Synthesis, molecular docking and biological evaluation of bis-pyrimidine Schiff base derivatives

Chemistry

The synthetic work is based on Claisen-Schmidt condensation (Scheme 1). Initially, the bis-chalcone was synthesized by the reaction of 1-(4-hydroxyphenyl)ethanone and terephthalaldehyde. The cyclization of bis-chalcone (intermediate-I) to yield bis-pyrimidine (intermediate-II) was effected with guanidine hydrochloride. The reaction of bis-pyrimidine (intermediate-II) with corresponding substituted aldehyde resulted in the formation of title compounds (q1–q20). The poor % yield of some of the synthesized compounds may be attributed to any one or more of the following reasons: (1) The reaction may be reversible and position of equilibrium is unfavorable to the product; (2) The incursion of side reactions leading to the formation of by-products; (3) The premature work-up of the reaction before its completion; (4) The volatilization of products during reaction or work-up; (5) The loss of product due to incomplete extraction, inefficient crystallization or other work-up procedures; (6) The presence of contaminants in the reactants or reagents leading to a less efficient reaction [17]. The synthesized compounds were characterized by the determination of their physicochemical and spectral characteristics. The chemical structures of the synthesized bis-pyrimidine Schiff bases (q1–q20) were established by ¹H/¹³C-NMR, FT-IR, mass spectral studies and elemental analysis. The IR spectrum of bis-chalcone (I) showed the characteristic band at 1693 cm⁻¹ which indicated the presence of a –C=O group and characteristic bands at 3088 and 1427 cm⁻¹ for the presence of C–H and C=C group in aromatic ring, respectively. The existence of Ar–OH group in bis-chalcone (I) was displayed by the existence Ar–OH stretches in the scale of 3363 cm⁻¹ and characteristic bands at 2864 and 1497 cm⁻¹ indicated the presence of C–H and C=C group in alkyl chain, respectively. Bis-pyrimidine (II) showed the characteristic IR bands at 3058 and 1537 cm⁻¹ for the presence of C–H and C=C group in aromatic ring, respectively and characteristic bands at 3331 and 1604 cm⁻¹ for the presence of –NH₂ and N=CH str. The structure of the bis-chalcone and its cyclized products were further confirmed by the corresponding ¹H-NMR spectra. The ¹H-NMR spectrum of bis-chalcone I showed two doublets at 7.59 ppm (J = 15.1 Hz) and 8.06 ppm (J = 15.1 Hz) indicating that the CH=CH group in the enone linkage is in a trans-conformation. The ¹H-NMR spectrum of intermediate-II showed a multiplet signals between 7.65 and 8.26 δ ppm confirming the cyclisation of the bis-chalcone to give bis-pyrimidine ring. The ¹H-NMR spectrum of compound intermediate-II showed a sharp singlet at 7.26 δ ppm due to the NH₂ protons and it also showed a sharp singlet at 7.60 δ ppm due to HC=C group, which confirmed the cyclization of the bis-chalcone into a bis-pyrimidine ring. The impression of IR absorption band at 3387 − 2237 cm⁻¹ in the spectral data of synthesized derivatives (q1–q20) displayed the presence of Ar–OH category on the aromatic nucleus substituted at the ortho, meta and para-position of the synthesized derivatives. The IR absorption band in the scale of 690–515 cm⁻¹ corresponds to the C–Br stretching of aromatic-bromo derivatives (q14, q15 and q16). The existence of Ar–NO₂ category in derivatives q3, q7 and q18 was displayed by the existence of symmetric and asymmetric Ar–NO₂ stretches in the scale of 1365 − 1335 and 1550 − 1510 cm⁻¹ respectively. The existence of an arylalkyl ether category (Ar–OCH₃) in derivatives, q2, q4, q10, q13 and q20 are established by the existence of an IR absorption band around 3150 − 3050 cm⁻¹. Further, the existence of halogen group in compounds q5 and q17 is indicated by the existence of Ar–Cl stretching vibrations at 600–800 cm⁻¹. The impression of IR stretching vibration at 3100–3000 and 1580–1600 cm⁻¹ in the spectral data of synthesized derivatives (q1–q20) specified the existence of C–H and C=C group, respectively. The appearance of IR stretching 1604–1700 cm⁻¹ in the spectral data of synthesized derivatives (q1–q20) specified the existence of N=CH group. The impression of IR stretching at 1630 cm⁻¹ in the spectra of intermediate specified the existence of C=O group. The multiplet signals between 6.75 and 8.22 δ ppm in ¹H-NMR spectra is indicative of aromatic proton of synthesized derivatives. The compounds, q2, q4, q10, q13 and q20 showed singlet at 3.71–3.82 δ ppm due to the existence of OCH₃ of Ar–OCH₃. All compounds showed singlet at 7.51–8.43 δ ppm due to the existence of N=CH in pyrimidine ring. Compounds showed singlet at 7.70–7.74 δ ppm due to the existence of –CH in pyrimidine ring. Compound q6 showed singlet at 2.89 δ ppm due to existence of –N(CH₃)₂ at the para position. The compound q19 showed quadrate at 3.41 δ ppm and triplet at 1.13 δ ppm due to presence of –N(C₂H₅)₂ at para position. The elemental analysis studies of the synthesized bis-pyrimidine Schiff bases were found within ±0.4% of the theoretical results. Finally, the ¹³C-NMR spectra of the bis-chalcone and the cyclized bis-pyrimidine were recorded in DMSO-d ₆ and the spectral signals were in good agreement with the proposed molecular structure of the synthesized compounds. ¹³C-NMR spectral interpretation details synthesized compounds are given in the experimental section.

In vitro antimicrobial activity

In vitro anticancer activity

The in vitro anticancer activity of synthesized bis-pyrimidine derivatives was carried out against human colorectal cancer cell line (HCT-116 (ATCC CCL-247) and the results are presented in Table 1. Anticancer screening results revealed that in general bis-pyrimidine Schiff bases exhibited good anticancer potential against human colorectal cancer cell line, especially, compounds q1 (IC₅₀ = 0.18 µmol/mL) displayed anticancer activity more than the reference drug 5-fluorouracil (IC₅₀ = 0.35 µmol/L).

Structure–activity relationship

From the antimicrobial and anticancer results, the structure–activity relationship of synthesized bis-pyrimidine Schiff bases (SAR, Fig. 3) can be deduced as follows:

1. 1.

   Compound q1 (synthesized using 2-OH naphthaldehyde) was found to be most potent antimicrobial agent against B. subtilis and C. albicans as well anticancer potential against HCT-116 (ATCC CCL-247) cancer cell line. From the molecular docking studies, compound q1 being the most active molecule has the maximum hydrogen bond interaction (four) and π–π stacking (three) network among the bis-pyrimidine Schiff bases.

    
2. 2.

   Electron withdrawing group [–N(C₂H₅)₂] on benzylidene portion of compound q19 increased the antifungal potential against C. albicans.

    
3. 3.

   Presence of electron releasing group (–OCH₃) on benzylidene portion of compound q20 enhanced the antibacterial potential against S. aureus.

    
4. 4.

   Compound q16 (synthesized using 5-bromo-2-hydroxy benzaldehyde) improved the antimicrobial potential against A. niger and E. coli.

    

From the aforementioned results, we may conclude that different structural requirements are required for a compound to be effective against different targets. The aforementioned facts are supported by the earlier research findings [18, 19].

Docking studies and binding mode analysis

Molecular modeling studies were accomplished to investigate the possible binding mode of the synthesized twenty bis-pyrimidine Schiff base derivatives targeting the crystal structure of cyclin-dependent kinase 8 using GOLD docking program. The Schiff bases were docked into the active site of cyclin-dependent kinase CDK8, using co-complex 5XG ligand as the reference with 12 A radius. The results were analyzed based on the ChemPLP scoring function obtained from GOLD. The docked binding mode was analyzed for the interactions between specific compounds and CDK8. Figure 4a shows the binding mode of the active four compounds into the active site of CDK8. While in Fig. 4b shows the binding mode of the co-complexed ligand 5XG and 5-fluorouracil (the standard inhibitor of cancer) is having a different binding mode to that of the four active compounds of bis-pyrimidine Schiff bases. In-depth analysis of the interaction pattern for the most active compounds, q1, q5, q8 and q13 are discussed in the following section.

The binding mode of the compound q1 positioned in the gorge of the CDK8 active site shows that one of the naphthalenol OH of compound q1 forms hydrogen bond with Glu66 side chain oxygen and with NH of Lys52 side chain, respectively. Additionally, the side chain NH of Lys52 also hydrogen bond with pyrimidinyl nitrogen. While the one of the hydroxyphenyl OH of compound q1 form a hydrogen bond with side chain oxygen and backbone HN of Glu357. While the Tyr32 phenyl ring forms π–π stacking with phenyl and pyrimidinyl ring of compound q1 and one of the naphthalene ring also form π–π stacking with indole ring of Trp105. Besides a pool of hydrophobic interaction between compound q1 and Phe97, Leu70, Ala172, Ile79, Leu158, Met174, Phe176, Ile54, Val35, Val27, Leu359 and Ala155 also stabilize the interaction (Fig. 5a). Compound 1 being the most active compound has the maximum hydrogen bond interaction (four) and π–π stacking (three) network among the bis-pyrimidine Schiff base derivatives. Figure 5b shows the docking orientation of compound q5, which is stabilized by the hydrogen bond interaction between one of the hydroxyphenyl OH of compound q5 forms hydrogen bond with side chain oxygen and backbone HN of Glu357. While the other hydroxyphenyl OH forms hydrogen bond with backbone oxygen of Ile79. Meanwhile, π–π stacking between hydroxyphenyl ring and imidazole ring of His 106, and between chlorophenyl ring and indole ring of Trp105, and π–π stacking between pyrimidinyl ring of compound q5 and Tyr32 phenyl ring is observed. Additionally hydrophobic contact between compound q5 and residues such as Val27, Val35, Ala172, Phe97, Leu70, Ile171, Ile79, Val78, Met174, Phe176, Ile54, Leu359 and Ala155 stabilize the complex. While in the case of compound q5, is the second most active compound with two hydrogen bond interactions and three π–π stacking network.

In compound q8, hydrogen bond interaction between the side chain NH of Lys52 forms hydrogen bond with pyrimidinyl nitrogen and the Glu357 side chain oxygen and backbone HN forms hydrogen bond with hydroxyphenyl OH of compound q8. Subsequently, π–π stacking between pyrimidinyl ring of compound q8 with the Tyr32 phenyl ring and other π–π stacking between hydroxyphenyl rings of compound q8 with imidazole ring of His106 is observed. Likewise, the presence of aliphatic ethyl group with aromatic rings of compound q8 forms hydrophobic contacts with Val27, Val35, Ala172, Phe97, Leu70, Ile79, Leu158, Met174, Ile54, Ala63, Phe176, Ala177, Trp105, Pro154, Ala155, Tyr199 and Leu359 is observed (Fig. 5c). While in the case of compound q8, is the third most active compound with two hydrogen bond interactions and two π–π stacking interaction.

In the case of compound q13, there is a stable hydrogen bond established between the hydroxyphenyl OH with the Glu357 side chain oxygen and backbone HN. While there is the presence of π–π stacking between pyrimidinyl and phenyl ring of compound q13 with the Tyr32 phenyl ring is noticed. The indole ring of Trp105 forms π–π stacking with the one of the methoxyphenyl ring. Additionally, hydrophobic contact is established between the aromatic groups of compound q13 with the key hydrophobic residues such as Val27, Val35, Ile54, Phe97, Leu158, Val78, Ala172, Leu70, Ile79, Met174, Phe176, Ala155, Trp198, Leu359 that stabilize the complex (Fig. 5d). whereas in the case of compound q8, is the fourth most active compound with two hydrogen bond interactions and two π–π stacking interaction. In the activity profile of the inhibitory assay there is no much difference among compounds, q5, q8 and q13. Therefore their binding mode interaction is more or less closer to each other. The hydrogen bonding network and the π–π stacking properties could be the key interactions established by the most active compounds that significantly contribute towards their activity profile. Despite, the fact that the hydrophobic interaction contribution is moderate among the series.

Mutations in adenomatous polyposis coli (APC)/β-catenin resulting in an aberrant activation of Wnt/β-catenin pathway are common in colorectal cancer (CRC), suggesting that targeting the β-catenin pathway with chemopreventive/anticancer agents could be a potential translational approach to control CRC. Recent literature revealed that β-catenin transcriptional activity is positively regulated by the kinase activity of CDK8 and identified it as a CRC oncogene. CDK8, along with cyclin C, Med12, and Med13, forms a “mediator complex” that is involved in the regulation of transcription [20]. The synthesized bispyrimidine Schiff bases may exert their anticancer effect by the inhibition of CDK8 mediated transcription. This was also supported by the observation of Mariaule and Belmont [21] who stated that the pyrimidine is one of the most potential heterocyclic molecules in inhibiting the cyclin dependent kinase as well by the results of molecular docking studies against CDK8 in the current study. Pyrimidines are found to be antagonists of folic acid; hence, a large number of substituted pyrimidines have been synthesized as antifolates and it was eventually proved that these pyrimidines are inhibitors of dihydrofolate reductase (DHFR) [19]. In light of above, the antimicrobial activity of bispyrimidines synthesized in the present study may be attributed to the inhibition of dihydrofolate reductase of the microbe.

Theoretical ADME prediction of twenty bis-pyrimidine Schiff base derivatives

General procedure of the synthesized compounds

In vitro antimicrobial assay

The in vitro antimicrobial study of the synthesized bis-pyrimidines was evaluated against Gram +ve bacterial species: S. aureus (MTCC 3160), B. subtilis (MTCC 441), Gram −ve species: E. coli (MTCC 443) and fungus species: A. niger (MTCC 281) and C. albicans (MTCC 227) by tube dilution technique [25]. Dilutions of test and reference drug in double strength nutrient broth media I.P. was used for antibacterial study and Sabouraud dextrose broth media I.P. was used for the antifungal study. The stock solution was prepared for the test compounds (q1–q20) and reference drugs (norfloxacin and fluconazole) in dimethyl sulfoxide (DMSO) to get a concentration of 100 µg/mL and this stock solution was used for further tube dilution with six concentration of 50, 25, 12.5, 6.25, 3.125 and 1.562 µg/mL for the antimicrobial study [26]. The MIC values of synthesized bis-pyrimidine Schiff base derivatives were recorded at different incubation period: 37 ± 1 °C (bacterial species) for 24 h, 37 ± 1 °C (C. albicans) for 48 h and 25 ± 1 °C (A. niger) for 7 days and the antimicrobial results have been recorded in terms of minimum inhibitory concentration values in µmol/mL.

In vitro cytotoxicity assay

The anticancer screening of synthesized compounds was determined against human colorectal carcinoma [HCT-116 (ATCC (American Type Culture Collection) CCL-247)] cancer cell line using sulforhodamine B (SRB) assay. The optimal cell count (2500 cells/180 μL/well) of HCT-116 was seeded onto the 96 flat-bottom well plates and incubated overnight to allow attachment. 20 μL of pure compounds at tenfold the final concentrations were added in quadruplicates. Both drug-free control and treated cells were then incubated for 72 h. The drug-induced cytotoxicity was assessed using the SRB assay as previously described by Skehan et al. [27], but with minor modifications. Briefly, upon removal of media, cells in each well were fixed with 200 μL of 10% cold TCA [Sigma-Aldrich, St Louis, Missouri, USA] (w/v; in deionised water). After incubation at 4 0 °C for 30 min, the individual wells were rinsed with water for five times. Cells in each well were allowed to stain in 100 µL of 0.4% SRB [Sigma-Aldrich, St Louis, Missouri, USA] (w/v; in 1% acetic acid) for 15 min. Unincorporated dye was rinsed off with 1% acetic acid [Fisher Scientific, Loughborough, Leicestershire, UK] (v/v; in deionised water) and plates were left to air-dry at room temperature overnight. The air-dried plates were placed on a plate shaker and bound SRB was solubilised in 100 µL of 10 mM Tris base solution [Sigma-Aldrich, St Louis, Missouri, USA]. Absorbance was measured by a computer-interfaced 96-well plate spectrophotometer at 570 nm. A dose–response curve (percentage of cell viability vs log concentration) was plotted from which the IC50 value of each molecule against each cell type was graphically determined.

Molecular docking protocol

In order to reveal the binding modes of synthesized twenty bis-pyrimidine Schiff base derivatives, docking simulation was performed targeting the crystal structure of cyclin-dependent kinase 8 (CDK8). Prior to docking, the crystal structure [PDB ID: 5FGK] was retrieved from the protein data bank (PDB) [28]. The CDK8 structure was prepared using protein preparation wizard and optimized by removing the water molecules, hetero atoms and co-factors. Hydrogen, missing atoms, bonds and charges were computed through Maestro [Schrodinger Release 2015-1: Maestro, version 10.1, Schrodinger, LLC, New York, 2015]. The synthesized twenty.

Bis-pyrimidine Schiff base derivatives were used for docking. Meanwhile, the bis-pyrimidine Schiff base derivatives were prepared and optimized using built and LigPrep module implemented in Schrodinger Maestro. Ligands preparation includes generating various tautomers, assigning bond orders, ring conformations and stereo chemistries. All the conformations generated were minimized using OPLS2005 force field prior to docking study.

Molecular docking studies were performed using GOLD (Genetic Optimization for Ligand Docking) program version 5.1. GOLD is an automated docking program that employs the genetic algorithm to search the ligand conformational flexibility with a partial flexibility of protein’s active site [29]. GOLD uses genetic algorithm method for protein–ligand docking and it is well-known for its performance and accuracy specifically for the protein targets with buried active site. The GOLD software has four scoring functions namely ChemPLP, GoldScore, ChemScore, and ASP (the Astex Statistical Potential) which take into account the terms of hydrogen bonding, van der Waal and intramolecular energies. In the present GOLD docking study targeting cyclin-dependent kinase CDK8, the ChemPLP scoring function was used as it outperformed the other scoring function. All the bis-pyrimidine derivatives were docked to cyclin-dependent kinase CDK8 active site, using co-complex 5XG ligand as the reference with 12 A radius. Further, the population size was set to (100); selection-pressure (1.1); number of operations (10,000); number of islands (1); niche size (2); operator weights for migrate (0), mutate (100), and cross-over (100) and with 100 GA run. Results divergent by less than 1.50 A in ligand-all atom RMSDs were clustered together. Best cluster poses and top ranked scores were saved and visually analyzed by Pymol [PyMOL Molecular Graphics System, Schrödinger L, NY, USA, 2010.]. Additional, Qikprop prediction of ADME properties were done for all the synthesized twenty bis-pyrimidine derivatives [Rapid ADME, QikProp, Schrödinger LLC, New York, 2012].