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Synthesis of novel fluorinated benzothiazol-2-yl-1,2,4-triazoles: Molecular docking, antifungal evaluation and in silico evaluation for SAR
⁎Corresponding author. anjalisidhu@pau.edu (Anjali Sidhu)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Lead hybridization concept was used to design and synthesize twenty novel hybrid compounds by combining fungicidal leads viz. 6-flouro-1,3-benzothiazol-2-amine and 1,2,4-triazoles in a single molecule, with the aim of discovery of high potential novel fungicides. Antifungal evaluation of synthesized 6-flourobenzothiazol-2-yl-1,2,4-triazoles against various phytopathogenic fungi revealed synergistic effect of combination of leads with one another in all the test compounds. Some of the synthesized compounds showed excellent fungitoxicity comparable with the standard fungicides used. In silico toxicity of all the compounds was equivalent to the standard fungicides used. Docking studies and Lipinski filtration were performed in order to present the rationale of structure activity relation. Compounds 2, 8, 15 and 18 were screened to act as leads for further modification and use.
Keywords
6-Flourobenzothiazol-2-yl-1,2,4-triazoles
Lipinski filtration
Toxtree analysis
Antifungal activity
14α-Demethylase
Docking studies
1 Introduction
The fungal kingdom encompasses an enormous diversity of taxa with varied ecological niches, life-cycle strategies and morphologies. Attack by these fungal pathogens causes extensive losses in crops (Oerke and Dehne, 2004) annually during harvesting and processing which occurs in almost all regions of the world. Chemical control strategies by combination of fungicides (Fravel, 2005) have long been recognized to eradicate the fungal attacks especially at the time of sudden and severe outbreaks but still further improvement is needed for simple and cost effective process for their large scale manufacturing. Incompatibility of different fungicides (Jyothsna et al., 2013) with each other generally leads to their physical inactivation after mixing. An alternate strategy to combat this problem is putting different leads in a single molecule i.e. the lead hybridization. It is a pertinent concept, missing in the literature, may result in the development of novel molecules with multisite mode of action, lower dose of usage, use of same molecule at different stage of growth, high bioavailability and low or zero resistance.
Triazoles, may be considered as a bioisostere of imidazole (Rezaei et al., 2009), have received overwhelming attention (Gaikwad et al., 2012) during the last few decades as fungicides (Wu et al., 2001) due to their well known inhibition effect on the cytochrome P450-dependent lanosterol 14α-demethylase (CYP51) which inhibits the sterol biosynthesis of fungal cell membrane (Chai et al., 2011). These compounds have become the most rapidly expanding group of antifungals with the advantages of low toxicity (Kumamoto et al., 1990), high oral bioavailability (Scholz et al., 2009), and broad spectrum of activity (Torres et al., 2005) against several fungi. They have also been extensively derivatized (Bentiss et al., 1999) and used on commercial scale (Hof, 2008), on patent scenario (Koparir et al., 2004), and in the reported literature (Naresh et al., 2013) but derivatization of 1,2,4-triazoles is still a demanding area of research. Thus, the triazole class of antifungal agents currently plays a leading role in the field of development. On the other hand, benzothiazoles have already been reported in the literature as a versatile scaffold with well known potent inhibitor of various enzymes (Fajkusova et al., 2012). Its nucleus constitutes the active part of several biologically active compounds, including antifungal (Huang and Yang, 2006), antibacterial (Abbs Fen Rejia and Rajasekharan, 2010), antiparasitic (Mpamhanga et al., 2009), antimicrobial (Alang et al., 2010), anticancer (Kini et al., 2012), antitubercular (Telvekar et al., 2012), analgesic (Praveen et al., 2012), antitumor (Hutchinson et al., 2002), antihelmintic (Rana et al., 2007), anti-inflammatory (Ban et al., 1998), anti-oxidant (Cressier et al., 2009) and also part of commercially available fungicides (Lee and Karve, 1995), herbicides (Li et al., 2004), plant desiccants and defoliant (Imramovsky et al., 2014) compounds.
Most of the novel bioactive molecules fail to reach commercialization owing to their poor bioavailability. Introduction of fluorine in the molecule improves its lipophilicity (Xu et al., 2014) and suppresses the metabolic detoxification by increasing the in vivo lifetime of molecules and increases tissue bioavailability (Chinoy et al., 1995). So the demand and share of fluorinated compounds in agriculture (El-Zemity et al., 2006) is increasing.
Continuing our efforts directed toward synthesis of novel hybrid compounds having potent systemic effect with broad spectrum of fungicidal activity, we have synthesized a series of fluorinated benzothiazol-2-yl-1,2,4-triazoles. The compounds were checked for their in silico toxicity using Toxtree software program. The docking studies of the compounds were performed in the active site of lanosterol 14α-demethylase using ArgusLab 4.0.1 (Xanthopoulou et al., 2009) program and Lipinski characters (Kaki et al., 2012) were evaluated to carry out structure–activity relationship (SAR) of the synthesized compounds.
2 Materials and methods
2.1 Chemistry
Melting points were determined in open capillary tubes and are uncorrected. The products were splendidly purified and the purity of all the compounds was checked on a silica gel-G plates and visualization was done using iodine lamp. All solvents and reagents were purchased from Sigma–Aldrich Company. Solvents used were of analytical reagent grade and purified by simple distillation. Elemental analyses were done on Vario EL, CHNOS elemental analyzer. The IR spectra were recorded on a Perkin Elmer FT-IR spectrometer using KBr disk. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance II 400 MHz spectrometer in CDCl3 and DMSO-d6 using TMS as an internal standard. Mass spectra were recorded on a Perkin Elmer Clarus 500 Mass Spectrometer.
2.1.1 Synthesis of 1
4-Fluoroaniline (0.1 mol) and potassium thiocyanate (0.4 mol) were dissolved in glacial acetic acid (40 ml) and cooled. Bromine (0.04 mol) mixed with glacial acetic acid (24 ml) was added from dropping funnel at such a rate that temperature does not rise beyond 5–6 °C. After all bromine had been added, the solution was stirred with glass rod for an additional 2 h at low temperature. The mixture was allowed to stand overnight, during which period orange–yellow precipitates get settled at the bottom. Water was added and slurry formed was heated at 85 °C on steam bath for 20 min and filtered hot. The filtrate was cooled and neutralized with ammonia solution to pH 6. Off white precipitates obtained were collected, washed with water and recrystallized from benzene to get crystals. IR (KBr): 3452, 3080, 3010, 1350, 1215, 900 cm−1; 1H NMR (DMSO, 400 MHz): δ 5.4 (2H, s, NH2), δ 6.5–7.4 (3H, m, Ar C—H); 13C NMR (DMSO, 400 MHz): δ 108.0, 113.9, 117.8, 131.6, 148.8, 158.5, 166.3.
2.1.2 Synthesis of 2
To equimolar mixture of 6-fluoro-1,3-benzothiazol-2-amine and pyridine in methanol, chloroacetyl chloride was added dropwise with constant stirring at 10–15 °C. The stirring was continued for 2–3 h in the same cooling condition and then for 1 h at the room temperature. On completion of the reaction, the reaction mixture was poured into cold water. The resulted precipitates were filtered, dried and recrystallized from methanol. After that 4-amino-1,2,4-triazole was allowed to react with the precipitates collected and the reaction mixture was refluxed for 2–3 h at 80–85 °C and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford a yellow solid. IR (KBr): 3349, 3048, 1639, 1482, 1362, 1215, 918, 719; 1H NMR (CDCl3, 400 MHz): δ 6.5–7.4 (3H, m, Ar C—H), δ 12.49 (1H, s, NH), δ 3.9–5.1 (2H, s, CH2), δ 7.5–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 52.4, 108.0, 113.9, 117.8, 131.6, 145.3, 145.9, 148.8, 158.5, 168.5, 174.5.
2.1.3 Synthesis of 3
To equimolar mixture of 4-amino-1,2,4-triazole and pyridine in methanol, chloroacetyl chloride was added dropwise with constant stirring at 10–15 °C. The stirring was continued for 2–3 h in the same cooling condition and then for 1 h at the room temperature. On completion of the reaction, the reaction mixture was poured into cold water. The resulted precipitates were filtered, dried and recrystallized from methanol. After that 6-fluoro-1,3-benzothiazol-2-amine was allowed to react with the precipitates collected and the reaction mixture was refluxed for 2–3 h at 80–85 °C and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford a dark yellow solid. IR (KBr): 3345, 3041, 1432, 1639, 1362, 1215, 918, 822, 719; 1H NMR (CDCl3, 400 MHz): δ 6.5–7.4 (3H, m, Ar C—H), δ 3–4 (1H, t, NH), δ 3.2–4.1 (2H, d, CH2), δ 7.5–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 57.0, 108.0, 113.9, 117.8, 131.6, 144, 144.8, 148.8, 158.5, 173, 174.5.
2.1.4 Synthesis of 4
6-Fluoro-1,3-benzothiazol-2-amine (0.168 g) was allowed to react with thionyl chloride (0.0720 ml) in the presence of a base under 15–20 °C. Then 4-amino-1,2,4-triazole (0.084 g) was added to the reaction mixture which was refluxed for 2–3 h and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford yellow solid. IR (KBr): 3349, 3048, 1479, 1201, 918, 866, 719, 674; 1H NMR (CDCl3, 400 MHz): δ 6.5–7.4 (3H, m, Ar C—H), δ 3–4 (1H, t, NH), δ 7.5–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 108.0, 113.9, 117.8, 131.6, 144.2, 144.8, 148.8, 158.5, 174.5.
2.1.5 Synthesis of 5
6-Fluoro-1,3-benzothiazol-2-amine (0.168 g) was allowed to react with 1-bromo-3-chloro-propane (0.090 ml) in the presence of Zinc dust in THF at 10–15 °C. Then 4-amino-1,2,4-triazole (0.084 g) was added to the reaction mixture which was refluxed for 2–3 h and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford off-white solid. IR (KBr): 3349, 3048, 2801, 1639, 1484, 1178, 1122, 918, 717, 677; 1H NMR (CDCl3, 400 MHz): δ 6.5–7.4 (3H, m, Ar C—H), δ 7.5–8.4 (1H, t, NH), δ 1.4–4.2 (2H, m, CH2), δ 7.5–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 26.4, 41.8, 46.2, 108.0, 113.9, 117.8, 131.6, 145.1, 145.9, 148.8, 158.5, 174.5.
2.1.6 Synthesis of 6
To equimolar mixture of 6-fluoro-1,3-benzothiazol-2-amine and pyridine in methanol, chloroacetyl chloride was added dropwise with constant stirring at 10–15 °C. The stirring was continued for 2–3 h in the same cooling condition and then for 1 h at the room temperature. On completion of the reaction, the reaction mixture was poured into cold water. The resulted precipitates were filtered, dried and recrystallized from methanol. After that 1,2,4-triazole was allowed to react with the precipitates collected and the reaction mixture was refluxed for 2–3 h at 80–85 °C and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford off white solid. IR (KBr): 3349, 3048, 1639, 1482, 1362, 1215, 918, 719; 1H NMR (CDCl3, 400 MHz): δ 6.5–7.4 (3H, m, Ar C—H), δ 12.49 (1H, s, NH), δ 4.4–5.1 (2H, s, CH2), δ 7.9–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 55.5, 108.0, 113.9, 117.8, 131.6, 143.9, 148.8, 151.2, 158.5, 168.2, 174.5.
2.1.7 Synthesis of 7
To equimolar mixture of 1,2,4-triazole and pyridine in methanol, chloroacetyl chloride was added dropwise with constant stirring at 10–15 °C. The stirring was continued for 2–3 h in the same cooling condition and then for 1 h at the room temperature. On completion of the reaction, the reaction mixture was poured into cold water. The resulted precipitates were filtered, dried and recrystallized from methanol. After that 6-fluoro-1,3-benzothiazol-2-amine was allowed to react with the precipitates collected and the reaction mixture was refluxed for 2–3 h at 80–85 °C and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford off white solid. IR (KBr): 3345, 3041, 1432, 1639, 1362, 1215, 918, 822, 719; 1H NMR (CDCl3, 400 MHz): δ 7.0–7.7 (3H, m, Ar C—H), δ 3.8–4.0 (2H, t, NH), δ 4.1–4.3 (2H, d, CH2), δ 8.1–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 50.3, 108.0, 113.9, 117.8, 131.6, 143.0, 148.8, 152.8, 158.5, 174.5, 200.
2.1.8 Synthesis of 8
6-Fluoro-1,3-benzothiazol-2-amine (0.168 g) was allowed to react with thionyl chloride (0.0720 ml) in the presence of a base under 15–20 °C. Then 1,2,4-triazole (0.069 g) was added to the reaction mixture and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford yellow solid. IR (KBr): 3349, 3048, 1479, 1201, 918, 866, 719, 674; 1H NMR (CDCl3, 400 MHz): δ 6.5–7.4 (3H, m, Ar C—H), δ 3–4 (1H, s, NH), δ 7.5–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 108.0, 113.9, 117.8, 131.6, 146.8, 148.8, 155, 158.5, 174.5.
2.1.9 Synthesis of 9
6-Fluoro-1,3-benzothiazol-2-amine (0.168 g) was allowed to react with 1-bromo-3-chloro-propane (0.090 ml) in the presence of Zinc dust in THF at 10–15 °C. Then 1,2,4-triazole (0.069 g) was added to the reaction mixture which was refluxed for 2–3 h and the resulting solid which separated out, washed with water, dried and recrystallized from benzene to afford off-white solid. IR (KBr): 3349, 3048, 2801, 1639, 1484, 1122, 918, 717, 677; 1H NMR (CDCl3, 400 MHz): δ 6.5–7.4 (3H, m, Ar C—H), δ 7.5–8.4 (1H, t, NH), δ 2.4–4.2 (2H, m, CH2), δ 7.5–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 29.2, 41.8, 46.2, 108.0, 113.9, 117.8, 131.6, 142.9, 148.8, 151, 158.5, 174.5.
2.1.10 Synthesis of 10
To a stirring solution of compound 2 in methanol, 6-fluoro-1,3-benzothiazol-2-amine (0.168 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of the reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford light yellow product. IR (KBr): 3349, 3048, 1621, 1484, 1178, 918, 717, 677; 1H NMR (CDCl3, 400 MHz): δ 7.0–7.9 (3H, m, Ar C—H), δ 3.4–4 (1H, s, NH), δ 1.3–2 (2H, d, CH2), δ 8.0–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 52.8, 107.3, 108, 113.1, 113.9, 117.3, 123.3, 131.9, 136.6, 144.1, 145.3, 145.9, 148.4, 158.3, 158.8, 164.3, 174.3, 174.9.
2.1.11 Synthesis of 11
To a stirring solution of compound 2 in methanol, 4-amino-1,2,4-triazole (0.084 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of the reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford light yellow product. IR (KBr): 3351, 3044, 1619, 1487, 1171, 920, 719, 675; 1H NMR (CDCl3, 400 MHz): δ 7.0–7.8 (3H, m, Ar C—H), δ 3.8–4 (1H, s, NH), δ 2.2–2.7 (2H, s, CH2), δ 1.8–2.1 (1H, m, NH), δ 8.2–8.9 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 50.8, 108.1, 113.3, 117.1, 131.0, 145, 145.9, 147.8, 148.1, 148.8, 158.8, 164.1, 175.
2.1.12 Synthesis of 12
To a stirring solution of compound 2 in methanol, 4-fluoro-aniline (0.09587 ml) along 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the dark green product. IR (KBr): 3361, 3044, 1620, 1480, 1190, 914, 717, 679; 1H NMR (CDCl3, 400 MHz): δ 7.2–7.9 (3H, m, Ar C—H), δ 3.8–4 (1H, s, NH), δ 2.7–3.0 (2H, m, CH2), δ 1.8–2.0 (1H, t, NH), δ 8.0–8.5 (1H, s, triazole) δ 7–7.5 (4H, m, Ar C—H); 13C NMR (CDCl3, 400 MHz): δ 53.1, 107.9, 113.4, 116.2, 116.7, 117.7, 123.3, 123.8, 131.1, 145.5, 145.9, 147.6, 148.2, 158, 161.1, 163.8, 174.4.
2.1.13 Synthesis of 13
To stirring solution of compound 3 in methanol, 6-fluoro-1,3-benzothiazol-2-amine (0.168 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the yellow product. IR (KBr): 3351, 3041, 1619, 1484, 1174, 918, 719, 674; 1H NMR (CDCl3, 400 MHz): δ 7.0–7.4 (3H, m, Ar C—H), δ 3.8–4 (1H, t, NH), δ 3.0–3.5 (2H, m, CH2), δ 1.8–2.0 (1H, t, NH), δ 7.8–8.5 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 46.7, 107.3, 108, 113.4, 113.9, 117.7, 123.3, 131.1, 136.4, 144.3, 144.1, 144.7, 148.9, 158, 158.4, 163.8, 174, 174.5.
2.1.14 Synthesis of 14
To stirring solution of compound 3 in methanol, 4-amino-1,2,4-triazole (0.08408 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the off white product. IR (KBr): 3354, 3051, 1622, 1480, 1190, 914, 719, 674; 1H NMR (CDCl3, 400 MHz): δ 7.2–7.8 (3H, m, Ar C—H), δ 3.7–4 (1H, m, NH), δ 2.9–3.5 (2H, m, CH2), δ 1.8–2.1 (1H, s, NH), δ 7.5–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 44.7, 107.9, 113.4, 117.7, 131.1, 144.3, 144.4, 148.1, 148.5, 149, 158, 163.8, 174.4.
2.1.15 Synthesis of 15
To stirring solution of compound 3 in methanol, 4-fluoro-aniline (0.09587 ml) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the off white product. IR (KBr): 3351, 3023, 1618, 1480, 1190, 914, 719, 671; 1H NMR (CDCl3, 400 MHz): δ 7.3–7.9 (3H, m, Ar C—H), δ 3.6–4 (1H, m, NH), δ 2.9–3.2 (2H, m, CH2), δ 1.7–2.0 (1H, s, NH), δ 7.5–8.6 (1H, s, triazole), δ 7–7.5 (4H, m, Ar C—H); 13C NMR (CDCl3, 400 MHz): δ 47.1, 108.1, 113.7, 116.3, 116.8, 117.9, 123.2, 123.8, 131.1, 144.5, 144.9, 147.5, 148.7, 158, 161.1, 163.9, 174.4.
2.1.16 Synthesis of 16
To a stirring solution of compound 6 in methanol, 6-fluoro-1,3-benzothiazol-2-amine (0.168 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of the reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford light yellow product. IR (KBr): 3349, 3048, 1621, 1484, 918, 717, 677; 1H NMR (CDCl3, 400 MHz): δ 7.2–7.9 (3H, m, Ar C—H), δ 3.4–4 (1H, s, NH), δ 3.5–3.9 (2H, s, CH2), δ 8.0–8.6 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 50.3, 107.6, 108, 113.1, 113.6, 117.3, 123.4, 131.9, 136.6, 143.3, 144.9, 148.4, 151.1, 158.5, 158.9, 164.3, 174.3, 174.9.
2.1.17 Synthesis of 17
To a stirring solution of compound 6 in methanol, 4-amino-1,2,4-triazole (0.084 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of the reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford light yellow product. IR (KBr): 3351, 3044, 1619, 1487, 920, 719, 675; 1H NMR (CDCl3, 400 MHz): δ 7.0–7.8 (3H, m, Ar C—H), δ 3.8–4 (1H, s, NH), δ 3.4–3.9 (2H, s, CH2), δ 8.2–8.4 (1H, s, triazole), δ 8.5–9.0 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 47.8, 108.5, 113.6, 117.3, 131.0, 143.9, 147.8, 148.1, 148.8, 151.1, 158.8, 164.1, 174.4.
2.1.18 Synthesis of 18
To a stirring solution of compound 6 in methanol, 4-fluoro-aniline (0.09587 ml) along 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the dark green product. IR (KBr): 3361, 3044, 1620, 1480, 914, 717, 679; 1H NMR (CDCl3, 400 MHz): δ 7.2–7.9 (3H, m, Ar C—H), δ 3.8–4 (1H, s, NH), δ 3.2–3.8 (2H, m, CH2), δ 8.0–8.5 (1H, s, triazole) δ 7–7.5 (4H, m, Ar C—H); 13C NMR (CDCl3, 400 MHz): δ 50.1, 108.1, 113.4, 116.2, 116.8, 117.7, 123.2, 123.7, 131.8, 143.5, 147.1, 148.2, 151.1, 158, 161.1, 163.8, 174.9.
2.1.19 Synthesis of 19
To a stirring solution of compound 7 in methanol, 6-fluoro-1,3-benzothiazol-2-amine (0.168 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the yellow product. IR (KBr): 3351, 3041, 1619, 1484, 918, 719, 674; 1H NMR (CDCl3, 400 MHz): δ 7.2–7.9 (3H, m, Ar C—H), δ 3.8–4 (1H, t, NH), δ 3.0–3.5 (2H, s, CH2), δ 8.0–8.4 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 50.7, 107.9, 108.2, 113.2, 113.4, 117.7, 123.3, 131.1, 136.4, 144.3, 147.7, 148.9, 152.3, 158, 158.4, 164, 174.4, 174.9.
2.1.20 Synthesis of 20
To a stirring solution of compound 7 in methanol, 4-amino-1,2,4-triazole (0.08408 g) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the off white product. IR (KBr): 3354, 3051, 1622, 1480, 914, 719, 674; 1H NMR (CDCl3, 400 MHz): δ 7.2–7.8 (3H, m, Ar C—H), δ 3.7–4 (1H, m, NH), δ 2.9–3.3 (2H, m, CH2), δ 7.9–8.2 (1H, s, triazole), δ 8.4–8.9 (1H, s, triazole); 13C NMR (CDCl3, 400 MHz): δ 42.7, 107.9, 113.4, 117.7, 131.1, 147.3, 148.1, 148.3, 149, 152, 158, 163.8, 174.4.
2.1.21 Synthesis of 21
To a stirring solution of compound 7 in methanol, 4-fluoro-aniline (0.09587 ml) along with 3–4 drops of concentrated sulfuric acid was added. The reaction mixture is refluxed for 3–4 h. After the completion of reaction, controlled by TLC, the reaction mixture was cooled to room temperature giving a solid product after 30–40 min, which was filtered, washed with distilled water and recrystallized from benzene to afford the off white product. IR (KBr): 3351, 3023, 1618, 1480, 914, 719, 671; 1H NMR (CDCl3, 400 MHz): δ 7.3–7.9 (3H, m, Ar C—H), δ 3.6–4 (1H, m, NH), δ 2.9–3.2 (2H, m, CH2), δ 8.0–8.3 (1H, s, triazole), δ 7–7.4 (4H, m, Ar C—H); 13C NMR (CDCl3, 400 MHz): δ 50.1, 108.1, 113.7, 116.2, 116.9, 117.9, 123.1, 123.8, 131.8, 143.5, 147.5, 148.7, 152.2, 158, 161.1, 163.9, 174.4.
2.2 Antifungal assays
The in vitro antifungal activity of all the compounds was performed by Poisoned food technique (Devi and Chhetry, 2013; Kumar and Tyagi, 2013) against one phytopathogenic fungi viz Drechslera oryzae and by Spore germination inhibition technique (Devi and Chhetry, 2012) against Ustilago tritici, Puccinia striiformis, Puccinia triticina, Ustilago hordei and Blumeria graminis in comparison with the standard fungicides Bavistin (methyl benzimidazol-2-ylcarbamate), Tilt (1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole) and Vitavax (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxamide). The isolates of phytopathogenic fungi were provided by the Plant Pathology Department of the Punjab Agricultural University and the standards Bavistin, Tilt and Vitavax which served as the positive control were obtained from their respective manufacturers.
2.3 Docking procedure
Compounds were built using the builder tool kit of the software package ArgusLab 4.0.1 and geometry was optimized using semi-empirical quantum mechanical method PM3. Due to non-availability of fungal lanosterol demethylase, the crystal coordinates of human lanosterol 14α-demethylase (PDB ID 2 VKU) were downloaded from protein data bank (www.rcsb.org) and the active site of the enzyme was located, carrying a 4,4′-dihydroxybenzophenone (DHBP) as a ligand in the active site. The molecule to be docked in the active site of the protein was pasted in the work space carrying the structure of the enzyme. The docking program implements an efficient grid based docking algorithm which approximates an exhaustive search within the free volume of the binding site cavity. The conformational space was explored by the geometry optimization of the flexible ligand (rings were treated as rigid) in combination with the incremental construction of the ligand torsions. Thus, docking occurs between the flexible ligand parts of the compound and enzyme. The final positions of the compounds were ranked by lowest interaction energy values (docking score in kcal/mol). H-bond interactions between the compound and enzyme were explored.
2.4 In silico analysis
In silico molecular modification was the most important preliminary step in the molecular designing of novel biomolecules. In the present study different proposed derivatives were screened for different physico-chemical properties by using different softwares. Toxtree v2.6.6 is an open-source software application that places chemicals into categories and predicts various kinds of toxic effect by applying various decision tree approaches. Toxtree was developed by IdeaConsult Ltd. (Sofia, Bulgaria) under the terms of an ECB contract. The software is made freely available by ECB as a service to scientific researchers and anyone with an interest in the application of computer-based estimation methods in the assessment of chemical toxicity. The new module with the revised list of SAs includes also quantitative structure–activity relationships (QSAR) models that enable the toxicity evaluations for a number of chemical classes to be fine-tuned.
In order to find out the toxic hazards of all the synthesized compounds, two dimensional models of the compounds were first converted into its simplified molecular-input line-entry system (SMILES format). Then simply putting the SMILES code into the chemical identifier row available in the Toxtree software we can easily get the toxic characters. Molinspiration, a web based software, was used to obtain parameters of Lipinski’s rule of five already referred to as the Pfizer’s rule of five or simply the Rule of five (RO5). This rule was formulated by Christopher A. Lipinski in 1997. In order to calculate the molecular properties, firstly we have either to draw the molecule online (www.molinspiration.com) or simply put the SMILES code then all the Lipinski characters along with bioactivity and 3D structure of the molecule were generated.
3 Result and discussion
3.1 Chemistry
The compounds described in this paper were synthesized by the multistep reaction protocol. 6-Fluoro-1,3-benzothiazol-2-amine (1) used as a starting material was prepared according to the literature procedure reported earlier (Malik et al., 2010).
The synthesis of 6-flourobenzothiazol-2-yl-1,2,4-triazoles (2–9) was carried out by reacting 6-fluoro-1,3-benzothiazol-2-amine with 4-amino-1,2,4-triazole and 1,2,4-triazole using an appropriate linking reagent.
Compounds containing carbonyl linker (2, 3, 6 and 7) were further modified into their Schiff bases (10–21) by refluxing them with appropriate amines (Scheme 1). The spectral data (IR, 1H NMR, 13C NMR and mass spectroscopic data) and elemental analysis (Table 1) of all the synthesized compounds were correlated with the proposed structure which confirmed the formation of the compounds.
| Compounds | Yield (%) | M.P (°C) | Mol. formula | Elemental analysis (calculated %/found %) | ||
|---|---|---|---|---|---|---|
| C | H | N | ||||
| 2 | 84 | 280–285 | C11H9FN6OS | (45.01/45.20) | (3.02/3.10) | (28.51/28.75) |
| 3 | 81 | 308–310 | C11H9FN6OS | (45.07/45.20) | (3.02/3.10) | (28.59/28.75) |
| 4 | 77 | 209–211 | C9H7FN6OS2 | (36.31/36.24) | (2.03/2.37) | (28.11/28.17) |
| 5 | 71 | 261–262 | C12H13FN6S | (49.22/49.30) | (4.23/4.48) | (28.44/28.75) |
| 6 | 81 | 302–306 | C11H8FN5OS | (47.51/47.65) | (2.77/2.91) | (25.22/25.26) |
| 7 | 80 | 299–302 | C11H8FN5OS | (47.10/47.65) | (2.15/2.91) | (25.12/25.26) |
| 8 | 73 | 212–214 | C9H6FN5OS2 | (38.01/38.16) | (2.09/2.13) | (24.11/24.72) |
| 9 | 71 | 272–274 | C12H12FN5S | (51.45/51.97) | (4.12/4.36) | (25.11/25.25) |
| 10 | 61 | 241–243 | C18H12F2N8S2 | (48.21/48.86) | (2.31/2.73) | (25.10/25.32) |
| 11 | 59 | 223–225 | C13H11FN10S | (43.10/43.57) | (3.01/3.09) | (38.88/39.09) |
| 12 | 69 | 220–221 | C17H13F2N7S | (52.28/52.98) | (3.12/3.40) | (25.11/25.44) |
| 13 | 62 | 208–209 | C18H12F2N8S2 | (48.12/48.86) | (2.12/2.73) | (25.11/25.32) |
| 14 | 63 | 204–205 | C13H11FN10S | (43.11/43.57) | (3.04/3.09) | (38.91/39.09) |
| 15 | 66 | 201–203 | C17H13F2N7S | (52.12/52.98) | (3.11/3.40) | (25.12/25.44) |
| 16 | 66 | 201–203 | C17H13F2N7S | (52.12/52.98) | (3.11/3.40) | (25.12/25.44) |
| 17 | 59 | 209–211 | C13H10FN9S | (45.18/45.48) | (2.15/2.94) | (36.19/36.72) |
| 18 | 64 | 209–210 | C17H12F2N6S | (54.99/55.13) | (2.79/3.27) | (22.12/22.69) |
| 19 | 59 | 210–212 | C18H11F2N7S2 | (50.23/50.58) | (2.11/2.59) | (22.71/22.94) |
| 20 | 54 | 207–209 | C13H10FN9S | (45.12/45.48) | (2.77/2.94) | (36.12/36.72) |
| 21 | 60 | 202–204 | C17H12F2N6S | (54.92/55.13) | (3.11/3.27) | (22.45/22.69) |
3.2 Antifungal assays
The synthesized 6-fluorobenzothiazol-2-yl-1,2,4-triazoles (2–9) and their Schiff bases (10–21) were screened for their in vitro antifungal potential against various phytopathogenic fungi and EC50 values were calculated (Table 2). These data are the mean of three replicate tests performed with each antifungal compound. The results of antifungal evaluation of the synthesized compounds along with standard fungicides used viz. Bavistin (methyl benzimidazol-2-ylcarbamate), Tilt (1-[[2-(2,4-dichlorophenyl)-4- propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole) and Vitavax (5,6-dihydro-2-methyl-1,4-oxathiin-3- carboxamide) against various phytopathogenic fungi, are reported in Table 2.
| Compd | U. tritici | P. striiformis | P. triticina | B. graminis | D. oryzae | U. hordei |
|---|---|---|---|---|---|---|
| 1 | 2.08 | 5.65 | 5.95 | 4.46 | 5.95 | 5.95 |
| 2 | 0.25 | 0.23 | 0.34 | 0.68 | 0.27 | 0.17 |
| 3 | 0.68 | 1.09 | 1.88 | 1.03 | 0.86 | 0.59 |
| 4 | 1.51 | 0.84 | 0.36 | 0.31 | 0.59 | 0.34 |
| 5 | 1.02 | 0.66 | 0.75 | 1.19 | 1.03 | 0.77 |
| 6 | 0.32 | 0.36 | 0.43 | 0.77 | 0.72 | 0.61 |
| 7 | 1.98 | 1.08 | 0.61 | 0.72 | 1.44 | 0.90 |
| 8 | 0.53 | 1.06 | 0.42 | 0.53 | 0.97 | 0.88 |
| 9 | 0.68 | 0.90 | 1.11 | 0.68 | 1.69 | 0.72 |
| 10 | 0.91 | 0.57 | 1.13 | 1.13 | 1.58 | 1.13 |
| 11 | 0.69 | 2.79 | 0.83 | 0.53 | 1.11 | 0.61 |
| 12 | 1.39 | 0.19 | 2.79 | 0.33 | 2.79 | 0.97 |
| 13 | 1.59 | 1.24 | 0.68 | 1.47 | 0.57 | 0.46 |
| 14 | 0.59 | 2.79 | 1.11 | 0.41 | 0.69 | 0.97 |
| 15 | 0.25 | 0.41 | 0.28 | 1.26 | 0.56 | 0.22 |
| 16 | 0.70 | 2.15 | 2.34 | 0.93 | 2.34 | 0.58 |
| 17 | 1.45 | 1.02 | 0.72 | 1.07 | 0.27 | 0.58 |
| 18 | 0.25 | 0.27 | 0.25 | 0.33 | 0.27 | 0.44 |
| 19 | 0.58 | 1.17 | 0.93 | 1.52 | 0.58 | 1.14 |
| 20 | 1.16 | 2.91 | 1.45 | 1.09 | 1.13 | 0.90 |
| 21 | 0.40 | 0.94 | 1.35 | 0.40 | 0.27 | 0.67 |
| Bavistina | – | – | – | – | 0.23 | – |
| Tiltb | – | 0.10 | 0.02 | 0.03 | – | – |
| Vitavaxc | 0.05 | – | – | – | – | 0.09 |
For the sake of comparison of fungitoxicity of the molecules with standard fungicides, it was appropriate to express the results in terms of millimoles/L. Investigation of antifungal screening revealed that most of the synthesized hybrids showed promising inhibition of germination against all the test fungi. Concerning 6-fluoro-1,3-benzothiazole derivatives, the 6-fluorobenzothiazol-2-yl-1,2,4-triazoles (2–9) exhibited activity manifold better than the precursor 1. This revealed the synergistic effect of combination of 6-fluoro-1,3-benzothiazol-2-amine with 4-amino-1,2,4-triazoles as well as with 1,2,4-triazoles. Compound 2 exhibited broad spectrum activity against all the test fungi followed by compounds 6, 15 and 18 which were active against most of the test fungi. Compounds 2 and 12 have inflicted antifungal potential of EC50 0.23 and 0.19 mmoles/L, respectively against P. striiformis which were comparable with standard fungicide – (EC50 value 0.10 mmoles/L). Against U. hordei compound 2 was the best with EC50 value 0.17 mmoles/L which was again comparable with standard fungicide Vitavax (EC50 value 0.09 mmoles/L). Against D. oryza, compound 2, along with compounds 17, 18 and 21 which are the Schiff bases of the compounds 2 and 3 had shown the best potential with EC50 value of 0.27 mmoles/L each. This value was almost equal to the EC50 value of standard fungicide Bavistin (EC50 value 0.23 mmoles/L) against the test fungi.
One most important trend observed against all the test fungi was the low activity of Schiff bases of all the amines except with 4-flouroaniline (12, 15 and 18) derivatives of the 6-fluorobenzothiazol-2-yl-1,2,4-triazoles. Compound 21 which was the Schiff base with 4-amino-1,2,4-triazole was quite active against D. oryzae. The Schiff base derivatives with bulkier bicyclic amines were among the compounds with lowest activity. The introduction of low steric —C⚌N— spacer between 6-fluoro-1,3-benzothiazol-2-amine and 1,2,4-triazoles in hybrid compounds was the favorable factor for the fungitoxicity of the compounds.
3.3 Docking studies
Since it was planned to scrutinize the mode of interaction of compounds under investigation, as they showed appreciable inhibition of the fungal activity. For this purpose, molecular docking of all the synthesized compounds in the active site of the enzyme lanosterol 14α-demethylase (PDB ID 2VKU) was performed, using ArgusLab 4.0.1. It was observed that some of these compounds exhibit H-bond interactions with active site of lanosterol 14α-demethylase but with significant difference in their respective docking score. As shown in Fig. 1, all the four compounds get concealed in the interior of the enzyme. Docking of compound 18 in the active site showed that this compound interacts more effectively than other compounds. Compounds 2, 6 and 15, on the other hand, had free energies of binding −9.47, −9.36 and −11.39 kcal/mol respectively. For comparing the molecular interactions of compounds with that of standards which were also docked in the same active site of the enzyme. However, H-bond interactions of standards as well as their respective docking scores were less in comparison with compounds under study. Noticeably, most of the Schiff base derivatives have shown poor docking score but some of them were still very active (compound 12).
3.4 In silico analysis
Estimation of toxic hazards by Cramer rules was carried out using Toxtree v.2.6.6 software (Puratchikody et al., 2012), which showed that compounds belong to class III level of toxicity, which was same as that of the toxicity level standards fungicides used.
Toxicity profile was further rationalized for their potential bioactivity as antifungals or proantifungals, by theoretically calculating Lipinski properties of molecules using the Molinspiration software (Ahsan et al., 2011). Docking score along with Lipinski parameters of the synthesized compounds are presented in Table 3. According to Lipinski’s rule-of-five, molecules should have log p values of ⩽5 in order to be readily bioavailable. Lipinski’s rule says that the molecular weight should be below 500, the number of hydrogen bond acceptors should not be more than 5 and also the number of hydrogen bond donors should not be more than 5. These numbers are the upper limits for the biomolecules to be able to penetrate through biomembranes.
| Compound | Final docked energy (kcal/mol) | log p | Molecular weight | n ON | n OHNH |
|---|---|---|---|---|---|
| 1 | −8.62 | 2.59 | 168.19 | 2 | 2 |
| 2 | −9.47 | 1.08 | 292.29 | 7 | 2 |
| 3 | −9.14 | 1.08 | 292.29 | 7 | 2 |
| 4 | −8.78 | 0.88 | 298.32 | 7 | 2 |
| 5 | −9.17 | 1.89 | 292.34 | 6 | 2 |
| 6 | −9.36 | 1.79 | 277.28 | 6 | 1 |
| 7 | −8.25 | – | 277.28 | 6 | 1 |
| 8 | −8.96 | – | 283.31 | 6 | 1 |
| 9 | −9.26 | 2.67 | 277.32 | 5 | 1 |
| 10 | 4.79 | 442.47 | 8 | 2 | |
| 11 | – | 358.36 | 10 | 2 | |
| 12 | 3.59 | 358.39 | 7 | 2 | |
| 13 | 4.79 | 442.47 | 8 | 2 | |
| 14 | – | 358.36 | 10 | 2 | |
| 15 | −11.39 | 3.59 | 358.39 | 7 | 2 |
| 16 | 5.57 | 427.45 | 7 | 1 | |
| 17 | −4.22 | – | 343.34 | 9 | 1 |
| 18 | −11.51 | 4.37 | 370.38 | 6 | 1 |
| 19 | −1.51 | 5.57 | 427.45 | 7 | 1 |
| 20 | – | 343.34 | 9 | 1 | |
| 21 | −7.99 | 4.37 | 370.38 | 6 | 1 |
indicates no suitable ligand pose energy.
– indicates large value of log p.
log p, logarithm of the octanol–water partition coefficient; n ON, number of hydrogen bond acceptors (sum of O and N atoms); n OHNH, number of hydrogen bond donors (sum of OH and NH groups).
As shown in Table 3, all the synthesized compounds had molecular weights below 500 and hydrogen bond donor index was in range of 1–2. The calculated log p values were in the range of 0.88–5.57. Compounds 16 and 19 had unfavorable log p values and fungitoxicity level of these molecules was also low, which reveals the significance of favorable value of log p for antifungal potential. Furthermore, the number of hydrogen bond acceptor value was beyond Lipinski limit in case of all the compounds. This value beyond the limit has not effected the bioactivity of the synthesized hybrids. Compounds 2 and 6 have followed three out of four limits (Veber et al., 2002) of Lipinski’s rule but they are highly effective against most of the fungi with broad spectrum activity.
It was obvious from the above results that low molecular weight along with docking scores favored the activity of the most potent synthesized compounds 2, 6, 15 and 18. Low molecular weight Schiff bases (15 and 18) were found to be more effective than their high weight analogues which were probably due to high diffusion of lighter molecules into the active site of the enzyme to which they bind.
4 Conclusion
In summary, 20 new compounds based on lead hybridization of 1,2,4-triazoles with fluorinated benzothiazol-2-yl were synthesized and evaluated for their antifungal potential. The synthesized hybrid compounds showed promising activity against D. oryzae, P. striiformis and U. hordei comparable with standard fungicides used which indicated their potential for further lead development. The in silico toxicity analysis indicated their toxicity comparable with standard fungicides used. High docking score and favorable molecular characteristics according to Lipinski rule, accounted for the overall high activity of the compounds (2, 6, 15 and 18). The inhibition of fungal spores by 2, 6 and their respective 4-flouroaniline derivatives 15 and 18 were quite high that indicated that these compounds can act as leads for the development of newer antifungal compounds.
Acknowledgment
The authors express their thanks to UGC, New Delhi, India for Financial Assistance.
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