Introduction

Oxazole and Thiazole moiety (Venkatasubramanian & Easwaramoorthy, 2019) is an important, pharmaceutical, chemical entity, which coexists without toxicity. It plays a significant role in antibacterial (Jayaprakash, Saroj, Hemalatha, & Easwaramoorthy, 2016), antifungal, and anticancer (Mathew, Hobrath, Connelly, Guy, & Reynolds, 2018) discovery areas, where it has been used as linker moiety. A series of oxazole moieties is present in many bio-natural products. Oxazole analogs revealed substantial activities, for instance, antimicrobial (Mohammed, 2019), antitumor, anti-HIV, analgesic, and anti-Inflammatory (Rolfe, 2014). Nicotinamides were part of a naturally occurring part of vitamin B₃ (Peng, Shi, & Ke, 2017). Niacin is another name for amide linkage that holds good to enhance the biological activity. The amide linkage (Montalbetti & Falque, 2005) has a biological significance, especially towards the antimicrobial area. So, superior novel antibiotic investigation expected further consideration among the researchers to implicate in the proposal besides preparation of the novel dynamic molecules. With the variety of the reactant molecules, this effort was well-known to fascinate the diamide functionality. Microbial infections were the main threat to mankind. Since ancient times, huge amounts of increasing events of microbial infections have been observed and the frequent use of antibacterial, antifungal, and cytotoxic drugs. Microbial species found resistance towards various existing antimicrobial agents. Superbugs were a huge threat that can inhibit all existing drugs. Slowly, we were increasing the dosage of drugs to control the infections. Afterwards, the existing drugs will not work against new infections, which were caused by superbugs, and they can rule the entire world. Hence, it is necessary to synthesize new antimicrobial agents (Sathish et al., 2018). Proton NMR, Mass spectra, and HPLC (Sathiyanarayanan, Venkatasubramanian, & Easwaramoorthy, 2019) techniques were used to identify new series of oxazole compounds. The categorized chemical analogs' preventing capacity was restrained by the broth dilution method against bacterial and fungal targets. The accumulated experimental conclusions were dignified in the area of inhibition with millimeter units.

Experimental Methods

Materials and Methods

Nicotinic acid, 1-Ethyl-3-(3’-Dimethylamino) Carbodiimide, Hydroxy benzotriazole, N, N-Diisopropyl ethylamine, and DMF were obtained from Sigma Chemicals. The solvent drying process was followed by standard procedures. TLC plates were purchased from Merck. A mixture of 50% n-Hexane and Ethyl acetate was used to monitor the reaction progress. Using Jeol-JMS D-300 spectrometer, Mass spectra of the analogs were recorded. Spectra of ¹H-NMR (400 MHZ) were analyzed using BRUCKER. Agar and dextrose agar medium nutrients were used for the antimicrobial studies.

Synthetic Scheme

Preparation of Ethyl 2-(Nicotinamido)oxazole-4-carboxylate (3)

To a stirred solution of Nicotinic acid (pyridine-3-carboxylic acid, 5g, 0.0406 mol, 1) in 50 mL dried DMF, 1-Ethyl-3-(3'-Dimethylamino) Carbodiimide hydrochloride salt (EDCI, 15.5g, 0.0812 mol) and Hydroxy Benzotriazole (HoBt, 5.48 g, 0.042 mol) were added under an inert environment at 0 °C. Ethyl-2-aminooxazole-4-carboxylate (2, 9 g, 0.0487 mol) and N, N-Diisopropyl ethyl amine (DIPEA, 21 ml, 0.125 mol) were slowly added. The mixture was agitated for 3 hours at room temperature. Ice cooled water (50 ml) was added and stirred to the reaction mixture. The filtered pale yellow solid was vacuum desiccated. 89% of the yield was obtained from this reaction. The synthetic route is represented in Figure 2.

Preparation of 2-(Nicotinamido)oxazole-4-carboxylic acid (4)

To a stirred solution of compound 3 (10 g, 0.034 mol) in THF (100ml), aqueous sodium hydroxide (2.72g) was added at 0 °C. After stirring for 3 hours at room temperature, TLC was used to observe the reaction development. The solvents were distilled out in condensed pressure. The aqueous reaction mass was cooled to about 10 °C. The filtered yellow precipitate was dried. YIELD: 7 gm (82%). The synthetic route is represented in Figure 3.

Preparation of N-Substituted 2-(nicotinamido)oxazole-4-carboxamide (5)

The compound 4 (1 g, 0.005 mol) was stirred in dry DMF (20 ml), to which EDCI (2.9g, 0.015 mol) and HOBt (1g, 0.0076 mol) were added in a nitrogen atmosphere at 0 °C for 0.5 hrs. Then, cyclohexylamine (0.7g, 0.006 mol) was added and followed by DIPEA (3.2 ml, 0.025 mol) and stirring was continued for about 4 hours at 25⁰ °C. The progress of the reaction was examined by TLC. Ice cooled water (30 ml) was added and stirred to the reaction mixture. The filtered pale white precipitate was dried in reduced pressure. YIELD: 1.3 gm (78%). Similarly, all other analogs were prepared with the same procedure by substituted amines. The synthetic route is represented in Figure 4.

Spectral Data

Ethyl 2-(Nicotinamido)oxazole-4-carboxylate (3)

Pale yellow solid, m/z: 262 (M+1). ¹H-NMR (400MHz, CDCl₃) δ (ppm) 12.0 (s, br, 1H, NH amide), 9.2 (d, 1H, Pyridine-H), 8.9 (d, 1H, Pyridine-H), 8.4 (d, 1H, Pyridine-H), 7.7 (t,2H, Pyridine-H and oxazole-H), 4.6 (q, 2H, -CH₂), 1.5 (t, 3H, -CH₃).

2-(nicotinamido) oxazole-4-carboxylic acid (4)

Pale white solid, m/z: 232(M-1), ¹H -NMR (400MHz, DMSO-d6) δ (ppm) 12.9(b,1H, -COOH), 9.2 (d, 1H, Pyridine-H), 8.8 (q, 1H, Pyridine-H), 8.4 (q, 1H, Pyridine-H), 7.7 (q,2H, Pyridine-H and oxazole-H). Deuterium exchange experiment was done using D₂O.

2-(nicotinamido)-N-phenyloxazole-4-carboxamide (5a)

Pale yellow solid, m/z:309(M+1), ¹H-NMR (400MHz, DMSO-d6) δ (ppm) 12.6 (b, 1H, -NH amide), 12.1 (b, 1H, -NH amide), 9.2 (d, 1H, Pyridine-H), 8.9 (t, 2H, Pyridine-H, Benzylic-H), 8.4 (d, 2H, Pyridine-H, Benzylic-H), 8.3 (d,1H, Benzylic-H), 7.6 (t,2H, Benzylic-H), 7.7 (t, 2H, Pyridine-H and oxazole-H).

N-cyclopropyl-2-(nicotinamido) oxazole-4-carboxamide (5b)

Pale white solid, m/z:273(M+1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.6 (b, 1H, -NH amide), 12.3 (b,1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (d,1H, Pyridine-H), 8.30 (d,1H, Pyridine-H), 7.5 (t,1H, oxazole-H), 3.0 (m,1H, Cyclopropyl-CH), 1.4 (m,2H, Cyclopropyl- CH₂), 1.0 (h,2H, Cyclopropyl- CH₂).

N-cyclobutyl-2-(nicotinamido) oxazole-4-carboxamide(5c)

Pale white solid, m/z:287(M+1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.6 (b, 1H, -NH amide), 12.3 (b,1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (d,1H, Pyridine-H), 8.30 (d,1H, Pyridine-H), 7.5 (t,1H, oxazole-H), 4.2 (m,1H Cyclobutyl-CH), 2.0 (m,2H, Cyclobutyl- CH₂), 1.7 (m,2H, Cyclobutyl- CH₂), 1.5 (m,2H, Cyclobutyl- CH₂).

N-cyclopentyl-2-(nicotinamido) oxazole-4-carboxamide (5d)

Pale white solid, m/z:301(M+1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.6 (b, 1H, -NH amide), 12.3 (b,1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (d,1H, Pyridine-H), 8.30 (d,1H, Pyridine-H), 7.5 (t,1H, oxazole-H), 4.15 (m,1H, Cyclopenyl-CH), 1.8 (m,4H, Cyclopenyl-2CH₂), 1.65 (m,2H, Cyclopenyl-CH₂), 1.62 (q,2H, Cyclopenyl-CH₂).

N-cyclohexyl-2-(nicotinamido) oxazole-4-carboxamide (5e)

Pale white solid, m/z:315(M+1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.6 (b, 1H, -NH amide), 12.3 (b,1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (d,1H, Pyridine-H), 8.30 (d,1H, Pyridine-H), 7.5 (t,1H, oxazole-H), 3.5 (d,1H, Cylohexyl-CH), 1.6 (m,2H Cylohexyl-CH₂), 1.4 (m,9H, Cylohexyl-CH₂).

N-((1r, 3r, 5r, 7r)-adamantan-2-yl)-2-(nicotinamido) oxazole-4-carboxamide (5f)

Brown solid, m/z:367(M+1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.6 (b, 1H, -NH amide), 12.3 (b,1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (d,1H, Pyridine-H), 8.30 (d,1H, Pyridine-H), 7.5 (t,1H, oxazole-H), 4.05 (q,1H, Adamantyl-CH), 2.1 (m,2H, Adamantyl-CH₂), 1.8 (m,10H, Adamantyl-CH₂), 0.5 (h,2H, Adamantyl-CH₂).

N-(4-(4-methylpiperazine-1-carbonyl) oxazol-2-yl)nicotinamide (5g)

Pale brown solid, m/z:316(M+1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.6 (b, 1H, -NH amide), 12.3 (b,1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (d,1H, Pyridine-H), 8.30 (d,1H, Pyridine-H), 7.5 (t,1H, oxazole-H), 3.7 (m,2H, N-Methylpiperazyl-CH₂), 3.2 (m,4H, N-Methylpiperazyl-CH₂), 2.7 (dd,2H, N-Methylpiperazyl-CH₂), 2.2 (s, 3H,Nitrogen-CH₃).

N-(4-(morpholine-4-carbonyl) oxazol-2-yl) nicotinamide (5h)

Pale brown solid, m/z:303(M+1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.3 (b,1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (d,1H, Pyridine-H), 8.30 (d,1H, Pyridine-H), 7.5 (t,1H, oxazole-H), 3.7 (m,8H. Morpholinyl-CH₂).

N-(4-(thiomorpholine-4-carbonyl) oxazol-2-yl)nicotinamide (5i)

Pale brown solid, m/z:319(M-1), 1H-NMR (400MHz, DMSO-d6) δ (ppm) 12.4 (s, 1H, -NH amide), 9.1 (s, 1H, Pyridine-H), 8.75 (s,1H, Pyridine-H), 8.30 (t,2H, Pyridine-H), 7.6 (q,1H, oxazole-H), 3.3 (q, 4H, Thiomorpholinyl-CH₂), 3.1 (q, 4H, Thiomorpholinyl-CH₂).

Results and Discussion

Chemistry

In the current investigation, we have aimed to synthesize novel nicotinamide analogs (Venkatasubramanian, Saroj, Hemalatha, & Easwaramoorthy, 2019). The first step is that pyridine-3-carboxylic acid (Nicotinic acid) was coupled with ethyl-2-amino oxazole-4-carboxylate. The second step is the hydrolysis of ethyl 2-(nicotinamido)oxazole-4-carboxylate (3), to get 2-(nicotinamido) oxazole-4-carboxylic acid (4), which was prime scaffold. The scaffold (4) coupled with various amines to get other derivatives (5a-i) were synthesized. The molecular formula, molecular weight, yield, and melting point of the synthesized compounds are presented in Table 1.

This effort identified that analogs' polarization and melting points are higher than that of the parent molecules because of the molecular weight and diamide group of the compounds. Generally, the amide bond will have a greater affinity towards polarity and temperature withstanding capacity. Therefore, the expected melting point and polarity of the novel derivatives are higher than the calculated values of the new derivatives. Established on the R_f value modification and physical properties, all prepared analogs were analyzed for Mass and ¹H-NMR spectra. The proton (singlet, which was besides neighbouring pyridine nitrogen) was recognized in the range of 12.6 to 12.1 ppm based on the exposed ¹H-NMR outcomes. Between 8.7 ppm and 8.3 ppm, amide protons were recognized in all analogs. Likewise, protons of analog 3 (triplet and doublet) disappeared as well as confirmed in analog 4. However, the number of protons improved near the aliphatic regions established because of another amide peak. Later, the analogs confirmed the molecular ion peak by mass spectroscopy. The modification detected among the analogs 3 and 4 is 30 a.m.u., which authorize the ester hydrolyzed carboxylic acid. Similarly, the analogs were successfully characterized using Mass and ¹H-NMR spectra. The categorized analogs were approved for biological studies.

Antimicrobial Activity

Paper disc diffusion technique was used to estimate the in vitro antimicrobial activity of New nicotinamide-Oxazole analogs (Kakkar & Narasimhan, 2019). They were tested against in vitro antibacterial (Ubaid & Hemalatha, 2017) and antifungal activity (Saroj, Ubaid, Mubarak, & Hemalatha, 2019). Among all, four compounds were taken to a detailed antimicrobial activity (R, K.S, S, & D, 2016) with various concentrations to get a clear picture of efficiency.

All the prepared analogs were screened against gram-positive organisms (Saroj et al., 2018) such as Staphylococcus aureus and Staphylococcus epidermidis and gram-negative organisms such as Escherichia coli and Klebsiella pneumonia, and fungi like Candida albican were bio-assayed. Ciprofloxacin (5 mcg/disc) was used as a standard antibacterial drug for biological activity. Ketoconazole (50mcg/disc) was used as a standard antifungal drug for the inhibition assay. But Analogs like 5a, 5e, 5h, and 5i were shown highly active towards microbial studies. The results are displayed in Table 2. This effort exposed that the diamide (thiomorphinyl attached) showed worthy inhibition capacity. The antifungal disc of the 5i is exhibited in Figure 1. Moreover, analog 5i exhibited excellent results counter to the fungus. Especially, derivatives (5a, 5e, 5h, and 5i) were potentially active and all the analogs exposed decent outcomes.

Conclusions

In this study of new analogs of Nicotinamide-Oxazole, derivatives were successfully prepared, characterized, and subjected to antimicrobial bio-assay. Totally, eight analogs were biologically effective, while analogs such as 5a, 5e, 5h, and 5i) were found to be highly active. On the other hand, Nicotinamide-oxazole ester (3) and carboxylic acid (4) were moderately active, and that need to be considered. Further investigation established that diamide holding heterocyclic analogs were found to be better biological potent. The present outcomes encouraged us towards new antifungal agents.