Synthesis of New [ 1 , 2 , 4 ] Triazolo [ 3 , 4-b ] [ 1 , 3 , 4 ] thiadiazines and Study of Their Anti-Candidal and Cytotoxic Activities

1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 2Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 3 Stem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital & Research Center, MBC-03, P. O. Box 3354, Riyadh 11211, Saudi Arabia

Pyridine ring is a prominent scaffold present in various bioactive molecules and has played a vital role in the development of different medicinal agents [25][26][27].The anti-Candidal activity of pyridine has also been reported [28,29].There are evidences that hybridization of two or more different bioactive molecules with complimentary pharmacophoric function or with different mechanism of action often renders synergistic effects [30].Encouraged by this, we designed a set of compounds by hybridizing two different pharmacophores with the aim of increasing their anti-Candidal activity.We then synthesized hybrid compounds by linking triazolothiadiazine ring system with the pyridine ring (Figure 1).The compounds were evaluated in vitro against various Candida species and investigated for their cytotoxic effects.

Material and Methods
2.1.Chemistry.All the solvents were of LR grade and were obtained from Merck.The elemental analysis of compounds was performed on the CHN Elementar (Analysensysteme GmbH, Germany) and Vario EL III (Elementar Americas Corporation).The elemental analysis (C, H, N) of compounds was found within a limit of ±0.4% of the theoretical values.The homogeneity of the compounds was checked by TLC performed on Silica gel G coated plates (Merck).Iodine chamber was used for visualization of TLC spots.The FT-IR spectra were recorded in KBr pellets on a (Spectrum BX) Perkin Elmer FT-IR spectrometer.Melting points were determined on a Gallenkamp melting point apparatus, and thermometer was uncorrected.NMR Spectra were scanned in DMSO- 6 on a Bruker NMR spectrophotometer operating at 500 MHz for 1 H and 125.76 MHz for 13 C at the Research Center, College of Pharmacy, King Saud University, Saudi Arabia.Chemical shifts are expressed in -values (ppm) relative to TMS as an internal standard and D 2 O was added to confirm the exchangeable protons.Mass spectra were measured on Agilent Triple Quadrupole 6410 QQQ LC/MS with ESI (Electrospray ionization) source.

Cytotoxicity Evaluation.
The MCF-12 cell line is a nontumorigenic epithelial cell line established from tissue taken at reduction mammoplasty from a nulliparous patient with fibrocystic breast disease that contained focal areas of intraductal hyperplasia.MCF-12 cells were cultured in DMEM/F-12 (Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12), GIBCO, and were supplemented with 10 g/mL insulin (Sigma), 500 ng/mL hydrocortisone (Sigma), 10% fetal bovine serum (Lonza), 10 ng/mL EGF (Epidermal growth factor), BD Biosciences, and 1% ABM (Autologous Bone Marrow), GIBCO.Cells were seeded into 96-well plates at 0.6 × 10 4 /well and incubated overnight.The medium was replaced with fresh one containing the desired concentrations of the compounds.After 48 h, 10 L of the WST-1 (water soluble tetrazolium salts) reagent was added to each well and the plates were reincubated for 4 h at 37 ∘ C. The amount of formazan was quantified using ELISA (enzymelinked immunosorbent assay) reader at 450 nm.

Results and Discussion
3.1.Synthesis.Treatment of isonicotinic hydrazide (1) with carbon disulfide, in the presence of potassium hydroxide, afforded the potassium salt of hydrazinecarbodithioate 2. Furthermore, treatment of the salt 2 with hydrazine hydrate in aqueous ethanol afforded the corresponding 1,2,4-triazole-5-thione derivative 3 [30].Reaction of the compound 3 with substituted hydrazonoyl chlorides 4a-h in refluxing ethanol, in the presence of triethylamine, afforded compounds identified as (7Z)-7-[2-(aryl)hydrazinylidene]-6-methyl-3-(pyridine-4-yl)-7H- [1,2,4]triazolo [3,4-b][1, 3, 4]thiadiazines (5a-h).These reactions are summarized in Scheme 1.The purity of the compounds was checked by TLC and elemental analysis.The compounds of the series were identified by spectral data.The physiochemical properties of all compounds are given in (Table 1).In the IR spectra of all compounds, NH, C=N, and C=C bands were observed at 3200-3000 cm −1 , 1650-1560 cm −1 , and 1462-1400 cm −1 , respectively.In the 1 H NMR spectra of the compounds, which were taken in DMSO- 6 , D 2 O exchangeable NH proton was seen as broad singlet at about 10.22-11.1 ppm.The pyridyl protons appear as doublets at 7.3-8.9ppm and 8.0-9.6 ppm with  value of 4 Hz each.The aromatic protons appear as multiplet at about 6.9-8.2 ppm.The signal due to methyl protons, present in all compounds, appeared at 2.0-2.6 ppm, as singlet.Mass spectra (ESI) of the compounds showed molecular ion peaks [M] + , in agreement with their molecular formula.In the 13 C NMR spectra of all compounds 5a-h, the peaks belonging to triazolothiadiazine carbons appeared at 140-160 ppm.
All other aromatic and aliphatic carbons were observed at expected regions.All compounds gave satisfactory elemental analysis.The structures of the compounds were in complete agreement with the previously reported products of similar reactions [34,35].

Anti-Candidal Activity.
All compounds were screened for their in vitro anti-Candidal activity against eleven strains of Candida species (Table 2).Minimum inhibitory concentration (MIC) for Candida isolates was determined by microdilution method as per the protocol of CLSI (M27-A3 CLSI 2008) [36].Compound A (MIC 20-370 g/mL against ketoconazole) was chosen as the lead compound for further modification [37].Replacement of cyclohexylethyl moiety by pyridine ring, addition of substituted phenylhydrazine moiety at 6th position of 1,3,4-thiadiazine, and preserving the pharmacophore (triazolothiadiazine ring) were studied.The

Cytotoxicity.
From a pharmacological point of view, it is important for the studied compounds to exhibit high bioactivity and at the same time show no or low cytotoxicity effects.All the compounds were evaluated against noncancer cell line, MCF-12 (nontumorigenic epithelial cell line) for their cytotoxic properties using WST-1 assay.The biological study indicated that compound 5g with p-sulfonamido phenyl substitution possessed the highest cytotoxicity, whereas compound 5d with p-bromophenyl substitution exhibited the lowest cytotoxicity against MCF-12 cells among the title compounds (Table 3).Three compounds 5d, 5a, and 5h were found to have same IC 50 value as that of standard drug ketoconazole against noncancer cell line MCF-12 (IC 50 ≥ 1.0 × 10 5 g/mL).The most potent compound 5e against Candidal spp. was found to have medium cytotoxicity in comparison to standard drug.The high cytotoxicity of compound 5d may be because of the sulfonamido group (-SO 2 NH 2 ), which has been reported to show cytotoxicity in literature [38].

Conclusions
In conclusion, we focused on the synthesis of new triazolothiadiazine derivatives 5a-h, which were tested in vitro against various Candida species.substituent on triazolthiadiazine ring may have a considerable influence on anti-Candidal activity.The cytotoxic effects of the compounds were also investigated.Compound 5g possessed the highest cytotoxicity, because of the sulfonamido group (-SO 2 NH 2 ), whereas compounds 5d, 5a, and 5h exhibited the lowest cytotoxicity against MCF-12 cells.Based on the reported results, 5e derivative could also be used in association with azole derivatives to enhance their antifungal activity.Moreover, compound 5e displaying a better activity against Candida spp.represents a good template for the development of novel broad-spectrum anti-Candida spp.agents.

Figure 1 :
Figure 1: Structure of the lead compound A and new synthesized compounds 5a-h.

Table 1 :
Some properties of the synthesized compounds 5a-h.
inductive effect of the chloro substituent.The biological results indicate that Candida sp.[urine] 300 was more susceptible to compounds 5a, 5e, and 5g.It is apparent that there is a positive correlation between anti-Candidal activity and chlorophenyl substitutions on the triazolothiadiazine ring.

Table 3 :
In vitro cytotoxicity of compounds (5a-h) on noncancer cell line (MCF-12).IC 50 : concentration of the compound (g/mL) producing 50% cell growth inhibition after 48 h of compound exposure, as determined by the WST-1 assay.Each experiment was run at least three times, and the results are presented as average values. a