Bioorganic & Medicinal Chemistry Letters 15 (2005) 18111814

Enhancement in antimicrobial activity of 2-(phenyl)-3(2-butyl-4-chloro-1H-imidazolyl)-5-butylate isoxazolidine

M. P. Sadashiva,a H. Mallesha,a K. Karunakara Murthyb and K. S. Rangappaa,*
a b

Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India Department of Microbiology, Central Food Technological Research Institute, Mysore 570020, India
Received 29 December 2004; revised 10 February 2005; accepted 12 February 2005

AbstractThe trans rich isomer, 2-(phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-butylate isoxazolidine A (>96% ee) was synthesized by the condensation of E isomer rich nitrone 4 (>98% ee) with butyl acrylate in an inert solvent. Obtained isoxazolidine was screened for its antifungal activity against Aspergillus niger, Cephalosporium acremonium, Fusarium moniliforme by using Nystatin as positive control. It was also tested for its antibacterial activity against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus by using Streptomycin as positive control. Enhanced antifungal activity was observed in isoxazolidine of >96% ee compared to the isoxazolidine of >69% ee (B), and enhancement was not observed in antibacterial activity. 2005 Elsevier Ltd. All rights reserved.

1. Introduction In recent years, there is an alarming increase in life threatening microbial infections especially in immunocompromised individuals suering from AIDS, cancer, etc.14 There has been a constant eort by the researchers to develop more eective and safe antimicrobial drugs to combat with microbial infections.59 Despite the development of several new antimicrobial agents, their clinical value is limited to treat an increasing array of life threatening systemic infections because of their relatively high risk of toxicity, emergence of drug resistant strains, pharmacokinetic dierences, and/or insuciencies in their antimicrobial activity.10 Therefore, a great need for a more potent and broad spectrum antimicrobial agents with reduced side eects.11 We previously reported1215 the synthesis and biological activities of isoxazolidines constructed via 1,3-dipolar cycloaddition reactions of nitrones with olens. We recently reported the synthesis and antimicrobial properties of a series of novel 5-imidazolyl substituted isoxazolidines of moderate regioselectivity (up to 69% ee).16 Among tested, 2-(phenyl)-3-(2-butyl-4-chloro1H-imidazolyl)-5-butylate isoxazolidine (B, Scheme 2)
Keywords: Antifungal activity; Synthesis; Nitrone; Isoxazolidine. * Corresponding author. Tel.: +91 821 2412191; fax: +91 821 2412191/ 2421263; e-mail: rangappaks@yahoo.com 0960-894X/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2005.02.034

showed comparatively promising antimicrobial activity. Therefore, we thought of preparing high regioselective isomer of B to screen its antimicrobial activity. We herein report the synthesis and antimicrobial activity of high regioselective isoxazolidine 5/5 0 (A, Scheme 1). 2. Chemistry Condensation of hydroxylamine 2 with aldehyde 1 in the presence of Bronsted acid catalyst17 3 to yield E isomer rich nitrone 4 (E > 98% ee). The 1,3-dipolar cycloaddition of nitrone 4 (E > 98% ee) with butyl acrylate gave a mixture of regioisomers, 5/5 0 and 6/6 0 . The major isomer 5/5 0 (A, >96% ee) was separated on silica gel column (Scheme 1). Nitrone 4 (E/Z = 65/35) was synthesized by the reduction of a mixture of 7 and imidazole aldehyde 118 with zinc dust using histidine as a catalyst (Scheme 2).19 The crude product of nitrone showed mixture of isomers, 4(E)/4 0 (Z) in 65/35 ratio evaluated by 400 MHz 1 H NMR: E and Z isomers showed singlet at d 7.97 and 7.32, respectively for CH@N bond. Thus poor selectivity in isomer ratio was observed in the method reported previously. In the present method (Scheme 1), 2 was condensed directly with 1 in the presence of Bronsted acid 3/MgCl2 catalyst17 and gave E isomer rich nitrone 4 (>98% ee) evaluated by 400 MHz. 1H NMR (E isomer showed singlet peak at d 7.97 for CH@N bond).

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Cl N Bu

NHOH

CHO H NH +

COOBu

3 a 2

Cl N

C NH

H2C

Cl N O N COOBu Bu N H Bu

Cl N O

O-

N HBuOOC

Bu

4 E>98%ee
C F3

5/5' (major) & 6/6' (minor) column

C F3

Cl
OH

Cl N N O COOBu N COOBu Bu N H O

OH

N
C F3

Bu

N H

A, 5/5' >96%ee (trans)

C F3

Scheme 1. Reagents and conditions: (a) MgCl2, methylene dichloride; (b) toluene, reux, 38 h.

COOBu
Cl N CHO NO2 H

NH
Bu

Cl N

N+ O-

NH
Bu

5/5' & 6/6'

column

5/5' >69% ee (trans)

E/Z=65/35 4

Scheme 2. Reagents and conditions: (a) H2O:ethanol:DMF (7:2:1), zinc dust, 5 C, pH 7.27.4; (b) toluene, reux, 38 h.

Thus, 3/MgCl2 catalytic system greatly enhanced the E isomeric ratio of 4 from >65% ee to >98% ee. The 1,3cycloaddition of 4 (E > 98% ee) with butyl acrylate in inert solvent gave a mixture of 5- and 4-substituted regioisomers, 5/5 0 and 6/6 0 . After separation on silica gel column, the major stereoisomers, 5/5 0 (A) ratio showed high stereoselectivity (>96% ee) evaluated by the integration from 400 MHz 1H NMR spectra. 3. Material and methods 3.1. Microorganisms used

used for the cultivation of bacteria. The composition of BHIA iscalf brain infusion (solids) 12.5 g/L, beef heart infusion (solids) 5 g/L, peptic digest of animal tissue 10.0 g/L, sodium chloride 5.0 g/L, dextrose 2.0 g/L, agar 20.0 g/L, distilled water 1 L, and pH 7.4 0.2 (25 C). Apart from agar, the composition of BHIB is similar to BHIA. The fungal species were cultured in potato dextrose agar (PDA) and potato dextrose broth (PDB) media. The composition of PDA is potato extracts 200.0 g/L, dextrose 2.0 g/L, agar 20.0 g/L and distilled water 1 L. Except agar, the composition of PDB is similar to PDA. 3.3. Antimicrobial assay

Bacteria: Bacillus subtilis (ATCC-23857D), Escherichia coli (ATCC-700926D), and Staphylococcus aureus (ATCC-10832D). Fungi: Aspergillus niger (ATCC-1004), Cephalosporium acremonium (ATCC-10141), and Fusarium moniliforme (ATCC-10052). 3.2. Culture media Media were prepared according to the manufacturers (Hi-media) instructions. Brain heart infusion agar (BHIA) and brain heart infusion broth (BHIB) were

For antimicrobial assays, the compounds were dissolved in dimethyl sulfoxide (DMSO) (1 mL) and the solution was diluted with distilled water (9 mL). Further progressive dilutions with test medium gave the required concentrations in lg/mL. Minimum inhibitory concentrations (MIC) were determined in the culture tube containing 4.5 mL of broth media. Final volume was adjusted to 5 mL after addition of culture media and the required dilution of the compound. Cell/spore suspensions were prepared and adjusted to a nal concentration of 2 103 colony forming units (CFU) per millilitre. After inoculation the culture tubes were sha-

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ken well and bacterial cultures were incubated at 37 C for 24 h, whereas fungal cultures were incubated at 28 C for 48 h. MIC of each compound for bacteria and fungi were determined after incubation period by turbidometric method.20 The absorbance of the culture tubes were recorded at 640 nm and percentage of inhibition was calculated according to the formula: % inhibition = 100(P Q)/P, where, P = absorbance without test sample, Q = absorbance with test sample.21 Then the MIC was recorded as lg/mL. The MIC was further conrmed by removing 10 lL of the contents from each culture tube showing no visible growth and spreading them on to BHIA plates in case of bacteria and PDA plates in case of fungi. The plates were incubated according to the specications and growth (if any) was carefully examined under microscope. The MIC was dened, as the lowest concentration of substance at which there was no growth. 4. Results and discussion The antimicrobial activity of A and B was evaluated and compared with controls as shown in Table 1. Compounds A and B showed antibacterial properties with varied MIC values and they were less eective when compared with reference control streptomycin. On an average, double the concentration of compounds (both A and B) was required for complete inhibition of bacteria when compared with control, which inhibited E. coli at 6 lg/mL and both B. subtilis and S. aureus at 3 lg/ mL. Both the compounds, A and B exhibited similar antibacterial properties and their dierence in MIC values with respect to individual species is not noteworthy. With respect to fungi, though both the compounds exhibited antifungal properties, their eectiveness varied considerably. A. niger was inhibited by A at 14 lg/mL and by control (nystatin) at 12 lg/mL concentration, whereas, for B it was 36 lg/mL which is three times higher than the control. C. acremonium was inhibited at 11 lg/mL concentration by both compound A and nystatin and for B it took a threefold increase in concentration when compared to control and A. In case of F. moniliforme, A was most eective with MIC value 13 lg/mL, which is even better than the nystatin (14 lg/mL). Here, the concentration of B had to be increased twofolds when compared to the control to get complete inhibition. In general, with respect to bacteria, both the compounds A and B exhibited almost similar MIC and are less eec-

tive when compared with the control as they showed approximately double the MIC values of control with corresponding bacteria. However, results with antifungal studies are interesting. A was found to be more eective when compared with B, which required two to 3-fold increase in concentration as compared with the control as well as A to be eective against the fungal species. The antifungal eectiveness of A was on par with the control and in case of F. moniliforme, it is even better than the control. The increase in antifungal activity of A when compared with B may be due to the stereo chemical structural dierence between the two compounds, which might play a vital role in exhibiting antifungal activity. In conclusion, the preparation of 2-(phenyl)-3-(2-butyl4-chloro-1H-imidazolyl)-5-butylate isoxazolidine of >96% ee has enabled the exploration of its antimicrobial activity. As expected, A showed greater antifungal activity than B because of its higher ee purity. Thus the results are encouraging to better dene and optimize the antifungal activity of 2-(phenyl)-3-(2-butyl-4-chloro1H-imidazolyl)-5-butylate isoxazolidine. Further improvement in ee purity of A is currently under progress.

5. Experimental 5.1. General Melting points were determined on SELACO-650 instrument and are uncorrected. The IR was recorded on a PerkinElmer model RX-1 FT-IR spectrophotometer. The 1H NMR spectra were recorded on Bruker Avance-400 spectrometer at 400 MHz using TMS as an internal standard and CDCl3 as solvent. The chemical shift values are on the d scale and the coupling constant j is in Hz. The elemental analyses were obtained on a Vario-EL 111 CHNS, instrument. The analytical TLCs were performed on a coated Merk silca gel 60 F254 plates; the spots were detected either under UV light or by charring with 4% alcoholic H2SO4, followed by evaporation in vacuum. Some of the products are known compounds and were identied by comparison of their spectra and physical data with those of authentic samples. 5.2. Synthesis 5.2.1. E/Z-C-(2-Butyl-4-chloro-1H-imidazolyl)-N-phenyl nitrone (4, E/Z = 65/35). This compound was synthesized according to the procedure reported earlier.16

Table 1. The MIC (lg/mL) values of isoxazolidine 5/5 0 of 96% ee (A) and 69% ee (B) Isoxazolidine 5/5 0 E. coli Controla A, >96% ee B, >69% ee
a

Bacteria B. subtilis 3.0 7.0 7.0 S. aureus 3.0 7.0 8.0 A. niger 12.0 14.0 36.0

Fungi C. acremonium 11.0 11.0 33.0 F. moniliforme 14.0 13.0 28.0

6.0 10.0 11.0

Streptomycin for bacteria and nystatin for fungi.

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The E/Z isomers mixture (yellow oil, 0.57 g, 51%) was obtained from nitrobenzene 7 (0.41 mL, 4.06 mmol) and 2-butyl-4-chloro-1H-imidazolyle aldehyde 1 (0.75 g, 4.02 mmol). 1H NMR (CDCl3, 400 MHz): d 0.76 (t, 3H, CH3), 0.93 (t, 3H, CH3), 1.11 (m, 2H, CH2), 1.38 (m, 2H, CH2), 1.49 (m, 2H, CH2), 1.75 (m, 2H, CH2), 2.36 (t, 2H, CH2), 2.75 (t, 2H, CH2), 7.32 (s, 1H, CH@N), 7.327.48 (Ar-H, merged with isomer), 7.97 (s, 1H, CH@N), 12.13 (br s, 1H, NH), 12.72 (br s, 1H, NH). IR (KBr): 3045, 2863, 2857, 1548, 1176, 893, 740, 660 cm1. Anal. Calcd CHN: 60.54, 5.81, 15.13. Found: 60.63, 5.84, 15.18. 5.2.2. E-C-(2-Butyl-4-chloro-1H-imidazolyl)-N-phenyl nitrone (4, E > 98% ee). This compound was obtained by condensation aldehyde 1 (0.5 g, 2.68 mmol) and phenyl hydroxylamine hydrochloride 2 (0.39 g, 2.68 mmol) in the presence of Bronsted acid/MgCl2 catalyst. The yellow solid (0.49 g, 67.2%) melted at 4244 C. 1H NMR (CDCl3, 400 MHz): d 0.93 (t, 3H, CH3), 1.38 (m, 2H, CH2), 1.75 (m, 2H, CH2), 2.75 (t, 2H, CH2), 7.327.72 (Ar-H, 5H), 7.97 (s, 1H, CH@N), 12.72 (br s, 1H, NH). IR (KBr): 3045, 2863, 2857, 1548, 1176, 893, 740, 660 cm1. Anal. Calcd CHN: 60.54, 5.81, 15.13. Found: 60.63, 5.84, 15.18. 5.2.3. Procedure for the synthesis of 2-(phenyl)-3-(2butyl-4-chloro-1H-imidazolyl)-5-butylate isoxazolidine. A mixture of 4 and butyl acrylate in toluene was reuxed until reaction completes monitored by TLC. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The major regioisomer 5/5 0 was separated on silica gel column by eluting with hexane/ethylacetate (9:1). The isomer A: 1H NMR (CDCl3, 400 MHz): d 0.741.08 (m, 6H, CH3), 1.111.49 (m, 4H, CH2), 1.591.75 (m, 4H,CH2), 2.66 (t, 2H, CH2), 2.43 (ddd, 1H, H4a, J = 13.4, 5.87, 4.7 Hz), 2.30 (ddd, H4b, J = 13.28, 7.96, 5.3 Hz), 3.08 (t, 2H, OCH2), 4.84 (dd, 1H, CH, J = 8.8, 5.4 Hz), 5.12 (dd, 1H, CH, J = 8.4, 4.7 Hz), 7.02 (t, 1H, Ar-H), 7.07 (d, 2H, Ar-H), 7.28 (t, 2H, Ar-H), 10.46 (s, 1H, NH). IR (KBr): 3247, 3062, 2959, 2873, 1730, 1598, 1258, 1085, 825, 757, 694, 598 cm1. Anal. Calcd CHN: 62.14, 6.95, 10.35. Found: 62.19, 7.06, 10.38. The isomer B: brownish thick liquid (59%). 1H NMR (CDCl3, 400 MHz): d 0.811.01 (m, 6H, CH3), 1.22 1.42 (m, 4H, CH2), 1.551.82 (m, 4H, CH2), 2.65 (t, 2H, CH2), 2.43 (ddd, 1H, H4a, J = 13.4, 5.87, 4.7 Hz), 2.99 (ddd, H4b, J = 13.28, 7.96, 5.3 Hz), 3.08 (t, 2H, OCH2), 4.57 (dd, 1H, CH, J = 8.8, 5.4 Hz), 4.82 (dd, 1H, CH, J = 8.4, 4.7 Hz), 6.957.15 (m, 3H, Ar-H), 7.227.35 (m, 2H, Ar-H), 10.62 (br s, 1H, NH). IR

(KBr): 3247, 3062, 2959, 2873, 1730, 1598, 1258, 1085, 825, 757, 694, 598 cm1. Anal. Calcd CHN: 62.14, 6.95, 10.35. Found: 62.19, 7.06, 10.38.

Acknowledgements We are grateful to the University of Mysore, Mysore, India for nancial support under the UPG-JRF order No. DV-5/57/U.P.G.F./20032004. The CHNS and UVVis data obtained from this department with equipments funded by DST-FIST (No. SR/FST/CSI-051/ 2002. Dated 28.7.2003) grant.

References and notes

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