Synthesis method of 16-hydroxycapsaicin and its marker

Abstract

The present invention provides a method for synthesizing 16-hydroxycapsaicin and isotope-labeled substances thereof. Compound 4 and compound d3-3 or d3-3 are used as raw materials to undergo olefin metathesis reaction to efficiently synthesize a target compound. The method is simple and easy to operate. The structural formula of compound 4 is: The structural formula of compound 3 is: The structural formula of compound d3-3 is: In addition, the present invention also provides a method for synthesizing 16-hydroxycapsaicin and isotope-labeled substances thereof. Starting from 6-n-heptenoic acid, the target molecule is efficiently synthesized through amidation reaction and olefin metathesis reaction. The entire synthetic route is reasonably designed, the raw materials are simple and easy to obtain, the operation is simple, the operability is strong, the purification is easy, and the obtained product has high purity. The synthesized target molecule provides a test sample for in vivo pharmacokinetic research of capsaicin and metabolite biological activity research, and has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of chemical synthesis, and in particular to a method for synthesizing 16-hydroxycapsaicin and a marker thereof. Background Art

[0002] Capsaicin, also known as trans-8-methyl-N-vanillyl-6-nonenamide, is the most important spicy ingredient in chili peppers and is widely used in pepper sprays and crop protection agents. As an active pharmaceutical ingredient, capsaicin is often used to make topical preparations to relieve pain. In addition, capsaicin has been found to have antioxidant, anti-cancer and anti-inflammatory activities, and is in Phase III clinical trials as a drug for the treatment of arthritis, postoperative pain, acute and chronic neuropathy, and musculoskeletal pain.

[0003] In the body, drugs are usually biotransformed through phase I and phase II metabolic pathways. Since phase I reactions mainly involve oxidation, reduction and hydrolysis, the metabolites produced may have pharmacological activity, so further safety evaluation is needed. The phase I metabolism of capsaicin in the body is mainly mediated by P450 enzymes. Its phase I metabolic process mainly occurs on alkyl chains, aromatic rings and amides, generating capsaicin hydroxylation, demethylation, and dehydrogenation metabolites, among which macrocyclic metabolites (M1), ω-(16-hydroxycapsaicin, M2) and ω-1 (17-hydroxycapsaicin, M3) alcohols and terminal dehydrogenated dienes (16, 17-dehydrocapsaicin, M4) account for 80%. Further research on the pharmacokinetics of capsaicin in vivo and the pharmacological activities of its main metabolites will undoubtedly help to more fully understand the various effects of capsaicin in the body. However, the above research is currently greatly limited due to the lack of sufficient capsaicin metabolite standards and effective chemical synthesis methods. Summary of the invention

[0004] In view of this, the present invention provides a method for synthesizing 16-hydroxycapsaicin and a marker thereof.

[0005] Specifically, the synthesis method of 16-hydroxy capsaicin provided by the present invention comprises using compound 3 and compound 4 as raw materials to undergo olefin metathesis reaction to synthesize 16-hydroxy capsaicin, wherein the structural formula of the compound 3 is The structural formula of compound 4 is The structural formula of 16-hydroxycapsaicin is: The reaction synthesis route is:

[0006] Based on the above synthesis method, it includes: in a nitrogen atmosphere and under the action of Grubbs second-generation catalyst, the compound 3 and the compound 4 undergo olefin metathesis at 25°C to 50°C to synthesize 16-hydroxycapsaicin. Preferably, the molar ratio of the compound 3, the Grubbs second-generation catalyst and the compound 4 is 1: (0.01 to 0.05): (2.0 to 4.0).

[0007] The method for synthesizing 16-hydroxycapsaicin by olefin metathesis reaction specifically includes: in a nitrogen atmosphere and under the action of Grubbs second-generation catalyst, the compound 3 and the compound 4 are refluxed at 40° C. to 50° C. until the compound 3 reacts completely to synthesize 16-hydroxycapsaicin.

[0008] Based on the above synthesis method, it comprises the following steps: after the olefin metathesis occurs to synthesize 16-hydroxy capsaicin, the 16-hydroxy capsaicin is purified by vacuum concentration and column chromatography to obtain pure 16-hydroxy capsaicin.

[0009] Based on the above synthesis method, the synthesis method of the compound 3 includes: using 6-n-heptenoic acid and compound 2 as raw materials to undergo amidation reaction to synthesize the compound 3, wherein the structural formula of the compound 2 is The reaction synthesis route is:

[0010] Based on the above synthesis method, the synthesis method of the compound 3 includes: under the action of diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 6-n-heptenoic acid and compound 2 undergo amidation reaction at 0°C to 30°C to synthesize the compound 3. Preferably, the molar ratio of 6-n-heptenoic acid, diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and compound 2 is 1:(2.0-3.0):(1.0-1.5):(1.0-1.5).

[0011] The synthesis method of the compound 3 specifically includes: in an N,N-dimethylformamide solution, firstly, uniformly stirring 6-n-heptenoic acid, diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate at -5°C to 5°C; then, adding compound 2 in batches to undergo an amidation reaction at room temperature to synthesize the compound 3; then, quenching the reaction with aqueous hydrochloric acid, extracting with ethyl acetate, washing the organic phase with water and brine, drying with sodium sulfate and concentrating under reduced pressure, and purifying the crude product by column chromatography to obtain pure compound 3.

[0012] The present invention also provides a method for synthesizing a 16-hydroxy capsaicin marker, comprising the steps of: using compound d3-3 and compound 4 as raw materials to undergo olefin metathesis reaction to synthesize d3-16-hydroxy capsaicin, wherein the structural formula of the compound d3-3 is The structural formula of the compound 4 is: The structural formula of d3-16-hydroxycapsaicin is:

[0013] Based on the above synthesis method, it includes: in a nitrogen atmosphere and under the action of a Grubbs second-generation catalyst, the compound d3-3 and the compound 4 undergo olefin metathesis at 25°C to 50°C to synthesize d3-16-hydroxycapsaicin, and the molar ratio of the compound d3-3, the Grubbs second-generation catalyst and the compound 4 is 1: (0.01 to 0.05): (2.0 to 4.0).

[0014] Based on the above synthesis method, it includes preparing d3-pure 16-hydroxy capsaicin by vacuum concentration and column chromatography purification after the olefin metathesis occurs to synthesize d3-16-hydroxy capsaicin.

[0015] Based on the above synthesis method, the synthesis method of the compound d3-3 comprises: using 6-n-heptenoic acid and compound d3-2 as raw materials to undergo an amidation reaction to synthesize the compound d3-3, wherein the structural formula of the compound d3-2 is:

[0016] Based on the above synthesis method, the synthesis method of the compound d3-3 includes: under the action of diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 6-n-heptenoic acid and compound d3-2 undergo amidation reaction at 0°C to 30°C to synthesize the compound 3; and the molar ratio of 6-n-heptenoic acid, diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and compound d3-2 is 1:(2.0~3.0):(1.0~1.5):(1.0~1.5).

[0017] Therefore, the synthesis method of the above-mentioned 16-hydroxy capsaicin and its isotope-labeled substance provided by the present invention can efficiently synthesize the target compound by using compound 4 and compound d3-3 or d3-3 as raw materials to undergo olefin metathesis reaction, and the method is simple and easy to operate. In addition, the present invention also provides a synthesis method of 16-hydroxy capsaicin and its isotope-labeled substance starting from 6-n-heptenoic acid, and efficiently realizes the synthesis of the target molecule through amidation reaction and olefin metathesis reaction. The whole synthesis route is reasonably designed, the raw materials are simple and easy to obtain, the operation is simple, the operability is strong, the purification is easy, and the purity of the obtained target product is high. The product purity reaches more than 95%, which meets the purity requirements of the capsaicin metabolite standard product, and provides a test sample for the in vivo metabolic mechanism study of capsaicin, which has important application value. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0019] Example 1

[0020] This embodiment provides a method for synthesizing 16-hydroxy capsaicin, comprising the following steps: under nitrogen protection, 6.60g of compound 3 and 0.53g of Grubbs second-generation catalyst are added to a dry three-necked flask, and then 125mL of anhydrous dichloromethane containing 6.50g of compound 4 is added by syringe. The reaction system is heated to reflux (removing byproduct ethylene) for 12 hours under a continuous nitrogen flow, and the temperature is 45°C; after compound III is completely consumed, the reaction mixture is vacuum concentrated, and the crude product is purified by a chromatographic column to obtain 5.30g of 16-hydroxy capsaicin, with a yield of 66% and a purity of about 98.5%; the synthetic route of this step is as follows:

[0021] Among them, 16-hydroxycapsaicin 1 H NMR (400MHz, CDCl3) δ6.86(d,J=8.0Hz,1H),6.80(d,J=1.9Hz,1H),6.76(dd,J=8.0,1.9Hz,1H),5.84–5.71(m, 2H),5.62-5.54(m,2H),4.35(d,J=5.6Hz,2H),3.87(s,3H),2.20(t,J=7.5Hz,2H),2.06-2.01(m,2H),1.77(br s, 1H), 1.66 (p, J = 7.6Hz, 2H), 1.40 (p, J = 7.5Hz, 2H), 1.29 (s, 6H).

[0022] Example 2

[0023] This embodiment provides a method for synthesizing 16-hydroxycapsaicin, which is basically the same as the method provided in Example 1, except that, in a nitrogen atmosphere and under the action of 0.42 g of Grubbs second-generation catalyst, 5.28 g of compound 3 and 5.20 g of compound 4 undergo olefin metathesis reaction at 40° C. to synthesize 4.69 g of 16-hydroxycapsaicin, with a yield of 70% and a purity of about 95.9%.

[0024] Example 3

[0025] This embodiment provides a method for synthesizing 16-hydroxycapsaicin, which is basically the same as the method provided in Example 1, except that, in a nitrogen atmosphere and under the action of 0.32 g of Grubbs second-generation catalyst, 3.95 g of compound 3 and 3.90 g of compound 4 undergo olefin metathesis reaction at 50° C. to synthesize 3.2 g of 16-hydroxycapsaicin, with a yield of 64% and a purity of about 96.7%.

[0026] Example 4

[0027] The present embodiment provides a method for synthesizing 16-hydroxycapsaicin, which starts from 6-n-heptenoic acid and synthesizes the target compound through amidation reaction and olefin metathesis reaction. The main difference between the synthesis method provided in this embodiment and that provided in Example 1 is that compound 3 in this embodiment is synthesized by amidation reaction of 6-n-heptenoic acid and compound 2. The specific synthesis method of compound 3 in this embodiment is as follows: 3.80g of 6-n-heptenoic acid is dissolved in 60mL of N,N-dimethylformamide, 9.70g of diisopropylethylamine (DIPEA) and 13.7g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) are added at 0°C, the reaction system is stirred at 0°C for 30 minutes, and then 6.80g of compound 2 is added in batches, and stirring is continued at room temperature for 2 hours; the reaction is quenched with 0.5M hydrochloric acid solution, extracted with ethyl acetate, the organic phase is dried with sodium sulfate and concentrated under reduced pressure, and the crude product is purified by chromatographic column to obtain 6.70g of compound 3 with a yield of 85%. Compound 3 1 H NMR (400MHz, CDCl3) δ 6.87-6.74 (m, 3H), 5.83-5.73 (m, 3H), 5.02-4.92 (m, 2H), 4.35 (d, J = 5.7 Hz, 2H), 3.87 (s, 3H), 2.21 (t, J = 7.6 Hz, 2H), 2.09-2.03 (m, 2H), 1.71-1.62 (m, 2H), 1.46-1.38 (m, 2H) ppm. The synthetic route of this step is as follows:

[0028] Example 5

[0029] This example provides a method for synthesizing 16-hydroxycapsaicin, which is basically the same as the method provided in Example 4, except that the synthesis parameters of compound 3 are different. Specifically, in this example, under the action of 12.9 g DIPEA and 18.35 g HBTU, 5.10 g 6-n-heptenoic acid and 9.12 g compound 2 undergo amidation reaction at room temperature to synthesize 8.68 g of compound 3, with a yield of 83%.

[0030] Example 6

[0031] This example provides a method for synthesizing a 16-hydroxycapsaicin marker, which is basically the same as the synthesis method provided in Example 1, except that this step mainly uses 6.68g of compound d3-3 and 6.50g of compound 4 to undergo olefin metathesis reaction to synthesize 5.35g of 16-hydroxycapsaicin isotope marker d3-16-hydroxycapsaicin, with a yield of 63% and a purity of about 98.2%. The synthesis route of this step is as follows:

[0032] Among them, d3-16-hydroxycapsaicin 1 H NMR (400MHz, CDCl3) δ6.86(d,J=8.0Hz,1H),6.80(d,J=1.9Hz,1H),6.76(dd,J=8.0,2.0Hz,1H),6.15–6.10(m,1H),5.72(br s,2H),5.66–5.58(m,1H),4.86(s,2H),4.35(d,J=5.6Hz,2H),2.21(t,J=7.6Hz ,2H),2.15–2.09(m,2H),1.82(s,3H),1.73–1.64(m,2H),1.48–1.41(m,2H)ppm.

[0033] Example 7

[0034] The present embodiment provides a method for synthesizing a 16-hydroxycapsaicin marker, wherein the target compound is synthesized from 6-n-heptenoic acid and compound d3-2 through an amidation reaction and an olefin metathesis reaction. The main difference between the present embodiment and the synthesis method provided in Example 1 is that the compound d3-3 in the present embodiment is synthesized by an amidation reaction between 6-n-heptenoic acid and compound d3-2, which is basically the same as the method for synthesizing compound 3 provided in Example 4, the main difference being that the synthesis parameter conditions of compound d3-3 are different. Specifically, in the present embodiment, under the action of 6.45g DIPEA and 9.18g HBTU, 2.55g 6-n-heptenoic acid and 4.62g compound d3-2 undergo an amidation reaction at room temperature to synthesize 4.63g of the compound d3-3, with a yield of 88%. The synthetic route of this step is:

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A method for synthesizing 16-hydroxycapsaicin, comprising using compound 3 and compound 4 as raw materials to undergo olefin metathesis reaction to synthesize 16-hydroxycapsaicin, wherein: The structural formula of the compound 3 is The structural formula of compound 4 is 2. The synthesis method according to claim 1, characterized in that It includes: In a nitrogen atmosphere and under the action of Grubbs second-generation catalyst, the compound 3 and the compound 4 undergo olefin metathesis at 25° C. to 50° C. to synthesize 16-hydroxycapsaicin.

3. The synthesis method according to claim 2, characterized in that The method comprises the following steps: after the olefin metathesis occurs to synthesize 16-hydroxy capsaicin, vacuum concentration and column chromatography purification are performed to obtain pure 16-hydroxy capsaicin.

4. The synthesis method according to claim 1, 2 or 3, characterized in that: The synthesis method of the compound 3 comprises: using 6-n-heptenoic acid and compound 2 as raw materials to undergo amidation reaction to synthesize the compound 3, wherein the structural formula of the compound 2 is 5. The synthesis method according to claim 4, characterized in that The synthesis method of the compound 3 comprises: under the action of diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 6-n-heptenoic acid and compound 2 undergo amidation reaction at 0° C. to 30° C. to synthesize the compound 3.

6. A method for synthesizing a 16-hydroxycapsaicin marker, comprising the steps of: using compound d3-3 and compound 4 as raw materials to undergo olefin metathesis reaction to synthesize d3-16-hydroxycapsaicin, wherein: The structural formula of the compound d3-3 is The structural formula of the compound 4 is: The structural formula of d3-16-hydroxycapsaicin is 7. The synthesis method according to claim 6, characterized in that It includes: In a nitrogen atmosphere and under the action of Grubbs second-generation catalyst, the compound d3-3 and compound 4 undergo olefin metathesis at 25°C to 50°C to synthesize d3-16-hydroxycapsaicin, and the molar ratio of the compound d3-3, Grubbs second-generation catalyst and compound 4 is 1:(0.01~0.05):(2.0~4.0).

8. The synthesis method according to claim 7, characterized in that The method comprises the following steps: after the olefin metathesis occurs to synthesize d3-16-hydroxy capsaicin, vacuum concentration and column chromatography purification are performed to obtain d3-pure 16-hydroxy capsaicin.

9. The synthesis method according to claim 6, 7 or 8, characterized in that: The synthesis method of the compound d3-3 comprises: using 6-n-heptenoic acid and compound d3-2 as raw materials to undergo amidation reaction to synthesize the compound d3-3, wherein the structural formula of the compound d3-2 is 10. The synthesis method according to claim 9, characterized in that The synthesis method of the compound d3-3 includes: under the action of diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 6-n-heptenoic acid and compound d3-2 undergo amidation reaction at 0°C to 30°C to synthesize the compound 3; and the molar ratio of 6-n-heptenoic acid, diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and compound d3-2 is 1:(2.0~3.0):(1.0~1.5):(1.0~1.5).

Images