INTRODUCTION:

Infections caused by bacteria and fungi lead to diseases and an enormous social burden as millions of people are infected by bacteria and fungi every year worldwide. Therefore, a large number of antimicrobial drugs have been listed, which play an important role in treating infections.[1] As the need for antifungal intervention has increased, so too has the prevalence of resistance.[2] With the irrational use of antibiotics, the resistance of microorganisms has become a very serious clinical problem.

Growing antifungal resistance poses the threat that there will be no available drugs for the treatment of common infections in the future, [3] so there is an urgent need for the discovery of new compounds with antibacterial and antifungal activities,[4] especially those with mechanisms of action that are distinct from the well-known classes of antibacterial, antifungal agents.[5]

Imidazole nucleus forms the main structure of some well known components of human organisms, that is, the amino acid histidine, Vit-B₁₂, a component of DNA base structure and purines, histamine, and biotin. Imidazole containing drugs have a broad scope in remedying various dispositions in clinical medicine.[6] Imidazole has become an important part of many pharmaceuticals are present in many antiprotozal, antifungal, fungicides and antihypertensive medications. It is also present in the structure of many natural or synthetic drug molecules, that is, cimetidine, azomycin, and metronidazole. N-Substituted imidazoles exhibit a variety of valuable pharmacological properties such as antiparasitic,[7] antibacterial, antifungal,[8] and antimicrobial [9] activity and the most simple structure posses inhibitory effects on microsomal oxidation,[10] cytotoxicity. [11] The length of the alkyl chain on the imidazole ring is of importance for biological activity, as 1-alkylimidazoles require a hydrocarbon chain of appropriate length, generally 12 carbons to illustrate antibacterial, fungal and cytotoxic activity^3,8 among them imidazole derivatives having a N-substituent with stereogenic centers have attracted apecial attention, because of their potential utility in a wide range of fields related to chiral recognition and symmetric catalysts.[12,13]

Recently, Kikugawa [14] reported the use of powdered potassium hydroxide for N-alkylation of imidazoles and benzimidazoles. Xianjin [15] reported the synthesis of 2,6-bis (N-allylbenz imidazolyl) pyridine. Further development and modification of antimicrobial agents remains crucial, since rapid mutation and subsequent drug resistance of new microbial strains continues.¹⁶ From literature survey indicates that compounds containing 2-(1H-imidazol-1-yl)-1-phenylethanone derivatives exhibit powerful activities against C. albicans and P. chrysogenum, but moderate activity against A. niger at a concentration of 10 μg/mL [17,18]Derivatizing the imidazole group with long alkyl chains has dramatically improved the antibacterial activity of simple imidazoles. [19] A few substituent’s at the 2-position of N-alkyl imidazole derivatives such as the methyl group and the ether moiety have also been investigated for their effect on the anti microbial activity. [20]

In this study, new biological active heterocyclic systems relating to 2,5-disubstituted N-alkyl imidazole derivatives were synthesized using L–proline as catalyst, which is efficient, bifunction al, eco-friendly useful in several organic transformations and the antimicrobial activity against several Gram-positive and Gram-negative organisms was investigated. In this paper Hanztch reaction involves knoevenagel condensation followed by Michael addition, from the literature L-proline enhancing knoevenagel condensation [21] and Michael addition. [22] Additionally, the target compounds were subjected to insilico molecular properties prediction and drug likeness by employing Molinspiration and Mol-Soft. Further, insilico study of the absorption, distribution, metabolism, and excretion (ADME) properties of the compounds was performed by investigating their match of Lipinski’s rules, topological polar surface area (TPSA) and percentage of absorption (% ABS). Insilico ADME is currently used widely to determine whether it is possible for a drug candidate to reach its site of action. The designed compounds revealed that none of them violated rule of five suggesting them as good antibacterial, antifungal inhibitors.

RESULTS AND DISCUSSIONS:

Chemistry:

There is always a growing demand for the synthesis of novel chemical entities in different therapeutic areas of medicinal chemistry research, to cater the needs of the scientific community for countering multi-drug resistant (MDR) microorganisms. With an aim to synthesize novel chemical entities, multi substituted imidazoles containing an aldehyde functionality (1) and (3a-g) were exploited by condensing them with ethyl cyanoacetate by following Knovenagel condensation protocol. The reactions were conducted without utilizing any hazardous reaction solvents or reagents. Moreover, all the analogues were synthesized under mild conditions by using ethanol as solvent in the presence of catalytic amount of proline, produced the desired N-alkyl imidazole containing cyano acrylates (5a-g) with good purity and yield.

The isolated compounds were well characterized by the spectroscopic data and are in full agreement with the proposed structures. The IR spectrum of 5a exhibited the characteristic peaks at 3259, 2226 and 1722 cm^-1 due to the presence of amine group, nitrile group, and carbonyl group respectively. Proton NMR contained a characteristic alkenyl group at δ 7.83 as singlet, triplets were visible at δ 0.79-0.84 and δ 2.69-2.74, quintet was observed at δ 1.59-1.63, due to the methylene, methyl moiety of the aliphatic chain. Multiplet can be seen at δ 1.23-1.33 due to the aliphatic chain and ester group; quartet at δ 4.22-4.29 due to the ester group. Singlet was observed at 12.72 due to the –liable NH proton. Further the ¹³C NMR of desired product showed signals at δ 164.3 for carbonyl group, δ 155.10 for C₅ carbon, δ 140.27 for methyne group, δ 126.12 for C₃ carbon, δ 86.34 for CN carbon and δ 26.21 for methyl groups. The molecular ion peak at m/z 282 (M+1)⁺ further confirmed its mass. And IR spectrum of 5b exhibited the characteristic peaks at 2230 cm^-1 and 1728 cm^-1 due to the presence of nitrile group, and carbonyl group respectively. Proton NMR contained a characteristic alkenyl group at δ 7.94 as singlet. Triplets were visible at δ 0.81-0.86 and δ 2.68-2.73, quintet was observed at δ 1.59-1.63, due to the aliphatic chain, multiplet was observed at δ 1.24-1.34 due to the aliphatic chain and ester group, quartet was observed at δ 4.24-4.26 due to the ester group, singlet was observed at δ 3.66 due to the N-methyl. The molecular ion peak at m/z 296 (M+1)⁺ further confirmed the structure.

Figure 2: Antifungal activity of target compounds

The antifungal activities of the target compounds exhibit the excellent activity with good minimum inhibitory concentrations (MICs) were determined based on standard guidelines described in Table 2, Fig. 3. Compounds 5a, substituted (N-ethyl) imidazole core moiety 5c exhibited outstanding antifungal activity against Candida albicans 205 (MIC’s 0.25, 8 μg/mL) and targets 5b, 5c showed better antifungal activity against Candida krusei ATCC 6528 with MIC’s 4, 6 μg/mL values respectively. Furthermore, 5d, 5f has shown very good activity against Candida glabrata 168 (6, 4 μg/mL) and compound 5a against on fungal strain Candida parapsilosis 27 (0.5 μg/mL) compared to fluconazole (8 μg/mL respectively). Compound 5d, 5f was excellent active against fungal strain Candida parapsilosis 27 with MIC’s 2, 1 μg/mL values respectively.

Table 2: Antifungal activity of final compounds

S. No.	*Candida albicans* 205	*Candida krusei* ATCC 6528	*Candida glabrata* 168	*Candida* *parapsilosis* 27
5a	0.25	>125	16	0.5
5b	16	4	8	20
5c	8	6	>125	32
5d	32	8	6	2
5e	>125	16	8	>125
5f	16	8	4	1
5g	32	>125	6	8
Fluconazole	2	0.5	4	8

n.t: Not tested

Figure 3: Antifungal sensitivity of final compounds

SAR of this class of compounds has been well studied through variation of the n-substituted methyl, ethyl, benzyl, sulphophenyl, sulphobenzyl, sulphomethyl substituent’s on the imidazole ring were varied to improve the antibacterial, antifungal activity of the compounds. Thus, the newly synthesized compounds by replacing the ethyl, methyl, benzyl (5c, 5b, 5d) from parent nuclei imidazole and from results of our study electron-donating groups increased the antibacterial, antifungal activity.

EXPERIMENTAL SECTION:

Chemistry:

MATERIALS AND METHODS:

Melting points for the materials synthesised in this section were determined by using a Buchi melting point B-545 instrument and are uncorrected. All the reactions were monitored by thin layer chromatography (TLC) using precoated silica 60 F254, 0.25 mm aluminium plates (Merck millipore). IR spectra were recorded using Perkin-Elmer 1000 instrument in potassium bromide phase, proton and carbon NMR on Varian 400 MHz instrument,Mass spectra on Agilent-LC/MS instrument giving only M+1 or M-1 values. Ethanols, L–proline, ethylcyano acetate, were procured from Sigma Aldrich Pvt Ltd., India.

General procedure for the preparation of 5

2-butyl-4-chloro-1H-imidazole-5-carbaldehyde (1) (1g, 10mmol) reaction with different alkylating agents (2a-2g) in presence of PEG-600, reflux at 50-60 ˚C for one hr to give intermediate 3. Further the compound 3 (1.5g, 12mmol), ethyl cynoacetate (4) (12mmol) and ethanol (20 mL) was stirred for 3 hr at ambient temperature, in the presence of catalytic amount of L-proline (15 mol). After completion of the reaction, (monitored by TLC analysis) the solid product was filtered, and washed with cold ethanol (2 x 7 mL) and dried to afford the target compounds 5a-g (Scheme 1, Fig. 1).

(E)-ethyl-3-(2-butyl-4-chloro-1H-imidazol-5-yl)-2-cyanoacrylate (5a)

Yield 6.8 gm (90%); mp 161-164 ˚C; IR (KBr, cm^-1): 2226, 3259, 1722; ¹H NMR (400 MHz, DMSO-d₆/TMS): δ 0.79-0.84 (t, 3H, CH₃), 1.23-1.33 (m, 5H, -CH₂& -CH₃), 1.59-1.63 (quin, 2H, -CH₂), 2.69-2.74 (t, 2H, -CH₂), 4.22-4.29 (q, 2H, -CH₂), 7.83 (s, 1H, vinyl proton), 12.72 (s, 1H, -NH); ¹³C NMR (100 MHz, DMSO-d₆/TMS): δ 165.12, 142.23, 138.46, 120.13, 118.78, 115.46, 87.12, 58.78, 31.45, 27.45, 22.18; MS (ESI) m/z: 282 (M⁺+ 1).

(E)-ethyl-3-(2-butyl-4-chloro-1-methyl-1H-imidazol-5-yl)-2-cyanoacrylate (5b)

Yield 6.1 gm (82%); IR (KBr, cm^-1): 2230, 1728; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ 0.81-0.86 (t, 3H, CH₃), 1.24-1.34 (m, 5H, -CH₂& -CH₃), 1.56-1.61 (quin, 2H, -CH₂), 2.68-2.73 (t, 2H, -CH₂), 3.66 (s, 3H, N-CH₃), 4.24-4.26 (q, 2H, -CH₂), 7.94 (s, 1H, vinyl proton); ¹³C NMR (100 MHz, DMSO-d₆/TMS): δ 163.20, 140.42, 137.20, 121.14, 119.80, 114.40, 89.18, 57.45, 31.40, 28.81, 25.45, 22.18; MS (ESI) m/z: 296 (M⁺+1).

(E)-ethyl-3-(2-butyl-4-chloro-1-ethyl-1H-imidazol-5-yl)-2-cyanoacrylate (5c)

Yield 5.9 gm (81%); IR (KBr, cm^-1): 2229, 1731; ¹H NMR (400 MHz, DMSO-d₆/TMS): δ 0.82-0.87 (t, 3H, CH₃), 1.1-1.3 (m, 8H, -CH₂& -CH₃& -CH₃), 1.58-1.63 (quin, 2H, -CH₂), 2.64-2.70 (t, 2H, -CH₂), 3.91-3.96 (q, 2H, N-CH₂), 4.24-4.26 (q, 2H, -CH₂), 8.17 (s, 1H, vinyl proton). ¹³C NMR (75 MHz, CDCl₃): δ 162.24, 141.79, 138.42, 122.15, 119.08, 115.01, 86.82, 58.68, 31.40, 29.41, 23.18; MS (ESI) m/z: 310 (M⁺+1).

(E)-ethyl-3-(1-benzyl-2-butyl-4-chloro1H-imidazol-5-yl)-2-cyanoacrylate (5d)

Yield 5.7 gm (84%); IR (KBr, cm^-1): 2232, 1726; ¹H NMR (400 MHz, DMSO-d₆/TMS): δ 0.81-0.86 (t, 3H, CH₃), 1.23-1.32 (m, 5H, -CH₂& -CH₃), 1.56-1.61 (quin, 2H, -CH₂), 2.65-2.71 (t, 2H, -CH₂), 4.12-4.17 (q, 2H, -CH₂), 5.31 (s, 2H, N- CH₂), 7.21-7.26 (m, 2H, Ar-H), 7.34-7.38 (m, 3H, Ar-H), 8.19 (s, 1H, vinyl proton). ¹³C NMR (100 MHz, DMSO-d₆/TMS): δ 165.20, 142.80, 140.25, 138.78, 137.05, 122.67, 120.45, 118.82, 116.47, 84.14, 57.60, 34.42, 28.42, 23.15; MS (ESI) m/z: 372 (M⁺+1).

(E)-ethyl-3-(2-butyl-4-chloro-1-(phenylsulfonyl)-1H-imidazol-5-yl)-2-cyanoacrylate (5e)

Yield 5.1 gm (79%); IR (KBr, cm^-1): 2245, 1734; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ 0.80-0.83 (t, 3H, -CH₃), 1.26-1.35 (m, 5H, -CH₂& -CH₃), 1.54-1.59 (quin, 2H, -CH₂), 2.66-2.72 (t, 2H, -CH₂), 4.14-4.18 (q, 2H, -CH₂), 7.41-7.46 (m, 2H, Ar-H), 7.54-7.58 (m, 3H, Ar-H), 8.24 (s, 1H, vinyl proton). ¹³C NMR (100 MHz, CDCl₃): δ 166.27, 143.78, 141.48, 139.43, 136.46, 122.67, 120.49, 118.22, 116.46, 84.04, 57.60, 34.42, 28.42; MS (ESI) m/z: 422 (M⁺+1).

(E)-Ethyl 3-(2-butyl-4-chloro-1p-toluene sulfonyl-1H-imidazol-5-yl)-2-cyanoacrylate (5f)

Yield 5.19 gm (81%); IR (KBr, cm^-1): 2241, 1729; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ 0.82-0.87 (t, 3H, -CH₃), 1.25-1.34 (m, 5H, -CH₂and -CH₃),1.57-1.61 (quin, 2H, -CH₂), 2.15 (s, Ar-CH₃),2.69-2.75 (t, 2H, -CH₂), 4.16-4.21 (q, 2H, -CH₂), 7.14-7.19 (m, 2H, Ar-H), 7.27-7.32 (m, 2H, Ar-H), 8.21 (s, 1H, vinyl proton). ¹³C NMR (100 MHz, CDCl₃): δ 165.89, 143.78, 140.29, 139.71, 135.40, 122.46, 120.34, 119.24, 115.40, 84.64, 60.79, 57.60, 34.42, 28.42, 20.46; MS (ESI) m/z: 436 (M⁺+1).

(E)-ethyl-3-(2-butyl-4-chloro-1-(methylsulfonyl)-1H-imidazol-5-yl)-2-cyanoacrylate (5g)

Yield 5.8 gm (85 %); IR (KBr, cm^-1): 2239, 1728; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ 0.79-0.83 (t, 3H, CH₃), 1.23-1.32 (m, 5H, -CH₂and -CH₃),1.54-1.59 (quin, 2H, -CH₂), 2.67-2.72 (t, 2H, -CH₂), 3.94 (s, 3H, sulfonyl methyl), 4.14-4.18 (q, 2H, -CH₂), 8.19 (s, 1H, vinyl proton). ¹³C NMR (100 MHz, CDCl₃): δ 165.89, 140.29, 135.40, 122.46, 120.34, 119.24, 115.40, 60.79, 57.60, 34.42, 28.42, 24.16; MS (ESI) m/z: 360 (M⁺+1).

BIOLOGY:

Antibacterial activity:

The micro dilution method [23,24] was performed according to the standard guidelines described in the Clinical and Laboratory for the antibacterial susceptibility assays. The desired working concentrations of the derivatives 5a-g, and two positive drugs (amikacin, erythromycin) were obtained by adding the secondary DMSO stocks to broth culture, as described for the antifungal susceptibility assays. Then, 100 μL of broth culture containing the appropriate concentrations (125 mg/L to 0.5 mg/L) of each compound was added to each well of a 96-well plate.

Gram-positive bacterial isolates Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcus aureus UA1758, Gram-negative bacterial isolates Klebsiella pneumonia UF222, Pseudomonas aeruginosa UA1024 obtained from Microbiology laboratory of Global Hospital, Hyderabad. After culture in a blood plate at 37 °C in a humidified atmosphere of 5 % CO2 in air, cells were suspended in normal saline at a density of 2 × 10⁸ CFU/ mL. Then, the solution was diluted 1000 times with culture broth, and 100 μL of diluted solution was added to the 96-well plate containing compounds. After incubation for 24 h at 37 °C, the MIC was read as the lowest concentration that produced a prominent decrease in growth (100 % inhibition) compared with the control cells (without compound).

Antifungal activity:

The prepared compounds and fluconazole (Microbiology laboratory of Global Hospital, Hyderabad) were dissolved in DMSO to prepare primary stocks. The stock was then gradually diluted to prepare secondary stocks with different concentrations. Finally, the working concentrations of the derivatives 5a-g were obtained by adding the appropriate amount of the secondary DMSO stocks to RPMI 1640 medium. The amount of DMSO in working solutions did not exceeded 1 %. Antifungal susceptibility tests were performed according to the standard guidelines described in the Clinical and Laboratory Standards, and the micro dilution reference method was used. [25] Next, 100 μL of RPMI 1640 medium containing the desired concentrations (125 mg/L to 0.5 mg/L) of the appropriate compound was added to each well of a 96-well plate.

Four strains of Candida spp. were used in the assay, including the quality control Candida albicans 205, Candida krusei ATCC 6528, Candida glabrata 168, and Candida parapsilosis 27 isolates. They were cultured in solid Yeast Extract Peptone Dextrose (YPD) medium at 37 °C in a humidified atmosphere of 5 % CO₂ in air. The cells were dissolved in normal saline at a density of 5 × 10⁶ CFU/mL. Then, the solution was diluted 1000 times with RPMI 1640 medium, and 100 μL of diluted solution was added to the 96-well plate containing the compounds. After incubation for 48 h at 37°C, the MIC was read as the lowest concentration that produced a prominent decrease in growth (inhibition ≥ 80 %) compared to the control cells (without compound).

Insilico molecular properties investigation and prediction of drug likeness:

Many potential drug candidates do not reach the clinics because of their poor absorption, distribution, metabolism, excretion and toxic liabilities (ADMET). [26] Good oral bio availability can be achieved by right balance between partitioning and solubility. A computational structural sensitivity for predicting the important molecular properties such as hydrophobicity, molecular size, flexibility, toxic liabilities, bioactivity and drug likeness has been studied in order to obtain better analogues of imidazole ring.

Molinspiration calculations:

Lipinski’s rule of five [27] which is generally used by pharmaceutical chemists for screening the potentiality of drug like candidates states that a molecule is orally active if the (i) molecular weight is under 500 da; (ii) calculated octanol/water partition coefficient (log P) ≤ 5; (iii) number of hydrogen bond acceptors ≤ 10; (d) number of hydrogen bond donors ≤ 5. The method used by Molinspiration [28] is very robust and is based on the sum of fragment contributions and handles most of the organometallic and organic molecules. Our results (Table 3) indicate that derivatives 5a-5g under study presented lipophilicities (log P) varied in the range of 0.94 to 2.21 suggesting their better permeability across cell membranes. Number of hydrogen bond acceptors and number of hydrogen bond donors in the products 5a-5g were in accordance with the rule i.e., less than 10 and 5 respectively. Hydrogen bonding is considered to be an important parameter for describing the permeability of drugs [29] Number of rotatable bonds (nrotb) is an important parameter for molecular flexibility and conformational change for binding to the receptors and should be in the range of ≤ 10 [30] and the compounds 5a-5g under study exhibited nrotb with in the said range. Molecular weights of all the title compounds was found to be less than 500 and thus these molecules are anticipated to be easily transported, diffused and absorbed as compared to large molecules. Volume, percentage of absorption (% ABS), for the compounds 5a-5g are presented in Table 3. Molecular polar surface area (PSA) contributed by the sum of polar atoms such as oxygen, nitrogen and attached hydrogens were calculated by Ertl et al. methodology [31] and is believed to be a very useful descriptor for drug absorption and transportation properties apart from intestinal absorption, bioavailability, human intestinal epithelial adenocarcinoma (Caco-2) cells permeability and blood-brain barrier (BBB) penetration. Percentage of absorption and is calculated by the expression: % ABS = 109-0.345 * PSA. PSA and volume are inversely related to % ABS. PSA and logP are considered to be the two most important parameters for predicting oral bioavailability of a drug though not sufficient criteria TPSA is very much correlated with the hydrogen bonding of a molecule and is associated with the transport properties of drug across the membranes, prediction in the BBB and intestinal crossing. Molecules with TPSA/PSA in the range ≤ 160 Å² have good intestinal absorption and ≤ 60 Å²has BBB penetration. [32] For the analyzed series all the derivatives have come out to be best intestinal absorbers. Compounds 5a-5g obeyed the rule of five suggesting their drug likeness Fig.4. The bioactivity scores of the synthesized analogues for drug targets were also predicted by Molinspiration and are presented in Table 4 by means of numerical assignment. A molecule having bioactivity score more than 0.00 is most likely to exhibit considerable biological activities, while values -0.50 to 0.00 are excepted to be moderately active and if score is less than -0.50 it is presumed to be inactive.

Table 3: Pharmacokinetic parameters important for good oral bioactivity scores^a of the synthesized compounds 5

Compd.	% Abs	miLogP	TPSA	natoms	MW	nON	Nviolations	nOHNH	Nrotb	volume
5a	81.8209	3.20	78.78	19	281.74	5	0	1	7	250.98
5b	85.5676	3.27	67.92	20	295.77	5	0	0	7	267.93
5c	85.5676	3.64	67.92	21	284.73	5	0	0	8	284.73
5d	85.5676	4.86	67.92	26	371.87	5	0	0	9	339.57
5e	73.7893	4.17	102.06	28	421.91	7	0	0	9	354.20
5f	73.7893	4.23	102.06	29	453.93	7	0	0	10	371.01
5g	73.7893	2.64	102.06	23	359.83	7	0	0	8	299.36
^b	83.2733	-0.70	74.57	24	331.35	6	0	2	3	285.46

^a% ABS: % of absorption; MiLogP: partition coefficient; TPSA: topological polar surface area; Nrotb: number of rotatable bonds; nON: Number of hydrogen bond acceptor; nOHNH: Number of hydrogen bond donars; ^bCPF.

Table 4: Bioactivity scores^a for the designed molecules 5

Compounds	GPCRL	ICM	KI	NRL	EI	PI	HBD	HBA	Drug-likeness
5a	-0.07	-0.37	-0.76	-0.58	-0.09	-0.53	1	4	-0.75
5b	-0.12	-0.43	-0.76	-0.36	-0.14	-0.43	0	4	-0.71
5c	-0.02	-0.38	-0.77	-0.35	-0.12	-0.42	0	4	-0.71
5d	0.19	-0.28	-0.56	-0.13	-0.00	-0.15	0	4	-0.38
5e	0.03	-0.23	-0.51	-0.27	-0.06	-0.23	0	6	-0.67
5f	0.06	-0.43	-0.43	-0.15	-0.02	-0.22	0	6	-0.45
5g	0.08	-0.54	-0.51	-0.09	-0.07	-0.36	0	6	-0.48
^b	0.12	-0.04	-0.07	-0.19	0.28	-0.21	2	4	0.93

^aGPCR: GPCR ligand; ICM: Ion channel modulator; KI: Kinase inhibitor; NRL: Nuclear receptor ligand; PI: Protease inhibitor; EI: Enzyme inhibitor; ^bCPF.

X-ray studies of compound 5b:

The authors present results of studies on commercially available silver nanoparticles fabricated by Amepox Microelectronics and delivered in the form of silver powder. The studies were carried out by scanning electron microscopy (SEM). Figure 5, 6 shows typical results of the studies of compound (5b) powder deposited on a carbon strip by means of SEM. Part (a) of the figure represents the view of the sample 475 at 300 × magnification which stands for examining the area of 800 × 800 μm² surface. Around the examined area, one can notice the presence of objects of sizes within 200 μm to 300 μm. Those objects consist of tiny particles, as can be proved by SEM studies results gathered on one of the particles.

Figure 5: Powder XRD pattern of Ag systems

Figure 6: SEM images of compound 5b in Ag nanoparticals

CONCLUSION:

In conclusion, a series of new chemical entities, cyanoacrylate derivatives (5a-5g) have been synthesized and characterised by using various spectral techniques. L–Proline was employed as a catalyst for reaction of ethyl cyano acetate with N-alkyl-2-butyl-4-chloro-1H-imidazole-5-carbaldehyde derivatives. The prepared compounds were tested using invitro antibacterial, antifungal activity assays, and several compounds exhibited improved to excellent activities compared to the lead compound. Among these compounds, compounds 5g, 5c, 5d, 5b and 5a, 5f exhibited strong inhibitory effects toward bacterial and fungal strains respectively. The designed products showed excellent drug-like properties and are expected to present good bioavailability profile. Further mechanic studies and structural modification of identified hits are ongoing for the generation of newer analogues with enhanced efficacy as anticancer and antibacterial agents.

ACKNOWLEDGMENT:

One of the authors (NRT) is thankful to our Research Supervisor Venkateswara Rao for providing us required facilities and motivation for completion of the research work. We also extend our gratitude towards Department of Chemistry, Koneru Lakshmaiah Education Foundation.

REFERENCES:

1. Danishuddin, M., Kaushal, L., Hassan Baig, M., and Khan, A.U. Genomics Proteomics Bioinform., 2012, vol. 10, pp. 360–363.

2. Lafleur, M.D., Sun, L., Lister, I., Keating, J., Nantel, A., Long, L., Ghannoum, M., North, J., Lee, R.E., and Coleman, K., et al. Antimicrob. Agents Chemother., 2013, vol. 57, pp. 3585–3591.

3. Lloyd, D.H., Vet. Dermatol., 2012, vol. 23, pp. 299–304.

4. Wang, X.L., Zhou, C.H., and Geng, R.X., Chin. J. New Drug., 2010, vol. 19, pp. 2050–2059.

5. MTunçbilek, M., Kiper, T., and Altanlar, N. Eur. J. Med. Chem., 2009, vol. 44, pp. 1024–1033.

6. Kleeman, A., Engel, J., Kutscher, B., and D. Reichert, Pharmaceutical Substances: Syntheses, Patents, Applications of the Most Relevant APIs, Thieme Medical, New York, NY, USA, 3^rd edition, 1999.

7. Mukherjee, A., Kumar, S., Seth, M., and Bhaduri, A.P., Ind. J.Chem., 1989, 28B, p. 391.

8. Norman, S. M.; Bennett, R. D.; Poling, S. M.; Maier, V. P.; Nelson, M. D. Plant Physiol., 1986, vol. 80, p. 122.

9. Purohit, M., and Srivastava, S. K., Proc. Natl. Acad. Sci. India 1991, 61A, p. 461.

10. Wilkinson, C. F., and Hetnarski, K., Biochem. Pharmacol., 1974, vol. 23, p. 2377.

11. Miller, D. K., Griffiths, E., Lenard, J., and Firestone, R. A., J. Cell Biol., 1983, 97, 1841.

12. Levillain J., Dubant, G., Abrunhosa I, Gulea M., Gaumont, A. C., Chem.Commun. 2003, pp. 2914-2915. B) Ding, J., Welton, T., and Armstrong, D.W., Anal. Chem., 2004, vol. 76, pp. 6819-6822.

13. Wang, Z., Wang, Q., Zhang, Y., and Bao, W., Tetrahedron Lett., 2005, vol. 46, pp. 4657-4660.

14. Kikugawa, Y., Synthesis., 1981, p. 124.

15. Xu, Xi., Xi, Z., Chen, W., Wang, D., J. Coord. Chem., 2007, vol. 60, p. 21.

16. Khabnadideh, S., Rezaei, Z., Khalafi-Nezhad, A., Bahrinajafi, R., Mohamadi R., and Farrokhroz, A., Bioorg. Med. Chem. Lett., 2003, vol. 13, pp. 2863–2865.

17. Lakshmanan, B., Mazumder, P.M., Sasmal, D., and Ganguly, S. Pharm. Lett., 2010, vol. 2, pp. 82–89.

18. Babu, K.S., Li, X.-C., Jacob, M.R., Zhang, Q., Khan, S.I., Ferreira, D., and Clark, A.M., J. Med. Chem., 2006, vol. 49, pp. 7877–7886.

19. Samant B.S., and Shukhthankar M.G., Bioorg. Med. Chem. Lett., 2011, vol. 21, pp. 1015–1018.

20. Sharma, D., Narasimhan, B., Kumar, P., Judge, V., Narang, R., De Clercq E., and Balzarini J., Eur. J. Med. Chem., 2009, vol. 44, pp. 2347–2353.

21. Nandkishore, K., Gampawar, N., Sumit shinde, V., Sandeep jadhav, V., Wamanrao, N., Chinese journal of Chemistry., 2007, vol. 25, p. 1686.

22. Kotrusz, P., Toma, S., Arkivoc., 2006, pp. 100-109.

23. Kavanagh, F., Analytical Microbiology (vol II), Academic Press, New York and London, 1972, pp. 11–23.

24. Miles, A.A, Misra, S.S. The estimation of the bactericidal power of the blood. J. Hyg., 1938, vol. 38, pp. 732-749.

25. Chunli Liu., Ce Shi., Fei Mao., Yong Xu., Jinyan Liu., Bing Wei., Jin Zhu., Mingjie Xiang., and Jian Li., Molecules., 2014, vol. 19, pp. 15653-15672

26. Bakht, M.A., Yar, M.S., Abdel-Hamid S.G., Qasoumi S.I.A, Samad, A. Eur. J. Med. Chem., 2010, vol. 45, pp. 5862-5869.

27. Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J. Adv. Drug Deliv. Rev., 1997, vol. 23, pp. 3-25.

28. http://www.molinspiration.com. v 2014.11, 20-03-2016

29. Rajanarendar, E., Rama Krishna, S., Nagaraju, D., Govardhan Reddy, K., Kishore, B., and Reddy, Y. N., Bioorg. Med. Chem. Lett., 2015, vol. 25, pp. 1630-1634.

30. Ertl, P., Rohde, B, Selzer, P. J. Med. Chem., 2000, vol. 43, pp. 3714-3717.

31. Alafeefy, A. M., Alqasoumi, S.I., Ashour, A.E., Masand, V., Al Jaber, N.A., Ben Hadda, T., and Mohamed, M.A., Eur. J. Med. Chem., 2012, vol. 53, pp. 133-140.

32. Da Silva, M.M., Comin, M., Duarte, T.S., Foglio, M.A., de Carvalho, J.E., do Vieira, M. C., and Formagio, A.S., Molecules., 2015, vol. 20, pp. 5360-5373.

Received on 03.04.2019 Modified on 15.05.2019

Asian J. Research Chem. 2019; 12(3):157-164.

DOI: 10.5958/0974-4150.2019.00032.4

Entry	Antibacterial Activity MIC (μg/mL)
Entry	*Enterococcus faecalis*	*Staphylococcus epidermidis*	*Staphylococcus aureus* *UA1758*	*Klebsiella pneumonia* *UF222*	*Pseudomonas aeruginosa* *UA1024*
5a	> 125	20	14	> 125	12
5b	16	125	4	4	32
5c	4	8	8	16	2
5d	8	1	32	12	15
5e	12	14	> 125	8	16
5f	125	8	16	8	32
5g	24	12	6	2	> 125
Amikacin	1.5	0.75	2	24	8
Erythromycin	3	6	n.t	12	> 125