Andrew Morrell

Synthesis and Biological Evaluation of the First Dual Tyrosyl- DNA Phosphodiesterase I (Tdp1) - Topoisomerase I (Top1) Inhibitors

Substances with dual tyrosyl-DNA phosphodiesterase I-topoisomerase I inhibitory activity in one low molecular weight compound would constitute a unique class of anticancer agents that could potentially have significant advantages over drugs that work against the individual enzymes. The present study demonstrates the successful synthesis and evaluation of the first dual Top1-Tdp1 inhibitors, which are based on the indenoisoquinoline chemotype. One bis(indenoisoquinoline) had significant activity against human Tdp1 (IC(50) = 1.52 ± 0.05 μM), and it was also equipotent to camptothecin as a Top1 inhibitor. Significant insights into enzyme-drug interactions were gained via structure-activity relationship studies of the series. The present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in this indenoisoquinoline class of inhibitors even though it was demonstrated to work well for the steroid NSC 88915 (7). The current study will facilitate future efforts to optimize dual Top1-Tdp1 inhibitors.

Synthesis and biological evaluation of indenoisoquinolines that inhibit both tyrosyl-DNA phosphodiesterase I (Tdp1) and topoisomerase I (Top1).

Tyrosyl-DNA phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures were used to establish structure-activity relationships. The potencies of the indenoisoquinolines against Tdp1 ranged from 5 μM to 111 μM, which places the more active compounds among the most potent known inhibitors of this target. The cytotoxicity mean graph midpoints ranged from 0.02 to 2.34 μM. Dual Tdp1-Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could theoretically work synergistically to create more effective anticancer agents.

Induction of Retinoid X Receptor Activity and Consequent Upregulation of p21WAF1/CIP1 by Indenoisoquinolines in MCF7 Cells

Retinoid X receptor (RXR) has been targeted for the chemoprevention and treatment of cancer. To discover potential agents acting through RXRs, we utilized an RXR response element (RXRE)-luciferase reporter gene assay. Following extensive screening, 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36) was found to induce RXRE-luciferase activities. AM6-36 inhibited COX-2 expression and anchorage-independent growth with 12-O-tetradecanoylphorbol 13-acetate-stimulated JB6 Cl41 cells, induced the expression of CD38 in HL-60 cells, and attenuated the growth of N-methyl-N-nitrosourea–induced mammary tumors in rats. Consistent with other reports describing the antiproliferative effects of RXR agonists in breast cancers, AM6-36 showed growth inhibition with cultured MCF7 breast cancer cells, accompanied by G2/M-phase arrest at lower concentrations and enhanced S-phase arrest at higher concentrations. On the basis of DNA microarray analysis, AM6-36 upregulated the expression of CDKN1A, a target gene of RXR, by 35-fold. In accord with this response, the expression of the corresponding protein, p21WAF1/CIP1, was increased in the presence of AM6-36. Induction of p21 by AM6-36 was abrogated following transient knockdown of RXRα, demonstrating that the effect of AM6-36 on the expression of p21 is closely related to modulation of RXRα transcriptional activity. Intestinal permeability was suggested with Caco-2 cells and limited metabolism resulted when AM6-36 was incubated with human liver microsomes. Oral administration with rats resulted in 0.8 μg/mL, 4.3 μg/g, and 0.3 μg/g in serum, liver, and mammary gland, respectively. In sum, these data suggest that AM6-36 is a promising lead for the treatment or prevention of breast cancer and provide a strong rationale for testing in more advanced antitumor systems. Cancer Prev Res; 4(4); 592–607. ©2011 AACR.

Activity of Indenoisoquinolines against African Trypanosomes

ABSTRACT African trypanosomiasis (sleeping sickness), caused by protozoan Trypanosoma brucei species, is a debilitating disease that is lethal if untreated. Available drugs are antiquated, toxic, and compromised by emerging resistance. The indenoisoquinolines are a class of noncamptothecin topoisomerase IB poisons that are under development as anticancer agents. We tested a variety of indenoisoquinolines for their ability to kill T. brucei. Indenoisoquinolines proved trypanocidal at submicromolar concentrations in vitro. Structure-activity analysis yielded motifs that enhanced potency, including alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Detailed analysis of eight water-soluble indenoisoquinolines demonstrated that in trypanosomes the compounds inhibited DNA synthesis and acted as topoisomerase poisons. Testing these compounds on L1210 mouse leukemia cells revealed that all eight were more effective against trypanosomes than against mammalian cells. In preliminary in vivo experiments one compound delayed parasitemia and extended survival in mice subjected to a lethal trypanosome challenge. The indenoisoquinolines provide a promising lead for the development of drugs against sleeping sickness.

Abstract 1934: Characterization of the anti-proliferative effect of 6-(3-aminopropyl)-9-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione in cultured PC-3 cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Previously, we reported interesting pharmacological responses mediated with indenoisoquinolines, including cytotoxic effects mediated by the topoisomerase I inhibitor 6-(3-aminopropyl)-9-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (AM9-79). We currently describe further mechanistic evaluations performed with cultured PC-3 cells. AM9-79 strongly inhibited proliferation in a dose- and time-dependent manner (IC50 = 9.77 nM; 72 h incubation period). As indicated by Annexin V/7-AAD double staining, an elevated population of cells was undergoing apoptosis. Accordingly, cellular levels of pro-apoptotic proteins such as Bcl-2 were decreased, and the protein cleavage and enzymatic activities of several caspases, which play pivotal roles in apoptosis, were induced. Effects on gene expression were evaluated with a cDNA microarray, and it was found that AM9-79 significantly up-regulated the expression of BRCA2, CDKN1A, GADD45A, SERTAD1, and TP53. Consistent with these results, the protein level of CDKN1A (p21WAF1/CIP1), and phosphorylation/activation of ERK1/2, a signaling molecule positively correlated to the expression of BRCA2, were up-regulated. Further investigation of a potential upstream mechanism revealed treatment with AM9-79 decreased the protein level of EGFR and reduced nuclear translocation. This is relevant since current evidence indicates EGFR functions not only as a transmembrane receptor but also as a transcriptional factor. In accord with this response, decreased expression levels of downstream molecules (iNOS and cyclin D1) were observed. In sum, these data suggest AM9-79 can effectively function by novel mechanisms of relevance for the treatment or prevention of cancer. (Supported by P01 CA48112 awarded by the NCI) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1934. doi:1538-7445.AM2012-1934

Abstract B91: Induction of RXR transcriptional activity and consequent upregulation of p21 by 3‐amino‐6‐(3‐aminopropyl)‐5,6‐dihydro‐5, 11‐dioxo‐11H‐indeno[1,2‐c]isoquinoline dihydrochloride in MCF‐7 breast cancer cells

Retinoid X receptor (RXR) is known to play various roles in embryogenesis, metabolic processes, differentiation, and apoptosis. Recently, RXR has been studied as a target for cancer chemoprevention and treatment. To discover potential cancer chemopreventive agents acting through RXRs, we developed a cell line‐based RXR response element (RXRE)‐luciferase reporter gene assay system. Following extensive screening, 3‐amino‐6‐(3aminopropyl)‐5,6‐dihydro‐5,11‐dioxo‐11H‐indeno[1,2‐c]isoquinoline dihydrochloride (AM6‐36) was found to induce RXRE‐luciferase activities in a dose‐dependent manner. Subsequently, the substance was found to inhibit the proliferation of MCF‐7 breast cancer cells in a concentration‐dependent manner. Prompted by these results, we examined the underlying mechanism of AM6‐36 anti‐proliferative activity using MCF‐cells as a model. In brief, at lower concentrations, G2/M arrest was observed, and S phase arrest was enhanced at higher concentrations. Based on DNA microarray analysis, AM6‐36 was found to up‐regulate the expression of CDKN1A, a target gene of RXR, by ∼40‐fold. In accordance with this response, the protein level of CDKN1A (p21WAF1/CIP1) was increased in the presence of AM6‐36. Induction of p21 by AM6‐36 was abrogated following transient transfection of RXR siRNA, demonstrating the effect of AM6‐36 on the expression of p21 is closely related to modulation of RXR transcriptional activity. These data suggest AM6‐36 is a promising lead compound for the treatment or prevention of breast cancer. A synthetic procedure has been devised for large‐scale production, moderate absorption and slow metabolism has been demonstrated with rats (p.o. administration), and more advanced anti‐tumor evaluation is underway. (Supported by program project P01 CA48112 awarded by the National Cancer Institute.) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B91.