Introduction

Morbidity and mortality because of enteric bacterial infection are the major health problems in some areas like Indian subcontinent, portions of South America and tropical fraction of Africa (Qadri et al., 2005; Devasia et al., 2006). Every year millions of people are being killed by some or the other Gram-positive and Gram-negative strains of bacteria. These bacteria mostly lead to food poisoning, rheumatic, salmonellosis and diarrhoea (Khan et al., 2008). In addition, drug resistance is being developed by these bacteria against the commonly used antimicrobial agents which are being extensively used for the treatment of above diseases. Furthermore, the pharmacological drugs available are either too expensive or have undesirable side effects or contraindications (Berger, 1985). Many traditional plant treatments for the antimicrobial infections exist, and there lies a hidden wealth of potentially useful natural products for the control of microbial diseases (Gray and Flatt, 1997). Natural plant drugs are frequently considered to be less toxic and free from side effects than synthetic ones (Morin, 1987). Among the most significant classes of natural compounds, an important position is occupied by oxygen-containing heterocyclic compounds. 7-Hydroxy coumarin (7-HC) is a benzopyrone in nature, which is a major human metabolite and plays a role as dietary antioxidant in the human diet (fruits and vegetables). 7-HC has been reported to have antitumor (Kofinas et al., 1998), aldose reductase inhibitor (Okada et al., 1995) and xanthine oxidase inhibitor (Mills and Bone 2000) activities. The parent compound coumarin has been reported to reduce blood glucose levels (Marles and Farnsworth, 1996). A number of natural and synthetic coumarin (2-oxo-2H-chromene) derivatives have been reported to exert antimicrobial (Czerpack and Skolska, 1982; Jund et al., 1971), antifungal (El-Ansary et al., 1992; Reddy and Somayojulu, 1981), antiamoebic (Iqbal et al., 2009) and tuberculostatic (Abd Allah, 2000) activities. Moreover, the antibiotic novobiocin belongs to the hydroxy coumarin series. Coumarin may be a prodrug and 7-HC is the pharmacologically active agent (Ritschel et al., 1981). Many seed oils, fatty acids (FA) and their derivatives are known for their antimicrobial (Rauf and Parveen, 2005; Kabara et al., 1972), antifungal (Ahmed et al., 1985) and pesticidal (Khan et al., 1983) activities. A number of investigations have demonstrated that a variety of modified FA are promising molecules in cancer prevention and have potential in the treatment of cancers (Mujeebur-Rahman et al., 2005; Nagao et al., 1991; Lie Ken Jie et al., 1990). FA-ester analogs have received very little attention despite the fact that such molecules have been found to be associated with diverse biological activities such as antioxidant (Viklund et al., 2003), antifeedant (Mallavadhani et al., 2003), anti-inflammatory (Feng et al., 2009), antiparasitic (Grunberg et al., 1973), antimicrobial (Mod et al., 1975) and neuroprotective (Takahashi et al., 2003). Some fatty esters have been found to be very effective for the treatment of dermatitis (Csóka et al., 2007) cardiovascular, hepatic and renal disorders (Greelings et al., 2003). Thus, FA-esters may lead to a new route to potential pharmaceutical molecules. Literature reveals that long-chain fatty acids have not been esterified with 7-HC in presence of DCC and DMAP.

The purpose of this study is to find the novel bioactivity of 7-HC esters. As discussed, the coumarin derivatives possess antimicrobial activity and in view of the significance of long-chain FA as potential pharmacophores; we herein report the synthesis and spectral studies of new coumarin derivatives containing C11 and C18 FA chain. These derivatives were further tested for their in vitro antimicrobial activity against a panel of Gram-positive, Gram-negative strains of bacteria and selected strains of fungus. FA incorporated at the C7 hydroxy group of coumarin are undec-10-enioc, (9Z)-octadec-9-enoic, (9Z, 12R)-12-hydroxyoctadec-9-enoic (ricinoleic) and (9R, 12Z)-9-hydroxyoctadec-12-enoic (isoricinoleic) acids (Scheme 1).

Scheme 1
scheme 1

Synthetic route for the target compounds

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Results and discussion

Chemistry

FA and their derivatives have been reported as antimicrobial agents. It is expected that the incorporation of the hydroxyl and non-hydroxyl FA chain may increase the antimicrobial activity of certain organic moieties. This study is based on the synthesis, characterization and evaluation of antimicrobial activities of 7-HC derivatives derived from different FA. 7-Hydroxy coumarin reacts with fatty acid in the presence of DCC and 4-dimethyl aminopyridine (DMAP) in dichloromethane by stirring at room temperature. The completion of the reaction was checked by thin layer chromatography (TLC). The reaction time varied from 2 to 3 h. The purity of the compounds was checked by TLC and the compounds under the study were characterized by the spectral data. In general, the IR spectra showed ester peak at 1729 cm−1 and CH2 (methylene) peak at 2921 and 2849 cm−1. In the 1H-NMR spectra, the signals of the respective protons of the prepared derivatives were verified on the basis of their chemical shifts, multiplicities and coupling constants. The spectra of all the compounds showed peaks at δ 6.39–7.69 ppm corresponding to the protons of 7-hydroxy coumarin. The 13C-NMR spectra of all the compounds were also in good agreement. Characteristic molecular ion peaks [M + Na] was observed for all the compounds under study. A detailed spectral description for compound 6 is discussed below.

IR spectrum of compound 6 revealed characteristic band at 1729 cm−1 (ester C–O stretching). In the 1H-NMR the olefinic protons, C11′H2=C10′H were observed at δH 5.81 (tdd, 1H, J 10′−9′ = 6.8, \( J_{{10^{\prime} - H_{Z} }} = 1 6. 8 \), \( J_{{10^{\prime} - H_{E} }} = 10. 4 \), CH2=CH–), 4.99 (dd, 1H, \( J_{{H_{Z} - 10^{\prime}}} = 1 6. 8 \), \( J_{{H_{Z} - H_{E} }} = 3. 2 \), H Z C=CH), 4.93 (dd, 1H, \( J_{{H_{E} - 10^{\prime}}} = 10. 4 \), \( J_{{H_{E} - H_{Z} }} = 3. 2 \), H E C=CH–) and were correlated with observations in the 13C-NMR which gave signals at δC 139.18 and 114.24, respectively. Besides these a characteristic carbon signal for the fatty acid chain at δC 171.68 (C1′, ester C=O) was recorded. Similarly other compounds were characterized from their spectral data. Spectral studies have illustrated that the change in the nature of FA at C7 has not significantly influenced the pattern of proton and carbon signals of the 7-HC moiety.

Pharmacology

A variety of acylated 7-hydroxycoumarins were investigated in the early 1970s for antibiotic and antifungal activities (Jund et al., 1971). Acyl groups in the study included acetyl, propanoyl and butanoyl. These compounds were found to possess activity against a number of fungal strains (MIC approximately 125 μg/ml) and Gram-negative bacteria (MIC sub 500 μg/ml). It was observed that the ester derivatives of 7-hydroxycoumarins increased the antimicrobial activity of 7-hydroxycoumarin. Thus, it is expected that the use of long-chain hydroxyl and non-hydroxyl fatty acid groups can further increase the antimicrobial potency of the coumarin ester derivatives.

The determination of MIC of synthesized compounds against bacterial and fungal strains was performed by macro dilution test and the results were recorded visually and spectrophotometrically. The investigation of antibacterial screening data (Table 5) revealed that all the tested compounds showed moderate to good bacterial inhibition. Compound 9 showed good inhibition against S. pyogens, S. aureus and E. coli species at 32 μg/ml where as compound 8 showed good inhibition against S. aureus at 32 μg/ml concentrations. Compounds 8 and 9 showed good antibacterial activity nearly equivalent to that of standard drug (Chloramphenicol) against most of the bacterial strains.

The investigation of antifungal screening data revealed that all the tested compounds showed moderate to good fungal inhibition. Compound 8 showed good antifungal activity against all strains of fungi. Compound 9 exhibited antifungal activities nearly equivalent to that of standard drug (Fluconazole) against C. parapsilosis at 8 μg/ml, against C. neoformans at 16 μg/ml and C. albicans at 2 μg/ml.

One of the reasons for activity difference may be based on the several unique characteristics of Gram-negative bacteria such as the structure of the outer membrane. The outer leaflet of the membrane comprises a complex lipopolysaccharide whose lipid portion acts as an endotoxin. This outer membrane protects the bacteria from several antibiotics, dyes and detergents which would normally damage the inner membrane or cell wall (peptidoglycan). The outer membrane provides these bacteria with resistance to lysozyme and penicillin. That is why most of the times Gram-negative bacteria have higher MIC values as compared to Gram-positive bacteria. In case of antifungal activity, Candida albicans are generally susceptible for most of the antifungals while non-albicans like Candida krusei, Candida parapsilosis, Cryptococcus neoformans are resistant to most of the antifungal drugs. Furthermore, the compounds having a hydroxyl group in the alkenyl side chain showed greater activity.

Experimental

Chemicals and instruments

Undec-10-enioc (purity 98%) and (9Z)-octadec-9-enoic (97%) acids were purchased from Fluka Chemicals (Bucks, Switzerland). (9Z, 12R)-12-Hydroxyoctadec-9-enoic (ricinoleic, 98%) acid and (9R, 12Z)-9-hydroxyoctadec-12-enoic (isoricinoleic, 98%) acid were isolated from Ricinus communis and Wrightia tinctoria seed oils, respectively, following Gunstone’s (1954) partition procedure. 7-Hydroxy-coumarin was purchased from S-d fine-chem. (Mumbai, India). Thin layer chromatography was done on glass plates (20 × 5 cm) with a layer of silica gel G (Merck, Mumbai, India, 0.5-mm thickness). Mixture of petroleum ether–ethyl acetate–acetic acid (50:50:1, v/v) were used as developing solvents. Column chromatography was carried out on silica gel (Merck, Mumbai, India, 60–120 mesh). 1H NMR was recorded with Bruker DRX 400 spectrometer at 400 MHz and 13C NMR was recorded at 100 MHz in CDCl3. Chemical shifts (δ) are quoted in ppm. Melting points were taken in open capillary and are uncorrected.

Chemistry: synthesis of fatty acid derivatives of 7-hydroxy-coumarin

A solution of FA (5 mmol), DCC (5.5 mmol) and 7-hydroxy-coumarin (5 mmol) in dichloromethane (50 ml) with catalytic amount of DMAP were stirred mechanically at room temperature until esterification was complete. The N,N-dicyclohexylurea was filtered off and the filtrate was washed with water (3 × 50 ml), 5% acetic acid (3 × 50 ml) again with water (3 × 50 ml) and then dried over anhydrous sodium sulphate. The solvent was removed under reduced pressure to give the esters 6–9 (Scheme 1) which were chromatographed over a column of silica gel using n-hexane–ethyl acetate (94:6, v/v) as eluent. All these novel compounds were characterized from their spectral data. 1H, 13C NMR and COSY spectra of synthesized compounds shown in Tables 1, 2, 3, 4.

Table 1 1H-NMR, 13C-NMR and COSY data of (6) in CDCl3
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Table 2 1H-NMR, 13C-NMR and COSY data of (7) in CDCl3
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Table 3 1H-NMR, 13C-NMR and COSY data of (8) in CDCl3
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Table 4 1H-NMR, 13C-NMR and COSY data of (9) in CDCl3
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7-O-Coumarinyl undec-10-enoate (6)

White powder; Yield: 91%; mp: 168°C; Rf: 0.59; IR (KBr): 3080 (C=C aromatic ring), 2921 (C–H asymm.), 2849 (C–H symm.), 1729 (C=O ester), 1624 (C=O coumarin carbonyl), 1400 (C–O), 1269 (C=C), 1122 (C–H aromatic ring) cm−1. ESI–MS found [M + Na]+ 351.2; C20H24O4 [M + Na]+ requires 351.17.

7-O-Coumarinyl (9Z)-octadec-9-enoate (7)

Viscous solid; Yield: 89%; Rf: 0.57; IR (KBr): 3120 (C=C aromatic ring), 2921 (C–H asymm.), 2849 (C–H symm.), 1728 (C=O ester), 1651 (C=O coumarin carbonyl), 1405 (C–O), 1270 (C=C), 1122 (C–H aromatic ring) cm−1. ESI–MS found [M + Na]+ 449.3; C27H38O4 [M + Na]+ requires 449.28.

7-O-Coumarinyl (9Z, 12R)-12-hydroxyoctadec-9-enoate (8)

Viscous solid; Yield: 85%; Rf: 0.52; IR (KBr): 3384 (O–H), 3080 (C=C aromatic ring), 2920 (C–H asymm.), 2858 (C–H symm.), 1732 (C=O ester), 1618 (C=O coumarin carbonyl), 1401 (C–O), 1267 (C=C), 1122 (C–H aromatic ring) cm−1. ESI–MS found [M + Na]+ 465.3; C27H38O5 [M + Na]+ requires 465.27.

7-O-Coumarinyl (12Z,9R)-9-hydroxyoctadec-12-enoate (9)

Viscous solid; Yield: 87%; Rf: 0.53; IR (KBr): 3421 (O–H), 3002 (C=C aromatic ring), 2920 (C–H asymm.), 2852 (C–H symm.), 1736 (C=O ester), 1639 (C=O coumarin carbonyl), 1396 (C–O), 1270 (C=C), 1122 (C–H aromatic ring) cm−1. ESI–MS found [M + Na]+ 465.3; C27H38O5 [M + Na]+ requires 465.27.

Pharmacology

Antibacterial studies

The minimum inhibitory concentration (MIC) was assessed by the macro dilution test using standard inoculums of 5 × 105 c.f.u./ml. Initially, the compounds were dissolved in DMSO after that serial dilution of the test compounds were set to final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg/ml. To each tube was added 100 μl of 24-h old inoculums. The growth was monitored visually and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as minimum inhibitory concentration (MIC). The minimum inhibitory concentrations are given in Table 5.

Table 5 Minimum inhibition concentration (MIC) fatty acid analogs of 7-hydroxy coumarin
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Antifungal studies

The minimum inhibitory concentration (MIC) was assessed by the macro dilution test using standard inoculums of 1.6 × 104-6 × 104 c.f.u./ml. Initially, the compounds were dissolved in DMSO after that serial dilution of the test compounds were set to final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 μg/ml. To each tube was added 100 μl of 48–72-h-old inoculums. The growth was monitored visually and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of fungi was regarded as minimum inhibitory concentration (MIC). The minimum inhibitory concentrations are given in Table 5.

Conclusion

It is conceivable that these derivatives showing antimicrobial activity can be further modified to exhibit better potency than the standard drugs. Compound 9 showed good antibacterial activity nearly equivalent to that of chloramphenicol. The varied divergence in the antimicrobial activity of these compounds validates the reason of this study. Compounds 8 and 9 showed good antifungal activity against all strains of fungi. The importance of such kind of work lies in the possibility that the new compounds might be more efficient against bacteria for which a thorough study regarding the structure–activity relationship, toxicity and in their biological effects would be helpful in designing more effective antimicrobial agents.