Synthesis , Docking Studies and Anticancer Activity of New Substituted Pyrimidine and Triazolopyrimidine Glycosides

1 Photochemistry Department, National Research Centre, Dokki, Giza, Egypt. 2 Chemistry Department, College of Science, Aljouf University, Sakaka, Al-Jouf, Kingdom of Saudi Arabia. 3 Applied Organic Chemistry Department, National Research Centre, Dokki, Cairo, Egypt. 4 Hormone Department, National Research Centre, Dokki, Cairo, Egypt. 5 Department of Pharmaceutical Chemistry, National Research Centre, Dokki, Cairo, Egypt.


INTRODUCTION
Although, recently, there have been developing advance in various therapeutic strategies, cytotoxic drugs remains the main backbone for cancer treatment (Butler et al., 2015).Drugs which affect DNA biosynthesis have received much attention and amongst them pyrimidine derivatives remain the most important (Cieplik, 1992;Pogorelcnik et al., 2015).Many pyrimidine incorporating compounds constitute an assortment of drugs with ability to hinder biosynthesis of pyrimidine nucleotides or act effectively as naturalist metabolites, thus interfering in substantial ellular processes, for example nucleic acids synthesis.
Synthesized glycosides and their derived analogs (Zhao et al., 2008;Zhao and Li, 2007) revealed considerable inhibitory activity of DNA-topoisomerase II (TopoII) that controls DNA topology by transitory cleavage of the DNA double helix, which was affirmed as important molecular target of anticancer drugs (Champoux, 2001;Pommier et al., 2010).Moreover, studied biological assays found that exotic sugar part confers a number of glycosides with best conformation possessing high binding affinities via hydrogen bonding to the entry of the ATPase pocket (Gui et al., 2011;Shi et al., 2012).In previous docking studies, it has been shown that acetylated and deprotected sugar moieties play very important roles in their found significant inhibition activity (Zhao et al., 2012).Nucleoside analogs have been investigated as important drugs for the treatment of different cancer types.Compounds such as Cytarabine, Fludarabine, Cladribine, Gemcitabine, Clofarabine, Capecitabine, Floxuridine, Deoxycoformycin, Azacitidine and Decitabine (fig. 1) which belong to the nucleoside analogs class, currently approved antitumoral activity (Jordheim et al., 2013).Different nucleoside analogs incorporating substituents at the C-5 position in the heterocyclic base, especially in the 2-deoxyuridine type, were investigated to have interesting biological activities as antiviral and anticancer agents (Srinivasan et al., 20101;McGuian et al., 2000;Yoo et al., 2002;Hannah et al., 2000;Khan and Grinstaff, 1998).The above findings and our interest in glycosyl heterocycles synthesis with biological activity research field (Mohamed et al., 2015;El-Sayed et al, 2009;Amr et al., 2006;El-Sayed et al., 2016) promoted us to design and synthesize new pyrimidine and triazolopyrimidine glycosides with acetylated or free hydroxyl sugar moieties and acyclic analogs bearing ether or terminal hydroxyl studying their anticancer activity against a number of cancer cells.

Instruments and reagents
Melting points were measured using a Böetius PHMK (VebAnalytik Dresden) instrument.TLC was implemented using aluminum plates pre-coated with silica gel 60 or 60 F254 (Merck) and visualized using UV light (254 nm).Nuclear Magnetic Resonance of compounds was carried out on a Varian Gemini 300 and Bruker DRX 400 spectrometer at 25 ºCwith TMS as a reference and solvent shift ((CD 3 ) 2 SO δH 2.50 and δC 39.5).Coupling constants are expressed in Hz without sign.Mass spectrometry was performed using a Varian FINNIGAN MAT 212 machine.The Infra-red spectra were investigated (KBr) by means of a Jasco FT/IR-410 apparatus.Elemental analysis was measured using the Perkin Elmer 240 instrument.The starting hydrazinyl pyrimidine compound 1 was prepared as previously reported (Libermann and Rouaix, 1955).

Synthesis of acetylated N-glycosides 6 and 7
A solution of compound 2 (0.01 mol) in DMF (15 mL) was added portion wise to a stirred suspension of sodium hydride (0.015 mol) at 0°C then stirring was continued for 1 h at room temperature.Acetylated bromo-sugar 5a, b (0.01 mol) dissolved in DMF (10 mL) was added slowly to the mixture then continual stirring was performed for 6-8 h (TLC eluent: pet.ether/hexane, 3:1).Ice cold water was added with vigorous stirring for 30 minutes and the resulting precipitate was filtered, dried and crystallized from ethanol.

Synthesis of deacetylated N-glycosides 8 and 9
A solution of the acetylated glycoside 6 and 7 (0.5 g) in saturated methanolic ammonia (20 mL) was stirred at room temperature for 5 h.After completion of the deacetylation process (TLC: petroleum ether/hexane, 2:1), the liquid was vaporized by means of rotatory evaporator and the remnant was triturated with diethyl ether (25 mL) leading to a solid which was filtered, dried and crystallized from ethanol.

Synthesis of the N-substituted triazolopyrimidine derivatives 11 and 12
Method A: To a well stirred suspension of sodium hydride (0.05 mol) in dry DMF (15 mL) at 0°C, a solution of the triazolopyrimidine 10 (0.01 mol) in DMF (20 mL) was added portion-wise through 15 minutes while the mixture was preserved in cooling ice.The ice was removed and the reaction contents were stirred for 1 hour.2-(2-Chloroethoxy) ethanol or chloroacetaldehyde dimethyl acetal (0.01 mol) was inserted then the mixture was stirred for 7-10 h at 70 °C.After completion of the reaction (TLC: petroleum ether/ethyl acetate, 3:1), ice-cold water was added and the resulting gum substance was further stirred with crushed ice for about 1 h.Filtration of the precipitate and recrystallization from ethanol resulted in the acyclic oxygenated compounds 11 or 12, respectively.

Method B (for compound 12)
Anhydrous sodium acetate (0.01 mol) was added to a solution of compound 4 (0.01 mol) in acetic acid (20 mL) and the resulting mixture was heated till dissolution of the solid material.The reaction flask was cooled by means of ice-water bath then acetic acid (5 mL) containing bromine (0.015 mol) was slowly added drop-wise during 10-15 minutes.The afforded oily material was shacked continuously at room temperature for 30 minutes followed by heating to100 °C for 1 hour.The mixture was cooled then poured onto crushed ice and the precipitated triazolopyrimidine was collected by filtration, and crystallized from cold methanol.

Synthesis of acetylated triazolopyrimidine N-glycoside derivatives 13 and 14
Sodium hydride (0.015 mmol) was added portion wise to a stirred solution of compound 10 (0.01 mol) in dry DMF (20 mL) at 0°C then stirring was continued for additional 1 h at room temperature.The sugar 5a, b (0.01 mol) in dry DMF (12 mL) has been added slowly followed by stirring for 6-7 h at 20-25 °C.Ice cold water was added with vigorous stirring for 30 minutes and the resulting precipitate was filtered, dried and crystallized from ethanol to afford 13 or 14, respectively.

Synthesis of deacetylated N-glycosides 15 and 16
The acetylatedglycoside 13 or 14 (5 mmol) was added to a saturated methanolic ammonia solution (15 mL) at 0°C was stirring over a period of 20 minutes then the reaction mixture was further stirred at room temperature for 7 h.After completion of the deacetylation process (TLC: petroleum ether/hexane, 2:1), removing the solvent under vacuum gave a yellowish residue.Trituration with cold diethyl ether (20 mL) with stirring afforded a solid has been filtered and recrystallized using cold ethanol giving compounds 15 and 16, respectively.

Material
All cell lines were brought from ATCC via Vacsera tissue culture laboratories.All media were purchased from Lonza, Belgium, serum from Gibco, trypsin and MTT from Biobasic Canada.

In vitro antitumor bioassay on human tumor cell lines
Cell culture MCF7, PC3 and HCT116 cell lines were maintained in DMEM high glucose with l-glutamine, 10% foetal bovine serum at 37°C in 5% CO 2 and 95% moisture.Cells were sub-cultured employing trypsin versene 0.15%.

Viability test
After nearly 24h of cultivating 20000 cells per well (in 96-well plates), when about 60-70% confluence have attained by cells, alteration of the medium into % serum-free medium, exhibiting a definitive concentration of compounds of 100 µM in triplicates, occurred.Treatment of cells was performed for 72h.100µM of Doxorubicin was used as a positive control and serum free medium was used as a negative control.

In vitro antitumor bioassay on human normal cell line Cell culture
Human retinal pigmented epithelial cell line RPE1 was maintained in DMEM F12 medium, in addition to the same applied conditions and reagents for the cancer cell lines in the present study.In addition the viability test was carried, by seeding 40000 cell lines, as previously mentioned methodology for such latter cell lines.
Prepared of the protein crystal structure for docking was carried out by excluding by water molecules, providing and elimination of polar hydrogen atoms then separation of the active pocket.The active site was believed as the site where Roscovitine (as co-crystalline ligand) may complexe (PDB ID: 2a4l).Roscovitine ligand was re-docked in such pocket to assure the docking methodology is effective in addition that the applied pocket was most convenient for docking emulation of the synthesized products (ligands).
The structure of the selected compounds (ligands) for docking was drawn in ChemDraw Ultra 10.0 (ChemOffice package) and saved.Preparation steps which should precede docking process, involve; a) Visualizing the 3D form of ligands through conversion of the their 2D structure; b) Insertion and excluding polar H atoms; c) Applying MMFF94x force field to minimize energy till a RMSD (Root-mean-square deviation) of atomic position gradient of 0.01 Kcal mol -1 Å-1 was reached and saved as moe (Kaminski et al., 1996).
The docking Algorithm was done by MOE-DOCK default.It uses flexible, rigid technique for posing the molecule inside the cavity.All rotatable bonds of ligands are allowed to undergo free rotation to be placed into the rigid receptor binding site.The docking scores were displayed in negative energy expression; low binding free energy is an indication of better binding affinity (Lensink et al., 2007), and the ligand interactions (hydrogen bonding and hydrophobic interaction) with CDK 2 was determined.
The IR spectrum of the N-substituted pyrimidine derivative 3 showed the presence of hydroxyl absorption band.The 1 H NMR spectra of the N-substituted oxygenated alkyl products 3 and 4 revealed the presence of the signals assigned for the oxygenated alkyl chain in these products.When the key substituted pyrimidine derivative 2 was allowed to react with Oacetylated glycopyranosyl bromide 5a,b in presence of sodium hydride at room temperature, the corresponding acetylated N 3pyrimidine glycosides 6 or 7 were obtained in 74-76% yield.Their 1 H NMR spectra possessed the signals which are assigned for the alkyl, aryl and sugar part protons.
The anomeric proton signal appeared as doublet with coupling constants 10.2 and 9.5 Hz indicating that the afforded glycosides in the β-conformation.Deacetylation of the latter glycosides was performed effectively by means of saturated methanolic ammonia solution at room temperature to give the congruent glycosides with unprotected hydroxyls 8 and 9, respectively (scheme 1).
Their IR spectra showed the sugar-hydroxyl absorption bands and the disappearance of the acetyl-methyl signals in the corresponding NMR data.On the other hand, compound 2 was used also for the preparation of a condensed pyrimidine derivative having a free NH in the pyrimidine nucleus.The reaction was performed by means of bromine in methanol to result in the [1,2,4]triazolo[4,3-a]pyrimidine derivative 10.The 1 H NMR spectrum indicated the absence of proton of the azomethine (N=CH) group and displayed one NH signal.Its 13 C NMR showed the disappearance of the CH=N and presence of the C-3 ay higher chemical shift.Formation of the triazolopyrimidine with this mode of cyclization was also confirmed by formation of cyclized product 12 by reaction of substituted pyrimidine 4 with bromine in acetic acid which is the same product of alkylation of triazolopyrimidine 10 with 2-chloro-1,1-dimethoxyethane.These results are in accordance with previously reported results for similar triazolpyrimine syntheses (Shaban et al., 1995;Turk et al., 1998) and also with their mode of preparation.The prepared triazolopyrimidine 10 was also used, like the substituted pyrimidine 3, to prepare another N-substituted triazolopyrimidine derivative of the triazolopyrimidne system by reaction with oxygenated hydroxyl alkyl; namely 2-(2-chloroethoxy)ethan-1-ol and gave the N-substituted derivative 11, in 77% yield.
Glycosylation of the key triazolopyrimidine 10 with the same α-glycosyl bromides used previously lead to the formation of the N 3 -triazolopyrimidine glycosides 13 and 14, respectively.The 1 H NMR spectra showed, in addition to signals of alkyl and aryl protons, the sugar moiety signals appeared at 4.12-5.88ppm.The coupling constants of the H-1 in the glycosyl moiety 9.4 and 9.8 Hz indicated β-glycosidic linkage naturein glycosides 13 and 14. Preparation of the free hydroxyl glycoside derivatives 15 and 16 was performed by the deacetylation reaction of the acetylated Nglycosides 13 and 14, respectively by means of ammonia solution in dry methanol at room temperature.The IR spectra of the resulting deprotected glycosides showed the hydroxyl bands in addition to the disappearance of the acetyl-carbonyl functions.Their 1 H NMR spectra indicated the presence of the hydroxyl and sugar protons signals in addition to the disappearance of the methyl of the acetyl groups.
Scheme 1 Synthesis of pyrimidine glycosides and acyclic analogs.
pyrimidine ring system afforded higher inhibition activity which is obvious from the higher activity of the triazolopyrimidine 10 derivative than its pyrimidine precursor.Furthermore, the substitution at N-1position in triazolpyrimidine nucleus with acyclic oxygenated alkyl, as triazolopyrimidine acyclic nucleoside analog, lead to raised inhibition activity on PC3 compared to the terminal free hydroxyl substituent.Moreover, it was concluded that the substituted pyrimidine glycosides were found to be more active than their derived triazolpyrimidine glucoside or xyloside.These results could be basis of further studies for design and synthesis of more modified pyrimidine nucleoside analogs.

Molecular docking study
Docking studies are coveted so as to comprehend the mechanisms of actions of drugs, modes of interactions with targets, and to integrate any experimental guide announced.These forms are needed to get a suitable and more accurate form of biologically active molecules at the atomic level and thus, provide new willfulness which might be applied to design novel therapeutic agents.Docking process was performed for the target compounds intocyclin-dependent kinase 2 (CDK2) using MOE 2008.10 program.
From the obtained results (table 3, fig.4-7) it was shown that, the studied compounds exhibited good fitting ability inside the binding site of the protein molecular surface with minimum binding energy ranged from -13.432 to -22.760 kJ mol -1 in comparison to the co-crystallized ligand.Co-crystallized ligand Roscovitine exhibited binding energy of -23.54kJmol -1 and it showed van der Waals interaction with arene-cationand between Lys 89 (Fig 3).
Compound 7 showed binding energy of-21.569kJ mol -1 and formed one hydrogen bond with the oxygen atom of the carbonyl group moiety as it acts as a hydrogen bond acceptor with the side chain of Lys 89 in distance 2.53 Å with strength of 19% (Fig 4).Substitution at C-2 in the pyrimidine ring with substituted amino group linked to oxygenated moiety provides some similarity to the structure of Roscovitine.The docking conformation of compound 8 in the active site of the protein revealed good interactions with the active site residues of this protein.Compound 8 formed hydrogen bond interaction between hydrogen of hydroxyl group moiety with the side chain of Glu 12 residue (2.60 Å) with a strength of 33%.Furthermore, it showed van der Waals interaction with Lys 89 (Figure 5).
The absence of the hydroxyls responsible for the Hbonds accounts for the binding energy of this compound compared to its acetylated form.Meanwhile, docking study of compound 10 showed hydrogen bond interaction between oxygen atom of the carbonyl group moiety and the side chain of Lys89 residue (2.51 Å) with strength of 35% (Figure 6).Docking studies of compound 12 into the active site of the enzyme showed two hydrogen bonds one between oxygen of carbonyl group moiety and Lys89 residue (2.32 Å) and the other between oxygen of the methoxy group and the same protein residue (2.71Å) with strength of 15% and 62% respectively (Figure 7).
The conformation of the acyclic substituent at N-3 in the pyrimidine ring with acyclic oxygenated alkyl having two symmetrical methoxy groups might be with effect allowing for such binding mode.

Fig 3 .
Fig 3. Docking of compound Roscovitineinto the active site of CDK2.Fig 4. Docking of compound 7into the active site of CDK2.

Fig 5 .
Fig 5. Docking of compound 8into the active site of CDK2.Fig 6.Docking of compound 10into the active site of CDK2.

Fig 7 .
Fig 7. Docking of compound 12into the active site of CDK2.

Table 2 :
IC50 values for compounds which showed more than 40% inhibition.