Synthesis and antibacterial activity of novel Schiff bases of thiosemicarbazone derivatives with adamantane moiety

Increased bacterial resistance to antibiotics is a major threat to human health, and it is particularly important to develop novel antibiotic drugs. Here, we designed a series of Schiff base thiosemicarbazone derivatives containing an adamantane moiety, and carried out the structural characterization of the compounds and in vitro antibacterial activity tests. Compound 7e was as effective as the commonly used antibiotic ampicillin against the Gram-negative bacterium Escherichia coli, and compound 7g had a good inhibitory effect against Gram-positive Bacillus subtilis. These findings provide data for the development of better thiosemicarbazone antibacterial agents.


Introduction
In the past 30 years, only limited progress has been made in research into antibacterial drugs with new mechanisms and core structures [1][2][3]. Amoxicillin, norfloxacin, and ciprofloxacin are the commonest drugs used to treat bacterial infection but are associated with severe side effects. Toxicity and bacterial resistance to the drugs play an important role in the failure of treatment [4,5]. The development of antibacterial drugs with novel structures is very important to research for clinical application [6].
Recently, considerable attention has been focused on substituted thiosemicarbazone derivatives because of their interesting biological activities. Compounds with a thiosemicarbazone structure are known to possess tranquilizing, muscle relaxing, psychoanaleptic, hypnotic, ulcerogenic, antidepressant, antibacterial, antifungal, analgesic, and antiinflammatory properties [7][8][9][10][11][12][13][14]. Thiosemicarbazones are a type of Schiff base formed by condensation of thiosemicarbazide and an aldehyde or ketone. Studies have shown that changing the structure of the aldehydes and ketones or introducing different active groups on position N(4) can improve the antibacterial and anticancer activities of those compounds [15]. Therefore, the synthesis of thiosemicarbazone compounds with various structures and the study of their structure-activity relationships have important theoretical significance and potential practical application value.
Adamantane compounds have shown relatively good antivirus, antitumor, and anti-Parkinson's syndrome activities [16][17][18][19][20][21]. The introduction of adamantyl groups into the molecular structure of other compounds often enhances the biological activity of the compound. The adamantyl group has relatively good fat solubility, so can greatly increase the membrane permeability of a compound [22][23][24][25]. Much research has been carried out on thiosemicarbazone derivatives, but no work has been done screening adamantyl thiosemicarbazone derivatives for their antibacterial activity.
In this paper, novel adamantyl thiosemicarbazone derivatives were synthesized by the condensation of an adamantyl phenyl aldehyde with a thiosemicarbazide. The chemical structure of the compounds was elucidated by infrared (IR), 1 H NMR, and 13 C NMR spectroscopies, and mass spectrometry. The activities of the compounds were screened in vitro against Bacillus subtilis (a Grampositive bacterium) and Escherichia coli (Gram-negative). To the best of our knowledge, this is the first report that thiosemicarbazone analogs having an adamantane moiety inhibits the growth of bacteria.

Results and discussion
Chemistry Scheme 1 shows the procedure for the synthesis of novel Schiff base derivatives of thiosemicarbazones with an adamantane moiety (compounds 7a-h). The Friedel-Crafts alkylation reaction of bromoadamantane and toluene in the presence of anhydrous potassium carbonate and palladium carbon was used to obtain 1-(p-toluene)adamantane (2). Compound 2 underwent free radical substitution reaction using NBS and BPO to yield benzyl bromide (3), followed by oxidation to give 4 [4-(1-adamantyl)benzaldehyde]. The aromatic amines 5a-h were used to prepare the thiotoluamides 6a-h in basic conditions. Finally, 4 was reacted with 6a-h to produce thiosemicarbazone Schiff base derivatives 7a-h.
The yields of all thiosemicarbazone products were 62-86%. The compounds obtained were stable in both the solid and solution states. Analytical data for these compounds were consistent with their composition. The chemical structure of thiosemicarbazone compounds 7a-h was confirmed on the basis of IR, 1 H nuclear magnetic resonance (NMR), 13  In the 1 H-NMR spectra, adamantyl group protons had peaks between 1.74 and 2.07 ppm. The chemical shift values of aromatic ring protons were between 6.92 and 8.37 ppm, depending on the substituent groups. The characteristic azomethine (-CH=N-) protons appeared between 6.77 and 7.22 ppm. Besides, the characteristic N-H protons were seen at 9.93-11.91 ppm. In the 13 C-NMR spectra, aliphatic and aromatic carbons were observed between 21.05-56.56 and 113.78-163.19 ppm, respectively. the azomethine (-CH=N-) and the thiocarbamoyl carbons were detected at 143.06-143.93 and 175.10-177.60 ppm, respectively. In mass spectrometry (MS) analysis, the mass spectral data were coherent with their molecular formulas.

Antimicrobial activity
The antibacterial activity of the synthesized compounds against B. subtilis (Gram-positive) and E. coli (Gramnegative) was evaluated using the disk inhibition method and the microdilution method to determine MICs Scheme 1 Synthetic procedures of compounds 7a-h [17,26,27]. Ampicillin was used as a positive control drug. The results are shown in Tables 1 and 2.
In the disk inhibition method (Table 1), a diameter of the inhibition zone is 20 mm or more indicates that the drug has a very strong antibacterial effect on the strain; the inhibition zone of 10-20 mm is categorized as strong antibacterial effect; diameter of 5-10 mm indicates a moderate antibacterial effect; and diameter of 5 mm or less indicates that there is little or no antibacterial effect of the compound on the strain [28].
Based on the growth inhibition response, the antibacterial inhibition of compounds 7a and 7g on B. subtilis with a concentration of 8 μg/mL (22 mm) had very strong activity and other concentrations (10-20 mm) including strong activity. The antibacterial inhibition of compounds 7d and 7h on B. subtilis with concentrations of 1-8 μg/mL (10-20 mm) had strong activity, while compounds 7c and 7e with concentrations of 2-8 μg/mL (10-20 mm) indicated strong activity. The antibacterial inhibition of compounds 7b and 7f on B. subtilis with concentrations of 1-8 μg/mL (5-10 mm) had moderate activity. The antibacterial inhibition of compounds 7a and 7e on E. coli with concentrations of 1-8 μg/mL (10-20 mm) had the best inhibitory effect, similar to the effect of positive control ampicillin. Meanwhile compounds 7b-c and 7g-h with concentrations of 1-8 μg/mL (10-20 mm) had strong activity against E. coli, but compound 7f with a concentration of 1-2 μg/mL (5-10 mm) including moderate activity and concentrates of 4-8 μg/mL (10-20 mm) are categorized as strong activity.
The MIC values of the test derivatives indicated that most of the tested candidates exhibited good activity against Gram-negative bacteria as shown in Table 2. Ampicillin inhibits Gram-negative and Gram-positive bacteria with MIC of 0.03-3 μg/mL and 0.02-1.5 mg/mL, respectively. Compounds 7b-d bearing methoxy group on the phenyl had MIC 1-2 µg/mL for E. coli and MIC 4-8 µg/mL for B. subtilis, which was demonstrated that they had potent antibacterial activity compared to ampicillin. While compound 7c bearing no substituent showed less activity against E. coli (MIC = 8 μg/mL) and potent activity against B. subtilis (MIC = 8 μg/ mL) compared with positive control. Meanwhile, derivatives 7e-g with electron-withdrawing group displayed potent activity against E. coli (MIC = 1-2 µg/mL) and B. subtilis (MIC = 1-4 µg/mL) which was compared to that of ampicillin. Compound 7h bearing methyl group showed good antibacterial with MIC 1 µg/mL for E. coli compared with that of compound 7a. According to the MIC values as shown in Table 2, it was generally demonstrated that substitution on phenyl moiety of thiosemicarbazone had good potency on Gram-negative bacteria.

Conclusions
In summary, we investigated the antibacterial activity of novel adamantyl thiosemicarbazones prepared by the reaction of benzaldehyde adamantane with different amine-substituted thiosemicarbazides. Of particular note, in vitro antibacterial activity tests showed that compound 7e was as effective against E. coli (a Gram-negative bacterium) as ampicillin, while 7g had good activity against the Gram-positive bacterium B. subtilis. These data lay a foundation for the development of improved thiosemicarbazone antibacterial agents.

General
The starting materials which include 4-(1-adamantyl) benzaldehyde was prepared according to the procedure

Chemistry
General procedure for synthesis of derivatives 6a-h The anilines (1.0 equiv) were dissolved in 20 mL of DMF, NaOH (1.2 equiv), and CS 2 (1.0 equiv) were added and stirred at room temperature for 2 h, hydrazine hydrate (3.0 equiv) was added, and the reaction was heated to 65°C. TLC monitored the reaction. The reaction was cooled to room temperature and poured the solution into crushed ice, filtered with suction, and recrystallized the filter cake with ethanol to obtain intermediates 6a-h.

In vitro antibacterial activity evaluation
Minimum inhibitory concentration (MIC) assays using standard microdilution methods were carried out in 96-well microplates based on a modified procedure described previously according to the guidelines of the Clinical and Laboratory Standards Institute [29,30]. All compounds were prepared as 5 mg/mL solutions in dimethyl sulfoxide (DMSO) and were tested in a final concentration range of 1-8 μg/mL. MICs for the reference antibiotic ampicillin against quality control strains were used to confirm the validity of the screen. All experiments were performed in duplicate and repeated three times.