Optically active capsaicin molecules, methods of synthesis and use thereof

Abstract

The application discloses a kind of optically active capsaicin molecules and its synthesis method and application.The optically active capsaicin molecules are (R)-N-(4-hydroxy-3-methoxybenzyl)-2-phenylpropanamide and (S)-N-(4-hydroxy-3-methoxybenzyl)-2-phenylpropanamide, which are prepared by amidation reaction of (R)-2-phenylpropanoic acid or (S)-2-phenylpropanoic acid with 4-(aminomethyl)-2-methoxyphenol at room temperature.The optically active capsaicin molecules have the characteristics of activating TRPV1, and are expected to be applied in the fields of drug research and pharmaceutical engineering.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis and relates to an optically active capsaicin molecule, its synthesis method, and its applications. Background Technology

[0002] Capsaicin is a class of chemical compounds commonly found in plants of the Capsicum genus, giving chili peppers and chili products their pungent, hot flavor. Capsaicin molecules possess a variety of interesting biological activities and sensory properties, making them a focus of attention in various fields. In the food industry, capsaicin is widely used as a flavoring agent, enhancing the taste and aroma of food. In biological and medical research, chiral capsaicin molecules are used as a tool in pain research, contributing to a deeper understanding of how the nervous system works. Furthermore, capsaicin molecules are also being explored in areas such as obesity treatment and cancer therapy, showing potential applications for improving human health. In the pharmaceutical field, capsaicin is used to prepare analgesics and topical ointments because they can activate nerve endings and relieve pain. In addition, capsaicin also possesses anti-inflammatory and antioxidant properties. In cosmetics manufacturing, capsaicin is used to stimulate the skin and provide a warming sensation, while also exhibiting antioxidant and anti-aging properties.

[0003] Currently, capsaicin used industrially is mainly extracted from natural chili peppers, and can also be obtained through chemical synthesis in the laboratory. Traditional capsaicin itself is not optically active because its stereoisomers are overlapping. This means that the capsaicin molecule lacks a chiral center, and therefore does not have left-handed or right-handed isomers (Yang F, Xiao X, Cheng W, et al. Structural mechanism underlying capsaicin binding and activation of the TRPV1ion channel. Nature chemical biology, 2015, 11, 518-524.).

[0004] Researchers face many unresolved questions regarding the biological effects of capsaicin molecules on activating the transient receptor potential 1 (TRPV1). TRPV1 is a receptor closely associated with pain perception, and capsaicin interacts with it to elicit heat and pain sensations. However, different optical isomers of capsaicin may interact with TRPV1 in different ways, leading to varying biological effects. Designing and synthesizing optically active capsaicin molecules has been a challenging scientific problem. Developing novel synthetic methods to synthesize chiral capsaicin molecules and conducting systematic bioactivity studies to reveal the activation mechanisms and effects of different chiral isomers on TRPV1 receptors will lead to a better understanding of the interaction between capsaicin and biological systems, providing more opportunities for future medical and biological applications and potentially guiding innovation in drug development, pain management, and other fields. Summary of the Invention

[0005] The purpose of this invention is to provide an optically active capsaicin molecule, its synthesis method, and its applications.

[0006] The technical solution for achieving the objective of this invention is as follows:

[0007] The optically active capsaicin molecule is (R)-N-(4-hydroxy-3-methoxybenzyl)-2-phenylpropionamide (R-capsaicin molecule), with the structural formula shown below:

[0008]

[0009] Alternatively, it could be (S)-N-(4-hydroxy-3-methoxybenzyl)-2-phenylpropionamide (S-capsaicin molecule), with the structural formula shown below:

[0010]

[0011] The method for synthesizing the above-mentioned optically active capsaicin molecules includes the following steps:

[0012] Under a dry, inert atmosphere, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used as catalysts for the amidation reaction of (R)-2-phenylpropionic acid or (S)-2-phenylpropionic acid with 4-(aminomethyl)-2-methoxyphenol at room temperature. After the reaction was completed, hydrochloric acid was added to neutralize NHS and unreacted EDC, and the mixture was extracted with ethyl acetate to obtain (R)-N-(4-hydroxy-3-methoxyphenylmethyl)-2-phenylpropionamide or (S)-N-(4-hydroxy-3-methoxyphenylmethyl)-2-phenylpropionamide.

[0013] Preferably, the molar ratio of (R)-2-phenylpropionic acid or (S)-2-phenylpropionic acid, 4-(aminomethyl)-2-methoxyphenol, EDC, and NHS is 1:1:1.2 to 1.5.

[0014] Preferably, the reaction time is 4 hours.

[0015] Preferably, the hydrochloric acid concentration is 0.5 mol / L.

[0016] The above-mentioned optically active capsaicin molecules are used in the preparation of drugs for the prevention or treatment of pain.

[0017] The application of the aforementioned optically active capsaicin molecules in the preparation of drugs for weight loss.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) This invention synthesizes for the first time an optically active capsaicin molecule with a half-maximal effective concentration (WMC) that differs from that of conventional capsaicin, and the WMC also differs for different chiral isomers. The WMC of R-capsaicin is approximately 0.3 μmol / L, while that of S-capsaicin and conventional capsaicin is approximately 1.1 μmol / L and 0.6 μmol / L, respectively. Furthermore, R-capsaicin and S-capsaicin molecules exhibit higher activation currents than conventional capsaicin at near-saturation concentrations, indicating that the optically active capsaicin possesses superior activation effects.

[0020] (2) The synthesis method of the present invention is simple and easy to implement. In the pharmaceutical field, capsaicin molecules with different chiral isomers can be used to prepare drugs for pain perception or treatment, obesity treatment and cancer treatment. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the R-capsaicin molecule;

[0022] Figure 2 This is a schematic diagram of the structure of the S-capsaicin molecule;

[0023] Figure 3 The 1H NMR spectra of R-capsaicin and S-capsaicin molecules;

[0024] Figure 4 The carbon NMR spectra of R-capsaicin and S-capsaicin molecules;

[0025] Figure 5 A schematic diagram of a whole-cell patch-clamp recording experiment;

[0026] Figure 6 The current curve generated by the activation of the capsaicin receptor TRPV1 by the R-capsaicin molecule;

[0027] Figure 7 The current curve generated by the activation of the capsaicin receptor TRPV1 by the S-capsaicin molecule;

[0028] Figure 8 Concentration-dependent curves for the activation of the capsaicin receptor TRPV1 by R-capsaicin and S-capsaicin molecules. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0030] Example 1

[0031] Synthesis of R-capsaicin: In a dry reaction vessel under nitrogen atmosphere, 1.0 mol of (R)-2-phenylpropionic acid and 1.0 mol of 4-(aminomethyl)-2-methoxyphenol were added. Then, 1.2-1.5 mol of EDC and NHS were added as catalysts. The reaction mixture was then stirred at room temperature for 4 hours. After the reaction was complete, the NHS and unreacted EDC in the reaction mixture were neutralized with 0.5 mol / L dilute hydrochloric acid. (R)-N-(4-hydroxy-3-methoxyphenylmethyl)-2-phenylpropionamide was extracted with ethyl acetate. Finally, pure (R)-N-(4-hydroxy-3-methoxyphenylmethyl)-2-phenylpropionamide, i.e., R-capsaicin, was obtained by column chromatography. Its structural diagram is shown below. Figure 1 As shown, the proton and carbon NMR spectra are respectively as follows: Figures 3-4 As shown.

[0032] 1 H NMR(800MHz,Chloroform-d)δ7.35-7.24(m,5H),6.80(d,J=7.8Hz,1H),6.66-6.61(m,2H),5.57(s,1H),5.54(s,1H), 4.34(dd,J=14.6,5.8Hz,1H),4.29(dd,J=14.7,5.4Hz,1H),3.78(s,3H),3.58(q,J=7.0Hz,1H),1.55(d,J=7.0Hz,3H). 13 C NMR (201MHz, CDCl3) δ174.1,146.7,145.1,141.5,130.4,129.1,127.7,127.4,120.5,114.4,110.2,55.9,47.3,43.5,18.6.

[0033] Example 2

[0034] Synthesis of S-capsaicin molecules: This example is basically the same as Example 1, except that (R)-2-phenylpropionic acid is replaced with (S)-2-phenylpropionic acid to obtain pure (S)-N-(4-hydroxy-3-methoxybenzyl)-2-phenylpropionamide, which is the S-capsaicin molecule. Its structural diagram is shown in Figure 1. Figure 2 As shown, the proton and carbon NMR spectra are respectively as follows: Figures 3-4 As shown.

[0035] Example 3

[0036] Biological function assays of R-capsaicin and S-capsaicin molecules: Electrophysiological experiments were performed using the HEK293 cell line. One day before transfection, HEK293 cells were digested and then seeded in 60 mm culture dishes containing coverslips (cell crawling smears). The next day, after the cells had stably adhered to the coverslips, transient overexpression of the gene was performed using the conventional calcium phosphate method. To facilitate the effective identification of TRPV1-positive cells in electrophysiological experiments, the fluorescent protein gene YFP was fused to the C-terminus of the TRPV1 gene, constructing the TRPV1-YFP plasmid (the rat TRPV1 (GenBank: AAC53398.1) sequence synthesized by the company and inserted into the pcDNA3.1 vector). The fluorescent protein YFP sequence was amplified by PCR, and the resulting PCR fragment contained NotI and XbaI restriction sites at both ends. Subsequently, the vector and PCR fragment were double-digested and ligated together using ligase, thus fusing YFP to the C-terminus of TRPV1, ultimately constructing the TRPV1-YFP plasmid. HEK293 cells were transfected with 3 μg TRPV1-YFP and expressed for 24 hours. Cell slices were then transferred from the culture dish to an electrophysiological platform for whole-cell patch-clamp recording. Figure 5 As shown. In electrophysiological testing, the electrode intraelectrode composition (mmol / L) was: 140 KCl, 10 HEPES, 0.5 EGTA, with osmolarity adjusted to 290 mOsm using glucose and pH adjusted to 7.5 using KOH. The extracellular fluid was designed to simulate physiological conditions, with the following composition (mmol / L): 135 NaCl, 5 KCl, 2 CaCl2, 10 HEPES, with osmolarity adjusted to 310 mOsm using glucose and pH adjusted to 7.5 using NaOH. Electrophysiological data were acquired using a Clampex 11.1. During electrophysiological recording, the cell membrane potential was clamped at -80 mV, and rapid perfusion systems were used to apply 10 μmol / L R-capsaicin and S-capsaicin molecular stimulation, respectively.

[0037] from Figure 6 and Figure 7The current curves show that the TRPV1 channel is rapidly activated and generates an inward current when stimulated by either R-capsaicin or S-capsaicin. This indicates that both R-capsaicin and S-capsaicin can rapidly and effectively bind to the ligand-binding pocket of the TRPV1 channel, producing an activation effect.

[0038] Example 4

[0039] Channel activation performance assays of R-capsaicin and S-capsaicin molecules: First, R-capsaicin, S-capsaicin, and normal capsaicin were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions with a final concentration of 100 mmol / L. For whole-cell electrophysiological assays of HEK293 cells overexpressing TRPV1-YFP, the stock solutions were diluted to 10 μmol / L with extracellular fluid, and then gradually diluted 10-fold to 1.0 μmol / L, 0.1 μmol / L, and 0.01 μmol / L. Different concentrations of R-capsaicin, S-capsaicin, and normal capsaicin were applied using a rapid perfusion system, and the activation current and cell capacitance of TRPV1-YFP were recorded. Finally, the correlation between current density (the ratio of current magnitude to cell capacitance) and the concentrations of R-capsaicin and S-capsaicin molecules was statistically analyzed and fitted to evaluate the channel activation performance of R-capsaicin and S-capsaicin molecules, and compared with the activation ability of normal capsaicin. Figure 8 The curve fitting results show that the activation intensity of TRPV1 increases with the increase of R-capsaicin and S-capsaicin molecule concentrations, manifested as an increase in current density. Boltzmann fitting estimations indicate that the half-maximal effective concentration (WMC) of R-capsaicin is approximately 0.3 μmol / L, lower than that of S-capsaicin (~1.1 μmol / L) and normal capsaicin (~0.6 μmol / L). Furthermore, both R-capsaicin and S-capsaicin exhibit larger activation currents at saturation concentrations, suggesting that optically active capsaicins may possess stronger activation capabilities.

Claims

1. An optically active capsaicin molecule, characterized in that, It is (R)-N-(4-hydroxy-3-methoxybenzyl)-2-phenylpropionamide, with the following structural formula: ； Alternatively, it may be (S)-N-(4-hydroxy-3-methoxybenzyl)-2-phenylpropionamide, with the structural formula shown below: 。 2. The method for synthesizing optically active capsaicin molecules according to claim 1, characterized in that, Includes the following steps: Under a dry, inert atmosphere, (R)-2-phenylpropionic acid or (S)-2-phenylpropionic acid reacts with 4-(aminomethyl)-2-methoxyphenol at room temperature using EDC and NHS as catalysts. After the reaction is complete, hydrochloric acid is added to neutralize NHS and unreacted EDC, and the mixture is extracted with ethyl acetate to obtain (R)-N-(4-hydroxy-3-methoxyphenylmethyl)-2-phenylpropionamide or (S)-N-(4-hydroxy-3-methoxyphenylmethyl)-2-phenylpropionamide.

3. The synthesis method according to claim 2, characterized in that, The molar ratio of (R)-2-phenylpropionic acid or (S)-2-phenylpropionic acid, 4-(aminomethyl)-2-methoxyphenol, EDC, and NHS is 1:1:1.2~1.

5.

4. The synthesis method according to claim 2, characterized in that, The reaction time is 4 hours.

5. The synthesis method according to claim 2, characterized in that, The hydrochloric acid concentration is 0.5 mol / L.

6. The use of the optically active capsaicin molecule according to claim 1 in the preparation of a medicament for the prevention or treatment of pain.

7. The use of the optically active capsaicin molecule according to claim 1 in the preparation of a drug for weight loss.

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