Molecular docking, synthesis and biological significance of pyrimidine analogues as prospective antimicrobial and antiproliferative agents

Pyrimidine nucleus is a significant pharmacophore that exhibited excellent pharmacological activities. A series of pyrimidine scaffolds was synthesized and its chemical structures were confirmed by physicochemical and spectral analysis. The synthesized compounds were evaluated for their antimicrobial potential towards Gram positive and negative bacteria as well as fungal species. They were also assessed for their anticancer activity toward a human colorectal carcinoma cell line (HCT116). Whilst results of antimicrobial potential revealed that compounds Ax2, Ax3, Ax8 and Ax14 exhibited better activity against tested microorganisms, the results of antiproliferative activity indicated that compounds Ax7 and Ax10 showed excellent activity against HCT116. Further, the molecular docking of pyrimidine derivatives Ax1, Ax9 and Ax10 with CDK8 (PDB id: 5FGK) protein indicated that moderate to better docking results within the binding pocket. Compounds Ax8 and Ax10 having significant antimicrobial and anticancer activities may be selected as lead compounds for the development of novel antimicrobial and anticancer agent, respectively.


Introduction
Drug designing is a technique of searching and developing new molecules that exert specific action on a human kind [1]. The figure of multidrug resistant microbial infections is growing day by day which indicated that it is crucial to develop new class of antimicrobial drugs [2]. Tumor is a severe health issue and 2nd leading/most reason for mortality in the globe. It is caused by deregulation of the cell cycle which results in failure of cellular differentiation and unrestrained cellular growth [3,4]. So, it is necessary to develop and synthesize new bioactive molecules whose chemical structure and mode of action are noticeably differing from the available agents [5].
Discovery of drug is a slow, lengthy costly and interdisciplinary procedure but the new developments have transformed the methods by which researchers generate new drug molecules e.g. CADD tool overcomes the cost of drug design up to 50% [1]. Molecular docking technique is used to understand the (i) drug-receptor interaction (ii) binding affinity (iii) orientation and approach of drug molecules to the target site. The main objectives of docking study are precise structural modeling, correct prediction of activity. It presents the most promising vision of drug-receptor interaction and generates a new rational approach to drug design [6]. RMSD is the average distance between the atoms of superimposed structures. This value is widely used parameter to rank the performance of docking methods. If the docked ligand shows < 2.0 Å RMSD value with the crystallographic ligand, it is considered as a successful docking. To calculate the relative free energy, an accurate MM-GBSA binding affinity computation can also be applied [7,8].

BMC Chemistry
*Correspondence: naru2000us@yahoo.com 1 Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India Full list of author information is available at the end of the article Cyclin-dependent kinases play a significant role in the control of cell cycle. These holoenzymes have both catalytic (CDK) and regulatory (cyclin) subunits but present as higher order complexes that include additional proteins and are arbitrated by two classes of enzymes i.e. cyclin D-and E. The D-type cyclins (D1, D2 and D3) bind with two different catalytic sites (CDK4 and CDK6) to yield six possible holoenzymes that articulated in tissuespecific models [9].
CDKs are a class of enzymes that controls the cell cycle and are novel targets for prospective anticancer drugs [10]. A series of pyrimidines bearing 2-arylamino substituents was developed and screened for CDK1 and CDK2 inhibitory effect by Sayle et al. [11]. The SAR of 4-cyclohexylmethoxy-pyrimidines (inhibitors of CDK2) was explored [12]. The progression, transcription and other related functions of cell cycle are regulated by CDK8 that is a heterodimeric kinase protein. The carboxyterminal domain of RNA polymerase II is also phosphorylated by CDK-8. Hence, the inhibition of CDK-8 protein may be essential for regulating tumor [6,13].
In the present study we have planned to synthesize heterocyclic pyrimidine analogues and evaluate their antimicrobial, antiproliferative and docking study.

Chemistry
Synthesis of heterocyclic pyrimidine analogues followed the general procedure discussed in synthetic Scheme 1. The reaction of p-substituted acetophenone with substituted benzaldehyde resulted in the formation of Int-I. The resulted compound was treated with guanidine nitrate to yield pyrimidine ring (Int-II), which on reaction with corresponding substituted benzaldehyde in presence of glacial acetic acid yielded the final derivatives (Ax1-Ax19). The molecular scaffolds of the developed pyrimidine derivatives (Ax1-Ax19) were established by physicochemical properties (Table 1) and NMR, FTIR, MS spectra and elemental analysis ( Table 2). The IR spectrum of synthesized compound showed bands around 2934-3093 cm −1 and 1462-1595 cm −1 which indicate the C-H and C=C group in aromatic nucleus, respectively. The Ar-Cl group in compounds Ax5, Ax12, Ax16 were displayed stretches in the scale of 712-757 cm −1 . The IR str. vibrations at 512-628 cm −1 in the spectral data of compounds displayed the Ar-Br group at p-position of the aromatic nucleus. The existence of Ar-OCH 3 in synthesized analogues is established by absorption band around 1177-1276 cm −1 . The appearance of IR str. 1550-1685 cm −1 in the compounds (Ax1-Ax19) specified the existence of N=CH group. The Ar-NO 2 group in compounds Ax1, Ax6 and Ax15-Ax19 were displayed by symmetric Ar-NO 2 str. in the scale of 1345-1462 cm −1 . The IR stretching 1270-1363 cm −1 of synthesized compounds specified the existence of C-N group. The impression of IR absorption band at 3231-3491 cm −1 in the spectral data of the molecules displayed the presence of Ar-OH group on the aromatic nucleus. The signals between 6.39 and 8.38 δ in NMR spectra are indicative of aromatic proton. The prepared derivatives exhibited singlet at 7.46-8.39 δ due to the presence of N=CH group in pyrimidine nucleus. Molecules displayed singlet at 7.56-7.91 δ due to the presence of -CH group in pyrimidine nucleus. The singlet at 3.71-3.87 δ indicated the presence Ar-OCH 3 . Compound Ax8 exhibited singlet at 2.67 δ due to presence of -N(CH 3 ) 2 at the p-position. The compound Ax14 exhibited quadrate at 3.38 δ and triplet at 1.14 δ due to presence of -N(C 2 H 5 ) 2 group at p-position.

Antimicrobial screening results
The pyrimidine compounds (Ax1-Ax19) were examined for their antimicrobial potency towards Gram −ve and Gram +ve bacteria as well as fungal species by tube dilution technique. Table 3   Ax7.  Ax19 respectively. The molecules may be used as the lead compounds for the development of new antimicrobial agents. Table 4 and Fig. 3 show the screening results of the developed pyrimidine compounds (Ax1-Ax19) towards human colorectal carcinoma cell line by SRB assay [23]. The synthesized compounds exhibited good anticancer activity, with some of the findings comparable or highly potent than 5-fluorouracil (standard drug). Compounds Ax2 (IC 50 = 2.70 µM), Ax7 (IC 50 = 1.90 µM), Ax8 (IC 50 = 2.20 µM) and Ax10 (IC 50 = 0.80 µM), in particular, were the four best compounds which elicited more potent anticancer activity when compared to the reference drug (IC 50 = 6.20 µM). They may be used as lead molecules for the development of new anticancer agent.

Molecular docking results
The CDKs is an enzyme family that plays an significant role in the regulation of the cell cycle and thus is an especially advantageous target for the development of small inhibitory molecules [13]. The crystal structure of cyclin dependent kinase 8 (PDB Id: 5FGK) which has a good resolution of about 2.36 Å was used for docking study. The binding site of the target was generated using co-crystallized ligand (5XG) as reference (X = − 0.138, Y = − 24.891, Z = 150.623). Root-mean square deviation (RMSD) value of docked pose of native co-crystallized ligand was calculated as 0.08 Å. The synthesized pyrimidine compounds were then docked to the active site of CDK8. The docking results were analysed based on the docking score obtained from GLIDE. Among the docked compounds, compounds Ax1, Ax9 and Ax10 displayed moderate to good docked score with anticancer potency against a HCT116 cancer cell line. Ligand interaction image and binding mode of compounds Ax1, Ax9 and Ax10 in the active site of CDK8 protein having co-crystallized ligand 5XG and 5-Fu is having a different binding mode to that of active compounds (Figs. 4, 5, 6 and 7). The molecular docking results depend on the statistical evaluation function according to which the interaction energy in numerical values as docking scores [24]. Molecular docking study of the selected compounds have good to better anticancer potency toward cancer cell line were displayed moderate to better docking score within binding pocket. Binding mode of active compounds Ax1, Ax9 and Ax10 within the binding region, compound Ax10 have moderate docked score (− 4.191) with better potency (0.80 μM) and formation of pi-cation interaction with amino acid residue Arg356; compound Ax1 have better docked score (− 5.668) with lowest potency (48.4 μM) and formation of H-bond with amino acid residues Val27 and Lys153, pi-cation interaction with Arg356 and salt bridge with Asp173, Lys52 and Glu66 within the binding pocket and compound Ax9 have moderate docked score (− 4.477) with moderate potency (16.7 μM) and formation of H-bond with amino acid residue Lys153 within the binding pocket and compared to 5-fluorouracil have better docked score (− 5.753) with good potency (6.20 μM) and formation of H-bond with amino acid residues Ala100 and Asp98 within binding pocket. The docking score results and interacting residues are showing in Table 5. Thus the docking analyses suggested that the pyrimidines can act as of great interest in successful chemotherapy. Cyclin dependent kinase-8 may be the target protein of pyrimidine derivatives for their antiproliferative activity.

SAR (structure activity relationship) study
The following SAR can be deduced from the antimicrobial and anticancer screening results of pyrimidine analogues (Fig. 8).

Antimicrobial activity
The presence of EWG (electron withdrawing group) (inductively)-Br at p-position of the substituted benzylidene aromatic nucleus of compound Ax2 improved the antifungal activity against A. niger and -N(CH 3 ) 2 ) (an electron donating group, by mesomeric affect) at p-position of the benzylidene nucleus of compound Ax8 enhanced the antibacterial activity towards S. aureus and B. subtilis.
On the other side, The presence of EWG (inductively)-Br at p-position of the substituted benzylidene   aromatic nucleus of compound Ax3 improved the antifungal activity toward C. albicans and -N(C 2 H 5 ) 2 ) (an electron donating group, by mesomeric affect) at p-position the substituted benzylidene aromatic ring of compound Ax14 enhanced the antibacterial activity towards E. coli.

Anticancer activity
The presence of EWG (inductively)-Br at p-position of the substituted benzylidene aromatic nucleus of compounds Ax2 and -N(CH 3 ) 2 ) (an electron donating group, by mesomeric affect) at p-position of the substituted benzylidene aromatic ring of compound Ax8 enhanced the anticancer activity towards a human colorectal carcinoma cell line (HCT116), however, electron releasing groups like p-OCH 3 and o-OH on substituted benzylidene aromatic ring of compounds Ax7 and Ax10, respectively showed significant role in improving the anticancer activity toward a HCT116 cell line. The SAR study is consistent the results of Kumar et al. [6,15] and Xu et al. [25].     to yield pyrimidine [Int-II] [27]. The Int-II (0.01 mol) was then refluxed with substituted benzaldehyde (0.01 mol) in methanol 50 mL in presence of glacial acetic acid for 2-3 h (RT). The precipitate generated by adding the reaction mixture to the ice cold water was filtered and recrystallised with methanol [28].

Biological evaluations (antimicrobial and anticancer)
The antimicrobial evaluation of developed derivatives (Ax1-Ax19) was carried out by tube dilution technique [29]

Molecular docking
The molecular docking study was performed of the synthesized pyrimidine derivatives by GLIDE docking program of maestro v11.5 (Schrodinger 2018-1). Among the docked compounds, compounds Ax1, Ax9 and Ax10 displayed moderate to good docked score within the binding pocket of the selected protein i.e. PDB Id: 5FGK with anticancer potency against a HCT116. The protein target for heterocyclic pyrimidine compounds was identified through the literature survey [6,31]. Pyrimidine moiety has wide spectrum of biological potential in medicinal filed [32]. CDK8 (PDB Id: 5FGK) having native ligand 5XG (co-crystallized) with good resolution about 2.36 Å for docking study. Method: X-ray diffraction, R-value free: 0.237 [33]. The root-mean-square deviation is a measure of the average distance between the atoms of superimposed structures. RMSD value of the co-crystallized native ligand (5XG) was calculated. First, Grid is generated using ATP binding site, then docking scores are calculated (Schrodinger 2018-1, maestro v11.5) [34]. Ligand preparation is done using LigPrep module of maestro v11.5. To give the best results, the molecular structures that are docked must be good representations of the actual ligand structures as they would appear in a protein-ligand complex [35].

Conclusion
In the present study, a series of heterocyclic pyrimidine compounds was synthesized in considerable yield and confirmed by FTIR, NMR, MS, CHN analysis. The synthesized compounds showed appreciable antimicrobial and antiproliferative activities. Structure activity relationship study indicated that compounds (Ax2, Ax3, Ax8 and Ax14) having electron withdrawing and compounds (Ax7 and Ax10) have electron releasing groups at substituted benzylidene aromatic nucleus exhibited significant antimicrobial and antiproliferative activities. Further, molecular docking study demonstrated that compound Ax1 showed best docked score with lowest anticancer potency and compound Ax10 showed the moderate docked score with better anticancer potency and compared to the 5-fluorouracil having better docked score with good anticancer potency. Cyclin dependent kinase-8 may be the target protein of heterocyclic pyrimidine compound for their antiproliferative potency. Based on the docking results it is suggested that more structural modifications are required in derivatives Ax1 and Ax10 to make them more potent anticancer agents and these compounds may be used as leads for the development of novel antimicrobial and anticancer agents.