Design and synthesis of novel tetrabromophthalimide derivatives as potential tubulin inhibitors endowed with apoptotic induction for cancer treatment.

Although various approaches exist for treating cancer, chemotherapy continues to hold a prominent role in the management of this disease. Besides, microtubules serve as a vital component of the cellular skeleton, playing a pivotal role in the process of cell division making it an attractive target for cancer treatment. Hence, the scope of this work was adapted to design and synthesize new anti-tubulin tetrabromophthalimide hybrids (3-17) with colchicine binding site (CBS) inhibitory potential. The conducted in vitro studies showed that compound 16 displayed the lowest IC50 values (11.46 µM) at the FaDu cancer cell lines, whereas compound 17 exhibited the lowest IC50 value (13.62 µM) at the PC3 cancer cell line. However, compound 7b exhibited the lowest IC50 value (11.45 µM) at the MDA-MB-468 cancer cell line. Moreover, compound 17 was observed to be the superior antitumor candidate against all three tested cancer cell lines (MDA-MB-468, PC3, and FaDu) with IC50 values of 17.22, 13.15, and 13.62 µM, respectively. In addition, compound 17 showed a well-established upregulation of apoptotic markers (Caspases 3, 7, 8, and 9, Bax, and P53). Moreover, compound 17 induced downregulation of the antiapoptotic markers (MMP2, MMP9, and BCL-2). Furthermore, the colchicine binding site inhibition assay showed that compounds 15a and 17 exhibited particularly significant inhibitory potentials, with IC50 values of 23.07 and 4.25 µM, respectively, compared to colchicine, which had an IC50 value of 3.89 µM. Additionally, cell cycle analysis was conducted, showing that compound 17 could prompt cell cycle arrest at both the G0-G1 and G2-M phases. On the other hand, a molecular docking approach was applied to investigate the binding interactions of the examined candidates compared to colchicine towards CBS of the ß-tubulin subunit. Thus, the synthesized tetrabromophthalimide hybrids can be regarded as outstanding anticancer candidates with significant apoptotic activity.

Design, synthesis, and biological evaluation of novel benzimidazole derivatives as anti-cervical cancer agents through PI3K/Akt/mTOR pathway and tubulin inhibition.

Phosphatidylinositol 3-kinase (PI3K) is one of the most attractive therapeutic targets for cervical cancer treatment. In this study, we designed and synthesized a series of benzimidazole derivatives and evaluated their anti-cervical cancer activity. Compound 4r exhibited strong antiproliferative activity in different cervical cancer cell lines HeLa, SiHa and Ca Ski, and relative lower cytotoxicity to normal hepatic and renal cell lines LO2 and HEK-293t (IC50 values were at 21.08 µM and 23.96 µM respectively). Its IC50 value was at 3.38 µM to the SiHa cells. Further mechanistic studies revealed that 4r induced apoptosis, arrested cell cycle in G2/M phase, suppressed PI3K/Akt/mTOR pathway and inhibit the polymerization of tubulin. Molecular docking study suggested that 4r formed key H-bonds action with PI3Kα (PDB ID:8EXU) and tubulin (PDB ID:1SA0). Zebrafish acute toxicity experiments showed that high concentrations of 4r did not cause death or malformation of zebrafish embryos. All these results demonstrated that 4r would be a promising lead candidate for further development of novel PI3K and tubulin dual inhibitors in cervical cancer treatment.

Design and synthesis of novel imidazole-chalcone derivatives as microtubule protein polymerization inhibitors to treat cervical cancer and reverse cisplatin resistance.

Using the licochalcone moiety as a lead compound scaffold, 16 novel imidazole-chalcone derivatives were designed and synthesized as microtubule protein polymerization inhibitors. The proliferation inhibitory activities of the derivatives against SiHa (human cervical squamous cell carcinoma), C-33A (human cervical cancer), HeLa (human cervical cancer), HeLa/DDP (cisplatin-resistant human cervical cancer), and H8 (human cervical epithelial immortalized) cells were evaluated. Compound 5a exhibited significant anticancer activity with IC50 values ranging from 2.28 to 7.77 µM and a resistance index (RI) of 1.63, while showing minimal toxicity to normal H8 cells. When compound 5a was coadministered with cisplatin, the RI of cisplatin to HeLa/DDP cells decreased from 6.04 to 2.01, while compound 5a enhanced the fluorescence intensity of rhodamine 123 in HeLa/DDP cells. Further studies demonstrated that compound 5a arrested cells at the G2/M phase, induced apoptosis, reduced colony formation, inhibited cell migration, and inhibited cell invasion. Preliminary mechanistic studies revealed that compound 5a decreased the immunofluorescence intensity of α-/ß-tubulin in cancer cells, reduced the expression of polymerized α-/ß-tubulin, and increased the expression of depolymerized α-/ß-tubulin. Additionally, the molecular docking results demonstrate that compound 5a can interact with the tubulin colchicine binding site and generate multiple types of interactions. These results suggested that compound 5a has anticancer effects and significantly reverses cervical cancer resistance to cisplatin, which may be related to its inhibition of microtubule and P-glycoprotein (P-gp) activity.

Design, synthesis, and biological evaluation of novel 2,3-Di-O-Aryl/Alkyl sulfonate derivatives of l-ascorbic acid: Efficient access to novel anticancer agents via in vitro screening, tubulin polymerization inhibition, molecular docking study and ADME predictions.

A series of novel l-ascorbic acid derivatives bearing aryl and alkyl sulfonate substituents were synthesized and characterized. In vitro anticancer evaluation against MCF-7 (breast) and A-549 (lung) cancer cell lines revealed potent activity for most of the compounds, with 2b being equipotent to the standard drug colchicine against MCF-7 (IC50 = 0.04 µM). Notably, compound 2b displayed 89-fold selectivity for MCF-7 breast cancer over MCF-10A normal breast cells. Derivatives with two sulfonate groups (2a-g, 3a-g) exhibited superior potency over those with one sulfonate (4a-c,5g, 6b). Compounds 2b and 2c potently inhibited tubulin polymerization in A-549 cancer cells (73.12 % and 62.09 % inhibition, respectively), substantiating their anticancer potential through microtubule disruption. Molecular docking studies showed higher binding scores and affinities for these compounds at the colchicine-binding site of α, ß-tubulin compared to colchicine itself. In-silico ADMET predictions indicated favourable drug-like properties, with 2b exhibiting the highest binding affinity. These sulfonate derivatives of l-ascorbic acid represents promising lead scaffolds for anticancer drug development.

Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Structure-based design and synthesis of BML284 derivatives: A novel class of colchicine-site noncovalent tubulin degradation agents.

Our previous studies have demonstrated that BML284 is a colchicine-site tubulin degradation agent. To improve its antiproliferative properties, 45 derivatives or analogs of BML284 were designed and synthesized based on the cocrystal structure of BML284 and tubulin. Among them, 5i was the most potent derivative, with IC50 values ranging from 0.02 to 0.05 µM against the five tested tumor cell lines. Structure-activity relationship studies verified that the N1 atom of the pyrimidine ring was the key functional group for its tubulin degradation ability. The 5i-tubulin cocrystal complex revealed that the binding pattern of 5i to tubulin is similar to that of BML284. However, replacing the benzodioxole ring with an indole ring strengthened the hydrogen bond formed by the 2-amino group with E198, which improved the antiproliferative activity of 5i. Compound 5i effectively suppressed tumor growth at an intravenous dose of 40 mg/kg (every 2 days) in paclitaxel sensitive A2780S and paclitaxel resistant A2780T ovarian xenograft models, with tumor growth inhibition values of 79.4% and 82.0%, respectively, without apparent side effects, showing its potential to overcome multidrug resistance. This study provided a successful example of crystal structure-guided discovery of 5i as a colchicine-targeted tubulin degradation agent, expanding the scope of targeted protein degradation.

Design and biological evaluation of dual tubulin/HDAC inhibitors based on millepachine for treatment of prostate cancer.

In this work, a novel of dual tubulin/HDAC inhibitors were designed and synthesized based on the structure of natural product millepachine, which has been identified as a tubulin polymerization inhibitor. Biological evaluation revealed that compound 9n exhibited an impressive potency against PC-3 cells with the IC50 value of 16 nM and effectively inhibited both microtubule polymerization and HDAC activity. Furthermore, compound 9n not only induced cell cycle arrest at G2/M phase, but also induced PC- 3 cells apoptosis. Further study revealed that the induction of cell apoptosis by 9n was accompanied by a decrease in mitochondrial membrane potential and an elevation in reactive oxygen species levels in PC-3 cells. Additionally, 9n exhibited inhibitory effects on tumor cell migration and angiogenesis. In PC-3 xenograft model, 9n achieved a remarkable tumor inhibition rate of 90.07%@20 mg/kg, significantly surpassing to that of CA-4 (55.62%@20 mg/kg). Meanwhile, 9n exhibited the favorable drug metabolism characteristics in vivo. All the results indicate that 9n is a promising dual tubulin/HDAC inhibitor for chemotherapy of prostate cancer, deserving the further investigation.

Structure-based approaches for the design of 6-aryl-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d][1,2,3]triazoles as tubulin polymerization inhibitors.

The colchicine binding site on tubulin has been widely acknowledged as an attractive target for anticancer drug exploitation. Here, we reported the structural optimization of the lead compound 4, which was proved in our previous work as a colchicine binding site inhibitor (CBSI). Based on docking researches for the active binding conformation of compound 4, a series of novel 6-aryl-1-(3,4,5-trimethoxyphenyl)-1H-benzo[d][1,2,3]triazole derivatives (9a-9x) were developed by replacing a CH group in the 1H-benzo[d]imidazole skeleton of compound 4 with a nitrogen atom as a hydrogen bond acceptor. Among them, compound 9a showed the strongest antiproliferative activity with IC50 values ranging from 14 to 45 nM against three human cancer cell lines (MCF-7, SGC-7901 and A549), lower than that of compound 4. Mechanistic studies indicated that compound 9a could inhibit tubulin polymerization, destroy the microtubule skeleton, block the cell cycle in G2/M phase, induce cancer cell apoptosis, prevent cancer cell migration and colony formation. Moreover, compound 9a significantly inhibited tumor growth in vivo without observable toxicity in the mice 4T1 xenograft tumor model. In conclusion, this report shows a successful case of the structure-based design approach of a potent tubulin polymerization inhibitor for cancer treatment.

Design, synthesis and mechanistic study of new dual targeting HDAC/tubulin inhibitors.

Aim: The purpose of this work is to create and synthesize a new class of chemicals: 3-cyano-2-substituted pyridine compounds with expected multitarget inhibition of histone deacetylase (HDAC) and tubulin. Materials & methods: The target compounds (3a-c, 4a-c and 5a-c) were synthesized utilizing 6-(4-methoxyphenyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-3-cyanopyridine, with various linkers and zinc-binding groups (ZBGs). Results: Most of the tested compounds showed promising growth inhibition, and hydroxamic acid-containing hybrids possessed higher HDAC inhibition than other ZBGs. Compound 4b possessed the highest potency; however, it showed the most tubulin polymerization inhibition. Docking studies displayed good binding into HDAC1 and six pockets and tubulin polymerization protein. Conclusion: Compound 4b could be considered a good antitumor candidate to go further into in vivo and clinical studies.

Design, synthesis and biological evaluation of novel podophyllotoxin derivatives as tubulin-targeting anticancer agents.

CONTEXT: Podophyllotoxin (PPT) derivatives, used in cancer therapy, require development toward enhanced efficacy and reduced toxicity. OBJECTIVE: This study synthesizes PPT derivatives to assess their anticancer activities. MATERIALS AND METHODS: Compounds E1-E16 antiproliferative activity was tested against four human cancer cell lines (H446, MCF-7, HeLa, A549) and two normal cell lines (L02, BEAS-2B) using the CCK-8 assay. The effects of compound E5 on A549 cell growth were evaluated through molecular docking, in vitro assays (flow cytometry, wound healing, Transwell, colony formation, Western blot), and in vivo tests in female BALB/c nude mice treated with E5 (2 and 4 mg/kg). E5 (4 mg/kg) significantly reduced xenograft tumor growth compared to the DMSO control group. RESULTS: Among the 16 PPT derivatives tested for cytotoxicity, E5 exhibited potent effects against A549 cells (IC50: 0.35 ± 0.13 µM) and exceeded the reference drugs PPT and etoposide to inhibit the growth of xenograft tumours. E5-induced cell cycle arrest in the S and G2/M phases accelerated tubulin depolymerization and triggered apoptosis and mitochondrial depolarization while regulating the expression of apoptosis-related proteins and effectively inhibited cell migration and invasion, suggesting a potential to limit metastasis. Molecular docking showed binding of E5 to tubulin at the colchicine site and to Akt, with a consequent down-regulation of PI3K/Akt pathway proteins. DISCUSSION AND CONCLUSIONS: This research lays the groundwork for advancing cancer treatment through developing and using PPT derivatives. The encouraging results associated with E5 call for extended research and clinical validation, leading to novel and more effective cancer therapies.

Design of balanced dual-target inhibitors of EGFR and microtubule.

Motivated by the clinical success of combining tyrosine kinase inhibitors with microtubule-targeted drugs in antitumor treatment, this paper presents a novel combi-targeting design for dual-target inhibitors, featuring arylformylurea-coupled quinazoline backbones. A series of target compounds (10a-10r) were designed, synthesized, and characterized. Biological assessments demonstrated that 10c notably potentiated ten tumor cell lines in vitro, with IC50 values ranging from 1.04 µM to 7.66 µM. Importantly, 10c (IC50 = 10.66 nM) exhibited superior inhibitory activity against EGFR kinases compared to the reference drug Gefitinib (25.42 nM) and reduced phosphorylated levels of EGFR, AKT, and ERK. Moreover, 10c significantly impeded tubulin polymerization, disrupted the intracellular microtubule network in A549 cells, induced apoptosis, led to S-phase cell cycle arrest, and hindered cell migration. In anticancer evaluation tests using A549 cancer-bearing nude mice models, 10c showed a therapeutic effect similar to Gefitinib, but required only half the dosage (15 mg/kg). These findings indicate that compound 10c is a promising dual-target candidate for anticancer therapy.

Synthesis and characterization of bis-amide SSE1917 as a microtubule-stabilizing anticancer agent.

Microtubule dynamics are critical for spindle assembly and chromosome segregation during cell division. Pharmacological inhibition of microtubule dynamics in cells causes prolonged mitotic arrest, resulting in apoptosis, an approach extensively employed in treating different types of cancers. The present study reports the synthesis of thirty-two novel bis-amides (SSE1901-SSE1932) and the evaluation of their antiproliferative activities. N-(1-oxo-3-phenyl-1-(phenylamino)propan-2-yl)benzamide (SSE1917) exhibited the most potent activity with GI50 values of 0.331 ± 0.01 µM in HCT116 colorectal and 0.48 ± 0.27 µM in BT-549 breast cancer cells. SSE1917 stabilized microtubules in biochemical and cellular assays, bound to taxol site in docking studies, and caused aberrant mitosis and G2/M arrest in cells. Prolonged treatment of cells with the compound increased p53 expression and triggered apoptotic cell death. Furthermore, SSE1917 suppressed the growth of both mouse and patient-derived human colon cancer organoids, highlighting its potential therapeutic value as an anticancer agent.

Design, synthesis, and bioevaluation of 1h-pyrrolo[3,2-c]pyridine derivatives as colchicine-binding site inhibitors with potent anticancer activities.

A new series of 1H-pyrrolo[3,2-c]pyridine derivatives were designed and synthesised as colchicine-binding site inhibitors. Preliminary biological evaluations showed that most of the target compounds displayed moderate to excellent antitumor activities against three cancer cell lines (HeLa, SGC-7901, and MCF-7) in vitro. Among them, 10t exhibited the most potent activities against three cancer cell lines with IC50 values ranging from 0.12 to 0.21 µM. Tubulin polymerisation experiments indicated that 10t potently inhibited tubulin polymerisation at concentrations of 3 µM and 5 µM, and immunostaining assays revealed that 10t remarkably disrupted tubulin microtubule dynamics at a concentration of 0.12 µM. Furthermore, cell cycle studies and cell apoptosis analyses demonstrated that 10t at concentrations of 0.12 µM, 0.24 µM, and 0.36 µM significantly caused G2/M phase cell cycle arrest and apoptosis. The results of molecular modelling studies suggested that 10t interacts with tubulin by forming hydrogen bonds with colchicine sites Thrα179 and Asnß349. In addition, the prediction of physicochemical properties disclosed that 10t conformed well to the Lipinski's rule of five.

PM534, an Optimized Target-Protein Interaction Strategy through the Colchicine Site of Tubulin.

Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure-Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the order Lithistida, family Theonellidae, genus Discodermia (du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model. Its nanomolar affinity and high retention time modulate a strikingly high antitumor activity that efficiently overrides two resistance mechanisms in cells (detoxification pumps and tubulin ßIII isotype overexpression). Furthermore, PM534 induces significant inhibition of tumor growth in mouse xenograft models of human non-small cell lung cancer. Our results present PM534, a highly effective new compound in the preclinical evaluation that is currently in its first human Phase I clinical trial.

Design and synthesis of novel 3-amino-5-phenylpyrazole derivatives as tubulin polymerization inhibitors targeting the colchicine-binding site.

As the basic unit of microtubules, tubulin is one of the most important targets in the study of anticarcinogens. A novel series of 3-amino-5-phenylpyrazole derivatives were designed and synthesized, and evaluates for their biological activities. Among them, a majority of compounds exerted excellent inhibitory activities against five cancer cell lines in vitro. Especially, compound 5b showed a strong antiproliferative activity against MCF-7 cells, with IC50 value of 38.37 nM. Further research indicated that compound 5b can inhibit the polymerization of tubulin targeting the tubulin colchicine-binding sites. Furthermore, 5b could arrest MCF-7 cells at the G2/M phase and induce MCF-7 cells apoptotic in a dose-dependent and time-dependent manners, and regulate the level of related proteins expression. Besides, compound 5b could inhibit the cancer cell migration and angiogenesis. In addition, 5b could inhibit tumor growth in MCF-7 xenograft model without obvious toxicity. All these results indicating that 5b could be a promising antitumor agent targeting tubulin colchicine-binding site and it was worth further study.

Natural-Product-Inspired Discovery of Trimethoxyphenyl-1,2,4-triazolosulfonamides as Potent Tubulin Polymerization Inhibitors.

An approach of natural product-inspired strategy and incorporation of an NP-privileged motif has been investigated for the discovery of new tubulin polymerization inhibitors. Two series, N-Arylsulfonyl-3-arylamino-5-amino-1,2,4-triazole derivatives, and their isomers were considered. The compounds were synthesized by construction of the N-aryl-1,2,4-triazole-3,5-diamine motif and sulfonylation. Although the chemo- and regioselectivity in sulfonylation were challenging due to multiple ring-tautomerizable-NH and exocyclic NH2 functionalities present in the molecular motifs, the developed synthetic method enabled the preparation of designed molecular skeletons with biologically important motifs. The approach also led to explore interesting molecular regio- and stereochemical aspects valuable for activity. The X-ray crystallography study indicated that the hydrogen bonding between the arylamine-NH and the arylsulfonyl-"O" unit and appropriate molecular-functionality topology allowed the cis-locking of two aryls, which is important for tubulin-binding and antiproliferative properties. All synthesized compounds majorly showed characteristic antiproliferative effects in breast cancer cells (MCF-7), and four compounds exhibited potent antiproliferative activity. One compound potently bound to tubulin at the colchicine site and inhibited tubulin polymerization in vitro. The compound significantly depolymerized microtubules in MCF-7 cells, arrested the cells at the G2/M phase, and induced cell death. This study represents the importance of the design strategy in medicinal chemistry and the molecular structural features relevant to anticancer anti-tubulin properties. The explored molecules have the potential for further development.

Discovery of novel coumarin-based derivatives as inhibitors of tubulin polymerization targeting the colchicine binding site with potent anti-gastric cancer activities.

In this work, a series of novel coumarin-based derivatives were designed and synthesized as tubulin polymerization inhibitors targeting the colchicine binding site, and their antiproliferative activities against MGC-803, HCT-116 and KYSE30 cells were evaluated. Among them, the compound I-3 (MY-1442) bearing a 6-methoxy-1,2,3,4-tetrahydroquinoline group exhibited most potent inhibitory activities on MGC-803 (IC50 = 0.034 µM), HCT-116 (IC50 = 0.081 µM) and KYSE30 cells (IC50 = 0.19 µM). Further mechanism studies demonstrated that compound I-3 (MY-1442) could directly bind to the colchicine binding site of ß-tubulin to inhibit tubulin polymerization and microtubules at the cellular level. The results of molecular docking indicated there were well binding interactions between compound I-3 (MY-1442) and the colchicine binding site of ß-tubulin. Compound I-3 (MY-1442) also exhibited effective anti-proliferation, pro-apoptosis, and anti-migration abilities against gastric cancer cells MGC-803. Additionally, compound I-3 (MY-1442) could regulate the expression of cell cycle- and apoptosis-related proteins. Importantly, compound I-3 (MY-1442) could significantly inhibit tumor growth in the MGC-803 xenograft tumor model with a TGI rate of 65.5 % at 30 mg/kg/day. Taken together, this work suggested that the coumarin skeleton exhibited great potential to be a key pharmacophore of tubulin polymerization inhibitors for the discovery of anticancer agents.

3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines inhibit tubulin polymerisation and act as anticancer agents.

A series of cis-restricted 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines as novel tubulin polymerisation inhibitors was designed based on molecular docking. Compound 9p, exhibited potent antiproliferative activity against HeLa, MCF-7, and A549 cell lines. Mechanism studies indicated that 9p potently inhibited tubulin polymerisation and disrupted the microtubule dynamics of tubulin in HeLa cells. Moreover, 9p could cause G2/M phase cell cycle arrest and apoptosis in HeLa cells. In addition, the prediction of physicochemical properties disclosed that 9p conformed well to the Lipinski's rule of five. The initial results suggest that the 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines could serve as a promising scaffold for the development of novel anticancer drugs.

Anthraquinones from the roots of Morinda scabrida Craib exhibit antiproliferative activity against A549 lung cancer cells and antitubulin polymerization.

Six anthraquinones were isolated from Morinda scabrida Craib, an unexplored species of Morinda found in the tropical forest of Thailand. All six anthraquinones showed cytotoxicity against A549 lung cancer cells, with the most active compound, nordamnacanthal (MS01), exhibiting the IC50 value of 16.3 ± 2.5 µM. The cytotoxic effect was dose-dependent and led to cell morphological changes characteristic of apoptosis. In addition, flow cytometric analysis showed dose-dependent apoptosis induction and the G2/M phase cell cycle arrest, which was in agreement with the tubulin polymerization inhibitory activity of MS01. Molecular docking analysis illustrated the binding between MS01 and the α/ß-tubulin heterodimer at the colchicine binding site, and UV-visible absorption spectroscopy revealed the DNA binding capacity of MS01.

Synthesis and biological evaluation of structurally diverse 6-aryl-3-aroyl-indole analogues as inhibitors of tubulin polymerization.

The synthesis and evaluation of small-molecule inhibitors of tubulin polymerization remains a promising approach for the development of new therapeutic agents for cancer treatment. The natural products colchicine and combretastatin A-4 (CA4) inspired significant drug discovery campaigns targeting the colchicine site located on the beta-subunit of the tubulin heterodimer, but so far these efforts have not yielded an approved drug for cancer treatment in human patients. Interest in the colchicine site was enhanced by the discovery that a subset of colchicine site agents demonstrated dual functionality as both potent antiproliferative agents and effective vascular disrupting agents (VDAs). Our previous studies led to the discovery and development of a 2-aryl-3-aroyl-indole analogue (OXi8006) that inhibited tubulin polymerization and demonstrated low nM IC50 values against a variety of human cancer cell lines. A water-soluble phosphate prodrug salt (OXi8007), synthesized from OXi8006, displayed promising vascular disrupting activity in mouse models of cancer. To further extend structure-activity relationship correlations, a series of 6-aryl-3-aroyl-indole analogues was synthesized and evaluated for their inhibition of tubulin polymerization and cytotoxicity against human cancer cell lines. Several structurally diverse molecules in this small library were strong inhibitors of tubulin polymerization and of MCF-7 and MDA-MB-231 human breast cancer cells. One of the most promising analogues (KGP591) caused significant G2/M arrest of MDA-MB-231 cells, disrupted microtubule structure and cell morphology in MDA-MB-231 cells, and demonstrated significant inhibition of MDA-MB-231 cell migration in a wound healing (scratch) assay. A phosphate prodrug salt, KGP618, synthesized from its parent phenolic precursor, KGP591, demonstrated significant reduction in bioluminescence signal when evaluated in vivo against an orthotopic model of kidney cancer (RENCA-luc) in BALB/c mice, indicative of VDA efficacy. The most active compounds from this series offer promise as anticancer therapeutic agents.