Journal of Chemistry

Volume 2014 (2014), Article ID 897141, 7 pages

http://dx.doi.org/10.1155/2014/897141

Research Article

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

Mashooq Ahmad Bhat,¹ Abdul Arif Khan,² Shahanavaj Khan,² and Abdullah Al-Dhfyan³

¹Department 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

³Stem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital & Research Center, MBC-03, P. O. Box 3354, Riyadh 11211, Saudi Arabia

Received 9 April 2014; Revised 21 May 2014; Accepted 26 May 2014; Published 18 June 2014

Academic Editor: Xinyong Liu

Copyright © 2014 Mashooq Ahmad Bhat et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

New triazolothiadiazine derivatives 5a–h were synthesized from 4-amino-3-(4-pyridyl)-5-mercapto-4H-1,2,4-triazole (3) with substituted aryl hydrazonoyl chlorides 4a–h. The compounds were tested in vitro against eleven Candida species and compared with standard drug ketoconazole. Among these compounds, the compounds bearing p-chlorophenyl 5e, p-methoxyphenyl 5c, phenyl 5a, and p-sulphonamidophenyl 5g substituents on triazolothiadiazine system were found to be the most effective derivatives against Candida species. Compound 5e was the most effective compound against C. parapsilosis (ATCC 22019), C. albicans (ATCC 66027), C. specie [blood] 12810, and C. specie [urine] 300 with MIC value of 6.25 μg/mL, whereas ketoconazole exhibits the inhibitory activity with MIC value of 3–30 μg/mL against all tested strains. It was clear that there is a positive correlation between anti-Candidal activity and p-chlorophenyl substitution on triazolothiadiazine ring. All the synthesized compounds were also investigated for their potential cytotoxicity on noncancer cell line (MCF-12) using WST-1 assay. Three compounds 5d, 5a, and 5h were found to have the same IC₅₀ value as that of standard drug ketoconazole against noncancer cell line MCF-12 (IC₅₀ ≥ 1.0 × 10⁵ μg/mL).

1. Introduction

Candida species have emerged as the most common cause of systemic fungal infections worldwide over the last two decades. The most frequently implicated risk factors include treatment with broad-spectrum antibiotics, use of central venous catheters and implantable prosthetic devices, parenteral nutrition, prolonged intensive care unit stay, hemodialysis, and immunosuppression [1]. Amphotericin and azole drugs are used, for the treatment of an infection with Candida spp. [2]. The prominent drugs bearing triazole rings are fluconazole, itraconazole, voriconazole, and posaconazole, all of which are widely used antifungal drugs for the treatment of systemic fungal infection [3]. Resistance has been developed due to widespread use of these agents in recent years. As a result of this situation, medicinal chemists have focused on the research of new anti-Candidal agents [4, 5]. The mercapto and thione which substituted 1,2,4-triazole ring system have been reported to possess a variety of biological activities such as antibacterial [6], antifungal [7], antitubercular [8], anticancer [9], diuretic [10], and hypoglycemic [11]. The amino and mercapto groups are readymade nucleophilic centers for synthesis of condensed heterocyclic rings [12]. The recent literature survey revealed that 1,2,4-triazolo[3,4-b]thiadiazine derivatives have promising biological activities such as anti-HIV [13], CNS stimulant [14, 15], antifungal [16], and anti-inflammatory [17]. Studies have also confirmed that triazolothiadiazine derivatives possess anti-Candidal activity [18–24].

Pyridine ring is a prominent scaffold present in various bioactive molecules and has played a vital role in the development of different medicinal agents [25–27]. 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.

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

2. 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- on a Bruker NMR spectrophotometer operating at 500 MHz for ¹H and 125.76 MHz for ¹³C at the Research Center, College of Pharmacy, King Saud University, Saudi Arabia. Chemical shifts are expressed in δ-values (ppm) relative to TMS as an internal standard and D₂O was added to confirm the exchangeable protons. Mass spectra were measured on Agilent Triple Quadrupole 6410 QQQ LC/MS with ESI (Electrospray ionization) source.

2.1.1. 4-Amino-3-(4-pyridyl)-5-mercapto-4H-1,2,4-triazole (3)

Isonicotinic acid hydrazide (1) (13.7 g, 0.1 mol) was dissolved in absolute ethanol (200 mL) containing potassium hydroxide (11.2 g, 0.1 mol) at room temperature. To this, carbon disulfide (12.5 mL) was added in parts and the reaction mixture was stirred for 16 h at room temperature. After the reaction completion, diethyl ether (100 mL) was added and the reaction mixture was stirred for a further 3 h. After this hydrazine hydrate (99%, 10.3 g, 0.1 mol) was added gradually to the potassium dithiocarbazinate salt 2 which dissolved in water (100 mL) with stirring and the content was refluxed for 8 h, during which hydrogen sulfide gas evolved and the color of the reaction mixture changed to deep green. It was then cooled and acidified with hydrochloric acid to pH 1. The yellow-colored solid separated out and was filtered and purified by recrystallization from DMF to give compound 3 [31].

2.1.2. (7Z)-7-[2-(Aryl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5a–h)

To a mixture of 4-amino-3-(4-pyridyl)-5-mercapto-4H-1,2,4-triazole (3), (0.49 g, 2 mmol), and the appropriate hydrazonoyl chloride (4a–h) (2 mmol) in ethanol (20 mL), triethylamine (2 mmol, 0.2 mL) was added. The reaction mixture was heated under reflux for 3 h then left to cool. The precipitated solid was collected by filtration, washed with ethanol, and dried. Recrystallization of the reaction products from ethanol afforded the corresponding compounds [32].

2.1.3. (7Z)-7-[2-(Phenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5a)

IR (KBr) v max (cm⁻¹): 3180 (NH str.), 1590 (C=N str.), 1450 (C=C str.), 1220 (N–N=C str.), 750 (C–S–C str.); ¹H NMR (DMSO-, 500 MHz)  δ ppm: 2.0 (3H, s, CH₃), 7.32–7.38 (5H, m, Ar–H), 8.02 (2H, d, J = 4 Hz, pyridyl H), 8.7 (2H, d, J = 4 Hz, pyridyl H), 10.36 (1H, bs, –NH, D₂O exch.). ¹³C NMR (DMSO-, 125.76 MHz) δ ppm: 21.09 (CH₃), 115.9, 121.7, 132.8, 150.2. MS (ESI) m/z: 335.1 [M]⁺. Anal. Calc. for C₁₇H₁₃N₇S: C, 57.30; H, 3.91; N, 29.23; S, 9.56%. Found: C, 57.5; H, 3.90; N, 29.20; S, 9.53%.

2.1.4. (7Z)-7-[2-(2-Methylphenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5b)

IR (KBr) v max (cm⁻¹): 3000 (NH str.), 1600 (C=N str.), 1450 (C=C str.), 1200 (N–N=C str.), 700 (C–S–C str.); ¹H NMR (DMSO-, 500 MHz) δ ppm: 2.26 (3H, s, CH₃), 2.51 (3H, s, Ar–CH₃), 7.16–7.28 (4H, m, Ar–H), 8.10 (2H, d, J = 4 Hz, pyridyl H), 8.79 (2H, d, J = 4 Hz, pyridyl H), 10.26 (1H, bs, –NH, D₂O exch.). ¹³C NMR (DMSO-; 125.76 MHz) δ ppm: 21.1 (CH₃), 114.3, 116.3, 121.69, 129.2, 132.9, 143.6, 143.6, 147.3, 148.7, 150.1, 167.6. MS (ESI) m/z: 350.1 [M]⁺. Anal. Calc. for C₁₇H₁₅N₇S: C, 58.44; H, 4.33; N, 28.06; S, 9.18%. Found: C, 58.67; H, 4.32; N, 28.15; S, 9.17%.

2.1.5. (7Z)-7-[2-(4-Methoxyphenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5c)

IR (KBr) v max (cm⁻¹): 3180 (NH str.), 1600 (C=N str.), 1420 (C=C str.), 1220 (N–N=C str.), 750 (C–S–C str.); ¹H NMR (DMSO-; 500 MHz) δ ppm: 2.51 (3H, s, CH₃), 3.73 (3H, s, –OCH₃), 6.93 (2H, d, J = 9.0 Hz, Ar–H), 7.31 (2H, d, J = 8.5 Hz, Ar–H), 8.09 (2H, d, J = 3.5 Hz, pyridyl H), 8.79 (2H, d, J = 3.5 Hz, pyridyl H), 10.22 (1H, bs, –NH, D₂O exch.); ¹³C NMR (DMSO-, 125.76 MHz) δ ppm: 21.0 (CH₃), 55.2, 62.9, 114.5, 115.6, 121.6, 132.8, 137.2, 138.7, 148.7, 150.2, 152.4, 154.9. MS (ESI) m/z: 366.1 [M]⁺. Anal. Calc. for C₁₇H₁₅N₇SO: C, 55.88; H, 4.14; N, 26.83; S, 8.78%. Found: C, 55.68; H, 4.13; N, 26.73; S, 8.76%.

2.1.6. (7Z)-7-[2-(4-Bromophenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5d) [33]

IR (KBr) v max (cm⁻¹): 3000 (NH str.), 1560 (C=N str.) 1462 (C=C str.), 1220 (N–N=C str.), 780 (C–S–C str.); ¹H NMR (DMSO-, 500 MHz) δ ppm: 2.6 (3H, s, CH₃), 7.0–7.3 (4H, m, Ar–H), 8.0 (2H, d, J = 4 Hz, pyridyl H), 8.7 (2H, d, J = 4 Hz, pyridyl H), 10.3 (1H, bs, –NH, D₂O exch.). ¹³C NMR (DMSO-, 125.76 MHz) δ ppm: 21.11 (CH₃), 114.3, 116.4, 121.6, 122.2, 129.2, 132.8, 138.7, 143.6, 148.7, 150.2, 152.3. MS (ESI) m/z: 413.90 [M]⁺, 415.0 [M+2]⁺. Anal. Calc. for C₁₆H₁₂N₇SBr: C, 46.39; H, 2.92; N, 23.67; S, 7.74%. Found: C, 46.47; H, 2.93; N, 23.76; S, 7.73%

2.1.7. (7Z)-7-[2-(4-Chlorophenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5e)

IR (KBr) v max (cm⁻¹): 3000 (NH str.), 1600 (C=N str.), 1400 (C=C str.), 1210 (N–N=C str.), 780 (C–S–C str.); ¹H NMR (DMSO-, 500 MHz) δ ppm: 2.1 (3H, s, CH₃), 7.48 (2H, d, J = 8.5 Hz, Ar–H), 7.79 (2H, d, J = 9.0 Hz, Ar–H), 8.10 (2H, d, J = 4 Hz, pyridyl H), 8.80 (2H, d, J = 4 Hz, pyridyl H), 10.6 (1H, bs, –NH, D₂O exch.). ¹³C NMR (DMSO-, 125.76 MHz) δ ppm: 21.0 (CH₃), 113.9, 119.1, 121.5, 121.7, 132.7, 137.1, 138.5, 146.2, 147.3, 148.7, 150.1, 150.2, 152.1, 167.6. MS (ESI) m/z: 370.1 [M]⁺, 371.0 [M+1]⁺. Anal. Calc. for C₁₆H₁₂N₇SCl: C, 51.96; H, 3.27; N, 26.51; S, 8.67%. Found: C, 51.75; H, 3.26; N, 26.40; S, 8.65%.

2.1.8. (7Z)-7-[2-(4-Fluorophenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5f)

IR (KBr) v max (cm⁻¹): 3060 (NH str.), 1600 (C=N str.), 1462 (C=C str.), 1240 (N–N=C str.), 700 (C–S–C str.); ¹H NMR (DMSO-, 500 MHz) δ ppm: 2.0 (3H, s, CH₃), 8.0–8.2 (6H, m, Ar–H), 8.9 (2H, d, J = 4.0 Hz, pyridyl H), 11.1 (1H, bs, –NH, D₂O exch.). ¹³C NMR (125.76 MHz, DMSO-) δ ppm: 62.9, 116.4, 121.5, 150.1, 167.6. MS (ESI) m/z: 354.1 [M]⁺. Anal. Calc. for C₁₆H₁₂N₇SF: C, 54.38; H, 3.42; N, 27.75; S, 9.07%. Found: C, 54.18; H, 3.43; N, 27.65; S, 9.08%.

2.1.9. (7Z)-7-[2-(4-Sulphonamidophenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5g)

IR (KBr) v max (cm⁻¹): 3200 (NH str.), 600 (C=N str.), 1450 (C=C str.), 1180 (N–N=C str.), 780 (C–S–C str.). ¹H NMR (DMSO-, 500 MHz) δ ppm: 1.9 (3H, s, CH₃), 2.1 (2H, s, –NH, D₂O exch.), 7.3–7.8 (4H, m, Ar–H), 8.0 (2H, d, J = 6 Hz, pyridyl H), 8.77 (2H, d, J = 6 Hz, pyridyl H), 10.30 (1H, bs, –NH, D₂O exch.). ¹³C NMR (125.76 MHz, DMSO-) δ ppm: 21.1, 113.9, 121.7, 127.2, 132.9, 137.1, 147.3, 148.7, 150.2, 167.6. MS (ESI) m/z: 415.1 [M]⁺. Anal. Calc. for C₁₆H₁₄N₈S₂O₂: C, 46.37; H, 3.40; N, 27.04; S, 15.47%. Found: C, 46.57; H, 3.41; N, 27.15; S, 15.51%.

2.1.10. (7Z)-7-[2-(4-Methylphenyl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[]triazolo[3,4-b][]thiadiazine (5h)

IR (KBr) v max (cm⁻¹): 3100 (NH str.), 1650 (C=N str.), 1450 (C=C str.), 1200 (N–N=C str.), 700 (C–S–C str.). ¹H NMR (DMSO-, 500 MHz) δ ppm: 2.0 (3H, s, CH₃), 2.2 (3H, s, CH₃), 7.14–7.26 (4H, m, Ar–H), 8.02 (2H, s, CH, d, J = 4 Hz, pyridyl H), 8.77 (2H, d, J = 4 Hz, pyridyl H), 10.26 (1H, bs, –NH, D₂O exch.); ¹³C NMR (DMSO-; 125.76 MHz) δ ppm: 21.1 (CH₃), 114.0, 115.3, 121.1, 128.0, 131.9, 142.2, 143.4, 147.0, 147.5, 150.0, 165.2. MS (ESI) m/z: 350.1 [M]⁺. Anal. Calc. for C₁₇H₁₅N₇S: C, 58.44; H, 4.33; N, 28.06; S, 9.18%. Found: C, 58.67; H, 4.32; N, 28.15; S, 9.17%.

2.2. Anti-Candidal Evaluation

The compounds 5a –h were tested in vitro against eleven Candida spp. including C. albicans, C. tropicalis, and C. parapsilosis. Among these, C. albicans ATCC 10231, 66027, C. tropicalis ATCC 66029, and C. parapsilosis 22019 were procured from American type culture collection. Moreover, other Candida isolates (Candida sp. [HVS] 13184; Candida sp. [HVS] 11972; Candida sp. [HVS] 178; Candida sp. [urine] 300; Candida sp. [urine] 12341; Candida sp. [urine] 12485; and Candida sp. [blood] 12810) were collected from Prince Salman Hospital, Riyadh, Saudi Arabia. Isolates were derived from high vaginal swabs, blood, and urine. All isolates were grown on Sabouraud dextrose agar media and were preserved at 4°C till the use. Standard drug ketoconazole was used as positive control. Dimethyl sulfoxide (DMSO) was used as negative control. Stock solution of ketoconazole was prepared in (DMSO) (16 mg/1 mL). Other compounds were also dissolved in DMSO (100 mg/1 mL).

2.3. 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 μg/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 /well and incubated overnight. The medium was replaced with fresh one containing the desired concentrations of the compounds. After 48 h, 10 μL 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 (enzyme-linked immunosorbent assay) reader at 450 nm.

3. 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-triazolo[3,4-b]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 ¹H NMR spectra of the compounds, which were taken in DMSO-, D₂O exchangeable NH proton was seen as broad singlet at about 10.22–11.1 ppm. The pyridyl protons appear as doublets at 7.3–8.9 ppm 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 ¹³C 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].

Table 1: Some properties of the synthesized compounds 5a–h.

Compound
Ar
Yield (%)
M.p. (°C)
Molecular formula
Molecular weight (g/mol)

5a
C₆H₅
50
295–297

335.38

5b
2-CH₃-C₆H₅
60
288–290
C₁₇H₁₅N₇S
349.41

5c
4-OCH₃-C₆H₅
65
273–275
C₁₇H₁₅N₇OS
365.41

5d
4-Br-C₆H₅
80
270–272
C₁₆H₁₂BrN₇S
414.28

5e
4-Cl-C₆H₅
75
293–295
C₁₆H₁₂ClN₇S
369.83

5f
4-F-C₆H₅
80
278–280
C₁₆H₁₂FN₇S
353.37

5g
4-SO₂NH₂-C₆H₅
70
320–322
C₁₆H₁₄N₈O₂S₂
414.46

5h
4-CH₃-C₆H₅
60
290–292
C₁₇H₁₅N₇S
349.41

Scheme 1: Synthesis of compounds 5a–h.

3.2. 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 μg/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 compounds with ortho methylphenyl 5b and p-methylphenyl 5h were devoid of activity up to 100 μg/mL. The compounds with p-bromophenyl 5d and p-fluorophenyl 5f substituents exhibit inhibitory activity against Candida species at (MIC, 12.5 μg/mL). Compounds 5a, 5c, 5e, and 5 g exhibit the highest inhibitory activity against the Candida species (MIC, 6.25 μg/mL). The compound with phenyl substitution on triazolothiadiazine ring 5a was found to be active against . specie [urine] 300, C. specie [HVS] 11972, and . specie [urine] 12341. The compound with p-methoxyphenyl substituent on triazolothiadiazine ring 5c was found to be active against C. species [urine] 12485 and C. species [urine] 12341 and the compound with p-sulphonamidophenyl substituent on triazolothiadiazine ring 5g was found to be active against . species [urine] 300 with MIC value of 6.25 μg/mL, whereas ketoconazole (standard drug) exhibits the inhibitory activity with a MIC value of 3–30 μg/mL against all tested strains. The compound with p-chlorophenyl substituent on triazolothiadiazine ring 5e was found to be the most potent derivative against C. parapsilosis (ATCC 22019), C. albicans (ATCC 66027), C. specie [blood] 12810, and . specie [urine] 300. This could result from increased lipophilicity associated with chlorophenyl group. It can be also attributed to the inductive effect of the chloro substituent. The biological results indicate that Candida sp. [urine] 300 was more susceptible to compounds 5a, 5e, and 5 g. It is apparent that there is a positive correlation between anti-Candidal activity and chlorophenyl substitutions on the triazolothiadiazine ring.

Table 2: Anti-Candidal activity of the compounds (5a–h) as MIC values (g/mL).

5a
5b
5c
5d
5e
5f
5g
5h
K#

C. tropicalis ATCC 66029
25
>100
12.5
25
12.5
25
>100
>100
3.0

C. parapsilosis ATCC 22019
12.5
>100
25
12.5
6.25
6.25
25
>100
3.0

C. albicans ATCC 10231
25
>100
25
>100
>100
>100
>100
>100
30.0

C. albicans ATCC 66027
>100
>100
25
12.5
6.25
50
12.5
>100
30.0

Candida sp. [HVS*] 13184
12.5
>100
12.5
>100
>100
>100
>100
>100
7.0

Candida sp. [HVS] 11972
6.25
>100
12.5
>100
>100
>100
>100
>100
7.0

Candida sp. [HVS] 178
>100
>100
50
>100
>100
>100
>100
>100
7.0

Candida sp. [urine] 300
6.25
>100
12.5
>100
6.25
25
6.25
>100
15.0

Candida sp. [urine] 12341
6.25
>100
6.25
>100
25
12.5
25
>100
15.0

Candida sp. [urine] 12485
>100
>100
6.25
50
25
25
25
>100
15.0

Candida sp. [blood] 12810
12.5
>100
25
>100
6.25
>100
25
>100
7.0

HVS: high vaginal swab; K#: ketoconazole.

3.3. 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 IC₅₀ value as that of standard drug ketoconazole against noncancer cell line MCF-12 ( μg/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 (–SO₂NH₂), which has been reported to show cytotoxicity in literature [38].

Table 3: In vitro cytotoxicity of compounds (5a–h) on noncancer cell line (MCF-12).

Compound
IC₅₀ (µg/mL)a

5a
>1.0 × 10⁵

5b
3.0 × 10⁴

5c
6.6 × 10⁴

5d
>1.2 × 10⁵

5e
5.4 × 10⁴

5f
5.4 × 10⁴

5g
2.4 × 10⁴

5h
>1.0 × 10⁵

Gentamicin
>1.0 × 10⁵

Ketoconazole
>1.0 × 10⁵

IC₅₀: 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.

4. Conclusions

In conclusion, we focused on the synthesis of new triazolothiadiazine derivatives 5a–h, which were tested in vitro against various Candida species. Compound 5e was the most effective compound against C. parapsilosis (ATCC 22019), C. albicans (ATCC 66027), C. specie [blood] 12810, and . specie [urine] 300 with MIC value of 6.25 μg/mL compared to ketoconazole with MIC value of 3–30 μg/mL against all tested strains. This outcome confirms that p-chlorophenyl 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 (–SO₂NH₂), 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.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research group no. (RG 1435-006).

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