Synthesis and microbial inhibition study of novel 5-imidazolyl substituted isoxazolidines

Article abstract of DOI:10.1016/j.bmc.2004.09.031

Cycloaddition of C-imidazolyl-N-phenylnitrones with monosubstituted alkenes afforded 5-imidazolyl substituted isoxazolidines with high regioselectivity. Novel isoxazolidines were screened for their antibacterial activities against Staphylococcus aureus, Escherichia coli and Bacillus subtilis by using streptomycin as a positive control. They were also tested for their antifungal activities against Fusarium moniliforme, Aspergillus niger and Cephalosporium acremonium by using nystatin as positive control. Isoxazolidines, 4a and 4f exhibited more potent inhibition towards antifungal activity than the other isoxazolidines prepared. Cycloaddition of C-imidazolyl-N-phenylnitrones with monosubstituted alkenes afforded 5-imidazolyl substituted isoxazolidines with high regioselectivity. Novel isoxazolidines were screened for their antibacterial activities against S. aureus, E. coli and B. subtilis by using streptomycin as a positive control. They were also tested for their antifungal activities against F. moniliforme, A. niger and C. acremonium by using nystatin as a positive control. Isoxazolidines, 4a and 4f exhibited more potent inhibition towards antifungal activity than the other isoxazolidines prepared.

Full text of DOI:10.1016/j.bmc.2004.09.031

Bioorganic & Medicinal Chemistry 12 (2004) 6389–6395
Synthesis and microbial inhibition study of novel
5-imidazolyl substituted isoxazolidines
M. P. Sadashiva,a H. Mallesha,a N. A. Hiteshb and K. S. Rangappaa,*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
bDepartment of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
Received 9 August 2004; revised 17 September 2004; accepted 17 September 2004
Available online 5 October 2004
Abstract—Cycloaddition of C-imidazolyl-N-phenylnitrones with monosubstituted alkenes afforded 5-imidazolyl substituted isoxazo-
lidines with high regioselectivity. Novel isoxazolidines were screened for their antibacterial activities against S. aureus, E. coli and B.
subtilis by using streptomycin as a positive control. They were also tested for their antifungal activities against F. moniliforme, A.
niger and C. acremonium by using nystatin as a positive control. Isoxazolidines, 4a and 4f exhibited more potent inhibition towards
antifungal activity than the other isoxazolidines prepared.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
alternative for strong antifungal activity in the substi-
tuted imidazolyl isoxazoline class.11 In our continuing
interest, we recognized an opportunity to apply our
knowledge of 1,3-dipolar species to the synthesis of a
series of novel 5-imidazolyl substituted isoxazolidines
through the 1,3-dipolar cycloaddition reactions of C-
imidazolyl-N-phenylnitrones with monosubstituted
alkenes. The resulting 5-imidazolyl substituted isoxazol-
idines were expected to differ in their chemical and anti-
microbial properties from the known azole derivatives,
thus providing valuable information of a new generation
of antimicrobial agents.
The frequency of microbial infection in man has in-
creased dramatically because of multi-drug resistant
microbial isolates (e.g., fungi and bacteria).1–4 Hence,
the search of novel and active antimicrobial agents to
treat serious microbial infection has increased signifi-
cantly over the last two decades. Currently, physicians
use three major classes of prescription therapies to treat
serious fungal infections:3,5,6 (a) amphotericin B, a poly-
ene; (b) azoles, including ketoconazole, fluconazole and
itraconazole; and (c) the allylamine terbinafine. Azoles
are safer and more water-soluble than amphotericin B.
Hence, the chemistry of imidazoles occupies an extre-
mely important niche possessing diverse pharmacologi-
cal activity7–12 within the family of five-membered
heterocyclic compounds.
2. Chemistry
2-n-Butyl-4-chloro-1H-imidazole-5-carboxaldehyde,
1
was synthesized according to the procedure reported
earlier.23 C-Imidazolyl-N-phenylnitrones, 3/30 were syn-
thesized by the reduction of a mixture of nitro com-
pounds, 2 and imidazoaldehyde, 1 with zinc dust using
histidine as a catalyst.18 The novel 5-imidazolyl substi-
tuted isoxazolidines, 4/40 were prepared by 1,3-dipolar
cycloaddition of nitrones, 3/30 with monosubstituted
olefins as briefed in Scheme 1.
In our previous studies,13–17 we have described the syn-
thesis of biologically active isoxazolidines via 1,3-dipo-
lar cycloaddition reactions of nitrones18–20 with olefins.
Literature survey reveals that imidazolyl moieties are
rarely included in the preparation of isoxazolidines.21,22
Recently, we have identified 3-(2-butyl-4-chloro-1H-imi-
dazolyl)-5-substituted d2-isoxazoline as a new structural
3. Results and discussion
Keywords: Antimicrobial properties; Synthesis; Nitrones; Isoxazolidine
derivatives; Cycloaddition.
*
Arylhydroxylamines of corresponding nitro compounds
were generated in situ and were condensed with 1 to
Corresponding author. Tel.: +91 821 2412191; fax: +91 821 2518835/
2421263; e-mail: rangappaks@chemistry.uni-mysore.ac.in
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.031

6390
M. P. Sadashiva et al. / Bioorg. Med. Chem. 12 (2004) 6389–6395
R
olefins was carried out in refluxing toluene as solvent
and obtained two regioisomeric 5-imidazolyl substituted
isoxazolidines 4 and 40 with high regioselectivity. Ratio
of regioisomers, 4 and 40 was evaluated by integration
H
O
H
N+
O
N+
Cl
N
CHO
+
Cl
Cl
R
NO2
1
NH
N
NH
NH
N
+
from 300MHz H NMR spectra of crude reaction mass
Bu
Bu
Bu
after passing through a short silica gel column using
H/EA (5:1) as eluent (Table 1). The major isomers, 4
were separated either by recrystallization or using
appropriate combination of solvents as eluent as shown
R
E - Isomer
Z - Isomer
3a': R = H
1
2a: R = H
2b: R = Cl
3a: R = H
3b: R = Cl
R
3b': R = Cl
1
R
in the Table 1. Isoxazolidines, 4 showed a set of H
NMR signals at d 2.43–2.74 (ddd), 2.69–3.17 (ddd),
4.32–5.50 (dd) and 4.56–5.93 (dd) for C-4Ha, C-4Hb,
C-3H and C-4H, respectively. The yields of 4 were in
the range of 59–76% with high purity, it is found to
R1
Cl
N
N
O
O
Cl
N
NH
R1
1
be absent of minor isomers, 40 supported by H NMR
N
NH
Bu
4 (Major)
R1
Bu
signals. All novel isoxazolidines 4a–j were structur-
ally characterized by 1H NMR, IR and elemental
analysis.
4' (Minor)
a : R = H
b : R = H
c : R = H
R1 = CO2Me
R1 = CO2Et
R1 = CO2Bu
f : R = Cl
g : R = Cl
h : R = Cl
R1 = CO2Me
R1 = CO2Et
R1 = CO2Bu
4. Antibacterial activity
d : R = H
R1 = Ph
R1 = CO2Ph
i : R = Cl
R1 = Ph
R1= CO2Ph
e : R = H
j : R = Cl
All novel isoxazolidines, 4a–j were screened for their
antibacterial activity in blotter disc method by using
streptomycin as a positive control. The minimum inhibi-
tory concentration (MIC) values of 4a–j against
Staphylococcus aureus, E. coli and B. subtilis are summa-
rized in Table 2. Antibacterial activity of 4c,d,h and 4i
showed about 50% inhibition when compared with
streptomycin and remaining were not effective against
any of the three strains.
Scheme 1.
yield a mixture of nitrones E (3) and Z (30) isomers. The
E- and Z-isomers were formed in the ratio of 65:35 (a:
R = H) and 60:40 (b: R = Cl). The mixtures, after eval-
uating by 300MHz 1H NMR, were directly used for
cycloaddition with olefins. The 1,3-dipolar-cyclo-
addition reaction of nitrones, 3/30 with monosubstituted
Table 1. Reaction conditions and physical data of 5-imidazolyl substituted isoxazolidines (4a–j)
Isoxazolidine
Solventa
Time (h)
Rf valueb
Ratioc of 4/40
Eluentd
Yieldf of 4 in %
Mp (ꢁC)
4a
4b
4c
4d
4e
4f
Toluene
Toluene
Toluene
Xylene
26
26
28
34
36
24
24
26
27
29
0.33
0.38
0.48
0.32
0.35
0.38
0.41
0.48
0.38
0.31
77/23
81/19
87/13
89/11
90/10
78/22
84/16
90/10
91/9
H:EA (7:1)
H:EA (9:1)
H:EA (9:1)
H:EA (8:1)
H:EA (4:1)e
H:EA (9:1)
H:EA (9:1)
H:EA (9:1)
H:EA (8:1)
H:EA (4:1)e
60
62
59
76
72
61
60
60
72
63
Oil
Oil
Oil
Oil
128
Oil
Oil
Oil
Oil
134
Xylene
Toluene
Toluene
Toluene
Xylene
4g
4h
4i
4j
Xylene
93/7
a Used for cycloaddition reaction.
b Used 7:1 ratio of H (hexane)/EA (ethylacetate).
c Regioisomeric ratio of crude ascertained by 1H NMR (300MHz) after passing through a short column using H/EA (4:1).
d Used for purification of isoxazolidines.
e Recrystallized in alcohol after passing through a short column.
f Calculated with respect to nitrone.
Table 2. The minimum antibacterial inhibition concentrations of 4a–j against S. aureus, E. coli and B. subtilis by Bolter disc method; concentration:
10mg/mL; vol. of sample taken from the stock soln ꢀ10lL
Micro-organisms
Zone of inhibition (1mm)
Compounds
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
Streptomycin
S. aureus
E. coli
—
—
2
6
5
9
21
19
20
17
19
19
2
3
7
—
2
5
4
8
18
16
21
16
18
17
—
2
35
36
35
B. subtilis
—
—

M. P. Sadashiva et al. / Bioorg. Med. Chem. 12 (2004) 6389–6395
6391
Table 5. The minimum antifungal inhibitory concentrations (MIC)
lM of (4a–j) by Turbidometric method
5. Antifungal activity
All novel isoxazolidines 4a–j were screened for their
antifungal activity by two methods, cup borer and tur-
bidometric, using nystatin as a positive control. The
MIC values of 4a–j against Fusarium moniliforme,
Aspergillus niger and Cephalosporium acremonium are
summarized in Tables 3–5.
Isoxazolidine
F. moniliforme
A. niger
C. acremonium
4a
4b
17 1 0.6
15 0.3
112 1.7
83 2.7
121 1.7
27 0.9
15 1.1
31 1.2
107 2.4
68 2.7
76 1.7
32
23 1.3
93 2.7
97 1.9
98 1.5
95 2.5
17 0.5
127 3.0
79 3.0
97 2.7
31 2.1
39
135 2.1
160 3.2
156 2.1
85 1.3
13 0.7
95 1.4
83 2.3
115 3.0
85 3.0
35
4c
4d
4e
4f
4g
Compounds 4a and 4f exhibited better antifungal activ-
ity than nystatin. The MIC values of 4a were 17, 15 and
23 while 4f were 13, 15 and 17 against F. moniliforme, A.
niger and C. acremonium, respectively. The MIC values
of these compounds were less than 50% when compared
with the corresponding values of nystatin (Table 5). The
antifungal potential of 4a and 4f are found to be dose
dependant (Tables 3 and 4). Compounds 4e and 4g
exhibited similar inhibition to that of nystatin against
A. niger. Compound 4j showed similar activity against
F. moniliforme but not effective against the other two
fungal strains. Therefore, compounds 4e,g and 4j are
strain dependant.
4h
4i
4j
Nystatin
recorded on a Bruker Avance-300 spectrometer at 300
and 75MHz, respectively, using TMS as internal stand-
ard. The chemical shift values are on the d scale and the
coupling constants (J) are in Hertz. The elemental analy-
ses were obtained on a Vario-EL instrument. The ana-
lytical TLCs were performed on a coated Merck silica
gel 60F254 plates; the spots were detected either under
UV light (or) by charring with 4% alcoholic H2SO4.
All extracted solvents were dried over Na2SO4, followed
by evaporation in vacuum.
6. Conclusion
Isoxazolidines, 4a–j were synthesized and screened for
their antimicrobial activities (antifungal and antibacte-
rial). Amongst those tested, 4a and 4f exhibited more
potent antifungal activity against F. moniliforme, A.
niger and C. acremonium.
7.2. Synthesis
Nitrones, 3a,a0 and 3b,b0 were prepared according to the
procedure reported earlier.18
A mixture of nitrones E (3a) and Z (3a0) isomers (yellow
oil, 0.42mg, 51%) was obtained from nitrobenzene 2a
(0.33mL, 2.68mmol) and 2-butyl-4-chloro-1H-imidaz-
ole formaldehyde 1 (0.5g, 2.68mmol).
7. Experiment
7.1. General
Melting points were determined on SELACO-650
instrument and are uncorrected. The IR was recorded
on a Perkin–Elmer model RX1 FT-IR spectrophoto-
meter. The 1H NMR and 13C NMR spectra were
7.2.1. E-C-(2-Butyl-4-chloro-1H-imidazolyl)-N-phenyl-
1
nitrone 3a. H NMR (CDCl3, 300MHz): d 0.93 (t, 3H,
CH3); 1.38 (m, 2H, CH2); 1.75 (m, 2H, CH2); 2.75 (t,
2H, CH2); 7.32–7.48 (m, 3H, Ar–H, merged with Z
Table 3. Determination of MIC of (4a–j) against F. moniliforme, A. niger C. acremonium by Cup borer method; concentration: 1mg/mL; vol. of
sample taken from the stock soln ꢀ50lL
Microorganism
Zone of inhibition (1mm)
Compounds
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
Nystatin
F. moniliforme
A. niger
21
21
23
1
2
1
—
2
1
—
2
8
7
3
22
23
23
1
2
2
5
6
5
—
—
—
1
7
3
2
12
13
13
—
—
—
—
C. acremonium
1
—
Table 4. Determination of MIC of (4a–j) against F. moniliforme, A. niger, C. acremonium by Cup borer method; concentration: 1mg/mL; vol. of
sample taken from the stock soln ꢀ100lL
Microorganism
Zone of inhibition (1mm)
Compounds
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
Nystatin
F. moniliforme
A. niger
31
37
35
1
1
1
4
7
6
2
2
3
1
5
37
39
43
1
1
1
7
11
8
1
2
15
5
1
12
13
13
—
—
15
7
1
—
—
—
1
C. acremonium
5

6392
M. P. Sadashiva et al. / Bioorg. Med. Chem. 12 (2004) 6389–6395
isomer); 7.76–7.78 (m, 2H, Ar–H, merged with Z iso-
mer); 7.97 (s, 1H, CH@N); 12.13 (s, 1H, NH). IR
(KBr): 3230, 3045, 2863, 2857, 1548, 1176, 893, 740,
660cmꢀ1. Anal. Calcd CHN: 60.54, 5.81, 15.13. Found
60.63, 5.84, 15.18.
J = 8.6 and 5.3Hz); 7.25–7.44 (m, 5H, Ar–H); 10.73
(br s, 1H, NH). Anal. Calcd CHN: 59.42, 6.05, 11.55.
Found 59.45, 6.11, 11.58.
7.3.2. 2-(Phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-
ethylate isoxazolidine 4b. This compound was obtained
by the cycloaddition of 3a (0.42g, 1.44mmol) with ethyl
acrylate (5.4mL, 50mmol). Brownish thick liquid, 0.26g
(62%). IR (KBr): 3258, 3071, 2950, 1730, 1250, 1075,
7.2.2. Z-C-(2-Butyl-4-chloro-1H-imidazolyl)-N-phenyl-
nitrone 3a0. d 0.76 (t, 3H, CH3); 1.11 (m, 2H, CH2);
1.49 (m, 2H, CH2); 2.36 (t, 2H, CH2); 7.32 (s, 1H,
CH@N); 7.32–7.48 (m, 3H, Ar–H, merged with E iso-
mer); 7.76–7.78; (m, 2H, Ar–H, merged with E isomer);
12.72 (br s, 1H, NH). IR (KBr): 3230, 3045, 2863, 2857,
1548, 1176, 893, 740, 660cmꢀ1. Anal. Calcd CHN:
60.54, 5.81, 15.13. Found 60.63, 5.84, 15.18.
828, 750, 692cmꢀ1 1H NMR (CDCl3, 300MHz): d
.
0.92 (t, 3H, CH3); 1.14 (t, 3H, CH3); 1.23–1.40 (m,
2H, CH2); 1.61–1.80 (m, 2H, CH2); 2.45 (ddd, 1H,
H4a, J = 13.3, 8.2 and 3.0Hz); 2.72 (t, 2H, CH2); 2.98
(ddd, 1H, H4b, J = 13.3, 7.6 and 5.1Hz); 4.18 (q, 3H,
OCH2); 4.85 (dd, 1H, H3, J = 8.6 and 5.1Hz); 4.99
(dd, 1H, CH, J = 8.5 and 4.8Hz); 6.96 (m, 3H, Ar–H);
7.15 (m, 2H, Ar–H); 10.55 (br s, 1H, NH). Anal. Calcd
CHN: 60.40, 6.40, 11.12. Found 60.43, 6.44, 11.09.
A mixture of nitrones E (3b) and Z (3b0) isomers (yellow
oil, 0.44mg, 48%) was obtained from 4-chloronitroben-
zene 2b (0.42g, 2.68mmol) and 2-butyl-4-chloro-1H-
imidazole formaldehyde 1 (0.5g, 2.68mmol).
7.3.3. 2-(Phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-
butylate isoxazolidines 4c. This compound was obtained
by the cycloaddition of 3a (0.44g, 1.5mmol) with butyl
acrylate (7.2mL, 50mmol). Brownish thick liquid 0.26g
(59%). IR (KBr): 3247, 3062, 2959, 2873, 173, 1598,
7.2.3. E-C-(2-Butyl-4-chloro-1H-imidazolyl)-N-(4-chloro-
phenyl) nitrone 3b. H NMR (CDCl3, 300MHz): d 0.88
1
(t, 3H, CH3); 1.35 (m, 2H, CH2); 1.73 (m, 2H, CH2);
2.68 (t, 2H, CH2); 7.71(d, 2H, Ar–H); 7.93 (s, 1H,
CH@N); 8.46 (d, 2H, Ar–H); 12.41 (s, 1H, NH); IR
(KBr): 3220, 3015, 2854, 2863, 1566, 1166, 903, 790,
688cmꢀ1. Anal. Calcd CHN: 53.86, 4.84, 13.46. Found
53.91, 4.89, 13.42.
1
1258, 1085, 825, 757, 694, 598cmꢀ1. H NMR (CDCl3,
300MHz): 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, H4b, J = 13.4 5,87 and 4.7Hz); 2.99
(ddd, 1H, H4b, J = 13.28, 7.96 and 5.3Hz); 3.08 (t, 2H,
OCH2); 4.57 (dd, 1H, CH, J = 8.8 and 5.4Hz); 4.82
(dd, 1H, CH, J = 8.4 and 4.7Hz); 6.95–7.15 (m, 3H,
Ar–H); 7.22–7.35 (m, 2H, Ar–H); 10.5 (br s, 1H, NH).
Anal. Calcd CHN: 62.14, 6.95, 10.35. Found 62.19
7.06, 10.38.
7.2.4. Z-C-(2-Butyl-4-chloro-1H-imidazolyl)-N-(4-chloro-
phenyl) nitrone 3b0. 1H NMR (CDCl3, 300MHz): d 0.76
(t, 3H, CH3); 1.12 (m, 2H, CH2); 1.51 (m, 2H, CH2);
2.37 (t, 2H, CH2); 7.30 (s, 1H, CH@N); 7.74 (d, 2H,
Ar–H); 8.53 (d, 2H, Ar–H); 12.71 (br s, 1H, NH). IR
(KBr): 3220, 3015, 2854, 2863, 1566, 1166, 903, 790,
688cmꢀ1. Anal. Calcd CHN: 53.86, 4.84, 13.46. Found
53.91, 4.89, 13.42.
7.3.4. 3-(2-Butyl-4-chloro-1H-imidazolyl)-2,5-diphenyliso-
xazolidine 4d. This compound was obtained by the cy-
cloaddition of 3a (0.48g, 1.65mmol) with styrene
(5.73mL, 50mmol). White solid 0.364g (76%). IR
(KBr): 3428, 3033, 2956, 2867, 2745, 2664, 1487, 1452,
7.3. General procedure for the synthesis of novel isoxazo-
lidines 4a–j
1598, 1238, 1023, 760, 697cmꢀ1 1H NMR (CDCl3,
.
To a solution of the corresponding nitrone (1mmol) in
toluene/xylene was added monosubstituted alkene
(50mmol) and the resulting solution was refluxed until
reaction completes, monitored by TLC. Reaction mix-
ture was cooled to room temperature and concentrated
under reduced pressure. The regioisomeric ratio (rr%)
300MHz): d 0.90 (t, 3H, CH3); 1.34 (m, 2H, CH3);
1.65 (m, 2H, CH2); 2.61 (t, 2H, CH2); 2.48–2.61
(ddd, 1H, H4a, J = 13.0, 6.9 and 2.7Hz); 3.07–3.16
(ddd, 1H, H4b, J = 13.66, 6.75 and 4.2Hz); 5.10 (dd,
1H, CH, J = 8.10 and 5.7Hz); 5.24 (dd, 1H, CH,
J = 8.4 and 6.90Hz); 7.0 (q, 2H, Ar–H); 7.11 (d, 2H,
Ar–H, J = 7.8Hz); 7.24–7.44 (m, 6H, Ar–H); 9.68 (br
s, 1H, NH). Anal. Calcd CHN: 69.20, 6.33, 11.00.
Found 69.28, 6.37,11.12.
1
of the residue was determined by H NMR analysis.
The crude material was purified by silica gel column
using appropriate eluent to give major product 4 with
96% purity analysed by H NMR.
1
7.3.5. 2-(Phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-
benzoate isoxazolidine 4e. This compound was obtained
by the cycloaddition of 3a (0.43g, 1.47mmol) with vinyl
benzoate (4.7mL, 50mmol). Brownish thick liquid
0.31g (71.9%). IR (KBr): 3019, 2963, 2875, 1732, 1597,
7.3.1. 2-(Phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-
methylate isoxazolidine 4a. This compound was obtained
by the cycloaddition of 3a (0.4g, 1.37mmol) with methyl
acrylate (4.7mL, 50mmol). Brownish thick liquid, 0.24g
(60%). IR (KBr): 3230, 3058, 2962, 1730, 1254, 1080,
1260, 1026, 1489, 1454, 866, 759, 659, 668cmꢀ1 1H
.
820, 754, 694cmꢀ1
.
1H NMR (CDCl3, 300MHz): d
NMR (CDCl3, 300MHz): d 1.02 (t, 3H, CH3); 1.41
(m, 2H, CH2); 1.68 (m, 2H, CH2); 2.66 (ddd, 1H, H4a,
J = 13.3, 7.8 and 5.6Hz); 2.72 (t, 2H, CH2); 2.92 (ddd,
1H, H4b, J = 13.3, 7.0 and 5.1Hz); 5.5 (dd, 1H, CH,
J = 7.3 and 5.4Hz); 5.93 (dd, 1H, CH, J = 8.2 and
6.1Hz); 7.05–8.0 (m, 10H, Ar–H); 10.8 (br s, 1H, NH).
0.87 (t, 3H, CH3); 1.21–1.38 (m, 2H, CH2), 1.65–1.83
(m, 2H, CH2); 2.51 (ddd, 1H, H4, J = 12.58, 8.70 and
5.10Hz); 2.61 (t, 2H, CH2); 3.03 (ddd, 1H, H4
J = 12.40, 6.3 and 4.0Hz); 3.78 (s, 3H, OCH3); 4.84
(dd, 1H, J = 8.40 and 5.12Hz); 4.98 (dd, 1H, CH,

M. P. Sadashiva et al. / Bioorg. Med. Chem. 12 (2004) 6389–6395
6393
Anal Calcd CHN: 64.86, 5.68, 9.87. Found 64.91, 5.70,
9.89.
(br s, 1H, NH). Anal. Calcd CHN: 63.47, 5.57, 10.10.
Found 63.50, 5.61,10.07.
7.3.6. 2-(4-Chlorophenyl)-3-(2-butyl-4-chloro-1H-imidazol-
yl)-5-methylate isoxazolidine 4f. This compound was
obtained by the cycloaddition of 3b (0.44g, 1.42mmol)
with methyl acrylate (4.7mL, 50mmol). Brownish col-
our 0.268g (61%). IR (KBr): 3260, 3088, 2862, 1740,
7.3.10. 2-(4-Chlorophenyl)-3-(2-butyl-4-chloro-1H-imi-
dazolyl)-5-benzoate isoxazolidine 4j. This compound
was obtained by the cycloaddition of 3b (0.46g,
1.5mmol) with vinylbenzoate (6.9mL, 50mmol).
Brownish colour 0.29g (63%). IR (KBr): 3023, 2973,
2877, 1742, 1607, 1268, 1036, 1497, 1476, 869, 767,
1260, 1060, 840, 754, 684cmꢀ1 1H NMR (CDCl3,
.
1
300MHz): 0.96 (t, 3H, CH3); 1.30 (m, 2H, CH2); 1.83
(m, 2H, CH2); 2.71 (t, 2H; CH2); 2.57 (ddd, 1H, H4a,
J = 13.6, 9.4 and 2.6Hz); 2.78 (ddd, 1H, H4b J = 13.6,
6.7 and 4.0Hz); 3.73 (s, 3H, OCH3); 4.42 (dd, 1H, CH
J = 8.8 and 5.5Hz); 4.56 (dd, 1H, CH, J = 8.8 and
5.8Hz); 7.05 (d, 2H, Ar–H, J = 8.0Hz); 7.39 (d, 2H,
Ar–H, J = 8.3Hz); 10.6 (br s, 1H, NH). Anal. Calcd
CHN: 54.28, 5.31, 10.55. Found 54.31, 5.38, 10.50.
688, 660cmꢀ1. H NMR (CDCl3, 300MHz): d 1.10 (t,
3H, CH3); 1.46 (m, 2H, CH2); 1.74 (m, 2H, CH2); 2.8
(t, 2H, CH2); 2.74 (ddd, 1H, H4a, J = 13.3, 7.8 and
5.6Hz); 2.99 (ddd, 1H, H4b, J = 13.3, 7.0 and 5.1Hz);
5.7 (dd, 1H, CH, J = 7.3 and 5.4Hz); 6.01 (dd, 1H,
CH, J = 8.2 and 6.1Hz); 7.05 (d, 2H, Ar–H,
J = 8.2Hz); 7.39 (d, 2H, Ar–H); 7.44–7.51 (m, 5H, Ar–
H). Anal. Calcd CHN: 60.00, 5.03, 9.13, found 60.09,
5.08, 9.12.
7.3.7. 2-(4-Chlorophenyl)-3-(2-butyl-4-chloro-1H-imidazol-
yl)-5-ethylate isoxazolidine 4g. This compound was
obtained by the cycloaddition of 3b (0.4g, 1.3mmol)
with ethyl acrylate (7.2mL, 50mmol). Brownish colour
0.24g (60%). IR (KBr): 3268, 3081, 2960, 1736, 1255,
8. Biological assay
8.1. Antibacterial activity test
1085, 838, 760, 698cmꢀ1
.
1H NMR (CDCl3,
300MHz): d 0.97 (t, 3H, CH3); 1.14 (t, 3H, CH3); 1.38
(m, 2H, CH2); 1.88 (m, 2H, CH2); 2.69 (t, 2H, CH2);
2.55 (ddd, 1H, H4a, J = 13.3, 8.2 and 3.0Hz); 2.69
(ddd, 1H, H4b, J = 13.3, 7.6 and 5.1Hz); 4.19 (s, 2H,
OCH2); 4.42 (dd, 1H, H3, J = 8.6 and 5.1Hz); 4.88
(dd, 1H, CH, J = 8.5 and 4.8Hz); 7.04 (d, 2H, Ar–H,
J = 8.0Hz); 7.49 (d, 2H, Ar–H, J = 8.3Hz); 10.53 (br
s, 1H, NH). Anal Calcd CHN: 55.35, 5.62, 10.20. Found
55.41, 5.66, 10.16.
1. Bacterial strains used for the antibacterial activity:
S. aureus (ATCC10832D), E. coli (ATCC700926D)
and B. subtilis (ATCC23857D).
2. Preparation of test sample: 10mg of each of the isoxazol-
idines were dissolved in 1mL of DMSO, which served
as a stock. From the stock, 10lL was loaded on 1mm
sterile filter discs (Sartorius-Germany) and then
dried.
3. Preparation of nutrient agar and broth: Nutrient agar
and broth (HiMedia) were prepared according to the
manufacturerÕs instruction.
7.3.8. 2-(4-Chlorophenyl)-3-(2-butyl-4-chloro-1H-imidazol-
yl)-5-butylate isoxazolidine 4h. This compound was
obtained by the cycloaddition of 3b (0.42g, 1.37mmol)
with butyl acrylate (7.2mL, 50mmol). Brownish colour
0.25g (60%). IR (KBr): 3239, 3042, 2966, 2822, 1738,
Formula of the nutrient agar:
Peptic digest of animal tissue
Sodium chloride
Beef extract
Yeast extract
Agar
5.0g/L
5.0g/L
1.5g/L
1.5g/L
20.0g/L
7.4 0.2
1608, 1260, 1088, 830, 767, 704, 618cmꢀ1 1H NMR
.
(CDCl3, 300MHz): d 1.00–1.06 (m, 6H, CH3); 1.24–
1.40 (m, 4H, CH2); 1.68–1.84 (m, 4H, CH2); 2.60 (t,
2H,CH2); 2.53 (ddd, 1H, H4b, J = 13.45, 8.7 and
4.7Hz); 3.07 (t, 2H, OCH2); 3.17 (ddd, 1H, H4b,
J = 13.28, 7.96 and 5.3Hz); 4.32 (dd, 1H, CH, J = 8.8
and 5.4Hz); 4.92 (dd, 1H, CH, J = 8.4 and 4.7Hz);
7.11 (d, 2H, Ar–H, J = 8.0Hz); 7.39 (d, 2H, Ar–H,
J = 8.23Hz); 10.66 (br s, 1H, NH). Anal. Calcd CHN:
57.28, 6.18, 9.54. Found 57.31, 6.21, 9.51.
pH at (25ꢁC)
Formula of the nutrient broth:
Peptic digest of animal tissue
Sodium chloride
Beef extract
Yeast extract
pH at (25ꢁC)
5.0g/L
5.0g/L
1.5g/L
1.5g/L
7.4 0.2
7.3.9. 2-(4-Chlorophenyl)-3-(2-butyl-4-chloro-1H-imidazol-
yl)-5-phenyl isoxazolidine 4i. This compound was
obtained by the cycloaddition of 3b (0.44g, 1.4mmol)
with styrene (5.7mL, 50mmol). Pure white solid 0.32g
(72%). IR (KBr): 3328, 3066, 2946, 2877, 2790, 2688,
1
1487, 1460, 1608, 1250, 1045, 770, 677cmꢀ1. H NMR
(CDCl3, 300MHz): d 0.94 (t, 3H, CH3); 1.36 (m, 2H,
CH2); 1.68 (m, 2H, CH2); 2.64 (t, 2H, CH2); 2.50–2.58
(ddd, 1H, H4a, J = 13.0, 6.99 and 2.7Hz); 3.17–3.16
(ddd, 1H, H4b, J = 13.66, 6.75, 4.2Hz); 5.14 (dd, 1H,
CH, J = 8.10 and 5.74Hz); 5.27 (dd, 1H, CH, J = 8.4
and 6.90Hz); 7.0 (d, 2H, Ar–H, J = 8.6Hz); 7.2 (d,
2H, Ar, J = 7.8Hz); 7.24–7.44 (m, 5H, Ar–H); 10.18
8.1.1. Blotter disc method. In order to maintain uniform
growth rate of each microorganism, a fresh inoculum
was prepared by sub culturing microorganisms in luria
bertanii (LB) broth media and incubated until optical
density (OD, turbidity at 600nm) of the culture reaches
0.2, which indicates that, the bacterial density of 107–
108 cfu/mL.

6394
M. P. Sadashiva et al. / Bioorg. Med. Chem. 12 (2004) 6389–6395
Single colony of each of the cultures were inoculated in
5mL of LB broth and incubated under shaking condi-
tion overnight at 37ꢁC. Five hundred milliliters of over-
night grown cultures were inoculated into 25mL of LB
and incubated at 37ꢁC. At 0.2 OD600 nm, 300lL of each
of the cultures was inoculated into seven flasks contain-
ing 30mL each of nutrient agar medium. The contents
of the flasks were poured into the petriplates and
allowed to solidify. On solidifying four filter discs were
appropriately labelled: first for negative control
(DMSO), second for positive control (streptomycin),
third and fourth for isoxazolidines 4a–j (100lL). The
plates were kept at room temperature for 30min and
then maintained 37ꢁC for 24h. After 24h, the zone of
inhibition caused by the compounds was recorded.1,24
including standard and controls, the fresh inoculum
was added with a spore concentration adjusted to
1 · 102 spores/mL. After incubating all tubes at 37ꢁC
for 24h, their absorbance was recorded at 640nm. Per-
centage of inhibition was calculated according to the
formula.24,25
% Inhibition ¼ 100ðP ꢀ QÞ=P
where, P = absorbance without test sample and Q =
absorbance with test sample. Then, the MIC was
recorded in lM.
Acknowledgements
We are grateful to the University of Mysore, Mysore,
India for financial support under the UPG-JRF order
No. DV-57/U.P.G.F./2003-2004. The CHNS and UV–
vis data obtained from this department with equipments
funded by DST-FIST (order no. SR/FST/CSI-051/2002.
Dated 28.7.2003) grant.
8.2. Antifungal activity test
1. Fungus used for the antifungal activity: F. moniliforme
(ATCC10052), A. niger (ATCC1004) and C. acremo-
nium (ATCC10141).
2. Preparation of test sample: Isoxazolidine (1mg) was
dissolved in 1mL of DMSO, which served as a stock.
3. Preparation of potato dextrose agar/broth: Potato dex-
trose agar and broth (HiMedia) were prepared
according to the manufacturerÕs instruction.
References and notes
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Potato extract
Dextrose
Agar
200g/L
20g/L
20g/L
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8.2.1. Cup borer method. The pathogen spore suspension
was prepared in 50mL sterile distilled water. The spore
concentration was adjusted to 1 · 103 spores/ml. Sus-
pension of 2mL was spread on PDA plates uniformly.
After solidifying, four wells were bored in each plate
using a cork borer (0.5cm diameter). DMSO (negative
control) in first well, nystatin (positive control) in second
well, and isoxazolidine of different concentrations in the
remaining two wells—third (50lL) and fourth (100lL)
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on zone of inhibition caused by the compounds.1,24
8.2.2. Turbidometric method. The isoxazolidines 4a–j
were screened for their antifungal activity at different
concentrations (50 and 100lM) against F. moniliforme,
A. niger and C. acremonium using nystatin as a positive
control and DMSO as a negative control. To the culture
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Synthesis 2001, 2415.

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