Research ArticleExperimental Studies
Open Access

Cytostatic Benzylidene-isophthaloylhydazides With Diminished Neurotoxicity

MOHAMMAD HOSSAIN, ROSS A. FILBRUN, SHAYNE M. ROTH, HIROSHI SAKAGAMI, SHIGERU AMANO, HARUNA NAGASE, KEITARO SATOH and JONATHAN R. DIMMOCK
Anticancer Research March 2026, 46 (3) 1183-1195; DOI: https://doi.org/10.21873/anticanres.18022

Abstract

Background/Aim: Benzylidene-isophthaloylhydrazides possess functional groups capable of thiol alkylation, hydrogen bonding, and van der Waals interactions, making them promising scaffolds for anticancer drug design. This study involved the synthesis of such derivatives (1a-o) and evaluation of their cytotoxicity, tumor selectivity, neurotoxicity, structure–activity relationships, and drug-like properties to identify candidates with high anticancer potency and diminished neuronal toxicity.

Materials and Methods: Compounds 1a-l were synthesized by condensation of isophthalohydrazide with substituted aryl aldehydes, while 1m-o were formed from benzoylhydrazide analogs. Cytotoxicity was measured using oral squamous cell carcinoma cells (Ca9-22, HSC-2), normal oral cells (HGF, HPC), and NGF-differentiated PC-12 neuronal cells using MTT assays. Selectivity index (SI) and anticancer/neurotoxicity ratio (RAN) were calculated. Cell-cycle progression and apoptosis induction were examined using flow cytometry and western blotting. Torsion angles (θA, θB) and other physicochemical parameters were determined using computational analysis, and QSAR correlations were assessed.

Results: Most of the compounds showed greater cytotoxicity towards neoplastic cells than non-malignant ones. Compound 1h displayed the highest tumor specificity (SI >59.5) and minimal neurotoxicity, outperforming doxorubicin, melphalan, and cisplatin. Compounds 1d, 1f, and 1h exhibited high RAN values, indicating reduced neuronal toxicity. Cell-cycle analysis revealed that 1h induced S-phase accumulation with a modest increase in apoptotic subG1 populations, accompanied by caspase-3 activation. The dimeric analogs were significantly more potent than their monomeric counterparts, and higher torsion angles generally correlated with enhanced activity. ADME (Absorption, Distribution, Metabolism, and Excretion) properties evaluation showed favorable drug-like characteristics across the series.

Conclusion: Benzylidene-isophthaloylhydrazides – particularly compound 1h – are promising antineoplastic agents with noteworthy tumor selectivity and low neurotoxicity accompanied by favorable ADME properties. Their robust activity, favorable ADME properties, and clear structural trends support further optimization and preclinical development.

Keywords:

Introduction

The development of new cytotoxic agents remains a central objective in anticancer drug discovery, particularly compounds capable of engaging multiple molecular targets within malignant cells. With this aim, we designed a series of benzylidene-isophthaloylhydrazides that incorporate functional groups capable of alkylating cellular thiols, forming hydrogen bonds, and participating in van der Waals interactions. Such features may facilitate productive interactions with key biomolecular sites and thereby elicit potent cytotoxic effects. The proposed cellular interactions of the compounds in series 1 are portrayed in Figure 1.

Figure 1.
Figure 1.

Design of compounds in series 1.

A preliminary investigation revealed that 1a inhibited the growth of human HCT116 and HT29 colon cancer cells but was less toxic to human non-neoplastic CRL1790 colon cells (1). Thus, 1a became the lead molecule for this study, and development took place in the following ways. First, a variety of substituents were placed in two of the aryl rings to give 1a-h (Figure 2). The biodata obtained may allow structure-activity relationships (SAR) and quantitative structure-activity relationships (QSAR) to be discerned. Second, the potencies of these molecules may be influenced by the torsion angles theta (θ) formed between the aryl rings A and B with the azomethine groups (2-4). The insertion of two methyl groups onto the aryl rings A and B of 1f led to 1i and 1j (Figure 3), which should increase the sizes of the theta value (Figure 4). The replacement of the methine proton by methyl and ethyl groups, as displayed by 1k and 1l, should lead to compounds with significant changes in theta values. Third, the compounds 1m-o were prepared in order to compare their cytotoxic potencies with those of 1f, b, and e, respectively (Figure 3). The biodata should indicate whether a second group in ring C (Figure 1) contributes significantly to cytotoxic potencies. Fourth, the bioevaluations were designed to detect potent cytotoxins towards a range of different neoplasms while being well tolerated by non-malignant cells. The discovery of some of the mechanisms of action of lead molecules was also contemplated.

Figure 2.
Figure 2.

Synthesis of 1a-h.

Figure 3.
Figure 3.

Structures of the compounds 1i-o. These molecules were prepared by reacting isophthaloylhydrazide with various aryl aldehydes and ketones.

Figure 4.
Figure 4.

Designation of the torsion angles θA and θB between aryl rings A and B with the adjacent azomethine groups.

Materials and Methods

Chemistry. General. Melting points of all synthesized compounds were determined using an Electrochemical digital melting point apparatus, model number 1A9100 (Cole-Parmer Ltd, Stone, Staffordshire, UK), and are uncorrected. 1H and 13C NMR spectra were obtained using a Bruker Avance III 500 MHz NMR spectrometer (Bruker Scientific LLC, Billerica, MA, USA) in dimethylsulfoxide (DMSO-d6), or CDCl3. Chemical shifts are reported in δ (ppm) units relative to the internal standard tetramethylsilane (TMS). High-resolution mass spectra (HRMS) were obtained using a Thermo Scientific Finnigan LTQ Orbitrap XL Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and all samples were run using ESI positive ion mode. The purity of all final compounds was established by Agilent 1200 HPLC (Agilent Technologies, Inc., Santa Clara, CA, USA) with a diode-array detector. All compounds were found to be >95% pure by HPLC analysis. All chemicals and solvents used were of reagent grade without being purified or dried before use. Isophthalohydrazide was synthesized from the dimethyl isophthalate following published procedures (5).

General procedure for the synthesis of compound 1a-l. To a solution of isophthalohydrazide (200 mg, 1 mmol) in 15 ml ethanol was added a solution of appropriate benzaldehyde (2.2 mmol) in ethanol (5 ml) and 2-3 drops of acetic acid. The mixture was then refluxed at 85°C for 24 h. After about half of the solvent was evaporated using a rotary evaporator, the mixture was cooled to room temperature and then placed in an ice-water bath. The precipitate formed was collected by vacuum filtration, washed with ice-cold ethanol (about 2 ml), and finally dried in the oven at 50ºC to give compound 1a-l.

N′1,N′3-Bis(4-hydroxybenzylidene)isophthalohydrazide (1a). Compound 1a appeared as a white powder, yield 89.0%, melting point (MP): 326.3-328.4°C, 1H NMR (DMSO-d6): δ 11.83 (s, 2H, CONH), 9.96 (s, 2H, Ar-OH), 8.44 (s, 1H, Ar-H), 8.39 (s, 2H, N=CH), 8.09 (d, 2H, Ar-H, 7.7 Hz), 7.66 (dd, 1H, Ar-H, 7.7 Hz), 7.58 (d, 4H, Ar-H, 8.2 Hz), 6.85 (d, 4H, Ar-H, 8.2 Hz) ppm. 13C NMR (DMSO-d6): δ 162.9, 160.0, 149.0, 134.4, 131.0, 129.4, 129.2, 127.3, 125.7, 116.2 ppm. HRMS (ESI): m/z 403.1413 ([M+H]+), calcd 403.1401 for C22H18N4O4.

N′1,N′3-Bis((E)-4-chlorobenzylidene)isophthalohydrazide (1b). Compound 1b was obtained as white solid powder, yield 90.3%, MP: 293.2-295.3°C, 1H NMR (DMSO-d6): δ 12.12 (s, 2H, CONH), 8.48 (s, 3H, N=CH & Ar-H), 8.13 (d, 2H, Ar-H, 7.3 Hz), 7.71 (d, 4H, Ar-H, 7.7 Hz), 7.70 (dd, 1H, Ar-H, 7.3 Hz), 7.53 (d, 4H, Ar-H, 7.9 Hz) ppm. 13C NMR (DMSO-d6): δ 163.1, 147.3, 135.1, 134.8, 134.1, 133.7, 131.4, 129.4, 129.3, 127.5 ppm. HRMS (ESI): m/z 439.0723 ([M+H]+), and 461.0542 ([M+Na]+), calcd 439.0723 for C22H17N4O2Cl2 and 461.0543 for C22H16N4O2Cl2Na.

N′1,N′3-Bis((E)-4-fluorobenzylidene)isophthalohydrazide (1c). Compound 1c appeared as fluffy white powder, yield 92.6%, MP: 271.1-273.0°C, 1H NMR (DMSO-d6): δ 12.06 (s, 2H, CONH), 8.49 (s, 3H, N=CH & Ar-H), 8.12 (δ, 2H, Ar-H, 7.3 Hz), 7.81 (s, 4H, Ar-H), 7.69 (dd, 1H, Ar-H, 7.3 Hz), 7.30 (dd, 4H, Ar-H, 8.5 Hz) ppm. 13C NMR (DMSO-d6): d 164.6, 163.1, 162.7, 147.5, 134.2, 131.3, 129.8, 129.3, 127.4, 116.5, 116.3 ppm. HRMS (ESI): m/z 407.1315 ([M+H]+), and 429.1134 ([M+Na]+), calcd 407.1314 for C22H17N4O2F2 and 429.1134 for C22H16N4O2F2Na.

N′1,N′3-Bis((E)-4-methylbenzylidene)isophthalohydrazide (1d). Compound 1d appeared as white crystalline powder, yield 95.6%, MP: 300.0-302.1°C, 1H NMR (DMSO-d6): δ 11.98 (s, 2H, CONH), 8.46 (s, 3H, N=CH & Ar-H), 8.11 (d, 2H, Ar-H, 7.5 Hz), 7.69 (dd, 1H, Ar-H, 7.6 Hz), 7.64 (d, 4H, Ar-H, 7.6 Hz), 7.28 (d, 4H, Ar-H, 7.6 Hz), 2.34 (s, 6H, Ar-CH3) ppm. 13C NMR (DMSO-d6): δ 163.0, 148.7, 140.5, 134.3, 132.0, 131.2, 130.0, 129.3, 127.6, 127.4, 21.5 ppm. HRMS (ESI): m/z 399.1816 ([M+H]+), and 421.1636 ([M+Na]+), calcd 399.1816 for C24H23N4O2 and 421.1635 for C24H22N4O2Na.

N′1,N′3-Bis((E)-4-methoxybenzylidene)isophthalohydrazide (1e). Compound 1e appeared as white powder, yield 93.3%, MP: 267.6-269.9°C, 1H NMR (DMSO-d6): δ 11.91 (s, 2H, CONH), 8.45 (s, 1H, Ar-H), 8.43 (s, 2H, N=CH), 8.10 (d, 2H, Ar-H, 7.6 Hz), 7.69 (m, 3H, Ar-H), 7.03 (d, 4H, Ar-H, 8.3 Hz), 3.81 (s, 6H, OCH3) ppm. 13C NMR (DMSO-d6): δ 162.9, 161.4, 148.6, 134.3, 131.1, 129.2, 128.8, 127.3, 127.2, 114.8, 55.8 ppm. HRMS (ESI): m/z 431.1714 ([M+H]+), and 453.1534 ([M+Na]+), calcd 431.1714 for C24H23N4O4 and 453.1533 for C24H22N4O4Na.

N′1,N′3-Di((E)-benzylidene)isophthalohydrazide (1f). Compound 1f appeared as white solid powder, yield 72%, MP: 248.1-250.0°C, 1H NMR (DMSO-d6): δ 12.05 (s, 2H, CONH), 8.50 (s, 2H, N=CH), 8.49 (s, 1H, Ar-H), 8.13 (d, 2H, Ar-H, 7.3 Hz), 7.76 (d, 4H, Ar-H, 7.3 Hz), 7.70 (dd, 1H, Ar-H, 7.5 Hz), 7.47 (br, s, 6H, Ar-H), ppm. 13C NMR (DMSO-d6): δ 163.1, 148.7, 134.7, 134.2, 131.3, 130.7, 129.8, 129.3 127.7, 127.4 ppm. HRMS (ESI): m/z 371.1503 ([M+H]+) and 393.1323 ([M+Na]+), calcd 371.1503 for C22H19N4O2 and 393.1322 for C22H18N4O2Na.

N′1,N′3-Bis((E)-4-(dimethylamino)benzylidene)isophthalohydrazide (1g). Compound 1g appeared as yellow powder, yield 94.0%, MP: 299.7-302.3°C, 1H NMR (DMSO-d6): δ 11.73 (s, 2H, CONH), 8.44 (s, 1H, Ar-H), 8.35 (s, 2H, N=CH), 8.08 (d, 2H, Ar-H, 7.6 Hz), 7.68 (dd, 1H, Ar-H, 6.8 Hz), 7.56 (d, 4H, Ar-H, 8.4 Hz), 6.77 (d, 4H, Ar-H, 8.5 Hz), 2.98 (s, 12H, N(CH3)2) ppm. 13C NMR (DMSO-d6): δ 162.7, 152.1, 149.5, 134.5, 130.9, 129.1, 129.0, 127.2, 122.0, 112.3, 40.2 ppm. HRMS (ESI): m/z 457.2346 ([M+H]+), and 479.2164 ([M+Na]+), calcd 457.2347 for C26H29N6O2 and 479.2166 for C26H28N6O2Na.

N′1,N′3-Bis((E)-4-nitrobenzylidene)isophthalohydrazide (1h). Compound 1h was obtained as yellow powder, yield 87.5%, MP: 317.1-318.1°C, 1H NMR (DMSO-d6): δ 12.34 (s, 2H, CONH), 8.58 (s, 2H, N=CH), 8.51 (s, 1H, Ar-H), 8.30 (d, 4H, Ar-H, 8.0 Hz), 8.16 (d, 2H, Ar-H, 6.8 Hz), 8.01 (d, 4H, Ar-H, 7.8 Hz), 7.73 (dd, 1H, Ar-H, 7.0 Hz) ppm. 13C NMR (DMSO-d6): δ 163.3, 148.4, 146.2, 141.0, 133.9, 131.6, 129.4, 128.6, 127.7, 124.5 ppm. HRMS (ESI): m/z 461.1203 ([M+H]+), calcd 461.1204 for C22H17N6O6.

N′1,N′3-Bis((E)-3,5-dimethylbenzylidene)isophthalohydrazide (1i). Compound 1i was obtained as white powder, yield 87.8%, MP: 309.3-312.4°C, 1H NMR (DMSO-d6): δ 12.00 (s, 2H, CONH), 8.45 (s, 1H, Ar-H), 8.40 (s, 2H, N=CH), 8.11 (d, 2H, Ar-H, 7.4 Hz), 7.69 (dd, 1H, Ar-H, 7.6 Hz), 7.36 (s, 4H, Ar-H), 7.08 (s, 2H, Ar-H), 2.32 (s, 12H, Ar-CH3) ppm. 13C NMR (DMSO-d6): δ 163.0, 148.8, 138.4, 134.7, 134.3, 132.2, 131.3, 129.3, 127.4, 125.4, 21.3 ppm. HRMS (ESI): m/z 427.2130 ([M+H]+), and 449.1949 ([M+Na]+), calcd 427.2129 for C26H27N4O2 and 449.1948 for C26H26N4O2Na.

N′1,N′3-Bis((E)-2,6-dimethylbenzylidene)isophthalohydrazide (1j). Compound 1j was obtained as white powder, yield 92.5%, MP: 305.1-307.1°C, 1H NMR (DMSO-d6): δ 11.97 (s, 2H, CONH), 8.82 (s, 2H, N=CH), 8.50 (s, 1H, Ar-H), 8.14 (d, 2H, Ar-H, 7.3 Hz), 7.72 (dd, 1H, Ar-H, 7.5 Hz), 7.18 (d, 4H, Ar-H, 7.6 Hz), 7.28 (dd, 2H, Ar-H, 7.2 Hz), 7.11 (d, 4H, Ar-H, 7.1 Hz), 2.47 (s, 12H, Ar-CH3) ppm. 13C NMR (DMSO-d6): δ 162.9, 148.7, 137.8, 137.6, 134.3, 131.5, 131.2, 129.3, 129.1, 127.2, 21.5 ppm. HRMS (ESI): m/z 427.2129 ([M+H]+), and 449.1949 ([M+Na]+), calcd 427.2129 for C26H27N4O2 and 449.1948 for C26H26N4O2Na.

N′1,N′3-Bis((E)-1-phenylethylidene)isophthalohydrazide (1k). Compound 1k was obtained as a white powder, yield 94.3%, MP: 273.8-275.7°C, 1H NMR (DMSO-d6): δ 10.92 (s, 2H, CONH), 8.39 (s, 1H, Ar-H), 8.07 (s, 2H, N=CH), 7.87 (s, 4H, Ar-H), 7.66 (s, 1H, Ar-H), 7.44 (s, 6H, Ar-H), 2.40 (s, 6H, N=CCH3) ppm. 13C NMR (DMSO-d6): δ 163.9, 156.3, 138.5, 134.5, 131.4, 130.1, 128.9, 127.0, 15.2 ppm. HRMS (ESI): m/z 399.1817 ([M+H]+), and 421.1636 ([M+Na]+), calcd 399.1816 for C24H23N4O2 and 421.1635 for C24H22N4O2Na.

N′1,N′3-Bis((E)-1-phenylpropylidene)isophthalohydrazide (1l). Compound 1l was obtained as a white powder, yield 81.8%, MP: 267.5-270.6°C, 1H NMR (DMSO-d6): δ 10.95 (s, 2H, CONH), 8.27 (s, br, 1H, Ar-H), 8.04 (s, br, 2H, N=CH), 7.84 (s, 4H, Ar-H), 7.66 (s, 1H, Ar-H), 7.45 (s, 6H, Ar-H), 2.93 (s, 4H, N=CCH2), 1.08 (s, 6H, CH3) ppm. 13C NMR (DMSO-d6): data could not be collected as the compound was not very soluble in the solvent. HRMS (ESI): m/z 427.2127 ([M+H]+), and 449.1947 ([M+Na]+), calcd 427.2129 for C26H27N4O2 and 449.1948 for C26H26N4O2Na.

General procedure for synthesis of compound 1m-o. To a solution of phenylhydrazide (200 mg, 1.5 mmol) in 8 ml ethanol were added a solution of appropriate benzaldehyde (1.6 mmol) in ethanol (6 ml) and 2-3 drops of acetic acid. The mixture was then refluxed at 85°C for 24 h. After about 80% of the solvent was evaporated using a rotary evaporator, the mixture was cooled to room temperature and then placed in an ice-water bath. The precipitate formed was collected by vacuum filtration, washed with ice-cold ethanol (about 2 ml), and finally dried in the oven at 50°C to give compound 1m-o.

(E)-N′-benzylidenebenzohydrazide (1m). Compound 1m was obtained as white crystal, yield 85.6%, MP: 212.4-214.6°C, 1H NMR (DMSO-d6): δ 11.87 (s, 1H, CONH), 8.48 (s, 1H, N=CH), 7.93 (d, 2H, Ar-H, 7.3 Hz), 7.74 (d, 2H, Ar-H, 6.5 Hz), 7.59 (dd, 1H, Ar-H, 7.1 Hz), 7.53 (dd, 2H, Ar-H, 7.4 Hz), 7.46-7.45 (br, 3H, Ar-H) ppm. 13C NMR (DMSO-d6): δ 163.6, 148.3, 134.8, 133.9, 132.2, 130.6, 129.3, 128.9, 128.1, 127.6 ppm. HRMS (ESI): m/z 247.0844 ([M+Na]+), calcd 247.0842 for C14H12N2ONa.

(E)-N′-(4-chlorobenzylidene)benzohydrazide (1n). Compound 1n was obtained as white crystal, yield 78.0%, MP: 176.6-179.7°C, 1H NMR (DMSO-d6): δ 11.93 (s, 1H, CONH), 8.46 (s, 1H, N=CH), 7.92 (d, 2H, Ar-H, 7.3 Hz), 7.76 (d, 2H, Ar-H, 8.0 Hz), 7.59 (dd, 1H, Ar-H, 7.0 Hz), 7.53 (dd, 2H, Ar-H, 7.4 Hz), 7.54-7.51 (m, br, 4H, Ar-H) ppm. 13C NMR (DMSO-d6): δ 163.7, 146.9, 135.0, 133.8, 133.7, 132.3, 129.4, 129.2, 128.9, 128.1 ppm. HRMS (ESI): m/z 281.0455 ([M+Na]+), calcd 281.0452 for C14H11N2OClNa.

(E)-N′-(4-methoxybenzylidene)benzohydrazide (1o). Compound 1o was obtained as white crystal, yield 85.8%, MP: 155.5-157.9°C, 1H NMR (DMSO-d6): δ 11.74 (s, 1H, CONH), 8.41 (s, 1H, N=CH), 7.91 (d, 2H, Ar-H, 7.4 Hz), 7.68 (d, 2H, Ar-H, 8.4 Hz), 7.58 (dd, 1H, Ar-H, 7.0 Hz), 7.52 (dd, 2H, Ar-H, 7.4 Hz), 7.02 (d, 2H, Ar-H, 8.4 Hz), 3.81 (s, 3H, OCH3) ppm. 13C NMR (DMSO-d6): δ 163.4, 161.3, 148.2, 134.1, 132.1, 129.2, 128.9, 128.0, 127.4, 114.8 ppm. HRMS (ESI): m/z 277.0950 ([M+Na]+), calcd 277.0947 for C15H14N2O2Na.

Cytotoxicity assays. Human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2), rat adrenal pheochromocytoma cell line (PC-12), (all purchased from Riken Cell Bank, Tsukuba, Japan) and human normal oral cells (gingival fibroblasts, HGF; pulp cell, HPC) (established from the first premolar extracted tooth in the lower jaw and periodontal tissues of a twelve-year-old girl, according to the guidelines of Meikai University Ethics Committee (No. A0808) were cultured at 37°C in DMEM supplemented with 10% heat (56°C, 30 min)-inactivated FBS, 100 U/ml, penicillin G and 100 μg/ml streptomycin sulfate under a humidified 5% CO2 atmosphere, as described previously (6). Neuronal cells that extend neurites, the marker of neuronal differentiation, were prepared from PC-12 cells by the repeated feeding with NGF-containing culture medium in the absence of FBS (7). In brief, near confluent cells were incubated in triplicate for 48 h at 37°C with the indicated different concentrations of the compounds; then, viable cell numbers were measured using the MTT method (6). From the dose–response curve, a 50% cytotoxic concentration (CC50) was determined. All samples were dissolved in DMSO. The toxicity of DMSO alone was calculated and subtracted. Cell morphology was visualized under light microscopy (EVOSfl; Thermo Fisher Scientific).

Calculation of selectivity index (SI). SI was calculated by dividing the average CC50 value (concentration of the compound reducing the viability by 50%) of the compound towards HGF and HPC cells (Table I) by the average CC50 value of the compound against a specific neoplastic cell line. These results are shown in Table I.

View this table:
Table I.

Tumor-specificity of 1a-o and anticancer drugs.

Cell cycle analysis. Cells (approximately 106 cells) were fixed with paraformaldehyde (Fujifilm Wako Pure Chemical Ind., Osaka, Japan) in PBS(−) and treated with ribonuclease (RNase) A (Sigma-Aldrich Inc., St. Louis, MO, USA). After staining with propidium iodide (PI; Fujifilm Wako Pure Chemical Ind.) in the presence of 0.01% Nonidet-40 (Nacalai Tesque, Kyoto, Japan) to prevent cell aggregation, the cells were filtered through Falcon cell strainers (Corning Inc., Corning, NY, USA) and then subjected to cell sorting (SH800 Series, SONY, Tokyo, Japan), analyzed with Cell Sorter Software version 2.1.2. (SONY) as described previously (6).

Calculation of the ratio of anticancer activity to neurotoxicity (RAN). RAN was calculated by dividing the average of CC50 values of the compound towards differentiated PC12 cells by the average CC50 value of the compound against a specific neoplastic cell line (Table II).

View this table:
Table II.

Neurotoxicity of 1a-o and anticancer drugs.

Western blot analysis. The cells were washed and lysed, and their protein extracts subjected to western blot (WB) analysis, as described previously (6). In brief, all protein samples of cell lysates (9 μg) were separated in SDS-PAGE, transferred onto a PVDF filter, blocked at room temperature in skim milk and then probed for 120 min with a primary antibody cocktail (1:250) using Apoptosis Western Blot Cocktail kit (Abcam, Cambridge, UK). The blots were washed three times and then probed for 90 min with a horseradish peroxidase-conjugated secondary antibody cocktail (1:100). Immunoreactivities were detected using Amersham ECL Select (Cytiva, Tokyo, Japan). Images were acquired using LuminoGraph III (ATTO, Tokyo, Japan) and CS Analyzer 4 (ATTO).

Statistical and QSAR analysis. Experimental data are presented as the mean standard deviations (8) of triplicate determinations. One-way ANOVA and Bonferroni’s post-test were performed using IBM SPSS 27.0 statistics (IBM Co., Armonk, NY, USA). The significance level was set at p<0.05.

The s, p, and MR constants used in the QSAR determinations were taken from the literature (9). Linear and semilogarithmic plots were made between the CC50 values for 1a-h in the different screens using the IBM-SPSS 17.0 software (10).

Determination of theta (θ) values. The θ values were obtained by measuring the dihedral angles using the Spartan Student v8.0.6 (11). Each dihedral angle was measured by navigating the “Geometry” menu of Spartan followed by selecting “Measure Dihedral” and then clicking on the four atoms in the order, for example, N3, C9, C11, and C13 to measure the dihedral angle between, with the central bond being the middle two atoms C9 and C11. A negative dihedral angle indicates that the sequence of selected atoms was in a “clockwise” direction, which is how the software is designed to calculate the dihedral angle (Table III).

View this table:
Table III.

Some physicochemical data of 1d, f, i-l.

Results

The compounds in series 1 were prepared by reacting methyl isophthalate with hydrazine. This intermediate reacted with various aryl aldehydes to give 1a-l (Figure 2 and Figure 3). Reaction of benzoylhydrazine with several aryl aldehydes led to the formation of 1m-o (Figure 3).

Compounds 1a-o were evaluated against non-malignant oral human gingival fibroblast (HGF) and human pulp cells (HPC), as well as differentiated neuronal cells (dPC-12), in comparison with human oral squamous cell carcinoma (OSCC) cell lines derived from the gingiva (Ca9-22) and tongue (HSC-2), since anticancer drugs can show potent neurotoxicity (12, 13). These data are presented in Table I and Table II.

The alignment of a cytotoxin at a binding site may be influenced by the interplanar angle theta(θ) between an aryl ring and an adjacent unsaturated group, as illustrated in Figure 4. Thus, the θA and the θB angles of six representative compounds 1d, f, i-l were determined, and the results are presented in Table III.

Discussion

In general, the benzylidene isophthalate analogs showed cytostatic growth inhibition against two human OSCC cell lines, namely, Ca9-22 (derived from gingiva) and HSC-2 cells (derived from the tongue), in addition to two human normal oral cells (HGF from gingiva, HPC from pulp) and dPC12 cells extending neurites (Figure 5). Among them, 1h showed the highest tumor-specific cytotoxicity (SI >59.5), followed by 1f (>24.1) and 1e (>8.3) (Table I), comparable with doxorubicin (SI >31.1), melphalan (>8.2), and cisplatin (>13.9). Cell cycle (A, B) and western blot (C) analyses demonstrated that 1h significantly (p<0.05) increased the proportion of S-phase cells, slightly but significantly (p<0.05) and increased the subG1 apoptotic cell population (Figure 6). It should be noted that a number of isophthalate analogs namely 1d, f, g, h, I showed good relative RAN (anticancer activity to neurotoxicity) values (RAN=9.6, 15.7, 25.1, 59.5, 30.9) which were greater than the values for doxorubicin, melphalan and cisplatin (RAN=1.3, 2.1, 4.2). Their clinical neurotoxicity has been well documented (14, 15). We also noticed that doxorubicin, melphalan, and cisplatin, but not 1f and 1h, disrupted neurites in dPC-12 cells (Figure 7). These results reveal that 1f and 1h not only show one order of magnitude lower neurotoxicity than three anticancer drugs tested here but also exhibit comparable tumor selectivity.

Figure 5.
Figure 5.

Dose–response curve of the cytotoxicity of 1a-o, and anticancer drugs. Cells were incubated for 48 h without (control) or with the indicated concentrations of test samples, and the viable cell number was determined using the MTT method and expressed as % of control. Each value is expressed as the mean±SD of triplicate determinations.

Figure 6.
Figure 6.

Induction of S phase accumulation and minor production of subG1 apoptotic cells by 1h. Ca9-22 cells were treated for 24 h with vehicle (control), actinomycin D (Act.D) (1 μM) of the indicated concentrations of 1h (1, 23 μM), and cell cycle (A, B) and western blot analysis (C) for detection of apoptosis induction were performed. Each value is expressed as the mean±SD of triplicate determinations. *p<0.05 vs. control (Bonferroni’s post-test) (B).

Figure 7.
Figure 7.

Doxorubicin, melphalan and cisplatin, but not 1f and 1h, disrupted neurites. Scale bars=200 μm.

An initial issue to resolve was the tolerability of non-malignant cells to 1a-o. These compounds were evaluated against human non-malignant HGF and HPC cells and the results are shown in Table I. The CC50 values of 1a, b, d, e, f, h, i, m, n, o were greater than 10 μM in HGF cells, whereas those of 1a, f, h, i, m, n, o were higher than 10 μM in HPC cells. In fact, 37% of the CC50 values of 1a-o were below 10 μM.

A comparison was made between the potencies of three representative “monomers” 1m, n, o, and the related “dimers” 1f, b, e. The average CC50 values of 1m, n, o towards Ca9-22 and HSC-2 cells were >200, 43.6 and 149.4, respectively. However, the average CC50 values of 1f, b, e were 4.4, 7.9, and 12.3 μM, respectively (Table I). In light of this observation, the dimers but not the monomers, were synthesized.

An attempt was made to determine the correlations between cytotoxic potencies and the interplanar angles θA and θB. Six representative compounds, namely 1d, f, i, j, k, and l, were selected for the following reasons. The θ figures of 1d, and f are predicted to be similar but the presence of methyl substitution in the meta analog (1i) and 2 and 6 position (1j) should increase the θ values. Furthermore, one would predict that the presence of a methyl group (1k) and ethyl function (1l) on the azomethine carbons should lead to increases in the θ values.

The θA and θB angles of 1d, f, i-l were determined and are presented in Table III. The data reveal that 1d, f, i have small θ values whereas 1j, k, l have greater ones. In the case of 1d, f, and i, there are no substituents in the ortho location or azomethine carbon while the additional alkyl groups in 1j, k, and l lead to compounds with higher theta values.

An attempt was made to discern if the magnitude of the θ data correlates with cytotoxic potency. The average CC50 values of 1d, f, i towards Ca9-22 and HSC-2 cells were 20.94, 4.36, and >101.49 μM, respectively, while the figures for 1j, k, l were 0.39, 1.04, and 7.21 μM, respectively (Table I). Thus, in general, potencies increase as the θ values rise. While further work needs to be undertaken to explore the generality of this relationship, the data established so far suggests further studies.

A key question when considering the advisability of pursuing further studies with compounds 1a-o is whether they possess drug-like properties. Hence various physicochemical properties of 1a-o were determined and are listed in Table IV. The data revealed that 1a-o have druglike properties according to 95% of the figures obtained. There are no violations in terms of the molecular weight and LogP values or with hydrogen bond acceptor and donor atoms. In four cases the number of rotatable atoms were in excess and hence in the future the formation of cyclic analogs might overcome this potential disadvantage.

View this table:
Table IV.

Evaluation of 1a-o for drug-like properties.

The magnitude of cytotoxic potencies may be influenced by the physicochemical properties of various aryl substituents. Linear plots were made between the CC50 values of 1a-h towards Ca9-22 cells and the Hammett s, Hansch p and molar refractivity (MR) values of the aryl substituents. Neither correlations (p<0.05) nor trends towards a correlation (p<0.1) were observed. The analysis was repeated using semilogarithmic plots, but again, no meaningful relationships were observed. When the same evaluations were performed using cytotoxicity data from HSC-2 cells, a slight but notable trend toward a negative correlation was identified between the π values of 1a-h and IC50 data (p=0.06). This suggests that increasing the hydrophilicity of future analogs may enhance their cytotoxic activity in this cell line.

Conclusion

This study revealed that in general the compounds in series 1 are potent novel cytotoxic agents. In addition, many of the compounds displayed greater potencies to neoplasms than to non-malignant cells thereby generating noteworthy Selectivity Index figures. A number of lead molecules were identified based on their cytotoxic potencies namely 1g, 1j, 1c, 1k and SI figures namely 1f, 1h. In addition, this series of compounds have favorable ADME properties.

Several modes of action studies were undertaken with Ca9-22 cells. The representative compound 1h led to an accumulation of cells in the S phase and a slight increase in the apoptotic subG1 population due to caspase-3 activation, as shown by western blot analysis. Neither 1f nor 1h disrupted the neurites of dPC-12 cells, suggesting their weak neurotoxicity compared with cancer cells.

In view of the novelty of the compounds, their greater toxicity towards some neoplasms than non-malignant cells and their favorable ADME properties, future work should be initiated to develop these compounds. In addition, the reasons why the dimeric compounds are more toxic than the monomeric analogs should be investigated.

Acknowledgements

The Authors thank Office of the Executive Vice Chancellor for Academic Affairs, Indiana University Kokomo for financial support; the Ministry of Education, Science, Sports and Culture, Japan; and the Maunders McNeil Foundation Inc. The Authors would also like to thank Dr. Praveen K. Roayapalley for his help with QSAR analysis.

Footnotes

  • Authors’ Contributions

    Conceptualization, M.H., H.S., J.R.D.; methodology, MH, H.N., R.A.F., S.M.R., H.S., K.S., S.A.; validation, M.H., H.S., J.R.D.; formal analysis, M.H., H.S., J.R.D.; investigation, M.H., R.A.F., S.M.R., H.N., H.S., K.S., S.A.; data curation, MH, H.S; writing – original draft preparation, J.R.D., H.S., M.H.; writing – review and editing, J.R.D., H.S., M.H.; supervision, J.R.D., H.S., M.H.; project administration, M.H., J.R.D., H.S.; funding acquisition, M.H., J.R.D., H.S.. All Authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    The Authors declare no competing interests in relation to this study.

  • Funding

    Financial support for this study was provided by the Office of the Executive Vice Chancellor for Academic Affairs, Indiana University Kokomo, which awarded a Summer Faculty Fellowship and Grant-in-Aid to M. Hossain; the Ministry of Education, Science, Sports and Culture, Japan, awarded a grant-in-aid to H. Sakagami; and the Maunders McNeil Foundation Inc. for a grant to J. R. Dimmock.

  • Artificial Intelligence (AI) Disclosure

    During the preparation of this manuscript, a large language model (Grammarly) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.

  • Received December 1, 2025.
  • Revision received December 16, 2025.
  • Accepted December 18, 2025.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Cytostatic Benzylidene-isophthaloylhydazides With Diminished Neurotoxicity
MOHAMMAD HOSSAIN, ROSS A. FILBRUN, SHAYNE M. ROTH, HIROSHI SAKAGAMI, SHIGERU AMANO, HARUNA NAGASE, KEITARO SATOH, JONATHAN R. DIMMOCK
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