Kuntebommanahalli N. Thimmaiah

Anti-Tumor Activity of a Novel HS-Mimetic-Vascular Endothelial Growth Factor Binding Small Molecule

The angiogenic process is controlled by variety of factors of which the vascular endothelial growth factor (VEGF) pathway plays a major role. A series of heparan sulfate mimetic small molecules targeting VEGF/VEGFR pathway has been synthesized. Among them, compound 8 (2-butyl-5-chloro-3-(4-nitro-benzyl)-3H-imidazole-4-carbaldehyde) was identified as a significant binding molecule for the heparin-binding domain of VEGF, determined by high-throughput-surface plasmon resonance assay. The data predicted strong binding of compound 8 with VEGF which may prevent the binding of VEGF to its receptor. We compared the structure of compound 8 with heparan sulfate (HS), which have in common the functional ionic groups such as sulfate, nitro and carbaldehyde that can be located in similar positions of the disaccharide structure of HS. Molecular docking studies predicted that compound 8 binds at the heparin binding domain of VEGF through strong hydrogen bonding with Lys-30 and Gln-20 amino acid residues, and consistent with the prediction, compound 8 inhibited binding of VEGF to immobilized heparin. In vitro studies showed that compound 8 inhibits the VEGF-induced proliferation migration and tube formation of mouse vascular endothelial cells, and finally the invasion of a murine osteosarcoma cell line (LM8G7) which secrets high levels of VEGF. In vivo, these effects produce significant decrease of tumor burden in an experimental model of liver metastasis. Collectively, these data indicate that compound 8 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of endothelial cell migration and angiogenesis mediated by VEGF. In conclusion, compound 8 may normalize the tumor vasculature and microenvironment in tumors probably by inhibiting the binding of VEGF to its receptor.

Synthesis and chemical characterization of 2-methoxy-N-10-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N(10)-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid. This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6N potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield. N-(omega-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary amines. Products were characterized by UV, IR, 1H and 13C NMR, mass-spectral data and elemental analysis. The lipophilicity expressed in log(10) P and pK(a) of compounds have been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 7, 10, 12, and 15-19 at 100 microM caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, verapamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the MDR cell line KBCh(R)-8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that VRP and each of the modulators (1-19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19 compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Cytotoxicity has been determined and the IC(50) values lie in the range 8.00-18.50 microM for propyl and 4-15 microM for butyl derivatives against KBCh(R)-8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to 4 carbons at N(10)-position. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14-16, and 19 like VRP, were able to completely reverse the 24-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-methoxyacridones.

Sensitization of multidrug resistant (MDR) cancer cells to vinblastine by novel acridones: correlation between anti-calmodulin activity and anti-MDR activity.

Multidrug resistance (MDR) of cancer cells remains to be an important cause of chemotherapy failure. Search for the new MDR reversal agents is still an unceasing challenge for the scientists. In an attempt to find clinically useful modulators of MDR, a series of 19 N(10)-substituted-2-bromoacridones has been synthesized. Parent compound 1, prepared by the Ullmann condensation of o-chlorobenzoic acid and p-bromoaniline, undergoes N-alkylation in the presence of a phase transfer catalyst. N-(omega-Chloroalkyl) analogues were subjected to iodide catalyzed nucleophilic substitution reaction with various secondary amines to get the products 3-10 and 12-19, which increased the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells to a greater extent (1.25 to 1.9-fold) than did a similar concentration of the standard modulator, verapamil (VRP). Results of the efflux experiment showed that each modulator significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. All the compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 3.8 to 34-fold. The study on the structure-activity relationship revealed that substitution of hydrogen atom at position C-2 in acridone nucleus by a bromine atom increased the cytotoxic and anti-MDR activities. The ability of acridones to inhibit calmodulin-dependent cyclic AMP phosphodiesterase has been determined and the results have shown a strong positive correlation between anti-calmodulin activity and cytotoxicity in KBCh(R)-8-5 cells or anti-MDR activity.

Plasmid loss in plasmid-carrying strains of Escherichia coli treated with phenoxazines and an approach to study their DNA binding properties.

The effect of subinhibitory concentrations of 2-trifluoromethyl-N10-substituted phenoxazines on plasmid-coded antibiotic resistance in Escherichia coli was investigated. Phenoxazine treatment resulted in the loss of resistance markers to an extent of 8-63% in all the strains tested, and the disappearance of plasmid DNA in phenoxazine sensitive colonies was evidenced by agarose gel electrophoresis. The resistant strains were sensitized in the presence of phenoxazines with a concomitant reduction in the MIC (minimum inhibitory concentration) values. The UV, fluorescence spectral, and ethidium bromide displacement agarose gel assay methods revealed that phenoxazines are intercalated with plasmid DNA. Progressive addition of DNA led to a significant reduction in the peak intensity of the absorption maximum of phenoxazine derivative. Further, destabilization of ethidium bromide-DNA complex as seen from fluorescence microscopy in the presence of phenoxazines was observed. The potency of phenoxazines to sensitize the resistant organisms follows the order butyl > propyl > acetyl derivatives.

Synthesis of azetidin-2-one derivatives as inhibitor of secretory phospholipase A(2) with anti-inflammatory activity

The title compounds have been synthesized and tested for structure activity relationship for Phospholipase A(2) (PLA(2)) E.C. 3.1.1.4] enzyme inhibition. The in vitro PLA(2) enzyme inhibitory activity of azetidin-2-one derivatives and in vivo anti-inflammatory activity studies using mice are highlighted. The results show that some azetidin-2-one derivatives are very good PLA(2) inhibitors and can also be used as anti-inflammatory drugs.

Anti-Tubercular and Anti-Inflammatory Activities of Azetidin-2-One Derivatives and Their Effects on the Activity of Phospholipase A2

The title compounds have been synthesized and tested for structure activity relationship for Phospholipase A2 (PLA2) [E.C. 3.1.1.4] enzyme inhibition. The in vitro anti-tubercular, PLA(2) enzyme inhibitory activities of azetidin-2-one derivatives and in vivo anti-inflammatory studies using mice are highlighted. The analogues of azetidin-2-one were prepared based on the initial activity against Mycobacterium tuberculosis (Mtb). Certain azetidin-2-one analogues described herein showed moderate to good anti-tubercular activity. In particular, two compounds (4f) and (4g) exhibited MIC values of 1.56 and 0.78 microg/mL respectively against the Mtb H(37)Rv strain. Chloro substitution on aryloxy acid apparently enhanced the antimycobacterial activity and also PLA2 inhibition in the azetidin-2-one series described herein. The ability of azetidin-2-one analogues as anti-inflammatory agents has also been determined. The results show some correlation between anti-inflammatory, anti-tubercular activity and expression of PLA2 enzyme.