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

Article abstract of DOI:10.1016/j.bmcl.2005.02.034

The 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.

Full text of DOI:10.1016/j.bmcl.2005.02.034

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1811–1814
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,*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
bDepartment of Microbiology, Central Food Technological Research Institute, Mysore 570020, India
Received 29 December 2004; revised 10 February 2005; accepted 12 February 2005
Abstract—The trans rich isomer, 2-(phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-butylate isoxazolidine A (>96% ee) was synthe-
sized 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 Nys-
tatin 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
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 5/50 (A, Scheme 1).
In recent years, there is an alarming increase in life
threatening microbial infections especially in immuno-
compromised individuals suffering from AIDS, cancer,
etc.1–4 There has been a constant effort by the research-
ers to develop more effective and safe antimicrobial
drugs to combat with microbial infections.5–9 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 resis-
tant strains, pharmacokinetic differences, and/or insuffi-
ciencies in their antimicrobial activity.10 Therefore, a
great need for a more potent and broad spectrum anti-
microbial agents with reduced side effects.11
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 cycloaddi-
tion of nitrone 4 (E > 98% ee) with butyl acrylate gave
a mixture of regioisomers, 5/50 and 6/60. The major iso-
mer 5/50 (A, >96% ee) was separated on silica gel column
(Scheme 1).
We previously reported12–15 the synthesis and biological
activities of isoxazolidines constructed via 1,3-dipolar
cycloaddition reactions of nitrones with olefins. We re-
cently reported the synthesis and antimicrobial proper-
ties 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-chloro-
1H-imidazolyl)-5-butylate isoxazolidine (B, Scheme 2)
Nitrone 4 (E/Z = 65/35) was synthesized by the reduc-
tion 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 iso-
mers, 4(E)/40(Z) in 65/35 ratio evaluated by 400 MHz
1H NMR: E and Z isomers showed singlet at d 7.97
and 7.32, respectively for CH@N bond. Thus poor selec-
tivity in isomer ratio was observed in the method re-
ported previously. In the present method (Scheme 1), 2
was condensed directly with 1 in the presence of Bron-
sted acid 3/MgCl2 catalyst17 and gave E isomer rich nit-
Keywords: Antifungal activity; Synthesis; Nitrone; Isoxazolidine.
*
1
rone 4 (>98% ee) evaluated by 400 MHz. H NMR (E
isomer showed singlet peak at d 7.97 for CH@N bond).
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

1812
M. P. Sadashiva et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1811–1814
NHOH
Cl
N
COOBu
CHO
NH
H
Cl
Cl
N
H2C
N
O
O
3
a
C
N+
Cl
+
N
N
O-
b
COOBu
Bu
N
NH
N
H
N
HBuOOC
Bu
Bu
1
2
Bu
5/5' (major) & 6/6' (minor)
4
E>98%ee
column
CF
3
CF
3
Cl
N
Cl
O
OH
OH
N
O
N
3
N
COOBu
COOBu
N
H
N
H
CF
Bu
3
Bu
A, 5/5' >96%ee (trans)
CF
3
Scheme 1. Reagents and conditions: (a) MgCl2, methylene dichloride; (b) toluene, reflux, 38 h.
COOBu
H
NO2
Cl
CHO
Cl
N+
O-
b
column
a
5/5' & 6/6'
N
>69%ee (trans)
5/5'
+
NH
N
NH
Bu
Bu
B
1
7
E/Z=65/35
4
Scheme 2. Reagents and conditions: (a) H2O:ethanol:DMF (7:2:1), zinc dust, ꢀ5 ꢁC, pH 7.2–7.4; (b) toluene, reflux, 38 h.
Thus, 3/MgCl2 catalytic system greatly enhanced the E
isomeric ratio of 4 from >65% ee to >98% ee. The 1,3-
cycloaddition of 4 (E > 98% ee) with butyl acrylate in in-
ert solvent gave a mixture of 5- and 4-substituted regio-
isomers, 5/50 and 6/60. After separation on silica gel
column, the major stereoisomers, 5/50 (A) ratio showed
high stereoselectivity (>96% ee) evaluated by the inte-
used for the cultivation of bacteria. The composition
of BHIA is—calf brain infusion (solids) 12.5 g/L, beef
heart infusion (solids) 5 g/L, peptic digest of animal tis-
sue 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 ex-
tracts 200.0 g/L, dextrose 2.0 g/L, agar 20.0 g/L and dis-
tilled water 1 L. Except agar, the composition of PDB is
similar to PDA.
1
gration from 400 MHz H NMR spectra.
3. Material and methods
3.1. Microorganisms used
3.3. Antimicrobial assay
Bacteria: Bacillus subtilis (ATCC-23857D), Escherichia
coli (ATCC-700926D), and Staphylococcus aureus
(ATCC-10832D).
For antimicrobial assays, the compounds were dissolved
in dimethyl sulfoxide (DMSO) (1 mL) and the solution
was diluted with distilled water (9 mL). Further progres-
sive 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 final con-
centration of 2 · 103 colony forming units (CFU) per
millilitre. After inoculation the culture tubes were sha-
Fungi: Aspergillus niger (ATCC-1004), Cephalosporium
acremonium (ATCC-10141), and Fusarium moniliforme
(ATCC-10052).
3.2. Culture media
Media were prepared according to the manufacturerÕs
(Hi-media) instructions. Brain heart infusion agar
(BHIA) and brain heart infusion broth (BHIB) were

M. P. Sadashiva et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1811–1814
1813
ken well and bacterial cultures were incubated at 37 ꢁC
for 24 h, whereas fungal cultures were incubated at
28 ꢁC for 48 h.
tive when compared with the control as they showed
approximately double the MIC values of control with
corresponding bacteria. However, results with anti-
fungal studies are interesting. A was found to be more
effective when compared with B, which required two
to 3-fold increase in concentration as compared with
the control as well as A to be effective against the fungal
species. The antifungal effectiveness 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 activ-
ity of A when compared with B may be due to the stereo
chemical structural difference between the two com-
pounds, which might play a vital role in exhibiting anti-
fungal activity.
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:
% inhibi-
tion = 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
confirmed 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 specifications and growth (if any) was
carefully examined under microscope. The MIC was de-
fined, as the lowest concentration of substance at which
there was no growth.
In conclusion, the preparation of 2-(phenyl)-3-(2-butyl-
4-chloro-1H-imidazolyl)-5-butylate isoxazolidine of
>96% ee has enabled the exploration of its antimicrobial
activity. As expected, A showed greater antifungal activ-
ity than B because of its higher ee purity. Thus the re-
sults are encouraging to better define and optimize the
antifungal activity of 2-(phenyl)-3-(2-butyl-4-chloro-
4. Results and discussion
1H-imidazolyl)-5-butylate
isoxazolidine.
Further
improvement in ee purity of A is currently under
The antimicrobial activity of A and B was evaluated and
compared with controls as shown in Table 1. Com-
pounds A and B showed antibacterial properties with
varied MIC values and they were less effective when
compared with reference control streptomycin. On an
average, double the concentration of compounds (both
A and B) was required for complete inhibition of bacte-
ria 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 difference in MIC val-
ues with respect to individual species is not noteworthy.
progress.
5. Experimental
5.1. General
Melting points were determined on SELACO-650
instrument and are uncorrected. The IR was recorded
on a Perkin–Elmer model RX-1 FT-IR spectrophoto-
1
meter. The H NMR spectra were recorded on Bruker
Avance-400 spectrometer at 400 MHz using TMS as
an internal standard and CDCl3 as solvent. The chemi-
cal shift values are on the d scale and the coupling con-
stant 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 identified by comparison
of their spectra and physical data with those of authentic
samples.
With respect to fungi, though both the compounds
exhibited antifungal properties, their effectiveness 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 concen-
tration when compared to control and A. In case of
F. moniliforme, A was most effective with MIC value
13 lg/mL, which is even better than the nystatin
(14 lg/mL). Here, the concentration of B had to be in-
creased twofolds when compared to the control to get
complete inhibition.
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 synthe-
sized according to the procedure reported earlier.16
In general, with respect to bacteria, both the compounds
A and B exhibited almost similar MIC and are less effec-
Table 1. The MIC (lg/mL) values of isoxazolidine 5/50 of 96% ee (A) and 69% ee (B)
Isoxazolidine 5/50
Bacteria
Fungi
E. coli
B. subtilis
S. aureus
A. niger
C. acremonium
F. moniliforme
Controla
6.0
10.0
11.0
3.0
7.0
7.0
3.0
7.0
8.0
12.0
14.0
36.0
11.0
11.0
33.0
14.0
13.0
28.0
A, >96% ee
B, >69% ee
a Streptomycin for bacteria and nystatin for fungi.

1814
M. P. Sadashiva et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1811–1814
The E/Z isomers mixture (yellow oil, 0.57 g, 51%) was
obtained from nitrobenzene 7 (0.41 mL, 4.06 mmol)
(KBr): 3247, 3062, 2959, 2873, 1730, 1598, 1258, 1085,
825, 757, 694, 598 cmꢀ1. Anal. Calcd CHN: 62.14,
6.95, 10.35. Found: 62.19, 7.06, 10.38.
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), 7.32–7.48 (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 cmꢀ1. Anal. Calcd CHN: 60.54, 5.81, 15.13.
Found: 60.63, 5.84, 15.18.
Acknowledgements
We are grateful to the University of Mysore, Mysore,
India for financial support under the UPG-JRF order
No. DV-5/57/U.P.G.F./2003–2004. The CHNS and
UV–Vis data obtained from this department with equip-
ments funded by DST-FIST (No. SR/FST/CSI-051/
2002. Dated 28.7.2003) grant.
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 phe-
nyl 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 42–44 ꢁ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),
7.32–7.72 (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 cmꢀ1. Anal. Calcd CHN: 60.54, 5.81,
15.13. Found: 60.63, 5.84, 15.18.
References and notes
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5.2.3. Procedure for the synthesis of 2-(phenyl)-3-(2-
butyl-4-chloro-1H-imidazolyl)-5-butylate isoxazolidine. A
mixture of 4 and butyl acrylate in toluene was refluxed
until reaction completes monitored by TLC. The reac-
tion mixture was cooled to room temperature and
concentrated under reduced pressure. The major
regioisomer 5/50 was separated on silica gel column by
eluting with hexane/ethylacetate (9:1).
11. Graybill, J. R. Clin. Infec. Dis. 1992, 22(Suppl 2), S166.
12. Ravikumar, K. R.; Mallesha, H.; Basappa; Rangappa,
K. S. Eur. J. Med. Chem. 2003, 38, 163.
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14. Ravikumar, K. R.; Mallesha, H.; Basappa; Rangappa,
K. S. J. Heterocycl. Chem. 2003, 40, 607.
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appa, K. S. Bioorg. Med. Chem. 2004, 12, 6389.
17. McDougal, Nolan T.; Schaus, Scott E. J. Am. Chem. Soc.
2003, 125, 12094–12095.
The isomer A: 1H NMR (CDCl3, 400 MHz): d 0.74–1.08
(m, 6H, CH3), 1.11–1.49 (m, 4H, CH2), 1.59–1.75 (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 cmꢀ1
.
Anal. Calcd CHN: 62.14, 6.95, 10.35. Found: 62.19,
7.06, 10.38.
18. Beeranahally Doreswamy, H.; Basappa; Madegowda, M.;
Mantelingu, K.; Sridhar Anandalwar, M.; Javaregowda
Prasad; Rangappa, K. S. Anal. Sci. 2003, 19, 31.
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Mantelingu, K.; Rangappa, K. S. Indian Acad. Sci. 2001,
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20. Collins, C. H.; Lyne, P. M. Microb. Methods; Butter
worth: London, 1970, p 21.
21. (a) Elhance, D. N. Fundamental of Statistics; 30th ed.,
1984; (b) Coltan, T. Statistics in Medicine; 1st ed., 1974.
1
The isomer B: brownish thick liquid (59%). H NMR
(CDCl3, 400 MHz): d 0.81–1.01 (m, 6H, CH3), 1.22–
1.42 (m, 4H, CH2), 1.55–1.82 (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), 6.95–7.15 (m, 3H, Ar-H),
7.22–7.35 (m, 2H, Ar-H), 10.62 (br s, 1H, NH). IR