Novel synthesis of new triazine sulfonamides with antitumor, anti-microbial and anti-SARS-CoV-2 activities

Novel approach for synthesizing triazine sulfonamide derivatives is accomplished via reacting the sulfaguanidine derivatives with N-cyanodithioiminocarbonate. Further reaction of the novel triazine sulfonamide analogues with various secondary amines and anilines generated various substituted triazine sulfonamide analogues of promising broad-spectrum activities including anti-microbial, anti-tumor, and anti-viral properties. The in vitro anti-proliferative activities of most of the novel compounds were evaluated on the NCI-60 cell line panel. The antifungal and antibacterial activities of the compounds were also estimated. The anti-viral activity against SARS CoV-2 virus was performed using MTT cytotoxicity assay to evaluate the half-maximal cytotoxic concentration (CC50) and inhibitory concentration 50 (IC50) of a representative compound from the novel triazine sulfonamide category. Compound 3a demonstrated potent antiviral activity against SARS-CoV-2 with IC50 = 2.378 µM as compared to the activity of the antiviral drug remdesivir (IC50 = 10.11 µM). Our results indicate that, upon optimization, these new triazine sulfonamides could potentially serve as novel antiviral drugs.


Introduction
Numerous antibiotics and other antimicrobials have been developed.However, the threat raised by antimicrobial resistance (AMR) is more recent and requires immediate attention [1,2].A significant increase in antibiotic resistance have been observed on a global level in the recent years.Almost seventeen million people die every year from infectious diseases, especially bacterial infections [3].Many commercially available antibiotics are considered to be ineffective for treating microorganisms that have developed resistance to them [4].Antibiotic resistance is a problem that has been related to antibiotic overuse, abuse, and a lack of new efficient drugs.Bacteria are considered major, urgent, and alarming concerns by the Centers for Disease Control and Prevention (CDC), many of which have a significant clinical and economic impact on the global population [5].Due to the rapid increase in resistance to currently accessible commercially available antibiotics, it is imperative to develop novel antibacterial treatments with increased action to combat drug-resistant conditions [6].
In addition, sulfonamides are known to be effective as antimicrobial drugs such as silver sulfadiazine drug (Fig. 1) (Flamazine, Silvadene, Ssd, Thermazene) which is considered as a topical sulfonamide antibiotic that acts on the bacterial cell wall and cell membrane; approved for treating burns [48].Another example, sulfathiazole (Fig. 1) which is a short-acting sulfa drug was a widely used oral and topical antimicrobial until less toxic alternatives were discovered.The use of it is still sporadic, occasionally in combination with sulfacetamide and sulfabenzamide [49].Another sulfonamide antibiotic called sulfamethizole (Brand Name: Urobiotic) (Fig. 1) also is used to treat a wide range of susceptible bacterial infections [50].Furthermore, sulfonamides have anti-viral characteristics that can be utilized to develop drugs against enteroviruses, coxsackievirus B, encephalomyocarditis viruses, human parainfluenza viruses, adenoviruses, Ebola virus, HIV, Marburg virus, SARS-CoV-2 among other viruses [51].

Chemistry
The reaction of the substituted sulfaguanidine 1 with N-cyanodithioiminocarbonate 2 furnished the novel analogues of the triazine sulfonamide 3. Cyclization of arylsulfonyl guanidine 1a-d with compound 2 occurs in the presence of potassium hydroxide in dioxane under reflux to afford the targeted products 3a-c (Scheme 1).The 1 H NMR spectrum of compound 3a revealed the presence of a singlet signal δ 2.29 ppm for the three protons of the methythio group, singlet at δ 7.35 for the NH 2 Protons, and in the range from δ 7.54 to δ 7.98 ppm the aromatic protons were detectable, additionally the NH protons was appeared at δ 11.83 ppm.The structure of the compound is confirmed via single X-ray diffraction analysis as depicted in Fig. 2 [60].
Further reaction of the latter compounds with sec.amines such as morpholine, piperidine, N-methyl piperazine in the presence of potassium carbonate in refluxing dioxane furnishes the substituted triazine sulfonamides 4. Additionally, the reaction of compound 3 with pyrrolidine generated compound 5.The desired compounds were characterized using spectral and elemental analysis.The 1 H NMR spectrum of compound 5a revealed the presence of four multiplet signals at δ 1.76.ppm, two multiptet signals at δ 3.2 ppm, and two multiplet signals at δ 3.34 ppm of the methylene groups of the pyrrolidine moiety.Owing to the NH 2 signal, it was appeared at δ 6.82 ppm, also the aromatic signals appeared at the range from δ 7.43 to δ 7.87 ppm, and the NH proton of the sulfonamide group was appeared at δ 11.19 ppm.In order to investigate the scope of this approach the triazine sulfonamides was reacted with aniline derivatives to afford a general methodology to the substituted triazine sulfonamides 7 (Scheme 2).

Biological activity In-vitro anti-proliferative activity
Estimation of in vitro antiproliferative activity was performed on the NCI-60 cell line panel.The US NIH's National Cancer Institute ("NCI") has selected the majority of structures for its Developmental Therapeutic Program (DTP).Various human tumor cell lines are used in screening procedures, including cell lines expressing brain, melanoma, leukemia, lung, ovarian, colon, kidney, prostate and breast malignancies.

Antimicrobial evaluation
Most of the novel compounds were estimated for their in vitro anti-bacterial efficacy against some species of Gram (− ve) bacteria, namely, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa, along with two Gram (+ ve) bacteria, namely, Staphylococcus aureus and Streptococcus mutans.Additionally, their effectiveness against the fungus Candida albicans was assessed.To estimate the preliminary anti-bacterial and anti-fungal potencies, the agar-diffusion method was utilized.
Nystatin, Ampicillin, and Gentamicin were also used as standard drugs against fungal, Gram + ve bacterial, and Gram − ve bacterial strains, respectively.The reports of the antimicrobial results were expressed as the average diameter of inhibition zones of the microbial growth around the disks in mm values, as accomplished in Table 2.The optimization of antimicrobial evaluation was performed utilizing a statistical experimental design [61][62][63].

SARS-CoV-2
The novel synthesized compound 3a was evaluated for its anti-viral potency against SARS CoV-2 virus to determine the half-maximal cytotoxic concentration (CC 50 ) and inhibitory concentration 50 (IC 50 ) (Fig. 6).The antiviral activity of the compound is identified using the MTT assay.The results revealed that compound 3a has high and potent antiviral activity against SARS-CoV-2.
The inhibition concentration (IC50) was calculated from the slope on graph pad prism for compound 3a and according to that value, the promising compound had low value can inhibit propagation of virus in the same time with low toxicity on the cell as compound 3a

Staphylococcus aureus Streptococcus mutans
Fig. 4 The antibacterial activities of compounds 3a-c, 4a-d, 5a,b, and 7b as compared with ampicillin as standard antibiotic against Gram (+ ve) bacteria had IC50 = 2.378 µM and CC50 = 577.2µM with safety index = 250.Thus, compound 3a showed potent antiviral activity against SARS-CoV-2 with IC 50 = 2.378 µM that is comparable to the activity of the antiviral drug remdesivir (IC 50 = 10.11µM) (Fig. 7).Compound 3a revealed a selectivity index (SI = (CC 50 /IC 50 ) = 250) that is much higher than the selectivity index of remdesivir as positive control (SI = 41.07).Cytotoxicity assay of compound 3a in Vero E6 cells is shown in Fig. 3.The determination of the cytotoxicity of compound 3a and remdesivir based on the dose response was performed utilizing MTT assay.The calculations of the 50% cytotoxic concentration (CC 50 ) for the compound is identified via non-linear regression analysis of GraphPad Prism software (version 5.01).The inhibitory concentration 50% (IC 50 ) values were also calculated utilizing non-linear regression analysis of GraphPad Prism software through plotting log inhibitor versus the normalized response known as the variable slope.

Conclusion
In conclusion, the synthesis of triazine sulfonamides and its analogues were achieved starting from sulfaguanidine derivatives.Our synthetic approach is expected to contribute in the provision of a wide range of triazine sulfonamide analogs starting from the crucial intermedi-

In vitro anti-proliferative activity
Primary anticancer assays were carried out in accordance with NCI procedures [64][65][66][67][68].The compounds were applied at a single concentration, and the cell culture was then incubated for 48 h.Sulforhodamine B (SRB), a protein-binding dye, was used to detect the endpoints.The compound's effects were displayed as a percentage growth (GP%) of the treated cells relative to the untreated cells in the control.The range of growth (%) displayed the maximum and lowest growth arising from the initial single high dosage (10 −5 M) sensitivity against the different cancer cell lines.

Antimicrobial activity
Using the agar well diffusion method, the synthesized compounds were separately evaluated against a panel of Gram (+ ve) and Gram (−ve) bacterial pathogens and the fungi [69].The compounds were evaluated against fungal and bacterial strains at a concentration of 15 mg/mL.In sterilized saline equivalent to 0.5 McFarland standard solution (1.5 × 10 5 cfu/ml), the microbial suspension was prepared, then the turbidity of the medium was adjusted to the optical density (OD) = 0.13 at 625 nm utilizing a spectrophotometer.A sterile cotton swab should ideally be dipped into the adjusted suspension within fifteen minutes of adjusting the turbidity of the inoculum suspension, flooded over the dried agar surface, and then allowed to dry for another 15 min.Using a sterile borer, 6 mm-diameter wells were prepared in the solidified media.Using a micropipette, 100 μL of the tested compound solution was added to each well.At 37 °C, the plates were then incubated.Measuring the zone of inhibition (mm) was carried out after 24 h incubation at 30 °C for bacterial plates and 48 h for fugal plates.The results were recorded for each tested substance as % inhibition ± SD, and the experiment was run in triplicate.The inhibition zone s' diameters were measured in millimeters.

Cytotoxicity assay SARS-CoV2
MTT cytotoxicity assay To identify the half maximum cytotoxic concentration (CC 50 ), stock solutions of the tested substances were prepared in DMSO (10% in ddH 2 O) and subsequently diluted to the employed concentrations using DMEM.By slightly altering the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) technique, the cytotoxic activity of the extracts was examined in VERO-E6 cells.Briefly, the cells were seeded in 96-well plates at a density of 3 × 10 5 cells per ml (100 µl /well) and then incubated for 24 h at 37 °C in 5% carbon dioxide.
After 24 h, the examined compounds were treated in triplicates to cells in a range of doses.The supernatant was removed twenty-four hours in advance, and cell mono-layers were then washed three times with sterile 1 × PBS before being incubated for four hours at 37 degrees Celsius with MTT solution (20 µl of a 5 mg/ml stock solution).The medium was then aspirated.
In each well, 200 µl of acidified isopropyl alcohol (0.04 M hydrochloric acid in isopropyl alcohol = 0.073 ml hydrochloric acid in 50 ml isopropyl alcohol) was used to dissolve the produced formazan crystals.Using a multiwall-plate reader, the absorbance of formazan solutions was calculated at max λ 540 nm and 620 nm.Using a plot of cytotoxicity versus sample concentration, the concentration (CC 50 ) that indicated 50% cytotoxicity was determined.
Estimation of the inhibitory Concentration 50% (IC 50) 2.4 × 10 4 Vero E6 cells were seeded onto tissue culture plates (96-well), and they were then exposed to 5% carbon dioxide at 37°C for the duration of the full night.The cell monolayers were then treated with hCoV-19/ Egypt/NRC-03/2020 (Accession No. on GSAID/ EPI ISL 430820) and allowed to remain there for an additional hour at ambient temperature.The cell monolayers were then covered with DMEM (100 μl) with various test drug doses.
The cells were then stained with 0.1% crystal violet in distilled water at ambient temperature for fifteen minutes, fixed with 100 μl polyoxymethylene (4%) for twenty minutes, and kept in a 5% carbon dioxide incubator at 37 °C for the ensuing 72 h.After being fixed with 100 μl polyoxymethylene (4%) for 20 min, the cells were stained with 0.1% crystal violet in DH 2 O at room temperature for 15 min.The crystal violet dye was then dissolved in 100 μl of methanol in each well (Anthos Labtec Instruments, Heerhugowaard, Netherlands) before the optical density of the color was determined at 570 nm using an Anthos Zenyth 200rt-plate reader.The amount of a chemical required to lower the virally-induced cytopathic effect (CPE) in contrast to virus control by 50% is known as the IC 50.

Fig. 5
Fig.5 The antifungal activities of compounds 3a-c, 4a-d, 5a,b, and 7b as compared with Nystatin as standard antibiotics against Candida albicans ate N-cyanodithioiminocarbonate.The insertion of several amines or aryl groups yielded the novel substituted triazine sulfonamides.The in vitro anti-proliferative activities, the antimicrobial activities and the antiviral activity against SARS-CoV-2 virus were evaluated.Compounds 4a, 4d & 5b showed some activities against the gram (-ve) and gram (+ ve) bacterial strains compared to Gentamicin and Ampicillin.Compounds 3a, 3c and 5a displayed potency against the fungal strain Candida albicans compared to Nystatin as standard anti-fungal drug.The anti-proliferative efficacy of the novel triazine sulfonamides was also estimated on NCI 60 cancer cell lines.Compound 3a is considered to be the most potent derivative among the estimated compounds in which it revealed remarkably lowest cell growth promotion against melanoma SK-MEL-5, CNS cancer SNB-75, and non-small cell lung cancer EKVX cell line.Additionally, the anti-viral activity against SARS CoV-2 virus was performed utilizing the MTT cytotoxicity assay.Compound 3a exhibited antiviral potency against SARS-CoV-2 with IC 50 = 2.378 µM as compared to the antiviral drug remdesivir (IC 50 = 10.11µM).This study showed promising results for developing these novel structures.Further studies concerning synthesizing other triazine sulfonamide analogs and the evaluation of their biological potency are currently in progress.ExperimentalMethodsOn the pre-coated silica gel 60 F245 aluminum plates, TLC was utilized for monitoring the reaction's development and the UV light was used for visualization.The Stuart SMP30 equipment was used to determine the melting point that was uncorrected.In the faculty of Pharmacy at the Drug Discovery, Research & Development Centre at Ain Shams University and in the National Research Center, Egypt, the spectroscopic analyses of the compounds were carried out.On Bruker Fourier 400 and 500 (operating at 400 MHz and 500 MHz, respectively) at 300 K, the NMR spectra were measured.The National Cancer Institute in Bethesda, Maryland, United States, conducted the anticancer screening.Antimicrobial evaluation carried out at the Cairo University's Faculty of Science's Microbiology Unit in the Biochemistry Central Lab, Cairo, Egypt.The Centre of Scientific Excellence for Influenza Viruses, National Research Centre (NRC), Dokki, Cairo 12622, Egypt, conducted the antiviral assays for the SARS-CoV-2 virus.

Table 1
Anti-tumor properties of the compounds at a dose of 10 μM using human tumor cell lines