Antimicrobial and Hypoglycemic Activities of Novel N-Mannich Bases Derived from 5-(1-Adamantyl)-4-substituted-1,2,4-triazoline-3-thiones

The reaction of 5-(1-adamantyl)-4-ethyl or allyl-1,2,4-triazoline-3-thione with formaldehyde solution and various 1-substituted piperazines yielded the corresponding N-Mannich bases. The newly synthesized N-Mannich bases were tested for in vitro inhibitory activities against a panel of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus Candida albicans. Six compounds showed potent antibacterial activity against one or more of the tested microorganisms, while two compounds exhibited moderate activity against the tested Gram-positive bacteria. None of the newly synthesized compounds were proved to possess marked activity against Candida albicans. The oral hypoglycemic activity of six compounds was determined in streptozotocin (STZ)-induced diabetic rats. Four compounds produced significant strong dose-dependent reduction of serum glucose levels, compared to gliclazide at 10 mg/kg dose level (potency ratio > 75%).


Chemistry
Adamantane-1-carbohydrazide 3, required as starting material, was obtained via esterification of adamantane-1-carboxylic acid 1 with methanol to get the methyl ester 2, which was subsequently heated with hydrazine to afford the target carbohydrazide 3 [35]. The reaction of compound 3 with ethyl or allyl isothiocyanate yielded the intermediate 1-(1-adamantylcarbonyl)-4-substituted thiosemicarbazides 4a and 4b, which were cyclized to the corresponding 5-(1-adamantyl)-4-substituted-1,2,4-triazoline-3-thiones 5a [35] and 5b [19] via heating in 10% aqueous sodium hydroxide. Compounds 5a,b were reacted with the corresponding 1-substituted piperazine and formaldehyde solution in ethanol to yield the corresponding N-Mannich bases 6a-l in good to moderate yields. The reaction was carried out by heating the reactants in ethanol for 15 min to enhance the solubility of compounds 5a,b (Scheme 1, Table 1). The structures of compounds 6a-l were confirmed by elemental analyses, in addition to the 1 H NMR, 13 C NMR, and electrospray ionization (ESI-MS) or electron impact (EI-MS) mass spectral data which were in full agreement with their structures, and the X-ray spectra of compounds 6c [42], 6d [43] and 6e [44].

In Vitro Antimicrobial Activity
The newly synthesized compounds 6a-l 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 [45]. The results of the preliminary antimicrobial testing of compounds 6a-l (200 μg/disc), the antibacterial antibiotics Ampicillin trihydrate, Gentamicin (100 μg/disc) and the antifungal drug Clotrimazole (100 μg/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 2. 3 The results revealed that the tested compounds showed varying degrees of inhibition against the tested microorganisms. Strong antibacterial activity was displayed by the compounds 6d, 6e, 6f, 6j, 6k and 6l which produced growth inhibition zones ≥18 mm against one or more of the tested microorganisms. Meanwhile, the compounds 6c and 6i showed moderate activity (growth inhibition zones 14-17 mm), the compounds 6g and 6h produced marginal activity (growth inhibition zones 10-13 mm) and compounds 6a and 6b were practically inactive (growth inhibition zones < 10 mm) against the tested microorganisms.
The Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and to a lesser extent Micrococcus luteus are considered the most sensitive among the tested microorganisms. The activity against the tested Gram-negative bacteria was generally lower than that against the Gram-positive bacteria; compound 6d and 6k were strongly active against Escherichia coli and weakly active against Pseudomonas aeuroginosa. The newly synthesized compounds 6a-l lacked inhibitory activity against Candida albicans.
It was observed that the antibacterial activity of the newly synthesized N-Mannich bases 6a-l was mainly dependent on the nature of 4-piperazine substituents regardless the nature of the 1,2,4-triazole substituent at position 4 whether an ethyl or allyl. The presence of 4-aryl, benzyl or 2-pyridyl at the piperazine N-4 was optimistic for antibacterial activity, whereas, the aliphatic substituents (C2H5, COOC2H5) deteriorated the antibacterial activity. The lipophilicity of the compounds 6a-l had limited effect on the antibacterial activity. The minimal inhibitory concentrations (MIC) [46] for the most active compounds 6d, 6e, 6f, 6j, 6k and 6l, which are shown in Table 2, were in accordance with the results obtained in the primary antimicrobial screening.

In Vivo Hypoglycemic Activity
The oral hypoglycemic activity of compounds 6a, 6c, 6f, 6g, 6j and 6l was determined in streptozotocin (STZ)-induced diabetic rats. The compounds were tested at 10 and 20 mg/kg dose levels. The diabetogenic effect of STZ is the direct result of irreversible damage to the pancreatic beta cells, resulting in degranulation and loss of insulin secretion [47,48].
The results of oral hypoglycemic activity of compounds 6a, 6c, 6f, 6g, 6j and 6l (10 and 20 mg/kg) and the potent hypglycemic drug gliclazide in STZ-induced diabetic rats (10 mg/kg) are listed in Table 3. The highest activity was shown by compounds 6a, 6f, 6g and 6l, which produced significant strong dose-dependent reduction of serum glucose levels in STZ-induced diabetic rats, compared to gliclazide at 10 mg/kg dose level (potency ratio > 75%). Compound 6j displayed good hypoglycemic activity at 20 mg/kg dose level and a weak activity at 10 mg/kg dose level. Compound 6c produced medium activity at 10 mg/kg dose level (potency ratio 40.66%), without significant increase at 20 mg/kg dose level. The structure-hypoglycemic activity relationship of the tested adamantyl 1,2,4-triazoles N-Mannich bases revealed that the hypoglycemic activity is mainly dependent on the nature of the piperazine N-4 substituents. The piperazine N-4 ethyl and 2-pyridyl substituents were optimistic (compounds 6a, 6f, 6g and 6l), while the aromatic substituents (phenyl and 2-methoxyphenyl) were less active at 10 mg/kg dose level and retained the good activity at 20 mg/kg doses (compounds 6c and 6j). In addition, the 4-triazole substituents (ethyl and allyl) showed no significant influence on the hypoglycemic activity. Table 3. Oral hypoglycemic activity of compounds 6a, 6c, 6f, 6g, 6j and 6l (10 and 20 mg/kg) and gliclazide (10 mg/kg) in streptozotocin (STZ)-induced diabetic rats.

Oral Acute Toxicity Testing
The method of Litchfield and Wilcoxon was adopted for measuring the acute oral toxicity of compounds 6a, 6f, 6g and 6l which possessed the highest hypoglycemic activity [49]. The acute toxicity results of compounds 6a, 6f, 6g and 6l in normal albino mice are listed in Table 4. The oral LD50 of gliclazide was reported to be >3000 mg/kg in mice [50]. Although the oral acute toxicity of the tested adamantyl 1,2,4-triazole derivatives is higher than that of gliclazide, the compounds induce their hypoglycemic activity at safe doses.

General
Melting points (°C) were measured in open glass capillaries using a Branstead 9100 Electrothermal melting point apparatus (Thermo Fisher Scientific, Waltham, MA, USA) and are uncorrected. NMR spectra were obtained on a Bruker AC 500 Ultra Shield NMR spectrometer (Bruker, Fällanden, Switzerland) operating at 500.13 MHz for 1 H and 125.76 MHz for 13

General Procedure for the Preparation of 5-(1-Adamantyl)-4-ethyl or allyl-2-(4-substituted piperazine-1-ylmethyl)-1,2,4-triazoline-3-thiones 6a-l
A mixture of compound 5a or 5b (2.0 mmol), the appropriate N-substituted piperazine (2.0 mmol) and 37% formaldehyde solution (0.5 mL), in ethanol (8 mL), was heated under reflux for 15 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 from ethanol or aqueous ethanol.

Determination of the in Vitro Antimicrobial Activity (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 °C, and the diameter of the growth inhibition zones were measured after 24 h in the case of bacteria and 48 h in the case of Candida albicans.
The MIC values were determined as the lowest concentration that completely inhibited visible growth of the microorganism as detected by unaided eye.

Determination of the in Vivo Hypoglycemic Activity
Animals: Locally bred male Sprague-Dawley rats (250 ± 30 g body weight) were obtained from Abu Rawash, Giza, Egypt. The rats were housed in wire-bottomed cages at 22 ± 2 °C. A standard pellet diet and tap water were supplied ad libitium. The animals were acclimatized to these conditions for 15 days before the experiment.
Induction of experimental diabetes: Rats were fasted for 16 h before the induction of diabetes with STZ (Sigma Chemical Co., St Louis, MO, USA). The animals were injected intraperitoneally with 0.22-0.25 mL of a freshly prepared solution STZ (60 mg/mL in 0.01 M citrate buffer, pH 4.5) at a final dose of 60 mg/kg body weight. Only rats with serum glucose levels greater than 250 mg/dL were used in experiments.
Design of the experiment: Uniform suspensions of the compounds 6a, 6c, 6f, 6g, 6j and 6l and the oral hypoglycemic drug gliclazide (positive control) in 0.5% (w/v) aqueous carboxymethyl cellulose (CMC) solution were prepared at specific concentrations of 10 mg/mL in case of the test compounds and gliclazide. 48 h post STZ injection, the hypoglycemic activity of the compounds 6a, 6c, 6f, 6g, 6j and 6l was assessed, the diabetic rats were fasted for 16 h and divided into 14 groups each of 5 animals (n = 5) and the serum glucose level was determined for each group and considered as initial fasting serum glucose (C0). Group 1, which served as the negative diabetic control group, received only a single oral dose of 0.5% (w/v) aqueous CMC solution (5 mL/kg). Groups 2 was treated with 10 mg/kg gliclazide in 0.5% (w/v) aqueous CMC (positive control). Groups 3-14 were treated with either a single oral dose of the 10 or 20 mg/kg of the test compounds. All treatments were administered by oral gavage. 24 h after treatment, the blood samples were collected and the serum glucose level (C24) was determined for each group.
Determination of serum glucose: Blood samples from the tail vein were collected, allowed to clot, and centrifuged at 2000 rpm for 10 min. The serum was separated and used on the same day for the measurement of serum glucose levels using a commercial glucose oxidase (GO) assay kit (Sigma-Aldrich). Blood glucose levels were expressed in mg/dL as mean ± standard error of mean (SEM). The data were statistically analyzed using ANOVA with Tukey's multiple comparison test. The values of p < 0.01 were considered as significant. The percentage of serum glucose reduction for each group was calculated in relation to the initial serum glucose level as follows: % Serum glucose reduction = [(C0−C24/C0)] × 100 where C0 is the mean initial fasting serum glucose level, C24 is the mean serum glucose level 24 h after treatment. 6a, 6f, 6g and 6l Freshly prepared suspensions of compounds 6a, 6f, 6g and 6l in concentrations of 1%, 3%, 4%, 6%, 8% and 12% in 0.5% aqueous carboxymethyl cellulose solution were prepared. Each compound was given to six groups each of six normal albino mice of both sexes by oral intubation in doses of 250, 500, 750, 1000, 1250 and 1500 mg/kg. The percentage mortality was recorded 24 h after compound administration and the oral lethal dose LD50 was calculated.

Conclusions
In this study, new N-Mannich bases of 5-(1-adamantyl)-4-substituted-1,2,4-triazoline-3-thiones 6a-l were synthesized and their in vitro antimicrobial activity was determined. Compounds 6d, 6e, 6f, 6j, 6k and 6l displayed potent antibacterial activity. In addition, the in vivo oral hypoglycemic activity of compounds 6a, 6c, 6f, 6g, 6j and 6l was determined in streptozotocin (STZ)-induced diabetic rats. Compounds 6a, 6f, 6g and 6l produced marked hypoglycemic activity compared gliclazide. Although, the active compounds are considered to be good candidates as newer antibacterial and hypoglycemic agents, further studies including the exploration of the mechanism of their biological activity are being undertaken.