Potent Tubulin Polymerization Inhibitors Research Articles

3-Vinylazetidin-2-Ones: Synthesis, Antiproliferative and Tubulin Destabilizing Activity in MCF-7 and MDA-MB-231 Breast Cancer Cells.

Microtubule-targeted drugs are essential chemotherapeutic agents for various types of cancer. A series of 3-vinyl-β-lactams (2-azetidinones) were designed, synthesized and evaluated as potential tubulin polymerization inhibitors, and for their antiproliferative effects in breast cancer cells. These compounds showed potent activity in MCF-7 breast cancer cells with an IC50 value of 8 nM for compound 7s 4-[3-Hydroxy-4-methoxyphenyl]-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2-one) which was comparable to the activity of Combretastatin A-4. Compound 7s had minimal cytotoxicity against both non-tumorigenic HEK-293T cells and murine mammary epithelial cells. The compounds inhibited the polymerisation of tubulin in vitro with an 8.7-fold reduction in tubulin polymerization at 10 μM for compound 7s and were shown to interact at the colchicine-binding site on tubulin, resulting in significant G2/M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that β-lactam 7s is targeting tubulin and resulted in mitotic catastrophe. A docking simulation indicated potential binding conformations for the 3-vinyl-β-lactam 7s in the colchicine domain of tubulin. These compounds are promising candidates for development as antiproiferative microtubule-disrupting agents.

Synthesis and Biological Evaluation of RGD⁻Cryptophycin Conjugates for Targeted Drug Delivery.

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvβ3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)–cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvβ3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

Tubulin colchicine site binding agent LL01 displays potent antitumor efficiency both in vitro and in vivo with suitable drug-like properties.

Through rational drug design, we previously identified an indenoprazole derivative, 2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide (LL01), as a potent tubulin polymerization inhibitor targeting the tubulin colchicine binding site. In this study, we further demonstrated that LL01 was not a P-gp substrate. It potently inhibited the growth of a variety of tumor cells, including those with multidrug resistance, with GI50 values in the low nanomole ranges. In vitro liver microsome stability assay, LL01 was modest stable in the liver microsomes of human, mouse and rat, but was fast metabolized in dog. After single oral administration of LL01 at a dose of 10mg/kg in SD male rats, LL01 showed acceptable PK properties with a mean bioavailability of 41%. In human HepG2 hepatoma xenograft, at the oral doses of 25mg/kg/day and 12.5mg/kg/day, LL01 inhibited the tumor growth by 61.27%, and 43.74%, respectively, which is much better than the positive drug sorafenib (29.45%; 30mg/kg/day). Therefore, LL01 might be a potential drug candidate for further investigation for hepatocellular carcinoma therapy.

An orally antitumor chalcone hybrid inhibited HepG2 cells growth and migration as the tubulin binding agent.

Liver cancer is a kind of high mortality cancer due to the difficulty of early diagnosis. It is necessary to develop the anticancer agents to treat liver cancer. Here, a novel chalcone derivative was synthesized and evaluated for anticancer activity in vitro against liver cancer cell lines (HepG2, SNU-423, SMMC7221, and SNU-398). The chalcone hybrid 9 displayed the antiproliferative effect against HepG2, SNU-423, SMMC7221 and SNU-398 cells with IC50 values of 0.9μM, 2.7μM, 6.2μM and 4.6μM, respectively. Cellular mechanisms showed that derivative 9 could obviously inhibit HepG2 cells growth and colony formation in a concentration-dependent manner. Analogue 9 inhibited the migration by regulating the expression levels of migration-releated markers and transcription factors (Snail and Slug). Tubulin polymerization inhibition assay illustrated that chalcone hybrid 9 might be a potent tubulin polymerization inhibitor. Importantly, compound 9 displayed the antitumor activity against liver cancer HepG2 cells in vivo with the low toxicity toward mice. Therefore, compound 9 as a novel tubulin polymerization inhibitor deserves further investigation to treat liver cancer. Graphical abstract Compound 9 displayed the antitumor activity against liver cancer HepG2 cells in vivo and low toxicity toward mice Figure: Orally antitumor chalcone hybrid 9 inhibited HepG2 cells growth and migration as the tubulin binding agent.

3,5-Diaryl-1H-pyrazolo[3,4-b]pyridines as potent tubulin polymerization inhibitors: Rational design, synthesis and biological evaluation

A series of novel 3,5-diaryl-1H-pyrazolo[3,4-b]pyridines as tubulin polymerization inhibitors targeting the colchicine site were designed via ring tethering strategy, which was supported by conformational analysis. The general, chemically unstable and rotational linker, carbanyl group, was locked by 1H-pyrazolo[3,4-b]pyridine to avoid carbonyl reduction and restrict the instability of molecular conformation caused by the rotation of the carbon-carbon single bond beside carbonyl group. All of target compounds were synthesized and evaluated for their antiproliferative activities against three human cancer lines (SGC-7901, A549 and HeLa) by MTT assay. Most of these compounds showed prominent in vitro potency and the most potent compound in this scaffold 13d (SGC-7901: IC50 = 13 nM) could significantly inhibit tubulin polymerization and strongly disrupt cytoskeleton. The results of molecular modeling study revealed that 13d interacts with tubulin by binding to the colchicine site.

Synthesis, biological evaluation and molecular docking of benzimidazole grafted benzsulfamide-containing pyrazole ring derivatives as novel tubulin polymerization inhibitors.

Tubulin-targeting drugs have increasingly become the focus of anticancer drugs research. Twenty-five novel benzimidazole grafted benzsulfamide-containing pyrazole ring derivatives were synthesized and evaluated for bioactivity as potential tubulin polymerization inhibitors. Among them, compound 30 showed the most excellent inhibition against tubulin assembly (IC50 = 1.52 μM) and in vitro growth inhibitory activity against a panel of four human cancer cell lines (IC50 = 0.15, 0.21, 0.33 and 0.17 μM, respectively for A549, Hela, HepG2 and MCF-7). It could also validly induce A549 cell apoptosis, cause cell cycle arrest in G2/M phase and disrupt the cellular microtubule network. These results, along with molecular docking data, provided an important basis for further optimization of compound 30 as a potential anticancer agent.

Efficient synthetic methodology for the construction of dihydronaphthalene and benzosuberene molecular frameworks

Benzosuberene analogues (1 and 2) and dihydronaphthalene analogues (3 and 4) function as potent inhibitors of tubulin polymerization, demonstrate pronounced cytotoxicity (low nM to pM range) against human cancer cell lines, and are promising vascular disrupting agents (VDAs). As such, these compounds represent lead anticancer agents with potential translatability towards the clinic. Methodology previously established by us (and others) facilitated synthetic access to a variety of structural and functional group modifications necessary to explore structure activity relationship considerations directed towards the development of these (and related) molecules as potential therapeutic agents. During the course of these studies it became apparent that the availability of synthetic methodology to facilitate direct conversion of the phenolic-based compounds to their corresponding aniline congeners would be beneficial. Accordingly, modified synthetic routes toward these target phenols (benzosuberene 1 and dihydronaphthalene 3) were developed in order to improve scalability and overall yield [45-57% (1) and 32% (3)]. Moreover, benzosuberene-based phenolic analogue 1 and separately dihydronaphthalene-based phenolic analogue 3 were successfully converted into their corresponding aniline analogues 2 and 4 in good yield (>60% over three steps) using a palladium catalyzed amination reaction.

Synthesis, biological evaluation, and molecular docking investigation of 3-amidoindoles as potent tubulin polymerization inhibitors

A series of novel 3-amidoindole derivatives possessing 3,4,5-trimethoxylphenyl groups were synthesized and evaluated for their antiproliferative and tubulin polymerization inhibitory activities. Some of them demonstrated moderate to potent activities in vitro against six cancer cell lines including MCF-7, MDA-MB-231, BT549, T47D, MDA-MB-468, and HS578T. The most active compound 27 inhibited the growth of T47D, BT549, and MDA-MB-231 cell lines with IC50 values at 0.04, 3.17, and 6.43 μM, respectively. Moreover, the flow cytometric analysis clearly revealed that compound 27 significantly inhibited growth of breast cancer cells through arresting cell cycle in G2/M phase via a concentration-dependent manner. In addition, the compound also exhibited the most potent anti-tubulin activity with IC50 values of 9.5 μM, which was remarkable, compared to CA-4. Furthermore, molecular docking analysis demonstrated the interaction of the compound 27 at the colchicine-binding site of tubulin. These preliminary results suggest that compound 27 is a very promising tubulin-binding agent and is worthy of further investigation aiming to the development of new potential anticancer agents.

Discovery and optimization of 3,4,5-trimethoxyphenyl substituted triazolylthioacetamides as potent tubulin polymerization inhibitors

Based on our previous research, three series of new triazolylthioacetamides possessing 3,4,5-trimethoxyphenyl moiety were synthesized, and evaluated for antiproliferative activities and inhibition of tubulin polymerization. The most promising compounds 8b and 8j demonstrated more significant antiproliferative activities against MCF-7, HeLa, and HT-29 cell lines than our lead compound 6. Moreover, analogues 8f, 8j, and 8o manifested more potent antiproliferative activities against HeLa cell line with IC50 values of 0.04, 0.05 and 0.16 μM, respectively, representing 100-, 82-, and 25-fold improvements of the activity compared to compound 6. Furthermore, the representative compound, 8j, was found to induce significant cell cycle arrest at the G2/M phase in HeLa cell lines via a concentration-dependent manner. Meanwhile, compound 8b exhibited the most potent tubulin polymerization inhibitory activity with an IC50 value of 5.9 μM, which was almost as active as that of CA-4 (IC50 = 4.2 μM). Additionally, molecular docking analysis suggested that 8b formed stable interactions in the colchicine-binding site of tubulin.

Improved Methodology for the Synthesis of a Cathepsin B Cleavable Dipeptide Linker, Widely Used in Antibody-Drug Conjugate Research

Antibody-drug conjugates (ADCs) represent an emerging class of biopharmaceutical agents that deliver highly potent anticancer agents (payloads) selectively to tumors or components associated with the tumor microenvironment. The linker, responsible for the connection between the antibody and payload, is a crucial component of ADCs. In certain examples the linker is composed of a cleavable short peptide which imparts an additional aspect of selectivity. Especially prevalent is the cathepsin B cleavable Mc-Val-Cit-PABOH linker utilized in many pre-clinical ADC candidates, as well as the FDA approved ADC ADCETRIS® (brentuximab vedotin). An alternative route for the synthesis of the cathepsin B cleavable Mc-Val-Cit-PABOH linker is reported herein that involved six steps from l-Citrulline and proceeded with a 50% overall yield. In this modified route, the spacer (a para-aminobenzyl alcohol moiety) was incorporated via HATU coupling followed by dipeptide formation. Importantly, this route avoided undesirable epimerization and proceeded with improved overall yield. Utilizing this methodology, a drug-linker construct incorporating a potent small-molecule inhibitor of tubulin polymerization (referred to as KGP05), was synthesized as a representative example.

Design, synthesis and cytotoxicity evaluation of indibulin analogs

Abstract Indibulin is one of the most potent tubulin polymerization inhibitors with minimal peripheral neuropathy. The design and synthesis of new indibulin analogs were carried out in order to investigate their anti-cancer activity. The target compounds 4a–i were synthesized in multistep reactions starting with the related indole derivatives. Compound 4f shows the highest cytotoxic activity on HT-29 and Caco-2 cell lines with the respective half maximal inhibitory concentration (IC50) values of 5.1 μm and 7.3 μm. In the case of the T47-D cell line, compound 4c exerts the best cytotoxic activity with an IC50 value of 11.5 μm. In the cell cycle analysis on HT-29 cells, compound 4f at 5.1 μm showed an increase in the percentage of cells in the sub-G1 phase. Altogether, nine target compounds were synthesized and characterized by infrared spectroscopy (IR), proton nuclear magnetic resonance (1H NMR), carbon-13 nuclear magnetic resonance (13C NMR), mass spectrometry (MS) and elemental analysis. Some of the compounds show good cytotoxic activity against cancerous cell lines.

Design, synthesis and antiproliferative evaluation of novel sulfanilamide-1,2,3-triazole derivatives as tubulin polymerization inhibitors.

Microtubule as an important target in the cancer therapy was used to design novel tubulin polymerization inhibitors. Sulfanilamide-1,2,3-triazole hybrids were designed by a molecular hybridization strategy and their antiproliferative activity against three selected cancer cell lines (BGC-823, MGC-803 and SGC-7901) were evaluated. All sulfanilamide-1,2,3-triazole hybrids displayed potent inhibitory activity against all cell lines. In particular, compound 10b showed the most excellent inhibitory effect against MGC-803 cells, with an IC50 value of 0.4 μM. Cellular mechanism studies elucidated that 10b induced apoptosis by decreasing the expression level of Bcl-2 and Parp and increasing the expression level of BAX. 10b inhibited the epithelial-mesenchymal transition process by up-regulating E-cadherin and down-regulating N-cadherin. Furthermore, the tubulin polymerization inhibitory activity in vitro of 10b was 2.4 μM. In vivo anticancer assay, 10b effectively inhibited MGC-803 xenograft tumor growth without causing significant loss of body weight. These sulfanilamide-1,2,3-triazole hybrids as potent tubulin polymerization inhibitors might be used as promising candidates for cancer therapy.

Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4.

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes ( isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

Synthesis, Evaluation of Cytotoxicity and Molecular Docking Studies of the 7-Acetamido Substituted 2-Aryl-5-bromo-3-trifluoroacetylindoles as Potential Inhibitors of Tubulin Polymerization.

The 3-trifluoroacetyl–substituted 7-acetamido-2-aryl-5-bromoindoles 5a–h were prepared and evaluated for potential antigrowth effect in vitro against human lung cancer (A549) and cervical cancer (HeLa) cells and for the potential to inhibit tubulin polymerization. The corresponding intermediates, namely, the 3-unsubstituted 7-acetyl-2-aryl-5-bromoindole 2a–d and 7-acetamido-2-aryl-5-bromoindole 4a–d were included in the assays in order to correlate both structural variations and cytotoxicity. No cytotoxicity was observed for compounds 2a–d and their 3-trifluoroacetyl–substituted derivatives 5a–d against both cell lines. The 7-acetamido derivatives 4–d exhibited modest cytotoxicity against both cell lines. All of the 3-trifluoroacetyl–substituted 7-acetamido-2-aryl-5-bromoindoles 5e–h were found to be more active against both cell lines when compared to the chemotherapeutic drug, Melphalan. The most active compound, 5g, induced programmed cell death (apoptosis) in a caspase-dependent manner for both A549 and HeLa cells. Compounds 5e–h were found to significantly inhibit tubulin polymerization against indole-3-carbinol and colchicine as reference standards. Molecular docking of 5g into the colchicine-binding site suggests that the compounds bind to tubulin by different type of interactions including pi-alkyl, amide-pi stacked and alkyl interactions as well as hydrogen bonding with the protein residues to elicit anticancer activity.

New 6- and 7-heterocyclyl-1H-indole derivatives as potent tubulin assembly and cancer cell growth inhibitors

We designed new 3-arylthio- and 3-aroyl-1H-indole derivatives 3–22 bearing a heterocyclic ring at position 5, 6 or 7 of the indole nucleus. The 6- and 7-heterocyclyl-1H-indoles showed potent inhibition of tubulin polymerization, binding of colchicine to tubulin and growth of MCF-7 cancer cells. Compounds 13 and 19 inhibited a panel of cancer cells and the NCI/ADR-RES multidrug resistant cell line at low nanomolar concentrations. Compound 13 at 50 nM induced 77% G2/M in HeLa cells, and at 20 nM caused 50% stable arrest of mitosis. As an inhibitor of HepG2 cells (IC50 = 20 nM), 13 was 4-fold superior to 19. Compound 13 was a potent inhibitor of the human U87MG glioblastoma cells at nanomolar concentrations, being nearly one order of magnitude superior to previously reported arylthioindoles. The present results highlight 13 as a robust scaffold for the design of new anticancer agents.

Mechanism of action of the third generation benzopyrans and evaluation of their broad anti-cancer activity in vitro and in vivo

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.

Drug-Clinical Agent Molecular Hybrid: Synthesis of Diaryl(trifluoromethyl)pyrazoles as Tubulin Targeting Anticancer Agents.

Twenty-three combretastatin A-4 (CA-4) analogues were synthesized by judiciously incorporating a functional N-heterocyclic motif present in Celecoxib (a marketed drug) while retaining essential pharmacophoric features of CA-4. Combretastatin-(trifluoromethyl)pyrazole hybrid analogues, i.e., 5-trimethoxyphenyl-3-(trifluoromethyl)pyrazoles with a variety of relevantly substituted aryls and heteroaryls at 1-position were considered as potential tubulin polymerization inhibitors. The cytotoxicity of the compounds was evaluated using MCF-7 cells. Analog 23 (C-23) was found to be the most active among the tested compounds. It showed pronounced cytotoxicity against HeLa, B16F10, and multidrug-resistant mammary tumor cells EMT6/AR1. Interestingly, C-23 displayed significantly lower toxicity toward noncancerous cells, MCF10A and L929, than their cancerous counterparts, MCF-7 and B16F10, respectively. C-23 depolymerized interphase microtubules, disrupted mitotic spindle formation, and arrested MCF-7 cells at mitosis, leading to cell death. C-23 inhibited the assembly of tubulin in vitro. C-23 bound to tubulin at the colchicine binding site and altered the secondary structures of tubulin. The data revealed the importance of (trimethoxyphenyl)(trifluoromethyl)pyrazole as a cis-restricted double bond-alternative bridging motif, and carboxymethyl-substituted phenyl as ring B for activities and interaction with tubulin. The results indicated that the combretastatin-(trifluoromethyl)pyrazole hybrid class of analogues has the potential for further development as anticancer agents.

Synthesis of dihydronaphthalene analogues inspired by combretastatin A-4 and their biological evaluation as anticancer agents.

The natural products colchicine and combretastatin A-4 (CA4) have provided inspiration for the discovery and development of a wide array of derivatives and analogues that inhibit tubulin polymerization through a binding interaction at the colchicine site on β-tubulin. A water-soluble phosphate prodrug salt of CA4 (referred to as CA4P) has demonstrated the ability to selectively damage tumor-associated vasculature and ushered in a new class of developmental anticancer agents known as vascular disrupting agents (VDAs). Through a long-term program of structure activity relationship (SAR) driven inquiry, we discovered that the dihydronaphthalene molecular scaffold provided access to small-molecule inhibitors of tubulin polymerization. In particular, a dihydronaphthalene analogue bearing a pendant trimethoxy aryl ring (referred to as KGP03) and a similar aroyl ring (referred to as KGP413) were potent inhibitors of tubulin polymerization (IC50 = 1.0 and 1.2 μM, respectively) and displayed low nM cytotoxicity against human cancer cell lines. In order to enhance water-solubility for in vivo evaluation, the corresponding phosphate prodrug salts (KGP04 and KGP152, respectively) were synthesized. In a preliminary in vivo study in a SCID-BALB/c mouse model bearing the human breast tumor MDA-MB-231-luc, a 99% reduction in signal was observed with bioluminescence imaging (BLI) 4 h after IP administration of KGP152 (200 mg kg-1) indicating reduced tumor blood flow. In a separate study, disruption of tumor-associated blood flow in a Fischer rat bearing an A549-luc human lung tumor was observed by color Doppler ultrasound following administration of KGP04 (15 mg kg-1).

Design, synthesis and anti-cancer evaluation of novel podophyllotoxin derivatives as potent tubulin-targeting agents

A series of podophyllotoxin derivatives (M1–M16) that were selectively acylated by various phenoxy acids at the C-4 position of podophyllotoxin were synthesized, and their biological activities were also evaluated. Among them, compound M4 showed the most potent anti-cancer activity against HeLa cells with an IC50 value of 1.64 ± 0.41 μM. Additionally, flow cytometry analysis results indicated that it could cause a remarkable cell cycle arrest at G2/M phase, but the effect on apoptosis inducing was not significant. Moreover, the expression of cell cycle relative protein CDK1 was up regulated while cyclin B1 and Cdc25C, two proteins required for mitotic initiation were down regulated. Furthermore, the confocal assay and extracellular polymerized tubulin expression analysis also demonstrated that M4 was a potent tubulin polymerization inhibitor and the effect was comparable to that of colchicine. Finally, docking simulation results showed that M4 could nicely bind to the colchicine binding site of tubulin which further comfirmed the tubulin inhibiton activity of M4.

Computational design and synthesis of novel fluoro-analogs of combretastatins A-4 and A-1

Combretastatin A-1 (CA-1) and combretastatin A-4 (CA-4) isolated from the African bush willow Combretum caffrum are highly potent tubulin polymerization inhibitors, possessing strong antitumor activities because of their vascular disrupting properties. Extensive SAR studies have been done for CA-4 analogs. Because of poor solubility, water-soluble prodrugs of CA-4 and CA-1 have been developed, which are currently in human clinical trials. Fluorine plays an important role in the current drug discovery and development due to its unique properties. Thus, several fluorine-containing analogs of CA-4/CA-1 have been studied. However, no analogs, which have a CF3O-, CF2HO- or CF3- group instead of the 4′-methoxy group in the B ring, have been investigated. Therefore, we set out to design and synthesize those novel fluoro-analogs of CA-4/CA-1. For the design of the new analogs, we took a structure-based design approach based on the X-ray crystal structure of colchicine-tubulin complex (PDB: 4O2B) and computational docking analysis using the AutoDock Vina program. A library of novel fluoro-analogs of CA-4/CA-1 was generated and their docking energy scores obtained. It was found that those novel fluoro-analogs exhibited better docking energy scores than CA-4/CA-1. Also, docking poses of all of these fluoro-analogs were virtually superimposable and very good fit to the colchine binding site. Among 15 compounds designed and analyzed, we have synthesized 5 compounds and evaluated their cytotoxicity against drug-sensitive and multidrug-resistant cancer cell lines. All fluoro-analogs exhibited strong cytotoxicity even against multidrug-resistant cell line. However, the critical activity of this class of compounds is its vascular disrupting activity. Thus, further biological evaluations are warranted for those novel fluoro-analogs of CA-4/CA-1.