Synthesis, Antimicrobial, and Anti-inflammatory Activities of Novel 5-(1-Adamantyl)-4-arylideneamino-3-mercapto-1,2,4-triazoles and Related Derivatives

The reaction of 5-(1-adamantyl)-4-amino-3-mercapto-1,2,4-triazole (5) with various aromatic aldehydes in ethanol or acetic acid yielded the corresponding 4-arylideneamino derivatives 6a–v. Treatment of the 4-(2,6-difluoro- and dichlorobenzylideneamino) derivatives 6o and 6q with 1-substituted piperazines, and formaldehyde solution in ethanol afforded good yields of the corresponding 5-(1-adamantyl)-4-(2,6-dihalobenzylideneamino-2-(4-substituted-1-piperazinylmethyl)-1,2,4-triazoline-3-thiones 7a–p. 5-(1-Adamantyl)-4-arylideneamino-2-(4-ethoxycarbonyl-1-piperidylmethyl)-1,2,4-triazoline-3-thiones 8a–n, were similarly prepared via the reaction of the corresponding arylideneamino derivative with ethyl 4-piperidinecarboxylate and formaldehyde solution in ethanol. Compounds 6a–v, 7a–p and 8a–n were tested for in vitro activities against a panel of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus Candida albicans. Several derivatives showed good or moderate activities, particularly against the tested Gram-positive bacteria. In addition, the in vivo anti-inflammatory activity of 21 compounds was determined using the carrageenan-induced paw oedema method in rats. Compounds 7d, 7g, 7i, 7j, 7l, 8c, 8e and 8l showed good or moderate dose-dependent activity in this area.


Antimicrobial Testing
The newly synthesized compounds 6a-v, 7a-p and 8a-n 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 (Grampositive 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 6a-v, 7a-p and 8a-n (200 μg/disc), the antibacterial antibiotic ampicillin trihydrate (100 μg/disc) and the antifungal drug clotrimazole (100 μg/disc) are shown in table 4. The antimicrobial activity results of the 4-arylideneaminotriazoles 6a-v revealed that the aryl substituents greatly influenced the antimicrobial activity. The halo and hydroxyl derivatives were highly active particularly against the tested Gram-positive bacteria, while the nitro and methoxy derivatives were generally inactive. In addition, the hydroxy derivatives 6g and 6h showed marked activity against the tested Gram-negative bacteria. The 4-fluoro 6c and 4-bromo 6f derivatives were significantly active against Candida albicans. Based on the antibacterial activity of the arylideneamino derivatives 6a-v, and the previously reported high chemotherapeutic activity of several 2,6-dihalophenyl derivatives [31][32][33], the 2,6-difluoro-and dichlorobenzylideneamino derivatives 6o and 6q were selected to prepare their 4-substituted-1-piperazinyl Mannich bases 7a-p. The results of the antimicrobial activity of the 4-substituted-1-piperazinyl Mannich bases 7a-p were generally lower than those of the arylideneamino derivatives 6a-v but the specificity was almost similar. On the other hand, the antimicrobial activity of the 4-ethoxycarbonyl-1-piperidyl Mannich bases 8a-n was higher than the 4-substituted-1-piperazinyl Mannich bases 7a-p. The most potent member of these derivatives were the 4-hydroxybenzylidene 8f and the 3,4-dimethoxybenzylidene 8m derivatives which displayed strong broad spectrum activity. The minimal inhibitory concentration (MIC) for the most active compounds 6h, 6o, 7n, 8a, 8e, 8f and 8m against the same microorganism used in the primary screening was determined using the microdilution susceptibility method in Müller-Hinton Broth and Sabouraud Liquid Medium [34]. The MIC of the most active compounds, the antibacterial antibiotic ampicillin trihydrate and the antifungal drug clotrimazole (Table 5) were in accordance with the results obtained in the primary screening. Table 4. Antimicrobial activity of compounds 6a-v, 7a-p and 8a-n (200 μg/8 mm disc), the broad spectrum antibacterial drugs gentamicin (100 μg/8 mm disc), ampicillin (100 μg/8 mm disc) and the antifungal drug clotrimazole ( * ND: Not determined.

Acute anti-inflammatory activity testing
The acute in vivo anti-inflammatory activity of 21 representative compounds (6b, 6e, 6h, 6j, 6r, 6t, 7b, 7d, 7g, 7j, 7l, 7m, 7p, 8a, 8c, 8e, 8f, 8h, 8k, 8l and 8n) was determined following the carrageenaninduced paw oedema method in rats [35]. The selection of the representative compounds and dose levels were made after carrying out pilot experiments which showed the absence of anti-inflammatory activity for some compounds and that the dose levels 20 and 40 mg/kg showed no signs of acute toxicity. The results of the anti-inflammatory activity of the tested compounds (20 & 40 mg/kg) and the potent anti-inflammatory drug Indomethacin (5 mg/kg) are listed in Table 6. The majority of the tested compounds showed varying degrees of activity. The highest activity was shown by compound 7l, which produced strong dose-dependent inhibition of carrageenan-induced paw oedema (>50%), while compounds 7d and 8c were moderately active (30-50%) at 20 & 40 mg/kg dose level.
* Inactive: Significantly different from Indomethacin at p < 0.05. ** Activity comparable to Indomethacin (significantly different from Indomethacin at p < 0.05. The N-2 unsubstituted adamantyltriazoles 6b, 6e, 6h, 6j, 6r and 6t were weakly active or completely inactive, while the N-2 piperazinomethyl derivatives 7b, 7d, 7g, 7j, 7l, 7m and 7p were generally active. The activity was also found to be dependent on the nature of the 4-arylideneamino and the 4-piperazinyl substituents. The activity of the 2,6-dichlorobenzylidene derivatives were slightly higher than their 2,6-difluoro-benzylidene analogues. It could be also concluded that the phenyl substituents are better compared with the ethyl, 4-fluorophenyl, 2-methoxyphenyl and benzyl substituents. The replacement of the 4-substituted-1-piperazinyl moiety with a 4-carbethoxy-1piperidyl moiety resulted in marked decrease in activity, only the chloro derivatives 8c and 8l and the 2-hydroxy derivative 8e exhibited moderate activity. There are in fact a high number of enzyme/receptors involved in the inflammatory process. Without specific tests it is quite difficult to hypothesize the mechanism of action of the tested compounds, they may exert their action via inhibition of the cyclooxygenase enzymes like other nonsteroidal anti-inflammatory agents. In addition, the recently reported activity of some adamantane derivatives as selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) [36,37] should be taken inconsideration. The 11β-hydroxysteroid dehydrogenase type 1 converts cortisone to the active glucocorticoid cortisol, which is responsible for various metabolic disorders including water retention, thus the inhibition of 11β-HSD1 would result in increasing the intracellular cortisone level.

General
Melting points (ºC) were measured in open glass capillaries using a Branstead 9001 Electrothermal melting point apparatus and are uncorrected. NMR spectra were obtained on a Bruker AC 500 Ultra Shield NMR spectrometer (Fällanden, Switzerland) operating at 500.13 MHz for 1 H and 125.76 MHz for 13 C; the chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane (TMS) as internal standard; coupling constants (J) are expressed in Hz. Electrospray ionization mass spectra (ESI-MS) were recorded on a Waters QuatroMicro triple quadrupole tandem mass spectrometer at 4.0 and 3.5 kV for positive and negative ions, respectively. Elemental analyses (C, H, N, S) were in full agreement with the proposed structures within ± 0.4% of the theoretical values. Monitoring the reactions and checking the purity of the final products were carried out by thin layer chromatography (TLC) using silica gel precoated aluminum sheets (60 F 254 , Merck) and visualization with ultraviolet light (UV) at 365 and 254 nm. The bacterial strains and Candida albicans fungus were obtained from the Institute of Fermentation of Osaka (IFO), Osaka, Japan. The reference drugs ampicillin trihydrate (CAS 7177-48-2), clotrimazole (CAS 23593-75-1) and indomethacin (CAS 53-86-1) were obtained from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany. The Sprauge-Dawley rats were purchased from local animal house (Abu-Rawash, Giza, Egypt). The animal experiments for the determination of the anti-inflammatory activity were carried out in agreement with the pertinent legal and ethical standards of the international guidelines.

5-(1-Adamantyl)-4-arylideneamino-2-(4-ethoxycarbonyl-1-piperidylmethyl)-1,2,4-triazoline-3-thiones 8a-n
A mixture of the 5-(1-adamantyl)-4-arylideneamino-3-mercapto-1,2,4-triazole 6 (1.0 mmol), ethyl 4-piperidinecarboxylate (0.16 g, 1.0 mmol) and 37% formaldehyde solution (1 mL), in ethanol (8 mL), was heated under reflux for 20 min when a clear solution was obtained. Stirring was continued for 12 h at room temperature and the mixture was allowed to stand overnight. Cold water (5 mL) was added and the reaction mixture was stirred for 20 min. The precipitated crude products were filtered, washed with water, dried, and crystallized (Table 3).     -1-piperidylmethyl)   Determination of the antimicrobial activity by the agar disc-diffusion method. Sterile filter paper discs (8 mm diameter) were moistened with the compound solution in dimethylsulphoxide of specific concentration (200 μg/disc), the antibacterial antibiotics Gentamicin and Ampicillin trihydrate (100 μg/disc) and the antifungal drug Clotrimazole (100 μg/disc) were carefully placed on the agar culture plates that had been previously inoculated separately with the microorganisms. The plates were incubated at 37 ºC, and the diameter of the growth inhibition zones were measured after 24 h in case of bacteria and 48 h in case of Candida albicans. Compounds 6h, 6o, 7n, 8a, 8e, 8f and 8m, Gentamicin, Ampicillin trihydrate and Clotrimazole were dissolved in dimethylsulphoxide at concentration of 128 μg/mL. The twofold dilutions of the solution were prepared (128, 64, 32, …, 0.5 μg/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 ºC for 24 and 48 h for the bacteria and Candida albicans, respectively. The MIC values were determined as the lowest concentration that completely inhibited visible growth of the microorganism as detected by unaided eye.

Determination of minimal inhibitory concentration (MIC).
Determination of the anti-inflammatory activity. Male Sprague-Dawley rats weighing 140-190 g were maintained at room temperature (20-23 ºC). The animals were randomly divided into 42 groups each of 5 animals. The animals were housed with food and water ad libitum and allowed to be accustomed to their environment for two days before testing. Each group was injected with the specific dose of the test compound (20 and 40 mg/kg), or Indomethacin (5 mg/kg) intraperitoneally as a uniform suspension in 1 ml of 0.5% (w/v) aqueous carboxymethyl cellulose solution, one hour before injection of 0.1 mL of carrageenan (1% solution in normal saline) into the plantar tissue of the right hind paw. The left hind paw was injected with 0.1 mL of normal saline solution. Four hours after carrageenan injection, the volume of paw oedema (mL) was determined using water plethysmometer. The percentage protection against inflammation was calculated as follows: Where V c is the mean percentage increase in paw volume in the absence of the test compound (control) and V d is the mean percentage increase in paw volume after injection of the test compound. The values are expressed as the mean percentage reduction ± S.E.M. Statistical significance between the control and treated groups was performed using the Student "t" test.