A Facile Synthesis and Molecular Characterization of Certain New Anti-Proliferative Indole-Based Chemical Entities

Cancer cells frequently develop drug resistance, which leads to chemotherapeutic treatment failure. Additionally, chemotherapies are hindered by their high toxicity. Therefore, the development of new chemotherapeutic drugs with improved clinical outcomes and low toxicity is a major priority. Several indole derivatives exhibit distinctive anti-cancer mechanisms which have been associated with various molecular targets. In this study, target compounds 4a–q were obtained through the reaction of substituted benzyl chloride with hydrazine hydrate, which produces benzyl hydrazine. Subsequently, the appropriate substituted benzyl hydrazine was allowed to react with 1H-indole-2-carboxylic acid or 5-methoxy-1H-indole-2-carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a coupling agent. All compounds exhibited cytotoxicity in three cell lines, namely, MCF-7, A549, and HCT. Compound 4e exhibited the highest cytotoxicity, with an average IC50 of 2 µM. Moreover, a flow cytometry study revealed a significantly increased prevalence of Annexin-V and 7-AAD positive cell populations. Several derivatives of 4a–q showed moderate to high cytotoxicity against the tested cell lines, with compound 4e having the highest cytotoxicity, indicating that it may possess potential apoptosis-inducing capabilities.


Introduction
Cancer, a major causes of mortality worldwide, affects billions of people annually [1]. Despite significant progress in the development of new anticancer drugs, several drawbacks, including low efficacy, high toxicity and drug resistance, affect the outcomes of treatment [2]. Therefore, many researchers aim to develop new anticancer drugs with superior clinical outcomes and a safer profile.
Heterocyclic compounds have a broad range of biological activities and play important roles in medicinal chemistry. In the field of drug discovery, the indole ring is considered an imperative building block owing to its versatile chemistry and wide range of biological functions, including antimicrobial, antiviral, anticonvulsant, analgesic, anti-inflammatory and anticancer activities [3][4][5][6][7][8][9]. Several commercially available potent anticancer drug molecules, such as the natural vinca alkaloids vinblastine and vincristine, contain the indole ring [10]. Additionally, Cediranib is a potent tyrosine kinases receptor inhibitor of vascular endothelial growth factor (VEGF) [11], and Panobinostat, a histone deacetylase inhibitor belonging to the hydroxamic acid class [12].
A literature review has revealed that the distinctive molecular mechanisms regulating the anticancer properties are associated with various molecular targets. Some of the indole derivatives inhibit tubulin polymerization, and induce apoptosis in cancer cells [13][14][15][16]. However, other derivatives were found that inhibit protein kinases, such as casein kinase 2 (CK2), tyrosine kinases (TrK), and cyclin-dependent kinases 4 and 6 (CDK), as well as vascular endothelial growth factor-2 (VEGFR). Furthermore, indoles can induce apoptosis The synthetic pathway used to prepare the target compounds 4a-q is depicted in Scheme 1. Commercially available substituted benzyl chloride 1 was allowed to react with hydrazine hydrate in ethanol to give benzyl hydrazine 2. Subsequently, the title compounds 4a-q were achieved by reacting the appropriate substituted benzyl hydrazine with 1Hindole-2-carboxylic acid or 5-methoxy-1H-indole-2-carboxylic acid in dichloromethane using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a coupling agent. The direct conversion of a carboxylic acid to an amide is difficult because amines are very basic and tend to convert carboxylic acids to their highly unreactive carboxylate ions. Therefore, EDCI as a coupling agent was used to drive this reaction by forming a good leaving group which could then be displaced by an amine during the addition elimination reaction [23]. The nucleophilic attack happened due to the secondary amine rather than the primary amine of the hydrazine terminal, which led to the formation of the sole product in the target compounds 4a-q. The assigned structures were confirmed by 1 HNMR, 13 CNMR, ESI-MS, and X-ray crystallography. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 2 of 12 apoptosis via the inhibition of Mcl-1, Bcl-2, and poly(ADP-ribose) polymerase 1 (PARP), which prevents DNA repair. It also exhibits anticancer effects by suppressing DNA topoisomerases, aromatases, and histone deacetylases (HDAC) [9,[17][18][19][20]. Therefore, compounds containing indole moieties exhibit different mechanisms of action involving multiple targets during cell replication and proliferation. Considering this, and in continuation of ousr previous research on new potent anti-proliferative compounds [21,22], in this study we aimed to synthesize indole backbone molecules tethered with free rotating substituted benzyl moieties 4a-q. The flexibility of the molecules is a modality for obtaining a number of bioactive confirmations, and hence increasing the likelihood of obtaining new bioactive compounds with effective anti-proliferative properties.

Chemistry
The synthetic pathway used to prepare the target compounds 4a-q is depicted in Scheme 1. Commercially available substituted benzyl chloride 1 was allowed to react with hydrazine hydrate in ethanol to give benzyl hydrazine 2. Subsequently, the title compounds 4a-q were achieved by reacting the appropriate substituted benzyl hydrazine with 1H-indole-2-carboxylic acid or 5-methoxy-1H-indole-2-carboxylic acid in dichloromethane using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a coupling agent. The direct conversion of a carboxylic acid to an amide is difficult because amines are very basic and tend to convert carboxylic acids to their highly unreactive carboxylate ions. Therefore, EDCI as a coupling agent was used to drive this reaction by forming a good leaving group which could then be displaced by an amine during the addition elimination reaction [23]. The nucleophilic attack happened due to the secondary amine rather than the primary amine of the hydrazine terminal, which led to the formation of the sole product in the target compounds 4a-q. The assigned structures were confirmed by 1 HNMR, 13 CNMR, ESI-MS, and X-ray crystallography. The 1 H-NMR spectra of the target compounds 4a-q showed D2O exchangeable singlet peaks integrated for two protons in the range of δ 4.81-5.03 ppm assigned for the NH2. Other singlets integrated for two protons in the range of δ 4.91-5.34 ppm were noticed for the benzylic CH2. Meanwhile, compounds 4b, 4d, 4f, 4h, 4j, 4l, 4n, 4p, and 4q showed singlets at around δ = 3.76 ppm, representing three protons of the indole -OCH3 group. Compound 4q exhibited a singlet at δ = 2.30 ppm, which was assigned to be for the The 1 H-NMR spectra of the target compounds 4a-q showed D 2 O exchangeable singlet peaks integrated for two protons in the range of δ 4.81-5.03 ppm assigned for the NH 2 . Other singlets integrated for two protons in the range of δ 4.91-5.34 ppm were noticed for the benzylic CH 2 . Meanwhile, compounds 4b, 4d, 4f, 4h, 4j, 4l, 4n, 4p, and 4q showed singlets at around δ = 3.76 ppm, representing three protons of the indole -OCH 3 group. Compound 4q exhibited a singlet at δ = 2.30 ppm, which was assigned to be for the three protons of the CH 3 group. The aromatic protons were observed in the region of δ = 6.84-8.25 ppm. Moreover, the indole NH appeared as singlets integrated for one proton in the range δ = 11.36-11.55 ppm, whereas the 13 C-NMR spectra of the target compounds 4a-q exhibited signals in the range of δ = 54.0-55.6 ppm, indicating carbons of the benzylic CH 2 . The methyl carbons of compound 4q were observed at δ = 21.2 ppm, and the indole methoxy carbon for compounds 4b, 4d, 4f, 4h, 4j, 4l, 4n, 4p, and 4q resonated at δ = 55.7 ppm. Moreover, the aromatic carbons appeared in the range of δ = 102.4-158.9 ppm while the amide carbonyl carbon appeared at about δ 163 ppm.

Antiproliferative Activity
The title compounds 4a-q were subjected to MTT assay to determine their cytotoxicity, and their in vitro antiproliferative activity was examined as well. The cancer cell growth inhibitory activity of the synthesized compound was tested against three human cancer cell lines, namely, breast cancer (MCF-7), colon cancer (HCT116) and lung cancer (A549), as well as non-tumorigenic human lung cell line (WI38) to estimate the selectivity for tumor cells. Staurosporine was used as a reference drug.

Flowcytometry
Since a paramount goal for anticancer agents is to induce apoptosis and cause malfunctions in the DNA, flowcytometry was conducted in this study for compound 4e and the results (Figure 3) depicted a significant increase in Annexin-V and 7-AAD positive cell populations, which indicated that compound 4e harbors potential apoptosis-inducing capabilities.

Flowcytometry
Since a paramount goal for anticancer agents is to induce apoptosis and cause malfunctions in the DNA, flowcytometry was conducted in this study for compound 4e and the results (Figure 3) depicted a significant increase in Annexin-V and 7-AAD positive cell populations, which indicated that compound 4e harbors potential apoptosis-inducing capabilities.

Cell Cycle Arrest
To evaluate if the antiproliferative effect of 4e was due to the disturbance of the cell cycle, cells were treated with 10 µm for 48 h. Results show that there was an increase in the cell population in the S phase (48.72%) compared to control (34.72%) in MCF-7 cells, about a 1.04-fold increase ( Figure 4). Therefore, it is possible that the antiproliferative effect of 4e is due to S-phase cell cycle arrest.

Cell Cycle Arrest
To evaluate if the antiproliferative effect of 4e was due to the disturbance of the cell cycle, cells were treated with 10 µm for 48 h. Results show that there was an increase in the cell population in the S phase (48.72%) compared to control (34.72%) in MCF-7 cells, about a 1.04-fold increase ( Figure 4). Therefore, it is possible that the antiproliferative effect of 4e is due to S-phase cell cycle arrest.

Flowcytometry
Since a paramount goal for anticancer agents is to induce apoptosis and cause malfunctions in the DNA, flowcytometry was conducted in this study for compound 4e and the results (Figure 3) depicted a significant increase in Annexin-V and 7-AAD positive cell populations, which indicated that compound 4e harbors potential apoptosis-inducing capabilities.

Cell Cycle Arrest
To evaluate if the antiproliferative effect of 4e was due to the disturbance of the cell cycle, cells were treated with 10 µm for 48 h. Results show that there was an increase in the cell population in the S phase (48.72%) compared to control (34.72%) in MCF-7 cells, about a 1.04-fold increase ( Figure 4). Therefore, it is possible that the antiproliferative effect of 4e is due to S-phase cell cycle arrest.

General
The melting points were measured using a Gallenkamp melting point device and are uncorrected. The NMR samples of the synthesized compounds 4a-q were dissolved in DMSO-d 6 , and the NMR spectra were recorded using a Bruker NMR spectrometer (Bruker, Reinstetten, Germany) at 500/700 MHz for 1 H and 125.76/175 MHz for 13 C at the Research Center, College of Pharmacy, King Saud University, Saudi Arabia. TMS was used as an internal standard, and chemical shift values were recorded in ppm on the δ scale. The 1 H NMR spectral data are represented as follows: chemical shifts, multiplicity (s, singlet; d, doublet; t, triplet; and m, multiplet), and number of protons. The 13 C NMR spectral data were represented as chemical shifts and type of carbon. Mass spectra were measured on an Agilent Quadrupole 6120 LC/MS with ESI (electrospray ionization) source (Agilent Technologies, Palo Alto, CA, USA). Elemental analysis was carried out at the Microanalysis Laboratory, Cairo University, Cairo, Egypt, using an Elemental C, H, N analyzer Vario EL III, Germany, and the results agreed favorably with the proposed structures within ±0.4% of the theoretical values. Silica gel thin layer chromatography (TLC) plates from Merck, Burlington, MA, USA (silica gel precoated aluminum plates with fluorescent indicator at 254 nm) were used for thin layer chromatography. Visualization was performed by illumination with a UV light source (254 nm). Cell line cells were purchased from American Type Culture Collection, and cells were cultured using DMEM (Invitrogen/Life Technologies, Carlsbad, CA, USA) supplemented with 10% Hyclone FBS, 10 ug/mL of insulin (Sigma, St. Louis, MO, USA), and 1% penicillin-streptomycin. All of the other chemicals and reagents were from Sigma or Invitrogen.

General Method for the Synthesis of Substituted Benzyl Hydrazine 3
The appropriate benzyl chloride (0.01 mol) dissolved in 8 mL absolute ethanol was added dropwise to a stirred solution of 98% hydrazine hydrate (6 mL, 0.12 mol) in 12 mL absolute ethanol, and the resulting mixture continued to be stirred at room temperature for 24 h. Then, the solvent was evaporated under reduced pressure, 0.12 mL of aqueous solution 50% NaOH was added to the residue, and the resulting mixture was extracted by diethyl ether (3 × 20 mL). The combined organic layer was dried over anhydrous Na 2 SO 4 and then concentrated to afford benzyl hydrazine with 60-80 yield %, which was used in the next step without further purification.

General Method for the Synthesis of the Target Compounds 4a-q
A mixture of indole-2-carboxylic acid 3 (0.25 g, 1.55 mmol) and EDCI (0.3 g, 1.55 mmol) in methylene chloride (5 mL) was stirred for about 10 min until a clear solution was obtained. Substituted benzyl hydrazine 2 (1.55 mmol) dissolved in methylene chloride (5 mL) was added to the mixture and stirred for 24 h. The mixture was washed successively with water (2 × 20 mL), 10% NaHCO 3 solution (2 × 15 mL), and water (2 × 15 mL). The combined organic layer was dried over anhydrous Na 2 SO 4 and concentrated. The formed precipitate was collected and re-crystallized from ethanol to yield the corresponding 4a-q.

Conclusions
Herein, we reported the design and synthesis of a series of substituted-N-benzyl-1H-indole-2-carbohydrazide 4a-q. All the synthesized compounds were tested for their antiproliferative activity against three cancer cell lines, namely, MCF-7, A549, and HCT using an MTT assay. Several compounds showed moderate to high cytotoxicities with IC50, similar or superior to the reference drug Staurosporine. In particular, compound 4e was the most active congener from the series, with IC50s as low as 0.57, 1.95, and 3.49 µM, respectively, against MCF7, HCT116, and A549 compared to the IC50s of Staurosporine (11.1, 7.02, and 8.42 µM, respectively). Additionally, the selectivity of all the series were evaluated against a non-tumorigenic cell line (WI38), and compound 4e exhibited selectivity toward cancer cells. Finally, flow cytometry suggested that compound 4e harbors potential apoptosis-inducing capabilities. Based on the findings, compound 4e seems to be a promising lead compound for further investigation.