Srinivasa R. Ramisetti

Design, synthesis and evaluation of Ospemifene analogs as anti-breast cancer agents.

The synthesis of some novel Ospemifene derived analogs and their evaluation as anti-breast cancer agents against MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) human breast cancer cell lines are described. Few of these analogs for instance, compounds 6, 7 and 8 are shown to be more effective than recent Selective Estrogen Receptor Modulators (SERMs) i.e. Ospemifene and Tamoxifen, against these cell lines. Compound 8 was relatively more cytotoxic to MCF-7 cells similar to Ospemifene and Tamoxifen, while most potent compounds 6 and 7 were equally effective in inhibiting growth of both ER-positive and ER-negative cell lines. The observed activity profiles were further supported by the docking studies performed against estrogen receptors (ERα and ERβ). Compounds 6, 7 and 8 exhibited stronger binding affinities with both ERα and ERβ compared to Ospemifene and Tamoxifen.

Design, synthesis, and anti-breast cancer evaluation of new triarylethylene analogs bearing short alkyl- and polar amino-/amido-ethyl chains.

The synthesis of novel triarylethylene analogs, designed based on well-known Selective Estrogen Receptor Modulators (SERMs), i.e., ospemifene and tamoxifen, as potential anti-breast cancer agents is described. The cytotoxic potential of these analogs against ER-positive (MCF-7) and ER-negative (MDA-MB-231) human breast cancer cell lines was determined and compared with the standards, ospemifene and tamoxifen. In initial screening, analogs 5, 14 and 15 were found to be much more effective than the standards against both the cell lines. The results showed that these novel analogs inhibit the expression of proteins involved in the migration and metastasis, compound 5 being most effective. Compound 5 inhibited the expression of MMP-9, c-Myc and Caveolin in both MCF-7 and MDA-MB-231 cells, and suppressed the invasion of ER-negative cells in a dose dependent manner. Finally, in silico docking simulations of the representative compounds in the binding sites of the estrogen receptors (ERs) indicated a good binding affinity of the compounds with the ERs, and supported their experimental toxicity against MCF-7 cancer cell lines.

An Efficient Synthesis of Dibenzo[a,l]tetracene and Dibenzo[a,j]tetracene and Their Identification in a Coal Tar Extract

ABSTRACT An efficient synthesis of potentially carcinogenic dibenzo[a,l]tetracene 1 and dibenzo[a,j]tetracene 2 has been developed and the synthetic standards were used to establish their environmental presence. The synthesis involves Diels–Alder reaction of in situ generated ortho-quinodimethane with phenanthrene-1,4-dione, followed by reductive aromatization as key steps. This synthetic strategy resulted in good overall yield and the mixture of 1 and 2 is easily separable by reversed-phase liquid chromatography (RP-LC). Compounds 1 and 2 were identified in a complex mixture of polycyclic aromatic hydrocarbons from coal tar (Standard Reference Material 1597a) and thus could be potential environmental hazards.

Abstract 3067: A novel tamoxifen analog, GA-11, as potential breast cancer therapeutic

Tamoxifen is the most commonly used treatment for patients with ER+ breast cancer (BC). Although many patients benefit from tamoxifen in the adjuvant and metastatic settings, resistance is an important clinical problem. We hypothesized that tamoxifen structure can be modulated to generate a more effective and a better drug-like compound. Over the past three years, extensive structure-activity relationship (SAR) studies were carried out in our laboratories to optimize tamoxifen structure. We generated over 50 tamoxifen analogs and evaluated their activity against MCF-7 (ER+) and the triple negative MDA-MB-231 (ER-, PR-, and HER2 -) human breast cancer cell lines following MTT assay. Both ER+ and triple negative cell lines were used to evaluate if the novel analogs were selectively cytotoxic to the ER+ cells similar to tamoxifen. These studies identified a novel compound GA-11 that non-selectively targeted both the ER+ and triple negative cells, interestingly being more cytotoxic to MDA-MB-231 cells. GA-11 exhibited remarkable activity with IC50 values 7 times lower against MDA-MB-231 (triple negative) and 3 times lower against MCF-7 (ER+) than tamoxifen. GA-11 contains a methoxy group in place of N,N-dimethylethyl functionality of tamoxifen, and an additional polar amine group, which makes it more cytotoxic than tamoxifen to both the cancer cell lines (ER+ and triple negative) while being non-toxic to normal mouse embryonic fibroblast (MEF) cells, suggesting that the cytotoxic response of GA-11 is selective for cancer cells. In addition, GA-11 inhibited the expression of MMP-9, c-Myc and Caveolin, the proteins associated with adhesion, migration and metastasis, in a dose-dependent manner. The response of GA-11 in triple negative MDA-MB-231 cells was more dramatic as compared to MCF-7 (ER+) cells, again suggesting its relative selectivity towards triple negative cells and its potential to target triple-negative breast cancer for which currently no effective chemotherapy options exist. Furthermore, GA-11 inhibited the migration and invasion of MDA-MB-231 cells clearly demonstrating its anti-metastatic properties. In silico evaluation showed that GA-11 with cLogP 4.8 and MW 329, fits better into the requirements of Lipinski9s Rule-of-Five, formulated to determine the drug-likeness of a small molecule; than tamoxifen (cLogP 7.34). GA-11 is thus expected to have a better oral bioavailability than tamoxifen. These in vitro studies thus indicate GA-11 to be an effective agent that is superior to tamoxifen both in potency and drug-likeness. Citation Format: Gurleen Kaur, Mohinder P. Mahajan, Manoj K. Pandey, Parvesh Singh, Srinivasa R. Ramisetti, Arun K. Sharma. A novel tamoxifen analog, GA-11, as potential breast cancer therapeutic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3067.

Abstract 4501: Development of novel Lithocholic acid carboxamides with antiproliferative and pro-apoptotic effects on human cancer cells

Bile acids, the products of cholesterol catabolism, are synthesized in the liver and subsequently excreted into the bile canaliculus and the digestive tract as N-acyl conjugates of glycine or taurine. Among all bile acids, Lithocholic acid (LCA) and Ursodeoxycholic acid (UDCA) conjugates have been reported to inhibit the growth of various human cancer cells both in vitro and in vivo. Most importantly LCA has been shown to inhibit growth of different cancer cell lines including prostate and neuroblastoma cells, while sparing normal cells. In addition, a report has shown LCA-amphiphiles to be effective anti-tumor agents in a colon cancer xenograft model. In contrast, there are reports indicating that LCA may promote dimethyl hydrazine (DMH) induced colon carcinogenesis and may also act as a carcinogen itself. This indicates that in spite of having anti-cancer properties, LCA may pose a risk of developing colon cancer. We hypothesized that LCA structure can be optimized to eliminate the carcinogenesis promoting properties, while enhancing its potency to create effective therapeutics against colon, prostate, and other cancers. Therefore, we designed and synthesized a series of novel cyclic and acyclic LCA-carboxamides by blocking the acid functionality of LCA. The cytotoxicity of the novel analogs was evaluated against colon (HT29) and prostate (DU145) cancer cells. Three analogs ASR-320, ASR-322 and ASR-339 inhibited the viability of these cells with an EC50 value of 5-10 μM, while LCA had no effect on cell viability at highest dose used (50 μM). The results suggests that the new analogs are at least 10 times more potent than the parent compound, LCA. One of the agents, induced apoptosis in both prostate (DU145) and colon (HT29) cancer cell lines, as indicated by Caspase 3 activation and PARP degradation. Currently, we are further optimizing the structure based on the initial leads and investigating efficacy and mechanistic studies in prostate and colon cancer models. Detailed results of these investigations will be presented. Citation Format: Srinivasa R. Ramisetti, Deepkamal Karelia, Melanie Neagley, Shantu Amin, Arun K. Sharma. Development of novel Lithocholic acid carboxamides with antiproliferative and pro-apoptotic effects on human cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2015-4501

Abstract B06: Development of novel thiobarbituric acid derivative compounds for treatment of lung cancers.

Previously, we reported the identification of a cytotoxic chemotype compound CC-I (a derivative of thiobarbituric acid) that is effective against chemotherapy resistant glioblastomas (GBMs) and neuroblastoma in an in vitro cell culture and in vivo mouse tumor models. In this study, we determined the cytotoxicity of CC-I and structurally similar compounds along in lung cancers to determine whether CC-I is toxic to lung cancers and to identify additional analog compounds that could be more toxic than CC-I. We designed several CC-I analog compounds by manipulating functional substitutions at N1-, N3- and C5-positions (either furan ring or benzene ring) of CC-I. For example, the novel compounds were synthesized starting from substituted thioureas, key precursors for the synthesis of substituted thiobarbituric acids, and finally by condensing cinnamaldehyde (CMC-2 series: benzene ring compound) or trans-3-(2-furyl)-acrolein (CC-I series: furan ring compound) with appropriately substituted thiobarbituric acids in the presence of catalytic amounts of pyridine. We used MTS cell proliferation or SRB cytotoxicity assay to determine the toxic level of compounds on the human lung cancer cell lines (e.g., A549, H520, H460, H69). The data suggest that the functional group at N1-, N3- and C5 sites is important for toxicity. Among the compounds with similar structures, CC-I-v3 and CC-I-v4 are the two most cytotoxic compounds to lung cancer cells and also the cisplatin resistant lung cancer cell lines such as H1993 and H520. In some of the cell lines we investigated, CC-I-v1 and CMC-2 compounds showed greater toxicity than CC-I compound. Currently, we are studying the anti-tumor effect of these compounds in an in vivo nude mouse tumor model. In summary, this structure-activity study clearly indicates that we can develop more efficacious compounds compared to our original compounds by rational modifications of the chemotype compounds. [This project is supported by the Elsa U. Pardee Foundation] Citation Format: Sang Y. Lee, Srinivasa Ramisetti, Becky Slagle-Webb, Arun K. Sharma, James R. Connor. Development of novel thiobarbituric acid derivative compounds for treatment of lung cancers. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B06.

Abstract 1639: Development of novel thiobarbituric acid derivatives as potential cancer therapeutics

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Chemotherapy drugs for the treatment of cancer are generally directed to inhibit the reproduction of malignant cells and killing cells, thereby preventing tumor growth or reducing tumor size. Many of these drugs develop resistance to tumor cells by different mechanisms. Therefore a significant clinical need exists for the development of additional chemotherapeutic agents that are toxic to a wide range of tumor cells, in particular tumors that are resistant to current treatments such as radiotherapy and chemotherapy. From recent past, it has been known that barbituric acid derivatives exhibit different biological activities such as anti-depressant, anti-convulsant, anti-viral and anti-cancer activities. Among them some thiobarbituric acid derivatives, such as merocyanine and merbarone have been proposed as possible modulators of apoptosis in several cancer cells including glioblastoma multiforme (GBM), lung, melanoma, leukemia, breast cancer, prostate cancer, and cervical cancer; albeit not particularly for cancers that are resistant to current therapy. We designed and synthesized novel thio- and seleno- barbituric derivatives by varying the substituents at N1, N3 (ethyl, methyl, allyl, and phenyl) and C5 tethered with dienyl and trienyl moieties attached with different substituents like phenyl, 2-furanyl, 2-thiophenyl, 1-naphthyl & 3-pyridyl. These compounds were tested against both chemotherapy sensitive and resistant, melanoma (UACC903 and CHL-1), GBM (U87-MG, T98G, and CCF-STTG1) , and lung cancer (A549, H460, H520and H69) cells. Most of these compounds reduced cell viability in a dose and time dependent manner. Among all the compounds ASR-198, ASR-199, ASR-243 and ASR-244 were most cytotoxic to both chemotherapy resistant and sensitive cells. In addition, these compounds triggered a dose dependent induction of apoptosis by activation of caspase-3 and PARP cleavage in melanoma cells. Overall compound ASR-198, having a 2-thiophen-dienyl substitution and C-5 position, emerged as the most efficacious compound in all three cancer cell lines. Interestingly its isosteric selenium analogs were relatively less cytotoxic. In summary, this structure-activity relationship study clearly indicates that more efficacious compounds, compared to existing thiobarbituric analogs, can be developed based on barbituric acid scaffold, by rational modifications of the chemo type compounds. Detailed results of these investigations will be presented. Citation Format: Srinivasa R. Ramisetti, Sang Y. Lee, Manoj K. Pandey, Shantu G. Amin, Arun K. Sharma. Development of novel thiobarbituric acid derivatives as potential cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2014-1639