Molecular design and synthesis of certain new quinoline derivatives having potential anticancer activity

Accepted Manuscript Molecular design and Synthesis of Certain New Quinoline Derivatives having Potential Anticancer Activity Diaa A. Ibrahim, Dalal A. Abou El Ella, Amira M. El-Motwally, Rasha M. Aly PII:

S0223-5234(15)30162-8

DOI:

10.1016/j.ejmech.2015.07.030

Reference:

EJMECH 8014

To appear in:

European Journal of Medicinal Chemistry

Received Date: 6 December 2014 Revised Date:

15 July 2015

Accepted Date: 16 July 2015

Please cite this article as: D.A. Ibrahim, D.A. Abou El Ella, A.M. El-Motwally, R.M. Aly, Molecular design and Synthesis of Certain New Quinoline Derivatives having Potential Anticancer Activity, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.07.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Graphical Abstract The present work explores the utility of 4-anilino quinolone-3-carboxamide, a privileged scaffold as inhibitors of protein kinases (EGFR) with high and selective anticancer activities.

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Molecular design and Synthesis of Certain New Quinoline Derivatives having Potential Anticancer Activity

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Diaa.A. Ibrahim, Dalal A. Abou El Ella, Amira .M. ElMotwally and Rasha. M. Aly

ACCEPTED MANUSCRIPT Molecular design and Synthesis of Certain New Quinoline Derivatives having Potential Anticancer Activity

Diaa A. Ibrahim*a,b, Dalal A. Abou El Ella c*, Amira M. El-Motwallyb and Rasha M. Alyb

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a) Organic Chemistry Department, Faculty of Science, Jazan University, Jazan, KSA. b) National Organization for Drug Control and Research, Cairo, Egypt. c) Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt.

Abstract

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EGFR, which plays a vital role as a regulator of cell growth, is one of the intensely studied TK targets of anticancer inhibitors. The most two common anticancer inhibitors are anilinoquiazolines and anilinoquinolines that inhibit EGFR kinase intracellularly. The present investigation dealt with design (pharmacophore, docking and binding energy) and synthesis of a new series of 4-anilinoquinoline-3carboxamide derivatives as potential anticancer agents targeting EGFR. All the newly synthesized compounds were screened for their anticancer activity against MCF-7 and compounds 4f, 7a and 7b showed significant activity with IC50 values 13.96 µM, 2.16 µM and 3.46 µM, respectively. Most of the synthesized compounds were subjected to enzyme assay (EGFR TK) for measuring their inhibitory activity with the determination of IC50 values and the preliminary results revealed that compound 7b, which had potent inhibitory activity in tumor growth and had potent activity on the EGFR TK enzyme with 67% inhibition compared to ATP would be a potential anticancer agent.

Keywords: 4-Anilinoquinoline-3-carboxamide, EGFR-TK inhibitors, Pharmacophore, Docking study, Binding energy, Anti-proliferative activity *Corresponding authors. Tel.: +966 0567580443. E-mail addresses:[email protected]; [email protected] (Diaa A. Ibrahim)

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1. Introduction

Cancer is a fatal disease. It is an abnormal growth of cells caused by multiple changes in gene expression leading to dysregulated balance of cell proliferation and cell death and ultimately evolving into a population of cells that can invade tissues and metastasize to distant sites, causing significant morbidity and, if untreated, death of the host [1].

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Cancer is caused by changes in a cell’s DNA. Some of these changes may be inherited from our parents (genetic factors, 5-10%), while others may be caused by outside exposures, which are often referred to as environmental factors (90-95%) [2-4]. The biological properties of malignant tumor cells involve acquisition of sustained angiogenesis, ability to invade neighboring tissues, ability to build metastases at distant sites and self-sufficiency in growth signals, and loss of sensitivity to anti-growth signals, capacity for apoptosis, capacity for senescence and capacity to repair genetic errors [5]. The goals of cancer treatment methods fall into three categories: curative, control and palliative; the most common modalities are surgery, radiation, chemotherapy, hormonal therapy, and biotherapy [6].

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Drug design in the cancer therapeutics is developing a trend toward more precise mechanisms of cancer cell destruction thereby minimizing adverse effects incurred during the course of cancer treatment (nausea, vomiting, hair loss, fatigue, organ toxicity, etc.). The key to selectively targeting cancer cells is to exploit some basic difference these cells have developed compared to their normal precursors. One such difference is the activity of the enzyme telomerase, topoisomerase and protein kinases [7-10].

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The complexity and the number of the protein kinases (PKs) being used as molecular targets in drug discovery have greatly increased. The sequencing effort of the human genome project has revealed that ~ 600 PKs and ~130 protein phosphates are probably present in the human genome [7]. About 30% of human protein contains covalently bound phosphate. Protein phosphorylation is considered as one of the main post-translated mechanisms used by cells to finally tune their metabolic and regulatory pathways. PKs catalyze the phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues of proteins using ATP or GTP as the phosphate donor, while phosphatases are responsible for dephosphorylation, the opposite reaction [11,12].

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Tyrosine kinases (TKs) are one of the most widely studied and important kinase families with respect to cancer biology. In humans, there are around 90 distinct TKs, which can broadly be divided into: (a) 58 receptor tyrosine kinases (RTKs), e.g. EGFR, PDGFR, FGFR and the VEGF and (b) 32 non-receptor tyrosine kinases (NRTKs) [13], e.g. SRC, ABL and FAK kinase. RTKs form a large superfamily of receptor molecules on the plasma membranes of eukaryotic cells. RTKs are specifically activated by growth factors, such as EGF, VEGF, FGF, PDGF and many others [14, 15]. A typical member of RTKs is a single-membrane-spanning protein consisting of extracellular ligand binding domain, a short membrane spanning α helix, and a cytoplasmic domain with TK activity. The intracellular kinase domains of RTKs can be further divided into those containing a stretch of amino acids separating the kinase domain, e.g. VEGFR and PDGFR, and those in which the kinase domain is continuous, e.g. EGFR and HER2 / neu, [14-16]. EGFR family of receptors consists of four structurally related receptors, HER1 (EGFR/ErbB1), HER2 (ErbB2), ErbB3, and ErbB4 [17], for which a variety of different ligands have been characterized 2

[18]. In response to extracellular growth factors, these receptors combine to form 1 of 4 possible ACCEPTED MANUSCRIPT homodimers (EGFR/EGFR, HER2/HER2, ErbB3/ErbB3 and ErbB4/ErbB4) or 6 possible heterodimers (EGFR/HER2, EGFR/ErbB3, EGFR/ErbB4, ErbB3/HER2, ErbB4/HER2 and ErbB3/ ErbB4) [17-20].

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EGFR can bind to several ligands including epidermal growth factor (EGF), transforming growth factorα (TGF-α), betacellulin (BTC), epiregulin (EPR), heparin-binding EGF like growth factor (HB-EGF) and amphiregulin (AR) [21, 22]. In absence of ligand, EGFR exist as monomers on the cell surface, while binding of ligand to EGFR leads to the formation of receptor homo- and heterodimers, depending on whether EGFR is dimerized with another EGFR or with other ErbB family members, respectively [22, 23]. Two different EGFR dimer structures occur, " back-to-back" configuration, in which the two receptors are linked by the dimerization loops so that the associated ligands are located at opposite sites on the dimer, and " head-to-head" configuration, in which subdomain I of each receptor interacts with subdomain III of its dimeric counterpart, so that the ligands are located at the center of the dimer. The back-to-back dimer has better conformational symmetry, a wider interface between the receptors, and a more conserved amino acid sequence at the dimer interface than the alternative head-to-head dimer. Therefore, the back-to-back dimer is favored as the biologically relevant conformation [15].

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Lapatinib, Anilinoquinazolines, also known as (GW-2016) had approved by FDA in 2007, as a dual inhibitor of EGFR and the closely related receptor ErbB2 (HER2). The latter receptor has been identified as an important therapeutic target in a number of cancers as it is overexpressed in around 20–30% of patients with aggressive breast cancer and other tumors. For this reason, Lapatinib is under clinical assays for several solid tumors [24, 25]. Recently, many of anilinoquinazolines had been discovered as EGFR inhibitors, e.g. [26], and Allitinib [27, 28]. Some of the anilinoquinolines (I) [29] act as anti-tumour agents by inhibiting CSF-1 kinase while few 3cyanoquinolines (II) [30] developed as inhibitors of insulin like growth factor receptors (IGF-1R). A few

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4-anilinoquinolines (III) [31] have been found to be TK inhibitors. HIK-272 [32] and EKB-569 [33] are also cyanoquinoline derivatives that inhibit irreversibly EGFR [34, 35] [Figure 1]. Fig.1

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In continuing our work [36, 37] strategy to develop new and potent antitumor agents, herein we carried out design, synthesis and biological evaluation of new class of quinoline-3-carboxamide derivatives as potent EGFR inhibitors with remarkable antitumor effect.

2. Result and Discussion 2.1. Rationale and design In this investigation, Lapatinib (IC50 = 10.8 nM) [38], and the biological active 3, 4, 6-trisubstituted quinoline, compound (IV) (IC50 = 0.65µM) [39] were used as a reference compounds. The design of targeted compounds was derived from the structure optimization of these reference compounds, which depends on the reported SAR of 4-anilinoquinazolines and the molecular modeling studies.

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2.1.1. The reported SAR of the proposed compounds:

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Quinazoline and quinoline-3-carbonitrile of Lapatinib and compound (IV), were bioisosterically replaced with cyanoacrylamide in compound 4 or quinoline-3-carboxamide in compounds 6, 7, 8 and 9 to occupy the adenine region of ATP binding pocket. The H-bond formed via N3 of quinazoline ring in Lapatinib and the mediated water molecule was replaced by a H-bond between the cyano [40] in compound 4 or carboxamide substituent in the other compounds and the amino acid (THR 854) in the ATP binding site. The anilino moiety at position-4 was retained and a small hydrophobic group at its pposition was added to occupy the hydrophobic region, which not occupied by ATP. Different extended side chains at position-6 were designed to mimic that of reference compounds to occupy the sugar/phosphate region. The design included the incorporation of isoxazole or thiazole ring in some targeted compounds to mimic the furan ring of Lapatinib and the other strategy was the introduction of α, β-unsaturated ketone to other targted compounds hoping to form covalent bond with EGFR TK enzyme [Figure 2].

2.2. Molecular modeling studies 2.2.1. Field Analysis

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We carried out molecular field analysis to estimate the similarities between our proposed compounds and the reference ones. Field analysis had approved that there is a high similarities between the 4anilinoquinazoline, 4-anilinoquinoline-3-carbonitrile and 4-anilinoquinoline-3-carboxamide moieties of Lapatinib, compound (IV) and our proposed compounds, respectively [Figure 3].

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2.2.2. Pharmacophore model development:

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The goal of this account is to develop 3D pharmacophore models based on the known EGFR inhibitors, which can correctly reflect the SAR of the existing EGFR inhibitors. Then, this model will be used as 3D search queries for searching the proposed compounds to identify new inhibitors of EGFR. The hit compounds will be subsequently subjected to filtering by Lipinski’s rule of five [41], docking studies and binding energy calculations to refine the retrieved hits. Finally, the promising compounds will be synthesized and will be subjected to an in-vitro inhibitory assay against EGFR protein kinase and antitumor inhibitory activity [42]. 2.2.2.1. Training set selection and conformational analysis: A set of 25 EGFR inhibitors were collected from different literature resources and were carefully chosen to form a training set which was based on the principles of structural diversity and wide coverage of activity range. The IC50 values of the inhibitors in the training set span a range of five orders of magnitude or more (IC50 values range from 29 pM to 2.7µM) [Figure 4]. All compounds were built in 4

2D/ 3D Visualizer within CATALYST4.1 and minimized to the closest local minimum using the ACCEPTED MANUSCRIPT CHARMm-like force field incorporated in the CATALYST program. A series of energetically reasonable conformational models, which represent the flexibility of each compound were generated by CatConf program within CATALYST. Conformations of all molecules were generated by using the ‘Best conformer generation with 20 kcal/mol as energy cutoff and 250 as maximum number conformers, while all other parameters were set to default.

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2.2.2.2. Common features pharmacophores:

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Before performing pharmacophore modeling for the EGFR kinase inhibitors, qualitative HIPHOP [43] models were first generated based on the most-active compounds (6 compounds) in the training set, where the purpose was to identify the common chemical features necessary for potent EGFR kinase inhibitors, as well as to provide some information for the development of quantitative pharmacophore model. In the HIPHOP run, Lapatinib was considered as the reference molecule.

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Structural information from the training set identified a set of features crucial for activity and was considered to represent a pharmacophore hypothesis. Based on our previous expertise [44], HypoGen module in Discovery Studios (DS) 2.5 [45] was used to generate our pharmacophore models wherein it evaluates a collection of conformational models for all compounds and maps them to the selected crucial features. Fig.4

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The best HIPHOP model generated contains three types of chemical features, namely, H-bond donors (HBD), H-bond acceptor (HBA) and hydrophobic features. The top ranked pharmacophore model is expected to identify the common binding features and the hypothetical orientation of the active compounds interacting with their target. Our model is represented by two hydrophobic centers (Hydrophobic 1, Hydrophobic 2; cyan color), one H-bond donor (HBD; magenta color) associated to its protein acceptor site and acceptor atom and two H-bond acceptors (HBA; green color) [Figure 5].

2.2.2.3. Mapping of the proposed compounds:

We proposed a library of 4-anilinoquinoline derivatives and all the proposed compounds (150 compounds) were mapped to the top ranked pharmacophore. The proposed compounds with high fitvalues (> 3, to be more than 50% of the produced common features) were selected for the docking and binding energy calculations [Figure 6, 7]. Fig.6

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2.2.3. Docking and binding energy calculations: ACCEPTED MANUSCRIPT

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Table (1)

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All the proposed compounds that met the fit value criteria were chosen for docking and binding energy calculations. The enzyme structure was obtained from protein data bank (PDB 1XKK) and corrected by DS 2.5. The hydrogen atoms and the missing residues were added and the structure was minimized to relax and correct the clashes. Finally, the binding site was defined and the selected compounds were docked using the default C-docker protocol. The proposed compounds that showed a good - C-docker interaction energy values (> 40) were chosen to carry out binding energy calculations [Table 1]. According to the binding energy values (> -25), the promising compounds were selected for synthesis [Figure 8, 9].

2.3. Chemistry

The synthesis of substituted quinolines has been a subject of great focus in organic chemistry and our paper introduces a new method for synthesis of quinoline-3-carboxamide derivatives. According to

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literature, it has been reported that 2-cyanoacrylamide derivatives were obtained by reacting cyanoacetamide with DMFDMA in toluene at 90 °C for 1-4 h with molar ratio 1:2 [49]. In the present investigation, the desired 2-cyano-acrylamides 2a-c were prepared according to the previously mentioned procedure, but with decreasing the quantity of DMFDMA to (1.1eq) and increasing the reaction time to 7

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h. The reaction mixture revealed the presence of a by-product (by TLC) which supposed to be the dimer 3a-c because its mass has almost twice as the desired product.

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The pure 2-cyanoacrylamides 2a-c were isolated by fractional crystallization from ethanol in about 70% yield. The structures of these compounds were confirmed by 1HNMR, which showed the appearance of singlet signal around 3.18-3.21 ppm due to the 6 protons attributed to N(CH3)2 group. The reaction of 2cyanoacrylamide derivatives 2a-c with the appropriate amines in glacial acetic acid afforded the corresponding 3-(4-substituted phenylamino)-2-cyanoacrylamides 4a-f in a good yield with little quantity of the acylated amine 5 as a by-product which was removed by washing with ethyl acetate or ethanol [Scheme1]. The structures of compounds 4a-f were substantiated by 1HNMR, which showed the disappearance of the singlet signal related to the 6 protons of the N(CH3)2 group around 3 ppm and revealed the presence of a new singlet signal corresponding to the 3 protons of (COCH3 or isoxazoleCH3) around 2.5 ppm. In addition, a new singlet signal attributed to the exchangeable NH protons of aromatic NH in 4a-f was appeared at around 10.5 and 11.3 ppm. All our attempts to cyclize cyanoacrylamide derivatives 4a-f to quinoline-3-carbonitrile derivatives in refluxing phosphoryl chloride were failed. Therefore, we decide to carry out a modification by using phosphoryl chloride as a reactant and carry out the reflux in another solvent as acetonitrile, the quinoline6

3-carbonitrile derivatives were obtained but in very low yield. After many unsuccessful experiments to ACCEPTED MANUSCRIPT improve the yield and to get pure products, we decide to use polyphosphoric acid (PPA). Finally, the cyclization of 3-(4-substituted phenylamineo)-2-cyanoacrylamides 4a-f was achieved by heating in PPA at 100-140 °C for several hours but quinoline-3-carboxamide 6a-f was obtained instead of the cyano derivatives . Compounds 6a-f were established by IR spectra that lacked the band at 2220 cm-1 attributed to the cyano group and showed the presence of NH2 bands at 3220 and 3150 cm-1. 1HNMR showed a

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new singlet signal around 7.6 ppm assigned to the two protons of the carboxamido group (CONH2) which was exchangeable with D2O [Scheme2].

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Reaction of 6-acetylquinoline-3-carboxamide 6a-c with the appropriate aldehyde to give α, βunsaturated keto derivatives 7a-f was carried out according to Claisen-Schmidt condensation conditions where two different reaction conditions were used for the preparation of compounds 7a-f. The first one included the stirring of the acetyl derivatives 6a-c with aldehydes in DMSO and in presence of NaOH at room temperature, while the second one involved the reflux of the acetyl derivatives 6a-c with aldehydes in DMF and in presence of anhydrous potassium carbonate for more than 24 hours. The first reaction condition was preferred as the 6-(3-(4-substituted phenyl)acryloyl)quinoline-3-carboxamide derivatives 7a-f were obtained in good yields and high purity. The structures of 7a-f were verified by 1HNMR that showed the disappearance of the singlet signal corresponding to the 3 protons of the (COCH3) group, also revealed an increase in integration of aromatic protons in addition to the doublet signals assigned to the CH=CH protons at the range of 6.5-8.5ppm.

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6-(2-Aminothiazole-4-yl) quinoline-3-carboxamide derivatives 8a-c were obtained by reaction of 6acetyl quinoline-3-carboxamide 6a-c with thiourea and iodine in refluxing alcohol. The desired compounds 8a-c were confirmed by 1HNMR that lacked the singlet signal attributed the 3 protons of the COCH3 group and revealed the presence of new singlet signal at 7.5 ppm due to 2 protons of the thiazoleNH2, which was exchangeable with D2O. Thiourea derivatives 9a-c were prepared in good yield by

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heating 6-(2-aminothiazol-4-yl) derivatives 8a-c with phenyl isothiocyanate in DMF. The structures of 9a-c were established by 1HNMR that revealed the appearance of a singlet signal around 11.5 ppm corresponding to NHCSNH that disappeared upon deuteration in addition to the increase in the integration of aromatic protons [Scheme3]. Scheme 1

Scheme 2 Scheme 3

2.4. Pharmacology Two assays of biological evaluation had been carried out on the newly synthesized target compounds, cytotoxicity assays and biochemical assays (EGFR protein kinase inhibitor activity). 2.4.1. Cytotoxicity assay:

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Cytotoxicity assays were conducted to determine the level of sensitivity as well as the selectivity ACCEPTED MANUSCRIPT (relative tumor specificity) of cancer and normal cells to the experimental compound. All the newly synthesized compounds were screened for their anticancer activity against MCF-7 cancer cell line using High Throughput Screening Technique and the results are presented in [Table 2].

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The analysis of the IC50 values in [Table 2] revealed that the compounds 4f, 7a and 7b exhibited high anticancer activity against MCF-7 cancer cell line with IC50 values 13.96, 2.16 and 3.46 µM, respectively. The anticancer activity was determined using Doxorubicin as a reference drug. Since these compounds exhibit the best, IC50 values so they can be considered as primary hits. Compounds 4d, 4e, 6f, 7c, 7d and 8a showed good to moderate proliferative activity against the cell line taken for the study with IC50 values ranging from 28.38 to 50.96 µM. The rest of the newly synthesized compounds were found to be inactive against MCF-7 cell line but it could be active against other cell lines so, we decided to carry out a full 60cell lines antitumor activity at the NCI (USA). From the previously mentioned study of the anticancer activity of the newly synthesized compounds against MCF-7 cell line, we could conclude that compound 7a and 7b exhibited the highest anticancer activity among all the tested compounds expressed by their IC50 values 2.16 and 3.46 µM, respectively compared to Doxorubicin IC50= 1.172 µM. These compounds can be used as a lead compounds and by further optimization they could have a high biological profile more than the reference compound. 2.4.2. Biochemical assay (EGFR protein kinase inhibitor activity):

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As it was discussed in the introductory part, quinoline containing molecules are very promising targets as EGFR kinase inhibitors. Therefore, the study of kinase inhibitory activity of these compounds was very important to us because many kinases have been found to be deeply involved in the processes leading to tumor cell growth. Accordingly, if quinoline-3-carboxamides have appeared to be successful EGFR kinase inhibitors, this will be the first reported class of quinoline-3-carboxamide derivatives with EGFR

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inhibitory activities. KINEXUS Corporation, Vancouver, British Columbia, Canada, has carried out the kinase inhibitory activity of the newly synthesized compounds at conc. 10 µM. All the newly synthesized compounds (4d-f, 6d-f, 7a-f, 8a-c and 9a, b) except compound 9c were tested as a competitive inhibitors regarding to the ATP. The in vitro enzyme inhibition determination for the synthesized compounds was

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estimated to evaluate their competition with respect to ATP by performing profiling of the compounds against protein kinase target EGFR using radiometric assay method. Inhibition of target activity by the compound gave negative (-) values, while activation of target activity gave positive (+) value. The results of percentage activity changes were summarized in [Table 2]. Table (2)

By analyzing the data in table (2), we found that two compounds of chalcone series 7b and 7f had competitive values more than ATP itself with percentage activity changes values -67 and -69, respectively. The chalcone series 7a, 7c, 6d, 6f, 9a and 9b derivatives of the quinoline-3-carboxamide showed low to moderate competitive inhibitory effect with % activity changes values ranging from -10 to -19. The rest of the newly synthesized compounds were found to be inactive.

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When we compared the results of the antitumor screening and biochemical assays we found that ACCEPTED MANUSCRIPT compound 7b has a high antitumor activity with IC50 3.46 and a high competitive inhibitory effect with 67 % which proves that its antitumor activity is due to the inhibition of EGFR enzyme. Furthermore, this compound can be used as a lead for further studies. Compound 7f has no activity as antitumor agent but it has a high competitive inhibitory effect with -69 % activity changes value. Compound 7a, showing the highest antitumor activity with IC50 value 2.16, has a moderate competitive inhibitory effect with %

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activity changes value -18. This can be explained that compound 7f may be active on another cell lines, also compound 7a may not be specific inhibitor to EGFR enzyme. 2.4.3. IC50 Determination for compound 7b and 7f against EGFR:

Compounds 7b and 7f profiling activity was undertaken by performing profiling of the two compounds against one protein kinase target EGFR at 6 concentrations (0.001 µM, 0.1 µM, 1 µM, 5 µM, 10 µM and

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25 µM) in singlicate using radiometric assay method [Table 3]. It was found that at 25 µM concentration, the two compounds gave >80% inhibition of EGFR activity. A graph of log inhibitor versus normalized

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response with variable slope [Figure 10] showed increased inhibition of EGFR activity with increasing compound concentration. An IC50 value for compound 7b and 7f were determined to be 5.283 µM and 4.824 µM, respectively.

Table (3)

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By analyzing the obtained results from [Table 3] and [Figure 10], we found that the profiling data for compounds 7b and 7f against EGFR showed a promising inhibition of EGFR target and these compounds can be used as promising lead compounds for further optimizations.

Quantum docking correlation:

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2.5.

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In attempt to understand the high difference between compound 7a, 7b and 7f in the enzymatic assay, we decide to carry out quantum docking to study the effect of electrostatic properties of these compounds.

The quantum docking was carried out by using Glid [50] to study the effect of electrostatic differences between compounds 7a, 7b and 7f. The results showed that these compounds had a different mode of interaction with the active site other than Lapatinib, substitution at position 6 of them overlay at 4substitution of Lapatinib and the substitution at their 4-position overlay at the position-6 of Lapatinib. From that, p-toluidine moiety at position 4 are exposed to a highly hydrophilic pocket so, there is an unfavorable interaction, therefore the enzymatic inhibitory are reduced dramatically [Figure 11]. On the other hand, due to the electrostatic properties of p-chloro and p-bromo derivatives [Figure 12, 13], these compounds achieved a high enzymatic activity. The p-bromo derivative had the highest enzymatic activity, which may be due to the bulk of bromine atom, which is favorable with the active site more than the chlorine, and the presence of chlorine, in the side chain of position-6, which is more favorable than dimethylamino group in the aldehyde part [Figure 14, 15 and 16]. 9

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Conclusion:

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Drug discovery is a complex and lengthy endeavor. Several lead finding strategies include the use of accumulated information for ligands of previously executed discovery programs. In this paper, the molecular modeling techniques, including pharmacophore model development, docking and binding energy calculations were used to design the targeted compounds. The targeted compounds having fitvalue (> 3), -C-docker interaction energy values (> 40) and binding energy values (> -25) were selected for synthesis.

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By comparing the results of antitumor and biochemical assays, we found that compound 7b had a high antitumor activity with IC50 3.46 µM and a high competitive inhibition activity on EGFR with IC50 5.283 µM, which prove that this compound exhibited its antitumor activity due to the inhibition of EGFR enzyme. Compound 7f, which had no activity as antitumor, exerted a high competitive inhibitory with IC50 4.824 µM on EGFR while compound 7a, which showed a high antitumor activity with IC50 value 2.16 µM, had a moderate competitive inhibitory with -18% of activity changes. This could indicate that compound 7a may not be specific inhibitor to EGFR enzyme and compound 7f may be active on another

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cell lines. We carried out quantum docking to study the effect of electrostatic differences between compounds 7a, 7b and 7f and understand the high difference between them in the enzymatic assay. The results showed that: p-toluidine moiety at position 4, in compound 7a, are exposed to a highly hydrophilic pocket so, there is an unfavorable interaction, and therefore, the enzymatic inhibitory activity are reduced dramatically. Due to the electrostatic properties of p-chloro and p-bromo derivatives, compounds 7b and 7f achieved a high enzymatic activity. The p-bromo derivative of compound 7f had the highest enzymatic activity that may be due to the bulk of bromine atom, which is favorable with the active site. Additionally, the presence of chlorine, in the side chain at position-6, is more favorable than dimethylamino group in the aldehyde part of compound 7b. To our knowledge, this will be the first reported class of quinoline-3carboxamide derivatives showing EGFR inhibition activities.

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4. Experimental: ACCEPTED MANUSCRIPT 4.1. Materials and methods:

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All chemicals used were purchased from Sigma-Aldrich (USA) and SD-fine (India) and used without further purification. Separation of the compounds by column chromatography was carried out using silica gel 60 (200–300 mesh ASTM, E. Merck). The quantity of silica gel used was 50–100 times the weight charged on the column. Then, the reactions were monitored using TLC and visualized under U.V. light (254 nm). Melting points (uncorrected) were determined on a XT4 MP apparatus (NODCAR, Egypt). Mass spectra were recorded on a Varian MAT 112 spectrometer AL-Azhar University. Analytical data (IR) were performed at NODCAR, the Micro-analytical Data Center of Cairo University and National Research Center. The 1H NMR spectra were recorded on a Varian spectrometer 300 MHz, at the Microanalytical Data Center of Cairo University and the main laboratories of the chemical war, or 500 MHz, at National Research Center, at 25°C with TMS and solvent signals allotted as internal standards. Chemical shifts were reported in ppm (δ). Elemental analyses were performed on a CHNS-O-Rapid instrument in the Micro-analytical Data Center at Cairo University and the main laboratories of the chemical war.

4.2. Chemistry

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4.2.1. 2-Cyano-N-(4-substituted aryl)-3-(dimethylamino) acrylamide 2a-c A mixture of N-(4-substituted aryl)-2-cyanoacetamide 1a-c (0.1 mol) and DMFDMA (0.11 mol, 14.6 mL) was refluxed in toluene (150 mL) for 7-10 h. The mixture was allowed to cool, filtered, washed with diethyl ether, and dried to yield a mixture of new enamine derivatives 2a-c and other product which may be a dimer 3a-c. The desired enamine derivatives 2a-c were isolated by fraction crystallization from ethyl alcohol in about 70% yield. 4.2.1.1. 2-Cyano-3-(dimethylamino)-N-p-tolylacrylamide (2a) Compound 2a was prepared according to general procedure above by using p-toluidine and refluxed for 7 h. Yellow powder. Yield 70%. M.p. 224-226 °C. IR (ύ max, cm-1): 3341 (NH), 2183 (CN), 1669 (amide CO). 1H-NMR (300 MHz)(DMSO-d6) δ: 2.24 (s, 3H, CH3), 3.21 (s, 6H, N(CH3)2, 7.07 (d, 2H, J= 7.5, Ar-H), 7.44 (d, 2H, J = 8.5, Ar-H), 7.80 ( s, 1H, CH), 8.90 (s, 1H, NH, exchangeable by D2O). ESI-MS: m/z, 229.65 (M+· , [100%]). Anal. Calcd. for C13H15N3O: C, 68.10; H, 6.59; N, 18.33; Found: C, 67.21; H, 6.89; N, 17.85. 4.2.1.2. N-(4-Chlorophenyl)-2-cyano-3-(dimethylamino) acrylamide (2b) Compound 2b was prepared according to general procedure above by using p-chloroaniline and refluxed for 9 h. Yellow powder. Yield 72%. M.p. 226-228 °C. IR (ύ max, cm-1): 3324 (NH), 2195 (CN), 1665 (amide CO). 1H-NMR (300 MHz)(DMSO-d6) δ: 3.21 (s, 6H, N(CH3)2, 7.31 (d, 2H, J= 6.8, Ar-H), 7.59 (d, 2H, J= 7.6, Ar-H), 7.81 (s,1H, CH), 9.20 (s, 1H, NH, exchangeable by D2O).

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DMSO-d6) δ 39.203, 39.757, 103.25, 115.243, 121.819, 128.199, 129.254, 133.622, 156.088, 163.898. ESI-

MS: m/z, 249.50 (M+· , [7.79%]), 251.35 (M+2, [2.95%]). Anal. Calcd. for C12H12ClN3O: C, 57.72; H, 4.84; N, 16.83; Found: C, 56.83; H, 4.76; N, 16.06. 4.2.1.3. N-(4-Bromophenyl)-2-cyano-3-(dimethylamino) acrylamide (2c) Compound 2c was prepared according to general procedure above by using p-bromoaniline and refluxed for 10 h. Yellow powder. Yield 71%. M.p. 216-220 °C. IR (ύ max, cm-1): 3332 (NH), 2227 (CN), 1666 (amide CO). 1H-NMR (300 MHz)(DMSO-d6) δ: 3.18 (s, 6H, N(CH3)2, 7.40 (d, 2H, J= 7.8, Ar-H), 7.49 (d, 2H, J= 8.3, Ar-H), 7.81 (s, 1H, CH), 9.12 (s, 1H, NH, exchangeable by D2O). ESI-MS: m/z, 293.80 (M+· , [6.4%]), 295.65 (M+1, [7.05%]). Anal. Calcd. for C12H12BrN3O: C, 49.00; H, 4.11; N, 14.29; Found: C, 49.98; H, 4.47; N, 13.55. 11

4.2.2. 3-(4-Substitutedphenylamino)-N-(4-substitutedphenyl)-2-cyanoacrylamide 4aACCEPTED MANUSCRIPT f A mixture of 2-cyano-N-(4-substituted aryl)-3-(dimethylamino) acrylamide 2a-c (0.1mol) and the appropriate 4-substituted aniline (0.11 mol) was refluxed while stirring in glacial acetic acid (50 mL) for 16-20 h. The mixture was allowed to cool, concentrated, poured onto water, filtered, washed with water then diethyl ether and dried to afford the target cyanoacrylamide derivatives 4a-f in good yield with a little byproduct of the acetanilides 5, which was removed by washing with ethyl acetate or alcohol.

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4.2.2.1. 3-(4-Acetylphenylamino)-2-cyano-N-p-tolylacrylamide (4a) Compound 4a was prepared according to general procedure above by refluxing p-aminoacetophenone and compound 2a for 18 h. Brownish yellow powder. Yield 68%. M.p. 218-220 °C. IR (ύ max, cm-1): 3410 (NH), 3163 (amide NH), 2211 (CN), 1663 (CH3CO), 1645 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.22 (s, 3H, Ar-CH3), 2.51 (s, 3H, COCH3), 7.08 (d, 2H, J= 6.6, Ar-H), 7.45 (d, 2H, J= 6.5, Ar-H), 7.52 (d, 2H, J= 8.3, Ar-H), 7.91 (d, 2H, J= 6.6,Ar-H), 8.49 (s, 1H, CH), 9.61 (s, 1H, amidic NH, exchangeable by D2O), 10.56 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 319.85 (M+· , [18.93%]). Anal. Calcd. for C19H17N3O2; C, 71.46; H, 5.37; N, 13.16; Found: C, 70.86; H, 5.74; N, 12.98.

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4.2.2.2. 3-(4-Acetylphenylamino)-N-(4-chlorophenyl)-2-cyanoacrylamide (4b) Compound 4b was prepared according to general procedure above by refluxing p-aminoacetophenone and compound 2b for 20 h. Yellow powder. Yield 67 %. M.p. 243-244 °C. IR (ύ max, cm-1): 3315 (NH), 3181 (amide NH), 2205 (CN), 1672 (CH3CO), 1658 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.46 (s, 3H, CH3), 7.34 (d, 2H, J= 7.1, Ar-H), 7.51 (d, 2H, J= 7.1, Ar-H), 7.61 (d, 2H, J= 6.5, Ar-H), 7.92 (d, 2H, J= 8.3, Ar-H), 8.51 (s,1H, CH), 9.79 (s,1H, amidic NH, exchangeable by D2O), 10.66 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 339.90 (M+· , [19.02%]), 341.95 (M+2, [6.55%]). Anal. Calcd. for C18H14ClN3O2; C, 63.63; H, 4.15; N, 12.37; Found: C, 62.9; H, 3.98; N, 12.29.

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4.2.2.3. 3-(4-Acetylphenylamino)-N-(4-bromophenyl)-2-cyanoacrylamide (4c) Compound 4c was prepared according to general procedure above by refluxing p-aminoacetophenone and compound 2c for 18 h. Yellow powder. Yield 72%. M.p. 246-248 °C. IR (ύ max, cm-1): 3321 (NH), 3180 (amide NH), 2204 (CN), 1673 (CH3CO), 1655 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.465 (s, 3H, CH3), 7.47 (d, 2H, J= 7.5, Ar-H), 7.54 (d, 2H, J= 7.8, Ar-H), 7.59 (d, 2H, J= 6.5, Ar-H), 7.91 (d, 2H, J= 8.3, Ar-H), 8.54 (s, 1H, CH), 9.72 (s, 1H, amidic NH, exchangeable by D2O), 10.62 (s, 1H, aromatic NH, exchangeable by D2O). 13C NMR (125 MHz, DMSO-d6) δ 26.421, 81.380, 115.003, 116.916 (d/2C), 118.187, 122.225 (d/2C), 123.409, 129.762 (d/2C), 131.185 (d/2C), 132.301, 149.143, 151.762, 162.019, 196.394. ESI-MS: m/z, 384.00 (M+· , [10.57%]), 385.95 (M+1, [10.04%]). Anal. Calcd. for C18H14BrN3O2; C, 56.27; H, 3.67; N, 10.94; Found: C, 57.18; H, 3.74; N, 10.88. 4.2.2.4. 2-Cyano-3-(4-(N-(5-methylisoxazol-3-yl) sulfamoyl) phenylamino)-N-ptolylacrylamide (4d) Compound 4d was prepared according to general procedure above by refluxing sulfamethoxazole and compound 2a for 17 h. Light brown powder. Yield 69%. M.p. 110-114 °C. IR (ύ max, cm-1): 3566 (NH), 3163 (SO2NH), 3075 (amide NH), 2201 (CN), 1653 (CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.23 (s,3H, isoxazole-CH3), 2.25 (s,3H, Ar-CH3), 6.12 (s, 1H, isoxazole-H), 7.08 (d, 2H, J= 7.9, Ar-H), 7.44 (d, 2H, J= 7.6, Ar-H), 7.57 (d, 2H, J= 7.6, Ar-H), 7.76 (d, 2H, J= 6.8, Ar-H), 8.43 (s,1H, CH), 9.53 (s,1H, amidic NH, exchangeable by D2O), 10.60 (s, 1H, aromatic NH, exchangeable by D2O), 11.44 (s, 1H, isoxazole NH, exchangeable by D2O). 13C NMR (125 MHz, DMSO-d6) δ 20.266, 20.539, 95.247, 100.872, 12

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115.998, 117.402, 120.442, 128.297, 128.783, 129.026, 132.400, 140.314, 148.676, 151.371, 164.220, ACCEPTED MANUSCRIPT 166.921. ESI-MS: m/z, 437.75 (M+· , [7.90%]). Anal. Calcd. for C21H19N5O4S; C, 57.66; H, 4.38; N, 16.01; S, 7.33 ; Found: C, 56.74; H, 4.46; N, 15.64; S, 6.87. 4.2.2.5. N-(4-Chlorophenyl)-2-cyano-3-(4-(N-(5-methylisoxazol-3-yl) sulfamoyl) phenylamino) acrylamide (4e) Compound 4e was prepared according to general procedure above by refluxing sulfamethoxazole and compound 2b for 16 h. Yellow powder. Yield 69%. M.p. 118-120 °C. IR (ύ max, cm-1): 3298 (NH), 3168 (SO2NH), 3105 (amide NH), 2208 (CN), 1654 (CO). 1HNMR (500 MHz)(DMSO-d6) δ: 2.20 (s, 3H, isoxazole-CH3), 6.11 (s, 1H, isoxazole-H), 7.34 (d, 2H, J= 7.6, Ar-H), 7.56 (d, 2H, J= 7.6, Ar-H), 7.60 (d, 2H, J=7.6, Ar-H), 7.77 (d, 2H, J= 6.8, Ar-H), 8.45 (s,1H, CH), 9.76 (s,1H, amidic NH, exchangeable by D2O), 10.67 (s, 1H, aromatic NH, exchangeable by D2O), 11.39 (s, 1H, isoxazole NH, exchangeable by D2O). ESI-MS: m/z, 457.65 (M+· , [0.42%]), 459.75 (M+2, [0.17%]). Anal. Calcd. for C20H16ClN5O4S; C, 52.46; H, 3.52; N, 15.29; S, 7.00 ; Found: C, 53.14; H, 3.09; N, 14.79; S, 6.02. 4.2.2.6. N-(4-Bromophenyl)-2-cyano-3-(4-(N-(5-methylisoxazol-3-yl) sulfamoyl)phenylamino)acrylamide (4f) Compound 4f was prepared according to general procedure above by refluxing sulfamethoxazole and compound 2c for 17 h. Yellow powder. Yield 73%. M.p. 116-120 °C. IR (ύ max, cm-1): 3350 (NH), 3163 (amide NH), 3098 (SO2NH), 2204 (CN), 1657 (CO). 1HNMR (500 MHz)(DMSO-d6) δ: 2.30 (s, 3H, isoxazole-CH3), 6.14 (s, 1H, isoxazole-H), 7.50 (d, 2H, J= 7.2, Ar-H), 7.61 (d, 2H, J= 7.5, Ar-H), 7.64 (d, 2H, J= 8.5, Ar-H), 7.78 (d, 2H, J= 8.3, Ar-H), 8.47 (s,1H, CH), 9.81 (s,1H, amidic NH, exchangeable by D2O), 10.67 (s, 1H, aromatic NH, exchangeable by D2O), 11.36 (s,1H, isoxazole NH, exchangeable by D2O). ESI-MS: m/z, 502.90 (M+· , [0.03%]), 503.60 (M+1, [0.03%]). Anal. Calcd. for C20H16BrN5O4S; C, 47.82; H, 3.21; N, 13.94; S, 6.38, Found: C, 46.94; H, 3.37; N, 12.98; S, 6.91. 4.2.3. 6-Substituted-4-(4-substituted phenylamino)quinoline-3-carbox-amide 6a-f A mixture of 3-(4-substitutedphenylamino)-N-(4-substituted phenyl)-2-cyanoacrylamide 4a-f (0.01 mol) and polyphosphoric acid (PPA, 50 g) was stirred at 100-140 °C for 2-3 h. The mixture was allowed to cool, poured onto ice/water, basified with dilute NaOH solution till PH 6-7 and allowed to stand till complete precipitation. The product was obtained by filtration, washing with water and air-drying. The crude product was purified with ethanol and column chromatography (ethyl acetate: n-hexane, 9: 1) to afford the pure titled compounds 6a-f in 62-70 % yield. 4.2.3.1. 6-Acetyl-4-(p-tolylamino) quinoline-3-carboxamide (6a) Compound 6a was prepared according to general procedure above by refluxing compound 4a for 2 h. at 110 °C. Yellow powder. Yield 67%. M.p. ˃ 300 °C. IR (ύ max, cm-1): 3377 (NH), 3174 (NH2), 1685 (CH3CO), 1649 (amide CO). 1HNMR (300 MHz) (DMSO-d6) δ: 2.36 (s, 3H, Ar-CH3), 2.50 (s, 3H, COCH3), 7.34 (dd, 4H, J= 8.2, Ar-H), 7.46 (d, 1H, J=7.8, quinoline-H), 7.48 (d, 1H, J= 7.8, quinoline-H), 7.69 (s, 1H, quinoline-H), 8.75 (s, 1H, quinoline-H), 7.50 (s, 2H, amide NH2 exchangeable by D2O), 9.09 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 318.85 (M+· , [70.43%]). Anal. Calcd. for C19H17N3O2; C, 71.46; H, 5.37; N, 13.16; Found: C, 70.91; H, 5.03; N, 12.65. 4.2.3.2. 6-Acetyl-4-(4-chlorophenylamino) quinoline-3-carboxamide (6b) Compound 6b was prepared according to general procedure above by refluxing compound 4b for 2.5 h. at 120 °C. Light brown powder. Yield 65%. M.p. 219-222 °C. IR (ύ max, cm-1): 3377 (NH), 3219 (NH2), 1650 (CH3CO), 1627 (amide CO). 1HNMR (300 MHz)(DMSO-d6) δ: 2.50 (s, 3H, COCH3), 7.63 (s, 2H, NH2 exchangeable by D2O), 7.72 (d, 2H, J= 8.3, Ar-H), 7.82 (d, 2H, J= 8.6, Ar-H), 8.07 (d, 1H, J= 7.5, quinoline-H), 8.30 (d , 1H, J= 8.5, quinoline-H), 8.66 (s, 1H, quinoline-H), 9.63 (s, 1H, quinoline-H), 9.81 (s, 1H, NH, exchangeable by D2O). ESI-MS: m/z, 340.00 (M+· , [25.47%]), 342.00 (M+2, [9.23%]). Anal. Calcd. for C18H14ClN3O2; C, 63.63; H, 4.15; N, 12.37; Found: C, 62.67; H, 3.58; N, 12.74. 4.2.3.3. 6-Acetyl-4-(4-bromophenylamino) quinoline-3-carboxamide (6c) 13

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Compound 6c was prepared according to general procedure above by refluxing compound 4c for 2.5 h. at ACCEPTED MANUSCRIPT 120 °C. Yellowish brown powder. Yield 68%. M.p. 206-210 °C. IR (ύ max, cm-1): 3364 (NH), 3215 (NH2), 1656 (CH3CO), 1621 (amide CO). 1HNMR (500 MHz)(DMSO-d6) δ: 2.69 (s, 3H, CH3), 7.88 (d, 2H, J= 8.3, Ar-H), 7.95 (d, 2H, J= 8.6, Ar-H), 8.03 (d, 1H, J= 8.2, quinoline-H), 8.27 (d, 1H, J= 8.5, quinoline-H), 8.55 (s, 2H, amide NH2, exchangeable by D2O), 8.77 (s, 1H, quinoline-H), 9.58 (s, 1H, quinoline-H), 9.77 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 384.45 (M+· , [3.32%]), 385.56 (M+1, [3.05%]). Anal. Calcd. for C18H14BrN3O2; C, 56.27; H, 3.67; N, 10.94; Found: C, 55.61; H, 3.74; N, 9.95. 4.2.3.4. 4-(p-Tolylamino)-6-(N-(5-methylisoxazol-3-yl) sulfamoyl) quinoline-3carboxamide (6d) Compound 6d was prepared according to general procedure above by refluxing compound 4d for 2 h. at 140 °C. Green powder. Yield 65%. M.p. 278-282 °C. IR (ύ max, cm-1): 3356 (NH), 3280 (NH), 3150 (NH2), 1679 (amide CO). 1HNMR (300 MHz) (DMSO-d6) δ: 2.29 (s, 3H, isoxazole-CH3), 2.32 (s, 3H, Ar-CH3), 7.29 (s, 1H, isoxazole-H), 7.28 (d, 1H, J = 8.4, quinoline-H), 7.64 (dd, 4H, J = 7.8, Ar-H), 7.44 (d, 1H, J = 8.4, quinoline-H), 7.70 (s, 2H, amidic NH2, exchangeable by D2O), 8.67 (s, 1H, quinoline-H), 9.14 (s, 1H, quinoline-H), 9.09 (s, 1H, aromatic NH, exchangeable by D2O), 12.3 (s, 1H, SO2NH, exchangeable by D2O). 13C NMR (125 MHz, DMSO-d6) δ 13.436, 20.391, 115.045, 115.170, 118.430, 121.982, 127.113, 128.768, 128.836, 129.811, 131.781, 134.031, 137.652, 143.690, 143.925, 151.054, 161.712, 164.137, 169.413.ESI-MS: m/z, 438.00 (M+. [19.95%]. Anal. Calcd. for C21H19N5O4S; C, 57.66; H, 4.38; N, 16.01; S, 7.33; Found: C, 58.1; H, 4.95; N, 15.34; S, 6.65. 4.2.3.5. 4-(4-Chlorophenylamino)-6-(N-(5-methylisoxazol-3-yl) sulfamoyl) quinoline3-carboxamide (6e) Compound 6e was prepared according to general procedure above by refluxing compound 4e for 2.5 h. at 130 °C. Brown powder. Yield 67%. M.p. 300 °C. IR (ύ max, cm-1): 3310 (NH), 3250 (NH), 3189

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(NH2), 1659 (amide CO). 1HNMR (500 MHz)(DMSO-d6) δ: 2.57 (s, 3H, CH3), 7.23 (s, 1H, isoxazole-H), 7.37 (d, 2H, J= 7.6, Ar-H), 7.41 (d, 2H, J=7.8, Ar-H), 7.64 (d, 1H, J= 8.2, quinoline-H), 7.79 (s, 2H, amidic NH2, exchangeable by D2O), 8.05 (d, 1H, J= 8.3, quinoline-H), 8.34 (s, 1H, quinoline-H), 8.99 (s, 1H, quinoline-H), 11.19 (s, 1H, aromatic NH, exchangeable by D2O), 11.49 (s, 1H, SO2NH, exchangeable by D2O). ESI-MS: m/z, 455.05 (M-2 [0.51%]). Anal. Calcd. for C20H16ClN5O4S; C, 52.46; H, 3.52; N, 15.29; S, 7.00; Found: C, 52.67; H, 3.04; N, 15.27; S, 6.16. 4.2.3.6. 4-(4-Bromophenylamino)-6-(N-(5-methylisoxazol-3-yl) sulfamoyl) quinoline3-carboxamide (6f) Compound 6f was prepared according to general procedure above by refluxing compound 4f for 3 h. at 120 °C. Green powder. Yield 69%. M.p. 270-272 °C. IR (ύ max, cm-1): 3310 (NH), 3192 (NH), 3110 (NH2), 1668 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.53 (s, 3H, CH3), 7.23 (s, 1H, isoxazole H), 7.25 (d, 2H, J = 7.8, Ar-H), 7.29 (d, 2H, J = 8.3, Ar-H), 7.56 (d, 1H, J = 8.2, quinoline-H), 7.75 (d, 1H, J = 8.3, quinoline-H), 7.79 (s, 2H, amidic NH2, exchangeable by D2O), 8.14 (s, 1H, quinoline-H), 8.75 (s, 1H, quinoline-H), 11.23 (s, 1H, aromatic NH, exchangeable by D2O), 11.48 (s, 1H, SO2NH, exchangeable by D2O). ESI-MS: m/z, 502.00 (M+· , [4.18%]), 503.00 (M+1, [3.36%]). Anal. Calcd. for C20H16BrN5O4S; C, 47.82; H, 3.21; N, 13.94; S, 6.38; Found: C, 46.92; H, 3.58; N, 14.83; S, 5.77. 4.2.4. 6-(3-(4-Substituted phenyl) acryloyl)-4-(4-substituted phenylamino) quinoline3-carboxamide derivatives 7a-f Method A: A mixture of 6-acetyl-4-(4-substituted phenyl amino) quinoline-3-carboxamide 6a-c (0.01mol) and the appropriate 4-substituted benzaldehyde (0.012 mol) was dissolved in DMSO (50 mL), then NaOH solution (10%, 5 mL) was added dropwise and the mixture was stirred at R. T. for 24 h. The reaction mixture was poured onto cold water, neutralized with glacial acetic acid, allowed to stand for 4 h, 14

filtered, washed with water and dried. The powder was washed with cold alcohol to afford the pure titled ACCEPTED MANUSCRIPT compounds 7a-f.

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Method B: A mixture of 6-acetyl-4-(4-substituted phenyl amino) quinoline-3-carboxamide 6a-c (0.01 mol) and the appropriate 4-substituted benzaldehyde (0.012 mol) in DMF (50 mL) was treated with anhydrous potassium carbonate (0.02 mole, 2.8 g) and was refluxed at 90 °C for 24 h. The reaction mixture was allowed to cool, poured onto cold water, neutralized with glacial acetic acid, allowed to stand for 4 h, filtered, washed with water and dried. The powder was washed with cold alcohol to afford the pure titled compounds 7a-f.

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4.2.4.1. 6-(3-(4-(Dimethylamino) phenyl)acryloyl)-4-(4-tolylamino)-quinoline-3carboxamide (7a) Compound 7a was prepared from compound 6a and 4-(dimethylamino) benzaldehyde according to general procedure A. Yellow powder. Yield 80%. M.p. ˃ 300 °C. IR (ύ max, cm-1): 3357 (NH), 3176 (NH2), 1684 (CO), 1647 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.32 (s, 3H, Ar-CH3), 3.32 (s, 6H, N(CH3)2, 7.26 (d, 1H, J = 15.5, CH), 7.28 (d, 2H, J = 8.2, Ar-H), 7.44 (d, 1H, J =8.5, quinoline-H), 7.62 (d, 1H, J = 15.5, CH), 7.65 (dd, 4H, J = 8.6, Ar-H), 7.66 (d, 2H, J = 7.6, Ar-H), 7.72 (s, 2H, amide NH2, exchangeable by D2O), 8.69 (d, 1H, J = 8.3, quinoline-H), 8.71 (s, 1H, quinoline-H), 9.1 (s, 1H, quinoline-H), 12.31 (s, 1H, aromatic NH, exchangeable by D2O). 13C NMR (125 MHz, DMSO-d6) δ 20.395, 46.838, 47.070, 115.041, 115.132, 118.442, 122.013, 128.775, 128.859, 129.753, 131.770, 134.020, 137.659, 143.686, 143.925, 150.320, 152.015, 164.118, 175.702ESI-MS: m/z, 449.45 (M-1, [0.01%]). Anal. Calcd. for C28H26N4O2; C,74.65; H, 5.82; N, 12.44; Found : C, 73.86; H, 4.95; N, 12.8. 4.2.4.2. 4-(4-Chlorophenylamino)-6-(3-(4-(dimethylamino) phenyl) acryloyl) quinoline-3-carboxamide (7b) Compound 7b was prepared from compound 6b and 4-(dimethylamino) benzaldehyde according to

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general procedure B. Brown powder. Yield 72%. M.p. ˃ 300 °C. IR (ύ max, cm-1): 3384 (NH), 3214 (NH2), 1631 (CO), 1628 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.97 (s, 6H, N(CH3)2), 6.73 (d, 1H, J = 15.8, CH), 7.63 (d, 1H, J = 15.8, CH), 7.80 (dd, 4H, J = 8.6, Ar-H),7.9 (s, 2H, amide NH2, exchangeable by D2O), 8.05 (dd, 4H, J = 8.2, Ar-H), 8.20 (d, 1H, J = 8.6, quinoline-H), 8.61 (s, 1H, quinoline-H), 9.6 (s, 1H, quinoline-H) , 9.69 (d, 1H, J = 8.5, quinoline-H), 9.76 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 472.80 (M+1, [0.12%]), 474.80 (M+3, [0.04%]). Anal. Calcd. for C27H23ClN4O2; C, 68.86; H, 4.92; N, 11.90; Found : C, 69.19; H, 3.97; N, 12.8. 4.2.4.3. 4-(4-Bromophenylamino)-6-(3-(4-(dimethylamino) phenyl) acryloyl) quinoline-3-carboxamide (7c) Compound 7c was prepared from compound 6c and 4-(dimethylamino) benzaldehyde according to general procedure B. Yellow powder. Yield 70%. M.p. 238-242 °C. IR (ύ max, cm-1): 3380 (NH), 3217 (NH2), 1629 (CO), 1617 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 3.00 (s, 6H, N(CH3)2), 6.98 (d, 1H, J = 16, CH), 7.36 (d, 1H, J = 16, CH), 7.45 (dd, 4H, J = 8.5, Ar-H ), 7.60 (dd, 4H, J = 8.6, Ar-H), 7.77 (d, 1H, J = 8.3, quinoline-H), 7.91 (s, 1H, quinoline-H), 8.27 (s, 2H, amide NH2, exchangeable by D2O), 8.40 (d, 1H, J = 8.3, quinoline-H), 8.54 (s, 1H, quinoline-H), 11.36 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 516.25 (M+1, [1.01%]), 517.20 (M+2, [0.91%]. Anal. Calcd. for C27H23BrN4O2; C, 62.92; H, 4.50; N, 10.87; Found : C, 62.05; H, 4.99; N, 10.04. 4.2.4.4. 6-(3-(4-Chlorophenyl) acryloyl)-4-(p-tolylamino)-quinoline-3-carboxamide (7d)

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Compound 7d was prepared from compound 6a and 4-chlorobenzaldehyde according to general ACCEPTED MANUSCRIPT procedure A. Yellow powder. Yield 85%. M.p. ˃ 300 °C. IR (ύ max, cm-1): 3358 (NH), 3180 (NH2), 1683 (CO), 1646 (amide CO). 1HNMR (500 MHz)(DMSO-d6) δ: 2.29 (s, 3H, Ar-CH3), 7.25 (d, 1H, J = 15.8, CH), 7.27 (dd, 4H, J = 8.6, Ar-H), 7.41 (d, 1H, J = 15.8, CH), 7.44 (dd, 4H, J = 8.3, Ar-H), 7.64 (d, 1H, J = 8.2, quinoline-H), 7.65 (s, 2H, amide NH2, exchangeable by D2O), 7.70 (d, 1H, J = 8.2, quinoline-H), 8.71 (s, 1H, quinoline-H), 9.1 (s, 1H, quinoline-H), 9.07 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 442.10 (M+· , [1.39%]), 444.20 (M+· , [0.91%]). Anal. Calcd. for C26H20ClN3O2; C, 70.67; H, 4.56; N, 9.51; Found : C, 70.16; H, 4.61; N, 9.01. 4.2.4.5. 6-(3-(4-Chlorophenyl) acryloyl)-4-(4-chlorophenylamino) quinoline-3carboxamide (7e) Compound 7e was prepared from compound 6b and 4-chlorobenzaldehyde according to general procedure A. Brown powder. Yield 85%. M.p. 246-250 °C. IR (ύ max, cm-1): 3382 (NH), 3183 (NH2), 1670 (CO), 1632 (amide CO). 1HNMR (500 MHz)(DMSO-d6) δ: 6.97 (d, 2H, J = 8.6, Ar-H), 7.40 (dd, 4H, J = 8.5, Ar-H), 7.88 (d, 2H, J = 8.3, Ar-H), 7.90 (s, 2H, amide NH2, exchangeable by D2O), 7.91 (d, 1H, J = 15.2, CH), 8.19 (d, 1H, J = 15.2, CH), 8.22 (d, 1H, J = 8.4, quinoline-H), 8.38 (d, 1H, J = 8.6, quinoline-H), 8.42 (s, 1H, quinoline-H), 11.32 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 461.10 (M-1, [9.81%]), 462.10 (M+· , [3.45%]). Anal. Calcd. for C25H17Cl2N3O2; C, 64.95; H, 3.71; N, 9.09; Found : C, 64.52; H, 4.12; N, 9.71. 4.2.4.6. 4-(4-Bromophenylamino)-6-(3-(4-chlorophenyl) acryloyl) quinoline-3carboxamide (7f) Compound 7f was prepared from compound 6c and 4-chlorobenzaldehyde according to general procedure A. Brown powder. Yield 82%. M.p. ˃ 300 °C. IR (ύ max, cm-1): 3384 (NH), 3216 (NH2), 1630 (CO), 1625 (amide CO). 1HNMR (500 MHz) (DMSO-d6) δ: 7.19 (d, 2H, J = 8.6, Ar-H), 7.38 (d, 2H, J = 8.5, Ar-H), 7.41 (d, 2H, J = 8.5, Ar-H), 7.50 (d, 2H, J = 8.6, Ar-H),7.93 (s, 2H, amide NH2, exchangeable by D2O), 7.94 (d, 1H, J = 15.5, CH), 8.16 (d, 1H, J = 15.5, CH), 8.22 (d, 1H, J = 8.2, quinoline-H), 8.38 (d, 1H, J = 8.3, quinoline-H), 8.42 (s, 1H, quinoline-H), 8.77 (s, 1H, quinoline-H), 9.99 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 506.15 (M+· , [2.08%]), 507.60 (M+1, [2.10%]). Anal. Calcd. for C25H17BrClN3O2; C, 59.25; H, 3.38; N, 8.29; Found : C, 58.83; H, 3.41; N, 8.62. 4.2.5. 6-(2-Aminothiazol-4-yl)-4-(4-substitutedphenylamino) quinoline-3carboxamide 8a-c A mixture of 6-acetyl-4-(4-substitutedphenylamino) quinoline-3-carboxamide (6a-c) (10 mmol), thiourea (40 mmol, 3.04 g) and I2 (11 mmol, 2.79 g) in methanol (200 mL) was refluxed in an oil bath at 100 °C for 12 h. The reaction mixture was allowed to cool and poured onto (ice/water) containing sodium thiosulphate solution to destroy excess I2. The mixture was allowed to stand for 24h, filtered, washed with water and dried. The product was purified with ethanol and column chromatography (ethyl acetate: nhexane, 9:1) to afford the pure titled compounds 8a-c. 4.2.5.1. 6-(2-Aminothiazol-4-yl)-4-(p-tolylamino)quinoline-3-carboxamide (8a) Compound 8a was prepared from compound 6a according to general procedure above. Yellow powder. Yield 83%. M.p. ˃ 300 °C. IR (ύ max, cm-1): 3376 (NH), 3144 (NH2), 3111 (CONH2), 1649 (CO). 1 HNMR (500 MHz) (DMSO-d6) δ: 2.31 (s, 3H, CH3), 7.26 (dd, 4H, J = 7.8, Ar-H), 7.28 (s, 2H, thiazoleNH2, exchangeable by D2O), 7.40 ( s, 1H, thiazole-H), 7.42 (d, 1H, J = 8.2, quinoline-H), 7.62 (d, 1H, J = 8.2, quinoline-H), 7.70 (s, 2H, amide NH2, exchangeable by D2O), 8.70 (s, 1H, quinoline-H), 9.1 (s, 1H, quinoline-H), 12.28 (s, 1H, aromatic NH, exchangeable by D2O). 13C NMR (125 MHz, DMSO-d6) δ 20.395, 115.053, 118.442, 122.017, 128.779, 131.773, 134.031, 135.935, 136.124, 137.659, 139.103, 143.690, 143.921, 148.160, 149.214, 161.720, 164.114. ESI-MS: m/z, 376.00 (M+· , [28.11%]). Anal. Calcd. for C20H17N5OS; C, 63.98; H, 4.56; N, 18.65; S, 8.54; Found : C, 63.19; H, 4.86; N, 18.48; S, 8.83. 16

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4.2.5.2. 6-(2-Aminothiazol-4-yl)-4-(4-chlorophenylamino) quinoline-3-carboxamide ACCEPTED MANUSCRIPT (8b) Compound 8b was prepared from compound 6b according to general procedure above. Brown powder. Yield 78%. M.p. 242-246 °C. IR 3363 (NH), 3182 (NH2), 3110 (NH2), 1668 (CO). 1HNMR (500 MHz) (DMSO-d6) δ: 7.36 (d, 2H, J = 7.8, Ar-H), 7.41 (d, 2H, J = 7.6, Ar-H), 7.44 (d, 1H, J = 8.3, quinoline-H), 7.66 (s, 1H, thiazole-H), 7.79 (s, 2H, amide NH2, exchangeable by D2O), 7.91 (d, 1H, J = 8.3, quinolineH), 8.05 (s, 2H, thiazole-NH2, exchangeable by D2O), 8.79 (s, 1H, quinoline-H), 8.99 (s, 1H, quinolineH), 11.34 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 396.00 (M+· , [18.24%]), 398.00 (M+2, [6.59%]). Anal. Calcd. for C19H14ClN5OS; C, 57.65; H, 3.56; N, 17.69; S, 8.10; Found : C, 57.85; H, 3.89; N, 17.14; S, 8.54.

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4.2.5.3. 6-(2-Aminothiazol-4-yl)-4-(4-bromophenylamino) quinoline-3-carboxamide (8c) Compound 8c was prepared from compound 6c according to general procedure above. Brown powder. Yield 85%. M.p. 204-208 °C. IR 3330 (NH), 3181 (NH2), 3112(NH2), 1668 (CO). 1HNMR (500 MHz) (DMSO-d6) δ: 7.30 (d, 2H, J = 8.2, Ar-H), 7.43 (d, 2H, J = 8.3, Ar-H), 7.45 (s, 1H, thiazole-H), 7.48 (d, 1H, J = 8.6 , quinoline-H), 7.75 (d, 1H, J = 8.6, quinoline-H), 7.79 (s, 2H, amide NH2, exchangeable by D2O), 8.2 (s, 2H, thiazole-NH2, exchangeable by D2O), 8.78 (s, 1H, quinoline-H), 8.98 (s, 1H, quinolineH), 11.66 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 440.00 (M+· , [23.73%]), 441.00 (M+1, [21.64%]). Anal. Calcd. for C19H14BrN5OS; C, 51.83; H, 3.20; N, 15.91; S, 7.28; Found : C, 51.95; H, 3.61; N, 16.13; S, 6.89. 4-(4-Substitutedphenylamino)-6-(2-(3-phenylthioureido) thiazol-4yl)quinoline-3-carboxamide 9a-c To a solution of 6-(2-aminothiazol-4-yl)-4-(4-substitutedphenylamino)quinoline-3-carbox-amide 8a-c (0.01mol) in DMF (25 mL) was added phenyl isothiocyanate (0.012 mol, 1.43 mL) and the reaction mixture was stirred at R.T. for 24 h, then heated at 60-65 °C for another 24 h. The reaction mixture was allowed to cool, poured onto (ice /water) and left at R. T. for further 24 h to complete precipitation. The solid precipitate was filtered, washed with water, air-dried and purified by column chromatography (ethyl acetate: n-hexane, 9: 1) to afford the pure titled compounds 9a-c. 4.2.6.1. 6-(2-(3-Phenylthioureido) thiazol-4-yl)-4-(p-tolylamino)-quinoline-3carboxamide (9a) Compound 9a was prepared from compound 8a according to general procedure above. Yellow powder. Yield 63 %. M.p. ˃ 300 °C. IR (ύ max, cm-1): 3381 (NH), 3178 (NH), 3130 (NH), 3112 (NH2), 1656 (CO). 1HNMR (500 MHz) (DMSO-d6) δ: 2.23 (s, 3H, CH3), 7.66 (dd, 4H, J = 8.2, Ar-H), 7.23 (t, 1H, J = 8.2, Ar-H), 7.27 (d, 1H, J = 8.4, quinoline-H), 7.40 (d, 2H, J = 8.4, Ar-H), 7.45 (d, 1H, J = 8.6, quinoline-H), 7.64 (d, 2H, J = 8.6 , Ar-H), 7.70 (s, 1H, thiazole-H), 7.71 (s, 2H, amide NH2, exchangeable by D2O), 8.69 (s, 1H, thiazole-NH, exchangeable by D2O), 8.71 (s, 1H, quinoline-H), 9.09 (s, 1H, quinoline-H), 12.22 (s, 1H, aromatic NH, exchangeable by D2O), 12.30 (s, 1H, aromatic NH, exchangeable by D2O). 13C NMR (125 MHz, DMSO-d6) δ 20.417, 115.144, 115.053, 118.442, 122.024, 127.684, 128.775, 131.770, 133.352, 134.020, 137.663, 139.235, 143.686, 143.952, 156.441, 159.011, 161.723, 164.114, 170.262, 171.097. ESI-MS: m/z, 510.10 (M+· , [25.17%]). Anal. Calcd. for C27H22N6OS2; C, 63.51; H, 4.34; N, 16.46; S, 12.56; Found: C, 62.35; H, 4.76; N, 15.81; S, 12.35. 4.2.6.2. 4-(4-Chlorophenylamino)-6-(2-(3-phenylthioureido) thiazol-4-yl)-quinoline-3carboxamide (9b) Compound 9b was prepared from compound 8b according to general procedure above. Brown powder. Yield 65 %. M.p. 210-214 °C. IR (ύ max, cm-1): 3339 (NH), 3220 (NH), 3197 (NH), 3115 (NH2), 1668

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(CO). 1HNMR (500 MHz) (DMSO-d6) δ: 7.09 (t, 1H, J = 8.2, Ar-H), 7.37 (d, 2H, J = 8.2, Ar-H), 7.41 ACCEPTED MANUSCRIPT (dd, 4H, J = 8.3, Ar-H), 7.43 (d, 1H, J = 8.6, quinoline-H), 7.59 (d, 2H, J = 8.3, Ar-H), 7.65 ( s, 1H, thiazole-H), 7.79 (s, 2H, amide NH2, exchangeable by D2O), 7.91 (d, 1H, J = 8.5, quinoline-H) 8.28 (s, 1H, thiazole-NH, exchangeable by D2O), 8.79 (s, 1H, quinoline-H), 8.99 (s, 1H, quinoline-H), 11.34 (s, 1H, aromatic NH, exchangeable by D2O), 11.66 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 532.00 (M+1, [7.59%]), 533.00 (M+3, [2.72%]). Anal. Calcd. for C26H19ClN6OS2; C, 58.80; H, 3.61; N, 15.83; S, 12.08; Found: C, 58.59; H, 3.79; N, 16.07; S, 12.42.

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4.2.6.3. 4-(4-Bromophenylamino)-6-(2-(3-phenylthioureido)thiazol-4-yl)-quinoline-3carboxamide (9c)

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Compound 9c was prepared from compound 8c according to general procedure above. Brown powder. Yield 64%. M.p. 242-244 °C. IR (ύ max, cm-1): 3420 (NH), 3350 (NH), 3222 (NH), 3110 (NH2), 1666 (CO). 1HNMR (500 MHz) (DMSO-d6) δ: 7.09 (t, 1H, J = 8.4, Ar-H), 7.31 (dd, 4H, J = 8.3 , Ar-H), 7.33 (d, 2H, J = 8.2, Ar-H), 7.42 ( s, 1H, thiazole-H), 7.48 (d, 1H, J = 8.6, quinoline-H), 7.56 (d, 2H, J = 8.2, Ar-H), 7.59 (s, 1H, thiazole NH, exchangeable by D2O), 7.75 (d, 1H, J = 8.4, quinoline-H), 7.79 (s, 2H, amide NH2, exchangeable by D2O), 8.78 (s, 1H, quinoline-H), 9.03 (s, 1H, quinoline-H), 11.20 (s, 1H, aromatic NH, exchangeable by D2O), 11.56 (s, 1H, aromatic NH, exchangeable by D2O); 12.48 (s, 1H, aromatic NH, exchangeable by D2O). ESI-MS: m/z, 575.70 (M+· , [10.76%]), 576.80 (M+1, [9.51%]). Anal. Calcd. for C26H19BrN6OS2; C, 54.26; H, 3.33; N, 14.60; S, 11.14; Found: C, 53.89; H, 3.75; N, 13.99 ; S, 10.56.

4.3. Biological evaluation of compounds

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4.3.1. Cytotoxicity assessment Cell culture: MCF-7 human breast cancer cells was grown in RPMI-1640 medium, supplemented with 10% heat inactivated FBS, 50 units/mL of penicillin and 50 mg/mL of streptomycin and maintained at 37 °C in a humidified atmosphere containing 5% CO2. The cells were maintained as “monolayer culture” by serial sub-culturing. SRB cytotoxicity assay: Cytotoxicity was determined using SRB method as previously described by Skehan et al. [51]. Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at 1000-2000 cells/well in RPMI-1640 supplemented medium. After 24 h, cells were incubated for 72 h with various concentrations of the tested compounds. Following 72 h treatment, the cells will be fixed with 10% trichloroacetic acid for 1 h at 4 ºC. Wells were stained for 10 min at R.T. with 0.4% SRB dissolved in 1% acetic acid. The plates were air dried for 24 h and the dye was solubilized with Tris-HCl for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300, FL, USA). The IC50 values were calculated according to the equation for Boltzman sigmoidal concentrationresponse curve using the nonlinear regression fitting models (Graph Pad, Prism Version 5). 4.3.2. Biochemical Assay (EGFR protein kinase inhibitor activity): 4.3.2.1. Evaluation of synthesized compounds at concentration 10 µM against EGFR TK target:

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The in vitro enzyme inhibition determination for the synthesized compounds was carried out in ACCEPTED MANUSCRIPT KINEXUS Corporation, Vancouver, British Columbia, Canada. Kinexus has developed an open-access, on-line resource called DrugKiNET, www.drugkinet.ca. The evaluation performed profiling of the compounds against PK target EGFR at 1 concentration (10 µM) in singlicate using radiometric assay method to evaluate the competition of the targeted compounds with respect to ATP. Kinexus evaluated the profile of various compounds against PK targets by employing the standardized assay methodology. The intra-assay variability was determined to be less than 10%. Inhibition of target activity by the compound gives negative (-) values while activation of target activity gives positive (+) value. Kinexus considers only values of >25% change in activity compared to control to be significant. Materials: Quality control and reagents: the PKs employed in the compound profiling process was cloned, expressed and purified using proprietary methods. Quality control testing is routinely performed to ensure compliance to acceptable standards. 33P-ATP was purchased from PerkinElmer. All other materials were of standard laboratory grade. The compounds were supplied by Kinexus as a powder and stock solution was made in DMSO. The stock solution was then diluted to form an assay stock solution and this was used to profile against the protein kinase targets. The assay condition for the PK targets was optimized to yield acceptable enzymatic activity. In addition, the assays were optimized to give high signal-to-noise ratio. Protein Kinase (PK) Assays: a radioisotope assay format was used for profiling evaluation of PK targets and all assays were carried out in a designated radioactive working area. PK assays (in singlicate) were performed at ambient temperature for 20-30 min in a final volume of 25 µL according to the following assay reaction recipe: component 1: 5 µl of diluted active PK target (~10-50 nM final concentration in the assay). Component 2: 5 µl of stock solution of substrate (1-5 µg of peptide substrate). Component 3: 5 µl of kinase assay buffer. Component 4: 5 µl of compound (50 µM) or 10%

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DMSO. Component 5: 5 µl of 33P-ATP (250 µM stock solution, 0.8 µCi). The assay was initiated by the addition of 33P-ATP and the reaction mixture incubated at ambient temperature for 20-30 minutes. After the incubation period, the assay was terminated by spotting 10 µL of the reaction mixture onto Multiscreen phosphocellulose P81 plate. The Multiscreen phosphocellulose P81 plate was washed 3 times for approximately 15 min each in a 1% phosphoric acid solution. The radioactivity on the P81 plate was counted in the presence of scintillation fluid in a Trilux scintillation counter. Blank control was set up that included all the assay components except the addition of the appropriate substrate (replaced with equal volume of assay dilution buffer). The corrected activity for PK target was determined by removing the blank control value. 4.3.3. IC50 Determination for compound 7b and 7f against EGFR: IC50 determination of the two compounds (7b and 7f) against EGFR was achieved by performing profiling of the two compounds against 1 PK target EGFR at 6 concentrations (0.001 µM, 0.1 µM, 1 µM, 5 µM, 10 µM and 25 µM) in singlicate using radiometric assay method. It was carried out in KINEXUS Corporation, Vancouver, British Columbia, Canada by the same radiometric assay method of PK assays, mentioned previously, using the 6 concentrations of each compound of 7b and 7f. A graph of log inhibitor versus normalized response with variable slope [Figure 9] was generated using the Prism software.

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5. References:

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[1] R. W. Ruddon, Cancer biology, Oxford university press, fourth ed., 2007, pp. 4. [2] N. James, Cancer a very short introduction, New York, NY: Oxford University Press, 2011, pp. 1-3. [3] A. Ali and S. Bhattacharya, Bioorg. Med. Chem. 22, (2014), 4506-4521. [4] B. Maji and S. Bhattacharya, Chem. Commun. 50, (2014), 6422-6438. [5] D. Hanahan and R. A. Weinberg, Review, The hallmarks of cancer, Cell 1 (2000) 57-70. [6] C. M. porth, K. J. Gaspard and K. A. Noble, Essential pathophysiology: Concepts of altered health states, third ed., 2007, pp.151. [7] D. Fabbro, , S. Ruetz, E. Buchdunger, S. W. Cowan-Jacob, G. Fendrich, J. Liebetanz, J. Mestan, T. O'Reilly, P. Traxler, B. Chaudhuri, H. Fretz, J. Zimmermann, T. Meyer, G. Caravatti, P. Furet and P. W. Manley, Protein kinases as targets for anticancer agents: from inhibitors to useful drugs, Pharmacol. Ther. 93 (2002) 79-98. [8] J. B. Leppard, J. J. Champoux, Chromosoma, 114 (2005) 75-85. [9] P. Chaudhuri, B. Ganguly, and S. Bhattacharya, J. Org. Chem. 72 (2007) 1912-1923. [10] P. Chaudhuri, H. K. Majumder, and S. Bhattacharya, J. Med. Chem. 50 (2007) 2536-2540. [11] L. A. Pinna and T.W. Patricia, Inhibitors of protein kinases and protein phosphates, 2005, pp. 48. [12] D. Secko, Protein phosphorykation: A global regulator of cellular activity, 2003. Through: http://www.scq.ubc.ca/protein-phosphorylation-a-global-regulator-of-cellular-activity. [13] D. J. Matthews and M. E. Gerritsen, Targeting protein kinases for cancer therapy, 2009, pp. 8-10. [14] A. Kleespies, K. W. Jauch and C. J. Bruns, Tyrosine kinase inhibitors and gemcitabine: New treatment options in pancreatic cancer?, Drug Resist Update 9, 2006, pp. 1-18. [15] Y. M. ueda, Cell signaling reactions, single molecular kinetic analysis, 2011, pp. 2-3. [16] M. Muller, Lecture Material Online: Lecture 6: Cellular Communication, 2003. http://www.uic.edu/classes/bios/bios100/summer2003/lect06.htm. [17] N. E. Hynes and H. A. Lane, ERBB receptors and cancer: the complexity of targeted inhibitors, Nat. Rev. Cancer 5 (2005) 341-354. [18] http://www.cellsignal.com/reference/kinase/tk.html [19] R. S. Herbst, J. V. Heymach and S. M. Lippman, Lung cancer, N. Engl. J. Med. 13 (2008) 13671380. [20] M. A. Olayioye, R. M. Neve, H. A. Lane and N. E. Hynes. The ErbB signaling network: receptor heterodimerization in development and cancer, EMBO J. 13 (2000) 3159-3167. [21] C. L. Arteaga, Epidermal growth factor receptor dependence in human tumors: more than just expression?, Oncologist 4 (2002) 31-39. [22] N. E. Hynes, K. Horsch, M. A. Olayioye and A. Badache, The ErbB receptor tyrosine family as signal integrators, Endocr- Relat. Cancer 8 (2001) 151-159. [23] J. Schlessinger, Cell signaling by receptor tyrosine kinases, Cell 103 (2000) 211-225. [24] D. W. Rusnak, K. Affleck, S. G. Cockerill, C. Stubberfield, R. Harris, M. Page, K. J. Smith, S. B. Guntrip, M. C. Carter, R. J. Shaw, A. Jowett, J. Stables, P. Topley, E. R. Wood, P. S. Brignola, S. H. Kadwell, B. R. Reep, R. J. Mullin, K. J. Alligood, B. R. Keith, R. M. Crosby, D. M. Murray, W. B. Knight, T. M. Gilmer and K. Lackey, The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: Potential therapy for cancer, Cancer Res. 61 (2001) 7196-7203. [25] P. J. Medina and S. Goodin, Lapatinib: A Dual Inhibitor of Human Epidermal Growth Factor Receptor Tyrosine Kinases, Clin. Ther. 30 (2008) 1426-1447. [26] http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm?fuseaction=Search.DrugDetails)

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[27] L. Lishan, G. Zhiwei, C. Xiaoyan and Z. Dafang, Development and validation of a sensitive LCACCEPTED MANUSCRIPT MS/MS assay for the simultaneous quantification of allitinib and its two metabolites in human plasma, J. Pharm. Biomed. Anal. 86 (2013) 49-55. [28] H. Xie , L. Lin, Tong L, Y. Jiang , M. Zheng , Z. Chen , X. Jiang , X. Zhang , X. Ren , W. Qu , Y. Yang , H. Wan , Y. Chen , J. Zuo , H. Jiang , M. Geng , J. Ding , AST1306, a novel irreversible inhibitor of the epidermal growth factor receptor 1 and 2, exhibits antitumor activity both in vitro and in vivo, PLoS. One 6 (2011) e21487. [29] Scott, D. A., Balliet, C. L., Cook, D. J., Davies, A. M., Gero, T. W., Omer, C. A., Poondru, S., Theoclitou, M. E., Tyurin, B., Zinda and M. J., Identification of 3-amido-4-anilinoquinolines as potent and selective inhibitors of CSF-1R kinase, Bioorg. Med. Chem. Lett. 19 (2009) 697-700. [30] Miller, L. M., Mayer, S. C., Berger, D. M., Boschelli, D. H., Boschelli, F., Di, L., Du, X., Dutia, M., Floyd, M. B., Johnson, M., Kenny, C. H., Krishnamurthy, G., Moy, F., Petusky, S., Tkach, D., Torres, N., Wu, B., Xu and W., Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment, Bioorg. Med. Chem. Lett. 19 (2009) 62-66. [31] Assefa, H., Kamath, S., Buolamwini and J. K., 3D-QSAR and docking studies on 4anilinoquinazoline and 4-anilinoquinoline epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, J. Comput. Aided. Mol. Des. 17 (2003) 475-493. [32] S. K. Rabindran , C. M. Discafani, E. C. Rosfjord , M. Baxter , M.B. Floyd , J. Golas , W. A. Hallett , B. D. Johnson , R. Nilakantan , E. Overbeek , M. F. Reich , R. Shen , X. Shi , H. R. Tsou , Y. F. Wang and A. Wissner, Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase, Cancer Res. 64 (2004) 3958-3965. [33] C. Erlichman, M. Hidalgo, J. P. Boni, P. Martins, S. E. Quinn, C. Zacharchuk, P. Amorusi, A. A. Adjei, and E. K. Rowinsky, Phase I Study of EKB-569, an Irreversible Inhibitor of the Epidermal Growth Factor Receptor, in Patients With Advanced Solid Tumors, J. Clin. Oncol. 24 (2006) 2252-2260. [34] H. R. Tsou , N. Mamuya , B. D. Johnson , M. F. Reich, B. C. Gruber, F. Ye, R. Nilakantan, R. Shen, C. Discafani, R. D. Blanc, R. Davis, F. E. Koehn, L. M. Greenberger, Y. F. Wang and A. Wissner, 6Substituted-4-(3-bromophenyl- amino)quinazolines as putative irreversible inhibitors of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases with enhanced antitumor activity, J. Med. Chem. 44 (2001) 2719-2734. [35] C. M. Discafani, M. L. Carroll, M. B. Floyd, I. J. Hollander, Z. Husain, B. D. Johnson, D. Kitchen, M. K. May, M. S. Malo, A. A. Minnick, R. Nilakantan, R. Shen, Y. F. Wang, A. Wissner and L. M. Greenberger. Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with in vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785), Biochem. Pharmacol. 57 (1999) 917-925. [36] M. M. Hamed, Dalal A. Abou El Ella, A. B. Keeton, G. A. Piazza, M. Engel, b R. W. Hartmann and A. H. Abadi. Quinazoline and tetrahydropyridothieno[2,3-d]-pyrimidine derivatives irreversible EGFR tyrosine kinase inhibitors: influence of the position 4 substituent, Med. Chem. Commun. 4 (2013) 12021207. [37] Diaa A. Ibrahim and Amira M. Elmetwally. Design, Synthesis, and Biological Evaluation of Novel Pyrimidine Derivatives as CDK2Inhibitors. Eur. J. Med Chem. 45 (2010) 1158–1166. [38] Zev A. Wainberg, Adrian Anghel, Amrita J. Desai, et al.Lapatinib, a Dual EGFR and HER2 Kinase Inhibitor, Selectively Inhibits HER2-Amplified Human Gastric Cancer Cells and is Synergistic with Trastuzumab In vitro and In vivo, Clin Cancer Res. 16 (2010) 1509-1519. [39] N. Akula, J. Sridhar and N. Pattabiraman, Binding modes of 6, 7 di-substituted4-anilinoquinoline3carbonitriles to EGFR, Bioorg. Med. Chem. Lett. 14 (2004) 3397-3400. [40] F. Pisaneschi, Q. Nguyen, E. Shamsaei, M. Glaser, E. Robins, M. Kaliszczak., G. Smith, A. C. Spivey and E. O. Aboagye, Development of a new epidermal growth factor receptor positron emission 21

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tomography imaging agent based on the 3-cyano-quinoline core: Synthesis and biological evaluation, ACCEPTED MANUSCRIPT Bioorg. Med. Chem. 18 (2010) 6634-6645. [41] C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug. Deliv. Rev. 23 (1997) 3-25. [42] P. S. Charifson, A. L. Grillot, T. H. Grossman, J. D. Parsons, M. Badia, S. Bellon, D. D. Deininger, J. E. Drumm, C. H. Gross, A. LeTiran, Y. Liao, N. Mani, D. P. Nicolau, E. Perola, S. Ronkin, D. Shannon, L. L. Swenson, Q. Tang, P. R. Tessier, S. K. Tian, M. Trudeau, T. Wang, Y. Wei, H. Zhang and D. Stamos, J. Med. Chem. 51 (2008) 5243-5263.

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[43] S. Chun, J. Y. Lee, S. G. Ro, K. W. Jeong, Y. Kim, and C. J. Yoon, 3D Quantitative and Qualitative Structure-Activity Relationships of the δ-Opioid Receptor Antagonists, Bull. Korean Chem. Soc. 29 (2008) 656-662. [44] Diaa. A. Ibrahim, J. Boucau, D. H. Lajiness, S. K. Veleti, K. R. Trabbic, S. S. Adam, D. R. Ronning, and S. J. Sucheck, Design, synthesis and X-ray analysis of a glycoconjugate bound to Mycobacterium tuberculosis antigen 85C, Bioconjucate chem. 23 (2012) 2403-2416. [45] Discovery Studio version 2.5 (DS 2.5) User Manual; in, Accelrys Inc.: San Diego, CA, 2009. [46] D. W. Fry , A. J. Kraker , A. McMichael , L. A. Ambroso , J. M. Nelson , W. R. Leopold , R. W. Connors and A. J. Bridges .A specific inhibitor of the epidermal growth factor receptor tyrosine kinase, Science 265 (1994) 1093-1095. [47] Y. Kitano, T. Suzuki, E. Kawahara and T. Yamazaki, Synthesis and inhibitory activity of 4-alkynyl and 4-alkenylquinazolines: Identification of new scaffolds for potent EGFR tyrosine kinase inhibitors, Bioorg. Med. Chem. Lett. 17 (2007) 5863-5867. [48] Arkin, M. and Moasser, M.M., HER-2-directed, small-molecule antagonists, Curr. Opin. Investig. Drugs 9 (2008) 1264-1276. [49] Y. Kobayashi, T. Nakatani, R. Tanaka, M. Okada, E. Torii, T. Harayama and T. Kimachi, aDimethylaminomethylenation-induced HoubeneHoesch-type cyclization of cyano-acetanilides: a practical synthesis of 3-formyl-4-hydroxyquinolin-2(1H)-ones, Tetrahedron 67 (2011) 3457-3463. [50] Glide New York: Schrodinger; 2012. [51] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H. Bokesch, S. Kenney, and M.R. Boyd, New colorimetric cytotoxicity assay for anticancer-drug screening, J. Nat. Cancer Inst. 82 (1990) 1107-1112.

22

-C-Docker

Binding

interaction Energy

Energy

45.9836

77.6742

-47.468

4.50162

20.3636

54.0045

-43.5268

4d

3.7296

28.2142

50.6694

-38.6957

4e

3.69332

31.1379

52.2855

-34.1397

4f

3.56844

28.4456

51.9906

-35.8542

6d

3.97321

16.3022

49.3521

-35.1634

6e

3.97008

17.7857

51.0738

-36.3098

6f

3.93832

16.1996

50.0022

-35.7868

7a

3.72949

23.7218

59.1896

-35.6041

7b

3.75976

24.0822

57.0524

-34.2012

7c

3.77084

18.9062

63.7652

-37.6808

7d

3.85343

19.9801

58.0604

-40.4646

7e

3.80202

19.7491

59.4547

-39.8478

7f

3.78797

20.2433

59.7126

-39.7814

8a

3.65428

16.4561

42.4096

-25.3996

8b

3.64623

17.2357

43.3076

-25.3862

8c

3.67366

15.3983

44.4899

-25.303

9a

3.94805

17.7823

58.2713

-36.1471

9b

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Table (1): The newly synthesized compounds with their promising fit-values, -C-docker energy, -CACCEPTED MANUSCRIPT docker interaction energy and binding energy compared with Lapatinib and compound (IV).

3.93835

18.1818

55.8579

-38.6939

14.0955

51.7318

-37.0541

-C-Docker Energy

Lapatinib

4.91551

Compound (IV)

3.94565

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Fit values

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Code

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Table (2): The results of EGFR TK inhibition activity and antitumor activity assays: ACCEPTED MANUSCRIPT IC50 µM(against EGFR % Activity CDK2 % Activity Code MCF-7 cell line) changes changes -------------99 -------------Lapatinib --------------

---------------

1.172

Staurosporin

---------------

-100

---------------

4d

4

0

50.85

4e

10

-4

28.38

4f

-1

-12

6d

-10

-11

6e

8

-10

6f

-18

-1

7a

-18

-5

7b

-67

-1

7c

-19

-8

7d

-7

7e

1

7f

-69

8a

-4

8b

9a

9c

119.6 145

SC

33.71 2.16

M AN U

3.46

50.96 41.24

-12

277.1

-3

327.5

-2

62.21

5

-7

315.3

3

-9

333.8

-12

0

385.7

-15

-1

326.5

NT

656.6

EP

9b

13.96

-7

TE D

8c

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Doxorubicin

NT

AC C

Table (3): % Activity change of EGFR target in the presence of compounds 7b and 7f and IC50 values of them. Compound ID

% Activity Change 0.001 µM

% Activity Change 0.1 Μm

% Activity Change 1 µM

% Activity Change 5 Μm

% Activity Change 10 µM

% Activity Change 25 µM

7b

1

3

-13

-48

-67

-89

7f

0

-3

-15

-50

-69

-90

2

IC50 (µM) 5.283 4.824

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Fig.1: Some of the anilinoquinolines act as anti-tumour

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ACCEPTED MANUSCRIPT

Fig.2: Similarities of pharmacophore features between target compounds and reference compounds.

1

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Fig.3: Molecular field analysis of 4-anilinoquinazoline, 4-anilinoquinoline-3-carbonitrile and 4-anilinoquinoline-

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3-carboxamide [cyan: quinazoline as a reference; pink: 3-cyanoquinoline (similarity = 0.823) and orange: quinoline-3-carboxamide (similarity = 0.868)].

2

Cl

ACCEPTED MANUSCRIPT

F CN

N

O

CN

HN N

O

N

O

HN

H N

N

Cl

O (0.13 µM)

HN

N

(30 nM)

Br

EP

Br

AC C

O

O O

Br

S

N (2.29 µM)

CN

O

N

(0.55 µM)

Br HN

H N

H3CO(H2C)2O

CN

O

N

(0.93 µM)

Br

Br H N

CN

HN CN

O

N

N (1.62 µM)

(0.96 µM)

Cl Cl

F

F

HN

CN

HN

H N

N

CN

F

N

N

N

CN

HN

Cl

H N

CN

(0.15 µM)

N

H N

H3CO

CN

(0.94 µM)

O

O

(0.33 µM)

N

HN

H N

N

HN

TE D O

(0.25 µM)

O

Br

(0.59 µM)

H N

H3COH2CO

N

HN

O

Lapatinib(10.8nM)

Br

HN

H N

O

N N

H N

HN

H N

O

(0.31 µM)

CN

O

Br

CN N

OS

CN

N

Br H N

HN

H N

N

N

O

O

Br

(0.037 nM)

N

H N

N

Erlotinib (2nM)

N

Cl CN

N

O

Cl N

HN

N

N

Cl

O

H N

O

F

F

N

F

HN

HN

O O

Gefitinib(26nM)

Canertinib(0.8 nM)

HN

N Cl

Cl N

O

HIK-272 (92nM)

O

O

Cl CN

RI PT

HN

O HN

HN

F

N

F HN

O

N PD153035(29 pM)

H N

EKB-569(39 nM)

O

O

H3C

N

Cl

H N O

Br

N

O

N

EKI-785 (250pM)

N

N CH3

O

N

O

IV (0.65 µM)

H3C

HN

Br

SC

O

HN

H N

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N

H 3C

HN

H N

F

H N

N O

HN CN N (2.64 µM)

O

H N

N O

HN CN N (2.7 µM)

Fig.4: The literature compounds used as a training set in the pharmacophore building with their IC50 values [29, 30, 32, 33, 35, 36, 43-45].

3

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ACCEPTED MANUSCRIPT

Fig.5: a) The common features pharmacophore generated from training set; two hydrophobic centers (1, 2; cyan

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color), one HBD (magenta color) and two HBA (green color).

Fig.5: b) The common features pharmacophore generated from training set including the distance between the five generated features.

4

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ACCEPTED MANUSCRIPT

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Fig.6: Mapping of compound 4d (fit value =3.7296) on the generated high ranked pharmacophore.

Fig.7: Mapping of compound 6d (fit value = 3.97321) on the generated high ranked pharmacophore.

5

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ACCEPTED MANUSCRIPT

Fig.8: H-Bond interactions of compounds 4d with the active site of EGFR (no H-bonds; binding energy

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-38.6957).

Fig.9: a) H-Bond interactions of compound 7d with the active site of EGFR (2H-bonds with MET 793; binding energy -40.4646).

6

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Fig.9: b) H-Bond interactions of compound 7a with the active site of EGFR (3H-bonds with MET 793,

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THR 854 and GLN 791; binding energy -35.6041).

Fig.10: Graph of log compounds 7b and 7f concentrations against % inhibition of activity.

7

SC

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ACCEPTED MANUSCRIPT

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Fig.11: Overlay of compound 7a (in grey color) on Lapatinib (in green color) according to the result of quantum

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docking.

Fig.12: Overlay of compound 7b (in grey color) on Lapatinib(in green color) according to the result of quantum docking.

8

SC

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ACCEPTED MANUSCRIPT

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Fig.13: Overlay of compound 7f (in grey color) on Lapatinib (in green color) according to the result of quantum

AC C

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docking.

Fig.14: Unfavorable interaction of 7a with the active site.

9

SC

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ACCEPTED MANUSCRIPT

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Fig.15: Favorable interaction of compound 7b with the active site of EGFR.

Fig.16: Favorable interaction of compound 7f with the active site of EGFR.

10

R1

R1 HN C

HN

N

C

O

1a-c

N

HN O O

+ R1

N

2a-c

NH

O

+

R2

O

N H 5 4 a : R1= p-CH3 , R2= COCH3 b : R1= p-Cl , R2= COCH3 c : R1= p-Br , R2= COCH3

N H 4a-f d : R1=p-CH3 , R2= , R2=

N O

2, 3 a : R1= p-CH3 b : R1= p-Cl c : R1= p-Br

N

O HN S O O S HN O

N O O O S HN

M AN U

e : R1=p-Cl

f : R1=p-Br , R2=

N O

Reagents and conditions: (i) DMFDMA, reflux; (ii) Aromatic amine, glacial acetic acid, reflux.

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Scheme1a

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Highlights

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We use computer aided drug design to discover site-specific inhibitors for EGFR. The proposed compounds were selecting due to their fit values, docking scores and binding energy. The selected compounds were synthesized and changes in the side chains were studied to yield notable tumor activity. The newly synthesized compounds were studies as EGFR inhibitors and as antitumor agents.

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Al-Azhar Univ-Mycol.Center

1HA.D00

12/08/06 11:06:26

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Dr .RashaMohamed-Dl-Hl-DMS0-D20- Ma in.Defence .Chemi cal .Laboratory

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Al·Azhar Univ-Mycol.Center

,. *** CLASS-5000 ***

Report No. = 1 Data : D5RASHA.DOO 12/08/06 13:34:27 Sample : Oil/ RASHA ID : D5 Operator Method File Nam,3 : DI.MET

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18

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%T 70

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Intensity 92.90765 93.46690 90.46324 91.42358 81.90576 95.14559 77.60425 65.25110 74.16739 74.10172

AC C

Position 3908.04 3588.88 3109.65 2859.92 2198.45 1746.23 ..1439.6. 1239.04 913.129 635.43

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No. Position 3 3854.04 3497.27 7 11 3062.41 15 2593.79 19 2030.68 23 1663.3 27 ...1.3"Z0.18 1183.11 31 35 879.381 568.898 39

Intensity 92.75737 93.01759 89.73765 92.70856 92.07459 59.85882 68.76503 64.50340 74.26440 68.33370

No. 4 8 12 16 20 24 28 32 36 40

40

29

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Position 3750.87 3408.57 2965.02 2503.15 1957.39 1591.95 1303.64 1116.58 821.527 494.652

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No. 2 6 10 14 18 22 26 .. 30 34 38

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[ Result of Peak Picking ] Intensity No. Position 1 3961.07 92.82048 5 3711.33 93.35388 90.14297 3173.29 9 2917.77 13 90.49728 17 2428.9 92.55170 21 92.21064 1904.36 1520.6 --- 61.15472 25 29 1267 59.56821 71.35889 958.448 33 68.35287 773.315 37 41 436.798 78.96593

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Peak Find - E1

Intensity 93.00979 89.41767 90.83929 92.64628 92.46417 58.18309 64.02718 69.62143 63.78024 66.63996

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