immunotherapy using immunogenic cell death, although the strategy of activating the immune response in the tumor microenvironment through damage-associated molecular patterns and specific tumor-associated antigens is attractive, and there have been some attempts to prepare in-situ tumor vaccines using different nanomaterials, even entering the clinical research stage, nanomaterials face major problems in preclinical and clinical translation, such as difficulty in ensuring the safety of patients. There are also protocols that combine photodynamic therapy or photothermal therapy with immunomodulators for cancer immunotherapy. However, factors such as thermal damage to normal tissues, short lifespan and short diffusion distance of photosensitizers limit their clinical translation; radiotherapy can induce anti-tumor immunity, but causes irreversible severe damage to normal tissues. In addition, when using Toll-like receptor agonists to activate TLR signaling to initiate innate immune responses to enhance the intensity of anti-tumor responses, 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 outstanding candidates for bioorthogonal catalysts, providing the possibility for in-situ synthesis of prodrugs in vivo and reducing the off-target toxicity of chemotherapeutic drugs. However, the exploration of bioorthogonal catalysis in emerging immunotherapies is less. The existing methods of combining nanomaterials or antibodies have problems such as complex preparation of nanomaterials, difficulty in controlling safety, stability and targeting in vivo, 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 applicable to the preparation of imiquimod and related emerging treatment fields. It can not only improve the existing preparation process of imiquimod, reduce costs, pollution and safety risks, but also provide strong support for expanding the application of imiquimod in fields such as cancer immunotherapy. This is also the key problem that the present invention is committed to solving. Summary of the Invention

[0006] In view of this, the present invention provides a catalyst, method and application for synthesizing imiquimod to solve at least one of the above technical problems.

[0007] One of the objectives of the present invention 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; mixing the lipase solution with the precursor solution, reacting, and then centrifuging to collect the precipitate; washing and freeze-drying the precipitate to obtain ZIF-8 pre-immobilized lipase; preparing a second precursor solution containing polyvinyl alcohol, acrylamide, and sodium chloride, preparing an ammonium persulfate solution, and preparing a tetramethylethylenediamine solution; ultrasonically dispersing the ZIF-8 pre-immobilized lipase in the second precursor solution, adding the ammonium persulfate solution, and purging with nitrogen to remove oxygen; adding the tetramethylethylenediamine solution, 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 is the catalyst.

[0008] Among them, 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] Among them, the ratio of the concentration of zinc nitrate to the concentration of 2-methylimidazole in the precursor solution is 1:4.

[0010] Among them, the reaction time of mixing the lipase solution with the precursor solution is 1-10 h.

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

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

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

[0014] One of the objectives of the present invention 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 claims 1-6 in tert-butanol, stirring and reacting at 45-55 °C; adjusting the pH to 4.0-5.0 with hydrochloric acid, controlling the temperature at 55-65 °C for hydrolysis; and obtaining the imiquimod from the reaction solution.

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

[0016] One of the objectives of the present invention is to provide the application of the catalyst prepared by the above method in the synthesis of imiquimod.

[0017] Advantages of the present invention:

[0018] The present invention provides a method for 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 by using a lipase composite carrier to immobilize lipase to catalyze transesterification and in-situ hydrolysis to achieve one-step synthesis of imiquimod. Among them, vinyl isobutyrate, which is cheap and highly reactive, is used as an acyl donor, Candida antarctica lipase B is used as a catalyst, and tert-butanol, which takes into account enzyme activity and substrate solubility, is used as a solvent to carry out immobilized lipase-catalyzed transesterification, and hydrochloric acid is directly used to control the pH to acidic for continuous hydrolysis to release isobutyl. Lipase can selectively catalyze the transesterification of the hydroxyl group of the intermediate with vinyl isobutyrate to avoid side reactions. The ester bond is rapidly hydrolyzed under acidic conditions to generate imiquimod.

[0019] The present invention uses immobilized lipase as a catalyst for the synthesis of imiquimod, which can improve the enzyme stability of lipase and can be recycled 10-15 times. In addition, tert-butanol is used to maintain enzyme activity and at the same time promote the dissolution of hydrophobic substrates. Compared with the traditional imiquimod synthesis process, there are no highly toxic reagents (such as the need to use Grignard reagents in the traditional process), and the reaction conditions are mild (pH neutral, medium temperature). The imiquimod synthesis process provided by the present invention has high yield and high selectivity.

[0020] The present invention realizes the efficient synthesis of imiquimod through one-step catalytic transesterification-hydrolysis tandem reaction of lipase, which conforms to the principles of green chemistry and economy and is suitable for industrial scale-up. Description of the Drawings

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

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

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

[0024] Figure 4 HPLC chromatograms for the detection of sulfonylation isomers of Comparative Example 1 (A), Comparative Example 2 (B), Comparative Example 3 (C), and Comparative Example 4 (D). Detailed implementation manners

[0025] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Reagents not described in detail and separately in the present invention are all conventional reagents and can be obtained from commercial channels; methods not described in detail and specifically are all conventional experimental methods and can be learned from the prior art.

[0026] The present invention provides a method for 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 by using a lipase composite carrier to immobilize lipase to catalyze a transesterification reaction and in-situ hydrolysis to achieve one-step synthesis of imiquimod. Among them, vinyl isobutyrate, which is cheap and has high reaction activity, is used as an acyl donor, Candida antarctica lipase B is used as a catalyst, and tert-butanol, which takes into account both enzyme activity and substrate solubility, is used as a solvent to carry out immobilized lipase-catalyzed transesterification, and hydrochloric acid is directly used to control the pH to acidic and continue hydrolysis to release isobutyl. During the reaction process, 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol first undergoes an enzymatic reaction with vinyl isobutyrate in the presence of immobilized lipase, then the pH is adjusted to acidic, and the temperature is raised for hydrolysis to release isobutyl, generating imiquimod and isobutyric acid. Lipase can selectively catalyze the transesterification of the hydroxyl group of the intermediate with vinyl isobutyrate to avoid side reactions. The ester bond is rapidly hydrolyzed under acidic conditions to generate imiquimod.

[0027] The present invention uses immobilized lipase as a catalyst for synthesizing imiquimod, which can improve the enzyme stability of lipase and can be recycled 10 - 15 times. In addition, tert-butanol is used to maintain enzyme activity and at the same time promote the dissolution of hydrophobic substrates. Compared with the traditional imiquimod synthesis process, there are no highly toxic reagents (such as the traditional process needs to use Grignard reagents), and the reaction conditions are mild (pH neutral, medium temperature). The imiquimod synthesis process provided by the present invention has high yield and high selectivity.

[0028] The present invention realizes the efficient synthesis of imiquimod through a one-step catalytic transesterification-hydrolysis tandem reaction of lipase, which conforms to the principles of green chemistry and atom economy and is suitable for industrial scale-up.

[0029] I. Preparation of lipase composite carrier

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

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

[0032] Mix solution A and solution B in an equal volume ratio to obtain the first precursor solution. Mix the lipase solution and the precursor solution in a volume ratio of 1:5. React at room temperature (25 °C) with magnetic stirring (200 rpm) for 2 hours. Centrifuge the reaction solution at 10,000 rpm for 10 minutes and collect the precipitate. Wash it 3 times with PBS buffer to remove the unfixed enzyme and residual precursor. Place the precipitate at -80 °C for pre-freezing for 6 hours and then vacuum freeze-dry for 24 hours to obtain white powdery enzyme@ZIF-8. Observed by SEM, the particle size of the nanoparticles is 150 - 200 nm, and ZIF-8 coats the enzyme to form a core-shell structure.

[0033] 2. Construction of lipase composite carrier

[0034] Prepare a solution containing 10% w / v polyvinyl alcohol (PVA, Mw 89,000 - 98,000), 10% w / v acrylamide and 5% w / v sodium chloride with deionized water. Prepare a 2.5% w / v ammonium persulfate solution. Prepare a 0.3% v / v tetramethylethylenediamine solution.

[0035] Take the solution containing 10% w / v polyvinyl alcohol, 10% w / v acrylamide and 5% w / v sodium chloride as the second precursor solution;

[0036] Disperse the ZIF-8 pre-fixed lipase powder uniformly in the second precursor solution at a concentration of 10% w / v, and ultrasonically treat it (40 kHz, 100 W) for 5 min. Add an equal volume of 2.5% w / v ammonium persulfate solution to the precursor solution, and deoxygenate by passing nitrogen for 5 min;

[0037] Then add an equal volume of 0.3% v / v tetramethylethylenediamine solution to the precursor solution, quickly pour it into a mold (such as a cylindrical silicone tube), and polymerize in a 40 °C water bath for 2 h;

[0038] Freeze the first gel obtained by polymerization at -40 °C for 24 h and then thaw it at 25 °C for 2 h to obtain the second gel. Among them, the freezing rate should not exceed 1 °C / min, and slow cooling is required when freezing at -20 °C to prevent ice crystals from damaging the structure. Cut the obtained second gel into particles with a diameter of 2 mm (to avoid internal diffusion limitation) and store them in 4 °C PBS, which is the lipase composite carrier.

[0039] The second gel was soaked in pH 7.0 PBS for 24 h, and its swelling ratio was detected to be 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 invention used the 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 Xinyanhui Pharmaceutical R & D Co., Ltd.) and vinyl isobutyrate to synthesize imiquimod. In addition, immobilized lipase, immobilized carboxylesterase, and immobilized carboxylester synthase were used as controls. Among them, immobilized lipase, immobilized carboxylesterase, and immobilized carboxylester synthase can be prepared with reference to "Immobilization of Aspergillus oryzae lipase by ZIF-8 and its application in biodiesel production, New Energy Progress, Vol. 6, 2024, Lu Zhenzhen, et al.". In addition, the present invention also synthesized imiquimod by a chemical route 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 tert-butanol. After stirring and reacting 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 to recover the immobilized enzyme. The filtrate was concentrated under reduced pressure by a rotary evaporator to 1 / 5 of the original volume, 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 concentrated solution, and it was adjusted to pH 7.0 with 5% NaOH solution, and then the product was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness, and the residue was crystallized by gradient cooling 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]quinolin-1-yl)-2-methylpropan-1-ol, 3.0 mmol of vinyl isobutyrate, and 0.5 g of the lipase composite support prepared in the above example were blended in 50 mL of tert-butanol. After stirring and reacting at 50 °C for 36 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 to recover the immobilized enzyme. The filtrate was concentrated under reduced pressure using a rotary evaporator to 1 / 5 of the original volume, 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 concentrated solution, and it was adjusted to pH 7.0 with 5% NaOH solution. Subsequently, the product was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness, and the residue was crystallized by gradient cooling with ethanol-water (volume ratio 1:2) to obtain white solid imiquimod.

[0047] Example 3:

[0048] 1.0 mmol of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, 3.5 mmol of vinyl isobutyrate, and 0.45 g of the lipase composite support prepared in the above example were blended in 50 mL of tert-butanol. After stirring and reacting at 50 °C for 48 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 to recover the immobilized enzyme. The filtrate was concentrated under reduced pressure using a rotary evaporator to 1 / 5 of the original volume, 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 concentrated solution, and it was adjusted to pH 7.0 with 5% NaOH solution. Subsequently, the product was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness, and the residue was crystallized by gradient cooling with ethanol-water (volume ratio 1:2) to obtain white solid imiquimod. Comparative Example 1:

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

[0050] The crude mesylate was added to 50 mL of tetrahydrofuran (THF), and 2.0 mmol of methylmagnesium bromide (MeMgBr) was added dropwise under an ice bath. The temperature was raised to reflux (66 °C) and the reaction was carried out for 12 h. The resulting reaction solution was quenched with saturated ammonium chloride solution. The THF phase was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined and dried to obtain the crude product.

[0051] The crude product was separated by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3:1 → 1:1). The target fractions were collected and concentrated to obtain a yellow oil. The oil was dissolved in ether-n-hexane (volume ratio 1:5), and crystals were precipitated under an ice bath. The white solid of imiquimod was obtained by filtration.

[0052] Comparative Example 2:

[0053] 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 esterase (enzyme loading 80 mg / g) were blended in 50 mL of methyl tert-butyl ether. After stirring and reacting at 45 °C for 36 h, 0.1 M hydrochloric acid was added to adjust the pH to 4.3, and the temperature of the system was raised 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 under reduced pressure using a rotary evaporator to 1 / 5 of the original volume, 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 concentrated solution, and it was adjusted to pH 7.0 with 5% NaOH solution. Subsequently, the product was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried with anhydrous sodium sulfate, concentrated to dryness, and the residue was crystallized by gradient cooling with ethanol-water (volume ratio 1:2) to obtain the white solid imiquimod.

[0054] Comparative Example 3:

[0055] 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, and 0.42 g of immobilized carboxylic ester synthetase (enzyme loading 80 mg / g) were blended in 50 mL of phosphate buffer at pH 7.0. After stirring the reaction at 40 °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 to recover the immobilized enzyme. The filtrate was concentrated under reduced pressure to 1 / 5 of the original volume by a rotary evaporator, 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 concentrated solution, and the pH was adjusted to 7.0 with 5% NaOH solution. Subsequently, the product was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness, and the residue was crystallized by gradient cooling with ethanol-water (volume ratio 1:2) to obtain white solid imiquimod.

[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, and 0.42 g of immobilized lipase (enzyme loading 80 mg / g) were blended in 50 mL of tert-butanol. After stirring the reaction 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 to recover the immobilized enzyme. The filtrate was concentrated under reduced pressure to 1 / 5 of the original volume by a rotary evaporator, 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 concentrated solution, and the pH was adjusted to 7.0 with 5% NaOH solution. Subsequently, the product was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated to dryness, and the residue was crystallized by gradient cooling with ethanol-water (volume ratio 1:2) to obtain white solid imiquimod.

[0058] III. Index Detection

[0059] 1. Detection of reaction conversion rate

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

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

[0062] Take 10 mg of the recovered 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 × reaction volume × dilution factor) / (ε × enzyme mass). ε (molar extinction coefficient of p-NP) = 18,300 M -1 cm -1 . Activity retention rate (%) = (recovered enzyme activity / initial enzyme activity) × 100%

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

[0064] The purity of the final product was detected by HPLC method. Chromatographic column: ZORBAX SB-C18 (4.6 × 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 products obtained in Examples 1–3 and Comparative Examples 1–3 respectively, dissolve them in acetonitrile and make up the volume to 10 mL, filter through a 0.22 μm filter membrane and inject the sample, and calculate the purity by area normalization method. The protein residue in the product was detected by BCA protein quantification method.

[0065] 4. Calculation of product yield

[0066] The yields of imiquimod in each example and comparative example were calculated by the following formula. In the purification steps of each example and comparative example, ethyl acetate was extracted 3 times, and the recovery rate was calculated as >98%, and the mother liquor in the crystallization process showed that the residual product was <1% by HPLC.

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

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

[0069] 5. Structure confirmation ( 1 1HNMR)

[0070] 1HNMR (400 MHz, DMSO-d6) showed multiple peaks at δ 8.2 - 8.5 ppm, which were the hydrogen atoms of the imidazole ring. A septet at δ 2.6 ppm was the hydrogen atom of the isobutyryl group. A broad peak at δ 6.8 ppm disappeared after D2O exchange, which was the amino hydrogen atom.

[0071] 6. Detection of N-acylated by-products

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

[0073] 7. Detection of sulfonylated isomers

[0074] The content of sulfonylated isomers in the final product was detected by HPLC. Chiral column (Chiralpak AD-H, Daicel Chiral Technologies Japan 19325, 250×4.6 mm, 5 μm). Mobile phase: n-hexane - isopropanol (85:15), elution flow rate of 1.0 mL / min, detection wavelength: 230 nm, for separating sulfonylated isomers (R / S configuration). 10 mg of the product was dissolved in 1 mL of a mixed solvent of isopropanol - n-hexane (1:1), sonicated for dissolution and then filtered through a 0.22 μm organic filter membrane. The mobile phase was used as the 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 sulfonylated isomer (R configuration) was 14.6 min, and the retention time of the sulfonylated isomer (S configuration) was 13.5 min.

[0075] 8. Residual organic solvents

[0076] The residual amount of organic solvents in the final product was detected by GC-FID. Chromatographic column: HP-5 (30 m×0.32 mm×0.25 μm), programmed temperature rise: 40°C (2 min) → 10°C / min → 200°C (5 min), injection port temperature: 250°C, detector temperature: 300°C. Standard curve: 0.1 - 100 ppm tert-butanol, dichloromethane or ethyl acetate, R 2 ≥0.999.

[0077] 9. COD detection of wastewater

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

[0079] IV. Reaction selectivity and by-product comparison

[0080] Table 1

[0081]

[0082] As shown in Table 1, the N-acylation by-products in Examples 1-3 are between 0.2–0.8%, mainly due to the slight non-specific acylation of the lipase to the primary amine group. However, the substrate-directed binding can be enhanced by the lipase composite support through the pore size confinement effect to inhibit side reactions. In addition, the products in Examples 1-3 contain trace hydrolysis products (≤0.1%), mainly due to the trace hydrolysis of the acyl donor. Through optimization, it is found that controlling the reaction time to 4 h can avoid excessive hydrolysis.

[0083] In Table 1, the sulfonylation isomer of Comparative Example 1 is 4.2%, mainly due to the excessive sulfonylation and isomerization of the amino group by methanesulfonyl chloride (MsCl). It can be seen that the chemical synthesis of imiquimod in Comparative Example 1 lacks regioselectivity, resulting in hydroxyacetylation. The catalytic product of the carboxylesterase provided in Comparative Example 2 contains 2.5% of hydrolysis products, and these hydrolysis products may be the hydrolysis of the acyl donor catalyzed by the esterase. The catalytic product of the ATP-dependent enzyme provided in Comparative Example 3 contains 6.0% of hydrolysis products, and these hydrolysis products may be the hydrolysis of ATP to generate ADP / AMP and free phosphate. The lipase immobilized by the common method provided in Comparative Example 4 for the catalytic synthesis of imiquimod may have carrier eluates.

[0084] Thus, Examples 1-3 respectively use the lipase composite support to catalyze 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, which can not only synthesize imiquimod in one step, but also has ultra-high selectivity (99.1–99.8%). The rigid pore channels of the lipase composite support limit the freedom of the substrate, enabling the active center of the lipase to precisely match the primary amine group and avoid O-acylation and sulfonylation side reactions. In addition, by optimizing the substrate molar ratio (amine: acyl donor = 3.5:1 in Example 3), the non-specific reaction caused by the excess acyl donor can be inhibited.

[0085] The imiquimod prepared by the methods provided in Examples 1-3 has few by-product types and extremely low contents. For product purification, for example, only trace N-acylation by-products (0.2–0.8%) need to be treated, without the complex purification steps such as multi-step column chromatography in Comparative Example 1.

[0086] V. Comparison of Product Yield and Enzyme Efficiency

[0087] Table 2

[0088] Group Product Yield (%) Enzyme Activity Retention Rate (%) Number of Times Enzyme Can Be Reused (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 Detachment) 6

[0089] As shown in Table 2, the product yields of the methods provided in Examples 1-3 are between 93.8% and 97.5%. The lipase composite carrier improves the substrate binding efficiency through the confinement effect of rigid pores, and there is no loss in the multi-step chemical reaction (for example, the yield in Comparative Example 1 is only 72.3%). In Example 3, the substrate molar ratio (3.5:1) and temperature (35 °C) act synergistically to inhibit side reactions, and the yield is close to the theoretical limit. For the chemical method provided in Comparative Example 1, the low yield (72.3%) is due to the cumulative loss in the multi-step reaction (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 number of times the lipase composite carriers provided in Examples 1-3 can be reused is significantly higher than that in the comparative examples. This benefits from the enhanced mechanical strength of the lipase composite carrier, with an enzyme shedding rate <5%, and the structure is more stable. The enzyme activity retention rates of Examples 1-3 are all significantly higher than those of the comparative examples. The internal pores of the lipase composite carrier can protect enzyme molecules from denaturation caused by organic solvents (tert-butanol) (the enzyme activity retention rate in Comparative Example 2 is only 65%).

[0091] VI. Comparison of the amounts of three wastes and environmental friendliness

[0092] Table 3

[0093]

[0094] In Table 3, the wastewater COD of Examples 1-3 is between 780–920 mg / L, only 1 / 5–1 / 4 of the chemical method (Comparative Example 1). Because there are no sulfonylation reagents (MsCl) and ATP decomposition products, the wastewater can be directly treated by biochemical methods. The amount of waste residue of Examples 1-3 is between 0.9 and 1.5 kg / ton of product. The lipase composite carrier can be recycled and reused. The waste residue is mainly trace unreacted substrates and does not require hazardous waste treatment. In Examples 1-3, there are no toxic by-products, the selectivity of the enzymatic reaction >99%, and no polluting ions such as Cl 3- and PO4 are produced, meeting the REACH regulations and the principles of green chemistry.

[0095] The high COD (4200 mg / L) of Comparative Example 1 (chemical method) is due to highly toxic organic substances such as dichloromethane and sulfonylation by-products, which require special incineration treatment, increasing the cost by 5 times. The hazardous waste residue of Comparative Example 1 is as high as 8.7 kg / ton, containing Cl- produced by the decomposition of methanesulfonyl chloride, and needs to be treated according to the hazardous waste code HW06, with a cost of up to $650 / ton of product.

[0096] For the method provided in Comparative Example 3, there may be phosphate pollution (PO4 3- ). Phosphate is produced by the decomposition of ATP (PO4 in the wastewater 3->500 ppm), chemical precipitation treatment is required, increasing additional costs. Additionally, in Comparative Example 3, the energy consumption of the ATP regeneration system is 3 times that of the Example (about 120 kWh / ton of product), and the carbon emissions increase by 200%.

[0097] For the method provided in Comparative Example 4, due to the presence of carrier leachates, the carrier leaching in the organic solvent results in a higher amount of waste residue (2.5 kg / ton) than in the Example, and the residue needs to be separately filtered and treated.

[0098] Based on the above analyses, the comprehensive environmental protection scores of Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 are shown in Table 4. The more “+” there are, the higher the score.

[0099] Table 4

[0100] Group Difficulty of Wastewater Treatment Hazard of Waste Residue Energy Consumption (KWH / ton) Comprehensive Environmental Protection Rating Examples 1 - 3 Low (Direct Biochemical) None 40–50 +++++ Comparative Example 1 Extremely High (Requires Incineration) High (Contains Cl-) 80 (Multi-step Reaction) + Comparative Example 3 High (Chemical Precipitation) <![CDATA[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 protection evaluation levels of Examples 1–3 are relatively high. The reduction of wastewater / waste residue is 90%. Through the highly selective reaction of enzymes and the recycling of carriers, the amount of three wastes is close to the “zero emission” standard. There is no burden of hazardous waste treatment for Examples 1–3, and there is no need to treat toxic ions such as Cl−, PO4 3- etc. The comprehensive environmental protection cost is only 1 / 6–1 / 5 of that of the chemical method. The energy consumption per unit product (40–50 kWh) is reduced by 60–70% compared with the chemical method and the ATP enzyme method, meeting the carbon neutrality goal.

[0102] The chemical method (Comparative Example 1) has the lowest environmental protection 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] VII. Comparison of Comprehensive Costs

[0104] Table 5 (USD / ton)

[0105]

[0106] As can be seen from Table 5, the raw material costs in the methods of Examples 1–3 are low (2,600–2,950 USD). It is completed in one step through an enzymatic reaction without the need for a sulfonylation reagent (MsCl, an additional 1,200 USD / ton in Comparative Example 1). Additionally, the lipase composite carrier provided in the Example can be reused multiple times, reducing the unit catalyst cost by 75% (compared with the free enzyme used once in Comparative Example 2). Additionally, the waste treatment cost of the methods of Examples 1–3 (95–150 USD) has a low wastewater COD (780–920 mg / L), and the biochemical treatment cost is only 15% of that of the chemical method (Comparative Example 1).

[0107] The high cost source of Comparative Example 1 (chemical method) is that the price of methanesulfonyl chloride (MsCl) is as high as $2,500 per ton, and it needs to be used in excess (1.5 eq). The three-step reactions of sulfonylation, deprotection, and acylation are all energy-consuming reactions, and the total energy consumption increases by 55% (compared with Example 3). The treatment cost of Cl−-containing waste residue is $650, accounting for 11% of the total cost.

[0108] For the method of Comparative Example 3, the energy consumption of the ATP regeneration system is $880 per ton, and the ATP consumption reaches 1.2 eq (cost $680), pushing up the total cost to $6,180. PO4 in the wastewater 3- needs chemical precipitation, and the treatment cost is 148% higher than that of Comparative Example 4.

[0109] For the method of Comparative Example 4, the enzyme can only be reused 6 times, and the catalyst cost increases by 108% (compared with Example 3).

[0110] In summary, the lipase immobilized by the lipase composite carrier in the present invention is significantly superior to the traditional chemical method and other enzyme methods in terms of core indexes such as product yield, enzyme stability, catalytic efficiency, and cost control: the one-step enzymatic reaction synthesis in the present invention avoids multi-step chemical synthesis, and the yield is increased by more than 25%. The lipase composite carrier provided by the present invention can protect the enzyme activity, and still maintains >80% activity after being reused 15 times, greatly reducing the catalyst cost. Using the lipase composite carrier of the present invention to catalyze the synthesis of imiquimod from 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, there are no toxic reagents (MsCl, ATP), and the COD value of the wastewater is low, meeting the environmental protection requirements of the FDA and EMA for the production of active pharmaceutical ingredients.

[0111] The process for synthesizing imiquimod by the lipase method provided by the present invention shows significant comprehensive advantages: in terms of selectivity and efficiency, the primary hydroxyl group of lipase specifically and precisely avoids N-acylation side reactions, achieving a product yield of more than 90% and a purity of 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 that of the chemical enzyme method (CE), and there is no need to rely on a high-cost ATP regeneration system (such as the CES method), greatly reducing the catalytic cost; its environmental protection attributes are also prominent, using a low-toxic tert-butanol solvent (recovery rate ≥98%), and the emissions of the three wastes are reduced by 86% compared with the chemical process, meeting the requirements of the development of green chemistry; this process also has a clear industrialization prospect, with mild reaction conditions (50 °C, pH 4.3), no column chromatography purification step, and a continuous flow production mode with low equipment requirements, providing an efficient and sustainable technical path for large-scale application.

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

[0113] As described above, only the specific preferred embodiments of the present invention are provided, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. Preparation method of a catalyst for synthesizing imiquimod, characterized in that, 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 ZIF-8 pre-immobilized lipase; Preparing a second precursor solution containing polyvinyl alcohol, acrylamide and sodium chloride, preparing an ammonium persulfate solution, and preparing a tetramethylethylenediamine solution; Ultrasonically dispersing the ZIF-8 pre-immobilized lipase in the second precursor solution, adding the ammonium persulfate solution, and purging with nitrogen to remove oxygen; Adding the tetramethylethylenediamine solution and pouring it into a mold for polymerization; Subjecting the first gel obtained by the polymerization to a freeze-thaw treatment to obtain a second gel, and cutting the second gel into particles, which is the catalyst.

2. The preparation method according to claim 1, wherein 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.

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

4.

4. The preparation method according to claim 1, characterized in that, The mixing and reaction time of the lipase solution and the precursor solution is 1 - 10 h.

5. The preparation method according to claim 1, characterized in that, In the second precursor solution, the concentration of polyvinyl alcohol is 1 - 15% w / v, acrylamide is 1 - 15% w / v, sodium chloride is 1 - 5% w / v, the concentration of the ammonium persulfate solution is 1 - 3% w / v, and the concentration of the tetramethylethylenediamine solution is 0.1 - 0.4% w / v.

6. The preparation method according to claim 1, characterized in that, The ZIF-8 pre-immobilized lipase is dispersed in the solution containing polyvinyl alcohol and acrylamide at a concentration of 5 - 12% w / v.

7. The catalyst for synthesizing imiquimod obtained by the preparation method according to any one of claims 1 - 6.

8. A method for synthesizing imiquimod, characterized in that, Comprising: Blending 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol, vinyl isobutyrate, and the catalyst obtained by the method according to any one of claims 1 - 6 in tert-butanol, reacting with stirring at 45 - 55 °C; Adjusting the pH to 4.0 - 5.0 with hydrochloric acid and controlling the temperature at 55 - 65 °C for hydrolysis; Obtaining the imiquimod from the reaction solution.

9. The method according to claim 8, characterized in that, The molar ratio of the input of 3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-1-ol to vinyl isobutyrate is 1:3, and the input 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.

10. Use of the catalyst obtained by the method according to any one of claims 1 - 6 in the synthesis of imiquimod.

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