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According to the statistics from American Cancer Society, the 5-year survival rate for patients with advanced melanoma is as low as 5%. Treatment of advanced melanoma, therefore, represents an unmet medical need. In this dissertation, I will show the effort to develop new generations of bioavailable tubulin inhibitors targeting the colchicine binding site and selective small-molecule survivin inhibitors for treating advanced melanoma. Extensive structure-activity relationship (SAR) studies of lead molecules ABI-231 and UC-112 have been performed. Chapter 1 will introduce the current situation of advanced or metastatic melanoma, its clinical drug treatments, as well as problems in current drug treatments. Microtubule dynamics and survivin will be discussed as promising therapeutic targets for developing anticancer drugs. 20S-hydroxyvitamin D3 (20S-OH-D3) will be introduced as a promising anti-inflammatory scaffold. Chapter 2 will disclose the SAR study of ABI-231, a previously reported potent tubulin inhibitor from our lab. In this chapter, a new synthetic method was developed to enable the synthesis of ABI-231 analogues modifying the indole moiety. The novel synthetic method involved the synthesis of a key diamine intermediate and imidazoline formation. From the new synthetic method, thirty ABI-231 analogues were synthesized and tested for activities. Among all analogues, 10ab with a 4-methyl-3-indole moiety and 10bb with a 4-indole moiety showed the most potent antiproliferative activities against a panel of melanoma cell lines. 10ab and 10bb had IC50s of 2.2 and 3.0 nM, respectively. The SAR result revealed that modification of the indole moiety in ABI-231 was beneficial to activity. In Chapter 3, we will describe our effort to develop the SAR study of ABI-231 focusing on modification of the 3,4,5-TMP moiety. This is selected since it is one of the most common moieties in current tubulin inhibitors targeting the colchicine binding site. To circumvent the use of potentially explosive azide reported in Chapter 2, an alternative was established to efficiently generate ABI-231 analogues. This new synthetic method involved Suzuki coupling and Grignard reactions to modify the 3,4,5-TMP moiety and to produce target compounds in gram-scale. Among the eight analogues synthesized, the one containing an unique 3-methoxybenzo[4,5]-dioxene moiety had the strongest antiproliferative activity against a panel of melanoma cell lines with an average IC50 of 1.9 nM. To our best knowledge, it represents the most successful instance of isosterically modifying the 3,4,5-TMP moiety in CA-4 derivatives. Chapter 4 will highlight our effort to synthesize reverse ABI (RABI) analogues for SAR study. In this chapter, a novel and concise synthetic route was established toaccess RABI scaffold. RABI scaffold was constructed through a Grignard reaction/Suzuki-Miyaura coupling reaction strategy. From this new synthetic method, twelve novel RABI analogues were synthesized. Compared to MX-RABI (the previously reported most potent RABI), several new RABI analogues showed significantly improved cytotoxicities. In particular, analogue 15i with a 4-indazole moiety showed the most potent antiproliferative activity against a panel of melanoma cell lines and had an average IC50 of 0.8 nM. This is the first sub-nM anti-tubulin compound in the related scaffolds. Chapter 5 will reveal our latest SAR study of UC-112, a previously reported selective survivin inhibitor. Fourteen UC-112 analogues modifying the benzyloxy moiety of UC-112 were synthesized. Their corresponding SAR result demonstrated that indole moiety was the most favorable (analogue 12a). Subsequent structural optimization of 12a by introducing either mono-substituent or di-substituent to the indole moiety led to the synthesis of another twenty-four new UC-112 analogues. Several substituted indole analogues showed equipotency to that of UC-112 and MX-106. Importantly, new indole analogues exhibited significant abilities to overcome multidrug-resistance mediated by Pgp overexpression. Chapter 6 is characterized by the establishment of a total synthetic method of 20SOH-D3 which showed comparable antiproliferative activity to 1,25α-dihydroxyvitamin D3 without hypercalcemic toxic effect upto a concentration of 60 μg/kg in vivo. The total synthesis of 20S-OH-D3 involved parallel generation of key intermediates from ergocalciferol. The vitamin D3 core structure was constructed through Wittig-Horner coupling reaction. Deprotection of SEM and TBS was achieved in one step. 20S-OH-D3 was furnished in sixteen steps with an overall yield of 0.4%.

The first part (Chapter 1 and 2) of this dissertation presents a novel combination study of melanoma therapy. Acquired clinical resistance to vemurafenib, a selective BRAFV600E inhibitor, arises frequently after short term chemotherapy. Since the inhibitions of targets in the RAFMEK-ERK pathway result in G0/G1 cell cycle arrest, vemurafenib-resistant cancer cells are expected to escape this cell cycle arrest and progress to subsequent G2/M phase. We hypothesized that a combined therapy using vemurafenib with a G2/M phase blocking agent will trap resistant cells and overcome vemurafenib resistance. To test this hypothesis, we first determined the combination index (CI) values of our novel tubulin inhibitor ABI-274 and vemurafenib on parental human A375 and MDA-MB-435 melanoma cell lines to be 0.32 and 0.1, respectively, suggesting strong synergy for the combination. We then developed an A375RF21 subline with significant acquired resistance to vemurafenib and confirmed the strong synergistic effect. Next we studied the potential mechanisms of overcoming vemurafenib resistance. Flow cytometry confirmed that the combination of ABI-274 and vemurafenib synergistically arrested cells in G1/G2/M phase, and significantly increased apoptosis in both parental A375 and the vemurafenib-resistant A375RF21 cells. Western blot analysis revealed that the combination treatment effectively reduced the level of phosphorylated and total AKT, activated the apoptosis cascade, and increased cleaved caspase-3 and cleaved PARP, but had no significant influence on the level of ERK phosphorylation. Finally, in vivo co-administration of vemurafenib with ABI-274 showed strong synergistic efficacy in the vemurafenib-resistant xenograft model in nude mice. Overall, these results offer a rational combination strategy to significantly enhance the therapeutic benefit in melanoma patients who inevitably become resistant to current BRAF inhibition therapy. The second part (Chapter 3 to 5) of this dissertation focuses on the discovery of a series of small molecule survivin inhibitors. Inhibitors of apoptosis (IAP) proteins are widely considered as promising cancer drug targets, especially for drug-resistant tumors. Mimicking the IAP-binding motif of second mitochondria-derived activator of caspases (Smac) is a rational strategy to design potential IAP inhibitors. In this report, we used the bioactive conformation of AVPI tetrapeptide in the N-terminus of Smac as a template and performed a shape-based virtual screening against a drug-like compound library to identify novel IAP inhibitors. Top hits were subsequently docked to available IAP crystal structures as a secondary screening followed by validation using in vitro biological assays. Four novel hit compounds were identified that potently inhibited cell growth in two human melanoma (A375 and M14) and two human prostate (PC-3 and DU145) cancer cell lines. The best compound, UC-112, has IC50 values ranging from 0.7 to 3.4 µM. UC-112 also potently inhibits the growth of P-glycoprotein (Pgp) overexpressed multidrug-resistant cancer cells, strongly activates capase-3/7 and caspase-9 activities, and selectively down-regulates survivin level at a concentration as low as 1 µM. Co-incubation of UC-112 with a known proteasome inhibitor (MG132) rescued survivin inhibition, consistent with the anticipated mechanism of action for UC-112. As a single agent, UC-112 strongly inhibits tumor growth and reduces both XIAP and survivin levels in an A375 human melanoma xenograft model in vivo. Three analogs generated from UC-112 structural modification along with template compound UC-112 were submitted to NCI-60 cancer cell line screening. The results indicated that structural modification of UC-112 to give our best compound MX106 has improved activity by four fold (2.2 µM for UC-112 vs. 0.5 µM for MX106, average GI50 values over all cancer cell lines in the NCI-60 panel).Western blot analyses demonstrated the new compounds maintained high selectivity for survivin inhibition over other members in the inhibitiors of apoptosis protein family. When tested in an A375 human melanoma xenograft model, the most active compound MX106 effectively suppressed tumor growth and strongly induced cancer cell apoptosis in tumor tissues. Taken together, this novel scaffold is promising for the development of selective survivin inhibitors as potential anticancer agents.