Pyrazoloanthrone and its analogues form the central core of the thesis and the work is focused on the evaluation of chemical and biological applications of pyrazoloanthrones. Selective and sensitive detection of biologically, environmentally and industrially important molecular species such as fluoride, cyanide and picric acid by using pyrazoloanthrones as sensors form the first part while the second part deals with selective and specific kinase inhibition by pyrazoloanthrones to moderate inflammation associated disorders like septic shock. All the investigations are based on extensive crystallographic studies of the participating molecules. Chapter 1 provides a brief review on the history and biological importance of 1,9-pyrazoloanthrones. The potential of these molecules as probes in sensor chemistry and protein kinase inhibition is envisaged. A short account of the techniques employed for the investigations along with a preamble is presented. Chapter 2 is divided into two parts. Part A deals with the design of a colorimetric and “turn-on” fluorescent chemosensor based on 1,9-pyrazoloanthrone specifically for cyanide and fluoride ion detection. A remarkable solid state reaction indicated by the development of intense red color occurs when crystals of tetrabutylammonium cyanide/fluoride are brought in physical contact with 1,9¬pyrazoloanthrone resulting in corresponding molecular complexes (Figure 1). X-ray crystal structures of these complexes and also of 1,9-pyrazoloanthrone have been determined and the ion sensing activity has been substantiated on the basis of spectroscopic (absorption, fluorescence and NMR) and structural analyses. The crystal structure of the parent compound exhibits a disorder as a consequence of tautomerism and the disorder gets carried on to the complexes as well with even the cyanide and the fluoride ions showing partial occupancy sites. The presence of the –NH group and associated intramolecular charge transfer upon complex formation is attributed to the extreme sensitivity of 1,9-pyrazoloanthrone for cyanide and fluoride (detection limits of 0.2 ppb and 2 ppb) ions respectively. Figure 1. Development of intense red color during the solid state reaction (shown on left) and the turn on fluorescence behavior (shown to the right) Part B demonstrates the utilization of electron rich N-alkyl substituted pyrazoloanthrones to design sensors for detecting explosive and electron deficient nitro aromatics such as picric acid (PA). The N-alkyl derivative of 1,9-pyrazoloanthrone has been synthesized, characterized by single crystal X-ray diffraction studies and evaluated as a potent sensor for picric acid. NMR and fluorescence lifetime measurements validate that the fluorescence quenching of sensor compound by PA (Figure 2) as due to the formation of excited state charge-transfer complex resulting in dynamic quenching. Figure 2. Fluorescence quenching measurements demonstrating the dynamic quenching in the charge transfer complex. Chapter 3 deals with the biological evaluation of 1,9-pyrazoloanthrone and its alkyl derivatives towards the inhibition of a decisive protein kinase called c-Jun N-terminal Kinase (JNK), an important member of MAP kinase family. JNK controls crucial cellular processes like apoptosis and cell proliferation and is implicated in disorders associated with inflammation such as septic shock, arthritis, inflammatory bowel disease, etc. Therapeutic inhibition of JNK activity by small molecules has proven to be advantageous in the treatment of diseases coupled with derailed inflammation. In this context, it is already established that 1,9-pyrazoloanthrone (SP600125) effectively and selectively inhibits JNK at concentrations beyond 10 M. A series of alkyl isomers of pyrazoloanthrone derivatives have been synthesized to evaluate the structural implications of inhibition and to elevate both selectivity and sensitivity at lower concentrations. The crystal structures of these isomers have been characterized and their utility as inhibitors has been tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). The minimum inhibitory concentrations required by these molecules to inhibit JNK was found to be lesser as compared to 1,9-pyrazoloanthrone (<5 µM; Figure 3). Critically, it turns out that among the various inhibitors synthesized, the lead candidates SPP1 and SPB1 display specific inhibition of JNK among other LPS activated MAP kinases like ERK1/2 and p38. These results suggest that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders. Figure 3. Inhibition of JNK in macrophages by the SPP1 and SPB1 compared to the known SP600125. Inspired by the results reported in the previous chapter, Chapter 4 is devoted to the generation of a library of compounds based on SPP1 and SPB1 with a purpose to design inhibitors of JNK which perform at the lowest possible concentrations and the consequent evaluation of their potential on endotoxin induced septic shock. Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. Figure 4. Two selected molecules for specific inhibition studies of JNK at lower concentrations. It is demonstrated that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Based on the results from both in vitro with macrophages and in vivo with the mouse model of septicemia, the potential role of two selected molecules D1 and D2 (Figure 4) in regulating endotoxin induced inflammation is firmly established. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the in vitro as well as the in vivo data clearly potentiates the selective inhibitory capacity of small molecule inhibitors like D1 and D2 which can facilitate the treatment of current inflammatory disorders when used in combination with the available drugs having varied efficacies. The results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text