Synthesis and characterization of novel 1,6-dihydropyrimidine derivatives for their pharmacological properties

Basavaraj Padmashali1*, Ballekere Nanjundaswamy Chidananda2, Banuprakash Govindappa3, Siddesh M. Basavaraj4, Sandeep Chandrashekharappa2*, Katharigatta N. Venugopala5,6 1Department of Chemistry, School of Basic Sciences, Rani Channamma University, Belagavi, India. 2Department of Chemistry, Sahyadri Science College (Kuvempu University), Shimoga, India. 3Department of Chemistry, SJB Institute of Technology, Bangalore, India. 4Department of Chemistry, KLE College of Engineering and Technology, Chikodi, Belagavi, India. 5Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Kingdom of Saudi Arabia. 6Department of Biotechnology and Food Technology, Faculty of Applied Science, Durban University of Technology, Durban, South Africa.


INTRODUCTION
Heterocyclic compounds play an important roles in biological and pharmaceutical process. As several drug molecules contain heterocycles as a core structure, great efforts have been made to develop improved synthetic methods for this structure (Chandrashekharappa et al., 2018a;2018b;Mallikarjuna et al., 2016;Nagesh et al., 2014;Sandeep et al., 2013a;2013b;2016a;2016b;Siddesh et al., 2014a). Among all heterocycles, pyrimidinebased heterocycles are more interesting in biological applications (Bairagi et al., 2018;Dharma Rao et al., 2017;Devi et al., 2009;Siddesh et al., 2013;Siddesh et al., 2014a;Venugopala et al., 2014). Pyrimidines linked to thiopheno moiety has been reported in the literature for many years (Ram et al., 1987;Ramesh and Bhalgat, 2011). They have been found to possess a wide spectrum of biological activities (Ghith et al., 2017;Noravyan et al., 2012;Wu, 2012), and many of them have been used as drugs in the market (Shishoo et al., 2009), and some of the structures of active pharmaceutical ingredients are highlighted in Figure 1.

Chemistry
All the chemicals and solvents used were of AR grade and procured from Sigma-Aldrich, India. The Scheme 1 chemical reactions were carried out under a nitrogen atmosphere using a dry solvent. The progress of the reaction was monitored by thin layer chromatography (TLC). TLC was performed on Merck silica gel on TLC aluminum foil with ethyl acetate and hexane as the solvent system and visualization in a UV chamber. IR spectra were recorded on Thermo scientific Fourier-transform infrared spectroscopy (FT-IR) spectrophotometer, and 1 H Nuclear magnetic resonance (NMR) spectra were recorded at ambient temperature using dimethyl sulfoxide (DMSO) and CDCl 3 as a solvent using Bruker AV 800 spectrometer. The chemical shifts are expressed in δ ppm and were reference with tetramethyl silane. The peak multiplicities were specified as follows: s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet. Liquid chromatography-mass spectrometry (LC-MS) were performed on a Jeol JMS-D 300 mass spectrometer operating at 70 eV. Elemental analysis was performed on a Thermo Finnigan FLASH FA 1112 CHN analyzer.

Antibacterial activity
The antibacterial activity of the synthesized compounds was performed using a cup plate method (Nagesh et al., 2015) employing Hi-Media agar medium against two Gram-positive bacteria Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923) and Gram-negative bacteria Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa (ATCC 10145). The antibacterial results of the studied compounds are summarized in Table 2. The tested compounds exhibited slight to moderate antibacterial activity against all microorganisms when compared to the standard compounds.

Antifungal activity
The antifungal activity (Nagesh et al., 2015) of the test compounds was tested against two different fungal strains, namely, Candida albicans and Aspergillus niger by a filter paper disc technique at 50 and 100 μg/ml concentrations. After 48 hours incubation, the zone of inhibition was measured in Table 1

Analgesic activity
Analgesic activity of the test compounds was tested by an acetic-acid-induced writhing method using Albino mice of either sex (20-30 g) (Siddesh et al., 2014b). A 0.6% Acetic acid solution was used to induce writhing in mice. Eleven groups of animals were prepared with six animals in each. The analgesic response was assessed by counting the number of abdominal constrictions for 20 minutes starting 3 minutes after the injection of acetic acid solution. The test compounds were administered to group 1-10 at 100 mg/kg body weight and eleventh group received the standard drug at 100 mg/kg body weight. After 1 hour, the acetic acid solution was administered intraperitoneally, and a number of abdominal constrictions were documented for 20 minutes starting 3 minutes after the injection of acetic acid solution. The analgesic activity was calculated as the percentage of maximum possible effect, and the results are given in Table 4. Animal ethical clearance to conduct in vivo analgesic activity was obtained from the ethical committee from the institution.

C. albicans
The IR spectrum of compound 3 exhibited stretching bands at 3,350, 2,220, and 1,670 cm −1 due to NH, CN, and C=O, respectively. 1 H NMR spectrum of parent compound 3 taken in DMSO exhibited multiplet at δ 8.2-7.2 and singlet at δ 3.2 toward three aromatic protons and N-methyl protons, respectively. Two characteristic signals for NH and NH 2 at δ 3.4 and 2.4, respectively. The mass spectrum of the compound exhibited its molecular ion peak at m/z 248 corresponding to its molecular weight.
Intermediate compound 3 on acetylation with acetyl chloride in an acetic acid solvent for 8 hours refluxation led to the formation of N′- [5-cyano-1-methyl-6-oxo-4-(thiophen-2-yl)-1,6dihydropyrimidin-2-yl] acetohydrazide (4a). The IR spectrum of compound 4a exhibited peaks at 3,074, 2,216, 1,679 cm −1 due to NH, CN, and C=O groups, respectively. 1 H NMR spectrum of the same compound was recorded in DMSO, the multiplet observed at 7.3-8.3 corresponding to three aromatic protons, two NH protons appeared at 2.4 and 4.0, COCH 3 proton appeared at 2.6, and a singlet at 3.5 belongs to three protons of N-methyl. The structure of 4a was further confirmed by the appearance of a molecular ion peak at m/z 290 (M+1) in its mass spectrum. Similarly, the compounds 4b-d were prepared by using the corresponding acid chlorides.
The compound 3 was also refluxed with the benzaldehyde in ethanol with a catalytic amount of acetic acid for 12 hours to yield 2-[(2E)-2-benzylidenehydrazinyl]-1-methyl-6-oxo-4-(thiophen-2-yl)-1,6-dihydropyrimidine-5-carbonitrile (5a). The IR spectrum of compound 5a exhibited peaks at 3,113, 2,224, 1,671, and 1,551 cm −1 due to NH, CN, C=O, and CH=N groups, respectively. 1 H NMR spectrum of the same compound recorded in DMSO, exhibited a peak at δ 3.4 corresponding to one proton of amine, at δ 9.9 for CH=N proton, multiplet between δ 7.1 and 8.5 corresponds to eight aromatic protons, singlet at 2.1 belongs to three protons of N-CH 3 . The structure of 5a was further confirmed by the appearance of a molecular ion peak at m/z 336 (M+1) in its mass spectrum. Similarly, compounds 5b-g were prepared by using the corresponding aldehydes.
The detailed experimental procedure, analysis data for the compounds mentioned above have been incorporated in the experimental section. The structures of all the synthesized compounds have been elucidated by IR, 1 H NMR, LC-MS, and elemental analysis data. Some of the selected compounds have been tested for antibacterial, antifungal, and analgesic activities, and the results have been discussed.

Pharmacology
The tested compounds exhibited significant to moderate antibacterial activity (Table 2) compared to the standard drugs against all microorganisms. Compounds 4a and 5b showed significant antibacterial activity against all the bacterial strains. Compound 4a exhibited equipotent antifungal activity as that of a standard compound (Table 3). Compounds 5a and 5b showed considerable analgesic activity when compared to a standard substance (Table 4).

CONCLUSION
Reactions performed to achieve Schiff bases, and carboxamides of parent compound 2-hydrazineyl-1-methyl-6-oxo-4-(thiophen-2-yl)-1,6-dihydropyrimidine-5-carbonitrile (3) were eco-friendly and yields obtained were satisfactory. Purification of the compounds was achieved by recrystallization method, and the purity was over 99% which was ascertained by Highperformance liquid chromatography (HPLC). Characterization of the compounds was completed by spectral analysis. Title compounds were screened for antibacterial, antifungal, and analgesic properties. Title compounds 4a and 5b showed significant antibacterial activity against all the bacterial strains. For antifungal activity, test compound 4a exhibited equipotent as that of standard compound. Title compounds 5a and 5b exhibited considerable analgesic activity when compared to a standard substance.

ACKNOWLEDGMENT
The authors would like to thank the Department of Chemistry, School of Basic Sciences, Rani Channamma University, National Research Foundation, South Africa and Table 4. Analgesic activity of test compounds 3a, 4a, 4b, 5a, 5b, 5c, and 5f in comparison with controls.