Chemical and biochemical transformations of 5-ethoxycarbonyl-5-phenyl-2- isoxazolines

Article abstract of DOI:10.1016/j.farmac.2005.07.003

The salts, 2-methyl-5,5-disubstituted 4,5-dihydroisoxazolium methylsulfates comprising various substituents at the C-3 carbon atom were subjected to transformations. The structure of applied compounds permitted to monitor the effect of this factor on the transformation course of the 2-isoxazoline ring. The nucleophilic addition of cyanide anion to the selected salts enabled the obtaining of a next heterocyclic system of changed physicochemical and biological properties in comparison to the starting 2-isoxazolines. The diastereoselective hydrolysis of the cyanide group in 2-isoxazolidines by the bacteria strain Rhodococcus rhodochrous PCM 909 leads to the obtaining of a racemic mixture of the trans-hydroxyacid. The introduction of new functional groups into the heterocyclic ring made these compounds attractive objects for further chemical and microbial transformations and to study their biological activity.

Full text of DOI:10.1016/j.farmac.2005.07.003

Il Farmaco 60 (2005) 948–954
http://france.elsevier.com/direct/FARMAC/
Chemical and biochemical transformations
of 5-ethoxycarbonyl-5-phenyl-2-isoxazolines
Irmina Zadroz˙na a,*, Joanna Kurkowska a, Hanna Kruszewska b
a Faculty of Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, 00-664 Warsaw, Poland
b National Institute of Public Health, ul. Chełmska 30/34, 00-725 Warsaw, Poland
Received 6 October 2004; received in revised form 6 July 2005; accepted 11 July 2005
Abstract
The salts, 2-methyl-5,5-disubstituted 4,5-dihydroisoxazolium methylsulfates comprising various substituents at the C-3 carbon atom were
subjected to transformations. The structure of applied compounds permitted to monitor the effect of this factor on the transformation course of
the 2-isoxazoline ring. The nucleophilic addition of cyanide anion to the selected salts enabled the obtaining of a next heterocyclic system of
changed physicochemical and biological properties in comparison to the starting 2-isoxazolines. The diastereoselective hydrolysis of the
cyanide group in 2-isoxazolidines by the bacteria strain Rhodococcus rhodochrous PCM 909 leads to the obtaining of a racemic mixture of the
trans-hydroxyacid. The introduction of new functional groups into the heterocyclic ring made these compounds attractive objects for further
chemical and microbial transformations and to study their biological activity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 4,5-Dihydroisoxazolium salts; Biological activity; Rhodococcus rhodochrous
1. Introduction
types of reactions in comparison with the initial 2-isoxazoline,
due to an increase in electrophility of the C-3 carbon atom,
especially when in this position is electroacceptor substitu-
ent. The problem of obtaining and transformation of 4,5-
dihydroisoxazolium 2-alkyl salts was dealt with in the seven-
ties of last century [23]. Studies of the reactivity of 2-methyl-
1,4-dihydroisoxazolium methylsulfates in the presence of the
cyanide anion presented by Zadroz˙na and Kornacka [17] is a
broadly described problem. The aim of this study was the
reactivity of 2-methyl-4,5-dihydroisoxazoline methylsulfates-
salts of cycloadducts of aliphatic nitryle oxides and methyl
and phenyl esters p-substituted cinnamic acids.
A number of compounds comprising in their structure a
2-isoxazoline ring or its hydrogenated derivative are known
to show biological activity [1–5]. These compounds are often
an intermediate in the total syntheses of ergot alkaloids (cha-
noclavine, lysergic acid), prostaglandins, macrolides and anti-
biotics (acivicine, sarkomicin, mitomycin, vermiculine, pali-
clavine, nicomycine) [6–13]. Thus, in recent years there is a
noticeable increase in the interest of the substituted
2-isoxazoline system in biochemical studies, leading to the
obtaining of biologically active compounds. This work, is a
continuation of studies on the substituted 2-isoxazoline sys-
tem [4,5,14–21]. The purpose of this study is examination of
the reactivity of selected 2-isoxazoline systems in various
chemical and biochemical reactions resulting in a change of
their physicochemical and biological properties.
2. Results and discussion
In this work the salts, 5,5-disubstituted 2-methyl-4,5-
dihydroisoxazolium methylsulfates comprising various sub-
stituents at the C-3 carbon atom (CH3, H, C6H5) were sub-
jected to transformations. The structure of applied compounds
permitted to monitor the effect of this factor on the transfor-
mation course of the 2-isoxazoline ring.
Studies on the reactivity of 2-isoxazoline systems are per-
formed by first obtaining from them 4,5-dihydroisoxazolium
salts [22], which are much more susceptible towards various
The nucleophilic addition of cyanide anion to the selected
salts enabled the obtaining of a new heterocyclic system of
* Corresponding author. Tel.: +48 22 660 7473; fax: +48 22 628 2741.
E-mail address: zadrozna@ch.pw.edu.pl (I. Zadroz˙na).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.07.003

I. Zadroz˙na et al. / Il Farmaco 60 (2005) 948–954
949
Table 1
changed physicochemical and biological properties in com-
parison to the starting 2-isoxazolines. On the other hand, the
introduction of new functional groups into the heterocyclic
ring made these compounds attractive objects for further
chemical and biochemical transformations and to study their
biological activity.
Percentage share of the products of the 2-methyl-4,5-dihydroisoxazolium
methylsulfate 4 reaction with a stoichiometric amount of sodium cyanide
Temperature
room
7-trans (%) 7-cis (%) 8 (%)
Yield (%)
34
34
22
66
52
7
–
67
59
44
60 °C
14
71
110 °C
2-Methyl-4,5-dihydroisoxazolium methylsulfates were
obtained from the methylation with methylsulfate of
2-isoxazolines formed in the 1,3-dipolar cycloaddition of
formyl-, acetyl-, and benzonitrile oxides with ethyl atropate
(Scheme 1). Two standard methods of the 1,3-dipolar cycload-
dition of nitrile oxides to a,b-unsaturated esters were utilized
for the synthesis of the starting 2-isoxazolines, the method of
Mukayama and Hoshino [24] for the synthesis of compounds
1 and 2 and that of Larsen and Torssell [25] for the synthesis
of compound 3, obtaining 5,5-disubstituted products.
The methylation reactions were carried out in toluene as
solvent, with a fivefold excess of dimethyl sulfate with respect
to the substrate used. The reaction mixture was maintained at
60–65 °C for 1 or 4 days depending on the structure of the
substrate used (Scheme 1). The reaction yields were practi-
cally quantitative. The crude products, after evaporating of
toluene, were subjected to further chemical transformations.
2-isoxazoline. The reactions were carried out at room tem-
perature as moderate reaction conditions, at 60 °C, i.e. the
temperature of obtaining the salt and at 110 °C temperature
of the heterocyclic ring cleavage, during 2 h, in DMSO.
2-Methyl-4,5-dihydroisoxazolium methylsulfate 4, ob-
tained from compound 1, was the first starting material used
for these studies (Scheme 2).
In the reaction carried out at room temperature with a sto-
ichiometric amount of sodium cyanide two diastereomeric
addition products, 7-cis and 7-trans, were obtained, the struc-
ture of which was determined on the basis of an analysis of
1H NMR spectra and 1H NMR spectra with applying the Over-
hauser nuclear effect (NOE DIFF) (Scheme 2).
When carrying out the reaction at 60 °C a third compound
8 (3-isoxazoline derivative) appeared besides the mixture of
products 7. Identical three compounds, but with a different
percentage share, were obtained during the reaction carried
out at 110 °C (Table 1). A considerable increase in the per-
centage share of compound 8 took place at this temperature.
No product of the cyanide anion addition reaction was
observed in the case of 4,5-dihydroisoxazolium salts bearing
a proton (5) or phenyl group (6) at position 3 of the hetero-
cyclic ring. In the case of the reaction of product 5, obtained
from 2-isoxazoline 2, b-oxyaminoaldehyde 9 was isolated in
a 40% yield, formed in the aqueous medium during process-
ing of the post-reaction mixture [26]. However, during the
reactions carried out at 60 and 110 °C only product 10 was
obtained, probably resulting from the proton abstraction from
position C-4 of the heterocyclic ring, where in this case the
cyanide anion acted as a base (Scheme 3). Compound 11 and
b-oxyaminophenylketone 12 were obtained during the reac-
tion of 2-isoxazoline 6 (Scheme 4 and Table 2).
2.1. Cyanide anion nucleophilic addition
Studies on the reactivity of 2-methyl-4,5-dihydro-
isoxazolium methylsulfates in the presence of cyanide anion
were carried out in a homogeneous phase system in an apro-
tic solvent DMSO and at various temperatures. The nucleo-
philic addition was carried out with the addition of a stoichio-
metric amount of the anion with respect to the starting
Compounds 7–12 were separated on a column chromatog-
raphy (hexane/diethyl ether = 7:2).
Scheme 1
.
Scheme 2
.

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I. Zadroz˙na et al. / Il Farmaco 60 (2005) 948–954
Scheme 3
.
2.2. Chemical reduction
or hydrolysis by whole microorganisms. Kakey at al. used
Rhodococcus butanica ATCC strain 21197 to carry out the
stereoselective hydrolysis of 3-substituted glutaronitriles and
obtained monoacids of S configuration [28,29]. Meth-Cohn
and Wang [30–32] described a microbiological hydrolysis
leading to carboxylic acids. The authors used Rhodococcus
rhodochrous strain AJ270 and carried out the reaction in a
phosphate buffer of pH 7.0 at 30 °C. With this approach, they
were able to obtain heterocyclic compounds. In one of the
latest works, Dadd et al. [33] reported regioselective trans-
formation of nitriles, namely, hydrolysis of 2-, 3- and
4-(cyanomethyl)-benzonitriles by R. rhodochrous strain
LL100-21.
In this report we describe microbiologically catalyzed
selective hydrolysis of the cyanide group in the mixture of
enantiomers of compound 7. Two strains of the Rhodococcus
species NCIB 10554 (Institute of Biotransformation andAnti-
biotics, Warsaw) and R. rhodochrous PCM 909 (Central Col-
lection of Microorganisms, Institute of Immunology and
Experimental Therapy of Polish Academy of Science,
Wrocław) were used. Despite the literature reporting easy,
selective hydrolysis of the cyanide group present in com-
pounds bearing other constituents capable of biotransforma-
tion [28–33], in compounds 7-cis and 7-trans the ester group
but the cyanide group underwent microbiological hydrolysis
when Rhodococcus species NCIB 10554 was applied. As a
result of biotransformation the mixture of diastereoisomeric
acids 14 (trans:cis = 65:35) was obtained (Scheme 6). Pro-
longation of the process from 48 h to 14 days resulted in the
increase of the yield of reaction (to 14%).
Basing on the results of studies concerning the reduction
of ester groups being substituents in 2-isoxazolines, the reduc-
tion of the mixture of compounds 7 was carried out with
sodium borohydride to check whether it will be a sufficiently
strong reducing agent to reduce the ester group also in this
compound [21,27]. Unfortunately, attempts to reduce 7 with
this reagent failed, even when used a 10-fold excess of NaBH4
with respect to the applied substrate, prolonged the reaction
time and raised the temperature. The unreacted ester was each
time quantitatively isolated from the post-reaction mixture.
Therefore, the diastereoisomeric products 7 were sub-
jected to the reduction with lithium aluminum hydride (LAH)
to check the behavior of the ester and cyanide functional
groups, presented at the heterocyclic ring. The reactions were
carried out in anhydrous diethyl ether at room temperature,
in an argon atmosphere during 1.5 h. From the analysis of the
products it was found that the reduction proceeded selec-
tively with respect to the ester group. The reaction resulted in
the obtaining of products in the form of racemic mixtures of
diastereoisomeric alcohols 13 with a cyanide group at the
C-3 position in an overall yield of 63% (Scheme 5). Prolon-
gation of the reaction time caused a cleavage of the hetero-
cyclic ring.
2.3. Microbial hydrolysis
According the literature [28–33], methods of hydrolysis
of nitriles to corresponding amides or carboxylic acids can
be divided into two groups, hydrolysis by isolated enzymes
R. rhodochrous PCM 909 was applied for the hydrolysis
of the mixture of diastereoisomeric alcohols 13-cis and
Scheme 4
.

I. Zadroz˙na et al. / Il Farmaco 60 (2005) 948–954
951
Table 2
chosen for the tests: Gram-positive bacteria Staphylococcus
aureus ATTC 6538, Gram-negative bacteria Eschericha coli
ATTC 8739 and Pseudomonas aeruginosaATTC 15442, and
yeast Candida albicans ATTC 10231.
Percentage share of the products of the 2-methyl-4,5-dihydroisoxazolium
methylsulfate 6 reaction with a stoichiometric amount of sodium cyanide
Temperature
Room
11 (%)
54
12 (%)
46
Yield (%)
27
6
The biological activity of the tested compounds was tested
by cylinder-plate method [5,34]. Briefly, Petri plates contain-
ing 15 ml of Muller–Hinton Agar were inoculated with a
respective strain. The aliquot 100 µl of each tested solution of
20 mM stock was added to the cylinder placed on the surface
of agar. Plates were incubated at 37 °C for 18 h for bacteria,
and at 30 °C for 24 h for yeast, respectively. Growth inhibi-
tion was visualized as a clear region around the cylinder con-
taining the studied compound. Diameter of the inhibition
region was taken as a measure of growth inhibition.
Tetracycline (1 mM) and miconazole (1 mM) were used
as a positive control for bacteria and fungi, respectively. The
following inhibition regions were obtained: tetracycline-
E coli = 19 mm, S. aureus = 27 mm, Ps. aeruginosa = 15 mm;
miconazole, C. albicans = 23 mm.
60 °C
88
12
Scheme 5
.
Of 15 compounds tested in this study, only compounds 3,
7, 13 and 14 considerably inhibited growth of C. albicans.
Respective inhibition regions were as follows: 11 mm, 10 mm,
13 mm and 12 mm.
3. Conclusions
In the reaction of various anions with the 2-methyl-4,5-
dihydroisoxazolium salt, the product of the nucleophilic addi-
tion to the double bond or a 3-isoxazoline derivative is formed.
The structure, percentage share and yield of products depend
on the structure of the selected salt as well as on the reaction
temperature.
Chemical reduction of the ester group present at the
2-isoxazoline ring, while preserving the heterocyclic system,
to a corresponding primary alcohol by means of sodium boro-
hydride is possible. The use of a stronger reducing agent, e.g.
lithium aluminum hydride, is required in the case of isoxazo-
lidines bearing an ester group.
Scheme 6
.
13-trans obtained by the reduction with LAH of compounds
7. The reaction catalyzed by this strain proceeded diastereo-
selectively during 48 h A racemic mixture of the hydroxy-
acid, trans-3-carboxy-2,3-dimethyl-5-hydroxymethyl-5-
phenylisoxazolidine 15 was obtained with an 11% yield
(Scheme 7).
2.4. Biological activity
The diastereoselective hydrolysis of the cyanide group in
isoxazolidines 7 by means the bacteria strain R. rhodochrous
PCM 909 leads to a racemic mixture of the hydroxyacid
-trans-3-carboxy-2,3-dimethyl-5-hydroxymethyl-5-phenyl-
isoxazolidine 15.
All the compounds described here were examined for their
biological activity. Strains of various cell wall structures were
Studies of the biological activity of the compounds
obtained in this work encourage to continue the search of
more effective derivatives in the isoxazole group influencing
the growth inhibition of microorganisms.
4. Experimental
4.1. General
IR spectra were measured by means of a Specord 71 IR
spectrophotometer, the band positions are expressed in cm–1.
Scheme 7
.

952
I. Zadroz˙na et al. / Il Farmaco 60 (2005) 948–954
1H and 13C NMR spectra were recorded on a Varian Gemini
200 spectrometer in chloroform-d. The chemical shift values
are expressed in d ppm. Flash column chromatography was
carried out using Merck Kieselgel 60 (230–400 meshASTM).
The reactions were controlled by TLC. Elemental analysis
was performed on a Perkin–Elmer CHNO analyzer.
4.3.1.2. cis-2,3-Dimethyl-3-cyano-5-ethoxycarbonyl-5-
phenylisoxazolidine (7-cis). H NMR: 1.21 (t, 3H, CH3,
1
J = 7 Hz), 1.46 (s, 3H, CH3), 2,63 and 3,82 (2 × d, 2H, CH2,
J = 13.2 Hz), 2.88 (s, 3H, CH3–N), 4.21 (q, 2H, CH2,
J = 7 Hz), 7.31–7.55 (m, 5H,Ar); 13C NMR: 13.84 (CH3CH2),
20.81 (CH3), 38.93 (CH3–N), 53.66 (C-4), 62.22 (C-3), 63.79
(CH3CH2), 83.92 (C-5), 117.81 (C≡N), 124.49, 125.12,
128.33, 137.35 (Ar), 172.46 (C=O); IR (film) cm−1: 2232
(C≡N),1732 (C=O).
4.2. General procedure for the preparation
of 2-methyl-4,5-dihydroisoxazolium methylsulfates
2-Isoxazoline, fivefold excess of dimethyl sulfate and a
small volume of toluene were placed in a dry flask. The con-
tent of the flask was heated for 1 or 4 days at 60 °C. After
cooling this amount of toluene was added to the mixture and
fell into layers. The upper toluene layer was decanted, and
the lower one of the salt (thick, dark-brown oil) was washed
with small portions of toluene. The remaining solvent was
then removed under reduced pressure. The thus prepared salt
was subjected to nucleophilic addition.
4.3.1.3. Analysis of mixture of compounds. 7-trans and 7-cis
C15H18N2O3 Calc.: C 65.69, N 10.22, H 6.57, Found: C 65.72,
N 10.23, H 6.53.
4.3.1.4. 2,3-Dimethyl-5-ethoxycarbonyl-5-phenyl-3-isoxazo-
line (8). 1H NMR: 1.37 (t, 3H, CH3, J = 7.4 Hz), 2.34 (s, 3H,
CH3), 2.91 (s, 3H, CH3-N), 4.23 (q, 2H, CH2, J = 7.2 Hz),
6.63 (s, 1H, CH), 7.31–7.55 (m, 5H, Ar); 13C NMR: 13.93
(CH3CH2), 21.99 (CH3), 39.06 (CH3–N), 62.07 (CH3CH2),
94.39 (C-5), 103.10 (C-4), 126.97, 127.50, 128.99, 144.82
(C-3), 146.17 (Ar), 175,90 (C=O); IR(film) cm−1: 1731
(C=O); Analysis of compound C14H17NO3 Calc.: C 68.02, N
5.67, H 6.88, Found: C 67.99, N 5.65, H 6.83.
4.3. General procedure for carrying out the reaction
of 2-methyl-4,5-dihydroisoxazolium methylsulfates with
the cyanide anion
4.3.2. Reactions of the 5-ethoxycarbonyl-2-methyl-5-phe-
nyl-4,5-dihydroisoxazolium methylsulfate (5) with sodium
cyanide
Products, yellow oils; room temperature: 9 yield: 40%;
60 °C: 10 yield: 33%; 110 °C: 10 yield: 12%.
To a solution of 2-methyl-4,5-dihydroisoxazolium meth-
ylsulfate dissolved in DMSO (the solvent was distilled from
above calcium hydride) sodium cyanide was added in a sto-
ichiometric amount with respect to the initial 2-isozoxazoline.
The reactions were carried out for 2 h at different tempera-
tures: room temperature, 60 and 110 °C.
After reaction completion the mixture was poured into
water; turbidity of the solution was noticed. The products were
then extracted with diethyl ether. The extracts were dried with
magnesium sulfate, and then ether was removed under reduced
pressure and a mixture of crude products was obtained. The
products obtained were purified by dissolution in hot hexane
and boiling with active carbon, which was then filtered off on
a fluted filter paper.After concentration the residue was chro-
matographed (hexane/diethyl ether = 7:2) to give products.
4.3.2.1. Ethyl 2-(N-methylamineoxy)-4-oxo-2-phenylbuta-
1
neate (9). H NMR: 1.43 (t, 3H, CH3, J = 7.0 Hz), 2.67 (s,
1H, NH), 2.95 (s, 3H, CH3–N), 3.08 and 3.89 (2 × d, 2H,
CH2, J = 13.5 Hz), 4.32 (q, 2H, CH2, J = 7.0 Hz), 7.35–7.41
(m, 5H,Ar), 9.95 (m, 1H, CHO); 13C NMR: 13.63 (CH3CH2),
34.23 (CH3–N), 47.67 (CH2), 60.88 (CH3CH2), 81.41 (CIV),
127.37 (p-Ar), 128.92 (m-Ar), 131.11 (o-Ar), 135.29 (Ar),
174.06 (C=O), 200.65 (CHO); IR(film) cm−1: 3432 (m N–H),
2856 (CHO), 1734 (C=O), 1724 (C=O ald), 1528 (d N–H);
Analysis of compound C12H17NO4 Calc.: C 6.25, N 5.86, H
7.11, Found: C 60.31, N 5.84, H 7.08.
4.3.1. Reactions of the 2,3-dimethyl-5-ethoxycarbonyl-5-
phenyl-4,5-dihydroisoxazolium methylsulfate (4) with
sodium cyanide
Products, yellow oils; room temperature: yield: 67%,
7-trans:7-cis = 34%:66%
4.3.2.2. 5-Ethoxycarbonyl-2-methyl-5-phenyl-3-isoxazoline
1
(10). H NMR: 1.21 (t, 3H, CH3, J = 7.0 Hz), 2.67 (s, 3H,
CH3–N), 3.47 (q, 2H, CH2, J = 7.0 Hz), 4.73 (d, 1H, CH-4,
J = 3.6 Hz), 5.81 (d, 1H, CH-3, J = 3.6 Hz), 7.35–7.39 (m,
5H, Ar); 13C NMR: 14.18 (CH3CH2), 39.78 (CH3–N), 61.62
(CH3CH2), 96.97 (C-5), 109.55 (C-4), 127.82 (o-Ar), 131.21
(m-Ar), 132.68 (p-Ar), 136.28 (Ar), 141.08 (C-3), 175.09
(C=O); IR(film) cm−1: 1728 (C=O); Analysis of compound
C12H15NO3 Calc.: C 65.16, N 6.33, H 6.79, Found: C 65.18,
N 6.32, H 6.81.
60 °C: yield: 59%, 7-trans:7-cis: 8 = 34%:52%:14%;
110 °C: yield: 44%, 7-trans:7-cis: 8 = 22%:7%:71%.
4.3.1.1. trans 2,3-Dimethyl-3-cyano-5-ethoxycarbonyl-5-
1
phenylisoxazolidine (7-trans). H NMR: 1.20 (t, 3H, CH3,
J = 7 Hz), 1.55 (s, 3H, CH3), 2.90 and 3.50 (2 × d, 2H, CH2,
J = 13.2 Hz), 2.89 (s, 3H, CH3–N), 4.20 (q, 2H, CH2,
J = 7 Hz), 7.31–7.55 (m, 5H,Ar); 13C NMR: 13.98 (CH3CH2),
21.86 (CH3), 39.11 (CH3–N), 52.98 (C-4), 61.92 (C-3), 63.97
(CH3CH2), 84.35 (C-5), 118.75 (C≡N), 124.93, 127.06,
128.47, 141.01 (Ar), 169,79 (C=O); IR (film) cm−1: 2232
(C≡N),1732 (C=O).
4.3.3. Reactions of the 3,5-diphenyl-5-ethoxycarbonyl-2-
methyl-4,5-dihydroisoxazolium methylsulfate (6) with
sodium cyanide
Products, yellow oils; room temperature: yield: 27%,
11:12 = 54%:46%; 60 °C: yield: 6%, 11:12 = 88%:22%.

I. Zadroz˙na et al. / Il Farmaco 60 (2005) 948–954
953
4.3.3.1. 3,5-Diphenyl-5-ethoxycarbonyl-2-methyl-3-isoxazo-
line (11). H NMR: 1.26 (t, 3H, CH3, J = 7.2 Hz), 2.98 (s,
4.4. General procedures of biotransformation
with Rhodococcus
1
3H, CH3–N), 4.25 (q, 2H, CH2, J = 7.2 Hz), 5.62 (s, 1H, CH),
7.32–7.51 (m, 10H, Ar); 13C NMR: 14.04 (CH3CH2), 37.28
(CH3–N), 59.62 (CH3CH2), 96.06 (C-5), 98.85 (C-4), 127.32
– 136.25 (Ar), 148.68 (C-3), 177.17 (C=O); IR(film) cm−1:
1740 (C=O) Analysis of compound C19H19NO3 Calc.: C
73.79, N 4.53, H 6.15, Found: C 73.77, N 4.55, H 6.14.
Culture of Rhodococcus from an agar slop with solid
medium (1.5% pancreatic digest of casein, 0.5% papaic digest
of soya bean, 0.5% NaCl, 2% glucose, 1,5% agar, pH
7.0 0.2) was used to inoculate 100 ml of liquid medium
(1.7% pancreatic digest of casein, 0.3% papaic digest of soya
bean, 0.5% NaCl, 2% glucose, 0.25% dipotassium hydrogen
phosphate, pH 7.0 0.2) in a 300 ml conical flask. Culture
was incubated at 30 °C, in a rotary shaker at 120 rpm for
24 h. Than it was centrifuged at 4000 rpm for 30 min, and
about 1 g of wet mass of Rhodococcus was resuspended in
50 ml of phosphate buffer solution (pH 7) in 250 ml flask.
The culture was activated at 30 °C for 0.5 h with rotary shak-
ing (120 rpm), and then 0.5 mmol of appropriate compound
was added (dissolved in 1 ml of suitable solvent). The mix-
ture was incubated at 30 °C with rotary shaking (120 rpm).
After reaction completion the post-reaction mixture was
extracted three times with 20 cm3 of CH2Cl2. The solvent
was then distilled off under reduced pressure. After prelimi-
nary analysis of IR and NMR spectra it was found that the
ester and water are present in the reaction product. The prod-
uct was dissolved in 5 cm3 of methylene chloride and treated
with 10 cm3 of a saturated NaHCO3 solution and thus the
acid was converted into a sodium salt. The layers were sepa-
rated and the organic layer was dried with magnesium sul-
fate, after which the solvent was distilled off under reduced
pressure and the unreacted ester was obtained. The aqueous
layer was neutralized with a diluted hydrochloric acid solu-
tion and also extracted with 20 cm3 of methylene chloride.
After drying the solution with magnesium sulfate the solvent
was distilled off under reduced pressure, and thus the prod-
uct, i.e. the acid, was obtained.
4.3.3.2. Ethyl 2,4-diphenyl-2-(N-methylamineoxy)-4-oxobuta-
neate (12). 1H NMR: 1.45 (t, 3H, CH3, J = 7.0 Hz), 2.63 (s,
1H, NH), 2.94 (s, 3H, CH3–N), 2.99 and 3.87 (2 × d, 2H,
CH2, J = 13.2 Hz), 4.39 (q, 2H, CH2, J = 7.0 Hz), 7.30–7.38
(m, 10H, Ar); 13C NMR: 13.62 (CH3CH2), 34.26 (CH3–N),
46.50 (CH2), 60.11 (CH3CH2), 89.45 (CIV), 126.23 (Ar),
126.98 (Ar); 127.37 (Ar), 128.92 (Ar), 129.11(Ar), 131.11
(Ar), 135.29 (Ar), 135.86 (Ar), 174.33 (C=O) 197.46 (C=O);
IR(film) cm−1: 3430 (m N–H), 1732 (C=O),1528 (d N–H);
Analysis of compound C19H21NO4 Calc.: C 69.72, N 4.28, H
6.42, Found: C 69.73, N 4.26, H 6.41.
4.3.4. Reduction of the trans and cis 3-cyano-2,3-
dimethyl-5-ethoxycarbonyl-5-phenylisoxazolidine (7)
with LAH
0.09 g (0.33 mmol) of the mixture of 7-cis and 7-trans in
20 ml of dry ethyl ether was added to the suspension of 0.07 g
(2 mmol) of LiAlH4 in 10 ml of ether at 5 °C in 5 min. After
90 min reflux to the stirred mixture was added slowly 10 ml
of water and the product was extracted with ethyl ether. The
extracts were dried with magnesium sulfate and ether was
removed under reduced pressure.
Product, yellow oil, yield 0.048 g (63%); 13-trans: 13-
cis = 42%: 58%.
4.3.4.1. trans 3-Cyano-2,3-dimethyl-5-hydroxymethyl-5-
phenylisoxazolidine (13-trans). 1H NMR: 1.57 (s, 3H, CH3),
2.89 and 3.47 (2 × d, 2H, CH2, J = 12.6 Hz), 2.87 (s, 3H,
CH3–N), 3.81 (s, 2H, CH2), 7.28–7.36 (m, 5H,Ar); 13C NMR:
21.32 (CH3), 39.01 (CH3–N), 50.96 (C-4), 64.03 (C-3), 70.51
(CH2), 83.43 (C-5), 118.47 (C≡N), 127.48, 128.04, 128.57,
140.22 (Ar); IR (film) cm−1: 3372 (OH), 2232 (C≡N).
4.4.1. Microbiological hydrolysis of 7-trans and 7-cis
using Rhodococcus species NCIB 10554
Product, yellow oil; 14 days, yield: 14%, 14-trans:14-
cis = 65%:35%.
4.4.1.1. trans 5-Carboxy-3-cyano-2,3-dimethyl-5-phenyl-
1
isoxazolidine (14-trans). H NMR: 1.56 (s, 3H, CH3), 2.92
(s, 3H, CH3–N), 2.98 and 3.40 (2 × d, 2H, CH2, J = 13.4 Hz),
7.35–7.48 (m, 5H, Ar), 9.18 (s, 1H, OH); 13C NMR: 20.76
(CH3), 38.42 (CH3–N), 53.38 (C-4), 64.08 (C-3), 83.60 (C-5),
117.97 (C≡N), 124.83, 125.56, 128.63, 140.60 (Ar), 173.22
(C=O); IR(KBr) cm−1: 3028 (OH), 2236 (C≡N), 1720 (C=O).
4.3.4.2. Cis-3-Cyano-2,3-dimethyl-5-hydroxymethyl-5-
phenylisoxazolidine (13-cis). H NMR: 1.47 (s, 3H, CH3),
1
2.56 and 3.70 (2 × d, 2H, CH2, J = 12.6 Hz), 2.97 (s, 3H,
CH3–N), 3.79 (s, 2H, CH2), 7.28–7.36 (m, 5H,Ar); 13C NMR:
21.93 (CH3), 38.83 (CH3–N), 51.24 (C-4), 62.36 (C-3), 67.52
(CH2), 84.74 (C-5), 118.99 (C≡N), 124.52, 125.10, 125.50,
143.35 (Ar); IR (film) cm−1: 3372 (OH), 2232 (C≡N).
4.4.1.2. cis 5-Carboxy-3-cyano-2,3-dimethyl-5--phenyl-
isoxazolidine (14-cis). 1H NMR: 1.47 (s, 3H, CH3), 2.70 and
3.77 (2 × d, 2H, CH2, J = 13.4 Hz), 2.89 (s, 3H, CH3–N),
7.35–7.48 (m, 5H, Ar), 8.25 (s, 1H, OH); 13C NMR: 21.74
(CH3), 38.99 (CH3–N), 52.83 (C-4), 63.95 (C-3), 84.16 (C-5),
118.52 (C≡N), 125.233, 125.59, 128.55, 136.50 (Ar), 176.23
(C=O); IR(KBr) cm−1: 3028 (OH), 2236 (C≡N), 1720 (C=O).
4.3.4.3. Analysis of mixture of compounds. 13-trans and 13-cis
C13H16N2O2 calc.: C 67.24, N 12.07H 6.90, Found: C 67.23,
N 12.06, H 6.87.

954
I. Zadroz˙na et al. / Il Farmaco 60 (2005) 948–954
[14] I. Zadroz˙na, E. Kornacka, 4,5-Dihydroisoxazoles. Regioselectivity in
Analysis of mixture of compounds 14-trans and 14-cis
C13H14N2O3 Calc.: C 63. 42, N 11.38, H 5.69, Found: C 63.43,
N 11.37, H 5.69.
the 1,3-Dipolar Cycloaddition of Nitrile Aliphatic Oxides to Cin-
namicAcid Esters, NMR Criteria, Bull. Pol.Acc. Chem. 45 (4) (1997)
363–372.
[15] I. Zadroz˙na, J. Kurkowska, “Microbial Biotransformation of the Sub-
stituted 4,5-Dihydroisoxazoles” –7th European Conference on Spec-
troscopy of Biological Molecules, Madrid, Spain, September 1997
(7–12).
4.4.2. Microbiological hydrolysis of 13-trans and 13-cis
using R. rhodochrous PCM 909
Product, yellow oil; 48 h; yield: 0.014 g (11%).
[16] I. Zadroz˙na, E. WalTg, D. Starzomska, J. Kurkowska, Chemoenzy-
matic synthesis of optically active acids and alcohols with a 4,5-
dihydroisoxazolic group, XVII-th European Colloquium on Hetero-
cyclic Chemistry B-112 (1998) 4–7 [Rouen, France].
[17] I. Zadroz˙na, E. Kornacka, Reactivity of methylsulfates of N-Methyl-
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[18] I. Zadroz˙na, J. Kurkowska, I. Makuch, Synthesis of optically active
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(1999) 111–118.
[19] I. Zadroz˙na, J. Kaniuk, J. Kurkowska, I. Makuch, H. Kruszewska,
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4.4.2.1. trans 3-Carboxy-2,3-dimethyl-5-hydroxymethyl-5-
phenylisoxazolidine (15-trans). 1H NMR: 1.59 (s, 3H, CH3),
3.00 and 3.45 (2 × d, 2H, CH2, J = 13.4 Hz), 2.96 (s, 3H,
CH3–N), 3.88 (s, 2H, CH2), 7.39–7.51 (m, 5H,Ar); 13C NMR:
21.18 (CH3), 38.77 (CH3–N), 51.28 (C-4), 71.59 (CH2), 77.92
(C-3), 89.96 (C-5), 125.57, 128.08, 128.91, 139.32 (Ar),
179.51 (C=O); IR(film) cm−1: 3340 (OH), 1708 (C=O);
Analysis of compound C13H16NO4 Calc.: C 62.15, N 5.58, H
6.77, Found: C 62.20, N 5.57, H 6.63.
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