In Vitro and In Silico Evaluation of New 1,3,4-Oxadiazole Derivatives of Pyrrolo[3,4-d]pyridazinone as Promising Cyclooxygenase Inhibitors

Szczęśniak-Sięga B.M., Wiatrak B., Czyżnikowska Ż., Janczak J., Wiglusz R.J., Maniewska J.

Novel arylpiperazine-1,2-benzothiazine derivatives have been designed and synthesized as potential anti-inflammatory agents. Their structure and properties have been studied using spectroscopic techniques (1H NMR, 13C NMR, FT-IR), MS, elemental analyses, and single-crystal X-ray diffraction (SCXRD, for compound 7b). This study aimed to evaluate the inhibitory activity of new derivatives against both cyclooxygenase isoforms COX-1 and COX-2 due to the similarity of new compounds to oxicams drugs from the NSAIDs group. All new compounds were divided into two series – A and B – with a different linker between thiazine and piperazines nitrogens. Series A included the three-carbon aliphatic linker and series B – two-carbon with a carbonyl group. According to in vitro and molecular docking studies all new compounds exhibited cyclooxygenase inhibitory activity. The series of A compounds included COX-1 inhibitors only. In contrast, the B series showed inhibition of both COX-1 and COX-2, which suggested the importance of the acetoxy linker for COX-2 inhibition. Moreover, the most selective compound 7b, towards COX-2, was non-toxic for the normal human cell line (in concentration of 10 µM) comparable to reference drug meloxicam. Additionally, investigation of influence on model membranes confirmed the ability of the compound 7b to penetrate lipid bilayers which seemed to be important to the influence with membrane protein-cyclooxygenase.

Szczukowski Ł., Krzyżak E., Zborowska A., Zając P., Potyrak K., Peregrym K., Wiatrak B., Marciniak A., Świątek P.

The long-term use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in treatment of different chronic inflammatory disorders is strongly restricted by their serious gastrointestinal adverse effects. Therefore, there is still an urgent need to search for new, safe, and efficient anti-inflammatory agents. Previously, we have reported the Mannich base-type derivatives of pyrrolo[3,4-d]pyridazinone which strongly inhibit cyclooxygenase, have better affinity to COX-2 isoenzyme and exert promising anti-oxidant activity. These findings encouraged us to perform further optimization of that structure. Herein, we present the design, synthesis, molecular docking, spectroscopic, and biological studies of novel pyrrolo[3,4-d]pyridazinone derivatives bearing 4-aryl-1-(1-oxoethyl)piperazine pharmacophore 5a,b–6a,b. The new compounds were obtained via convenient, efficient, one-pot synthesis. According to in vitro evaluations, novel molecules exert no cytotoxicity and act as selective COX-2 inhibitors. These findings stay in good correlation with molecular modeling results, which additionally showed that investigated compounds take a position in the active site of COX-2 very similar to Meloxicam. Moreover, all derivatives reduce the increased level of reactive oxygen and nitrogen species and prevent DNA strand breaks caused by oxidative stress. Finally, performed spectroscopic and molecular docking studies demonstrated that new compound interactions with bovine serum albumin (BSA) are moderate, formation of complexes is in one-to-one ratio, and binding site II (subdomain IIIA) is favorable.

Glomb T., Wiatrak B., Gębczak K., Gębarowski T., Bodetko D., Czyżnikowska Ż., Świątek P.

Numerous studies have confirmed the coexistence of oxidative stress and inflammatory processes. Long-term inflammation and oxidative stress may significantly affect the initiation of the neoplastic transformation process. Here, we describe the synthesis of a new series of Mannich base-type hybrid compounds containing an arylpiperazine residue, 1,3,4-oxadiazole ring, and pyridothiazine-1,1-dioxide core. The synthesis was carried out with the hope that the hybridization of different pharmacophoric molecules would result in a synergistic effect on their anti-inflammatory activity, especially the ability to inhibit cyclooxygenase. The obtained compounds were investigated in terms of their potencies to inhibit cyclooxygenase COX-1 and COX-2 enzymes with the use of the colorimetric inhibitor screening assay. Their antioxidant and cytotoxic effect on normal human dermal fibroblasts (NHDF) was also studied. Strong COX-2 inhibitory activity was observed after the use of TG6 and, especially, TG4. The TG11 compound, as well as reference meloxicam, turned out to be a preferential COX-2 inhibitor. TG12 was, in turn, a non-selective COX inhibitor. A molecular docking study was performed to understand the binding interaction of compounds at the active site of cyclooxygenases.

Szczukowski Ł., Redzicka A., Wiatrak B., Krzyżak E., Marciniak A., Gębczak K., Gębarowski T., Świątek P.

Novel Mannich base analogues of pyrrolo[3,4-d]pyridazinone 7a,b-13a,b are designed and synthesized as potential anti-inflammatory agents. The title compounds are obtained via convenient one-pot synthesis with good yields. Their structures and properties are described by spectroscopic techniques and elemental analyses. The aim of this study is to evaluate the inhibitory activity of the new derivatives against both cyclooxygenase isoforms COX1 and COX2 as well as their cytotoxicity. The results clearly indicate that the tested compounds 7a,b-13a,b are not toxic, all show better affinity towards isoform COX-2, and some of them act as selective COX-2 inhibitors. Moreover, every examined derivative of pyrrolo[3,4-d]pyridazinone demonstrates better inhibitory activity towards COX-2 and a superior COX-2/COX-1 selectivity ratio compared to the reference drug meloxicam. Molecular docking studies confirm that compounds 7a,b-13a,b preferably bind COX-2 and all of them bind to the active site of cyclooxygenase in a way very similar to meloxicam. Subsequently, taking into account that inflammation is strongly correlated with oxidative stress and both of these processes can potentiate each other, synthesized Mannich bases are evaluated for potential antioxidant activity. Most of the investigated derivatives reduce induced oxidative and nitrosative stress. Moreover, compounds 7a,b, 8a, 10a,b, 11b, 12a,b-13a,b protect chromatin from oxidative stress and decrease the number of DNA strand breaks caused by intracellular growth of free radicals. Finally, a study of the binding mechanism between compounds 7a,b-13a,b and bovine serum albumin (BSA) was carried out. According to spectroscopic and molecular docking studies, all examined derivatives interact with BSA, which suggests their potential long half-life in vivo.

Caballero J., Núñez-Vivanco G., Valdés-Jiménez A., Durán-Verdugo F., Velázquez-Libera J.L.

Abstract Motivation Root mean square deviation (RMSD) is one of the most useful and straightforward features for structural comparison between different conformations of the same molecule. Commonly, protein-ligand docking programs have included some utilities that allow the calculation of this value; however, they only work efficiently when exists a complete atom label equivalence between the evaluated conformations. Results We present LigRMSD, a free web-server for the automatic matching and RMSD calculations among identical or similar chemical compounds. This server allows the user to submit only a pair of identical or similar molecules or dataset of similar compounds to compare their three-dimensional conformations. Availability and implementation LigRMSD can be freely accessed at https://ligrmsd.appsbio.utalca.cl. Supplementary information Supplementary data are available at Bioinformatics online.

Redzicka A., Szczukowski Ł., Kochel A., Wiatrak B., Gębczak K., Czyżnikowska Ż.

• Pyrrolo[3,4- c ]pyrrole derivatives were synthesized and their COX-1/COX-2 inhibition activities were evaluated. • All compounds exhibited higher analgesic activity than the reference drug. • The new pyrrolo[3,4- c ]pyrroles were not toxic (LD 50 ≥ 2000 mg/kg). • Three of the compounds may be promising inhibitors of COX-2. In the present paper we describe the biological activity of newly designed and synthesized series of pyrrolo[3,4- c ]pyrrole Mannich bases ( 7a - n ). The Mannich bases were obtained in good yields by one-pot, three-component condensation of pyrrolo[3,4– c ]pyrrole scaffold ( 6a - c ) with secondary amines and an excess of formaldehyde solution in C 2 H 5 OH. The chemical structures of the compounds were characterized by 1 H NMR, 13 C NMR, FT-IR, and elemental analysis. Moreover, single crystal X-ray diffraction has been recorded for compound 7l . All synthesized derivatives were investigated for their potencies to inhibit COX-1 and COX-2 enzymes by colorimetric inhibitor screening assay. In order to analyse the intermolecular interactions between the ligands and cyclooxygenase, experimental data were supported with the results of molecular docking simulations. According to the results, all of the tested compounds inhibited the activity of COX-1 and COX-2.

Świątek P., Strzelecka M., Urniaz R., Gębczak K., Gębarowski T., Gąsiorowski K., Malinka W.

One of the main challenges for nowadays medicine is drugs selectivity. In COX-1 and COX-2, the active sites are composed of the same group of amino acids with the exception of the only one residue in position 523, in COX-1 is an isoleucine, while in COX-2 is a valine. Here, we presented a series of isothiazolopyridine/benzisothiazole derivatives substituted differently into an isothiazole ring, which were synthesized and investigated for their potencies to inhibit COX-1 and COX-2 enzymes by colorimetric inhibitor screening assay. All the tested compounds inhibited the activity of COX-1, the effect on COX-2 activity was differential. The mode of binding was characterized by a molecular docking study. Comparing biological activity of the investigated compounds, it was observed that compounds sharing the most similar position to flurbiprofen and meloxicam, representing the two main enzyme subdomains, achieved higher biological activity than others. It is directly related to the fit to the enzyme's active site, which prevents too early dissociation of the compounds.

Palkar M.B., Singhai A.S., Ronad P.M., Vishwanathswamy A.H., Boreddy T.S., Veerapur V.P., Shaikh M.S., Rane R.A., Karpoormath R.

A novel series of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-(substituted)-1,3,4-oxadiazol-2(3H)-thione (4a-k) derivatives have been synthesized by the Mannich reaction of 5-[2-(2,6-dichlorophenylamino)benzyl]-1,3,4-oxadiazol-2(3H)-thione (3) with an appropriately substituted primary/secondary amines, in the presence of formaldehyde and absolute ethanol. Structures of these novel compounds were characterized on the basis of physicochemical, spectral and elemental analysis. The title compounds (4a-k) were screened for in vivo acute anti-inflammatory and analgesic activities at a dose of 10mg/kg b.w. Compound 4k exhibited the most promising and significant anti-inflammatory profile while compounds 4a, 4d, 4e, 4i, and 4j showed moderate to good inhibitory activity at 2nd and 4thh, respectively. These compounds were also found to have considerable analgesic activity (acetic acid induced writhing model) and antipyretic activity (yeast induced pyrexia model). In addition, the tested compounds were also found to possess less degree of ulcerogenic potential as compared to the standard NSAIDs. Compounds that displayed promising anti-inflammatory profile were further evaluated for their inhibitory activity against cyclooxygenase enzyme (COX-1/COX-2), by colorimetric COX (ovine) inhibitor screening assay method. The results revealed that the compounds 4a, 4e, 4g and 4k exhibited effective inhibition against COX-2. In an attempt to understand the ligand-protein interactions in terms of the binding affinity, docking studies were performed using Molegro Virtual Docker (MVD-2013, 6.0) for those compounds, which showed good anti-inflammatory activity. It was observed that the binding affinities calculated were in agreement with the IC50 values.

Xu S., Hermanson D.J., Banerjee S., Ghebreselasie K., Clayton G.M., Garavito R.M., Marnett L.J.

Background: The oxicams are anti-inflammatory drugs targeting the cyclooxygenase enzymes. Results: Crystal complexes of mCOX-2·isoxicam, mCOX-2·meloxicam, and oCOX-1·meloxicam are solved. Conclusion: Oxicams bind to the cyclooxygenase active sites in a novel mode. Significance: The first structural description of cyclooxygenase-oxicam complexes reveal a new binding pocket of inhibitors to cyclooxygenases. Oxicams are widely used nonsteroidal anti-inflammatory drugs (NSAIDs), but little is known about the molecular basis of the interaction with their target enzymes, the cyclooxygenases (COX). Isoxicam is a nonselective inhibitor of COX-1 and COX-2 whereas meloxicam displays some selectivity for COX-2. Here we report crystal complexes of COX-2 with isoxicam and meloxicam at 2.0 and 2.45 angstroms, respectively, and a crystal complex of COX-1 with meloxicam at 2.4 angstroms. These structures reveal that the oxicams bind to the active site of COX-2 using a binding pose not seen with other NSAIDs through two highly coordinated water molecules. The 4-hydroxyl group on the thiazine ring partners with Ser-530 via hydrogen bonding, and the heteroatom of the carboxamide ring of the oxicam scaffold interacts with Tyr-385 and Ser-530 through a highly coordinated water molecule. The nitrogen atom of the thiazine and the oxygen atom of the carboxamide bind to Arg-120 and Tyr-355 via another highly ordered water molecule. The rotation of Leu-531 in the structure opens a novel binding pocket, which is not utilized for the binding of other NSAIDs. In addition, a detailed study of meloxicam·COX-2 interactions revealed that mutation of Val-434 to Ile significantly reduces inhibition by meloxicam due to subtle changes around Phe-518, giving rise to the preferential inhibition of COX-2 over COX-1.

Takeuchi K.

This article reviews the pathogenic mechanism of non-steroidal anti-inflammatory drug (NSAID)-induced gastric damage, focusing on the relation between cyclooxygenase (COX) inhibition and various functional events. NSAIDs, such as indomethacin, at a dose that inhibits prostaglandin (PG) production, enhance gastric motility, resulting in an increase in mucosal permeability, neutrophil infiltration and oxyradical production, and eventually producing gastric lesions. These lesions are prevented by pretreatment with PGE₂ and antisecretory drugs, and also via an atropine-sensitive mechanism, not related to antisecretory action. Although neither rofecoxib (a selective COX-2 inhibitor) nor SC-560 (a selective COX-1 inhibitor) alone damages the stomach, the combined administration of these drugs provokes gastric lesions. SC-560, but not rofecoxib, decreases prostaglandin E₂ (PGE₂) production and causes gastric hypermotility and an increase in mucosal permeability. COX-2 mRNA is expressed in the stomach after administration of indomethacin and SC-560 but not rofecoxib. The up-regulation of indomethacin-induced COX-2 expression is prevented by atropine at a dose that inhibits gastric hypermotility. In addition, selective COX-2 inhibitors have deleterious influences on the stomach when COX-2 is overexpressed under various conditions, including adrenalectomy, arthritis, and Helicobacter pylori-infection. In summary, gastric hypermotility plays a primary role in the pathogenesis of NSAID-induced gastric damage, and the response, causally related with PG deficiency due to COX-1 inhibition, occurs prior to other pathogenic events such as increased mucosal permeability; and the ulcerogenic properties of NSAIDs require the inhibition of both COX-1 and COX-2, the inhibition of COX-1 upregulates COX-2 expression in association with gastric hypermotility, and PGs produced by COX-2 counteract the deleterious effect of COX-1 inhibition.

Smith W.L., Urade Y., Jakobsson P.

Prostanoids are cyclic, oxygenated products of ω3 and ω6 20- and 22-carbon essential fatty acids (FAs) that are formed enzymatically through “cyclooxygenases”. Prostaglandin endoperoxide synthases -1 and -2 (PGHS-1 and -2)a, which are also known as cyclooxygenases -1 and -2 (COX-1 and -2), catalyze the committed step in the biosynthesis of prostanoids (Figure 1). These compounds include what are sometimes referred to as the “classical” prostaglandins (PGs) PGD, PGE, and PGF as well as prostacyclins denoted as PGI's and the thromboxanes abbreviated Tx's; monohydroxy acids can also be formed via PGHSs, but information on the possible physiologic importance of such compounds is incomplete.1-3 The most abundant prostanoids are the “2-series” compounds (e.g. PGE2) that are formed from arachidonic acid (AA; 5Z, 8Z, 11Z, 14Z- eicosatetraenoic acid; 20:4 ω6; Figure 1). The “2” denotes the number of carbon-carbon double bonds in the product. Figure 1 Biosynthetic pathway for the formation of prostanoids PGHSs catalyze two distinct reactions that occur at physically distinct but functionally interacting sites. The cyclooxygenase (COX) reaction is a bis-oxygenation in which two O2 molecules are inserted into the carbon backbone of AA to yield PGG2 (Figure 1). The peroxidase (POX) reaction is a transformation in which the 15-hydroperoxyl group of PGG2 undergoes a net two electron reduction to PGH2 plus water. The POX reaction is important in the enzyme mechanism, but other peroxidases such as glutathione peroxidase may contribute importantly to the reduction of PGG2 to PGH2 in vivo. PGH2 is thought not to accumulate in cells but rather to be converted quickly to what are considered the biologically relevant, downstream products. There are specific synthases involved in forming PGD2, PGE2, PGF2α, PGI2 and TxA2 from PGH2. Except for the case of PGF2α, which is formed by a two electron reduction of PGH2, these enzymes catalyze non-oxidative rearrangements. Finally, there is a PGH 19-hydoxylase that converts PGHs to their corresponding 19-hydroxy derivatives which themselves are substrates for PGE synthase(s). There are one or more specific G protein-linked receptors for each prostanoid, and in some cases prostanoids may also act through nuclear peroxisome proliferator activated receptors. AA and other 20- and 22-carbon, highly unsaturated FAs are found esterified at the sn2 position of glycerophospholipids. Basal prostanoid formation generally occurs at a low rate relative to stimulated formation. A major factor limiting prostanoid formation is AA availability, which is controlled through the net rates of deacylation and reacylation of glycerophospholipids. Prostanoid formation is enhanced when phospholipase A2 (PLA2) activity is increased, and thus PLA2s play a substrate-limiting role in regulating prostanoid biosynthesis. Although reacylation may also be important, its possible role in regulating prostanoid biosynthesis is largely unexplored. In this chapter we review the biochemistry and the biochemical pharmacology of the enzymes involved in converting AA to various prostanoid products. These enzymes include PGHS-1, PGHS-2, hematopoietic PGD synthase (H-PGDS), lipocalin-type PGD synthase (L-PGDS), microsomal PGE synthase-1 (mPGES-1), microsomal PGE synthase-2 (mPGES-2), cytosolic PGE synthase (cPGES), PGF synthase (PGFS), PGI synthase (PGIS) and TXA synthase (TXAS). The PLA2s involved in mobilizing AA and the receptors through which prostanoids function are surveyed in other chapters of this volume.

Gautam R., Jachak S.M., Kumar V., Mohan C.G.

Stellatin (4), isolated from Dysophylla stellata is a cyclooxygenase (COX) inhibitor. The present study reports the synthesis and biological evaluation of new stellatin derivatives for COX-1, COX-2 inhibitory and anti-inflammatory activities. Eight derivatives showed more pronounced COX-2 inhibition than stellatin and, 17 and 21 exhibited the highest COX-2 inhibition. They also exhibited the significant anti-inflammatory activity in TPA-induced mouse ear edema assay and their anti-inflammatory effects were more than that of stellatin and indomethacin at 0.5mg/ear. The derivatives were further evaluated for antioxidant activity wherein 16 and 17 showed potent free radical scavenging activity against DPPH and ABTS radicals. Molecular docking study revealed the binding orientations of stellatin and its derivatives into the active sites of COX-1 and COX-2 and thereby helps to design the potent inhibitors.

Akhter M., Akhter N., Alam M.M., Zaman M.S., Saha R., Kumar A.

Dual cyclooxygenase/lipoxygenase (COX/LOX) inhibitors constitute a valuable alternative to classical nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors for the treatment of inflammatory diseases. A series of 3-(5-phenyl/phenylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-one and N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]oxadiazol-2-yl]-benzamide derivatives were synthesized and screened for anti-inflammatory, analgesic activity. All the derivatives prepared are active in inhibiting oedema induced by carrageenan. Compound 4e was found more potent with 89% of inhibition followed by compound 4b (86%). Compounds with >70% of anti-inflammatory activity were tested for analgesic, ulcerogenic, and lipid peroxidation profile. Selected compounds were also evaluated for inhibition of COXs (COX-1 and COX-2) and LOXs (LOX-5, LOX-12, and LOX-15). Compound 4e was comparatively selective for COX-2, LOX-5, and LOX-15. Study revealed that these derivatives were more effective than ibuprofen with reduced side effects. It can be suggested that these derivatives could be used to develop more potent and safer NSAIDs.

Manjunatha K., Poojary B., Lobo P.L., Fernandes J., Kumari N.S.

The acid hydrazides (2) derived from ibuprofen and 4-methylthiophenyl acetic acids have been subjected to cyclization with carbon disulphide under basic conditions to yield 1,3,4-oxadiazol-2-thiones (3) which on aminomethylation with formaldehyde and secondary amines afforded a series of Mannich bases (4 and 5). Purity of the compounds has been confirmed by TLC. Structures of these compounds were established on the basis of elemental analyses and spectral studies. The newly synthesized compounds were evaluated for their anti-inflammatory, analgesic, ulcerogenic and antimicrobial activities.

Vecchio A.J., Simmons D.M., Malkowski M.G.

The cyclooxygenases (COX-1 and COX-2) are membrane-associated heme-containing homodimers that generate prostaglandin H(2) from arachidonic acid (AA). Although AA is the preferred substrate, other fatty acids are oxygenated by these enzymes with varying efficiencies. We determined the crystal structures of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) bound to Co(3+)-protoporphyrin IX-reconstituted murine COX-2 to 2.1, 2.4, and 2.65 A, respectively. AA, EPA, and docosahexaenoic acid bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel. The interactions identified between protein and substrate when bound to COX-1 are conserved in our COX-2 structures, with the only notable difference being the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared. Unlike COX-1, mutating this residue to Ala, Phe, Pro, or Thr did not result in a significant loss of activity or substrate binding affinity. Determination of the L531F:AA crystal structure resulted in AA binding in the same global conformation in each monomer. We speculate that the mobility of the Leu-531 side chain increases the volume available at the opening of the cyclooxygenase channel and contributes to the observed ability of COX-2 to oxygenate a broad spectrum of fatty acid and fatty ester substrates.

Yilmaz Y.B., Güngör T., Dönmez S., Atalay H.N., Siyah P., Durdağı S., Ay M., Boyunegmez Tumer T.

New isothiocyanate derivatives (I1–3 and I1a–e) were synthesized and screened for their anti-inflammatory activities and H2S-releasing capacities. Compounds I1 and I1c demonstrated the most potent and selective COX-2 inhibition.

Szczęśniak-Sięga B.M., Maniewska J., Wiatrak B., Janek T., Nowotarska P., Czyżnikowska Ż.

The design of novel anti-inflammatory drugs remains a critical area of research in the development of effective treatments for inflammatory diseases. In this study, a series of 1,2-benzothiazine was evaluated through a multifaceted approach. In particular, we investigated the potential interactions of the potential drugs with lipid bilayers, an important consideration for membrane permeability and overall pharmacokinetics. In addition, we evaluated their ability to inhibit cyclooxygenase 1 and cyclooxygenase 2 activity and selectivity using both a cyclooxygenase inhibition assay and molecular docking simulations. To evaluate their therapeutic potential, we performed in vitro assays to measure cytokine mRNA expression in inflamed cells. The antioxidant activity was evaluated using both in vitro assays, such as 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid scavenging, to determine the compounds’ capacity to neutralize free radicals and reduce oxidative stress. Theoretical calculations, including density functional theory, were used to predict the reactivity profiles of the compounds.

Tylińska B., Janicka-Kłos A., Gębarowski T., Nowotarska P., Plińska S., Wiatrak B.

Pyrimidine derivatives exhibit a wide range of biological activities, including anti-inflammatory properties. The aim of this study was to investigate the effects of tested pyrimidine derivatives on the activity of cyclooxygenase isoenzymes (COX-1 and COX-2), antioxidant properties, and their ability to inhibit the growth of inflammatory cells. In vitro tests were conducted to assess the ability of pyrimidine derivatives L1–L4 to inhibit COX-1 and COX-2 activity using the TMPD oxidation assay (N,N,N',N'-tetramethyl-p-phenylenediamine). The compounds’ ability to inhibit the growth of lipopolysaccharide (LPS)-stimulated THP-1 (human leukemia monocytic) monocyte cells and their impact on reactive oxygen species (ROS) levels in an inflammatory model were also evaluated. The binding properties of human serum albumin (HSA) were assessed using UV–Vis spectroscopy, circular dichroism (CD), and isothermal titration calorimetry (ITC). Among the tested pyrimidine derivatives, L1 and L2 showed high selectivity towards COX-2, outperforming piroxicam and achieving results comparable to meloxicam. In the sulforhodamine B (SRB) assay, L1 and L2 demonstrated dose-dependent inhibition of LPS-stimulated THP-1 cell growth. Additionally, ROS assays indicated that these compounds reduced free radical levels, confirming their antioxidant properties. Binding studies with albumin revealed that L1 and L2 formed stable complexes with HSA. These results suggest that these compounds could serve as a basis for further research into anti-inflammatory and anticancer drugs with reduced toxicity.

Mishra A.C., Upadhyay J., Dixit P.P., Baheti K., Thore S.N.

A series of novel trans-3-substituted aminomethyl-5-(4-(4-chlorophenyl)cyclohexyl)-1,3,4-oxadiazole-2(3H)-thiones was synthesized successfully from achiral trans-4-(4-chlorophenyl)cyclohexane-1-carboxylic acid. To investigate their potential binding interactions with proteins at the active site, molecular docking studies were conducted using CDOCKER module (Biovia Discovery Studio 2022) against Penicillin Binding Protein 2 of Escherichia coli (PDB: 6G9F) and Pseudomonas aeruginosa (PDB: 7KIS). The results of the docking studies indicate that the compounds exhibit limited binding efficacy. Molecular Dynamics simulations were carried out for Ceftazidime to predict the ligand binding status in the physiological environment. The antibacterial in-vitro inhibitory potential was evaluated against a panel of microorganisms consisting of two Gram-positive bacterial strains, Bacillus subtilis (ATCC6633) and Staphylococcus aureus (ATCC6538), as well as three Gram-negative bacterial strains, Pseudomonas aeruginosa (ATCC9027), Escherichia coli (ATCC8739), and Salmonella typhi (ATCC9207). The N-Mannich bases displayed promising antibacterial activity against both the Gram-positive microorganisms and demonstrated effective inhibition of Escherichia coli. However, their activity against Pseudomonas aeruginosa was moderate. The binding affinity to Penicillin Binding Proteins was evaluated by observing morphological changes in Escherichia coli rods under an optical microscope. The results revealed a notable decrease in cell count without observable morphological changes, indicating that the N-Mannich bases do not bind strongly to Penicillin Binding Proteins and likely operate through an alternative mechanism. The antifungal activity against Saccharomyces cerevisiae (ATCC9763) and Aspergillus niger (ATCC16404) was not observed.

Ewieda S.Y., Ahmed E.M., Hassan R.A., Hassan M.S.

AbstractSelective cyclooxygenase (COX)‐2 inhibitors have several advantages over nonselective COX inhibitors (nonsteroidal anti‐inflammatory drugs [NSAIDs]), including the absence of adverse effects (renal and hepatic disorders) associated with the long‐term use of standard NSAIDs, as well as an improved gastrointestinal profile. The pyridazine nucleus is regarded as a promising scaffold for the development of powerful COX‐2 inhibitors, particularly when selectively functionalized. This article summarizes some methods for the synthesis of pyridazine derivatives. Furthermore, it covers all of the pyridazine derivatives that have appeared as selective COX‐2 inhibitors, making it useful as a reference for the rational design of novel selective COX‐2 inhibitors.

Du J., Wu W., Zhu B., Tao W., Liu L., Cheng X., Zhao M., Wu J., Li Y., Pei K.

The pathogenesis of coronary heart disease is a highly complex process, with lipid metabolism disorders being closely linked to its development. Therefore, this paper analyzes the various factors that influence lipid metabolism, including obesity, genes, intestinal microflora, and ferroptosis, through a comprehensive review of basic and clinical studies. Additionally, this paper delves deeply into the pathways and patterns of coronary heart disease. Based on these findings, it proposes various intervention pathways and therapeutic methods, such as the regulation of lipoprotein enzymes, lipid metabolites, and lipoprotein regulatory factors, as well as the modulation of intestinal microflora and the inhibition of ferroptosis. Ultimately, this paper aims to offer new ideas for the prevention and treatment of coronary heart disease.

Mikus J., Świątek P., Przybyła P., Krzyżak E., Marciniak A., Kotynia A., Redzicka A., Wiatrak B., Jawień P., Gębarowski T., Szczukowski Ł.

Secure and efficient treatment of diverse pain and inflammatory disorders is continually challenging. Although NSAIDs and other painkillers are well-known and commonly available, they are sometimes insufficient and can cause dangerous adverse effects. As yet reported, derivatives of pyrrolo[3,4-d]pyridazinone are potent COX-2 inhibitors with a COX-2/COX-1 selectivity index better than meloxicam. Considering that N-acylhydrazone (NAH) moiety is a privileged structure occurring in many promising drug candidates, we decided to introduce this pharmacophore into new series of pyrrolo[3,4-d]pyridazinone derivatives. The current paper presents the synthesis and in vitro, spectroscopic, and in silico studies evaluating the biological and physicochemical properties of NAH derivatives of pyrrolo[3,4-d]pyridazinone. Novel compounds 5a-c–7a-c were received with high purity and good yields and did not show cytotoxicity in the MTT assay. Their COX-1, COX-2, and 15-LOX inhibitory activities were estimated using enzymatic tests and molecular docking studies. The title N-acylhydrazones appeared to be promising dual COX/LOX inhibitors. Moreover, spectroscopic and computational methods revealed that new compounds form stable complexes with the most abundant plasma proteins–AAG and HSA, but do not destabilize their secondary structure. Additionally, predicted pharmacokinetic and drug-likeness properties of investigated molecules suggest their potentially good membrane permeability and satisfactory bioavailability.

Redzicka A., Wiatrak B., Jęśkowiak-Kossakowska I., Kochel A., Płaczek R., Czyżnikowska Ż.

In the present study, we characterize the biological activity of a newly designed and synthesized series of 15 compounds 2-[2-hydroxy-3-(4-substituted-1-piperazinyl)propyl] derivatives of pyrrolo[3,4-c]pyrrole 3a–3o. The compounds were obtained with good yields of pyrrolo[3,4-c]pyrrole scaffold 2a–2c with secondary amines in C2H5OH. The chemical structures of the compounds were characterized by 1H-NMR, 13C-NMR, FT-IR, and MS. All the new compounds were investigated for their potencies to inhibit the activity of three enzymes, i.e., COX-1, COX-2, and LOX, by a colorimetric inhibitor screening assay. In order to analyze the structural basis of interactions between the ligands and cyclooxygenase/lipooxygenase, experimental data were supported by the results of molecular docking simulations. The data indicate that all of the tested compounds influence the activity of COX-1, COX-2, and LOX.

Shankara S.D., Isloor A.M., Kudva A.K., Raghu S.V., Jayaswamy P.K., Venugopal P.P., Shetty P., Chakraborty D.

In the present work, a series of new 1-{5-[2,5-bis(2,2,2-trifluoroethoxy)phenyl]-1,3,4-oxadiazol-3-acetyl-2-aryl-2H/methyl derivatives were synthesized through a multistep reaction sequence. The compounds were synthesized by the condensation of various aldehydes and acetophenones with the laboratory-synthesized acid hydrazide, which afforded the Schiff’s bases. Cyclization of the Schiff bases yielded 1,3,4-oxadiazole derivatives. By spectral analysis, the structures of the newly synthesized compounds were elucidated, and further, their anti-cancer and anti-diabetic properties were investigated. To examine the dynamic behavior of the candidates at the binding site of the protein, molecular docking experiments on the synthesized compounds were performed, followed by a molecular dynamic simulation. ADMET (chemical absorption, distribution, metabolism, excretion, and toxicity) prediction revealed that most of the synthesized compounds follow Lipinski’s rule of 5. The results were further correlated with biological studies. Using a cytotoxic assay, the newly synthesized 1,3,4-Oxadiazoles were screened for their in vitro cytotoxic efficacy against the LN229 Glioblastoma cell line. From the cytotoxic assay, the compounds 5b, 5d, and 5m were taken for colony formation assay and tunnel assay have shown significant cell apoptosis by damaging the DNA of cancer cells. The in vivo studies using a genetically modified diabetic model, Drosophila melanogaster, indicated that compounds 5d and 5f have better anti-diabetic activity among the different synthesized compounds. These compounds lowered the glucose levels significantly in the tested model.

Hamouzová P., Stehlíková Š., Řeháková K., Jekl V., Chloupek J., Doubek J.

Meloxicam is a commonly used analgesic in rabbits. However, its possible impact on lymphocyte subpopulations remained unknown. The aim of the study was to investigate a possible effect of long-term administration of meloxicam on rabbit lymphocyte subpopulations. The study included 8 rabbits given meloxicam orally once daily (1 mg/kg BW) for 14 days and 8 rabbits as a control group. Peripheral blood samples were collected on day 0 (before the first dose of meloxicam), day 3, 7 and 14. Samples were evaluated with a haematology analyser and a flow cytometer. A significant decrease in T: B cell ratio was found in all samples taken during meloxicam administration compared to day 0, as well as in comparison with the control group (P < 0.01). A significant increase (P < 0.05) in proportion of CD5 +CD8 + lymphocytes occurred by day 3. Subsequently, although the values slightly decreased, they still remained elevated throughout all the experiment compared to the values from day 0 (P < 0.05). A slight decrease in T and B cell activation (CD5 +CD25 + and IgM+CD25 +) noticed by day 3, declined during the next days of administration and became more and more significant (finally, P = 0.0078). Since a high significant decrease (P < 0.01) in both T and B cell activation as well as a significant increase (P < 0.05) in CD5 +CD8 + T cells proportion were observed after meloxicam administration, a predicted effect of long-term administration of meloxicam on rabbit lymphocytes was confirmed.

Mashood Ahamed F.M., Shakya B., Shakya S.

The novel Mannich base benzimidazole derivative (CB-1), 1-((1H-benzo[d]imidazol-1-yl)(3-chlorophenyl)methyl)-3-phenylurea) has been designed and synthesized by reacting benzimidazole, 3-chloro benzaldehyde, and N-Phenyl urea. CB-1 has been characterized by UV- Visible, FTIR, and 1H NMR. CB-1 was explored to study the interaction with the most abundant blood protein which involved in the role of transport of molecules (drugs), human serum albumin (HSA). Fluorescence results are evident for the presence of both dynamic and static quenching mechanisms in the binding of CB-1 to HSA. Antimicrobial screening were carried out against three bacteria and three fungi pathogens employing disc diffusion method. Molecular docking using AutoDock Vina tool further confirms the experimental binding interactions obtained from fluorescence. Density functional theory (DFT) with B3LYP/6-311G++ basis set was used for correlating theoretical data and obtaining optimized structures of CB-1 along with reactants with molecular electrostatic potential (MEP) map and HOMO→LUMO energy gap calculation. HIGHLIGHTSThe novel Mannich base benzimidazole derivative (CB-1) has been designed and synthesized by Mannich reaction.CB-1 has been characterized by UV- Visible, FTIR, and 1H NMR.Fluorescence quenching reveals that HSA binds to CB-1 via aromatic residues, which is corroborated by molecular docking.Antifungal and antibacterial activity was evaluated in comparison to Nystatin and Gentamicin standard drugs, respectively.DFT calculations support experimental data and provide HOMO-LUMO energy gap.

Cantini N., Schepetkin I.A., Danilenko N.V., Khlebnikov A.I., Crocetti L., Giovannoni M.P., Kirpotina L.N., Quinn M.T.

Persistent inflammation contributes to a number of diseases; therefore, control of the inflammatory response is an important therapeutic goal. In an effort to identify novel anti-inflammatory compounds, we screened a library of pyridazinones and structurally related derivatives that were used previously to identify N-formyl peptide receptor (FPR) agonists. Screening of the compounds for their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor κB (NF-κB) transcriptional activity in human THP1-Blue monocytic cells identified 48 compounds with anti-inflammatory activity. Interestingly, 34 compounds were FPR agonists, whereas 14 inhibitors of LPS-induced NF-κB activity were not FPR agonists, indicating that they inhibited different signaling pathways. Further analysis of the most potent inhibitors showed that they also inhibited LPS-induced production of interleukin 6 (IL-6) by human MonoMac-6 monocytic cells, again verifying their anti-inflammatory properties. Structure–activity relationship (SAR) classification models based on atom pair descriptors and physicochemical ADME parameters were developed to achieve better insight into the relationships between chemical structures of the compounds and their biological activities, and we found that there was little correlation between FPR agonist activity and inhibition of LPS-induced NF-κB activity. Indeed, Cmpd43, a well-known pyrazolone-based FPR agonist, as well as FPR1 and FPR2 peptide agonists had no effect on the LPS-induced NF-κB activity in THP1-Blue cells. Thus, some FPR agonists reported to have anti-inflammatory activity may actually mediate their effects through FPR-independent pathways, as it is suggested by our results with this series of compounds. This could explain how treatment with some agonists known to be inflammatory (i.e., FPR1 agonists) could result in anti-inflammatory effects. Further research is clearly needed to define the molecular targets of pyridazinones and structurally related compounds with anti-inflammatory activity and to define their relationships (if any) to FPR signaling events.

Hassan M.S., Ahmed E.M., El‐Malah A.A., Kassab A.E.

The pyridazinone core has emerged as a leading structure for fighting inflammation, with low ulcerogenic effects. Moreover, easy functionalization of various ring positions of the pyridazinone core structure makes it an attractive synthetic and therapeutic target for the design and synthesis of anti-inflammatory agents. The present review surveys the recent advances of pyridazinone derivatives as potential anti-inflammatory agents to provide insights into the rational design of more effective anti-inflammatory pyridazinones.

Palumbo Piccionello A.

Oxadiazoles are electron-poor, five-membered aromatic heterocycles that contain one oxygen and two nitrogen atoms [...]