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

Authors: Mashooq Ahmad Bhat; Abdul Arif Khan; Shahanavaj Khan; Abdullah Al-Dhfyan
Journal: Journal of Chemistry
 (2014)

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## Abstract

New triazolothiadiazine derivatives5a–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).


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## Body

## 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 withCandida 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][
1,3
,
4
]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 evaluatedin vitroagainst variousCandida species and investigated for their cytotoxic effects.Figure 1
Structure of the lead compound A and new synthesized compounds5a–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-d
6 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 salt2 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 compound3 [31].


#### 2.1.2. (7Z)-7-[2-(Aryl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6; 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6; 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6) 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6) 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6; 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 compounds5a–h were testedin vitro against elevenCandida spp. includingC. albicans, C. tropicalis, andC. parapsilosis. Among these,C. albicans ATCC 10231, 66027, C. tropicalis ATCC 66029, and C. parapsilosis 22019 were procured from American type culture collection. Moreover, otherCandida 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 10μg/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 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.


## 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-d
6 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 salt2 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 compound3 [31].


### 2.1.2. (7Z)-7-[2-(Aryl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6; 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6; 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6) 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6) 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6; 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%.


## 2.1.1. 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 salt2 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 compound3 [31].


## 2.1.2. (7Z)-7-[2-(Aryl)hydrazinylidene]-6-methyl-3-(pyridin-4-yl)-7H-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6; 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6; 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6, 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6) 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6) 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-[1,2
,
4]triazolo[3,4-b][1,3
,
4]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-d
6, 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-d
6; 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 compounds5a–h were testedin vitro against elevenCandida spp. includingC. albicans, C. tropicalis, andC. parapsilosis. Among these,C. albicans ATCC 10231, 66027, C. tropicalis ATCC 66029, and C. parapsilosis 22019 were procured from American type culture collection. Moreover, otherCandida 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 10μg/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 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 hydrazinecarbodithioate2. Furthermore, treatment of the salt2 with hydrazine hydrate in aqueous ethanol afforded the corresponding 1,2,4-triazole-5-thione derivative3[30]. Reaction of the compound3 with substituted hydrazonoyl chlorides4a–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 1H NMR spectra of the compounds, which were taken in DMSO-d
6, 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 J 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 compounds5a–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 compounds5a–h.
Compound
Ar
Yield (%)
M.p. (°C)
Molecular formula
Molecular weight (g/mol)
5a
C6H5
50
295–297
C
16
H
13
N
7
S
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
349.41Scheme 1
Synthesis of compounds5a–h.


### 3.2. Anti-Candidal Activity

All compounds were screened for theirin vitro anti-Candidal activity against eleven strains ofCandida species (Table 2). Minimum inhibitory concentration (MIC) forCandida 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 methylphenyl5b andp-methylphenyl5h were devoid of activity up to 100 μg/mL. The compounds withp-bromophenyl5dandp-fluorophenyl5f substituents exhibit inhibitory activity againstCandida species at (MIC, 12.5 μg/mL). Compounds5a,5c,5e, and5 gexhibit the highest inhibitory activity against theCandida species (MIC, 6.25 μg/mL). The compound with phenyl substitution on triazolothiadiazine ring5awas found to be active against C
. specie [urine] 300,C. specie [HVS] 11972, and C
. specie [urine] 12341. The compound with p-methoxyphenyl substituent on triazolothiadiazine ring5c was found to be active against C. species [urine] 12485 andC. species [urine] 12341 and the compound with p-sulphonamidophenyl substituent on triazolothiadiazine ring5g was found to be active against C
. 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 withp-chlorophenyl substituent on triazolothiadiazine ring5e was found to be the most potent derivative againstC. parapsilosis (ATCC 22019),C. albicans (ATCC 66027),C. specie [blood] 12810, and C
. 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 thatCandidasp. [urine] 300 was more susceptible to compounds5a,5e, and5 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 (μg/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
Candidasp. [HVS*] 13184
12.5
>100
12.5
>100
>100
>100
>100
>100
7.0
Candidasp. [HVS] 11972
6.25
>100
12.5
>100
>100
>100
>100
>100
7.0
Candidasp. [HVS] 178
>100
>100
50
>100
>100
>100
>100
>100
7.0
Candidasp. [urine] 300
6.25
>100
12.5
>100
6.25
25
6.25
>100
15.0
Candidasp. [urine] 12341
6.25
>100
6.25
>100
25
12.5
25
>100
15.0
Candidasp. [urine] 12485
>100
>100
6.25
50
25
25
25
>100
15.0
Candidasp. [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 compound5g withp-sulfonamido phenyl substitution possessed the highest cytotoxicity, whereas compound5d withp
-bromophenyl substitution exhibited the lowest cytotoxicity against MCF-12 cells among the title compounds (Table 3). Three compounds5d,5a, and5h were found to have same IC50 value as that of standard drug ketoconazole against noncancer cell line MCF-12 (IC
50
≥
1.0
×
10
5 μg/mL). The most potent compound5e againstCandidal spp. was found to have medium cytotoxicity in comparison to standard drug. The high cytotoxicity of compound5d 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
aIC50: concentration of the compound (μg/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.


## 3.1. Synthesis

Treatment of isonicotinic hydrazide (1) with carbon disulfide, in the presence of potassium hydroxide, afforded the potassium salt of hydrazinecarbodithioate2. Furthermore, treatment of the salt2 with hydrazine hydrate in aqueous ethanol afforded the corresponding 1,2,4-triazole-5-thione derivative3[30]. Reaction of the compound3 with substituted hydrazonoyl chlorides4a–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 1H NMR spectra of the compounds, which were taken in DMSO-d
6, 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 J 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 compounds5a–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 compounds5a–h.
Compound
Ar
Yield (%)
M.p. (°C)
Molecular formula
Molecular weight (g/mol)
5a
C6H5
50
295–297
C
16
H
13
N
7
S
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
349.41Scheme 1
Synthesis of compounds5a–h.


## 3.2. Anti-Candidal Activity

All compounds were screened for theirin vitro anti-Candidal activity against eleven strains ofCandida species (Table 2). Minimum inhibitory concentration (MIC) forCandida 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 methylphenyl5b andp-methylphenyl5h were devoid of activity up to 100 μg/mL. The compounds withp-bromophenyl5dandp-fluorophenyl5f substituents exhibit inhibitory activity againstCandida species at (MIC, 12.5 μg/mL). Compounds5a,5c,5e, and5 gexhibit the highest inhibitory activity against theCandida species (MIC, 6.25 μg/mL). The compound with phenyl substitution on triazolothiadiazine ring5awas found to be active against C
. specie [urine] 300,C. specie [HVS] 11972, and C
. specie [urine] 12341. The compound with p-methoxyphenyl substituent on triazolothiadiazine ring5c was found to be active against C. species [urine] 12485 andC. species [urine] 12341 and the compound with p-sulphonamidophenyl substituent on triazolothiadiazine ring5g was found to be active against C
. 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 withp-chlorophenyl substituent on triazolothiadiazine ring5e was found to be the most potent derivative againstC. parapsilosis (ATCC 22019),C. albicans (ATCC 66027),C. specie [blood] 12810, and C
. 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 thatCandidasp. [urine] 300 was more susceptible to compounds5a,5e, and5 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 (μg/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
Candidasp. [HVS*] 13184
12.5
>100
12.5
>100
>100
>100
>100
>100
7.0
Candidasp. [HVS] 11972
6.25
>100
12.5
>100
>100
>100
>100
>100
7.0
Candidasp. [HVS] 178
>100
>100
50
>100
>100
>100
>100
>100
7.0
Candidasp. [urine] 300
6.25
>100
12.5
>100
6.25
25
6.25
>100
15.0
Candidasp. [urine] 12341
6.25
>100
6.25
>100
25
12.5
25
>100
15.0
Candidasp. [urine] 12485
>100
>100
6.25
50
25
25
25
>100
15.0
Candidasp. [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 compound5g withp-sulfonamido phenyl substitution possessed the highest cytotoxicity, whereas compound5d withp
-bromophenyl substitution exhibited the lowest cytotoxicity against MCF-12 cells among the title compounds (Table 3). Three compounds5d,5a, and5h were found to have same IC50 value as that of standard drug ketoconazole against noncancer cell line MCF-12 (IC
50
≥
1.0
×
10
5 μg/mL). The most potent compound5e againstCandidal spp. was found to have medium cytotoxicity in comparison to standard drug. The high cytotoxicity of compound5d 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
aIC50: concentration of the compound (μg/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 derivatives5a–h, which were testedin vitro against variousCandida species. Compound5e was the most effective compound againstC. parapsilosis (ATCC 22019),C. albicans (ATCC 66027),C. specie [blood] 12810, and C
. 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 thatp-chlorophenyl substituent on triazolthiadiazine ring may have a considerable influence on anti-Candidal activity. The cytotoxic effects of the compounds were also investigated. Compound5g possessed the highest cytotoxicity, because of the sulfonamido group (–SO2NH2), whereas compounds5d, 5a, and5h 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, compound5e displaying a better activity againstCandida spp. represents a good template for the development of novel broad-spectrum anti-Candida spp. agents.



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*Source: 897141-2014-06-18.xml*