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\def\RunningAuthor{Hemalatha K et al.}
\firstPage{7559}
\articleType{Original Article}
\receivedDate{20 Oct 2020}
\acceptedDate{23 Nov 2020}
\revisedDate{10 Nov 2020}
\journalVolume{2020, 11}
\journalIssue{4}
\journalDoi{ijrps.v11i4.3962}
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\title{\textbf{4-Anilino Quinazoline Derivatives: Molecular Docking and Evaluation \textit{of In vitro }Cytotoxic Activity\textit{}}}
\author{Hemalatha~K\textsuperscript{*}\textsuperscript{1},  
            Sujatha~K\textsuperscript{2},  
            Panneerselvam~P\textsuperscript{3}, 
            Girija~K\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Pharmaceutical Chemistry, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, (A Government of Puducherry Institution), Indira Nagar, Gorimedu, Puducherry-06, India}~\\{\textsuperscript{2}Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sri Ramachandra Institute of Higher Education and Research, (Deemed to be University), Porur, Chennai-116, Tamil Nadu, India}~\\{\textsuperscript{3}Faculty of Pharmacy, (Medical Campus), Bharath Institute of Higher Education and Research, Chrompet, Chennai-44, Tamil Nadu, India}}

\begin{abstract}
A novel scaffold of 4-anilino quinazoline derivatives was designed on the basis of known inhibitor of quinazoline based drugs. The designed derivatives were synthesized using optimized reaction condition. Their structures were confirmed by FT-IR, \ensuremath{^{1}}H-NMR, \ensuremath{^{13}}C-NMR and Mass spectral data. The structures of synthesized compounds were subjected to \textit{in silico } molecular docking using AutoDock software against the target Poly (ADP-ribose) polymerase-1 (PARP-1) enzyme. The compounds were evaluated for their \textit{in vitro} cytotoxic activity against Daltons Lymphocyte Ascites (DLA) Cell lines. Molecular docking study of the newly synthesized compounds showed good binding mode in the active site of PARP-1. The docking results were compared with the 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 {\AA}. 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.A\ensuremath{^\circ} Compound DMUQ5 showed binding energy of -11.42 kcal/mol and produced two hydrogen bonds with Arg 878 and Asn 767. Among the synthesized compounds, compounds SMOQ2 and DMUQ5 showed significant binding affinity compared to the standard drug Doxorubicin. The \textit{in vitro} cytotoxic evaluation indicated that compounds SMOQ2 and DMUQ5 showed significant cytotoxic activity against Daltons Lymphoma Ascites cell line. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Anilino quinazoline,\newline AutoDock software,\newline cytotoxic activity,\newline PARP 1,\newline Daltons Lymphoma Ascites cell line
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Hemalatha~K~\\ Phone:\ 9787627805~\\  Email:\ hemalathampharm@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
        \textsc{DOI:}\ \href{https://doi.org/10.26452/\@journalDoi}{\textcolor{blue}{\underline{\smash{https://doi.org/10.26452/\@journalDoi}}}}\par%
        \vspace*{-11pt}\hrulefill\\{\fontsize{9.12}{10.12}\selectfont Production and Hosted by}\par{IJRPS |~\href{https://ijrps.com}{https://ijrps.com}}\par%
        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
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\section{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 \unskip~\citep{939509:20820968}. 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 \unskip~\citep{939509:20820954,939509:20820965}. 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 \unskip~\citep{939509:20820959}. The various hetero compounds such as Quinazoline, Phthalimide, Phthalazine and Benzimidazole were developed as PARP inhibitor scaffolds \unskip~\citep{939509:20820955,939509:20820953,939509:20820961}. Quinazoline nucleus is a versatile heterocyclic nucleus having a broad spectrum of biological activities such as Analgesic \unskip~\citep{939509:20820952}, Anti-inflammatory \unskip~\citep{939509:20820960}, Anti-bacterial \unskip~\citep{939509:20820963}, anti-tubercular \unskip~\citep{939509:20820958}, anti-diabetic \unskip~\citep{939509:20820966}, anti-HIV \unskip~\citep{939509:20820964}, Anti-Cancer activities \unskip~\citep{939509:20820956,939509:20820962,939509:20820957} etc.  An effort has been made to test the quiazoline derivatives as potential anticancer properties, we designed a series of 4-anilino quinazoline (Figure~\ref{f-41f6b65565f9}), molecular docking study were performed against the target Poly (ADP-Ribose) Polymerase-1 (PARP-1) by evaluating their binding interaction and screened for their \textit{in vitro } cytotoxic evaluation.  
    
\section{Materials and Methods}
\textbf{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.

\textit{\textbf{In-vitro }} \textbf{Cytotoxic activity of synthesized compounds}

The \textit{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 \ensuremath{\mu}g/mL. 
    
\section{Results and Discussion}
\textbf{Synthesis}

Anthanilic acid (1) on treatment with benzoyl chloride (2) in the presence of dry pyridine yielded 2-phenyl-4\textit{H}-3,1-benzoxazin-4-one (3),  which was refluxed with formamide to obtain 2-phenyl quinazoline-4(3\textit{H})-one (4). 2-phenyl quinazoline-4(3\textit{H})-one reacted with the chlorinating agent using POCl\ensuremath{_{3}}/PCl\ensuremath{_{5}} under reflux conditions produced 4-chloro quinazoline derivatives\textbf{\space }(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~\ref{f-83aa7e391ee8}. The structure of synthesized compounds and its code were given in Figure~\ref{f-07f486484762}.


\begin{table*}[!htbp]
\caption{\boldmath {Molecular Interactions of synthesized compounds with amino acid residue in the active site of PARP-1 Enzyme} }
\label{tw-0a829f5be174}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0687\linewidth-2\tabcolsep}p{\dimexpr.2817\linewidth-2\tabcolsep}p{\dimexpr.16969999999999995\linewidth-2\tabcolsep}p{\dimexpr.1628\linewidth-2\tabcolsep}p{\dimexpr.15719999999999997\linewidth-2\tabcolsep}p{\dimexpr.1599\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Sl.No. & Compound Code & Binding energy (Kcal/mol) & No. of Hydrogen bonds formed & Residue involved in H-Bond & Distance between the donor and acceptor \mbox{}\protect\newline (A\ensuremath{^{0}})\\
\tblmidrule 
1 &
  PNAQ1 &
  -9.6 &
  0 &
  ------- &
  -----\\
2 &
  SMOQ2 &
  -11.87 &
  1 &
  Arg 878(NH) &
  1.895\\
3 &
  SNAQ3 &
  -10.96 &
  2 &
  Asn767 (NH) \mbox{}\protect\newline Arg 865 (NH) &
  1.708 \mbox{}\protect\newline 1.957\\
4 &
  SGQ4 &
  -10.94 &
  2 &
  Asn767 (NH) \mbox{}\protect\newline Arg 865 (NH) &
  1.895 \mbox{}\protect\newline 1.991\\
5 &
  DMUQ5 &
  -11.42 &
  2 &
  Arg 878 (NH) \mbox{}\protect\newline Asn 767 (NH) &
  1.914 \mbox{}\protect\newline 1.895\\
6 &
  6AUQ6 &
  -9.75 &
  1 &
  Arg 865 (NH) &
  2.038\\
7 &
  4AAQ7 &
  -8.54 &
  1 &
  Arg 865 (NH) &
  2.228\\
8 &
  PTQ8 &
  -9.36 &
  1 &
  Arg 878 (NH) &
  1.959\\
9 &
  2APQ9 &
  -8.87 &
  1 &
  Tyr 896 (NH) &
  2.218\\
10 &
  PABAQ10 &
  -9.61 &
  0 &
  ----- &
  ------\\
11 &
  Standard (Doxorubicin) &
  -8.94 &
  1 &
  Val 833 (NH) &
  1.527\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Cytotoxic activity against DLA Cell Lines} }
\label{tw-f50b4adafe37}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.08079999999999998\linewidth-2\tabcolsep}p{\dimexpr.1992\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.0961\linewidth-2\tabcolsep}p{\dimexpr.2039\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{Sl. No.}} & {\multirow{2}{*}{Compound Code}} & \multicolumn{5}{p{\dimexpr(.72\linewidth-2\tabcolsep)}}{Percentage Cell Death at the various Concentration (\ensuremath{\mu}g/mL)}\\
\rowcolor{kwdboxcolor} &  & 50 & 100 & 200 & 500 & 1000\\
\tblmidrule 
1 &
  PNAQ1 &
  20 &
  36 &
  42 &
  52 &
  65\\
2 &
  SMOQ2 &
  17 &
  22 &
  35 &
  55 &
  75\\
3 &
  SNAQ3 &
  11 &
  26 &
  34 &
  50 &
  68\\
4 &
  SGQ4 &
  11 &
  32 &
  60 &
  65 &
  70\\
5 &
  DMUQ5 &
  15 &
  24 &
  35 &
  55 &
  72\\
6 &
  6AUQ6 &
  15 &
  32 &
  58 &
  62 &
  70\\
7 &
  4AAQ7 &
  14 &
  25 &
  33 &
  46 &
  65\\
8 &
  PTQ8 &
  9 &
  20 &
  50 &
  58 &
  70\\
9 &
  2APQ9 &
  11 &
  28 &
  40 &
  48 &
  66\\
10 &
  PABAQ10 &
  10 &
  22 &
  35 &
  48 &
  62\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Characterization of synthesized compounds}

N\textbf{-(4-nitrophenyl)-2-phenylquinazolin-4-amine ( PNAQ1) }

Yield 70 \%; MF: C\ensuremath{_{20}}H\ensuremath{_{14}}N\ensuremath{_{4}}O\ensuremath{_{2;\ }} mp 136 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.6; IR (KBr, cm\ensuremath{^{-1}}): \ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 8.72 {\textendash} doublet (2H) in benzene C1 and C6 proton / 8.70 {\textendash} singlet (1H) C8 proton in quinazoline/ 7.95 {\textendash} doublet (2H) C5 and C7 proton in quinazoline/ 7.58 {\textendash} doublet (2H), in benzene C5 \& C3 proton / 7.56 {\textendash} singlet (1H) benzene C4 proton /7.58 {\textendash} singlet (1H) C6 in quinazoline/ 8.06 {\textendash} doublet (2H) C5 \& C3 proton in nitro benzene/ 7.18 - doublet (2H) C2 \& C6 proton in nitro benzene/ 3.51 {\textendash} singlet (1H) secondary amine proton.\ensuremath{^{13}}C-NMR:\ensuremath{\delta }\textbf{ = } 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\ensuremath{^{+}}).


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\caption{\boldmath {Structure of Quinazoline}}
\label{f-41f6b65565f9}
\end{figure}
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\begin{figure}[!htbp]
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\makeatother 
\caption{\boldmath {Scheme for the synthesis of Title compounds}}
\label{f-83aa7e391ee8}
\end{figure}
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\makeatother 
\caption{\boldmath {Structure of the synthesized compounds and its code}}
\label{f-07f486484762}
\end{figure}
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\makeatother 
\caption{\boldmath {Crystal structure of Poly (ADP-Ribose) Polymerase-1 along with inhibitor }}
\label{f-bb7a7c3d5ac3}
\end{figure}
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\caption{\boldmath {Docking interaction of Compound SMOQ2 in the active site of PARP-1 enzyme}}
\label{f-b1990cebfdd8}
\end{figure}
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\makeatother 
\caption{\boldmath {Docking interaction of Doxorubicin in the active site of PARP-1 enzyme}}
\label{f-ba2a6425a6c6}
\end{figure}
\egroup
\textbf{N-(5-methyl-1, 2-oxazol-3-yl)-4-\{(2-phenyl quinazolin-4-yl)amino] benzene-1-sulphonamide (SMOQ2)}

Yield 85 \%; MF: C\ensuremath{_{24}}H\ensuremath{_{19}}N\ensuremath{_{5}}O\ensuremath{_{3}}S; mp 144 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.56; \ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 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 {\textendash} triplet (3H) methyl/ 6.53 {\textendash} singlet (1H) isoxazole/ 4.72 {\textendash} doublet (2H) secondary amine. \ensuremath{^{13}}C-NMR: \ensuremath{\delta }\textbf{ = } 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\ensuremath{^{+}}) .

\textbf{ 4-[(2-phenylquinazolin-4 yl)amino] benzene sulfonamide (SNAQ3)}

Yield 73 \%; MF: C\ensuremath{_{20}}H\ensuremath{_{16}}N\ensuremath{_{4}}O\ensuremath{_{2}}S ; mp 170 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.62; \ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 8.06 doublet (2H) aromatic/ 8.04 {\textendash} singlet (1H) aromatic/ 7.94 {\textendash} doublet (2H) aromatic / 7.66 {\textendash} triplet (3H) aromatic/ 7.64 {\textendash} doublet (2H) aromatic/ 7.62 {\textendash} singlet (1) aromatic/ 7.20 {\textendash} doublet (2H)/ 2.50 {\textendash} singlet (1H) secondary amine/ 2.06 {\textendash} doublet (2H) primary amine. \ensuremath{^{13}}C-NMR: \ensuremath{\delta } =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\ensuremath{^{+}}). 

\textit{\textbf{ N}} \textbf{-(diaminomethylidene)-4-[(2-phenylquinazolin-4-yl)amino] benzenesul formamide (2SGQ4):}

 Yield 75 \%; MF: C\ensuremath{_{21}}H\ensuremath{_{18}}N\ensuremath{_{6}}O\ensuremath{_{2}}S ; mp 158 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.59; IR (KBr, cm\ensuremath{^{-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) \ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 8.72 - quartet (4H) two primary amine/ 8.70 {\textendash} doublet (2H) in benzene C1 and C6 proton / 8.07{\textendash} singlet (1H) C8 proton in quinazoline/ 7.95 {\textendash} doublet (2H) C5 and C7 proton in quinazoline/ 7.58 {\textendash} doublet (2H), in benzene C5 \& C3 proton / 7.56 {\textendash} singlet (1H) benzene C4 proton /7.59 {\textendash} singlet (1H) C6 in quinazoline/ 4.7 - singlet (1H) secondary amine proton/ 7.61 {\textendash} doublet (2H) 3\ensuremath{^{rd}} and 5\ensuremath{^{th}} carbon next to sulfonyl group in sulfanilamide ring/ 7.21 {\textendash} doublet (2H) 2\ensuremath{^{nd}} and 6\ensuremath{^{th}} carbon in sulfanilamide ring\textbf{.}\ensuremath{^{13}}C-NMR: \ensuremath{\delta } =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\ensuremath{^{+}}). 

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

Yield 80 \%; MF: C\ensuremath{_{20}}H\ensuremath{_{17}}N\ensuremath{_{5}}O\ensuremath{_{2;\ }} mp 168 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.78; IR (KBr, cm\ensuremath{^{-1}}): 3354.57 (N-H), 3276.47 (CH-arom),  1642.1 (C=O), 1571.7 (C=N)'\ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 8.71 {\textendash} doublet (2H) in benzene/ 8.69 {\textendash} singlet (1H) C8 proton in quinazoline/ 7.93 {\textendash} doublet (2H) C5 and C7 proton in quinazoline/ 7.57 {\textendash} doublet (2H), in benzene/ 7.55 {\textendash} singlet (1H) benzene/7.59 {\textendash} singlet (1H) C6 in quinazoline/ 4.70{\textendash} singlet (1H) CH proton in uracil/ 4.00 {\textendash} singlet (1H) NH proton in b/w quinazoline and uracil/ 3.17 {\textendash} triplet (3H) N methyl proton present in b/w two carbonyl group/ 3.06 {\textendash} tiplet (3H) N methyl in uracil ring.\ensuremath{^{13}}C-NMR: \ensuremath{\delta } = 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\ensuremath{^{+}}). 

\textbf{6-[(2-phenylquinazolin-4-yl)amino]pyrimidine-2,4(1\textit{H},3\textit{H})-dione (6AUQ6)}

Yield 79 \%; MF: C\ensuremath{_{18}}H\ensuremath{_{13}}N\ensuremath{_{5}}O\ensuremath{_{2;\ }} mp 162 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.72; IR (KBr, cm\ensuremath{^{-1}}): 3395.07 (N-H), 2920.66 (CH-arom),  1642.1 (C=O), 1623.77 (C=N);\ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 10.17 {\textendash} singlet (1H) NH proton present in between two carbonyl carbon/ 8.71 {\textendash} doublet (2H) in benzene/ 8.16 {\textendash} singlet (1H) C8 proton in quinazoline/ 7.95 {\textendash} doublet (2H) C5 and C7 proton in quinazoline/ 7.60 {\textendash} triplet (3H), in benzene/ 7.20 {\textendash} singlet (1H) C6 in quinazoline/ 6.22 {\textendash} singlet (1) NH proton in uracil/ 4.42 {\textendash} singlet (1H) CH proton in uracil/ 4.18 {\textendash} sinlet (1H) NH proton in b/w quinazoline and uracil. \ensuremath{^{13}}C-NMR: \ensuremath{\delta } =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\ensuremath{^{+}}).

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

Yield 75 \%; MF: C\ensuremath{_{22}}H\ensuremath{_{17}}N\ensuremath{_{3}}O ;mp 150 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.52; IR (KBr, cm\ensuremath{^{-1}}): 3322.75 (N-H), 2921.63 (CH-arom), 1573.63 (C=N). 

\textit{\textbf{ N}} \textbf{-(4-methylphenyl)-2-phenylquinazolin-4-amine (PTQ8)}

Yield 76 \%; MF: C\ensuremath{_{21}}H\ensuremath{_{17}}N\ensuremath{_{3;\ }} mp 156 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.28; IR (KBr, cm\ensuremath{^{-1}}): 3322.75 (N-H), 2921.63 (CH-arom), 1573.63 (C=N). \ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 8.73 {\textendash} doublet (2H) in benzene/ 8.71 {\textendash} singlet (1H) C8 proton in quinazoline/ 7.95 {\textendash} doublet (2H) C5 and C7 proton in quinazoline/ 7.67 {\textendash} doublet (2H), in benzene/ 7.56 {\textendash} singlet (1H) benzene/7.59 {\textendash} singlet (1H) C6 in quinazoline/ 7.21 {\textendash} doublet (2H) in C2 and C6 proton in toluene/ 7.17 {\textendash} doublet (2H) in C3 and C5 in toluene/4.0 - singlet (1H) NH proton in b/w quinazoline and toluene/ 2.22 {\textendash} triplet (3H) methyl proton in toluene.\ensuremath{^{13}}C-NMR: \ensuremath{\delta } =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\ensuremath{^{+}}).

\textbf{2-phenyl-\textit{N}-(pyridin-2-yl)quinazolin-4-amine (2APQ9)}

Yield 81 \%; MF: C\ensuremath{_{19}}H\ensuremath{_{14}}N\ensuremath{_{4;\ }} mp 148 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.28; \ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 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.\ensuremath{^{13}}C-NMR: \ensuremath{\delta } =116.68, 119, 122, 127, 129, 131, 132, 134, 141, 164, 170. MS: m/z: 298 (M\ensuremath{^{+}}).

\textbf{4-[(2-phenylquinazolin-4-yl)amino]benzoic acid (PABAQ10)}

Yield 85 \%; MF: C\ensuremath{_{21}}H\ensuremath{_{15}}N\ensuremath{_{3}}O\ensuremath{_{2;\ }} mp 172 \ensuremath{^{0}}C; R\ensuremath{_{f}} value 0.64; \ensuremath{^{1}}H-NMR (CDCl\ensuremath{_{3}}): 8.72 {\textendash} doublet (2H) in benzene C1 and C6 proton / 8.70 {\textendash} singlet (1H) C8 proton in quinazoline/ 7.95 {\textendash} doublet (2H) C5 and C7 proton in quinazoline/ 7.58 {\textendash} doublet (2H), in benzene C5 \& C3 proton / 7.56 {\textendash} singlet (1H) benzene C4 proton /7.58 {\textendash} singlet (1H) C6 in quinazoline/ 12.2 {\textendash} singlet (1H) may be Acid proton not confirm (11.00)/ 7.94 {\textendash} doblet (2H) C5 \& C3 proton in PABA/ 7.89 - doublet (2H) C2 \& C6 proton in PABA/ 3.72 {\textendash} singlet (1H) secondary amine proton. \ensuremath{^{13}}C-NMR:\textbf{ \ensuremath{\delta } = } 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\ensuremath{^{+}}).

\textbf{Molecular Docking}

The crystal structure of the protein Poly (ADP-Ribose) Polymerase-1 (PDB Code: 1UK1) with resolution 3{\AA} was chosen as the protein model (Figure~\ref{f-bb7a7c3d5ac3}). 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 A\ensuremath{^{0}}. 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.A\ensuremath{^{0}} 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~\ref{tw-0a829f5be174}. The binding mode of compound SMOQ2 and Doxorubicin were given in Figures~\ref{f-b1990cebfdd8} and~\ref{f-ba2a6425a6c6}.

\textit{\textbf{In-vitro}} \textbf{ Cytotoxic activity}

The \textit{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 \ensuremath{\mu}g/mL. The number of stained (Dead cells) and unstained (Live cells) cells was counted separately and the results were shown in Table~\ref{tw-f50b4adafe37}.
    
\section{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 \ensuremath{\mu}g/mL. 
\section*{Acknowledgement}The authors wish to place heartfelt thanks to Amala Cancer Research Centre, Thrissur, Kerala for providing the facilities to carry out the \textit{in vitro} cytotoxic activity.

\textbf{Funding Support}

The authors declare that they have no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest for this study.
    

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