Introduction

The World Health Organization estimates revealed that “Cancer is a disease responsible for major morbidity and mortality worldwide”. Nearly 8.2 million deaths were reported due to cancer in the year 2012 (WHO & International Agency for Research on Cancer, 2014). Cancer is identified as a massive disease of human society because of its high morbidity and mortality rates. The drugs used in the treatment of cancer have a narrow therapeutic index and showed a high incidence of untoward side effects (Arora & Scholar, 2005; Mousavi, Tayarani-Najaran, & Hersey, 2008). The DNA damage repair process in the cell was activated by the enzyme Poly (ADP-Ribose) Polymerase-1 (PARP-1). The inhibition of PARP-1 will cause lethal effects in cells, which makes PARP-1 as a key target in anti-cancer therapy (Lord & Ashworth, 2017). The various hetero compounds such as Quinazoline, Phthalimide, Phthalazine and Benzimidazole were developed as PARP inhibitor scaffolds (Almahli et al., 2018; Bürkle, 2001; Malyuchenko, Kotova, Kulaeva, Kirpichnikov, & Studitskiy, 2015). Quinazoline nucleus is a versatile heterocyclic nucleus having a broad spectrum of biological activities such as Analgesic (Alafeefy, Kadi, Al-Deeb, El-Tahir, & Al-jaber, 2010), Anti-inflammatory (Laddha & Bhatnagar, 2009), Anti-bacterial (McLaughlin & Evans, 2010), anti-tubercular (Jampilek et al., 2009), anti-diabetic (Wang, 2008), anti-HIV (Selvam, Kumar, Kumar, & Emerson, 2010), Anti-Cancer activities (Connell, 2004; Kumar, Shakya, Gupta, Sarkar, & Sahu, 2009; Marvania et al., 2011) etc. An effort has been made to test the quiazoline derivatives as potential anticancer properties, we designed a series of 4-anilino quinazoline (Figure 1), molecular docking study were performed against the target Poly (ADP-Ribose) Polymerase-1 (PARP-1) by evaluating their binding interaction and screened for their in vitro cytotoxic evaluation.

Materials and Methods

Molecular Docking Study

The Novel quinazoline derivatives were subjected to molecular docking in the active site of Poly (ADP-Ribose) Polymerase-1 (PARP-1) enzyme using Autodock 4 software. We investigated the theoretical binding mode of ten ligands along with standard Doxorubicin using molecular docking. Molecular docking studies were performed for these ligands to understand the various intermolecular interactions between the designed derivatives and the target.

In-vitro Cytotoxic activity of synthesized compounds

The in vitro cytotoxic activities were screened for all the compounds against DLA cell lines by Trypan blue dye exclusion method using various concentrations such as 50, 100, 200, 500 and 1000 µg/mL.

Results and Discussion

Synthesis

Anthanilic acid (1) on treatment with benzoyl chloride (2) in the presence of dry pyridine yielded 2-phenyl-4H-3,1-benzoxazin-4-one (3), which was refluxed with formamide to obtain 2-phenyl quinazoline-4(3H)-one (4). 2-phenyl quinazoline-4(3H)-one reacted with the chlorinating agent using POCl₃/PCl₅ under reflux conditions produced 4-chloro quinazoline derivatives (5) which were followed by condensation with various amino group substituted compounds yield 4-anilino quinazolines (6). The scheme for the synthesis of title compounds was given in Figure 2. The structure of synthesized compounds and its code were given in Figure 3.

Characterization of synthesized compounds

N-(4-nitrophenyl)-2-phenylquinazolin-4-amine ( PNAQ1)

Yield 70 %; MF: C₂₀H₁₄N₄O_2; mp 136 ⁰C; R_f value 0.6; IR (KBr, cm^-1): ¹H-NMR (CDCl₃): 8.72 – doublet (2H) in benzene C1 and C6 proton / 8.70 – singlet (1H) C8 proton in quinazoline/ 7.95 – doublet (2H) C5 and C7 proton in quinazoline/ 7.58 – doublet (2H), in benzene C5 & C3 proton / 7.56 – singlet (1H) benzene C4 proton /7.58 – singlet (1H) C6 in quinazoline/ 8.06 – doublet (2H) C5 & C3 proton in nitro benzene/ 7.18 - doublet (2H) C2 & C6 proton in nitro benzene/ 3.51 – singlet (1H) secondary amine proton.¹³C-NMR:δ = 170.11, 164.78, 155.76, 127.08, 116.51, 132.25, 134.39, 131.36, 129.05, 126.46, 141.18, 135.68, 123.02, 119.94. MS: m/z: 342 (M⁺).

N-(5-methyl-1, 2-oxazol-3-yl)-4-{(2-phenyl quinazolin-4-yl)amino] benzene-1-sulphonamide (SMOQ2)

Yield 85 %; MF: C₂₄H₁₉N₅O₃S; mp 144 ⁰C; R_f value 0.56; ¹H-NMR (CDCl₃): 8.70 doublet (2H) aromatic/ 8.05 singlet (1H) aromatic/ 7.75 doublet (2H) aromatic/ 7.58, 7.56, 7.54 triplet (3H) aromatic/ 7.21 doublet (2H) aromatic/ 2.24 – triplet (3H) methyl/ 6.53 – singlet (1H) isoxazole/ 4.72 – doublet (2H) secondary amine. ¹³C-NMR: δ = 170.16, 169.97, 153.36, 95.35, 12.15, 164.77, 141.25, 134.61, 132.39, 131.39, 129.05, 127.10, 122.9, 119.92, 116.53. MS: m/z: 457 (M⁺) .

4-[(2-phenylquinazolin-4 yl)amino] benzene sulfonamide (SNAQ3)

Yield 73 %; MF: C₂₀H₁₆N₄O₂S ; mp 170 ⁰C; R_f value 0.62; ¹H-NMR (CDCl₃): 8.06 doublet (2H) aromatic/ 8.04 – singlet (1H) aromatic/ 7.94 – doublet (2H) aromatic / 7.66 – triplet (3H) aromatic/ 7.64 – doublet (2H) aromatic/ 7.62 – singlet (1) aromatic/ 7.20 – doublet (2H)/ 2.50 – singlet (1H) secondary amine/ 2.06 – doublet (2H) primary amine. ¹³C-NMR: δ =116.53, 119.92, 122.9, 127.10, 129.05, 131, 132.39, 132.24, 134.24, 141.25, 164.77, 170.16. MS: m/z: 376.0 (M⁺).

N -(diaminomethylidene)-4-[(2-phenylquinazolin-4-yl)amino] benzenesul formamide (2SGQ4):

Yield 75 %; MF: C₂₁H₁₈N₆O₂S ; mp 158 ⁰C; R_f value 0.59; IR (KBr, cm^-1): 3397.96 (N-H), 3050.83 (CH-arom), 1566.88 (C=N), 1155.2 (S=O str), 814.0 (C-S str), 932.5 (S-N str) ¹H-NMR (CDCl₃): 8.72 - quartet (4H) two primary amine/ 8.70 – doublet (2H) in benzene C1 and C6 proton / 8.07– singlet (1H) C8 proton in quinazoline/ 7.95 – doublet (2H) C5 and C7 proton in quinazoline/ 7.58 – doublet (2H), in benzene C5 & C3 proton / 7.56 – singlet (1H) benzene C4 proton /7.59 – singlet (1H) C6 in quinazoline/ 4.7 - singlet (1H) secondary amine proton/ 7.61 – doublet (2H) 3^rd and 5^th carbon next to sulfonyl group in sulfanilamide ring/ 7.21 – doublet (2H) 2^nd and 6^th carbon in sulfanilamide ring. ¹³C-NMR: δ =170.20, 164.76, 157.86, 141.47, 134.17, 132.47, 131.39, 130.84, 129.06, 127.10, 122.9, 117.92, 112.53. MS: m/z:418 (M⁺).

1,3-dimethyl-6-[(2-phenylquinazolin-4-yl)amino]pyrimidine-2,4(1H,3H)-dione (DMUQ5):

Yield 80 %; MF: C₂₀H₁₇N₅O_2; mp 168 ⁰C; R_f value 0.78; IR (KBr, cm^-1): 3354.57 (N-H), 3276.47 (CH-arom), 1642.1 (C=O), 1571.7 (C=N)’¹H-NMR (CDCl₃): 8.71 – doublet (2H) in benzene/ 8.69 – singlet (1H) C8 proton in quinazoline/ 7.93 – doublet (2H) C5 and C7 proton in quinazoline/ 7.57 – doublet (2H), in benzene/ 7.55 – singlet (1H) benzene/7.59 – singlet (1H) C6 in quinazoline/ 4.70– singlet (1H) CH proton in uracil/ 4.00 – singlet (1H) NH proton in b/w quinazoline and uracil/ 3.17 – triplet (3H) N methyl proton present in b/w two carbonyl group/ 3.06 – tiplet (3H) N methyl in uracil ring.¹³C-NMR: δ = 170.14, 141.19, 119.85, 116.68, 132.25, 127.67, 134.55, 131.36, 129.04, 164.72. MS: m/z:359(M⁺).

6-[(2-phenylquinazolin-4-yl)amino]pyrimidine-2,4(1H,3H)-dione (6AUQ6)

Yield 79 %; MF: C₁₈H₁₃N₅O_2; mp 162 ⁰C; R_f value 0.72; IR (KBr, cm^-1): 3395.07 (N-H), 2920.66 (CH-arom), 1642.1 (C=O), 1623.77 (C=N);¹H-NMR (CDCl₃): 10.17 – singlet (1H) NH proton present in between two carbonyl carbon/ 8.71 – doublet (2H) in benzene/ 8.16 – singlet (1H) C8 proton in quinazoline/ 7.95 – doublet (2H) C5 and C7 proton in quinazoline/ 7.60 – triplet (3H), in benzene/ 7.20 – singlet (1H) C6 in quinazoline/ 6.22 – singlet (1) NH proton in uracil/ 4.42 – singlet (1H) CH proton in uracil/ 4.18 – sinlet (1H) NH proton in b/w quinazoline and uracil. ¹³C-NMR: δ =170.12, 151.06, 132.26, 128.67, 127.82, 116.68, 134.59, 131.36, 129, 127.08, 164.77, 155.25, 74.18. MS: m/z: 331 (M⁺).

1-{4-[(2-phenylquinazolin-4-yl)amino]phenyl}ethanone (4AAQ7)

Yield 75 %; MF: C₂₂H₁₇N₃O ;mp 150 ⁰C; R_f value 0.52; IR (KBr, cm^-1): 3322.75 (N-H), 2921.63 (CH-arom), 1573.63 (C=N).

N -(4-methylphenyl)-2-phenylquinazolin-4-amine (PTQ8)

Yield 76 %; MF: C₂₁H₁₇N_3; mp 156 ⁰C; R_f value 0.28; IR (KBr, cm^-1): 3322.75 (N-H), 2921.63 (CH-arom), 1573.63 (C=N). ¹H-NMR (CDCl₃): 8.73 – doublet (2H) in benzene/ 8.71 – singlet (1H) C8 proton in quinazoline/ 7.95 – doublet (2H) C5 and C7 proton in quinazoline/ 7.67 – doublet (2H), in benzene/ 7.56 – singlet (1H) benzene/7.59 – singlet (1H) C6 in quinazoline/ 7.21 – doublet (2H) in C2 and C6 proton in toluene/ 7.17 – doublet (2H) in C3 and C5 in toluene/4.0 - singlet (1H) NH proton in b/w quinazoline and toluene/ 2.22 – triplet (3H) methyl proton in toluene.¹³C-NMR: δ =170.14, 164.74, 141.20, 119.89, 116.61, 132.22, 127.07, 134.59, 134.59, 134.33, 129.25, 131.36, 129.05, 122.96. MS: m/z:311(M⁺).

2-phenyl-N-(pyridin-2-yl)quinazolin-4-amine (2APQ9)

Yield 81 %; MF: C₁₉H₁₄N_4; mp 148 ⁰C; R_f value 0.28; ¹H-NMR (CDCl₃): 7.5 triplet (3H), 7.6 singlet (1H), 7.6 singlet (1H), 7.9 doublet (2H), 7.9 singlet (1H), 8.7 doublet (2H), 7.1 singlet (1H), 7.1 singlet (1H), 8.7 S (1), 4.0 S (1H) amine.¹³C-NMR: δ =116.68, 119, 122, 127, 129, 131, 132, 134, 141, 164, 170. MS: m/z: 298 (M⁺).

4-[(2-phenylquinazolin-4-yl)amino]benzoic acid (PABAQ10)

Yield 85 %; MF: C₂₁H₁₅N₃O_2; mp 172 ⁰C; R_f value 0.64; ¹H-NMR (CDCl₃): 8.72 – doublet (2H) in benzene C1 and C6 proton / 8.70 – singlet (1H) C8 proton in quinazoline/ 7.95 – doublet (2H) C5 and C7 proton in quinazoline/ 7.58 – doublet (2H), in benzene C5 & C3 proton / 7.56 – singlet (1H) benzene C4 proton /7.58 – singlet (1H) C6 in quinazoline/ 12.2 – singlet (1H) may be Acid proton not confirm (11.00)/ 7.94 – doblet (2H) C5 & C3 proton in PABA/ 7.89 - doublet (2H) C2 & C6 proton in PABA/ 3.72 – singlet (1H) secondary amine proton. ¹³C-NMR: δ = 170.12, 164.79, 141.18, 127.09, 116.51, 132.51, 134.58, 134.40, 129.05, 119.94, 131.37. MS: m/z: 341(M⁺).

Molecular Docking

The crystal structure of the protein Poly (ADP-Ribose) Polymerase-1 (PDB Code: 1UK1) with resolution 3Å was chosen as the protein model (Figure 4). The binding features of ten synthesized compounds with PARP-1 were evaluated in the same manner of binding of standard drug Doxorubicin. The results of molecular docking interactions of synthesized compounds were compared with the docking interactions of standard drug Doxorubicin. Doxorubicin showed binding energy of -8.94 kcal/mol and formed one hydrogen bond with Asn767 with a distance of 1.98 A⁰. Compound SMOQ2 showed the least binding energy, i.e., 11.87kcal/mol and formed one hydrogen bond with Arg (878) with a distance of 1.895.A⁰ Compound DMUQ5 showed binding energy of -11.42 kcal/mol and produced two hydrogen bonds with Arg 878 and Asn 767. Molecular interactions of synthesized compounds with amino acid residue in the active site of PARP-1 Enzyme were given in Table 1. The binding mode of compound SMOQ2 and Doxorubicin were given in Figure 6; Figure 5.

In-vitro Cytotoxic activity

The in vitro cytotoxic activities were screened for all the compounds against DLA cell lines by Trypan blue dye exclusion method using various concentrations such as 50, 100, 200, 500 and 1000 µg/mL. The number of stained (Dead cells) and unstained (Live cells) cells was counted separately and the results were shown in Table 2.

Conclusion

In the present study, a series of novel 4-anilino Quinazoline derivatives were designed and synthesized by fragment replacement and lipophilic group insertion. The structure of the synthesized compounds was characterized by spectral analysis. The data were in correlation with the expected structure. The designed derivatives were docked into the active site of PARP-1Enzymes (Cancer target). The results were compared with the standard Doxorubicin. Compound SMOQ2 and DMUQ5 were found to have least binding energy -11.87kcal/mol and -11.42 kcal/mol respectively compared to the standard drug Doxorubicin The data for the cytotoxic activity screening revealed that compounds SMOQ2 and DMUQ5 showed significant cytotoxic activity against the DLA cell lines at the concentration of 1000 µg/mL.