Synthesis, Antitumor Evaluation and Molecular Docking of New Morpholine Based Heterocycles

A series of new morpholinylchalcones was prepared and then used as building blocks for constructing a series of 7-morpholino-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-ones via their reaction with 6-aminothiouracil. The latter thiones reacted with the appropriate hydrazonoyl chloride to give the corresponding pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-ones. The assigned structures for all the newly synthesized compounds were confirmed on the basis of elemental analyses and spectral data and the mechanisms of their formation were also discussed. Most of the synthesized compounds were tested for in vitro activity against human lung cancer (A-549) and human hepatocellular carcinoma (HepG-2) cell lines compared with the employed standard antitumor drug (cisplatin) and the results revealed that compounds 8, 4e and 7b have promising activities against the A-549 cell line (IC50 values of 2.78 ± 0.86 μg/mL, 5.37 ± 0.95 μg/mL and 5.70 ± 0.91 μg/mL, respectively) while compound 7b has promising activity against the HepG-2 cell lines (IC50 = 3.54 ± 1.11 μg/mL). Moreover, computational studies using MOE 2014.09 software supported the biological activity results.


Chemistry
The required starting compounds, namely 1-morpholino-1-(2-phenylhydrazono)propan-2-one (2a) and 1-morpholino-1-(2-(p-tolyl)hydrazono)propan-2-one (2b) were prepared by a previously reported method (Scheme 1) [34]. The morpholinohydrazonopropanone derivatives 2a,b were next used as starting compounds for preparation of a number of novel chalcone derivatives. Thus stirring a mixture of 1-morpholino-1-(2-arylhydrazono)propan-2-ones 2a,b and the appropriate benzaldehyde derivatives 3a-c in glacial acetic acid in the presence of a catalytic amount of concentrated H2SO4, gave the morpholinylchalcone derivatives 4a-f in good yield (Scheme 1). The assigned structures of the products 4a-f was confirmed based on both elemental analyses and spectral data (IR, 1 H-NMR and MS). The IR spectra of compounds 4a-f revealed in each case two absorption bands in the regions υ 3250-3236, 1683-1676 cm −1 attributed to the NH and C=O groups. The 1 H-NMR spectra of compounds 4a-f showed in each case three signals assigned for the CH=CH and NH in addition to the expected signals for the morpholine and aromatic protons (see Experimental). For example, the 1 H-NMR spectrum of compound 4a taken as a typical example of the products 4, revealed three signals at δ = 7.45 (d, J = 8 Hz, 1H, CH=CH), 7.79 (d, J = 8 Hz, 1H, CH=CH), 10.58 (brs, 1H, NH) and morpholine protons ppm in addition to the characteristic signals of the aromatic protons. The 13 C-NMR spectrum of compound 4a showed three signals at δ = 25.76, 68.04, 188.22 ppm assignable for the morpholine-C and the carbonyl-C, in addition to twelve aromatic and olifinic carbons. Moreover, the mass spectrum of 4a revealed a molecular ion peak at m/z = 335 which is consistent with its expected molecular weight.
The structures assigned for products 4 were further evidenced via alternative method. Thus, condensation of 2-oxo-N-phenylpropanehydrazonoyl chloride (1) with benzaldehyde 3a in glacial acetic acid in the presence of catalytic amount of concentrated H2SO4, afforded 2-oxo-N,4-diphenylbut-3-enehydrazonoyl chloride (5). Refluxing an equimolar amounts of 5 and morpholine in ethanol for 3 h, gave a product identical in all respects (m.p., mixed m.p. and IR spectra) with compound 4a obtained from the 2a + 3a reaction (Scheme 1).
The morpholinylchalcone derivatives 4a-e were then used for the preparation of novel series of 2-thioxo-2,3-dihydropyrido [2,3-d]pyrimidin-4(1H)-one derivatives bearing morpholine moieties. Thus, reaction of 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6) and the appropriate chalcones 4a-e in ethanol under reflux for 8-12 h led to the formation of one isolable product as evidenced by TLC analysis of the crude product (Scheme 2). The structures of the products was identified to be 7a-e based on elemental analysis and spectral data. For example, the IR spectrum of 7a-e revealed three absorption bands near υ max 3125, 3243, 3447 cm −1 due to the three NH groups, and another absorption band near υ max 1679 cm −1 attributed to the carbonyl group. The 1 H-NMR spectra displayed three singlet signals near δ 9.67, 10.65 and 11.17 ppm attributed to the three NH protons (disappeared by D 2 O), in addition to the expected signals due to the morpholine and aryl protons. Also, the 13 C-NMR spectra showed the expected number of aliphatic and aromatic signals. The mass spectra of the products 7a-e revealed in each case a molecular ion peak m/z at the expected molecular weight calculated for each compound (see Experimental).
For a much more rigorous identification of the structures of compounds 7a-e, a comparison with authentic material prepared from the reaction between thione 8 (obtained from reaction of chalcone 5 and compound 6) with morpholine was achieved, the product obtained from this reaction was identical to that from the reaction of 4a and 6 (Scheme 2). The morpholinylchalcone derivatives 4a-e were then used for the preparation of novel series of 2-thioxo-2,3-dihydropyrido [2,3-d]pyrimidin-4(1H)-one derivatives bearing morpholine moieties. Thus, reaction of 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6) and the appropriate chalcones 4a-e in ethanol under reflux for 8-12 h led to the formation of one isolable product as evidenced by TLC analysis of the crude product (Scheme 2). The structures of the products was identified to be 7a-e based on elemental analysis and spectral data. For example, the IR spectrum of 7a-e revealed three absorption bands near υmax 3125, 3243, 3447 cm −1 due to the three NH groups, and another absorption band near υmax 1679 cm −1 attributed to the carbonyl group. The 1 H-NMR spectra displayed three singlet signals near δ 9.67, 10.65 and 11.17 ppm attributed to the three NH protons (disappeared by D2O), in addition to the expected signals due to the morpholine and aryl protons. Also, the 13 C-NMR spectra showed the expected number of aliphatic and aromatic signals. The mass spectra of the products 7a-e revealed in each case a molecular ion peak m/z at the expected molecular weight calculated for each compound (see Experimental).
For a much more rigorous identification of the structures of compounds 7a-e, a comparison with authentic material prepared from the reaction between thione 8 (obtained from reaction of chalcone 5 and compound 6) with morpholine was achieved, the product obtained from this reaction was identical to that from the reaction of 4a and 6 (Scheme 2).

Cytotoxic Activity
The in vitro growth inhibitory activity of the newly synthesized compounds 4a,c,e,f, 5, 7a-c, 8 and 10a-d was investigated against two carcinoma cell lines, a human lung cancer cell line (A-549) and a human hepatocellular carcinoma cell line (HepG-2), in comparison with a well-known anticancer standard drug (cisplatin) under the same conditions using a colorimetric MTT assay. Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC50) was determined. The results are depicted in Table 1 revealed that the descending order of activity of the newly synthesized compounds towards the lung carcinoma cell line (A-549) were as follows: The descending order of activity of the newly synthesized compounds towards the human hepatocellular carcinoma cell line (HepG-2) were as follows: 7b > 7c > 10d > 4f > 5 > 7a > 10c > 4e > 10b > 4c > 4a > 10a > 8.

Cytotoxic Activity
The in vitro growth inhibitory activity of the newly synthesized compounds 4a,c,e,f, 5, 7a-c, 8 and 10a-d was investigated against two carcinoma cell lines, a human lung cancer cell line (A-549) and a human hepatocellular carcinoma cell line (HepG-2), in comparison with a well-known anticancer standard drug (cisplatin) under the same conditions using a colorimetric MTT assay. Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC 50 ) was determined. The results are depicted in Table 1 revealed that the descending order of activity of the newly synthesized compounds towards the lung carcinoma cell line (A-549) were as follows: The descending order of activity of the newly synthesized compounds towards the human hepatocellular carcinoma cell line (HepG-2) were as follows: (substituted with COOEt group at position 3) have more in vitro inhibitory activity than compounds 10a and 10b (substituted with a COCH 3 group at position 3). Also compound 10d is more active than 10c where the p-substitution with a methyl group increases the activity via its +I effect. (substituted with COOEt group at position 3) have more in vitro inhibitory activity than compounds 10a and 10b (substituted with a COCH3 group at position 3). Also compound 10d is more active than 10c where the p-substitution with a methyl group increases the activity via its +I effect.

Molecular Docking
A major problem in chemotherapy is resistance to the chemotherapeutic agents. There are generally two major forms of resistance encountered in the clinic. One is intrinsic resistance, which is a property of the tumor cells and is not triggered by drug exposure. The other is known as acquired resistance, which occurs following exposure to the drug(s). Anti-folates such as methotrexate (MTX) and fluoropyrimidines such as 5-fluorouracil (5-FU) have been used in the clinic for the management of childhood acute lymphoblastic leukemias (ALL) and colorectal cancer, respectively, with modest success. Among the several enzymes that participate in the synthesis of nucleic acid precursors, DHFR is an important target for several human dis-eases, namely, protozoal, bacterial and fungal infections, psoriasis, autoimmune diseases and neoplastic diseases. Traditionally, several DHFR inhibitors are reported as potential drug candidates in various diseases.
In the late 1950s DHFR was discovered as a ubiquitous enzyme with respect to drug design due to its central role in the synthesis of DNA. Most eukaryotic organisms synthesize the essential metabolite thymidylate via the thymidylate cycle, which consists of three enzymes: serine hydroxymethyl transferase; thymidylate synthase (TS) and the much promising DHFR. It reduces the NADPH-dependent 7,8-DHF to 5,6,7,8-THF utilizing NADPH 2+ as cofactor. This THF acts in the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase. Inhibition of TS or of DHFR leads to thymineless death, which has found clinical utility as antitmalarial, antiprotozoal and antimicrobial agents.
The MOE 2014.010 package software was used to analyze all docking poses and binding energies between compound 7b and the enzyme dihydrofolate reductase (DHFR) to evaluate the affinity of 7b according to its binding energy with the enzyme.
From Figures 2 and 3 which represent all the binding energies of the two compounds.it is clear

Molecular Docking
A major problem in chemotherapy is resistance to the chemotherapeutic agents. There are generally two major forms of resistance encountered in the clinic. One is intrinsic resistance, which is a property of the tumor cells and is not triggered by drug exposure. The other is known as acquired resistance, which occurs following exposure to the drug(s). Anti-folates such as methotrexate (MTX) and fluoropyrimidines such as 5-fluorouracil (5-FU) have been used in the clinic for the management of childhood acute lymphoblastic leukemias (ALL) and colorectal cancer, respectively, with modest success. Among the several enzymes that participate in the synthesis of nucleic acid precursors, DHFR is an important target for several human dis-eases, namely, protozoal, bacterial and fungal infections, psoriasis, autoimmune diseases and neoplastic diseases. Traditionally, several DHFR inhibitors are reported as potential drug candidates in various diseases.
In the late 1950s DHFR was discovered as a ubiquitous enzyme with respect to drug design due to its central role in the synthesis of DNA. Most eukaryotic organisms synthesize the essential metabolite thymidylate via the thymidylate cycle, which consists of three enzymes: serine hydroxymethyl transferase; thymidylate synthase (TS) and the much promising DHFR. It reduces the NADPH-dependent 7,8-DHF to 5,6,7,8-THF utilizing NADPH 2+ as cofactor. This THF acts in the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase.
Inhibition of TS or of DHFR leads to thymineless death, which has found clinical utility as antitmalarial, antiprotozoal and antimicrobial agents.
The MOE 2014.010 package software was used to analyze all docking poses and binding energies between compound 7b and the enzyme dihydrofolate reductase (DHFR) to evaluate the affinity of 7b according to its binding energy with the enzyme.
From Figures 2 and 3 which represent all the binding energies of the two compounds.it is clear that the total binding energy of compound 7b equals −1.6 E (Kcal/mol), showing good affinity with the DHFR enzyme by forming four pi-hydrogen interactions with binding energy −1.4 E (kcal/mol) , one hydrogen acceptor interaction with binding energy −0.2 E (Kcal/mol) and one pi-pi interaction with almost zero binding energy.on the other hand compound comp showing affinity to the DHFR enzyme by −1.3 E (Kcal/mol) by making one hydrogen donor interaction with −0.6 E (Kcal/mol) and tow pi-hydrogen interactions with −0.7 E (Kcal/mol).   Bioactivity and ADME Toxicity Due to its impact on society, the design of new drugs has the potential to interest a wide audience, and provides a rare opportunity to introduce several concepts in chemistry and biochemistry. Drug design can be seen as a multi-objective cyclic optimization process. Indeed, it is important to develop the understanding not only that a drug is generally an effective ligand for a protein of therapeutic interest, but also that these molecules need to have drug-like properties. Computer-aided drug design and bioinformatics approaches play a fundamental role in addressing these different challenges. Basically, drug design consists of the conception of molecules that are complementary to   Bioactivity and ADME Toxicity Due to its impact on society, the design of new drugs has the potential to interest a wide audience, and provides a rare opportunity to introduce several concepts in chemistry and biochemistry. Drug design can be seen as a multi-objective cyclic optimization process. Indeed, it is important to develop the understanding not only that a drug is generally an effective ligand for a protein of therapeutic interest, but also that these molecules need to have drug-like properties. Computer-aided drug design and bioinformatics approaches play a fundamental role in addressing these different

Bioactivity and ADME Toxicity
Due to its impact on society, the design of new drugs has the potential to interest a wide audience, and provides a rare opportunity to introduce several concepts in chemistry and biochemistry. Drug design can be seen as a multi-objective cyclic optimization process. Indeed, it is important to develop the understanding not only that a drug is generally an effective ligand for a protein of therapeutic interest, but also that these molecules need to have drug-like properties. Computer-aided drug design and bioinformatics approaches play a fundamental role in addressing these different challenges. Basically, drug design consists of the conception of molecules that are complementary to the protein target in terms of 3D-shape and charge distribution, to optimize molecular recognition and binding. On the contrary, ligand based approaches rely on the knowledge implicitly contained in the chemical structure or physical properties of other molecules that bind to the biological target of interest.
Molecular properties in relation to lipophilicity, drug likeness, or pharmacokinetics (PK), for example. These molecular properties are fundamental in drug design. Indeed, although a high affinity for the protein target is essential, it is not sufficient for the designed small molecule to become a drug: to obtain a therapeutic effect, the molecule needs to reach its target in the body, and stay there long enough for the expected biological events to occur. Therefore, to support efficiently the design of new drugs, it is important to predict their PK behaviors with computer-aided approaches.
A computational study was also carried out including prediction of pharmacokinetic properties, toxicity and bioactivity studies. In Table 2 Molecular properties were calculated on the basis of Lipinski's rule and its components, Furthermore, TPSA values of the tested compounds, the prediction of bioactivity scores of the compounds were recorded by calculating the activity scores of GPCR, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme inhibitor.
Physicochemical properties, with an emphasis on lipophilicity computed by a variety of methodologies to enable a consensus approach by calculating the molecular weight and TPSA of the tested compounds. Druglikeness, estimated by simple rules to evaluate oral bioavailability through evaluating the compounds according to Lipinski rule five and also Pharmacokinetics, which predicts several ADME behaviors (e.g., substrate of P-glycoprotein, cytochromes P450, gastrointestinal absorption, brain blood barrier) by binary classification models relying on physicochemical descriptors. While medicinal chemistry that gives a score for synthetic accessibility, leadlikeness and pan-assay interference structure of molecules together with structural alerts for problematic fragments. Topological polar surface area (TPSA), gastrointestinal absorption (GI absorption), blood brain barrier (BBB) permeant, P-glycoprotein substrate (P-gp substrate), cytochrome P50 1A2 inhibitor (CYP1A2 inhibitor), pan-assay interference structure (PAINS).
The docking study was performed using the MOE 2014.010 software. The crystal structure of the enzyme dihydrofolate reductase (DHFR, PDB ID (3NU0)) was downloaded out from Protein Data Bank website. Regularization and optimization for protein and ligand were performed. Determination of the essential amino acids in the binding site was carried out and compared with that presented in the literature. The performance of the docking method was evaluated by re-docking the crystal ligand into the assigned active dihydrofolate reductase (DHFR) enzyme to determine a RMSD value. Interactive docking to the selected active site was carried out for all the conformers of interesting compounds. Each docked compound was assigned a score according to its fit in the ligand binding pocket (LBP) and its binding mode.

General Information
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). IR spectra were measured on Shimadzu FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan) in potassium bromide discs. NMR spectra were measured on a Varian Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV. Elemental analyses were measured by using an Elementar Vario LIII CHNS analyzer (GmbH & Co.KG, Hanau, Germany). Antitumor activity of the products was measured at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt. Hydrazonoyl halides were prepared following a literature method [35].

Synthesis of Chalcones 4a-f
A mixture of 1-morpholino-1-(2-arylhydrazono)propan-2-ones 2a,b (10 mmol) and the appropriate benzaldehyde derivatives 3a-c (10 mmol) in glacial acetic acid (20 mL) containing a few drops of concentrated H 2 SO 4 was stirred at room temperature for 4 h (monitored by TLC). The formed precipitate after cooling was isolated by filtration, washed with ethanol, dried and recrystallized from ethanol to give products 4a-f.

Alternative Synthesis of 4a
Equimolar amounts of 5 (0.284 g, l mmol) and morpholine (0.87 g, 1 mmol) in ethanol (15 mL) was refluxed for 3 h, gave product identical in all respects (m.p., mixed m.p. and IR spectra) with compound 4a which obtained from reaction of 2a + 3a.

Alternative Synthesis of 7a
Equimolar amounts of 8 (0.407 g, l mmol) and morpholine (0.87 g, 1 mmol) in ethanol (15 mL) was refluxed for 3 h, gave product identical in all respects (m.p., mixed m.p. and IR spectra) with compound 7a which obtained from reaction of 4a + 6.

Anticancer Activity
The cytotoxic evaluation of the synthesized compounds was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt according to the reported method [36,37].

Molecular Modeling
Docking studies were performed using the MOE 2014.09 software. Regularization and optimization for protein and ligand were performed. Each docked compound was assigned a score according to its fit in the ligand binding pocket (LBP) and its binding mode [38][39][40].

Conclusions
In our present work, we present an efficient synthesis of novel morpholinylchalcones, which have not been hitherto reported. These chalcones were used as building blocks for constructing a series of pyridopyrimidinethiones and pyrido [2,3-d]