Design and synthesis of pyrimido[4,5-b]indoles and furo[2,3-d]pyrimidines as single agents with combination chemotherapy potential or as inhibitors of tubulin or thymidylate synthase

This dissertation describes an introduction, background and research progress in the areas of multitargeted single agents and tubulin inhibitors in cancer chemotherapy and selective Toxoplasma gondii TS inhibitors for the treatment of toxoplasmosis. Tubulin inhibitors are important antitumor agents that disrupt microtubule dynamics. Thymidylate synthase (TS) inhibitors prevent cell division by interfering with de novo thymidylate synthesis. Antiangiogenic agents target tumor angiogenesis crucial for tumor growth and metastasis. Under normal circumstances, angiogenesis is typically limited to tumor cells and is mediated by receptor tyrosine kinases (RTKs). Combination chemotherapies of RTK inhibitors with cytotoxic agents that target either TS or tubulin have shown significant promise and several preclinical and clinical studies with such combinations are in progress. Multitargeted single agents with dual antiangiogenic and cytotoxic mechanisms could avoid the major limitations associated with cancer chemotherapy: multidrug resistance and dose limiting toxicities. This dissertation focuses on the design and synthesis of pyrimido[4,5-b]indoles and furo[2,3-d]pyrimidines as potential single agents with dual antiangiogenic and cytotoxic activities. These efforts led to the identification of structural features that are necessary for inhibition of RTKs and/or tubulin polymerization. Novel synthetic strategies were developed for efficient synthesis of 2,4-diamino-5-thioaryl-pyrimido[4,5-b]indoles and 4-anilino-5-methyl-furo[2,3-d]pyrimidines. Taxanes and vinca alkaloids are widely used tubulin inhibitors in cancer chemotherapy. However, their clinical use is compromised by two major mechanisms of drug resistance: the overexpression of Pgp and bIII-tubulin. This dissertation describes the design and synthesis of pyrimido[4,5-b]indoles as tubulin inhibitors that circumvent Pgp and bIII-tubulin mediated resistance. This work identified the structural features crucial for tubulin inhibition for the pyrimido[4,5-b]indole scaffold. Infection by Toxoplasma gondii can lead to toxoplasmosis in immune compromised patients such as organ transplant, cancer and AIDS patients. Current therapy involving combination of sulfadiazine and pyrimethamine is limited by drug resistance and treatment failures. The thymidylate synthase‒dihydrofolate reductase enzyme is important for thymidylate synthesis in T. gondii, and hence can be targeted to treat T. gondii infection. TS is highly conserved across species and selectivity for tgTS over human TS is significantly more challenging. The present work provides an efficient synthesis of 2-diamino-4-oxo-5-thioaryl-pyrimido[4, 5-b]indoles as selective tgTS inhibitors

Design, synthesis, and structure–activity relationships of pyrimido[4,5-b]indole-4-amines as microtubule depolymerizing agents that are effective against multidrug resistant cells

To identify the structural features of 9H-pyrimido[4,5-b]indoles as microtubule depolymerizers, pyrimido[4,5-b]indoles 2–8 with varied substituents at the 2-, 4- and 5-positions were designed and synthesized. Nucleophilic displacement of 2,5-substituted-4-chloro-pyrimido[4,5-b]indoles with appropriate arylamines was the final step employed in the synthesis of target compounds 2–8. Compounds 2 and 6 had two-digit nanomolar potency (IC50) against MDA-MB-435, SK-OV-3 and HeLa cancer cells in vitro. Compounds 2 and 6 also depolymerized microtubules comparable to the lead compound 1. Compounds 2, 3, 6 and 8 were effective in cells expressing P-glycoprotein or the βIII isotype of tubulin, mechanisms that are associated with clinical drug resistance to microtubule targeting drugs. Proton NMR and molecular modeling studies were employed to identify the structural basis for the microtubule depolymerizing activity of pyrimido[4,5-b]indoles

Synthesis of N\u3csup\u3e4\u3c/sup\u3e-(substituted phenyl)-N\u3csup\u3e4\u3c/sup\u3e-alkyl/ desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines and identification of new microtubule disrupting compounds that are effective against multidrug resistant cells

A series of fourteen N4-(substituted phenyl)-N 4-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cyclization of 2-amino-6-hydrazinylpyrimidin-4(3H)-one with cyclohexanone, followed by oxidation, chlorination and displacement with appropriate anilines. Compounds 6, 14 and 15 had low nanomolar potency against MDA-MB-435 tumor cells and depolymerized microtubules. Compound 6 additionally had nanomolar GI50 values against 57 of the NCI 60-tumor panel cell lines. Mechanistic studies showed that 6 inhibited tubulin polymerization and [3H]colchicine binding to tubulin. The most potent compounds were all effective in cells expressing P-glycoprotein or the βIII isotype of tubulin, which have been associated with clinical drug resistance. Modeling studies provided the potential interactions of 6, 14 and 15 within the colchicine site. © 2012 Elsevier Ltd. All rights reserved

Janus Compounds, 5-Chloro-N4-methyl-N4-aryl-9H-pyrimido[4,5-b]indole-2,4-diamines, Cause Both Microtubule Depolymerizing and Stabilizing Effects

While evaluating a large library of compounds designed to inhibit microtubule polymerization, we identified four compounds that have unique effects on microtubules. These compounds cause mixed effects reminiscent of both microtubule depolymerizers and stabilizers. Immunofluorescence evaluations showed that each compound initially caused microtubule depolymerization and, surprisingly, with higher concentrations, microtubule bundles were also observed. There were subtle differences in the propensity to cause these competing effects among the compounds with a continuum of stabilizing and destabilizing effects. Tubulin polymerization experiments confirmed the differential effects and, while each of the compounds increased the initial rate of tubulin polymerization at high concentrations, total tubulin polymer was not enhanced at equilibrium, likely because of the dueling depolymerization effects. Modeling studies predict that the compounds bind to tubulin within the colchicine site and confirm that there are differences in their potential interactions that might underlie their distinct effects on microtubules. Due to their dual properties of microtubule stabilization and destabilization, we propose the name Janus for these compounds after the two-faced Roman god. The identification of synthetically tractable, small molecules that elicit microtubule stabilizing effects is a significant finding with the potential to identify new mechanisms of microtubule stabilization

Janus Compounds, 5-Chloro-N4-methyl-N4-aryl-9H-pyrimido[4,5-b]indole-2,4-diamines, Cause Both Microtubule Depolymerizing and Stabilizing Effects

While evaluating a large library of compounds designed to inhibit microtubule polymerization, we identified four compounds that have unique effects on microtubules. These compounds cause mixed effects reminiscent of both microtubule depolymerizers and stabilizers. Immunofluorescence evaluations showed that each compound initially caused microtubule depolymerization and, surprisingly, with higher concentrations, microtubule bundles were also observed. There were subtle differences in the propensity to cause these competing effects among the compounds with a continuum of stabilizing and destabilizing effects. Tubulin polymerization experiments confirmed the differential effects and, while each of the compounds increased the initial rate of tubulin polymerization at high concentrations, total tubulin polymer was not enhanced at equilibrium, likely because of the dueling depolymerization effects. Modeling studies predict that the compounds bind to tubulin within the colchicine site and confirm that there are differences in their potential interactions that might underlie their distinct effects on microtubules. Due to their dual properties of microtubule stabilization and destabilization, we propose the name Janus for these compounds after the two-faced Roman god. The identification of synthetically tractable, small molecules that elicit microtubule stabilizing effects is a significant finding with the potential to identify new mechanisms of microtubule stabilization

Design, Synthesis, and Preclinical Evaluation of 4‑Substituted-5-methyl-furo[2,3‑<i>d</i>]pyrimidines as Microtubule Targeting Agents That Are Effective against Multidrug Resistant Cancer Cells

The design, synthesis, and biological evaluations of eight 4-substituted 5-methyl-furo­[2,3-<i>d</i>]­pyrimidines are reported. Synthesis involved <i>N</i>⁴-alkylation of <i>N</i>-aryl-5-methylfuro­[2,3-<i>d</i>]­pyrimidin-4-amines, obtained from Ullmann coupling of 4-amino-5-methylfuro­[2,3-<i>d</i>]­pyrimidine and appropriate aryl iodides. Compounds <b>3</b>, <b>4</b>, and <b>9</b> showed potent microtubule depolymerizing activities, while compounds <b>6</b>–<b>8</b> had slightly lower potency. Compounds <b>4</b>, <b>6</b>, <b>7</b>, and <b>9</b> inhibited tubulin assembly with IC₅₀ values comparable to that of combretastatin A-4 (CA-4). Compounds <b>3</b>, <b>4</b>, and <b>6</b>–<b>9</b> circumvented Pgp and βIII-tubulin mediated drug resistance, mechanisms that can limit the efficacy of paclitaxel, docetaxel, and the vinca alkaloids. In the NCI 60-cell line panel, compound <b>3</b> exhibited GI₅₀ values less than 10 nM in 47 of the cell lines. In an MDA-MB-435 xenograft model, compound <b>3</b> had statistically significant antitumor effects. The biological effects of <b>3</b> identify it as a novel, potent microtubule depolymerizing agent with antitumor activity