Morpholine substituted quinazoline derivatives as anticancer agents against MCF-7, A549 and SHSY-5Y cancer cell lines and mechanistic studies

Abstract

A series of morpholine substituted quinazoline derivatives have been synthesized and evaluated for cytotoxic potential against A549, MCF-7 and SHSY-5Y cancer cell lines. These compounds were found to be non-toxic against HEK293 cells at 25 μM and hence display anticancer potential. In these series compounds, AK-3 and AK-10 displayed significant cytotoxic activity against all the three cell lines. AK-3 displayed IC₅₀ values of 10.38 ± 0.27 μM, 6.44 ± 0.29 μM and 9.54 ± 0.15 μM against A549, MCF-7 and SHSY-5Y cancer cell lines. Similarly, AK-10 showed IC₅₀ values of 8.55 ± 0.67 μM, 3.15 ± 0.23 μM and 3.36 ± 0.29 μM against A549, MCF-7 and SHSY-5Y, respectively. In the mechanistic studies, it was found that AK-3 and AK-10 inhibit the cell proliferation in the G1 phase of the cell cycle and the primary cause of death of the cells was found to be through apoptosis. Thus, morpholine based quinazoline derivatives have the potential to be developed as potent anticancer drug molecules.

A series of morpholine substituted quinazoline derivatives have been synthesized and evaluated for cytotoxic potential against A549, MCF-7 and SHSY-5Y cancer cell lines.

1. Introduction

Cancer is one of the most serious health problems and leading causes of death worldwide. Human health is critically compromised by malignant tumors, and drug resistance is one of the major causes of relapse of the disease.^1,2 Chemotherapy remains the central treatment mode for different types of cancers. However, cancer cells develop resistance against chemotherapeutic agents and become nonresponsive to them.³ The multidrug resistance of cancerous cells necessitates exploration of alternative and/or complementary treatment modalities.^4,5 In recent years, the rate of recovery from malignancy has marginally improved with the emergence of novel antitumor drugs.⁶

One of the strategies in clinical oncology is the development of drug candidates that promote effective elimination of tumor cells through apoptosis.⁷ Apoptosis is a programmed cell death which plays a crucial role in controlling the proliferation of tumor cells.⁸ Apoptosis proceeds through extrinsic and intrinsic pathways, and both the pathways involve activation of caspases (cysteine aspartate protease enzymes).^9,10 The main caspases involved in apoptosis are caspase-8 and caspase-9 in the extrinsic and intrinsic pathways, respectively, and both of these congregate to caspase-3, which is also known as an executioner caspase and its activation results in irreversible cell apoptosis.¹¹ The intrinsic pathway is mainly regulated by the Bcl-2 family of proteins, which consists of both types of proteins, one that induces apoptosis such as Bax and another that prevents apoptosis such as Bcl-2.¹² Thus, the Bcl-2 proteins act as a barrier to apoptosis and inhibitors of these proteins may activate a cascade of effects that ultimately leads to apoptosis.¹³ In the last few years, a number of nonpeptidic and cell permeable small organic compounds have been developed as potent Bcl-2 inhibitors.^14,15 A number of structurally different scaffolds such as navitoclax, venetoclax, GX15-070, HA14-1, AT101, etc. (Fig. 1) have been identified as potent Bcl-2 inhibitors.¹⁶

Fig. 1. Molecules reported as potent Bcl-2 inhibitors.

Quinazoline is a versatile heterocyclic scaffold that shows a variety of pharmaceutical applications including anticancer activity.^17–19 A comprehensive review of the literature from PubMed, as depicted in Fig. 2, suggests that from 1986 to 2021, a large number of research publications were reported on the anticancer potential of quinazoline derivatives.

Fig. 2. Year-wise research publications on quinazoline based anticancer agents (source: PubMed).

A number of quinazoline based anticancer drugs such as erlotinib, gefitinib, vandetanib, etc. have been approved by the US FDA (Fig. 3).^20,21 These quinazoline based compounds target various proteins such as kinases, tubulins, Bcl-XL, BAX, Bcl-2 and many more.²² Thus, a quinazoline nucleus is considered as an important scaffold for the development of novel anticancer agents. Elena Cubedo et al. synthesized symmetrical quinazoline derivatives and evaluated their cytotoxic activity against T24, HT-29 and MDA-MB-231 cancer cell lines. Time dependent apoptosis studies were performed for the potent compounds obtained in the series, and it was found that compound I (Fig. 4) induces apoptosis in all the cancer cell lines.²³ Mohamed Fares et al. synthesized isatin and quinazoline based hybrid compounds and assessed their cytotoxic potential against different cancer cell lines, and compound II (Fig. 4) was tested for the expression of proapoptotic and antiapoptotic proteins and it induces apoptosis by modulating the expression of these proteins.²⁴ Similarly, Jantova et al. reported quinazoline based compounds and compound III (Fig. 4), which displayed significant cytotoxic activity against L1210 and NIH-3T3 cells and induces apoptosis.²⁵

Fig. 3. Quinazoline based drug molecules in clinical practice for the treatment of cancer.

Fig. 4. Quinazoline based cytotoxic and apoptosis inducing agents.

A morpholine nucleus²⁶ is incorporated as an important pharmacophore^27–29 in various ligands being explored for anticancer, antiviral, anti-inflammatory, and antitubercular drugs. In comparison to other six membered nitrogen containing heterocycles, a morpholine ring imparts better pharmacokinetics and a pharmacodynamic profile to the compounds.²⁶ Various modified natural products^27–29 and synthetic chemical scaffolds^30,31 with a morpholine ring such as gefitinib (Fig. 3) displayed potent cytotoxic activities. Thus, in the current studies, a morpholine ring is incorporated as a pharmacophore in all the target molecules. Taking leads from the literature studies and in continuation of our work on anticancer drug development,³² in this research article we have designed, synthesized and evaluated various morpholine substituted quinazoline derivatives against MCF-7, A549 and SHSY-5Y cancer cell lines. Most of the compounds displayed significant anticancer activities against the three cancer cell lines. In this series, compounds AK-3 and AK-10 were found to be effective against all the three cancer cell lines. In the cell cycle and apoptosis studies, AK-3 and AK-10 showed cell cycle arrest values of 59% and 62%, respectively, at the G0/G1 phase and apoptosis was found to be the primary cause of cell death.

2. Results and discussion

2.1. Chemistry

Target compounds were synthesized as described in Scheme 1. Differently substituted quinazolinones (3) were synthesized by reacting different aromatic aldehydes with anthranilamide in the presence of iodine. These synthesized quinazolinones were chlorinated, using thionyl chloride that leads to formation of intermediate 4. The target compounds (5) were synthesized by an SNAr type of reaction in which chlorine was substituted with morpholine. All the synthesized compounds were characterized by ¹H NMR, ¹³C NMR, ESI-MS and HRMS. In the first step, formation of quinazolinones is confirmed through TLC and the presence of aromatic peaks in the ¹H and ¹³C NMR spectra. The second intermediate formed through chlorination and aromatization of the ring is confirmed with the help of TLC and EIMS. The third step involves the incorporation of the morpholine ring in the final product. All the morpholine containing compounds showed two broader triplet peaks for –NCH₂ and –OCH₂ and these peaks don't appear as a sharp triplet due to the interconversion of axial and equatorial hydrogens of the chair and boat conformations of morpholine. These interconversions take place at a very high speed at room temperature and result in slightly broader peaks for the –NCH₂ and –OCH₂ protons of the morpholine ring. All the final compounds were characterized by the characteristic broad peaks for –NCH₂ and –OCH₂ of the morpholine moiety located at 3.85 to 3.90 ppm and 3.98 to 4.11 ppm, respectively. In the ¹³C NMR spectra, in addition to aromatic peaks, two peaks were observed for the morpholine ring at 50.6 ppm (NCH₂) and 66.8 ppm (OCH₂).

Scheme 1. Synthesis of morpholine substituted quinazoline based apoptosis inducers.

2.2. Biological evaluation of compounds and discussion

2.2.1. Synthesized compounds displaying significant cytotoxicity and selectivity to cancer cell lines

All the synthesized compounds (AK-1 to AK-13) were investigated for their in vitro cytotoxicity against human MCF-7 (breast cancer), A549 (lung cancer) and SHSY-5Y (neuroblastoma) cancer cell lines using the standard protocol for MTT assays as reported by us²⁸ (Table 1) where colchicine was used as a positive control. Four different concentrations (1 μM, 5 μM, 10 μM and 25 μM in triplicate) of the compounds were used and the results were analyzed after 48 h of drug treatment. The IC₅₀ values for all the compounds are reported in micromolar concentration.

In vitro cytotoxic activity (IC₅₀ in μM) of compounds AK-1 to AK-13 against A549, MCF-7 and SHSY-5Y cancer cell lines.

Compound	R	A549	MCF-7	SHSY-5Y	HEK293
AK-1		9.47 ± 0.36	>25	>25	NT
AK-2		17.74 ± 0.17	10.13 ± 0.49	24.3 ± 0.63	NT
AK-3		10.38 ± 0.27	6.44 ± 0.29	9.54 ± 0.15	>25
AK-4		11.52 ± 0.18	>25	14.51 ± 0.19	NT
AK-5		12.48 ± 0.41	5.57 ± 0.39	20.65 ± 0.49	>25
AK-6		11.07 ± 0.47	>25	>25	NT
AK-7		9.5 ± 0.63	10.51 ± 0.38	15.22 ± 0.27	NT
AK-8		20.84 ± 0.28	5.01 ± 0.39	>25	>25
AK-9		10.81 ± 0.46	>25	>25	NT
AK-10		8.55 ± 0.17	3.15 ± 0.23	3.36 ± 0.29	>25
AK-11		17.84 ± 0.31	>25	22.01 ± 0.28	NT
AK-12		18.94 ± 0.17	4.93 ± 0.65	18.02 ± 0.37	NT
AK-13		9.83 ± 0.53	6.69 ± 0.26	15.81 ± 0.43	>25
COL		5.13 ± 0.57	5.19 ± 0.57	7.89 ± 0.23	>25

From the data reported in Table 1, it is obvious that most of the compounds showed good cytotoxic activity against all the cancer cells (A549, MCF-7 and SHSY-5Y) and some of the compounds showed better anticancer activity when compared with colchicine. All the compounds in the series were found to be non-toxic against the HEK293 cell line thereby displaying selectivity for anticancer potential. In particular, compounds AK-3 and AK-10 were found to be the most active compounds in the series. In general, all the compounds were found to be effective against A549 with IC₅₀ values ranging from 8.55 to 20.84 μM. Some of the compounds such as AK-8, AK-10 and AK-12 displayed better activity against MCF-7 cells as compared to colchicine. In this series, compound AK-10 was found to be the most potent against all the three cancer cell lines with IC₅₀ values of 8.55 μM, 3.15 μM, and 3.36 μM against A549, MCF-7 and SHSY-5Y, respectively. AK-10 exhibited more than 2-fold potency against MCF-7 and SHSY-5Y as compared to the positive control colchicine. In general, it was observed that compounds with para substitution on the phenyl ring were more active as compared to unsubstituted and ortho substituted derivatives.

2.2.2. Cell cycle and apoptosis studies of the lead compounds

Amongst the synthesized compounds, AK-3 and AK-10 displayed the most promising anticancer activity in all the three cancer cell lines. So, these compounds were selected for cell cycle analysis and apoptosis study to understand their mechanism of action. The cell cycle and apoptosis studies were performed on SHSY-5Y neuroblastoma cells. The cells were grown in a 6-well treated corning plate, and treated with 10.0 μM concentration of the compounds. It was observed that AK-3 and AK-10 showed cell cycle arrest values of 59%, and 62%, respectively, at the G0/G1 phase. Non-treated cells showed a cell arrest value of 53.0% at the G1 phase, 16% at the S phase, and 31% at the G2 phase of the cell cycle. (Fig. 5). Thus, it was concluded that AK-3 and AK-10 might be acting on some proteins such as Bcl-2 or other antiapoptotic proteins that arrest the cell cycle at the G1 phase.

Fig. 5. Cell cycle assay with propidium iodide at 10.0 μM concentration of the test compounds AK-3 and AK-10 treated for 48 h.

In addition, apoptotic study was also performed to understand the mechanism of the cell death induced by AK-3 and AK-10. A cytofluorimetric analysis method was used on the SHSY-5Y cells with a Muse™ Annexin V and Dead Cell kit to detect the phosphatidylserine membrane translocation. This is considered as a major hallmark of the late-stage apoptosis. The SHSY-5Y cells were treated with AK-3 and AK-10 as test compounds at 10 μM concentrations. It was observed that AK-3 and AK-10 showed apoptotic cell death values of 78.2% and 77.6%, respectively, as compared to the 1.16% apoptosis of the non-treated cells after 48 h treatment (Fig. 6). Hence, it was concluded that the primary mode of cell death by the test compounds is through apoptosis.

Fig. 6. Quantitative analysis of the apoptotic cells after treatment with AK-3 and AK-10 using AnnexinV FITC/PI double staining and flow-cytometry calculations.

2.3. Docking studies

In order to investigate the interaction of the active compounds in the binding pockets of the Bcl-2 protein, AK-3 and AK-10 were docked on Bcl-2, Bcl-xL, CDK-4 and BAX; however, these compounds showed a significant interaction with the Bcl-2 protein (PDB ID: 4LVT) only. The Bcl-2 protein consists of a hydrophobic alpha helix surrounded by six or seven amphipathic α helices.³⁴ The hydrophobic groove is made up of conserved Bcl-2 homology domain 1 (BH1) and domain 2 (BH2) and a cloistered BH3 domain. Most of the anti-apoptotic small molecules bind to one or more sub-pockets (P1–P4) of this domain (BH3). These compounds are also known as BH3 mimetics which activate proapoptotic BAX and BAK proteins that lead to apoptosis.^35,36 The interactions between the ligands and receptor sites were scored using Glide (GLIDE 12.5 module of Schrödinger Suite). In this series, AK-3 and AK-10 were found to be the most potent compounds and in the cell cycle and apoptosis studies, these two compounds showed G1 phase arrest and cell death due to apoptosis. Hence, these two compounds were selected for the docking studies with the Bcl-2 protein and compared with HA14-1, a standard antagonist against antiapoptotic Bcl-2 proteins. In the docking studies, both the compounds and standard fit well in the active cavity of the protein (Fig. 7). The chromene ring and ester chain of HA14-1 were surrounded by amino acid residues Ala97, Asp100, Phe101, Arg104, and Tyr105 and Asn140, Trp141, Gly142, Arg143, Val145, and Ala146. Also, the carbonyl group directly attached to the chromene ring showed a hydrophilic interaction with Tyr105 bridged via a water molecule. Compound AK-3 displayed pi–pi stacking between the pyrimidine ring and phenyl ring of Tyr199 whereas the quinazoline ring was surrounded by the amino acid residues Tyr105, Arg104, Asp100 and Tyr199. Similarly, in AK-10, the quinazoline and morpholine rings were surrounded by amino acid linings Ala97, Asp100, Phe101, Arg104, and Tyr105 and Asn140, Trp141, Gly142, Arg143, Val145, and Ala146. The methoxy group of AK-10 showed a similar hydrophilic interaction as shown by the standard inhibitor with Asn140 bridged via a water molecule. Thus, both AK-3 and AK-10 bind with the same amino acid residues of the active site as done by standard inhibitor HA14-1 (Fig. 7).

Fig. 7. Docking poses of AK-3 (a), AK-10 (b) and HA14-1 (c) in the Bcl-2 binding site. AK-3, AK-10 and HA14-1 showed dock scores of −4.409 kcal mol⁻¹, −5.299 kcal mol⁻¹ and −5.470, respectively.

2.4. ADME studies

Schrödinger's QikProp module was used for ADME studies of the synthesized compounds. The QikProp module utilizes different parameters of Lipinski's rule of five for the analysis of drug like properties. Lipophilicity of compounds is used to determine whether the molecule will cross the biological membrane or not, and for that log P (<5) is an important physicochemical property. For standard HA14-1, it is 2.87 and AK-3 and AK-10 displayed lipophilicity within the range of 4.14 to 4.17. These two compounds displayed log P values of less than 5 which is in the limit for druggable compounds indicating that the molecules are lipophilic in nature. Solubility of a drug candidate in a biological system is also an important parameter that must be considered and log S values for HA14-1, AK-3 and AK-10 were found to be −4.93, −4.89, and −4.81, respectively. This indicates that both the compounds display optimum aqueous solubility and there should be no issue of bioavailability during in vivo studies. Low values of the docking score indicate that these molecules are binding strongly in the active site of the protein. The molecular weights of these compounds were less than 500 and the number of hydrogen bond donors and hydrogen bond acceptors also lies within the acceptable range (Table 2). Both the compounds showed 100% human oral absorption as compared to HA14-1 and compounds AK-3 and AK-10 undergo a smaller number of metabolic reactions. Thus, both AK-3 and AK-10 showed drug like characteristics and may be considered for next stage evaluation.

Drug like characteristics of compounds AK-3 and AK-10 as determined by the QikProp application of Schrödinger.

Name	Mol. wt.	Log P	Log S	HB donor	HB acceptor	% human oral absorption	Dipole	#metb
AK-3	334.42	4.17	−4.89	0	5	100	3.648	3
AK-10	430.45	4.14	−4.87	0	5	100	6.165	4
HA14-1	409.23	2.87	−4.93	1	5	89	11.25	3

2.5. SAR studies

In the current study, a series of morpholine based quinazoline based compounds were synthesized and evaluated for cytotoxic activities against A549, MCF-7 and SHSY-5Y cancer cell lines. Most of the compounds displayed cytotoxic activities against these cancer cell lines in a low to submicromolar range. The target compounds composed of three rings which include a quinazoline ring (ring A), a morpholine ring (ring B), and a differently substituted aromatic ring (ring C). Aromatic ring C is optionally substituted with electron withdrawing and electron releasing groups to analyze their impact on the cytotoxic activity. AK-1 with an unsubstituted phenyl ring was found to be effective against A549 cells but showed less potency against MCF-7 and SHSY-5Y cells. A thiomethyl substitution at the para position of the phenyl ring increased the potency against the two cell lines (MCF-7 and SHSY-5Y) while activity against A549 cells decreased by half. AK-3 with N,N-dimethyl amine substitution was found to be effective against all the three cell lines with IC₅₀ values of 10.38 ± 0.27 μM, 6.44 ± 0.29 μM and 9.54 ± 0.15 μM against A549, MCF-7 and SHSY-5Y cancer cell lines, respectively. An indole moiety (AK-4) as aromatic ring C displayed moderate activity while hydroxy (AK-5) and isopropyl (AK-7) substituents at the para position of the phenyl ring were found to be favorable for the cytotoxic activity. However, para-cyno (AK-6) and ortho-fluoro (AK-9) substituted phenyl rings were found to be active against A549 cells and did not show any activity against the other two cell lines. In this series, AK-10 with a 3,4,5-trimethoxy substituent was found to be the most effective against all the three cancer cell lines with IC₅₀ values of 8.55 ± 0.67 μM, 3.15 ± 0.23 μM and 3.36 ± 0.29 μM against A549, MCF-7 and SHSY-5Y, respectively. It showed similar or better activity when compared with the standard inhibitor colchicine. AK-11 with a 2-chloro-6-fluoro substituent displayed moderate activity while AK-12 (with a para-methyl substituent) and AK-13 (with a para-trifluoromethyl substituent) were also found to be effective against all the three cell lines. The substitution pattern in compounds like AK-3, AK-7, AK-10, AK-12 and AK-13 indicates that a bulkier substituent at the para position increases the cytotoxic activity. In general, it has been observed that the compounds with electron donating substituents showed higher cytotoxic activity. It was interesting to observe that most of the compounds were found to be more potent against the glioblastoma cell line (SHSY-5Y) as compared to the other cell lines. Thus, it was concluded that some of the promising compounds from the current series especially AK-3 and AK-10 can act as leads for further developments.

3. Conclusion

Thirteen morpholine substituted quinazoline based compounds were synthesized and evaluated for cytotoxic activities against A549, MCF-7 and SHSY-5Y cancer cell lines. These compounds were found to be non-toxic against HEK293 cells at 25 μM and hence display anticancer potential. In this series, some of the compounds displayed promising cytotoxic activity even better than standard colchicine. AK-3 and AK-10 were found to be the most active compounds in this series and hence various mechanistic studies were performed on these compounds. It was found that compounds AK-3 and AK-10 inhibit the cell proliferation in the G1 phase of the cell cycle and the primary cause of death of the cells was found to be through apoptosis. From the mechanistic studies, it was concluded that these compounds might be binding to the Bcl-2 proteins and induce apoptosis. In the docking studies, it was found that AK-3 and AK-10 fit well in the active cavity of Bcl-2 and showed an interaction with the surrounding amino acids. It was concluded that the current series of morpholine substituted quinazoline derivatives have the potential to be developed as anticancer ligands and AK-3 and AK-10 were identified as potent leads suitable for further studies.

4. Experimental

4.1. General

The chemicals, reagents and solvents were procured from different sources like Sigma-Aldrich, S.D. Fine Chemicals, Spectrochem, Sisco Research Laboratory and Avra Synthesis Ltd. and were used without any purification. The reactions were monitored using thin layer chromatography in a solvent mixture of pet. ether/ethyl acetate and chloroform/methanol on glass TLC plates prepared using F254 UV grade silica and a UV/fluorescence analysis cabinet and/or iodine chamber. Melting points of the intermediate and final products were determined using an open glass capillary tube in Stuart melting point apparatus (SMP-30). ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of the compounds were recorded in CDCl₃/d₆-DMSO on a Bruker Avance II (400 MHz) NMR spectrometer using TMS (δ = 0) as the internal standard at IIT Ropar. Mass spectra were recorded on a Shimazdu GC-MS (ESI), at the Central University of Punjab, Bathinda, Punjab, India.

4.2. General procedure for the synthesis of 3

In methanol (10 ml), substituted aldehyde (1 eq.), anthranilamide (1 eq.) and iodine (1 eq.) were added as an oxidative catalyst. The reaction mixture was refluxed for 4 h. The completion of the reaction was monitored via thin layer chromatography (TLC). After the completion of the reaction, the excess solvent was evaporated from the mixture under vacuum using a rotary evaporator. Chilled water was poured in the reaction mixture and the precipitates obtained were filtered and dried.

4.3. General procedure for the synthesis of 4

In a 100 ml round bottom flask, compound 3 (differently substituted quinazolinones) was dissolved in dichloromethane, then thionyl chloride (1.2 eq.) was added dropwise under an ice cold condition and a catalytic amount of DMF was added. The reaction mixture was refluxed for 5 to 6 h at 40 °C. The progress of the reaction was monitored via TLC and on the completion of the reaction, ice was added into the reaction mixture, neutralized by sodium bicarbonate, extracted with chloroform (25 ml × 3), and washed with brine, dried over anhydrous sodium sulphate. The obtained organic layer was then concentrated under vacuum using a rotary evaporator.

4.4. General procedure for the synthesis of AK-1 to AK-13

In a 50 ml reaction vial, the compounds were dissolved in DMF, and K₂CO₃ (2.5 eq.) and morpholine (1.5 eq.) were added and the reaction mixture was heated at 80 °C for 4 h. The progress of the reaction was monitored with TLC. After the completion of the reaction, water (5 ml) was poured and the mixture was extracted with ethyl acetate (15 ml × 3), washed with brine, and dried over anhydrous sodium sulphate, and the obtained organic layer was then concentrated under vacuum using a rotatory evaporator. The crude product was further purified with column chromatography to afford a pure product. Final product formation was confirmed by EIMS, NMR and HRMS.

AK-1 (4-(2-phenylquinazolin-4-yl)morpholine)

Yield: 71%, white in colour, m.p.: 168–170 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.59 (2H, d, J = 8 Hz), 8.03 (1H, d, J = 8 Hz), 7.93 (1H, d, J = 8 Hz), 7.78 (1H, t, J = 8 Hz), 7.47 (4H, m,), 3.98 (4H, t, J = 4 Hz), 3.89 (4H, t, J = 4 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.96, 159.47, 152.84, 138.49, 132.54, 130.27, 129.18, 128.41, 128.37, 125.09, 124.63, 115.36, 66.83, 50.41. HRMS: m/z [M + H]⁺ for C₁₈H₁₇N₃O, calculated 292.1458; observed: 292.1450.

AK-2 (4-(2-(4-(methylthio)phenyl)quinazolin-4-yl)morpholine)

Yield: 60%, pale white in colour, m.p.: 171–173 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.5 (2H, d, J = 8 Hz), 8.0 (1H, d, J = 8 Hz), 7.8 (1H, d, J = 8 Hz), 7.75 (1H, t, J = 8 Hz), 7.4 (1H, t, J = 8 Hz), 7.35 (2H, d, J = 8 Hz), 4.03 (4H, t, J = 4 Hz), 3.8 (4H, t), 3.94 (3H, t, J = 8 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.91, 159.06, 152.83, 141.39, 135.17, 132.55, 129.04, 128.76, 125.72, 124.96, 124.65, 115.31, 66.82, 50.39, 15.40. HRMS: m/z [M + H]⁺ for C₁₉H₁₉N₃OS, calculated 338.1327; observed: 338.1333.

AK-3 (N,N-dimethyl-4-(4-morpholinoquinazolin-2-yl)aniline)

Yield: 67%, off white in colour, m.p.: 169–171 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.25 (1H, d, J = 4 Hz), 8.04 (2H, d, J = 4 Hz), 7.72 (2H, d, J = 4 Hz), 7.31 (1H, t, J = 4 Hz), 6.74 (2H, m,), 7.51 (1H, t, J = 16 Hz), 3.99 (4H, t, J = 4 Hz), 3.91 (4H, t, J = 4 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 186.54, 163.48, 152.55, 151.71, 150.05, 134.67, 128.35, 127.47, 126.36, 125.70, 120.32, 119.19, 111.70, 66.80, 50.39, 40.14, 33.35, 29.31, HRMS: m/z [M + H]⁺ for C₂₀H₂₂N₄O, calculated 335.4310; observed: 335.4338.

AK-4 (4-(2-(1H-indol-2-yl)quinazolin-4-yl)morpholine)

Yield: 62%, brown in colour, m.p.: 174–176 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.8 (1H, s), 8.7 (1H, d, J = 4 Hz), 8.25 (1H, d, J = 4 Hz), 7.9 (1H, d, J = 4 Hz), 7.75 (1H, d, J = 4 Hz), 7.65 (1H, t, J = 8 Hz), 7.35 (1H, d, J = 4 Hz), 7.3 (1H, t, J = 8 Hz), 7.23–7.15 (2H, m): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 186.56, 164.92, 159.07, 136.98, 132.63, 128.84, 127.94, 127.65, 126.23, 124.71, 124.15, 122.64, 122.57, 121.23, 114.64, 111.40, 66.88, 50.62. HRMS: m/z [M + H]⁺ for C₂₀H₈N₄O, calculated 331.1559; observed: 331.1566.

AK-5 (4-(4-morpholinoquinazolin-2-yl)phenol)

Yield: 65%, white in colour, m.p.: 175–177 °C ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.5 (2H, d, J = 8 Hz), 7.98 (1H, d, J = 8 Hz), 7.91 (1H, d, J = 8 Hz), 7.76 (1H, t, J = 8 Hz), 7.44 (1H, t, J = 8 Hz), 7.28 (1H, s), 6.97 (2H, d, J = 8 Hz), 3.98 (4H, t, J = 4 Hz), 3.86 (4H, t,J = 4 Hz): HRMS: m/z [M + H]⁺ for C₁₈H₁₇N₃O₂, calculated 308.1399; observed: 308.1405.

AK-6 (4-(4-morpholinoquinazolin-2-yl)benzonitrile)

Yield: 63%, off white in colour, m.p.: 168–170 °C ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.70 (2H, d, J = 8 Hz), 8.04 (1H,t, J = 8 Hz), 7.95 (1H, d, J = 8 Hz), 7.83 (1H, d, J = 8 Hz), 7.81 (2H, d, J = 8 Hz), 7.53 (1H, d, J = 8 Hz), 3.99 (4H, t, J = 8 Hz), 3.91 (4H, t, J = 8 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.95, 157.51, 152.62, 142.75, 132.90, 132.17, 129.33, 128.84, 125.88, 124.72, 119.08, 115.48, 113.35, 66.77, 50.35,. HRMS: m/z [M + H]⁺ for C₁₉H₁₆N₄O, calculated 317.1402; observed: 317.1409.

AK-7 (4-(2-(4-isopropylphenyl)quinazolin-4-yl)morpholine)

Yield: 65%, white in colour, m.p.: 164–166 °C ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.50 (2H, d, J = 8 Hz), 8.01 (1H, d, J = 8 Hz), 7.92 (1H, d, J = 8 Hz), 7.77 (1H, t, J = 8 Hz), 7.46 (1H, t, J = 8 Hz), 7.39 (2H, d, J = 8 Hz), 3.98 (4H, t, J = 8 Hz), 3.88 (4H, t, J = 8 Hz), 1.33 (6H, d, J = 4 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.95, 159.63, 152.88, 151.33, 136.19, 132.45, 129.11, 128.47, 128.62, 126.49, 124.87, 124.61, 115.30, 66.83, 50.42,. HRMS: m/z [M + H]⁺ for C₂₁H₂₃N₃O, calculated 334.4512; observed: 334.4529.

AK-8 (4-(2-(4-methoxyphenyl)quinazolin-4-yl)morpholine)

Yield: 76%, pale white in colour m.p.: 174–176 °C ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.85 (2H, d, J = 12 Hz), 7.99 (1H, d, J = 8 Hz), 7.90 (1H, d, J = 8 Hz), 7.76 (1H, t, J = 16 Hz), 7.44 (1H, t, J = 8 Hz), 7.04 (2H, d, J = 8 Hz), 3.97 (4H, t, J = 4 Hz), 3.91 (3H, s), 3.87 (4H, t, J = 4 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.91, 161.55, 159.26, 152.88, 132.48, 131.15, 130.02, 128.92, 124.69, 124.63, 115.13, 113.68, 66.83, 55.38, 50.41 HRMS: m/z [M + H]⁺ for C₁₉H₁₉N₃O₂, calculated 322.1556; observed: 322.1561.

AK-9 (4-(2-(2-fluorophenyl)quinazolin-4-yl)morpholine)

Yield: 58%, off white colour, m.p.: 172–174 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.69 (2H, d, J = 8 Hz), 8.04 (1H, d, J = 8 Hz), 7.94 (1H, d, J = 8 Hz), 7.81 (1H, t, J = 8 Hz), 7.77 (2H, d, J = 8 Hz), 7.51 (1H, t, J = 4 Hz), 3.99 (4H, t, J = 8 Hz), 3.91 (4H, t, J = 8 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.95, 159.63, 152.88, 151.33, 136.19, 132.45, 129.11, 128.47, 126.49, 124.87, 124.61, 115.30, 66.83, 50.42. HRMS: m/z [M + H]⁺ for C₁₈H₁₆FN₃O, calculated 310.1408; observed: 310.1409.

AK-10 (4-(2-(3,4,5-trimethoxyphenyl)quinazolin-4-yl)morpholine)

Yield: 72%, off-white in colour, m.p.: 176–178 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.01 (1H, d, J = 8 Hz), 7.91 (2H, d, J = 12 Hz), 7.91 (1H, d, J = 8 Hz), 7.78 (1H, t, J = 8 Hz), 7.46 (1H, t, J = 8 Hz), 4.03 (6H, s), 3.99 (4H, t, J = 8 Hz), 3.95 (3H, s), 3.86 (4H, t, J = 8 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.99, 158.99, 153.16, 152.77, 140.27, 134, 132.99, 129.09, 125.10, 124.65, 115.29, 105.66, 66.77, 60.96, 56.22, 50.42. HRMS: m/z [M + H]⁺ for C₂₁H₂₃N₃O₄, calculated 382.1770; observed: 382.1767.

AK-11 (4-(2-(2-chloro-6-fluorophenyl)quinazolin-4-yl)morpholine)

Yield: 67%, pale yellow in colour, m.p.: 178–180 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.0 (1H, d, J = 4 Hz), 7.8 (1H, d, J = 4 Hz), 7.75 (1H, d, J = 4 Hz), 7.5 (1H, d, J = 4 Hz), 7.25 (1H, t, J = 4 Hz), 7.20 (1H, t, J = 4 Hz), 7.0 (1H, t, J = 8 Hz), 3.91 (4H, d, J = 4 Hz), 3.8 (4H, d, J = 4 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 186.54, 164.65, 161.74, 159.75, 156.48, 152.22, 134.12, 134.08, 132.78, 129.93, 129.86, 129.08, 125.93, 125.47, 125.44, 124.67, 115.78, 114.60, 114.42, 66.81, 50.32. HRMS: m/z [M + H]⁺ for C₁₈H₁₅ClFN₃O, calculated 344.0966; observed: 344.0972.

AK-12 (4-(2-(p-tolyl)quinazolin-4-yl)morpholine)

Yield: 59%, m.p.: 163–165 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.48 (2H, d, J = 8 Hz), 8.01 (1H, d, J = 8 Hz), 7.90 (1H, d, J = 4 Hz), 7.75 (1H, t, J = 8 Hz), 7.45 (1H, t, J = 8 Hz), 7.43 (2H, d, J = 8 Hz), 3.99 (4H, t, J = 12 Hz), 3.88 (4H, t, J = 8 Hz), 2.45 (3H, s): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.93, 159.56, 152.87, 140.42, 135.76, 132.47, 129.19, 129.13, 129.08, 128.42, 128.37, 124.88124.61, 115.31, 66.83, 50.41, 21.53. HRMS: m/z [M + H]⁺ for C₁₉H₁₉N₃O, calculated 306.1606; observed: 306.1614.

AK-13 (4-(2-(4-(trifluoromethyl)phenyl)quinazolin-4-yl)morpholine)

Yield: 68%, white in colour, m.p.: 168–170 °C, ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): 8.69 (2H, d, J = 8 Hz), 8.04 (1H, d, J = 8 Hz), 7.94 (1H, d, J = 8 Hz), 7.81 (1H, t, J = 8 Hz), 7.77 (2H, d, J = 8 Hz), 7.51 (1H, t, J = 16 Hz), 3.99 (4H, t, J = 8 Hz), 3.91 (4H, t, J = 8 Hz): ¹³C NMR (CDCl₃, 100 MHz, δ with TMS = 0) δ: 164.95, 159.63, 152.88, 151.33, 136.19, 132.45, 129.11, 128.47, 126.49, 124.87, 124.61, 115.30, 66.83, 50.42. HRMS: m/z [M + H]⁺ for C₁₉H₁₆F₃N₃O, calculated 360.1324; observed: 360.1330.

4.5. Biological studies

4.5.1. Cytotoxic studies

MTT is a colorimetric assay used for the measurement of cell viability relying on the cellular reduction of the tetrazolium salt to formazan crystals.

For performing MTT bioassay,³³ the different cancer cell lines (A549, MCF7 and SHSY5Y) were grown in DMEM containing 10% serum. The cell lines were procured from the National Centre for Cell Science (NCCS), Pune, India. Approximately 8000 cells were seeded in each well of the 96 well plate. The plate was incubated at 37 °C with 5% CO₂ for 24 h followed by serum starvation for 8 h for synchronization and replenishing with complete media. Then the treatment of synthesized compounds was given to the cells in triplicate at concentration of 1 μM, 5 μM, 10 μM and 25 μM and incubated for 48 h. An MTT solution (5 mg/10 mL) was added after removing the media from each well and incubated in the dark for 4 h. After incubation, the MTT solution was removed from each well, and the intracellular formazan crystals were dissolved in DMSO solution, and the absorbance is measured at 590 nm using a spectrophotometer, which was expressed as % cell viability (mean ± S.D).

4.5.2. Cell cycle assay

SHSY5Y cells were seeded in 6-well plates at a cell density of 2.5 × 10⁵ cells per well. The cells were treated with compounds AK-3 and AK-10 for 48 h. Then, the cells were harvested, fixed in cold 70% ethanol overnight, treated with RNase A at 37 °C for 30 min, and incubated with propidium iodide (PI) solution (50 μg ml⁻¹) at 4 °C for 15 min. Cell cycle distribution was analysed using a FACS flow cytometer.

4.5.3. Apoptosis

Apoptosis utilizing a Muse™ Annexin V and Dead Cell kit (catalogue no. 15-0180) was performed using a Muse™ cell analyzer. The predefined protocol of the Muse™ Annexin V and Dead Cell kit was used for the preparation of the cellular samples (SHSY5Y) previously treated with AK-3 and AK-10 for 48 h. The cells and media were centrifuged at 1200 rpm for 5 min and washed with 1× PBS. 50 μL of kit reagent was added and incubated for 30 min at room temperature in the dark before the analysis.

4.5.4. Docking studies

To determine the mode of interaction between synthesized ligands AK-3 and AK-10 and the Bcl-2 protein (4LVT), docking studies were performed. Chem BioDraw Ultra15 software and Maestro 12.3 (Schrödinger) software were used for the preparation of 2D and 3D structures of the compounds. The X-ray crystal structure of tubulin (PDB ID: 4LVT) co-crystallized with ABT-263 was downloaded from the Protein Data Bank (https://www.rcsb.org). Before the molecule is docked into the protein, the ligand and protein were separately prepared using LigPrep and the protein preparation wizard. In LigPrep, all the different possible stable conformations of the ligands were formed and the protein was prepared by adding a missing polar hydrogen, adding side chains and removing water molecules that are present other than the active site. The prepared protein was energy minimized using the OPLS3e force field. After the ligand and protein preparation, a grid is generated using the receptor grid generation panel at the site in the protein where the ligand is to be docked by replacing the crystallized ligand. The binding site was defined around the co-crystallized ligand. The top-score docking poses were selected for the final ligand–target interaction analysis employing the XP interaction visualizer of Maestro 11.1 software. The QikProp application of the Schrödinger suite was used to determine the drug like and ADME properties of the compounds.

Abbreviations

QPlogBB

Qualitatively predicted logarithmic ratio between the concentration of a compound in the brain and blood

Log P

Partition coefficient of a molecule between an aqueous phase and lipophilic phase (octanol and water)

Log S

Aqueous solubility

#metab

Number of likely metabolic reactions

PDB ID codes 4LVT

Coordinates for the 4LVT binding site x = 8.00, y = −2.46, z = −8.64.

Conflicts of interest

The authors declare no potential conflict of interest.