Class Of Tubulin Polymerization Inhibitors Research Articles

Rational design, synthesis and biological evaluation of novel 2-(substituted amino)-[1,2,4]triazolo[1,5-a]pyrimidines as novel tubulin polymerization inhibitors

Following our previously reported compound 3, we designed and synthesized a series of new 2-(substituted amino)- [1,2,4]triazolo[1,5-a]pyrimidines as potential tubulin polymerization inhibitors. Among them, analogue 4k, having a 3-hydroxy-4-methoxyphenylamino group, was observed to display excellent antiproliferative activity toward HeLa, HCT116, A549, and T47D with the IC50 values of 0.31, 1.28, 3.99 and 10.32 μM, respectively, which were approximately 32, 48, 4, and 5-fold improvement compared with 3. Importantly, 4k possessed significant selectivity in inhibiting cancer cell lines over the normal HEK293 cells. Moreover, futher mechanism analysis demonstrated that 4k caused G2/M arrest, induced cells apoptosis in HeLa cells, and manifested significant tubulin polymerization inhibitory activity with the IC50 value of 4.9 μM, which is comparable to CA-4 (IC50 = 4.2 μM). The observations performed in this study reveal that 2-arylamino- [1,2,4]triazolo[1,5-a]pyrimidines represent a novel class of tubulin polymerization inhibitors with potent antiproliferative efficacy.

Cinnamide derived pyrimidine-benzimidazole hybrids as tubulin inhibitors: Synthesis, in silico and cell growth inhibition studies

An approach in modern medicinal chemistry to discover novel bioactive compounds is by mimicking diverse complementary pharmacophores. In extension of this strategy, a new class of piperazine-linked cinnamide derivatives of benzimidazole-pyrimidine hybrids have been designed and synthesized. Their in vitro cytotoxicity profiles were explored on selected human cancer cell lines. Specifically, structural comparison of target hybrids with tubulin-DAMA-colchicine and tubulin-nocodazole complexes has exposed a deep position of benzimidazole ring into the αT5 loop. All the synthesized compounds were demonstrated modest to interesting cytotoxicity against different cancer cell lines. The utmost cytotoxicity has shown with an amine linker of benzimidazole-pyrimidine series, with specificity toward A549 (lung cancer) cell line. The most potent compound in this series was 18i, which inhibited cancer cell growth at micromolar concentrations ranging 2.21–7.29 µM. Flow cytometry studies disclosed that 18i inhibited the cells in G2/M phase of cell cycle. The potent antitumor activity of 18i resulted from enhanced microtubule disruption at a similar level as nocodazole on β-tubulin antibody, explored using immunofluorescence staining. The most active compound 18i also inhibited tubulin polymerization with an IC50 of 5.72 ± 0.51 µM. In vitro biological analysis of 18i presented apoptosis induction on A549 cells with triggering of ROS generation and loss of mitochondrial membrane potential, resulting in DNA injury. In addition, 18i displayed impairment in cellular migration and inhibited the colony formation. Notably, the safety profile of most potent compound 18i was revealed by screening against normal human pulmonary epithelial cells (L132: IC50: 69.25 ± 5.95 μM). The detailed binding interactions of 18i with tubulin was investigated by employing molecular docking, superimposition and free energy analyses. Thus remarks made in this study established that pyrimidine-benzimidazole hybrids as a new class of tubulin polymerization inhibitors with significant anticancer activity.

Novel [1,2,4]triazolo[1,5-a]pyrimidine derivatives as potent antitubulin agents: Design, multicomponent synthesis and antiproliferative activities

As restricted CA-4 analogues, a novel series of [1,2,4]triazolo[1,5-a]pyrimidines possessing 3,4,5-trimethoxylphenyl groups has been achieved successfully via an efficient one-pot three-component reaction of 3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine, 1,3-dicarbonyl compounds and aldehydes. Initial biological evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro against three cancer cell lines. Among them, the most highly active analogue 26 inhibited the growth of HeLa, and A549 cell lines with IC50 values at 0.75, and 1.02 μM, respectively, indicating excellent selectivity over non-tumoural cell line HEK-293 (IC50 = 29.94 μM) which suggested that the target compounds might possess a high safety index. Moreover, cell cycle analysis illustrated that the analogue 26 significantly induced HeLa cells arrest in G2/M phase, meanwhile the compound could dramatically affect cell morphology and microtubule networks. In addition, compound 28 exhibited potent anti-tubulin activity with IC50 values of 9.90 μM, and molecular docking studies revealed the analogue occupied the colchicine-binding site of tubulin. These observations suggest that [1,2,4]triazolo[1,5-a]pyrimidines represent a new class of tubulin polymerization inhibitors and well worth further investigation aiming to generate potential anticancer agents.

Design, synthesis and biological evaluation of 2-phenylquinoline-4-carboxamide derivatives as a new class of tubulin polymerization inhibitors

A novel series of 2-phenylquinoline-4-carboxamide derivatives was synthesized, characterized and evaluated for its antiproliferative activity against five cancer cell lines, Hela, SK-OV-3, HCT116, A549 and MDA-MB-468, and a normal human fetal lung fibroblastic cell line, MRC-5. Among them, compound 7b displayed potent cytotoxic activity in vitro against SK-OV-3 and HCT116 cell lines with IC50 values of 0.5 and 0.2μM, respectively. In general, the antiproliferative activity was correlated with the binding property of the colchicine binding site and inhibitory effect on tubulin polymerization. In addition, immunofluorescence and flow cytometry analysis revealed that selected compounds caused disruption of the mitotic spindle assembly and G2/M phase arrest of the cell cycle, which correlated with proliferation inhibitory activity. Molecular docking analysis demonstrated the interaction of 7b at the colchicine binding site of tubulin. These results indicate these compounds are promising inhibitors of tubulin polymerization for the potent treatment of cancer.

Quinolin-6-Yloxyacetamides Are Microtubule Destabilizing Agents That Bind to the Colchicine Site of Tubulin.

Quinolin-6-yloxyacetamides (QAs) are a chemical class of tubulin polymerization inhibitors that were initially identified as fungicides. Here, we report that QAs are potent anti-proliferative agents against human cancer cells including ones that are drug-resistant. QAs act by disrupting the microtubule cytoskeleton and by causing severe mitotic defects. We further demonstrate that QAs inhibit tubulin polymerization in vitro. The high resolution crystal structure of the tubulin-QA complex revealed that QAs bind to the colchicine site on tubulin, which is targeted by microtubule-destabilizing agents such as colchicine and nocodazole. Together, our data establish QAs as colchicine-site ligands and explain the molecular mechanism of microtubule destabilization by this class of compounds. They further extend our structural knowledge on antitubulin agents and thus should aid in the development of new strategies for the rational design of ligands against multidrug-resistant cancer cells.

Application of GA-MLR for QSAR Modeling of the Arylthioindole Class of Tubulin Polymerization Inhibitors as Anticancer Agents.

Microtubules are dynamic filamentous cytoskeletal proteins which have used widely in cancer chemotherapy. Generally, the action of these compounds depends on binding to the tubulin proteins which are α, β -heterodimers that form the core of the microtubules. Arylthioindoles (ATIs) inhibit tubulin polymerization by binding to the colchicine site, inhibiting the binding of colchicine to tubulin. QSAR modeling play an important role in modern medicinal chemistry, presenting a unique potential for transforming the early phases of drug research, in terms of time and money savings. In this study we are intending to achieve some useful information about the structural properties of mentioned ATIs using the QSAR modeling method. In this research, the QSAR modeling has been employed to evaluate the efficacy of ATIs derivatives in the inhibition of tubulin polymerization. The best multivariate linear model for each training and test data subset calculated using both genetic algorithm-multiple linear regressions (GA-MLR) and stepwise-multiple linear regressions (S-MLR) methods. The best RS trial set has been selected by comparing the two different QSAR modeling results in all train and test subsets. Finally, the S-MLR and GA-MLR results were compared for finding the best random selection (RS) subset. The molecular structures and experimental activity values for inhibition of tubulin polymerization obtained from the literature. Here, using Dragon package over 1185 molecular descriptors such as RDF, GETAWAY, WHIM, 3D-MoRSE descriptors, and functional group descriptors derived for proper characterizing the arylthioindole derivatives structures. These molecular descriptors reduced to 437 one by eliminating the constant variables, collinear descriptors and the variables having low correlation with response. In fact in order to obtain the best RS subsets in the mentioned method, the random samplings of the training subset (80% of data) were carried out 20 times and the remaining molecules (20% of data) considered as external validation set. The QSAR models were constructed using stepwise-MLR and GA-MLR. In order to select important variables the GA-MLR and S-MLR methods applied on the training subsets after data splitting. The best MLR models (models with high fitness function values) with four, five and six variables built to obtain the best QSAR model. The best MLR model had four parameters in both GA-MLR and S-MLR methods. The best significant relationships, using comparison of Q2 of models, for the IC50 values of tubulin assembly in the models obtained in S-MLR and GA-MLR methods are presented for all of the random sets and SOM set. The result of external validation indicates that in the study of inhibition of tubulin polymerization, the Q2test values are 0.7242 and 0.3215 for GA-MLR and S-MLR methods respectively. Considering the results which obtained in both methods it can be concluded that in variable selecting the GA-MLR method is more powerful than the S-MLR method.

Design, Synthesis and Molecular Docking Studies of Novel Indole-Pyrimidine Hybrids as Tubulin Polymerization Inhibitors.

A series of novel hybrids of indole-pyrimidine-containing piperazine moiety were designed, synthesized and evaluated for their antiproliferative and tubulin polymerization inhibitory activities. The results indicated that most of these compounds possessed significant cytotoxic potency against four cancer cell lines, HT-29, A549, MDA-MB-231 and MCF-7. Particularly, the most promising compound 34 showed more potent and broad-spectrum cytotoxic activities with the IC50 values ranged from 5.01 to 14.36μm against A549, MDA-MB-231 and MCF-7 cell lines. Meanwhile, 34 also displayed the most potent tubulin polymerization inhibitory activity with IC50 value of 11.2μm. Furthermore, molecular docking analysis demonstrated 34 interacts and binds efficiently with the tubulin protein at the colchicine-binding site. It was worth noting that the compound did not affect the normal human embryonic kidney cells, HEK-293. These results suggest that this novel class of indole-pyrimidine hybrids may have potential to be developed as new a class of tubulin polymerization inhibitors.

Synthesis and biological evaluation of novel millepachine derivatives as a new class of tubulin polymerization inhibitors.

Twenty-one novel derivatives of millepachine were synthesized and evaluated for their in vitro antiproliferative activity. Among them, 8 exhibited the most potent activity, with IC50 values of 8-27 nM against panel of cancer cell lines and retained full activity in multidrug resistant cancer cells. Treated cells were arrested in G2/M phase and resulted in cellular apoptosis. Microtubule dynamics confirmed 8 was a novel tubulin polymerization inhibitor by binding at the colchicine site. 8 also exhibited antivascular activity because it concentration dependently reduced the cell migration and disrupted capillary like tube formation in HUVEC cells. Furthermore, the hydrochloride salt of 8 (8·HCl) significantly improved the bioavailability up to 47% while retaining the antiproliferative activity. Importantly, 8·HCl significantly inhibited tumor growths in four xenograft models including resistance tumor-cell-bearing mice models without causing significant loss of body weight, suggesting that 8 is a promising new orally anticancer agent to be developed.

Synthesis and fungicidal activity of quinolin-6-yloxyacetamides, a novel class of tubulin polymerization inhibitors

A novel class of experimental fungicides has been discovered, which consists of special quinolin-6-yloxyacetamides. They are highly active against important phytopathogens, such as Phytophthora infestans (potato and tomato late blight), Mycosphaerella graminicola (wheat leaf blotch) and Uncinula necator (grape powdery mildew). Their fungicidal activity is due to their ability to inhibit fungal tubulin polymerization, leading to microtubule destabilization. An efficient synthesis route has been worked out, which allows the diverse substitution of four identified key positions across the molecular scaffold.

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.

Synthesis, biological evaluation and molecular docking studies of trans-indole-3-acrylamide derivatives, a new class of tubulin polymerization inhibitors

In this study, we synthesized a series of trans-indole-3-acrylamide derivatives (3a–k) and investigated their activity for inhibition of cell proliferation against five human cancer cell lines (HeLa, MCF7, MDA-MB-231, Raji and HL-60) by MTT assay. Compound 3e showed significant antiproliferative activity against both the Raji and HL-60 cell lines with IC50 values of 9.5 and 5.1μM, respectively. Compound 3e also exhibited moderate inhibitory activity on tubulin polymerization (IC50=17μM). Flow cytometric analysis of cultured cells treated with 3e also demonstrated that the compound caused cell cycle arrest at the G2/M phase in HL-60 and HeLa cells. Moreover, 3e, the most active compound, caused an apoptotic cell death through the activation of caspase-3. Docking simulations suggested that 3e binds to the colchicine site of tubulin.

N-Aryl-6-methoxy-1,2,3,4-tetrahydroquinolines: A novel class of antitumor agents targeting the colchicine site on tubulin

Structural optimizations of the prior lead 1a led to the discovery of a series of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinoline derivatives as a novel class of tubulin polymerization inhibitors targeted at the colchicine binding site. The most active compound 6d showed extremely high cytotoxicity against a human tumor cell line panel (A549, KB, KBvin, and DU145) with GI50 values ranging from 1.5 to 1.7 nM, significantly more potent than paclitaxel, especially against the drug-resistant KBvin cell line, in the same assays. Analogs 5f, 6b, 6c, and 6e were also quite potent, with a GI50 range of 0.011–0.19 μM. In further studies, active compounds 6b–e and 5f significantly inhibited tubulin assembly, with IC50 values of 0.92–1.0 μM and strongly inhibited colchicine binding to tubulin, with inhibition rates of 75–99% (at 5 μM), comparable with or more potent than combretastatin A-4 (IC50 0.96 μM). Current studies included design, synthesis, and biological evaluations of 24 new compounds (series 3–6). Related SAR analysis, molecular modeling, and evaluation of essential drug-like properties, i.e. water solubility, log P, and in vitro metabolic stability, were also performed.

Design, synthesis and biological evaluation of millepachine derivatives as a new class of tubulin polymerization inhibitors

A series of novel tubulin polymerization inhibitors (9a–9p) have been synthesized and evaluated for their in vitro and in vivo biological activities. Among these compounds, 9e displayed strong antiproliferative activity against several tumor cell lines (IC50=0.15–0.62μM). Compound 9e was also shown to arrest cells in the G2/M phase of the cell cycle and inhibit the polymerization of tubulin. Molecular docking studies suggested that 9e binds into the colchicine binding site of tubulin. In xenograft experiments, 9e exerted more potent anticancer effect than anticancer drug taxol against the H460 Human lung carcinoma in BALB/c nude mice. In summary, these findings suggest that 9e is a promising new antimitotic compound for the potential treatment of cancer.

Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin-2-amines as a new class of tubulin polymerization inhibitors

Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin-2-amines as a new class of tubulin polymerization inhibitors

Toward highly potent cancer agents by modulating the C-2 group of the arylthioindole class of tubulin polymerization inhibitors.

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC₅₀ = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes.

Synthesis of tetrazole–isoxazoline hybrids as a new class of tubulin polymerization inhibitors

A new series of tetrazole based isoxazolines (4a–l) was synthesized and evaluated for their anticancer potential against two cancer cell lines. All these compounds exhibited profound cytotoxicity with IC50 values ranging from 1.22 to 3.62 μM and compounds 4h, 4i showed prominent anticancer efficacy with IC50 values of 1.51, 1.49 μM in A549 and 2.83, 2.40 μM in MDA-MB-231 cell lines. Further, these compounds (4h, 4i) induced apoptotic cell death by inhibition of tubulin polymerization leading to cell cycle arrest at G2/M phase of the cell cycle followed by caspase-3 activity. Moreover, the level of tubulin inhibition by these compounds was examined by in vitro HTS tubulin polymerization assay. Docking of compound 4h and 4i to the active site of tubulin revealed that the trimethoxy ring of the compounds occupies the colchicine binding site of tubulin, whereas the isoxazoline moiety moves towards the interface of α–β tubulin and involves a series of hydrogen bonds with αTyr224 and αSer178.

Synthesis and Biological Evaluation of 2,4,5-Substituted Pyrimidines as a New Class of Tubulin Polymerization Inhibitors

Members of a series of 2,4,5-substituted pyrimidine derivatives were synthesized, and their interactions with tubulin and their antiproliferative activities against the human hepatocellular carcinoma cells of liver (BEL-7402) were evaluated. One member of this family, the indole-pyrimidine 4k, having an indole-aryl-substituted aminopyrimidine structure, was observed to be an excellent inhibitor of tubulin polymerization (IC(50) = 0.79 μM) and to display significantly high antiproliferative activities against several cancer cell lines with IC(50) values ranging from 16 to 62 nM. This substance displayed a high propensity to arrests cells at the G(2)/M phase of the cell cycle (EC(50) = 20 nM). In addition, 4k was found to competitively inhibit colchicine binding to tubulin, indicating that it binds to the colchicine-binding site of tubulin. The observations made in this investigation demonstrate that 2,4,5-substituted pyrimidines represent a new class of tubulin polymerization inhibitors with significant antiproliferative activity.

Recent Development and SAR Analysis of Colchicine Binding Site Inhibitors

Microtubules are cytoskeletal components that play important roles in a number of cellular processes. Colchicine binding site inhibitors (CSIs) is one major class of tubulin polymerization inhibitors, inhibiting microtubule polymerization and blocking cell proliferation at metaphase during mitosis. Many CSIs were discovered or designed and synthesized as anticancer agents in the past several years and great progress had been made. Here, we discuss the insights gained so far relevant to the mechanism of CSIs and their common pharmacophore. The recent development of CSIs with their biological activity and structure-activity relationship (SAR) are also reviewed.

Molecular modelling evaluation of the cytotoxic activity of podophyllotoxin analogues

Podophyllotoxin and its structural derivatives, a class of tubulin polymerization inhibitors, have been the objective of numerous studies to prepare better and safer anti-cancer drugs. A library of podophyllotoxin analogues has been designed consisting of 154 analogues. Their molecular interactions and binding affinities with tubulin protein (1SA1) have been studied using the docking-molecular mechanics based on generalized Born/surface area (MM-GBSA) solvation model. Quantitative structure activity relationships were developed between the cytotoxic activity (pIC(50)) of these compounds and molecular descriptors like docking score and binding free energy. For both the cases the r(2) was in the range of 0.642-0.728 indicating good data fit and r(cv)(2) was in the range of 0.631-0.719 indicating that the predictive capabilities of the models were acceptable. In addition, a linear correlation was observed between the predicted and experimented pIC(50) for the validation data set with correlation coefficient r(2) of 0.806 and 0.887, suggesting that the docked structure orientation and the interaction energies are reasonable. Low levels of root mean square error for the majority of inhibitors establish the docking and Prime/MM-GBSA based prediction model as an efficient tool for generating more potent and specific inhibitors of tubulin protein by testing rationally designed lead compounds based on podophyllotoxin derivatization.

Novel Antitumor Prodrugs Designed for Activation by Matrix Metalloproteinases-2 and -9

Enzyme prodrug monotherapy takes advantage of the selectivity and specificity of enzymes that are differentially active in the immediate environment of tumor cells. Matrix metalloproteinases-2 and -9 (MMP-2 and -9, respectively) are cell-surface Zn-dependent endoproteases associated with diverse processes throughout tumor formation and progression. These enzymes have demonstrated high ratios of tumor- to nontumor-associated activity and may represent candidates for antitumor prodrug activation. Our MMP targeting strategy was to prepare and evaluate two classes of enzyme prodrugs, peptides and sequence-similar peptidomimetics, and determine which would be substrates for the enzymes and thus suitable for further in vitro and in vivo evaluation. We selected representatives of three structurally and mechanistically distinct classes of compounds for delivery, doxorubicin, several auristatins (novel synthetic members of the dolastatin class of tubulin polymerization inhibitors), and CBI-TMI (a duocarmycin class minor groove binder). The drugs were acylated on available amines with the broadly recognized MMP substrate P3-P1' sequence acetyl L-prolyl-L-leucyl-glycyl-L-leucine, or with a peptidomimetic analogue. From a panel of four peptides and four peptidomimetics, two compounds, both peptides, were found to be substrates, with specific activities in the range of 1-20 nmol min(-1) mg(-1). For MMP-9, complete conversion took place in 4-16 h; proteolysis by MMP-2 was considerably slower. Cleavage occurred, as predicted, at the Gly-Leu bond to liberate a leucyl drug, and no other intermediates or cleavage products were observed. Although the MMP-9 proteolysis products were equipotent with the parent leucyl drugs, the prodrugs were not differentially active against MMP-2 or -9-expressing versus nonexpressing cell lines during a 4 h exposure. Our data can be interpreted in light of the current understanding of the structural and mechanistic factors governing MMP-2 and -9 proteolysis.