Anticancer and Antiviral Activity of the Pyridine-Biphenyl Glycoside System

Ahmed I. Khodair1*, Adel M. Attia1, Eman A. Gendy1, Yaseen A. M. M. Elshaier2, Mohammed A. El-Magd3 1Chemistry Department, Faculty of Science, kafrelshiekh University, El-Geish Street, kafrelshiekh, Post Box 33516, Egypt 2Department of Organic and Medicinal chemistry, Faculty of Pharmacy, University of Sadat City, Menoufiya, 32897, Egypt 3Anatomy Department, Faculty of Veterinary Medicine, Kafrelshiekh University, El-Geish Street, kafrelshiekh, Post Box 33516, Egypt

According to the report published recently by the World Health Organization, the number of cancer cases in the world will increase to 22 million by 2030. So, the anticancer drug research and development is taking place in the direction where the new entities are developed which are low in toxicity and are with improved activity. Pyridine and their pyridine-biphenyl system derivatives represent a very important class of heterocyclic compounds, which have a diverse therapeutic area. Recently, many active compounds synthesized are very effective; natural products isolated with pyridine moiety have also shown to be potent towards cancer.
In the last few years, many research groups have designed and developed many novel compounds with pyridine as their backbone and checked their anticancer activity. In this short communication, the recent developments made in the direction of design and synthesis of new scaffolds with very potent anticancer activity are briefly described. The effect of various heterocycles attached to the pyridinebiphenyl system and their effect on the anticancer activity are thoroughly studied and recorded in this short communication.
Cancer is a one of the major health problems for human beings with the leading mortality rate [1]. Natural, synthetic, or biological and chemical substances are the cancer-causing agents [2]. Many drugs are used to cure it, but they have their own toxic side effects [3]. Hence, there are lots of research carried out to synthesize new [4][5][6], effective, and affordable anticancer drugs with more selectivity, minimum dosage, and lesser side effects.
Drug discovery over the years have focussed more on the heterocyclic chemistry due to their huge success rate in forming active pharmaceutical intermediate. Among the heterocyclic compounds, pyridine-biphenyl system is one of the most important heterocyclic compounds which exhibit remarkable pharmacological activities [7][8][9][10][11]. There are many reputations of synthetic compounds and naturally occurring compounds with pyridine-biphenyl system backbone showing a very active anti-cancer activity. Different research groups have done much progress in designing compounds with pyridine-biphenyl system, synthesizing them, and collecting anticancer activity data of those against various human cancer cell lines. An attempt has been made to see how various heterocyclic moiety attached with pyridine-biphenyl system have an effect on the anticancer activity of the various pyridinebiphenyl system compounds synthesized by different groups. A compiled data of all these recent articles helps in providing a direction towards further research.
In view of the above observations, our research interest focused on the design and synthesis of small heterocyclic nucleosides targeting cancer especially MCF-7 cell lines, rotavirus WA, HAVHM175, and herpes simplex 1. The elaboration of pyridine-biphenyl system linked with glucopyranose sugars (Figure 1) to form the target nucleosides was our task [49].  Figure 1: N-glycosides of dihydropyridine as P-glycoprotein inhibitors.  The aglycone part is a biphenyl system (ring A and ring B) linked by benzotriazole moiety. The glycone part originated from the two epimerise monosaccharides, glucose, galactose acetylated or deacetylated and examining the effect of N-glycoside or S-glycoside in activity relationship ( Figure 2). Their anticancer activities against breast cancer (MC7) including apoptosis studies were evaluated. The cytotoxicity of these compounds against the normal cells and their antiviral activities were also determined. Docking studies and shape similarity studies were also investigated. Glycosides of structurally similar heterocyclic systems have been reported before .
The cytotoxicity of the synthesized compounds was evaluated in vitro against MCF7 cells by MTT assay [99]. The results of novel compounds showed IC 50 values compared to the reference cisplatin are shown in Table 1. The results showed compounds 1f, 1g, 3f, 3h moderate activity against MCF7 cells with IC 50 = 30.63, 24.39, 27.24, 20.49 respectively. Considering the glycone part, it was observed that compounds contain galactose moiety were more active than others which contain glucose part and this also stratify on their free acetyl of moiety sugar; compound 1f against 1b, compound 1g against to 1c, compound 3f against 3b, and compound 3h against 3d. Regarding aglycone part, compounds contain R 1 = CN, R 2 = NH 2 or OH are least active. We hope that the synthesized compounds serve as lead chemical entities for further modification to render them clinically useful drug agents. N-glycosides are less active in comparison to S-glycosides.
To check whether the cytotoxic effect of compounds 1f, 1g, 3f, and 3h occurred through induction of apoptosis in MCF7 cells, changes in gene expression of apoptotic genes Bax and p53 and the anti-apoptotic gene BCl 2 were detected by qPCR before and after addition of these compounds. Addition of these compounds resulted in a significant (P ≤ 0.05) increase in the expression of the Bax and p53 genes in MCF7 cells, with highest expression in 3h followed by 3g, then 3f and finally 1f, as compared to vehicle (DMSO)treated MCF7 cells ( Figure 4). However, BCl 2 expression was significantly decreased in MCF7 cells treated by the 4 compounds, with the following order 3h>1g>3f>1f, as compared to vehicle-treated MCF7 cells ( Figure 3).   showed activity less than others are shown in Table 3.

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In general, the deacetylated glycosides are more reactive than acetylated analogues. The deacetylated analogues are more reactive while in considering the aglycone part, the compounds contain R 1 = nitrile or acetyl functionality and R 2 = OH, or NH 2 in pyridine part is more reactive.
P53 is an endogenous protein which acts as a tumor suppressor that stop cancer cell from growing and multiplying. The over expression of MDM2 has been observed in a wide range of tumor types. MDM2 interact P53 and so cancer cell blocked the suppression effect of P53 and so avoid apoptosis [101][102][103][104]. As our compounds induce apoptosis and increase P53, the goal was directed to examine the docking of these compounds with MDM2 and examine their activity to inhabit P53-MDM2 interactions.   In this regard, a library of target compounds was energy minimized using MMFF94 force field calculations.
The catalytic domain of MDM2 (PDB code 5law) [105] was prepared for docking using Open Eye ® (Fast Rigid Exhaustive Docking (FRED) Receptor, OMEGA; VIDA. Open Eye Omega application was used to generate different conformations of each ligand. Docking was conducted using FRED, and the data were visualized by the Veda application. This software package generates consensus scoring, a filtering process, to obtain virtual binding affinity; the lower the consensus score, the better the binding affinity of the ligand toward the receptor.
The most active compound 3h binds with the specific receptor of MDM (ID: 5law) with best consensus score 1 and forms hydrogen bonding (HB) through its OH of C-5 of galactose moiety with Tyr 100 AA and through OH of C-4 with both Gly 24 AA and Ala 21 AA. The triazoe ring forms HB with Gly 58 AA, ( Figure 4A). The pyridine and the aryl ring from biphenyl system are adopted perpendicularly in the receptor through formation of hydrophobic-hydrophobic interactions. Compound 3f docks with consensus score 5 and overlays completely with 3h but with formation of one HB through its triazole ring with Gly 58 AA ( Figure 4B). However, compound 1g showed consensuses score 23 and docked with different pose and mode through formation of HB with Ala 21 AA. Acetylated galactose (1g) forms hydrophobic-hydrophobic interaction with the receptor ( Figure 4C). In order to understand the effect of galactose and glucose moiety, the docking pose and mode for compound 1f was compared to 1b, compound 1g was compared to 1c, compound 3f was compared to 3b and compound 3h was compared to 3d. It was clear that the axial position of epimeric hydroxyl in galactose moiety switch the molecule to form HB interactions.
The final compounds behave special manners as the epimeric isomers are not near each other in activity (compound 1g versus 1c, compound 1f versus 1b; and  compound 3h versus 1d, compound 3f versus 1b) and also variations of substituent on pyridine ring play an important role in activity. To gain insight about structure activity relationship and understand the compounds activity, ROCS was employed [106][107][108]. ROCS are a shape-based superposition method and used to perceive similarity between molecules based on their three-dimensional shape. Shape similarity is as a fundamental descriptor in drug design. ROCS alignment requires a) query molecules and those queries here are the most active compounds in both acetylated (1g) and deacetylated (3h) sugar and; b) the database molecules that our final compounds.
The quality of alignment between database and query was calculated using Tanimoto Combo. Tanimoto Combo is the summation of Shape Tanimoto and Colour Tanimoto. Shape Tanimoto represents the shared volume and mismatch volume and has scale from 0 to 1. Colour Tanimoto (also scale from 0 to 1) is reflective of the degree of matching or mismatching of light chemical features in 3diminssions. From ROCs model (shape and colour), quires volume showed many points acceptors, donors and rings. Quality of alignment, using ROCS, between compounds 1g and 3h (queries) Figure 5A, Figure 5C respectively, and database molecules (final compounds) was calculated using Tanimoto Combo (Table 5). Compound 1f overlay complete within the query volume shape  Figure 5D. Based on the ROCs data, Shape Tanimoto data revealed good correlation with biological activities. For examples compounds 4f and 1b exhibited highly Shape Tanimoto score using 1g as query and compounds 3d and 3f using 3h as query.
Structure activity relationship studies revealed the following features:1) S-glycosides pyridine are more active than N-gycosides probably due to strength and rigidity of N-glycosides; 2) compounds contain deacetylate sugar moiety are more reactive than acetylated derivatives. This hypothesis is clear because the alcoholic hydroxyl groups form HB with the receptor amino acids; 3) the galactosyl compounds are more reactive than glucosyl anaslogs because the epimeric OH forms HB when it located axially; compounds contain glycosides of acetyl galactose is more active than acetylated glucose; 4) the pyridine ring perpendicular to methoxyphenyl ring allowing this biphenyl system to from hydrophobic-hydrophobic interaction.

Conclusion
A biphenyl system from poly functionalized pyridine tethered with benzotriazole moiety was synthesized in very simple method. This system linked to glycoside formation with glucose and galactose epimers. The glycoside side chain was either S-glycosides or N-glycosides. S-Glycosides are more reactive than N-glycosides analogues. In case of antiviral activity, the deacetylated sugars are more active which indicate to importance of lipophilicity. Finally, we recommend further in vivo cancer models for this compound so that it can be developed as chemotherapeutic anti-cancer agent.