Dwayaja H. Nadkarni

In vitro and In vivo Anticancer Activity of Novel Synthetic Makaluvamine Analogues

Purpose: The present study was designed to determine biological structure-activity relationships for four newly synthesized analogues of natural compounds (makaluvamines). The compounds, 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ); 7-(phenethylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (PEA-TPQ); 7-(3,4-methylenedioxyphenethylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (MPA-TPQ); and 7-(3,4-dimethoxyphenethylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (DPA-TPQ), were synthesized and purified, and their chemical structures were elucidated on the basis of physicochemical constants and nuclear magnetic resonance spectra. Experimental Design: The structure-activity relationship of the compounds was initially evaluated by comparing their in vitro cytotoxicity against 14 human cell lines. Detailed in vitro and in vivo studies were then done in MCF-7 and MDA-MB-468 breast cancer cell lines. Results: The in vitro cytotoxicity was compound, dose, and cell line dependent. Whereas all of the compounds exerted some activity, FBA-TPQ was the most potent inducer of apoptosis and the most effective inhibitor of cell growth and proliferation, with half maximal inhibitory concentration values for most cell lines in the range of 0.097 to 2.297 μmol/L. In MCF-7 cells, FBA-TPQ exposure led to an increase in p53/p-p53, Bax, ATM/p-ATM, p-chk1 and p-chk2, and p-H2AX; and cleavage of poly(ADP)ribose polymerase, caspase-3, caspase-8, and caspase-9. It also decreased the levels of MDM2, E2F1, Bcl-2, chk1/2, and proteins associated with cell proliferation [cyclin-dependent kinase (Cdk)2, Cdk4, Cdk6, cyclin D1, etc.]. Moreover, FBA-TPQ inhibited the growth of breast cancer xenograft tumors in nude mice in a dose-dependent manner. Western blot analysis ofthe xenograft tumors indicated that similar changes in protein expression also occur in vivo. Conclusion: Our preclinical data indicate that FBA-TPQ is a potential therapeutic agent for breast cancer, providing a basis for the development of the compound as a novel anticancer agent.

A novel synthetic iminoquinone, BA-TPQ, as an anti-breast cancer agent: in vitro and in vivo activity and mechanisms of action

Herein, we report our examination of the anti-breast cancer activity of a novel synthetic compound, 7-(benzylamino)-1, 3, 4, 8-tetrahydropyrrolo [4, 3, 2-de]quinolin-8(1H)-one (BA-TPQ). This agent is an analog of a naturally occurring marine compound, and was found to be the most active out of more than 40 related compounds. We investigated the in vitro activity of BA-TPQ on the survival, proliferation, and apoptosis of breast cancer cells using the MTT and BrdUrd assays, and Annexin/Annexin-PI staining and flow cytometry. The in vivo anti-cancer effects of BA-TPQ were evaluated in xenograft models of breast cancer. Finally, the mechanisms of action of the compound were also assessed by cDNA microarrays, RT–PCR and Western blotting. In a dose-dependent manner, BA-TPQ inhibited cell growth and induced apoptosis and cell cycle arrest in human MCF-7 and MDA-MB-468 breast cancer cells in vitro, and showed in vivo efficacy in mice bearing MCF-7 or MDA-MB-468 xenograft tumors. We demonstrated that BA-TPQ modifies the expression of numerous molecules involved in cell cycle progression and apoptosis. Similar changes in protein expression were observed in vitro and in vivo, as determined by examination of cells and excised xenograft tumors. Our preclinical data indicate that BA-TPQ is a potential therapeutic agent for breast cancer that has multiple hormone-, Her2-, and p53-independent mechanisms of action, providing a basis for further development of the compound as a novel anticancer agent.

FBA-TPQ, a novel marine-derived compound as experimental therapy for prostate cancer

SummaryWe recently synthesized a series of novel makaluvamine compounds, and found that the most potent was FBA-TPQ. The effects of FBA-TPQ on human (LNCaP and PC3) and murine (TRAMP C1) prostate cancer cells were evaluated. Potential mechanisms of action of the compound were also determined. FBA-TPQ exhibited dose-dependent cytotoxicity in the low micromolar range, inhibited proliferation, caused cell cycle arrest, and induced apoptosis in prostate cancer cell lines. The compound also decreased the expression of the androgen receptor and PSA. The results presented herein support the further development of FBA-TPQ as a novel agent for prostate cancer.

Preclinical Evaluation of Anticancer Efficacy and Pharmacological Properties of FBA-TPQ, a Novel Synthetic Makaluvamine Analog

We have recently designed and synthesized a novel iminoquinone anticancer agent, 7-(4-fluorobenzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one (FBA-TPQ) and initiated its preclinical development. Herein we investigated its efficacy, safety, and pharmacokinetics in in vitro and in vivo models of human pancreatic cancer. Our results demonstrated that FBA-TPQ inhibited pancreatic cancer cell growth, induced apoptosis, and caused cell cycle arrest in vitro. It inhibited the growth of xenograft tumors with minimal host toxicity. To facilitate future preclinical and clinical development of the agent, we also developed and validated a Rapid Resolution Liquid Chromatography (RRLC) method for quantitative analysis of FBA-TPQ in plasma and tissue samples. The method was found to be precise, accurate, and specific. Using this method, we carried out in vitro and in vivo evaluations of the pharmacological properties of FBA-TPQ, including stability in plasma, plasma protein binding, metabolism by S9 enzymes, plasma pharmacokinetics, and tissue distribution. Our results indicate that FBA-TPQ is a potential therapeutic agent for pancreatic cancer, providing a basis for future preclinical and clinical development.

Synthesis and In Vitro Anti-Lung Cancer Activity of Novel 1, 3, 4, 8- Tetrahydropyrrolo (4, 3, 2-de)quinolin-8(1H)-o ne Alkaloid Analogs

The high mortality rate and lack of effective therapies make lung cancer an ideal target for novel therapeutic agents. The present study was designed to implement a novel chemical synthesis pathway and to determine the biological activities of synthetic makaluvamine analogs in human lung cancer. Seventeen compounds were synthesized and purified, and their chemical structures were elucidated on the basis of physicochemical constants and NMR spectra. Their in vitro activity was determined in human lung cancer cell lines. Based on initial screens, compound Ic was found to be the most potent, and was therefore used as a model for further studies in lung cancer cells. Ic induced both apoptosis and S-phase cell cycle arrest. Furthermore, it activated p53 and induced cleavage of PARP and caspases 8 and 9. Our preclinical data indicate that the makaluvamine analogs are potential therapeutic agents against lung cancer, providing a basis for further development of Ic (and perhaps other analogs) as a novel anti-cancer agent.

Antibacterial and Antibiofilm Activities of Makaluvamine Analogs

Streptococcus mutans is a key etiological agent in the formation of dental caries. The major virulence factor is its ability to form biofilms. Inhibition of S. mutans biofilms offers therapeutic prospects for the treatment and the prevention of dental caries. In this study, 14 analogs of makaluvamine, a marine alkaloid, were evaluated for their antibacterial activity against S. mutans and for their ability to inhibit S. mutans biofilm formation. All analogs contained the tricyclic pyrroloiminoquinone core of makaluvamines. The structural variations of the analogs are on the amino substituents at the 7-position of the ring and the inclusion of a tosyl group on the pyrrole ring N of the makaluvamine core. The makaluvamine analogs displayed biofilm inhibition with IC50 values ranging from 0.4 μM to 88 μM. Further, the observed bactericidal activity of the majority of the analogs was found to be consistent with the anti-biofilm activity, leading to the conclusion that the anti-biofilm activity of these analogs stems from their ability to kill S. mutans. However, three of the most potent N-tosyl analogs showed biofilm IC50 values at least an order of magnitude lower than that of bactericidal activity, indicating that the biofilm activity of these analogs is more selective and perhaps independent of bactericidal activity.

Identification of the ZAK-MKK4-JNK-TGFβ Signaling Pathway as a Molecular Target for Novel Synthetic Iminoquinone Anticancer Compound BA-TPQ

Identification and validation of molecular targets are considered as key elements in new drug discovery and development. We have recently demonstrated that a novel synthetic iminoquinone analog, termed [7-(benzylamino)- 1,3,4,8-tetrahydropyrrolo [4,3, 2-de]quinolin-8(1H)-one] (BA-TPQ), has significant anti-breast cancer activity both in vitro and in vivo, but the underlying molecular mechanisms are not fully understood. Herein, we report the molecular studies for BA-TPQs effects on JNK and its upstream and downstream signaling pathways. The compound up-regulates the JNK protein levels by increasing its phosphorylation and decreasing its polyubiquitination-mediated degradation. It activates ZAK at the MAPKKK level and MKK4 at the MAPKK level. It also up-regulates the TGFβ2 mRNA level, which can be abolished by the JNK-specific inhibitor SP600125, but not TGFβ pathway-specific inhibitor SD-208, indicating that both JNK and TGFβ signaling pathways are activated by BA-TPQ and that the JNK pathway activation precedes TGFβ activation. The pro-apoptotic and anti-growth effects of BA-TPQ are significantly blocked by both the JNK and TGFβ pathway inhibitors. In addition, BA-TPQ activates the ZAK-MKK4-JNK pathway in MCF7 cells, but not normal MCF10A cells, demonstrating its cancer-specific activities. In conclusion, our results demonstrate that BA-TPQ activates the ZAK-MKK4-JNK-TGFβ signaling cascade as a molecular target for its anticancer activity.

Synthesis of Pyrroloquinones via a CAN Mediated Oxidative Free Radical Reaction of 1,3-Dicarbonyl Compounds with Aminoquinones

Pyrroloquinone ring systems are important structural units present in many biologically active molecules including a number of marine alkaloids. For example, they are found in a series of marine metabolites, such as tsitsikammamines, zyzzyanones, wakayin, and terreusinone. Several of these alkaloids have exhibited antimicrobial, antimalarial, antifungal, antitumor, and photoprotecting activities. Synthesis of pyrroloquinone unit is the key step in the synthesis of many of these important organic molecules. Here, we present a ceric (IV) ammonium nitrate (CAN) mediated oxidative free radical cyclization reaction of 1,3-dicarbonyl compounds with aminoquinones as a facile methodology for making various substituted pyrroloquinones. 1,3-dicarbonyl compounds used in this study are ethyl acetoacetate, acetylacetone, benzoyl acetone, and N,N-dimethyl acetoacetamide. The aminoquinones used in this study are 2-(benzylamino)naphthalene-1,4-dione and 6-(benzylamino)-1-tosyl-1H-indole-4,7-dione. The yields of the synthesized pyrroloquinones ranged from 23–91%.