Chalcone compounds as TRPV3 inhibitors

Abstract

The invention discloses a chalcone compound serving as a TRPV3 inhibitor, the general formula of the chalcone compound is shown in the specification, and in the general formula, R1, R2 and R3 are respectively and independently selected from hydrogen, hydroxyl, halogen, C1-8 alkyl or alkoxy; and R4 is hydrogen, hydroxyl, halogen, trifluoromethyl, C1-8 alkyl or alkoxy. The chalcone compound provided by the invention has a high-selectivity inhibition effect on the TRPV3 (Transient Receptor Physical Vapor Deposition 3). The invention also provides a preparation method of the derivative, and the preparation method is scientific, reasonable, simple and easy to implement. The chalcone compound disclosed by the invention is used for preparing medicines for chronic itching, allergy, skin diseases related to inflammation and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine, and particularly relates to a chalcone derivative as a highly selective TRPV3 inhibitor. Background Art

[0002] Transient receptor potential vanilloid 3 (TRPV3) is a non-selective cation channel protein that is most abundantly expressed in skin keratinocytes and is involved in physiological processes such as keratinocyte proliferation, skin barrier formation, sensory conduction, and hair growth, playing an important role in maintaining skin homeostasis. The overactivation of TRPV3 has been found to be related to the development of some diseases, such as pruritus, inflammation, and skin diseases. TRPV3 has been proven to be one of the important pathways leading to pruritus. In addition, genetic and pharmacological studies have shown that TRPV3 plays an important role in the occurrence and development of skin diseases such as atopic dermatitis, Olmsted syndrome, and psoriasis.

[0003] Chalcone is a kind of active substance widely present in plants and has a relatively high content in traditional Chinese medicines such as Angelica keiskei, Glycyrrhiza glabra, and Carthamus tinctorius. Research shows that this kind of compound is the main secondary metabolite of plants and serves as a defense mechanism of plants to resist reactive oxygen species (ROS) and prevent damage to themselves by microorganisms, insects, or herbivores. Its classical structure is mainly composed of an α,β-unsaturated ketone structure containing a 3C unit connecting two aryl groups. This structure enables chalcone to meet the conditions of a receptor, easily combine with free radicals, making it tolerant to different biomolecules, and at the same time also showing a wide range of biological activities, such as anti-inflammatory, anti-cancer, anti-tumor, and inhibiting hyperglycemia. Due to the convenience of synthesis, chalcone derivatives are easy to prepare. Natural products and synthetic compounds have shown biological activities and have clinical potential for treating various diseases.

[0004] Currently, there are only a few reports on the inhibition of TRPV1 by chalcone (European Journal of Pharmacology, 2024, 985(15), 177093; The International Journal of Biochemistry & Cell Biology, 2019, 112, Pages 18 - 23). TRPV1 is another ligand-gated ion channel subtype mainly distributed in sensory neurons of the peripheral nervous system and can be stimulated by physical and chemical stimuli such as high temperature (>43 °C) and low pH (<5.9), and is a therapeutic target for preclinical models of various chronic pains. However, there is no report on the inhibition of TRPV3 by chalcone compounds. Summary of the Invention

[0005] The object of the present invention is to provide a chalcone compound which has a highly selective inhibitory effect on TRPV3. The present invention also provides a preparation method for such derivatives, which is scientific, reasonable, simple and feasible. The use of the chalcone compound described in the present invention is to prepare drugs for skin diseases related to chronic pruritus, allergy and inflammation, etc.

[0006] The present invention first provides a chalcone compound. The chalcone compound has the following general formula (I):

[0007] In the general formula: R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, halogen, C 1-8 alkyl or alkoxy; R4 is: hydrogen, hydroxyl, halogen, trifluoromethyl, C 1-8 alkyl or alkoxy.

[0008] Preferably, in the general formula, R1 is: hydrogen, hydroxyl; R2 is: hydrogen; R3 is: hydrogen, hydroxyl, halogen or alkoxy; R4 is: hydrogen, halogen, trifluoromethyl or alkoxy.

[0009] More preferably, in the general formula, when R1 is hydroxyl and R2 and R4 are hydrogen, R3 is hydroxyl or alkoxy; or, when R1, R2 and R4 are hydrogen, R3 is halogen; or, when R1, R2 and R3 are hydrogen, R4 is halogen, trifluoromethyl or alkoxy; or, R1, R2, R3 and R4 are all hydrogen.

[0010] Even more preferably, the halogen is Cl.

[0011] The present invention secondly provides a preparation method for the above-mentioned chalcone compound. According to whether there is a phenolic hydroxyl group to be protected in the substituted acetophenone, the preparation method includes: 1) If there is a phenolic hydroxyl group substitution to be protected among R1, R2, R3 and R4, referring to the preparation method of compound A, both 4-hydroxybenzaldehyde and the substituted acetophenone are first protected with 2-pyranyl, and then through condensation and deprotection, the target product is obtained. 2) If there is no phenolic hydroxyl group substitution to be protected among R1, R2, R3 and R4, it is directly condensed with 2-pyranyl-protected 4-hydroxybenzaldehyde and deprotected to obtain the target product. It should be noted that the phenolic hydroxyl group substitution at the 4'-position on the A ring in the structure has a significant influence on the TRPV3 inhibitory activity of this type of compound. If there is no such substitution, its inhibitory activity will be greatly reduced.

[0012] The synthetic route of the chalcone compound described in the present invention is as follows:

[0013]

[0014]

[0015] The specific synthesis steps are as follows: (1) Dissolve p-hydroxybenzaldehyde 1 and 3,4-dihydro-2H-pyran in dichloromethane. Add pyridinium p-toluenesulfonate (PPTS) under stirring, and react at room temperature for 2 - 10 hours. Then add 10% sodium carbonate and stir until the reaction is complete. Extract the reactant with dichloromethane, separate the dichloromethane layer, dry it with anhydrous sodium sulfate, and distill off the organic solvent under reduced pressure to obtain product 2. Among them, the molar ratio of p-hydroxybenzaldehyde, 3,4-dihydro-2H-pyran, and PPTS is 1:1.1 - 1.5:0.09 - 1; (2) If the substituted acetophenone contains phenolic hydroxyl groups that need to be protected, the substituted acetophenone 3 can be used to prepare intermediate 4 according to the method in step (1); among them, the molar ratio of the number of phenolic hydroxyl groups to be protected in the substituted acetophenone, 3,4-dihydro-2H-pyran, and PPTS is 1:1.1 - 1.5:0.09 - 1. Dissolve compound 2 in absolute ethanol, stir until clear, add intermediate 4, then add sodium hydroxide, and react at 20 - 30 °C for 12 h until the reaction ends. Dilute with water, extract with ethyl acetate, dry with anhydrous sodium sulfate, distill off the organic solvent under reduced pressure to obtain product 5. If the substituted acetophenone does not contain phenolic hydroxyl groups that need to be protected, then directly carry out a condensation reaction between the substituted acetophenone 3' and intermediate 2 to obtain product 5'. Among them, the molar ratio of the substituted acetophenone to intermediate 2 is 1:1.

[0016] It should be noted that the above 4-hydroxyacetophenone is only an example. If there are multiple phenolic hydroxyl groups that need to be protected, each phenolic hydroxyl group needs to be protected.

[0017] (3) Dissolve the compound obtained in step (2) in absolute ethanol, add 10% hydrochloric acid, stir at 25 °C until the reaction ends, dilute with water, adjust the pH to 5 - 6 with 10% NaOH solution, extract with ethyl acetate, dry with anhydrous sodium sulfate, distill off the organic solvent under reduced pressure, and purify by column chromatography to obtain the target product.

[0018] In the specific examples of the present invention, the chalcone compounds shown in the following formula were synthesized: .

[0019] Pharmacological experiments have confirmed that the chalcone compounds shown in formula (I) prepared by the present invention have high selectivity and excellent inhibitory activity against TRPV3, and can be used as selective TRPV3 inhibitors.

[0020] In view of the above pharmacological effects, the present invention provides the use of the compound of formula (I) in the preparation of a TRPV3 inhibitor.

[0021] Modern pharmacological studies have found that the over-activation of TRPV3 is related to the development of some diseases, such as pruritus, inflammation and skin diseases, etc., and it plays an important role in the occurrence and development of skin diseases such as atopic dermatitis, Olmsted syndrome, psoriasis, etc.

[0022] Furthermore, the present invention provides the use of the compound of formula (I) in the preparation of a drug for treating diseases related to TRPV3 over-activation. The diseases related to TRPV3 over-activation include skin diseases with clinical manifestations such as skin pruritus and skin inflammation.

[0023] The present invention provides a pharmaceutical composition, which contains the compound of formula (I).

[0024] The present invention also provides a pharmaceutical preparation, which comprises the above pharmaceutical composition and a pharmaceutically acceptable carrier. The dosage form of the pharmaceutical preparation can be a spray, liniment, cream, tablet, injection, suspension, liposome, etc.; the administration form of the pharmaceutical preparation can be topical administration or systemic administration.

[0025] The chalcone compound with the structure shown in general formula I provided by the present invention has a simple and easily accessible structure and good TRPV3 inhibitory activity, and can be used for the preparation of drugs for treating skin diseases related to chronic pruritus, allergy and inflammation, etc. Detailed implementation mode

[0026] The technical solution of the present invention will be described in detail below in conjunction with examples. The following examples are used to illustrate rather than limit the synthesis method of the compound of general formula I.

[0027] Example 1: Synthesis of compound a (2,4-dihydroxy-4'-hydroxychalcone)

[0028]

[0029] (1) Preparation of intermediate 2 Add p-hydroxybenzaldehyde (1.22 g, 1 mmol), 3,4-dihydro-2H-pyran (1.26 g, 1.5 mmol), and 75 ml of dichloromethane to a three-necked flask. While stirring, add pyridinium p-toluenesulfonate (0.23 g, 0.09 mmol). React at room temperature for 2 hours. After the reaction is complete, add 100 ml of 10% sodium carbonate solution to quench the reaction. Separate the dichloromethane layer, dry it over anhydrous sodium sulfate, and evaporate the organic solvent under reduced pressure to obtain intermediate 2 (1.72 g, yield: 83.3%). Without further purification, directly use it in the next reaction.

[0030] (2) Preparation of intermediate 4 Add 2,4-dihydroxyacetophenone (1.52 g, 1 mmol), 3,4-dihydro-2H-pyran (1.26 g, 1.5 mmol), and 75 ml of dichloromethane to a three-necked flask. While stirring, add pyridinium p-toluenesulfonate (0.23 g, 0.09 mmol). React at room temperature for 2 hours. After the reaction is complete, add 100 ml of 10% sodium carbonate solution to quench the reaction. Separate the dichloromethane layer, dry it over anhydrous sodium sulfate, and evaporate the organic solvent under reduced pressure to obtain intermediate 4 (2.07 g, yield: 87.9%). Without further purification, directly use it in the next reaction.

[0031] (3) Preparation of intermediate 5 Place intermediate 2 (2.06 g, 1 mmol) in a three-necked flask. After dissolving it completely in 40 ml of absolute ethanol, add intermediate 4 (2.36 g, 1 mmol). Then add 1.25 g of NaOH and react at 30 °C for 12 hours. After the reaction ends, add 100 ml of water for dilution, extract with 150 ml of ethyl acetate, and wash with saturated brine 2 - 3 times. Separate the ethyl acetate layer, dry it over anhydrous sodium sulfate, and evaporate the organic solvent under reduced pressure to obtain product 5 (2.93 g, yield: 69.2%). Without further purification, directly use it in the next reaction.

[0032] (4) Preparation of compound a Place product 5 (4.24 g, 1 mmol) in a round-bottom flask. Sequentially add 20 ml of absolute ethanol and 5 ml of 10% hydrochloric acid, and stir at room temperature for 2 hours. After the reaction is complete, add 80 ml of water for dilution, extract with 100 ml of ethyl acetate, separate the ethyl acetate layer, wash with saturated brine 2 - 3 times, dry it over anhydrous sodium sulfate, and evaporate the organic solvent under reduced pressure. Purify by column chromatography to obtain compound a (2.01 g, yield: 78.6%). ESI MS: 257.3 [M + H] +1 , 1 H NMR (400 MHz, DMSO-d 6) δ 13.63(s, 1H), 10.71 (s, 1H), 10.16 (s, 1H), 8.23 (d, J = 15.2 Hz, 1H), 7.83 - 7.74(m, 4H), 6.86 (d, J = 8.1 Hz, 2H), 6.42 (d, J = 15.2 Hz, 1H), 6.29 (m, 1H).

[0033] Example 2: Preparation of Compound b (2 - Hydroxy - 4 - methoxy - 4'-hydroxychalcone)

[0034] Synthesis was carried out with reference to the preparation method of Compound a in Example 1. In step (2), no hydroxyl protection was required, and 2 - hydroxy - 4 - methoxyacetophenone was directly condensed with Intermediate 2. The total yield based on p - hydroxybenzaldehyde was 39.2%. ESI MS: 271.2[M + H] +1 , 1 H NMR (400 MHz, DMSO - d 6) δ 13.69 (s, 1H), 10.20 (s, 1H), 8.27 (d, J = 15.1 Hz, 1H), 7.87 - 7.74 (m, 4H), 6.86 (d, J = 8.2 Hz, 2H), 6.61 - 6.49 (m,2H), 3.86 (s, 3H).

[0035] Example 3: Preparation of Compound c (2 - Dihydroxy - 4 - n - propyloxy - 4'-hydroxychalcone)

[0036] Synthesis was carried out with reference to the preparation method of Compound b in Example 2. In step (2), 2 - hydroxy - 4 - n - propyloxyacetophenone was used instead of 2 - hydroxy - 4 - methoxyacetophenone for condensation with Intermediate 2. The total yield based on p - hydroxybenzaldehyde was 35.5%. ESI MS: 299.6 [M + H] +1 , 1 H NMR (400 MHz, DMSO - d 6) δ 13.63 (s, 1H), 10.19 (s, 1H),8.21 (d, J= 15.3 Hz, 1H), 7.88 - 7.74 (m, 4H), 6.90 - 6.82 (m, 2H), 6.56 (m, 1H), 6.50 (m, 1H), 4.04 (m, 2H), 1.76 (m, 2H), 1.01 (m, 3H).

[0037] Example 4: Preparation of Compound d (4 - Hydroxy - 4'-hydroxychalcone)

[0038] Synthesis was carried out with reference to the preparation method of Compound a in Example 1. In step (2), 4 - hydroxyacetophenone was used instead of 2,4 - dihydroxyacetophenone for condensation with Intermediate 2. The total yield based on p - hydroxybenzaldehyde was 46.5%. ESI MS: 241.7 [M + H] +1 , 1 H NMR (400 MHz, DMSO - d 6) δ 10.37 (s, 1H), 10.01 (s, 1H), 8.05 (m, 2H), 7.73 - 7.61 (m, 4H), 6.90 - 6.83 (m, 4H).

[0039] Example 5: Preparation of Compound e (4 - Chloro - 4'-hydroxychalcone)

[0040] Synthesis was carried out with reference to the preparation method of Compound b in Example 2. In step (2), 4 - chloroacetophenone was used instead of 2 - hydroxy - 4 - methoxyacetophenone for condensation with Intermediate 2. The total yield based on p - hydroxybenzaldehyde was 36.6%. ESI MS: 259.8[M + H] +1 , 1 H NMR (400 MHz, DMSO - d 6) δ 10.14 (s, 1H), 8.15 (m, 2H), 7.81 - 7.65 (m, 4H), 7.65 - 7.53 (m, 2H), 6.83 (m, 2H).

[0041] Example 6: Preparation of Compound f (3 - Chloro - 4'-hydroxychalcone)

[0042] Synthesis was carried out according to the preparation method of compound b in Example 2. In step (2), 3-chloroacetophenone was used instead of 2-hydroxy-4-methoxyacetophenone for condensation with intermediate 2. The total yield based on p-hydroxybenzaldehyde was 33.2%. ESI MS: 259.6 [M + H] +1 , 1 H NMR (300 MHz, DMSO- d 6) δ 10.16 (s, 1H), 8.17 (m, 1H), 8.07 (m,1H), 7.80-7.68 (m, 5H), 7.59 (m, 1H), 6.86 (m, 2H). Example 7: Preparation of compound g (3-trifluoromethyl-4'-hydroxychalcone)

[0043] Synthesis was carried out according to the preparation method of compound b in Example 2. In step (2), 3-trifluoromethylacetophenone was used instead of 2-hydroxy-4-methoxyacetophenone for condensation with intermediate 2. The total yield based on p-hydroxybenzaldehyde was 27.6%. ESI MS:293.8 [M + H] +1 , 1 H NMR (400 MHz, DMSO- d 6) δ 10.21 (s, 1H), 8.45 (m, 2H), 8.03(m, 1H), 7.89-7.73 (m, 5H), 6.87 (d, J = 7.8 Hz, 2H). Example 8: Preparation of compound h (3-n-propyloxy-4'-hydroxychalcone)

[0044] Synthesis was carried out according to the preparation method of compound b in Example 2. In step (2), 3-n-propoxyacetophenone was used instead of 2-dihydroxy-4-methoxyacetophenone for condensation with intermediate 2. The total yield based on p-hydroxybenzaldehyde was 30.7%. ESI MS:283.1 [M + H] +1 , 1 H NMR (400 MHz, DMSO- d 6) δ 10.12 (s, 1H),7.77-7.63 (m, 5H),7.58 (m, 1H), 7.46 (m, 1H), 7.21 (m, 1H), 6.84 (d, J= 8.2 Hz, 2H), 4.02 (m, 2H), 1.77 (m, 2H), 1.01 (m, 3H).

[0045] Example 9: The TRPV3 inhibitory activity of the compounds obtained in Examples 1-8 was studied. Experimental method: Referring to the method of a Chinese patent (a small molecule allosteric inhibitor of TRPV3 and its preparation method, CN112480018A), the inhibitory activity of the compounds on the TRPV3 channel was tested by the whole cell patch clamp technique. In HEK-293 cells transiently expressing the TRPV3 channel, green fluorescent EGFP cells selected as surface markers for gene expression were used for patch clamp studies. The TRPV3 channel current needs to be activated by a channel agonist, and then the pharmacological effects of the inhibitor were observed. The target compound was dissolved in an appropriate amount of 100% DMSO to prepare a 30 mM stock solution and stored frozen at 4°C; during the experiment, the stock solution was serially diluted with extracellular fluid. First, the extracellular fluid containing 2-APB (50 μM) was perfused, and then the extracellular fluid containing different concentrations of the target compound and 2-APB (50 μM) was perfused. The maximum outward current mediated by the hTRPV3 channel induced by 2-APB and the outward current mediated by the hTRPV3 channel under the simultaneous action of the inhibitor and 2-APB were recorded, and the inhibition rate of the compound at different concentrations was calculated based on the outward current of the whole cell current mediated by the hTRPV3 channel. In addition, the channel selectivity of these compounds was determined on cells expressing rTRPV1. The IC50 curve was plotted from the results obtained by administering compounds at different concentrations (0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM). Each concentration was tested 3 times, and the IC 50 value (Table 1) was calculated by non-linear regression.

[0046] Table 1 Inhibitory activity of compounds on TRPV3 / 1 IC 50 (μM) .

[0047] From the specific IC 50 value data in the above table, for compounds a-h and 4'-hydroxychalcone, they showed strong inhibitory effects on TRPV3 (IC 50 < 20 μM), while only weak inhibitory effects on TPVV1, and have potential for treating diseases such as skin itching and inflammatory pain. However, chalcone compounds without 4'-hydroxy substitution such as 2'-hydroxychalcone, 3-chloro-chalcone, and 2-hydroxy-4-methoxychalcone lost their TRPV3 inhibitory activity (IC50 > 100 μM).

[0048] Although the present invention has been illustrated by the previous specific embodiments, it should not be construed as being limited thereto; rather, the present invention encompasses the general aspects disclosed previously. Various modifications and various embodiments can be made without departing from the spirit and scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements can be made without departing from the principles of the present invention, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A chalcone compound as a TRPV3 inhibitor has a structure shown in formula (I): ， Wherein: R1, R2, and R3 are each independently selected from hydrogen, hydroxy, halogen, C 1-8 alkyl or alkoxy; R4 is hydrogen, hydroxy, halogen, trifluoromethyl, C 1-8 alkyl or alkoxy.

2. The chalcone compound according to claim 1, wherein R1 is hydrogen or hydroxyl; R2 is hydrogen; R3 is hydrogen, hydroxyl, halogen or alkoxy; R4 is hydrogen, halogen, trifluoromethyl or alkoxy.

3. The chalcone compound according to claim 2, wherein When R1 is hydroxyl and R2 and R4 are hydrogen, R3 is hydroxyl or alkoxy; or when R1, R2 and R4 are hydrogen, R3 is halogen; or when R1, R2 and R3 are hydrogen, R4 is halogen, trifluoromethyl or alkoxy; or R1, R2, R3 and R4 are all hydrogen.

4. A method for preparing a chalcone compound according to any one of claims 1 to 3, characterized in that, According to whether the substituted acetophenone has a phenolic hydroxyl group that needs to be protected, its preparation method includes: if there is a phenolic hydroxyl group that needs to be protected among R1, R2, R3 and R4, 4-hydroxybenzaldehyde and the substituted acetophenone are respectively protected with 2-pyranyl, and then the target product is obtained through condensation and deprotection; if there is no phenolic hydroxyl group that needs to be protected among R1, R2, R3 and R4, the substituted acetophenone is condensed with 2-pyranyl-protected 4-hydroxybenzaldehyde and deprotected to obtain the target product.

5. Use of the chalcone compound according to any one of claims 1-3 in the preparation of a selective TRPV3 inhibitor.

6. Use of the chalcone compound according to any one of claims 1-3 in the preparation of a drug for treating diseases related to TRPV3 overactivation.

7. The application according to claim 6, characterized in that, The diseases related to TRPV3 overactivation are skin diseases, and the clinical manifestations of the skin diseases include itching and inflammation.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the chalcone compound according to any one of claims 1-3.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation includes the pharmaceutical composition according to claim 8 and a pharmaceutically acceptable carrier.

10. The pharmaceutical preparation according to claim 9, wherein, The preparation form of the pharmaceutical preparation includes spray, liniment, cream, tablet, injection, suspension, liposome; the administration form of the pharmaceutical preparation includes topical administration or systemic administration.

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