Remarkable utilization of quinazoline-based homosulfonamide for in vitro cytotoxic effects with triple kinase inhibition activities: cell cycle analysis and molecular docking profile

Abstract

We tested newly synthesized compounds 1–13 on 59 cancer cell lines and found that acylhydrazones 5, 6, 7, 9, and 12 showed the best in vitro cytotoxic activity. They stopped the mean growth percentage (MG%) by an average of 23.5, 55.2, 89.4, 88.5, and 88.4%, respectively. Compound 5 was subjected to NCI tests at five-dose dilutions on 59 tumor cells. It is more effective in killing tumor cells than gefitinib (mean GI₅₀: 7.7 μM) and erlotinib (mean GI₅₀: 2.1 μM). Its mean GI₅₀ value was 1.0 μM, and its LC₅₀ value was over 100 μM, whereas gefitinib's was 95.6 μM and erlotinib's was 14.3 μM. Its TGI was 89.2 μM, while those drugs were 66.3 and 14.3 μM, respectively. We evaluated acylhydrazones 5, 6, 7, 9, and 12 for dose-dependent enzymatic inhibition of EGFR, HER2, and CDK9 kinases to study the mechanism of the in vitro cytotoxicity. With IC₅₀ values of 84.4 and 51.5 nM, compounds 5 and 6 are the most potent EGFR inhibitor analogs, similar to Gefitinib (IC₅₀ of 53.1 nM). Compounds 5, 6, and 12 blocked HER2 like Gefitinib did (IC₅₀ = 38.8 nM); their IC₅₀ values were 53.9, 44.1, and 110.6, respectively. Compounds 5, 6, and 7 had IC₅₀ values of 146.9, 96.1, and 155.4 nM, which means they blocked CDK9 activity almost as well as Dinaciclib (IC₅₀ 53.1 nM). Flow cytometers count the amount of DNA in T-47D and MOLT4 cells treated with compounds 5 and 6. The IC₅₀ value of compound 5 increases from 6.6% for the DMSO/T-47D control to 26.3% in the G2-M phase, while compound 6 goes from 61.4 for the DMSO/MOLT4 control to 89.0% in the G1 phase. The tested compounds cause early death, ranging from 0.4% and 0.6% (a DMSO control sample) to 9.3% and 19.2%, respectively. Derivatives 5 and 6 also increased late death from 0.1 to 14.8% and 12.6 to 0.3%, respectively, favoring the apoptotic route over the necrotic one for cell death to 50.5 μM. When tested for cell death against the standard WI-38 fibroblast cell line, imines 5 and 6 were less toxic than doxorubicin.

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1 Introduction

A failure of certain enzymes and proteins that control cell division and transformation causes cancer, characterized by abnormal cell growth.^1,2 Cancer is one of the most critical causes of death, with about 10 million deaths on average during the last few years.³ The quinazoline nucleus is one of the primary heterocyclic compounds with cytotoxic activity.^4–7 Multikinase inhibitors with high selectivity and efficacy contain quinazoline moieties like erlotinib (I), afatinib (II), and gefitinib (III), which are effective chemotherapeutic agents (Fig. 1). Epidermal growth factor receptors (EGFRs) belong to the family of sizeable transmembrane growth factor receptors (PTKs), including homologous receptors such as HER1, HER2, HER3, and HER4.⁸ Many malignancies, such as colon, breast, ovarian, and NSC lung cancers, are characterized by overexpression of EGFRs.⁸ Designing a cytotoxic molecule that binds to the target enzyme's catalytic domain and treats different types of human cancers that compete with ATP, like an EGFR inhibitor, could be a primary method for a cytotoxic molecule.⁸ Chemotherapy has been one of the best methods for treating cancer. Hence, synthesizing new cytotoxic molecules with potential bioactivity and a high therapeutic index is the main goal for many pharmaceutical and medicinal researchers. Compounds containing a quinazoline core, on the other hand, exhibit inhibitor activities, such as COX-2,^9–12 and carbonic anhydrase inhibitors,^13–17 as well as tyrosine kinase inhibitor activities, such as EGFRs, CDK-9,^18–27 anticonvulsant^28–31 cytotoxic activity,^32–39 and antimicrobial.^40–43 Schiff base derivatives, such as aldimines or ketamine,^44,45 can bind to and block EGFRs by interacting with their ATP-binding site, showing cytotoxic activities.^45,46 Sulfonamides also showed many pharmacologic activities.^47–60 Fig. 2 also shows several hydrazones that kill cancer cells, such as quinazolinylhydrazone (V) and PAC-1 (VI); arylhydrazone (VII and VIII) showed cytotoxic activity higher than erlotinib, gefitinib, and sorafenib against several human cancer cells.^61–65 Some hydrazones stop cancer cells from growing by blocking EGFR, HER2, and COX-2 receptors.^61–65 Here, aldimines and ketimines (4–13) are formed (Fig. 3) by combining various aldehydes or ketones with 4-((4-oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)benzenesulfonamide (1). We investigated the in vitro cytotoxic efficacy of target compounds in vitro in 59 human cancer cell lines, revealing the structure–activity relationship (SAR). We then used an enzymatic assay on effective cytotoxic acylhydrazones to determine EGFR, HER2, CDK9, and COX-2 inhibitions. We triggered apoptosis and examined cell cycles on the most active compounds to assess their in vitro cytotoxic potential. We performed molecular docking in the binding pockets of the EGFR, HER2, and CDK9 kinases to explore how the potential variants might line up.

Fig. 1 The EGFR and HER2 inhibitors antitumor agent (I–IV).

Fig. 2 The reported hydrazones with antitumor activities.

Fig. 3 The designed target quinazolinylhydrazone derivative (compounds 4–13) are based on the chemical structures of compounds I–XI.

2 Result and discussion

4-((4-Oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)benzenesulfonamide (1) was obtained in excellent yield by the heating of 2-aminobenzoic acid with 4-isothiocyanatobenzenesulfonamide in ethanol (Scheme 1). Confirmation of the compound 1 structure was provided by the presence of the thioamide (N̲HCS) singlet peak at 13.15 ppm and doublet (NH₂) peak of the sulfonamide moiety at 7.34 ppm, together with the singlet methylene peak of the phenylmethansulfonamide moiety at 5.37 ppm in ¹H NMR. At the same time, ¹³C NMR showed a thioketone (NHCS) at 175.89 ppm and benzylic methylene peaks at 49.11 ppm, respectively. The reaction of compound 1 with ethyl 2-bromoacetate in acetone and potassium carbonate was a crucial step in our research. This reaction led to the formation of the ethyl 2-((4-oxo-3-(4-sulfamoylbenzyl)-3,4-dihydroquinazolin-2-yl)thio)acetate (2). The singlet thiomethylencarbonyl peak (–SC̲H₂CO–) at 4.11 ppm in ¹H NMR, the quartet and triplet peaks of the ethoxide moiety (OCH₂CH₃) at 4.14 and 1.21 ppm, respectively, and fading of the thioamide (NH) singlet peak at 13.15 ppm confirming ester 2. ¹³C NMR of the ester 2 reveals the presence of thiomethylene peak (–SCH₂) at 47.13 ppm and carbonyl (C=O) peaks at 168.58 ppm of the thiomethylenecarbonyl group (–SC̲H₂C=O), the ethoxide peaks (OCH₂CH₃) at 61.58 and 14.61 ppm, respectively, and the disappearance of the thione group (C=S) at 175.89 ppm, which supported the structure conformation. 4-((2-((2-Hydrazineyl-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (3) are obtained by stirring ester derivative 2 with hydrazine hydrate in ethanol. The acid hydrazide 3 was confirmed by the disappearance of ethoxide peaks (OCH₂CH₃) at (4.14 and 1.21 ppm) and (61.58 and 14.61 ppm) in ¹H NMR and ¹³C NMR spectra, respectively. Additionally, it strongly supported the structure confirmation presence of the amidic proton (CONH–) peak at 9.40 ppm and (NH₂) singlet peaks at 4.38 and 4.32 ppm due to an acid hydrazide moiety (CONHNH₂) in ¹H NMR and the carbonyl peak of an amide group (CONH) at 156.62 ppm in ¹³C NMR. The aldimines 4–9 and ketimines 10–13 were obtained by the heating of 4-((2-((2-hydrazineyl-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (3) in ethanol with various aldehydes and ketones. The formation of these imines 4–13 was confirmed by the disappearance of the singlet peaks of the amino group (NH₂) at 4.38 and 4.32 ppm in the ¹H NMR spectrum, which is attributed to the acid hydrazide moiety (CONHN̲H₂). Furthermore, the presence of the E and Z isomer peaks at 11.86–11.76 and 10.85–11.36 ppm of the imide group (CON̲H–), together with the olefinic protons of the imine group (CONH–N=C–H–) at 8.25–8.13 and 8.16–8.00 ppm in ¹H NMR, strongly supported the confirmation of aldimines 4–9. Ketimines 10–13 were characterized by E and Z isomeric peaks at 10.97–10.93 and 10.80–10.77 ppm of the imide group (CON̲H–) in ¹H NMR, and the methyl peaks of the ethylidenehydrazineyl group (CONH–N=C–CH₃) at 2.34–2.30 and 2.30–2.27 ppm and 15.0–14.71 and 14.36–14.15 ppm in ¹H NMR and ¹³C NMR respectively as isomeric mixtures.

Scheme 1 Synthesis of ester 2, acidhydrazide 3, acylhydrazones 4–13 based on quinazolines incorporating benzylulfonamide.

2.1 Biological evaluation

2.1.1 In vitro cytotoxic activity. The NCI, Bethesda, MD, USA, assessed thirteen compounds 1–13 for their in vitro cytotoxic activity against the imatinib reference drug. They determined the growth inhibition percentage (GI%) against 59 cancer cell lines from various human tissues at a single treatment concentration of 10 μM (Table 1). The chemo-cytotoxicity of compounds 1–13 ranged from moderate to potent activity at a dose of 10 μM, with a mean growth rate (MG) of 104.4–23.5% compared to imatinib and the number of cell lines with growth inhibition > 10% (PCE) ranging from 6/59 to 59/59 (Table 1). Compounds 5, 6, 7, 9, and 12 had the highest PCE at 59/59, 58/59, 29/59, 28/59, and 25/59, respectively, with MG values of 23.5, 55.2, 89.4, 88.5, and 88.4%. Compounds 8 and 11 had PCEs of 15/59 and 17/59, respectively (MG = 96.8 and 94.5%), compared to imatinib PCE of 20/55 and MG value of 92.6%. Compounds 1–4 and 10 have the lowest PCE (0-9/59; MG = 104.42–97.1%). Derivatives 1–13 (Table 1) were evaluated against 59 cancer cells. Schiff bases 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 showed GI values greater than 10–>100% against most cancer cell lines, whereas imatinib had GI values ranging from 10 to 47. Quinazolines 5, 6, 7, 9, and 12 exhibited substantial in vitro cytotoxic activity against leukemia (GI% = 13–120), NSCLC (GI% = 10–126), colon cancer (GI% = 11–80), CNS cancer (GI% = 11–82), melanoma (GI% = 11–99), ovarian cancer (GI% = 11–88), renal cancer (GI% = 11–87), prostate cancer (GI% = 11–62), and breast cancer (GI% = 11–110). In leukemia (GI% = 13–18), NSCLC (GI% = 11–17), colon cancer (GI% = 12–47), CNS cancer (GI% = 11–25), melanoma (GI% = 12–22), ovarian cancer (GI% < 10), renal cancer (GI% < 10–14), prostate cancer (GI% = 11–14), and breast cancer (GI% = 11–29), imatinib exhibited moderate antitumor activity.

Table 1 In vitro cytotoxic activity of 2-substituted-mercaptoquinazoline derivatives 1–13 presented as growth inhibition percentages (GI%) for 59 subpanel tumor cell lines^a

Compound no.	PCE*	Cancer cell line assays (10.0 μM in one dose, GI %)	**MG%
a *Most sensitive cell lines PCE: positive cytotoxic effect is defined as the ratio between the number of cell lines with percentage growth inhibition of >10% and the total number of cell lines **: mean growth percent.
1	4/59	Non-small cell Lung (HOP-92, 14%), C.N.S. (SNB-75, 23%), and Renal (CAKI-1, 11%; UO-31, 19%)	97.13
2	2/59	CNS (SNB-75, 15%), and Renal (UO-31, 16%)	100.32
3	0/59	—	104.42
4	9/59	Non-Small Cell Lung (EKVX, 14%; NCI–H522, 18%), CNS (SNB-75, 13%), Melanoma (UACC-62, 11%), Renal (CAKI-1, 16%; UO-31, 21%), Breast (MCF-7, 14%; MDA-MB-231/ATCC, 12%; HS-578T, 11%)	99.39
5	59/59	Leukemia (CCRF-CEM, 90%; HL-60(TB), 120%; K-562, 94%, MOLT-4, 89%; RPMI-8226, 83%; SR, 90%), Non-Small Cell Lung (A549/ATCC, 71%; EKVX, 65%; HOP-62, 79%; HOP-92, 42%; NCI-H226, 69%; NCI-H23, 61%; NCI-H322M, 67%; NCI-H460, 94%; NCI-H522, 126%), Colon (COLO-205, 102%; HCC-2998, 80%; HCT-116, 79%; HCT-15, 53%; HT-29, 68%; KM-12, 85%; SW-620, 79%), CNS (SF-268, 82%; SF-295, 80%; SF-539; 72%; SNB-19, 59%; SNB-75, 62%; U-251, 76%), Melanoma (LOX IMVI, 99%; MALME-3M, 69%; M14, 96%; MDA-MB-435, 87%; SK-MEL-2, 90%; SK-MEL-28, 67%; SK-MEL-5, 95%; UACC-257, 87%; UACC-62, 94%), Ovarian (IGROV1, 74%; IGROV-3, 85%; IGROV-4, 88%; IGROV-5, 45%; OVCAR-8, 80%; NCI/ADR-RES, 13%; SK-OV-3, 79%), Renal (786-0, 68%; ACHN, 67%; CAKI-1, 69%; RXF 393, 58%; SN-12C, 68%; TK-10, 85%; UO-31, 54%), Prostate (PC-3, 51%; DU-145, 62%), Breast (MCF-7, 87%; MDA-MB-231/ATCC, 75%; HS-578T, 37%; BT-549, 67%; T-47D, 94%; MDA-MB-468, 110%).	23.46
6	58/59	Leukemia (CCRF-CEM, 77%; HL-60(TB), 60%; K-562, 64%, MOLT-4, 80%; RPMI-8226, 51%; SR, 76%), Non-Small Cell Lung (A549/ATCC, 31%; EKVX, 40%; HOP-62, 65%; HOP-92, 46%; NCI-H226, 43%; NCI-H23, 37%; NCI-H322M, 53%; NCI-H460, 51%; NCI-H522, 101%), Colon (COLO-205, 71%; HCC-2998, 50%; HCT-116, 58%; HCT-15, 25%; HT-29, 14%; KM-12, 61%; SW-620, 40%), CNS (SF-268, 62%; SF-295, 35%; SF-539; 46%; SNB-19, 45%; SNB-75, 15%; U-251, 57%), Melanoma (LOX IMVI, 84%; MALME-3M, 41%; M14, 58%; MDA-MB-435, 29%; SK-MEL-2, 64%; SK-MEL-28, 43%; SK-MEL-5, 42%; UACC-257, 30%; UACC-62, 62%), Ovarian (IGROV1, 38%; IGROV-3, 87%; IGROV-4, 60%; IGROV-5, 32%; OVCAR-8, 56%; SK-OV-3, 37%), Renal (786-0, 29%; ACHN, 28%; CAKI-1, 46%; RXF 393, 28%; SN-12C, 44%; TK-10, 47%; UO-31, 39%), Prostate (PC-3, 33%; DU-145, 28%), Breast (MCF-7, 50%; MDA-MB-231/ATCC, 70%; HS-578T, 29%; BT-549, 44%; T-47D, 67%; MDA-MB-468, 48%).	55.22
7	29/59	Leukemia (CCRF-CEM, 46%; HL-60(TB), 16%; K-562, 40%, MOLT-4, 36%; RPMI-8226, 56%; SR, 17%), Non-Small Cell Lung (A549/ATCC, 14%; HOP-62, 14%; NCI-H23, 16%; NCI-H522, 30%), Colon (HCT-15, 26%; KM-12, 11%), CNS (SF-268, 15%; SF-295, 23%; SNB-75, 18%), Melanoma (LOX IMVI, 21%; SK-MEL-2, 19%; 67%; SK-MEL-5, 32%; UACC-62, 33%), Ovarian (IGROV1, 15%; IGROV-4, 10%; OVCAR-8, 23%), Renal (CAKI-1, 16%; SN-12C, 11%; UO-31, 29%), Prostate (PC-3, 18%), Breast (MCF-7, 17%; MDA-MB-231/ATCC, 13%; T-47D, 36%).	89.35
8	15/59	Leukemia (RPMI-8226, 13%), Non-Small Cell Lung (EKVX, 15%; NCI-H226, 11%; NCI-H322M, 15%; NCI-H522, 48%), CNS (SF-268, 15%; SF-539, 12%;), Renal (CAKI-1, 14%; RXF 393, 10%; SN-12C, 10%; UO-31, 25%), Breast (MCF-7, 11%; MDA-MB-231/ATCC, 15%; HS-578T, 15%; MDA-MB-468, 19%).	96.77
9	28/59	Leukemia (CCRF-CEM, 13%; K-562, 37%, MOLT-4, 14%; SR, 51%), Non-Small Cell Lung (A549/ATCC, 15%; EKVX, 10%; NCI-H226, 16%; NCI-H522, 58%), Colon (HCT-15, 11%; HT-29, 14%; KM-12, 14%), CNS (SF-268, 22%; SNB -19, 11%; SNB-75, 29%), Melanoma (LOX IMVI, 18%; MDA-MB-435, 54%; SK-MEL-5, 26%; UACC-62, 22%), Ovarian (OVCAR-3, 21%), Renal (CAKI-1, 36%; RXF 393, 14%; UO-31, 20%), Prostate (PC-3, 11%), Breast (MCF-7, 41%; MDA-MB-231/ATCC, 11%; HS-578T, 12%; T-47D, 19%; MDA-MB-468, 19%).	88.50
10	6/59	Non-small cell Lung (EKVX, 10%; NCI-H322M, 14%), CNS (SF-268, 14%; U-251, 15%), and Renal (CAKI-1, 16%; UO-31, 20%).	100.92
11	17/59	NSC Lung (NCI-H522, 52%), Colon (HCT-116, 20%), C.N.S. (SF-268, 25%; SF-539; 28%; SNB-19, 15%; SNB-75, 22%; U-251, 20%), Melanoma (SK-MEL-5, 10%), Ovarian (OVCAR-5, 12%), Renal (CAKI-1, 21%; RXF 393, 16%; UO-31, 21%), Breast (M.C.F.-7, 10%; MDA-MB-231/ATCC, 25%; HS-578T, 19%; T-47D, 11%; MDA-MB-468, 19%).	94.54
12	25/59	Leukemia (SR, 28%), N.S.C. Lung (NCI-H23, 17%; NCI-H522, 62%), Colon (HCT-116, 17%; KM-12, 30%), CNS (SF-268, 64%; SF-539; 64%; SNB-19, 49%; SNB-75, 39%; U-251, 26%), Melanoma (LOX IMVI, 20%; MALME-3M, 30%; M14, 11%; MDA-MB-435, 30%; SK-MEL-5, 32%; UACC-62, 17%), Ovarian (OVCAR-5, 14%), Renal (786-0, 15%; CAKI-1, 16%; RXF 393, 30%), Breast (MDA-MB-231/ATCC, 28%; HS-578T, 11%; BT-549, 57%; T-47D, 62%; MDA-MB-468, 27%).	88.40
13	8/59	Leukemia (S.R., 12%), C.N.S. (SNB-75, 13%), Renal (CAKI-1, 15%; UO-31, 24%), Breast (M.C.F.-7, 11%; MDA-MB-231/ATCC, 11%; T-47D, 15%; MDA-MB-468, 16%)	97.44
Imatinib	20/59	Leukemia (MOLT-4, 18%; PRMI-8226, 12.6%; SR, 14.6%), N.S.C. Lung (EKVX, 15.7%; NCI-H226, 10.6%; NCI-H23, 17.1%), Colon (HCT-116, 18.6%; HCT-15, 11.5%; HT-29, 47.1%), CNS (SF-295, 15.1%; SF-539, 24.5%; U251, 10.6%), Melanoma (LOX IMVI, 11.6%; SK-MEL-5, 22.3%), Renal (A-498, 13.7%), Prostate (PC-3, 10.6%; DU-145, 14.4%), Breast (MDA-MB-231/ATCC, 11.2%; T-47D, 18.6%; MDA-MB-468, 29.1%)	92.62%

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2.1.2 Structure–activity relationship. Structure correlation analysis revealed that quinazolinthione 1, ester 2, and acid-hydrazide 3 had little tumor-killing efficacy (PCE = 4/59, 2/59, and 0/59, respectively). The conversion of acid-hydrazide 3 into hydrazones 4–9 resulted in different levels of in vitro cytotoxic activity, each higher than hydrazide 3 (PCE = 9/59–59/59). This group of hydrazones, including a 2-hydroxyphenyl moiety, like 5 and 6 (PCE = 59/59 and 58/59), was much better at killing tumor cells than compound with an unsubstituted phenyl 4 and methoxyphenyl derivative, including 7 and 8, as well as the 3-tolyl derivative 9 (PCE = 9/59, 29/59, 15/59, and 28/59, respectively). Adding three methoxy groups to the phenyl ring, as in compound 8, made it more effective against tumor cells (PCE = 15/59) than parent phenyl 4 (PCE = 9/59); however, it wasn't as effective as methoxyphenyl 7, which had a PCE of 29/59. The conversion of acid-hydrazide 3 into ethylidene hydrazones 10–13 resulted in varying levels of in vitro cytotoxic activity higher than parent hydrazide 3 (PCE = 6/59–25/59).

Among ethylidene hydrazones, those with a methoxyphenyl group, like 11 and 12 (PCE = 17/59 and 25/59), were much better at killing tumors than those with an unsubstituted phenyl 10 (PCE = 6/59). The introduction of more than two methoxy groups to the phenyl ring, as in compound 13, slightly increased its effectiveness against tumors (PCE = 8/59) compared to unsubstituted phenyl 10 (PCE = 6/59); however, it was not as potent as methoxyphenyl 11 and 12, which had PCEs of 17/59 and 25/59, respectively. Hydrazones 4, 5, and 8 kill tumor cells more effectively (PCE = 9/59, 29/59, and 15/59, respectively) than the comparable ethylidene hydrazones 10, 11, and 13 (PCE = 6/59, 17/59, and 8/59, respectively).

2.1.3 GI₅₀, TGI, and LC₅₀ of compound 5. Compound 5 is the most effective in vitro cytotoxic agent among the newly synthesized compounds, inhibiting the mean growth percentage (MG%) by an average of 23.5% (Table 1). NCI evaluated this compound on 59 tumor cells at five dosage dilutions.^62,63 For each cell, NCI assessed the mean 50% cell growth inhibition (GI₅₀), the total cell growth inhibition (TGI), and the median lethal concentration (LC₅₀) (Table 2 & Chart 1). With a mean (GI₅₀ MG_MID) value of 1.096 μM, compound 5 is more effective at killing cells than erlotinib and gefitinib, which have (GI₅₀ MG_MID) values of 7.7 and 2.1 μM, respectively. Additionally, compound 5 had a median lethal dose (LC₅₀) of more than 100 μM, which was higher than both gefitinib (95.6 μM) and erlotinib (14.3 μM). It also had a total cell growth inhibition (TGI) of 89.2 μM compared to those drugs (66.3 and 14.3 μM), respectively. Compound 5 had a GI₅₀ value of 0.4 μM, which was higher cytotoxicity than erlotinib and gefitinib (27.9 and 2.6 μM) against leukemia, non-small cell lung cancer (1.1, 13.1, and 2.1 μM), colon cancer (1.6, 51.7, and 5.2 μM), CNS cancer (1.5, 17.0, and 5.6 μM), melanoma cancer (1.5, 23.7, and 3.7 μM), ovarian cancer (15.1, 20.9, and 3.3 μM), renal cancer (1.9, 2.5, and 1.4 μM), prostate cancer (0.9, 20.9, and 3.3 μM), and breast cancer (1.2, 24.7, and 4.7 μM) (Table 2 & Chart 1). In general, compound 5 could kill tumor cells with GI₅₀ values (μM) that were about the same as or higher than erlotinib and gefitinib against most cell lines (Table 3 & Chart 2).

Table 2 Median growth-inhibitory (GI₅₀, μM), total growth-inhibitory (TGI, μM), and median lethal (LC₅₀, μM) concentrations of compound 5 and reference drugs erlotinib (718 781) and gefitinib (759 856) on in vitro subpanel tumor cell lines μm^a

Compound	Subpanel tumor cell lines										MG_MID^a
	Activity	Leukemia	NSC lung cancer	Colon cancer	CNS cancer	Melanoma	Ovarian cancer	Renal cancer	Prostate cancer	Breast cancer
a ^aFull panel mean-graph midpoint (μM); ^c Compounds showed values > 100 μM; mean 50% cell growth inhibition (GI50); total cell growth inhibition (TGI); median lethal concentration (LC50); Mean GI50 graph midpoints (GI50 MG_MID).
5	GI50	0.384	1.059	1.557	1.494	1.501	15.11	1.917	0.886	1.23	1.096
	TGI	68.371	c	c	c	59.09	c	91.54	c	84.17	89.24
	LC50	c	c	c	c	c	c	c	c	c	c
Erlotinib	GI50	27.85	13.11	51.68	16.99	23.74	5.52	2.46	20.90	24.72	7.68
	TGI	96.57	73.76	c	82.11	77.89	74.41	42.59	c	70.53	66.3
	LC50	c	97.71	c	c	93.31	97.06	89.15	c	96.43	95.6
Gefitinib	GI50	2.56	2.05	5.23	5.64	3.68	3.05	1.41	3.29	4.67	2.10
	TGI	12.07	13.86	18.47	19.62	12.49	33.29	12.50	31.62	18.62	14.3
	LC50	93.85	94.68	51.74	50.56	36.40	83.58	52.82	89.72	52.47	51.9

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Chart 1 Median growth-inhibitory (GI₅₀, μM), total growth-inhibitory (TGI, μM), and median lethal (LC₅₀, μM) concentrations of compound 5 and reference drugs erlotinib (718 781) and gefitinib (759 856) on in vitro subpanel tumor cell lines.

Table 3 The GI₅₀ values (μM) of compound 5 on in vitro subpanel tumor cell lines, compared to erlotinib and gefitinib^a

Subpanel tumor cell lines	GI50 (μM)
	5	Erlotinib	Gefitinib
a nt = not tested; ^c compounds showed values > 100 μM.
Leukemia
CCRF-CEM	0.196	15.84	5.01
HL-60(TB(K-562))	0.412	5.01	5.01
	0.592	15.48	2.51
MOLT-4	0.372	5.01	3.98
RPMI-8226	0.521	5.01	1.58
SR	0.212	6.30	3.16
Non-small cell lung cancer
A549/ATCC	1.57	7.94	7.94
EKVX	0.650	0.005	0.005
HOP-62	0.961	12.58	10.00
HOP-92	nt	6.30	7.94
NCI-H226	0.894	6.30	15.84
NCI-H23	1.96	19.95	15.84
NCI-H322M	0.575	0.05	0.08
NCI-H460	0.424	5.01	6.30
NCI-H522	2.40	1.00	6.30
Colon cancer
COLO 205	1.51	31.62	6.30
HCC-2998	0.882	79.34	10.00
HCT-116	0.363	5.01	7.94
HCT-15	3.62	3.16	5.01
HT29	3.01	50.11	3.98
KM12	0.569	63.09	7.94
SW-620	0.949	5.01	7.94
CNS cancer
SF-268	0.766	19.95	7.94
SF-295	1.47	15.84	1.99
SF-539	1.26	12.58	10.00
SNB-19	2.10	3.98	12.58
SNB-75	2.86	12.58	6.30
U251	0.510	19.95	10.00
Melanoma
LOX IMVI	0.387	5.01	7.94
MALME-3M	1.09	5.01	3.16
M14	0.452	6.30	5.01
MDA-MB-435	2.07	15.84	3.16
SK-MEL-2	2.41	12.58	12.58
SK-MEL-28	1.62	31.62	0.31
SK-MEL-5	1.57	15.84	3.98
UACC-257	3.03	100	6.30
UACC-62	0.886	1.25	5.01
Ovarian cancer
IGROV1	0.785	0.25	0.20
OVCAR-3	0.287	3.16	5.01
OVCAR-4	0.311	19.95	7.94
OVCAR-5	2.09	19.95	10.00
OVCAR-8	0.932	7.94	10.00
NCI/ADR-RES	c	6.30	12.58
SK-OV-3	1.32	0.39	0.63
Renal cancer
786-0	1.84	5.01	7.94
A498	nt	1.58	0.40
ACHN	1.58	0.15	0.20
CAKI-1	1.02	0.10	0.16
RXF 393	2.50	6.30	5.01
SN12C	1.93	6.3	6.30
TK-10	1.66	0.10	0.10
UO-31	2.89	1.99	1.25
Prostate cancer
PC-3	0.829	50.11	0.79
DU-145	0.944	1.58	2.51
Breast cancer
MCF7	0.457	100	10.00
MDA-MB-231/ATCC	0.643	1.99	12.58
HS 578T	2.11	6.30	10.00
BT-549	2.68	39.81	7.94
T-47D	0.925	3.16	6.30
MDA-MB-468	0.524	0.20	0.01

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Chart 2 The GI₅₀ values (μM) of compound 5 on in vitro subpanel tumor cell lines, compared to erlotinib and gefitinib.

2.1.4 EGFR, HER2 and CDK9 kinase enzyme inhibition assay. We selected candidates 5, 6, 7, 9, and 12 because they had the highest in vitro cytotoxic activity against the tested cell lines. We conducted dose-related enzymatic inhibition against EGFR, HER2, and CDK9 kinases at different concentrations to determine their IC₅₀ values in the nanomolar range. Table 4 demonstrates that compounds 5 and 6 have intense EGFR inhibitory action, with IC₅₀ values in the nanomolar range. The most potent analogs, 5 and 6, had IC₅₀ values of 84.4 and 51.5 nM, which showed that they blocked EGFR similarly or much more effectively than the standard Gefitinib (IC₅₀ of 53.1 nM). Compounds 7, 9, and 12 exhibited modest efficacy against EGFR with IC₅₀ values of (377.5–594.5 nM). Also, compounds 5, 6, and 12 were relatively similar at inhibiting HER2 than Gefitinib (IC₅₀ 38.8 nM), with IC₅₀ values of 53.9, 44.1, and 110.6, respectively. Compounds 7 and 9 have less action than Gefitinib, with IC₅₀ values of 387.0 and 228.5 nM, respectively. Also, compounds 5, 6, and 7 had IC₅₀ values of 146.9, 96.1, and 155.4 nM, which were almost as strong at stopping CDK9 activity as Dinaciclib (IC₅₀ 53.1 nM). Compounds 9 and 12 are less active than Dinaciclib, having IC₅₀ values of 316.4 and 319.8 nM, respectively. Compared to Celecoxib, which has an IC₅₀ value of 0.15 μM, Schiff bases 5, 6, 7, 9, and 12 did not exhibit any inhibition activity against COX-2, with IC₅₀ values ranging from 3.3 to 50.5 μM (Table 4).

Table 4 In vitro inhibitory effects of the quinazolines 5, 6, 7, 9, and 12 against COX-2, EGFR, HER2, and CDK9: the cytotoxicity of standard cell line (WI-38) for compounds 5 and 6

Compound no.	IC50^a nM				IC50^a (μM)
	COX-2 inhibition	EGFR inhibition	HER2 inhibition	CDK9 inhibition	WI138
5	3.32 ± 0.06 (μM)	84.39 ± 2.07	53.91 ± 1.32	146.9 ± 3.60	45.326 ± 2.66
6	4.47 ± 0.09 (μM)	51.52 ± 1.26	44.13 ± 1.08	96.07 ± 2.35	27.772 ± 1.63
7	50.48 ± 1.06 (μM)	594.45 ± 14.5	387.01 ± 9.50	155.4 ± 3.81	—
9	15.41 ± 0.32 (μM)	377.53 ± 9.26	228.53 ± 5.61	316.40 ± 7.76	—
12	21.27 ± 0.44 (μM)	492.27 ± 12.1	110.64 ± 2.71	319.8 ± 7.85	—
Celecoxib	0.15 ± 0.003 (μM)	—	—	—	—
Gefitinib		53.12 ± 1.30	38.81 ± 0.95		—
Dinaciclib	—	—	—	53.12 ± 1.30
Doxorubicin	—	—	—	—	9.57 ± 0.59

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2.2 Structure–activity relationship

Based on the data, we can conclude that the Schiff base series with a 2-hydroxyphenyl group on the acylhydrazone moiety is the most potent kinase inhibitor against EGFR (IC₅₀; 84.4 and 51.5 nM), HER2 (IC₅₀; 53.9 and 44.1 nM), and CDK9 (IC₅₀; 146.9 and 96.1 nM), as compounds 5 and 6, respectively. Schiff base with a 3-methoxyphenyl group on the acylhydrazone moiety, such as compound 7, showed a potent CDK9 kinase inhibitor with an IC₅₀ value of 155.4 nM, while compound 12 with the (3,5-dimethoxyphenyl)ethylidene)hydrazineyl) moiety showed vigorous HER2 kinase inhibitor activity with an IC₅₀ value of 110.6 nM.

2.3 In vitro cytotoxicity against WI-38 fibroblast cell line

The most active kinase inhibitors, acylhydrazones 5 and 6, were less toxic than doxorubicin when measured for their safety margin cytotoxicity against the standard WI-38 fibroblast cell line with IC50 values of 45.3 and 27.8 μM, respectively, compared to 9.6 μM for doxorubicin Table 4.

2.4 Cell cycle arrest analysis and apoptosis detection

We are studying cell cycle arrest and apoptosis in T-47D and MOLT4 cells to determine the role of our promising derivatives 5 and 6 in the cell cycle (Tables 5 and 6). Flow cytometry assays quantify the DNA content. After being treated with compounds 5 and 6, the number of cells in the S phase dropped from 32.3 percent for DMSO-control cells to 20.9 percent for T-47D cells and 22.5 percent for MOLT4 cells. Also, compound 5 stopped the cells in the G2/M stage, raising the percentage of T-47D cells in the G2-M phase from 6.6% in the control cell to 26.3%. Acylhydrazone 6 stopped the cells in the G1 stage, lowering the percentage of MOLT4 cells in the G0-G1 phase from 89.0% in the control cell to 61.4%. Acylhydrazone 6 lowered the percentage of MOLT4 cells in the G0-G1 phase from 89.0% in the control cell to 61.4% at the G1 stage. Furthermore, compounds 5 and 6 boosted early apoptosis from 0.4 and 0.6 (DMSO control sample) to 9.3% and 19.2, respectively; also, derivatives 5 and 6 increased late apoptosis from 0.1 to 14.8% and 12.6 to 0.3%, respectively, when stained with annexin-5/PI in T-47D and MOLT4 cells compared to the control group treated with DMSO. These investigated that compounds 5 and 6 preferred the apoptotic pathway over the necrotic pathway for cell death (Fig. 4 and 5).

Table 5 Compounds 5 and 6 affect the cell cycle of T-47D and MOLT4 cells at their IC₅₀ concentration levels compared to their DMSO control

Compound no.	%G0-G1	%S	%G2-M	Comment
Five/T-47D	46.31	27.42	26.27	Cell growth arrest at the G2/M phase
DMSO/T-47D	61.11	32.29	6.6	—
6/MOLT4	89.04	9.56	1.40	Cell growth arrest at the G1 phase
DMSO/MOLT4	61.37	22.54	16.09	—

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Table 6 Compounds 5 and 6 change the stained positive for annexin V-FITC of T-47D and MOLT4 cells at different IC₅₀ concentration percentages values

Compound no.	Apoptosis			Necrosis
	Total	Early	Late
5/T-47D	31.07	9.28	14.75	7.04
DMSO/T-47D	2.37	0.37	0.14	1.86
6/MOLT4	35.06	19.22	12.61	3.23
DMSO/MOLT4	1.71	0.55	0.31	0.85

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Fig. 4 Effect of DMSO (upper right panel) and 5 (lower right panel) on the percentage of annexin V-FITC-positive staining in MCF-7 cell. Cell cycle analysis of T47D cells treated with DMSO (upper left panel) and cell cycle analysis of T47D cells treated with compound 5 (lower left panel).

Fig. 5 Effect of DMSO (upper right panel) and 6 (lower right panel) on the percentage of annexin V-FITC-positive staining in MCF-7 cells. Cell cycle analysis of MOLT4 cells treated with DMSO (upper left panel) and MOLT4 cells treated with compound 6 (lower left panel).

2.5 Molecular docking

Molecular docking simulations of compound 5 and compound 6 with the HER2 receptor (PDB: 7PCD), EGFR kinase domain (PDB: 1XKK), and CDK9 (PDB: 3RCD) reveal significant insights into their potential as inhibitors, highlighting key hydrogen bond and hydrophobic interactions contributing to their binding affinities.

2.6 HER2 receptor interactions

Compound 5 exhibits a strong binding affinity of −9.2 kcal mol⁻¹ with the HER2 receptor, attributed to a network of interactions within the active site (Table 7). It forms hydrogen bonds with PHE864, LYS753, THR862, and MET801 as an acceptor and LEU796 as a donor. These hydrogen bonds, particularly those with THR862 and MET801 within the critical ATP-binding pocket, likely underpin its strong affinity. Additionally, compound 5 engages in hydrophobic interactions with LEU726, VAL734, ALA751, LYS753, GLU770, ALA771, THR798, LEU800, and PHE864, further stabilizing its binding within the hydrophobic environment of the active site (Fig. 6A). Compound 6 shows a weaker binding affinity of −7.9 kcal mol⁻¹, forming fewer hydrogen bonds within the active site. It acts as a hydrogen bond acceptor with MET801, LYS753, LYS860, and ARG784. While the interaction with MET801 in the ATP-binding site is promising, the fewer hydrogen bonds suggest a weaker overall interaction than compound 5. Derivative 6 also benefits from hydrophobic interactions with LEU726, ILE767, THR798, LEU800, THR862, and PHE864, contributing to its binding stability (Fig. 6B).

Table 7 Compound 5 and Compound 6 hydrogen bond interactions with HER2, EGFR, and CDK9

Compound	Ligand	Receptor	Interaction	Distance	Binding affinities (kcal mol^−1)
EGFR (PDB: 1XKK)
5	C 15	OD2 ASP 855 (A)	H-donor	3.42	−8.513
	O 40	O SER 720 (A)	H-donor	3.01
	O 26	N MET 793 (A)	H-acceptor	3.54
	O 36	HA ASP 855 (A)	H-acceptor	3.33
6	N 1	O LEU 788 (A)	H-donor	3.22	−8.109
	O 20	N MET 793 (A)	H-acceptor	3.67
HER2 (PDB: 7PCD)
5	O 38	O PHE 864 (A)	H-acceptor	3.83	−9.159
	N 21	HZ1 LYS 753 (A)	H-acceptor	3.84
	O 14	OG1 THR 862 (A)	H-acceptor	3.35
	O 26	N MET 801 (A)	H-acceptor	3.38
	N 38	O LEU 796 (A)	H-donor	3.1
6	O 20	N MET 801 (A)	H-acceptor	3.23	−7.854
	O 24	NZ LYS 753 (A)	H-acceptor	3.56
	N 27	O LYS 860 (A)	H-acceptor	3.51
	O 35	N ARG 784 (A)	H-acceptor	4.09
CDK9 (PDB: 3BLR)
5	O 26	N CYS 106 (A)	H-acceptor	3.47	−7.425
	N 38	OD1 ASP 109 (A)	H-donor	2.96
6	N 1	OD2 ASP 109 (A)	H-donor	2.92	−7.656
	O 20	N CYS 106 (A)	H-acceptor	3.12
	O 33	N LYS 151 (A)	H-acceptor	4.09

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Fig. 6 3D interaction poses of compound 5 and compound 6 within the active sites of (A and B) HER2, (C and D) EGFR, and (E and F) CDK9. Compound 5 is depicted in orange, and compound 6 is in yellow.

2.7 EGFR kinase domain interactions

Compound 5 demonstrates a strong binding affinity of −8.5 kcal mol⁻¹ with the EGFR kinase domain, engaging in a network of hydrogen bonds and hydrophobic interactions within the active site (Table 7). It forms hydrogen bonds with ASP855 as both a donor and acceptor, SER720 as a donor, and MET793 as an acceptor. The interaction with MET793, a key residue within the hinge region of the ATP-binding pocket, is particularly noteworthy. Moreover, compound 5 engages in hydrophobic interactions with LEU718, THR790, LEU792, ARG841, and LEU844, further enhancing its binding stability within the hydrophobic environment of the active site (Fig. 6C).

Quinazoline 6 exhibits a slightly weaker binding affinity of −8.1 kcal mol⁻¹, forming fewer hydrogen bonds within the active site. It acts as a hydrogen bond donor with LEU788 and an acceptor with MET793. The interaction with MET793 in the ATP-binding pocket is favorable, but the lower number of hydrogen bonds suggests a slightly weaker interaction than compound 5. Schiff base 6 also benefits from hydrophobic interactions with a broader range of residues, including LEU718, VAL726, ALA743, LYS745, THR790, LEU792, ARG841, and THR854, contributing to its overall binding stability (Fig. 6D).

2.8 CDK9 kinase interactions

Derivative 5 exhibits a binding affinity of −7.4 kcal mol⁻¹ with CDK9, primarily driven by two key hydrogen bonds. It acts as a hydrogen bond acceptor with CYS106 and a donor with ASP109. These interactions likely position compound 5 within the active site to effectively interfere with ATP binding or catalytic activity. Furthermore, compound 5 benefits from hydrophobic interactions with ILE25, PHE30, ALA46, VAL79, PHE103, ASP109, LEU156, and THR191, contributing to its overall binding stability within the hydrophobic environment of the active site (Table 7, Fig. 6E). Compound 6 demonstrates a slightly stronger binding affinity of −7.7 kcal mol⁻¹, attributed to more hydrogen bonds. It forms a hydrogen bond as a donor with ASP109 and an acceptor with CYS106 and LYS151. The interaction with LYS151, while at a longer distance (4.09 Å), could provide additional binding energy. Compound 6 also engages in hydrophobic interactions with ILE25, PHE30, ALA46, VAL79, PHE103, ASP109, and LEU156, further stabilizing its binding within the active site (Fig. 6F).

3 Conclusion

The NCI, Bethesda, MD, USA, evaluated the newly synthesized quinazolines 1–13 for their in vitro cytotoxic activity against 59 cell lines. Acylhydrazones 5, 6, 7, 9, and 12 were the most effective at killing cancer cells, lowering the mean growth percentage (MG%) by an average of 23.5, 55.2, 89.4, 88.5, and 88.4%, in that order. Compound 5 was selected for other investigation by NCI, Bethesda, MD, USA, at five-dose dilutions on 59 tumor cells. Regarding tumor cell death, compound 5 compared to gefitinib (mean GI₅₀: 7.7 μM) and erlotinib (mean GI₅₀: 2.1 μM), with a mean GI₅₀ value of 1.1 μM. It had an LC₅₀ value above 100 μM, whereas the values for gefitinib and erlotinib were 95.6 μM and 14.3 μM, respectively. Those medications' TGIs were 14.3 μM and 66.3 μM, respectively, compared to compound 5, with a TGI value of 89.2 μM. acylhydrazones 5, 6, 7, 9, and 12 exhibited the highest cytotoxic activity of the newly synthesized compounds 1–13 evaluated against the cell lines by reducing the mean growth percentage (MG%) by an average of 23.5, 55.2, 89.4, 88.5, and 88.4%, respectively. The acylhydrazones 5, 6, 7, 9, and 12 exhibit superior cytotoxic efficacy against the cell lines under investigation. The dose-dependent enzymatic inhibition experiments were conducted on EGFR, HER2, and CDK9 kinases at different dosages to determine their IC₅₀ magnitudes in the nanomolar range. The Schiff base series with 2-hydroxyphenyl moiety, represented by compounds 5 and 6, is the most effective kinase inhibitor against EGFR (IC₅₀; 84.4 and 51.5 nM), HER2 (IC₅₀; 53.9 and 44.1 nM), and CDK9 (IC₅₀; 146.9 and 96.1 nM). Compound 12 with the (3,5-dimethoxyphenyl)ethylidene)hydrazineyl) moiety demonstrated intense HER2 kinase inhibitor activity with an IC₅₀ value of 110.6 nM. Schiff bases 7, with a 3-methoxyphenyl group on the acylhydrazone moiety, demonstrated a potent CDK9 kinase inhibitor with an IC50 value of 155.4 nM. Throughout the investigation of cell cycle analysis for acylhydrazones 5 and 6, the S phase cell count decreased from 32.3% and 9.56 in the DMSO-control cells to 20.9% in T-47D cells and 22.5% in MOLT4 cells. Additionally, compound 5 increased the proportion of T-47D cells from 6.6% in the control cell to 26.3% by halting the G2/M stage cells. After adding substance 6, the percentage of MOLT4 cells in the G0-G1 phase decreased from 89.0% in the control cell to 61.4%. Compounds 5 and 6 enhanced early death from 0.4 and 0.6% (DMSO control sample) to 9.3% and 19.2%, respectively, as measured by flow cytometers using annexin-5/PI staining of T-47D and MOLT4 cells, respectively. The late death rate increased from 0.1 to 14.8% with derivative 5 and 12.6 to 0.3% with derivative 6. These results demonstrate that acylhydrazones 5 and 6 preferred the apoptotic pathway than the necrotic pathway. Acylhydrazones 5 and 6 were less toxic than doxorubicin, with IC₅₀ values of 45.3 and 27.8 μM, respectively, compared to 9.6 μM for doxorubicin when testing the cell death against the standard WI-38 fibroblast cell line. 2-Hydroxyacylhydrazones 5 and 6 showed potential inhibition of HER2, EGFR, and CDK9, due to their potential interaction. Due to the greater number of hydrogen bonds, 2-hydroxylamine 5 shows stronger binding affinities within the ATP-binding pockets, and the hydrophobic interactions contribute to their stability within the active sites.

4 Experimental section

4.1 Chemistry

Melting points were recorded on a Barnstead 9100 Electrothermal melting apparatus. IR spectra (KBr) were recorded on an FT-IR PerkinElmer spectrometer (n cm⁻¹). ¹H and ¹³C NMR were recorded on Bruker 700 MHz spectrometers using DMSO-d₆ as the solvent. We obtained microanalytical data (C, H, and N) using a PerkinElmer 240 analyzer, and the proposed structures were within approximately 0.4% of the theoretical values. Acquity UPLC machine (the UPH model with serial number H10UPH) and the Acquity TQD MS instrument (the TQD model with serial number QBB1203) are used for mass spectra recording. Compound 1 was prepared according to the reported method,⁶⁶ with a melting point of 301–303 °C compared to 286–287 °C.

4.2 4-((4-Oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)benzenesulfonamide (1)

A mixture of 2-aminobenzoic acid (411 mg, 3.0 mmol) with 4-isothiocyanatobenzenesulfonamide (642.00 mg, 3.00 mmol) in ethanol (19.0 ml) and trimethylamine (404.00 mg, 4.00 mmol) was heated for 5 hours. The quinazoline-2-thione 3 was obtained by filtration, washing with iced ethanol (70%), and drying. M.p 301–303°, 90% yield; ν: 3203, 3134 (NH), 1777 (C=O), 1320, 1155 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 13.15 (s, 1H), 7.97 (dd, J = 7.8, 1.6 Hz, 1H), 7.87–7.65 (m, 3H), 7.47 (dd, J = 14.4, 8.3 Hz, 3H), 7.34 (d, J = 17.8 Hz, 3H), 5.73 (s, 2H). ¹³C NMR (176 MHz, DMSO) δ 175.89, 160.07, 143.09, 141.21, 140.08, 136.10, 127.81, 127.68, 126.15, 125.01, 116.57, 116.00, 49.11; Ms; [M+1, 348].

4.2.1 Ethyl 2-((4-oxo-3-(4-sulfamoylbenzyl)-3,4-dihydroquinazolin-2-yl)thio)acetate (2). A mixture of (695.0 mg, 2.00 mmol) 4-((4-oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)methyl)benzenesulfonamide (1), (351.0 mg, 2.10 mmol) ethyl 2-bromoacetate in propane-2-one (13.0 ml) with potassium carbonate (290.0 mg, 2.10 mmol) was heated in ethanol for 6 hours. The ester 3 was obtained by filtration, washing with iced ethanol (70%), and drying. M.p 189–190°, 92% yield; ν: 3334, 3243 (NH), 1741, 1678 (C=O), 1340, 1154 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 8.12 (dd, J = 8.0, 1.5 Hz, 1H), 7.87–7.79 (m, 3H), 7.52–7.33 (m, 6H), 5.42 (s, 2H), 4.14 (q, J = 7.1 Hz, 2H), 4.11 (s, 2H), 1.21 (t, J = 7.1 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 168.63, 161.24, 156.43, 147.14, 143.78, 139.91, 135.60, 127.57, 127.19, 126.81, 126.51, 126.34, 119.11, 61.61, 47.15, 34.67, 14.62; Ms; [M+1, 434].

4.2.2 4-((2-((2-Hydrazineyl-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (3). A mixture of (867 mg, 2.0 mmol) ethyl 2-((4-oxo-3-(4-sulfamoylbenzyl)-3,4-dihydroquinazolin-2-yl)thio)acetate (2) and hydrazine hydrate (96 mg, 3.0 mmol), was heated in ethanol for 8 hours. The acid hydrazide 3 was obtained by filtration, washing with iced ethanol (70%), and drying. M.p 220–221°, 91% yield; ν: 3288, 3182, 3135 (NH), 1654, 1603 (C=O), 1329, 1159 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 9.40 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.86–7.84 (m, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 8.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.37 (s, 2H), 5.42 (s, 2H), 4.38 & 4.32 (ss, 2H), 3.97 (s, 2H), ¹³C NMR (176 MHz, DMSO) δ 166.48, 161.38, 156.62, 147.26, 143.65, 140.06, 135.47, 127.57, 127.11, 126.70, 126.62, 126.53, 119.14, 47.08, 34.68; Ms; [M+1, 420].

4.2.3 4-((2-((2-(Substituted-2-benzylidenehydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (4–9). An equimolar mixture of (210.0 mg, 0.5 mmol) 4-((2-((2-hydrazineyl-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (3)and appropriate aldehyde (0.05 mmol) was heated in ethanol for 9 hours. The aldimine 4–9 was obtained by filtration, washing with iced ethanol (70%), and drying.
4.2.3.1 4-((2-((2-(2-Benzylidenehydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (4). M.p 267–268°, 93% yield; ν: 3363, 3249 (NH), 1667, 1602 (C=O), 1333, 1152 (O=S=O); ¹H NMR (500 MHz, DMSO) δ 11.85 & 11.68 (s, 1H), 8.28 & 8.08 (s, 1H), 8.12 (dd, J = 7.9, 1.7 Hz, 1H), 7.83–7.78 (m, 3H), 7.71 (td, J = 6.2, 2.0 Hz, 2H), 7.57–7.41 (m, 6H), 7.37 (d, J = 2.2 Hz, 2H), 5.45 (d, J = 3.0 Hz, 2H), 4.60 & 4.12 (s, 2H). ¹³C NMR (176 MHz, DMSO) δ 169.10, 163.89, 161.33, 161.31, 156.85, 156.70, 147.22, 147.15, 143.91, 143.70, 143.65, 140.08, 140.03, 135.52, 134.57, 134.54, 130.61, 130.42, 129.31, 127.63, 127.60, 127.58, 127.33, 127.17, 126.74, 126.67, 126.53, 126.49, 126.47, 126.43, 119.17, 119.12, 47.18, 35.61, 34.49; Ms; [M+1, 508].
4.2.3.2 4-((2-((2-(2-(4-(Diethylamino)-2-hydroxybenzylidene)hydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (5). M.p 270–271°, 92% yield; ν: 3429 (OH), 3303, 3201 (NH), 1780, 1636 (C=O), 1331, 1153 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 11.76 & 11.36 (s, 1H), 11.14 & 9.98 (s, 1H), 8.25 & 8.16 (s, 1H), 8.12 (ddd, J = 8.3, 4.4, 1.6 Hz, 1H), 7.85–7.76 (m, 3H), 7.59–7.53 (m, 1H), 7.51–7.44 (m, 3H), 7.40–7.32 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H), 6.25 & 6.18 (dd, J = 8.8, 2.5 Hz, 1H), 6.12 & 6.08 (d, J = 2.4 Hz, 1H), 5.43 (d, J = 5.8 Hz, 2H), 4.50 (s, 1H), 4.08 (s, 1H), 3.35–3.31 (m, 4H), 1.10–1.07 (m, 6H), ¹³C NMR (176 MHz, DMSO) δ 167.18, 162.34, 160.79, 159.42, 158.26, 156.35, 156.14, 150.09, 149.97, 148.74, 146.71, 146.69, 143.14, 143.07, 139.55, 139.48, 134.99, 134.96, 131.38, 127.10, 127.04, 126.64, 126.60, 126.20, 126.11, 125.99, 125.95, 125.93, 125.91, 118.62, 118.58, 107.04, 106.11, 103.86, 103.59, 97.29, 97.16, 46.61, 43.74, 43.71, 34.80, 34.29, 12.44; Ms; [M+1, 595].
4.2.3.3 4-((2-((2-(2-(2-Hydroxybenzylidene)hydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (6). M.p 288–290°, 94% yield; ν: 3372, 3265, 3183 (NH), 1671, 1611 (C=O), 1338, 1156 (O=S=O); ¹H NMR (500 MHz, DMSO) δ 11.80 & 11.62 (s, 1H), 9.66 (s, 1H), 8.17 & 8.00 (s, 1H), 8.13–8.10 (m, 1H), 7.85–7.76 (m, 3H), 7.59–7.43 (m, 4H), 7.38 (d, J = 2.7 Hz, 2H), 7.28–7.06 (m, 3H), 6.84–6.82 (m, 1H), 5.45 (d, J = 5.8 Hz, 2H), 4.58 & 4.10 (s, 2H); ¹³C NMR (176 MHz, DMSO) δ 168.74, 163.87, 161.35, 161.33, 157.70, 156.89, 156.84, 156.62, 147.37, 147.21, 143.67, 143.62, 141.63, 140.10, 140.02, 135.55, 135.52, 131.97, 131.72, 129.63, 127.65, 127.59, 127.18, 127.16, 126.84, 126.78, 126.69, 126.54, 126.50, 126.46, 126.42, 120.51, 119.91, 119.88, 119.15, 119.10, 116.82, 116.64, 47.19, 35.34, 34.61; Ms; [M+1, 524].
4.2.3.4 4-((2-((2-(2-(3-Methoxybenzylidene)hydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (7). M.p 279–280°, 91% yield; ν: 3362, 3246 (NH), 1669, 1590 (C=O), 1351, 1151 (O=S=O); ¹H NMR (500 MHz, DMSO) δ 11.86 & 11.70 (s, 1H), 8.24 & 8.05 (s, 1H), 8.12 (dt, J = 8.0, 2.0 Hz, 1H), 7.80 (qd, J = 7.9, 1.6 Hz, 3H), 7.56–7.43 (m, 4H), 7.37 (q, J = 2.4 Hz, 2H), 7.35–7.25 (m, 3H), 7.03–6.98 (m, 1H), 5.45 (d, J = 3.9 Hz, 2H), 4.61& 4.12 (s, 2H), 3.79 (d, J = 2.6 Hz, 3H), ¹³C NMR (176 MHz, DMSO) δ 169.10, 163.94, 161.33, 161.30, 160.01, 159.98, 156.86, 156.69, 147.21, 147.03, 143.76, 143.69, 143.65, 140.08, 140.03, 136.00, 135.96, 135.53, 135.50, 130.43, 127.63, 127.61, 127.17, 126.74, 126.66, 126.53, 126.50, 126.47, 126.39, 120.47, 119.93, 119.16, 119.12, 116.78, 116.33, 112.01, 111.70, 55.64, 55.60, 49.08, 47.18, 35.59, 34.57; Ms; [M+1, 538].
4.2.3.5 4-((4-Oxo-2-((2-oxo-2-(2-(3,4,5-trimethoxybenzylidene)hydrazineyl)ethyl)thio)quinazolin-3(4H)-yl)methyl)benzenesulfonamide (8). M.p 210–212°, 91% yield; ν: 3300, 3249 (NH), 1655, 1611 (C=O), 1331, 1149 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 11.82 & 11.71 (ss, 1H), 9.39 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 7.89–7.78 (m, 3H), 7.65–7.59 (m, 1H), 7.52–7.45 (m, 3H), 7.36 (s, 2H), 7.07–6.98 (m, 1H), 6.79 (s, 1H), 5.47–5.39 (m, 2H), 4.50–4.11 (m, 2H), 4.69–3.66 (m, 9H); ¹³C NMR (176 MHz, DMSO) δ 166.47, 161.38, 156.64, 153.64, 153.48, 147.27, 143.66, 140.06, 138.67, 137.51, 135.47, 132.58, 127.57, 127.12, 126.70, 126.62, 126.52, 119.15, 106.06, 104.78, 104.58, 102.73, 60.50, 56.42, 56.35, 56.18, 47.07, 34.69; \Ms; [M+1, 598].
4.2.3.6 4-((2-((2-(2-(4-Methylbenzylidene)hydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (9). M.p 273–274°, 95% yield; ν: 3302, 3236 (NH), 1665, 1610 (C=O), 1334, 1153 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 11.77 & 11.61 (s, 1H), 8.23 & 8.04 (s, 1H), 8.13–8.12 (m, 1H), 7.84–7.79 (m, 3H), 7.61–7.46 (m, 6H), 7.36 (s, 2H), 7.27–7.23 (m, 2H), 5.45 (d, J = 4.5 Hz, 2H), 4.59 & 4.11 (s, 2H), 2.34 (s, 3H); ¹³C NMR (176 MHz, DMSO) δ 168.95, 163.73, 161.34, 161.31, 156.88, 156.71, 147.23, 143.99, 143.66, 140.21, 140.08, 135.51, 131.85, 129.91, 127.63, 127.59, 127.56, 127.31, 127.17, 126.66, 126.53, 126.49, 126.47, 119.13, 47.18, 35.61, 34.52, 21.50; Ms; [M+1, 522].

4.2.4 4-((4-Oxo-2-((2-oxo-2-(2-(1-(substituted-phenyl)ethylidene)hydrazineyl)ethyl)thio)quinazolin-3(4H)-yl)methyl)benzenesulfonamide (10–13). An equimolar mixture of (210.0 mg, 0.5 mmol) 4-((2-((2-hydrazineyl-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (3) and appropriate ketone (0.05 mmol) was heated in ethanol for 11.0 hours. The ketimine 4–13 was obtained by filtration, washing with iced ethanol (70%), and drying.
4.2.4.1 4-((4-Oxo-2-((2-oxo-2-(2-(1-(p-tolyl)ethylidene)hydrazineyl)ethyl)thio)quinazolin-3(4H)-yl)methyl)benzenesulfonamide (10). M.p 279–280°, 90% yield; ν: 3353, 3201 (NH), 1668, 1610 (C=O), 1338, 1156 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 10.93 & 10.80 (s, 1H), 8.13 (t, J = 9.3 Hz, 1H), 7.84–7.78 (m, 5H), 7.61–7.38 (m, 9H), 5.45 & 5.42 (s, 2H), 4.65 (d, J = 3.1 Hz, 1.3H), 4.25 (d, J = 3.2 Hz, 0.7H), 2.33 (s, 1H), 2.30 (d, J = 2.7 Hz, 2H), ¹³C NMR (176 MHz, DMSO) δ 169.96, 161.35, 156.92, 152.64, 148.52, 147.26, 147.23, 143.68, 143.63, 140.09, 140.05, 138.45, 135.55, 129.84, 129.66, 128.87, 128.81, 127.62, 127.57, 127.17, 126.81, 126.66, 126.56, 126.49, 126.40, 119.12, 47.15, 35.52, 35.20, 14.71, 14.15; Ms; [M+1, 522].
4.2.4.2 4-((2-((2-(2-(1-(3-Methoxyphenyl)ethylidene)hydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (11). M.p 263–265°, 88% yield; ν 3356, 3255, 3200 (NH), 17 674, 1619 (C=O), 133, 1157 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 10.95 & 10.80 (s, 1H), 8.13 (t, J = 9.5 Hz, 1H), 7.83 (td, J = 17.3, 7.5 Hz, 3H), 7.59–7.29 (m, 9H), 7.00 (d, J = 7.9 Hz, 1H), 5.46 (s, 2H), 4.67 & 4.25 (s, 2H), 3.78 (s, 3H), 2.32 & 2.29 (s, 3H); ¹³C NMR (176 MHz, DMSO) δ 169.97, 161.35, 159.74, 159.65, 156.94, 156.90, 148.31, 147.25, 143.70, 143.64, 140.10, 140.05, 139.93, 135.52, 129.96, 129.88, 127.59, 126.51, 126.40, 119.35, 119.13, 119.07, 115.42, 115.01, 112.20, 112.14, 55.59, 47.18, 35.44, 14.88, 14.27; Ms; [M+1, 552].
4.2.4.3 4-((2-((2-(2-(1-(3,5-Dimethoxyphenyl)ethylidene)hydrazineyl)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)methyl)benzenesulfonamide (12). M.p 295–296°, 89% yield; ν: 3331, 3261 (NH), 1674, 1591 (C=O), 1336, 1158 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 10.95 & 10.80 (s, 1H), 8.13 (dd, J = 12.3, 7.4 Hz, 1H), 7.84–7.79 (m, 3H), 7.48 (td, J = 15.1, 7.1 Hz, 4H), 7.38 (d, J = 4.0 Hz, 2H), 6.99 & 6.92 (s, 2H), 6.62–6.51 (m, 1H), 5.45 (s, 2H), 4.66 & 4.24 (s, 2H), 3.77 (d, J = 2.9 Hz, 6H), 2.30 (s, 1H), 2.27 (d, J = 2.7 Hz, 2H), ¹³C NMR (176 MHz, DMSO) δ 169.97, 160.88, 160.77, 156.94, 148.27, 147.24, 143.62, 140.53, 140.10, 135.48, 127.63, 127.58, 127.15, 126.66, 126.51, 126.38, 119.11, 105.02, 104.97, 101.63, 101.17, 55.73, 47.18, 35.56, 35.48, 14.95, 14.31; Ms; [M+1, 582].
4.2.4.4 4-((4-Oxo-2-((2-oxo-2-(2-(1-(3,4,5-trimethoxyphenyl)ethylidene)hydrazineyl)ethyl)thio)quinazolin-3(4H)-yl)methyl)benzenesulfonamide (13). M.p 283–284°, 90% yield; ν: 3322, 3243, 3168 (NH), 1659, 1612 (C=O), 1333, 1162 (O=S=O); ¹H NMR (700 MHz, DMSO) δ 10.91 & 10.77 (t, J = 4.2 Hz, 1H), 8.13 (p, J = 6.7 Hz, 1H), 7.90–7.79 (m, 3H), 7.59–7.42 (m, 4H), 7.39–7.38 (m, 2H), 7.13–7.06 (m, 2H), 5.46 (s, 2H), 4.66 & 4.24 (t, J = 4.1 Hz, 2H), 3.81 (s, 6H), 3.70 (s, 3H), 2.34–2.30 (m, 3H); ¹³C NMR (176 MHz, DMSO) δ 169.81, 164.22, 161.34, 156.99, 156.91, 153.16, 153.09, 148.60, 147.25, 143.64, 140.10, 139.17, 135.52, 134.08, 133.99, 127.65, 127.55, 127.19, 126.67, 126.52, 126.35, 119.11, 104.41, 104.31, 60.60, 56.38, 47.20, 35.56, 15.00, 14.36; Ms; [M+1, 612].

4.3 Biological evaluation

4.3.1 In vitro cytotoxic activity. The in vitro cytotoxicity assay was evaluated following the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda, MD^66,67 ESI.†

4.3.2 In vitro cyclooxygenase (COX) inhibition assay. The colorimetric COX (ovine) inhibitor screening assay kit (kit catalog number 560101, Cayman Chemical, Ann Arbor, MI) was utilized according to the manufacturer's instructions to examine the ability of the test compounds and the reference drug to inhibit the COX-2 isozymes^68,69 ESI.†

4.3.3 Kinases assay. In vitro luminescent EGFR tyrosine kinase assay using Kinase-Glo^® MAX as a detection reagent⁷⁰ and In vitro HER2 tyrosine kinase assay using DP-Glo™ reagent^71,72 that measures ADP formed from a kinase reaction, this luminescent signal positively correlates with ADP amount and kinase activity. In vitro, luminescent CDK9 kinase assay using Kinase-Glo^® MAX as a detection reagent⁷³ ESI.†

4.4 Apoptosis assay

The apoptosis of cancer cell lines T-47D and MOLT4 cells was measured by Annexin 5-FITC/PI apoptosis detection kit using FACSCalibur flow cytometer for analysis⁷⁴ ESI.†

4.5 Cell cycle analysis

Cancer cell lines T-47D and MOLT4 were stained with the DNA fluorochrome PI and analyzed by FACSCalibur flow cytometer.⁷⁵

4.6 Docking methodology

We performed molecular docking simulations using AutoDock 4.2,⁷⁶ which employed the Lamarckian Genetic Algorithm (LGA) with defined parameters. We used Accelrys Discovery Studio Visualizer (version 4.0) to visualize binding poses and interactions and analyze the binding dynamics. We further characterized intermolecular interactions using the PLIP web server.^77–82

Data availability

Data supporting this study are included in the article and ESI.†

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors extend their appreciation to the Researchers Supporting Project number (RSPD2024R1049), King Saud University, Riyadh, Saudi Arabia, for funding this research.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra07174c

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