Sulfadiazine Salicylaldehyde-Based Schiff Bases: Synthesis, Antimicrobial Activity and Cytotoxicity

The resistance among microbes has brought an urgent need for new drugs. Thus, we synthesized a series of Schiff bases derived from the sulfa drug sulfadiazine and various salicylaldehydes. The resulting 4-[(2-hydroxybenzylidene)amino]-N-(pyrimidin-2-yl)benzene-sulfonamides were characterized and evaluated against Gram-positive and Gram-negative bacteria, yeasts, moulds, Mycobacterium tuberculosis, nontuberculous mycobacteria (M. kansasii, M. avium) and their cytotoxicity was determined. Among bacteria, the genus Staphylococcus, including methicillin-resistant S. aureus, showed the highest susceptibility, with minimum inhibitory concentration values from 7.81 µM. The growth of Candida sp. and Trichophyton interdigitale was inhibited at concentrations starting from 1.95 µM. 4-[(2,5-Dihydroxybenzylidene)amino]-N-(pyrimidin-2-yl)-benzenesulfonamide was identified as the most selective Schiff base for these strains with no apparent cytotoxicity and a selectivity index higher than 16. With respect to M. tuberculosis and M. kansasii that were inhibited within the range of 8 to 250 µM, unsubstituted 4-[(2-hydroxy-benzylidene)amino]-N-(pyrimidin-2-yl)benzenesulfonamide meets the selectivity requirement. In general, dihalogenation of the salicylic moiety improved the antibacterial and antifungal activity but also increased the cytotoxicity, especially with an increasing atomic mass. Some derivatives offer more advantageous properties than the parent sulfadiazine, thus constituting promising hits for further antimicrobial drug development.


Introduction
Schiff bases are condensation products of primary (aromatic) amines with aldehydes or ketones carrying the azomethine (imino) moiety (-CR=N-). They are considered versatile pharmacophores for various pharmacological activities where the azomethine group has been demonstrated to be critical to the bioactivity. For example, Schiff bases, whether of natural or non-natural origin, have exhibited promising antibacterial, antitubercular, antifungal, antiparasitic, antiviral, antioxidant, anticancer, analgesic, anti-inflammatory properties etc. [1][2][3]. Altogether, they represent very frequently used and useful scaffold in medicinal chemistry. Schiff bases of substituted salicylaldehydes (2-hydroxybenzaldehydes) are well-known antimicrobial agents in "free" form or as ligands in metallic complexes [4][5][6][7]. Similar biological action have been reported for Schiff bases of various sulfonamides [4,8,9].

Chemistry
Sulfadiazine-derived Schiff bases 2 were prepared by reactions of SDZ 1 and the corresponding aldehydes (salicylaldehydes, 5-nitrofuran-2-carbaldehyde) in boiling methanol (MeOH) for 5 h (Scheme 1). Aldehydes were added to a hot methanolic suspension of SDZ. The poor solubility of SDZ in MeOH is a reason for an extended reaction time in contrast to previous work [4].
The yields were good, within the range of 85-95%, with 4-[(2-hydroxy-5-nitrobenzylidene)amino]-N-(pyrimidin-2-yl)benzenesulfonamide (2f) being an exception (74 %). In this case, it was beneficial to dissolve 5-nitrosalicylaldehyde in hot MeOH and then add the sulfonamide; the reaction time was extended to 8 h. pharmacophore for antimicrobial activity (e.g., [14,15]). This report is focused on chemical synthesis, characterization and initial biological evaluation (antimicrobial screening, toxicity for eukaryotic cells) to identify the most promising candidates for advanced microbiological and pharmacological tests.

Chemistry
Sulfadiazine-derived Schiff bases 2 were prepared by reactions of SDZ 1 and the corresponding aldehydes (salicylaldehydes, 5-nitrofuran-2-carbaldehyde) in boiling methanol (MeOH) for 5 h (Scheme 1). Aldehydes were added to a hot methanolic suspension of SDZ. The poor solubility of SDZ in MeOH is a reason for an extended reaction time in contrast to previous work [4]. The yields were good, within the range of 85-95%, with 4-[(2-hydroxy-5-nitrobenzylidene)amino]-N-(pyrimidin-2-yl)benzenesulfonamide (2f) being an exception (74 %). In this case, it was beneficial to dissolve 5nitrosalicylaldehyde in hot MeOH and then add the sulfonamide; the reaction time was extended to 8 h. All of the compounds (Table 1) were characterized by 1 H-, 13 C-NMR, IR spectra and melting points; their purity was checked additionally by elemental analysis. In the 1 H-NMR spectra of Schiff bases 2, an azomethine (CH=N) singlet appeared at 9.16-8.89 ppm. The phenolic hydrogen was observed in a comparatively broader range of 13.93-11.68 ppm. IR spectroscopy displayed a typical sharp and strong azomethine (-CH=N-) band observed at 1582-1570 cm −1 .

Antimicrobial Activity
The sulfadiazine derivatives 1-2 were screened in vitro for their antimicrobial properties.  (Table 3) [17]. This panel of twenty microbial species covers a wide range of important human pathogens including those with an acquired resistance. It is a useful screening tool for an initial identification of potential antimicrobial activity of novel compounds. All of the compounds (Table 1) were characterized by 1 H-, 13 C-NMR, IR spectra and melting points; their purity was checked additionally by elemental analysis. In the 1 H-NMR spectra of Schiff bases 2, an azomethine (CH=N) singlet appeared at 9.16-8.89 ppm. The phenolic hydrogen was observed in a comparatively broader range of 13.93-11.68 ppm. IR spectroscopy displayed a typical sharp and strong azomethine (-CH=N-) band observed at 1582-1570 cm −1 .

Antimicrobial Activity
The sulfadiazine derivatives 1-2 were screened in vitro for their antimicrobial properties.  (Table 3) [17]. This panel of twenty microbial species covers a wide range of important human pathogens including those with an acquired resistance. It is a useful screening tool for an initial identification of potential antimicrobial activity of novel compounds.     Bacitracin (BAC), fluconazole (FLU) and isoniazid (INH) were employed as the comparative drugs for antibacterial, antifungal and antimycobacterial activity, respectively. The parent sulfadiazine 1 showed only a weak action against both strains of Staphylococcus aureus (Table 1), and no antifungal properties, but it is an antimycobacterial agent ( Table 2). Four Schiff bases (compounds 2b, 2j-l) showed no significant antibacterial activity (Table 1). In general, the action against Gram-negative species is only negligible (MICs starting from 125 µM). MICs tend to be even lower for MRSA when compared to the methicillin-susceptible strain (e.g., 2g). For S. aureus, the 3,5-dihalogen substitution is translated into an enhanced activity (MICs of 7.81-62.5 µM) where heavier atoms contribute to more intense action (7.81-62.5 µM). Another substituent decreasing MIC values is a second hydroxyl group (i.e., 2i) in the para-position with respect to the salicylic hydroxyl with a uniform MIC of 31.25 µM. The rest of sulfadiazines 2 share MICs of 125-500 µM. The analysis of the effect of the chlorine position on the salicylic ring revealed that the activity is conferred by its presence at the position 5 while its shift to another carbon hampers antibacterial activity completely (2c vs. 2k-l). 3,5-Diiodosalicylidene sulfadiazine 2p was also superior for the inhibition of S. epidermidis (7.81 µM), followed by 5-OH (2i, 15.62 µM), other 3,5-dihalogen (2m-o) and 5-nitrofuran 2q (31.25 µM) derivatives. The diiodo derivative 2p was superior to bacitracin against S. epidermidis, and five compounds (2i, 2m-o, 2q)  Structure-activity relationships for the substitution of the salicylidene fragment are reported in Table 4. In general, the introduction of any substituent does not offer any benefit when compared to unsubstituted Schiff base 2a. Among single halogens, bromine (compound 2d) is favourable, in the case of NTM strains concomitantly with 5-and 3-chloro isomers (compounds 2c, 2l). 5-Methoxy (compound 2h) and tert-butyl (compound 2j) groups contribute to the improved action, too. On the other hand, halo 3,5-disubstitution (compounds 2m-p), and a nitro group (compound 2f) are detrimental for the inhibition of all mycobacteria. For NTM, a fluorine atom (compound 2b), 5-methyl (compound 2g), or 5-nitrofurylidene (compound 2q) abrogate the activity. Although lipophilicity represents an important factor influencing antimycobacterial action, no direct correlation between lipophilicity and MIC values was identified.   compounds (2a, 2c, 2h, 2j, and 2l) were comparable after 7 and/or 14 d of incubation (± one dilution). In contrast, eleven derivatives (2a, 2c-e,  2h, 2j-n, 2p)  Unfortunately, the modification of the parent sulfadiazine 1 to the derivatives 2 did not bring any significant improvement of antimycobacterial properties but the most active Schiff bases (2a, 2d, 2h, 2j, and 2l) exhibited activity comparable to that of 1 (± one dilution).
Seeking insights into the potential mechanism of action, we evaluated the inhibition potency of selected salicylidenesulfonamides against isocitrate lyase, but they were virtually inactive in spite of the substitution pattern (i.e., their inhibition rates were lower than 10% at the single concentration of 100 µM).

Cytotoxicity and Selectivity
The cytotoxicity of the investigated compounds 1 and 2 was measured using the standard hepatic cell line HepG2 (Table 5) [16]. The used CellTiter 96 assay is based on the reduction of tetrazolium dye in living cells to formazan. This reduction is related to availability of NADH or NADPH. The parameter IC 50 , i.e., the concentration that reduces the viability of the cells to 50% of the maximal viability, was used as a measure of cytotoxicity. It was not possible to determine the exact IC 50 values of two derivatives (compounds 2a, 2b) due to their limited solubility in the cell culture medium used in this experiment. We can divide the investigated Schiff bases 2 into three groups according to their cytotoxicity for HepG2 cells. The first one covers comparatively non-toxic compounds 1, 2a, 2b, and 2g-i (IC 50 values > 250 µM). The group of the compounds with an intermediate toxicity and IC 50 s in the range of 102.2 to 160.5 µM consists of 2d-f, and 2k-m. The last group includes sulfonamides with an increased cytotoxicity (IC 50 ≤ 100 µM: 2j, 2n-q). 4-{[(5-Nitrofuran-2-yl)methylene]amino}-N-(pyrimidin-2-yl)benzene-sulfonamide (2q) was found to be the most toxic substance (IC 50 = 16.0 µM). Obviously, the replacement of the salicyl ring by 5-nitrofuran-2-yl produced a more pronounced toxicity for mammalian cells. The majority of the modifications of the parent sulfadiazine 1 (IC 50 > 1500 µM) increased the cytotoxicity by up to two orders of magnitude for the most toxic compounds. Generally, the enhanced cytotoxicity is observed in the presence of 3,5-halogen disubstitution and a tert-butyl moiety. Among particular halogens, the cytotoxicity correlates positively with an increased atomic mass. The presence of a non-bulky electron-donating group (CH 3 , CH 3 O, OH) or hydrogen results in less cytotoxic compounds (i. e., 2a, 2g-i).
We calculated the selectivity indices (SIs) for M. tuberculosis, both strains of M. kansasii, MRSA, S. epidermidis, all of the Candida species and T. interdigitale ( Table 5). The SI is defined as the ratio of IC 50 to MIC, and values higher than 10 indicate rather acceptable toxicity (based on the analogy of the therapeutic index). Considering mycobacteria, in addition to the parent sulfadiazine 1, two compounds exhibited targeted selectivity:

General Information
All of the reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Penta Chemicals (Prague, Czech Republic) and were used as received. The reactions and the purity of the products were monitored by thin-layer chromatography (TLC) with a mixture of dichloromethane with n-hexane and methanol (4:3:1, v/v) as the eluent. The plates were coated with 0.2 mm Merck 60 F254 silica gel (Merck Millipore, Darmstadt, Germany) and were visualised by UV irradiation (254 nm). The melting points were determined using a B-540 melting point apparatus (Büchi, Flawil, Switzerland) with open capillaries, and the reported values are uncorrected. The elemental analysis (C, H, N) was performed with an automatic microanalyser instrument CHNS-O CE (Fisons EA 1110, Milano, Italy). The infrared spectra (ATR) were recorded on a Nicolet 6700 FT-IR spectrometer (ThermoFisher Scientific, Waltham, MA, USA) in the range of 600 to 4000 cm −1 . The NMR spectra were measured in DMSO-d 6 at ambient temperature using a Varian VNMR S500 instrument (500 MHz for 1 H and 125 MHz for 13 C; Varian Inc., Palo Alto, CA, USA). The chemical shifts, δ, are given in ppm with respect to tetramethylsilane as the internal standard. The coupling constants (J) are reported in Hz. The calculated logP values (ClogP), which are the logarithms of the partition coefficients for octan-1-ol/water, were determined using the CS ChemOffice Ultra version 16.0 program (CambridgeSoft, Cambridge, MA, USA).

Synthesis
Sulfadiazine 1 (1 mmol, 250.3 mg) was suspended in methanol (MeOH, 10 mL), and 1.1 mmol of the appropriate aldehyde was added in one portion under vigorous stirring. The solution was refluxed for 5 h and then stirred at room temperature overnight. The resulting crystals were filtered off, washed with a small amount of MeOH and then acetonitrile, and dried. The crystals were recrystallized from MeOH or tetrahydrofuran/n-hexane mixture if necessary. The identity of known compounds (i. e., 2a, 2c, 2d, and 2m) was confirmed by NMR ( 1 H and 13 C) and IR spectroscopy. All spectroscopic characteristics were in accordance with previously reported data. The purity was checked additionally by melting points measurement and elemental analysis. The numbering of hydrogen atoms in the 1 H-NMR spectra is depicted in Figure 3.    After 24 h incubation in a humidified atmosphere containing 5% CO 2 at 37 • C, the reagent from the kit CellTiter 96 was added. After 2 h incubation at 37 • C, absorbance of samples was recorded at 490 nm (Infinita M200, TECAN, Grödig, Austria). A standard toxicological parameter IC 50 was calculated by nonlinear regression from a semilogarithmic plot of incubation concentration versus percentage of absorbance relative to untreated controls using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA). IC 50 is the inhibitory concentration that reduces the cell viability to 50% of the maximal (control) viability.

Conclusions
In conclusion, we identified several interesting structure-activity relationships in some novel sulfadiazine Schiff bases. Our findings suggested that several were selective for microbial pathogens (M. tuberculosis, M. kansasii, Staphylococcus spp. including one MRSA strain, yeasts and Trichophyton interdigitale). 4-[(2-Hydroxybenzylidene)amino]-N-(pyrimidin-2-yl)benzenesulfonamide (2a) is active in the inhibition of tuberculous and atypical mycobacteria. The 2,5-dihydroxybenzaldehyde-derived Schiff base 2i exhibited the highest selectivity for staphylococci and fungi. For these molecules, the desirable action is separated from an unwanted toxicity, thus constituting a promising hit for further structure optimization and development of potential antimicrobial agents. Further studies are required to gain a deeper understanding of their properties (especially mechanism of action and interactions with eukaryotic cells).