Next Article in Journal
From Extraction to Valorization: Unlocking the Potential of Bark-Derived Extraction Residues for Sustainable Material Development
Previous Article in Journal
Locust Bean Gum: A Natural Polysaccharide as an Eco-Friendly Corrosion Inhibitor for N80 Carbon Steel in CO2-Saturated Saline Solution, Useful for the Oil and Gas Industry
Previous Article in Special Issue
Structural and Computational Insights into Transketolase-like 1 (TKTL-1): Distinction from TKT and Implications for Cancer Metabolism and Therapeutic Targeting
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 30
Issue 23
10.3390/molecules30234538
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Design, Synthesis and Anticancer Activity of 6-Substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole Against Tubulin Polymerisation

by
Yuanna Gu
1,†,
Conghao Gai
2,†,
Sijie Zou
3,
Yan Song
3,
Juan Zhang
3,
Qingjie Zhao
2,*,
Xiaoyun Chai
2,* and
Peipei Wang
1,4,*
1
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2
Department of Organic Chemistry, School of Pharmacy, Naval Medical University, Shanghai 200433, China
3
Department of Pharmacy, PLA Naval Medical Center, Naval Medical University, Shanghai 200052, China
4
Marine Biomedical Science and Technology Innovation Platform of Lin-Gang Special Area, Shanghai 201306, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2025, 30(23), 4538; https://doi.org/10.3390/molecules30234538
Submission received: 20 October 2025 / Revised: 18 November 2025 / Accepted: 19 November 2025 / Published: 24 November 2025
(This article belongs to the Special Issue Small-Molecule Drug Design and Discovery)
Browse Figures
Versions Notes

Abstract

Using virtual FragLites screening and a fragment-based drug discovery (FBDD) strategy, we designed and synthesized a series of 6-substituted-1-(3,4,5-trimethoxyphe-nyl)-1H-indole derivatives as potential tubulin polymerization inhibitors. Among them, compound 3g exhibited the best antiproliferative activity within this series and affected microtubule dynamics in a concentration-dependent manner. Further studies indicated that 3g induced G2/M cell-cycle arrest and triggered apoptosis in MCF-7 cells. In vivo, 3g achieved tumor growth inhibition rates of 23.3% and 44.2% at 20 mg/kg and 50 mg/kg, respectively, without evident systemic toxicity. These results suggest that 3g shows preliminary antitumor efficacy and may serve as a starting point for further mechanistic and structural studies. Further optimization and detailed pharmacokinetic and toxicity studies are merited to advance these inhibitors in preclinical development.

Graphical Abstract

1. Introduction

Tubulin polymerizes into a dynamic microtubule network via α/β-tubulin heterodimers, which play essential roles in cell division, migration, and signal transduction of the eukaryotic cytoskeleton [1,2]. Microtubules form the spindle apparatus to mediate chromosome segregation during mitosis [3]. Dysregulated tubulin is closely associated with malignant progression in cancer cells [4,5,6]. Due to dysregulated cell cycle control, cancer cells display uncontrolled proliferation and genomic instability, leading to an increased dependence on microtubule function [7,8].
Microtubule-targeting agents (MTAs) interfere with microtubule dynamics by inhibiting either polymerization or depolymerization, thereby blocking mitosis in cancer cells and inducing G2/M phase arrest and apoptosis [9,10,11]. MTAs are typically classified into two classes: microtubule-stabilizing agents such as taxanes and microtubule-destabilizing agents, including colchicine and combretastatin A-4 [12,13,14]. The former stabilize microtubules by binding to the taxane site on β-tubulin, whereas the latter inhibit microtubule assembly by occupying the colchicine-binding site (CBS), a well-characterized allosteric pocket on β-tubulin [11,12]. However, the clinical efficacy of existing MTAs is limited by drug resistance, primarily due to P-glycoprotein (P-gp) overexpression leading to enhanced drug efflux, or β-tubulin mutations that reduce drug-binding affinity [15,16,17,18]. Consequently, the development of colchicine-binding site inhibitors (CBSIs) has emerged as a promising strategy for overcoming drug resistance [12,19,20]. Fragment-based drug discovery (FBDD) has emerged as a powerful strategy in early-stage lead identification [21,22]. FragLites is an innovative and highly effective technique in this field [23]. It employs small halogenated fragments (e.g., Cl and Br) and leverages their anomalous scattering signals in X-ray crystallography to rapidly identify potential binding sites on the protein surface, followed by structural optimization through fragment linking or growth [24,25,26]. In this study, guided by virtual FragLites screening and the FBDD strategy, we designed and synthesized a series of 6-substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole derivatives as potential tubulin polymerization inhibitors. Although the 3,4,5-trimethoxyphenyl–indole scaffold is a known pharmacophore in CBSIs [27,28,29], our work focuses on optimizing substitution patterns to evaluate their preliminary antitumor activities both in vitro and in vivo. This work provides new insight into the structural optimization of 3,4,5-trimethoxyphenyl–indole CBSIs and lays the groundwork for the development of improved microtubule inhibitors.

2. Results and Discussion

2.1. Chemistry

Initially, a virtual fragment library was employed to explore potential ligandable sites on β-tubulin, particularly around the nucleotide-binding cleft. Preliminary molecular docking (Figure 1B) identified two fragment hits, 6-chloro-1H-indole and 5-iodo-1,2,3-trimethoxybenzene, both exhibiting favorable binding conformations within this region (PDB ID:3E22).
The trimethoxybenzene fragment established hydrogen-bonding interactions with Asn101 and Lys252, while the indole fragment (Fr1) fitted well into a nearby hydrophobic pocket composed of Lys352 and Ile347. Interestingly, the spatial complementarity between the iodine atom of the trimethoxybenzene fragment and the NH group of the indole scaffold suggested a feasible fragment-merging strategy. Guided by this model, the merged compound exhibited a measurable inhibitory effect on tubulin polymerization with an IC50 value of 101.3 μM, validating the design concept.
In the predicted binding pose, the 6-chloro substituent of the indole ring was solvent-exposed, indicating that further modification at this position would be structurally tolerated. Therefore, a series of 6-substituted derivatives (3a–3p) was designed and synthesized, aiming to optimize the binding affinity for the colchicine-binding pocket and evaluate its anti-tubulin inhibitory activities by interfering with microtubule dynamics.
The synthetic route to the target compounds 3a–3p is outlined in Scheme 1. The synthesis commenced with a Suzuki–Miyaura cross-coupling reaction between 6-substituted indole boronic acids and 5-iodo-1,2,3-trimethoxybenzene. The crude biaryl intermediates 2 were used directly in the subsequent step without further purification. Then, the Ullmann-type N-arylation was employed to form a new C-N bond, which constructed the N1-substituted indole scaffold, affording the desired 6-substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole derivatives (3a–3p) in decent yields. All final compounds were characterized by 1H NMR, 13C NMR, and HRMS, and their structures were consistent with the expected molecular frameworks.

2.2. Biological Evaluation

2.2.1. Binding Affinity to Tubulin Protein

To detect the direct interaction between the synthesized compounds and tubulin, biolayer interferometry (BLI) experiments were performed using purified porcine brain tubulin immobilized on Super Streptavidin (SSA) biosensor [30]. Compounds 3a–3p were tested at concentrations ranging from 0 to 200 μM. Association and dissociation kinetics were monitored in real time, and equilibrium dissociation constants (KD) were determined by global fitting of the sensorgrams to a 1:1 binding model. All measurements were conducted in triplicate, and results are reported as mean ± SD.
Among the tested derivatives, compound 3g, bearing a 3-cyano-4-methyl substituent at the 6-position of the indole ring, exhibited the strongest binding affinity toward tubulin, with a KD value of 13 μM. In contrast, most other compounds displayed moderate binding with KD values in the range of 20–50 μM (e.g., 3a, 3b, 3c, 3d, 3f, 3l, 3o), or undetectable binding (e.g., 3h, 3i, 3j, 3k, 3m, 3p) under the tested conditions (Table 1). These results validated tubulin as the primary molecular target of 3g.
The binding affinity analysis provides direct biophysical evidence that compound 3g interacts with tubulin at sub-micromolar levels, supporting its mechanism of action as a microtubule-targeting agent.

2.2.2. Antiproliferative Activity Against Cancer Cell Lines

To evaluate the anticancer potential of the synthesized compounds, a panel of six human and murine cancer cell lines representing different tissue origins was selected, including MCF-7 (human breast adenocarcinoma), MDA-MB-231 (human triple-negative breast cancer), A549 (human non-small cell lung cancer), HeLa (human cervical cancer), A375 (human melanoma), and B16-F10 (murine melanoma) (Table 2). The antiproliferative activity of compounds 3a–3p was assessed using the standard CCK-8 assay after 48 h of drug treatment at varying concentrations. DMSO was used as the vehicle control, and all experiments were performed in triplicate to ensure reproducibility.
Among the tested derivatives, compound 3g also exhibited the most potent and broad-spectrum antiproliferative activity across all six cell lines. The IC50 values of 3g were determined to be 2.94 ± 0.56 μM (MCF-7), 1.61 ± 0.004 μM (MDA-MB-231), 6.30 ± 0.30 μM (A549), 6.10 ± 0.31 μM (HeLa), 0.57 ± 0.01 μM (A375), and 1.69 ± 0.41 μM (B16-F10), respectively, indicating sub-micromolar to low micromolar efficacy in multiple cancer types.
These results highlight compound 3g as a promising lead candidate for further development as a tubulin-targeting anticancer agent with potential application in both breast cancer and melanoma-derived malignancies.

2.2.3. Inhibition of Tubulin Polymerization

The ability of compound 3g to disrupt microtubule dynamics was assessed using an in vitro tubulin polymerization assay [31]. As shown in Figure 2, compound 3g inhibited tubulin polymerization in a concentration-dependent manner over the range of 2.5–50 μM. At 50 μM, 3g markedly suppressed tubulin assembly, indicating potent inhibition at higher concentrations. As expected, colchicine and paclitaxel exhibited characteristic depolymerizing and stabilizing effects, respectively, while 0.1% DMSO had no significant influence on polymerization. These findings confirm that compound 3g effectively interferes with tubulin polymerization in vitro, supporting its proposed mechanism of action as a microtubule-targeting agent.

2.2.4. Inhibition of Cancer Cell Colony Formation

To assess the long-term antiproliferative potential of compound 3g, a colony formation assay was conducted using MCF-7 breast cancer cells. Treatment with compound 3g for 24 h significantly suppressed colony-forming capacity in a dose-dependent manner (Figure 3). Notably, low concentrations (0.1 μM) led to a moderate reduction in clonogenic survival, while higher doses (1 μM and 10 μM) produced progressively greater inhibition, culminating in near-complete suppression of colony formation at 10 μM. These results indicate that compound 3g effectively impairs the self-renewal and long-term survival of cancer cells, supporting its potential as a potent antiproliferative agent.

2.2.5. Suppression of MCF-7 Cell Migration

The impact of compound 3g on MCF-7 cell migration was assessed using wound healing and transwell migration assays. As shown in Figure 4A, treatment with 3g markedly inhibited wound closure compared to the untreated control, indicating impaired lateral motility. In parallel, the transwell migration assay further confirmed the antimigratory effect of 3g, with a significant reduction in the number of cells traversing the membrane after 24 h exposure (Figure 4C). Both assays consistently demonstrated that compound 3g suppressed the migratory capacity of MCF-7 cells in a dose-dependent manner.

2.2.6. Antimicrotubule Activity in MCF-7 Cells

Given the potent in vitro antiproliferative activity of compound 3g, its effects on microtubule organization were further investigated using immunofluorescence staining in MCF-7 cells. The results are shown in Figure 5. Untreated control cells exhibited an intact and well-organized microtubule network, with fine, fibrous microtubules (red) distributed throughout the cytoplasm and surrounding the nucleus (blue). In contrast, treatment with compound 3g at concentrations of 0.1, 1, and 10 μM for 24 h resulted in progressive disruption of the microtubule architecture. The network appeared disassembled and fragmented in a dose-dependent manner, indicating significant cytoskeletal destabilization. Compound 3g potently suppressed tubulin polymerization in a concentration-dependent manner. These results, together with the immunofluorescence data, strongly suggest that compound 3g exerts its antiproliferative effects through microtubule destabilization and disruption of cytoskeletal integrity in cancer cells.

2.2.7. Analysis of Cell Cycle Analysis

Given that microtubule polymerization inhibitors are known to disrupt mitosis and induce cell cycle arrest, we evaluated the effect of compound 3g on cell cycle progression in MCF-7 cells by flow cytometry. As shown in Figure 6A,B, treatment with 3g (10 μM, 48 h) resulted in a marked accumulation of cells in the G2/M phase, indicating that 3g effectively induces cell cycle arrest at this checkpoint.

2.2.8. Analysis of Cancer Cell Apoptosis Analysis

Given that mitotic arrest induced by tubulin polymerization inhibitors is commonly associated with apoptosis, an Annexin V-FITC/propidium iodide (PI) assay was conducted to evaluate the pro-apoptotic effect of compound 3g in MCF-7 cells. Treatment with increasing concentrations of 3g (0.1, 1, and 10 μM) for 48 h led to a dose-dependent increase in apoptotic cell populations, with 10 μM 3g significantly inducing apoptosis in MCF-7 cells (Figure 7A,B).

2.2.9. In Vivo Antitumor Efficacy

The in vivo antitumor activity of compound 3g was evaluated using a mouse melanoma tumor model in BALB/c mice. Tumors were established by subcutaneous inoculation of B16F10 cells, and treatment was initiated when tumors reached a measurable size. Mice were administered vehicle control, paclitaxel (PTX, 10 mg/kg), or compound 3g (20 and 50 mg/kg) via intraperitoneal injection once daily for 14 consecutive days. As shown in Figure 8A, compound 3g significantly inhibited tumor growth in a dose-dependent manner, achieving tumor growth inhibition (TGI) rates of 23.34% and 44.18% at 20 and 50 mg/kg, respectively, compared to 59.84% TGI with PTX at 10 mg/kg.
Importantly, treatment with compound 3g did not induce significant body weight loss throughout the study period (Figure 8B), suggesting good systemic tolerability. In addition, histopathological analysis of major organs by hematoxylin and eosin (H&E) staining did not reveal obvious morphological abnormalities in the liver or kidneys of treated animals (Figure 9). As no serum biochemical analyses were performed, these results should be interpreted as preliminary indications of tolerability based solely on histological assessment.

3. Materials and Methods

3.1. General Chemistry Methods

Unless otherwise specified, all chemicals and reagents were obtained from commercial suppliers and were used without further purification. 1H and 13C NMR spectra were recorded on a Bruker Avance (Billerica, MA, USA) at 500 MHz, using CDCl3 or DMSO-d6 as solvent and tetramethylsilane as internal standard. Chemical shifts are reported in parts per million (ppm). Multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), and multiplet (m). Compound purities were estimated by reversed-phase C18 high-performance liquid chromatography, with UV detection at 254 nm. The major peak area of each tested compound was ≥95% of the combined total peak area. High-resolution mass spectra (HRMS) were measured with an Agilent Technologies 6538 UHD accurate-Mass Q-TOF MS spectrometer (Santa Clara, CA, USA) using ESI. Column chromatography was carried out using commercially available silica gel (200–300 mesh) under pressure. Flash column chromatography was performed on silica gel (60 Å, 200–300 mesh) under positive pressure. Elution was carried out using petroleum ether/ethyl acetate mixtures (from 50:1 to 5:1, v/v) as the mobile phase unless otherwise stated.

3.2. Docking Studies

The molecular modeling studies of the household Virtual FragLites library were carried out using Molecular Operating Environment MOE version 2019.01. The crystal structure of tubulin (PDB ID: 3E22) complex was chosen for the docking study, which was downloaded from the protein data bank website (http://www.rcsb.org). The protein structures were prepared after downloading using the MOE quick preparation tool. The compound data was prepared by minimizing energy, adding hydrogen atoms, calculating partial charges and calculating potential energy. The GBVI/WSA ΔG is a force field-based scoring function that estimates the free energy of binding of the ligand from a given pose. Ligand poses were ranked according to their binding free energy. Figures were prepared using the CCP4MG 2.10.6 suite.

3.3. Synthesis of 6-Phenyl-1H-indoles (Intermediate 1)

In a 100 mL round-bottom flask containing indole-6-boronic acid (1 eq.), iodobenzenes (1 eq.), Pd(PPh3)4 (0.06 eq.), and Na2CO3 (2 eq.) were added to 1,4-dioxane (8 mL) and water (4 mL). The reaction mixture was stirred at 80 °C for 12 h under an argon atmosphere. Reaction progress was monitored by TLC. The mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo, which afforded the crude intermediates 2a–2p.

3.4. Synthesis of 6-Phenyl-1-(3,4,5-trimethoxyphenyl)-1H-indoles (Target Compound)

To a solution of the above crude intermediates 2a–2p (1 eq.), 5-iodo-1,2,3-trimethoxybenzene (0.8 eq.), CuI (0.15 eq.), and Cs2CO3 (0.6 eq.) in DMF (6 mL) were mixed in a round-bottom flask under argon. The mixture was stirred at 150 °C for 16 h, and the reaction was monitored by TLC. The reaction was quenched by water and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography yielded the target compounds 3a–3p.
  • 6-phenyl-1-(3,4,5-trimethoxyphenyl)-1H-indole (3a)
Pink solid; yield: 57.3%. m.p. 127.1–127.7 °C. 1H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.0 Hz, 2H), 7.64 (dd, J = 8.0, 1.4 Hz, 2H), 7.46–7.43 (m, 3H), 7.34 (d, J = 3.0 Hz, 2H), 6.76 (s, 2H), 6.70 (d, J = 3.0 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 154.01, 142.41, 137.03, 136.86, 136.14, 135.63, 128.98, 128.85, 128.52, 127.51, 126.85, 121.49, 120.40, 109.12, 103.27, 102.66, 61.18, 56.49. HRMS (ESI): calcd. for C23H21NO3, [M + H]+ 360.1594; found: 360.1603.
  • 3-(1-(3,4,5-trimethoxyphenyl)-1H-indol-6-yl)benzonitrile (3b)
White solid; yield: 35.7%. m.p. 134.6–135.0 °C. 1H NMR (500 MHz, CDCl3) δ 7.88 (d, J = 1.5 Hz, 1H), 7.84 (dt, J = 7.5, 1.5 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.69–7.65 (m, 2H), 7.59 (dt, J = 7.5, 1.5 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.40–7.36 (m, 2H), 6.73 (s, 2H), 6.71 (dd, J = 3.5, 1.0 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 154.10, 143.65, 137.34, 136.87, 135.28, 133.57, 131.79, 131.00, 130.19, 129.76, 129.66, 129.22, 121.95, 119.93, 119.14 (C≡N), 112.99, 109.18, 103.30, 102.82, 61.19, 56.52. HRMS (ESI): calcd. for C24H20N2O3, [M + H]+ 385.1547; found: 385.1550.
  • 6-(m-tolyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3c)
White solid; yield: 84.2%. m.p. 131.0–131.4 °C. 1H NMR (500 MHz, CDCl3) δ 7.74 (d, J = 8.5 Hz, 2H), 7.45–7.42 (m, 3H), 7.37–7.30 (m, 2H), 7.15 (d, J = 7.5 Hz, 1H), 6.76 (s, 2H), 6.70 (d, J = 3.5 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 6H), 2.43 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 154.01, 142.41, 138.41, 136.99, 136.83, 136.29, 135.68, 128.91, 128.76, 128.47, 128.35, 127.62, 124.62, 121.42, 120.47, 109.10, 103.27, 102.64, 61.19, 56.48, 21.72. HRMS (ESI): calcd. for C24H23NO3, [M + H]+ 374.1751; found: 374.1764.
  • 6-(2-ethylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3d)
Yellow solid; yield: 75.6%. m.p. 87.3–87.8 °C. 1H NMR (500 MHz, CDCl3) δ 7.71 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 0.6 Hz, 1H), 7.35 (d, J = 3.2 Hz, 1H), 7.31 (dd, J = 6.0, 1.6 Hz, 2H), 7.25–7.21 (m, 2H), 7.15 (dd, J = 8.1, 1.6 Hz, 1H), 6.74–6.71 (m, 3H), 3.90 (s, 3H), 3.88 (s, 6H), 2.66 (q, J = 7.5 Hz, 2H), 1.11 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 153.94, 142.63, 141.96, 136.97, 136.62, 136.22, 135.65, 130.49, 128.60, 127.90, 127.35, 125.57, 122.41, 120.61, 111.16, 103.27, 102.61, 61.15, 56.41, 26.40, 15.89. HRMS (ESI): calcd. for C25H25NO3, [M + H]+ 388.1907; found: 388.1913.
  • 6-(4-ethylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3e)
Pink solid; yield: 22.9%. m.p. 100.1–100.5 °C. 1H NMR (500 MHz, CDCl3) δ 7.75–7.73 (m, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.44 (dd, J = 8.0, 1.4 Hz, 1H), 7.33 (d, J = 3.0 Hz, 1H), 7.28 (d, J = 8.5 Hz, 2H), 6.76 (s, 2H), 6.69 (dd, J = 3.0, 0.5 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 6H), 2.71 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 154.00, 142.96, 139.79, 136.96, 136.87, 136.14, 135.68, 128.82, 128.39, 128.32, 127.42, 121.41, 120.35, 108.91, 103.27, 102.64, 61.18, 56.49, 28.62, 15.75. HRMS (ESI): calcd. for C25H25NO3, [M + H]+ 388.1907; found: 388.1915.
  • 6-(2-isopropylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3f)
Yellow solid; yield: 65.4%. m.p. 112.9–112.5 °C. 1H NMR (500 MHz, CDCl3) δ 7.71 (d, J = 7.5 Hz, 1H), 7.49–7.44 (m, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.38–7.31 (m, 2H), 7.25–7.18 (m, 2H), 7.13 (d, J = 8.0 Hz, 1H), 6.74 (s, 2H), 6.72 (d, J = 3.0 Hz, 1H), 3.90 (s, 3H), 3.87 (s, 6H), 3.16 (p, J = 7.0 Hz, 1H), 1.17 (d, J = 6.8 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 153.93, 146.73, 142.09, 136.94, 136.77, 136.19, 135.64, 130.41, 128.58, 127.86, 127.57, 125.55, 125.28, 122.48, 120.53, 111.19, 103.29, 102.58, 61.15, 56.39, 29.57, 24.46. HRMS (ESI): calcd. for C26H27NO3, [M + H]+ 402.2064; found: 402.2069.
  • 2-methyl-5-(1-(3,4,5-trimethoxyphenyl)-1H-indol-6-yl)benzonitrile (3g)
Yellow solid; yield: 71.6%. m.p. 157.4–158.0 °C. 1H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 2.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.72 (dd, J = 8.0, 2.0 Hz, 1H), 7.66–7.64 (m, 1H), 7.40–7.33 (m, 3H), 6.73 (s, 2H), 6.70 (dd, J = 3.5, 1.0 Hz, 1H), 3.95 (s, 3H), 3.90 (s, 6H), 2.58 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 154.09, 140.77, 140.11, 137.27, 136.85, 135.34, 133.59, 131.62, 131.19, 130.81, 129.57, 129.01, 121.86, 119.83 (C≡N), 113.33, 108.93, 103.30, 102.76, 61.19, 56.51, 20.18. HRMS (ESI): calcd. for C25H22N2O3, [M + H]+ 399.1703; found: 399.1720.
  • 6-(3-(trifluoromethoxy)phenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3h)
Yellow solid; yield:28.3%. m.p. 81.7–82.3 °C. 1H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 8.0 Hz, 1H), 7.73–7.71 (m, 1H), 7.55 (dt, J = 8.0, 1.5 Hz, 1H), 7.48–7.46 (m, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.41 (dd, J = 8.5, 1.5 Hz, 1H), 7.37 (d, J = 3.0 Hz, 1H), 7.19–7.15 (m, 1H), 6.75 (s, 2H), 6.71 (dd, J = 3.0, 0.5 Hz, 1H), 3.94 (s, 3H), 3.94 (s, 3H), 3.90 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 154.08, 149.82 (d, J = 1.7 Hz), 144.55, 137.12, 136.76, 135.45, 134.48, 130.13, 129.42, 129.05, 125.79, 121.75, 120.16, 119.98, 119.67, 119.07, 109.23, 103.35, 102.57, 61.20, 56.48. HRMS (ESI): calcd. for C24H20F3NO4, [M + H]+ 444.1417; found: 444.1444.
  • 6-(4-butylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3i)
Yellow solid; yield: 37.3%. m.p. 77.1–77.8 °C. 1H NMR (500 MHz, CDCl3) δ 7.74–7.72 (m, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.44 (dd, J = 8.5, 1.5 Hz, 1H), 7.33 (d, J = 3.2 Hz, 1H), 7.24 (s, 1H), 6.75 (s, 2H), 6.69 (d, J = 3.2 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 6H), 2.66 (t, J = 7.5 Hz, 2H), 1.65 (p, J = 7.5 Hz, 2H), 1.42–1.36 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 153.98, 141.63, 139.71, 136.94, 136.86, 136.15, 135.68, 129.93, 128.81, 128.30, 127.32, 121.40, 120.34, 108.89, 103.26, 102.61, 61.18, 56.48, 35.40, 33.82, 22.55, 14.11. HRMS (ESI): calcd. for C27H29NO3, [M + H]+ 416.2220; found: 416.2248.
  • 6-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3j)
Pink solid; yield: 57.8%. m.p. 110.7–111.4 °C. 1H NMR (500 MHz, CDCl3) δ 7.75–7.70 (m, 2H), 7.57–7.54 (m, 2H), 7.42 (dd, J = 8.2, 2.0 Hz, 1H), 7.35–7.33 (m, 2H), 7.32 (d, J = 2.0 Hz, 1H), 6.75 (s, 2H), 6.69 (dd, J = 3.2, 0.8 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 6H), 2.52 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 154.02, 139.31, 137.03, 136.96, 136.85, 135.59, 135.42, 129.00, 128.50, 127.82, 127.23, 121.55, 120.0, 108.77, 103.30, 102.62, 61.17, 56.49, 16.17. HRMS (ESI): calcd. for C24H23NO3S, [M + H]+ 406.1471; found: 406.1498.
  • 6-(4-isopropylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3k)
White solid; yield: 24.3%. m.p. 115.8–116.3 °C. 1H NMR (500 MHz, CDCl3) δ 7.75–7.72 (m, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.44 (dd, J = 8.5, 1.5 Hz, 1H), 7.32 (d, J = 3.2 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 6.76 (s, 2H), 6.69 (d, J = 3.2 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 6H), 2.96 (p, J = 6.9 Hz, 1H), 1.30 (d, J = 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 154.00, 147.57, 139.93, 136.99, 136.88, 136.15, 135.68, 128.81, 128.32, 127.42, 126.95, 121.40, 120.38, 108.94, 103.27, 102.65, 61.18, 56.50, 33.90, 24.17. HRMS (ESI): calcd. for C26H27NO3, [M + H]+ 402.2064; found: 402.2073.
  • 2-(1-(3,4,5-trimethoxyphenyl)-1H-indol-6-yl)benzonitrile (3l)
White solid; yield: 54.8%. m.p. 163.2–163.8 °C. 1H NMR (500 MHz, CDCl3) δ 7.82–7.78 (m, 1H), 7.79 (dd, J = 8.0, 1.0 Hz, 1H), 7.75 (ddd, J = 7.8, 1.4, 0.6 Hz, 1H), 7.62 (td, J = 7.7, 1.4 Hz, 1H), 7.56 (ddd, J = 7.9, 1.4, 0.6 Hz, 1H), 7.43–7.36 (m, 2H), 7.30 (dd, J = 8.0, 1.5 Hz, 1H), 6.80 (s, 2H), 6.73 (dd, J = 3.0, 1.0 Hz, 1H), 3.93 (s, 6H), 3.92 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 154.09, 146.79, 137.03, 136.01, 135.35, 133.79, 132.78, 132.53, 130.51, 129.64, 129.42, 127.08, 121.63, 121.38, 119.40 (C≡N), 111.57, 111.48, 103.36, 102.49, 61.12, 56.62. HRMS (ESI): calcd. for C24H20N2O3, [M + H]+ 385.1547; found: 385.1564.
  • 4-(1-(3,4,5-trimethoxyphenyl)-1H-indol-6-yl)benzonitrile (3m)
Pink solid; yield: 50.2%. m.p. 137.8–138.6 °C. 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 1.5 Hz, 1H), 7.71 (s, 4H), 7.42 (dd, J = 8.2, 1.6 Hz, 1H), 7.38 (d, J = 3.2 Hz, 1H), 6.73 (s, 2H), 6.71 (dd, J = 3.0, 1.0 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 154.10, 146.89, 137.32, 136.84, 135.26, 133.80, 132.68, 129.92, 129.4, 127.93, 121.93, 120.02, 119.24 (C≡N), 110.28, 109.38, 103.36, 102.78, 61.18, 56.53. HRMS (ESI): calcd. for C24H20N2O3, [M + H]+ 385.1547; found: 385.1547.
  • 3-fluoro-4-(1-(3,4,5-trimethoxyphenyl)-1H-indol-6-yl)benzonitrile (3n)
White solid; yield: 15.6%. m.p. 164.7–165.4 °C. 1H NMR (500 MHz, CDCl3) δ 7.79–7.77 (m, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.51 (dd, J = 8.0, 2.0 Hz, 1H), 7.45 (dd, J = 10.0, 1.5 Hz, 1H), 7.41 (d, J = 3.0 Hz, 1H), 7.34 (dt, J = 8.0, 1.5 Hz, 1H), 6.74 (s, 2H), 6.72 (d, J = 3.0 Hz, 1H), 3.92 (s, 3H), 3.90 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 160.36, 158.37, 154.08, 137.15, 136.18, 135.53, 135.43, 135.19, 132.0, 131.99, 129.94, 129.67, 128.47, 128.44, 127.87, 121.64, 121.30, 120.16, 119.94, 117.91, 111.68, 111.64, 111.60, 111.53, 103.46, 102.43, 61.17, 56.46. HRMS (ESI): calcd. for C24H19FN2O3, [M + H]+ 403.1452; found: 403.1467.
  • 6-(4-Phenoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-indole (3o)
White solid; yield: 18.6%. m.p. 140.2–140.9 °C. 1H NMR (500 MHz, CDCl3) δ 7.74 (dd, J = 8.0, 1.0 Hz, 1H), 7.71 (p, J = 1.0 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.42 (dd, J = 8.0, 1.5 Hz, 1H), 7.38–7.32 (m, 3H), 7.14–7.05 (m, 5H), 6.75 (s, 2H), 6.69 (dd, J = 3.0, 1.0 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 157.39, 156.53, 154.02, 137.53, 137.06, 136.83, 135.62, 135.50, 129.90, 128.95, 128.72, 128.35, 123.42, 121.52, 120.23, 119.23, 119.06, 108.83, 103.28, 102.69, 61.18, 56.51. HRMS (ESI): calcd. for C29H25NO4, [M + H]+ 452.1856; found: 452.1876.
  • 2-(1-(3,4,5-trimethoxyphenyl)-1H-indol-6-yl)benzo[d]thiazole (3p)
White solid; yield: 16.6%. m.p. 162.3–162.7 °C. 1H NMR (500 MHz, CDCl3) δ 8.34–8.30 (m, 1H), 8.07–8.01 (m, 1H), 7.91 (dd, J = 8.5, 1.5 Hz, 1H), 7.89–7.87 (m, 1H), 7.77 (dd, J = 8.0, 0.5 Hz, 1H), 7.47 (ddd, J = 8.2, 7.2, 1.2 Hz, 1H), 7.43 (d, J = 3.0 Hz, 1H), 7.35 (ddd, J = 8.2, 7.2, 1.1 Hz, 1H), 6.76 (s, 2H), 6.72 (dd, J = 3.2 Hz, 0.8 Hz, 1H), 3.97 (s, 3H), 3.93 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 169.37, 154.32, 154.03, 137.24, 136.29, 135.04, 134.97, 131.41, 130.66, 128.09, 126.23, 124.82, 122.87, 121.61, 121.51, 120.22, 110.08, 103.63, 102.60, 61.09, 56.43. HRMS (ESI): calcd. for C24H20N2O3S, [M + H]+ 417.1267; found: 417.1295.

3.5. Biological Assays

3.5.1. BLI Analysis

BLI assays were performed on an Octet RED96 instrument (FortéBio, Pall Life Sciences, Menlo Park, CA, USA) at 25 °C in PBS buffer supplemented with 0.02% (v/v) Tween-20 as the running buffer. Biotinylated tubulin proteins (3 μg/mL) were immobilized onto Super Streptavidin (SSA) biosensors. Association and dissociation were monitored for 60 s each. Compounds were tested at serial concentrations of 200, 100, 50, 25, 12.5, and 6.25 μM. Reference subtraction was performed using sensors incubated in running buffer without protein loading to correct for baseline drift. Data were analyzed using Octet Data Analysis software (version 9.0), and equilibrium dissociation constants (KD) were determined by fitting the curves to a 1:1 binding model. All measurements were performed in triplicate, and results are reported as mean ± SD.

3.5.2. Cell Lines and Cell Culture

Human breast cancer cells (MCF-7), human lung cancer cells (A549), human cervical cancer cells (HeLa), human triple-negative breast cancer cells (MDA-MB-231), and human malignant melanoma cells (A375) were cultured in DMEM. Mouse melanoma cells (B16F10) were maintained in RPMI-1640 medium. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA) and maintained at 37 °C in a humidified atmosphere with 5% CO2.

3.5.3. Cytotoxicity Assay

The cytotoxicity of the test compounds was evaluated against MCF-7, A549, HeLa, MDA-MB-231, A375, and B16F10 using the CCK-8 assay. Cells were seeded into 96-well plates at a density of 5000 cells/well. After removing the medium, 100 μL of medium with 0.1% DMSO containing test compounds at different concentrations was added to each well and incubated at 37 °C for another 48 h. The CCK-8 solution was added and incubated for another 1 h, and then, the absorbance was detected with a microplate reader at a wavelength of 450 nm. The IC50 values were calculated by nonlinear regression analysis using GraphPad Prism 8.0.2. All the experiments were repeated at least three times.

3.5.4. In Vitro Antiproliferative Assay

Cells were seeded into 96-well plates at a density of 5000 cells/well. After being treated for 48 h with compound 3g at the indicated concentrations or time points, the CCK-8 was added and incubated for another 1 h. Cell viability was detected with a microplate reader at a wavelength of 450 nm.

3.5.5. Colony Formation Assay

MCF-7 cells (1000 cells/well) were counted and seeded in 6-well plates. After being cultured for 24 h at 37 °C, the medium was replaced with medium added with compound 3g at the indicated concentration. After 24 h of treatment, the medium was changed to normal. After being cultured for another 10–14 days, the colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 15 min. Following washing with PBS, the colonies were then photographed and quantified using Image J 1.54g.

3.5.6. In Vitro Tubulin Polymerization Assay

Porcine brain tubulin protein was dissolved in EM buffer containing 5% glycerol (80 mM PIPES, 2 mM GTP, 2 mM MgCl2, and 0.5 mM EGTA, pH 6.9). Compounds were diluted in EM buffer to final concentrations of 50, 25, 10, 5, and 2.5 μM and added to a pre-warmed 384-well plate, followed by pre-incubation at 37 °C. Subsequently, 14 μL of the tubulin-GTP mixture was added to each well containing the test compounds. Tubulin polymerization was monitored by measuring absorbance at 340 nm every minute for 60 min. Colchicine and paclitaxel were used as positive controls, and 0.1% DMSO served as the negative control. Each assay was performed in triplicate.

3.5.7. Wound Healing Assay

MCF-7 cells were seeded and incubated in a 6-well plate overnight. Scratches were made in confluent monolayers with a 200 μL pipette tip. Then, wounds were washed twice with media to remove debris and uprooted cells. Cells were treated with various concentrations (0, 0.1, 1, and 10 μM) of compound 3g. Images were obtained using phase contrast microscopy at 0 and 24 h. The migration distance of cells in the wound area was quantified using ImageJ.

3.5.8. Transwell Assay

MCF-7 cells were seeded into six-well culture plates at a density of 5 × 105 cells per well for 24 h. After treatment with compounds 3g at different concentrations for 24 h, cells were harvested and inoculated on transwell filters (8 μM pore size, Millipore, Billerica, MA, USA) in 24-well culture plates. The cells in the top chamber were cultured in the FBS-free medium, and those in the bottom chamber were cultured in the medium containing 10% FBS. The plates were cultivated for an additional 48 h, and the cells that had migrated to the bottom of the top chambers were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. Images of migrated cells were obtained using an inverted microscope (Olympus IX73, Olympus Corporation, Tokyo, Japan).

3.5.9. Immunofluorescence Staining

MCF-7 cells were seeded on glass coverslips in 24-well plates and then treated with vehicle control 0.1% DMSO, 3g (0.1, 1, 10 μM) for 24 h. The cells were fixed with 4% paraformaldehyde and then permeabilized with PBS containing 0.2% Triton X-100. After blocking for 30 min by adding 100 μL of goat serum albumin at room temperature, cells were incubated with a β-tubulin polyclonal antibody (Proteintech, Wuhan Sanying Biotechnology, Wuhan, China, Cat# 10094-1-AP, 1:500 dilution) at 37 °C for 12 h. Then, the cells were washed three times with PBS and incubated with a secondary antibody (Beyotime, Shanghai, China, Cat# A0516, 1:500 dilution) for 1 h at room temperature. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI). Cells were finally visualized using a fluorescence microscope (Nikon TI2-E+A1R, Nikon Corporation, Tokyo, Japan).

3.5.10. Cell Cycle Analysis

MCF-7 cells were seeded in 6-well plates and cultured overnight. Cells were then treated with compound 3g at concentrations of 0.1, 1, and 10 μM for 48 h. After treatment, cells were collected and fixed in 70% ethanol at 4 °C overnight. The fixed cells were washed and resuspended in staining buffer containing RNase A and propidium iodide (PI), followed by incubation in the dark at 37 °C for 30 min. DNA content was subsequently analyzed by flow cytometry.

3.5.11. Cancer Cell Apoptosis Analysis

MCF-7 cells were seeded into 6-well plates and cultured overnight. After treating with compound 3g (0.1, 1, and 10 μM) for 48 h, cells were harvested and suspended in binding buffer containing annexin V-FITC (0.5 mg/mL) and PI (0.5 mg/mL). After that, samples were incubated for 20 min in the dark and analyzed with a flow cytometer.

3.5.12. In Vivo Antitumor Evaluation

All animal procedures were approved by the Animal Ethics Committee of the Naval Medical University (permit number: EC11-055). Male BALB/c mice (6–8 weeks old, average initial body weight approximately 18–20 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The inhibitory effect of compound 3g was evaluated using a subcutaneous B16-F10 melanoma xenograft model. Log-phase B16F10 cells were suspended in PBS and subcutaneously injected into mice (200 μL containing 8 × 105 cells per mouse) to establish tumors. Mice were randomly divided into control and treatment groups (n = 8). Compound 3g and paclitaxel were dissolved in a vehicle consisting of DMSO:PEG:Tween-80:saline (v/v/v/v, 5:35:10:50) to the desired concentrations. Treatments (paclitaxel, 10 mg/kg; compound 3g, 20 and 50 mg/kg, doses calculated on a body-weight basis) and vehicle were administered once daily via intraperitoneal injection for 14 consecutive days. Body weights were monitored throughout the study to assess acute toxicity. Tumor volumes were measured using calipers and calculated with the formula: a × b2 × 0.5, where a and b represent the larger and smaller tumor diameters, respectively. On day 14, mice were sacrificed and tumors were excised and weighed. TGI was calculated using the formula: TGI (%) = [1 − Wt/Wv] × 100%, where Wt and Wv are the average tumor weights of the treatment and vehicle groups, respectively. Major organs (liver and kidneys) were collected, fixed in 4% paraformaldehyde, processed into paraffin sections, stained with hematoxylin and eosin (H&E), and examined microscopically.

4. Conclusions

Using a virtual FragLites screening workflow combined with an FBDD strategy, we successfully designed and synthesized a series of 6-substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole derivatives as potential tubulin inhibitors. BLI assays revealed that most compounds exhibited measurable binding to tubulin, with compounds 3g, 3c, 3d, 3l, and 3b showing the strongest affinities (KD values in the low micromolar range). The enhanced binding of 3g can be attributed to the 3-cyano-4-methyl substitution at the indole 6-position, which likely strengthens hydrophobic and halogen bonding interactions within the colchicine-binding site. The correlation between KD values and IC50 in cell viability assays indicates that cellular antiproliferative effects are largely mediated through tubulin engagement.
Compound 3g demonstrated the most potent antiproliferative activity among the tested derivatives, with low micromolar IC50 values across multiple cancer cell lines, including MCF-7, MDA-MB-231, A549, HeLa, A375, and B16-F10. Mechanistic studies showed that 3g disrupted tubulin polymerization in vitro in a concentration-dependent manner, resulting in G2/M cell-cycle arrest and apoptosis in MCF-7 cells. These cellular responses align with the established mechanism of microtubule-destabilizing agents, such as colchicine and combretastatin A-4, which perturb spindle formation, activate the mitotic checkpoint, and trigger apoptotic cell death [32,33].
In vivo evaluation in the B16-F10 melanoma xenograft model demonstrated that 3g produced dose-dependent tumor growth inhibition (TGI = 23.34% and 44.18% at 20 and 50 mg/kg, respectively). Although slightly less efficacious than paclitaxel, which stabilizes microtubules via a different mechanism, the antitumor activity of 3g is comparable to that of other colchicine-binding site inhibitors reported in the literature [34]. Importantly, histopathological examination of major organs revealed no overt toxicity at the tested doses, suggesting favorable systemic tolerability. However, more comprehensive pharmacokinetic and toxicological studies, including biochemical enzyme assays, are warranted to fully evaluate the safety profile.
Taken together, our results indicate that the 6-substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole scaffold, particularly compound 3g, is a promising lead for the development of new tubulin polymerization inhibitors. The observed correlation between structural modification, binding affinity, and cellular activity provides guidance for further optimization. Future work will focus on improving pharmacokinetic properties, expanding the structure–activity relationship, and performing detailed preclinical toxicity evaluations to further characterize compound 3g.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules30234538/s1. Figures S1–S16: NMR, HRMS, HPLC Spectra of All Products; Figures S17–S33: Analysis of the affinity of 3a–3g and colchicine to tubulin by bio-layer interferometry (BLl); Figure S34: Superposition of the docking poses of 3g (sea green) and the original hit compound (pink) in the colchicine-binding site of tubulin; Figure S35: Predicted binding pose of compound 3g in the colchicine-binding site of tubulin.

Author Contributions

Investigation, Y.G., S.Z. and J.Z.; Methodology, Q.Z. and X.C.; Project administration, P.W.; Supervision, Q.Z., X.C. and P.W.; Writing—original draft, C.G. and Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No. 82204347 and 82473778). This research was also funded by Open Research Project of Marine Biomedical Science and Technology Innovation Platform of Lin-Gang Special Area (RWS-2024-002).

Institutional Review Board Statement

All animal procedures were approved by the Animal Ethics Committee of the Naval Medical University (permit number: EC11-055).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We thank all authors for their help and efforts with writing and data collection. We thank the staff members of the Large-scale Protein Preparation System (https://cstr.cn/31129.02.NFPS.LSPS (accessed on 15 August 2025)) at the National Facility for Protein Science in Shanghai (https://cstr.cn/31129.02.NFPS (accessed on 15 August 2025)) for providing technical support and assistance in data collection and analysis (NFPSS2025P0405).

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Binarová, P.; Tuszynski, J. Tubulin: Structure, Functions and Roles in Disease. Cells 2019, 8, 1294. [Google Scholar] [CrossRef]
  2. Gudimchuk, N.B.; McIntosh, J.R. Regulation of microtubule dynamics, mechanics and function through the growing tip. Nat. Rev. Mol. Cell Biol. 2021, 22, 777–795. [Google Scholar] [CrossRef] [PubMed]
  3. McIntosh, J.R.; Grishchuk, E.L.; West, R.R. Chromosome-microtubule interactions during mitosis. Annu. Rev. Cell Dev. Biol. 2002, 18, 193–219. [Google Scholar] [CrossRef] [PubMed]
  4. Borys, F.; Joachimiak, E.; Krawczyk, H.; Fabczak, H. Intrinsic and Extrinsic Factors Affecting Microtubule Dynamics in Normal and Cancer Cells. Molecules 2020, 25, 3705. [Google Scholar] [CrossRef] [PubMed]
  5. Halim, C.E.; Deng, S.; Crasta, K.C.; Yap, C.T. Interplay Between the Cytoskeleton and DNA Damage Response in Cancer Progression. Cancers 2025, 17, 1378. [Google Scholar] [CrossRef]
  6. Lopes, D.; Maiato, H. The Tubulin Code in Mitosis and Cancer. Cells 2020, 9, 2356. [Google Scholar] [CrossRef]
  7. Dominguez-Brauer, C.; Thu, K.L.; Mason, J.M.; Blaser, H.; Bray, M.R.; Mak, T.W. Targeting Mitosis in Cancer: Emerging Strategies. Mol. Cell 2015, 60, 524–536. [Google Scholar] [CrossRef]
  8. Parker, A.L.; Kavallaris, M.; McCarroll, J.A. Microtubules and their role in cellular stress in cancer. Front. Oncol. 2014, 4, 153. [Google Scholar] [CrossRef]
  9. Rieder, C.L.; Maiato, H. Stuck in division or passing through: What happens when cells cannot satisfy the spindle assembly checkpoint. Dev. Cell 2004, 7, 637–651. [Google Scholar] [CrossRef]
  10. Prota, A.E.; Bargsten, K.; Diaz, J.F.; Marsh, M.; Cuevas, C.; Liniger, M.; Neuhaus, C.; Andreu, J.M.; Altmann, K.H.; Steinmetz, M.O. A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs. Proc. Natl. Acad. Sci. USA 2014, 111, 13817–13821. [Google Scholar] [CrossRef]
  11. Abal, M.; Andreu, J.M.; Barasoain, I. Taxanes: Microtubule and centrosome targets, and cell cycle dependent mechanisms of action. Curr. Cancer Drug Targets 2003, 3, 193–203. [Google Scholar] [CrossRef]
  12. McLoughlin, E.C.; O’Boyle, N.M. Colchicine-Binding Site Inhibitors from Chemistry to Clinic: A Review. Pharmaceuticals 2020, 13, 8. [Google Scholar] [CrossRef] [PubMed]
  13. Karatoprak, G.; Küpeli Akkol, E.; Genç, Y.; Bardakci, H.; Yücel, Ç.; Sobarzo-Sánchez, E. Combretastatins: An Overview of Structure, Probable Mechanisms of Action and Potential Applications. Molecules 2020, 25, 2560. [Google Scholar] [CrossRef] [PubMed]
  14. Steinmetz, M.O.; Prota, A.E. Microtubule-Targeting Agents: Strategies To Hijack the Cytoskeleton. Trends Cell Biol. 2018, 28, 776–792. [Google Scholar] [CrossRef] [PubMed]
  15. Waghray, D.; Zhang, Q. Inhibit or Evade Multidrug Resistance P-Glycoprotein in Cancer Treatment. J. Med. Chem. 2018, 61, 5108–5121. [Google Scholar] [CrossRef]
  16. Hari, M.; Wang, Y.; Veeraraghavan, S.; Cabral, F. Mutations in alpha- and beta-tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine. Mol. Cancer Ther. 2003, 2, 597–605. [Google Scholar]
  17. Hari, M.; Loganzo, F.; Annable, T.; Tan, X.; Musto, S.; Morilla, D.B.; Nettles, J.H.; Snyder, J.P.; Greenberger, L.M. Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules. Mol. Cancer Ther. 2006, 5, 270–278. [Google Scholar] [CrossRef]
  18. Ganguly, A.; Cabral, F. New insights into mechanisms of resistance to microtubule inhibitors. Biochim. Biophys. Acta 2011, 1816, 164–171. [Google Scholar] [CrossRef]
  19. Ning, N.; Yu, Y.; Wu, M.; Zhang, R.; Zhang, T.; Zhu, C.; Huang, L.; Yun, C.H.; Benes, C.H.; Zhang, J.; et al. A Novel Microtubule Inhibitor Overcomes Multidrug Resistance in Tumors. Cancer Res. 2018, 78, 5949–5957. [Google Scholar] [CrossRef]
  20. Weng, H.; Li, J.; Zhu, H.; Carver Wong, K.F.; Zhu, Z.; Xu, J. An update on the recent advances and discovery of novel tubulin colchicine binding inhibitors. Future Med. Chem. 2023, 15, 73–95. [Google Scholar] [CrossRef]
  21. Chan, B.; Lynch, N.B.; Tran, W.; Joyce, J.M.; Savage, G.P.; Meutermans, W.; Montgomery, A.P.; Kassiou, M. Fragment-based drug discovery for disorders of the central nervous system: Designing better drugs piece by piece. Front. Chem. 2024, 12, 1379518. [Google Scholar] [CrossRef] [PubMed]
  22. Mallakuntla, M.K.; Togre, N.S.; Santos, D.B.; Tiwari, S. Implications of Fragment-Based Drug Discovery in Tuberculosis and HIV. Pharmaceuticals 2022, 15, 1415. [Google Scholar] [CrossRef] [PubMed]
  23. Wood, D.J.; Lopez-Fernandez, J.D.; Knight, L.E.; Al-Khawaldeh, I.; Gai, C.; Lin, S.; Martin, M.P.; Miller, D.C.; Cano, C.; Endicott, J.A.; et al. FragLites-Minimal, Halogenated Fragments Displaying Pharmacophore Doublets. An Efficient Approach to Druggability Assessment and Hit Generation. J. Med. Chem. 2019, 62, 3741–3752. [Google Scholar] [CrossRef]
  24. Lamoree, B.; Hubbard, R.E. Current perspectives in fragment-based lead discovery (FBLD). Essays Biochem. 2017, 61, 453–464. [Google Scholar] [CrossRef]
  25. Hope, I.; Martin, M.P.; Jiang, Z.; Waring, M.J.; Noble, M.E.M.; Endicott, J.A.; Tatum, N.J. Crystallographic fragment screening of CDK2-cyclin A: FragLites map sites of protein-protein interaction. Structure 2025, 33, 1971–1983. [Google Scholar] [CrossRef]
  26. Martin, M.P.; Endicott, J.A.; Noble, M.E.M.; Tatum, N.J. Crystallographic fragment screening in academic cancer drug discovery. Methods Enzymol. 2023, 690, 211–234. [Google Scholar] [CrossRef]
  27. Tang, S.; Zhou, Z.; Jiang, Z.; Zhu, W.; Qiao, D. Indole-Based Tubulin Inhibitors: Binding Modes and SARs Investigations. Molecules 2022, 27, 1587. [Google Scholar] [CrossRef]
  28. Wang, Q.; Arnst, K.E.; Wang, Y.; Kumar, G.; Ma, D.; Chen, H.; Wu, Z.; Yang, J.; White, S.W.; Miller, D.D.; et al. Structural Modification of the 3,4,5-Trimethoxyphenyl Moiety in the Tubulin Inhibitor VERU-111 Leads to Improved Antiproliferative Activities. J. Med. Chem. 2018, 61, 7877–7891. [Google Scholar] [CrossRef]
  29. Li, L.; Jiang, S.; Li, X.; Liu, Y.; Su, J.; Chen, J. Recent advances in trimethoxyphenyl (TMP) based tubulin inhibitors targeting the colchicine binding site. Eur. J. Med. Chem. 2018, 151, 482–494. [Google Scholar] [CrossRef]
  30. Bates, T.A.; Gurmessa, S.K.; Weinstein, J.B.; Trank-Greene, M.; Wrynla, X.H.; Anastas, A.; Anley, T.W.; Hinchliff, A.; Shinde, U.; Burke, J.E.; et al. Biolayer interferometry for measuring the kinetics of protein-protein interactions and nanobody binding. Nat. Protoc. 2025, 20, 861–883. [Google Scholar] [CrossRef]
  31. Zhu, T.; Wang, S.H.; Li, D.; Wang, S.Y.; Liu, X.; Song, J.; Wang, Y.T.; Zhang, S.Y. Progress of tubulin polymerization activity detection methods. Bioorg. Med. Chem. Lett. 2021, 37, 127698. [Google Scholar] [CrossRef]
  32. Jędrzejczyk, M.; Morabito, B.; Żyżyńska-Granica, B.; Struga, M.; Janczak, J.; Aminpour, M.; Tuszynski, J.A.; Huczyński, A. Novel Combretastatin A-4 Analogs—Design, Synthesis, and Antiproliferative and Anti-Tubulin Activity. Molecules 2024, 29, 2200. [Google Scholar] [CrossRef]
  33. Abdul Hussein, S.A.; Razzak Mahmood, A.A.; Tahtamouni, L.H.; Balakit, A.A.; Yaseen, Y.S.; Al-Hasani, R.A. New Combretastatin Analogs as Anticancer Agents: Design, Synthesis, Microtubules Polymerization Inhibition, and Molecular Docking Studies. Chem. Biodivers. 2023, 20, e202201206. [Google Scholar] [CrossRef] [PubMed]
  34. Romagnoli, R.; Oliva, P.; Salvador, M.K.; Manfredini, S.; Padroni, C.; Brancale, A.; Ferla, S.; Hamel, E.; Ronca, R.; Maccarinelli, F.; et al. A facile synthesis of diaryl pyrroles led to the discovery of potent colchicine site antimitotic agents. Eur. J. Med. Chem. 2021, 214, 113229. [Google Scholar] [CrossRef]
Molecules 30 04538 g001
Figure 1. (A) Design rationale for the hit compound (H1) and its optimized derivatives (3a–3p). (B) Predicted binding pose of two fragments 6-chloro-1H-indole (Fr1) in orange and 5-iodo-1,2,3-trimethoxybenzene (Fr2) in green, respectively, within the colchicine-binding site of β-tubulin (PDB ID: 3E22). (C) Predicted binding pose of the merged hit compound H1 in pink at the colchicine-binding site. (D) Two-dimensional schematic of the detailed interactions between hit H1 and the colchicine-binding pocket. The computational docking was achieved by MOE. The image was generated by CCP4MG 2.10.6.
Figure 1. (A) Design rationale for the hit compound (H1) and its optimized derivatives (3a–3p). (B) Predicted binding pose of two fragments 6-chloro-1H-indole (Fr1) in orange and 5-iodo-1,2,3-trimethoxybenzene (Fr2) in green, respectively, within the colchicine-binding site of β-tubulin (PDB ID: 3E22). (C) Predicted binding pose of the merged hit compound H1 in pink at the colchicine-binding site. (D) Two-dimensional schematic of the detailed interactions between hit H1 and the colchicine-binding pocket. The computational docking was achieved by MOE. The image was generated by CCP4MG 2.10.6.
Molecules 30 04538 g001
Molecules 30 04538 sch001
Scheme 1. Synthesis of 6-substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole species. Reaction condition: (a) iodobenzene analogs, Pd(PPh3)4 (0.06 eq.), Na2CO3 (2 eq.), 1,4-Dioxane, H2O, 80 °C, and overnight; (b) 5-iodo-1,2,3-trimethonxybenzene, CuI (0.15 eq.), Cs2CO3 (0.6 eq.), DMF, 150 °C, and 16 h.
Scheme 1. Synthesis of 6-substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole species. Reaction condition: (a) iodobenzene analogs, Pd(PPh3)4 (0.06 eq.), Na2CO3 (2 eq.), 1,4-Dioxane, H2O, 80 °C, and overnight; (b) 5-iodo-1,2,3-trimethonxybenzene, CuI (0.15 eq.), Cs2CO3 (0.6 eq.), DMF, 150 °C, and 16 h.
Molecules 30 04538 sch001
Molecules 30 04538 g002
Figure 2. Compound 3g disturbs microtubule polymerization. Tubulin polymerization assay with 2.5, 5, 10, 25, and 50 μM of compound 3g; 0.1% DMSO; 10 μM paclitaxel; and 10 μM colchicine. Absorbance at 340 nm was determined every 1 min.
Figure 2. Compound 3g disturbs microtubule polymerization. Tubulin polymerization assay with 2.5, 5, 10, 25, and 50 μM of compound 3g; 0.1% DMSO; 10 μM paclitaxel; and 10 μM colchicine. Absorbance at 340 nm was determined every 1 min.
Molecules 30 04538 g002
Molecules 30 04538 g003
Figure 3. Inhibition of MCF-7 Cell Colony Formation by Compound 3g. (A) 3g inhibited the colony formation of MCF-7 cells at different concentrations in a dose-dependent manner. (B) Colony units in the different groups after treatment with 3g. Number of colonies was counted after treating MCF-7 cells with the indicated concentration of compound 3g for 24 h (n = 3, * p < 0.05, ** p < 0.01 vs. DMSO control, one-way ANOVA).
Figure 3. Inhibition of MCF-7 Cell Colony Formation by Compound 3g. (A) 3g inhibited the colony formation of MCF-7 cells at different concentrations in a dose-dependent manner. (B) Colony units in the different groups after treatment with 3g. Number of colonies was counted after treating MCF-7 cells with the indicated concentration of compound 3g for 24 h (n = 3, * p < 0.05, ** p < 0.01 vs. DMSO control, one-way ANOVA).
Molecules 30 04538 g003
Molecules 30 04538 g004
Figure 4. Effect of compound 3g on the migration of MCF-7 breast cancer cells. (A) 3g inhibited the migration of MCF-7 cells at different concentrations in a dose-dependent manner. Representative images of wound healing were captured by an inverted microscope at 0 h and 24 h treatment. Scale bar = 200 μm. (B) Wound closure percentages in the different groups after treatment with 3g for 24 h. (C) Results of the transwell migration assay. Representative images of the stained migrated cells after incubation with 3g at different concentrations for 48 h. Scale bar = 200 μm. Data are presented as the mean ± SD of three independent experiments. Bar values represent the standard deviation SD. ** p < 0.01, and *** p < 0.001 (one-way ANOVA).
Figure 4. Effect of compound 3g on the migration of MCF-7 breast cancer cells. (A) 3g inhibited the migration of MCF-7 cells at different concentrations in a dose-dependent manner. Representative images of wound healing were captured by an inverted microscope at 0 h and 24 h treatment. Scale bar = 200 μm. (B) Wound closure percentages in the different groups after treatment with 3g for 24 h. (C) Results of the transwell migration assay. Representative images of the stained migrated cells after incubation with 3g at different concentrations for 48 h. Scale bar = 200 μm. Data are presented as the mean ± SD of three independent experiments. Bar values represent the standard deviation SD. ** p < 0.01, and *** p < 0.001 (one-way ANOVA).
Molecules 30 04538 g004
Molecules 30 04538 g005
Figure 5. Effects of compound 3g on microtubules. MCF-7 cells were treated with vehicle control 0.1% DMSO, compound 3g (0.1μM, 1μM, and 10 μM for 24 h). Microtubules were visualized with an anti-β-tubulin antibody (red), and the cell nucleus was visualized with DAPI (blue).
Figure 5. Effects of compound 3g on microtubules. MCF-7 cells were treated with vehicle control 0.1% DMSO, compound 3g (0.1μM, 1μM, and 10 μM for 24 h). Microtubules were visualized with an anti-β-tubulin antibody (red), and the cell nucleus was visualized with DAPI (blue).
Molecules 30 04538 g005
Molecules 30 04538 g006
Figure 6. Cell cycle arrest induced by compound 3g. (A) Compound 3g induced G2/M arrest in MCF-7 cells. MCF-7 cells were incubated with varying concentrations of compound 3g (0.1, 1, and 10 μM) for 48 h. The percentages of cells in different phases of the cell cycle were analyzed by FlowJo 10.8.1. (B) Histograms display the percentage of cell cycle distribution after treatment with compound 3g. Data are expressed as mean ± standard deviation from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.
Figure 6. Cell cycle arrest induced by compound 3g. (A) Compound 3g induced G2/M arrest in MCF-7 cells. MCF-7 cells were incubated with varying concentrations of compound 3g (0.1, 1, and 10 μM) for 48 h. The percentages of cells in different phases of the cell cycle were analyzed by FlowJo 10.8.1. (B) Histograms display the percentage of cell cycle distribution after treatment with compound 3g. Data are expressed as mean ± standard deviation from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.
Molecules 30 04538 g006
Molecules 30 04538 g007
Figure 7. Effects of compound 3g on apoptosis in MCF-7 cells. (A) Apoptosis induction by compound 3g in MCF-7 cells. Cells were treated with increasing concentrations of 3g (0.1, 1, and 10 μM) for 48 h, followed by Annexin V-FITC/PI staining and flow cytometric analysis. Cell populations were categorized as necrotic (upper left quadrant), late apoptotic (upper right), viable (lower left), and early apoptotic (lower right). (B) Quantification of apoptotic populations. Bar graphs show the percentage of cells in each apoptotic stage after treatment. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.
Figure 7. Effects of compound 3g on apoptosis in MCF-7 cells. (A) Apoptosis induction by compound 3g in MCF-7 cells. Cells were treated with increasing concentrations of 3g (0.1, 1, and 10 μM) for 48 h, followed by Annexin V-FITC/PI staining and flow cytometric analysis. Cell populations were categorized as necrotic (upper left quadrant), late apoptotic (upper right), viable (lower left), and early apoptotic (lower right). (B) Quantification of apoptotic populations. Bar graphs show the percentage of cells in each apoptotic stage after treatment. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test.
Molecules 30 04538 g007
Molecules 30 04538 g008
Figure 8. Compound 3g inhibits melanoma tumor growth in vivo. Murine melanoma B16-F10 xenograft tumors were established in mice, which were then administered vehicle, PTX (10 mg/kg per day), compound 3g (20 mg/kg per day), and compound 3g (50 mg/kg per day) for 14 days. The mice were sacrificed, and the tumors were weighed. (A) Images of tumors from mice at 14 days after initiation of treatment. (B) Body weight changes in mice during treatment. (C) Weight of the excised tumors of each group. * p < 0.05, and *** p < 0.001 vs. control group. (D) Tumor volume changes in mice during treatment. Statistical analyses were performed using one-way ANOVA (tumor weight) or two-way repeated-measures ANOVA (tumor volume).
Figure 8. Compound 3g inhibits melanoma tumor growth in vivo. Murine melanoma B16-F10 xenograft tumors were established in mice, which were then administered vehicle, PTX (10 mg/kg per day), compound 3g (20 mg/kg per day), and compound 3g (50 mg/kg per day) for 14 days. The mice were sacrificed, and the tumors were weighed. (A) Images of tumors from mice at 14 days after initiation of treatment. (B) Body weight changes in mice during treatment. (C) Weight of the excised tumors of each group. * p < 0.05, and *** p < 0.001 vs. control group. (D) Tumor volume changes in mice during treatment. Statistical analyses were performed using one-way ANOVA (tumor weight) or two-way repeated-measures ANOVA (tumor volume).
Molecules 30 04538 g008
Molecules 30 04538 g009
Figure 9. Pathological sections of major tissues (liver and kidney) were obtained from mice bearing melanoma tumors. Organs were stained with H&E, and the representative images were captured.
Figure 9. Pathological sections of major tissues (liver and kidney) were obtained from mice bearing melanoma tumors. Organs were stained with H&E, and the representative images were captured.
Molecules 30 04538 g009
Table 1. Binding Affinities of Compounds to Tubulin Protein (KD Values [μM] Were Determined from the Biolayer Interferometry Assay).
Table 1. Binding Affinities of Compounds to Tubulin Protein (KD Values [μM] Were Determined from the Biolayer Interferometry Assay).
IDKD (μM)IDKD (μM)
3a39 ± 2.603iNB
3b29 ± 4.103jNB
3c23 ± 2.123kND
3d24 ± 2.133l26 ± 3.38
3e110 ± 14.463mNB
3f47 ± 5.623n220 ± 30.80
3g13 ± 1.173o31 ± 3.41
3hND3pNB
Colchicine8.60 ± 1.20
ND = not detected (no specific binding detected); NB = no binding or very weak binding.
Table 2. In Vitro Antiproliferative Activities of Compounds against Six Cancer Cell Lines.
Table 2. In Vitro Antiproliferative Activities of Compounds against Six Cancer Cell Lines.
Compd.IC50 (μM)
MCF-7MDA-MB-231A549HelaA375B16-F10
3a10.13 ± 0.6878.92 ± 0.5736.60 ± 1.78>1001.16 ± 0.1245.36 ± 1.11
3b3.33 ± 0.2842.93 ± 3.1341.20 ± 3.4238.52 ± 2.2527.53 ± 2.503.78 ± 0.14
3c9.15 ± 0.2137.72 ± 0.8992.44 ± 8.5866.50 ± 6.2153.16 ± 4.0659.21 ± 5.86
3d34.22 ± 1.6223.37 ± 0.4460.24 ± 4.1746.60 ± 3.6524.84 ± 0.166.49 ± 0.40
3e>100>100>100>100>100>100
3f17.39 ± 0.3521.37 ± 0.1287.43 ± 3.4541.80 ± 1.1121.07 ± 1.176.67 ± 0.29
3g2.94 ± 0.561.61 ± 0.0046.30 ± 0.306.10 ± 0.310.57 ± 0.011.69 ± 0.41
3h>100>100>100>100>100>100
3i>100>100>100>100>100>100
3j>100>100>100>100>100>100
3k>100>100>100>100>100>100
3l>100>100>100>100>100>100
3m>100>100>100>100>100>100
3n14.17 ± 0.5141.37 ± 1.2148.01 ± 2.5665.00 ± 3.2016.10 ± 0.3868.94 ± 3.17
3o>100>100>100>100>100>100
3p>100>100>100>100>100>100
Colchicine0.26 ± 0. 080.48 ± 0.030.082 ± 0.0060.027 ± 0.0020.110 ± 0.020.013 ± 0.001
IC50: Values [μM] Were Determined from the Cell Viability Assay for 48 h.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Gu, Y.; Gai, C.; Zou, S.; Song, Y.; Zhang, J.; Zhao, Q.; Chai, X.; Wang, P. Design, Synthesis and Anticancer Activity of 6-Substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole Against Tubulin Polymerisation. Molecules 2025, 30, 4538. https://doi.org/10.3390/molecules30234538

AMA Style

Gu Y, Gai C, Zou S, Song Y, Zhang J, Zhao Q, Chai X, Wang P. Design, Synthesis and Anticancer Activity of 6-Substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole Against Tubulin Polymerisation. Molecules. 2025; 30(23):4538. https://doi.org/10.3390/molecules30234538

Chicago/Turabian Style

Gu, Yuanna, Conghao Gai, Sijie Zou, Yan Song, Juan Zhang, Qingjie Zhao, Xiaoyun Chai, and Peipei Wang. 2025. "Design, Synthesis and Anticancer Activity of 6-Substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole Against Tubulin Polymerisation" Molecules 30, no. 23: 4538. https://doi.org/10.3390/molecules30234538

APA Style

Gu, Y., Gai, C., Zou, S., Song, Y., Zhang, J., Zhao, Q., Chai, X., & Wang, P. (2025). Design, Synthesis and Anticancer Activity of 6-Substituted-1-(3,4,5-trimethoxyphenyl)-1H-indole Against Tubulin Polymerisation. Molecules, 30(23), 4538. https://doi.org/10.3390/molecules30234538

Article Metrics

Back to TopTop