Method for highly selective, ultrasensitive fluorimetric detection of Cu2+ and Al3+ by Schiff bases containing o-phenylenediamine and o-aminophenol.

Schiff base probes (1 and 2) made from o-phenylenediamine and o-aminophenol were appeared as highly selective fluorimetric chemosensor of Cu2+ and Al3+ ions respectively. Strong fluorescence emission of probe 1 at 415 nm (excitation at 350 nm) was instantly turned off on addition of Cu2+. Very weak fluorescence of probe 2 at 506 nm (excitation at 400 nm) was immediately turned on specifically by Al3+. Job's plot and ESI-MS results suggested 1:1 molar stoichiometric ratio of metal ion and probe in their respective complexes. Probe 1 and 2 had demonstrated very low detection limit (9.9 and 2.5 nM respectively). Binding of Cu2+ with probe 1 was found chemically reversible on addition of EDTA, while complexation between Al3+ and probe 2 was not reversible. On the basis of density functional theory (DFT) and spectroscopic results, probable mode of sensing of the metal ions by the probes were proposed. Quenching of the fluorescence of probe 1 by Cu2+ was attributed to the extensive transfer of charge from the probe molecule to paramagnetic copper ion. Whereas, in the Al3+-complex of probe 2, photo-induced electron transfer (PET) process from the imine nitrogen to salicylaldehyde moiety was restricted and thereby the weak emission intensity of probe 2 was enhanced significantly. Effective pH range of sensing the metal ions by probe 1 and 2 were 4 to 8 and 6 to 10 respectively. Probe 1 was also applied in the design of a logic gate for Cu2+ detection. Moreover, probe 1 and 2 was also used in water sample analysis for quantitative estimation of Cu2+ and Al3+ respectively.

A comparative study of diaryl urea molecules with and without sulfonamide group on Carbonic anhydrase IX and XII inhibition and its consequence on breast cancer cells.

To investigate the intrinsic relation between carbonic anhydrase inhibition and anticancer activity, we have prepared four sets of diaryl urea molecules and tested for the inhibition of hCA-IX and XII on two breast cancer cell lines. Among 21 compounds, compound J2 (with -SO2NH2 group) and J16 (without -SO2NH2 group) showed the best activity under normoxic and hypoxic conditions. The IC50 values of J16 for MDA-MB-231 and MCF-7 cells, under normoxic condition were 6.3 and 3.7 µM respectively, which are 1.9/3.3 and 15.8 times better than U-4-Nitro and SLC-0111 respectively. Whereas, under the hypoxic condition the corresponding values were 12.4 and 1.1 µM (MDA-MB-231 and MCF-7 cells respectively), which are equal/8 times better than U-4-Nitro. Whereas, J2 showed better IC50 value than U-4-Nitro (6.3 µM) under normoxic condition for both MDA-MB-231 and MCF-7 cells (1.9/2.7 times). Compound J2 inhibits the activity of hCA-IX and XII in nanomolar concentration [Ki values 4.09 and 9.10 nM respectively with selectivity ratio of 1.8 and 0.8 with hCA-II]. The crystal structure and modelling studies demonstrates that the inhibition of CAs arises due to the blocking of the CO2 coordination site of zinc in its catalytic domain. However, J16 was found to be unable to inhibit the activity of hCAs (Ki > 89000 nM). qPCR and western blot analysis showed a significant reduction (1.5 to 20 fold) of the transcription and expression of HIF1A, CA9 and CA12 genes in presence of J2 and J16. Both J2 and J16 found to reduce accumulation of HIF-1α protein by inhibiting the chaperone activity of hHSP70 with IC50 values of 19.4 and 15.3 µM respectively. Perturbation of the hCA-IX and XII activity by binding at active site or by reduced expression or by both leads to the decrease of intracellular pH, which resulted in concomitant increase of reactive oxygen species by 2.6/2.0 (MCF-7) and 2.9/1.8 (MDA-MB-231) fold for J2/J16. Increased cyclin D1 expression in presence of J2 and J16 was presumed to be indirectly responsible for the apoptosis of the cancer cells. Expression of the other apoptosis markers Bcl-2, Bim, caspase 9 and caspase 3 substantiated the apoptosis mechanism. However, decreased transcription/expression of HIF1A/HIF-1α and hCA-IX/XII also implies the inhibition of the extracellular signal-regulated kinase pathway by J2 and J16.

Oral combination of eugenol oleate and miltefosine induce immune response during experimental visceral leishmaniasis through nitric oxide generation with advanced cytokine demand.

Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.

Oral administration of eugenol oleate cures experimental visceral leishmaniasis through cytokines abundance.

Visceral leishmaniasis (VL) is an endemic fatal infectious disease in tropical and subtropical nations. The limited treatment options, long treatment regimens, invasive mode of administration of drugs, and lack of effective vaccination are the main reasons for the search of new alternative therapeutics against VL. On this quest, from a series of eugenol derivatives, we had demonstrated eugenol oleate as a lead immunomodulatory anti-VL molecule earlier. In this report, the oral efficacy and mechanism of eugenol oleate in inducing immunomodulatory anti-VL activity has been studied in BALB/c mice model. The plasma pharmacokinetic and acute toxicity studies suggested that the eugenol oleate is safe with an appreciable pharmacokinetic profile. Eugenol oleate (30 mg/kg B.W.) showed 86.5% of hepatic and 84.1% of splenic parasite clearance. The increased Th1 cytokine profile and decreased Th2 cytokine profile observed from ELISA and qRTPCR suggested that the eugenol oleate induced the parasite clearance through the activation of the host immune system. Subsequently, the mechanistic insights behind the anti-leishmanial activity of eugenol oleate were studied in peritoneal macrophages in vitro by inhibitor response study and immunoblotting. The results inferred that eugenol oleate activated the PKC-ßII-p38 MAPK and produced IL-12 and IFN-γ which intern activated the iNOS2 to produce NO free radicals that cleared the intracellular parasite.

2-Aryl benzazole derived new class of anti-tubercular compounds: Endowed to eradicate mycobacterium tuberculosis in replicating and non-replicating forms.

The high mortality rate and the increasing prevalence of Mtb resistance are the major concerns for the Tuberculosis (TB) treatment in this century. To counteract the prevalence of Mtb resistance, we have synthesized 2-aryl benzazole based dual targeted molecules. Compound 9m and 9n were found to be equally active against replicating and non-replicating form of Mtb (MIC(MABA) 1.98 and 1.66 µg/ml; MIC(LORA) 2.06 and 1.59 µg/ml respectively). They arrested the cell division (replicating Mtb) by inhibiting the GTPase activity of FtsZ with IC50 values 45 and 64 µM respectively. They were also capable of kill Mtb in non-replicating form by inhibiting the biosynthesis of menaquinone which was substantiated by the MenG inhibition (IC50 = 11.62 and 7.49 µM respectively) followed by the Vit-K2 rescue study and ATP production assay.

Inhibition of copper-mediated aggregation of human γD-crystallin by Schiff bases.

Protein aggregation, due to the imbalance in the concentration of Cu2+ and Zn2+ ions is found to be allied with various physiological disorders. Copper is known to promote the oxidative damage of ß/γ-crystallins in aged eye lens and causes their aggregation leading to cataract. Therefore, synthesis of a small-molecule 'chelator' for Cu2+ with complementary antioxidant effect will find potential applications against aggregation of ß/γ-crystallins. In this paper, we have reported the synthesis of different Schiff bases and studied their Cu2+ complexation ability (using UV-Vis, FT-IR and ESI-MS) and antioxidant activity. Further based on their copper complexation efficiency, Schiff bases were used to inhibit Cu2+-mediated aggregation of recombinant human γD-crystallin (HGD) and ß/γ-crystallins (isolated from cataractous human eye lens). Among these synthesized molecules, compound 8 at a concentration of 100 µM had shown ~95% inhibition of copper (100 µM)-induced aggregation. Compound 8 also showed a positive cooperative effect at a concentration of 5-15 µM on the inhibitory activity of human αA-crystallin (HAA) during Cu2+-induced aggregation of HGD. It eventually inhibited the aggregation process by additional ~20%. However, ~50% inhibition of copper-mediated aggregation of ß/γ-crystallins (isolated from cataractous human eye lens) was recorded by compound 8 (100 µM). Although the reductive aminated products of the imines showed better antioxidant activity due to their lower copper complexing ability, they were found to be non-effective against Cu2+-mediated aggregation of HGD.

Flexible and biomimetic analogs of triple uptake inhibitor 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol: Synthesis, biological characterization, and development of a pharmacophore model.

In this study we have generated a pharmacophore model of triple uptake inhibitor compounds based on novel asymmetric pyran derivatives and the newly developed asymmetric furan derivatives. The model revealed features important for inhibitors to exhibit a balanced activity against dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). In particular, a 'folded' conformation was found common to the active pyran compounds in the training set and was crucial to triple uptake inhibitory activity. Furthermore, the distances between the benzhydryl moiety and the N-benzyl group as well as the orientation of the secondary nitrogen were also important for TUI activity. We have validated our findings by synthesizing and testing novel asymmetric pyran analogs. The present work has also resulted in the discovery of a new series of asymmetric tetrahydrofuran derivatives as novel TUIs. Lead compounds 41 and 42 exhibited moderate TUI activity. Interestingly, the highest TUI activity by lead tetrahydrofuran compounds for example, 41 and 42, was exhibited in a stereochemical preference similar to pyran TUI for example, D-161.

Amino and carboxy functionalized modified nucleosides: a potential class of inhibitors for angiogenin.

The 3'-amino and carboxy functionalize thymidines execute their ribonucleolytic inhibition activity for angiogenin. These modified nucleosidic molecules inhibit the ribonucleolytic activity of angiogenin in a competitive manner like the other conventional nucleotidic inhibitors, which have been confirmed from kinetic experiments. The improved inhibition constant (Ki) values 427 ± 7, 775 ± 6 µM clearly indicate modified nucleosides are an obvious option for the designing of inhibitors of angiogenesis process. The chorioallantoic membrane (CAM) assay qualitatively suggests that amino functionalized nucleosides have an effective potency to inhibited angiogenin-induced angiogenesis. Docking studies further demonstrate the interaction of their polar amino group with the P1 site residues of angiogenin, i.e., His-13 and His-114 residues.

Dinucleosides with non-natural backbones: a new class of ribonuclease A and angiogenin inhibitors.

Ribonuclease A (RNase A) serves as a convenient model enzyme in the identification and development of inhibitors of proteins that are members of the ribonuclease superfamily. This is principally because the biological activity of these proteins, such as angiogenin, is linked to their catalytic ribonucleolytic activity. In an attempt to inhibit the biological activity of angiogenin, which involves new blood vessel formation, we employed different dinucleosides with varied non-natural backbones. These compounds were synthesized by coupling aminonucleosides with dicarboxylic acids and amino- and carboxynucleosides with an amino acid. These molecules show competitive inhibition with inhibition constant (K(i)) values of (59±3) and (155±5) µM for RNase A. The compounds were also found to inhibit angiogenin in a competitive fashion with corresponding K(i) values in the micromolar range. The presence of an additional polar group attached to the backbone of dinucleosides was found to be responsible for the tight binding with both proteins. The specificity of different ribonucleolytic subsites were found to be altered because of the incorporation of a non-natural backbone in between the two nucleosidic moieties. In spite of the replacement of the phosphate group by non-natural linkers, these molecules were found to selectively interact with the ribonucleolytic site residues of angiogenin, whereas the cell binding site and nuclear translocation site residues remain unperturbed. Docked conformations of the synthesized compounds with RNase A and angiogenin suggest a binding preference for the thymine-adenine pair over the thymine-thymine pair.

Synergic effect of eugenol oleate with amphotericin B augments anti-leishmanial immune response in experimental visceral leishmaniasis in vitro and in vivo.

Present treatment regimen on visceral leishmaniasis has multiple limitations including severe side effects, toxicity, and resistance of Leishmania strains. Amphotericin B is a well-established pharmacologically approved drug; however, mainly toxicity is a foremost issue with that drug. Recently, our group identified eugenol oleate as an anti-leishmanial immunomodulatory compound. The important objectives of this present study was to evaluate the possible synergistic effect of eugenol oleate with amphotericin B to reduce the toxicity of this approved drug. Results obtained from this study signified that combination of eugenol oleate and amphotericin B showed indifferent combinatorial effect against promastigotes with xΣFIC 1.015, while, moderate synergistic activity with xΣFIC 0.456 against amastigotes. It was also notable that eugenol oleate (2.5 µM) with low concentrations of amphotericin B (0.3125 µM) showed 96.45% parasite reduction within L. donovani-infected murine macrophages. Furthermore, eugenol oleate and amphotericin B significantly (p < 0.01) enhanced the nitrite generation, and pro-inflammatory cytokines (IL-12, IFN-γ and TNF-α) in infected macrophages in vitro and in BALB/c mice in vivo. Eugenol oleate (10 mg/Kg b. wt.) with amphotericin B (1 mg/Kg b.wt.) significantly (p < 0.01) controlled the parasite burden in liver by 96.2% and in spleen by 93.12%. Hence, this study strongly suggested the synergic potential of eugenol oleate with low concentration of amphotericin B in experimental visceral leishmaniasis through anti-leishmanial immune response.

Comparative inhibitory activity of 3'- and 5'-functionalized nucleosides on ribonuclease A.

Modified nucleosides, molecules, functionalized with various polar groups at different positions have been synthesized to rationalize the impact of structural modification on their inhibitory activity. Agarose gel and precipitation assays indicate their improved inhibitory activity on ribonuclease A (RNase A). Kinetic experiments clearly categorize them as competitive inhibitors of RNase A with improved inhibition constant (K(i)) values (37±9, 67±6, and 193±7µM for compounds 10, 3, and 7, respectively). The preferential hydrogen bonding network formation between His-12 and His-119 of RNase A with the polar carboxylic and amino groups of these compounds has been evidenced from the docking studies. The relationship between structural modifications and inhibitory activity of these compounds is further justified in terms of energetics using PEARLS.

Inhibition of copper-induced aggregation of human γD-crystallin by a diimine molecule and investigations on their molecular interactions.

Communicated by Ramaswamy H. Sarma.

1,3-Oxazine-2-one derived dual-targeted molecules against replicating and non-replicating forms of Mycobacterium tuberculosis.

The high mortality rate and increasing prevalence of resistant Mtb are the major concerns for the Tuberculosis (TB) treatment in this century. To curtail the prevalence of resistant Mtb, we have prepared 1,3-oxazine-2-one based dual targeted molecules. Compound 67 and 68 were found to be equally active against replicating and non-replicatiing form of Mtb (MICMABA 3.48 and 2.97 µg/ml; MICLORA 2.94 and 2.15 µg/ml respectively). They had found to suppress the biosynthesis of alfa, methoxy and keto-mycolate completely, as well as inhibit enzymatic activity of MenG (IC50 = 9.11 and 6.25 µg/ml respectively for H37Ra; IC50 = 11.76 and 10.88 µg/ml respectively for M smegmatis).

Nucleoside-amino acid conjugates: An alternative route to the design of ribonuclease A inhibitors.

Nucleoside-amino acid conjugates have been employed to inhibit the ribonucleolytic activity of ribonuclease A (RNase A) and affect the protonation/deprotonation equilibrium of its active site histidine residues. Agarose gel and precipitation assays indicate inhibition of RNase A activity by these molecules with a possible role of the polar side chains of the amino acids in RNase A inhibition. Kinetic experiments demonstrated that the mode of inhibition is competitive in nature with inhibition constants (K(i)) in the micromolar range. The nucleoside-serine conjugate occupies the active site of RNase A and preferential perturbs the pK(a) value of His-119 by its 'free amino group' as found from (1)H NMR studies. Docking studies revealed that the free amino groups of the most active compounds are within hydrogen bonding distance of His-119 in inhibitor-RNase A complexes.

Inhibition of ribonuclease A by nucleoside-dibasic acid conjugates.

We report the inhibition of the ribonucleolytic activity of ribonuclease A (RNase A) by nucleoside-dibasic acid conjugates for the first time. Agarose gel and precipitation assays show that the spacer length and the pK(a) of the carboxylic group have an important role in the inhibitory capacity. Kinetic experiments indicate a competitive mode of inhibition with inhibition constant (K(i)) value of 132+/-3 microM for Oxa-aT. Docking studies revealed that the carboxylic group of the most active compounds is within hydrogen bonding distance of His-12, Lys-41 and His-119.

Using proton nuclear magnetic resonance to study the mode of ribonuclease A inhibition by competitive and noncompetitive inhibitors.

The C(2) proton resonances of the active site histidines (His 12 and His 119) of ribonuclease A have been exploited to study the inhibition pattern of both noncompetitive (four green tea polyphenols and their copper complexes) and competitive (3'-O-carboxy esters of thymidine and 3'-amino derivatives of uridine) inhibitors. Competitive inhibitors devoid of any phosphate group have the ability to change the pK(a) of the histidine residues at the active site. Their mode of inhibition, albeit competitive, is found to be different compared to known phosphate inhibitors 2'-CMP and 3'-CMP as revealed by changes in the pK(a) values. We find a correlation between the changes in the chemical shift of His 12 and the corresponding inhibition constants (K(i)).

Exploring the potential of 3'-O-carboxy esters of thymidine as inhibitors of ribonuclease A and angiogenin.

In this study, compounds with a carboxy ester in lieu of the phosphate ester at the 3'-position have been employed to inhibit the ribonucleolytic activity of ribonuclease A (RNase A). Phosphates at the 3'-position of pyrimidine bases are well-known inhibitors of the protein. We have investigated the inhibition of RNase A by 3'-O-carboxy esters of thymidine. The compounds behave as competitive inhibitors with inhibition constants ranging from 42 to 95 microM. The mode of inhibition has also been confirmed by (1)H NMR studies of the active site histidines of RNase A. Docking studies have further substantiated the experimental results. The compounds are also found to inhibit the ribonucleolytic activity of angiogenin, a homologous protein and potent inducer of blood vessel formation.

Inhibition of Ribonuclease A by polyphenols present in green tea.

We report the effect of the natural polyphenolic compounds from green tea on the catalytic activity of Ribonuclease A (RNase A). The compounds behave as noncompetitive inhibitors of the protein with inhibition constants ranging from 80-1300 microM. The dissociation constants range from 50-150 microM for the RNase A-polyphenol complexes as determined by ultraviolet (UV) and circular dichroism (CD) studies. We have also investigated the changes in the secondary structure of RNase A on complex formation by CD and Fourier transformed infrared (FTIR) spectroscopy. The presence of the gallate moiety has been shown to be important for the inhibition of enzymatic activity. Docking studies for these compounds indicate that the preferred site of binding is the region encompassing residues 34-39 with possible hydrogen bonding with Lys 7 and Arg 10. Finally we have also looked at changes in the accessible surface area of the interacting residues on complex formation for an insight into the residues involved in the interaction.

Eugenol derived immunomodulatory molecules against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a life threatening infectious disease caused by Leishmania donovani. It leads to the severe immune suppression in the host defense system. Higher cytotoxicity, rigorous side effects and lower therapeutic indexes (TI) of current antileishmanial drugs have created a necessity to develop new molecules with better antileishmanial activity and high TI value. In this study, we have synthesized 36 derivatives of eugenol and screened them for their activity against promastigote and amastigote forms of L. donovani. Among the synthesized derivatives, comp.35 showed better antileishmanial activity against extra cellular promastigotes (IC50- 20.13 ± 0.91 µM) and intracellular amastigotes (EC50-4.25 ± 0.26 µM). The TI value (82.24 ± 3.77) was found to improve by 10-13 fold compared to Amphotericin B and Miltefosine respectively. Treatment with comp.35 (5 µg/ml) enhanced the nitric oxide (NO) generation, iNOS2 mRNA expression (∼8 folds increase) and decreased the arginase-1 activity (∼4 folds) in L. donovani infected peritoneal macrophages. Comp.35 had also increased the IL-12 (∼6 folds) and decreased the IL-10 (∼3 folds) mRNA expression and release in vitro. Results of in vivo studies revealed that comp.35 treatment at 25 mg/kg body weight efficiently cleared the hepatic and splenic parasite burden with enhanced Th1 response in L. donovani infected BALB/c mice. Hence, this study clearly represents comp.35, as an immunomodulatory molecule, can induce host protective immune response against visceral leishmaniasis through enhanced NO generation and Th1 response, which are essentials against this deadly disease.

Attenuation of Mycobacterium species through direct and macrophage mediated pathway by unsymmetrical diaryl urea.

Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivatives targeting the inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46, 57, 58 and 86 showed MIC values ≤ 10 µg/ml against H37Rv and mc26030 strains. The best molecule with a methyl at ortho position of the first aromatic ring and prenyl group at the meta position of the second aromatic ring showed the MIC value of 5.2 µg/ml and 1 µg/ml against H37Rv and mc26030 respectively, with mammalian cytotoxicity of 163.4 µg/ml. The effective compounds showed selective inhibitory effect on mycolic acid (epoxy mycolate) biosynthesis in 14C-radiolabelled assay. At the same time these molecules also executed their potent immunomodulatory activity by up-regulation of IFN-γ and IL-12 and down-regulation of IL-10.