Uses of Anthranilic Acid for the Synthesis of Dihydroquinazolin Derivatives with Antitumor , Antiproliferative and Pim-1 kinase Activities

The reaction of anthranilic acid with ethoxycarbonylisothiocyanate gave the ethyl 4-oxo-2-thioxo-1,2-dihydroquinazoline-3(4H)-carboxylate (4). The reaction of compound 4 with hydrazine hydrate and α-halocarbonyl derivatives was found to give either hydrazono or S-alkylated products. Heterocyclization reactions of some of the S-alkylated derivatives 8 and 12 were carried out to afford thiazole, pyran and pyridine derivatives. The cytotoxicity of the newly synthesized compounds towards the six cancer cell lines NUGC, DLD-1, HA22T, HEPG-2, HONE-1 and MCF-7 showed that compounds 6, 8, 13, 19c–f, 21b-f, 24a and 24c with the highest cytotoxicity. The c-Met kinase inhibition for some of the selected compounds showed that compounds 8, 13, 19d, 21e, 21f and 24a were the most active compounds. Screening toward tyrosine kinases revealed that compounds 13, 21e and 24a exhibit the highest inhibitions and therefore their molecular modeling was described. In addition, compounds 13 and 24a showed the highest activities towards Pim-1 kinase.


Introduction
2-Thioxoquinazolin-4(1H)-ones are important heterocyclic compounds that are widely present in natural products as well as medicinal, and pharmacological compounds. 1In addition, several thioxoquinazolin analogues have been developed as antitumor, antibiotic, antidefibrillator and antipyretic agents (Fig. 1).Furthermore, they display a broad range of applications against diabetes, 2 cancer, 3 and as selective plant grow regulators. 4,54][15][16][17][18][19][20][21][22][23][24][25][26][27] Recently, our research group was involved in the synthesis and determination of the anti-proliferative and anti-inflammatory activity of a large number of heterocyclic compounds. 28,29In the continuation of this pro-gram, in this context, we aimed to develop an efficient and facile approach to synthesize and measure the cytotoxicity of a series of thioxoquinazolin-4(1H)-ones from anthranilic acid and ethoxycarbonylisothiocyanate.

Results and Discussion
Quinazoline derivatives showed a wide range spectrum of biological applications, especially in the field of anti-cancer therapy 30 which encouraged us to direct our work towards their synthesis.Thus, in the present work we aimed to synthesis a series of heterocyclic compounds derived from dihydroquinazoline derivative.The reaction of anthranilic acid (1) with ethoxycarbonylisothiocyanate (2) in 1,4-dioxane gave the ethyl 4-oxo-2-thioxo-1,2-dihydroquinazoline-3(4H)-carboxylate (4).The formation of the latter product took place through intermediate formation of the thiourea derivative followed by cyclization.Compound 4 was used for the synthesis of different heterocyclic compounds through its reaction with various chemi-Mohareb and Halim: Uses of Anthranilic Acid for the Synthesis ... cal reagents.Thus, the reaction of compound 4 with hydrazine hydrate (5) gave the hydrazone derivative 6. Compound 4 was capable to form thioether derivatives through its reaction with α-halocarbonyl compounds.Thus, the reaction of compound 4 with any of the following: ethyl chloroacetate (7), chloroacetone (9) or 2-bromo-1-(4-methoxyphenyl)ethanone (11), gave the thioether derivatives 8, 10 and 12, respectively (Scheme 1).The structures of compounds 8, 10 and 12 were established on the basis of their analytical and spectral data.Thus, the 1 H NMR spectrum of compound 12 (as an example) showed the presence of a triplet at δ 1.13 and a quartet at δ 4.22 ppm showing the presence of an ester CH 3 and CH 2 , respectively, a singlet at δ 3.73 ppm showing the OCH 3 group, a singlet at δ 5.49 ppm for the CH 2 group and a multiplet at δ 7.23-7.42ppm for the two C 6 H 4 groups.In addition, the 13  The reaction of compound 8 with hydrazine hydrate (5) gave the hydrazino derivative 13.On the other hand, the reaction of compound 8 with either of malononitrile (14a) or ethyl cyanoacetate (14b) gave the dihydro [1,3,4]  thiadiazino [3,2-a]quinazolin-6(1H)-one derivatives 16a and 16b, respectively (Scheme 2).Formation of compounds 16a and 16b took place through the intermediate formation of 15a,b followed by cyclization.The structures of compounds 16a and 16b were confirmed on the basis of analytical and spectral data.Thus, the 1 H NMR spectrum of 16a showed, besides the expected signals, two triplets at δ 1.12, 1.15 and two signals at δ 4.16, 4.20 ppm indicating the presence of two ester CH 3 and CH 2 groups, respectively, a singlet at δ 6.01 ppm belonging to the pyrimidine H-2 and a singlet at δ 8.28 ppm for the NH group.In addition its 13  In continuation of our recent interest to conduct multi-component reactions using aromatic aldehydes and cyanomethylene reagents [31][32][33] we studied the multi-component reactions of compound 12.Thus, the multi-component reaction of compound 12 with any of the aromatic On the other hand, the multi-component reaction of compound 12 with any of benzaldehyde (17a), 4-chlorobenzaldehyde (17b) or 4-methoxybenzaldehyde (17c) and either of malononitrile (14a) or ethyl cyanoacetate (14b) in 1,4-dioxane containing ammonium acetate gave the pyridine derivatives 21a-f, respectively (Scheme 4).Formation of the latter products took place through the intermediate formation of 20a-f.
5][36] Products of the re- actions were either thiophene or thiazole derivatives or a mixture of both depending on the reaction conditions and the nature of the α-halocarbonyl compound.In continuation of this program we carried out the reaction of compound 8 with phenylisothiocyanate (22) in dimethylformamide containing potassium hydroxide to give the intermediate potassium salt 23.The reaction of the intermediate 23 with any of ethyl chloroacetate (7), chloroacetone (9) or 2-bromo-1-(4-methoxyphenyl)ethanone (11)  afforded the thiazole derivatives 24a-c, respectively (Scheme 5).All synthesized compounds were obtained in good yields and their cytotoxicity against cancer cell lines was measured.

Cell Cultures
Were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF-7), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt).They grow as a monolayer and were routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 µM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL), at 37 °C in a humidified atmosphere containing 5% CO 2 .Exponentially growing cells were obtained by plating 1.5 × 10 5 cells/mL for the six human cancer cell lines including cells derived from 0.75 × 10 4 cells/mL followed by 24 h of incubation.The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.
The heterocyclic compounds, prepared in this study, were evaluated according to standard protocols for their in vitro cytotoxicity against six human cancer cell lines, including cells derived from human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharynge-al carcinoma (HONE1) and a normal fibroblast cells (WI38).All of IC 50 values are listed in Table 1.Some heterocyclic compounds were observed to display significant cytotoxicity against most of the cancer cell lines tested (IC 50 = 10-1000 nM).Normal fibroblasts cells (WI38) were affected to a much lesser extent (IC 50 >10,000 nM).The reference compound used was the foretinib anti-tumor agent.

1. Structure Activity Relationship
It is clear from Table 1 that most of the tested compounds showed cytotoxicity against the selected cancer cell lines.Compound 4 showed high cytotoxicity against HEPG-2 and MCF-7 cell lines with IC 50 values of 683 and 460 nM, respectively.The reaction of compound 4 with hydrazine hydrate gave the hydrazone derivative 6 which showed high potency against the six cancer cell lines, this was attributed to the high nitrogen content in this compound.It is worthy to note that compound 6 showed higher cytotoxicity than foretinib against DLD-1 and HEPG-2 cell lines with IC 50 values of 69 and 125 nM, respectively.On the other hand, the reaction of compound 4 with ethyl α-chloroacetate gave the thioether derivative 8 with high cytotoxicity against the six cancer cell lines but its potency is a little bit less than that of compound 6.It is obvious that compound 8 showed higher cytotoxicity than foretinib against DLD-1 and HEPG-2 cell lines with IC 50 values of 230 and 64 nM, respectively.On the other hand, the thioether 10 showed high potency against HONE-1 and MCF-7 cell lines but compound 12 showed low potency against the six cancer cell lines.The reaction of compound 8 with hydrazine hydrate gave the hydrazino derivative 13 with a high nitrogen content, showing relatively high potency against the six cancer cell lines.On the other hand, the dihydro- [1,3,4]thiadiazino [3,2-a]

1. 3. Cell Proliferation Assay
The anti-proliferative activity of compounds 6, 8, 13, 19c, 19d, 19e, 21b, 21d, 21e, 21f, 24a and 24c was evaluated (Table 2) against the five c-Met-dependent cancer cell lines (A549, HT-29, MKN-45, U87MG, and SMMC-7721) and one c-Met-independent cancer cell line (H460) using the standard MTT assay in vitro, with foretinib as the positive control. 37,38The cancer cell lines were cultured in the minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS).Approximate 4 × 10 3 cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO 2 at 37 °C for 24 h.The compounds tested at the indicated final concentrations were added to the culture medium and the cell cultures were continued for 72 h.Fresh MTT was added to each well at a terminal concentration of 5 mg/mL, and incubated with cells at 37 °C for 4 h.The formazan crystals were dissolved in 100 µL of DMSO for each well, and the absorbance at 492 nM (for absorbance of MTT formazan) and 630 nM (for the reference wavelength) was measured with an ELISA reader.All of the compounds were tested three times in each cell line.The results expressed as IC 50 (inhibitory concentration 50%) are the averages of three determinations and were calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.

1. 4. In vitro Cell Assays
The antitumor evaluations 39 of the synthesized compounds were measured against A549 (non-small cell lung

1. 5. HTRF Kinase Assay
The c-Met kinase activities (Table 3) of the most potent compounds 6, 8, 13, 19c, 19d, 19e, 21b, 21d, 21e, 21f, 24a and 24c were measured using homogeneous time-resolved fluorescence (HTRF) assay as previously reported. 40,41In addition, the most potent compounds toward c-Met kinase were further evaluated against other five tyrosine kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDG-FR) using the same method (Table 4).Briefly, 20 mg/mL poly (Glu, Tyr) 4:1 (Sigma) was used as a substrate in 384well plates.Then, 50 µL of 10 mMATP (Invitrogen) solution diluted in kinase reaction buffer (50 mM HEPES, Ph 7.0, 1 M DTT, 1 M MgCl 2 , 1 M MnCl 2 , and 0.1% NaN 3 ) was added to each well.Various concentrations of the tested compounds diluted in 10 µL of 1% DMSO (v/v) were used as the negative control.The kinase reaction was start-ed by the addition of the purified tyrosine kinase proteins diluted in 39 µL of kinase reaction buffer solution.The incubation times for the reactions were 30 min at 25 °C and were ceased by the addition of 5 µL of Streptavidin-XL665 and 5 µL Tk Antibody Cryptate working solution added to all of wells.The plates were read using Envision (Perkin-Elmer) at 320 and 615 nM.The inhibition rate (%) was calculated using the mathematical equation: % inhibition = 100 -[(Activity of enzyme with tested compounds -Min)/(Max -Min)] × 100 (Max: the observed enzyme activity measured in the presence of enzyme, substrates, and cofactors; Min: the observed enzyme activity in the presence of substrates, cofactors and in the absence of enzyme).IC 50 values were calculated using the inhibition curves.
According to Table 3 it is clear that compounds 8, 13, 19d, 21e, 21f and 24a were the most active compounds towards c-Met kinase.Moreover, compounds 13, 19d and 24a showed activity towards c-Met enzymatic activity higher than that of foretinib.Mohareb and Halim: Uses of Anthranilic Acid for the Synthesis ...

1. 6. Inhibitory Effects of the Most Active Compounds Towards Tyrosine Kinases
The most potent compounds 8, 13, 19d, 21e, 21f and 24a towards c-Met enzymatic activity of the five tyrosine kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR) were determined using the same method described for c-Meth enzymatic activity and the data are shown in Table 4.Here foretinib was also used as the reference drug.It is clear from Table 4 that compounds 13, 21e and 24a showed the highest inhibitory effect towards the five tyrosine kinases.

1. 7. Pim-1 Kinase Inhibition of Compounds 13, 21e and 24a
Compounds 13, 21e and 24a were selected to examine their Pim-1 kinase inhibition activity 42 at a range of 10 concentrations and the IC 50 values were calculated.Our slection for 13, 21e and 24a was based on their relative activity towards c-Met kinase, together with their inhibition towards tyrosine kinases; the more potent to inhibit Pim-1 activity were 13 and 24a with IC 50 value of 0.36 and 0.28 µM, while 21e was less effective (IC 50 > 10 µM).SGI-1776 was used as the positive control with IC 50 0.048 µM in the assay.These profiles in combination with cell growth inhibition data of compounds 13, 21e and 24a are listed in Table 5 and indicate that Pim-1 is a potential target of these compounds.

Experimental Protocol of Docking Study
All the molecular modeling studies were carried out on an Intel Pentium 2.3 GHz processor, 8 GB memory with Windows 7 operating system using Molecular Operating Environment (MOE, 10.2008;Chemical Computing Group, Canada) software.The X-ray crystallographic structure of c-Met kinase enzyme with its co-crystallized ligand XL880 (Foretinib) in the file (PDB ID: 3LQ8) was obtained from RCSB Protein Data Bank with a 2.02 Å resolution.All water of crystallization was deleted from the active site except the one involved in interactions with the ligand.Hydrogens and partial charges were added to the system using protonate 3D application.Isolation of the active site, recognition of the amino acids and the backbone was hidden.The docking algorithm was validated via docking of the native ligand (Foretinib) into its c-Met kinase active site where the docking procedure was able to    Mohareb and Halim: Uses of Anthranilic Acid for the Synthesis ... retrieve the co-crystallized pose with RMSD value of 0.55 Å.The three-dimensional structures of the most active compounds 13, 21e & 24a were built using MOE molecular builder, then their energy was minimized by Merk Molecular Force Field (MMFF94x).Hydrogens and partial charges were added to the system using protonate 3D application.

Docking Results
For each docked compound, only one pose was selected based on number of binding interactions, superposition with the original ligand, docking score and the formed H-bonds were measured.The docking results obtained from the docking study are summarized in Table 6.

Discussion of Molecular Modeling
The X-ray crystallographic structure of XL880 (foretinib) in complex with c-Met kinase shows that the inhibitor forms two hydrogen bonds between quinoline N and Met 1160 , CO of malonamide moiety and Lys 1110 .Phe 1223 of the activation loop has relocated from the position in the active conformation to stack underneath the fluorophenyl ring (π-π interaction), placing the kinase in a pseudo-unactivated conformation 43 (Fig. 2).Compounds 13, 21e and 24a showed good fitting to the active binding site of c-Met kinase by interaction with Asp 1222 , Lys 1110 , Asp 1164 , Asn 1171 and His 1094 amino acid residues (Fig. 3-5).

1. General
All melting points were determined on an electrothermal apparatus (Büchi 535, Switzerland) in an open capillary tube and are uncorrected. 13C NMR and 1 H NMR spectra were recorded on Bruker DPX200 instrument in DMSO with TMS as internal standard for proton spectra and solvent signals as internal standard for carbon spectra.Chemical shift values are given in δ (ppm).Mass spectra were recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Daltonics instrument.Elemental analyses were carried out by the Microanalytical Data Unit at Cairo University.The progress of all reactions was monitored by TLC on 2 × 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck).

1. Synthesis of the thioether derivatives 8, 10
To a solution of compound 4 in ethanol (40 mL) any of compounds 7 (1.22 g, 0.01 mol), 9 (0.92 g, 0.01 mol) or 11 (2.29 g, 0.01 mol) was added.The reaction mixture, in each case, was heated under reflux for 3 h then poured into ice/water mixture containing a few drops of sodium hydroxide solution and the formed solid product was collected by filtration.
C NMR spectrum showed two signals at δ 16.3, 16.5 for the two ester CH 3 groups and two quartets at δ 52.1, 53.8 for the two ester CH 2 groups as well as three signals at δ 163.8, 164.4,165.2 belonging to the three CO groups.
quinazoline deriva-Mohareb and Halim: Uses of Anthranilic Acid for the Synthesis ... tives 16a,b showed low potency.The multi-component reactions of compound 12 with any of the aromatic aldehydes 17a-c and either of malononitrile or ethyl cyanoacetate gave the pyran derivatives 19a-f.Compounds 19c (X = Cl, Y = NH 2 ), 19d (X = Cl, Y = OH) and 19e (X = OCH 3 , Y = NH 2 ) showed the highest cytotoxicity among this series of compounds.On the other hand, the pyridine derivatives 21a-f where the four compounds 21b (X = H, Y = OH), 21d (X = Cl, Y = OH), 21e (X = OCH 3 , Y = NH 2 ) and 21f (X = OCH 3 , Y = OH) showed the highest potency.Compound 21f showed cyctotoxicity higher than foretinib gainst the DLD-1cell line with IC 50 79 nM.Considering the thiazole derivatives 24a-c, it is obvious that compounds 24a (Y = OH) and 24c (Y = 4-OCH 3 -phenyl) showed higher potency than 24b (Y = CH 3 ).It is clear that throughout the synthesized compounds the presence of the electronegative groups, like the Cl and OH groups, or the electron-rich NH 2 groups enhance the potency of the compound.

Figure 2 .Figure 3 .
Figure 2. Interactions of XL880 (foretinib) with the amino acid residues of the active site of c-Met 3D(a) and 2D(b)

Figure 5 .
Figure 5. (a) The superposition of foretinib (blue) and compound 24a (red) docked in the binding site of c-Met, the dotted lines represent H-bonding interactions; (b) 2D ligand interaction of 24a in binding site of c-Met.

Figure 4 .
Figure 4. (a) The superposition of foretinib (blue) and compound 21e (red) docked in the binding site of c-Met, the dotted lines represent H-bonding interactions; (b) 2D ligand interaction of 21e in binding site of c-Met.

Table 1 :
Cytotoxicity of the newly synthesized products against a variety of cancer cell lines [IC 50 a (nM)] a Drug concentration required to inhibit tumor cell proliferation by 50% after continuous exposure of 48 h.b NUGC, gastric cancer; DLD-1, colon cancer; HA22T, liver cancer; HEPG-2, liver cancer; HONE-1, nasopharyngeal carcinoma; MCF-7, breast cancer; WI-38, normal fibroblast cells.NA:Not Active.cancer),H460(humanlungcancer), HT-29 (human colon cancer) and MKN-45 (human gastric cancer), U87MG (human glioblastoma) and SMMC-7721 (human liver cancer) cancer cell lines.Foretinib was used as the positive control by a MTT assay.The results are expressed as IC 50 after three different experiments were summarized and are shown in Table2.The data listed in Table2reveal that the compounds possess moderate to strong cytotoxicity against the six tested cell lines in the single-digit µ M range, and high selectivity for inhibition of A549, H460 and MKN-45 cells.The promising compounds were 13, 19c, 19e, 21d, 21e and 24c, these were the most active with IC 50 values of 0.09 and 0.93 µM, respectively.Moreover, compound 13 showed potency higher that foretinib towards H460 with IC 50 0.06 µM.Compounds 6, 13, 19e, 21d, 21e showed activities toward U87MG cell line higher that foretinib with IC 50 values of 0.39, 0.69, 0.26, 0.70 and 0.42 µM, respectively.It is of great value to note that compound 6 showed higher potency than foretinib with IC 50 0.29 µM against SMMC-7721.

Table 3 .
c-Met enzymatic activity and line of the most potent compounds

Table 2 .
In vitro growth inhibitory effects IC 50 ± SEM (µM) of the most potent compounds

Table 6 .
Docking study data showing amino acid interactions and the hydrogen bond lengths of target compounds and foretinib on c-Met kinase enzyme.