Novel Derivatives of 4-Methyl-1,2,3-Thiadiazole-5-Carboxylic Acid Hydrazide: Synthesis, Lipophilicity, and In Vitro Antimicrobial Activity Screening

: Bacterial infections, especially those caused by strains resistant to commonly used antibiotics and chemotherapeutics, are still a current threat to public health. Therefore, the search for new molecules with potential antimicrobial activity is an important research goal. In this article, we present the synthesis and evaluation of the in vitro antimicrobial activity of a series of 15 new derivatives of 4-methyl-1,2,3-thiadiazole-5-carboxylic acid. The potential antimicrobial effect of the new compounds was observed mainly against Gram-positive bacteria. Compound 15 , with the 5-nitro-2-furoyl moiety, showed the highest bioactivity: minimum inhibitory concentration (MIC) = 1.95–15.62 µ g/mL and minimum bactericidal concentration (MBC)/MIC = 1–4 µ g/mL.


Introduction
Antibiotics have been one of the most dynamically developing groups of medicines of the last decade [1]. The first use of an antibiotic (penicillin) in the early 20th century became a landmark in treating infections. Thanks to this, it was possible to reduce the mortality and the risk of postinfectious complications [2]. Unfortunately, as a result of the common use of penicillin, a strain of Staphylococcus aureus bacterium appeared, which produced the penicillinase enzyme, giving it resistance to penicillin [3]. In response to this fact, newer antibiotics were introduced as treatments. Additionally, strains of bacteria resistant to these new antibacterial agents were also isolated [4]. This was an important signal that the golden age of antibiotics would not last forever. This century has seen that the problem of antibiotic resistance poses a real threat to patients and global public health [5]. Alarm pathogens are particularly dangerous due to therapeutic limitations. These include multidrug-resistant pathogens (MDR), extended resistance pathogens (XDR), as well as pathogens resistant to all available antibacterial drugs (PDR) [6,7]. This group includes extremely virulent strains of methicillin-resistant S. aureus (MRSA) [3,8,9]. This etiological agent is thought to be one of the most common causes of life-threatening infections in palliative care facilities and during inpatient treatment [10]. This problem has been noticed by key institutions and global and European public health organizations [1]. The EAAD (European Antibiotic Awareness Day) and the WAAW (World Antibiotic Awareness Week) information campaigns, organized by the European Centre for Disease Prevention and Control (ECDC) and the World Health Organization (WHO), aim to raise awareness of global antimicrobial resistance and encourage general public healthcare professionals to follow best practices to avoid the further emergence and spread of drug-resistant infections [11].
We assumed that the combination of these two pharmacophores-the hydrazidehydrazone and 1,2,3-thiadiazole moieties-could have a beneficial biological effect.
Based on the results obtained so far and on literature reports on the biological potential of hydrazide-hydrazones, in this study, we synthesized novel compounds in order to obtain substances with significant antimicrobial activity.

Microbiology
The in vitro bioactivity screening of compounds 2-16 (Table 1) was performed according to the procedure described earlier by our group [31,32,34], as well as by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute guidelines [38,39]. In microbiology assays, we used a panel of reference and clinical or saprophytic strains of microorganisms from the American Type Culture Collection (ATCC). All the experiments were repeated three times and representative data are presented. All the stock solutions of the tested compounds were dissolved in DMSO.
Among the microorganisms that were taken into account in the evaluation of the activity of these substances, Gram-positive bacteria turned out to be the most sensitive. The tested compounds showed a similar effect to them, with the exception of hydrazidehydrazone 15. This compound had particular effectiveness. The minimum inhibitory concentration (MIC) of substance 15 which inhibited bacterial growth ranged from 1.95 µg/mL (for some Staphylococcus spp.) to 15.62 µg/mL (for Enterococcus faecalis ATCC 29212). This proves the strong or very strong activity of this substance against these microorganisms. The minimum bactericidal concentration (MBC) values of hydrazide-hydrazone 15, which showed a lethal effect against Gram-positive bacteria, were in the range of 3.91-62.5 µg/mL. The MBC/MIC values for this hydrazide-hydrazone were within the range of 1-4, which indicates the lethal effect of this compound. The activity of compound 15 against the Staphylococcus aureus strains ATCC 25923 and ATCC 43300 was twofold greater than the reference compound-nitrofurantoin. On the other hand, against the S. aureus ATCC 6538 strain, the activity of this substance was seven times higher than for nitrofurantoin. Compound 15 also showed good activity in comparison to nitrofurantoin against strains of S. epidermidis ATCC 12228 and Micrococcus luteus ATCC 10240-two and eight times greater, respectively.
Among Gram-negative bacteria, the antimicrobial effect was only demonstrated by compound 15 (MIC = 125-1000 µg/mL, MBC > 1000 µg/mL). Escherichia coli ATCC 25922 was the most sensitive strain against this substance (MIC = 125 µg/mL). In contrast, Pseudomonas aeruginosa ATCC 9027 did not have any sensitivity. Compound 2 was also found to have a weak effect against Bordetella bronchiseptica ATCC 4617. No antibacterial effect was observed for the remaining Gram-negative rods. In relation to yeast-like fungi of the genus Candida, a slight antifungal effect of compounds 2, 3, 5, 8, 15, and 16 was demonstrated (weak or moderate antimycotic effect). The minimum fungicidal concentration (MFC) values of the tested substances exceeded 1000 µg/mL. The Candida parapsilosis ATCC 22019 strain was the most sensitive to the tested substances (2, 3, 5, 8, 15, and 16). The remaining compounds (4, 6, 7, and 9-14) did not show an inhibitory effect on the growth of the fungi in the tested concentration ranges.

Lipophilicity
It is well known that chromatographic methods allow for establishing the experimental lipophilicity. In this research, the standardization procedure with six reference substances covering the range of lipophilicity of 0.46-3.80 was used. As a result, their log P values were highly correlated with their R M0 values in four organic modifiers, namely, acetone, acetonitrile, 1,4-dioxane, and methanol solvent systems, and appropriate calibration curves for further lipophilicity study were obtained: (1) acetone: log P EXP = 1.250R M0 − 0.256; r 2 = 0.7265 (2) acetonitrile: log P EXP = 2.563R M0 − 2.402; r 2 = 0.8984 (3) 1,4-dioxane: log P EXP = 0.902R M0 + 0.662; r 2 = 0.9457 (4) methanol: log P EXP = 1.101R M0 − 0.242; r 2 = 0.9821 The obtained coefficients of determination (r 2 ) for the mentioned equations were above 0.89 for three organic modifiers (i.e., acetonitrile, 1,4-dioxane, and methanol). Moreover, the r 2 values were rather high (>0.72) for all organic modifiers used, including acetone. Similarly, for six reference compounds, the correlations between the R F and R M0 values for 2-6 and 8-16 were sufficiently high (r 2 ≥ 0.91) for all solvents used. In addition, even better correlations (i.e., r 2 ≥ 0.98 for 12 or 11 compounds) were obtained for acetonitrile, acetone, and 1,4-dioxane solvent systems, providing accuracy for further lipophilicity determination ( Table 2). Experimental lipophilicity (log P EXP ) of the synthesized 14 hydrazide-hydrazones (2-6 and 8-16) was calculated on the basis of the above calibration equations and respective R M0 values (Table 3). Generally, the highest log P EXP values were calculated for the methanol-water solvent systems, except for compounds 4 and 13, while the lowest values were obtained for acetonitrile, except for compound 14. As far as more detailed differences were concerned, the highest log P EXP values were calculated for compounds 2-4 containing monochloro(phenyl) substituents in ortho-, meta-, or para-positions. This was observed for all solvent systems used in our experiments. Thus, it can be stated that the position of the chloride atom in a phenyl ring did not affect the lipophilicity of these three derivatives (2)(3)(4). On the contrary, clearly seen differences were observed for dimethoxyphenyl-substituted hydrazide-hydrazones (compounds 12-14). Between them, compound 13, with a 2,4dimethoxyphenyl substituent, showed the highest log P values for all solvent systems used in our experiments, illustrating the importance of positional isomerism in the lipophilicity of these compounds. At the same time, two chlorophenyl substituted derivatives (i.e., 2 and 3) showed higher log P values than similar fluorophenyl-substituted compounds (5 and 6). When compounds 8 and 10 were compared, the 4-hydroxy-3-iodo-5-methoxyphenylsubstituted hydrazide-hydrazone (10) was found to be more lipophilic than compound 8 with a 4-hydroxy-5-ethoxyphenyl substituent. This was observed for almost all solvent systems used in the present study (i.e., for the mixtures with acetonitrile, 1,4-dioxane, and methanol). Bearing in mind all the tested hydrazide-hydrazones (2-6 and 8-16), the lowest lipophilicity was shown for compound 15 substituted with the 5-nitro-2-furyl moiety.

Discussion
Condensation reaction of 4-methyl-1,2,3-thiadiazole-5-carboxylic acid hydrazide (1) with appropriate aldehydes allowed us to obtain a series of new hydrazide-hydrazone derivatives (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). The reaction efficiency was in the range of 57-98%, what can be considered as satisfactory. The most active compound was the hydrazide-hydrazone numbered 15, which had a 5-nitro-2-furyl moiety in its structure (i.e., a moiety analogous to a group of medicines, such as nitrofurazone, furazolidone, and nitrofurantoin). Due to this, it can be concluded that this fragment of compound 15 is essential for its bioactivity. It can also be seen that compounds 2, 3, 5, and 6, with the halogen atom in the orthoand meta-position of the phenyl ring, had greater antimicrobial activity in comparison to compounds 4 and 7 with the halogen atom in the para-position, which did not show any activity.
The performed activity assays of the hydrazide-hydrazones showed that the best activity was attributed to substance 15 with the 5-nitro-2-furyl moiety, which showed activity especially against all tested Gram-positive bacterial strains. Based on the lipophilicity measured in methanol, because in this case the correlation coefficient is the highest, it can be concluded that despite the differences in the structure of Gram-negative bacteria and Gram-positive bacteria, the most desirable lipophilicity is within the limits of 2.25 (compound 15). Above this value, activity decreases. On the other hand, lipophilicity in the range of 3.63-4.09 causes activity against Gram-positive bacteria (compounds 2, 3, 5, and 16).

Conflicts of Interest:
The authors declare no conflict of interest.