Chien-Ming Li

Discovery of 4-Substituted Methoxybenzoyl-Aryl-Thiazole as Novel Anticancer Agents: Synthesis, Biological Evaluation and Structure-Activity Relationships

A series of 4-substituted methoxybenzoyl-aryl-thiazoles (SMART) have been discovered and synthesized as a result of structural modifications of the lead compound 2-arylthiazolidine-4-carboxylic acid amides (ATCAA). The antiproliferative activity of the SMART agents against melanoma and prostate cancer cells was improved from muM to low nM range compared with the ATCAA series. The structure-activity relationship was discussed from modifications of A, B, and C rings and the linker. Preliminary mechanism of action studies indicated that these compounds exert their anticancer activity through inhibition of tubulin polymerization.

Discovery of novel 2-aryl-4-benzoyl-imidazoles targeting the colchicines binding site in tubulin as potential anticancer agents.

A series of 2-aryl-4-benzoyl-imidazoles (ABI) was synthesized as a result of structural modifications based on the previous set of 2-aryl-imidazole-4-carboxylic amide (AICA) derivatives and 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART). The average IC(50) of the most active compound (5da) was 15.7 nM. ABI analogues have substantially improved aqueous solubility (48.9 μg/mL for 5ga vs 0.909 μg/mL for SMART-1, 0.137 μg/mL for paclitaxel, and 1.04 μg/mL for combretastatin A4). Mechanism of action studies indicate that the anticancer activity of ABI analogues is through inhibition of tubulin polymerization by interacting with the colchicine binding site. Unlike paclitaxel and colchicine, the ABI compounds were equally potent against multidrug resistant cancer cells and the sensitive parental melanoma cancer cells. In vivo results indicated that 5cb was more effective than DTIC in inhibiting melanoma xenograph tumor growth. Our results suggest that the novel ABI compounds may be developed to effectively treat drug-resistant tumors.

Biological Activity of 4-Substituted Methoxybenzoyl- Aryl-Thiazole: An Active Microtubule Inhibitor

Formation of microtubules is a dynamic process that involves polymerization and depolymerization of αβ-tubulin heterodimers. Drugs that enhance or inhibit tubulin polymerization can destroy this dynamic process, arresting cells in the G(2)/M phase of the cell cycle. Although drugs that target tubulin generally demonstrate cytotoxic potency in the subnanomolar range, resistance due to drug efflux is a common phenomenon among the antitubulin agents. We recently reported a class of 4-substituted methoxybenzoyl-aryl-thiazoles (SMART) that exhibited great in vitro potency and broad spectrum cellular cytotoxicity. Evaluation of the in vitro and in vivo anticancer activities of 3 SMART compounds, SMART-H (H), SMART-F (F), and SMART-OH (OH), with varying substituents at the 4-position of aryl ring, demonstrated that they bind potently to the colchicine-binding site in tubulin, inhibit tubulin polymerization, arrest cancer cells in G(2)/M phase of the cell cycle, and induce their apoptosis. The SMART compounds also equipotently inhibit the growth of parental and MDR-overexpressing cells in vitro, indicating that they can overcome multidrug resistance. In vivo antitumor efficacy studies in human prostate (PC-3) and melanoma (A375) cancer xenograft models demonstrated that SMART-H and SMART-F treatments resulted in %T/C values ranging from 4% to 30%. In addition, in vivo SMART-H treatment for 21 days at the higher dose (15 mg/kg) failed to produce any apparent neurotoxicity. These studies provide the first in vivo evidence and proof-of-concept that SMART compounds are similarly efficacious to currently FDA approved antitubulin drugs for cancer treatment, but they can circumvent P-glycoprotein-mediated drug resistance.

Synthesis, in vitro structure-activity relationship, and in vivo studies of 2-arylthiazolidine-4-carboxylic acid amides as anticancer agents.

A series of (2RS,4R)-2-arylthiazolidine-4-carboxylic acid amide (ATCAA) was synthesized. Antiproliferative activity against melanoma and prostate cancer cells compared with control cells (fibroblast and RH7777, respectively) was evaluated. Compound 3id showed the best selectivity and growth-inhibition activity against three melanoma cell lines (B16-F1, A375, and WM-164). Compounds 15b and 3ac had good selectivity and potency against four prostate cancer cell lines (DU 145, PC-3, LNCaP, and PPC-1). The structure-activity relationship (SAR) of the side chain, the thiazolidine ring, and phenyl substituents is discussed. Cell cycle analysis showed that the percentage of cancer cells undergoing apoptosis (sub-G1 phase) increased after treatment with 1b and 3ad, which also strongly inhibited melanoma colony formation. In vivo studies on nude mice bearing A375 melanoma tumors showed that compound 1b inhibited tumor growth in a dose-dependent manner. At a dose of 10mg/kg, 1b significantly inhibited melanoma tumor growth and showed higher efficacy than did dacarbazine at 60mg/kg.

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non-radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine-, vinblastine- and paclitaxel-binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin-binding site of 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART-H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART-H specifically and reversibly bound only to the colchicine-binding site, but not to vinblastine- or paclitaxel sites. This new non-radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors.

I-387, a Novel Antimitotic Indole, Displays a Potent In vitro and In vivo Antitumor Activity with Less Neurotoxicity

(3-(1H-indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel synthetic compound that inhibits tubulin action and exhibits potent antitumor activity in various preclinical models. I-387 inhibited the in vitro growth of several human cancer cell lines with IC50 values in the range of 15 to 39 nmol/L. Nanomolar concentrations of the compound induced apoptosis and caused phosphorylation of the antiapoptotic protein Bcl-2. I-387 induced a strong and concentration-dependent G2-M arrest in PC-3 cells by constitutive activation of Cdc2/cyclin B1 complex and destabilized polymerization of purified tubulin in vitro by binding to the colchicine-binding site. In vivo, I-387 treatment effectively inhibited tumor growth in mice bearing PC-3 tumor xenografts. In vitro studies of nerve growth factor–dependent neurite outgrowth in PC12 pheochromocytoma cells and in vivo studies of mouse behavior showed that I-387 was less neurotoxic than vinblastine and vincristine, tubulin destabilizers with known neurotoxicity. Interestingly, multidrug-resistant cell lines that overexpressed P-glycoprotein (P-gp), multidrug resistance–associated proteins, and breast cancer resistance protein were rendered resistant to docetaxel, vinblastine, SN-38, and doxorubicin, but not to I-387. I-387 dosed at 10 mg/kg was equally effective with 76% tumor growth inhibition in xenograft models using MES-SA uterine sarcoma cells and MES-SA/DX5 cells overexpressing P-gp. In contrast, docetaxel and vinblastine were not effective in MES-SA/DX5 xenograft models. The potent in vitro and in vivo antitumor activity of I-387 suggests that it may represent a new antimitotic agent for management of various malignancies, particularly for patients with drug-resistant cancer. Mol Cancer Ther; 9(11); 2859–68. ©2010 AACR.

Drug Metabolism and Pharmacokinetics of 4-Substituted Methoxybenzoyl-aryl-thiazoles

Tubulins are some of the oldest and most extensively studied therapeutic targets for cancer. Although many tubulin polymerizing and depolymerizing agents are known, the search for improved agents continues. We screened a class of tubulins targeting small molecules and identified 4-(3,4,5-trimethoxybenzoyl)-2-phenyl-thiazole (SMART-H) as our lead compound. SMART-H inhibited the proliferation of a variety of cancer cells in vitro, at subnanomolar IC50, and in vivo, in nude mice xenografts, with near 100% tumor growth inhibition. Metabolic stability studies with SMART-H in liver microsomes of four species (mouse, rat, dog, and human) revealed half-lives between <5 and 30 min, demonstrating an interspecies variability. The clearance predicted based on in vitro data correlated well with in vivo clearance obtained from mouse, rat, and dog in vivo pharmacokinetic studies. SMART-H underwent four major metabolic processes, including ketone reduction, demethylation, combination of ketone reduction and demethylation, and hydroxylation in human liver microsomes. Metabolite identification studies revealed that the ketone and the methoxy groups of SMART-H were most labile and that ketone reduction was the dominant metabolism reaction in human liver microsomes. We designed and tested four derivatives of SMART-H to improve the metabolic stability. The oxime and hydrazide derivatives, replacing the ketone site, demonstrated a 2- to 3-fold improved half-life in human liver microsomes, indicating that our prediction regarding metabolic stability of SMART-H can be extended by blocking ketone reduction. These studies led us to the next generation of SMART compounds with greater metabolic stability and higher pharmacologic potency.

Abstract 730: Targeting colchicine binding site in tubulin for the treatment of advanced melanoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnInhibition of tubulin dynamics has been a fruitful drug target for cancer treatment. Out the three ligand binding sites in tubulin, drugs targeting taxane binding site (e.g. taxol and epithilones) and vinca alkaloids binding site (e.g. vinblastine) are clinically very successful drugs. However, no FDA-approved cancer drugs targeting the colchicine binding site in tubulin currently exist. In our ongoing efforts to search for effective drugs for the treatment of advanced melanoma that are highly resistant to all existing cancer treatment, we have discovered a new class of compounds that are not substrates of P-glycoprotein (Pgp) and are highly efficacious against human melanoma tumors in A375 xenograft models. At relatively low doses (5∼15 mg/kg), these compounds inhibit melanoma tumor growth more effectively than DTIC (the gold standard drug for advanced melanoma) at 60 mg/kg. Extensive biological and molecular modeling studies suggest that these compounds work by competitively binding to the colchicine site in tubulin. Further optimization of these compounds hold great promise in providing an effective agent for the treatment of advanced melanoma.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 730.