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Triazole hybrids as new type of anti-fungal agents
⁎Corresponding author. Address: Department of Pharm. Chem., Sinhgad Institute of Pharmacy, Narhe, Pune 411041, India. Mobile: +91 09822677423; fax: +91 020 66831816. pankajmpharm@yahoo.co.in (Pankaj B. Miniyar)
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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
The present work involves synthesis of novel anti-fungal agents containing triazole scaffold. The newly designed compounds were synthesized on the trails of ketoconazole using the molecular hybridization approach. A series of 10 compounds having (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl esters (4a–j) were prepared by conventional synthetic approach. The synthesized compounds were subjected for in vitro anti-fungal screening against Aspergillus niger, Aspergillus fumigatus, Candida albicans and Penicillium notatum. Out of 10 newly synthesized compounds, six compounds (4b–f and j) showed remarkable anti-fungal activity (MIC range 6.5–25 μg/ml), whereas compound 4d (MIC 6.5 μg/ml) was more potent than standard drug ketoconazole (MIC 12.5 μg/ml). These triazole hybrids can be considered as potential anti-fungal agents.
Keywords
1,2,4-Triazole
Dioxolane
Hybrid molecule
Anti-fungal
1 Introduction
Fungal infections pose a continuous and serious threat to human life. The severity of infection ranges from minor irritations such as athlete’s foot to life-threatening systemic infections caused by Aspergillus fumigates (Uchida et al., 2008). Hence the development of a potent, safe and selective antifungal agent is of prime importance for medicinal chemist in the quest for effective chemotherapeutic treatment for fungal diseases (Rezaei et al., 2009). At present antifungal treatment with existing drugs proved to be less effective, due to drug toxicity and drug resistance against a wide variety of fungal species (Behbehani et al., 2011).
Triazole is an antifungal scaffold because of its high potency and low toxicity (Xu et al., 2004). Hence many potent antifungal compounds were developed in recent past by bioisosteric replacement of imidazole moiety with triazole (Acetti et al., 2009). Triazole derivatives competitively inhibit lanosterol 14α-demethylase (CYP51), a key enzyme in sterol biosynthesis of fungi. Based on the structure of the active site of CYP51 and the extensive investigation of azole antifungals, triazole inhibitors are able to fit in the active site by H-bonding, hydrophobic interactions and π–π stacking within the heme environment of the enzyme (Upadhayaya et al., 2009; Kamal et al., 2011).
The literature survey revealed that, many researchers developed potent anti-fungal agents by the molecular hybridization approach using dioxolone with diazole/triazole moiety (Hiromichi and Yasushi, 2000; Dariusz and Andrzej, 2007; Fringuelli and Giacche, 2009; Xu and Cao, 2011). Well known drugs viz. terconazole and itraconazole were developed as molecular hybrids of triazole and dioxolone, whereas triazole moiety was replaced by diazole in ketoconazole. The three crucial features of clinically active anti-fungal agents are diazole/triazole ring, aromatic ring and side chains (Fig. 1). Hence novel compounds were developed on the trails of the above mentioned molecular hybridization approach (Fig. 1).
Comparison of ketoconazole and targeted anti-fungal agent. A = Iron coordinating group (diazole/triazole moiety); B = Aromatic group; C = Second aromatic ring; D = Additional hydrophobic group (side chain).
2 Results and discussion
2.1 Chemistry
The targeted compounds were synthesized by a four-step reaction process (Scheme 1). In the first step, glycerol and 2,4-dichloroacetophenone were used to synthesize (2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methanol (1) in the presence of p-toluene sulfonic acid. Compound (1) was brominated by using bromine and glacial acetic acid to obtain (2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methanol (2). Intermediate (2) was converted into (2-(bromomethyl)-2-(2,4-dichloroacetophenyl)-1,3-dioxolan-4-yl) methyl ester (3) by reaction with corresponding aliphatic/substituted and unsubstituted aromatic acid chlorides. Finally, compounds (3) was stirred overnight with 50% sodium hydride and 1,2,4-triazole to obtain (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl esters (4a–j) (Table 1).
Synthesis scheme for 4a–j compounds. R = Aliphatic/aromatic acid chloride; a = n-BuOH, Benzene, p-TsOH; b = Br2, 25 °C;c = Pyridine, 5 °C, substituted acid chlorides; d = NaH, Triazole, DMSO, 130 °C.
Molecule code
R
Molecular formula
4a
–COCH3
C15H15Cl2N3O4
4b
–COCH2Cl
C15H14Cl3N3O4
4c
–COCH⚌CH–C6H5
C22H19Cl2N3O4
4d
–SO2C6H5
C19H17Cl2N3O5S
4e
–COC6H5
C20H17Cl2N3O4
4f
–COC6H4Cl(o)
C20H16Cl3N3O4
4g
–COC6H4Cl(m)
C20H16Cl3N3O4
4h
–COC6H4NH2(o)
C20H18Cl2N4O4
4i
–COC6H4NH2(p)
C20H18Cl2N4O4
4j
–COC6H4–CH3(o)
C21H19Cl2N3O4
2.2 Biological activity
The anti-fungal activities of 4a–j were performed against Aspergillus niger, Penicillium notatum, A. fumigatus and Candida albicans (Odds and Vanden Bossche, 2000) and results with respect to zone of inhibition and MIC are summarized in Tables 2 and 3. Compound 4d which is benzenesulfonyl methylester (MIC 6.5 μg/ml) was found more potent than reference standard ketoconazole (MIC 12.5 μg/ml), while compound 4c, cinnamoyl methylester (MIC 12.5 μg/ml) was at par with the reference standard. It was observed that in case of compounds 4a and b esterification with aliphatic acid chlorides and o and m substituted aromatic methyl esters viz. 4f–i exhibited less anti-fungal activity as compared with standard drug. However, compound 4j formed by esterification with o-methylbenzoyl chloride (MIC 12.5 μg/ml) was equally active as that of standard drug against P. notatum and A. fumigates.
Compound code
A. niger
P. notatum
A. fumigatus
C. albicans
100
50
100
50
100
50
100
50
4a
5
3
4
3
6
4
7
4
4b
8
6
5
3
7
5
6
4
4c
10
8
9
7
9
5
8
6
4d
17
12
24
18
14
10
25
19
4e
9
7
6
5
10
8
9
7
4f
9
6
8
5
11
8
10
7
4g
7
4
6
3
7
6
8
6
4h
6
4
6
5
6
3
7
6
4i
5
4
6
4
7
5
7
5
4j
7
4
9
7
10
8
8
6
Ketoconazole
10
7
18
14
18
12
20
15
Molecule code
MICa (μg/ml)
A. niger
P. notatum
A. fumigatus
C. albicans
4a
>50
>50
>50
>50
4b
25
>50
25
25
4c
12.5
12.5
12.5
12.5
4d
6.5
6.5
6.5
6.5
4e
25
25
25
25
4f
25
25
25
25
4g
>50
>50
>50
>50
4h
>50
>50
>50
>50
4i
>50
>50
>50
>50
4j
25
12.5
12.5
25
Ketoconazole
12.5
12.5
12.5
12.5
The present series could be developed as a novel class of anti-fungal agents by further structural optimization.
3 Experimental
3.1 General
Melting points were determined in open capillaries using Veego VMP-1 melting point apparatus and are uncorrected. The IR spectra were recorded on JASCO FTIR 4100 series spectrometer. 1H NMR and 13C spectra were recorded on Varian Mercury YH-300 at 300 MHz, in CDCl3 as a solvent and TMS as internal standard. Peak values are shown in δ ppm. The mass spectra were recorded on Waters Q-ToF micro by the electron spray ionization (ESI) method. Progress of the reaction and purity of the compounds were confirmed by pre-coated TLC plates (Merck, 60F-254) and spots were visualized using iodine vapor or UV light.
3.2 Chemistry
3.2.1 Synthesis of (2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methanol (1) (Heeres and Hendickx, 1984)
Glycerol 1 g (0.02 mol) and 2,4-dichloroacetophenone 1.89 g (0.01 mol) in 4 ml of toluene and 2 ml of n-butanol were refluxed in the presence of p-toluene sulfonic acid monohydrate (0.06 g) for 24 h with azeotropic removal of water. The excess amount of toluene was removed on a rota evaporator and the oily product 2-(2,4-dichlorophenyl)-1,3-dioxolane-4-methanol (1) was obtained. Yield: 70%; B.P.: 130–132 °C.
3.2.2 Synthesis of (2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methanol (2) (Furniss et al., 1989)
The oily product 2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methanol (0.01 mol) was dissolved in glacial acetic acid and cooled to 20 °C, then into it bromine was added (1.92 g, 0. 012 mol) over a period of 2 h. The mixture was stirred for 0.5 h and then evaporated in vacuum to obtain product (2) with 74% yield; B.P.: 182–184 °C.
3.2.3 General procedure for the synthesis of (2-(bromomethyl)-2-(2,4-dichloroacetophenyl)-1,3-dioxolan-4-yl) methyl ester (3a–j) (Heeres and Hendickx, 1984)
The substituted acid chlorides (1.40 g, 0.01 mol) were added dropwise at 5 °C to a solution of compound 2 (3.1 g, 0.091 mol) in 6 ml of dry pyridine over a period of 1 h. The mixture was stirred for 2.5 h and diluted with water. After extraction with CHCl3, the organic layer was washed with 6 N HCl, dried (MgSO4) and evaporated in vacuum to leave an oily residue which solidified on stirring with CH3OH and obtained product 3a–j with yield 75–85%; B.P.: 194–196 °C.
3.2.4 General procedure for the synthesis of aliphatic/substituted aromatic esters of (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl esters (4a–j)
Furniss et al., 1989 A suspension of 50% sodium hydride (0.53 g, 0.011 mol) in dry DMSO 5 ml was added to triazole (0.69 g, 0.01 mol) and compound 3 was mixed into it. It was stirred at 130 °C overnight. After the mixture was cooled, diluted with water and extracted with dichloromethane the organic extract was washed with water, dried and evaporated in vacuum. An oily residue of the final product (4) was obtained with 60–85% yield; B.P.: 146–148 °C.
Spectral data for 4a–j compounds are as follows where MS (c) = calculated and (f) = found.
3.2.4.1 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methylacetate (4a)
Yield: 65%; B.P.: 180–182 °C; FTIR (cm−1): 814, 1290, 1600, 1732, 3092; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 2.0–2.3 (s, 3H, methyl), 4.5–4.6 (d, 2H, J = 9.6, 5.1, methylene), 4.0 (m, 1H, d, 2H, J = 8.2, 6.1; dioxolane), 4.8 (d, 2H, J = 14.6; methylene), 7.0–7.9 (m, 3H, aromatic), 8.2–8.5 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 170.3 (C⚌O), 151.2, 143.6 (C-triazole), 135.7, 134.6, 134.9, 130.4, 126.6 (C-aromatic), 105.1, 72.8, 67.6 (C-dioxolane), 65.7, 63.3 (C-methylene), 20.3 (C-methyl); MS (ESI): (c) 372.2033, (f) 373.3690 [M+1]+.
3.2.4.2 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl-2-chloro acetate (4b)
Yield: 62%; B.P.: 200–202 °C; FTIR (cm−1): 752, 1049, 1501, 1671, 3062; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 4.3 (s, 2H, methylene), 4.5–4.6 (d, 2H, J = 9.8, 5.0, methylene), 4.3 (m, 1H, d, 2H, J = 8.0, 6.3; dioxolane), 4.5 (d, 2H, J = 13.6, methyl), 7.2–7.9 (m, 3H, aromatic), 8.1–8.3 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.8 (C⚌O), 151.3, 143.5 (C-triazole), 135.7, 134.6, 134.4, 130.5, 126.3 (C-aromatic), 105.0, 72.3, 67.7 (C-dioxolane), 64.4, 40.3 (C-methylene); MS (ESI): (c) 406.6484, (f) 408.4762 [M+2]+.
3.2.4.3 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl- cinnamate (4c)
Yield: 71%; B.P.: 250–253 °C; FTIR (cm−1): 759, 1200, 1693, 1789, 3021; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 6.4–6.5 (d, 2H, J = 10.2, vinyl), 4.5–4.9 (d, 2H, J = 9.8, 5.0, methylene), 3.9–4.0 (m, 1H, d, 2H, J = 7.8.0, 6.1; dioxolane), 4.5–4.9 (d, 2H, J = 14.2, methyl), 7.2–7.9 (m, 8H, aromatic), 8.1–8.2 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.4 (C⚌O), 151.3, 145.9 (C-triazole), 143.4, 135.4, 134.6, 134.2, 130.2, 128.6, 126.7 (C-aromatic), 105.1, 72.2, 67.6 (C-dioxolane), 65.7, 63.1 (C-methylene); MS (ESI): (c) 460.3100, (f) 483.2231 [M+Na]+.
3.2.4.4 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl- benzene sulfonate (4d)
Yield: 73%; B.P.: 217–219 °C; FTIR (cm−1): 723, 1259, 1592, 1683, 3122; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 4.3–4.7 (d, 2H, J = 10.0, 5.2, methyl), 3.7–3.9 (m, 1H, d, 2H, J = 8.0, 5.9; dioxolane), 4.5–4.9 (d, 2H, J = 14.0, methyl), 7.0–7.8 (m, 8H, aromatic), 8.1–8.5 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 151.2 (C⚌O), 143.5, 141.4 (C-triazole), 134.2, 130.3, 129.6, 126.8 (C-aromatic), 105.3, 71.4, 67.5 (C-dioxolane), 63.0 (C-methylene); MS (ESI): (c) 470.3264, (f) 471.3304 [M+1]+.
3.2.4.5 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl-benzoate (4e)
Yield: 75%; B.P.: 160–162 °C; FTIR (cm−1): 723, 1259, 1590, 1690, 3122; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 4.2–4.6 (d, 2H, J = 10.1, 5.2, methylene), 3.7–3.9 (m, 1H, d, 2H, J = 8.4, 5.9; dioxolane), 4.7–4.9 (d, 2H, J = 14.4, methylene), 7.1–7.4 (m, 8H, aromatic), 8.1–8.3 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.1 (C⚌O), 151.3, 143.5 (C-triazole), 135.5, 134.8, 133.2, 130.2, 129.9, 128.6, 126.7 (C-aromatic), 105.1, 72.1, 67.5 (C-dioxolane), 65.2, 63.1(C-methylene); MS (ESI): (c) 434.2727, (f) 435.1697 [M+1]+.
3.2.4.6 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl-2-chloro- benzoate (4f)
Yield: 78%; B.P.: 190–192 °C; FTIR (cm−1): 740, 1041, 1506, 1702, 2312; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 4.1–4.4 (d, 2H, J = 9.6, 4.8, methylene), 3.7–3.9 (m, 1H, d, 2H, J = 8.2, 5.9; dioxolane), 4.7–4.9 (d, 2H, J = 14.0, methylene), 7.0–7.5 (m, 7H, aromatic), 8.0–8.2 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.1 (C⚌O), 151.3, 143.4 (C-triazole), 134.4, 131.7, 130.1, 128.6, 126.9 (C-aromatic), 105.1, 72.4, 67.4 (C-dioxolane), 63.1, 60.3 (C-methylene); MS (ESI): (c) 468.7177, (f) 470.0233 [M+2]+.
3.2.4.7 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl-3-chloro- benzoate (4g)
Yield: 60%; B.P.: 287–289 °C; FTIR (cm−1): 759, 1020, 1583, 1693, 3087; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 4.0–4.3 (d, 2H, J = 9.4, 4.6, methylene), 3.5–3.7 (m, 1H, d, 2H, J = 8.0, 5.7; dioxolane), 4.5–4.7 (d, 2H, J = 14.2, methylene), 7.1–7.6 (m, 7H, aromatic), 8.0–8.2 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.0 (C⚌O), 151.3, 134.4 (C-triazole), 133.2, 131.5, 130.3, 128.8, 126.6 (C-aromatic), 105.0, 72.4 (C-dioxolane), 65.1, 63.2 (C-methylene); MS (ESI): (c) 468.7177, (f) 469.0277 [M+1]+.
3.2.4.8 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl-2-amino- benzoate (4h)
Yield: 80%; B.P.: 228–232 °C; FTIR (cm−1): 1259, 3122, 1730, 1685, 758, 3413; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 4.0 (s, 2H, NH), 4.1–4.2 (d, 2H, J = 9.2, 4.4, methylene), 3.6–3.8 (m, 1H, d, 2H, J = 8.2, 5.8; dioxolane), 4.4–4.6 (d, 2H, J = 14.0, methylene), 7.1–7.7 (m, 7H, aromatic), 8.1–8.3 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.1 (C⚌O), 151.3, 150.3 (C-triazole), 135.6, 134.6, 133.7, 130.4, 126.8 (C-aromatic), 118.6, 116.4, 105.0 (C-dioxolane), 72.2, 65.2, 63.0 (C-methylene); MS (ESI): (c) 449.2873, (f) 451.3545 [M+2]+.
3.2.4.9 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl-4-amino- benzoate (4i)
Yield: 85%; B.P.: 270–272 °C; FTIR (cm−1): 757, 1256, 1594, 1673, 2929; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 4.0 (s, 2H, NH), 4.2–4.4 (d, 2H, J = 9.3, 4.5, methylene), 3.7–3.9 (m, 1H, d, 2H, J = 8.0, 5.6; dioxolane), 4.4–4.6 (d, 2H, J = 14.2, methylene), 7.1–7.4 (m, 7H, aromatic), 8.0–8.4 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.3 (C⚌O), 152.5, 151.1 (C-triazole), 143.6, 135.4, 134.1, 130.6, 126.9 (C-aromatic), 120.2, 116.3, 105.2, 72.0, 67.6, 65.1, 63.0 (C-methylene); MS (ESI): (c) 449.2873, (f) 450.2034 [M+1]+.
3.2.4.10 (2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl-2-benzoate (4j)
Yield: 76%; B.P.: 290–292 °C; FTIR (cm−1): 869, 1259, 1592, 1633, 3122; 1H NMR (300 MHz, CDCl3, J Hz) δ ppm: 2.2 (s, 3H, CH3), 4.0–4.3 (d, 2H, J = 9.1, 4.4, methylene), 3.7–3.9 (m, 1H, d, 2H, J = 8.1, 5.5; dioxolane), 4.4–4.6 (d, 2H, J = 14.0, methylene), 7.0–7.5 (m, 7H, aromatic), 8.1–8.2 (s, 2H, triazole); 13C NMR (75 MHz, CDCl3) δ ppm: 166.0 (C⚌O), 151.2, 143.6 (C-triazole), 139.4, 135.0, 134.6, 133.1, 130.5, 129.6, 126.9 (C-aromatic), 120.6, 105.1, 72.1, 67.5, 65.4, 63.1 (C-methylene); MS (ESI): (c) 448.2993, (f) 449.0711 [M+1]+.
3.2.5 Biological activity
A. niger (NCIM 592), P. notatum (NCIM 745), A. fumigatus (NCIM 535) and C. albicans (NCIM 3471) were obtained from National Collection of Industrial Microorganism (NCIM), National Chemical Laboratory (NCL), Pune, India. The different concentrations of all test compounds were prepared in DMSO at 5–50 μg/ml. Ketoconazole was used as standard. Micro dilutions for control experiments were performed as described by Galgiani (1993) and National Committee for Clinical Laboratory Standards (1997).
Acknowledgement
Authors are thankful to Prof. M.N. Navale, President, Sinhgad Technical Education Society, Pune and Dr. K.G. Bothara, Principal, Sinhgad Institute of Pharmacy, Pune for constant encouragement and providing infrastructure.
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