Compound for regulating TRPV1 ion channel and preparation method and application thereof

Abstract

The invention provides a compound for regulating and controlling a TRPV1 ion channel. The structural formula of the compound is as shown in formula I. The invention also provides application of the compound in preparation of a medicine for regulating and controlling the TRPV1 ion channel. The invention provides a pharmaceutical composition with an anti-inflammatory effect, the pharmaceutical composition comprises the compound, and pharmaceutically acceptable auxiliary materials or auxiliary components are added to prepare a pharmaceutically common preparation. Compared with azobenzene, the compound has the advantages that the illumination wavelength is increased, the illumination wavelength is 660 nm, a TRPV1 targeted photosensitizer is used, a channel is activated by using a low-energy near-infrared light'precise key ', depth, safety and reversibility are considered, and the anti-inflammatory effect of the compound is obvious.

Description

Technical Field

[0001] This invention relates to a compound that regulates TRPV1 ion channels, its preparation method, and its uses. Background Art

[0002] The TRPV1 (Transient Receptor Potential Vanilloid 1) ion channel is a non-selective cation channel belonging to the vanillic acid subtype of the transient receptor potential (TRP) channel family. It is widely distributed in sensory neurons, particularly those associated with pain perception and heat sensitivity. TRPV1 channels can be activated by a variety of physical and chemical stimuli, including high temperatures (>43°C), protons (acidic environments), vanillic acid compounds (such as capsaicin), and certain lipid metabolites (such as endocannabinoids).

[0003] The activity of TRPV1 channels is regulated by a variety of factors, including chemical ligands, physical stimulation, phosphorylation modification, and interactions with other proteins. In recent years, optical control technology has attracted much attention as an emerging regulatory method due to its high spatiotemporal precision and reversibility.

[0004] In recent years, photosensitive technology has been widely used in the regulation of TRPV1 channels. By designing photosensitive molecules (such as azobenzene derivatives), researchers can change the conformation of these molecules under specific wavelengths of light, thereby achieving reversible regulation of TRPV1 channel activity, for example:

[0005] Fatty acids (FAs) and their derivatives play important roles in cellular signaling, including regulating ion channels such as TRPV1. The research team aimed to develop photocontrolled fatty acid analogs (FAAzos) to achieve optical modulation of the TRPV1 channel. A series of azobenzene-based photocontrolled fatty acid analogs (AzCAs) were synthesized, and their photocontrolled effects on the TRPV1 channel in HEK293T cells and primary sensory neurons were evaluated. AzCA2, AzCA3, and AzCA4 significantly activated the TRPV1 channel under ultraviolet light (350 nm) irradiation and lost activity under blue light (450 nm) irradiation. AzCA4 achieved photocontrolled activation in dorsal root ganglion (DRG) neurons and C-fiber nociceptors of wild-type mice, with effects comparable to the natural agonist capsaicin (CAP), but with higher temporal precision and reversibility. This study demonstrates the potential of photoswitchable fatty acid analogs to achieve high-precision spatiotemporal control of TRPV1 channels in complex neural systems (Frank JA, Moroni M, Moshourab R, Sumser M, Lewin GR, Trauner D. Photoswitchable fatty acids enable optical control of TRPV1[J]. Nature Communications, 2015, 6(1):7118.)

[0006] TRPV1 channels are non-selective cation channels that are widely expressed in sensory neurons and are involved in pain perception and thermal sensitivity.

[0007] Traditional chemical methods cannot precisely control its activity, and conventional azobenzene molecules require ultraviolet light to trigger isomerization, which may damage biological tissues. Therefore, efforts are being made to develop redshifted azobenzene molecules, enabling them to function in the visible light range.

[0008] A series of tetrachloro-substituted azobenzene molecules (such as red-AzCA-4) were synthesized using a palladium-catalyzed CH activation reaction via a late-stage functionalization strategy. These molecules are isomerized under green light (560 nm) and violet light (400 nm). red-AzCA-4, as a photo-controlled agonist of the TRPV1 channel, activates the TRPV1 channel under green light irradiation and loses its activity under violet light irradiation. This method provides a simple and universal approach for synthesizing photo-switching molecules that operate under biocompatible light conditions. (Konrad DB, Frank JA, Trauner D. Synthesis of Redshifted Azobenzene Photoswitches by Late-Stage Functionalization[J]. Chemistry–A European Journal,2016,22(13):4364–4368.)

[0009] Although red-shifted azobenzenes (such as red-AzCA-4) can work in the visible light range, improving biocompatibility, the penetration of light in biological tissues is still limited, especially in deep tissues or organs, where light may not be able to effectively reach the target area, thus affecting the regulatory effect. Moreover, at low wavelengths, using high-energy photochemical "hard-prying" to target TRPV1 results in poor selectivity, significant damage, and shallow penetration. Summary of the Invention

[0010] This invention provides a compound for regulating TRPV1 ion channels, its preparation method, and its uses.

[0011] This invention provides a compound for regulating TRPV1 ion channels, with the structural formula shown in Formula I:

[0012]

[0013] The present invention provides a method for synthesizing the aforementioned compound, which uses methyl 4-amino-3-fluorophenylcarboxylate and 3-(tert-butoxycarbonylamino)propionic acid as starting compounds, wherein methyl 4-amino-3-fluorophenylcarboxylate is synthesized by selective electrophilic chlorination of the aromatic ring, C–H directed chlorination, and ester hydrolysis.

[0014] 3-(tert-Butoxycarbonylamino)propionic acid was subjected to TBTU / DIPEA-mediated amidation and Boc deprotection to yield compound 7;

[0015] Compound 4 was then semi-selectively amidated under the action of HATU / DIPEA, and then synthesized with compound 7 to obtain the bisamide-azo product shown in Formula I.

[0016] Its synthetic route is as follows:

[0017]

[0018] Specifically, the steps include the following:

[0019] (1) Synthesize compound 2:

[0020] DBU was added to a solution of methyl 4-amino-3-fluorophenylcarboxylate dissolved in DCM; the mixture was stirred at room temperature and then cooled to -78°C; N-chlorosuccinimide (NCS) was added as a solid to the reaction mixture; the orange solution was stirred at -78°C and then quenched by adding a saturated bicarbonate solution; the organic layer was separated, washed successively with water and HCl, dried with anhydrous sodium sulfate, concentrated to dryness under vacuum, and purified to obtain the product;

[0021] (2) Synthesis of compound 3: Compound 2 synthesized in step (1), Pd(OAc)2 and NCS were dissolved in AcOH and heated to 120°C for 6 hours; after cooling to room temperature, the solvent was removed under vacuum and the brown residue was dissolved in CH2Cl2; the organic layer was washed successively with saturated NaHCO3 aqueous solution and saturated NaCl aqueous solution and dried with Na2SO4; the residue was purified by rapid column chromatography (hexane-ethyl acetate) as eluent to obtain product compound 3;

[0022] (3) Synthesis of compound 4: Compound 3 and LiOH were dissolved in an aqueous solution of methanol / tetrahydrofuran and stirred at room temperature; the reaction solution was dissolved in an aqueous solution of CH2Cl2 and the aqueous layer was extracted; ethyl acetate was added to the aqueous layer in sequence, and diluted concentrated hydrochloric acid was added dropwise to extract the ethyl acetate layer, which was then dried under vacuum to obtain the product;

[0023] (4) Synthesize compound 6:

[0024] Compound 5, TBTU (404 mg, 1.26 mmol, 1.2 equivalents) was dissolved in DMF and added sequentially.

[0025] DIPEA, 4-(aminomethyl)-2-methoxyphenol: The reaction solution was stirred at room temperature; ethyl acetate was added sequentially to the reaction solution, and the mixture was washed with saturated NaCl aqueous solution to extract the ethyl acetate layer, which was then dried over Na2SO4. The residue was purified by rapid column chromatography (dichloromethane-methanol) as the eluent to obtain the product.

[0026] (5) Synthesis of compound 7: Compound 6 was dissolved in a trifluoroacetic acid / dichloromethane solution and stirred at room temperature; the solvent was removed from the reaction solution under vacuum to obtain product compound 7;

[0027] (6) Synthesis of compound 8: Compound 4 and HATU were dissolved in DMF, and DIPEA and butan-1-amine were added sequentially. The reaction solution was stirred at room temperature, and ethyl acetate was added sequentially. The ethyl acetate layer was obtained by washing with saturated NaCl aqueous solution and dried with Na2SO4. The residue was purified by rapid column chromatography (dichloromethane-methanol) as eluent to obtain product compound 8.

[0028] (7) Synthesize compound 9:

[0029] Compound 8 and HATU were dissolved in DMF, and DIPEA and compound 7 were added sequentially. The reaction solution was stirred at room temperature. Ethyl acetate was added sequentially to the reaction solution, and the ethyl acetate layer was extracted by washing with saturated NaCl aqueous solution and dried over Na2SO4. The residue was purified by rapid column chromatography (dichloromethane-methanol) as eluent to obtain product compound 9 (formula I).

[0030] The molar ratio of compound 1, DBU, and DCM is 1:2:2.

[0031] The molar ratio of compound 2, Pd(0Ac), and NCS is 1:0.2:5;

[0032] The molar ratio of compound 3 to LiOH is 1:3.5;

[0033] The molar ratio of compound 5, TBTU, and 4-(aminomethyl)-2-methoxyphenol is 1:1.2:1.

[0034] Compound 4: The molar ratio of HATU and butan-1-amine is 1:1.2:1:1;

[0035] The molar ratio of compound 8:HATU:DIPEA:compound 7 is 1:1.2:3.5:1.1.

[0036] This invention provides the use of the compound in the preparation of drugs that regulate TRPV1 ion channels.

[0037] This invention provides the use of the compound in the preparation of a medicament with anti-inflammatory effects.

[0038] This invention provides a pharmaceutical composition with anti-inflammatory effects, comprising the aforementioned compound, and prepared into a pharmaceutically commonly used formulation by adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0039] The auxiliary material mentioned above is a silk fibroin film.

[0040] Compound 1: ethyl 4-amino-3-fluorobenzoate;

[0041] Compound 2: Ethyl 4-[(1E)-[4-(ethoxycarbonyl)-2-fluorophenyl]ethazenoyl]-3-fluorobenzoate;

[0042] Compound 3: Ethyl 5-chloro-4-[(1E)-[6-chloro-4-(ethoxycarbonyl)-2-fluorophenyl]ethazenoyl]-3-fluorobenzoate;

[0043] Compound 4: 4-[(1E)-(4-carboxy-6-chloro-2-fluorophenyl)ethazenoyl]-5-chloro-3-fluorobenzoic acid;

[0044] Compound 5: 3-({[(2-methylprop-2-yl)oxy]carbonyl}amino)propionic acid;

[0045] Compound 6: [(3-{[(4-hydroxy-3-methoxyphenyl)methyl]amino}-3-oxoylidenepropyl)amino]methane-2-methylpropyl-2-yl ester;

[0046] Compound 7: 3-Amino-N-[(4-hydroxy-3-methoxyphenyl)methyl]propionamide;

[0047] Compound 8: 4-[(1E)-{4-[(butylamino)carbonyl]-2-chloro-6-fluorophenyl}ethazenoyl]-3-chloro-5-fluorobenzoic acid;

[0048] Compound 9: N-Butyl-5-chloro-4-[(1E)-{6-chloro-2-fluoro-4-[7-(4-hydroxy-3-methoxyphenyl)-1,5-dioxane-2,6-diazahept-1-yl]phenyl}ethazenyl]-3-fluorobenzamide.

[0049] Azobenzene regulates the switching effect of TRPV1 ion channels in cells, based on the design of a switching unit with TRPV1 ion channel switching regulation function. The design principle involves 2F-2Cl ortho-substitution in the azobenzene core structure, with vanillin groups and alkane chains attached to both ends of the azobenzene core structure, endowing it with the function of targeting TRPV1 ion channels and binding to the cell membrane. Simultaneously, upon contact with cells, azobenzene binds to the vicinity of TRPV1 ion channels on the cell surface.

[0050] Molecular design, such as Figure 1As shown, a photoisomerized azobenzene group serves as the core group, with a TRPV1 channel membrane porin-binding vanillin ligand and a lipophilic group capable of interacting with the cell membrane covalently attached to its two ends. Under 660 nm light irradiation, the azobenzene group undergoes photoisomerization, thereby opening or closing the ion flow of the channel. Therefore, this study will first synthesize azobenzene based on the designed route of the azobenzene molecule, and verify it using 1H NMR spectroscopy. Subsequent explorations will use UV absorber to verify the photoisomerization properties and fatigue resistance of the azobenzene molecule. Using macrophages as a template, the degree of calcium ion influx will be tested to verify the on / off effect of the azobenzene molecule regulating the TRPV1 ion channel.

[0051] Compared with azobenzene, the compound of this invention improves the light wavelength to 660nm. With the help of TRPV1 targeting photosensitizer, the channel is activated by a "precise key" of low-energy near-infrared light, taking into account depth, safety and reversibility. The compound of this invention has a significant anti-inflammatory effect. Attached Figure Description

[0052] Figure 1 Molecular design diagram;

[0053] Figure 2 NMR spectrum of compound 2;

[0054] Figure 3 NMR spectrum of compound 3;

[0055] Figure 4 NMR spectrum of compound 4;

[0056] Figure 5 NMR spectrum of compound 6;

[0057] Figure 6 NMR spectrum of compound 7;

[0058] Figure 7 NMR spectrum of compound 8;

[0059] Figure 8 NMR spectrum of compound 9;

[0060] Figure 9 The chemical structure reveals the photoisomerization of compound 9 between the trans and cis configurations under irradiation with visible light at 405 nm and 660 nm.

[0061] Figure 10 UV-Vis absorption spectra of compound 9 after irradiation with 405 nm and 660 nm light;

[0062] Figure 11 The absorbance of compound 9 at 430 nm as a function of time under illumination at 405 nm and 660 nm, respectively;

[0063] Figure 12 1H NMR spectroscopy revealed photoisomerization of compound 9 at δ8.3 ppm and δ8.65 ppm after visible light irradiation;

[0064] Figure 13 AZO light-induced Ca in macrophages 2+ Inflow fluorescence imaging. Green: Fluo-4 fluorescence;

[0065] Figure 14 Immunofluorescence M1 / M2 polarization;

[0066] Figure 15 Flow cytometry analysis of M1 / M2 polarization;

[0067] Figure 16 Fluorescent images of IL-10 and TNF-α in normal skin wounds of rats on day 3;

[0068] Figure 17 Images of HE and Sirius red on normal skin wounds of rats on day 14. DETAILED DESCRIPTION

[0069] Example 1: Synthesis process of the compound of the present invention

[0070] Synthesis of Compound 2: DBU (8.28 g, 62 mmol, 2 equivalents) was added to a solution of Compound 1 (5.7 g, 31 mmol) dissolved in 300 mL of DCM. The solution was stirred at room temperature for 5 min and then cooled to -78 °C. NCS (9.42 g, 62 mmol, 2 equivalents) was added as a solid to the reaction mixture. The orange solution was stirred at -78 °C for 10 min and then quenched by adding a saturated bicarbonate solution. The organic layer was separated, washed successively with 50 mL of water and 50 mL of 1N HCl, dried over anhydrous sodium sulfate, and concentrated to dryness under vacuum. The residue was purified by silica gel rapid chromatography (ethyl acetate-hexane) as eluent to give the product (3.5 g, 9.66 mmol).

[0071] Synthesis of Compound 3: Compound 2 (3.5 g, 9.66 mmol), Pd(0Ac)2 (433 mg, 1.93 mmol, 0.2 equivalents), and NCS (6.42 g, 48.3 mmol, 5 equivalents) were dissolved in AcOH (90 mL) and heated to 120 °C for 6 hours. After cooling to room temperature, the solvent was removed under vacuum, and the brown residue was dissolved in...

[0072] CH2Cl2. The organic layer was washed successively with saturated NaHCO3 aqueous solution and saturated NaCl aqueous solution, and dried over Na2SO4. The residue was purified by rapid column chromatography (hexane-ethyl acetate) as eluent to give the product (2.5 g, 5.78 mmol).

[0073] Synthesis of compound 4: Compound 3 (2.5 g, 5.78 mmol) and LiOH (483 mg, 20.25 mmol, 3.5 equivalences) were dissolved in an aqueous methanol / tetrahydrofuran solution and stirred at room temperature for 2 h. The reaction solution was dissolved in an aqueous CH2Cl2 solution, and the aqueous layer was extracted. Ethyl acetate was added to the aqueous layer sequentially, followed by dropwise addition of diluted concentrated hydrochloric acid. The resulting ethyl acetate layer was extracted and dried under vacuum to give the product (1.5 g, 4.0 mmol).

[0074] Synthesized compound 6:

[0075] Compound 5 (200 mg, 1.05 mmol) and TBTU (404 mg, 1.26 mmol, 1.2 equivalents) were dissolved in 5 ml of DMF, and DIPEA (542 mg, 4.2 mmol, 4 equivalents) was added sequentially.

[0076] 4-(aminomethyl)-2-methoxyphenol (200 mg, 1.05 mmol, 1 equivalent) was added, and the reaction solution was stirred at room temperature for 2 h. Ethyl acetate was added sequentially to the reaction solution, and the mixture was washed with saturated NaCl aqueous solution to obtain an ethyl acetate layer, which was then dried over Na₂SO₄. The residue was purified by rapid column chromatography (dichloromethane-methanol) as the eluent to give the product (300 mg, 0.925 mmol).

[0077] Synthesis of compound 7: Compound 6 (300 mg, 0.925 mmol) was dissolved in a trifluoroacetic acid / dichloromethane solution and stirred at room temperature for 1 h. The solvent was removed from the reaction solution under vacuum to obtain product compound 7 (200 mg, 0.89 mmol).

[0078] Synthesis of compound 8: Compound 4 (200 mg, 0.536 mmol) and HATU (244 mg, 0.643 mmol, 1.2 equivalences) were dissolved in 5 mL of DMF. DIPEA (276 mg, 2.144 mmol, 4 equivalences) and butan-1-amine (43 mg, 0.589 mmol, 1.1 equivalences) were added sequentially, and the reaction mixture was stirred at room temperature for 2 h. Ethyl acetate was added sequentially, and the mixture was washed with saturated NaCl aqueous solution to obtain the ethyl acetate layer, which was then dried over Na2SO4. The residue was purified by rapid column chromatography (dichloromethane-methanol) as the eluent to give product compound 8 (100 mg, 0.232 mmol).

[0079] Synthesized compound 9:

[0080] Compound 8 (100 mg, 0.232 mmol) and HATU (106 mg, 0.279 mmol, 1.2 equivalents) were dissolved in 5 mL of DMF. DIPEA (105 mg, 0.812 mmol, 3.5 equivalents) and compound 7 (57 mg, 0.256 mmol, 1.1 equivalents) were added sequentially, and the reaction mixture was stirred at room temperature for 2 h. Ethyl acetate was added sequentially to the reaction mixture, and the mixture was washed with saturated NaCl aqueous solution to obtain an ethyl acetate layer, which was then dried over Na₂SO₄. The residue was purified by rapid column chromatography (dichloromethane-methanol) as the eluent to give product compound 9 (30 mg, 0.047 mmol) (i.e., the compound of this invention).

[0081] The compound spectral characterization of this invention is as follows: Figures 2-8 As shown:

[0082] Compound 2: ¹H NMR (400 MHz, Chloroform-d) δ 7.99 (dd, J = 10.8, 1.7 Hz, 2H), 7.96–7.89 (m, 2H), 7.85 (dd, J = 8.4, 7.0 Hz, 2H), 4.45 (q, J = 7.1 Hz, 4H), 1.46 (t, J = 7.1 Hz, 6H).

[0083] Compound 3: 1 H NMR (400MHz, Chloroform-d) δ8.05 (s, 2H), 7.82 (d, J = 10.5Hz, 2H), 4.43 (q, J = 7.1Hz, 4H), 1.43 (t, J = 7.1Hz, 6H).

[0084] Compound 4: 1H NMR (400MHz, DMSO-d6) δ8.03 (t, J = 1.5 Hz, 2H), 7.92 (dd, J = 10.8, 1.6 Hz, 2H).

[0085] Compound 6: ¹H NMR (400 MHz, DMSO-d6) δ 8.90 (s, ¹H), 8.24 (t, J = 5.9 Hz, ¹H), 6.83 (d, J = 8.2 Hz, ¹H), 6.76 (d, J = 5.6 Hz, ¹H), 6.67 (d, J = 2.1 Hz, ¹H), 6.62 (dd, J = 8.2, 2.1 Hz, ¹H), 4.10 (d, J = 5.8 Hz, 2H), 3.73 (s, 3H), 3.14 (q, J = 6.9 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H).

[0086] Compound 7: 1 H NMR (400MHz, DMSO-d6) δ8.49(t,J=5.8Hz,1H),6.85(d,J=8.2Hz,1H),6.70(d,J=2.1Hz,1H),6.65(dd ,J=8.2,2.1Hz,1H),4.15(d,J=5.8Hz,2H),3.74(s,3H),3.01(p,J=6.3Hz,2H),2.48(d,J=6.9Hz,2H).

[0087] Compound 8: 1 H NMR(400MHz,DMSO-d6)δ8.78(t,J=5.6Hz,1H),8.04(dt,J=14.9,1.4Hz,2H),8.01–7.87(m ,2H),3.32–3.29(m,2H),1.55(q,J=7.0Hz,2H),1.40–1.31(m,2H),0.92(t,J=7.4Hz,3H).

[0088] Compound 9: 1H NMR (400MHz, DMSO-d6) δ8.93(t,J=5.7Hz,1H),8.89(s,1H),8.79(t,J=5.7Hz,1H),8.34(q,J =6.3Hz,1H),8.05(s,2H),7.93–7.86(m,2H),6.76(d,J=8.4Hz,1H),6.69(s,1H),6.61(d,J= 8.3Hz,1H),4.15(d,J=5.9Hz,2H),3.71(s,3H),3.53(q,J=6.3Hz,2H),3.30(d,J=6.4Hz,2H) ,2.47(t,J=6.8Hz,2H),1.53(q,J=7.2Hz,2H),1.35(q,J=7.4Hz,2H),0.92(t,J=7.5Hz,3H).

[0089] The following experiments demonstrate the beneficial effects of the present invention.

[0090] Experimental Example 1: Monomerization of Compound 9 under Visible Light Irradiation

[0091] 1. Test method:

[0092] To determine the photoisomerization potential of compound 9, ultraviolet absorption spectra and proton NMR spectra were used for verification.

[0093] 1) UV Absorption Spectroscopy: A 200 μm solution was prepared in dimethyl sulfoxide. The UV-Vis absorption spectrum of the sample in the 350–600 nm range was measured using a UV-Vis spectrometer. The sample was then irradiated with light at 660 nm and 405 nm, respectively, to induce cis and trans isomerization. Irradiation times ranged from 0 seconds to 20 minutes. Absorption spectra were measured intermittently throughout the irradiation period to monitor the progress of the photoisomerization reaction.

[0094] 2) 1H NMR spectrum: Compound 9 was dissolved in deuterated DMSO to prepare a 5mg mL solution. -1 Solution. Irradiate the solution with 660 nm and 405 nm light, and immediately acquire 1H NMR spectra using a 400 MHz spectrometer.

[0095] 2. Test Results:

[0096] Figure 9 The chemical structure reveals the photoisomerization of compound 9 between the trans and cis configurations under irradiation with visible light at 405 nm and 660 nm.

[0097] Figure 10 , Figure 11Upon exposure to 660 nm red light, an increase in the characteristic absorbance of azobenzene at 430 nm was observed, indicating effective conversion to the cis isomer. This photoinduced isomerization process was reversible, as subsequent irradiation with 405 nm blue light completely restored the initial trans configuration, achieving 100% recovery of the azobenzene absorbance. The degree of photoisomerization showed a significant dependence on the duration of light irradiation, with the cis isomer reaching near-steady-state after 1 hour of 660 nm irradiation. In contrast, the trans isomer reached the same equilibrium within a shorter time of 1 minute under 405 nm blue light irradiation.

[0098] Figure 12 ¹H NMR analysis provided direct structural evidence of the isomerization process. The formation of the cis isomer was confirmed by different chemical shifts at δ 8.3 ppm and δ 8.65 ppm. Red light irradiation led to a significant increase in peak intensity, confirming successful trans-to-cis photoisomerization. Subsequent blue light irradiation indicated partial reversal to the trans isomer.

[0099] 3. Analysis of Experimental Results:

[0100] The results show that the ability of compound 9 to undergo reversible conversion via safer visible light, as well as the robust stability of its cis configuration, opens up new possibilities for innovative applications in the biomedical field.

[0101] Experiment Example 2: Calcium Influx Experiment

[0102] Experimental method: Macrophage cells were seeded at a density of 3 × 10⁶ cells per well. 4 –4×10 4 Cells were cultured in 35mm glass-bottomed culture dishes for 24 hours. Then, 20 μm of AZO was added to each well and incubated for 2 hours. Afterwards, Ca... 2+ The indicator (Fluo-4) was incubated in the dark at 37°C for 20 min, and then the cells were washed three times with PBS. The cells were then irradiated with a 660 nm laser for 5 min, and the fluorescence images were observed using a Zeiss confocal microscope.

[0103] Figure 13 Under 660 nm irradiation, azobenzene molecules undergo isomerization, thereby activating the opening of TRPV1 channels in macrophages. In macrophages infused with azobenzene molecules, 660 nm laser irradiation resulted in a sharp increase in flou-4 green fluorescence, while cells incubated without laser irradiation and those irradiated only with laser showed negligible changes in green fluorescence. These results indicate that azobenzene molecules can effectively open TRPV1 channels in macrophages under 660 nm light irradiation.

[0104] Example 3: Anti-inflammatory phenotype of macrophages after SFAZO light exposure (immunofluorescence and flow cytometry)

[0105] 1. Experimental materials:

[0106] AZO: Compound 9

[0107] SF: A 5 wt% SF solution was prepared by dissolving SF (silk fibroin) in deionized water. Then, 1 wt% PEG-DE was mixed in. The mixture was stirred at 60°C for 20 minutes, after which 1 wt% CaCl2 was added. Each milliliter of solution was then poured into a 4×4 cm square weighing dish and air-dried to form a cross-linked SF film (SF).

[0108] SFAZO: The film was made by adding 20 μM AZO to the final solution, mixing thoroughly, pouring the mixture into a mold, and allowing it to air dry.

[0109] SF-laser: Silk fibroin membrane plus light exposure;

[0110] SFAZO-laser: An SF film containing AZO is exposed to light;

[0111] 2. Experimental Methods:

[0112] RAW 264.7 cells were plated (5×10⁻⁶ cells per plate). 4 RAW 264.7 cells were seeded in 5 × 10⁶ wells and stimulated with LPS (1 μg / ml, 6 hours post-seeding) to drive M1 polarization. After adding material and irradiating at 660 nm, the cells were fixed for immunofluorescence staining after 48 hours. The expression of inflammatory cytokines (TNF-α and IL-10) was visualized using confocal imaging. Furthermore, RAW 264.7 cells were seeded in 5 × 10⁶ wells. 4 Cells were co-cultured (wells) and stimulated with LPS (1 μg / ml, 6 h, 6 h post-seeding) to drive M1 polarization. After adding material and irradiating at 660 nm, cells were co-cultured for 48 h. Cells were then stained with fluorescently labeled antibodies against CD86 and CD206 for 30 min to assess macrophage polarization. Fluorescence signals were analyzed by flow cytometry (Invitrogen).

[0113] 3. Experimental Results:

[0114] To assess the effect of SFAZO on macrophage polarization

[0115] Figure 14 Immunofluorescence staining was performed to detect inflammation-related cytokines TNF-α and IL-10. Compared with other groups, the SFAZO+LASER-treated groups showed decreased TNF-α expression, which was combined with upregulation of IL-10.

[0116] Figure 15 Compared with the SF and SF+LASER and SF+AZO groups, the SFAZO+LASER group showed the highest CD206 expression (28.4%) and the lowest CD86 expression (7.45%).

[0117] 4. Experimental Analysis: These results confirm that SFAZO+LASER can significantly promote macrophage polarization from M1 to M2, reduce pro-inflammatory factors and promote the secretion of anti-inflammatory factors, thereby creating a regenerative microenvironment that is conducive to healing progress.

[0118] Experimental Example 4: The in vivo therapeutic effect of SFAZO on the healing of normal skin wounds.

[0119] 1. Experimental Method:

[0120] Twenty female SD rats (180-220g, Chengdu, China) were used to establish a skin wound model. Female rats were chosen as the experimental model primarily because they provided better wound visualization compared to male rats. A full-thickness wound model was established by creating a circular wound with a diameter of 8mm on the rat's back using a tissue drill. The rats were randomly assigned to four groups: SF group, SF+laser group, SFAZO group, and...

[0121] SFAZO+laser team. Subsequent data collection will take place over 3 and 14 days.

[0122] 2. Experimental Results:

[0123] Figure 16 In a rat model of normal skin wound, immunofluorescence analysis of inflammatory factors (TNF-α and IL-10) on day three showed that SFAZO-laser had a significant regulatory effect on inflammation. Staining results indicated that the SFAZO-laser group significantly downregulated the expression of the pro-inflammatory factor TNF-α and upregulated the expression of the anti-inflammatory factor IL-10 by opening the TRPV1 ion channel. This regulatory effect suggests that SFAZO-LIGHT effectively modulates the polarization of macrophages towards the M2 anti-inflammatory phenotype, reducing the persistence of inflammation and paving the way for subsequent re-epithelialization and wound healing.

[0124] Figure 17 On day 14, skin wounds were examined using Sirius red staining under a polarized light microscope to assess Col I and Col III deposition post-treatment. The SF-AZO-laser group exhibited the highest Col I and Col III deposition compared to other groups.

[0125] SFAZO, when exposed to 660nm light, has the effect of regulating the anti-inflammatory properties of macrophages, thereby achieving the effect of skin wound repair.

Claims

1. A compound that regulates TRPV1 ion channels, characterized in that: The structural formula is shown in Formula I:

2. A method for synthesizing the compound according to claim 1, characterized in that: It is based on methyl 4-amino-3-fluorophenylcarboxylate and 3-(tert-butoxycarbonylamino)propionic acid as starting compounds. Among them, methyl 4-amino-3-fluorophenylcarboxylate is synthesized by selective electrophilic chlorination of the aromatic ring, C-H directed chlorination, and ester hydrolysis. 3-(tert-Butoxycarbonylamino)propionic acid was subjected to TBTU / DIPEA-mediated amidation and Boc deprotection to yield compound 7; Compound 4 was then semi-selectively amidated under the action of HATU / DIPEA, and then synthesized with compound 7 to obtain the bisamide-azo product shown in Formula I.

3. The method for synthesizing the compound according to claim 2, characterized in that: The synthetic route is as follows:

4. The method for synthesizing the compound according to claim 3, characterized in that: Includes the following steps: (1) Synthesize compound 2: DBU was added to a solution of compound 1 dissolved in DCM; the mixture was stirred at room temperature and then cooled to -78°C; N-chlorosuccinimide NCS was added to the reaction mixture as a solid; the orange solution was stirred at -78°C and then quenched by adding a saturated bicarbonate solution; the organic layer was separated, washed successively with water and HCl, dried with anhydrous sodium sulfate, concentrated to dryness under vacuum, and purified to obtain the product; (2) Synthesis of compound 3: Compound 2 synthesized in step (1), Pd(OAc)2 and NCS were dissolved in AcOH and heated to 120°C for 6 hours; after cooling to room temperature, the solvent was removed under vacuum and the brown residue was dissolved in CH2Cl2; the organic layer was washed successively with saturated NaHCO3 aqueous solution and saturated NaCl aqueous solution and dried with Na2SO4; the residue was purified by rapid column chromatography (hexane-ethyl acetate) as eluent to obtain product compound 3; (3) Synthesis of compound 4: Compound 3 and LiOH were dissolved in an aqueous solution of methanol / tetrahydrofuran and stirred at room temperature; the reaction solution was dissolved in an aqueous solution of CH2Cl2 and the aqueous layer was extracted; ethyl acetate was added to the aqueous layer in sequence, and diluted concentrated hydrochloric acid was added dropwise to extract the ethyl acetate layer, which was then dried under vacuum to obtain the product; (4) Synthesize compound 6: Compound 5 and TBTU were dissolved in DMF, and DIPEA was added sequentially. 4-(aminomethyl)-2-methoxyphenol: The reaction solution was stirred at room temperature; ethyl acetate was added sequentially to the reaction solution, and the ethyl acetate layer was extracted by washing with saturated NaCl aqueous solution and dried over Na2SO4; the residue was purified by rapid column chromatography (dichloromethane-methanol) as eluent to obtain the product; (5) Synthesis of compound 7: Compound 6 was dissolved in a trifluoroacetic acid / dichloromethane solution and stirred at room temperature; the solvent was removed from the reaction solution under vacuum to obtain product compound 7; (6) Synthesis of compound 8: Compound 4 and HATU were dissolved in DMF, and DIPEA and butan-1-amine were added sequentially. The reaction solution was stirred at room temperature, and ethyl acetate was added sequentially. The ethyl acetate layer was obtained by washing with saturated NaCl aqueous solution and dried with Na2SO4. The residue was purified by rapid column chromatography (dichloromethane-methanol) as eluent to obtain product compound 8. (7) Synthesize compound 9: Compound 8 and HATU were dissolved in DMF, and DIPEA and compound 7 were added sequentially. The reaction solution was stirred at room temperature. Ethyl acetate was added sequentially to the reaction solution, and the ethyl acetate layer was extracted by washing with saturated NaCl aqueous solution and dried over Na2SO4. The residue was purified by rapid column chromatography (dichloromethane-methanol) as eluent to obtain product compound 9 (formula I).

5. The method for synthesizing the compound according to claim 4, characterized in that: The molar ratio of compound 1, DBU, and DCM is 1:2:

2. The molar ratio of compound 2, Pd(0Ac), and NCS is 1:0.2:5; The molar ratio of compound 3 to LiOH is 1:3.5; The molar ratio of compound 5, TBTU, and 4-(aminomethyl)-2-methoxyphenol is 1:1.2:

1. Compound 4: The molar ratio of HATU and butan-1-amine is 1:1.2:1:1; The molar ratio of compound 8:HATU:DIPEA:compound 7 is 1:1.2:3.5:1.

1.

6. Use of the compound of claim 1 in the preparation of a drug that regulates the TRPV1 ion channel.

7. Use of the compound of claim 1 in the preparation of a medicament having anti-inflammatory effects.

8. A pharmaceutical composition having anti-inflammatory effects, characterized in that: It comprises the compound of claim 1, and is prepared into a pharmaceutically commonly used formulation by adding pharmaceutically acceptable excipients or auxiliary ingredients.

9. The pharmaceutical composition with anti-inflammatory effect according to claim 8, characterized in that: The auxiliary material mentioned is silk fibroin film.

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