Updates in pharmacotherapy for non-small cell lung cancer: a focus on emerging tubulin inhibitors.

INTRODUCTION: The treatment landscape of non-small cell lung cancer (NSCLC) has seen significant advancements in recent years, marked by a shift toward target agents and immune checkpoint inhibitors (ICIs). However, chemotherapy remains a cornerstone of treatment, alone or in combination. Microtubule-targeting agents, such as taxanes and vinca alkaloids, play a crucial role in clinical practice in both early and advanced settings in NSCLC. AREA COVERED: This review outlines the mechanisms of action, present significance, and prospective advancements of microtubule-targeting agents (MTAs), with a special highlight on new combinations in phase 3 trials. The online databases PubMed, Web of Science, Cochrane Library, and ClinicalTrials.gov were searched using the terms 'Microtubule-targeting agents' and 'non-small cell lung cancer' or synonyms, with a special focus over the last 5 years of publications. EXPERT OPINION: Despite the emergence of immunotherapy, MTA remains crucial, often used alongside or after immunotherapy, especially in squamous cell lung cancer. Next-generation sequencing expands treatment options, but reliable biomarkers for immunotherapy are lacking. While antibody-drug conjugates (ADCs) show promise, managing toxicities remain vital. In the early stages, MTAs, possibly with ICIs, are standard, while ADCs may replace traditional chemotherapy in the advanced stages. Nevertheless, MTAs remain essential in subsequent lines or for patients with contraindications.

Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Corrigendum: The role of microtubules in pancreatic cancer: therapeutic progress.

[This corrects the article DOI: 10.3389/fonc.2021.640863.].

Effects of Eribulin on the RNA Content of Extracellular Vesicles Released by Metastatic Breast Cancer Cells.

Extracellular vesicles (EVs) are small lipid particles secreted by almost all human cells into the extracellular space. They perform the essential function of cell-to-cell communication, and their role in promoting breast cancer progression has been well demonstrated. It is known that EVs released by triple-negative and highly aggressive MDA-MB-231 breast cancer cells treated with paclitaxel, a microtubule-targeting agent (MTA), promoted chemoresistance in EV-recipient cells. Here, we studied the RNA content of EVs produced by the same MDA-MB-231 breast cancer cells treated with another MTA, eribulin mesylate. In particular, we analyzed the expression of different RNA species, including mRNAs, lncRNAs, miRNAs, snoRNAs, piRNAs and tRNA fragments by RNA-seq. Then, we performed differential expression analysis, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, and miRNA-target identification. Our findings demonstrate the possible involvement of EVs from eribulin-treated cells in the spread of chemoresistance, prompting the design of strategies that selectively target tumor EVs.

mTOR inhibitor introduce disitamab vedotin (RC48-ADC) rechallenge microtubule-chemotherapy resistance in HER2-low MBC patients with PI3K mutation.

This study aimed to explore the efficacy and potential mechanisms of rechallenge therapy with microtubule-targeting agents (MTAs) in patients with HER2-low metastatic breast cancer (MBC). We performed a systematic review to investigate the rechallenge treatment concept in the field of HER2-low MBC treatment and utilized a series of cases identified in the literature to illustrate the concept. Here we reported two clinical cases of HER2-low MBC patients whose disease progressed after prior treatment with MTAs such as docetaxel and vincristine. When rechallenged with disitamab vedotin ((RC48-antibody-drug conjugate (ADC), a monomethyl auristatin (MMAE) MTA)), both patients achieved a partial response and the final progression-free survival (PFS) was 13.5 and 9 months, respectively. Genomic profiling detected a PIK3CA H1047R mutation in the patients. The patients were treated with everolimus before being rechallenged with RC48, which may lead to a better response. This study further summarizes and analyzes the potential mechanism of the PI3K-AKT signaling pathway in MTA resistance and reveals that the PIK3CA H1047R mutation may be a potential molecular marker for the efficacy prediction of mTOR inhibitors, providing new insights and potential therapeutic strategies for the application of MTAs to MBC patients.

The discovery of water-soluble indazole derivatives as potent microtubule polymerization inhibitors.

Taking a previously discovered indazole derivative 1 as a lead, systematic structural modifications were performed with an indazole core at the 1- and 6-positions to improve its aqueous solubility. Among the designed indazole derivatives, 6-methylpyridin-3-yl indazole derivative 8l and 1H-indol-4-yl indazole derivative 8m exhibited high potency in the low nanomolar range against A549, Huh-7, and T24 cancer cells, including Taxol-resistant variant cells (A549/Tax). As a hydrochloride salt, 8l exhibited much improved aqueous solubility, and its log P value fell into a favorable range. In mechanistic studies, 8l impeded tubulin polymerization through interacting with the colchicine site, resulting in cell cycle arrest and cellular apoptosis. In addition, compared to lead compound 1, 8l reduced cell migration and led to more potent inhibition of tumor growth in vivo without apparent toxicity. In summary, indazole derivative 8l could work as a potential anticancer agent and deserves further investigation for cancer therapy.

Design, synthesis and anticancer activity of N-aryl indolylsulfoximines: Identification of potent and selective anticancer agents.

A facile and efficient approach utilizing copper-mediated cross-coupling reaction of N-boc-3-indolylsulfoximines with aryl iodides was developed to synthesize a diverse range of N-arylated indolylsulfoximines 11a-m in excellent yields (up to 91%). The key precursors, free NH sulfoximines 9 were readily prepared by the treatment of N-boc-3-methylthioindoles 8 with a combination of IBD and ammonium carbamate. Under similar conditions NH-free indolylsulfoximine 9a was successfully prepared in gram-scale quantities. The reaction is highly chemoselective and tolerant of a wide range of functional groups. The process is environmentally friendly and is amenable to scale-up. Among the prepared N-arylated indolylsulfoximines 11a-m, compounds 11i-j (2.68-2.76 µM), 11f-g (1.9-3.7 µM) and 11k (1.28 µM) showed potent and selective cytotoxicity against 22Rv1, C4-2 and MCF7 cells, respectively. Indolylsulfoximine derivative 11l displayed a broad spectrum of activity (1.7-8.2 µM) against the tested cancer cell lines. These compounds were found to be non-cytotoxic to normal HEK293 cells, indicating their potential selectivity for cancer cells. We analysed the impact of 11l on various cellular assays to uncover its mechanism of action. Cellular assay shows that 11l increases the endogenous level of ROS, leading to the increased level of p-53 and c-jun inducing apoptosis. 11l also induced mitochondrial dysfunction, further promoting apoptotic pathways. Besides, 11l also restricts cell invasiveness, indicating that it could serve as an effective anti-metastatic agent. As oxidative stress severe F actin causing tubulin depolymerization, we examined the impact of 11l on tubulin dynamics. Accordingly, 11l treatment decreased the levels of polymerized tubulin in 22Rv1 and C4-2 cells. Although future studies are needed to determine their exact molecular target(s), our data shows that N-aryl indolylsulfoximines could serve as effective anti-cancer agents.

The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4.

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the anti-inflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomain-containing protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent anti-inflammatory effects of depolymerizing microtubule-targeting agents.

Exploration of the Pharmacophore for Cytoskeletal Targeting Ruthenium Polypyridyl Complexes.

Ruthenium(II) trisdiimine complexes of the formula, [Ru(dip)n (L-L)3-n ]2+ , where n=0-3; dip=4,7-diphenyl-1,10-phenanthroline; L-L=2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen) were prepared and tested for cytotoxicity in two cell lines (H358, MCF7). Cellular uptake and subcellular localization were determined by harvesting treated cells and determining the ruthenium concentration in whole or fractionated cells (cytosolic, nuclear, mitochondrial/ ER/Golgi, and cytoskeletal proteins) by Ru ICP-MS. The logP values for the chloride salts of these complexes were measured and the data were analyzed to determine the role of lipophilicity versus structure in the various biological assays. Cellular uptake increased with lipophilicity but shows the biggest jump when the complex contains two or more dip ligands. Significantly, preferential cytoskeletal localization is also correlated with increased cytotoxicity. All of the RPCs promote tubulin polymerization in vitro, but [Ru(dip)2 phen]2+ and [Ru(dip)3 ]2+ show the strongest activity. Analysis of the pellet formed by centrifugation of MTs formed in the presence of [Ru(dip)2 phen]2+ establish a binding stoichiometry of one RPC per tubulin heterodimer. Complexes of the general formula [Ru(dip)2 (L-L)]2+ possess the necessary characteristics to target the cytoskeleton in live cells and increase cytotoxicity, however the nature of the L-L ligand does influence the extent of the effect.

Organotin benzohydroxamate derivatives (OTBH) target colchicine-binding site exerting potent antitumor activity both in vitro and vivo revealed by quantitative proteomic analysis.

The activity of four typical organotin benzohydroxamate compounds (OTBH) with the different electronegativity of fluorine and chlorine atoms was assessed both in vitro and in vivo, revealing that they all exhibited notable antitumor effects. Furthermore, it was discovered that the biochemical capacity against cancer was influenced by their substituents' electronegativity and structural symmetry. For instance, benzohydroxamate derivatives with single chlorine at the fourth site on the benzene ring, two normal­butyl organic ligands, a symmetrical structure, and so on ([n-Bu2Sn[{4-ClC6H4C(O)NHO}2] (OTBH-1)) had stronger antitumor activity than others. Furthermore, the quantitative proteomic analysis discovered 203 proteins in HepG2 cells and 146 proteins in rat liver tissues that were differently identified before and after administration. Simultaneously, bioinformatics analysis of differentially expressed proteins demonstrated that the antiproliferative effects involved in the microtubule-based process, tight junction and its downstream apoptosis pathways. As predicted analytically, molecular docking indicated that ''-O-'' were the target docking atoms for the colchicine-binding site; meanwhile, this site was additionally verified by the EBI competition experiment and the microtubule assembly inhibition test. In conclusion, these derivatives promising for developing microtubule-targeting agents (MTAs) were shown to target the colchicine-binding site, impair cancer cell microtubule networks, and then halt mitosis and trigger apoptosis.

Destabilization of microrchidia family CW-type zinc finger 2 via the cyclin-dependent kinase 1-chaperone-mediated autophagy pathway promotes mitotic arrest and enhances cancer cellular sensitivity to microtubule-targeting agents.

BACKGROUND: Microtubule-targeing agents (MTAs), such as paclitaxel (PTX) and vincristine (VCR), kill cancer cells through activtion of the spindle assembly checkpoint (SAC) and induction of mitotic arrest, but the development of resistance poses significant clinical challenges. METHODS: Immunoblotting and RT-qPCR were used to investigate potential function and related mechanism of MORC2. Flow cytometry analyses were carried out to determine cell cycle distribution and apoptosis. The effect of MORC2 on cellular sensitivity to PTX and VCR was determined by immunoblotting, flow cytometry, and colony formation assays. Immunoprecipitation assays and immunofluorescent staining were utilized to investigate protein-protein interaction and protein co-localization. RESULTS: Here, we identified microrchidia family CW-type zinc finger 2 (MORC2), a poorly characterized oncoprotein, as a novel regulator of SAC activation, mitotic progression, and resistance of cancer cells to PTX and VCR. Mechanically, PTX and VCR activate cyclin-dependent kinase 1, which in turn induces MORC2 phosphorylation at threonine 717 (T717) and T733. Phosphorylated MORC2 enhances its interation with HSPA8 and LAMP2A, two essential components of the chaperone-mediated autophagy (CMA) mechinery, resulting in its autophagic degradation. Degradation of MORC2 during mitosis leads to SAC activation through stabilizing anaphase promoting complex/cyclosome activator protein Cdc20 and facilitating mitotic checkpoint complex assembly, thus contributing to mitotic arrest induced by PTX and VCR. Notably, knockdown of MORC2 promotes mitotic arrest induced by PTX and VCR and enhances the sensitivity of cancer cells to PTX and VCR. CONCLUSIONS: Collectively, these findings unveil a previously unrecognized function and regulatory mechanism of MORC2 in mitotic progression and resistance of cancer cells to MTAs. These results also provide a new clue for developing combined treatmentstrategy by targeting MORC2 in combination with MTAs against human cancer.

Drug-repurposing screen on patient-derived organoids identifies therapy-induced vulnerability in KRAS-mutant colon cancer.

Patient-derived organoids (PDOs) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here, we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify hidden vulnerabilities within therapy-induced phenotypes. Using a microscopy-based screen that accurately scores drug-induced cell killing, we have tested 414 putative anti-cancer drugs for their ability to switch the EGFRi/MEKi-induced cytostatic phenotype toward cytotoxicity. A majority of validated hits (9/37) are microtubule-targeting agents that are commonly used in clinical oncology, such as taxanes and vinca-alkaloids. One of these drugs, vinorelbine, is consistently effective across a panel of >25 different CRC PDOs, independent of RAS mutational status. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle and shows tolerability and effective anti-tumor activity in vivo, setting the basis for a clinical trial to treat patients with metastatic RAS-mutant CRC.

Simple monocyclic pyrimidine analogs as microtubule targeting agents binding to the colchicine site.

Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC50 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC50 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the ßIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.

cGAS-dependent proinflammatory and immune homeostatic effects of the microtubule-targeting agent paclitaxel.

Taxanes are Microtubule-Targeting Agents (MTAs) that exert potent anticancer activity by directly killing cancer cells. However, recent evidence suggests that they may also stimulate inflammation and anticancer adaptive immunity and that these actions strongly contribute to their therapeutic efficacy. Details on how Taxanes may modulate inflammation and anticancer immunity are, nevertheless, still missing. We show here that at very low doses the Taxane Paclitaxel (Pxl) indeed induces a potent proinflammatory response in various cancer cell types in a cyclic GMP-AMP (cGAMP) synthase (cGAS)- and Stimulator of Interferon Genes (STING)-dependent manner, leading to interferon (IFN) signaling. However, we find that Pxl treatment also strongly upregulates the expression of the immune checkpoint protein Programmed Death-Ligand 1 (PD-L1) in cancer cells, therefore, inducing an inhibitory response to adaptive immunity potentially attenuating anticancer immunity and therapeutic success. These observations provide a mechanistic explanation of why clinical benefit may derive from the combination of Pxl with Immune Checkpoint Inhibitors (ICIs) and suggest that more accurately tailoring dosage and schedule of this combination therapy may provide benefit in the management of a larger number of cancer types and stages.

Computational Approaches to the Rational Design of Tubulin-Targeting Agents.

Microtubules are highly dynamic polymers of α,ß-tubulin dimers which play an essential role in numerous cellular processes such as cell proliferation and intracellular transport, making them an attractive target for cancer and neurodegeneration research. To date, a large number of known tubulin binders were derived from natural products, while only one was developed by rational structure-based drug design. Several of these tubulin binders show promising in vitro profiles while presenting unacceptable off-target effects when tested in patients. Therefore, there is a continuing demand for the discovery of safer and more efficient tubulin-targeting agents. Since tubulin structural data is readily available, the employment of computer-aided design techniques can be a key element to focus on the relevant chemical space and guide the design process. Due to the high diversity and quantity of structural data available, we compiled here a guide to the accessible tubulin-ligand structures. Furthermore, we review different ligand and structure-based methods recently used for the successful selection and design of new tubulin-targeting agents.

Structural Perspective of Benzophenones Targeting Tubulin as Anticancer Agents.

Cancer is the leading cause of death and the most significant determinant of life expectancy in almost every country in this twenty-first century. According to the World Health Organization (WHO), cancer is responsible for the leading cause of death globally. Benzophenone derivatives are found in a variety of naturally occurring compounds which are known to be pharmacologically efficacious against a variety of diseases, including cancer. Microtubules are thought to be a good target for cancer chemotherapies. Microtubule polymerization and depolymerization are induced by a variety of natural, synthetic, and semisynthetic chemicals having a benzophenone nucleus, affecting tubulin dynamics. Several medications that affect microtubule dynamics are in various stages of clinical trials, including Combretastatins (phase II), Vincristine (clinically approved), Paclitaxel (in clinical usage), and epothilone (phase III), and only a few have been patented. Benzophenone derivatives target the colchicine binding site of microtubules, damage them and cause cell cycle arrest in the G2-M phase. Belonging to this class of molecules, phenstatin, a potent inhibitor of tubulin polymerization, has shown strongly inhibit cancer cell growth and arrest the G2/M phase of the cell cycle by targeting the colchicine binding site of microtubules. In the present manuscript, we described the benzophenone as tubulin polymerization inhibitors, their Structure-Activity Relationships (SARs) and molecular docking studies that reveal its binding affinity with the colchicine binding site.

ß-III Tubulin Levels Determine the Neurotoxicity Induced by Colchicine-Site Binding Agent Indibulin.

Indibulin, a microtubule-depolymerizing agent, produces minimal neurotoxicity in animals. It is also less cytotoxic toward differentiated neuronal cells than undifferentiated cells. We found that the levels of ß-III tubulin, acetylated tubulin, and polyglutamylated tubulin were significantly increased in differentiated neuroblastoma cells (SH-SY5Y). Since neuronal cells express ß-tubulin isotypes differently from other cell types, we explored the binding of indibulin to different ß-tubulin isotypes. Our molecular docking analysis suggested that indibulin binds to ß-III tubulin with lower affinity than to other ß-tubulin isotypes. We therefore studied the implications of different ß-tubulin isotypes on the cytotoxic effects of indibulin, colchicine, and vinblastine in differentiated SH-SY5Y cells. Upon depletion of ß-III tubulin in the differentiated cells, the toxicity of indibulin and colchicine significantly increased, while sensitivity to vinblastine was unaffected. Using biochemical, bioinformatics, and fluorescence spectroscopic techniques, we have identified the binding site of indibulin on tubulin, which had not previously been established. Indibulin inhibited the binding of colchicine and C12 (a colchicine-site binder) to tubulin and also increased the dissociation constant of the interaction between tubulin and colchicine. Indibulin did not inhibit the binding of vinblastine or taxol to tubulin. Interestingly, indibulin antagonized colchicine treatment but synergized with vinblastine treatment in a combination study performed in MDA-MB-231 cells. The results indicate that indibulin is a colchicine-site binder and that the efficacy of colchicine-site binders is affected by the ß-III tubulin levels in the cells.

Conformation and energy investigation of microtubule longitudinal dynamic instability induced by natural products.

The natural products plinabulin, docetaxel, and vinblastine are microtubule targeting agents (MTAs). They have been used alone or in combination in cancer treatment. However, the exact nature of their effects on microtubule (MT) polymerization dynamics is poorly understood. To elucidate the longitudinal conformational and energetic changes during MT dynamics, a total of 140 ns molecular dynamic simulations combined with binding free energy calculations were performed on seven tubulin models. The results indicated that the drugs disrupted MT polymerization by altering both MT conformation and binding free energy of the neighboring tubulin subunits. The combination of plinabulin and docetaxel destabilized MT polymerization due to bending MT and weakening the polarity of tubulin polymerization. The new combination of docetaxel and vinblastine synergistically enhanced MT depolymerization and bending, while plinabulin and vinblastine had no synergistic inhibitory effects. The results were verified by the tubulin assembly assay. Our study obtained a comprehensive understanding of the action mechanisms of three natural drugs and their combinations on MT dynamic, provided theoretical guidance for new MTA combinations, and would promote the optimal use of MTA and contribute to developing new MTAs as anticancer agents.

The Microtubule Destabilizer Eribulin Synergizes with STING Agonists to Promote Antitumor Efficacy in Triple-Negative Breast Cancer Models.

Eribulin is a microtubule destabilizer used in the treatment of triple-negative breast cancer (TNBC). Eribulin and other microtubule targeted drugs, such as the taxanes, have shared antimitotic effects, but differ in their mechanism of microtubule disruption, leading to diverse effects on cellular signaling and trafficking. Herein, we demonstrate that eribulin is unique from paclitaxel in its ability to enhance expression of the immunogenic cytokine interferon beta (IFNß) in combination with STING agonists in both immune cells and TNBC models, including profound synergism with ADU-S100 and E7766, which are currently undergoing clinical trials. The mechanism by which eribulin enhances STING signaling is downstream of microtubule disruption and independent of the eribulin-dependent release of mitochondrial DNA. Eribulin did not override the requirement of ER exit for STING activation and did not inhibit subsequent STING degradation; however, eribulin significantly enhanced IRF3 phosphorylation and IFNß production downstream of the RNA sensing pathway that converges on this transcription factor. Additionally, we found that eribulin enhanced the population of activated CD4+ T-cells in vivo when combined with either a STING agonist or tumor, demonstrating the ability to function as an immune adjuvant. We further interrogated the combination of eribulin with ADU-S100 in the MMTV-PyVT spontaneous murine mammary tumor model where we observed significant antitumor efficacy with combination treatment. Together, our findings demonstrate that microtubule targeted chemotherapeutics have distinct immunological effects and that eribulin's ability to enhance innate immune sensing pathways supports its use in combination with immunotherapies, such as STING agonists, for the more effective treatment of TNBC and other malignancies.

Design, Synthesis and Biological Investigation of 2-Anilino Triazolopyrimidines as Tubulin Polymerization Inhibitors with Anticancer Activities.

A further investigation aiming to generate new potential antitumor agents led us to synthesize a new series of twenty-two compounds characterized by the presence of the 7-(3',4',5'-trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine pharmacophore modified at its 2-position. Among the synthesized compounds, three were significantly more active than the others. These bore the substituents p-toluidino (3d), p-ethylanilino (3h) and 3',4'-dimethylanilino (3f), and these compounds had IC50 values of 30-43, 160-240 and 67-160 nM, respectively, on HeLa, A549 and HT-29 cancer cells. The p-toluidino derivative 3d was the most potent inhibitor of tubulin polymerization (IC50: 0.45 µM) and strongly inhibited the binding of colchicine to tubulin (72% inhibition), with antiproliferative activity superior to CA-4 against A549 and HeLa cancer cell lines. In vitro investigation showed that compound 3d was able to block treated cells in the G2/M phase of the cell cycle and to induce apoptosis following the intrinsic pathway, further confirmed by mitochondrial depolarization and caspase-9 activation. In vivo experiments conducted on the zebrafish model showed good activity of 3d in reducing the mass of a HeLa cell xenograft. These effects occurred at nontoxic concentrations to the animal, indicating that 3d merits further developmental studies.