CN115957218B - A CCR5 inhibitor and its application - Google Patents

Abstract

The present invention belongs to the field of medical technology, and discloses a CCR5 inhibitor and its application, especially the application in the preparation of a drug for preventing or treating CCR5-related diseases. The compound can bind to the CCR5 receptor, inhibit its receptor function, have a significant protective effect on the damage caused by ischemia, significantly improve the neurobehavioral disorder of experimental animals with cerebral ischemia, reduce the rate of cerebral infarction, promote the recovery of cognitive behavioral ability, reduce liver ischemic damage, etc., and therefore can be used as a new CCR5 inhibitor for the prevention and treatment of CCR5-related diseases (especially ischemic diseases), and has very good application prospects and development value.

Description

A CCR5 inhibitor and its application

Technical Field

The present invention relates to the field of medical technology, and in particular to a compound used as a novel CCR5 inhibitor in the preparation of a drug for preventing or treating CCR5-related diseases (especially ischemic diseases).

Background Art

Chemokine receptors are G protein-coupled receptors. Under inflammatory conditions, secreted cytokines or chemokines interact with chemokine receptors to control the activation of leukocytes and other cell types, playing an important role in physiological and pathological processes such as cancer, transplantation, diabetes and obesity, atherosclerosis, skin diseases and inflammatory bowel disease.

As an important chemokine receptor, CCR5 can be expressed on the surface of many types of cells, especially blood, dendritic cells, pancreatic islet cells, T cells in primary and secondary lymphoid organs, monocytes and macrophages. Its ligands include chemokines CCL5/RANTES, monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1α (MIP-1α), and macrophage inflammatory protein β (MIP-1β), which regulate the occurrence and development of many diseases. Among them, CCR5 plays the role of auxiliary receptor in the process of HIV entering host immune cells, which has attracted widespread attention. Based on this, in 2007, the FDA approved the first small molecule CCR5 inhibitor Maraviroc (C ₂₉ H ₄₁ F ₂ N ₅ O) for the treatment of AIDS. In addition, CCR5, as an important receptor for leukocyte activation, is closely related to the development of cancer and inflammation. The diseases involved include but are not limited to various infections, liver fibrosis, non-alcoholic fatty liver disease, hepatitis B, hepatitis C, acute lung injury, cardiovascular and cerebrovascular diseases, atherosclerosis, ischemic stroke, subcortical hemorrhagic stroke, endothelial dysfunction, cognitive dysfunction, etc. Therefore, the development of CCR5 inhibitors has a relatively broad range of indications.

Ischemic disease is one of the above diseases. Ischemic disease is caused by insufficient blood supply to tissues due to various reasons, which in turn causes hypoxia, nutrient deficiency, etc. Vasoactive substances, oxidative stress response, pro-inflammatory mediators and profibrotic factors can all participate in the process of tissue ischemia injury, including cerebral ischemia, myocardial ischemia, liver ischemia, renal ischemia, etc. Cerebral ischemia, also known as ischemic stroke, is the main type of stroke. In 2018, 81.9% of stroke hospitalized patients were ischemic stroke, mainly due to embolism or arteriosclerosis leading to insufficient cerebral blood flow, which causes the brain to lack oxygen, glucose and other essential nutrients, thereby causing further damage to the brain parenchyma. The pathogenesis and pathological process of stroke are complex, including calcium overload, excitotoxic injury, oxidative stress injury, inflammatory injury, etc., accompanied by high disability rate and high morbidity rate. It has become the leading cause of death among Chinese residents. Patients with acute cerebral ischemia are often accompanied by serious sequelae, which affects the quality of life of patients and brings huge economic burden to families and society. At present, there are limited clinical anti-cerebral ischemia drugs and intervention measures, mainly intravenous thrombolytic therapy and intravascular thrombectomy. Intravenous thrombolytic therapy uses tissue plasminogen activator (t-PA), which has a narrow treatment time window of only 4.5 hours, limiting its use in 95% of clinical patients. In addition, t-PA has a high risk of bleeding, and a small number of patients are still at risk of hemorrhagic transformation after receiving t-PA thrombolysis. Intravascular thrombectomy has few indications. Therefore, the above measures can only benefit a small number of clinical patients, and the development of safe and effective new drugs for the treatment of stroke has great social significance.

CCR5 is the first gene confirmed to be associated with neurological recovery after human stroke. CCR5 expression increases specifically in cortical neurons after stroke. Studies have found that CCR5 delta32 gene carriers (CCR5 functional loss) have significantly better recovery of motor and cognitive functions after stroke than non-carriers. During global cerebral ischemia and reperfusion injury, CCR5 knockout mice have reduced cerebral infarction area, fewer ischemic neurons, and overexpression of the nutritional factor BDNF compared with WT mice, which has potential neuroprotective effects. During the limited recovery period after the first stroke, CCR5 knockdown treatment of neurons from the motor cortex to the premotor cortex can promote early motor recovery, which is related to the stabilization of dendritic spines in the premotor cortex adjacent to the motor center. The above research results show that blocking CCR5 function or inhibiting CCR5 expression can significantly improve stroke-induced movement disorders and learning and memory decline. Currently, the only CCR5-related drug Maraviroc used in ischemic research (clinical phase II) has a large molecular weight (513.7) and poor blood-brain barrier permeability. Large-scale use can cause drug resistance, so it is very necessary to develop new CCR5 inhibitors.

Summary of the invention

To overcome the deficiencies of the prior art, the present invention provides a compound or a pharmaceutically acceptable salt thereof as a novel CCR5 inhibitor for use in the preparation of a drug for preventing or treating CCR5-related diseases (especially ischemic diseases).

To this end, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof for use in preparing a CCR5 inhibitor or a drug for preventing or treating a CCR5-related disease, wherein the compound has the following structure:

Among them, the compound represented by formula I is 5-{[(3,4-dimethylphenyl)sulfonyl]amino}-2-{methyl[3-(1,3,5-trimethyl-1H-pyrazol-4-yl)propyl]amino}nicotinic acid (PubChem CID: 53024879); the compound represented by formula II is 1-butyl-4-{1-[4-(2,3-dimethylphenoxy)butyl]-1H-benzimidazol-2-yl}pyrrolidin-2-one (PubChem CID: 16016262).

Specifically, the CCR5-related diseases are diseases known to be associated with CCR5, including but not limited to various inflammatory diseases, immune diseases, infectious diseases, allergic diseases, cardiovascular and cerebrovascular diseases, tumors, diabetes and its complications, etc.

Specifically, the above-mentioned inflammatory diseases can be asthma, nephritis, hepatitis, arthritis, rhinitis, conjunctivitis, inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD), spondyloarthritis, scleroderma, psoriasis, dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, vasculitis, fasciitis, etc.

Specifically, the immune disease mentioned above may be autoimmune disease, transplant rejection (including allogeneic transplant rejection or graft-versus-host disease), immunosuppression, and the like.

Specifically, the above autoimmune diseases may be rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, myasthenia gravis, juvenile-onset diabetes, glomerulonephritis, autoimmune thyroiditis, and the like.

Specifically, the infectious diseases mentioned above may be human immunodeficiency virus (HIV) infection, acquired immune deficiency syndrome, respiratory syncytial virus (RSV) infection, hepatitis B, hepatitis C, and the like.

Specifically, the above-mentioned cardiovascular and cerebrovascular diseases can be atherosclerosis, coronary heart disease, ischemic reperfusion injury, and ischemic diseases.

Specifically, the above-mentioned ischemic diseases include various tissue ischemias, which may be cerebral ischemia, liver ischemia, myocardial ischemia, renal ischemia, etc.; ischemic diseases include, for example, ischemic cerebrovascular disease, ischemic heart disease, ischemic hepatitis, ischemic nephropathy, lower limb artery ischemic disease; more specifically, ischemic cerebrovascular disease includes ischemic stroke; ischemic heart disease includes myocardial infarction, angina pectoris, heart failure, arrhythmia, thrombosis and embolism.

Specifically, the above-mentioned tumor may include a tumor of the brain, breast, prostate, lung, digestive system, reproductive organ or hematopoietic tissue, for example, breast cancer, prostate cancer, lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ovarian cancer, cervical cancer, melanoma, glioma, bladder cancer, leukemia, etc. Specifically, the above-mentioned tumor is metastatic. Specifically, the above-mentioned tumor is refractory to chemotherapy or radiotherapy and/or resistant to chemotherapy or radiotherapy.

Specifically, the above-mentioned diabetic complications may be diabetic nephropathy, diabetic cardiovascular and cerebrovascular disease, diabetic retinopathy, diabetic neuropathy, and the like.

Specifically, in the above-mentioned drugs, the compound or its pharmaceutically acceptable salt can be used as the sole active ingredient, or can be used in combination with other active ingredients for the same or different indications (such as Maraviroc).

Specifically, the above-mentioned medicine further comprises one or more pharmaceutically acceptable excipients.

Specifically, the pharmaceutically acceptable excipients may be selected from: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, and the like. Fillers include starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; flavoring agents include sweeteners and various flavors; preservatives include parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include PEG6000, PEG4000, insect wax, etc.

Specifically, the above-mentioned drugs can be administered by any suitable route, such as gastrointestinal administration or parenteral administration (for example, intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, rectal, etc.); the above-mentioned drugs can be in any suitable dosage form, such as a dosage form for gastrointestinal administration, for example, including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.; a dosage form for parenteral administration, for example, an injection, Dosage forms: such as injections (for example, for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), respiratory tract administration forms: such as sprays, aerosols, powder mists, etc., skin administration forms, such as external solutions, lotions, ointments, plasters, pastes, patches, etc., mucosal administration forms: such as eye drops, eye ointments, nasal drops, gargles, sublingual tablets, etc., cavity administration forms: such as suppositories, aerosols, effervescent tablets, drops, pills, etc., used for the rectum, vagina, urethra, nasal cavity, ear canal, etc.

Specifically, various dosage forms of the above-mentioned drugs can be prepared according to conventional production methods in the pharmaceutical field.

Specifically, the above-mentioned drugs can be made into ordinary preparations, or they can be made into sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle drug delivery systems.

In a second aspect, the present invention further provides use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and/or treating ischemic diseases.

In particular, the compound and the ischemic disease have the corresponding definitions in the first aspect of the present invention.

In particular, the compound has the structure of Formula I.

In particular, ischemic diseases include various tissue ischemias, for example, cerebral ischemia, liver ischemia, myocardial ischemia, renal ischemia, etc.; ischemic diseases include, for example, ischemic cerebrovascular disease, ischemic heart disease, ischemic hepatitis, ischemic nephropathy, lower limb artery ischemic disease; more specifically, ischemic cerebrovascular disease includes ischemic stroke; ischemic heart disease includes myocardial infarction, angina pectoris, heart failure, arrhythmia, thrombosis and embolism.

Specifically, the above-mentioned ischemic disease may be acute, transient or chronic blood supply insufficiency.

Specifically, the stage of the ischemic disease may be an early stage, a late stage, or a sequelae stage.

Specifically, cerebral ischemia can be acute ischemic stroke, transient ischemic attack or chronic cerebral ischemia.

Specifically, the symptoms of ischemic diseases are selected from one or more of: motor dysfunction, cognitive dysfunction, and cerebral infarction.

The beneficial effects of the present invention are as follows: the present invention is respectively subjected to virtual screening of CCR5 receptors, verification of CCR5 receptor activity, ischemia-reperfusion models of different tissues, in vitro and in vivo verification, and the compounds (especially compounds having the structure shown in Formula I) are screened, and the inhibitory effects of the compounds on CCR5 receptors and anti-ischemic injury are investigated. The results show that the compound can bind to the CCR5 receptor and inhibit its receptor function; the in vitro oxygen-glucose deprivation/reoxygenation injury model shows that the compound has a significant protective effect on the damage caused by ischemia. In vivo experiments use rat cerebral ischemia-reperfusion models and liver ischemia-reperfusion models. By injecting the compound, the neurobehavioral disorders of rats with cerebral ischemia can be significantly improved; the cerebral infarction rate of rats with cerebral ischemia can be reduced; the cognitive behavioral ability of rats with cerebral ischemia-reperfusion can be promoted to recover; and liver ischemic injury can be reduced. Therefore, it is expected that the compound or its pharmaceutically acceptable salts, esters, prodrugs, solvates, stereoisomers can be used as a new CCR5 inhibitor for the prevention and treatment of CCR5-related diseases, especially ischemic diseases, and have very good application prospects and development value.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the interaction model between the target compound F293-0916 and CCR5. In the upper figure, the protein CCR5 is represented by the surface, and the small molecule F293-0916 is represented by a stick. In the lower figure, the protein CCR5 is represented by a cartoon, and the small molecule F293-0916 is represented by a stick in the middle of the figure. The other sticks around represent the key amino acid residues on CCR5, which are involved in the binding with the small molecule compound F293-0916.

Figure 2 shows the screening and detection of compound F293-0916, where (A) the top 30 compounds with the highest virtual screening scores were compared for CCR5 inhibitory activity at a concentration of 10 μM; (B) IC ₅₀ detection of F293-0916 and positive control Maraviroc. The concentrations of F293-0916 and Maraviroc were 0.01 μM, 0.1 μM, 1 μM, 10 μM and 100 μM, and the horizontal axis is the logarithm of the concentration. IC ₅₀ was obtained by Prism nonlinear regression analysis and dose-response analysis.

Figure 3 shows the effect of compound F293-0916 on the oxygen glucose deprivation/reoxygenation (OGD/R) model of SH-SY5Y-CCR5 cells ( n＝4). Control group; oxygen glucose deprivation/reoxygenation model group; different concentrations of drug test groups: F293-0916 0.01μM group, F293-0916 0.1μM group, F293-0916 1μM group, F293-0916 10μM group; positive drug Maraviroc 1μM group. ^#### p<0.001 compared with the control group; **, ***, ***p<0.01, p<0.001, p<0.0001 compared with the model group, respectively.

Figure 4 shows the effect of compound F293-0916 on the Zea Longa score of rats ( n＝7-10). Sham operation group; middle cerebral artery occlusion/reperfusion (MCAO/R) model group prepared by suture embolism; drug administration groups: F293-0916 low-dose group (2.5 mg/kg group), F293-0916 medium-dose group (5.0 mg/kg group), F293-0916 high-dose group (10.0 mg/kg group); positive drug edaravone group (10.0 mg/kg); ^#### p<0.0001 compared with sham operation group; ****p<0.0001 compared with model group.

Figure 5 shows the effect of compound F293-0916 on cerebral infarction rate in rats ( n＝7-10). Grouping was the same as in Example 4, ^#### compared with the sham operation group, p<0.0001; ** compared with the model group, p<0.01; **** compared with the model group, p<0.0001.

Figure 6 shows the effect of compound F293-0916 on cognitive behavior in the recovery period of rats with cerebral ischemia-reperfusion ( n＝5). The experimental grouping was the same as that in Example 4; D2: the discrimination index between the same objects on the second day of the experiment; D3: the discrimination index between different objects on the third day of the experiment; (A) Experimental schematic diagram; (B) Comparison of the discrimination ability on the second and third days in each group; *p<0.05 compared with D2; ***p<0.001 compared with D2; (C) Comparison of the discrimination ability of the new objects in each test group on the third day; ^### p<0.001 compared with the sham operation group; **p<0.01 compared with the model group; *p<0.05 compared with the model group.

Figure 7 shows the effect of compound F293-0916 on ALT and AST activities after ischemia-reperfusion in rat liver ( n＝6, U/L). The experimental groups were sham operation control group, model group, F293-0916 low-dose group (2.5mg/kg), medium-dose group (5mg/kg), and high-dose group (10mg/kg). (A) ALT, alanine aminotransferase; (B) AST, aspartate aminotransferase; ^#### p<0.0001 compared with sham operation group; **p<0.01 compared with model group; ****p<0.0001 compared with model group.

DETAILED DESCRIPTION

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Virtual screening of small molecule inhibitors targeting CCR5 and molecular docking results of compound F293-0916 and CCR5

Experimental materials: Small molecule compound library: Small molecule databases include Chemdiv and Targetmol (including bioactive compounds, natural compounds and approved drugs). The "Compoud Filtering" module of sybyl-X2.1 was used to select compounds in Chemdiv, and compounds that met the following rules were selected: 1) molecular weight less than 700; 2) cLogP in the range of -4–6; 3) the number of hydrogen bond acceptors is not more than 15; 4) the number of hydrogen bond donors is not more than 6; 5) the number of rotatable bonds is less than 11.

Select and prepare target protein: Based on the three-dimensional structure of CCR5, select the A chain and use the "Prepare Protein Structure" module of sybyl-X2.1 to process the CCR5 protein. Select the region where the allosteric site inhibitor Maraviroc is located as the small molecule inhibitor binding domain, and use "Analyze Selected Structure" to analyze and modify the protein, complete operations such as adding hydrogen atoms to the protein and repairing amino acid side chains, and generate a small molecule inhibitor binding site file.

Perform the first round of virtual screening calculations: Use the Surflex module in the Sybyl-X2.1 software for screening, modify some molecular docking parameters to speed up the first round of virtual screening, and finally screen out small molecule compounds with molecular score values in the top 1%. Specifically, reduce the "Max conformations per Fragment" from the default 20 to 10, and reduce the "Max number of rotatable bonds per molecule" from the default 100 to 50. Cancel the default "Per-DockMinimization" and "Post-Dock Minimization" options. Reduce the "Maximum number of poses perligand" from the default 20 to 3, that is, only retain the top 3 molecular conformations of each ligand molecule to speed up docking. Use the default values for other parameters. Finally, the top 1% compounds are obtained.

Perform the second round of virtual screening calculations: Use the Surflex module in the Sybyl-X2.1 software to perform the second round of screening. Based on the top 1% hits from the first round of screening, restore the default parameters. Select the top 500 target compounds for manual screening.

Manual screening and review: 500 target compounds obtained in the second round of screening were manually screened, with comprehensive consideration: they can form stable interactions (hydrogen bonds and hydrophobic interactions) with CCR5, that is, they can form multiple hydrogen bond interactions with the side chain hydroxyl groups of Ser180 and Tyr37 of CCR5, the main chain carbonyl groups of Trp86, Cys178, Lys191, and the main chain amino group of Ser180; they can have strong hydrophobic interactions with multiple hydrophobic amino acids on CCR5, such as Trp86, Tyr89, Thr105, Tyr108, Phe109, Phe112, Asn163, Thr167, Phe182, Gln194, Ile198, and Tyr251 (or the hydrophobic part of the side chain of polar amino acids). The structure of the compound should be relatively rigid, that is, the number of rotatable bonds should not be too many. Finally, 150 compounds were selected from the Chemdiv library as potential CCR5 inhibitors, and the top 30 compounds were selected for activity screening at the cellular level.

Results: Figure 1 shows the molecular docking results, showing the interaction pattern of compound F293-0916 and CCR5. Compound F293-0916 can form a hydrogen bond interaction with the main chain amino group of Ser180 (distance is 2.94 angstroms). The five-membered nitrogen heterocyclic ring of compound F293-0916 extends into the hydrophobic cavity composed of amino acids such as Thr82, Trp86, Tyr89, Trp94, Tyr108, and Phe109 to interact. At the same time, the benzene ring group of compound F293-0916 also has a strong hydrophobic interaction with another hydrophobic cavity of the binding site (composed of Phe182, Try187, Thr195, Ile198, Tyr251, Leu255, Thr259, Met279, etc.). In addition, the five-membered nitrogen heterocyclic ring of compound F293-0916 forms a π-π stacking interaction with Trp86. It can be seen that hydrogen bonding, hydrophobic and π-π stacking interactions jointly maintain the binding of compound F293-0916 to the target CCR5.

Table 1 ChemDiv numbers of the top 30 compounds in virtual screening

The structural formula of compound D347-4205 is shown below:

The structural formula of compound F293-0916 is shown below:

Example 2: Inhibitory activity of compound F293-0916 on CCR5

Experimental materials: SH-SY5Y-CCR5 stable overexpression cell line was constructed by the inventor and recorded in patent application 202011396951.7. CCR5 ligand recombinant human chemokine RANTES/CCL5 was purchased from MCE (catalog number: HY-P7283), positive control drug Maraviroc (catalog number: T6016, CAS: 376348-65-1) and the top 30 compounds were purchased from Shanghai Taosu Biochemical Technology Co., Ltd., among which compound No. 14 was compound F293-0916 (ChemDiv). Fluo-4 AM calcium ion fluorescent probe (S1060) and calcium ion carrier ionomycin (S1672) were purchased from Bio-Tech.

CCL5-induced Ca ²⁺ signal was used to detect CCR5 receptor inhibition: SY5Y-CCR5 stable overexpression cell line was seeded in 96-well plates at 4×10 ⁵ cells/well. After adhesion, 1 μM Fluo-4 fluorescent calcium indicator dye was added and incubated at 37°C for 40 minutes for fluorescent probe loading. Then, the cells were washed three times with HBSS. After washing, the cells were incubated in HBSS balanced salt solution containing 20 mM HEPES. Then, the cells were incubated with the test compound or positive drug and incubated at 37°C for 20 minutes, while ensuring that Fluo-4 AM was completely converted to Fluo-4 in the cells. Then, the CCR5 receptor stimulator RANTES/CCL5 was added and the fluorescence was measured by a fluorescence microplate reader. Ionomycin was added 80 seconds after the addition of CCL5 to determine the total calcium load. The fluorescence measurements were normalized relative to the untreated control wells. The first 30 compounds were initially screened and the 10 μM concentration was selected to detect the inhibition rate. IC ₅₀ was determined for F293-0916, which had the best effect. The concentration of the test compound required to inhibit the calcium signal induced by CCR5 agonist CCL5 by 50% relative to the untreated control wells was the 50% inhibition concentration (IC ₅₀ value). The tested drug concentrations were 0.01 μM, 0.1 μM, 1 μM, 10 μM and 100 μM.

Experimental results: The intracellular Ca ²⁺ concentration increases at the moment of CCL5 production. As shown in FIG2A , compound No. 14 F293-0916 has the highest inhibitory activity on the Ca ²⁺ flow produced by CCL5. IC ₅₀ detection was performed on it. As shown in FIG2B , F293-0916 exhibits a dose-dependent inhibition on the Ca ²⁺ flow produced by CCL5. In this experimental test, the IC ₅₀ of compound F293-0916 is 239.3 nM, and the IC ₅₀ of the positive drug Maraviroc is 722.9 nM.

Example 3: Effect of compound F293-0916 on SH-SY5Y-CCR5 cell oxygen glucose deprivation/reoxygenation model

Experimental groups: control group, oxygen glucose deprivation/reoxygenation (OGD/R) group, OGD/R+0.01μM F293-0916, OGD/R+0.1μM F293-0916, OGD/R+1μM F293-0916, OGD/R+10μM F293-0916, OGD/R+1μM Maraviroc group, independent cell experiments were repeated 4 times, n=4.

OGD/R modeling: SH-SY5Y-CCR5 cells were seeded into 96-well culture plates at a density of 3×10 ⁴ cells/mL. After 24 hours of culture, the cells were washed once with PBS and 100 μL of Earle's solution without glucose was added to each well. The culture plate was placed in a hypoxic chamber and pure N ₂ was continuously introduced into the chamber at a low flow rate until the oxygen meter reading dropped below 0.5% to confirm that the oxygen was emptied. The air inlet and outlet were clamped and the hypoxic chamber was transferred to a 37°C incubator for culture. SH-SY5Y-CCR5 cells were hypoxic for 3 hours. At the same time, a group with only medium replacement and no glucose and hypoxia was set as a control. After reoxygenation, the culture medium was replaced with normal serum-containing culture medium to terminate glucose deprivation, and the cells were restored to a normal oxygen content environment in a 37°C incubator as reperfusion. Cell survival rate was detected 24 hours after reoxygenation.

In vitro screening of compounds: After OGD, serum-containing medium containing different concentrations of compounds was added, with the concentrations being 0.01 μM, 0.1 μM, 1 μM, and 10 μM. MTT assay was performed after reoxygenation in a 37°C incubator for 24 h.

Determination of cell viability by MTT method: The cell survival rate was determined using the thiazolyl blue (MTT) assay. The cells were seeded onto a 96-well plate. After the corresponding treatment, 10 μL of MTT solution (5 mg/mL) was added to each well. The reaction was allowed to proceed at 37°C for 4 hours until blue-purple crystals appeared. Then 100 μL/well of the triple solution was added and incubated at 37°C for 8 hours to dissolve the formazan crystals. The optical density value (OD value) was measured at 570 nm using a microplate reader. The relative cell survival rate was calculated by taking the average value. Relative cell survival rate (%) = (OD value of the experimental group/OD value of the control group) × 100%.

Statistics and analysis: All data are The data were analyzed statistically using one-way analysis of variance (Prism 8.0), Dunnett's test was used as a post hoc test, and p < 0.05 was set as the standard of statistical difference.

Results: As shown in Table 2 and Figure 3, the results of SH-SY5Y-CCR5 model detection showed that compared with the blank group, oxygen-glucose deprivation and reoxygenation caused significant cell damage (71.27% ± 1.00%, p < 0.0001). The Maraviroc treatment group significantly increased the cell survival rate compared with the model group (85.5% ± 4.00%, p < 0.001), indicating that the model can be used to evaluate drugs. Compared with the model group, 0.1μM, 1μM and 10μM F293-0916 significantly increased the cell survival rate (83.57% ± 1.46%, p < 0.01; 88.21% ± 3.09%, p < 0.0001; 84.73% ± 3.33%, p < 0.001), and 0.01μM F293-0916 had no significant effect. The above results suggest that F293-0916 has anti-ischemic and hypoxic therapeutic effects in in vitro experiments at a concentration of 0.1-10 μM.

Table 2 Effect of compound F293-0916 on SH-SY5Y-CCR5 cell OGD/R model

####, p<0.0001 vs control group; **p<0.01, ***p<0.001, ****p<0.0001 vs model group

Example 4: Effects of compound F293-0916 on neurobehavior in rats with cerebral ischemia-reperfusion

Experimental materials: SPF male SD rats, weighing 240-260 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., certificate number: SCXK (Beijing) 2019-0010. The positive drug edaravone was purchased from Shanghai Taosu Biochemical Technology Co., Ltd.

Experimental groups: 60 adult SD rats were randomly divided into sham operation control group, model group, F293-0916 low-dose group (2.5 mg/kg), medium-dose group (5 mg/kg), high-dose group (10 mg/kg), and positive drug edaravone group (10 mg/kg), with 10 rats in each group, and the drugs were dissolved in normal saline. Except for the sham operation group, all animals underwent MCAO surgery modeling, and the suture was removed after 90 minutes of ischemia for reperfusion. After the suture was removed, the F293-0916 and edaravone groups were immediately given the corresponding drug treatment through the tail vein, and the sham operation group and the model group were given the same volume of solvent control saline.

Establishment of the animal model of cerebral ischemia-reperfusion: The middle cerebral artery occlusion/reperfusion (MCAO/R) model was prepared by suture embolization. Rats were anesthetized with isopentane and fixed on the operating table in a supine position. The neck was disinfected, and a 2-cm incision was made in the middle of the anterior neck. The subcutaneous soft tissue and muscle were bluntly separated to expose and separate the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA and its branches were ligated with No. 0 surgical thread, and artery clamps were placed before the bifurcation of the CCA and near the cranial end of the ICA. A micro-incision was made in the ECA 3 to 4 mm away from the bifurcation of the CCA. The artery clamp of ICA was loosened, and a special nylon thread (40 mm long, 0.26 mm in diameter) was inserted and pushed along the ICA to the beginning of the middle cerebral artery (MCA), about 18.5±0.5 mm, and stopped when resistance was encountered, resulting in blockage of the beginning of MCA. The nylon thread was fixed with No. 1 surgical thread, and the wound was covered with cotton balls soaked in saline. The rats were placed on an electric blanket for warmth. After 90 minutes, the nylon thread was withdrawn and reperfused, and the wound was sutured. The sham operation group did not insert the nylon thread, and the rest of the operations were the same as the cerebral ischemia model group. During the experiment, the rats were placed on a temperature-controlled blanket, and the temperature controller was used to control the rat rectal temperature to maintain within the range of 36.5-37°C.

Neurological function score: Neurobehavioral score is one of the most intuitive external indicators of animal brain damage. This study used the Zea Longa method for scoring. Animal behavioral scoring was observed 24 hours after surgery. Referring to the Longa method, the rat's tail was lifted about 1 foot off the ground and the condition of the two forelimbs was observed; the rat was placed on a horizontal ground and its shoulders were pushed to observe whether there was any difference in resistance on both sides; the rat was placed on the ground and its walking was observed. A five-point method (0-4 points) was used to score the behavior of each group. The higher the score, the more severe the neurobehavioral damage. (1) Those with completely normal behavior were scored 0 points; (2) Those with the tail lifted off the ground and the forelimb on the opposite side of the surgery rotated inward were scored 1 point; (3) The rat was placed on the ground and the resistance was checked by squeezing both sides with hands. Those with reduced resistance were scored 2 points; (4) The rat was placed on the ground and its walking was observed. Those who circled around the opposite side of the surgery were scored 3 points; (5) Those with extremely severe damage and no independent movement were scored 4 points.

Statistics and analysis: All data are The data were analyzed statistically using one-way analysis of variance (Prism 8.0), Dunnett's test was used as a post hoc test, and p < 0.05 was set as the standard of statistical difference.

Results: As shown in Table 3 and Figure 4, the effect of compound F293-0916 on the Zea Longa score of rats, the sham group had no motor disorder, and the model group had obvious motor dysfunction (3.14±0.437). Compared with the model group, the positive drug treatment group can significantly improve the motor ability of rats (1.48±0.299), with a significant difference (p<0.0001), indicating that this experimental system can be used to evaluate the effect of F293-0916 on the motor dysfunction caused by ischemic stroke. Compared with the model group, F293-0916 5.0mg/kg and 10.0mg/kg can significantly reduce the Zea Longa score of rats, which are 1.63±0.491 and 1.37±0.391, respectively, with statistical differences (p<0.0001, 5mg/kg; p<0.0001, 10mg/kg). The above results indicate that compound F293-0916 has the efficacy of treating movement disorders caused by ischemic stroke at 5.0 mg/kg and 10.0 mg/kg.

Table 3 Effects of compound F293-0916 on Zea Longa scores in rats

####, p<0.0001 vs sham operation group; ****p<0.0001 vs model group. n=7-10

Example 5: Effect of compound F293-0916 on cerebral infarction rate in rats with cerebral ischemia-reperfusion

Determination of rat cerebral infarction rate: After the neurological function score was completed, the animals were immediately killed and the brain tissue was separated. After the brain tissue was separated, it was quickly placed in a -20℃ refrigerator and returned to room temperature after 30 minutes. The rat brain was placed in a slice mold, and after removing the olfactory bulb, cerebellum and lower brainstem, coronal slices were made at intervals of 2mm, for a total of 6 slices. Then the brain slices were quickly placed in 5ml of PBS solution containing 1% TTC, and incubated at 37℃ for 30 minutes in the dark. The brain slices were turned over every 5 minutes. After TTC staining, normal tissue was rose red, and infarcted tissue was not stained but white. The brain slices were taken out and fixed in 10% formalin. Each group of brain slices was arranged neatly and photographed for preservation. Imagepro Plus image analysis software was used for processing and statistics, and the infarction area of each brain slice was calculated. It was assumed that the infarction area of each brain slice was A, the area of each brain slice on the affected side was B, and the area of each slice on the healthy side was C. Infarction rate = (A1+A2+A3+A4+A5+A6)/(B1+B2+B3+B4+B5+B6+C1+C2+C3+C4+C5+C6)×100%.

Statistics and analysis: All data are The data were analyzed statistically using one-way analysis of variance (Prism 8.0), Dunnett's test was used as a post hoc test, and p < 0.05 was set as the standard of statistical difference.

Results: As shown in Table 4 and Figure 5, the effect of F293-0916 on the cerebral infarction rate of rats can directly reflect the degree of ischemia-reperfusion injury. TTC staining can make normal brain tissue red and the infarction area white. One-way analysis of variance and Dunnett's test showed that compared with the model group (22.98% ± 2.252%), the infarction rate of rats in the positive drug group was significantly reduced (16.81% ± 2.205%), with statistical significance (p < 0.01), indicating that the experimental system is established and can be used to detect the pharmacodynamic evaluation of F293-0916 in the treatment of ischemic stroke. F293-0916 5.0mg/kg (14.00% ± 4.637%) and 10.0mg/kg (10.75% ± 5.445%) can reduce the infarction rate, with statistical difference (5.0mg/kg, p < 0.0001; 10.0mg/kg, p < 0.0001); there is no significant difference between the 2.5mg/kg group and the model group. The above results suggest that compound F293-0916 has the efficacy of treating ischemic stroke and reducing the infarction rate at a dose of 5.0mg/kg and 10.0mg/kg.

Table 4 Effect of compound F293-0916 on cerebral infarction rate in rats

####, p<0.0001 vs sham operation group; **p<0.01, ****p<0.0001, vs model group. n=7-10.

Example 6: Effects of compound F293-0916 on cognitive and behavioral abilities in rats during the recovery period after cerebral ischemia-reperfusion

Novel object recognition experiment: Novel object recognition is a delicate and sensitive behavioral method that uses the rodents' natural instinct to approach and explore novel objects to detect animal recognition memory. The novel object recognition experiment is used to detect the neurological repair and cognitive behavior ability of stroke rats during the recovery period. One day before the test, the rats were placed in a square black-bottom box (40×40×45cm) and allowed to explore freely in the box for 5 minutes. On the second day, two identical objects were placed in the box, and the rats were put back in and allowed to explore freely in the box for 5 minutes. The number of times the rats touched the two novel objects in the box was recorded. On the third day, one of the old objects in the box was replaced with a new object that the test animal had never seen before, and the rats were put back in the environment to explore freely for 5 minutes. The number of times the rats touched the two objects was recorded, and the percentage of interest in the new and old objects was compared. In order to avoid the influence of rat odor during the period, the box was wiped with 70% alcohol after each rat finished exploring. The time to explore the new object was recorded as T _new , and the time to explore the new object was recorded as T _old . The discrimination index (DI) = (T _new / (T _new + T _old )) is used to evaluate the exploration of the target object.

Statistics and analysis: All data are The discrimination index of each group on D2 and D3 was statistically analyzed by t-test, and the novel object discrimination ability of each group on the third day was statistically analyzed by one-way analysis of variance and Dunnett's test (Prism 8.0), with p < 0.05 as the statistical difference standard.

Results: As shown in Figure 6, the effect of compound F293-0916 on the cognitive and behavioral abilities of rats in the recovery period after cerebral ischemia and reperfusion showed that the model group rats did not show interest in exploring novel objects, and the positive drug group had significant differences in the exploration of novel objects (p<0.05), indicating that the experimental system is established and can be used to detect the pharmacodynamic evaluation of F293-0916 on the cognitive and behavioral abilities of rats in the recovery period after cerebral ischemia and reperfusion. F293-0916 10.0mg/kg (p<0.01) has statistical differences; the 2.5mg/kg and 5mg/kg groups have no significant differences compared with the model group. The above results suggest that compound F293-0916 at a dose of 10.0mg/kg promotes the recovery of cognitive and behavioral abilities in rats in the late stage of cerebral ischemia and reperfusion.

Example 7: Protective effect of compound F293-0916 on rats with hepatic ischemia-reperfusion

Experimental materials: SPF male SD rats, weighing 200-220 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., certificate number: SCXK (Beijing) 2019-0010.

Hepatic ischemia-reperfusion grouping and experiment: 30 adult SD rats were randomly divided into sham control group, model group, F293-0916 low-dose group (2.5 mg/kg), medium-dose group (5 mg/kg), and high-dose group (10 mg/kg), with 6 rats in each group, and the drug was dissolved in normal saline. Except for the sham group, all animals underwent liver ischemia-reperfusion modeling, and the whole liver was ischemic for 30 minutes by clamping the liver pedicle. The rat liver ischemia-reperfusion model was established by reperfusion. In the sham group, only the liver pedicle was exposed, and the liver blood flow was not blocked. The drug-treated group was given the corresponding drug treatment through the tail vein immediately after reperfusion, and the sham group and the model group were given the same volume of solvent control saline. Blood was collected from the abdominal aorta 6 hours after reperfusion, and the activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), sensitive markers of acute hepatocyte damage, was detected by biochemical analyzer.

Statistics and analysis: All data are The data were analyzed statistically using one-way analysis of variance (Prism 8.0), with Dunnett's test as the Posthoc test and p < 0.05 as the standard of statistical difference.

Results: As shown in Table 5 and Figure 7, compared with the sham operation group, the serum ALT and AST levels of the rats with liver ischemia-reperfusion were significantly increased 6 hours after reperfusion (ALT, 122.45±3.18 ^#### ; AST, 389±15.19 ^#### ). Compared with the model group, compound F293-0916 5.0 mg/kg and 10.0 mg/kg could significantly reduce the serum ALT and AST levels of rats with liver ischemia-reperfusion, which were ALT, 87.46±3.92****; AST, 289.45±13.96**** and ALT, 93.52±4.02****; AST, 318.45±14.54****, respectively, with statistical differences (p<0.01, 5 mg/kg; p<0.0001, 10 mg/kg). The above results indicate that compound F293-0916 has the efficacy of treating liver ischemia-reperfusion injury at 5.0 mg/kg and 10.0 mg/kg.

Table 5 Effect of compound F293-0916 on ALT and AST activities after liver ischemia-reperfusion in rats (U/L)

####, p<0.0001 vs sham operation group; **p<0.01, ****p<0.0001, vs model group. n=6.

In summary, the present invention found that compound F293-0916 can bind to CCR5, inhibit receptor activation, increase the survival rate of cells in the ischemic model, reduce ischemic tissue damage in animals, and improve post-ischemic behavioral ability through CCR5 receptor virtual screening, CCR5 receptor activity verification, different tissue ischemia-reperfusion models, in vitro and in vivo verification.

Therefore, compound F293-0916 has the effect of inhibiting CCR5 and its related diseases. Compound F293-0916 can be used as an active substance alone or/and combined with other pharmacologically active compounds and/or extracts to form a compound for use, and can be prepared into various dosage forms for anti-CCR5 related diseases, especially ischemic diseases, according to conventional preparation methods in the pharmaceutical field, or combined with other drugs to form a compound preparation for reducing adverse reactions in drug action while maintaining the therapeutic effect, which can provide an effective solution for the prevention and treatment of CCR5 related diseases, especially ischemic diseases.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing ischemic stroke or ischemic hepatitis, wherein the compound has the following structure: 2. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating ischemic stroke or ischemic hepatitis, characterized in that the pharmaceutical composition contains the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 3. The use according to claim 2, characterized in that the pharmaceutical composition is selected from oral preparations, injection preparations or skin and mucosal administration preparations. 4. The use according to claim 3, characterized in that the oral preparations include tablets, capsules, sustained-release agents, controlled-release agents, pills, powders, granules, solutions, emulsions, and suspensions; the injectable preparations include intramuscular injection preparations and intravenous injection preparations; the skin and mucosal administration preparations include lotions, liniments, ointments, plasters, pastes, patches, eye drops, nasal drops, gargles, sublingual tablets, adhesive sheets, and film patches.