Use of beta-lactam compounds for the preparation of anti-inflammatory medicaments

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of a beta-lactam compound in preparation of anti-inflammatory drugs. The application is found through research and verified through experiments that the beta-lactam compound is combined with the surface of alpha and beta tubulin proteins, inhibits tubulin aggregation, and thus inhibits various cell processes in which microtubules participate; the beta-lactam compound inhibits inflammatory reactions through the following pathways: inhibiting immune activation, NLRP3 inflammasome activation and Caspase-1 activation; inhibiting neutrophil recruitment, adhesion and chemokine release; inhibiting vascular endothelial growth factor secretion and endothelial cell proliferation, and thus can be used for preparing anti-inflammatory drugs, including for treating local inflammatory reaction diseases such as acute gout, familial Mediterranean fever, chronic kidney disease, pancreatitis, sarcopenia and the like caused by inflammation, and inflammation-related cardiovascular diseases such as ischemia-reperfusion injury, diabetes, atherosclerosis, pericarditis, acute coronary syndrome and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the novel application of β-lactam compounds in the preparation of anti-inflammatory drugs. Background Technology

[0002] Inflammation is a protective response involving immune cells, blood vessels, and molecular mediators. When the body is exposed to harmful external stimuli, such as burns, chemical irritants, pathogens, physical damage, or cytokine stimulation, a series of complex biological reactions, including inflammation, occur. The main symptoms of inflammation are redness, swelling, heat, and pain. Acute inflammation can rapidly recruit immune cells to eliminate invading pathogens and initiate wound repair processes after tissue damage and infection, providing protection for the body. However, the accumulation of potassium and hydrogen ions in local inflammatory lesions, stimulation by inflammatory mediators such as prostaglandins, serotonin, and bradykinin, and the compression of exudates within inflammatory lesions can all cause persistent and severe pain, seriously affecting the patient's quality of life and even potentially endangering life. Clinical trials have shown that chronic inflammation is a risk factor for cardiovascular disease, familial Mediterranean fever, chronic kidney disease, diabetes, cancer, depression, dementia, and sarcopenia.

[0003] Gout is a typical localized inflammatory response, primarily caused by excessive uric acid accumulation in the body, typically occurring in the first metatarsophalangeal joint, ankle joint, and arch of the foot. Acute gouty arthritis manifests as acute pain, redness, swelling, and tenderness in the joints, severely impacting quality of life. Monosodium urate (MSU) crystal deposition can activate inflammatory cells such as macrophages and neutrophils, producing pro-inflammatory factors like IL-1β, IL-6, and TNF-α, leading to the onset and progression of gout. The overall incidence of gout in China is as high as 1.3%, and the prevalence is rapidly increasing at an annual rate of 9.7%, far exceeding the population growth rate. During a gouty joint attack, patients suffer from severe and persistent pain. Commonly used medications for the acute phase of gouty joint inflammation include colchicine (COL), nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids. However, colchicine, as one of the first-line drugs for acute gout attacks, can cause gastrointestinal reactions such as vomiting and diarrhea, leukopenia, bone marrow suppression, and abnormal liver function; nonsteroidal anti-inflammatory drugs (NSAIDs) may increase the risk of gastrointestinal ulcers and bleeding, and may further damage kidney function; while corticosteroids have side effects including skin problems, immune system dysfunction, and impaired bone development. Therefore, the development of more effective and less toxic novel anti-gout drugs remains a hot topic in clinical research.

[0004] Ischemia / reperfusion (I / R) injury is a pathophysiological process widely observed in organ transplantation and surgical procedures. It primarily manifests as hypoxic injury occurring during the ischemic phase of an organ or tissue, which is then exacerbated upon reperfusion. Ischemia / reperfusion induces tissue cell damage, releasing injury-related molecular patterns, which further activate various immune cells through pattern recognition receptors, leading to aseptic inflammation and exacerbating tissue damage. The inflammatory response and its mediators induced by reperfusion can worsen ischemia / reperfusion injury and are one of the important pathological mechanisms of this injury. Therefore, inhibiting the development of inflammation can effectively reduce vascular endothelial damage, decrease myocardial necrosis after reperfusion, and improve ischemia / reperfusion injury.

[0005] In our previous research, we obtained a novel class of microtubule aggregation inhibitors that exhibited good inhibitory activity against various tumor cells, including paclitaxel-resistant ovarian cancer cells, with an IC50 score of [missing value]. 50 These compounds reached nM levels, while exhibiting weak activity against normal ovarian epithelial cells, demonstrating good selectivity (J.Med.Chem. 2016, 59, 10329-10334. Eur.J.Med.Chem. 2018, 144, 817-842.). Pharmacological mechanisms revealed that these compounds inhibited protein and intracellular tubulin aggregation, arrested the cell cycle at the G2 / M phase, induced apoptosis in a concentration-dependent manner, and inhibited angiogenesis both in vitro and in vivo. In vivo experiments showed that intraperitoneal administration of these compounds significantly reduced the volume and weight of A2780 ovarian cancer cell xenografts in nude mice. H&E staining showed extensive necrosis of tumor cells in the treated group, while the weight of the nude mice did not decrease significantly, and there were no obvious toxic side effects on the liver, kidneys, spleen, or other internal organs. Acute toxicity experiments showed that intraperitoneal injection of the compounds in mice exhibited a good therapeutic window and did not inhibit the potassium ion channel hERG. X-ray single-crystal diffraction of the compound and its microtubule crystal complex also confirmed their binding to the colchicine site of microtubules. Preliminary in vitro stability experiments showed that the compound exhibited good stability at pH 3.0 and 7.4. 1 / 2 >24h. Preliminary pharmacokinetic and water solubility studies showed that the compound possesses good pharmacokinetic properties and good water solubility. Preliminary rat pharmacokinetic experiments indicated that the elimination half-life of the compound was essentially the same after intravenous and oral administration, approximately 6 hours, and its absolute bioavailability was approximately 56-58%. However, no reports have been found regarding the anti-inflammatory properties and applications of this type of β-lactam compound. Summary of the Invention

[0006] The purpose of this invention is to provide a new application of β-lactam compounds in the preparation of anti-inflammatory drugs.

[0007] The β-lactam compounds of this invention have the structure shown in formula (I):

[0008]

[0009] Wherein, R is a monosubstituted or polysubstituted group, and the substituent is independently selected from alkyl, hydroxymethyl, hydroxyethyl, hydrogen atom, alkoxy, acyloxy, hydroxyl, halogen, amino, substituted amino, methanesulfonyloxy, methoxymethyl, N,N-dimethylaminomethyl, 4-hydroxybenzyl, trimethylsilylethyl, ethoxycarbonylmethyl; X is a hydroxyl, amino, acyloxy, alkoxy, substituted amino, halogen, alkyl, hydroxymethyl or hydrogen atom.

[0010] This invention, through further research on β-lactam compounds and experimental demonstration, reveals that β-lactam compounds inhibit tubulin aggregation by binding to the surface of α and β tubulin, thereby suppressing various microtubule-mediated cellular processes, including cell shape maintenance, intracellular transport, cytokine and chemokine secretion, cell migration, and regulation of ion channels and cell division. β-lactam compounds may inhibit inflammatory responses through several pathways: suppression of immune activation, NLRP3 inflammasome activation, and Caspase-1 activation; inhibition of neutrophil recruitment, adhesion, and chemokine release; and inhibition of vascular endothelial growth factor secretion and endothelial cell proliferation.

[0011] Therefore, β-lactam compounds can inhibit tubulin aggregation, suppress inflammatory responses, and inhibit neutrophil function, thus they can be used to prepare anti-inflammatory drugs for the treatment of local inflammatory diseases caused by inflammation, such as acute gout, familial Mediterranean fever, chronic kidney disease, pancreatitis, sarcopenia, and depression, as well as inflammatory cardiovascular diseases such as ischemia-reperfusion injury, diabetes, atherosclerosis, pericarditis, and acute coronary syndrome.

[0012] Specifically, including:

[0013] β-lactam compounds, as inhibitors of tubulin aggregation, are used to treat local inflammatory responses, cardiovascular diseases, familial Mediterranean fever, and other conditions.

[0014] β-lactam compounds, as inhibitors of tubulin aggregation, are used to inhibit inflammation-induced NLRP3 activation and Caspase-1 activation, inhibit neutrophil recruitment and chemokine release, and inhibit vascular endothelial growth factor secretion and endothelial cell proliferation.

[0015] β-lactam compounds, as inhibitors of tubulin aggregation, are used to treat or prevent acute gouty arthritis, etc.

[0016] β-lactam compounds, as inhibitors of tubulin aggregation, are used to treat or prevent ischemia-reperfusion injury.

[0017] β-lactam compounds, as inhibitors of tubulin aggregation, are used to treat or prevent other local inflammatory responses and cardiovascular diseases caused by inflammation.

[0018] This invention uses THP-1 cells to detect the effect of β-lactam compounds on the release of the inflammatory cytokine IL-1β. The results show that these compounds have moderate to potent inhibitory activity against LPS-induced IL-1β release, indicating that they possess good anti-inflammatory activity.

[0019] This invention uses Raw 264.7 cells to detect the effects of selected β-lactam compounds 14b and C9 on the expression of various inflammation-related cytokines, including IL-1β, and the inflammasome NLRP3 induced by MSU and LPS. The results showed that the compounds, even at low concentrations, significantly inhibited the expression of the inflammasome NLRP3 and the genes of inflammatory factors (IL-1β, IL-6, IL-18, Caspase-1, and iNOS) in Raw 264.7 cells, while also inhibiting the secretion of the inflammatory factors IL-1β and IL-6.

[0020] This invention uses an MSU-induced mouse model of gouty arthritis to detect the in vivo anti-inflammatory activity of preferred β-lactam compounds 14b and C9. Results showed that treatment with the compounds inhibited paw edema in mice; simultaneously, the compounds inhibited the expression of the inflammasome NLRP3 and inflammatory cytokine (IL-1β, IL-6, and Caspase-1) genes in the mouse paw induced by MSU. These results indicate that the compounds significantly inhibit the MSU-induced inflammatory response in mice and possess a good anti-acute gouty inflammatory effect.

[0021] This invention uses a mouse ischemia-reperfusion injury model to detect the in vivo efficacy of β-lactam compounds 14b and C9 in improving ischemia-reperfusion injury. The results show that compounds 14b and C9 improved the decline in cardiac function and ventricular dilation caused by ischemia-reperfusion injury, exerting a good cardioprotective effect and exhibiting good activity in improving myocardial ischemia-reperfusion injury. Attached Figure Description

[0022] Figure 1 The compound inhibits LPS-induced IL-1β release activity.

[0023] Figure 2 Compound 14b significantly inhibited the expression and secretion of the inflammasome NLRP3 and inflammatory cytokine genes.

[0024] Figure 3Compound 14b significantly suppressed paw swelling in mice after treatment; it also inhibited the expression of inflammasome NLRP3 and inflammatory factor genes in MSU-induced swollen paws of mice.

[0025] Figure 4 Compound 14b was shown to inhibit inflammatory infiltration and NLRP3 expression in mouse tissues.

[0026] Figure 5 Treatment with the representative compound C9 significantly suppressed paw swelling in mice.

[0027] Figure 6 Compounds 14b and C9 are representative of compounds that improve ischemia-reperfusion injury in mice. Detailed Implementation

[0028] The invention is further illustrated below with reference to specific embodiments. These embodiments are merely illustrative and do not alter the scope of protection of the invention.

[0029] Example 1: Establishing an in vitro inflammation model and testing the anti-inflammatory activity of compounds.

[0030] After treating THP-1 cells with phorbol ester (PMA, 100 ng / mL) for 48 hours, the cells were pretreated with the test compounds for 2 hours, followed by the addition of lipopolysaccharide (LPS) (1 μg / mL). After 16 hours, the supernatant was collected, and the IL-1β cytokine release level was detected using an ELISA kit. Several compounds showed moderate to potent inhibitory activity against IL-1β release at a concentration of 50 nM, with cell viability greater than 80%. The activities of compounds 14b and C9 were comparable to those of the positive control colchicine (Table 1). Figure 1 ).

[0031] Table 1. Activity of compounds in inhibiting LPS-induced IL-1β release.

[0032]

[0033]

[0034] Example 2: Establishing an in vitro acute gout model and testing compound activity

[0035] RAW264.7 cells were treated with lipopolysaccharide (LPS) (1 μg / mL) for 24 hours, followed by treatment with MSU (100 μg / mL), MSU+COL (10 μM), and MSU+14b (6 nM). qPCR was used to detect the mRNA expression levels of NLRP3, IL-1β, IL-6, IL-18, iNOS, and Caspase-1 in the four groups of cells. ELISA was used to detect the secretion levels of IL-1β and IL-6 in the supernatant. The results showed that treatment with compounds 14b or C9 significantly reduced the gene expression levels of the inflammasome NLRP3 and inflammation-related factors (IL-1β, IL-6, IL-18, Caspase-1, and iNOS) in RAW 264.7 cells induced by LPS and MSU, and also significantly reduced the secretion of cytokines IL-1β and IL-6. Figure 2 ).

[0036] Example 3: Establishment of an in vivo acute gout model in mice and testing the in vivo anti-acute gout inflammatory response activity of compound 14b.

[0037] Six- to eight-week-old C57 mice were divided into four groups: CON, MSU, MSU+COL, and MSU+14b. The MSU+COL and MSU+14b groups were administered colchicine (1 mg / kg) or 14b (7.5 mg / kg) daily, respectively, while the CON and MSU groups were administered an equal volume of PBS by gavage. One week after administration, the MSU, MSU+COL, and MSU+14b groups were injected into the paws with MSU (50 mg / mL, 0.2 mL) to induce an acute gouty inflammatory response, while the control group was injected with 0.2 mL of PBS. Paw swelling and pathological damage were observed in the treatment groups, MSU group, and control group. Immunohistochemistry and Western blotting were used to detect the expression of Caspase-1 and NLRP3. Blood was collected from the eyes, and IL-1β and IL-6 levels were detected by ELISA in each group. The results showed that treatment with compound 14b (7.5 mg / kg) significantly suppressed paw edema in mice; simultaneously, compound 14b inhibited the expression of the inflammasome NLRP3 and inflammatory cytokines (IL-1β, IL-6, and Caspase-1) genes in the swollen paws of mice. These results indicate that 14b significantly inhibits MSU-induced inflammatory responses in mice and possesses good in vivo activity. Figure 3 Furthermore, HE staining, immunohistochemistry, and Western blotting experiments showed that treatment with compound 14b significantly reduced inflammatory infiltration in mouse tissues and significantly decreased NLRP3 expression in the paw tissue. Figure 4 ).

[0038] Example 4: Establishment of an in vivo acute gout model in mice and testing the in vivo anti-acute gout inflammatory response activity of compound C9.

[0039] Six- to eight-week-old C57 mice were divided into five groups: CON, MSU, MSU+COL, MSU+C9 (7 mg / kg), and MSU+C9 (3.5 mg / kg). The MSU+COL, MSU+C9 (7 mg / kg), and MSU+C9 (3.5 mg / kg) groups were administered colchicine (1 mg / kg) or C9 (7 mg / kg or 3.5 mg / kg) daily, respectively. The CON and MSU groups were administered an equal volume of PBS by gavage. One week after administration, the MSU, MSU+COL, MSU+C9 (7 mg / kg), and MSU+C9 (3.5 mg / kg) groups were injected into the paws with MSU (50 mg / mL, 0.2 mL) to induce an acute gouty inflammatory response. The control group was injected with 0.2 mL of PBS. Eight hours later, the paw swelling and pathological damage in the administered groups, MSU group, and control group were observed. The results showed that compound C9 at a concentration of 7 mg / kg reduced MSU-induced paw edema in mice. Figure 5 ).

[0040] Example 5: Establishing a mouse model of ischemia-reperfusion injury and testing the activity of compounds.

[0041] Six- to eight-week-old C57 mice were divided into four groups: CON, COL (0.2 mg / kg), 14b (4 mg / kg), and C9 (10 mg / kg) for model establishment. The specific model establishment procedure was as follows: Mice were anesthetized with isoflurane by inhalation. The left chest was shaved and disinfected. A transverse incision was made along the 3rd-4th intercostal space, and the muscles were bluntly dissected to open the thoracic cavity and expose the heart. The chest wall was gently pressed to partially evert the heart, exposing the left ventricle and left anterior descending artery (LAD). Sutures were threaded through the distal segment of the LAD and ligated to induce ischemia. After 30 minutes of ischemia, the ligatures were released to restore blood flow, and the myocardial color was observed to change from pale to congested. The muscles and skin were sutured layer by layer. Mice were administered medication by gavage for one week starting on the day of model establishment. Echocardiography was used to assess cardiac function, and the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic diameter (LVEDV), and left ventricular end-systolic volume (LVESV) were calculated. Mice were dissected, and Masson staining was used to assess the degree of myocardial fibrosis. The results showed that, compared to the CON group, mice in the compound 14b and C9 groups had significantly increased left ventricular ejection fraction and left ventricular fractional shortening, and significantly decreased left ventricular end-diastolic diameter and volume. This indicates that compounds 14b and C9 improved cardiac function decline and ventricular dilation caused by ischemia-reperfusion injury, exerting a good cardioprotective effect. Finally, histological analysis using Masson staining showed that the degree of myocardial fibrosis (blue area) was significantly reduced after compound treatment. These results indicate that compounds 14b and C9 have good activity in improving myocardial ischemia-reperfusion injury. Figure 6 ).

Claims

1. β The use of β-lactam compounds in the preparation of medicaments for the treatment or prevention of acute gouty arthritis, wherein... β - Lactam compounds have the structure shown in formula (I): ， in, R is independently selected from alkyl or hydroxymethyl; X is hydroxyl.

2. β The use of β-lactam compounds in the preparation of medicaments for the treatment or prevention of ischemia-reperfusion injury, the β - Lactam compounds have the structure shown in formula (I): ， in, R is independently selected from alkyl or hydroxymethyl; X is hydroxyl.

3. The application according to claim 1 or 2, wherein the drug is used to inhibit inflammation-induced NLRP3 activation and Caspase-1 activation.

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