Potent Tubulin Polymerization Inhibitors Research Articles

Design, synthesis and anti-cancer activity evaluation of podophyllotoxin-norcantharidin hybrid drugs

In this study, we designed and synthesized eighteen podophyllotoxin-norcantharidin hybrid drugs which could exhibit more potent anti-cancer activity than the parent drugs. Through the anti-proliferation assay, the most potent anti-cancer agent was screened out, namely Q9 (IC50=0.88±0.18μM against MCF-7 cell line), and it showed lower cytotoxicity against non-cancer cells, human embryonic kidney cells (293T) (IC50=54.38±3.78μM). Additionally, based on the flow cytometry analysis result, it can cause a remarkable cell cycle arrest at G2/M phase and induce apoptosis in MCF-7 cells more significantly than podophyllotoxin or norcantharidin per se. Moreover, the expression of cell cycle relative protein CDK1 was up regulated while a protein required for mitotic initiation, Cyclin B1 was down regulated. Furthermore, according to the confocal microscopy observation results, it was shown that Q9 was a potent tubulin polymerization inhibitor and the effect is comparable to that of colchicine. For further investigation on the aforementioned mechanisms, we performed western blot experiments, thus finding the increase of the cleavage of PARP. Consistent with these new findings, molecular docking observations suggested that compound Q9 could be developed as a potential anticancer agent.

Design, Synthesis, and Biological Evaluation of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazole Analogues as Potential Anticancer Agents Targeting Tubulin Colchicine Binding Site.

By targeting a new binding region at the interface between αβ-tubulin heterodimers at the colchicine binding site, we designed a series of 7-substituted 1-methyl-1,4-dihydroindeno[1,2-c]pyrazoles as potential tubulin polymerization inhibitors. Among the compounds synthesized, 2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide 6a and 2-(6-ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yloxy)-N-hydroxyacetamide 6n showed noteworthy low nanomolar potency against HepG2, Hela, PC3, and MCF-7 cancer cell lines. In mechanism studies, 6a inhibited tubulin polymerization and disorganized microtubule in A549 cells by binding to tubulin colchicine binding site. 6a arrested A549 cells in G2/M phase that was related to the alterations in the expression of cyclin B1 and p-cdc2. 6a induced A549 cells apoptosis through the activation of caspase-3 and PARP. In addition, 6a inhibited capillary tube formation in a concentration-dependent manner. In nonsmall cell lung cancer xenografts mouse model, 6a suppressed tumor growth by 59.1% at a dose of 50 mg/kg (ip) without obvious toxicity, indicating its in vivo potential as anticancer agent.

Synthesis and Biological Evaluation of 1-Methyl-1H-indole-Pyrazoline Hybrids as Potential Tubulin Polymerization Inhibitors.

A series of 1-methyl-1H-indole-pyrazoline hybrids were designed, synthesized, and biologically evaluated as potential tubulin polymerization inhibitors. Among them, compound e19 [5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide] showed the most potent inhibitory effect on tubulin assembly (IC50 =2.12 μm) and in vitro growth inhibitory activity against a panel of four human cancer cell lines (IC50 values of 0.21-0.31 μm). Further studies confirmed that compound e19 can induce HeLa cell apoptosis, cause cell-cycle arrest in G2 /M phase, and disrupt the cellular microtubule network. These studies, along with molecular docking and 3D-QSAR modeling, provide an important basis for further optimization of compound e19 as a potential anticancer agent.

Indole based Tubulin Polymerization Inhibitors: An Update on Recent Developments

The exploration of cancer microenvironment and its physiology have exposed a number of potential molecular targets for selective therapeutic intervention by anti-cancer agents. Microtubules are basic cell components formed by polymerization of αβ heterodimers which play a pivotal role in cellular functions as well as cell division. Drugs that can control the microtubule assembly either by hindering tubulin polymerization or by obstructing microtubule disassembly are of great importance in anti-cancer therapy. Diverse classes of naturally occurring as well as synthetic and semi-synthetic compounds with an indole nucleus induce microtubule polymerization and depolymerization and thereby change tubulin dynamics. Rapid development of several novel tubulin polymerization inhibitors has been observed over the past few years and some of them have associated vascular disrupting properties too. The present review starts with the structure, function and importance of microtubules in a eukaryotic cell. The well characterized tubulin binding domains and the corresponding inhibitors including their mechanism of action is also a part of this article. The report mainly focuses on the brief synthetic methodology with the relevant SAR studies of different indole derived molecules that have been reported in the past few years as potential inhibitors of tubulin polymerization is discussed. This review will provide the up-to-date evidence-base for synthetic chemists as well as biologists to design and synthesize new active molecules to inhibit tubulin polymerization.

Synthesis and biological evaluation of quinoline analogues of flavones as potential anticancer agents and tubulin polymerization inhibitors

A new series of 2-aryl-trimethoxyquinoline analogues was designed and synthesized as tubulin inhibitors using methoxylated flavones as the lead compounds. The cytotoxic activity of the synthesized compounds was evaluated against four human cancer cell lines including MCF-7, MCF-7/MX, A-2780, and A-2780/RCIS. All the alcoholic derivatives (6a–6e) showed significant cytotoxic activity with IC50 in the range of 7.98–60 μM. The flow cytometry analysis of the four human cancer cell lines treated with 6e and 5b showed that 6e induced cell cycle arrest at G2/M phase and apoptosis as well. The effect of quinolines on tubulin polymerization was also evaluated. Compound 6e that demonstrated the best antiproliferative activity in the series was identified as the most potent inhibitor of tubulin polymerization as well. Molecular docking studies of 6e into the colchicine-binding site of tubulin displayed possible mode of interaction between this compound and tubulin.

Combretastatin linked 1,3,4-oxadiazole conjugates as a Potent Tubulin Polymerization inhibitors

A new class of combretastatin linked 1,3,4-oxadiazoles were designed, synthesized and screened for their cytotoxic activity against five human cancer cell lines such as HeLa, DU-145, A549, MDA-MB-231 and B16. These compounds showed significant cytotoxicity with IC50 values in the range 0.118–54.32μM. Conjugate 5m displayed potent antiproliferative activity against DU-145 cell line. Flow cytometric analysis revealed that these compounds arrested the cell cycle in G2/M phase. Moreover, the tubulin polymerization assay and immunofluorescence analysis indicate that 5m exhibits potent inhibitory effect on the tubulin assembly. Further, DNA fragmentation and Hoecst staining assays confirm that 5m induces apoptosis. Molecular docking studies and competitive binding assay indicated that 5m effectively bind at the colchicine binding site of the tubulin.

Antiproliferative and proapoptotic effects of a pyrrole containing arylthioindole in human Jurkat leukemia cell line and multidrug-resistant Jurkat/A4 cells

Recently, a series of novel arylthioindole compounds, potent inhibitors of tubulin polymerization and cancer cell growth, were synthesized. In the present study the effects of 2-(1H-pyrrol-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (ATI5 compound) on cell proliferation, cell cycle progression, and induction of apoptosis in human T-cell acute leukemia Jurkat cells and their multidrug resistant Jurkat/A4 subline were investigated. Treatment of the Jurkat cells with the ATI5 compound for 48 hrs resulted in a strong G2/M cell cycle arrest and p53-independent apoptotic cell death accompanied by the induction of the active form of caspase-3 and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage. ATI5 treatment also caused non-cell death related mitotic arrest in multidrug resistant Jurkat/A4 cells after 48 hrs of treatment suggesting promising opportunities for the further design of pyrrole-containing ATI compounds as anticancer agents. Cell death resistance of Jurkat/A4 cells to ATI5 compound was associated with alterations in the expression of pro-survival and anti-apoptotic protein-coding and microRNA genes. More importantly, findings showing that ATI5 treatment induced p53-independent apoptosis are of great importance from a therapeutic point of view since p53 mutations are common genetic alterations in human neoplasms.

Exploring the size adaptability of the B ring binding zone of the colchicine site of tubulin with para-nitrogen substituted isocombretastatins

We have synthesized and assayed dimethylaminophenyl, pyrrolidin-1-ylphenyl and carbazole containing phenstatins and isocombretastatins as analogues of the highly potent indoleisocombretastatins with extended or reduced ring sizes. This is an attempt to explore beyond the structural constraints of the X-ray crystal structures the zone of the colchicine site where the tropolone ring of colchicine binds to tubulin (zone 1). The isocombretastatins display up to 30 fold increased water solubility when compared with combretastatin A-4, potent inhibition of tubulin polymerization, and nanomolar cytotoxicities against several human cancer cell lines irrespective of the size of the B ring. On the other hand, substitutions ortho to the nitrogen cause an important reduction in potency. We have also shown that representative compounds inhibit autophagy. These results show that zone 1 can adapt to systems of different size as far as they stay in a common plane, but does not tolerate substituents protruding above or below it. These results can help in the understanding of the binding modes of structures with similar systems and in the design of new colchicine site ligands.

Synthesis, biological evaluation, and molecular docking studies of novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives as potential tubulin polymerization inhibitors

A series of 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives were designed, synthesized and evaluated as potential tubulin polymerization inhibitors and for the cytotoxicity against anthropic cancer cell lines. Among the novel compounds, compound 11f was demonstrated the most potent tubulin polymerization inhibitory activity (IC50 = 1.5 μM) and antiproliferative activity against A549, HepG2 and MCF-7 (GI50 = 2.4, 3.8 and 5.1 μM, respectively), which was compared with the positive control colchicine and CA-4. We also evaluated that compound 11f could effectively induce apoptosis of A549 associated with G2/M phase cell cycle arrest. Docking simulation and 3D-QSAR model in these studies provided more information that could be applied to design new molecules with more potent tubulin inhibitory activity.

Synthesis and Biological evaluation of novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxins as anticancer compounds.

A series of novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxin derivatives were synthesized by employing azide-nitrile click chemistry approach. All the derivatives were evaluated for their cytotoxicity against a panel of four human cancer cell lines and their IC50 values were found to be in the range of 2.4-29.06 μM. The cytotoxicity exhibited by the majority of test compounds were found to comparable and often more effective than doxorubicin and all compounds exhibited higher cytotoxicity on A-549 cell lines. Cell cycle analysis showed that the novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxins resulted in cell cycle arrest at G2/M phase and were also found to be the potent inhibitors of tubulin polymerization in vitro.

Discovery of a potent microtubule-targeting agent: Synthesis and biological evaluation of water-soluble amino acid prodrug of combretastatin A-4 derivatives.

Amino acid prodrugs are known to be very useful for improving the aqueous solubility of sparingly water soluble drugs (Drug Discovery Today 2013, 18, 93). Therefore, we synthesized eleven novel combretastatin A-4 amino acid derivatives and evaluated their anti-tumor activities in vitro and in vivo. Among them, compound 15 (valine attached to compound 3, which was shown to be a potent tubulin polymerization inhibitor in our previous study) exhibited high efficacy in tumor-bearing mice, and pharmacokinetic analysis in rats indicated that compound 15 was an effective prodrug as well. Besides, compound 15 significantly inhibited tubulin polymerization in vitro and in vivo by binding to the colchicine binding site. In addition, compound 15 induced cell cycle arrest in the G2/M phase and triggered apoptosis in a caspase-dependent manner. In conclusion, our study showed that compound 15 could have significant anti-tumor activity as a novel microtubule polymerization disrupting agent with improved aqueous solubility.

Synthesis and biological evaluation of 5,6,7-trimethoxy-1-benzylidene-3,4-dihydro-naphthalen-2-one as tubulin-polymerization inhibitors

A series of new combretastatin-A4 analogs were synthesized, in which a six-membered ring connects the linking bridge and A ring, and their tumor cell growth and tubulin-polymerization inhibitory activity were evaluated. These compounds appear to be potential tubulin-polymerization inhibitors. Compounds 1b with amino substituted on position 3 of B ring conferred optimal bioactivity, higher than that of the lead compound 22b and equivalent to that of CA-4. The binding modes of these compounds to tubulin were obtained by molecular docking, which can explain the structure–activity relationship. The studies presented here provide a new structural type for the development of novel antitumor agents.

Synthesis, biological evaluation and 3D-QSAR studies of novel 5-phenyl-1H-pyrazol cinnamamide derivatives as novel antitubulin agents

A series of novel 5-phenyl-1H-pyrazol derivatives (5a–5x) containing cinnamamide moiety were synthesized and their biological activities as potential tubulin polymerization inhibitors were evaluated. Among them, compound 5j exhibited the most potent inhibitory activity with an IC50 value of 1.02 μM for tubulin, which was superior to that of Colchicine (IC50 = 1.34 μM). Docking simulation was performed to insert compound 5j into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. 3D-QSAR model was also built to provide more pharmacophore understanding that could be used to design new agents with more potent tubulin inhibitory activity.

Synthesis of phenstatin/isocombretastatin-chalcone conjugates as potent tubulin polymerization inhibitors and mitochondrial apoptotic inducers.

A series of phenstatin/isocombretastatin–chalcones were synthesized and screened for their cytotoxic activity against various human cancer cell lines. Some representative compounds exhibited significant antiproliferative activity against a panel of sixty human cancer cell lines of the NCI, with GI50 values in the range of 0.11 to 19.0 μM. Three compounds (3b, 3c and 3e) showed a broad spectrum of antiproliferative efficacy on most of the cell lines in the sub-micromolar range. In addition, all the synthesized compounds (3a–l and 4a–l) displayed moderate to excellent cytotoxicity against breast cancer cells such as MCF-7 and MDA-MB-231 with IC50 values in the range of 0.5 to 19.9 μM. Moreover, the tubulin polymerization assay and immunofluorescence analysis results suggest that some of these compounds like 3c and 3e exhibited significant inhibitory effect on the tubulin assembly with an IC50 value of 0.8 μM and 0.6 μM respectively. A competitive binding assay suggested that these compounds bind at the colchicine-binding site of tubulin. A cell cycle assay revealed that these compounds arrest at the G2/M phase and lead to apoptotic cell death. Furthermore, this was confirmed by Hoechst 33258 staining, activation of caspase 9, DNA fragmentation, Annexin V-FITC and mitochondrial membrane depolarization. Molecular docking studies indicated that compounds like 3e occupy the colchicine binding site of tubulin.

New ligands of the tubulin colchicine site based on X-ray structures.

Colchicine site ligands have proved to be potent inhibitors of tubulin polymerization, which leads them not only to display cytotoxic effects but also vascular disrupting effects on tumour neovasculature. In recent years, many compounds have been designed, synthesized and evaluated in order to improve the potency, stability and physicochemical properties of these agents with the aim of developing an agent that could reach the clinical assay level. Here we analyze the eleven X-ray structures of tubulin in complex with ligands at the colchicine site by dividing it into four different zones of interaction, we review the new compounds that have appeared in the literature since 2008 and that were designed based on any of these X-ray structures and, finally, we describe our latest results in the design of new potent antimitotic indole derivatives that have confirmed the flexibility of one of the zones described for the colchicine site.

Synthesis and structure–activity relationships of pyridinyl-1H-1,2,3-triazolyldihydroisoxazoles as potent inhibitors of tubulin polymerization

Three series of compounds; pyridinyl-1H-1,2,3-triazoles, pyridinyl-1H-1,2,3-triazolylisoxazoles and pyridinyl-1H-1,2,3-triazolyldihydroisoxazoles with TMP moiety were designed, synthesized and screened for their anti-cancer and anti-tubulin properties. By sequentially designing three series of compounds comprising of dihydroisoxazole in the linker, a small substituent like chlorine on one side (R(1)) and aromatic group (R) on the pyridine ring, we have optimized the anti-cancer as well as anti-tubulin activity. Pyridinyl-1H-1,2,3-triazolyldihydroisoxazoles 28b and 28c were found to be potent anti-cancer agents against all the cell lines tested with a concomitant accumulation of cells in the G2/M phase of the cell cycle. Molecular modeling suggests that the trimethoxyphenyl ring in 28b and 28c occupies the cholchicine binding domain of β-tubulin, whereas, the dihydroisoxazole extends towards the interface of α,β-tubulin.

Pharmacokinetic Evaluation of a Novel Benzopyridooxathiazepine Derivative as a Potential Anticancer Agent

Background/Aims: The in vivo metabolic profile of a benzopyridooxathiazepine (BPT) derivative, a potent tubulin polymerization inhibitor with a promising in vitro activity, was investigated. Methods: The quantification of the BPT derivative and the identification of metabolites in the plasma of Wistar rats after i.p. and oral administration of 10 mg/kg were performed by the HPLC-mass spectrometry method. Results: Following a single i.p. dose of the BPT derivative, the plasma concentrations showed a biexponential decay (with a rapid decline) followed by a slow decay with a terminal half-life of 77.90 min. The area under the concentration-time curve from time 0 to infinity (AUC_0-_∞) was 18.90 µg/ml·min. After oral administration, the plasmatic concentrations reached a peak of 0.06 μg/ml at 35 min and then decayed with a half-life of 108 min. The AUC_0-_∞ was 10.25 µg/ml·min, representing 54.2% of the relative bioavailability. The compound was well distributed in the body, and its elimination seemed to be fast, regardless of the administration route. The major metabolic pathways were demethylation and hydroxylation reactions, both followed by conjugation with glucuronic acid. Conclusion: In rats, the BPT derivative is well distributed and undergoes extensive metabolism, leading to several metabolites. With promising in vitro activity and very good oral bioavailability, this compound seems to be an attractive candidate for further development as an anticancer agent.

Discovery and molecular modeling of novel 1-indolyl acetate – 5-Nitroimidazole targeting tubulin polymerization as antiproliferative agents

A series of 18 novel 1-indolyl acetate-5-nitroimidazole 3a–3r were designed, synthesized, and evaluated for their in vitro biological activities as potential tubulin polymerization inhibitors. Among these compounds, 3p displayed strong antitumor activity with IC50 of 2.00, 1.05, 0.87 μM against A549, Hela and U251 respectively, and also showed the most potent PLK1 inhibitory activity with IC50 of 2.4 μM. Molecular docking studies within the colchicine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and confirmed the importance of the configuration of the synthesized 1-indolyl acetate-5-nitroimidazolefor potential tubulin polymerization inhibitors.

New pyrrole derivatives with potent tubulin polymerization inhibiting activity as anticancer agents including hedgehog-dependent cancer.

We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway.

Design, synthesis and biological evaluation of (E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine derivatives as a new class of tubulin polymerization inhibitors

A series of novel (E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine derivatives had been synthesized and evaluated their biological activities as potential tubulin polymerization inhibitors. Among these compounds, compound 3q exhibited potent antiproliferative activities against three cancer cell lines in vitro, and antitubulin polymerization activity with IC50 of 0.92μM, which was superior to that of colchicine (IC50=1.34μM). Docking simulation was performed to insert compound 3q into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. These results suggested that compound 3q may be a promising antitubulin agent for the potential treatment of cancer.