Synthesis,p38α MAP kinase inhibition,anti‐inflammatory activity,and molecular docking studies of 1,2,4‐triazole‐based benzothiazole‐2‐amines

Recent studies have demonstrated that inhibition of p38α MAP kinase could effectively inhibit pro‐inflammatory cytokines including TNF‐α and interleukins. Thus, inhibition of this enzyme can prove greatly beneficial in the therapy of chronic inflammatory diseases. A new series of N‐[3‐(substituted‐4H‐1,2,4‐triazol‐4‐yl)]‐benzo[d]thiazol‐2‐amines ( 4a–n ) were synthesized and subjected to in vitro evaluation for anti‐inflammatory activity (BSA anti‐denaturation assay) and p38α MAPK inhibition. Among the compounds selected for in vivo screening of anti‐inflammatory activity ( 4b , 4c , 4f , 4g , 4j , 4m , and 4n ), compound 4f was found to be the most active with an in vivo anti‐inflammatory efficacy of 85.31% when compared to diclofenac sodium (83.68%). It was also found to have a low ulcerogenic risk and a protective effect on lipid peroxidation. The p38α MAP kinase inhibition of this compound (IC₅₀ = 0.036 ± 0.12 μM) was also found to be superior to the standard SB203580 (IC₅₀ = 0.043 ± 0.27 μM). Furthermore, the in silico binding mode of the compound on docking against p38α MAP kinase exemplified stronger interactions than those of SB203580.

     

Synthesis and Anti‐neuroinflammatory Activity of Lactone Benzoyl Hydrazine and 2‐nitro‐1‐phenyl‐1H‐Indole Derivatives as p38α MAPK Inhibitors

Inhibition of p38 mitogen‐activated protein kinases (MAPKs) would allow significant modulation of the neuroinflammation condition associated with Alzheimer's disease (AD). Inspired from the pharmacophore of natural NF‐κB and p38α MAPK inhibitor 5,6‐dehydrokawain and p38α MAPK inhibitors 1a, 1‐pyrazolyl‐3‐(4‐((2‐anilinopyrimidin‐4‐yl)oxy)napththalen‐1‐yl)ureas, and 1b , a class of indole–pyrimidinyl compounds which were patented respectively, we designed, de novo synthesized, and evaluated two kinds of novel series of lactone benzoyl hydrazine derivatives and 2‐nitro‐1‐phenyl‐1H‐indole derivatives in an effort to develop pharmacologically tractable agents to alleviate the progression of AD. Fourteen of the seventeen synthesized compounds exhibit significant inhibitory effect on the nitric oxide (NO) production induced by lipopolysaccharide (LPS)‐induced microglia activation with IC₅₀ less than the control 5,6‐dehydrokawain. Notably, compound 27 , 6‐methoxy‐2‐nitro‐1‐(1H‐1, 2, 3‐triazol‐1‐yl)‐1H‐indole, with IC₅₀ values of 1.6  μ m can markedly inhibit p38 α MAPK and NO release in BV‐2 microglial cells. The molecular dynamic (MD) simulations demonstrate that compound 27 inhibits p38 α MAPK through binding to the Glu71 and Asp168 residues. Moreover, in vitro study shows that all compounds can easily cross the blood–brain barrier (BBB) and did not exhibit any acute cellular toxicity checked by MTT assay. These investigations provide promising chemical lead candidate as anti‐neuroinflammatory agents for AD.     

The Pyrazolobenzothiazine Core as a New Chemotype of p38 Alpha Mitogen‐Activated Protein Kinase Inhibitors

The identification, synthesis, biological activity, and binding mode prediction of a series of pyrazolobenzothiazines as novel p38α MAPK inhibitors are reported. Some of these compounds showed interesting activity in both p38α MAPK and TNF‐α release assays. Derivative 6 emerged as the most interesting compound with IC₅₀ (p38 α ) = 0.457  μ m , IC₅₀ (TNF‐ α ) = 0.5  μ m and a promising kinase selectivity profile. The obtained results strongly indicate the pyrazolobenzothiazine core as a new p38α inhibitor chemotype worthy of future chemical optimization efforts directed toward identifying a new generation of anti‐inflammatory agents.     

Andrographolide inhibits hypoxia‐induced hypoxia‐inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP‐5 pathways in EA.hy926 cells

Andrographolide is a potent anti‐inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET‐1) is an endothelium‐derived vasoconstrictor with pro‐inflammatory properties secreted in response to hypoxia. Mitogen‐activated protein kinase phosphatase 5 (MKP‐5) is a dual‐specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia‐induced HIF‐1α expression and ET‐1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP‐5 plays in andrographolide's inhibition of hypoxia‐induced expression of HIF‐1α and ET‐1. Hypoxic conditions were created using the hypoxia‐mimetic agent CoCl₂. Andrographolide enhanced HO‐1 and MKP‐5 expression and cellular cGMP content in addition to inhibiting hypoxia‐induced ROS generation. Concomitantly, the HO‐1 byproduct CO and the cGMP analogue 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) increased MKP‐5 expression, and pretreatment with CO and 8‐Br‐cGMP inhibited hypoxia‐induced HIF‐1α and ET‐1 expression. Transfection of HO‐1 siRNA or pretreatment with the HO‐1 inhibitor ZnPP‐9 or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide‐induced MKP‐5 expression. Moreover, silencing MKP‐5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia‐induced p38 MAPK activation and HIF‐1α expression. The inhibition of hypoxia‐induced HIF‐1α and ET‐1 expression by andrographolide is likely associated with HO‐1/CO/cGMP/MKP‐5 pathways, which is involved in inhibiting hypoxia‐induced p38 MAPK activation.     

Daphnetin inhibits TNF‐α and VEGF‐induced angiogenesis through inhibition of the IKKs/IκBα/NF‐κB,Src/FAK/ERK1/2 and Akt signalling pathways

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti‐angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti‐arthritic and anti‐inflammatory. The present study was performed to investigate the anti‐angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti‐angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)‐induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8 ng/mL)‐induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10 ng/mL) and VEGF‐induced HUVECs by extracting the mRNA and protein levels using RT‐qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor‐κB signalling together including TNF‐α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF‐κB‐p65 protein. Furthermore, western blotting revealed that DAP significantly down‐regulated the VEGF‐induced signalling such as c‐Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF‐stimulated HUVECs by the caspase‐3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi‐targeted medication for the anti‐angiogenesis and cancer therapy.     

Curcumin‐loaded poly(ε‐caprolactone) nanofibres: Diabetic wound dressing with anti‐oxidant and anti‐inflammatory properties

• 1 Curcumin is a naturally occurring poly‐phenolic compound with a broad range of favourable biological functions, including anti‐cancer, anti‐oxidant and anti‐inflammatory activities. The low bioavailability and in vivo stability of curcumin require the development of suitable carrier vehicles to deliver the molecule in a sustained manner at therapeutic levels.
• 2 In the present study, we investigated the feasibility and potential of poly(caprolactone) (PCL) nanofibres as a delivery vehicle for curcumin for wound healing applications. By optimizing the electrospinning parameters, bead‐free curcumin‐loaded PCL nanofibres were developed.
• 3 The fibres showed sustained release of curcumin for 72 h and could be made to deliver a dose much lower than the reported cytotoxic concentration while remaining bioactive. Human foreskin fibroblast cells (HFF‐1) showed more than 70% viability on curcumin‐loaded nanofibres.
• 4 The anti‐oxidant activity of curcumin‐loaded nanofibres was demonstrated using an oxygen radical absorbance capacity (ORAC) assay and by the ability of the fibres to maintain the viability of HFF‐1 cells under conditions of oxidative stress.
• 5 The curcumin‐loaded nanofibres also reduced inflammatory induction, as evidenced by low levels of interleukin‐6 release from mouse monocyte–macrophages seeded onto the fibres following stimulation by Escherichia coli‐derived lipopolysaccharide.
• 6 The in vivo wound healing capability of the curcumin loaded PCL nanofibres was demonstrated by an increased rate of wound closure in a streptozotocin‐induced diabetic mice model.
• 7 These results demonstrate that the curcumin‐loaded PCL nanofibre matrix is bioactive and has potential as a wound dressing with anti‐oxidant and anti‐inflammatory properties.

     

3‐Acetyl‐11‐keto‐β‐boswellic acid loaded‐polymeric nanomicelles for topical anti‐inflammatory and anti‐arthritic activity

Objectives The aim of this study was to develop 3‐acetyl‐11‐keto‐β‐boswellic acid (AKBA)‐loaded polymeric nanomicelles for topical anti‐inflammatory and anti‐arthritic activity. Methods Polymeric nanomicelles of AKBA were developed by a radical polymerization method using N‐isopropylacrylamide, vinylpyrrolidone and acrylic acid. The polymeric nanomicelles obtained were characterized by Fourier transform infrared (FTIR), transmission electron microscopy (TEM) and dynamic light scattering (DLS). In‐vitro and in‐vivo evaluations of AKBA polymeric nanomicelles gel were carried out for enhanced skin permeability and anti‐inflammatory and anti‐arthritic activity. Key findings TEM and DLS results demonstrated that polymeric nanomicelles were spherical with a mean diameter approximately 45 nm. FTIR data indicated a weak interaction between polymer and AKBA in the encapsulated system. The release of drug in aqueous buffer (pH 7.4) from the polymeric nanomicelles was 23 and 55% after 2 and 8 h, respectively, indicating sustained release. In‐vitro skin permeation studies through excised abdominal skin indicated a threefold increase in skin permeability compared with AKBA gel containing the same amount of AKBA as the AKBA polymeric nanomicelles gel. The AKBA polymeric nanomicelle gel showed significantly enhanced anti‐inflammatory and anti‐arthritic activity compared with the AKBA gel. Conclusions This study suggested that AKBA polymeric nanomicelle gel significantly enhanced skin permeability, and anti‐inflammatory and anti‐arthritic activity.     

Discovery of Novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole Derivatives as Potential Anti‐Inflammatory Agents

A novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 5 with good anti‐inflammatory activity was identified from our in‐house library. Based on hit compound 5 , two series of 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 6a – g and 7a – h were designed and synthesized as novel anti‐inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on NO and TNF‐α production in LPS‐stimulated RAW 264.7 macrophages, in which the compound 6e showed most potent inhibitory activity on NO (IC₅₀ = 0.86 μm ) and TNF‐α (IC₅₀ = 1.87 μm ) production. Further evaluation revealed that compound 6e displayed more potent in vivo anti‐inflammatory activity than ibuprofen did on xylene‐induced ear oedema in mice. Additionally, Western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and protein expression of p65 NF‐κB in LPS‐stimulated RAW 264.7 macrophages.     

Arylazopyrazole AAP1742 Inhibits CDKs and Induces Apoptosis in Multiple Myeloma Cells via Mcl‐1 Downregulation

Inhibitors of cyclin‐dependent kinases 9 have been developed as potential anticancer drugs for the treatment of multiple myeloma. We have previously prepared a library of arylazo‐3,5‐diaminopyrazole inhibitors of CDKs. Here, we describe a novel member, AAP1742 (CDK9 inhibition with IC₅₀ = 0.28 μm ), that reduces the viability of multiple myeloma cell lines in low micromolar concentrations. Consistent with inhibition of CDK9, AAP1742 decreases the phosphorylation of RNA polymerase II and inhibits mRNA synthesis of anti‐apoptotic proteins Mcl‐1, Bcl‐2, and XIAP, followed by apoptosis in the RPMI‐8226 cell line in a dose‐ and a time‐dependent manner. These results are consistent with the biochemical profile of AAP1742 and further suggest cellular inhibition of CDK9 as a possible target for anticancer drugs.     

Investigation of anti‐inflammatory,nitric oxide donating,vasorelaxation and ulcerogenic activities of 1, 3‐diphenylprop‐2‐en‐1‐one derivatives in animal models

The main aim of this work is to find out novel chemical moieties with potent anti‐inflammatory and vasorelaxant activities with reduced gastric toxicities. For fulfilling the above aim, here we investigated novel chalcones (1, 3‐diphenylprop‐2‐en‐1‐one derivatives) with nitric oxide (NO) and hydrogen sulphide (H₂S) donating potency for anti‐inflammatory activity by carrageenan‐induced rat paw oedema. These molecules then further evaluated for in‐vitro NO‐releasing potency and vasorelaxation effect on isolated adult goat aortic tissue. The promising molecules were further screened for ulcerogenic activity in the rat model. The tested compounds produced % inhibition in paw oedema ranging from 29.16% to 79.69% and standard drug Diclofenac sodium produced 85.30% reduction in paw oedema after 5 hours. Out of this dataset, compounds AI1, AI7, Ca1, B2, B10, D2, and E8 showed 73.01%, 79.69%, 75.02%, 75.46%, 74.35%, 73.9% and 74.35% reduction in paw oedema respectively, which is approximately 80%–90% to that of standard Diclofenac sodium. The compound Ca1 was found to release 0.870 ± 0.025 mol/mol of NO and standard Glyceryl trinitrate (GTN) was found to release 0.983 ± 0.063 mol/mol of NO. The compound Ca1 produced 950.2 μmol/L of EC₅₀ whereas standard GTN produced 975.8 μmol/L of EC₅₀ for aortic smooth relaxation. The compounds Ca1 produced 0.1117 of ulcer index which is far less than that of standard Diclofenac sodium (1.148). The potent lead molecules were further evaluated to understand the mechanism of vasorelaxation by using specific antagonists or blockers of NO and H₂S.     

Design,Synthesis, and Biological Evaluation of 1‐(thiophen‐2‐yl)‐9H‐pyrido[3,4‐b]indole Derivatives as Anti‐HIV‐1 Agents

A novel series of 1‐(thiophen‐2‐yl)‐9H‐pyrido [3,4‐b]indole derivatives were synthesized using DL‐tryptophan as starting material. All the compounds were characterized by spectral analysis such as ¹H NMR, Mass, IR, elemental analysis and evaluated for inhibitory potency against HIV‐1 replication. Among the reported analogues, compound 7g exhibited significant anti‐HIV activity with EC₅₀ 0.53 μm and selectivity index 483; compounds 7e , 7i , and 7o displayed moderate activity with EC₅₀ 3.8, 3.8, and 2.8 μm and selectivity index >105, >105, and 3.85, respectively. Interestingly, compound 7g inhibited p24 antigen expression in acute HIV‐1_IIIB infected cell line C8166 with EC₅₀ 1.1 μm . In this study, we also reported the Lipinski rule of 5 parameters, predicted toxicity profile, drug‐likeness, and drug score of the synthesized analogues.     

Tris (1,3‐dichloro‐2‐propyl) phosphate induces toxicity by stimulating CaMK2 in PC12 cells

Tris (1,3‐dichloro‐2‐propyl) phosphate (TDCIPP) is one of the widely used organophosphorus flame retardants (OPFRs), which are regarded as suitable substitutes for brominated flame retardants (BFRs). Previously, we have validated the toxicity of TDCIPP in PC12 cells owing to the induced alterations in GAP43, NF‐H, CaMK2a/2b, and tubulin α/β proteins; however, limited information is currently available on the toxicity and mechanism of TDCIPP. In the present study, cytotoxicity effects were evaluated by exposing PC12 cells to different concentrations of TDCIPP (0–50 μM) for 4 days. To explore the possible mechanisms through which cytotoxicity is induced, changes in intracellular [Ca²⁺]_i levels and the activation of calmodulin dependent protein kinase 2 (CaMK2), c‐Jun N‐terminal kinase (JNK), extracellular regulated protein kinases (ERK1/2), and p38 mitogen‐activated protein kinases (MAPK) pathways were evaluated. Furthermore, PC12 cells were pretreated with CaMK2 inhibitor KN93 to investigate the relationship between TDCIPP‐induced phosphorylation of CaMK2 and activation of JNK, ERK1/2, and p38 MAPK pathways. Our results indicate that TDCIPP‐induced toxicity might be associated with the overload of [Ca²⁺]_i levels, increased phosphorylation of CaMK2, and activation of the JNK, ERK1/2, and p38 MAPK pathways, the lattermost of which was further demonstrated to be partially elicited by the CaMK2 phosphorylation.     

The Design,Synthesis and Potential Utility of Fluorescence Probes that Target DFG‐out Conformation of p38α for High Throughput Screening Binding Assay

The design, synthesis and utility of fluorescence probes that bind to the DFG‐out conformation of p38α kinase are described. Probes that demonstrate good affinity for p38α, have been identified and one of the probes, PF‐04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non‐classical p38α inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non‐classical kinase inhibitors to the unactive form of the enzyme.     

Cyanidin‐3‐glucoside inhibits inflammatory activities in human fibroblast‐like synoviocytes and in mice with collagen‐induced arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint tissue inflammation. Cyanidin‐3‐glucoside (C3G) is a major component in the flavonoid family and has shown anti‐inflammatory, anti‐oxidant and anti‐tumour activity. In this study, we investigated the effects of C3G on lipopolysaccharides (LPS)‐induced inflammation on human rheumatoid fibroblast‐like synoviocytes (FLS) and on collagen‐induced arthritis (CIA) mice model. We treated FLS with C3G followed by LPS induction, the expressions of tumour necrosis factor alpha (TNF‐α), interleukin 1 beta (IL‐1β) and IL‐6 and the activation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and mitogen‐activated protein kinase (MAPK) signalling pathway were analyzed. CIA was induced in mice and the arthritic mice were treated with C3G for 3 weeks. The disease severity was compared between control and C3G treated mice. The serum levels of TNF‐α, IL‐1β and IL‐6 were analyzed by ELISA. C3G inhibited LPS‐induced TNF‐α, IL‐1β and IL‐6 expression in FLS. Moreover, C3G inhibited LPS‐induced p65 production and IκBa, p38, ERK and JNK phosphorylation. Administration of C3G significantly attenuated disease in mice with CIA and decreased the serum level of TNF‐α, IL‐1β and IL‐6. C3G inhibited LPS‐induced inflammation in human FLS by inhibiting activation of NF‐κB and MAPK signalling pathway. C3G exhibited therapeutic effects in mice with CIA.     

Design,Synthesis and Biological Evaluation of Peptidyl Epoxyketone Proteasome Inhibitors Composed of β‐amino Acids

A series of novel di‐ and tripeptidyl epoxyketone derivatives composed of β‐amino acids were designed, synthesized and evaluated for their proteasome inhibitory activities and anti‐proliferation activities against two multiple myeloma cell lines RPMI 8226 and NCI‐H929 and normal cells (peripheral blood mononucleated cells). Among these tested compounds, tripeptidyl analogues showed much more potent activities than dipeptides, and four tripeptidyl compounds exhibited proteasome inhibitory activities with IC₅₀ values ranging from 0.97 ± 0.05 to 1.85 ± 0.11 μm . In addition, all the four compounds showed anti‐proliferation activities with IC₅₀ values at low micromolar levels against two multiple myeloma cell lines and weak activities against normal cells. Furthermore, Western blot analysis was performed to verify the proteasome inhibition induced by compounds 21d and 21e . All the experimental results validated that the β‐amino acid building block has the potential for the development of proteasome inhibitors.     

Hypoxia inducible factor‐1α inhibition produced anti‐allodynia effect and suppressed inflammatory cytokine production in early stage of mouse complex regional pain syndrome model

Complex regional pain syndrome (CRPS) is related to microcirculation impairment associated with tissue hypoxia and peripheral cytokine overproduction in the affected limb. Previous studies suggest that the pathogenesis involves hypoxia inducible factor‐1α (HIF‐1α) and exaggerated regional inflammatory response. 1‐methylpropyl 2‐imidazolyl disulfide (PX‐12) acts as the thioredoxin‐1 (Trx‐1) inhibitor and decreases the level of HIF‐1α, and can rapidly be metabolized for Trx‐1 redox inactivation. This study hypothesized that PX‐12 can decrease the cytokine production for nociceptive sensitization in the hypoxia‐induced pain model. CD1 mice weighing around 30 g were used. The animal CRPS model was developed via the chronic post‐ischaemic pain (CPIP) model. The model was induced by using O‐rings on the ankles of the mice hind limbs to produce 3‐h ischaemia–reperfusion injury on the paw. PX‐12 (25 mg/kg, 5 mg/kg) was given through tail vein injection immediately after ischaemia. Animal behaviour was tested using the von Frey method for 7 days. Local paw skin tissue was harvest from three groups (control, 5 mg/kg, 25 mg/kg) 2 h after injection of PX‐12. The protein expression of interleukin‐1β (IL‐1β) and HIF‐1α was analysed with the Western blotting method. Mice significantly present an anti‐allodynia effect in a dose‐related manner after the PX‐12 administration. Furthermore, PX‐12 not only decreased the expression of HIF‐1α but also decreased the expression of IL‐1β over the injured palm. This study, therefore, shows the first evidence of the anti‐allodynia effect of PX‐12 in a CPIP animal model for pain behaviour. The study concluded that inhibition of HIF‐1α may produce an analgesic effect and the associated suppression of inflammatory cytokine IL‐1β in a CPIP model.