Utility of 2-Methyl-quinazolin-4 ( 3 H )-one in the Synthesis of Heterocyclic Compounds with Anticancer Activity

Quinolino[2,1-b]quinazolines 3 and 4, pyrrolo[2,1-b]quinazoline 5 and various substituted 2-(4chlorostyryl)quinazoline derivatives including: 4-amino derivative 8, 4-hydrazino derivative 9, thiourea derivative 10, thiosemicarbazide derivative 19, 4-benzylidene hydrazinyl derivative 21, 4-amino thiazolidene derivatives 11, 12, 13, 22, imidazoquinazolines 15, 16, quinazolinium chloride 14, triazino[4,3-c]quinazolines 17, 18, tetrazino[1,6-c]quinazoline 20, 4-amino azetidinyl derivative 23, triazolo[4,3-c]quinazoline 24, 4-amino substituted quinazolines 25, 26, 27, 29 and quinazolino quinazoline 28 were synthesized through different chemical reactions. The obtained compounds were evaluated for their in vitro antitumor activity against HEPG2 and MCF-7 cell lines compared to the reference drug (doxorubicin). Compounds 18, 19, 20, 23 and 24 were found to be the most active against both cell lines exhibiting IC50 values ranging from 10.82 29.46 μM/L and 7.09 31.85 μM/L against Hep-G2 and MCF-7 cell lines, respectively, in addition to docking study of these five compounds against thymidylate synthase and dihydrofolate reductase enzymes active sites.


Introduction
Quinazoline-4(3H)-one ring system has been consistently regarded as promising privileged structural icon owing to its pharmacodynamic versatility in many of its synthetic derivatives as well as in several naturally occurring alkaloids isolated from animals, families of plant kingdoms and micro-organisms [1]- [3].
A systematic perpusal of literature, reveals that quinazoline derivatives encompass a broad spectrum of pharmaceutical activity profile such as antitumor [4]- [10], anti-HIV [11]- [13], antimicrobial [14]- [16], antibacterial, anti-inflammatory [17]- [20], CNS activity [21]- [23] and cardiovascular activity [24] [25].The quinazoline nucleus is the scaffold of many antitumor drugs mainly acting as inhibitors of tyrosine kinase receptor (TKR) [26].Over expression of the receptors has been observed in a number of cancers such as breast, ovarian, colon and prostate cancer.Therefore, blocking of tyrosine kinase activity represents a rational approach to cancer therapy [27]- [34].Also quinazoline derivatives have a therapeutic potential as an anti-invasive agent with potential activity in early and advanced solid tumors, metastatic bone disease and leukemia [35].So the quinazoline ring is considered as one of the major classes of benzoheterocyclic compounds that have drawn much attention in the field of cancer chemotherapy besides other pharmacological activities [27]- [29].Moreover, many 4-quinazoline derivatives revealed potent anticancer activity mediated through the inhibition of dihydrofolate reductase enzyme (DHFR) [30] [31] or through inhibition of chki kinase [32].

Chemistry
2-Methyl-quinazolin-4-one was synthesized from the fusion of anthranilic acid with thioacetamide [36] which was utilized as a building unit for novel quinazoline and fused quinazolinone compounds "(Scheme 1)".The extent of the pharmacological effect of a quinazolinone derivative depends on the active group which is attached to it.Recently, several scientists have elucidated that in quinazolinone system sites like position 2 and 3, can be suitably modified by the introduction of various heterocyclic moieties to show excellent pharmacological results [37] [38].A known procedure for building up such heterocyclic system is based on cyclization of o-chlorobenzoyl chloride with 2-methyl-quinazolin-4(3H)-one 1 in dry THF.The reaction stopped at the stage of formation of 2-[2-(2-chlorophenyl)-2-oxoethyl]quinazolin-4(3H)-one 2. This could be explained in the first place by steric hindrance of the o-substituent for optimal position of the reacting groups.We attempted to decrease the effect of the latter by heating compound 2 in THF for 2h.However under this reaction condition, cyclization occurred readily to yield 5H-quinolino[2,1-b]quinazolin-5, 12(6H)-dione 3.
The 1 H-NMR spectrum of compound 2 revealed a singlet at δ 4.33 ppm attributed to CH 2 protons and a deuterium oxide exchangeable singlet at δ 7.73 ppm due to the NH proton.While, 1 H-NMR spectrum of compound 3 showed a singlet at δ 2.49 ppm assigned to CH 2 protons and lacked any deuterium oxide exchangeable signals of its precursor.
Attempts to carry out a similar conversion of compound 1 into 4 by refluxing in THF are unsuccessful.However, the cyclic quinolono [2,1-b]quinazoline 4 was obtained through the reaction of compound 1 with 2-chlorobenzaldehyde in glacial acetic acid/sodium acetate.In this case, isolation of the intermediate 4' was difficult because of its high reactivity which led directly to the cyclic product 4.
Pyrrolo[2,1-b]quinazolindione derivative 5 was prepared by the reaction of compound 1 with chloroacetyl chloride in dry THF.The reaction is assumed to proceed through chloroacetylation of compound 1 at the 2-position followed by intramolecular cyclization on N 3 -NH function with elimination of a hydrochloride molecule to give the target compound 5. 1 H-NMR spectrum of compound 5 showed two singlets at δ 2.08, 2.49 ppm each integrated for two protons attributed to two pyrrolo methylene protons.
Our attention was drawn to the earlier discovery by scientists that modification of methyl group present at the second position of 4(3H)-quinazolinone into other chemical intities yielded structural analogues with appreciable biological activity [39] [40] "(Scheme 2)".Therefore, compound 1 was condensed with 4-chlorobenzaldehyde in glacial acetic acid/sodium acetate to afford the chalcone derivative 6 [41].Chlorination of compound 6 with phosphorous oxychloride was reported to give [42] poor yields of impure chlorinated product.However, the addition of triethylamine to the reaction mixture accelerated the reaction speed [43] and afforded 4-chloroquinazoline derivative 7 [41].Furthermore, amination of compound 7 was carried out by refluxing in pyridine and ammonium acetate to afford the corresponding 4-aminoquinazoline derivative 8.Moreover, hydrazinolysis of compound 7 was accomplished by the use of hydrazine hydrate 99% in ethanol to afford 4-hydrazinylquinazoline derivative 9.The 1 H-NMR spectrum of compound 9 showed two deuterium oxide exchangeable singlets at δ 4.15 and 12.26 ppm due to NH 2 and NH protons; respectively.The work was extended to shed more light on the activity and synthetic potential of the amino group in compound 8 "(Scheme 3)".Thus, compound 8 was reacted with phenyl isothiocyanate in boiling pyridine to give the corresponding thiourea derivative 10.The 1 H-NMR spectrum of compound 10 revealed two deuterium oxide exchangeable singlets at δ 7.96 and 12.13 ppm due to thiourea N 3 and N 1 -NH protons; respectively.Furthermore, the thiourea derivative was used as a good starting material for preparation of several polyfunctional thiazolyl derivatives with expected biological activities as the thiazolyl moieties were reported to possess a wide range of pharmacological actions especially anti cancer activity [44].Therefore, compound 10 was reacted with ethyl chloroacetate as well as phenacyl chloride in boiling ethanol/sodium acetate to yield 3-phenylthiazolidin-4-one derivative 11 and 3,4-diphenylthiazolylidene derivative 12; respectively.It was also fused with chloroacetone to give 4-methyl-3-phenyl thiazol-ylidene dreivative 13.
The reaction mechanisms are expected to proceed via elimination of hydrochloride molecules followed by cyclization through ethanol elimination or dehydration.The 1 H-NMR spectrum of compound 11 revealed a singlet at δ 3.46 ppm due to SCH 2 protons.The 1 H-NMR spectrum of compounds 12 revealed a singlet at δ 7.49 ppm due to CH-thiazole proton.
However, tricyclic systems in which the quinazoline ring was condensed with imidazole ring have not been adequately studied "(Scheme 4)".Therefore our goal was directed to synthesize imidazoquinazoline derivatives through condensation of compound 8 with phenacyl chloride in ethanol, which afforded quinazolin-3-ium chloride derivative 14 and imidazo[1,2-c]quinazoline derivative 15 according to the mechanism suggested in the literature [45].The first step of this process involved the addition of a phenacyl chloride molecule on N-3 position to form the corresponding quinazolinium derivative 14 which underwent cyclization under the same experimental conditions to give the imidazoquinazoline derivative 15.The 1 H-NMR spectrum of compound 14 showed a singlet at δ 3.63 ppm due to CH 2 protons, in addition to two deuterium oxide exchangeable singlets at δ 5.18 and 12.33 ppm due to NH 2 protons and OH tautomer proton; respectively.While the 1 H-NMR spectrum of compound 15 revealed a singlet integrated for one proton at δ 7.96 ppm due to CH-imidazole.Moreover, acylation of the amino group of compound 8 with chloroacetyl chloride under anhydrous conditions which underwent intramolecular cyclization on the ring to yield the cyclized product imidazo[1,2-c]quinazolinone derivative 16.In the case of chloroacetyl chloride, the isolation of the intermediate 16' was difficult due to its high reactivity which led directly to the cyclic product 16.The 1 H-NMR spectrum of compound 16 revealed a singlet at δ 3.55 ppm due to CH 2 protons of imidazole moiety and another singlet at δ 7.50 ppm attributed to the imidazole = CH-tautomer.In addition to a deuterium oxide exchangeable singlet at δ 8.51 ppm corresponding to OH tautomer.
Recently, several biological activities have been reported for [1,2,4]triazino [4,3-c]quinazolines [46], so our aim was extended to the development of different synthetic methods for novel [1,2,4]triazino [4,3-c]quinazolines 17 and 18 and investigation of their cytotoxicity and antitumor activity "(Scheme 5)".Therefore, condensation of compound 9 with benzoin and with chloroacetyl chloride were applied to furnish the target compounds 17 and 18; respectively.The 1 H-NMR spectrum of compound 17 revealed a singlet at δ 6.02 ppm due to the triazine C 5 -proton.While, the 1 H-NMR spectrum of compound 18 showed a singlet at δ 4.25 ppm due to the triazine CH 2 protons, in addition to a deuterium oxide exchangeable singlet at δ 12.20 ppm attributed to NH proton.Furthermore, from our survey in the literature, it was found that, a new series of quinazoline compounds was designed in such a way to accommodate the isothiocyanates, Schiff's bases and amine bridges at positions 2, 3 or 4 of the quinazoline ring.Isothiocyanate, Schiff's base and amide functions are known to contribute in the enhancement of the antitumor activity [4].The reaction of phenyl isothiocyanate with compound 9 afforded hydrazine carbothioamide derivative 19.The 1 H-NMR spectrum of compound 19 showed a deuterium oxide exchangeable singlet at δ 3.98 ppm due to two NH protons that are attached to C=S function and a deuterium oxide exchangeable singlet at δ 11.08 ppm due to NH proton attached to the quinazoline ring which is more deshielded by the nitrogens of the quinazoline ring.The thiosemicarbazide derivative 19 underwent oxidative cyclization by stirring with bromine to yield [1,2,4,5]tetrazino [1,6-c]quinazoline thione derivative 20.The 1 H-NMR spectrum of compound 20 showed a deuterium oxide exchangeable singlet at δ 4.00 ppm due to NH proton.Moreover, the reaction of compound 9 with 4-chlorobenzaldehyde in presence of piperidine furnished the Schiff's base 21 "(Scheme 6)".The Schiff's base 21 was used as a good starting material for preparation of several quinazoline derivatives.Among which its reaction with thioglycolic acid in presence of anhydrous zinc chloride to furnish the thiazolidinone derivative 22. Furthermore, the reaction of the Schiff's base 21 with chloroacetyl chloride in presence of triethylamine as a base afforded azetidinone derivative 23.The 1 H-NMR spectrum of compound 23 revealed two doublets at δ 4.06 and 4.30 ppm attributed to CH-C 6 H 4 -Cl and CH-Cl of the azetidine ring; respectively.In addition to a deuterium oxide exchangeable singlet at δ 10.68 ppm attributed to NH proton.Moreover, a group of Slovak's scientists reported that [1,2,4]triazolo [4,3-c]quinazoline derivatives possess considerable in vitro antitumor activity [46].So we synthesized [1,2,4]triazolo [4,3-c]quinazoline compound 24 through the cyclization of the Schiff's base 21 in glacial acetic acid/sodium acetate.
Finally, our work was extended to react 4-chloroquinazoline derivative 7 with several primary and secondary amines preparing a series of promising derivatives in order to compare their biological activities "(Scheme 7)".Focusing on setting up a simple, cheap, fast and efficient synthesis; the reaction of compound 7 with the primary amine, 2-aminoethanol was accomplished by fusion with excess amine reducing as much as possible both reaction time and temperature so as to preserve the styryl group from reacting.This rapid reaction protocol resulted in the formation of the secondary aminoquinazoline derivative 25 in good yields.The 1 H-NMR spectrum of compound 25 showed a multiplet at δ 3.66 -3.71 ppm integrated for four protons corresponding to two CH 2 protons and two deuterium oxide exchangeable singlets at δ 4.40 and δ 12.25 ppm attributed to OH and NH protons, respectively.Moreover, the substrate 7 was also reacted very easily with secondary amines such as dimethylamine and pyrrole by the same manner as aminoethanol to yield tertiary amino quinazolines 26 and 27; respectively in very good yields.Furthermore, compound 7 was reacted with anthranilic acid in glacial acetic acid to afford the polycyclic quinazoline derivative 28 while hot, while the reaction filtrate yielded the open chain secondary aminoquinazoline derivative 29 that bears a free carboxylic group which could possibly alter the biological properties including cytotoxic activity [47].However, the reaction is expected to proceed through formation of the secondary aminobenzoic acid derivative 29 first which carried out intramolecular dehydration reaction leading to the cyclic quinazolinoquinazoline derivative 28.The 1 H-NMR spectrum of compound 29 showed two deuterium oxide exchangeable singlets at δ 8.76 and δ 12.18 ppm attributed to NH and carboxylic OH protons; respectively.While the 1 H-NMR spectrum of compound 28 lacked any deuterium oxide exchangeable singlets due to NH and OH protons.

Biology
The six dose growth inhibition percent and the IC 50 values of the tested compounds (2 -29) against liver HepG2 and breast MCF-7 cell lines are represented in Tables 1 and 2; respectively.The thiourea derivative 10 exhibited strong anticancer activity against liver cancer cell line about half the activity of doxorubicin (IC 50 = 8.55 µM/L) with IC 50 16.35µM/L.However, it showed moderate activity against breast cancer MCF-7 with IC 50 68.75µM/L.Fusion of [1,2,4]triazine or [1,2,4,5]tetrazine to the quinazoline back bone as in compounds 18 and 20 resulted in marked increase in activity against both HepG2 and MCF-7 cell lines exerting IC 50 31.85and 27.04 µM/L against HepG2 cell line and 29.46 and 20.98 µM/L against MCF-7 cell line; respectively.It is to be noted that, the presence of a phenyl thiosemicarbazide side chain attached to the C 4 -quinazoline ring as in compound 19 exhibited marked anticancer activity against both HepG2 and MCF-7 cell lines exerting IC 50 18.79 and 13.46 µM/L; respectively.However, incorporating N-azetidinone-4-amino moiety to the quinazoline back bone as in compound 23 resulted in improvement in activity against both HepG2 and MCF-7 cell lines which exhibited slightly better anticancer activity against HepG2 (IC 50 7.09 µM/L) than the reference drug doxorubicin (IC 50 8.55 µM/L).While, it showed comparable activity to that of doxorubicin (IC 50 8.90 µM/L) against MCF-7 exerting IC 50 11.94µM/L.Furthermore, fusion of triazole ring to quinazoline nucleus as in compound 24 resulted in marked increase in activity against both HepG2 and MCF-7 cell lines exerting IC 50 10.58 and 10.82 µM/L; respectively which represent a strong anticancer agent against these two cell lines (Figures 1 and 2).Moreover, the presence of  2-amino benzoic acid moiety on the C 4 -quinazoline ring as in compound 29 exhibited remarkable increase in activity towards HepG2 cell line showing IC 50 19.95µM/L.

Docking on the Active Site of Thymidylate Synthase
MOE docking studies of the inhibitors were performed using thymidylate synthase co-crystallized with the sub-

Docking on the Active Site of Dihydrofolate Reductase (DHFR)
MOE docking studies of the inhibitors were performed using dihydrofolate reductase co-crystallized with me thotrexate (PDB ID: 4DFR) as a template.
1) Docking of MTX into DHFR active site revealed that: Hydrogen bond interactions beside hydrophobic interactions were considered to be responsible for the observed affinity as it acts as a hydrogen bond donor to the backbone Ile 5 and Ile 94 residues and the side chain Asp 27 residue.It also acts as a hydrogen bond accepter to Arg 52 and Arg 57 residues.This beside many hydrophobic interactions with various amino acid residues: ILe 5, Ala 6, Ala 7, Asp 27, Leu 28, Phe 31, Lys 32, Ser 49, Ile 50, Arg 52, Leu 54, Arg 57, Ile 94, Tyr 100 and Thr 113 as shown in Figure 4(a).
2) Docking of compound 18 into DHFR active site: Revealed the interaction of quinazoline-N 1 atom as a hydrogen bond acceptor with the side chain residue Ser 59 (2.93 Å) at a strength of 43.7%, in addition to the interaction of carbonyl group oxygen as a hydrogen bond acceptor with the side chain residue Tyr 121 (2.86 Å) at a strength of 18.9% beside many hydrophobic interactions among quinazoline-C 6 atom, chlorine atom, styryl moiety and triazine-C 6) Docking of compound 24 into DHFR active site revealed: The interaction of quinazoline-N 1 atom as a hydrogen bond acceptor with the side chain residue Ser 59 (3.00 Å) at a strength of 5.6% as well as the interaction of triazole-N 2 atom as a hydrogen bond acceptor with the side chain residue Tyr 121 (3.66 Å) at a strength of 3.9 %, besides many hydrophobic interactions among the chlorine atom, quinazoline-C 6 carbon, triazole-C 3 carbon, in addition to triazole-C 3 -4-chlorophenyl moiety and the following amino acid residues: Ile 16, Leu 22, Phe 31, Phe 34, Lys 55, Thr 56, Ser 59, Ile 60, Pro 61, Val 115, Gly 117, and Tyr 146 as shown in Figure 4(f).

Chemistry
All melting points were measured on Electro thermal LA 9000 SERIS, Digital Melting point Apparatus and are uncorrected.IR spectra (KBr) were recorded on FT-IR 200 spectrophotometer (ύ cm −1 ), pharmaceutical analytical unit, Faculty of Pharmacy, Al-Azhar University. 1 H-NMR spectra were recorded in (DMSO-d 6 ) at 300 MHz on a Varian Gemini NMR spectrometer (δ ppm) using TMS as an internal standard, Resarch Service Unit, Faculty of Science, Cairo University.Mass spectra were recorded on GC Ms-QP 5050A mass spectrometer at 70 eV and microanalytical data were performed on Elementar Vario EI III CHN analyzer at the microanalytical unit, in Regional center for Mycology and Biotechnology, Al-Azhar University.Thin layer chromatography was performed on precoated (0.25 mm) silica gel GF 254 plates (E.Merck, Germany).Compounds were detected with 254 nm UV lamp.

Pyrrolo[2,1-b]quinazolin-2,9(1H, 3H)-dione; 5
A solution of compound 1 (1.6 g, 0.01 mol) in dry THF (20 mL) was added dropwise while stirring to a solution of chloroacetyl chloride (2.26 g, 1.6 mL, 0.02 mol) in dry THF (10 mL) at 0˚C and stirring was continued for 2 h.The reaction mixture was further stirred for 4h at room temperature then the excess solvent was evaporated.The reaction mixture was cooled and poured onto crushed ice.The solid obtained was filtered off, washed with water, dried and recrystallized from ethanol.

General Procedure for Synthesis of Compounds 11 & 12
A solution of compound 10 (0.4 g, 0.01 mol) in ethanol (30 mL) and in presence of a catalytic amount of sodium acetate (1 g, 0.01 mol) was heated under reflux for 6 h with an equimolar amount of the appropriate chloro compound namely; ethyl chloroacetate ( 0.6 g, 0.5 mL, 0.01mol) or phenacyl chloride ( 0.5 g, 0.01 mol).The solid obtained after cooling the reaction mixture was filtered off and washed with water to yield compounds 11 and 12 respectively.

General Procedure for Synthesis of Compounds 14 & 15
An equimolar mixture of compound 8 (1.4 g, 0.01 mol) and phenacyl chloride (0.8 g, 0.01 mol) in ethanol (30 mL) was refluxed for 18 h.The reaction mixture was then concentrated, allowed to cool and the solid product obtained was filtered off to yield compound 14.The filtrate was poured onto crushed ice and the obtained solid was collected to give compound 15.  2 mL, 0.15 mol) were stirred in dry DMF (30 mL) for 2 h at room temperature, then refluxed for another 5 h.The reaction mixture was allowed to cool then poured onto crushed ice.The solid separated was filtered, washed with water, dried and recrystallized from ethanol.

Anticancer Screening
The synthesized compounds were screened for their in vitro cytotoxic activity against human hepatocellular liver carcinoma (Hep-G2) and human breast cancer cell line (MCF-7).Doxorubicin was used as the reference drug.
Mammalian cell lines MCF-7 cells (human breast cancer cell line) were obtained from VACSERA Tissue culture unit.Hep-G2 cells (human cell line of a well differentiated hepatocellular carcinoma isolated from a liver biopsy of a male Caucasian aged 15 years) were obtained from the American type culture collection (ATCC).

Cell line Propagation
The cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50 µg/mL gentamycin.All cells were maintained at 37˚C in a humidified atmosphere with 5% CO 2 and were subcultured two times a week.

Cytotoxicity Evaluation Using Viability Assay
For cytotoxicity assay, the cells were seeded in 96-well plate at a cell concentration of 1 × 10 4 cells per well in 100 µL of growth medium.Fresh medium containing different concentrations of the test sample was added after 24 h of seeding.Serial two-fold dilutions of the tested chemical compound were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette.The microtiter plates were incubated at 37˚C in a humidified incubator with 5% CO 2 for a period of 48 h.Three wells were used for each concentration of the test sample.Control cells were incubated without test sample and with or without DMSO.The little percentage of DMSO present in the wells (maximal 0.1%) was found not to affect the experiment.After incubation of the cells for 24 h at 37˚C, various concentrations of sample (50, 25, 12.5, 6.25, 3.125 & 1.56 µg/L) were added, and the incubation was continued for 48 h and viable cells yield was determined by a colorimetric method.After the end of the incubation period, media were aspirated and the crystal violet solution (1%) was added to each well for at least 30 minutes.The stain was removed and the plates were rinsed using tap water until all excess stain is removed.Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, and then the absorbance of the plates were measured after gently shaken on Microplate reader (TECAN, Inc.), using a test wavelength of 490 nm.All results were corrected for background absorbance detected in wells without added stain.Treated samples were compared with the cell control in the absence of the tested compounds.All experiments were carried out in triplicate.The cell cytotoxic effect of each tested compound was calculated [49] [50].The cytotoxicity of the tested compounds was estimated in terms of percent growth inhibition compared to untreated control cells and their IC 50 in µM/L which is the concentration of the compound that inhibits the tumor cell growth by 50%.

Materials
All the molecular studies were carried out on an Intel pentium 1.6 GHz processor, 512 MB memory with windows XP operating system using Molecular Operating Environment (MOE 2005.06;Chemical Computing Group, Montreal, Canada) as the computational software.All the minimizations were performed with MOE until a RMSD gradient of 0.05 Kcal mol -1 A 0−1 with MMFF94X forcefield and the partial charges were automatically calculated.

General Methodology
The coordinates of the X-ray crystal structure of 2'-deoxyuridine-5'-monophosphate (DUMP) bound to thymidylate synthase enzyme were obtained from Protein Data Bank (PDB ID: 1BID) as well as the coordinates of the X-ray crystal structure of methotrexate (MTX) bound to dihydrofolate reductase (DHFR) enzyme (PDB ID: 4DFR).Enzyme structures were checked for missing atoms, bonds and contacts.Hydrogen atoms were added to the enzyme structure.Water molecules and bound ligands were manually deleted.The ligand molecules were constructed using the builder molecule and were energy minimized.The active site was generated using the MOE-Alpha site finder.Dummy atoms were created from the obtained alpha spheres.Ligands were docked within the thymidylate synthase and dihydrofolate reductase active sites using the MOE-Dock with simulated annealing used as the search protocol and MMFF94X molecular mechanics forcefield for 8000 interactions.The lowest energy conformation was selected and subjected to an energy Minimization using MMFF94X force field.

Conclusion
Compounds 18, 19, 20, 23 and 24 exhibited very potent anticancer activity against both Hep-G2 and MCF-7 cell lines.However, compounds 10, 15 and 29, showed remarkable anticancer activity against liver Hep-G2 cell line, while compound 22 exhibited potent anticancer activity against MCF-7 cell line.Docking was performed for the five most active anticancer compounds 18, 19, 20, 23 and 24 on the two enzymes; thymidylate synthase and dihydrofolate reductase in a trial to predict their mode of action as anticancer drugs.Also, the compounds show several interactions with both enzymes but they exhibit strong interactions with dihydrofolate reductase enzyme, mainly compounds 18, 19, 23 and 24, giving rise to the conclusion that they might exert their action through inhibition of both enzymes but mainly DHFR enzyme.

Figure 1 .
Figure 1.Growth inhibition curves of most active compounds against HepG2 cell line.

Figure 2 .
Figure 2. Growth inhibition curves of most active compounds against MCF-7 cell line.
DUMP(PDB ID: IBID) as a template.1) Docking of DUMP into TS active site revealed that: Several interactions were considered to be responsible for the observed affinity of the compound.As it acts as a hydrogen bond acceptor with the backbone Asp 169 residue and the side chain residues; Arg 166, Ser 167, Asn 177, His 207 and Tyr 209 as well as hydrogen bond donor with the side chain residues; Asn 177, His 207 and Tyr 209.In addition to hydrophobic interactions with: Arg 21, Cys 146, His 147, Gln 165, Arg 166, Ser 167, Cys 168, Asp 169, Gly 173, Asn 177, His 207 and Tyr 209 as shown in Figure 3(a).

Figure 3 . 3 ) 4 ) 5 ) 6 )
Figure 3. (a) Docking of DUMP in the active site of TS; (b) Docking of compound 18 in the active site of TS; (c) Docking of compound 19 in the active site of TS; (d) Docking of compound 20 in the active site of TS; (e) Docking of compound 23 in the active site of TS; (f) Docking of compound 24 in the active site of TS.

Table 1 .
Six dose growth inhibition percent and IC 50 values of the test compounds against HepG2 cell line.

Table 2 .
Six dose growth inhibition percent and IC 50 values of the test compounds against MCF-7 cell line.