Design, synthesis, antimicrobial and cytotoxicity study on human colorectal carcinoma cell line of new 4,4′-(1,4-phenylene)bis(pyrimidin-2-amine) derivatives

Background Pyrimidine molecules attracted organic chemists very much due to their biological and chemotherapeutic importance. Their related fused heterocycles are of interest as potential bioactive molecules so, we have designed and prepared a new class of 4,4′-(1,4-phenylene)bis(pyrimidin-2-amine) molecules and screened for their in vitro antibacterial, antifungal and cytotoxicity studies. Results The structures of synthesized bis-pyrimidine molecules were confirmed by physicochemical and spectral means. The synthesized compounds were further evaluated for their in vitro biological potentials i.e. antimicrobial activity using tube dilution method and anticancer activity against human colorectal carcinoma (HCT116) cancer cell line by Sulforhodamine B assay. Conclusions The biological study demonstrated that compounds s7, s8, s11, s14, s16, s17 and s18 have shown more promising antimicrobial activity with best MIC values than the cefadroxil (antibacterial) and fluconazole (antifungal) and compound s3 found to have better anticancer activity against human colorectal carcinoma (HCT116) cancer cell line.


Open Access
*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 Background Among a wide variety of heterocyclic that have been explored for developing medicinally important molecules [1]. Pyrimidine derivatives attracted organic chemists very much due to their biological and chemotherapeutic importance especially the fused heterocycles are of interest as potential bioactive molecules. Pyrimidine derivatives are known to exhibit biological activities i.e. anticancer [2,3], antiviral [4], anti-inflammatory [5], antimalarial [6], antibacterial [1,7] and antifungal [8] etc. As pathogenic bacteria continuously evolve mechanisms of resistance to currently used antibacterial, so the discovery of novel and potent antibacterial drugs is the best way to overcome bacterial resistance and develop effective therapies [9].
Cancer is one of the most serious health problems all over the world and one of the leading causes of death. Thus, in the past for several decades, researchers have been struggling to find effective clinical approaches for the treatment of cancer and search for novel anticancer agents. Recently, accumulating evidences have illustrated that heterocyclic derivatives are considered to be the most promising molecules as leads for the discovery of novel synthetic drugs. In particular, substituted pyrimidines, present in the cores of many physiologically active molecules, display interesting therapeutic properties, especially antitumor activities with different bio targets and mechanisms by means of inhibiting several enzymes as well as modulating the activity of many receptors [10]. Pyrimidine is found as a core structure in a large variety of compounds that exhibit important biological activity, specifically pyrimidines known to inhibit Pneumocystis carinii (pc), Toxoplasma gondii (tg) of tumour cell lines in culture and the activity is attributed to inhibition of dihydrofolate reductase (DHFR) [11]. 2,4-Disubstituted and 2,4,6-trisubstituted pyrimidines have shown potent anticancer activity as CDK inhibitors, TNF-α inhibitors, Abl tyrosine protein kinase inhibitors, PI-3 kinase inhibitors, Akt kinase inhibitors and cytokines inhibitors [12]. Design of pyrimidine molecules for antimicrobial and anticancer potentials based on literature is presented in Fig. 1. Selected marketed drug contains pyrimidine ring presented in Fig. 2 [13].

Chemistry
Synthesis of the intermediate and target molecules was performed according to the reactions outlined in Scheme 1 (based on Claisen-Schmidt condensation). Initially, the bis-chalcone was prepared by the reaction of 1-(2,4-dichlorophenyl)ethanone and terephthalaldehyde. The cyclization of bis-chalcone (int-I) to yield bis-pyrimidine (int-II) was effected with guanidine hydrochloride. The reaction of bis-pyrimidine (int-II) with corresponding substituted aldehyde resulted in the formation of title compounds (s1-s18). The synthesized compounds were characterized by the determination of their physicochemical properties (Table 1) and spectral characteristics. The chemical structures of the synthesized 4,4′-(1,4-phenylene)bis(pyrimidin-2-amine) molecules (s1-s18) were established by 1 H/ 13 C-NMR, FT-IR, mass spectral studies and elemental analysis. The IR spectrum of bis-chalcone (I) showed the characteristic IR band at 1692.29 cm −1 which indicated the presence of -C=O group and characteristic bands at 3089.33 and 1596.22 cm −1 indicated the presence of C-H and C=C group in aromatic ring, respectively. The existence of Ar-Cl group in 3,3′- (1,4-phenylene) bis (1-(2,4-dichlorophenyl)prop-2-en-1-one) (I) was displayed by the existence Ar-Cl stretches in the scale of 752.59 cm −1 and characteristic bands at 2829.17 and 1461.71 cm −1 indicated the presence of C-H and C=C group in alkyl chain, respectively. 6,6′-(1,4-Phenylene)bis (4-(2,4-dichlorophenyl)pyrimidin-2-amine) (II) showed the characteristic bands at 3089.99 and 1598.31 cm −1 for the presence of C-H and C=C group in aromatic ring, respectively and characteristic bands at 3363.97 and 1692.49 cm −1 indicated the presence of -NH 2 and N=CH str. The molecular structure of the intermediate-I and its cyclized products were further confirmed by proton-NMR spectral data. The 1 H-NMR spectrum of intermediate-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 1 H-NMR spectrum of (II) showed a multiplet signals between 7.42 and 8.01 δ ppm confirming the cyclisation of the 3,3′- (1,4-phenylene)bis (1-(2,4-dichlorophenyl)prop-2-en-1-one) (I) to give 6,6′- (1,4-phenylene)bis (4-(2,4dichlorophenyl)pyrimidin-2-amine) (II). The 1 H-NMR spectrum of intermediate-II showed a sharp singlet at 7.09 δ ppm due to the NH 2 protons and it also showed a sharp singlet at 7.85 δ ppm due to HC=C group, which confirmed the cyclization of the bis-chalcone into a bis-pyrimidine ring. The IR stretching vibrations at 733.88-750.53 cm −1 in the spectral data of synthesized derivatives (s1-s18) displayed the presence of halogen group (Ar-Cl) on the aromatic nucleus substituted at the ortho, meta and para-position. The existence of Ar-NO 2 functional group in compounds s3, s6 and s7 was displayed by the existence of symmetric Ar-NO 2 stretches in the scale of 1372.55-1373.82 cm −1. The existence of an arylalkyl ether group (Ar-OCH 3 ) in compounds, s5, s9, s10, s12 and s13 are established by the existence of an IR absorption band around 3088.33-3089.60 cm −1 . The impression of IR stretching vibration at 3088.93-2972.97 cm −1 and 1599.67-1595.05 cm −1 in the spectral data of synthesized derivatives (s1-s18) specified the existence of C-H and C=C group, respectively. The appearance of IR stretching 1698.99-1663.17 cm −1 in the spectral data of synthesized derivatives (s1-s18) specified the existence of N=CH group. The impression of IR absorption band at 3461.41-3345.04 cm −1 in the spectral data s2, s4, s13 and s15 displayed the presence of Ar-OH group on the aromatic ring at ortho and para position. The multiplet signals between 6.77 and 8.34 δ ppm in proton-NMR spectra is indicative of aromatic proton of synthesized derivatives. The compounds, s5, s9, s10, s12 and s13 showed singlet at 3.84-3.85 δ ppm due to the existence of OCH 3 of Ar-OCH 3 . The synthesized compounds showed singlet at 9.01-10.05 δ ppm due to the existence of N=CH in pyrimidine ring. Compounds showed singlet at 10.00-10.15 δ ppm due to the existence of -CH in pyrimidine ring. Compound s8 showed singlet at 3.04 δ ppm due to existence of -N(CH 3 ) 2 at the para position. The compound s16 showed quadrate at 3.38-3.49 δ ppm and triplet at 1.07-1.15 δ ppm due to presence of -N(C 2 H 5 ) 2 at para position. The 13 C-NMR spectral data and elemental analysis studies of the synthesized pyrimidine derivatives were found within ± 0.4% of the theoretical results of synthesized compounds are given in the "Experimental section".

In vitro antimicrobial activity
Antimicrobial screening of synthesized 4,4′-(1,4-phenylene)bis(pyrimidin-2-amine) molecules against Gram positive and Gram negative bacterial and fungal strains was done by tube dilution technique. Antimicrobial activity results indicated ( Table 2) particularly; compounds, s7, s8, s11, s14, s16, s17 and s18 have shown more promising antimicrobial activity as compared to standard drugs cefadroxil (antibacterial) and fluconazole (antifungal) while other derivatives are moderately active. In the case of Gram positive bacterial study, compound s11 (MIC sa = 0.14 µmol/mL, MIC bc = 0.07 µmol/mL) was found to be most potent one against S. aureus and B. cereus. In the case of Gram negative bacterial study, compound s7 and s18 displayed appreciable antibacterial activity against Providencia rettgeri with MIC value of 0.08 µmol/mL. Compound s8 (MIC pa = 0.15 µmol/ mL) exhibited excellent activity against Pseudomonas aeruginosa and compound s11 showed good antibacterial activity against Salmonella typhi and Escherichia coli with the MIC values of 0.29 and 0.23 µmol/mL, respectively. The antifungal screening results demonstrated that compounds s11 displayed appreciable antifungal activity against Aspergillus niger and Aspergillus flavus with the MIC values of 0.58 and 0.14 µmol/mL, respectively. Compounds, s14 and s17 (MIC af = 0.31 µmol/mL) were found to be most potent ones against Aspergillus fumigatus and compound s16 (MIC af = 0.14 µmol/mL) was found to be most effective one against Aspergillus flavus. The antimicrobial screening results of synthesized molecules (s7, s8, s11, s14, s16, s17 and s18) have more than standard drugs and may be used as a lead compound to discover novel antimicrobial scaffolds.
From structure activity relationship study, 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 [3,14,16,17].

Conclusion
In conclusion, we have described a simple and efficient protocol for the synthesis of new bis-pyrimidine molecules (s1-s18) with appreciable yields. The in vitro antibacterial, antifungal and anticancer potential of all the synthesized compounds were investigated. It is evident that synthesized compounds, s7, s8, s11, s14, s16, s17 and s18 have excellent antimicrobial activity and compound s3 exhibited good anticancer activity. For the above compounds a significant improvement in their antibacterial and antifungal activities has been examined over the earlier reported compounds. The 4,4′-(1,4-phenylene)bis(pyrimidin-2-amine) molecules reported have a probability to emerge as a valuable lead series with great potential to be used as antibacterial, antifungal and anticancer agents and as promising candidates for further efficacy evaluation.
Authors' contributions BN and SK have designed, synthesized and carried out the antimicrobial activity and SML, KR, MV and SAAS have carried out the spectral analysis, interpretation and cytotoxicity study of synthesized compounds. All authors read and approved the final manuscript.