Synthesis and anti-HBV activity of carbocyclic nucleoside hybrids with salient features of entecavir and aristeromycin.

Modified carbocyclic nucleosides (4a-g) constituting 7-deazapurine, 4'-methyl, exocyclic double bond and 2',3'-hydroxy were synthesized. NOE and X-ray studies of 4c confirmed the α-configuration of 4'-methyl. The anti-HBV assay demonstrated 4e (IC50 = 3.4 µM) without notable cytotoxicity (CC50 = 87.5 µM) as a promising lead for future exploration.

Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock protein 90 (HSP90) inhibitors with significant anti-HIV activity.

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.

First Insight on Small Molecules as Cardiac Calsequestrin Stabilizers.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations of cardiac calsequestrin (CASQ2) that impair its characteristic ability of Ca2+-induced polymerization-depolymerization. However, stabilizing the CASQ2 polymer by pharmacological agents to treat CPVT has not been reported so far. Here, we tested whether small molecules can stabilize CASQ2 polymers. We synthesized 24 glycinate/alaninate/acetate α-pyranone analogs and conducted the CASQ2 depolymerization assay. Most of the molecules of this class of compounds inhibited the depolymerization of the protein upon Ca2+ chelation by ethylene glycol tetraacetic acid. Structure-activity relationship studies revealed that the compounds with the 4-fluoro-phenyl group at the C-6 position of the pyranone ring and open-chain primary amine at C-4 are the most active of the class. This is the first report of an α-pyranone class of compounds with the ability to stabilize CASQ2 polymers and opens up the possibility to target Ca2+-release disorders via modulation of CASQ2 polymerization.

Rhodium catalyzed stereospecific reductive carbocyclization of 1,6-enynes and synthesis of 4'-methyl-6'-substituted aristeromycins.

The need of long-term treatment for chronic HBV, emergence of drug-resistant viruses and inefficiency of currently approved therapies to eliminate covalently closed circular DNA (cccDNA), mandates identification of potent and selective inhibitors of HBV replication with novel mechanisms of action. Entecavir, a carbocyclic guanosine nucleoside analog, is the most potent inhibitor of HBV replication on the market. Moreover, the naturally occurring carbocyclic nucleosides aristeromycin are known for their wide range of antiviral activities. In this research, we have utilized BINAP directed rhodium catalyzed reductive carbocyclization of 1,6-enynes (8a-b) through asymmetric hydrogenation which is an approach, not yet explored in carbocyclic sugar synthesis. Interestingly, we obtained exclusive anti-(9a) and Z-anti (9b) carbocyclic sugars. The new aristeromycin analogs (10a-b) with scaffold combination of entecavir and aristeromycin were then synthesized using the Mitsunobu reaction followed by deprotection.

Synthesis and anti-HBV activity of α-stereoisomer of aristeromycin based analogs.

The potential antiviral activity of aristeromycin type of derivatives (I) is limited by associated toxicity due to its possible 5'-O-phosphorylation and S-adenosyl-l-homocysteine hydrolase (SAHase) inhibitory activity. Aristeromycin structure has major pharmacophoric motif as 5'-OH and adenosine base, which may have significant role in enzyme binding followed by activity and or toxicity. Thus, the structural optimization to alter this major motif by replacing with its bioisostere and changing the 5'-O conformation through stereochemistry reversal was of interest. Thus, the inverted stereochemistry at 4'-position coupled with bioisostere of adenosine base in the target compounds (6-7) to access antiviral potential. The stereoselective formation of a key stereoisomer (2a) was achieved exclusively from neplanocin sugar (1a) by reduction in a single step. The novel target molecules (6-7) were synthesized in 4 steps with 55-62% yield. Compound 6 was analyzed by single crystal X-ray diffraction, which confirms the stereoselective formation of α-analogs with highly puckered cyclopentane ring and 2'-endo conformation. The compound 6 shown significant anti-hepatitis B virus activity of 6.5µM with CC50>100µM and yielded a promising lead with novel structural feature.

Synthesis and Anti-HCV Activity of 4-Methoxy-7H-Pyrrolo[2,3-d] Pyrimidine Carbocyclic Nucleosides.

The present study includes the exploration of new possible nucleoside mimetics based on 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleosides (4a-g), which were synthesized by 10-15 synthetic steps and characterized adequately. We report the anti-HCV activities and cytotoxicities of 4a-g. Compound 4a was analyzed by single crystal X-ray diffraction which showed some puckering in the cyclopentene ring with a 2'-endo conformation and anti-base disposition (χ = -125.7°).

Regioselective Synthesis of Pyrazolo[3,4-D]Pyrimidine Based Carbocyclic Nucleosides as Possible Antiviral Agent.

Carbocyclic nucleosides are considered as nucleoside mimetic having high therapeutic potentials, however diverse exploration is still limited due to their synthetic difficulties. The major challenges are associated with the preparation of new base and carbocyclic sugar key intermediates. The modified base may provide conformational advantage to achieve better nucleoside mimetics and may also help in increasing the drug-like properties. In this manuscript, we report the use of acetamidine hydrochloride to synthesize 6-methyl-4-amino-pyrazolo[3,4-d]pyrimidine base and regioselective synthesis of six new carbocyclic nucleosides (6a-f) for antiviral evaluation. Theoretical investigations were carried out on the basis of thermodynamic and kinetic stability using MM based energy optimizations and QM based transition state search for the significant regioselectivity, which was further experimentally analyzed by NOE and UV spectroscopy.

Synthesis and anti-HCV determinant motif identification in pyranone carboxamide scaffold.

Hepatitis C Virus exhibits high genetic diversity. The current treatment for genotype-1 with ∼80% sustained virologic responses is a combination of pegylated interferon, ribavirin and boceprevir/telaprevir/simeprevir which is associated with several side effects and need close monitoring. Therefore, novel therapies are invited for safer and more efficient treatment. This study was designed for synthesis of new α-pyranone carboxamide analogs for evaluation of anti-HCV activity to delineate structure-activity relationship (SAR) and to identify anti-HCV determinant motif on this new scaffold. Forty four new α-pyranone carboxamide analogs were synthesized. Six potential anti-HCV candidates 11a (EC50=0.35 µM), 11e (EC50=0.48 µM), 12f (EC50=0.47 µM), 12g (EC50=0.39 µM), 12h (EC50=0.20 µM) and 12j (EC50=0.25 µM) with lower cytotoxicity (CC50>20 µM) were discovered through cell based HCV replicon system. The activity profile of forty four new α-pyranone carboxamide analogs suggests the role of an aromatic motif in the B region to add a synergistic effect to NHOH motif at 4-position and revels an anti-HCV activity determinants motif under this scaffold. The biochemical assay against most promising HCV target protein 'NS3 protease and NS5B polymerase' showed no activity and open a scope to explore new mechanism inhibitor.

Synthesis of multi ring-fused imidazo [1,2- a]isoquinoline-based fluorescent scaffold as anti-Herpetic agent.

BACKGROUND: Natural product-inspired synthesis is a key incorporation in modern diversity-oriented synthesis to yield biologically novel scaffold. Inspired by ß-carboline fused system, we have designed molecules with multi ring fused scaffold by modifying the tricyclic pyrido[3,4- b]indole ring with imidazo[1,2- a]isoquinoline. METHODS: A highly convergent approach with new C-N and C-C bond formation to synthesize multiring fused complex scaffold imidazo[1,2- a]isoquinolinies as fluorophores. N-nucleophile-induced ring transformation of 2 H-pyran-2-one followed by in situ cis-stilbene-type oxidative photocyclization yielded new C-C bond formation without additional oxidant. The cytotoxicity, effective concentrations, and the mode of action of the synthesized analogs were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),, plaque reduction, time of addition, and reverse transcriptase Polymerase Chain Reaction (PCR). RESULTS: Novel imidazo[1,2- a]isoquinoline analogs were prepared, and the results revealed that trans isomer of cyclopropyl analog (EC50 35 and 37.5 µg/ml) and trans isomer of citric acid salt of phenyl analog (EC50 38.2 and 39.8 µg/ml) possess significant anti-Herpes Simplex Virus (HSV) activity with selectivity index of >10. The kinetic study demonstrated that both the analogs inhibited HSV-1F and HSV-2G at 2-4 h postinfection. Finally, western blot and reverse transcriptase PCR assays revealed that both the analogs suppressed viral immediate early transcription. CONCLUSION: Novel imidazo[1,2- a]isoquinoline analogs were synthesized from pyranone with appropriate amines. Two compounds showed better antiviral profile on HSV-infected Vero cells, compared to the standard drug acyclovir (ACV). Overall, we discovered a promising scaffold to develop a nonnucleoside lead targeting the viral immediate early transcription for the management of HSV infections.

Synthesis and Anti-HCV Activity of 4-Hydroxyamino α-Pyranone Carboxamide Analogues.

High genetic variability in hepatitis C virus (HCV), emergence of drug resistant viruses and side effects demand the requirement for development of new scaffolds to show an alternate mechanism. Herein, we report discovery of new scaffold I based on 4-hydroxyamino α-pyranone carboxamide as promising anti-HCV agents. A comprehensive structure-activity relationship (SAR) was explored with several newly synthesized compounds. In all promising compounds (17-19, 21-22, 24-25, and 49) with EC50 ranging 0.15 to 0.40 µM, the aryl group at C-6 position of α-pyranone were unsubstituted. In particular, 25 demonstrated potential anti-HCV activity with EC50 of 0.18 µM in cell based HCV replicon system with lower cytotoxicity (CC50 > 20 µM) and provided a new scaffold for anti-HCV drug development. Further investigations, including biochemical characterization, are yet to be performed to elucidate its possible mode of action.

Skeletal hybridization and PfRIO-2 kinase modeling for synthesis of α-pyrone analogs as anti-malarial agent.

The pharmacophoric hybridization and computational design approach were applied to generate a novel series of α-pyrone analogs as plausible anti-malarial lead candidate. A putative active site in flexible loop close to wing-helix domain of PfRIO2 kinase was explored computationally to understand the molecular basis of ligand binding. All the synthesized molecules (3a-g) exhibited in vitro antimalarial activity. Oxidative stress induced by 3a-d were calculated and found to be significantly higher in case of 3b. Therefore, 3b, which shown most significant result was identified as promising lead for further SAR study to develop potent anti-malarials.

A conformational mimetic approach for the synthesis of carbocyclic nucleosides as anti-HCV leads.

Computer-aided approaches coupled with medicinal chemistry were used to explore novel carbocyclic nucleosides as potential anti-hepatitis C virus (HCV) agents. Conformational analyses were carried out on 6-amino-1H-pyrazolo[3,4-d]pyrimidine (6-APP)-based carbocyclic nucleoside analogues, which were considered as nucleoside mimetics to act as HCV RNA-dependent RNA polymerase (RdRp) inhibitors. Structural insight gained from the modeling studies revealed the molecular basis behind these nucleoside mimetics. The rationally chosen 6-APP analogues were prepared and evaluated for anti-HCV activity. RdRp SiteMap analysis revealed the presence of a hydrophobic cavity near C7 of the nucleosides; introduction of bulkier substituents at this position enhanced their activity. Herein we report the identification of an iodinated compound with an EC50 value of 6.6 µM as a preliminary anti-HCV lead.

Targeting peroxisome proliferator-activated receptor gamma for generation of antidiabetic drug.

The incidence of Diabetes Mellitus (DM) has increased to alarming levels not only in developed countries but also in developing and underdeveloped countries. Scientific data have made it clear by now that patients with DM are predisposed to many other diseases. One of the worst associations of DM is with obesity and the number of DM patients with obesity is increasing at a very fast pace due to dramatic change in life style around the world in last few decades. This necessitates the discovery of new drugs to treat increasing numbers of people with both DM and obesity. Peroxisome Proliferator activated receptor gamma (PPARγ) is a well known target for DM and thiazolidiniones (TZDs; a common class of antidiabetic drug) which includes rosiglitazone and pioglitazone act through PPARγ. Recent studies have demonstrated that PPARγ apart from being important in glucose utilization also plays a critical role in lipid metabolism and energy homeostasis affecting long-term metabolic status. The possibility of selective modulation of PPARγ has opened up a whole new avenue of research and has the potential of producing some drug which can simultaneously fight both DM and obesity, without the side-effects of the currently available PPARγ modulators. Here, we discuss various aspects of selective modulation of PPARγ action.

Identification of calcium binding sites on calsequestrin 1 and their implications for polymerization.

Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70-80 Ca(2+) ions. However, the nature of Ca(2+)-binding sites has not yet been fully characterized. In this study, we employed in silico approaches to identify the Ca(2+) binding sites and to understand the molecular basis of CASQ1-Ca(2+) recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: ) and adding the missing C-terminal residues (aa350-364). Using this model we performed extensive 30 ns molecular dynamics simulations over a wide range of Ca(2+) concentrations ([Ca(2+)]). Our results show that the Ca(2+)-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca(2+)-binding sites share a similar geometry and interestingly, the majority of them were found to be induced by increased [Ca(2+)]. We also found that the system shows maximal Ca(2+)-binding to the CAS (consecutive aspartate stretch at the C-terminus) before the rest of the CASQ1 surface becomes saturated. Simulated data show that the CASQ1 back-to-back stacking is progressively stabilized by the emergence of an increasing number of hydrophobic interactions with increasing [Ca(2+)]. Further, this study shows that the CAS domain assumes a compact structure with an increase in Ca(2+) binding, which suggests that the CAS domain might function as a Ca(2+)-sensor that may be a novel structural motif to sense metal. We propose the term "Dn-motif" for the CAS domain.

Molecular and structural insight into plasmodium falciparum RIO2 kinase.

Among approximately 65 kinases of the malarial genome, RIO2 (right open reading frame) kinase belonging to the atypical class of kinase is unique because along with a kinase domain, it has a highly conserved N-terminal winged helix (wHTH) domain. The wHTH domain resembles the wing like domain found in DNA binding proteins and is situated near to the kinase domain. Ligand binding to this domain may reposition the kinase domain leading to inhibition of enzyme function and could be utilized as a novel allosteric site to design inhibitor. In the present study, we have generated a model of RIO2 kinase from Plasmodium falciparum utilizing multiple modeling, simulation approach. A novel putative DNA-binding site is identified for the first time in PfRIO2 kinase to understand the DNA binding events involving wHTH domain and flexible loop. Induced fit DNA docking followed by minimization, molecular dynamics simulation, energetic scoring and binding mode studies are used to reveal the structural basis of PfRIO2-ATP-DNA complex. Ser105 as a potential site of phosphorylation is revealed through the structural studies of ATP binding in PfRIO2. Overall the present study discloses the structural facets of unknown PfRIO2 complex and opens an avenue toward exploration of novel drug target.

Structure based medicinal chemistry approach to develop 4-methyl-7-deazaadenine carbocyclic nucleosides as anti-HCV agent.

The structure-based approaches were implemented to design and rationally select the molecules for synthesis and anti-HCV activity evaluation. The systematic structure-activity relationships of previously discovered molecules (types I, II, III) were analyzed to design new molecules (type IV) by bioisosteric replacement of the amino group. The ligand conformation, binding mode studies and drug like properties were major determinant for selection of molecules for final synthesis. The replacement of amino group with methyl restored the interactions with RNA-template (Tem 799) through bifurcated weak H-bond (C-H...O). This is an interesting finding observed from molecular modeling studies. It was found that 6c-e has anti-HCV activity (EC(50) in 37-46 µM) while 6a, 6b and 6g were inactive. The compound 6f (EC(50) 28 µM) was the most active among the series however it also showed some cytotoxicity (CC(50) 52.8 µM). Except 6f, none of the compounds were found to be cytotoxic (CC(50)>100 µM). The present study discloses structure-based approach for novel anti-HCV lead discovery and opens a future scope of lead optimization.

Structure based molecular design, synthesis and biological evaluation of α-pyrone analogs as anti-HSV agent.

Several options for treating Herpes Simplex Virus type 1 and type 2 are available. However, non-specific inhibition and drug resistance warrants the discovery of new anti-herpetic compounds with better therapeutic profile or different mode of action. The non-nucleoside inhibitors of HSV DNA polymerase target the site that is less important for the binding of a natural nucleoside or nucleoside inhibitors. In the present study, we have explored the possibility to find a new lead molecule based on α-pyrone analogs as non-nucleoside inhibitors using structure based modeling approach. The designed molecules were synthesized and evaluated for anti-HSV activity using MTT assay. The compound 5h with EC(50) 7.4µg/ml and CC(50) 52.5µg/ml was moderately active against HSV when compared to acyclovir. A plaque reduction assay was also carried out and results reveal that 5h is more effective against HSV-1 with better selective index of 12.8 than against HSV-2 (SI=3.6). The synthesized compounds were also evaluated for anti-HIV activity, but none were active.

Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants.

CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.

Synthesis and anti-hepatitis B virus and anti-hepatitis C virus activities of 7-deazaneplanocin A analogues in vitro.

A series of 7-deazaneplanocin A (7-DNPA, 2) analogues were synthesized and evaluated for in vitro antiviral activity against HBV and HCV. The syntheses of target carbocyclic nucleosides were accomplished via a convergent procedure. 7-Substitutions were introduced by using 7-substituted-7-deaza heterocyclic base precursors (F, Cl, Br, and I) or via substitution reactions after the synthesis of the carbocyclic nucleosides. Among the synthesized compounds, 2, 13-15, 24, and 27 exhibited significant anti-HCV activity (EC(50) ranged from 1.8 to 20.1 microM) and compounds 2, 15, 22, and 24 demonstrated moderate to potent anti-HBV activity (EC(50) = 0.3-3.3 microM). In addition, compound 24 also showed activity against lamivudine- and adefovir-associated HBV mutants.

Unusual molecular conformation in dissymmetric propylene-linker compounds containing pyrazolo[3,4-d]pyrimidine and phthalimide moieties.

The crystal structure of 4,6-bis(methylsulfanyl)-1-phthalimidopropyl-1H-pyrazolo[3,4-d]pyrimidine, C(18)H(17)N(5)O(2)S(2), (VI), reveals an unusual folded conformation due to an apparent intramolecular C-H.pi interaction between the 6-methylsulfanyl and phenyl groups. However, the closely related compound 6-methylsulfanyl-1-phthalimidopropyl-4-(pyrrolidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine, C(21)H(22)N(6)O(2)S, (VII), exhibits a fully extended structure, devoid of any intramolecular C-H.pi or pi-pi interactions. The crystal packing of both molecules involves intermolecular stacking interactions due to aromatic pi-pi interactions. In addition, (VI) exhibits intermolecular C-H.O hydrogen bonding and (VII) exhibits dimerization of the molecules through intermolecular C-H.N hydrogen bonding.