L-Proline catalysed synthesis and in silico studies of novel α-cyano bis(indolyl)chalcones as potential anti-cancer agents

Abstract

A diverse range of α-cyano bis(indolyl)chalcones (21a–r) were synthesized in high yields (90–95%) through the L-proline catalysed reaction of appropriate aldehydes and 3-cyanoacetylindoles. Formation of α-cyano bis(indolyl)chalcones involves readily available starting materials, catalytic L-proline, environmentally benign and metal-free conditions. The prepared eighteen α-cyano bis(indolyl)chalcones 21a–r were screened against prostate, breast, epithelial cancer cells and found to be non-cytotoxic to normal HEK293 cells. The α-cyano bis(indolyl)chalcones 21a (3.9 μM), 21c (7.5 μM), 21i (2.2 μM) and 21o (5.9 μM) displayed good cytotoxicity against C4-2 cells, whereas, derivatives 21c (1.23 μM), 21h (5.23 μM), and 21l (2.5 μM) showed selective cytotoxicity against 22Rv1 cells. With broad spectrum of activity (0.98–5.6 μM), the compound 21j was found to increase the endogenous level of ROS, upregulate the level of p-53 and c-jun besides mitochondrial dysfunction, cause apoptosis.

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1. Introduction

The indole motif is a key building block of many naturally occurring and synthetic compounds endowed with different biological activities. Heterocyclic compounds are a significant source of pharmacologically active compounds, and can be obtained from organic synthesis or isolated from natural products.¹ Heterocyclic compounds with one or more nitrogen, oxygen or sulphur, besides at least one carbon in the ring could be used as potential hydrogen bond donors and acceptors. With wider presence among a large number of heterocyclic compounds endowed with interesting medicinal properties, indole and derived compounds have been pursued with greater interest.² Particular attention has been paid to bisindole containing compounds which are known to exhibit interesting anticancer activities by affecting numerous biological targets.³ Additionally, bisindole alkaloids, isolated from marine sources, continue to inspire for the development of novel anticancer agents. Most of the bisindole alkaloids possess two indole units connecting either through a linear or heterocyclic ring spacer as shown in Fig. 1. Isolated from marine sponges Topsentia genitrix and Spongosorites, bisindole alkaloids, Topsentin (1) and Nortopsentin (2) with an imidazole linker, have been reported to display potent cytotoxicity against diverse cancers.⁴ Another emerging class of marine alkaloid, Dragmacidin B (3) with a piperazine linker, was isolated from the deep-water marine sponge Hexadella sp. and found to display good anticancer activity (IC₅₀ = 15 μg mL⁻¹, P388; 1–10 μg mL⁻¹, A-549, HCT-8 and MDA-MB-231).⁵

Fig. 1 Indole analogues as anticancer agents.

Despite the interesting anticancer activity exerted by bisindole alkaloids, in the recent past, researchers have identified several synthetic analogues of bisindole alkaloids with improved anticancer properties.^6,7 Kumar et al. identified bis(indolyl)-1,3,4-oxadiazoles (4) as apoptosis inducing cytotoxic agents (IC₅₀ = 20 nM; HeLa).⁸ Several synthetic analogues of bisindole alkaloids with variety of cyclic spacers such as thiazole (5), have been reported for their cytotoxic properties (Fig. 1).⁹ The inhibition of tubulin polymerization is a well-established strategy for anticancer drug development, as microtubules play an essential role in cellular processes such as mitosis, intracellular transport, and cell motility.¹⁰ Tubulin targeting substances are broadly categorized into microtubule stabilizing (taxanes, epothilones and discodermolide) and destabilizing (colchicine, vinca alkaloids, and CA-4P) agents. Microtubules targeting agents (MTAs) (6) that bind to the colchicine binding site (also called CBSI) have the potential to overcome the drawbacks associated with taxanes and vinca alkaloids, owing to their structural simplicity, non-substrate nature for the multidrug resistance protein 1 (P-glycoprotein) and reasonable physicochemical properties.^11,12

Kamal and Nagesh group explored chalcone linked β-carboline hybrids (7) as anti-topoisomerase-I, DNA-interactive, and apoptosis inducing anticancer agents.¹³ The α-methylated chalcones (8) were prepared by Kamal and Pal-Bhadra group showed 10-folds enhanced activity when compared to their parent derivatives.¹⁴ Natural and synthetic chalcones have been reported to show diverse biological activities such as antiinflammatory,¹⁵ antimalarial,¹⁶ antiviral, antifungal, antibacterial¹⁷ and anticancer.¹⁷ The N,N-dimethylamino substituted acrylonitrile bearing N-isobutyl and cyano substituents placed on the benzimidazole nuclei (9), showed strong and selective antiproliferative activity in submicromolar range of inhibitory concentrations (IC₅₀ = 0.2–0.6 μM), while being significantly less toxic than reference systems docetaxel and staurosporine, thus promoting them as lead compounds.¹⁸ Coscinamides A–C (10–12) with a linear α-keto enamide spacer from the extract of marine sponge Coscinoderma sp., were reported to exhibit antitumor activity against human prostate cancer cell line (IC₅₀ = 7.6 μg mL⁻¹).¹⁹ Isolated from a red alga Chondria sp. two cytotoxic bis-indole amides, chondriamides A–B, were found to be cytotoxic against KB and LOVO cell lines (IC₅₀ = 0.5–10 μg mL⁻¹).²⁰ In 2023, Jan et al. conducted a study focused on synthesizing novel bis-indole analogues containing a phenyl linker derived from indole phytoalexins. The synthesis involved the reaction of [1-(tert-butoxycarbonyl)indol-3-yl]methyl-isothiocyanate with p-phenylenediamine to obtain the target bis-indole thiourea (13) linked with a phenyl linker.⁷ Literature reports show that the indole scaffold frequently encounters in anticancer drug discovery research as illustrated in Fig. 1. On the other hand, the α,β-unsaturated ketones also known as chalcones are reported to play a vital role in the identification of bioactive molecules. Indolyl chalcones (14) as potential cytotoxic agents.²¹ Boumendjel et al. first reported that α-substituted chalcones are more potent than their unsubstituted analogs.^22a Li and Huang group reported (15), exhibited most potent activity, with IC₅₀ values of 3–9 nM and also displayed excellent tubulin polymerization inhibitory activity with an IC₅₀ of 2.68 μM.^22b

Inspired from the interesting anticancer activities of naturally occurring bis-indoles with linear chain spacers, Kumar et al. report ed bis(indolyl)hydrazide-hydrazones (16) as potent cytotoxic agents (IC₅₀ = 1 μM; MDA-MB-231).²³ Over a period of time, several chalcones have been reported with structural modifications around the basic enone template.¹⁴ Liu X. et al. reported the synthesis of hybrid molecules containing indole and 3,4,5-trimethoxy-phenyl moieties as tubulin targeting agents. Among them, a fluorine-containing derivative (17) (Fig. 2) exhibited significant inhibitory activity toward HCT116 and CT26 cells.^24a Additionally, the presence of a α-cyano moiety in enone framework likely to plays an important role in view of its several advantageous properties, including enhanced binding affinity, improved pharmacokinetic profiles and reduced drug resistance.^25,26a Trans-indol-3-ylacrylamide (18) exhibited antiproliferative activity, with an IC₅₀ value of 5 μM in Huh7 cells.^24b In 2011, Venkatanarayana and Dubey reported triphenylphosphine (40 mol%) promoted synthesis of α-cyano bis(indolyl)chalcones involving the reaction of indole-3-carboxaldehydes with 3-cyanoacetylindole.^26b Cluskey et al. reported a library of substituted acrylonitriles via piperidine catalysed reaction of 3-oxo-3-(1H-pyrrol-2-yl)propanenitrile with various substituted benzaldehydes at 70 °C.^26c In 2014, Kumar et al. prepared different α-cyano bis(indolyl)chalcones by employing microwave-assisted reaction of indole-3-carboxaldehyde with 3-cyanoacetylindole in presence of piperidine (0.2 mL for 1 mmol) at 80 °C. Some of the α-cyano bis(indolyl) chalcones exhibited cytotoxicity against lung cancer cells and their preliminary mechanism of action studies indicated weak enhancement of tubulin polymerization.^26d In our current work, the L-proline catalysed economical and practical synthesis of α-cyano bis(indolyl)chalcones is advantageous due to high product yields, easy isolation and ambient reaction conditions. The heterocyclic pharmacophores in medicinal chemistry plays a crucial role in developing potent moieties towards successful cancer drug design. Among them all bis-indole derivatives are the leading structural fragments which play an important role in synthetic and medicinal chemistry as well as various other fields. With the observed side effects and high resistance rate against available drugs; identification of new and potent chemical entities is desirable to tackle the increasing disease problem.^9,27 In efforts to develop eco-friendly protocol and identify potent and selective tubulin interacting agents, in present work, we prepared a new set of α-cyano bis(indolyl)chalcones with improved colchicine binding site affinity, via proline catalysed reaction of readily available indole-3-carboxaldehyde with 3-cyanoketones in ethanol at room temperature.

Fig. 2 Rational design of 21.

2. Results and discussion

2.1 Synthesis

Synthesis of α-cyano bis(indolyl)chalcone (21a) involves the reaction of 3-cyanoacetyl indole (19a) with N-methyl indole-3-carboxaldehyde 20a in presence of a base as described in Table 1. Initially, the reaction of 19a with 20a in presence of either piperidine or triethylamine resulted in lower product yield (Table 1, entries 1 and 2). Next, with the use of KOH as a base at 25 °C produced the expected α-cyano bis(indolyl)chalcone 21a in 80% yield (Table 1, entry 3). With the increase in reaction time from 2.5 h to 3 h, the product yield was increased to 93% (Table 1, entry 4). When a neat mixture of 3-cyanoacetylindole 19a and N-ethylindole-3-carboxyaldehyde (20a) in the presence of KOH was grinded at room temperature for 0.5 h, bis-indole 21a was formed in 88% yield (Table 1, entry 5). No product was obtained when the same reaction was performed in absence of base (Table 1, entry 6). The use of L-tryptophan as a catalyst at 25 °C produced the expected α-cyano bis(indolyl)chalcone 21a in 42% yield (Table 1, entry 7).

Table 1 Optimization of reaction conditions for 21a^a,b

Entry	Solvent	Base	Temp.	Time (h)	Yield^b (%)
a Reagents and conditions: 19a (1.0 mmol, 1.0 equiv.), 20a (1.0 equiv.), L-proline or L-tryptophan (0.1 equiv.), EtOH (3 mL) at 25 °C.b Isolated yield.c Grinding.d NR; no reaction.
1	EtOH	Piperidine	25 °C	2.5	48
2	EtOH	Et3N	25 °C	2.5	45
3	EtOH	KOH	25 °C	2.5	80
4	EtOH	KOH	25 °C	3.0	93
5	—	KOH	25 °C	0.5	88^c
6	EtOH	—	25 °C	2.5	NR^d
7	EtOH	L-Tryptophan	25 °C	2.5	42
8	EtOH	L-Proline	25 °C	2.5	67
9	EtOH	L-Proline	25°C	5.5	95
10	EtOH : H2O (1 : 1)	L-Proline	25 °C	5.5	78
11	EtOH	—	25°C	5.5	NR^d

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In the recent past, the natural and inexpensive L-proline has been widely used as a prominent organocatalyst for carbon–carbon bond formation in various organic transformations.^26a In view of success of proline catalysed transformations along with its inexpensive and environmentally benign nature, next we performed the reaction of 3-cyanoacetylindole 19a and N-ethyl indole-3-carboxyaldehyde 20a in the presence of readily available and metal-free catalytic L-proline, in ethanol at 25 °C, to obtain 21a in 95% yield (Table 1, entry 9) as illustrated in Scheme 1. Using a mixture of solvents (EtOH : H₂O), desired product 21a obtained in 78% yield (Table 1, entry 10). However, in absence of L-proline, the reaction was failed to produce 21a (Table 1, entry 11). The structure of 21a was confirmed by the NMR (¹H & ¹³C) and mass analysis. The proton NMR of 21a displayed characteristic singlets at 8.50 ppm (alkenyl proton, =CH), 4.44 ppm (CH₂) while CH₃ protons resonated at 1.46 ppm.

Scheme 1 Synthesized α-cyano bis(indolyl)chalcones 21a–r.

In ¹³C NMR spectrum signals due to carbonyl, nitrile carbon, alkenyl (=CH), CH₃ and CH₂ carbons were observed at about 181.26, 121.07, 144.96, 42.18 and 15.60 ppm, respectively. In IR spectra, a characteristic sharp peak at ∼2200 cm⁻¹ was observed due to the presence of a nitrile functional group. Mass spectrum of 21a showed a peak at m/z 340.1595 in agreement with the calculated mass at m/z 340.1405 (M + H)⁺ for C₂₂H₁₇N₃O. Similarly, using the optimized reaction conditions, compounds 21b–r were prepared in high yields (90–95%) and characterized the products by their NMR and mass spectral data (Scheme 1).

The formation of 21 may be rationalized by the in situ generation of enamine intermediate I by the condensation of 3-cyanoacetyl-indole 19a and L-proline. Further, the nucleophilic addition of enamine I to the carbonyl group of indole-3-carboxaldehyde (20) and followed by dehydration is likely to produce 21 as illustrated in Scheme 2.

Scheme 2 Plausible mechanistic pathway for the formation of α-cyano bis(indolyl)chalcones.

2.2 Biological evaluation

2.2.1 Anticancer activity. The cytotoxicity of the synthesized α-cyano bis(indolyl)chalcones derivatives 21a–r were evaluated against five human cancer cell lines; prostate (C4-2, PC3 and 22Rv1), breast (MCF7), epithelial cancer (MIAPACA) and normal human kidney (HEK293) cell lines by using MTT assay. The anticancer activity is expressed in terms of IC₅₀ values for inhibition of tumor cell growth as provided in Table 2. A structure–activity relationship (SAR) study was conducted by synthesizing a diverse set of α-cyano bis(indolyl)chalcones with various substituents on both indole rings (Fig. 3).

Table 2 IC₅₀ (μM) values of α-cyano bis(indolyl)chalcones (21a–r)

Compd	MCF7	PC3	C4-2	22Rv1	MIAPACA	HEK293
21a	10.2	13	3.9	>40	>40	>40
21b	6.9	10.6	13.5	21.5	4.9	40
21c	25.3	15.45	7.5	1.23	4.5	>40
21d	10.9	>40	8	25	15.5	39.2
21e	12.47	7.61	15.7	28	>40	>40
21f	37	24	12.5	>40	>40	40
21g	10.5	14.3	13.3	34	7.9	>40
21h	16.75	27.3	10	5.23	1.35	40
21i	7.38	2.63	2.2	8.9	5.8	>40
21j	1.2	5.6	0.98	2.9	5.3	>40
21k	18.6	16.1	13.8	28	28.9	39.8
21l	7.95	4.6	26.4	2.5	37.6	37.9
21m	2.98	11.7	17.6	16.4	1.6	>40
21n	5.61	7.1	1.02	20.5	15.6	>40
21o	31.5	19	5.9	26	12.95	>40
21p	>40	>40	31	>40	>40	>40
21q	25	37	>40	28	15.4	>40
21r	>40	25	>40	34	37	31

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Fig. 3 SAR for the α-cyano bis(indolyl)chalcones (21a–r).

Most of the synthesized compounds are not inhibiting the normal cells (HEK293). Initial bis-indole 21a was found to be selectively cytotoxic against C4-2 (IC₅₀ = 3.9 μM). Next, protection of indole-NH with p-chlorobenzyl moiety produced 21b with moderate activity. Incorporation of a C5-methoxy group in indole moiety led to compounds 21c and 21d. Particularly, compound 21c with 5-methoxyindole and N-methylated indole displayed selective cytotoxicity against 22Rv1 (1.23 μM). With the change in position of methoxy group in indole ring from C-5 to C-6 and protection of indole NH with p-chlorobenzyl group led to compounds 21e, 21f and 21g with reduced activity. To optimize the size of C5-alkoxy group, ethoxy and propyloxy derivatives 21h, 21i and 21j were prepared with significantly enhanced cytotoxicity against the tested cancer cells. By the introduction of 5-isopropyloxy group led to 21l with improved selectivity against prostate cancer cells (2.5 μM, 22Rv1 cells). The presence of an additional methoxy group in indole ring and protection of indole NH as N-Me/N-Et (compounds 21n and 21o with 5,6-dimethoxyindole moiety) improved the selectivity against prostate cancer cell lines. The protection of indole nitrogen with p-chlorobenzyl moiety (compounds 21p) or replacement of the indole ring with a phenyl group (compounds 21q and 21r) or was found to be detrimental for the activity. These activity results suggest that both the indole rings are necessary for the potency of α-cyano bis(indolyl)chalcones. Protection of second indole ring as N-Et (21j) instead of N-Me (21k) or N-chlorobenzyl (21b) is also beneficial for the activity. Particularly, 21j with C-5 propyloxy substituent and N-ethylindole was found to be the best compound of series with an IC₅₀ value of 0.98 μM against C4-2 (prostate) cancer cells.

2.2.2 Ethidium bromide (EB)/acridine orange (AO) assay for simultaneously ascertaining cell viability and death. Our results showed that 21j was the most effective compound across all cancer cell lines tested, with no notable impact on non-cancerous HEK293 cells, prompting us to investigate the mechanism of cell death. We initially employed EB/AO staining to ascertain the percentage of dead and live cells. AO stains both live and dead cells. In contrast, ethidium bromide (EB) stains only dead cells. Treatment of compound 21j in C4-2 cells for 48 h caused robust cell death as indicated by enhanced red fluorescence as compared to control DMSO-treated cells (Fig. 4).

Fig. 4 Fluorescent AO images of C4-2 cells treated with DMSO or 21j for 48 h, followed by AO–EB staining. Control cells are viable as they show green fluorescence. Both 10 and 20 μM concentrations of 21j induced substantial apoptosis as visualized by the red fluorescence.

2.2.3 21j triggers mitochondrial depolarization in C4-2 cells. Mitochondria are known to generate significant amount of ROS from at least ten different sites, which in turn increases intracellular oxidative stress. As oxidative stress triggers mitochondrial damage, we investigated whether 21j exposure could cause mitochondrial depolarization. C4-2 cells were treated with 10 μM or 20 μM of compound 21j for 48 h, both of which induced significant mitochondrial depolarization, thereby confirming that 21j cytotoxicity at least partly arises due to mitochondrial dysfunction (Fig. 5).

Fig. 5 21j induces mitochondrial depolarization in C4-2 cells. Cells were treated with either DMSO or 21j at concentrations of 10 μM and 20 μM for 48 h, and then stained with JC-1. Images were captured using a fluorescence microscope (Keyence) with FITC (green) and TRITC (red) channels at a 20× magnification objective.

2.2.4 Induces reactive oxygen species (ROS) accumulation. Increased oxidative stress induces cell death. Therefore, we examined whether compound 21j promotes ROS using DCFDA staining in C4-2 cells. Both 10 μM and 20 μM concentrations of 21j induced robust increase in ROS levels (Fig. 6). These results confirm that 21j induces cytotoxicity at least in part by increasing oxidative stress.

Fig. 6 21j raises the levels of ROS in C4-2 cells. C4-2 cells were treated with either 10 μM and 20 μM of 21j for 48 h. Following treatments, they were stained using H2-DCFDA. The bar graph depicts relative ROS levels (green signal) using control DMSO, 10 μM 21j-treated and 20 μM 21j-treated cells. Data analysis was performed using three independent replicates, and statistical significance between DMSO (control), 10 and 20 μM 21j-treated cells were performed using Student's T-test. *P < 0.05.

2.2.5 Induces tubulin depolymerization. Low levels of ROS levels promote cytoskeleton polymerization, but high levels of ROS levels inhibit microtubule polymerization. As 21j increases ROS levels, we examined whether 21j has an effect on tubulin polymerization in C4-2 cells. Accordingly, C4-2 cells were exposed to 21j for 48 h and then analysed using β-tubulin antibody. These data showed significant loss of tubulin assembly in 21j-exposed group. These cells resembled colchicine-treated cells, which is a highly potent tubulin polymerization inhibitor (Fig. 7).

Fig. 7 21j increases tubulin depolymerization in C4-2 cells. C4-2 cells were exposed to either DMSO (negative control) or 21j at two concentrations (10 μM and 20 μM) for 48 h and stained with tubulin antibody. Colchicine was used as the positive control. Photographs were taken in FITC (green) and DAPI (blue) channels at 20× objective with the fluorescence microscope (Keyence).

To further validate a potential impact of 21j on tubulin dynamics, we measured the relative concentrations of polymerized versus depolymerized tubulin in control and 21j-treated C4-2 cells. As a positive control, colchicine was employed. While colchicine treatment significantly decreased the levels of polymerized tubulin by ∼30% as compared to control. 21j treatment also resulted in ∼20% less polymerized tubulin. These results suggest that at least some percentage of 21j's anti-cancer effect stems from its tubulin-depolymerizing activity (Fig. 8).

Fig. 8 Tubulin polymerization is increased by 21j in C4-2 cells. (a) C4-2 cells were exposed to colchicine (100 nM) or compound 21j (20 μM) for 24 h. DMSO was used as a negative control. Following cell lysis in hypotonic buffer, pellet and supernatant were separated using ultracentrifugation. Equal amounts of proteins were loaded in each lane. (b) Data analysis was performed using 3 independent replicates, and statistical significance between DMSO (control), colchicine, and 21j-treated cells was determined using Student's T-test. *P < 0.05.

2.2.6 21j reduces the number and size of colonies. Clonogenic assay is an in vitro cell based technique that is often used to determine the tumorigenic potential of cells in vivo. Therefore, we determined the effect of 21j on colony forming ability of C4-2 cells. 21j treatment significantly inhibited the number and size of colonies as compared to the control group (Fig. 9) indicating that 21j should serve as an effective anti-cancer agent.

Fig. 9 Compound 21j inhibits colony formation in C4-2 cells. An equal number of cells (1000 cells per well) were plated in a 6-well plate. They were treated with 0.05% DMSO (control) or compound 21j at 10 μM or 20 μM. 200 nM colchicine was used as a positive control. After 10 days cells were fixed and photographed.

2.3 Molecular docking studies

Tubulin–colchicine complex (PDB code: 1SA0) structure was provided by the Protein Data Bank (https://www.rcsb.org/).²⁸ To explore the potential interactions between tubulin and α-cyano bis(indolyl)chalcones, including compound 21j, we compared their orientations with the reference drug colchicine. To better understand the potency of 21j, we examined its interaction with the tubulin crystal structure (PDB code: 1SA0) using AutoDock 1.5.6 software (The Scripps Research Institute, USA). The X-ray crystallographic structure of the tubulin–colchicine complex reveals a crucial hydrogen bonding interaction with CYS241, along with additional hydrophobic interactions. The selected pose of the 21j out of eight poses that showed similarity to the binding mode of DAMA colchicine is considered the best pose with binding energy is −7.7 kcal mol⁻¹ (Fig. 10). As shown in Fig. 10, C₅-propyloxy and NH moieties of indole in 21j form hydrogen bonding interactions with CYS241 and LEU255, respectively, in addition to hydrophobic interactions (ALA250, ALA354, LEU248, ALA316, LYS352).

Fig. 10 (a) 21j in the binding pocket of the protein 1SA0. (b) 3D view of 21j with β-tubulin. (c) 2D overview of the molecular interactions of 21j in colchicine binding site of 1SA0.

Molecular docking results further highlight that 21j as a novel tubulin polymerization inhibitor that displayed interactions in the colchicine binding site of the tubulin. The structures were visualised and analysed in Discovery studio 2021 software (Fig. 10).

2.4 Molecular dynamics simulation studies

To further elucidate the procedural binding mode of 21j and tubulin, a 100 ns molecular dynamics (MD) study was performed based on the docked conformation of 21j with tubulin (PDB:1SA0). Through MD simulations, root mean square deviation (RMSD) plot for the protein–ligand complex 21j-1SA0 was determined between 6–8 Å and the complex was centred without significant scattering (Fig. 11a). This indicates that the ligand formed a stable binding with protein throughout the simulation. The protein-ligand contact map in Fig. 11c indicated that CYS241, ALA251, ASN258, and LYS352 were highly involved in H-bond formation with the ligand 21j during the MD simulation. Additionally, the RMSF plot along with the heatmap simulation trajectory point out the stability of the acquired docking model, as illustrated in Fig. 11. Moreover, a 2D diagram of the ligand–protein contacts of 21j-1SA0 (Fig. S1, ESI†), shows important interactions of 21j with the protein residues. A detailed description of the MD study of the complex is given in the ESI.†

Fig. 11 Statistical analysis of the MD simulation trajectory. (a) RMSD plot showing the ligand-bound state (red) and the Cα atoms of the target protein (blue), illustrating system stability over the 100 ns simulation. (b) RMSF plot of the protein-ligand complex, with green spikes highlighting the ligand binding site residues during the simulation. (c) Protein–ligand contact mapping in a bar plot, indicating the frequency and duration of interactions of the protein with the ligand 21j. (d) Heatmap illustrating the consistency and strength of interactions between the protein and ligand over the simulation trajectory.

2.5 In silico ADMET evaluation of α-cyano bis(indolyl)chalcone 21a–r

As a result, numerous in silico models have been developed to predict chemical ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties and it has become advantageous as it reveals a pharmacokinetics-related failure of drugs before proceeding to the clinical phase.^29a Lipophilicity is generally considered a key determinant of permeability across tissue membranes, while water solubility is another physicochemical property that determines a drug's ADMET behaviours. Here, we evaluate the ADME properties of the synthesized compounds by using in silico SwissADME server to see the pharmacokinetic properties such as lipophilicity, water-solubility, drug-likeness, medicinal chemistry of the compounds (Fig. 12).^29b

Fig. 12 Oral bioavailability radar charts for the studied potent compounds 21a–r. In bioavailability radar, the pink area represents the optimal range for each physicochemical property of oral bioavailability (LIPO-lipophilicity, SIZE-size, POLAR-polarity, INSOLU-solubility, INSATU-saturation and FLEX-flexibility), while the red lines represent compounds: 21a–r.

Orally administered drugs typically exhibit high lipophilicity, which facilitates their absorption through the intestinal lining, penetration of target cell membranes, and transport within the bloodstream. There is a direct relationship between the log P value and lipophilicity, but this negatively correlates with water solubility.^26a The calculated log P values of test compounds 21a–r ranges between 3.17 and 5.16. Drug-likeness is established based on chemical structures and physicochemical properties and is a qualitative assessment of oral bioavailability.³⁰ Moreover, Lipinski's rule states that for an orally active drug, the following conditions must be obeyed: ≤5 H-bond donors, ≤10 H-bond acceptors, a molecular weight ≤500 g mol⁻¹, and a log P ≤ 5.43; a ligand is considered orally inactive if it violates two or more of Lipinski's rules.³¹ Considering these criteria, all the compounds 21a–r meet the requirements for oral bioavailability (Table 3). Moreover, none of the test compounds violated Veber's rule, whose criteria are the presence of rotatable bonds ≤10 and polar surface (TPSA) area ≤140 Ų.³¹ Moreover, evident from the bioavailability score of 0.55, all the selected test compounds 21a–r are orally suitable.

Table 3 Physicochemical properties of 21a–r

Compd	MW^a (g mol^−1)	C log Po/w^b	nHBA^c	nHBD^d	nRB^e	TPSA (Å^2)^f	Log S^g	Druglikeness
a Molecular weight.b Lipophilicity.c No. of H-bond acceptors.d No. of H-bond donor.e No. of rotatable bonds.f Topological surface area.g water solubility.
21a	339.39	3.70	2	1	4	61.58	−4.97	Yes
21b	435.90	5.16	2	1	5	61.58	−6.58	Yes
21c	355.39	2.91	3	1	4	70.81	−4.67	Yes
21d	369.42	3.17	3	1	5	70.81	−4.86	Yes
21e	355.29	3.36	3	1	4	70.81	−4.67	Yes
21f	369.42	3.68	3	1	5	70.81	−4.86	Yes
21g	465.93	5.14	3	1	6	70.81	−6.65	Yes
21h	399.44	3.68	4	1	6	80.04	−4.93	Yes
21i	383.44	4.03	3	1	6	70.81	−5.09	Yes
21j	397.47	4.40	3	1	7	70.81	−5.43	Yes
21k	383.44	3.37	3	1	6	70.81	−5.24	Yes
21l	354.40	3.92	2	1	4	47.02	−4.86	Yes
21r	332.35	3.18	4	1	5	75.11	−4.34	Yes

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Table 4 shows the results of the pharmacokinetics prediction of the potent compounds 21a–r. As highlighted in the table, the skin permeation values (log K_p in cm s⁻¹) of the test compounds ranged from −5.54 (more permeant) to −5.07 (most permeant). Compounds 21c and 21j are the most skin permeant among the prepared compounds; however, the range of values of each test compound suggested that they are permeable compared to the values from the standard ligand. All the test compounds possess high gastrointestinal (GI) absorption potential, and the compounds (21b, 21c, 21d, 21k and 21r) displayed the ability to penetrate the blood–brain barrier (BBB). According to the pharmacokinetic predictions, except for 21a, all the test compounds were predicted to be inhibitors of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 (Table 4). Cytochrome P450 (CYP) is an isoenzyme superfamily that catalyzes various biochemical processes in phase I of drug metabolism (Hollenberg, 2002). The inhibition of the five main isoforms CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 from eventually becoming the substrates of medications is a primary cause of pharmacokinetics-related drug interactions.³²

Table 4 Selected pharmacokinetic parameters of 21a–r

Compd	GI absorption	BBB permeant	Log Kp (cm s^−1)	CYP1A2	CYP2C19	CYP2C9	CYP2D6	CYP3A4
21a	Low	No	−5.07	No	Yes	Yes	No	Yes
21b	High	Yes	−5.45	Yes	Yes	Yes	No	Yes
21c	High	Yes	−5.54	Yes	Yes	Yes	No	Yes
21d	High	Yes	−5.45	Yes	Yes	Yes	No	Yes
21e	High	No	−5.25	Yes	Yes	Yes	No	Yes
21f	High	No	−5.25	Yes	Yes	Yes	No	Yes
21g	High	No	−5.25	Yes	Yes	Yes	No	Yes
21h	High	No	−5.38	Yes	Yes	Yes	No	Yes
21i	High	No	−5.47	Yes	Yes	Yes	No	Yes
21j	High	No	−5.18	Yes	Yes	Yes	No	Yes
21k	High	Yes	−5.07	Yes	Yes	Yes	No	Yes
21l	High	No	−5.25	Yes	Yes	Yes	No	Yes
21r	High	Yes	−5.45	Yes	Yes	Yes	No	Yes

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Moreover, according to Table 4, the bioavailable radar charts (in Fig. 12), and the BOILED-Egg plot in Fig. 13, the investigated compounds were predicted to possess high gastrointestinal tract (GI) absorption and blood–brain barrier (BBB) permeability.

Fig. 13 BOILED-Egg plot for α-cyano bis(indolyl)chalcones 21j.

2.6 The BOILED-Egg is of great support for lead optimization

The BOILED-Egg model offers a rapid, and easily reproducible yet statistically robust method for predicting the passive gastrointestinal absorption and brain access of small molecules, which is valuable for drug discovery and development.

Highest probability of absorption by the gastrointestinal tract, while the yellow region (yolk) indicates the highest probability of permeating to the brain.

It's important to note that the yolk and white areas are not mutually exclusive. This means that some compounds can simultaneously have high gastrointestinal absorption and brain permeation. Such compounds are particularly desirable in drug development as they can effectively reach both systemic circulation and the central nervous system. This dual capability can lead to more efficient treatments for diseases that affect multiple organs, including the brain.

According to the determined parameters related to the absorption of the drug substance, it can be said that the synthesized compounds 21a–r were characterized by good intestinal absorption (HIA) and bioavailability. Distribution analysis predicted the location of the tested compounds in the mitochondria and did not reveal permeability through the blood–brain barrier (BBB). In addition, the compounds were identified as P-glycoprotein substrates but not inhibitors. In silico toxicity and carcinogenicity are assessed and are given in Table 5. Furthermore, the computed rat acute toxicity, that is, LD₅₀ in mol kg⁻¹ seems to be sufficiently safe in the range 2.23–2.66 mol kg⁻¹. The LD₅₀ and other bioactivity score of 21j is similar to that of the standard colchicine drug shown in Table 5.

Table 5 Physiochemical and ADME parameters

Toxicity target	Toxicity probability of compounds
	Colchicine	21i	21j	21m	21n
Caco-2 permeability	0.65	0.53	0.53	0.58	0.51
Rat acute toxicity (LD50) mol kg^−1	2.37	2.63	2.61	2.56	2.66
Carcinogens	Non-carcinogens
	0.81	0.88	0.91	0.91	0.93
Distribution
Subcellular localization	Nucleus	Mitochondria
P-glycoprotein substrate	0.59	0.52	0.55	0.62	0.60
Absorption	Log P app, cm s^−^1
Caco-2 permeability	1.17	1.29	1.20	1.29	1.64
Human intestine absorption	0.98 high	1.00 high	1.00 high	1.00 high	1.00 high

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3. Conclusions

The high yielding synthesis of indolyl α-cyano bis(indolyl)chalcones was achieved from the L-proline catalysed reaction of appropriate aldehydes with 3-cyanoacetylindoles. Of the prepared eighteen α-cyano bis(indolyl)chalcones, compound 21j demonstrated remarkable potency against the C4-2 prostate cancer cell line (IC₅₀ = 0.9 μM). With broad spectrum of activity (0.98–5.6 μM), the compound 21j was found to increase the endogenous level of ROS, besides mitochondrial dysfunction, causes apoptosis. Additionally, moderate tubulin activity of 21j suggest that at least some percentage of 21j's anti-cancer effect stems from its tubulin de-polymerization. The molecular docking study of 21j displayed important interactions in the colchicine binding site of the tubulin which supports its observed tubulin activity.

4. Experimental section

4.1 Chemistry

4.1.1 General methods. All laboratory reagents were purchased from Sigma-Aldrich, BLD Pharma, Alfa Aesar and Spectrochem India Pvt. Ltd and used without additional purification. Reaction progress was monitored using thin layer chromatography and performed on Merck pre-coated plates (silica gel 60 F254, 0.2 mm). Column chromatographic purification of products was carried out using silica gel (100–200 mesh) and ethyl acetate/hexane mixture was used for elution. NMR (¹H and ¹³C) spectra were recorded at 400 MHz and 100 MHz using CDCl₃ and DMSO-d₆ solvents. Chemical shifts are given in ppm relative to the residual solvent peak (¹H NMR: CDCl₃ δ 7.26; DMSO-d₆ δ 2.50; ¹³C NMR: CDCl₃ δ 77.0; DMSO-d₆ δ 39.52) with multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants (J, in Hz) and integration. Melting points were determined by using E–Z melting point apparatus and are uncorrected. High-resolution mass data (HRMS) were obtained on an Agilent 6545 Q-TOF LC/MS (ESI).

4.1.2 General procedure for the synthesis of 3-cyanoacetyl indoles³³ (19). Methane sulfonyl chloride (0.13 mL, 0.17 mmol) and corresponding indoles (0.20 g, 0.17 mmol) were added to a stirred solution of potassium cyanoacetate (0.42 g, 0.34 mmol) in acetonitrile (3 mL). The resulting solution was stirred at room temperature for 1 h. The progress of the reaction was monitored by TLC. After the consumption of the starting material, contents were allowed to cool and a white solid thus obtained was collected by filtration, washed with methanol and dried to obtain pure products (19a–g) as mentioned below:

4.1.3 Procedure for the synthesis of 5-alkoxyindoles and 5,6-dimethoxyindole³⁴. A mixture of 5-hydroxyindole/5,6-hydroxyindole (0.50 g, 3.8 mmol) and K₂CO₃ (1.52 g, 11.0 mmol), dissolved in 5 mL ethanol and heated to reflux. The ethyliodide/propylbromide/isopropylbromide (0.77 g, 4.9 mmol) was added and the reaction mixture was allowed to reflux for 1 h and then concentrated the mixture at reduced pressure. Water (20 mL) was added and the aqueous layer was extracted with ethyl acetate (3 × 30 mL). The combined organic layer was dried with MgSO₄ and concentrated. The crude product was purified by flash column chromatography using 5% ethyl acetate/hexane (v/v) as eluent.

4.1.4 General procedure for the synthesis of indole-3-carboxaldehyde^26d. A round bottomed flask containing freshly distilled dimethylformamide (DMF) (10 mL) was cooled in an ice–salt bath for about 0.5 h and freshly distilled phosphorus oxychloride was added with stirring to DMF (5 mL) over a period of 0.5 h. A solution of indole (2 g, 85.47 mmol) in DMF (130 mmol) was added to the yellow solution over a period of 1 h. The solution was stirred at 35 °C till it became a yellow paste. At the end of the reaction, 30 g of crushed ice was added to the paste with stirring to obtain a clear cherry-red aqueous solution. To this solution, sodium hydroxide (10 g, 94 mmol) in 100 mL of water was added dropwise with stirring. The resulting suspension was heated rapidly to 90 °C and allowed to cool at room temperature, after which it was placed in refrigerator for overnight. The product was filtered, washed with water (2 × 100 mL) and air dried to afford the pure indole-3-carboxaldehyde in 93% yield, mp 196–197 °C.

4.1.5 Procedure for alkylation of indole-3-carboxaldehydes (20a–c)³⁵. In a reaction flask, indole-3-carboxaldehyde (1 g, 1.0 equiv.) was dissolved in THF (15 mL), followed by the addition of sodium hydride (0.3 g, 2.5 equiv.) and methyl iodide/ethyl iodide/4-chlorobenzylchloride (3.0 equiv.) at 0 °C to room temperature. Reaction was monitored by the TLC. After the completion of reaction, organic phase was washed twice with aqueous NaHCO₃ (50 mL), water and saturated brine (100 mL), and then dried over anhydrous Na₂SO₄. The solvent was evaporated under vacuum and residue was purified by the column chromatography with ethylacetate and hexane led to pure product (20a–c) as follows: 1-methyl-1H-indole-3-carbaldehyde (20a), 95% yield (mp 68–70 °C), 1-ethyl-1H-indole-3-carbaldehyde (20b), 96% yield, (mp 98–100 °C), 1-(4-chlorobenzyl)-1H-indole-3-carbaldehyde (20c), 98% yield, (mp 117–119 °C).

4.1.6 General procedure for the preparation of α-cyano bis(indolyl)chalcones (21a–r). A mixture containing 3-cyanoacetylindole derivative 19 (0.1 g, 1 mmol) and appropriate aldehyde 20 (0.065 g, 1 mmol) in ethanol (10 mL) was stirred at 25 °C. Catalytic amount of L-proline (10 mol%) was added to reaction mixture and it was stirred for 5 h at 25 °C. The reaction progress was monitored via TLC using a developing solvent system of n-hexane : ethyl acetate. The resulting yellow solid was then recrystallized from ethanol to obtain pure α-cyano bis(indolyl)chalcones 21a–r in 90–95% yields.
4.1.6.1 (E)-3-(1-Ethyl-1H-indol-3-yl)-2-(1H-indole-3-carbonyl)acrylonitrile (21a). Pale yellow solid, 95% yield, mp 221–222 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.13 (s, 1H), 8.63 (d, J = 10.5 Hz, 2H), 8.50 (s, 1H), 8.26–8.22 (m, 1H), 7.97 (dt, J = 7.7, 1.0 Hz, 1H), 7.69 (dt, J = 8.2, 0.9 Hz, 1H), 7.59–7.54 (m, 1H), 7.38–7.23 (m, 4H), 4.44 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 181.26, 144.96, 136.88, 136.34, 134.34, 133.43, 128.50, 126.96, 124.01, 123.74, 122.63, 122.50, 122.07, 121.08, 119.38, 114.81, 112.83, 111.76, 110.12, 102.20, 42.18, 15.60. HRMS (ESI) m/z calcd for C₂₂H₁₈N₃O: 340.1405 (M + H)⁺, found: 340.1595.
4.1.6.2 3-(1-(4-Chlorobenzyl)-1H-indol-3-yl)-2-(1H-indole-3-carbonyl)acrylonitrile (21b)^26d. Yellow solid, 90% yield, mp 222−225 °C (lit mp 219–221 °C); ¹H NMR (400 MHz, DMSO-d₆) δ 12.17 (s, 1H), 8.75 (s, 1H), 8.63 (s, 1H), 8.51 (s, 1H), 8.26 (d, J = 6.5 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.59 (d, J = 14.8 Hz, 2H), 7.42 (s, 2H), 7.31 (d, J = 10.0 Hz, 6H), 5.68 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 181.31, 144.92, 136.82, 136.44, 135.84, 134.45, 134.10, 133.09, 129.62, 129.22, 128.41, 126.78, 124.33, 123.95, 122.89, 122.71, 122.01, 120.86, 119.31, 114.75, 112.88, 111.96, 110.51, 102.80, 49.87.
4.1.6.3 2-(5-Methoxy-1H-indole-3-carbonyl)-3-(1-methyl-1H-indol-3-yl)acrylonitrile (21c). Yellow solid, 95% yield, mp 220–221 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.03 (s, 1H), 8.62 (s, 1H), 8.59 (s, 1H), 8.45 (d, J = 3.3 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 2.8 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.40–7.28 (m, 2H), 6.91 (dd, J = 8.7, 2.6 Hz, 1H), 4.01 (s, 3H), 3.81 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.90, 156.10, 144.84, 137.34, 134.92, 134.35, 131.66, 128.31, 127.83, 124.03, 122.68, 121.10, 119.11, 114.65, 113.64, 113.59, 111.75, 109.89, 103.84, 101.87, 55.74, 34.22. HRMS (ESI) m/z calcd for C₂₂H₁₈N₃O₂: 356.1354 (M + H)⁺, found: 356.1373.
4.1.6.4 3-(1-Ethyl-1H-indol-3-yl)-2-(5-methoxy-1H-indole-3-carbonyl)acrylonitrile (21d). Yellow solid, 91% yield, mp 214–215 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 8.63 (d, J = 3.8 Hz, 2H), 8.47 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.80 (d, J = 2.6 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.38–7.26 (m, 2H), 6.92 (dd, J = 8.7, 2.6 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 3.82 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.87, 156.10, 144.81, 136.31, 134.43, 133.30, 131.73, 128.52, 127.88, 123.99, 122.62, 121.18, 119.30, 114.69, 113.62, 113.58, 111.73, 110.12, 103.87, 102.04, 55.73, 42.17, 15.57. HRMS (ESI) m/z calcd for C₂₃H₂₀N₃O₂: 370.1511 (M + H)⁺, found: 370.1721.
4.1.6.5 2-(6-Methoxy-1H-indole-3-carbonyl)-3-(1-methyl-1H-indol-3-yl)acrylonitrile (21e). Yellow solid, 95% yield, mp 217–220 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (s, 1H), 8.59 (d, J = 6.2 Hz, 2H), 8.37 (d, J = 3.1 Hz, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.41–7.27 (m, 2H), 7.04 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.8, 2.3 Hz, 1H), 4.00 (s, 3H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ ¹³C NMR (101 MHz, DMSO) δ 180.94, 157.16, 144.81, 137.77, 137.34, 134.95, 133.34, 128.29, 124.02, 122.75, 122.67, 121.08, 120.87, 119.13, 114.92, 112.33, 111.74, 109.88, 101.88, 95.81, 55.74, 34.22. HRMS (ESI) m/z calcd for C₂₂H₁₈N₃O₂: 356.1354 (M + H)⁺, found: 356.3691.
4.1.6.6 3-(1-Ethyl-1H-indol-3-yl)-2-(6-methoxy-1H-indole-3-carbonyl)acrylonitrile (21f). Yellow solid, 94% yield, mp 228–230 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.93 (s, 1H), 8.62 (d, J = 8.3 Hz, 2H), 8.40 (s, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.4 Hz, 1H), 7.05 (s, 1H), 6.90 (d, J = 11.1 Hz, 1H), 4.41 (q, J = 7.3 Hz, 2H), 3.82 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.92, 157.17, 144.79, 137.80, 136.31, 133.34, 128.51, 123.98, 122.78, 122.61, 121.15, 120.91, 119.32, 114.96, 112.31, 111.72, 110.12, 102.05, 95.81, 55.73, 42.16, 15.56. HRMS (ESI) m/z calcd for C₂₃H₂₀N₃O₂: 370.1511 (M + H)⁺, found: 370.1538.
4.1.6.7 3-(1-(4-Chlorobenzyl)-1H-indol-3-yl)-2-(6-methoxy-1H-indole-3-carbonyl)acrylonitrile (21g). Yellowish solid, 93% yield, mp 215–217 °C.^{36 1}H NMR (400 MHz DMSO-d₆) δ 12.07 (s, 1H), 8.74 (s, 1H), 8.63 (s, 1H), 8.48 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.80 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.45 (m, J = 17.1, 8.4 Hz, 3H), 7.31 (m, J = 15.3, 7.8 Hz, 4H), 6.93 (d, J = 9.0 Hz, 1H), 5.68 (s, 2H), 3.81 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.91, 156.15, 144.65, 136.52, 136.16, 134.61, 133.96, 133.01, 131.72, 129.67, 129.26, 128.56, 127.84, 124.20, 122.74, 120.88, 119.39, 114.64, 113.67, 113.61, 112.06, 110.57, 103.87, 103.01, 55.74, 49.80. HRMS (ESI) m/z calcd for C₂₈H₂₁ClN₃O₂: 466.1578 (M + H)⁺, found: 466.1525.
4.1.6.8 2-(5-Ethoxy-1H-indole-3-carbonyl)-3-(1-methyl-1H-indol-3-yl)acrylonitrile (21h). Yellow solid, 93% yield, mp 217–220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.02 (s, 1H), 8.61 (s, 1H), 8.57 (s, 1H), 8.45 (s, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 6.9 Hz, 1H), 6.90 (dd, J = 8.8, 2.6 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.99 (s, 3H), 1.37 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.79, 155.31, 144.76, 137.31, 134.84, 134.32, 131.65, 128.33, 127.89, 123.97, 122.62, 121.16, 119.08, 114.69, 114.05, 113.54, 111.69, 109.92, 104.72, 101.88, 63.75, 34.18, 15.31. HRMS (ESI) m/z calcd for C₂₃H₂₀N₃O₂: 370.1511 (M + H)⁺, found: 370.1722.
4.1.6.9 2-(5-Ethoxy-1H-indole-3-carbonyl)-3-(1-ethyl-1H-indol-3-yl)acrylonitrile (21i). Yellow solid, 91% yield, mp 272–273 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.06 (s, 1H), 8.62 (d, J = 11.4 Hz, 2H), 8.44 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 2.6 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.37 (t, J = 7.1 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 6.90 (dd, J = 8.8, 2.4 Hz, 1H), 4.44 (q, J = 7.2 Hz, 2H), 4.06 (q, J = 7.0 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.91, 155.29, 144.80, 136.32, 134.46, 133.33, 131.69, 128.50, 127.86, 124.00, 122.62, 121.13, 119.34, 114.63, 114.05, 113.57, 111.77, 110.10, 104.66, 102.10, 63.75, 42.17, 15.61, 15.32. HRMS (ESI) m/z calcd for C₂₄H₂₂N₃O₂: 384.1667 (M + H)⁺, found: 384.1676.
4.1.6.10 3-(1-Ethyl-1H-indol-3-yl)-2-(5-propoxy-1H-indole-3-carbonyl)acrylonitrile (21j). Yellow solid, 93% yield, mp 294–295 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.02 (s, 1H), 8.63 (s, 1H), 8.62 (s, 1H), 8.45 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 9.2 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.38–7.27 (m, 2H), 6.91 (dd, J = 8.8, 2.4 Hz, 1H), 4.43 (q, J = 7.2 Hz, 2H), 3.96 (t, J = 6.6 Hz, 2H), 1.77 (h, J = 7.4 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.90, 155.47, 144.80, 136.31, 134.38, 133.31, 131.65, 128.52, 127.86, 123.99, 122.62, 121.15, 119.30, 114.66, 114.09, 113.53, 111.75, 110.11, 104.77, 102.06, 69.83, 42.17, 22.67, 15.58, 10.94. HRMS (ESI) m/z calcd for C₂₅H₂₄N₃O₂: 398.1824 (M + H)⁺, found: 398.1839.
4.1.6.11 3-(1-Methyl-1H-indol-3-yl)-2-(5-propoxy-1H-indole-3-carbonyl)acrylonitrile (21k). Yellow solid, 95% yield, mp 224–225 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.56 (s, 1H), 8.50 (s, 1H), 8.39 (s, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 2.6 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.38–7.26 (m, 2H), 6.89 (dd, J = 8.8, 2.6 Hz, 1H), 3.93 (s, 3H), 3.74 (s, 3H), 1.73 (h, J = 7.2 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 181.11, 155.47, 145.00, 137.33, 135.06, 134.19, 131.45, 128.20, 127.69, 124.17, 122.82, 121.12, 119.02, 114.57, 113.59, 111.74, 109.85, 104.80, 101.63, 69.94, 34.20, 22.57, 10.88. HRMS (ESI) m/z calcd for C₂₄H₂₂N₃O₂: 384.1667 (M + H)⁺, found: 384.1676.
4.1.6.12 3-(1-Ethyl-1H-indol-3-yl)-2-(5-isopropoxy-1H-indole-3-carbonyl)acrylonitrile (21l). Yellow solid, 91% yield, mp 196–197 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.00 (s, 1H), 8.64 (s, 1H), 8.61 (s, 1H), 8.45 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.39–7.27 (m, 2H), 6.89 (dd, J = 8.8, 2.4 Hz, 1H), 4.57 (quintet, J = 6.0 Hz, 1H), 4.43 (q, J = 7.2 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H), 1.31 (d, J = 6.1 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.92, 154.07, 144.83, 136.32, 134.49, 133.32, 131.77, 127.90, 123.99, 122.62, 121.15, 119.34, 115.32, 114.60, 113.56, 111.75, 110.12, 106.97, 102.08, 70.46, 42.17, 22.44, 15.59. HRMS (ESI) m/z calcd for C₂₄H₂₂N₃O₂: 384.1667 (M + H)⁺, found: 384.1676.
4.1.6.13 2-(5-Isopropoxy-1H-indole-3-carbonyl)-3-(1-methyl-1H-indol-3-yl)acrylonitrile (21m). Yellow solid, 94% yield, mp 220–221 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.00 (s, 1H), 8.60 (d, J = 11.1 Hz, 2H), 8.44 (s, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 7.39–7.27 (m, 2H), 6.89 (dd, J = 8.7, 2.5 Hz, 1H), 4.57 (p, J = 6.1 Hz, 1H), 4.00 (s, 3H), 1.31 (d, J = 6.1 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) ¹³C NMR (101 MHz, DMSO) δ 184.84, 180.89, 154.06, 144.82, 137.34, 134.88, 134.45, 131.75, 128.32, 127.89, 124.00, 122.65, 121.11, 119.14, 115.32, 114.58, 113.57, 111.74, 109.91, 106.95, 101.91, 70.46, 34.22, 22.44, 22.40. HRMS (ESI) m/z calcd for C₂₅H₂₄N₃O₂: 398.1824 (M + H)⁺, found: 398.2055.
4.1.6.14 2-(5,6-Dimethoxy-1H-indole-3-carbonyl)-3-(1-methyl-1H-indol-3-yl)acrylonitrile (21n). Yellow solid, 95% yield, mp 286–289 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (s, 1H), 8.56 (s, 1H), 8.33 (s, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.76 (s, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.40–7.26 (m, 2H), 7.08 (s, 1H), 3.98 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.75, 147.94, 146.94, 144.73, 137.32, 134.87, 132.28, 131.06, 128.29, 124.03, 122.69, 121.18, 119.86, 119.01, 114.96, 111.71, 109.88, 103.95, 101.75, 96.05, 6.15, 34.19. HRMS (ESI) m/z calcd for C₂₃H₂₀N₃O₃: 386.1460 (M + H)⁺, found: 386.1488.
4.1.6.15 2-(5,6-Dimethoxy-1H-indole-3-carbonyl)-3-(1-ethyl-1H-indol-3-yl)acrylonitrile (21o). Yellow solid, 94% yield, mp 250–251 °C. ¹H NMR (400 MHz, DMSO) δ 8.54 (d, J = 7.0 Hz, 2H), 8.30 (s, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.72 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.35–7.24 (m, 2H), 7.07 (s, 1H), 4.33 (q, J = 7.2 Hz, 2H), 3.79 (s, 9H), 1.41 (t, J = 7.3 Hz, 3H).·¹³C NMR (100 MHz, DMSO-d₆) δ 180.84, 147.86, 146.86, 144.84, 136.24, 133.27, 132.10, 130.84, 128.40, 122.73, 121.27, 119.71, 119.09, 114.90, 111.66, 110.03, 103.78, 101.56, 95.88, 56.04, 42.18, 15.38. HRMS (ESI) m/z calcd for C₂₄H₂₂N₃O₃: 400.1616 (M + H)⁺, found: 400.1651.
4.1.6.16 3-(1-(4-Chlorobenzyl)-1H-indol-3-yl)-2-(5,6-dimethoxy-1H-indole-3-carbonyl)-acrylonitrile (21p). Yellow solid, 95% yield, mp 294–295 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 1H), 8.74 (s, 1H), 8.62 (s, 1H), 8.36 (s, 1H), 7.97 (d, J = 7.2 Hz, 1H), 7.77 (s, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 8.4 Hz, 3H), 7.32 (dd, J = 15.9, 8.3 Hz, 5H), 7.08 (s, 1H), 5.69 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 180.73, 147.95, 146.97, 144.51, 136.51, 136.23, 133.90, 132.98, 132.58, 131.11, 129.68, 129.26, 128.57, 124.18, 122.72, 120.91, 114.90, 112.08, 110.55, 103.93, 102.96, 96.05, 56.13, 49.77. HRMS (ESI) m/z calcd for C₂₉H₂₃ClN₃O₃: 496.1424 (M + H)⁺, found: 496.1405.
4.1.6.17 2-(1H-Indole-3-carbonyl)-3-(4-methoxyphenyl)acrylonitrile (21q)³⁷. Yellow solid, 92% yield, mp 257–259 °C (lit mp 256–258 °C). ¹H NMR (400 MHz, DMSO-d₆) δ 12.25 (s, 1H), 8.45 (s, 1H), 8.20 (s, 2H), 8.09 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 7.7 Hz, 1H), 7.28 (s, 2H), 7.15 (d, J = 8.6 Hz, 2H), 3.87 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 181.93, 163.21, 152.48, 137.09, 135.83, 133.33, 126.69, 125.38, 123.96, 122.79, 121.89, 118.93, 115.22, 114.20, 112.93, 108.41, 56.12.
4.1.6.18 3-(3,4-Dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (21r)³⁷. Yellow solid, 94% yield, mp 258−260 °C (lit mp 260–261 °C). ¹H NMR (400 MHz, DMSO-d₆) δ 12.27 (s, 1H), 8.47 (s, 1H), 8.22 (d, J = 9.5 Hz, 2H), 7.80 (s, 1H), 7.57 (d, J = 6.8 Hz, 1H), 7.32–7.24 (m, 2H), 7.15 (d, J = 8.6 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 181.87, 153.14, 152.88, 149.12, 137.08, 135.74, 126.74, 126.38, 125.45, 123.94, 122.76, 121.93, 119.14, 114.24, 113.20, 112.91, 112.22, 108.33, 56.26, 55.96.

4.2 Biology protocols

4.2.1 MTT assay. C4-2, PC-3, and 22Rv1 prostate cancer cells were cultured in RPMI 1640 media, while HEK293 (human kidney) and MCF7 (human breast cancer) cells were maintained in Dulbecco's modified Eagle's medium (DMEM). Both media were supplemented with 10% fetal bovine serum (FBS), 100 μg per mL streptomycin, and 100 I.U. per mL penicillin. For the MTT assay, 5000 cells per well were plated in 96-well plates. After 12 h of incubation, the cells were treated with various concentrations of 21a–r, ranging from 0.1 μM to 40 μM. A 0.1% DMSO solution served as the vehicle control. Following 48 h of treatment, the media were removed, and the cells were washed with PBS. Then, 100 μL of serum-free media and an MTT cocktail (5 mg mL⁻¹) in a 4 : 1 ratio was added to each well. The cells were incubated for 4 h at 37 °C. Afterward, the MTT solution was aspirated, cells were washed with PBS, and 100 μL of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a Tecan Spectrafluor Plus plate reader. Relative inhibition was calculated by dividing the mean absorbance of treated cells by the mean absorbance of DMSO-treated control cells. The IC₅₀ values and dose–response curves were generated through nonlinear regression analysis using the sigmoidal dose–response model with variable slope in GraphPad Prism, version 6.0 (GraphPad Software Inc., CA, USA).

4.2.2 Acridine orange/ethidium bromide assay. To investigate the effects of the 21j on plasma membrane permeability, chromatin condensation, and nuclear morphology, AO/EB assay was performed. C4-2 cells (0.25 × 10⁵) were seeded on 12 mm coverslips in 24-well plates for 12 h. The cells were subsequently treated with DMSO control or 21j (10 and 20 μM) for 48 h. Following treatments and media removal, the cells were washed with PBS. The cells were then incubated with a mixture containing AO (100 μg mL⁻¹) and EB (100 μg mL⁻¹) in PBS for 20 min. The cells were washed with PBS and imaged using a Nikon fluorescence microscope, with green fluorescence observed using the FITC channel and red fluorescence observed using the TRITC channel.

4.2.3 JC-1 staining. To assess the impact of compound 21j on mitochondrial health, JC-1 staining was performed. C4-2 cells (0.25 × 10⁵) were seeded onto 12 mm coverslips for 12 h, following which they were exposed to DMSO or 21j (10 or 20 μM) for 48 h. The cells were afterwards washed with PBS and incubated with JC-1 dye (2 μM) in PBS for 20 min. Following incubation, the cells were washed, and images were captured using a Nikon fluorescence microscope with FITC (green) and TRITC (red) channels. Healthy mitochondria emit orange fluorescence in the TRITC channel due to aggregation, while depolarized mitochondria show green color due to reduced JC-1 accumulation.

4.2.4 Measurement of intracellular reactive oxygen species (ROS) levels. To assess intracellular ROS levels, the fluorogenic dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) was employed. C4-2 cells seeded on coverslips were treated with DMSO, 21j (10 μM), or H₂O₂ (10 μM) for 48 h. Following treatment, the cells were washed with PBS and subsequently incubated with 100 μL of H2DCFDA in PBS at 2 μM final concentration for 40 min at 37 °C. Following incubation, the cells were washed with PBS, and intracellular ROS levels were visualized and imaged using the FITC channel on a Nikon inverted fluorescence microscope.

4.2.5 Western blot analysis. C4-2 cells were treated with DMSO, 20 μM of 21j, or 100 nM of colchicine for 48 h at 37 °C. The cells were washed and lysed using a hypotonic buffer. The proteins were separated using SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in 0.1% TBST. It was then incubated overnight at 4 °C with a tubulin primary antibody (1 : 5000 dilution), obtained from the Developmental Studies Hybridoma Bank (DSHB). Following washes with 0.1% TBST, the membrane was incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using a chemiluminescence detection reagent (Pierce Biotechnology) and imaged with a GeneGnomeXRQ chemiluminescence imager.

4.2.6 Tubulin polymerization assay. C4-2 cells were plated for 16 h. The cells were then treated with either 10 μM of compound 21j or 100 nM of colchicine for 48 h at 37 °C. The cells were lysed using a buffer containing 1 mM MgCl₂, 2 mM EGTA, 0.5% NP40, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 20 mM Tris–HCl (pH 6.8). Ultracentrifugation was employed to separate the polymerized tubulin (pellet) from the depolymerized tubulin (supernatant). The pellet was resuspended in the lysis buffer, and protein concentration was determined using the Bradford assay. Equal amounts of protein from both pellet and supernatant fractions were loaded onto a 12% SDS-PAGE gel, followed by immunoblotting to detect tubulin levels in the different fractions.

4.2.7 Immunofluorescence. C4-2 cells were seeded onto coverslips for 16 h that had been coated with poly-lysine. The cells were treated with DMSO or 21j at concentrations of 10 μM and 20 μM for 24 h. The cells were fixed, permeabilized using ice-cold methanol. To block non-specific binding, the cells were incubated for one hour with a blocking solution containing 2% BSA and 1% Triton X-100 in 1× PBS. The coverslips were incubated overnight at 4 °C with tubulin antibody, followed by FITC-conjugated goat anti-mouse secondary antibody for 3 h in the dark. The images were captured using a BZ-X810 Keyence fluorescence microscope.

4.2.8 Clonogenic assay. 1000 cells per well were plated in a 6-well plate. The plates were incubated for 10–12 days in the incubator. After incubation, cells were washed with 1× PBS and fixed with cold solution of methanol and acetic acid (3 : 1) for 5–10 min and subsequently photographed. The assay was performed in duplicates.

5. Computational methods

The detailed methodology of MD simulation study is mentioned in the ESI.†

Data availability

The authors confirm that the data supporting the findings of this study are available within the article and/or its ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors are highly grateful to DST, New Delhi (CRG/2022/007389), [NIH R01-CA237660] and NIH 1RF1NS124779 USA for funding. Monika Malik, acknowledges her fellowship grant under the Purdue-India research collaboration program through Science and Engineering Research Board (SERB)-India and BITS Pilani, Pilani campus. We also thank Prof. Atish T. Paul and Mr Sanket Rathod, Department of Pharmacy, BITS Pilani, Pilani Campus, for their help in performing MD simulations.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra06796g

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