Use of an indole derivative for the preparation of a medicament for the prevention and / or treatment of pain

Abstract

The application provides use of an indole derivative in preparation of a medicine for preventing and / or treating pain, and belongs to the technical field of chemical medicines. The indole derivative is a compound shown in formula I, a pharmaceutically acceptable salt of the compound, a stereoisomer of the compound or a solvate of the compound. The compound can effectively treat pain, has small side effects, and has a very good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, specifically relating to the use of an indole derivative in the preparation of a medicament for the prevention and / or treatment of pain. Background Technology

[0002] Pain is an unpleasant sensory and emotional experience, or a similar experience, associated with actual or potential tissue damage (as defined by the International Association for the Study of Pain). In my country, the number of people suffering from chronic pain exceeds 300 million and is increasing by 10 to 20 million annually. The pain population is becoming increasingly younger, and it has become the third largest health problem after cardiovascular disease and cancer, seriously affecting people's health and quality of life. Based on the causes of chronic pain, it can be mainly divided into inflammatory pain, neuropathic pain, and cancer pain. Neuropathic pain (also called neuropathic pain) is often accompanied by comorbidities of emotional and cognitive impairment, but its underlying mechanisms are unclear. In animal models of neuropathic pain, the behavioral and functional changes of the chronic sciatic nerve ligation (CCI) model and the selective sciatic nerve branch injury (SNI) model are more similar to clinical neuropathic pain symptoms, and can induce late-stage neuropsychiatric disorders (anxiety-like, depression-like, and cognitive impairment, etc.), and are often used in research on cognitive function in neuropathic pain. Inflammatory pain, also known as inflammatory pain, is caused by inflammatory factors stimulating tissues and cells in the body, damaging and destroying them, thereby causing pain.

[0003] Currently, pain management drugs are mainly opioids, but also include other drugs such as cannabis-based medications. The therapeutic targets of pain medications primarily include analgesic targets of the opioid system, the analgesic effect of cannabinoid receptors themselves and their synergistic analgesic effects, the mechanisms of action of various other protein-coupled receptors (GPCRs), and ion channel K7.2, among others. However, existing drugs still suffer from problems such as a limited number of analgesics, poor analgesic efficacy, adverse reactions and social harms associated with opioids, necessitating the urgent development of new drugs.

[0004] The Compendium of Materia Medica first reported that the plant cannabis (Cannabis sativa L.) can be used to treat pain. The main active ingredient in cannabis is Δ9-tetrahydrocannabinol (THC), which works by targeting cannabinoid receptors CB1 and CB2 (both G protein-coupled receptors, an important target for pain treatment). As the receptor with the highest expression in the central nervous system, CB1 is involved in regulating important physiological and pathological processes, including memory, mood, pain, inflammation, and appetite. Therefore, CB1 has become an attractive target for developing treatments for a variety of diseases. After activation by endocannabinoids or exogenous ligands, CB1 activates intracellular Gi / o proteins or recruits β-arrestin effector proteins to mediate various cellular signaling events. Gi / o protein activation inhibits adenylate cyclase activity, reducing intracellular cAMP concentration; β-arrestin recruitment causes receptor internalization, weakening or blocking G protein signaling. It has been reported that CB1 relieves pain by de-inhibiting GABAergic neurons ("OFF" cells) and inhibiting GABAergic neurons ("ON" cells). Furthermore, β-arrestin2 knockout mice exhibited enhanced THC-mediated analgesia. Therefore, drug development targeting CB1 should aim to minimize β-arrestin recruitment to improve analgesic efficacy.

[0005] Traditional CB1 drug development strategies typically focus solely on endogenous ligand binding (ortho-alignment sites) of the receptor. Because the ortho-alignment sites of cannabinoid receptors CB1 and CB2 are extremely similar, ligands targeting CB1 often suffer from low subtype selectivity, leading to off-target effects. Allosteric ligands for CB1 can overcome the limitations associated with targeting only the ortho-alignment sites of the receptor. Furthermore, allosteric ligands exhibit biased signal transduction characteristics. Therefore, developing allosteric ligand drugs targeting CB1 biased signal transduction can not only increase therapeutic efficacy but also reduce or eliminate unnecessary adverse reactions.

[0006] However, many drugs reported so far activate β-arrestin signaling along with G protein signaling, leading to significant side effects. On the other hand, many drug developments target orthotopic sites, lacking specificity and acting on most receptor subtypes, resulting in off-target effects and posing potential risks. Summary of the Invention

[0007] The purpose of this invention is to provide the use of indole derivatives in the preparation of medicaments for the prevention and / or treatment of pain. The specific technical solution is as follows:

[0008] This invention provides the use of compounds of Formula I, pharmaceutically acceptable salts thereof, stereoisomers thereof, or solvates thereof in the preparation of medicaments for the prevention and / or treatment of pain:

[0009]

[0010] in,

[0011] R1 is selected from hydrogen and C1-C6 alkyl groups;

[0012] R2 is a substituent at any position on the benzene ring, and n is the number of substituents; n is selected from integers from 0 to 5; each R2 is independently selected from C1 to C6 alkoxy, halogen, substituted or unsubstituted C1 to C6 alkyl; the substituents of the alkyl group are selected from halogens.

[0013] Furthermore, the compound is as shown in Formula II:

[0014]

[0015] R2 is selected from C1-C6 alkoxy, halogen, substituted or unsubstituted C1-C6 alkyl groups; the substituents of the alkyl group are selected from halogens.

[0016] Furthermore, the compound is the following compound:

[0017]

[0018] Furthermore, the drug is a drug for the prevention and / or treatment of chronic pain.

[0019] Furthermore, the drug is a drug for the prevention and / or treatment of neuropathic pain and inflammatory pain.

[0020] Furthermore, the drug is a drug that targets human cannabinoid receptors.

[0021] Furthermore, the drug is a drug that targets the human cannabinoid receptor CB1.

[0022] Furthermore, the drug activates the G protein signaling pathway but does not activate the β-arrestin signaling pathway.

[0023] Furthermore, the drug is prepared by using the aforementioned compound, its pharmaceutically acceptable salt, its stereoisomer or its solvate as the active ingredient, plus pharmaceutically acceptable excipients.

[0024] The present invention also provides a medicament for the prevention and / or treatment of pain, said medicament being prepared by using the aforementioned compound, its pharmaceutically acceptable salt, its stereoisomer or its solvate as the active ingredient, plus pharmaceutically acceptable excipients.

[0025] This invention relates to CB-05, an indole derivative that is an allosteric regulator of G protein-coupled receptors that activates only the G protein signaling pathway (the therapeutic pathway) without activating the β-arrestin signaling pathway. As a biased allosteric ligand of CB1, CB-05 activates only the G protein signaling pathway and not the β-arrestin2 signaling pathway. The G protein signaling pathway is beneficial for pain treatment. In neuropathic pain models and inflammatory pain models, CB-05 exhibits significant analgesic effects without causing adverse reactions such as hypothermia or motor dysfunction.

[0026] In this invention, CB-05 activates only Gi1, Gi2, and Gz in the downstream signaling pathways within cells, without activating other Gi / o subtypes, thus clearly targeting the target.

[0027] This invention is the first to discover that CB-05 can effectively and selectively activate the cannabinoid receptor CB1, a traditional non-opioid receptor analgesic target, without activating β-arrestin signaling, thereby exerting excellent pain treatment effects with few side effects and promising clinical application prospects.

[0028] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0029] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0030] Figure 1 Results of activation of downstream G protein signaling and β-arrestin signaling mediated by cannabinoid receptor CB1 by compounds CB-05 and ZCZ011: a is β-arrestin signaling, b is downstream G protein signaling.

[0031] Figure 2 The effects of compounds CB-05 and ZCZ011 on the activation of different Gi / o protein subtypes: a is compound CB-05, b is compound ZCZ011.

[0032] Figure 3 The results of compounds CB-05 and ZCZ011 modulating inflammatory pain.

[0033] Figure 4 The results of compounds CB-05 and ZCZ011 modulating neuropathic pain.

[0034] Figure 5Results of the study on adverse reactions after injection of compound CB-05: a) Effect of compound CB-05 on body temperature; b) Results of drug tolerance to compound CB-05.

[0035] Figure 6 The results of cytotoxicity assays for compounds CB-05 and ZCZ011 are shown: a) compound CB-05, b) compound ZCZ011. Detailed Implementation

[0036] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0037] The compound used in this embodiment of the invention is compound CB-05, whose structure is as follows:

[0038]

[0039] ZCZ011 is an existing drug for treating pain, and its structural formula is:

[0040]

[0041] Example 1: Study on the target of compound CB-05 of the present invention

[0042] 1. Experimental Methods

[0043] 1) Effects on downstream G proteins and β-arrestin signaling activated by G protein-coupled receptor CB1

[0044] 1~5×10 5CHO-K1 mammalian cells were seeded into 6-well plates and incubated at 37°C for 24 hours. Following the instructions of the polyethylenimine linear (PEI) transfection kit, plasmids Gαi-Nluc, Gγ-mVenus, Gβ-WT, and CB1 were transfected into the cells at a mass ratio of 1:1:1:1 to investigate the activation of the CB1-mediated G protein signaling pathway by compound CB-05. Plasmids CB1–Lg, β-arrestin-Sm, and GRK2 were transfected into the cells at a mass ratio of 3:3:1 to investigate the activation of the CB1-mediated β-arrestin signaling pathway by compound CB-05. After 24 hours of cell transfection, the cell pellet was collected, resuspended in complete culture medium, and evenly seeded into 96-well plates for another 24 hours of incubation. After removing the supernatant culture medium, the cells were incubated with buffer containing coelenterazine H (Gibco, #14175095). After 10 minutes, different concentrations of CB-05 and ZCZ011 (DMSO solvent) were added to the cells, and the signals were immediately detected using a multi-functional microplate reader (detection wavelengths of 460 / 535 nm). Finally, the data were analyzed and plotted.

[0045] 2) Activation of different G protein-coupled receptors

[0046] The procedures are similar to those described above. The only difference is the use of different types of plasmids to infect the cells. G i1 -Rluc、G i2 -Rluc、G i3 -Rluc、G oa -Rluc、G ob -Rluc and G z -Rluc was used to infect cells with Gγ-mVenus, Gβ-WT, and CB1 at a mass ratio of 1:1:1:1. After 48 hours, different concentrations of CB-05 and ZCZ011 (DMSO solvent) were added to the cells, and the signals were immediately detected in a multi-functional microplate reader (detection wavelengths of 460 / 535 nm). Finally, the data were analyzed and plotted.

[0047] 2. Experimental Results

[0048] 1) Experimental results are as follows Figure 1 As shown: From Figure 1 As can be seen, compound CB-05 of this invention does not activate β-arresin signaling, while compound ZCZ011 does. Therefore, compound CB-05 can avoid side effects (such as drug tolerance) caused by β-arresin signal activation. Figure 1As can be seen from b, the compound CB-05 of this invention can significantly activate the G protein signaling pathway mediated by the cannabinoid receptor CB1. Therefore, compound CB-05 of this invention is a G protein biased agonist.

[0049] 2) Experimental results are as follows Figure 2 As shown, the compound CB-05 of this invention can only activate Gi1, Gi2, and Gz; while the existing drug ZCZ011 can activate almost all Gi / o subtype receptors.

[0050] Experimental results show that the compound CB-05 of this invention can effectively activate the G protein signaling pathway mediated by the cannabinoid receptor CB1 without activating β-arrestin signaling, resulting in minimal side effects. Among the activated G protein subtypes, it only activates Gi1, Gi2, and Gz, without activating other Gi / o subtypes, indicating a clear target.

[0051] Example 2: Animal experimental verification of the effect of compound CB-05 of the present invention in relieving inflammatory pain.

[0052] 1. Experimental Methods

[0053] 1) Modeling method

[0054] 25 μL of 1% w / v carrageenan solution was drawn using a microsyringe and inserted into the toe of the hind paw on one side of a C57BL / 6 experimental animal mouse. The carrageenan solution was slowly injected into the subcutaneous area of ​​the mouse's palm, forming a wheal. The needle was stopped for 20 seconds and then slowly rotated out to avoid leakage.

[0055] 2) Administration method

[0056] Mice were intraperitoneally injected with the test drugs: ZCZ011 (40 mg / kg), CB-05 (40 mg / kg), or placebo (10 μL / g). The solvents for dissolving ZCZ011 and CB-05 consisted of 10% DMSO, 1% Tween-80, 40% PEG400, and 49% physiological saline; the placebo was a solvent for dissolving ZCZ011 and CB-05 that contained no other compounds. The injection volume of placebo was the same as that of ZCZ011 and CB-05.

[0057] For the specificity test, the CB1 antagonist Rimonabant (CAS No.:168273-06-1) was administered intraperitoneally for 10 minutes, followed by an intraperitoneal injection of the test drug to investigate the specificity of the test drug in targeting CB1.

[0058] 3) Detection methods

[0059] To assess the analgesic effect of the test drug, model mice were intraperitoneally injected with ZCZ011 (40 mg / kg), CB-05 (40 mg / kg), or placebo (10 μL / g) 80 minutes before the measurement.

[0060] Five hours after injecting carrageenan into the paws of mice, mechanical abnormal pain was assessed using a plantar sensory analyzer. The animals were placed on a mechanical pain-inducing metal mesh and kept still for one hour until they were relatively quiet and no longer looking around or exploring. Testing began by slowly and gently stimulating the hind paws with Von-Frey fibers, causing the fibers to bend, and observing the withdrawal response for 2-3 seconds. The hind paws were stimulated sequentially with fibers of increasing weight, five times for each fiber weight, with at least 10 seconds between each stimulation. If fewer than three positive responses (marked as paw lifting, avoidance, or licking due to stimulation) were observed, the procedure was repeated with a larger fiber weight. When three or more positive responses were observed on the first test, that fiber was considered the animal's pain threshold (two tests were conducted per animal, and the average was used). The experiment included a control group, a vehicle group, and an experimental group. The control group consisted of normal mice, the model group consisted of mice with the model but without drug administration, and the experimental group consisted of mice with the model and drug administration.

[0061] 2. Experimental Results

[0062] The results are as follows Figure 3 As shown in Table 1, the experimental results demonstrate that the compound CB-05 of this invention can effectively treat inflammatory pain, and its effect is significantly better than that of the existing drug ZCZ011.

[0063] Table 1. Results of the inflammatory pain test in each group

[0064]

[0065] Example 3: Animal experimental verification of the effect of compound CB-05 of the present invention in relieving neuropathic pain.

[0066] 1. Experimental Methods

[0067] 1) Modeling method

[0068] Spinal nerve injury (SNI) model: C57BL / 6J mice were anesthetized with isoflurane, and the hair in the lateral thigh to femur region of the left hind limb was shaved with an electric shaver. The animals were placed on the operating table and the legs were secured with tape. A 1 cm longitudinal incision was made proximal to the knee joint, and the skin was bluntly dissected with the tip of sterile scissors. Near the clearly visible blood vessels close to the femur, the muscle layer was dissected by blunt dissection to expose the sciatic nerve directly beneath the muscle, and the L5 transverse process was removed to expose the L3 and L4 spinal nerves. The L4 spinal nerve was then carefully dissected and cut without damaging the L3 spinal nerve. In a sham operation, the L4 spinal nerve was exposed but not cut. The surgical area was rinsed with saline or PBS, and the muscles, subcutaneous tissue, and skin were sutured sequentially. The wound was cleaned and disinfected with alcohol.

[0069] 2) Administration method

[0070] Two days after modeling, the drug to be tested was injected, and pain was measured four hours after the drug injection.

[0071] Mice were intraperitoneally injected with the test drugs: ZCZ011 (40 mg / kg), CB-05 (40 mg / kg), or placebo (10 μL / g). The solvents for dissolving ZCZ011 and CB-05 consisted of 10% DMSO, 1% Tween-80, 40% PEG400, and 49% physiological saline; the placebo was a solvent for dissolving ZCZ011 and CB-05 that contained no other compounds. The injection volume of placebo was the same as that of ZCZ011 and CB-05.

[0072] For the specificity test, the CB1 antagonist Rimonabant (CAS No.:168273-06-1) was administered intraperitoneally for 10 minutes, followed by an intraperitoneal injection of the test drug to investigate the specificity of the test drug in targeting CB1.

[0073] 3) Detection methods

[0074] The experimental animals were placed on a mechanical pain-inducing metal mesh frame and left to rest for one hour until they stopped looking around and became relatively calm before the test began. The soles of the hind limbs were slowly and gently stimulated with Von-Frey fibers, causing the fibers to bend. The mice's paw withdrawal response was observed for 2-3 seconds. The soles of the hind limbs were stimulated sequentially according to the fiber weight, from smallest to largest, with each fiber weight being stimulated five times consecutively, with an interval of at least 10 seconds between each stimulation. If fewer than three positive responses (marked as a positive response due to stimulation, including paw lifting, avoidance, or licking) occurred, the above procedure was repeated using a larger fiber weight. When three or more positive responses occurred on the first test, that fiber was considered the animal's pain threshold (two tests were conducted per animal, and the average value was used). The experiment included a sham group, a vehicle group, and an experimental group.

[0075] 2. Experimental Results

[0076] The results are as follows Figure 4 As shown in Table 2, the experimental results demonstrate that the compound CB-05 of this invention can effectively treat neuropathic pain, and its effect is comparable to that of the existing drug ZCZ011.

[0077] Table 2. Results of neuropathic pain experiments in each group

[0078]

[0079] Example 4: Animal experiments verifying that compound CB-05 of the present invention has no obvious side effects.

[0080] 1. Experimental Methods

[0081] 1) Body temperature detection

[0082] Mice were intraperitoneally injected with CB-05 (40 mg / kg), ZCZ011 (40 mg / kg), CP55940 (3 mg / kg, the positive ligand targeting CB1), or placebo (10 μl / g). The solvents for dissolving ZCZ011, CB-05, and CP55940 consisted of 10% DMSO, 1% Tween-80, 40% PEG400, and 49% physiological saline; the placebo was a solvent containing no other compounds for dissolving ZCZ011, CB-05, and CP55940; the placebo injection volume was the same as the injection volume for ZCZ011, CB-05, and CP55940. CP55940 is a CB1 agonist, and its compound number is CAS No.: 83002-04-4.

[0083] The rectal temperature of C57BL / 6J mice was measured at 0, 0.5, 1, 2 and 4 hours after intraperitoneal injection.

[0084] 2) Drug tolerance

[0085] The animal model used was the SNI model, with modeling methods and administration routes similar to the neuropathic pain model (Example 3). The difference was that mechanical abnormal pain was measured on day 6 after continuous intraperitoneal injection of ZCZ011 (40 mg / kg), CB-05 (40 mg / kg), or placebo (10 μL / g) for 6 days. The analgesic effect of repeated administration of CB1 agonists on neuropathic pain was evaluated.

[0086] 2. Experimental Results

[0087] 1) Results are as follows Figure 5 As shown in a, the positive ligand CP55940 caused a decrease in body temperature of about 8°C (the literature reports that Δ9-THC caused a decrease in body temperature of about 6°C). Compared with CP55940, the compound CB-05 of the present invention did not have a significant hypothermic effect within 4 hours after administration.

[0088] 2) Results are as follows Figure 5 As shown in b, repeated administration of CB-05 did not cause drug tolerance, and there was no significant difference compared with the single intraperitoneal injection experimental group (only one injection of the drug), and it still had analgesic effect.

[0089] Example 5: Experimental verification that compound CB-05 of the present invention has no obvious cytotoxicity.

[0090] 1. Experimental Methods

[0091] Human cell lines, including the Müller cell line (MIO-M1), human bronchial epithelial cell line (BEAS-2B), and human umbilical vein endothelial cells (HUVEC), were seeded in 96-well plates and treated for 3 days with different concentrations of CB-05 or ZCZ011 (the buffer-treated group served as the control). On day 4, 20 μL of MTT solution (5 mg / mL) was added to the cells, and the cells were incubated for another 2–4 hours. Subsequently, DMSO was added to the cells to dissolve formazan. Finally, the absorbance was measured at 570 nm using a multi-plate reader, and the inhibition rate of cell viability was calculated.

[0092] 2. Experimental Results

[0093] The results are as follows Figure 6 As shown, ZCZ011, at concentrations exceeding 3 μM, significantly inhibited the activity of Müller cell line (MIO-M1), human bronchial epithelial cell line (BEAS-2B), and human umbilical vein endothelial cells (HUVEC). Figure 6 b). Conversely, the compound CB-05 of the present invention did not exhibit significant cell-inhibiting activity. Figure 6a). The results showed that CB-05 was safer and less toxic than ZCZ011.

[0094] In summary, this invention is the first to discover that CB-05 can effectively and selectively activate the cannabinoid receptor CB1, a traditional non-opioid receptor analgesic target, without activating β-arrestin signaling, thereby exerting excellent pain treatment effects with few side effects and promising clinical application prospects.

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of pain, characterized in that: The compound is the following compound: 。 2. The use according to claim 1, characterized in that: The drug is a medication for the prevention and / or treatment of chronic pain.

3. The use according to claim 2, characterized in that: The drug is a drug for the prevention and / or treatment of neuropathic pain and inflammatory pain.

4. The use according to any one of claims 1 to 3, characterized in that: The drug is prepared by using the compound of claim 1 or a pharmaceutically acceptable salt thereof as the active ingredient, plus pharmaceutically acceptable excipients.

5. A medicine for preventing and / or treating pain, characterized in that: The drug is prepared by using the compound of claim 1 or a pharmaceutically acceptable salt thereof as the active ingredient, plus pharmaceutically acceptable excipients.

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