Use of anagalline a in the preparation of analgesic drugs

Abstract

The application relates to application of anagirine A in preparation of analgesic drugs, and relates to an anagirine A compound separated from anagallis arvensis, which is an analgesic drug with inhibitory effects on transient receptor potential cation channel subfamily M member 8 (TRPM8), voltage-gated potassium channel 1.2 (Kv1.2), voltage-gated potassium channel 1.3 (Kv1.3), transient receptor potential cation channel protein 6 (TRPC6) and voltage-gated calcium channel 2.1 (Cav2.1), and is specifically a drug for neuropathic pain, traumatic pain or inflammatory pain. The compound anagirine A in the application can be combined with multiple analgesic related target points, so that a synergistic effect is generated, the treatment effect is enhanced, and side effects and drug resistance are reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of anhalonidine A in preparation of analgesic drugs. BACKGROUND

[0002] It is estimated that about 600 million people worldwide suffer from neuropathic pain and cancer-related pain. At present, opioid drugs are still indispensable in the treatment of these diseases, especially in the treatment of advanced cancer pain. However, its addiction, respiratory depression, cold allodynia and harm to public health need to be paid attention to. Therefore, it is crucial to develop non-addictive and high-efficiency non-opioid analgesics. At present, three main strategies have been reported for the development of non-opioid analgesics, such as developing spinal inhibitory synaptic enhancers like gamma-aminobutyric acid (GABA) modulators, implementing neuro-immune axis blockers like CC chemokine receptor 2 (CCR2) and CC chemokine receptor 5 (CCR5) antagonists, and exploring new peripheral analgesic targets like TRPM8 and voltage-gated sodium ion channel 1.8 (Nav1.8).

[0003] TRPM8 is an ion channel that mediates cold sensation and pain sensation, and is distributed in sensory neurons of the dorsal root ganglion, trigeminal ganglion and prostate. Under physiological conditions, low temperature below 28℃ or menthol activates TRPM8 to trigger calcium influx, which mediates cold sensation transmission. However, under pathological conditions such as nerve injury and chemotherapy neuropathy, excessive activation of TRPM8 leads to cold allodynia, which causes severe pain even under slight cold stimulation. TRPM8 antagonists can directly inhibit abnormal neuron firing by selectively blocking the channel and inhibiting calcium influx, and down-regulate central sensitization mediated by spinal metabotropic glutamate receptor (mGluR) pathway, thereby reversing cold allodynia and blocking neuropathic, cancerous and inflammatory pain signals. However, existing antagonists (such as VBJ103) are limited in clinical research due to potential hypothermia side effects and low bioavailability. Notably, Kv1.2 potassium channels regulate cold sensation threshold by molecular interaction with TRPM8, and play a key role in action potential conduction triggered by cold stimulation. Simultaneous targeting of TRPM8 and Kv1.2 can synergistically inhibit calcium influx and potassium efflux, significantly reducing neuronal excitability and peripheral nociceptive signal transmission. This strategy not only effectively inhibits pain transmission and provides non-opioid analgesia, but also avoids adverse reactions such as hypothermia caused by single inhibition of TRPM8.

[0004] Therefore, it is of great value to develop new non-opioid multi-target potent analgesics that act on TRPM8 and Kv1.2. It is a hot spot in drug research to explore lead compounds with significant pharmacological activity from traditional medicinal plants. Therefore, it is an effective way to develop new analgesics to find novel ion channel inhibitors and compounds with analgesic effects from medicinal plants.

[0005] Anagirine A is a pair of highly conjugated diaminated 6 / 6 / 6 / 6 / 5 pentacyclic alkaloid enantiomers with a unique 6,13-diazapentacyclo[10.3.3 1,3 .1.0 2,7 .0 8,12 ]nonadecane ring system structure, studies have shown that the compound has certain anti-inflammatory effect, patent number: ZL 2023 1 1104612.0 Compound with Anti-inflammatory Effect in Anagiroots and Preparation and Application. In the process of continuing to study the biological activity of the compound, it also has good analgesic activity, and plays a role in analgesia by inhibiting multiple analgesia-related targets such as TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1. The compound anagirine A is used to inhibit TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 ion channels. SUMMARY

[0006] The purpose of the present application is to provide the application of anagirine A in the preparation of analgesic drugs, which is an anagirine A compound isolated from anagiroots. The compound has inhibitory effect on TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1, and is used in the preparation of analgesic drugs. Specifically, it is used in the preparation of drugs for neuropathic pain, traumatic pain or inflammatory pain. The compound anagirine A in the present application can bind to multiple analgesia-related targets, thereby producing a synergistic effect, enhancing the therapeutic effect, and reducing side effects and drug resistance, to solve the technical problems of low safety and large adverse reactions of analgesic drugs in the prior art.

[0007] The application of anagirine A in the preparation of analgesic drugs.

[0008] The application of anagirine A in the preparation of analgesic drugs described in the present application is further research based on patent number: ZL 2023 1 1104612.0 Compound with Anti-inflammatory Effect in Anagiroots and Preparation and Application, wherein the structural formula of anagirine A is: dextrogyre formula (+)-1, levogyre formula (-)-1, or a mixture of racemic formula (+)-1 and formula (-)-1.

[0009]

[0010] The application of anagirine A compound (dextrogyre formula (+)-1, levogyre formula (-)-1, or a mixture of racemic formula (+)-1 and formula (-)-1) in the preparation of analgesic drugs.

[0011] The application of the anagalline A compound in inhibiting ion channels of receptor potential cation channel subfamily M member 8 (TRPM8), voltage-gated potassium ion channel 1.2 (Kv1.2), voltage-gated potassium ion channel 1.3 (Kv1.3), transient receptor potential cation channel protein 6 (TRPC6) and voltage-gated calcium ion channel 2.1 (Cav2.1).

[0012] The application of the anagalline A compound in preparing analgesic drugs has the following technical advantages compared with the prior art:

[0013] (1) In terms of analgesic activity, the anagalline A compounds (±)-1, (+)-1 and (-)-1 provided in the application have significant analgesic activity on acetic acid-induced mouse writhing at a dose of 5 mg / kg. And at lower doses of 1 mg / kg, 0.2 mg / kg and 0.02 mg / kg, the analgesic activity of (±)-1 and (-)-1 is stronger than that of morphine (a first-line analgesic drug in clinical use), which can be used as a new drug component or a lead compound of analgesic agents and has a broad development prospect.

[0014] (2) The patch clamp experiment shows that the anagalline A compounds (+)-1 and (-)-1 play their analgesic activity by inhibiting multiple analgesic targets such as TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 ion channels, and the computer molecular docking technology is used to analyze the action sites between the anagalline A compounds (+)-1 and (-)-1 and TRPM8 and Kv1.2, which provides a theoretical basis for further designing and developing new multi-target analgesic agents acting on TRPM8 and Kv1.2 ion channels.

[0015] The application of the anagalline A compound in analgesic drugs, specifically relates to analgesic activity experiments, action target verification and molecular docking of the anagalline A (formula (+)-1, formula (-)-1).

[0016] The application provides that the anagalline A has the following right-handed formula (+)-1, left-handed formula (-)-1, or is a mixture of the racemic formula (+)-1 and formula (-)-1.

[0017]

[0018] Compound (±)-1: N-(1-(2,2,5,5,8,12,12-heptamethyl-3,7-diketo-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopentano[7,8]azepino[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide;

[0019] Compound (+)-1: N-(1-((8R,9aS,10aS)-2,2,5,5,8,12,12-heptamethyl-3,7-diketo-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopentano[7,8]azepino[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide;

[0020] Compound (-)-1: N-(1-((8S,9aR,10aR)-2,2,5,5,8,12,12-heptamethyl-3,7-diketo-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopentano[7,8]azepino[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide.

[0021] The present application provides experimental data of analgesic activity of the anagalline A compound (±)-1, (+)-1 and (-)-1; the analgesic activity of the anagalline A compound (±)-1, (+)-1 and (-)-1 is determined by an acetic acid-induced mouse writhing test. The results show that the compounds (±)-1, (+)-1 and (-)-1 have significant analgesic activity at a dose of 5 mg / kg; and at lower doses of 1 mg / kg, 0.2 mg / kg and 0.02 mg / kg, the analgesic activity of (±)-1 and (-)-1 is stronger than that of morphine (a first-line analgesic drug clinically used).

[0022] The present application screens possible target points of the anagalline A compound (+)- / (-)-1, and proves that the compound (+)- / (-)-1 is an inhibitor of TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 through a patch clamp experiment.

[0023] The present application uses molecular docking software such as Autodock vina and Discovery Studio to study the action mode of the anagalline A compound (+)- / (-)-1 and TRPM8 (8E4Q) and Kv1.2 (5WIE); and further confirms the binding mechanism of the compound (-)-1 and TRPM8 through molecular dynamics simulation. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 6 is a graph showing the analgesic activity of the anaferine A compound (±)-1, (+)-1 and (-)-1 in the acetic acid-induced mouse writhing model at doses of 5.0, 1.0, 0.2 and 0.04 mg / kg, wherein (A) is the number of writhing times of mice within 30 minutes at different concentrations of the compound, (B) is the percentage inhibition of analgesia at different concentrations of the compound, and NT represents not tested.

[0025] Figure 2 Figure 7 is a graph showing the TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 ion channel blocking experiment results of the anaferine A compound (+)- / (-)-1. (A) is the peak current inhibition curve of different concentrations of (+)-1 on TRPM8, (B) is the IC 50 value curve of the inhibition of (+)-1 on TRPM8, (C) is the peak current inhibition curve of different concentrations of (-)-1 on TRPM8, (D) is the IC 50 value curve of the inhibition of (-)-1 on TRPM8, (E) is the peak current inhibition curve of different concentrations of (-)-1 on Kv1.2, (F) is the IC 50 value curve of the inhibition of (-)-1 on Kv1.2.

[0026] Figure 3 Figure 8 is a graph showing the molecular docking results of the anaferine A compound (+)- / (-)-1 and TRPM8. (A) is a schematic diagram of the compound (+)-1 embedded in the entrance pore of the TRPM8 channel by electrostatic surface interaction, (B) is a three-dimensional structure schematic diagram of the interaction between the compound (+)-1 and the key residues of the TRPM8 channel, (C) is a two-dimensional structure schematic diagram of the interaction between (+)-1 and the key residues of the TRPM8 channel, (D) is a schematic diagram of the compound (-)-1 embedded in the entrance pore of the TRPM8 channel by electrostatic surface interaction, (E) is a three-dimensional structure schematic diagram of the interaction between the compound (-)-1 and the key residues of the TRPM8 channel, and (F) is a three-dimensional structure schematic diagram of the interaction between (-)-1 and the key residues of the TRPM8 channel. The green, light green and purple circles and lines represent hydrogen bonds, van der Waals forces and π-alkyl interactions, respectively.

[0027] Figure 4This diagram shows the molecular docking results of anachygen base A compound (+)- / (-)-1 with Kv1.2. In the diagram, (A) is a schematic diagram of compound (+)-1 embedding into the entrance pore of the Kv1.2 channel via electrostatic surface interaction; (B) is a three-dimensional structural schematic diagram of the interaction between compound (+)-1 and key residues of the Kv1.2 channel; (C) is a two-dimensional structural schematic diagram of the interaction between (+)-1 and key residues of the Kv1.2 channel; (D) is a schematic diagram of (-)-1 embedding into the entrance pore of the Kv1.2 channel via electrostatic surface interaction; (E) is a three-dimensional structural schematic diagram of the interaction between (-)-1 and key residues of the Kv1.2 channel; and (F) is a three-dimensional structural schematic diagram of the interaction between (-)-1 and key residues of the Kv1.2 channel. The green, light green, and purple circles and lines represent hydrogen bonds, van der Waals forces, and π-alkyl interactions, respectively. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Example 1

[0030] The analgesic activity of anachymosis base A compounds (±)-1, (+)-1, and (-)-1 was evaluated using an acetic acid-induced mouse writhing model.

[0031] Half male and half female Kunming mice were randomly divided into three groups (n=10 / group). The control group was injected intraperitoneally with 0.1 ml / 10 g of physiological saline, the morphine group was injected intraperitoneally with 5, 1, 0.2 or 0.04 mg / kg of morphine, and the test compound group was injected intraperitoneally with 5, 1, 0.2 or 0.04 mg / kg of the compound in physiological saline solution. Thirty minutes after administration, each group of mice was injected intraperitoneally (ip) with 0.8% (v / v) acetic acid solution (0.1 ml / 10 g), and the cumulative number of writhing responses within 30 minutes was immediately recorded. The writhing inhibition rate was calculated using the formula: Writhing inhibition rate (%) = [(number of writhing responses in the control group - number of writhing responses in the drug group) / number of writhing responses in the control group] × 100%.

[0032] Conclusion: The analgesic activity of anachine A compounds (±)-1, (+)-1, and (-)-1 was evaluated as follows: Figure 1The results showed that all the tested anagalline A compounds exhibited significant analgesic activity at the milligram / kilogram dose, with the writhing inhibition rate ranging from 54.8% to 96.6%; at the 1 mg / kg dose, the inhibition rates of (±)-1 (74.9% ± 0.8%), (-)-1 (88.1% ± 0.8%) and (+)-1 (89.9% ± 1.7%) were significantly higher than that of morphine (73.8% ± 1.3%); notably, at the clinically equivalent dose of 0.2 mg / kg (the morphine treatment dose), the analgesic activities of (±)-1 (59.8% ± 0.8%) and (-)-1 (74.9% ± 0.8%) were significantly superior to that of morphine (56.5% ± 2.3%); even at the lowest test dose of 0.04 mg / kg, (±)-1 (51.6% ± 1.8%) and (-)-1 (57.7% ± 1.1%) still maintained higher inhibition rates than morphine (44.2% ± 2.5%); the chiral selectivity analysis showed that the analgesic activity of the left isomer (-)-1 was significantly stronger than that of the right isomer (+)-1 and the racemate (±)-1 at all doses.

[0033] Example 2

[0034] The TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 inhibitory activities of the anagalline A compounds (+)- / (-)-1 were evaluated by patch clamp electrophysiological experiments:

[0035] The experiments were performed using HEK293T cells (ATCC Cell Bank), which were cultured in a 37°C incubator with a 5% carbon dioxide environment, and the culture medium was DMEM / Ham's F-12 basal medium supplemented with 10% fetal bovine serum; during the electrophysiological detection, the experimental data were collected by an EPC-10 amplifier (HEKA) and stored in the PatchMaster (HEKA) software; an inverted microscope (Mshot, China) and an MP225 microelectrode manipulator (Sutter Instruments, USA) were used for patch clamp recording; the recording electrode was contacted with the cell to form a GΩ seal, and then the whole-cell recording mode was used; all experiments were performed at room temperature;

[0036] Conclusion: At a concentration of 10 micromolar (μmol / L), the anagalline A compound (+)-1 exhibited significant inhibition of TRPM8, with an inhibition percentage of 68.76% ± 0.50%, moderate inhibition of Kv1.2, with an inhibition percentage of 30.95% ± 1.17%, and weak inhibition of TRPC6, Cav2.1 and Kv1.3, with inhibition rates of 3.77% ± 0.62%, 9.40% ± 6.12% and 4.50% ± 2.02%, respectively (see Table 2);

[0037] Table 2. The percentage of inhibition of five ion channels related to analgesia by the anaferine A compound (+)-1 and (-)-1 (2 replicates, concentration of 10 μmol / L)

[0038]

[0039] In contrast, the anaferine A compound (-)-1 showed significant inhibition of TRPM8 and Kv1.2 channels, with inhibition percentages of 68.37% ± 5.88% and 46.20% ± 1.19%, respectively (see Table 2); moderate inhibition of Cav2.1, with an inhibition rate of 21.20% ± 6.08%; weak inhibition of Kv1.3, with an inhibition rate of 12.26% ± 5.07%; and weak inhibition of TRPC6, with an inhibition rate of 3.94% ± 0.76%. The anaferine A compounds (+)-1 and (-)-1 inhibited the peak current of TRPM8 in a dose-dependent manner, with half-maximum inhibitory concentrations (IC 50 ) of 1.90 ± 0.09 and 1.40 ± 0.17 μmol / L Figure 3 ), respectively; and the anaferine A compound (-)-1 also blocked the peak current of Kv1.2 in a dose-dependent manner, with a half-inhibitory concentration (IC 50 ) value of 14.40 ± 0.62 μmol / L; indicating that the anaferine A compounds (+)-1 and (-)-1 exert their analgesic activity by inhibiting various analgesic targets such as the TRPM8, Kv1.2, Kv1.3, TRPC6 and Cav2.1 ion channels.

[0040] Example 3

[0041] This example uses the Autodock 4.2.6 molecular docking software (The Scripps Research Institute, USA) to study the mode of action of the anaferine A compounds (+)-1 and (-)-1 with TRPM8 (8E4Q) and Kv1.2 (5WIE), and to reflect the dynamic conformational changes and movement trajectories of the compound (-)-1 binding to the TRPM8 protein through molecular dynamics simulation;

[0042] Conclusion: The molecular docking results show that the compounds (+)-1 and (-)-1 of anarcardine A occupy almost the same position and have almost the same binding mode in the TRPM8 pocket; both of them form hydrophobic interactions with residues such as phenylalanine 846 (Phe846), isoleucine 746 (Ile746), leucine 842 (Leu842), tyrosine 835 (Tyr835), threonine 839 (Thr839), alanine 879 (Ala879), and valine 878 (Val878), and form π-alkyl interactions with residues such as leucine 749 (Leu749), leucine 750 (Leu750), alanine 874 (Ala874), phenylalanine 87 (Phe87), and valine 875 (Val875); however, there is a certain chiral difference between them, and the main difference lies in the orientation of the acetamide C-17 carbonyl group; the C-17 carbonyl group in compound (-)-1 of anarcardine A extends to a hydrophobic region, forms a hydrogen bond with the tyrosine 907 (Tyr907) residue, and forms a hydrophobic interaction with the methionine 877 (Met877) residue; while there is no such interaction in compound (+)-1 of anarcardine A; this difference can be used to reasonably explain the reason why compound (-)-1 of anarcardine A has a stronger inhibitory effect on TRPM8; Similar to TRPM8, the binding mode of compounds (+)-1 and (-)-1 of anarcardine A to the active pocket of Kv1.2 is almost the same, and they both form hydrophobic interactions with three residues such as valine 89 (Val89), tyrosine 90 (Tyr90), tryptophan 121 (Trp121), and tryptophan 243 (Trp243), form π-alkyl interactions with two residues such as tryptophan 57 (Trp57) and tryptophan 272 (Trp272), and form hydrogen bonds with two residues such as arginine 189 (Arg189) and lysine 276 (Lys276). The chiral difference is manifested in the spatial orientation of the C-17 carbonyl group. In (-)-1, the extended conformation of the C-17 carbonyl group forms two hydrogen bonds with the Arg189 residue. While in compound (+)-1 of anarcardine A, the C-17 carbonyl group only forms one hydrogen bond with the Arg189 residue. This difference may explain why (-)-1 has a stronger inhibitory effect on the Kv1.2 channel than (+)-1.

[0043] In order to further confirm the binding mechanism of the anagalline A compound (-)-1 and the TRPM8 ion channel, molecular dynamics simulation of the complex of (-)-1 and TRPM8 is carried out by using Discovery Studio software; the results show that the conformation of the anagalline A compound (-)-1 in the TRPM8 binding site has high stability, and no obvious conformation change is observed. The root mean square deviation (RMSD) of the complex always remains below 0.25 nanometers, and the maximum root mean square fluctuation (RMSF) is not more than 1.2 nanometers, thereby confirming the dynamic stability of the complex structure.

[0044] Those skilled in the art will easily understand that the above description is only the preferred embodiment of the present application, and is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of anagalline A in the preparation of an analgesic medicament, said anagalline A being the dextrorotatory form (+)-1, the levorotatory form (-)-1, or a mixture of the dextrorotatory form (+)-1 and the levorotatory form (-)-1, having the structural formula: ###0001### (+)-1 (-)-1

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