Tubulin Polymerization Inhibitors Research Articles

Design, Synthesis and Biological Evaluation of Imidazole-Substituted/Fused Aryl Derivatives Targeting Tubulin Polymerization as Anticancer Agents

AbstractThe development of new pharmacologically active molecules targeting tubulin polymerization has recently attracted great interest in research groups. In efforts to develop new potent anticancer compounds, imidazole-tethered/fused pharmacologically active aryl derivatives possessing different substitution patterns targeting tubulin polymerization have been rationally designed and synthesized. The target molecules (P1-5 and KG1-5) were synthesized by multistep syntheses involving the reaction of intermediate 2-aminophenyl-tethered imidazoles with appropriate reactants in the presence of p-TsOH under different conditions. The synthesized compounds displayed moderate to good cytotoxicity, comparable to that of colchicine, against four cancer cell lines (MCF-7, MD-MBA-231, A549, and HCT-116). Compounds P2 and P5, with an imidazoloquinoxaline moiety, emerged as potential leads with cytotoxicity profiles against these cell lines similar to colchicine. Compounds P2 and P5 arrested cell division at the G2/M phase and prevented cancerous cell growth through induced apoptosis. These results favored the hypothesis that the compounds might act by binding to the colchicine binding site, which was further confirmed with the help of a tubulin polymerization inhibition assay. The results encourage the further exploration of imidazoloquinoxalines as promising leads that deserve advanced clinical investigation.

SB226, an inhibitor of tubulin polymerization, inhibits paclitaxel-resistant melanoma growth and spontaneous metastasis

Extensive preclinical studies have shown that colchicine-binding site inhibitors (CBSIs) are promising drug candidates for cancer therapy. Although numerous CBSIs were generated and evaluated, but so far the FDA has not approved any of them due to undesired adverse events or insufficient efficacies. We previously reported two very potent CBSIs, the dihydroquinoxalinone compounds 5 m and 5t. In this study, we further optimized the structures of compounds 5 m and 5t and integrated them to generate a new analog, SB226. X-ray crystal structure studies and a tubulin polymerization assay confirmed that SB226 is a CBSI that could disrupt the microtubule dynamics and interfere with microtubule assembly. Biophysical measurements using surface plasmon resonance (SPR) spectroscopy verified the high binding affinity of SB226 to tubulin dimers. The in vitro studies showed that SB226 possessed sub-nanomolar anti-proliferative activities with an average IC50 of 0.76 nM against a panel of cancer cell lines, some of which are paclitaxel-resistant, including melanoma, breast cancer and prostate cancer cells. SB226 inhibited the colony formation and migration of Taxol-resistant A375/TxR cells, and induced their G2/M phase arrest and apoptosis. Our subsequent in vivo studies confirmed that 4 mg/kg SB226 strongly inhibited the tumor growth of A375/TxR melanoma xenografts in mice and induced necrosis, anti-angiogenesis, and apoptosis in tumors. Moreover, SB226 treatment significantly inhibited spontaneous axillary lymph node, lung, and liver metastases originating from subcutaneous tumors in mice without any obvious toxicity to the animals’ major organs, demonstrating the therapeutic potential of SB226 as a novel anticancer agent for cancer therapy.

Molecular Target Interactions of Quinoline Derivatives as Anticancer Agents: A Review.

One of the leading causes of death worldwide is cancer, which poses substantial risks to both society and an individual's life. Cancer therapy is still challenging, despite developments in the field and continued research into cancer prevention. The search for novel anticancer active agents with a broader cytotoxicity range is therefore continuously ongoing. The benzene ring gets fused to a pyridine ring at two carbon atoms close to one another to form the double ring structure of the heterocyclic aromatic nitrogen molecule known as quinoline (1-azanaphthalene). Quinoline derivatives contain a wide range of pharmacological activities, including antitubercular, antifungal, antibacterial, and antimalarial properties. Quinoline derivatives have also been shown to have anticancer properties. There are many quinoline derivatives widely available as anticancer drugs that act via a variety of mechanisms on various molecular targets, such as inhibition of topoisomerase, inhibition of tyrosine kinases, inhibition of heat shock protein 90 (Hsp90), inhibition of histone deacetylases (HDACs), inhibition of cell cycle arrest and apoptosis, and inhibition of tubulin polymerization.

Design, Synthesis, and Biological Evaluation of Novel Dihydropyrimidinone Derivatives as Potential Anticancer Agents and Tubulin Polymerization Inhibitors

The severity and prevalence of cancer in modern time are a huge global health burden. Continuous efforts are being made toward the development of newer therapeutic candidates to treat and manage this ailment. The dihydropyrimidinone scaffold is one of the key nuclei that have been highly explored and investigated against cancer. It has the potential to combat the consequences of cancer by interacting with several biological targets. Tubulin polymerization inhibition is one such strategy to prevent the progression of cancer. In the presented work, we have synthesized a series of sixteen dihydropyrimidinone derivatives by following a rational drug design. The synthesized compounds have been characterized by 1H NMR and 13C NMR and were further evaluated for cytotoxic activity against breast cancer cell lines (MCF-7 and MDA-MB-231), lung cancer cell lines (A549), and colon cancer cell lines (HCT-116). Compounds 5D and 5P were found most potent and revealed a better cytotoxic activity compared with the standard drug colchicine. Furthermore, the tubulin polymerization inhibition assay revealed that compound 5D showed better inhibition than colchicines, whereas compound 5P revealed an almost equal inhibition to that of colchicine. Furthermore, to investigate the possible mode of action and binding patterns, compounds 5P and 5D were subjected to molecular docking against tubulin (Protein Data Bank ID: ISA0). The results showed that compounds revealed significant interactions and were well occupied inside the cavity of tubulin. The compounds 5D and 5P may serve as new leads in drug development against cancer.

Discovery of Novel Acridane-Based Tubulin Polymerization Inhibitors with Anticancer and Potential Immunomodulatory Effects.

A series of novel acridane-based tubulin polymerization inhibitors were designed, synthesized, and bioevaluated as anticancer agents. The most potent compound NT-6 exhibited high tubulin polymerization inhibitory activity (IC50 = 1.5 μM) and remarkable antiproliferative potency against four cancer cell lines with an average IC50 of 30 nM, better than colchicine and the hit compound 1f (IC50 of 65 and 126 nM, respectively). In addition, NT-6 (10 mg/kg) exerted excellent antitumor efficacy in a melanoma tumor model with a tumor growth inhibition (TGI) of 65.1% without apparent toxicity. Importantly, the combination of NT-6 with a small-molecule PD-L1 inhibitor NP-19 decreased tumor burden significantly (TGI% = 77.6%). Moreover, the combination of NT-6 with NP-19 enhanced the antitumor immune response, mediated by a decrease of PD-L1 expression levels and increased infiltration of antitumor CD8+ effector T cells in tumor tissues. Collectively, NT-6 represents a novel tubulin polymerization inhibitor with immunopotentiating effects.

Optimization of Benzamide Derivatives as Potent and Orally Active Tubulin Inhibitors Targeting the Colchicine Binding Site.

Targeting the colchicine binding site on tubulin is a promising strategy to develop cancer therapeutics. Herein, we describe our systematic structure-activity relationship studies of benzamide derivatives that lead to an identification of a potent and orally active tubulin inhibitor 48, which occupied all three zones of the colchicine binding site in the X-ray co-crystal structure, inhibited tubulin polymerization, promoted mitotic blockade and apoptosis, and exhibited significant antiproliferative activities against various cancer cell lines. Compound 48 demonstrated favorable pharmacokinetic profiles, robust in vivo antitumor efficacies, and acceptable safety profiles. Furthermore, 48 overcame drug resistance in the paclitaxel-resistant A549 xenograft model. Collectively, 48 has been advanced into further preclinical evaluation for the development of next-generation microtubule-targeting drugs.

Discovery of Novel N-Heterocyclic-Fused Deoxypodophyllotoxin Analogues as Tubulin Polymerization Inhibitors Targeting the Colchicine-Binding Site for Cancer Treatment.

Natural products are a major source of anticancer agents and play critical roles in anticancer drug development. Inspired by the complexity-to-diversity strategy, novel deoxypodophyllotoxin (DPT) analogues were designed and synthesized. Among them, compound C3 exhibited the potent antiproliferative activity against four human cancer cell lines with IC50 values in the low nanomolar range. Additionally, it showed marked activity against paclitaxel-resistant MCF-7 cells and A549 cells. Moreover, compound C3 can inhibit tubulin polymerization by targeting the colchicine-binding site of tubulin. Further study revealed that compound C3 could arrest cancer cells in the G2/M phase and disrupt the angiogenesis in human umbilical vein endothelial cells. Meanwhile, C3 remarkably inhibited cancer cell motility and migration, as well as considerably inhibited tumor growth in MCF-7 and MCF-7/TxR xenograft model without obvious toxicity. Collectively, these results indicated that compound C3 may be a promising tubulin polymerization inhibitor development for cancer treatment.

RS6077 induces mitotic arrest and selectively activates cell death in human cancer cell lines and in a lymphoma tumor in vivo

We synthesized a new inhibitor of tubulin polymerization, the pyrrole (1-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrrol-3-yl)(3,4,5-trimethoxy-phenyl)methanone 6 (RS6077). Compound 6 inhibited the growth of multiple cancer cell lines, with IC50 values in the nM range, without affecting the growth of non-transformed cells. The novel agent arrested cells in the G2/M phase of the cell cycle in both transformed and non-transformed cell lines, but single cell analysis by time-lapse video recording revealed a remarkable selectivity in cell death induction by compound 6: in RPE-1 non-transformed cells mitotic arrest induced was not necessarily followed by cell death; in contrast, in HeLa transformed and in lymphoid-derived transformed AHH1 cell lines, cell death was effectively induced during mitotic arrest in cells that fail to complete mitosis. Importantly, the agent also inhibited the growth of the lymphoma TMD8 xenograft model. Together these findings suggest that derivative 6 has a selective efficacy in transformed vs non-transformed cells and indicate that the same compound has potential as novel therapeutic agent to treat lymphomas. Compound 6 showed good metabolic stability upon incubation with human liver microsomes.

Synthesis of Some New Indole-1,3,4-Oxadiazole Hybrids as Tubulin Polymerization Inhibitors

Synthesis of Some New Indole-1,3,4-Oxadiazole Hybrids as Tubulin Polymerization Inhibitors

Development of hydrazide hydrazone-tethered combretastatin-oxindole derivatives as antimitotic agents

A series of new combretastatin-oxindole derivatives via hydrazide hydrazone linker was synthesized by condensation using (E)-bis(phenyl) acrylohydrazides and substituted isatins as synthetic precursors. To determine the potential of these new derivatives, in vitro cytotoxic activity was evaluated using different cancer cell lines, particularly of lung, colorectal, breast and skin origin. A viability study also performed for the most potent compound 9o on HaCaT cells showed its non-toxic nature toward non-malignant cell line. Cell viability assay revealed that derivative 9o with trimethoxy substitution on ring A and N-benzyl substitution on isatin ring exhibited potent anti-cancer activity against lung cancer (A549) cell line with IC50 value 1.26±1.03 µM. Further, staining studies were performed using DAPI, AO/EB to determine induction of changes in nuclear morphology, DNA damage and apoptosis which was also affirmed by annexin V-FITC/PI assay, suggesting involvement of apoptotic mechanism in inhibition of cell growth. Additionally, inducing effect of compound 9o on ROS production indicates ROS-mediated cell death of unwanted cells as well as regulation of apoptosis. The antimitotic attribute was affirmed from cell cycle arrest at G2/M phase and tubulin polymerization inhibition property of compound 9o (IC50 0.17 µM). It is worth mentioning that the pattern of all results was alike standard colchicine. Furthermore, molecular modeling studies were also conducted to determine the binding interaction and ADMET profile of 9o on 3E22 protein.

Discovery of pyrazole-carbohydrazide with indole moiety as tubulin polymerization inhibitors and anti-tumor candidates.

In this work, a series of indole-containing pyrazole-carbohydrazide derivatives A1-A25 were synthesized, and their biological activity on tubulin polymerization inhibition and mitotic catastrophe was evaluated. For introducing indole group to CA-4 pattern, the carbohydrazide linker was used for the first time. As the top hit, A18 suggested notable antiproliferation efficacy and tubulin polymerization inhibitory activity. Inferring comparable antitubulin effect with the positive control Colchicine, A18 indicated obviously lower cyto-toxicity. The cell scratch test showed that A18 could block the cell migration, while the confocal imaging depicted that A18 could induce the mitotic catastrophe via a Colchicine-like approach. The docking simulation visualized the probable binding pattern of A18. With the information in this work, some new hints on modification might be involved in further tubulin-related investigations.

Discovery of potent microtubule-destabilizing agents targeting for colchicine site by virtual screening, biological evaluation, and molecular dynamics simulation

Microtubule has been considered as attractive therapeutic target for various cancers. Although numerous of chemically diverse compounds targeting to colchicine site have been reported, none of them was approved by Food and Drug Administration. In this investigation, the virtual screening methods, including pharmacophore model, molecular docking, and interaction molecular fingerprints similarity, were applied to discover novel microtubule-destabilizing agents from database with 324,474 compounds. 22 compounds with novel scaffolds were identified as microtubule-destabilizing agents, and then submitted to the biological evaluation. Among these 22 hits, hit4 with novel scaffold represents the best anti-proliferative activity with IC50 ranging from 4.51 to 14.81 μM on four cancer cell lines. The in vitro assays reveal that hit4 can effectively inhibit tubulin assembly, and disrupt the microtubule network in MCF-7 cell at a concentration-dependent manner. Finally, the molecular dynamics simulation analysis exhibits that hit4 can stably bind to colchicine site, interact with key residues, and induce αT5 and βT7 regions changes. The values of ΔGbind for the tubulin-colchicine and tubulin-hit4 are -172.9±10.5 and -166.0±12.6 kJ·mol−1, respectively. The above results indicate that the hit4 is a novel microtubule destabilizing agent targeting to colchicine-binding site, which could be developed as a promising tubulin polymerization inhibitor with higher activity for cancer therapy.

Palladium-Catalyzed Directed Aldehyde C-H Arylation of Quinoline-8-carbaldehydes: Exploring the Reactivity Differences between Aryl (Pseudo) Halides.

We have developed a method for Pd-catalyzed direct C-H arylation of quinoline-8-carbaldehydes with either aryl iodides or aryl diazonium salts for the synthesis of aryl quinolinyl ketones. Aryl iodide substituted with an electron-donating group favors the reaction, whereas aryl diazonium salt substituted with an electron-withdrawing group showed excellent reactivity. A range of aryl quinolinyl ketones were synthesized in good-to-excellent yields, with very good functional group tolerance. Our methodology was successfully applied to synthesize highly potent tubulin polymerization inhibitors and can be easily scaled up to a gram scale.

Design, synthesis, and bioevaluation of imidazo [1,2–a] pyrazine derivatives as tubulin polymerization inhibitors with potent anticancer activities

Through structural optimization and ring fusion strategy, we designed a series of novel imidazo[1,2–a]pyrazine derivatives as potential tubulin inhibitors. These compounds displayed potent anti-proliferative activities (micromolar to nanomolar) against a panel of cancer cell lines (including HepG-2, HCT-116, A549 and MDA-MB-231 cells). Among them, compound TB-25 exhibited the strongest inhibitory effects against HCT-116 cells with an IC50 of 23 nM. Mechanism studies revealed that TB-25 could effectively inhibit tubulin polymerization in vitro, and destroy the dynamic equilibrium of microtubules in HCT-116 cells. In addition, TB-25 dose-dependently induced G2/M phase cell cycle arrest and apoptosis in HCT-116 cells. Furthermore, TB-25 suppressed HCT-116 cell migration in a concentration-dependent manner. Finally, molecular docking showed that TB-25 fitted well in the colchicine binding site of tubulin and overlapped nicely with CA-4. Collectively, these results suggest that TB-25 represents a promising tubulin inhibitor deserving further investigation.

Novel amino analogs of the trimethoxyphenyl ring in potent colchicine site ligands improve solubility by the masked polar group incorporation (MPGI) strategy

The low aqueous solubility of colchicine site antimitotic agents, of which the trimethoxyphenyl (A ring) is a heavy contributor, is a serious drawback in their clinical development. We have designed new A ring analogs with chameleonic masked polar amino groups able to increase aqueous solubility and also behave as non-polar through intramolecular hydrogen bonds when bound to tubulin. We have incorporated these new A rings in several scaffolds (sulfonamides, combretastatins, phenstatins, isocombretastatins), synthesized, and assayed 43 representatives. The amino analogs show improved aqueous solubility and some of them (8, 60Z, and 67) nanomolar anti-proliferative potencies against human cancer cell lines, with the most favorable substituent being a 3-methylamino group. The antiproliferative effect relates to tubulin inhibition as shown by in vitro tubulin polymerization inhibition, immunofluorescence microscopy, and cell cycle and apoptosis analysis by flow cytometry. The compounds arrest the cell cycle of treated cells in G2/M and later develop an apoptotic response. Docking studies suggested binding at the colchicine site of tubulin with good agreement with the DFT models of the new structural variations made. The 3-methylamino-4,5‑dimethoxyphenyl moiety is an example of the masked polar group incorporation (MPGI) strategy for soluble ligands binding to hydrophobic sites and a good trimethoxyphenyl ring replacement for the development of new colchicine site ligands.

Crystal structure of tubulin-barbigerone complex enables rational design of potent anticancer agents with isoflavone skeleton

BackgroundIsoflavones possess many biological activities, including anti-inflammatory and anticancer effects. Microtubules (composed of αβ-tubulin heterodimers) are described as one possible cellular target of some of these isoflavones. However, the binding of tubulin to isoflavones has not been extensively studied, and until now, no crystal structure of the tubulin-isoflavone complex has been solved, and details of the isoflavone-tubulin interaction remain elusive. PurposeBarbigerone is an isoflavone mainly found in the genus Milletti, such as the edible leguminous plant Millettia ferruginea, with anticancer activity. This study aims to confirm the cellular target of barbigerone and to study its anticancer mechanism. Method: Surface plasmon resonance assays and X-ray crystallography were used to study the interaction of barbigerone with tubulin protein. Immunofluorescence, in-cell and in vitro tubulin polymerization assays were employed to investigate the mechanism. MTT assays, cell clonal formation assays, wound healing assays, tube formation assays and H460 xenograft models were conducted to evaluate the in vitro and in vivo anticancer activities of barbigerone and one of its derivatives, 0412. ResultsHere, we found that barbigerone binds to tubulin to inhibit tubulin polymerization. Moreover, we solved the X-ray crystal structure of the tubulin-barbigerone complex at 2.33 Å resolution, which unambiguously determined the orientation and position of barbigerone in the colchicine-binding site. Illuminated by the X-ray data, we synthetized and obtained a more active isoflavone, 0412. Both barbigerone and 0412 inhibit cancer cell proliferation, tubulin polymerization, migration of HeLa cells and capillary-like tube formation of HUVECs, induce G2/M phase cell cycle arrest and apoptosis, and exhibit anticancer activity in an H460 xenograft model. ConclusionIn all, through biochemical and X-ray crystal structure results, we identified tubulin as the cellular target of one isoflavone, barbigerone, and proved that the tubulin-barbigerone complex plays a guiding role in obtaining a more active compound, 0412. These studies provide a crucial research basis for the development of isoflavones as anticancer candidate compounds.

Pyridazinones: A versatile scaffold in the development of potential target‐based novel anticancer agents

AbstractPyridazinones are important nitrogen‐rich heterocyclic core that has been driving the substantial medicinal interest due to their wide range of biological activities. This privileged scaffold forms a central core in numerous potent compounds, which results in the development of novel anticancer drugs with fruitful biological activities. The current review article summarizes the progressive development of novel pyridazinone derivatives that are targets for numerous receptors such as C‐met kinase inhibitors, PARP inhibitors, Tubulin polymerization inhibitors, Dihydro folate reductase inhibitors, B‐Raf inhibitors, Bruton tyrosine kinase inhibitors, FER tyrosine kinase inhibitors, and fibroblast growth factors receptors. It features the various simple techniques for the synthesis of pyridazinones and also highlights the mechanistic insights into the anticancer properties of pyridazinone derivatives.

C118P exerted potent anti-tumor effects against melanoma with induction of G2/M arrest via inhibiting the expression of BUB1B

BackgroundThe incidence of melanoma rapidly increased in the past decades, and the clinical treatment of melanoma met huge challenges because of tumor heterogeneity and drug resistance. C118P, a novel tubulin polymerization inhibitor, exhibited strong anticancer effects in many tumors. However, there was no data regarding the potential effects of C118P in melanoma cells. ObjectiveTo investigate of the efficacy and potential target of C118P in melanoma cells. MethodsHuman melanoma cells were treated with C118P, followed by assessments of proliferation, apoptosis and cell cycle distribution. Subsequently, RNA sequencing was performed to further identify the drug targets of C118P in melanoma cells. GO analysis and protein-protein interaction networks analysis were used to screen the potential targets, and verified by a series of assays. Finally, the anti-growth activity of C118P was evaluated in A375-xenografted nude mice, and the expression of BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B), Ki67 and Tunel were determined. ResultsWe found that C118P concentration-dependently inhibited proliferation of melanoma cells. Moreover, C118P simultaneously triggered dramatic G2/M arrest and apoptosis via independent mechanisms in melanoma cells in vitro. C118P exerted anti-melanoma effects by inducing potent G2/M arrest, which was mechanistically related to downregulation of the expression of BUB1B. Importantly, C118P inhibited the tumor growth in A375-xenografted nude, and increased the staining of Ki-67 and Tunel and suppressed the expression of BUB1B in melanoma tissues, which was consistent with in vitro study. ConclusionC118P might provide a novel strategy for the clinical treatment of melanoma by inhibition of BUB1B.

Tirbanibulin for Actinic Keratosis: Insights into the Mechanism of Action.

Actinic keratosis (AK) is a common pre-neoplastic skin lesion constituted by uncontrolled proliferation of atypical keratinocytes that may evolve to squamous cell carcinoma. With global prevalence increasing, AK is expected to be the most common carcinoma of the skin. Tirbanibulin is a reversible tubulin polymerization inhibitor with potent anti-proliferative and anti-tumoral effects. In-vivo and in-vitro studies have shown that tirbanibulin significantly inhibits cell proliferation, tumor growth and downregulates Src signaling with no overt toxicity. Early phase and Phase III trials have shown high lesion clearance, compliance, and few side effects of once daily tirbanibulin treatment. This review discusses tirbanibulin anti-cancer activity, focusing on tubulin polymerization and Src signaling inhibitory effects, highlighting relevant literature and novel preclinical results from the ATNXUS-KX01-001 study. Furthermore, we address the relevant findings obtained in recent clinical trials to evaluate the safety, efficacy, pharmacokinetics, clearance efficacy, and side effects of the 1% tirbanibulin ointment applied once daily. In summary, we highlight preclinical and clinical evidence on the use of tirbanibulin as an effective and safe treatment option for AK.

Design, synthesis, and biological evaluation of diarylpyrazole derivatives as antitumor agents targeting microtubules

A new series of novel diarylpyrazole derivatives as microtubule destabilizers were synthesized and evaluated for the anti-proliferative activities. Anti-proliferative assays were performed on the human cervix adenocarcinoma cell line (HeLa) and human gastric adenocarcinoma cell line (SGC-7901), and the compound 9s containing indole ring showed great anti-proliferative activity against HeLa cells with IC 50 value of 1.9 ± 0.11 μM. Further biological studies showed that 9s was able to inhibit tubulin polymerization, disrupt the cytoskeleton, block the cell cycle in the G2/M phase, and induce cell apoptosis in a concentration-dependent manner. In addition, the results of molecular docking studies showed that compound 9s could bind tightly to the colchicine binding site of tubulin through hydrogen bonding interaction. These preliminary results recommend that compound 9s is likely to be a microtubule destabilizer that deserves further investigation.