Naphthalene-triazolopyrimidine hybrid compounds as potential multifunctional anti-Alzheimer's agents.

In an attempt to construct potential anti-Alzheimer's agents Naphthalene-triazolopyrimidine hybrids were synthesized and screened in vitro against the two cholinesterases (ChE)s, amyloid ß aggregation and for antioxidation activity. Single-crystal X-ray crystallography was utilized for crystal structure determination of one of the compounds. In vitro study of compounds revealed that most of the compounds are capable of inhibiting acetylcholinesterase and Butyrylcholinesterase activity. Particularly, the compounds 4e and 4d exhibited IC50 values ranging from 8.6 to 14 nM against AChE lower than the standard drug Donepezil (IC50 49 nM). Best result was found for compound 4e with IC50 of 8.6 nM (for AChE) and 150 nM (for BuChE). Selectivity upto that of Donepezil and even more was observed for 4a, 4c and 4h. Investigation by electron microscopy, transmission electron microscopy and ThT fluorescence assay unveils the fact that synthesized hybrids exhibit amyloid ß self-aggregation inhibition. The compounds 4i and 4j revealed highest inhibitory potential, 85.46% and 72.77% at 50 µM respectively; above the standard Aß disaggregating agent, Curcumin. Their antioxidation profile was also analyzed. Studies from DPPH free radical scavenging assay and ORAC assay depicts molecules to possess low antioxidation profile. Results suggest that triazolopyrimidines are potential candidate for Acetylcholinesterase (AChE), Butyrylcholinesterase (BuChE), and amyloid ß aggregation inhibition. In silico ADMET profiling indicates drug-like properties with a very low toxic influence. Such synthesized compounds provide a strong vision for further development of potential anti-Alzheimer's agents.

A multifunctional therapeutic approach: Synthesis, biological evaluation, crystal structure and molecular docking of diversified 1H-pyrazolo[3,4-b]pyridine derivatives against Alzheimer's disease.

2-(piperazin-1-yl)N-(1H-pyrazolo[3,4-b]pyridin-3-yl)acetamides are described as a new class of selective and potent acetylcholinesterase (AChE) inhibitors and amyloid ß aggregation inhibitors. Formation of synthesized compounds (P1P9) was justified via H1 NMR, C13 NMR, mass spectra and single crystal X-Ray diffraction study. All compounds were evaluated for their acetylcholinesterase and butyrylcholinesterase inhibitory activity, inhibition of self-mediated Aß aggregation and Cu(II)-mediated Aß aggregation. Also, docking study carried out was in concordance with in vitro results. The most potent molecule amongst the derivatives exhibited excellent anti-AChE activity (IC50 = 4.8 nM). Kinetic study of P3 suggested it to be a mixed type inhibitor. In vitro study revealed that all the compounds are capable of inhibiting self-induced ß-amyloid (Aß) aggregation with the highest inhibition percentage to be 81.65%. Potency of P1 and P3 to inhibit self-induced Aß1-42 aggregation was ascertained by TEM analysis. Compounds were also evaluated for their Aß disaggregation, antioxidation, metal-chelation activity.

Design, synthesis, in-silico and biological evaluation of novel donepezil derivatives as multi-target-directed ligands for the treatment of Alzheimer's disease.

A novel series of donepezil based multi-functional agents "(E)-5,6-dimethoxy-2-(4-(4-substituted piperazin-1-yl)benzylidene)-2,3-dihydro-1H-inden-1-ones" have been designed and synthesized as potential anti-Alzheimer's agents. In-vitro studies revealed that these compounds demonstrated moderate to good AChE and Aß aggregation inhibitory activity. These derivatives are also endowed with admirable antioxidant activity. Among the entire series compounds IP-9, IP-13 and IP-15 appeared as most active multi-functional agents and displayed marked AChE inhibitory, Aß disaggregation and antioxidant activity. Studies indicate that IP-13 and IP-15 showed better AChE inhibitory activity than the standard drug donepezil and IP-9, IP-13 as well as IP-15 exhibited better Aß aggregation inhibitory activity than curcumin. These compounds (IP-9, IP-13 and IP-15) successfully diminished H2O2 induced oxidative stress in SH-SY5Y cells and displayed excellent neuroprotective activity against H2O2 as well as Aß induced toxicity in SH-SY5Y cells in a concentration dependent manner. Moreover, these derivatives did not exert any significant toxicity in neuronal SH-SY5Y cells in cytotoxicity assay. To elucidate the plausible binding mode of the compounds IP-9, IP-13 and IP-15, molecular docking studies and molecular dynamics (MD) simulation studies were also performed and the results indicate their significant interactions with the active sites of AChE as well as Aß1-42 peptide. Thus, the present study evidently showed that IP-9, IP-13 and IP-15 are potent multi-functional agents against Alzheimer's disease and might serve as promising lead candidates for anti-Alzheimer drug development.

Design, synthesis and evaluation of novel indandione derivatives as multifunctional agents with cholinesterase inhibition, anti-ß-amyloid aggregation, antioxidant and neuroprotection properties against Alzheimer's disease.

A series of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)-1H-indene-1,3(2H)-diones were designed, synthesized and appraised as multifunctional anti-Alzheimer agents. In vitro studies of compounds 27-38 showed that these compounds exhibit moderate to excellent AChE, BuChE and Aß aggregation inhibitory activity. Notably, compounds 34 and 38 appeared as most active multifunctional agents in the entire series and exhibited excellent inhibition against AChE (IC50=0.048µM: 34; 0.036µM: 38), Aß aggregation (max% inhibition 82.2%, IC50=9.2µM: 34; max% inhibition 80.9%, IC50=10.11µM: 38) and displayed significant antioxidant potential in ORAC-FL assay. Both compounds also successfully diminished H2O2 induced oxidative stress in SH-SY5Y cells. Fascinatingly, compounds 34 and 38 showed admirable neuroprotective effects against H2O2 and Aß induced toxicity in SH-SY5Y cells. Additionally, both derivatives showed no considerable toxicity in neuronal cell viability assay and represented drug likeness properties in the primarily pharmacokinetics study. All these results together, propelled out that compounds 34 and 38 might serve as promising multi-functional lead candidates for treatment of AD in the future.

Synthesis and screening of triazolopyrimidine scaffold as multi-functional agents for Alzheimer's disease therapies.

In present study a series of triazolopyrimidine-quinoline and cyanopyridine-quinoline hybrids were designed, synthesized and evaluated as acetylcholinesterase inhibitors (AChEIs). Molecular docking and scoring was utilized for the design of inhibitors. The molecules were synthesized via an easily accessible, convergent synthetic route. Three triazolopyrimidine based compounds showed nanomolar activity towards acetylcholinesterase. Among them, Ethyl 6-fluoro-4-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl)quinoline-3-carboxylate (10d), strongly inhibited AChE with IC50 value of 42 nM. Furthermore compound 10d was identified as most promising compound with 12 fold selectivity against butyrylcholinesterase (BuChE). This compound displayed a composed multitargeted profile with promising inhibition of self-induced and AChE - induced Aß aggregation and antioxidant activity.

DADS Analogues Ameliorated the Cognitive Impairments of Alzheimer-Like Rat Model Induced by Scopolamine.

The development of agents that affect two or more relevant targets has drawn considerable attention in treatment of AD. Diallyl disulfide (DADS), an active principle of garlic, has been reported to prevent APP processing by amyloidogenic pathway. Recently, we have reported a new series of DADS derivatives and our findings revealed that compound 7k and 7l could provide good templates for developing new multifunctional agents for AD treatment. Thus, the present study was constructed to investigate the neuroprotective effect of DADS analogues (7k and 7l) against Aß-induced neurotoxicity in SH-SY5Y human neuroblastoma cells and in ameliorating the cognition deficit induced by scopolamine in rat model. The results indicated that compound 7k and 7l significantly inhibited Aß1-42-induced neuronal cell death by inhibiting ROS generation. Moreover, they prevented apoptosis, in response to ROS, by restoring normal Bax/Bcl-2 ratio. Furthermore, it was observed that scopolamine-induced memory impairment was coupled by alterations in neurotransmitters, acetylcholinesterase activity and oxidative stress markers. Histological analysis revealed severe damaging effects of scopolamine on the structure of cerebral cortex and hippocampus. Administration of compounds 7k and 7l at 5 mg/kg significantly reversed scopolamine-induced behavioural, biochemical, neurochemical and histological changes in a manner comparable to standard donepezil. Together the present findings and previous studies indicate that compounds 7k and 7l have neuroprotective and cognition-enhancing effects, which makes them a promising multi-target candidate for addressing the complex nature of AD.

Synthesis, biological evaluation and molecular docking study of novel piperidine and piperazine derivatives as multi-targeted agents to treat Alzheimer's disease.

Development of Multi-Target Directed Ligands (MTDLs) has emerged as a promising approach for targeting complex etiology of Alzheimer's disease (AD). Following this approach, a new series of N'-(4-benzylpiperidin-/piperazin-/benzhydrylpiperazin-1-yl)alkylamine derivatives were designed, synthesized and biologically evaluated as inhibitors of cholinesterases (ChEs), amyloid-beta (Aß) self aggregation and also for their radical scavenging activity. The in vitro studies showed that the majority of synthesized derivatives strongly inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC50 values in the low-nanomolar range, and were clearly more potent than the reference compound donepezil in this regard. Among them, inhibitors 5h and 5k, strongly inhibited AChE, with IC50 value of 6.83nM and 2.13nM, respectively, and particularly, compound 5k was found to be highly selective for AChE (∼38-fold). Moreover, both kinetic analysis of AChE inhibition and the docking study suggested that 5k binds simultaneously to catalytic active site and peripheral anionic site of AChE. Besides, these compounds also exhibited greater ability to inhibit self-induced Aß1-42 aggregation at 25µM with percentage inhibition from ∼54% to 89% and specially compound 5k provided highest inhibition (88.81%). Also, the derivatives containing methoxy and hydroxy groups showed potent oxygen radical absorbance capacity (ORAC) ranging from 2.2- to 4.4-fold of the Trolox value. Furthermore, results of ADMET studies suggested that all compounds exhibited appropriate drug like properties. Taken together, these results suggest that 5k might be a promising lead compound for further AD drug development.

Supplementation of Convolvulus pluricaulis attenuates scopolamine-induced increased tau and amyloid precursor protein (AßPP) expression in rat brain.

AIM: Scopolamine is known to produce amnesia due to blockade of the cholinergic neurotransmission. The present study investigated the potential of Convolvulus pluricaulis (CP) to attenuate scopolamine (2 mg/kg, i.p) induced increased protein and mRNA levels of tau, amyloid precursor protein (AßPP), amyloid ß (Aß) levels and histopathological changes in rat cerebral cortex. MATERIALS AND METHODS: The study was conducted on male Wistar rats (250 ± 20 g) divided into four groups of eight animals each. Groups 1 and 2 served as controls receiving normal saline and scopolamine for 4 weeks, respectively. Group 3 received rivastigmine (standard) and group 4 received aqueous extract of CP simultaneously with scopolamine. Western blot and RT-PCR analysis were used to evaluate the levels of protein and mRNA of amyloid precursor protein (AßPP) and tau in rat cortex and ELISA was used to measure the amyloid ß (Aß) levels. Histopathology was also performed on cortical section of all groups. RESULT: Oral administration of CP extract (150 mg/kg) to scopolamine treated rats reduced the increased protein and mRNA levels of tau and AßPP levels followed by reduction in Aß levels compared with scopolamine treated group. The potential of extract to prevent scopolamine neurotoxicity was reflected at the microscopic level as well, indicative of its neuroprotective effects. CONCLUSION: CP treatment alleviated neurotoxic effect of scopolamine reflects its potential as potent neuroprotective agent.

The action of Semaphorin7A on thalamocortical axon branching.

Developing axons form extensive branches to make synaptic contacts with their target cells. Despite the important role of axon branching in neural circuit formation, its underlying molecular mechanism is still largely unknown. In this study, we investigated the involvement of Semaphorin7A (Sema7A) in thalamocortical (TC) axon branching. In situ hybridization demonstrated that sema7a was expressed specifically in layer 4, the TC recipient layer, when TC axons form extensive arbors. A similar protein expression pattern was observed by immunohistochemistry with an anti-Sema7A antibody. The effect of Sema7A on axon branching was investigated in dissociated cell cultures from embryonic rat thalamus. TC axon branching increased dramatically on Sema7A-coated dishes. We further studied the activity of Sema7A in vivo using loss- and gain-of-function analyses. The number of vesicular glutamate transporter 2-positive puncta was markedly reduced in the Sema7A-deficient cortex. In contrast, their number increased significantly when Sema7A was over-expressed in layer 4 cells by in utero electroporation. Taken together, these findings suggest that Sema7A acts as a positive regulator for TC axon branching and/or pre-synaptic puncta formation.

Neuroprotective role of Convolvulus pluricaulis on aluminium induced neurotoxicity in rat brain.

AIM OF THE STUDY: Convolvulus pluricaulis (Convolvulaceae) has long been used as traditional herbal medicine in India as nerve tonic. We investigated neuroprotective effects of aqueous extract from Convolvulus pluricaulis (CP) against aluminium chloride induced neurotoxicity in rat cerebral cortex. MATERIAL, METHOD AND RESULT: Daily administration of CP (150 mg/kg) for 3 months along with aluminium chloride (50 mg/kg) decreased the elevated enzymatic activity of acetylcholine esterase and also inhibited the decline in Na(+)/K(+)ATPase activity which resulted from aluminium intake. Beside, preventing accumulation of lipid and protein damage, changes in the levels of endogenous antioxidant enzymes associated with aluminium administration were also rectified. Oral administration of CP preserved the mRNA levels of muscarinic receptor 1 (M1 receptor), choline acetyl transferase (ChAT) and Nerve Growth Factor-Tyrosine kinase A receptor (NGF-TrkA). It also ameliorated the upregulated protein expression of cyclin dependent kinase5 (Cdk5) induced by aluminium. The potential of CPE to inhibit aluminium induced toxicity was compared with rivastigmine tartrate (1mg/kg), which was taken as standard. The potential of the extract to prevent aluminium-induced neurotoxicity was also reflected at the microscopic level, indicative of its neuroprotective effects. CONCLUSION: Convolvulus pluricaulis possesses neuroprotective potential, thus validating its use in alleviating toxic effects of aluminium.

Inhibitory effect of novel diallyldisulfide analogs on HMG-CoA reductase expression in hypercholesterolemic rats: CREB as a potential upstream target.

AIMS: Diallyldisulfide (DADS), an active principle of garlic (Allium sativum) is known for its antihyperlipidemic properties. The present study was designed to evaluate the effect of novel synthesized DADS analogs, on the lipid profile of hypercholesterolemic rats and to investigate the molecular correlates of their activity at the cellular level. MAIN METHODS: Wistar rats, weighing 100-120 g each, were administered with 5% cholesterol for one week, and subsequently administered with lovastatin, allicin and DADS (20 mg/kg wt) analogs in the second week along with 5% cholesterol. All animals were sacrificed after overnight starvation. KEY FINDINGS: Our results show that DADS analogs are effective in reducing the total lipid levels which could be correlated with a significant decrease in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity. DADS analogs strongly inhibit HMGR activity in vivo but not in vitro. These results can be attributed to the significant decrease in the mRNA levels and protein expression of HMGR. Further, we show that DADS analogs significantly inhibited the activation of sterol regulatory element-binding protein-2 (SREBP-2) and interfered with DNA binding activity of cAMP response element-binding protein (CREB) but not nuclear factor-Y (NF-Y), with upstream regulatory sequences of HMGR. Moreover, DADS analogs are also effective in reducing the levels of oxidized low-density lipoprotein (ox-LDL), lipid peroxidation as well as NF-kappaB activity, showing good anti-inflammatory and antioxidant properties. SIGNIFICANCE: The unique anti-inflammatory effect and hypolipidemic action of DADS analogs may be beneficial in preventing the vascular complications that are induced by hyperlipidemia and provide a new therapeutic approach for the treatment of cardiovascular and related diseases.

The effect of the aqueous extract of the roots of Asparagus racemosus on hepatocarcinogenesis initiated by diethylnitrosamine.

Histopathological studies of the hepatic tissues of Wistar rats treated with diethylnitrosamine (DEN) (200 mg/kg b wt, i.p.) once a week for 2 weeks, followed by treatment with DDT, a tumor promoter (0.05% in diet) for 2 weeks and kept under observation for another 18 weeks, demonstrated the development of malignancy. Pretreatment of Wistar rats with the aqueous extract of the roots of Asparagus racemosus prevented the incidence of hepatocarcinogenesis. Immunohistochemical staining of the hepatic tissues of rats treated with DEN showed the presence of p53+ foci (clusters of cells expressing the mutated p53 protein), whereas an absence of p53+ foci was observed in Wistar rats pretreated with the aqueous extract of the roots of Asparagus racemosus. The microsections of the hepatic tissue of rats treated with DEN followed by treatment with the aqueous extract of Asparagus racemosus showed an absence of p53+ foci. The results of the biochemical determinations also show that pretreatment of Wistar rats with the aqueous extract of Asparagus racemosus leads to the amelioration of oxidative stress and hepatotoxicity brought about by treatment with DEN. These results prove that the aqueous extract of the roots of Asparagus racemosus has the potential to act as an effective formulation to prevent hepatocarcinogenesis induced by treatment with DEN.

Synthesis, DNA binding, and cytotoxic evaluation of new analogs of diallyldisulfide, an active principle of garlic.

Diallyldisulfide (DADS), an active principle of garlic (Allium sativum) is known for its antihyperlipidemic properties. However, its use is limited due to its extreme volatility. In the present study, we have synthesized and characterized a series of six new DADS analogs and investigated their interactions with different DNA duplexes. The spectroscopic and circular dichroism (CD) analyses revealed that DADS analogs bind preferentially with GC rich sequences. Thermodynamic parameters suggest that DADS analogs stabilize the calf thymus (CT) DNA and GC rich duplex by favorable enthalpic gains and follow the hierarchy, d(GC)(7)>CT DNA>d(AT)(10). Further, DADS analogs are less toxic and equally effective as the statins. The analogs therefore have a good potential to provide a new therapeutic approach for the treatment of cardiovascular and related diseases.

Effect of hyperhomocysteinemia on cardiovascular risk factors and initiation of atherosclerosis in Wistar rats.

Hyperhomocysteinemia is considered an independent risk factor for atherosclerosis. The present study was designed to assess the effect of high level of serum homocysteine on other cardiovascular risk factors and markers in rats and to study its mode of action in initiating atherosclerosis. To address this issue, four different doses of methionine (0.1 g/kg, 0.25 g/kg, 0.5 g/kg, 1 g/kg) were orally administered to four groups (Group II, III, IV, V respectively) of rats (6 rats in each group) for a period of 8 weeks to get different level of homocysteine in serum. Group I was administered with saline and served as control. Our results revealed that the level of Total cholesterol, Triglyceride, and Oxidized low-density lipoproteins increased significantly with the increase in the level of serum homocysteine. The levels of Resistin, C-reactive protein and cysteinyl-leukotrienes were found to be significantly high in Group IV (P<0.001 vs Group I) and Group V (P<0.001 vs Group I) at 8 weeks. Total antioxidant capacity and nitrite/nitrate level in serum showed negative correlation with the increased dose of methionine. The mRNA expression and the enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase significantly increased only in livers of rats of Group V. Furthermore, high mRNA expression of P2 receptors and caveolin were found in aorta of rats administered with high dose of methionine (Group IV and V at 8 weeks). Data obtained from in-vitro effect of homocysteine on isolated aortic arch also showed induction in P2 receptors and caveolin with the increase in the concentration of homocysteine. These findings collectively suggest that hyperhomocysteinemia initiates atherosclerosis by modulating the cholesterol biosynthesis and by significantly inducing the level of other cardiovascular risk factors and markers, which play important role in initiating atherosclerosis.

The use of riboflavin and metalloporphyrins in cytochrome P-450 content in Wistar rats.

Phototherapy is commonly used for the treatment of neonatal jaundice. Riboflavin is a photosensitizer that generates singlet oxygen, which promotes bilirubin photodecomposition. Metalloporphyrins are also effective photosensitizers. The effect of a combined dosing regimen of riboflavin and metalloporphyrins was studied, with the aim of increasing the efficiency of the phototherapeutic treatment of hyperbilirubinemia. It was envisaged that riboflavin and the metalloporphyrins, by promoting the photodecomposition of bilirubin, would thereby lead to a reduction of the toxic side effects associated with phototherapy. The results shows that a phototherapeutic treatment, in which riboflavin and metalloporphyrins were co-administered, was effective in reducing Heme Oxygenase activity. However, a comprehensive study of the possible side effects of metalloporphyrin treatment in the wavelength under consideration is essential prior to utilizing these compounds for any clinical applications.

Effect of 1-oxo-5beta, 6beta-epoxy-witha-2-ene-27-ethoxy-olide isolated from the roots of Withania somnifera on stress indices in Wistar rats.

OBJECTIVE: Isolation of biologically active fractions and compounds from the roots of Withania somnifera, a plant used extensively as a constituent of rasayana, in Ayurveda and to test their adaptogenic activity on stress indices using the cold-hypoxia-restraint (C-H-R) model. DESIGN: Bioactivity-guided fractionation of an aqueous extract of the roots of Withania somnifera led to the isolation of a new species of withanolide 1-oxo-5beta, 6beta-epoxy-witha-2-ene-27-ethoxy-olide. Structure elucidation, was carried out using proton nuclear magnetic resonance, infrared (IR), ultraviolet (UV), and mass spectroscopic analysis. Stress-related indices were evaluated, namely serum creatine phosphokinase (CPK) activity, serum lactate dehydrogenase (LDH) activity, serum corticosterone levels, and serum lipid peroxidation (LPO) levels. RESULTS: There was a significant decrease in a serum CPK, LDH, and LPO levels in animals pretreated with (1) fraction-I (20 mg/kg body weight), (2) 1-oxo-5beta, 6beta-epoxy-witha-2-ene-27-ethoxy-olide (2.5 mg/kg body weight) in comparison to control when subjected to C-H-R stress. CONCLUSIONS: The results show that the a new species of withanolide, 1-oxo-5beta, 6beta-epoxy-witha-2-ene-27-ethoxy-olide (compound-1) could prove to be an effective agent to counteract C-H-R stress.