Synthesis, Antimicrobial and Hypoglycemic Activities of Novel N-(1-Adamantyl)carbothioamide Derivatives

The reaction of 1-adamantyl isothiocyanate 4 with the various cyclic secondary amines yielded the corresponding N-(1-adamantyl)carbothioamides 5a–e, 6, 7, 8a–c and 9. Similarly, the reaction of 4 with piperazine and trans-2,5-dimethylpiperazine in 2:1 molar ratio yielded the corresponding N,N'-bis(1-adamantyl)piperazine-1,4-dicarbothioamides 10a and 10b, respectively. The reaction of N-(1-adamantyl)-4-ethoxycarbonylpiperidine-1-carbothioamide 8c with excess hydrazine hydrate yielded the target carbohydrazide 11, in addition to 4-(1-adamantyl)thiosemicarbazide 12 as a minor product. The reaction of the carbohydrazide 11 with methyl or phenyl isothiocyanate followed by heating in aqueous sodium hydroxide yielded the 1,2,4-triazole analogues 14a and 14b. The reaction of the carbohydrazide 11 with various aromatic aldehydes yielded the corresponding N'-arylideneamino derivatives 15a–g. The compounds 5a–e, 6, 7, 8a–c, 9, 10a, 10b, 14a, 14b and 15a–g were tested for in vitro antimicrobial activity against certain strains of pathogenic Gram-positive and Gram-negative bacteria and the yeast-like fungus Candida albicans. The compounds 5c, 5d, 5e, 6, 7, 10a, 10b, 15a, 15f and 15g showed potent antibacterial activity against one or more of the tested microorganisms. The oral hypoglycemic activity of compounds 5c, 6, 8b, 9, 14a and 15b was determined in streptozotocin (STZ)-induced diabetic rats. Compound 5c produced significant reduction of serum glucose levels, compared to gliclazide.


Chemical Synthesis
1-Adamantyl isothiocyanate 4, required as starting material, was prepared in good yield via modification of the previously described methods [39,40]. Thus, 1-adamantylamine 1 was reacted with carbon disulfide and trimethylamine, in ethanol, to yield the dithiocarbamate salt 2, followed by addition of di-tert-butyl dicarbonate (Boc2O) to yield the intermediate 3, which was converted to the target product 4 via stirring with catalytic amount of 4-dimethylaminopyridine (DMAP). 1-Adamantyl isothiocyanate 4 was reacted with the cyclic secondary amines namely, 1-substituted piperazines, morpholine, pyrrolidine, 4-substituted piperdines and 1,2,3,4-tetrahydroisoquinoline, in boiling ethanol, to yield the corresponding N-(1-adamantyl)carbothioamides 5a-e, 6, 7, 8a-c and 9, respectively. The reaction was found to proceed smoothly and the products were precipitated from the reaction mixture in good yields after two hours. 1-Adamantyl isothiocyanate 4 was similarly reacted with piperazine and trans-2,5-dimethylpiperazine in 2:1 molar ratio to yield the corresponding N,N'-bis(1adamantyl)piperazine-1,4-dicarbothioamide derivatives 10a and 10b in high yields (Scheme 1, Table 1). The structures of compounds 5a-e, 6, 7, 8a-c, 9, 10a and 10b were confirmed by elemental analyses, in addition to the 1 H-NMR, 13 C-NMR, and ESI-MS mass spectral data which were in full agreement with their structures, in addition to the X-ray spectrum of compound 9 [41]. N-(1-Adamantyl)-4-ethoxycarbonylpiperidine-1-carbothioamide 8c was reacted with excess hydrazine hydrate, in ethanol, at reflux temperature, to get the target carbohydrazide derivative 11. On monitoring the reaction with thin layer chromatography (TLC), it was observed that the target product was formed after few minutes in addition to a minor product which was further identified as 4-(1-adamantyl)thiosemicarbazide 12. It was also observed that prolongation of the reaction time results in higher ratios of the side product 12. The reaction time was optimized at 20 min to yield 72% of 11 and 18% of 12. The formation of the side product could be explained as a result of hydrazinolysis of the thiocarboxamide function of the major product 11. The structure of the side product 12 was assigned based on the 1 H-NMR and 13 C-NMR data, in addition to ESI-MS mass spectra and elemental analyses.
The reaction of the carbohydrazide 11 with equimolar amount of methyl or phenyl isothiocyanate, in ethanol for 6 h yielded the intermediate 1,4-disubstituted-3-thiosemicarbazides 13a or 13b. Dehydrative cyclization of compounds 13a and 13b was achieved by heating in 10% aqueous sodium hydroxide solution for 2 h, followed by acidification with hydrochloric acid to yield the target 1,2,4-triazole derivatives 14a and 14b in 38% and 44% overall yields, respectively. Attempted reaction of the carbohydrazide 11 with various aromatic aldehydes via prolonged heating in ethanol yielded fair yields of the corresponding arylideneamino derivatives. On the other hand, carrying out the reaction in the higher boiling solvent N,N-dimethylformamide (DMF) greatly improved the yield. Thus, the reaction of the carbohydrazide 11 with certain aromatic aldehydes via heating in DMF for two hours yielded the target compounds 15a-g in relatively higher yields (43%-71%).
The structures of compounds 11, 12, 14a, 14b and 15a-g (Scheme 2, Table 1) were confirmed by elemental analyses, in addition to the 1 H-NMR, 13 C-NMR, and ESI-MS mass spectral data, which were in full agreement with their structures.

Comp.
No. The antimicrobial activity results revealed that the tested compounds exhibited various degrees of inhibition against the tested microorganisms. Potent antibacterial activity was displayed by the compounds 5c-e, 6, 7, 10a, 10b, 15a, 15f and 15g which produced growth inhibition zones ≥ 18 mm against one or more of the tested bacteria. In addition, the derivatives 8a, 8b and 15c showed moderate activity (growth inhibition zones 14-17 mm), the derivatives 8c and 15b produced weak activity (growth inhibition zones 10-13 mm) and the derivatives 5a, 5b, 9, 14a, 14b, 15d and 15e 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. Meanwhile, the activity against the tested Gram-negative bacteria was generally lower than that of the Gram-positive bacteria, only compound 10a and 10b were found strongly active against Escherichia coli and moderately or weakly active against Pseudomonas aeuroginosa. The inhibitory activity of the compounds against Candida albicans was rather lower than their antibacterial activity, only compounds 15f and 15g displayed marginal activity compared to Clotrimazole. In addition, the antimicrobial activity of the compounds were not correlated to their lipophilicity.
The N-(1-adamantyl)carbothioamides 5c-e, 6, 7 and 8a-c showed marked activity against the tested Gram-positive bacteria and weak to moderate activity against the tested Gram-negative bacteria, in addition to the absence of antifungal activity. The antibacterial activity was dependent on the nature of the precursor cyclic secondary amine. Among the piperazine derivatives 5a-e, the 4-aryl and benzyl derivatives 5c, 5d and 5e were highly active and the ethyl and the ethoxycarbonyl derivatives 5a and 5b were inactive. The morpholine and pyrrolidine derivatives 6 and 7 were highly active against the Gram-positive bacteria and retained moderate activity against Escherichia coli and weak activity against Pseudomonas aeuroginosa. The antibacterial activity of the piperidine derivatives 8a-c was lower than their morpholine and pyrrolidine analogues with moderate to weak activity against the tested Gram-positive bacteria. The tetrahydroisoquinoline derivative 9 totally lacked antimicrobial activity.
The minimal inhibitory concentrations (MIC) [43] for the most active compounds 5c, 5d, 5e, 6, 7, 10a, 10b, 15a, 15f and 15g which are shown in Table 2, were in accordance with the results obtained in the primary screening. Despite the potent broad-spectrum antibacterial activity of compounds 10a and 10b, the MIC values were higher than expected. The high MIC values of compounds 10a and 10b may be attributed high lipophilicity and the poor water solubility in the aqueous Müller-Hinton Broth and Sabouraud Liquid Medium.

In Vivo Hypoglycemic Activity
The oral hypoglycemic activity of compounds 5c, 6, 8b, 9, 14a and 15b 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 [44,45].
The results of oral hypoglycemic activity compounds 5c, 6, 8b, 9, 14a and 15b (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 compound 5c, which produced significant strong dose-independent reduction of serum glucose levels in STZ-induced diabetic rats, compared to gliclazide at 10 mg/kg dose level (Potency ratio 92.48%). Compound 6 displayed good hypoglycemic at 20 mg/kg dose level and weak activity at 10 mg/kg dose level. Table 3. Oral hypoglycemic activity of compounds 5c, 6, 8b, 9, 14a, 15b (10 and 20 mg/kg) and gliclazide (10 mg/kg) in STZ-induced diabetic rats. The hypoglycemic activity of the tested N-adamantyl carbothioamides 5c, 6, 8b, 9, 14a and 15c greatly influenced by the nature of the carbothioamide moiety. The piperazine and morpholine carbothioamides 5c and 6 retained good potency, while the corresponding piperidine and tetrahydroisoquinoline analogs 8b, 9, 14a and 15c were almost inactive.

Oral Acute Toxicity Testing of Compound 5c
The method of Litchfield and Wilcoxon was adopted for measuring the acute oral toxicity of compound 5c which possessed the highest hypoglycemic activity [46]. The oral LD50 of compound 5c in normal albino mice was found to be 298 ± 15.50 mg/kg. The oral LD50 of gliclazide was reported to be >3000 mg/kg in mice [47]. Although the oral acute toxicity of compound 5c is higher than that of gliclazide, the compound induces its hypoglycemic activity at safe doses.

General
Melting points (°C) were measured in open glass capillaries using a Branstead 9100 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

Synthesis of
Aqueous sodium hydroxide solution (10%, 10 mL) was added to the crude product 13a or 13b and the mixture was heated under reflux for 2 h, then filtered hot. The filtrate was acidified with 37% HCl to pH 1-2 and the precipitated crude products 14a,b were filtered, washed with water and crystallized.

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 case of bacteria and 48 h in case of Candida albicans.

Determination of the Minimal Inhibitory Concentration (MIC)
Compounds 5c, 5d, 5e, 6, 7, 10a, 10b, 15f and 15g Gentamicin and Ampicillin trihydrate 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 °C for 24 h. 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 Sprauge-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 5c, 6, 8b, 9, 14a and 15b and the oral hypoglycemic drug gliclazide (positive control) in 0.5% (w/v) aqueous carboxymethyl cellulose (CMC) solution were prepared at specific concentration of 10 mg/mL in case of the test compounds and gliclazide. 48 h post STZ injection, the hypoglycemic activity of the compounds 5c, 6, 8b, 9, 14a and 15b 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, centrifuged at 2000 r.p.m. for 10 min. The serum was separated and used in the same day for the measurement of serum glucose levels using commercial glucose oxidase (GO) assay kit (Sigma-Aldrich Co., St. Louis, MO, USA). Blood glucose levels were expressed in mg/dL as 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.

Determination of the Oral Acute Toxicity of Compound 5c
Freshly prepared suspensions of compound 5c 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 6 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.
Compound 5c produced significant hypoglycemic activity compared gliclazide at a safe dose. The active compounds are considered to be good candidates as newer antibacterial and hypoglycemic agents, further studies such as molecular docking for the exploration of the mechanism of their biological activity are required for optimization of the activity are being undertaken.