The Antiproliferative and Apoptotic Effects of a Novel Quinazoline Carrying Substituted-Sulfonamides: In Vitro and Molecular Docking Study

Abstract

In order to investigate for a new effective and safe anticancer drug, we synthesized a novel series of quinazoline containing biologically active substituted-sulfonamide moiety at 3- position 4a–n. The structure of the newly prepared compounds was proved by microanalysis, IR, ¹H-NMR, ¹³C-NMR and mass spectral data. All the synthesized compounds were evaluated for their in vitro cytotoxic activity in numerous cancer cell lines including A549, HepG-2, LoVo and MCF-7 and normal HUVEC cell line. The two most active compounds 4d and 4f were then tested for their apoptosis induction using DNA content and Annexin V-FITC/PI staining. Moreover, apoptosis initiation was also confirmed using RT-PCR and Western blot. To further understand the binding preferences of quinazoline sulfonamides, docking simulations were used. Among the fourteen new synthesized compounds, we found that compounds 4d and 4f exerted the strongest cytotoxicity against MCF-7 cells with an IC₅₀ value of 2.5 and 5 μM, respectively. Flow cytometry data revealed the ability of compounds 4d and 4f to mediate apoptosis and arrest cell cycle growth at G1 phase. Furthermore, RT-PCR and Western blot results suggested that both 4d and 4f activates apoptotic cell death pathway in MCF-7 cells. Molecular docking assessments indicated that compounds 4d and 4f fit perfectly into Bcl2’s active site. Based on the biological properties, we conclude that both compounds 4d and 4f could be used as a new type of anticancer agent, which provides a scientific basis for further research into the treatment of cancer.

1. Introduction

Quinazoline and sulfonamide moieties have been identified as classes of chemotherapeutic agents with substantial therapeutic value against solid tumors [1,2,3,4]. Gefitinib (small-molecule inhibitor) being the original quinazoline molecule to be approved as an antitumor agent for treatment of Non-Small Cell Lung Cancer [5,6,7]. Due to the great performances of quinazoline compounds in antitumor applications, the enhancement of new quinazoline candidates as anticancer agents is a promising topic. In fact, several selective derivatives of quinazoline-containing drugs such as lapatinib, erlotinib, and vandetanib have been approved as anticancer drugs [8,9,10] (Figure 1). On the other hand, various biological activities have been attributed to sulfonamide-containing compounds, including antitumor, carbonic anhydrase inhibition, antimalarial, and antimicrobial activities [4,11]. In the new drug design, the development of hybrid derivatives through the combination of several pharmacophores may assign compounds with remarkable biological profiles [12]. Previously, various quinazoline sulfonamide derivatives were evaluated as anticancer agents against numerous cell lines, and many compounds had displayed a promising activity [3,13,14]. Apoptosis evasion is a remarkable feature of most cancer cells; therefore, designing drugs that induce apoptosis has emerged as a promising candidate for anti-cancer optimization [15]. In fact, several previous studies have demonstrated that quinazoline scaffolds had a remarkable antiproliferative capacity against a variety of cancer cells, highlighting their efficiency in the development of apoptosis inducers drugs. Moreover, numerous novel quinazoline-derived compounds which exert effective anticancer activity via apoptosis induction have been synthesized [10,16]. Lapatinib and erlotinib are among these quinazoline-containing drugs that have been demonstrated to exert cell cycle arrest and apoptosis initiation in various cancer cells [17,18]. Consequently, efforts have been in use to synthesize a hybrid of quinazoline–sulfonamide in expectation of a promising synergistic consequence on the inhibition of cancer cell propagation. Although, there are no literature reports on substituted quinazoline–sulfonamide hybrid synthesis and its anticancer activity on human breast cancer cell line and adenocarcinoma cell line. Hybridization has confirmed to be valuable for the synthesis of new antitumor agents and overcoming the problems of the traditionally used drugs [19]. Therefore, sulfonamides were included with quinazolin-4(3H)-ones to obtain more effective candidates. As a continuation of our previous efforts [13,20,21].

In this respect, we designed novel quinazoline derivatives bearing substituted-sulfonamide moiety, these derivatives were subjected to in vitro cytotoxic evaluation against numerous cell lines including A549 (lung), LoVo (colon), HepG2 (liver) and MCF-7 (breast) cancer cells as well as HUVEC (normal) cell line. Moreover, the most active compounds were selected to explore its apoptotic effects against the most responsive cancer cells. Molecular docking was also performed to determine their binding mode and their ability to satisfy the pharmacophoric features required to induce the desired inhibition.

2. Results and Discussion

2.1. Chemistry

Scheme 1 shows the synthesis of quinazoline carrying biologically active substituted- benzenesulfonamide moieties 4a–n. The required strategic starting material 4-(2-mercapto-8-methoxy-4- oxoquinazolin-3(4H)-yl) benzenesulfonamide 3 was synthesized in quantitative yield by cyclocondensation of 2-amino-3-methoxybenzoic acid 1 with 4-isothiocyanatobenzenesulfonamide 2 [19], in refluxing 1,4-dioxan containing triethylamine (Scheme 1). IR spectrum of compound 3 exhibited a characteristic band for NH₂, CH aromatic, C=O, C=N and SO₂ groups. ¹H-NMR spectrum revealed signals at 3.9 ppm assigned to OCH₃ and 12.3 ppm attributed to SH proton. ¹³C-NMR showed signals at 55.7 ppm for OCH₃ and 160.9 ppm due to C=O of quinazoline ring. The reaction of 3 with 2-chloro-N-arylacetamide derivatives was investigated. Thus, reaction of 3 with the excess of 2-chloro-N-substituted acetamide (2 mol) in dry acetone containing excess anhydrous K₂CO₃ (2 mol) gave the unexpected novel quinazoline incorporated substituted-sulfonamide moieties at 3-position 4a–n, rather than the expected quinazoline- containing free sulfonamide moiety at 3-position 5a–n [22] (Scheme 2). The formation of compounds 4a–n was explained in (Scheme 3). On the other hand, compounds 4a–n were obtained also in good yield via reaction of compounds 5a–n with 2-chloro-N-arylacetamide derivatives (1:1 mol) in presence of dry acetone containing (1 mol) anhydrous K₂CO₃. All the synthesized compounds 4a–n were established by elemental analysis, IR, ¹H-NMR, ¹³C-NMR and mass spectral data. IR spectra of 4a–n displayed additional 2NH and 2C=O bands at their assigned regions. ¹H-NMR spectra of 4a–n revealed signals at 3.7–3.9 ppm assigned to OCH₃, two singlet signals, one at 3.9–4.1 ppm referring to the SCH_2, 4.1–4.3 ppm due to N-CH₂, and 7.5–8.0 ppm for aromatic protons (AB system), 8.0–8.2 ppm attributed for SO₂NH proton and 8.8–10.5 ppm for 2NH protons. ¹³C-NMR of 4a–n displayed two signals peculiar to the SCH₂, NCH₂ and 3CO carbons. ¹H-NMR spectrum of 4a showed singlet signals at 3.8 ppm for OCH₃, 4.1 ppm for SCH₂, 4.2 ppm for NCH₂, 8.0 ppm for SO₂NH and 10.3 ppm due to 2NH protons. ¹³C-NMR spectrum of compound 4a revealed signals at 31.6 ppm assigned to SCH₂, 44.0 ppm attributed to NCH₂, 56.7 ppm for OCH₃, 161.4, 165.0, 165.6 ppm for 3C=O. ¹H-NMR spectrum of 4b showed signals at 2.1 ppm for 2CH₃ groups at the ortho positions of the phenyl ring, 3.8 ppm for OCH₃, 4.1, 4.3 ppm for 2CH₂, 8.1 ppm for SO₂NH and 9.6 ppm due to 2NH protons. ¹³C-NMR spectrum of compound 4b exhibited signals at 18.1 ppm due to 2CH₃ protons. ¹H-NMR spectrum of 4c revealed signals at 2.3 ppm for 2CH₃ group at the meta positions of the phenyl ring; ¹³C-NMR spectrum of compound 4c exhibited signals at 21.6 ppm due to 2CH₃ protons. ¹H-NMR spectrum of 4d revealed signals at 2.2 ppm for 2CH₃ groups at the para positions of the phenyl ring. ¹³C-NMR spectrum of compound 4d showed singlet at 20.9 ppm for 2CH₃ protons, 28.6, 44.0 ppm due to 2CH₂ protons. ¹H-NMR spectrum of 4e revealed triplets at 1.1 ppm attributed to the 2CH₃ ethyl, and quartet at 2.5 ppm referring to the 2CH₂ ethyl at the ortho positions of phenyl ring. ¹³C-NMR spectrum of compound 4e exhibited signals at 14.5 ppm for 2CH₃ ethyl group, 24.0 ppm due to 2CH₂ ethyl protons. ¹H-NMR spectrum of 4f revealed a triplet at 1.1 ppm attributed to the 2CH₃ ethyl, and quartet at 2.6 ppm referring to the 2CH₂ ethyl at the meta positions. ¹³C-NMR spectrum of compound 4f exhibited signals at 15.9 ppm due to 2CH₃ protons, 28.6 ppm for 2CH₂ ethyl group. ¹H-NMR spectrum of 4g revealed triplets at 1.1 ppm attributed to the 2CH₃ ethyl, and quartet at 2.6 ppm referring to the 2CH₂ ethyl at the para positions. ¹³C-NMR spectrum of compound 4g exhibited signals at 16.1 ppm due to 2CH₃ protons, 28.0 ppm for 2CH₂ ethyl groups. ¹H-NMR spectrum of 4h revealed signals at 3.72, 3.73, 3.80 ppm for 3 OCH₃ groups. ¹³C-NMR spectrum of compound 4h exhibited signals at 55.6 ppm due to 3 OCH₃ protons. ¹H-NMR spectrum of 4i revealed triplets at 1.3 ppm attributed to the 2CH₃ ethoxy groups, and quartet at 4.0 ppm referring to the 2CH₂ ethoxy groups at the para positions. ¹³C-NMR spectrum of compound 4i exhibited signals at 15.1 ppm for 2CH₃ ethoxy groups, 63.5 ppm due to 2CH₂ ethoxy groups. ¹H-NMR spectrum of 4j revealed signals at 3.81, 3.84, 3.90 ppm for 5 OCH₃ groups. ¹³C-NMR spectrum of 4j exhibited signals at 56.3 ppm due to 5 OCH₃ protons. ¹H-NMR spectrum of 4k revealed signals at 3.74, 3.77, 3.81, 3.91 ppm for 7 OCH₃ groups. ¹³C-NMR spectrum of 4k exhibited signals at 55.3, 56.8, 62.7 ppm due to 7 OCH₃ protons. IR spectra of compounds 4l–n showed bands of NO₂ groups at their specified regions. ¹H-NMR spectrum of 4l revealed singlet at 2.1 ppm and attributed to the 2CH₃ at the ortho positions. ¹³C-NMR spectrum of 4l exhibited signals at 18.0 ppm for 2CH₃ protons. ¹H-NMR spectrum of 4m revealed singlet at 2.2 ppm attributed to the 2CH₃ at the ortho positions. ¹³C-NMR spectrum of 4m exhibited signals at 18.0 ppm for 2CH₃ protons. ¹H-NMR spectrum of 4n exhibited singlet at 3.8 ppm attributed to the OCH₃ moiety, 3.9, 4.2 ppm for 2CH₂ protons. ¹³C-NMR spectrum of 4n showed signals at 28.6, 40.4 ppm assigned to 2CH₂ protons, 57.3 ppm for OCH₃ protons.

2.2. MTT Antiproliferative Evaluation

Compounds 4a–n were subjected to the cytotoxicity screening of the cell lines MCF-7 (breast), LoVo (colon), HepG2 (liver) and A549 (lung) carcinoma cells as well as HUVEC normal cell lines. The MTT assay was employed to assess cytotoxicity after 48 h of treatment with different concentrations of each compound and doxorubicin was used as a reference drug. The results were presented in

Table 1. Cytotoxic activity, expressed as IC₅₀, of synthetic compounds 4a–n and doxorubicin against different cancer cell lines.

Compound No.	Cell Lines and IC50 (µM)
	A549	HepG2	LoVo	MCF-7	HUVEC
4a	19.6 ± 1.7	35 ± 1.2	37.5 ± 1.8	14.2 ± 0.4	20.9 ± 1
4b	11.36 ± 1.6	33.7 ± 0.8	35.75 ± 1	36.2 ± 2	32.7 ± 1.2
4c	-	-	-	-	-
4d	6.6 ± 0.22	9 ± 0.3	6.87 ± 0.62	2.5 ± 0.2	7.8 ± 0.6
4e	-	-	-	-	-
4f	10.14 ± 0.24	11.7 ± 2	9.76 ± 1.68	5 ± 0.24	17.9 ± 0.6
4g	-	-	-	-	-
4h	13.3 ± 0.85	25.4 ± 0.95	31.7 ± 1	31.62 ± 1.3	24.3 ± 0. 56
4i	13.8 ± 0.5	20.6 ± 1.6	22.04 ± 1.01	26.4 ± 0.79	18.2 ± 2.2
4j	-	-	-	-	-
4k	-	-	-	-	-
4l	-	-	-	-	-
4m	-	-	-	-	-
4n	-	-	-	-	-
Doxorubicin	1.1 ± 0.01	2.8 ± 0. 01	1.5 ± 0.05	1.4 ± 0.3	3.7 ± 0.2

(-) Inactive at 40 µM (highest concentration tested). The values represented as the average ± SD (n = 3).

as growth inhibitory concentration (IC₅₀ values). Our results demonstrate that six compounds (4a, 4b, 4d, 4f, 4h and 4i) out of fourteen exhibited promising antiproliferative activities. A remarkable antiproliferative activity was observed for compound 4d with an IC₅₀ values of 2.5, 5.6, 6.87 and 9 µM against MCF-7, A549, LoVo and HepG2 cells, respectively. Additionally, compound 4f revealed a promising activity against all tested cell lines with an IC₅₀ value of 5, 9.76, 10.14 and 11.7 µM against MCF-7, LoVo, A549 and HepG2 cells, respectively. Interestingly, compound 4f had a lower cytotoxic effect in terms of IC₅₀ values against normal HUVEC cells (IC₅₀ = 17.9). Structure activity relationship revealed that 4-tolyl 4d and 3-ethylphenyl 4f (IC₅₀ value 2.5 and 5.0 µM) showed a demonstrated superior cytotoxic activity and are nearly as active as a reference drug doxorubicin (IC₅₀ value 1.4 µM) against MCF-7 breast cancer cell line. It is obvious that replacement of 4-tolyl moiety 4d (IC₅₀ value 2.5 µM) with 2-tolyl fragment 4b (IC₅₀ value 36.2 µM) led to increase the antiproliferative activity against MCF-7 cell line, while incorporation of unsubstituted phenyl moiety as in compound 4a (IC₅₀ value 14.2 µM) with a 4-tolyl fragment 4d (IC₅₀ value 2.5 µM) exhibited intermediate activity. On the contrary, insertion of a methyl group at 3-position 4c abolishes the cytotoxicity, as opposed to compound 4d (IC₅₀ value 2.5 µM) containing the methyl group in 4-position. On the other hand, the orientation of ethyl group significantly affected the cytotoxic activity of compounds 4e, 4f, and 4g where the presence of it at 3-position as in compound 4f enhanced the cytotoxic activity (IC₅₀ value 5.0 µM) compared to compounds 4e and 4g carrying the ethyl group at 2-position and 4-position, respectively, which assigned no antiproliferative completely. Additionally, we observed that 4d and 3-ethylphenyl moiety 4f had a considerable potent cytotoxic activity compared to the corresponding unsubstituted phenyl 4a (IC₅₀ value 14.2 µM) and 2-tolyl 4b (IC₅₀ value 36.2 µM). Based on this result, compounds 4d and 4f were selected for further investigations.

2.3. Compounds 4d and 4f Induces G1-Phase Cell Cycle Arrest on MCF-7 Cells

Because compounds 4d and 4f displayed the highest activity towards MCF-7 cells, we looked into their cellular effects with respect to cell cycle progression and cell death mode. Indeed, designing of new chemotherapeutic drugs targeting cell cycle progression and apoptosis is considered as an attractive strategy [23,24]. To explore the antiproliferative effect of compounds 4d and 4f, cell-cycle analysis was performed in MCF-7 cells were examined. The cells were treated with various concentrations of both compound for 24 h, stained with propidium iodide (PI) and analyzed by flow cytometry. As shown in (Figure 2), the incubation of MCF-7 cells with (1.25, 2.5, and 5 μM) of compound 4d for 24 h increased the percentage of cells at G1 (from 57.91 ± 0.61% in control) to 62.51 ± 0.14%, 64.95 ± 0.21% and 66.55 ± 0.78% in treated cells, respectively. At the same conditions, the percentage of cells in S and G2/M phases were obviously decreased. These results revealed that compound 4d arrested the cell cycle at the G1 stage. In the same manner, DNA content analyses also showed a statistically significant increase in the number of cells in the G1 phase of MCF-7 cells treated with compound 4f (increased to 61.65 ± 0.21% at 1.25 μM, 62.65 ± 0.35% at 2.5 μM and 65.15 ± 0.36% at 5 μM) when compared to control (57.9 ± 0.61%) (Figure 3). Collectivity, our data indicated that both compounds 4d and 4f inhibit MCF-7 cells proliferation by modulating cell cycle progress at G1 phase which is a critical stage in the cell cycle as well as a worth target for drug action [25].

2.4. Apoptosis Quantification

In addition to cell cycle arrest, apoptosis is another important anticancer process that inhibits cancer cell proliferation [26]. Therefore, we further used a double labeling staining with Annexin V-FITC and PI to investigate apoptotic cell death. Phosphatidylserine exposure on the cell surface is a hallmark of early and late apoptotic cells, which can be easily identified with Annexin V-FITC [27]. As shown in (Figure 4), early apoptotic cells were increased along with the concentrations increment of compound 4d from 1.05 ± 0.07% in untreated MCF-7 cells to 44.45 ± 1.06% at 1.25 µM, (p < 0.05), 67.55 ± 1.34% at 2.5 µM (p < 0.01), and 83.75 ± 2.19% at 5 µM (p < 0.001). For compound 4f (Figure 5), it was noticed that most treated cells were in a late apoptotic stage which were increased in a dose-dependent manner (from 1.75 ± 0.07% in control to 15.4 ± 0.28%, 31.05 ± 1.06% and 52.4 ± 0.28% at 1.25, 2.5 and 10 µM, respectively). Interestingly, we noticed that most treated cells were in early apoptotic stage after 4d compound treatment while most cells populations were in a late apoptotic stage with compound 4f and these different effects of both compounds could be due to their difference attached functional groups (4-tolyl in 4d and 3-ethylphenyl in 4f) which may be targeting different sites for inducing apoptosis in different time periods which need further investigation. Overall, the findings obviously showed that both compounds effectively triggered apoptosis in MCF-7 cells.

2.5. Effect of Compounds 4d and 4f on Apoptosis Regulatory Markers

We next used reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis to look at Bax (proapoptotic), Bcl-2 (antiapoptotic) and caspase 7 (executioner caspase) expression at gene and protein level as an additional marker of apoptosis occurrence [28]. In fact, apoptotic cell death is mostly directed by the levels of Bcl-2 family, whether they are pro-apoptotic or antiapoptotic and targeting these molecules as a therapeutic strategy is continuously under development [29]. As predicted, we observed an upregulation in the expression of Bax in MCF-7 treated cells to (1.7-, 2- and 2.4-fold) with compound 4d and to (1.2-, 1.4- and 1.6-fold) with compound 4f at (1.25, 2.5 and 5 µM) respectively (Figure 6). In contrast, Bcl-2 expression was downregulated into (0.88-, 0.68- and 0.49-fold) with 4d and into (0.92, 0.68 and 0.62) with 4f at indicated concentration in compare to DMSO-vehicle treated cells. Our results also showed that both 4d and 4f caused a significant increase in the expression of caspase 7 to (1.13-, 1.35- and 1.58-fold) relative to untreated MCF-7 cells (Figure 6). Consistently, Western blot analysis revealed that compounds 4d and 4f treatment markedly increased the expression of Bax whereas Bcl-2 was downregulated in a dose-dependent manner compared to that of control cells (Figure 7). This shifting in Bax to Bcl-2 ratio usually started caspases activation which plays a critical role in apoptosis execution [26,30]. Among all caspases, caspase 7 is considered as one of the most crucial executioner caspases that mediate cell death, and its expression strongly correlates with apoptosis stimulation [31,32]. Hence, we evaluated whether compounds 4d and 4f (1.25, 2.5 and 5 μM) promote the caspase 7 expression at protein level. We found that caspase 7 protein expression significantly increased after cell treatment with compound 4d (3-, 3.3- and 3.9-fold) and (1.14-, 1.2- and 1.5-fold) with compound 4f in comparison to the control, which suggested involvement of caspase 7 activation during compounds treatment. In line with our findings, several studies have demonstrated the apoptotic behavior of quinazoline derivatives through upregulating the proapoptotic genes and downregulating the antiapoptotic genes [33,34,35]. In particular, quinazoline-based anticancer drugs such as lapatinib and gefitinib were found to inhibit cancer cells growth through increasing the Bax and caspases gene levels and downregulating Bcl-2 levels, which is consistent with our finding in this study [36,37].

2.6. Molecular Docking

Overexpression of anti-apoptotic Bcl-2 protein has been linked to cancer progression and cell death avoidance. Hence, developing novel Bcl2 inhibitors is considered a promising field in designing chemotherapeutic agents [38]. The molecular docking procedure executed in this study was authenticated as reported by [39]. The X-ray bound ligand, i.e., 1-(2-{[(3S)-3-(aminomethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}phenyl)-4-chloro-5-methyl-N,N-diphenyl-1H-pyrazole-3-carboxamide (DRO), was extracted from the crystal structure and re-docked to Bcl-2. The poses of the bound and re-docked ligand were compared, and the root mean square deviation (RMSD) was calculated. The RMSD of the re-docked DRO was found to be 0.3842 Å (Figure S1). Because the calculated RMSD was within the acceptable limit (2.0 Å), we were confident in adopting the docking protocol to predict the binding of compounds 4d and 4f with Bcl-2.

The results of molecular docking between ligands 4d and 4f and Bcl-2 were compared with that of DRO, and the results are given in (

Table 2. Molecular docking of compound 4d and 4f with Bcl-2.

Donor Atoms	Acceptor Atom	Distance (Å)	Type of Interaction	Binding Free Energy, ΔG (kcal mol−1)	Binding Affinity, Kd (M−1)
Bcl-2 and DRO *
LIG:N ASP70:OD2 MET74:CE LEU96:CD1 LIG:C PHE63 PHE71 LIG:C LIG LIG LIG LIG LIG	ASP70:OD2 LIG LIG LIG TYR67 LIG LIG LEU96 ARG105 ALA108 LEU96 ALA108 VAL92	2.9553 4.8135 3.5674 3.8681 3.5199 5.2880 4.9682 4.9539 5.2941 4.4414 5.1674 4.4935 5.0873	Conventional Hydrogen Bond Electrostatic (Pi-Anion) Hydrophobic (Pi-Sigma) Hydrophobic (Pi-Sigma) Hydrophobic (Pi-Sigma) Hydrophobic (Pi-Pi T-shaped) Hydrophobic (Pi-Pi T-shaped) Hydrophobic (Alkyl) Hydrophobic (Pi-Alkyl) Hydrophobic (Pi-Alkyl) Hydrophobic (Pi-Alkyl) Hydrophobic (Pi-Alkyl) Hydrophobic (Pi-Alkyl)	−10.2	3.03 × 107
Bcl-2 and Compound (4d)
LIG: H MET74:CE LEU96:CD1 LIG:C TYR67 ALA108 LIG:C LIG	GLU95:OE1 LIG LIG PHE71 LIG LIG:C MET74 VAL92	2.0244 3.4311 3.9269 3.6605 3.8844 3.7959 4.8241 5.3864	Conventional Hydrogen Bond Hydrophobic (Pi-Sigma) Hydrophobic (Pi-Sigma) Hydrophobic (Pi-Sigma) Hydrophobic (Pi-Pi Stacked) Hydrophobic (Alkyl) Hydrophobic (Alkyl) Hydrophobic (Pi-Alkyl)	−7.8	5.25 × 105
Bcl-2 and Compound (4f)
TYR67:HH PHE63 TYR67 GLU95:C,O;LEU96:N ALA108 LIG:C LIG:C LIG LIG	LIG:O LIG LIG LIG LIG:C LEU96 MET74 ARG105 ALA108	2.4126 4.8823 5.1361 3.9675 3.7719 4.6857 4.0247 5.4994 5.1493	Conventional Hydrogen Bond Hydrophobic (Pi-Pi T-shaped) Hydrophobic (Pi-Pi T-shaped) Hydrophobic (Amide-Pi Stacked) Hydrophobic (Alkyl) Hydrophobic (Alkyl) Hydrophobic (Alkyl) Hydrophobic (Pi-Alkyl) Hydrophobic (Pi-Alkyl)	−7.5	3.17 × 105

* Chemically DRO is 1-(2-{[(3S)-3-(aminomethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}phenyl)-4-chloro-5-methyl-N,N-diphenyl-1H-pyrazole-3-carboxamide.

, Figure 8 and Figure 9). We found that both compounds 4d and 4f occupied a similar position at the Bcl-2 binding site as occupied by control ligand DRO (Figure 8A,B). The DRO-Bcl-2 complex was stabilized by a carbon interaction and an electrostatic interaction with the Asp70 residue (Figure 8C), as well as ten hydrophobic interactions with Phe63, Tyr67, Phe71, Met74, Val92, Leu96, and Ala108 (Table 2). The DRO-Bcl-2 complex was further stabilized by van der Waals’ interactions of DRO with the Arg88, Glu95, Gly104, Arg105, and Phe112 residues of Bcl-2. Analysis of the interactions between compound 4d and Bcl-2 revealed that the protein–ligand complex was stabilized by one conventional hydrogen bond with Glu95, and seven hydrophobic interactions with Tyr67, Phe71, Met74, Val92, Leu96, and Ala108 (Table 2). The compound 4d-Bcl-2 complex was further stabilized by van der Waals’ interactions of the compound with several other residues, including Phe63, Asp70, Gly104, and Arg105 (Figure 9A). Similarly, the compound 4f-Bcl-2 complex was stabilized by one conventional hydrogen bond with Tyr67, and eight hydrophobic interactions with Phe63, Tyr67, Met74, Glu95, Leu96, Arg105 and Ala108 (Table 2). Additionally, the Asp70, Phe71, Arg98, Asp99, Gly104, and Phe112 residues of Bcl-2 formed van der Waals’ interactions to stabilize the compound 4f-Bcl-2 complex (Figure 9B). In comparison, the binding energies and the corresponding binding affinities of DRO, compounds 4d and 4f towards Bcl-2, were estimated to be −10.2 kcal mol⁻¹ and 3.03 × 10⁷ M⁻¹, −7.8 kcal mol⁻¹ and 5.25 × 10⁵ M⁻¹, and −7.5 kcal mol⁻¹ and 3.17 × 10⁵ M⁻¹, respectively (Table 1). It is interesting to note that Phe63, Tyr67, Asp70, Phe71, Met74, Val92, Glu95, Leu96, Gly104, Arg105, Ala108, and Phe112 residues of Bcl-2 showed interactions with both compounds 4d and DRO. Similarly, Phe63, Tyr67, Asp70, Phe71, Met74, Glu95, Leu96, Gly104, Arg105, Ala108, Phe112 residues of Bcl-2 showed interactions with compound 4f as well as the control ligand DRO.

3. Experimental

3.1. Chemistry

Melting points (uncorrected) were determined in open capillary on a Gallen Kamp melting point apparatus (Sanyo Gallen Kamp, Southborough, UK). Precoated silica gel plates (Kieselgel 0.25 mm, 60 F254, Merck, Darmstadt, Germany) were used for thin-layer chromatography. A developing solvent system of chloroform/methanol (8:2) was used and the spots were detected by ultraviolet light. IR spectra (KBr disc) were recorded using an FT-IR spectrophotometer (Perkin Elmer, Norwalk, CT, USA). ¹H-NMR spectra were scanned on an NMR spectrophotometer (Bruker AXS Inc., Flawil, Switzerland), operating at 500 MHz for ¹H- and 125.76 MHz for ¹³C. Chemical shifts are expressed in δ-values (ppm) relative to TMS as an internal standard, using DMSO-d₆ as a solvent. Elemental analyses were done on a model 2400 CHNSO analyser (Perkin Elmer, Norwalk, CT, USA). All the values were within ±0.4% of the theoretical values. All reagents used were of AR grads.

• 4-(2-Mercapto-8-methoxy-4- oxoquinazolin-3(4H)-yl) benzenesulfonamide (3)

A mixture of 2-amino-3-methoxybenzoic acid 1 (1.67 g, 0.01 mol) and 4-isothiocyanatobenzenesulfonamide 2 (2.14 g, 0.01 mol) in dioxan (50 mL) containing 3 drops of triethylamine, was refluxed for 8 h. The solid product formed while hot was collected by filtration and recrystallized from ethanol to give 3. 3: Yield, 89%; m.p. 342–344 °C. IR: 3321, 3262, 3181 (NH₂), 3055 (arom.), 1691 (CO), 1620 (CN), 1381, 1156 (SO₂). ¹HNMR: 3.9 (s, 3H, OCH₃), 7.0–7.7 [m, 5H, Ar-H + SO₂NH₂], 7.8, 8.0 (2d. 4H, AB system, Ar-H), 12.3 [s, 1H, SH]. ¹³CNMR: 55.7, 116.2, 118.6, 119.8, 120.6 (2), 127.0, 128.9 (2), 130.1, 134.6, 135.3, 150.4, 160.5, 160.9. MS m/z (%): 363 (M⁺) (14.54), 155 (100). Anal. Calcd. for C₁₅H₁₃N₃O₄S₂ (363.41): C, 49.57; H, 3.61; N, 11.56. Found: C, 49.22; H, 3.86; N, 11.31. Quinazolin-3(4H)-yl)phenylsulfonamido)-N-phenylacetamide derivatives (4a-n).

3.2. General Procedure

3.2.1. Method A

A mixture of 3 (4.59 g, 0.01 mol) and 2-chloro-N-substitutedacetamide derivatives (0.02 mol) in dry acetone (50 mL) and anhydrous K₂CO₃ (2.56 g, 0.02 mol) was stirred at room temperature for 24 h., filtered and the solid product formed was recrystallized from ethanol to give 4a–n.

3.2.2. Method B

A mixture of 5a–n [20] (0.01 mol) and 2-chloro-N-substitutedacetamide derivatives (0.01 mol) in dry acetone (50 mL) and anhydrous K₂CO₃ (1. 38 g, 0.01 mol) was stirred at room temperature for 18 h, filtered and the solid product formed was recrystallized from ethanol to give 4a–n.

• 2-(4-(8-Methoxy-4-oxo-2-((2-oxo-2-(phenylamino)ethylthio)quinazolin-3(4H)-yl)phenylsul-fonamido)-N-phenylacetamide (4a).

5: Yield, 90%; m.p. 165–167 °C. IR: 3300, 3141 (NH), 3066 (arom.), 2965, 2844 (aliph.), 1700, 1693, 1665 (3CO), 1612 (CN), 1377, 1154 (SO₂). ¹HNMR: 3.8 [s, 3H, OCH₃], 4.1 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 7.5, 7.6 (2d, 4H, AB system, Ar-H), 7.0- 7.3 [m, 13H, Ar-H), 8.0 (s, 1H, SO₂NH], 10.3 [s, 2H, 2NH]. ¹³CNMR: 31.6, 44.0, 56.7, 116.2, 116.8, 119.8 (4), 120.6, 124.3 (2), 129.3 (2), 129.4 (4), 129.8 (2), 131.7 (2), 137.0, 138.9 (2), 145.7, 154.0, 158.8, 161.4, 165.0, 165.6. MS m/z (%): 629 (M⁺) (31.43), 231 (100). Anal. Calcd. for C₃₁H₂₇N₅O₆S₂ (629.71): C, 59.13; H, 4.32; N, 11.12. Found: C, 59.45; H, 4.65; N, 10.83.

• 2-(4-(8-Methoxy-4-oxo-2-((2-oxo-2-(o-tolylamino)ethylthio)quinazolin-3(4H)-yl)phenylsul-fonamido)-N-(o-tolyl)acetamide (4b).

4b: Yield, 90%; m.p. 102–104 °C. IR: 3354, 3154 (NH), 3036 (arom.), 2972, 2873 (aliph.), 1698, 1668, 1660 (3CO), 1610 (CN), 1386, 1166 (SO₂). ¹HNMR: 2.1 [s, 3H, CH₃], 3.8 [s, 3H, OCH₃], 4.1 [s, 2H, SCH₂], 4.3 [s, 2H, NCH₂], 7.1, 7.7 [m, 11H, Ar-H), 7.9, 8.0 (2d, 4H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 9.6 [s, 2H, 2NH]. ¹³CNMR: 18.1 (2), 31.6, 43.6, 57.8, 116.6, 117.8, 121.0, 124.8 (2), 125.6 (2), 126.1 (2), 126.5, 129.6 (2), 130.8 (2), 132.4 (2), 132.6 (2), 136.0, 137.9, 138.2 (2), 139.8, 147.6, 155.6, 161.2, 165.6, 165.9. MS m/z (%): 657 (M⁺) (11.84), 188 (100). Anal. Calcd. for C₃₃H₃₁N₅O₆S₂ (657.76): C, 60.26; H, 4.75; N, 10.65. Found: C, 60.60; H, 4.39; N, 10.36.

• 2-(4-(8-Methoxy-4-oxo-2-((2-oxo-2-(m-tolylamino)ethylthio)quinazolin-3(4H)-yl)phenylsul-fonamido)-N-(m-tolyl)acetamide (4c).

4c: Yield, 84%; m.p. 112–114 °C. IR: 3309, 3144 (NH), 3078 (arom.), 2954, 2863 (aliph.), 1692, 1677, 1661 (3CO), 1618 (CN), 1377, 1151 (SO₂). ¹HNMR: 2.3 [s, 6H, 2CH₃], 3.9 [s, 3H, OCH₃], 4.0 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 6.8–7.7 [m, 11H, Ar-H), 7.6, 8.0 (2d, 4H, AB system, Ar-H), 9.6 (s, 1H, SO₂NH], 10.1 [s, 2H, 2NH]. ¹³CNMR: 21.6 (2), 26.7, 40.3, 56.7, 116.8, 117.1 (2), 117.4, 120.1 (2), 120.7, 124.5 (2), 124.8 (2), 127.4, 128.9 (2), 129.0 (2), 130.7, 138.2, 138.3 (2), 138.7 (2), 141.3, 155.6, 161.2, 165.0, 168.5, 171.3. MS m/z (%): 657 (M⁺) (3.76), 154 (100). Anal. Calcd. for C₃₃H₃₁N₅O₆S₂ (657.76): C, 60.26; H, 4.75; N, 10.65. Found: C, 60.01; H, 4.43; N, 10.89.

• 2-(4-(8-Methoxy-4-oxo-2-((2-oxo-2-(p-tolylamino)ethylthio)quinazolin-3(4H)-yl)phenylsul-fonamido)-N-(p-tolyl)acetamide (4d).

4d: Yield, 84%; m.p. 160–162 °C. IR: 3353, 3132 (NH), 3064 (arom.), 2976, 2845 (aliph.), 1695, 1676, 1660 (3CO), 1620 (CN), 1387, 1155 (SO₂). ¹HNMR: 2.2 [s, 6H, 2CH₃], 3.8 [s, 3H, OCH₃], 4.0 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 7.1–7.4 [m, 3H, Ar-H), 7.6, 8.0 (2d, 12H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 10.2 [s, 2H, 2NH]. ¹³CNMR: 20.9 (2), 28.6, 44.0, 56.7, 118.0, 119.8, 120.8 (4), 120.9 (3), 129.6 (5), 129.7 (2), 129.9, 133.2 (2), 136.4, 138.0 (2), 139.2, 153.8, 158.6, 164.8, 165.2 (2). MS m/z (%): 657 (M⁺) (17.54), 224 (100). Anal. Calcd. for C₃₃H₃₁N₅O₆S₂ (657.76): C, 60.26; H, 4.75; N, 10.65. Found: C, 60.50; H, 4.55; N, 10.44.

• N-(2-ethylphenyl)-2-((3-(4-(N-(2-((2-ethylphenyl)amino)-2-oxoethyl)sulfamoyl)-phe-nyl)- 8-methoxy-4-oxo-3,4-dihydroquinazolin-2yl) thio)acetamide (4e).

4e: Yield, 64%; m.p. 246–248 °C. IR: 3409, 3256 (NH), 3075 (arom.), 2944, 2856 (aliph.), 1694, 1683, 1665 (3CO), 1617 (CN), 1398, 1155 (SO₂). ¹HNMR: 1.0 (t, 6H, 2 CH₃ ethyl), 2.5 (q, 4H, 2CH₂ ethyl), 3.8 [s, 3H, OCH₃], 3.9 [s, 2H, SCH₂], 4.1 [s, 2H, NCH₂], 7.1–7.7 [m, 11H, Ar-H), 7.9, 8.0 (2d, 4H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 9.6 [s, 2H, 2NH]. ¹³CNMR: 14.5 (2), 24.0 (2), 31.0, 40.3, 56.7, 116.3, 118.1, 120.9, 126.3 (2), 126.4 (2), 127.1 (2), 127.4 (2), 128.9 (2), 130.3 (2), 130.7 (2), 135.8 (2), 138.0 (2), 139.1 (2), 139.2, 153.7, 161.0, 166.4 (2). MS m/z (%): 685 (M⁺) (23.62), 288 (100). Anal. Calcd. for C₃₅H₃₅N₅O₆S₂ (685.81): C, 61.30; H, 5.14; N, 10.21. Found: C, 61.00; H, 5.37; N, 10.46.

• N-(3-ethylphenyl)-2-((3-(4-(N-(2-((3-ethylphenyl)amino)-2-oxoethyl)sulfamoyl)-phe-nyl)-8-methoxy-4-oxo-3,4-dihydroquinazolin-2yl) thio)acetamide (4f).

4f: Yield, 57%; m.p. 239–241 °C. IR: 3321, 3123 (NH), 3084 (arom.), 2975, 2866 (aliph.), 1691, 1685, 1660 (3CO), 1619 (CN), 1376, 1151 (SO₂). ¹HNMR: 1.2 (t, 6H, 2CH₃ ethyl), 2.6 (q, 4H, 2CH₂), 3.8 [s, 3H, OCH₃], 4,0 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 6.9–7.6 [m, 11H, Ar-H), 7.6, 8.0 (2d, 4H, AB system, Ar-H), 9.5 (s, 1H, SO₂NH], 10,4 [s, 2H, 2NH]. ¹³CNMR: 15.9 (2), 28.6 (2), 28.7, 40.3, 56.7, 116.4, 117.0 (2), 117.2, 118.1 (2), 119.1, 120.9 (2), 123.8 (2), 127.0, 127.4 (2), 129.2 (2), 130.7, 138.0, 139.3 (2), 144.7, 145.9 (2), 153.7, 155.6, 160.9, 165.0, 166.0. MS m/z (%): 685 (M⁺) (10.74), 412 (100). Anal. Calcd. for C₃₅H₃₅N₅O₆S₂ (685.81): C, 61.30; H, 5.14; N, 10.21. Found: C, 61.03; H, 4.87; N, 10.06.

• N-(4-ethylphenyl)-2-((3-(4-(N-(2-((4-ethylphenyl)amino)-2-oxoethyl)sulfamoyl)-phenyl)-8-methoxy-4-oxo-3,4-dihydroquinazolin-2yl)thio)acetamide (4g).

4g: Yield, 64%; m.p. 219–221 °C. IR: 3409, 3256 (NH), 3075 (arom.), 2944, 2856 (aliph.), 1694, 1683, 1665 (3CO), 1617 (CN), 1398, 1155 (SO₂). ¹HNMR: 1.1 (t, 6H, 2CH₃ ethyl), 2.5 (q, 4H, 2CH₂ ethyl₎, 3.8 [s, 3H, OCH₃], 4.0 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 7.1–7.5 [m, 3H, Ar-H), 7.6, 8.0 (2d, 12H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 10.2 [s, 2H, 2NH]. ¹³CNMR: 16.1 (2, 28.0 (3), 44.0, 58.6, 115.6, 117.8, 119.9 (4), 120.1, 121.6 (2), 126.7 (4), 127.8, 128.5 (2), 129.8, 136.6 (2), 136.8, 139.7, 140.2 (2), 153.9, 161.2, 164.8, 165.6 (2). MS m/z (%): 685 (M⁺) (23.62), 288 (100). Anal. Calcd. for C₃₅H₃₅N₅O₆S₂ (685.81): C, 61.30; H, 5.14; N, 10.21. Found: C, 61.66; H, 5.39; N, 10.04.

• 2-(4-(8-Methoxy-2-((2-((4-methoxyphenyl)amino)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)phenylsulfonamido)-N-(4-methoxy-phenyl)acetamide (4h).

4h: Yield, 71%; m.p. 117–119 °C. IR: 3350, 3231 (NH), 3054 (arom.), 2986, 2891 (aliph.), 1703, 1692, 1664 (3CO), 1611 (CN), 1364, 1152 (SO₂). ¹HNMR: 3.72, 3.73, 3.88 [3s, 9H, 3OCH₃], 4.1 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 6.9, 7.5 (2d, 8H, AB system, Ar-H), 7.5–7.6 (m, 3H, Ar-H), 7.6, 8.0 (2d, 4H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 10.5 [s, 2H, 2NH]. ¹³CNMR: 31.3, 43.9, 55.6 (3), 114.4 (4), 116.8, 118.6, 121.4, 123.7 (2), 125.8 (4), 125.9, 128.6 (2), 129.1 (2), 132.0, 137.2, 137.6, 155.9, 156.0 (2), 162.6, 164.6, 165.2 (2). MS m/z (%): 689 (M⁺) (9.86), 312 (100). Anal. Calcd. for C₃₃H₃₁N₅O₈S₂ (689.76): C, 57.46; H, 4.53; N, 10.15. Found: C, 57.13; H, 4.23; N, 10.35.

• N-(4-Ethoxyphenyl)-2-((3-(4-N-(2((4-ethoxyphenyl)amino)-2-oxoethyl)sulfamoyl)-phenyl-8-methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (4i).

4i: Yield, 66%; m.p. > 350 °C. IR: 3343, 3222 (NH), 3077 (arom.), 2965, 2836 (aliph.), 1698, 1685, 1666 (3CO), 1611 (CN), 1367, 1155 (SO₂). ¹HNMR: 1.3 (t, 6H, 2CH₃ ethyl), 3.9 [s, 3H, OCH₃], 4.0 (q, 4H, 2CH₂ ethyl), 4.1 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 6.8, 7.4 (2d, 8H, AB system, Ar-H), 7.6–7.8 (m, 3H, Ar-H), 7.9, 8.0 (2d, 4H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 10.1 [s, 2H, 2NH]. ¹³CNMR: 15.1 (2), 31.3, 43.9, 57.2, 63.5 (2), 114.9 (4), 116.0, 118.6, 121.3, 121.4 (2), 126.2 (4), 126.3, 129.7 (2), 131.9 (2), 132.0, 138.9, 139.0, 155.1, 157.4 (2), 161.6, 164.5, 165.1 (2). MS m/z (%): 717 (M⁺) (51.82), 365 (100). Anal. Calcd. for C₃₅H₃₅N₅O₈S₂ (717.81): C, 58.56; H, 4.91; N, 9.76. Found: C, 58.21; H, 4.65; N, 9.45.

• N-(3,5-Dimethoxyphenyl)-2-((3-(4-N-(2((3,5-dimethoxyphenyl)amino)-2-oxoethyl)-sulfa-moyl)phenyl-8-methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (4j).

4j: Yield, 65%; m.p. 239–241 °C. IR: 3384, 3198 (NH), 3045 (arom.), 2978, 2865 (aliph.), 1700, 1681, 1662 (3CO), 1612 (CN), 1356, 1154 (SO₂). ¹HNMR: 3.81, 3.84, 3.90 [3s, 15H, 5OCH₃], 4.0 [s, 2H, SCH₂], 4.1 [s, 2H, NCH₂], 6.2–7.6 [m, 9H, Ar-H), 7.8, 8.0 (2d, 4H, AB system, Ar-H), 8.2 (s, 1H, SO₂NH], 10.1 [s, 2H, 2NH]. ¹³CNMR: 27.6, 41.8, 56.3 (5), 97.2 (2), 101.0 (4), 114.0, 117.2, 120.3, 120.9 (2), 127.4, 128.3 (2), 131.9, 134.6, 139.7, 140.6 (2), 151.8, 158.6, 159.0 (4), 161.2, 167.4, 168.2, MS m/z (%): 749 (M⁺) (1.85), 147 (100). Anal. Calcd. for C₃₅H₃₅N₅O₁₀S₂ (749.81): C, 56.06; H, 4.70; N, 9.34. Found: C, 56.29; H, 4.45; N, 9.66.

• 2-(4-(8-Methoxy-4-oxo-2-((2-oxo-2-(3,4,5 trimethoxyphenyl)amino)ethyl)thio)quinazolin-3(4H)-yl)phenylsulfonamido)-N-(3,4,5-trimethoxyphenyl)acetamide (4k).

4k: Yield, 78%; m.p. 260–262 °C. IR: 3377, 3154 (NH), 3043 (arom.), 2933, 2875 (aliph.), 1690, 1672, 1665 (3CO), 1623 (CN), 1376, 1151 (SO₂). ¹HNMR: 3.74, 3.77, 3.81, 3.91 [4s, 21H, 7OCH₃], 4.0 [s, 2H, SCH₂], 4.1 [s, 2H, NCH₂], 6.9–7.7 [m, 7H,), 7.8, 8.0 (2d, 4H, AB system, Ar-H), 8.3 (s, 1H, SO₂NH], 10.2 [s, 2H, 2NH]. ¹³CNMR: 28.3, 42.7, 55.3, 56.8 (4), 62.7 (2), 97.0 (4), 114.6, 120.6, 120.8, 121.2 (2), 129.8, 130.5 (2), 131.7, 133.0, 134.2, 136.4 (2), 137.1, 139.6, 151.8, 155.1 (4), 158.3, 161.0, 168.1, 168.9. MS m/z (%): 809 (M⁺) (3.42), 364 (100). Anal. Calcd. for C₃₇H₃₉N₅O₁₂S₂ (809.86): C, 54.87; H, 4.85; N, 8.65. Found: C, 54. 49; H, 4.58; N, 8.28.

• 2-(4-(8-Methoxy-2-((2-((2-methyl-4-nitrophenyl)amino)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)phenylsulfonamido)-N-(2-methyl-4-nitrophenyl)acetamide (4l).

4l: Yield, 61%; m.p. 127–129 °C. IR: 3325, 3245 (NH), 3076 (arom.), 2976, 2843 (aliph.), 1694, 1682, 1660 (3CO), 1608 (CN), 1387, 1161 (SO₂). ¹HNMR: 2.1 (s, 6H, 2CH₃), 3.7 [s, 3H, OCH₃], 3.9 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 7.4–7.8 (m, 9H, Ar-H), 7.9, 8.0 (2d, 4H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 9.9 [s, 2H, 2NH]. ¹³CNMR: 18.0 (2), 28.6, 40.3, 56.5, 112.2 (2), 116.3, 118.0, 120.9 (2), 122.2, 125.8 (2), 127.1 (2), 127.4 (2), 130.7 (2), 130.9, 138.0, 139.1 (2), 143.2 (2), 145.9 (2), 153.7, 161.0, 166.9 (2), 172.0. MS m/z (%): 747 (M⁺) (16.66), 384 (100). Anal. Calcd. for C₃₃H₂₉N₇O₁₀S₂ (747.75): C, 53.01; H, 3.91; N, 13.11. Found: C, 53.33; H, 3.62; N, 13.43.

• 2-(4-(8-Methoxy-2-((2-((2-methyl-6-nitrophenyl)amino)-2-oxoethyl)thio)-4-oxoquinazolin-3(4H)-yl)phenylsulfonamido)-N-(2-methyl-6-nitrophenyl)acetamide (4m).

4m: Yield, 85%; m.p. 272–274 °C. IR: 3340, 3212 (NH), 3055 (arom.), 2954, 2865 (aliph.), 1695, 1684, 1661 (3CO), 1618 (CN), 1377, 1151 (SO₂). ¹HNMR: 2.2 (s, 6H, 2CH₃), 3.8 [s, 3H, OCH₃], 3.9 [s, 2H, SCH₂], 4.1 [s, 2H, NCH₂], 7.4–7.5 (m, 9H, Ar-H), 7.6, 8.0 (2d, 4H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 10.2 [s, 2H, 2NH]. ¹³CNMR: 18.0 (2), 31.6, 40.4, 56.7, 115.3, 118.1, 120.9, 122.6 (2), 123.0 (2), 126.7 (2), 127.1 (2), 127.4, 127.5 (2), 128.8 (2), 130.3, 135.4 (2), 137.7, 137.9 (2), 139.0, 153.7, 161.0, 162.5, 166.4 (2). MS m/z (%): 747 (M⁺) (28.14), 341 (100). Anal. Calcd. for C₃₃H₂₉N₇O₁₀S₂ (747.75): C, 53.01; H, 3.91; N, 13.11. Found: C, 52.76; H, 4.22; N, 13.35.

• N-(2,4-dinitrophenyl)-2-((3-(4-N-(2((2,4-dinitrophenyl)amino)-2-oxoethyl)sulfamoyl)phen-yl-8-methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (4n).

4n: Yield, 76%; m.p. 248–250 °C. IR: 3342, 3200 (NH), 3093 (arom.), 2984, 2876 (aliph.), 1696, 1681, 1669 (3CO), 1608 (CN), 1398, 1166 (SO₂). ¹HNMR: 3.8 [s, 3H, OCH₃], 3.9 [s, 2H, SCH₂], 4.2 [s, 2H, NCH₂], 7.1–7.6 (m, 9H, Ar-H), 7.7, 7.9 (2d, 4H, AB system, Ar-H), 8.1 (s, 1H, SO₂NH], 8.8 [s, 2H, 2NH]. ¹³CNMR: 28.6, 40.4, 57.3, 115.1, 120.2, 123.9 (2), 124.0, 125.6 (2), 126.9 (2), 129.1, 129.8 (2), 130.6 (2), 135.5, 136.7, 138.0 (2), 138.7, 138.9, 143.1 (2), 147.3, 150.3, 156.4, 161.0, 163.5, 164.6. MS m/z (%): 809 (M⁺) (16.42), 294 (100). Anal. Calcd. for C₃₁H₂₃N₉O₁₄S₂ (809.70): C, 45.98; H, 2.86; N, 15.57. Found: C, 45.64; H, 2.56; N, 15.25.

3.3. Biological Evaluations

3.3.1. MTT Cell Viability Assay

Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as stated previously [40]. In brief, A549 (lung), HepG2 (liver), LoVo (colon), MCF-7 (breast) cancer cells as well as normal cells (HUVEC) were seeded in 96-well plates (10⁵ per well) and compounds were added in a different final concentration. All cells were treated for 48 h, and then 10μL of MTT reagent (5 mg/mL) was added to each well and further incubated for 4 h. Thereafter, 0.1 mL of acidified isopropanol was added to solubilize formazan product and the developed color was measured at 570 nm in Microplate ELISA Reader (BioTek, Elx-800, Winooski, VT, USA). The IC₅₀ values (concentration required for 50% inhibition of cell viability) were calculated using dose response curve. Cell viability was calculated as = (Ab Treated/Ab Control) × 100.

The A549, HepG2, LoVo and MCF-7 cancer cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany). For Human umbilical vein endothelial cells (HUVEC cells), it was purchased from American Type Culture Collection (16549; Cat no. PCS-100-010; ATTCC, Manassas, VA, USA).

3.3.2. Cell Cycle Analysis

Cell cycle distribution was evaluated by PI staining as previously described [41]. Briefly, MCF-7 cells were seeded and incubated for a period of 24 h to allow for attachment. Subsequently, cells were exposed to different concentrations of compounds 4d and 4f. After 24 h, cells were harvested and fixed in 70% ethanol. Thereafter, cells were washed with PBS, resuspended in 1 mL of PBS containing PI (50 μg/mL), RNase A (0.5 mg/mL), sodium citrate (0.1%) and Triton X-100 (0.1%), then incubated for 15 min in the dark at room temperature. Flow cytometry was carried out by a FACS Scan Flow Cytometer (Cytomics FC 500; Beckman Coulter, Brea, CA, USA). The data were analyzed using CXP software V. 3.0.

3.3.3. Annexin V-FITC/PI Apoptosis Detection

Apoptotic cells were detected using Annexin V-FITC/PI staining protocol (BioLegend, San Diego, CA, USA) according to the manufacturer’s recommendations. Briefly, MCF-7 cells were incubated for 24 h with the different doses of promising compounds 4d and 4f. The cells were then harvested, washed twice with cold PBS and re-suspended with 1× Annexin V binding buffer (100 µL). Thereafter, cells were transferred into flow cytometry tube and stained with 5 μL of Annexin V- FITC and 5 μL PI for 15 min at room temperature in the dark. The cells were processed for apoptosis using a FACS Scan Flow Cytometer (Cytomics FC 500; Beckman Coulter, Brea, CA, USA) and analyzed using CXP software V. 3.0.

3.3.4. Analysis of Apoptotic Genes by RT-PCR

The MCF-7 cells (0.1 × 10⁶) in 12 well plates were treated with different concentrations of compounds 4d and 4f for 24 h and untreated cells were used as a control. After 24 h of treatment, cell culture media was removed and the cells were lysed with Trizol (Invitrogen, Waltham, MA, USA), and total RNA was extracted from all the samples [42]. The RNA was estimated by Nano-drop and 1 µg of RNA was used in the cDNA synthesis. The cDNA was synthesized from RNA by using a cDNA synthesis kit (Invitrogen, Thermo Fisher Scientific, lnc., Waltham, MA, USA) according to the manufacturer’s instruction. After Reverse transcription at 42 °C for 60 min, RT-PCR was performed using 5× Firepol Master Mix ready to load (Solis BioDyne) according to manufacturer’s instruction. RT-PCR was used to analyze the gene expression level of P53, Bcl2, Bax and caspase 7 using specific sets of primers for each gene [22], while β-actin was utilized as a housekeeping gene. The program was run as follows, initial denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s; annealing at 55 °C for 45 s; elongation at 72 °C for 45 s (30 Cycles) and final extension at 72 °C for 10 min. The RT-PCR products were electrophoresed on a 1.2% agarose gel containing ethidium bromide and the gel was imaged on a Licor machine.

3.3.5. Western Blotting to See the Effect of 4d and 4f Compounds

MCF-7 cells were plated in six well plates at the density of 0.5 × 10⁶ and kept for adherence for overnight. On the next day, complete media was replaced with the fresh one and treated the cells with compounds 4d and 4f for 24 h at various concentrations. After 24 h of treatment, cells were harvested and washed twice with 1× PBS before adding the lysis buffer (20 mM Tris pH 7.5, 150 mM Nacl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X100, 1 μg/mL Leupeptin and 100 µM phenylmethylsulfonyl fluoride—PMSF) and kept it for 1 h on ice, with intermittent vortex mixing every 10 min. The samples were centrifuged at 13,000 rpm at 4 °C for 15 min and then the supernatants were transferred into new tubes. Total protein was measured by using Bio-Rad Bradford kit and then 35 µg of the total protein was run on a 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA in TBST (150 mM Nacl, 20 mM Tris base and 0.1% Tween 20) for 2 h at room temperature and then washed three times with TBST buffer. Then the membrane was incubated with the desired antibodies (p53, Bcl-2, Bax, caspase7 and β-actin) (1:200, Santa Cruz Biotecnology, Inc., Dallas, TX, USA) and kept on a rocker at 4 °C overnight. On the next day, the membrane was washed three times with TBST buffer and then incubated with horseradish peroxidase-conjugated secondary antibody (1:3000) for 1 h at room temperature. After incubation, membranes were washed three times with TBST buffer and then were ready for immunodetection. For immunodetection, membranes were incubated with ECL Western blotting detecting reagents (Amersaham Pharmacia Biotech Inc., Zurich, Switzerland) and then bands were obtained by exposing to X-ray films (Amersham).

3.3.6. Molecular Docking

Molecular docking between potential compounds 4d and 4f with the target protein Bcl-2 was performed as reported earlier [43]. The X-ray crystal structure Bcl-2 bound with DRO (2.10 Å resolution) was used to retrieve 3D coordinates of the target protein [44]. The protein was processed to remove non-essential water molecules and other heterogeneous atoms. Hydrogen atoms were added, bond orders were defined, and rotatable bonds were assigned, as reported by [45]. Prior to molecular docking, hydrogen bonds were redefined, and the energy of the protein was minimized using MMFF (Merck Molecular Force Field) force field. The 2D structures of compounds 4d and 4f were drawn using ChemDraw, and their energies were minimized using Universal Forcefield (UFF). Molecular docking between Bcl-2 and ligands (4d and 4f) along with the control ligand (DRO) was performed inside a grid box of 27 × 30 × 25 Å, placed at 39 × 28 × −12 Å, keeping 0.375 Å spacing between the grid points, using Autodock 4.2 (Scripps Research, San Diego, CA, USA), as reported by Rabbani and coworkers [46]. Lamarckian Genetic Algorithm (LGA) and Solis and Wets local search methods were used to perform molecular docking. Initial torsions, positions, and orientations of ligands were set arbitrarily. A maximum of 2,500,000 calculations was for each docking run. The population size, translational step, quaternion and torsion were set to 150, 0.2 Å, 5 and 5, respectively. The docked protein-ligand complex with the lowest binding energy was selected for molecular interaction analysis in Discovery Studio (BIOVIA, San Diego, CA, USA). The binding affinity (K_d) and binding energy (ΔG) of compounds 4d and 4f towards Bcl-2 were evaluated using the following relation [47]

$$∆G=-RTln{K}_d$$

where, R and T are the universal gas constant (1.987 kcal mol⁻¹ K⁻¹) and temperature (298 K), respectively.

3.3.7. Statistical Analysis

OriginPro 8.5 software was used to conduct the statistical analysis. The mean and standard deviation of the data were displayed. Student’s t-test was used to examine differences, and p < 0.05 was regarded statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

4. Conclusions

Synthesizing a novel quinazoline incorporating substituted sulfonamide moieties in order to obtain anticancer activity with lower toxicity has attracted attention on the beneficial effect of this group to overcome one of the most serious problems in cancer chemotherapy, which is the lower efficacy and higher toxicity. Therefore, we aimed in this study to synthesize a novel series of quinazolinone derivatives carrying biologically active substituted-sulfonamide moieties at 3-position to achieve new compounds with the anticancer effect. Herein, a novel unexpected series of quinazoline incorporating substituted sulfonamide moieties was synthesized and evaluated in vitro for their anticancer activity. Some compounds were found to exhibit high cytotoxicity in the MCF-7 breast cancer cell line. Compounds 4d and 4f were appeared as the most effective derivatives against MCF-7 cells. Flow cytometry data showed that compounds 4d and 4f successfully triggered apoptosis in MCF-7 cells. Furthermore, changes in the expression of apoptosis-related markers at the gene and protein level were also indicative of apoptotic activity. Molecular docking of compounds 4d and 4f also suggested that they possess good binding affinity for Bcl-2. Overall, screening of the tested compounds could offer an encouraging framework in this field, which may lead to develop more potent, selective, clinically useful analogues anticancer agents. Furthermore, the existing compounds are currently under investigations for their in vivo anti-cancer activity in different transplantable mouse tumors model. A series of toxicity studies are also underway to determine the safety of these existing compounds.