Introduction

Fungi are widely distributed in nature and frequently appear as pathogens in the animal and plant kingdoms. The onset of the the acquired immunodeficiency syndrome (AIDS) epidemic (Plattener, 2003) combined with increased use of immunosuppressive drugs for organ transplants and cancer therapy have resulted in increased incidence of life-threatening fungal infections.

Chalcones are reported to have an array of important therapeutic activities such as antihypertensive and cardiovascular activity, antiprotozoal, anti-inflammatory, antidiabetic, nitric oxide inhibitory activity, anticancer activities as well as antifungal and antitubercular activities (Opletalova et al., 2003; Mei et al., 2003; Chun et al., 2001; Satyanarayana et al., 2004; Horng et al., 2003; Ruby et al., 1995; Opletalova and Sedivy, 1999; Lopez et al., 2001; Tsuchiya et al., 1994; El Sohly et al., 2001; Kulkarni 2003; Lin et al., 2002).

Elemental sulfur has long been known to act as an antifungal agent. Also, a well-known antifungal agent, tolnaftate has sulfur in organically combined form. Sulfur is also present in antifungal agents of natural origin, e.g., Allium sativum (garlic), which is known to inhibit Candida albicans (Lemar et al., 2002).

Antifungal activity of chalcones has been investigated by a number of researchers. Recently Nowakowska reviewed the antimicrobial and ant-iinflammatory activity of chalcones (Nowakowska, 2007). Sato et al. reported growth inhibitory properties of hydroxyl chalcones to Candida (Sato et al., 1994). Tomar et al. have reported synthesis and antimicrobial activity of chalcones containing the piperazine or 2,5-dichlorothiophene moiety (Tomar et al., 2007). Lahtchev et al. reported a mechanistic study on chalcones using various yeast strains (Lahtchev et al., 2008). Nowakowska et al. have reported antibacterial and antifungal activities of chalcones (Nowakowska et al., 2008).

Earlier work in our laboratory had indicated that, when a thiophene ring was incorporated into a chalcone structure, the molecule exhibited antifungal activity (Javeri, 2004).

figure b

1 was found to have activity against Candida albicans at concentration of 25 mg/ml while 2 was inactive at 400 mg/ml.

This led us to explore chalcones incorporating sulfur either as part of a heteroaromatic ring (thiophene) or as a side chain (thiomethyl group). Thus, in this study, various compounds were synthesized and tested for antifungal activity. Some of these chalcones have been reported earlier; however they have not been screened for antimicrobial activity (Hayashi et al., 1989; Tafi et al., 2002; Joo et al., 2003; Sivakumar et al., 2007; Meng et al., 2004; Basaif et al., 2005; Dhar, 1972; Taylor et al., 1980; Goto et al., 1991). The general scheme is shown in Scheme 1.

Scheme 1
scheme 1

Synthesis of various chalcones

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Results and discussions

Chemistry

A series of α,β-unsaturated ketones incorporating sulfur were synthesized and characterized spectroscopically. Their biological activity against fluconazole-sensitive as well as fluconazole-resistant strains of Candida albicans was evaluated.

The typical procedure involved the reaction of various substituted ketones with various aromatic or heteroaromatic aldehydes in equimolar ratio in basic conditions. All the synthesized compounds were characterized spectroscopically. In general, infrared (IR) spectra showed a C=O peak at 1643.9–1655.8 cm−1. The 1H nuclear magnetic resonance (NMR) spectra showed unsaturated and aromatic protons at δ 6.743–7.927. Literature survey revealed that compounds 14, 16, and 18 are novel. These were characterized spectroscopically by 1H-NMR, 13C-NMR, mass spectroscopy (MS), and IR spectroscopy. Physicochemical characterizations of the synthesized chalcones are shown in Table 1.

It was observed that compounds with unsubstituted thiophene ring (Table 1b) and thiomethyl substitution at the para position of benzaldehyde (Table 1b) exhibited good antifungal activity. High activity was found when both thiomethyl and thiophene ring were present (compound 13). Bromine substitution on the thiophene ring decreased antifungal activity (Table 1b).

In the first series (Table 1a), maximum activity was obtained with p-fluoro substitution (compound 1). Activity decreased with increasing halogen size (compounds 1, 2, and 3). Presence of p-methoxy (compound 6) or hydroxy groups at the ortho, meta or para position also resulted in good activity (compounds 8, 9, and 10) while the p-nitro group as well as the bulky p-phenyl substitution decreased activity (compounds 5 and 11) as compared with the unsubstituted compound 7.

Table 1a Synthesized compounds with their biological activities
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In the second series (Table 1b), meta and para disubstitution with methoxy (compound 15) led to increased activity while again the p-phenyl-substituted compounds (17 and 18) exhibited considerably decreased activity. All compounds with the bromo thiophene ring (14, 16, 18, and 20) exhibited less activity compared with those with the unsubstituted thiophene ring (13, 15, 17, and 19).

Table 1b Synthesized compounds with their biological activities
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Experimental

General procedures

Melting points were recorded on Thermomik Compbell electronics with an oil-heating system and were uncorrected. Fourier-transform infrared (FTIR) spectra were recorded on Buck Scientific infrared spectroscopy M500 spectrophotometer using KBr pellets. All NMR spectra were recorded on a FT-NMR JEOL, 60 MHz or JEOL AL 300 MHz spectrometer. The mass spectrum was recorded on Waters Micromass spectrometer. Chemical shifts are expressed in δ units relative to tetramethyl silane (TMS) as internal reference using CDCl3 as solvent.

Procedures for the synthesis of 1-(5-bromothien-2-yl)-3-(4-methylthiophenyl) prop-2-en-1-one (14)

To a solution of 4-(methylthio) benzaldehyde (0.25 g, 0.00164 mol) in methanol (20 ml) was added 10% aqueous NaOH solution (2.0 ml) in a 50-ml round-bottomed flask immersed in an ice bath. 2-Acetyl 5-bromothiophene (0.337 g, 0.00164 mol) was added dropwise over a period of 15–30 min and the reaction mixture was stirred for 3–4 h. The precipitate so obtained was filtered and washed with distilled water until the filtrate was neutral to litmus. The final washing with cold methanol gave 14 (0.50 g, 89.76 %), m.p. 124–125°C. IR (KBr) 3061.3, 1641.8, 1578.6, 1414.4, 1076, 809 cm−1. 1H-NMR (300 MHz, CDCl3): δ 7.13338 – 7.8096 (m, 8 H, Ar–H and HC=CH), δ 2.5139 (s, 3H, SCH3). 13C-NMR (300 MHz, CDCl3): δ 15.0603, 119.3151, 122.6691, 125.8834, 128.8922, 130.8898, 130.9227, 131.3337, 131.6132, 142.8345, 144.0758, 147.1832, 180.7812. MS (m/z): 339.13 [M]+, 341.14 [M + 2]+.

Procedures for the synthesis of 1-(5-bromothiophen-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (16)

3,4-Dimethoxybenzaldehyde (0.25 g, 0.0015 mol) was reacted with 2-acetyl-5-bromothiophene (0.189 g, 0.0015 mol) using the general procedure to obtain chalcone 16 (0.456 g, 86.08%), m.p. 130–132°C, IR (KBr) 1655.9, 1607.8, 1076.2, 811.6 cm−1. 1H-NMR (300 MHz, CDCl3): δ 7.755–7.806 (d, 1 H, Ar–H), 7.580–7.593 (d, 1 H, Ar–H), 7.124–7.261 (m, 4H, Ar–H and HC=CH), 6.874–6.902 (d, 1H, HC=CH), 3.924–3.943 (s, 6 H, OCH 3). 13C-NMR (300 MHz, CDCl3): δ 55.983, 56.024, 110.165, 111.127, 118.296, 122.439, 123.368, 127.486, 131.293, 131.506, 144.766, 142.274, 149.255, 151.663, 180.847. MS (m/z): 353.18 [M]+, 355.19 [M + 2]+.

Procedures for the synthesis of 3-(biphenyl-4-yl-1-(5-bromothien-2-yl)-)prop-2-en-1-one (18)

Biphenyl-4-carboxaldehyde (0.25 g, 0.00137 mol) was reacted with 2-acetyl 5-bromothiophene (0.28 g, 0.00137 mol) using the general procedure to obtain chalcone 18 (0.43 g, 87.22 %), m.p. 176–178°C. IR (KBr): 3050.9, 1643.3, 1581.0, 1412.6, 763.1 cm–1. 1H-NMR (300 MHz, CDCl3): δ 7.1436–7.9279 (m, 13 H, Ar–H and HC=CH), 13C-NMR (300 MHz, CDCl3): 120.2358, 120.2687, 122.8335, 127.0590, 127.6262, 127.9797, 128.9415, 129.0730, 131.3748, 131.7612, 133.4464, 140.0230, 143.5579, 144.1580, 147.1421, 180. 8058. MS: MS (m/z): 369.24 [M]+, 371.19 [M + 2]+.

Biological activity

The synthesized compounds were evaluated for their in vitro antifungal activity against fluconazole-sensitive and fluconazole -resistant strains using broth dilution technique. Dimethyl sulfoxide (DMSO) was used for initial dilution. The organisms employed for the in vitro testing were Candida albicans ATCC 10231 (fluconazole sensitive) and Candida albicans NCIM 3446 (fluconazole resistant). The inoculum was prepared according to the standard procedure (Barry 1976). The medium was prepared using appropriate quantities of Saboraud’s dextrose broth. The optical density of the compounds, standard drug, and positive and negative controls of DMSO was also recorded. Different concentrations of the test compound were prepared and added to the inoculated test tubes. The inoculated test tubes containing different concentration of drugs were maintained at room temperature for 48 h in a dark room at 37°C. At the end of the incubation period, the results were interpreted by comparison with negative controls.

Conclusion

A series of α,β-unsaturated ketones (chalcones) were successfully synthesized and characterized spectroscopically by 1H-NMR and IR. Few of the chalcones showed appreciable antifungal activity against fluconazole-sensitive and fluconazole-resistant organisms, with chalcone 13 exhibiting the highest activity. The information obtained in this study provides a tool for designing better compounds, and for guiding further structural modification and synthesizing potent new antifungal agents.