Synthesis and Antimicrobial Activity of N′-Heteroarylidene-1-adamantylcarbohydrazides and (±)-2-(1-Adamantyl)-4-acetyl-5-[5-(4-substituted phenyl-3-isoxazolyl)]-1,3,4-oxadiazolines

The reaction of adamantane-1-carbohydrazide (1) with heterocyclic aldehydes, namely 5-(4-chlorophenyl)isoxazole-3-carboxaldehyde (2a), 5-(4-methylphenyl)isoxazole-3-carboxaldehyde (2b), 5-(4-methoxyphenyl)isoxazole-3-carboxaldehyde (2c), 1H-imidazole-2-carboxaldehyde and 2-butyl-4-chloro-1H-imidazole-5-carboxaldehyde, in ethanol, yielded the corresponding N′-heteroarylidene-1-adamantylcarbohydrazides 3a, 3b, 3c, 4 and 5, respectively, in good yields. The 4-acetyl-1,3,4-oxadiazoline analogues 6a‑c were prepared in 48–55% yields by heating their corresponding N′-heteroarylidene-1-adamantylcarbohydrazides 3a–c with acetic anhydride for two hours. Compounds 3a–c, 4, 5 and 6a–c were tested for in vitro activities against a panel of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus Candida albicans. Compounds 4 and 5 displayed potent broad-spectrum antimicrobial activity, while compounds 3a–c showed good activity against the Gram-positive bacteria.


Antimicrobial Testing
The newly synthesized compounds 3a-c, 4, 5 and 6a-c were tested for their in vitro growth inhibitory activity against the standard strains of the Institute of Fermentation of Osaka (IFO) namely; Staphylococcus aureus IFO 3060, Bacillus subtilis IFO 3007, Micrococcus luteus IFO 3232 (Gram-positive bacteria), Escherichia coli IFO 3301, Pseudomonas aeuroginosa IFO 3448 (Gram-negative bacteria), and the yeast-like pathogenic fungus Candida albicans IFO 0583. The primary screening was carried out using the agar disc-diffusion method using Müller-Hinton agar medium [30]. The results of the preliminary antimicrobial testing of compounds 3a-c, 4, 5 and 6a-c (200 g/8 mm disc), the antibacterial antibiotics gentamicin (100 g/8 mm disc), ampicillin (100 g/8 mm disc) and the antifungal drug clotrimazole (100 g/8 mm disc) and the calculated log P values (Clog P) of the tested compounds (calculated using the CS ChemOffice Ultra, version 8.0, CambridgeSoft, Cambridge, MA, USA) are shown in Table 1.
The antimicrobial activity results of the synthesized compounds (Table 1) revealed that the tested compounds showed varying degrees of inhibition against the tested microorganisms. In general, it was observed that the antibacterial activity is not dependent on their lipophilicity. The antibacterial activity of the tested N'-heteroarylidene-1-adamantylcarbohydrazides 3a-c, 4 and 5 mainly depended on the heterocyclic nucleus. The imidazole derivatives 4 and 5 displayed higher and broad-spectrum activity than the isoxazole derivatives 3a-c, which exhibited potent or moderate activity against the tested Gram-positive bacteria. The cyclization of the N'-heteroarylidene analogues 3a-c to their 4-acetyl-1,3,4-oxadiazoline analogues 6a-c resulted in a dramatic decrease in the antibacterial activity, and only compound 6a retained marginal activity against the tested Gram-positive bacteria ( Table 1). The minimal inhibitory concentration (MIC) for the active compounds 3a-c, 4 and 5 against the same microorganism used in the primary screening was carried out using the microdilution susceptibility method in Müller-Hinton Broth and Sabouraud Liquid Medium [31]. The MIC of the compounds 3a-c, 4 and 5, the antibacterial antibiotics gentamicin and ampicillin trihydrate which are shown in Table 1, were in accordance with the results obtained in the primary screening.

General
Melting points (°C) were measured in open glass capillaries using a Branstead 9001 electrothermal melting point apparatus and are uncorrected. NMR spectra were obtained on a Brüker AC 500 Ultra Shield NMR spectrometer (Brüker BioSpin AG, Fällanden, Switzerland) operating at 500.

N′-Heteroarylidene-1-adamantylcarbohydrazides 3a-c, 4 and 5
The appropriate heterocyclic aldehyde 2a-c (0.01 mol) was added to a solution of adamantane-1carbohydrazide 1 (1.94 gm, 0.01 mol) in ethanol (10 mL), and the mixture was heated under reflux for 3 hours and allowed to stand at room temperature for overnight. The separated precipitate was filtered, washed with water, dried and crystallized from ethanol (compounds 3a-c) or aqueous ethanol (compounds 4, 5).

(±)-2-(1-Adamantyl)-4-acetyl-5-[5-(4-substituted phenyl-3-isoxazolyl)]-1,3,4-oxadiazolines 6a-c
A mixture of the appropriate N'-heteroarylidene-1-adamantylcarbohydrazides 3a-c (5.0 mmol) and acetic anhydride (8 mL) was heated under reflux for 2 hours. The excess acetic anhydride was then evaporated under reduced pressure and ice-water (50 mL) was added to the resulted oily or sticky residue and refrigerated for 2 hours. The separated solid was filtered, washed with water, dried and crystallized from aqueous ethanol to yield the products 6a-c in 48-55% yield.  [30] Sterile filter paper discs (8 mm diameter) were moistened with the compound solution in dimethylsulphoxide of specific concentration (200 g/disc), the antibacterial antibiotics gentamicin and ampicillin trihydrate (100 g/disc) and the antifungal drug clotrimazole (100 g/disc) were carefully placed on the agar culture plates that had been previously inoculated separately with the microorganisms. The plates were incubated at 37 °C, and the diameter of the growth inhibition zones were measured after 24 hours in case of bacteria and 48 h in case of Candida albicans.

Determination of Minimal Inhibitory Concentrations (MICs) [31]
Compounds 3a-c, 4, 5, gentamicin and ampicillin trihydrate were dissolved in dimethylsulphoxide at the concentration of 128 g/mL. The twofold dilutions of the solution were prepared (128, 64, 32, 16, 8, 4, 2, 1 and 0.5 g/mL). The microorganism suspensions at 106 CFU/mL (colony forming unit/mL) concentrations were inoculated to the corresponding wells. The plates were incubated at 36 °C for 24. The MIC values were determined as the lowest concentration that completely inhibited visible growth of the microorganism as detected by unaided eye.