In vitro multitarget activity of sulfadiazine substituted triazenes as antimicrobial, cytotoxic, and larvicidal agents

Multidrug resistance (MDR) causes difficulties in the treatment of infections and cancer. Research and development studies have become increasingly important for the strategy of preventing MDR. There is a need for new multitarget drug research and advancement to reduce the development of drug resistance in drug‐drug interactions and reduce cost and toxic effects. This study aimed to determine the effects of multi‐target triazene compounds on antibacterial, antifungal, antiviral, cytotoxic, and larvicidal activities were investigated in vitro. A series of 12 novel of 1,3‐diaryltriazene‐substituted sulfadiazine (SDZ) derivatives were synthesized, and the obtained pure products characterized in detail by spectroscopic and analytic methods (FT‐IR, 1H‐NMR, 13C‐NMR, and melting points). The antibacterial and antifungal activities of these derivatives (AH1‐12) were determined by broth microdilution method. All derivatives have been evaluated in cell‐based assays for cytotoxic and antiviral activities against Modified Vaccinia Virus Ankara. The larvicidal efficacy of these chemical compounds was also investigated by using Lucilia sericata (L. sericata) larvae. Twelve 1,3‐diaryltriazene‐substituted SDZ derivatives (AH1‐12) were designed and developed as potent multitargeted compounds. Among them, the AH1 derivative showed the most antibacterial and antifungal activity. Besides, synthesized derivatives AH2, AH3, AH5, and AH7 showed higher antiviral activity than SDZ. All synthesized derivatives showed higher cytotoxic activity than SDZ. Also, they showed larvicidal activity at 72 h of the experiment. As a result, these compounds might be great leads for the development of next‐generation multitargeted agents.


| INTRODUCTION
Multidrug resistance (MDR) causes difficulties in the treatment of infections and cancer. [1]The worldwide spread of MDR raises the risk for morbidity and mortality from diseases caused by bacteria, viruses, fungi, ectoparasites, [2] and neoplasms in humans and animals. [1,3,4]It creates a risk of MDR, especially in Escherichia coli, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Salmonella typhimurium, [5] Candida infections, neoplasia, [1] and miasis (larvae infestations). [2]Research and development studies have become increasingly essential for the strategy of preventing MDR. [6]It has been stated that the costs and adverse effects of multitargeted drugs can be reduced by using a single drug. [7]iazenes are a significant class of compounds studied for over 150 years for their interesting structural, pharmacological, and chemical properties. [8,9][12][13][14][15][16][17][18] Triazenesubstituted compounds has many applications in several fields, such as organometallic chemistry, polymer technology, and oligomer synthesis.[21] These reactive functional group chemistry (-N═N-NH-) forms a cornerstone of small lipophilic alkylating agents such as temozolomide (TMZ), dacarbazine (DTIC), and mitozolomide where their biological activities are mainly based on their ability to form diazonium salts that can alkylate DNA. [22,23] recent years, the triazene moiety has attracted widespread attention, especially when combined with some important sulfonamide drugs.[26][27] Because this type of incorporation of triazene moiety with sulfonamides structure could improve existing biological properties or contribute to the emergence of new valuable properties, novel 1,3diaryltriazene-substituted sulfadiazines (SDZs) were synthesized in an attempt to obtain more effective, less toxic and multitarget antimicrobial, cytotoxic, and larvicidal agents.The pathogens (S. aureus, MRSA, E. coli 180, E. coli, P. aeruginosa and S. typhimurium; Candida albicans [ATTC 64548] and C. albicans [ATTC 90028]) used in our study were chosen for comparison with other studies because they are the most commonly used pathogens in antibacterial and antifungal studies.Modified vaccinia virus Ankara for antiviral activity was used in this study because it is a virus from the Poxviridae family and is a highly attenuated strain.In this study, it is aimed to synthesize novel-substituted triazenes and investigate their multitargeted activities (antimicrobial, cytotoxic, and larvicidal).

| General synthetic procedure for the preparation of 1,3-diaryltriazene-substituted SDZ derivatives AH1-12
In our previous studies, we have successfully synthesized a triazine-substituted compounds with our optimized reaction conditions.In the present study, we applied the same reaction conditions as indicated in our previous studies. [24,25]Briefly, a mixture of SDZ (5 mmol) in 2 mL of conc.HCl and 10 mL of water were cooled to 0°C-5°C, and NaNO 2 (7 mmol) in 5 mL of water was added dropwise to this solution for about 15-20 min under continuous stirring.The mixture was stirred for about 30 min at 0°C-5°C, and diazonium solution was added to aniline solution (prepared by 5 mmol substituted anilines in 5 mL of MeOH) by adjusting the pH around 6-7 with simultaneous addition of saturated sodium acetate in water.Then, the reaction mixture was stirred for 3 h at 0°C-5°C and overnight at room temperature in the dark.Reactions were monitored through infrared spectroscopy and thin-layer chromatography.The obtained colorful solid was filtered off, washed several times with cold water, crystallized from ethanol, and dried under vacuum.The obtained pure products AH1-12 were characterized in detail by spectroscopic and analytic methods (FT-IR, 1 H-NMR, 13 C-NMR, and melting points).[30] Minimum inhibitory concentration (MIC) values were interpreted according to European Committee for Antimicrobial Susceptibility Testing (EUCAST) criteria with Mueller Hinton Broth (Boilife, Milano, Italia). [28]The broth microdilution test was performed in microdilution plates (96-wells microplate).Briefly, the tested Growth and sterility controls were included for each assay.The microplates were covered with the sterile lid, then agitated to mix the contents of the wells using a plate shaker, and incubated in ambient air at 35°C for 24 h.MIC values were assayed as a reduction of growth compared with the control.The results were analyzed visually. [28,30]

| In vitro antifungal activity
The antifungal activities were evaluated against C. albicans (ATTC 64548) and C. albicans (ATTC 90028).The antifungal activities of newly synthesized SDZ derivatives (AH1-12) were determined by the broth microdilution method. [31]MIC values were interpreted according to EUCAST criteria. [32]e broth microdilution test was performed in microdilution plates (96-well microdilution plates with flat-bottomed wells).SDZ 37°C and 5% CO 2, the supernatant was discarded and DMSO (100 µL) was added to wells.The microplate was gently shaken to solubilize the formazan crystals and read at a wavelength of 570 nm (Absorbance 96, Byonoy).CC 50 of all compounds was calculated.
For the dose-dependent assay, BHK-21 cells grown in 24-well cell culture plates (3 × 10 5 cell/well) and kept at 37°C and 5% CO 2 for 24 h to confluence at least 90%.After incubation, MVA (5.5 TCID 50 mL −1 ) was added at 0.1 MOI to the plates with fourreplicated wells.The plates were incubated to adsorb the virus for 1 h in the incubator.Then, the unbound virus was discarded by washing the wells with PBS.Then (The two-fold dilutions 500 µg mL −1 ) the compounds were added to six-replicated wells and the plates were incubated at 37°C and 5% CO 2 for 72 h.After incubation, the virus titration was investigated in the 96-well microplates and calculated by Spearman-Kärber [33,34] endpoint method for all dilutions of the compounds.The negative (cell control), positive control (virus control), and reference drug (TNF, tenofovir disoproksil fumarate) were conducted as well.An inert microscope (Olympus ix71) was used to observe the morphological changes and CPE on the wells.Inhibition concentrations 50 and selectivity index = Cytotoxic concentration 50 /Inhibition concentrations 50 (SI = CC 50 /IC 50 ) of all compounds were calculated.

| Larvicidal activity
The larvicidal efficacy of compounds AH1-12 were investigated by using L. sericata larvae.The sterile larvae of the fly were obtained from the Maggot Production Unit, Department of Veterinary Parasitology, Faculty of Veterinary Medicine, Selcuk University.7] In the study, seven serial dilutions were prepared from each of the AH1-12 chemical compounds from 1/2 to 1/128.All chemical compounds were dissolved in DMSO and diluted with the same solution.300 µL of each dilution was transferred onto 5 g chicken liver-agar medium in sterile petri dishes.300 µL malathion was separately added to another petri dish containing chicken liver-agar medium as a positive control.After the petri dishes were prepared, 100 first instars of L. sericata were left in each petri dish.For larval development, petri dishes were kept at 25°C in 55 ± 5% humidity.
The viability of the larvae was checked under a stereo zoom microscope (Nikon SMZ745T) for 3 days, 24 h intervals, and the numbers of live/dead larvae were recorded.

| Data 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 were distributed normally, and Levene's test was used for the homogeneity test of group variances.Groups were compared using the one-way analysis of variance.As a result of these analyses, significance between group means was identified using 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 the column

| Chemistry
In the present study, a series of 12 1,3-diaryltriazene-substituted SDZ derivatives AH1-12 was successfully synthesized with the procedure of our previous studies [24,25] as detailed in Scheme 1.
Briefly, the diazonium salt of SDZ was obtained with concentrate HCl and NaNO 2 at 0°C-5°C.Then, this diazonium salt was reacted with substituted primary aromatic amines to produce final 1,3diaryltriazene-substituted SDZ derivatives AH1-12.The chemical structures of the obtained pure products were confirmed by several analytical and spectral techniques (see experimental part for detail).

| In vitro antibacterial activity
The 1,3-diaryltriazene-substituted SDZ derivatives AH1-12 exhibited MIC between 15.62 and 125 µg.The MIC values of SDZ derivatives for bacteria are listed in Table 1.
All SDZ derivatives showed antibacterial activity against one or more gram-positive and gram-negative bacteria.Only AH1 showed antibacterial activity against all gram-positive and gram-negative bacteria tested.On the other hand, the compounds AH6-10 and AH12 showed antibacterial effects only on gram-negative bacteria.Among these compounds, AH1 showed great antibacterial activity against MRSA (15.62 µg), and AH4 against MRSA (15.62 µg) and E. coli (ATTC) (15.62 µg).It has been observed that the antibacterial effect of AH1 against S. aureus is the same as SDZ.More interestingly, the antibacterial effect of AH1 and AH4 on MRSA was found to be better than reference drug SDZ.

T A B L E 1
The MIC values (µg) of the SDZ derivatives for bacteria.As a result of antifungal activity tests, the antifungal effect of SDZ derivatives tested in our study was found to be lower than the standard drug Fluconazole.
The selectivity index (SI) is a predictive factor for the future application of antiviral compounds and is expected to be as high as possible.The highest SI was respectively determined as 9.84, 8.06, 5.88, and 4.16 with SDZ, AH3, AH2, and AH7.

| Larvicidal activity
The mortality of sterile larvae at different dilutions of each SDZ derivative is demonstrated in Table 4.In the study, it was determined that the larvae in all Petri dishes were dead on the 48th hour, except for the AH2 group.In this group, it was determined that only a few larvae lived at the 48th hour, and these larvae were dead at 72 h.

| DISCUSSION
The process of drug development and discovery has become increasingly important in the prevention of MDR. [6]It has been claimed that utilizing a single medicine can lower the price and side effects of multi-targeted drugs. [7]The triazene moiety has received a lot of attention in recent years, especially when paired with some key sulfonamide medicines.These triazene-sulfonamide hybrids demonstrated a wide range of intriguing biological, pharmacological, and chemical features. [8,9]Novel 1,3-diaryltriazene-substituted SDZs have been developed in the hope to produce safer, less toxic, and multitarget drugs.
Abdel-Galil et al. [38] reported that compound AG10 (4-((1′,6′dihydro-[1,1′:3′,1″-terphenyl]-5′(2′ H)-ylidene)triaz-1-en-1-yl)-1Hpyrazole-3,5-diamine) had a potent antibacterial effect against S. aureus (62.5 µg MIC) and E. coli (125 µg MIC) in their study with synthesized novel triazene derivatives.In this study, AH2, AH4, AH6-10, and AH12 (31.5 µg MIC) showed great antibacterial effects against E. coli.AH1 and AH5 showed the same antibacterial effect against S. aureus (31.5 µg MIC) and E. coli (62.5 µg MIC).The antibacterial effect of the derivatives used in this study was found to be higher than that of Abdel-Galil et al. [38] Abdel-Galil et al. [38] reported that compounds AG10, AG12, and AG14 showed the best antifungal activity against C. albicans in their study with triazene compounds.In this study, however, AH1 showed the best antifungal activity against C. albicans (ATTC 64548).Using the SDZ moiety instead of cyclohexanone derivatives in the target molecules drastically improved the antibacterial and antifungal The posttreatment virus titers (TCID 50 mL −1 ) after 72-h incubation and IC 50 of compounds against MVA.were evaluated in vitro in parallel cell-based assays for cytotoxicity and antiviral activity.In one of the previous studies, 46 triazene derivatives (MT1-46) were evaluated in vitro assays for antiviral activity against MVA (Poxviridae) by Tonelli et al. [39] None of the compounds exhibited antiviral activity.This is most probably because of they used primer and seconder amines on the one side of the triazene linker and primer aromatic amine on the other side of the triazine linker.In our case, we used the SDZ on the one site of the triazine linker to improve the biological properties of the newly synthesized compounds.And so we successfully reached to our aim with this strategy.In this study, derivatives were tested against MVA, representative of the DNA virus family.TNF was used as a reference inhibitors of DNA virus and all compounds except AH6 presented antiviral activity.Besides, Tonelli et al. [39] found that the triazene derivatives they tested were relatively nontoxic on BHK- effective, but mortality reached 100% at the 72nd h.Although the literature review showed that some triazine compounds, which have molecular structures similar to triazene compounds, are used in larvicidal activity studies, [40][41][42][43][44] studies on the use of triazene derivatives in this direction have not been found.Nagaa et al [44] determined that 1,2,4-triazine derivates have larvicidal potency against third larval instars of Culex pipiens mosquitos and supposed that these derivates can be an alternative to other commercial insecticides.In another larvicidal efficacy study, Castelino et al. [41] reported that novel thiadiazolotriazin-4-ones compounds exhibited moderate mosquito-larvicidal activities against a malarial vector, Anopheles stephensi.Similarly, the effectiveness of various triazine compounds, especially on mosquito larvae, was evaluated, and promising results were obtained. [42,43]However, no study on calliphorid fly larvae, especially on L. sericata, which is one of the primary etiological agents of myiasis cases frequently encountered in veterinary medicine and medicine, has been encountered. [45]Therefore, this study suggests that triazene derivatives, which have similar molecular structures with triazine compounds, whose larvicidal activity has been tested before, have a high level of larvicidal activity against L.
sericata larvae and that these triazene compounds can be among the alternative insecticides in larval control.

| CONCLUSION
In the present study, 12 new 1,3-diaryltriazene-substituted SDZ derivatives were synthesized by coupling of diazonium salt of SDZ and primary aromatic aniline derivatives.All the synthesized derivatives were tested for antibacterial, antifungal, antiviral, larvicidal, and cytotoxic activities.All derivatives presented antibacterial activity against one or more bacteria compared to the reference drug.
AH1 showed the most antibacterial activity among the synthesized derivatives.Also, AH1 derivative showed the strongest antifungal activity, but the antifungal activity of SDZ derivatives tested was found to be lower than the reference drug Fluconazole.Synthesized derivatives AH2, AH3, AH5, and AH7 showed higher antiviral activity than SDZ.All synthesized derivatives showed higher cytotoxic activity than SDZ.All synthesized derivatives showed larvicidal activity at 72 h of the experiment.It has been determined that triazene derivatives have a larvicidal effect.The results obtained in this study may be promising for the development of potential multitarget compounds in the treatment of different disorders.
derivatives were serially diluted in two-fold fashion and finally obtained a concentration range of 1.95-250 μg mL −1 .DMSO served as a diluent for all the derivatives.Fluconazole (Selfleks Flukosel 100 mg/50 mL, Haver Farma) was used in this study as a reference drug.The turbidity of fungal inoculum has been adjusted to the McFarland reference 0.5 scale.The final fungal inoculums were adjusted to a concentration of 5 × 10 5 CFU mL −1 .Growth and sterility controls were included for each assay.The microplates were covered with the sterile lid and incubated in ambient air at 35 ± 2°C for 24 h.The assay was repeated thrice.MIC values were assayed as a reduction of growth compared to the control.The results were analyzed visually.2.2.3 | In vitro cytotoxicity assayDetermination of cell viabilityThe compounds were two-fold serially diluted in the medium supplemented fetal bovine serum (FBS) and antibiotics with the final concentration of DMSO not exceeding 1% (v/v).Then, 100 µL of each dilution was added to six-replicated wells of the 96-well microplate confluent with the cell.Eagle's minimum essential medium (EMEM) solution was only added to cell control wells.The microplates were incubated at 37°C and 5% CO 2 for 24 h.An inert microscope (Olympus ix71) was used to observe the morphological changes of the cells.The cell viability and inhibition were determined by the tetrazolium-based colorimetric (MTT) assay.Briefly, 10 µl MTT in PBS (5 mg/mL) was added to each well.After a 4 h incubation at chart.A probability level of p < 0.05 was used to indicate the statistical significance of the results.CC 50 and IC 50 were calculated by the linear regression analysis with the formula y = mx + b S C H E M E 1 General synthetic route for the synthesis of the novel 1,3-diaryltriazene-substituted SDZ derivatives AH1-12.Reagent and conditions: (i) H 2 O, HCl (conc.),NaNO 2 , 0°C-5°C, 30 min, (ii) Substituted aromatic anilines 1-12, MeOH, H 2 O, sodium acetate (saturated), 0°C-5°C 3 h, r.t.overnight.
T A B L E 4 Larvicidal activity of AH1-12 compounds, % mortality of sterile larvae.Note:The numbers written indicate the number of dead larvae counted.