Evaluation of cytotoxic, antifungal, and larvicidal activities of different bis‐sulfonamide Schiff base compounds

Schiff bases (imines or azomethines) are versatile ligands synthesized from the condensation of amino compounds with active carbonyl groups and used for many pharmaceutical and medicinal applications. In our study, we aimed to determine the cytotoxic, antifungal and larvicidal activities of biologically potent bis‐sulfonamide Schiff base derivatives that were re‐synthesized by us. For this aim, 16 compounds were re‐synthesized and tested for their cytotoxic, antifungal and larvicidal properties. Among this series, compounds A1B2, A1B4, A4B2, A4B3, and A4B4 were shown to have cytotoxic activity against tested cancer lung cell line (A549). The most potent antifungal activity was observed in compounds A2B1 and A2B2 against all fungi. A1B1 showed the strongest larvicidal effect at all concentrations at the 72nd h (100% mortality). These obtained results demonstrate that these type of bis‐substituted compounds might be used as biologically potent pharmacophores against different types of diseases.

anticonvulsant, [7] potent carbonic anhydrase inhibitors and activators, [8][9][10][11][12][13] antitumor, [14][15][16] antitubercular, [17] anthelmintic, [18] antioxidant, [19,20] larvicidal, [21] and so forth. Morever, several studies have shown that the metal complexes of the Schiff bases can be obtained easily through the complexation of different metal ions via azomethine nitrogen and used as biologically more active derivatives. [22][23][24] Sulfonamides and their derivatives have been extensively researched since 1940s for many different types of pharmaceutical applications and have been used as an important chemical motif to improve the biological potential of the discovered compounds. Among these studies, many drugs come out and used as antibiotic, antibacterial, diuretic, anticancer, antiinfective, anti-glaucoma, and so forth agents. [25] More recently, bis-substituted compounds have been started to be investigated as a potent and biologically active derivatives against different enzymes targeting many types of diseases. [8,13,[26][27][28] By this strategy, the biological activities of the compounds were improved due to the more tight interactions of the compounds on the active site of enzyme(s). Also, the hybrid molecules obtained with one linker and two pharmacophores on the both sites might improve the pharmaceutical properties of the compounds in drug design and discovery approaches. [26][27][28] In one of our previous study, bis-sulfonamide Schiff base derivatives were designed, synthesized and fully characterized. [8] Also, the potent carbonic anhydrase (CA), antioxidant and cholinesterase (AChE and BChE) inhibition properties of these compounds were successfully presented. [8,26] Due to the potent biological activities of these compounds, in the present study, these derivatives were freshly re-synthesized as previously reported [8] and evaluated for their cytotoxic, antifungal and larvicidal activities.

| Chemistry
The general reaction procedure for the structurally diverse bissulfonamide Schiff bases was described in Scheme 1. These compounds were previously designed, synthesized and fully characterized by our research group as potent carbonic anhydrase (CA I, II, IX, and XII) inhibitors, antioxidant properties, acetylcholinesterase and butyrylcholinesterase inhibitors. [8,26] These compounds were resynthesized freshly in the present study to determine cytotoxicity, antifungal and larvicidal activities. Briefly, the compounds were prepared by condensing aromatic and heterocyclic bis-aldehydes and benzenesulfonamide derivatives in ethanol and formic acid as a catalyst. The physicochemical and spectroscopic evaluation of the compounds AnBm was reported by Akocak et al. [8] The chemical structures and codes of the re-synthesized compounds are demonstrated in Table 1. S C H E M E 1 General synthetic way for the bis-sulfonamide Schiff base compounds (AnBm). [8,26] T A B L E 1 The chemical structures and codes of the bis-sulfonamide Schiff base compounds. [8] AYDIN ET AL. | 3 of 10 Subsequently, MTT solution was removed and dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used to dissolve the absorbed dye.

| Cytotoxicity
Immediately, plates were read at 570 nm using an EZ read 400 microplate reader (Biochrom). The experiment was repeated four times. [29] 2.3 | Statistical analysis Statistical analysis was performed with SPSS 15.0 for Windows (SPSS Inc.). The one sample Kolmogorov-Smirnov test was used to determine whether the data was distributed normally and Levene's test were used for the homogeneity test of group variances.
Groups were compared using the One-way analysis of variance. As a result of these analysis, Significance between group means was identified using the Tukey's multiple range test. Mean differences were considered significant at p < 0.05. The results were reported as mean ± SD and were exhibited in column chart. A probability level of p < 0.05 was used to indicate the statistical significance of the results. To determine growth, passages were taken from each well using sterile plastic ring loops on Sabouraud Dextrose Agar (Merck 1.07315) plates and incubated under the same conditions. The previous value of the dilution titer where growth occurred was determined as the minimal inhibitory concentration value of that compound. In addition, fluconazole and sulfanilamide were used as reference drugs in our study. [30,31] 2.5 | Larvicidal activity there are egg, larva (feeding larvae I. II. III. stage and wandering larvae), pupa and adult forms. L. sericata was reared in a 12 m 2 room under laboratory conditions of 25°C-30°C and 50 ± 5% relative humidity (RH) in a particular light source. [32][33][34][35][36] Adults were fed on sucrose and water, but larvae were reared on chicken liver-agar medium recommended by Mumcuoğlu, [33] Uslu, [34] Aydın & Uslu, [35] and Uslu et al. [36]

| Pretreatment
The 2 mg were taken from all compounds and homogenized with 1 mL of 0.01% DMSO for 5 min with the help of a vortex.

| Treatment
100 µL of each dilution obtained was taken and transferred onto 5 g chicken liver-agar medium in sterile petri dishes. The same procedure was repeated for each dilution step.
For control purposes, 100 µL of DMSO solution was placed in an eighth petri dish containing chicken liver-agar medium. After these procedures, 100 first-stage larvae of sterile L. sericata fly, produced in the laboratory, were left in each petri dish. Petri dishes were kept at 25°C in 50% humidity for the development of the released larvae.
The viability of the larvae was checked under a stereo zoom microscope (Nikon SMZ745T) for 3 days, 24 h apart, and the numbers of live/dead larvae were recorded.
F I G U R E 1 100 first-stage larvae of sterile Lucilia sericata fly.  Although the cytotoxic effect of A3B1 and A3B4 compounds on lung cancer cells was statistically significant when evaluated between dose groups, no significant change was detected when compared to the control ( Figure 5).
The cytotoxic activities of bis-sulfonamide compounds (A4B1, A4B2, A4B3, and A4B4) are shown in Figure 6. All compounds excepting A4B1 appear to reduce the viability of A549 cells. It was determined that the 100 and 200 µg/mL groups of A4B2, A4B3 and A4B4 significantly reduced the viability of the cells compared with the control. These results show us that the 1,3-thiadiazole containing sulfonamide (Acetazolamide like compound) is an essential pharmacophore group in the better cytotoxic results. This is also well agreement in literature studies. [37,38] In general, the furan group was found to be more important linker as compared with benzene groups in the cytotoxic compound design against the tested cancer cell line (A549). Also, the cytotoxic activity was significantly affected by the position of F I G U R E 6 Cytotoxic activities of bis-sulfonamide compounds (A4B1, A4B2, A4B3, and A4B4) on lung cancer cell line A549. the used sulfonamides and aldehydes. More specifically, 4-aminobenzenesulfonamide and a 1,3-thiadiazole group from sulfonamides and the furan linker from the aldehydes were found the best combination for designing biologically more potent compounds against lung cancer cell lines. These results agree with the previous study conducted by us in which these compounds were tested against cancer related carbonic anhydrase isozymes (CA IX and CA XII) and as a result, it found once again that the 1,3-thiadiazole derivatives are the most effective among the tested compounds.

| Antifungal activity
The antifungal activity results of the re-synthesized compounds are summarized in Table 2 and compared with standard drugs Fluconazole and Sulfanilamide. In general aspect, none of the compounds were more active than the used standard drugs. The most potent inhibition was observed in compounds A2B1 and A2B2 with the lowest MIC (12.5 μg/mL) against all fungi. This demonstrates that the 3-aminobenzenesulfonamide is an important pharmacophore group to design more active antifungal agents as compared to counterparts used in the current study. On the other hand, the compounds A2B3 and A4B2 had the low antifungal activity of Candida albicans (ATCC 90028, ATCC 64548) and Candida albicans (ATCC 645489), respectively. Some of the compounds, including, A1B3, A3B3, and A4B1 have no inhibitory effect on all tested fungi.

| Larvicidal activity
The larvicidal activity results of the bis-sulfonamide Schiff bases were recorded in Table 3  mortality was observed at all concentrations of sulfanilamide. As a result, the bis-substitution on the sulfanilamide drastically changed the larvicidal activity. One of the most important findings in the present study was observed in larvicidal activity assays, in which 4-aminobenzenesulfonamide groups were shown to be the more important pharmacophore group for these types of tests among the used sulfonamide groups.

| CONCLUSION
In the present work, we report a series of 16 bis-sulfonamide Schiff bases, which were freshly re-synthesized from the condensation of aromatic/heterocyclic primary sulfonamides and bis-aldehydes. The   derivatives regarding cytotoxic activities against the tested cell line. Also, the best antifungal activity was observed with 3-aminobenzenesulfonamide containing bis-sulfonamide Schiff bases (A2B1 and A2B2) with the lowest MIC (12.5 μg/mL) value. Besides, 4-aminobenzenesulfonamide substituted compounds were more susceptible to larvicidal activity at all concentrations in the 72nd h (100% mortality) and the larvicidal activity of these compounds was much more potent than the standard drug sulfanilamide which is a starting material of these compounds. As a result, these compounds might be improved and discovered as potent cytotoxic and antifungal agents with some chemical modifications.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.