A trpv1 agonist and a method of preparing the same

Abstract

The application belongs to the field of pharmaceutical chemistry, and specifically discloses a TRPV1 agonist and a preparation method thereof. The structural feature of the TRPV1 agonist is that a OH or F substituent is introduced at the 3-position of a benzene ring of capsaicin, and the TRPV1 agonist is prepared by reacting vanillylamine hydrochloride and an acyl chloride compound with OH or F at the 3-position of the benzene ring. The reaction is carried out in a two-phase system composed of an aqueous solution of an inorganic base and an organic solvent at room temperature. The melting point of the TRPV1 agonist compound provided by the application is obviously improved, which is of great significance for developing capsaicin micro-powder type medicaments. The preparation method of the capsaicin derivative provided by the application uses a two-phase system composed of an aqueous solution of an inorganic base and an organic solvent for reaction, avoids the use of an organic base, and improves the selectivity of the acylation reaction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a TRPV1 agonist and a preparation method thereof. BACKGROUND

[0002] The transient receptor potential (TRP) channel is a kind of non-selective cation channel with diverse structures and functions, which plays an important role in the transmission of sensory information such as vision, temperature sensation, pain sensation, touch sensation, hearing and taste, and the regulation of intracellular Ca 2+ So far, there are 28 members of the TRP channel superfamily found in mammals. According to the homology difference of amino acid sequences, they can be divided into 7 subfamilies: TRPA, TRPC, TRPM, TRPML, TRPN, TRPP and TRPV, wherein TRPV1 is a member of the TRPV subfamily.

[0003] TRPV1 was first found to be a receptor that can be activated by capsaicin, so it is called capsaicin receptor, and it is also called vanilloid receptor 1 (VR1) in the early stage, which is a non-selective and heat-sensitive cation channel. TRPV1 is widely distributed and expressed in various tissues and organs, and it has complex biological functions and participates in various physiological and pathological processes.

[0004] Capsaicin is a vanillyl amide alkaloid compound in natural plant capsicum, which has a pungent odor and is difficult to dissolve in water. Capsaicin is the active ingredient of capsicum and is a representative compound of TRPV1 receptor agonists, and has a wide range of uses. In the medical field, capsaicin has pharmacological effects such as anti-inflammatory analgesia, rheumatism, and prevention of cardiovascular diseases. In addition, due to its strong stimulating characteristics, it can be used as a ship antifouling coating, a cable ant termite and rodent repellent, and a violence control agent.

[0005] The melting point of capsaicin compounds is generally low, and the melting point of capsaicin is 62-65℃, and the melting point of nonanoyl vanillylamine is 57-60℃. When capsaicin is prepared into a micro powder for use as various micro powder type medicaments, the low melting point can easily cause the micro powder to melt and agglomerate due to temperature rise during production, storage and use, thereby seriously affecting the dispersion effect of the micro powder. In addition, capsaicin compounds have strong lipophilicity and poor water solubility, and are difficult to absorb orally, which further limits their application.

[0006] Therefore, it is of great significance to develop micro powder type medicaments to chemically modify and modify capsaicin based on its structure, to improve the melting point of the compound or to improve the water solubility. SUMMARY

[0007] The technical problem solved by the present application is to provide a series of capsaicin derivatives with TRPV1 agonistic activity and a preparation method thereof.

[0008] The technical scheme adopted by the present application is as follows:

[0009] In a first aspect, the present application provides a TRPV1 agonist, a capsaicin derivative having the structure shown in formula 1, 2 and 3 and a pharmaceutically acceptable salt thereof:

[0010]

[0011] wherein R is hydroxyl or fluorine.

[0012] Further, the capsaicin derivative is specifically:

[0013]

[0014] In a second aspect, the present application provides a preparation method of the capsaicin derivative of the first aspect, specifically comprising the following steps:

[0015] The compound shown in formula 4 is dissolved in water, an inorganic base and an organic solvent are added, and then reacted with an acyl chloride compound shown in formula 5, 6 or 7 to generate a compound shown in formula 1, 2 or 3;

[0016]

[0017] wherein R is hydroxyl or fluorine;

[0018] The inorganic base is any one or a mixture of two or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, sodium phosphate, disodium hydrogen phosphate, potassium phosphate or dipotassium hydrogen phosphate;

[0019] The organic solvent is any one of diethyl ether, tert-butyl methyl ether, toluene, dichloromethane, chloroform or ethyl acetate.

[0020] Preferably, the molar ratio of the compound shown in formula 4, the acyl chloride compound shown in formula 5, 6 or 7 and the inorganic base is 1:(1-2.5): (1-1.5).

[0021] Preferably, the volume ratio of water for dissolving the compound shown in formula 4 to the volume of the added organic solvent is 1:1-1:2.

[0022] Preferably, the concentration of the aqueous solution of the compound shown in formula 4 is 0.2-0.8 mol / L.

[0023] Preferably, the reaction condition is at room temperature.

[0024] Preferably, the compound shown in formula 4 is specifically as follows:

[0025]

[0026] Preferably, the purification method of the product after the reaction is as follows: after the reaction is stopped, the organic phase is separated, dried, filtered and evaporated to dryness to obtain a solid crude product; the crude product is recrystallized by PE / EA to obtain a pure product.

[0027] In a third aspect, the application provides an application of the capsaicin derivative of the first aspect in the preparation of a capsaicin micro-powder type medicament.

[0028] Compared with the prior art, the application has the following advantages and beneficial effects:

[0029] The capsaicin derivative provided by the application introduces an OH or F substituent at the 3-position of the benzene ring of capsaicin, so that the melting point of the compound is obviously improved on the basis of maintaining a certain TRPV1 agonistic activity, which is of great significance for developing a capsaicin micro-powder type medicament; the preparation method of the capsaicin derivative provided by the application uses a two-phase system composed of an aqueous solution of an inorganic base and an organic solvent for reaction, avoids the use of an organic base, and improves the selectivity of the acylation reaction. DETAILED DESCRIPTION

[0030] The content of the application will be further described below in combination with examples.

[0031] Example 1

[0032] Synthesis of N-(3,4-dihydroxy-5-methoxybenzyl)nonanamide (compound 1a):

[0033]

[0034] The 3,4-dihydroxy-5-methoxybenzylamine hydrochloride 4a (2.06 g, 10 mmol) was dissolved in 20 mL of water, sodium carbonate (1.17 g, 11 mmol) was added, and after stirring uniformly, 20 mL of dichloromethane was added, then nonanoyl chloride 5 (1.94 g, 11 mmol) was added, and stirring was performed at room temperature for 2 h. The reaction was stopped, the organic phase was separated, dried with anhydrous sodium sulfate, then filtered and evaporated to dryness to obtain a light yellow solid crude product. The crude product can be recrystallized by PE / EA to obtain a pure product of N-(3,4-dihydroxy-5-methoxybenzyl)nonanamide (compound 1a), which is a white solid 2.7 g, with a crystallization yield of 87% and a melting point of 96-97℃. 1H NMR (300 MHz, CDC13) δ 6.50 (s, 1H), 6.39 (s, 1H), 5.79 (br, 1H), 5.57 (br, 1H), 4.30 (d, J = 5.6 Hz, 2H), 3.84 (s, 3H), 2.20 (t, J = 7.6 Hz, 2H), 1.71 - 1.54 (m, 2H), 1.37 - 1.16 (m, 10H), 0.86 (t, J = 6.6 Hz, 3H). 13 C NMR (75 MHz, CDC13) δ 173.28, 147.15, 144.15, 131.99, 129.93, 108.27, 103.03, 56.15, 43.68, 36.82, 31.78, 29.28, 29.12, 25.77, 22.61, 14.06.

[0035] Example 2

[0036] Synthesis of N-(3,4-dihydroxy-5-methoxybenzyl)-8-methylnonanamide (Compound 2a):

[0037]

[0038] N-(3,4-dihydroxy-5-methoxybenzyl)-8-methylnonanamide (Compound 2a) was synthesized by dissolving 3,4-dihydroxy-5-methoxybenzylamine hydrochloride 4a (2.06 g, 10 mmol) in 20 mL of water, adding sodium bicarbonate (1.85 g, 22 mmol), stirring well, then adding 8-methylnonanoyl chloride 6 (2.1 g, 11 mmol), and stirring at room temperature for 3 h. The reaction was stopped, the organic phase was separated, dried using anhydrous sodium sulfate, then filtered and evaporated to dryness to obtain a light yellow solid crude product. The crude product was recrystallized from PE / EA to obtain pure N-(3,4-dihydroxy-5-methoxybenzyl)-8-methylnonanamide (Compound 2a) as a white solid 2.5 g, with a crystallization yield of 77%, and a melting point of 107-109 °C. 1 H NMR (300 MHz, CDC13) δ 6.50 (s, 1H), 6.39 (s, 1H), 5.79 (br, 1H), 5.57 (br, 1H), 4.30 (d, J = 5.6 Hz, 2H), 3.84 (s, 3H), 2.20 (t, J = 7.6 Hz, 2H), 1.71 - 1.54 (m, 2H), 1.37 - 1.16 (m, 10H), 0.86 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDC13) δ 173.23, 147.13, 144.11, 131.94, 129.96, 108.25, 103.00, 56.14, 43.67, 38.91, 36.84, 29.58, 29.33, 27.91, 27.22, 25.78, 22.60.

[0039] Example 3

[0040] Synthesis of (E)-N-(3,4-dihydroxy-5-methoxybenzyl)-8-methyl-6- nonenamide (Compound 3a):

[0041]

[0042] Synthesis of (E)-N-(3,4-dihydroxy-5-methoxybenzyl)-8-methyl-6- nonenamide (Compound 3a): 1 H NMR (300 MHz, CDC13) δ 6.50 (s, 1H), 6.39 (d, J = 1.2 Hz, 1H), 6.13 (br, 1H), 5.78 (br, 1H), 5.59 (br, 1H), 5.42 - 5.22 (m, 2H), 4.31 (d, J = 5.6 Hz, 2H), 3.84 (s, 3H), 2.31 - 2.12 (m, 3H), 1.98 (dd, J = 13.2, 6.9 Hz, 2H), 1.65 (dt, J = 15.3, 7.5 Hz, 2H), 1.44 - 1.29 (m, 2H), 0.94 (d, J = 6.7 Hz, 6H). 13 C NMR (75 MHz, CDC13) δ 173.23, 147.13, 144.11, 131.94, 129.96, 108.25, 103.00, 56.14, 43.67, 38.91, 36.84, 29.58, 29.33, 27.91, 27.22, 25.78, 22.60.

[0043] Example 4

[0044] Synthesis of N-(3-fluoro-4-hydroxy-5-methoxybenzyl)nonanamide (Compound 1b):

[0045]

[0046] The 3-fluoro-4-hydroxy-5-methoxybenzylamine hydrochloride 4b (2.08 g, 10 mmol) was dissolved in 20 mL of water, potassium carbonate (1.52 g, 11 mmol) was added, after stirring well, 30 mL of toluene was added, then nonanoyl chloride 5 (1.94 g, 11 mmol) was added, stirring at room temperature for 2 h. The reaction was stopped, the organic phase was separated, dried with anhydrous sodium sulfate, then filtered and evaporated to dryness to obtain a light yellow solid crude product. The crude product can be recrystallized from PE / EA to obtain pure N-(3-fluoro-4-hydroxy-5-methoxybenzyl)nonanamide (Compound 1b) as a white solid 2.9 g, crystallization yield 90%, melting point 94-95 °C. 1 H NMR (300 MHz, CDC13) δ 6.57 (d, J = 11.7 Hz, 1H), 6.56 (s, 1H), 6.12 (br, 1H), 6.02 (br, 1H), 4.28 (d, J = 5.6 Hz, 2H), 3.82 (s, 3H), 2.19 (t, J = 7.5 Hz, 2H), 1.72 - 1.50 (m, 2H), 1.35 - 1.15 (m, 10H), 0.91 - 0.75 (m, 3H). 13 C NMR (75 MHz, CDC13) δ 173.35, 150.49 (d, J = 241.4 Hz), 148.39 (d, J = 6.1 Hz), 132.91 (d, J = 13.9 Hz), 129.66 (d, J = 7.7 Hz), 108.11 (d, J = 19.1 Hz), 106.15 (d, J = 2.4 Hz), 56.26, 43.02, 36.64, 31.72, 29.24, 29.08, 25.71, 22.55, 13.99.

[0047] Example 5

[0048] Synthesis of N-(3-fluoro-4-hydroxy-5-methoxybenzyl)-8-methylnonanamide (Compound 2b):

[0049]

[0050] To a solution of 3-fluoro-4-hydroxy-5-methoxybenzylamine hydrochloride 4b (2.08 g, 10 mmol) in 20 mL of water was added sodium phosphate (2.46 g, 15 mmol), stirred well and then chloroform 20 mL was added, followed by 8-methylnonanoyl chloride 6 (2.1 g, 11 mmol) and stirred at room temperature for 3 h. The reaction was stopped, the organic phase was separated, dried over anhydrous sodium sulfate and then filtered and evaporated to dryness to give a light yellow solid crude. The crude was recrystallized from PE / EA to give pure N-(3-fluoro-4-hydroxy-5-methoxybenzyl)-8-methylnonanamide (compound 2b) as a white solid 2.7 g, 83% yield of crystallization, melting point 75-77 °C. 1 H NMR (300 MHz, CDC13) δ 6.58 (d, J = 11.5 Hz, 2H), 6.56 (s, 1H), 6.09 (br, 1H), 6.00 (br, 1H), 4.29 (d, J = 5.8 Hz, 2H), 3.83 (s, 3H), 2.20 (t, J = 7.6 Hz, 2H), 1.70-1.56 (m, 2H), 1.46 (td, J = 13.1, 6.6 Hz, 1H), 1.37-1.16 (m, 6H), 1.16-1.03 (m, 2H), 0.83 (d, J = 6.6 Hz, 6H). 13 C NMR (75 MHz, CDC13) δ 173.34, 150.47 (d, J = 241.5 Hz), 148.37 (d, J = 6.1 Hz), 132.91 (d, J = 13.9 Hz), 129.64 (d, J = 7.7 Hz), 108.13 (d, J = 19.2 Hz), 106.14 (d, J = 2.4 Hz), 56.27, 43.01, 38.86, 36.66, 29.55, 29.29, 27.85, 27.17, 25.73, 22.54.

[0051] Example 6

[0052] Synthesis of (E)-N-(3-fluoro-4-hydroxy-5-methoxybenzyl)-8-methyl-6- nonenamide (compound 3b):

[0053]

[0054] To a solution of 3-fluoro-4-hydroxy-5-methoxybenzylamine hydrochloride 4b (2.08 g, 10 mmol) in 20 mL of water, dipotassium hydrogen phosphate (4.4 g, 25 mmol) was added, after stirring homogeneously, 20 mL of ethyl acetate was added, then 8-methyl-6-nonenoic acid chloride 7 (2.1 g, 11 mmol) was added, stirring at room temperature for 3 h. The reaction was stopped, the organic phase was separated, dried with anhydrous sodium sulfate, then filtered and evaporated to dryness to give a yellowish solid crude. The crude was recrystallized from PE / EA to give pure N-(3-fluoro-4-hydroxy-5-methoxybenzyl)-8-methyl-6-nonenoic amide (compound 3b) as a white solid 2.5 g, 77% of crystallization yield, melting point 72-75 °C. 1 H NMR (300 MHz, CDC13) δ 6.60 (d, J = 11.9 Hz, 2H), 6.58 (s, 1H), 5.99 (br, 1H), 5.40-5.22 (m, 2H), 4.30 (d, J = 5.8 Hz, 2H), 3.84 (s, 3H), 2.32-2.08 (m, 3H), 1.96 (dd, J = 13.2, 6.9 Hz, 2H), 1.63 (dt, J = 15.3, 7.5 Hz, 2H), 1.46-1.26 (m, 2H), 0.93 (d, J = 6.7 Hz, 6H). 13 C NMR (75 MHz, CDC13) δ 173.23, 150.43 (d, J = 241.6 Hz), 148.35 (d, J = 6.1 Hz), 138.05, 132.94 (d, J = 13.8 Hz), 129.63 (d, J = 7.7 Hz), 126.36, 108.22 (d, J = 19.1 Hz), 106.16 (d, J = 2.5 Hz), 56.32, 43.10, 36.53, 32.15, 30.90, 29.20, 25.20, 22.57.

[0055] Test results

[0056] The agonistic effect of the compounds on the TRPV1 receptor was detected using the FLIPR Calcium 6 Assay kit

[0057] The calcium flux assay kit is a commonly used method for measuring intracellular calcium changes, mainly used in drug discovery and basic research. The detection principle is that the lipophilic acetyloxy methyl (AM) carries a Ca 2+ sensitive indicator dye into the cell, which is cleaved by cytoplasmic enzymes to release free Ca 2+ sensitive indicator dye, when the channel is open, a large amount of calcium ions flow into the cell and bind to the indicator dye to emit a strong fluorescence signal, which reflects the agonistic / blocking effect of the drug on the channel.

[0058] 1 Stock solution preparation method of administration preparation

[0059] Agonist: Weigh an appropriate amount of Capsaicin and prepare a 100 mM stock solution with DMSO. After aliquoting, store at -20°C.

[0060] Test substance: Weigh an appropriate amount of test substance, calculate the required volume of DMSO according to the formula: DMSO volume = actual amount x purity / (molecular weight x theoretical concentration), and then pipette the corresponding volume of DMSO. After that, dissolve the weighed test substance with the pipetted DMSO, and weigh the mass of DMSO. According to the final DMSO usage, calculate the actual stock solution concentration.

[0061] 2 Working solution preparation method of administration preparation

[0062] Before TRPV1 receptor testing, the agonist stock solution and test substance stock solution are taken out from -20°C and diluted into an appropriate amount of Buffer as an intermediate solution. The highest test concentration of the test substance is directly diluted with Buffer to the stock solution concentration.

[0063] The test substance stock solution and control stock solution are stored at -20°C, and the test substance working solution and control working solution are prepared on the day of testing and stored at room temperature.

[0064] 3 Concentration selection basis

[0065] The test concentration of agonist Capsaicin is 100 μM starting, 5-fold dilution, 10 concentrations, 2 repeats; the test concentration of test substance is 10 μM, 2-fold or 3-fold dilution, 10 concentrations, 2 repeats.

[0066] 4 Cell culture

[0067] HEK-293 cell line stably expressing TRPV1 receptor is used, hTRPV1 gene information: TRPV1: NM_080704.

[0068] HEK-293 cell line stably expressing TRPV1 receptor is cultured in DMEM medium containing 10% fetal bovine serum, 10 μg / mL Blasticidin, 100 μg / mL Zeocin, the culture temperature is 37°C, and the carbon dioxide concentration is 5%.

[0069] Cell passage: Remove old medium and wash once with PBS, then add 0.5 mL 0.25% -Trypsin-EDTA solution, incubate at 37°C for about 0.5 min. When cells are detached from the dish bottom, add about 3 mL 37°C preheated complete medium. Gently pipette the cell suspension to dissociate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and collect the cells by centrifugation at 1000 rpm for 5 min. Expand or maintain the culture by seeding the cells to 6 cm cell culture dishes, with 2.5 x 10 5 cells (final volume: 5 mL).

[0070] To maintain the physiological activity of the cells, the fusion degree of the experimental cells is 80%-90%.

[0071] Before the FLIPR detection test, the cells are dissociated with 0.25% -Trypsin-EDTA, and the required cell suspension is calculated according to the density of 8000 cells per well, and tetracycline induction (the final concentration of tetracycline is 2 μg / ml) is added to the 384-well plate, and the test is detected after 12 hours of culture in the 384-well plate (final volume: 25 μL).

[0072] 5. FLIPR detection

[0073] 5.1 FLIPR detection method of TRPV1 target

[0074] Dissociate and collect the cells, and then seed the cells in a black-bottom transparent 384-well plate and culture overnight after counting. Prepare 1x buffer according to the kit instructions, and prepare 2x dye with 1x buffer. Remove the culture medium in the 384-well plate by inverted centrifugation, and immediately add 20 μL 1x buffer. Take 20 μL prepared dye and add it to the corresponding experimental wells, and incubate at 37°C in the dark for 2 h. Prepare 5x agonist and test intermediate, and transfer to the corresponding 384 source plate. After the cell plate is incubated, load the instrument, read the 10 s baseline value, and add 10 μL of the prepared test substance in step 5 to the test wells, and collect data for 5 min. The excitation light for calcium flow detection is 470-515 nm, and the emission light is 515-575 nm.

[0075] 5.2 Data analysis

[0076] 1) Z' factor = 1-3*(SD Max + SD Min ) / (AVG Max -AVG Min );

[0077] 2) PC (Positive Control) = Ave (100 μM Capsaicin)

[0078] 3) VC (Vehicle Control) = Ave (1% DMSO)

[0079] 4) CV Max = (SD Max / AVG Max )* 100%;

[0080] 5) CV Min = (SD Min / AVG Min )* 100%;

[0081] 6) S / B = Singal / Background;

[0082] 7) EC 50 50 calculated using GraphPad Nonlinear Regression Equation

[0083] 8) Y = Bottom + (Top-Bottom) / (1+10^((LogEC 50 -X)* Hill Slope))

[0084] 6 The TRPVl agonist activity of the compounds prepared in the present application was tested using the above assay method, and the results are shown in Table 1.

[0085] Table 1 In vitro screening results of the compounds of the present application

[0086] Compound No. Ca 2+ influx EC 50 (nM)]]> Capsaicin 7.4 Nonanoyl vanillylamide 24.9 1a 60.2 2a 96.0 3a 87.3 1b 95.6 2b 134.3 3b 119.1

[0087] The above examples of the present application are merely illustrative for clearly explaining the present application, and are not intended to limit the embodiments of the present application. Other different forms of changes or variations can be made on the basis of the above description by those of ordinary skill in the art. It is not necessary or possible to exhaust all the embodiments here. Any modifications, equivalent replacements and improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A TRPV1 agonist, characterized in that, Capsaicin derivatives having the structures shown in formulas 1a, 1b, 2a and 2b below, and their pharmaceutically acceptable salts: 、 、 、 。 2. A method for preparing the TRPV1 agonist according to claim 1, characterized in that, Specifically, the steps include the following: The compound shown in Formula 4 is dissolved in water, an inorganic base and an organic solvent are added, and then it is reacted with the acyl chloride compound shown in Formula 5 or 6 to generate the compound shown in Formula 1 or 2. 、 、 ； 、 ； Wherein, R is a hydroxyl group or fluorine; The inorganic base is any one or a mixture of two or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, sodium phosphate, disodium hydrogen phosphate, potassium phosphate, or dipotassium hydrogen phosphate. The organic solvent is any one of diethyl ether, tert-methyl ether, toluene, dichloromethane, chloroform, or ethyl acetate; The reaction was carried out at room temperature.

3. The method for preparing the TRPV1 agonist according to claim 2, characterized in that, The molar ratio of the compound shown in Formula 4, the acyl chloride compound shown in Formula 5 or 6, to the inorganic base is 1:(1~2.5):(1~1.5).

4. The method for preparing the TRPV1 agonist according to claim 2, characterized in that, The volume ratio of water to added organic solvent in dissolving the compound shown in Formula 4 is 1:1 to 1:

2.

5. The method for preparing the TRPV1 agonist according to claim 2, characterized in that, The aqueous solution concentration of the compound shown in Formula 4 is 0.2~0.8 mol / L.

6. The method for preparing the TRPV1 agonist according to claim 2, characterized in that, The compound represented by Formula 4 is specifically: or .

7. The method for preparing the TRPV1 agonist according to any one of claims 2 to 6, characterized in that, The purification method of the product after the reaction is as follows: after the reaction stops, the organic phase is separated, dried, filtered, and evaporated to obtain a crude solid product; the crude product is recrystallized by PE / EA to obtain a pure product.

8. The application of the TRPV1 agonist according to claim 1, characterized in that, Used for the preparation of capsaicin-based micronized powder formulations.

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