Synthesis, crystal structure, and in vitro antiprotozoal activity of some 5-phenyl(methyl)sulfonyl-substituted dihydroisoxazoles

Article abstract of DOI:10.1007/s00706-012-0866-6

4,5-Dihydroisoxazole derivatives are interesting synthetic targets that exhibit various biological activities, including anti-infective. Taking account of the principle of bioisosterism, a number of 4,5-dihydroisoxazoles carrying a phenyl- (or methyl-)sul

Full text of DOI:10.1007/s00706-012-0866-6

Monatsh Chem (2013) 144:707–716
DOI 10.1007/s00706-012-0866-6
ORIGINAL PAPER
Synthesis, crystal structure, and in vitro antiprotozoal activity
of some 5-phenyl(methyl)sulfonyl-substituted dihydroisoxazoles
•
•
•
˘
Yas¸ar Du¨ru¨st Cevher Altug Marcel Kaiser
•
Frank R. Fronczek Deniz Tasdemir
Received: 8 May 2012 / Accepted: 24 September 2012 / Published online: 9 January 2013
Ó Springer-Verlag Wien 2012
Abstract 4,5-Dihydroisoxazole derivatives are interest-
ing synthetic targets that exhibit various biological
activities, including anti-infective. Taking account of the
principle of bioisosterism, a number of 4,5-dihydroisox-
azoles carrying a phenyl- (or methyl-)sulfonyl group at
position 5 were designed and synthesized by 1,3-dipolar
cycloaddition of nitrolic acid-generated nitrile oxides with
electron-deficient phenyl (or methyl) vinyl sulfones. The
structures of all the cycloadducts were elucidated by means
of spectroscopic methods (NMR, MS), X-ray diffraction,
and physical characteristics. The in vitro antiprotozoal and
cytotoxic activities of these heterocyclic compounds were
investigated.
Introduction
Protozoal diseases such as malaria, trypanosomiasis, and
leishmaniasis are responsible for significant morbidity and
mortality in underdeveloped regions of the world. These
diseases are caused by single cell parasites and transmitted
to humans by different types of insects. With around 300
million new cases worldwide each year, and around one
million deaths [1], malaria is by far the most important
parasitic infection. Particularly falciparum malaria caused
by Plasmodium falciparum and spread from person to
person by Anopheles mosquitoes poses a high risk for
young children and pregnant women in Africa. American
trypanosomiasis (also known as Chagas’ disease) is caused
by Trypanosoma cruzi and transmitted to humans by tri-
atomine bugs. It is endemic in South America and has an
acute phase and a chronic stage resulting in heart or gas-
trointestinal complications. It is estimated that 10 million
people are infected with American trypanosomiasis
worldwide [2]. Human African trypanosomiasis (sleeping
sickness, HAT) is transmitted by the bite of the tsetse fly.
Trypanosoma brucei rhodesiense and T. b. gambiense are
responsible for East and West African trypanosomiasis,
respectively. The number of actual HAT cases estimated by
the World Health Organization (WHO) is 30,000, and the
number of new cases reported in 2009 is around 10,000 [3].
If left untreated, HAT is usually fatal. Human visceral
leishmaniasis (HVL), a potentially fatal parasitic disease
caused by Leishmania donovani, L. infantum, and L. chag-
asi, occurs via the bite of phlebotomine sandflies. HVL
accounts for more than 50,000 deaths every year [4]. In the
absence of a protective vaccine, antiparasitic drugs are
essential to addressing the health and economic burdens
caused by these diseases. Unfortunately, most current med-
ications are generally very old with limited effectiveness or
Keywords 1,3-Dipolar cycloaddition Á Nitrile oxide Á
Nitrolic acid Á Bioacitivity Á Toxicity
˘
Y. Du¨ru¨st (&) Á C. Altug
˙
Department of Chemistry, Abant Izzet Baysal University,
Bolu, Turkey
e-mail: yasardurust@ibu.edu.tr
M. Kaiser
Department of Medical Parasitology and Infection Biology,
Swiss Tropical and Public Health Institute, 4002 Basel,
Switzerland
F. R. Fronczek
Department of Chemistry, Louisiana State University,
Baton Rouge, LA 70803-1804, USA
D. Tasdemir
School of Chemistry, National University of Ireland,
Galway, University Road, Galway, Ireland
e-mail: deniz.tasdemir@nuigalway.ie
123

708
Y. Du¨ru¨st et al.
Scheme 1
O
O
H
R1
C
R1
C
N
S
O
O
R2
-HNO2
OH
O
S
R1
R2
O2N
N
N
Toluene
Toluene
H
O
Δ
Δ
H
3
4
5
R1
serious adverse reactions. Moreover, parasite resistance to
existing drugs has become a serious problem, indicating the
need for new drugs in this field. Owing to lack of financial
returns, however, antiprotozoal drug discovery and devel-
opment largely depends on funding by public authorities
and universities.
C
N
N
O
R1
N
R1
O
C
4,5-Dihydroisoxazoles are an interesting class of hetero-
cycles that are stable toward nucleophiles such as thiols and
are easily derived from 1,3-dipolar cycloaddition of nitrile
oxides with an acrylate or a substituted ethylene via an inter-
or intramolecular pathway [5–7]. 4,5-Dihydroisoxazoles are
often used as intermediates in the preparation of or as pro-
tecting groups for a wide variety of difunctionalized
compounds. For example, cleavage of the 4,5-dihydroisox-
azole can give rise to b-hydroxyketones, a,b-unsaturated
ketones, and c-aminoalcohols [8–14]. The dihydroisoxazole
moiety is also found as the end product in many pharma-
ceutically active compounds. Diverse biological activities
have been reported for this class of compounds. Antibacterial,
antitrypanosomal, and antitrichomoniasis activities of isox-
azole-substituted nitroimidazoles have been known for a long
time [15, 16]. Dihydroisoxazoles, particularly 4,5-dihyd-
roisoxazole derivatives, also display calcium channel
blocking, estrogen receptor agonist, anticancer, herbicidal,
neuroprotective, human transglutaminase 2 inhibitory activ-
ity, and binding affinity at glutamic acid receptors [17–25].
N-Hydroxy- or N-oxy-benzamidino-isoxazoles and dihy-
droisoxazolyl-oxadiazoles have been shown to exhibit
insecticidal, acaricidal, nematicidal, or molluscicidal poten-
tial [26–28]. Recent studies were devoted to the assessment of
the anti-inflammatory [29] and anti-infective (such as anti-
viral [30], antiprotozoal, antifungal [17, 28, 31], and
antibacterial [17]) properties of 4,5-dihydroisoxazole and
other isoxazoles. For example, 3-bromoacivicin ((aS,5S)-
a-amino-3-bromo-4,5-dihydroisoxazol-5-acetic acid) shows
very potent in vitro trypanocidal and in vivo trypanostatic
activity and inhibits CTP synthetase [32]. Dihydroisoxazolyl-
6-chloropurine and adenine are effective against HIV-1 in
acutely infected human lymphocytes [33], whereas phos-
phonated dihydroisoxazoles inhibit the herpes simplex virus
(HSV) [34]. Owing to their wide range of biological activi-
ties, dihydroisoxazoles have been constructed by numerous
synthetic approaches [35–42].
H
O
O
H
H
S
H
R2
O
H
5-regioisomer
O
S
R2
H
O
R1
C
N
O
R1
N
O
R1
C
N
H
O
O
H
H
S
H
O
R2
H
O
H
S
4-regioisomer
O
R2
Scheme 2
interest in 1,3-dipolar cycloaddition chemistry [44], a
number of 4,5-dihydroisoxazoles carrying phenyl(methyl)-
sulfonyl group at position 5 (5a–5p, Scheme 1) were
designed. Since there is no report on the synthesis of di-
hydroisoxazoles by using nitrolic acid as the precursor of
the nitrile oxide, we focused on the 1,3-dipolar cycload-
dition reaction of 4, which are generated in situ from
nitrolic acids 3 (Schemes 1, 2). The nitrile oxides reacted
regioselectively with phenyl or methyl vinyl sulfones to
afford 3,5-disubstituted 4,5-dihydroisoxazoles 5a–5p.
Nitrolic acids (a-nitro oximes), some of which show bio-
logical activities [45], were first reported by Meyer [46].
Since then, a limited number of studies on their synthesis,
stability, and use were reported in the literature [47–52].
Nitrolic acids are well known to undergo 1,3-dipolar
cycloadditions; however, to the best of our knowledge,
synthetic applications of this process are rarely found in
the literature in contrast to the functionally equivalent
In continuation of our interest in the synthesis of
novel synthetic antiprotozoal agents [43] plus our ongoing
123

Synthesis, crystal structure, and in vitro antiprotozoal activity
709
Table 1 Synthesis of
dihydroisoxazoles 5a–5p
(Scheme 1)
Comp.
R1
R2
Yield/%
Solvent
Time/h
5a
5b
5c
5d
5e
5f
2-Pyridyl
Ph
Ph
Ph
Ph
Me
Me
Me
Ph
Ph
Ph
Ph
Ph
Me
Ph
Me
Ph
55
71
49
72
88
80
92
70
69
65
60
69
75
58
78
58
Toluene
Toluene
Toluene
Toluene
THF
2
3-Pyridyl
2
4-Pyridyl
2
6-Cl-3-pyridyl
2-Pyridyl
2
0.5
0.5
0.5
2
3-Pyridyl
THF
5g
5h
5i
6-Cl-3-pyridyl
4-NO2-C6H4
2-NO2-C6H4
3-NO2-C6H4
4-Cl-C6H4
4-MeO-C6H4
4-MeO-C6H4
4-F-C6H4
THF
Toluene
Toluene
Toluene
Toluene
DCM
2
5j
2
5k
5l
2
2
5m
5n
5o
5p
DCM
2
DCM
2
4-F-C6H4
DCM
2
2-Furanyl
Toluene
2
cycloadditions of nitrile oxides [53–57]. This report deals
with their synthetic methodology and antiprotozoal activ-
ities evaluated against a small panel of protozoan parasites,
T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum.
Selective toxicity of all compounds was also determined
against L6 cells, a primary cell line derived from rat
skeletal myoblasts.
The structure elucidations of the new cycloadducts,
namely phenyl (or methyl) sulfonyl-substituted dihydrois-
oxazoles, were performed by 1H NMR, 13C NMR, MS (low
and high resolution), X-ray diffraction, and physical char-
acteristics. On the basis of the relative positions of the
peaks and coupling constants of the relevant protons in the
1H NMR spectra, the 5-regioisomers were the major iso-
mers formed, in all but one instance. In the case of
p-nitrophenyl-substituted 5h (Fig. 1), a mixture of the
5- and 4-regioisomers was found in a 3.46:1 ratio, calcu-
Results and discussion
1
lated from the H NMR spectrum of the crude reaction
Synthesis of nitrolic acids 3a–3g and 3h–3p
mixture. Attempts to separate the regioisomers failed
owing to their very close physical characteristics.
Pyridine-substituted nitrolic acids 3a–3g were prepared
from the reaction of aromatic aldoximes, which are easily
obtained from the corresponding aromatic aldehyde and
hydroxyl amine [47] with fuming nitric acid in acetic acid,
whereas aryl-substituted nitrolic acids 3h–3p were pre-
pared from the interaction of aryl aldoximes with
dinitrogen tetraoxide gas in diethyl ether. All nitrolic acids
were obtained as a single isomer and kept in a refrigerator
without decomposition over months.
Synthesis of methyl sulfonyl-substituted dihydroisox-
azoles required less severe reaction conditions than phenyl
sulfonyl-substituted derivatives and yields were also found
to be relatively higher than those of phenyl sulfonyl-
substituted products. This may be attributed to the electron-
releasing and less bulky nature of the methyl group.
Theoretically, the 1,3-dipolar nitrile oxide species may
approach the unsymmetrical alkene with the phenyl (or
methyl) vinyl sulfone oriented in two different ways
leading to the formation of 5-regioisomer or 4-regioisomer
regioselectively, depending on the transition state energy of
the 1,3-dipolar cycloaddition reaction as a result of the
nature of the groups existing both in the 1,3-dipole and
dipolarophile as depicted in Scheme 2 [52]. The regiose-
lectivity and configurations of the stereocenters for the
compounds 5a and 5b were also confirmed by single-
crystal X-ray diffraction data (Fig. 2).
Synthesis and structure determination
of dihydroisoxazoles 5a–5p
Reaction of nitrolic acid with the dipolarophiles phenyl
vinyl sulfone and methyl vinyl sulfone to yield dihydrois-
oxazoles 5a–5p (Scheme 1; Table 1) was carried out under
neutral conditions, and in this regard, this synthetic route
can be regarded as one of the main advantages of using
nitrolic acids in comparison to the other nitrile oxide
precursors.
As for the representative spectral characteristics of the
cycloaddition products phenyl (methyl) sulfonyl-substi-
tuted 4,5-dihydroisoxazoles, in the IR spectra of these
123

710
Y. Du¨ru¨st et al.
1
Fig. 1 Expanded partial H NMR spectrum of compound 5h showing proton signals related to both regioisomers
Fig. 2 X-ray crystal structures
of compounds 5a and 5b
heterocycles, the C=N stretching vibration arises at around
1,602–1,541 cm-1 and the stretching vibration for the SO2
group is found between 1,155 and 1,126 cm-1 indicating
asymmetric and symmetric stretching of the sulfonyl
and 2D NMR measurements, heteronuclear 2D NMR
experiments, i.e., HMBC and HSQC, were also performed
to establish the exact positions of the carbon atoms in the
isoxazole ring.
1
group, respectively. In the H NMR spectra, the hydrogen
In the EI mass spectra of the isoxazolines, the base
peaks were found to be due to ionic species, M-PhSO2, as
would be predicted because extrusion of PhSO2 from the
molecule produces an aromatic stabilized species, i.e., an
isoxazole (Scheme 3).
(Ha) on the sp3 carbon attached to the sulfonyl group is
most deshielded and resonates at around 5.60 ppm as a
doublet of doublets. Methylene protons in the isoxazoline
ring, in most cases, show a separate doublet of doublets
splitting pattern at around 4.10 and 3.80 ppm (Fig. 3).
The interrelationship between the Ha proton and adja-
cent CH2 hydrogens is clearly seen by COSY and also by
DEPT-135 and DEPT-90 spectra. The DEPT spectra
allowed the assignment of the number 4 and 5 carbons in
the isoxazole ring. In addition to the aforementioned 1D
Bioactivity assessments
Table 2 displays the in vitro antiprotozoal activity of syn-
thesized compounds against T. b. rhodesiense (bloodstream
forms), T. cruzi (intracellular amastigotes in L6 rat skeletal
123

Synthesis, crystal structure, and in vitro antiprotozoal activity
711
5m showed some toxic potential whereas the others were
nontoxic even at the highest test concentrations
(100 lg cm-3). These results indicate that a nitro sub-
stituent in either ortho or para positions of R1, as found in
compounds 5h and 5i, is favorable for antiprotozoal
activity, although this is associated with some toxicity.
Interestingly, a nitro substituent in the meta position of the
phenyl ring is not favored, as the antiprotozoal potency of
5j is much lower than that of 5h and 5i. As seen in com-
pound 5l, the combination of a p-methoxy substituent on
the phenyl ring (R1) and an aromatic substituent (phenyl) at
R2 results in a relatively good activity against L. donovani.
However, when the substituent at R2 is replaced by a
methyl group (5m), leishmanicidal activity drops signifi-
cantly. This tendency is clear for all other antiprotozoal
activity for this entry (5m). Among those compounds with
a pyridyl substituent at R1, the most significant activity was
obtained against L. donovani by 5d. This compound bears a
3-pyridyl substituent with a chlorine atom at the 6-position
at R1. The pyridyl- and fluorine-substituted compounds
with a methyl group at R2 generally display low or no
antiprotozoal potential, indicating the necessity of an aro-
matic function at R2 in general. The only furanyl-
substituted dihydroisoxazole, 5p, showed some activity
against Trypanosoma species, but was inactive towards
other remaining parasites.
Fig. 3 COSY spectrum of 5j indicating the couplings of C-4 (Hb,
Hc) and C-5 (Ha) hydrogens in the isoxazoline ring
+
N
N
N
N
-PhSO2
O
O
H
H
H
H
H
SO2Ph
Conclusions
Scheme 3
We have demonstrated a simple and practical new syn-
thetic route for the preparation of a series of aryl- and
phenyl(methyl)sulfonyl-substituted dihydroisoxazoles, some
of which were previously reported through oxime-generated
nitrile oxide cycloadditions, by 1,3-dipolar cycloaddition of
nitrolic acid-generated nitrile oxides to phenyl (or methyl)
vinyl sulfones and determined their structures by means of
spectroscopic methods including X-ray crystallography.
Owing to the reported diverse biological activities such
as antibacterial, antitrypanosomal, and antitrichomoniasis
for this class of compounds [15, 16] and the fact that they
also display calcium channel blocking, estrogen receptor
agonist, anticancer, herbicidal, neuroprotective, and human
transglutaminase 2 inhibitory activity and binding affinity
at glutamic acid receptors [17–25], isoxazole derivatives
5a–5p were efficiently designed, prepared, and assayed for
their antiprotozoal activity as well as for general cytotox-
icity. Among ten compounds that showed antileishmanial
potential, 5i, 5h, 5l, and 5d were found to be the most
myoblasts), L. donovani (axenic amastigotes), and P. fal-
ciparum (blood stage forms of multi-drug resistant K1
strain). Compounds 5h and 5i were the most potent against
the T. b. rhodesiense parasite (IC50 values 6.9 and
9.0 lg cm-3). All remaining compounds also possessed
some activity with IC50 values ranging between 27.2 and
53.1 lg cm-3
.
Towards the American trypanosomes (T. cruzi), low
activity was observed for all compounds (IC50 values
40.0–87.8 lg cm-3). Among ten compounds that showed
antileishmanial potential, 5i, 5h, 5l, and 5d were the most
active ones with IC50 values between 3.4 and 9.9 lg cm-3
.
A moderate leishmanicidal activity was exhibited by 5k
and 5n with IC50 values below 20 lg cm-3. The growth
inhibitory potential was modest towards the malaria para-
site (P. falciparum) and again 5h and 5i appeared to have
the lowest IC50 values (9.3 and 13.9 lg cm-3, respec-
tively). The IC50 values of the remaining eight active
active ones with IC50 values between 3.4 and 9.9 lg cm-3
.
molecules were within the range of 20.8–49.8 lg cm-3
When tested for toxicity against L6 cells, a primary cell
.
Nitro substitution in the ortho or para positions of R1, as
found in compounds 5h and 5i, is favorable for antiproto-
zoal activity, but associated with some toxicity. However,
line derived from rat skeletal myoblasts, only 5h, 5i, 5l, and
123

712
Y. Du¨ru¨st et al.
Table 2 Antiprotozoal
activities of compounds 5a–5p
Comp.
T. b. rhodesiense
T. cruzi
L. donovani
P. falciparum
Cytotoxicity L6 cells
Standarda
5a
0.005b
42.0
41.7
42.9
48.3
42.4
36.9
36.6
6.9
0.464c
44.6
50.7
43.4
48.2
50.0
53.2
50.4
42.3
44.2
55.4
48.1
40.0
87.8
50.3
73.3
58.0
0.171d
91.5
0.073e
49.8
36.3
41.7
[50
[50
33.3
44.6
9.3
0.007f
[100
[100
[100
[100
[100
[100
[100
28.5
5b
5c
97.5
[100
9.9
5d
5e
[100
[100
[100
4.2
5f
5g
5h
5i
9.0
3.4
13.9
43.1
28.9
20.8
38.6
[50
[50
[50
42.3
5j
28.7
29.3
27.2
43.4
27.4
41.8
53.1
53.3
[100
[100
52.9
IC50 values are in lg cm-3. The
significant activities are shown
in bold
5k
5l
15.7
8.8
5m
5n
5o
91.2
83.0
a
Standard compounds (control
b
16.6
[100
[100
[100
drugs): melarsoprol,
c
e
f
d
benznidazole, miltefosine,
[100
[100
chloroquine,
podophyllotoxin
5p
General procedure for synthesis of dihydroisoxazoles
5a–5p
a nitro substituent in the meta position of the phenyl ring is
not favored, interestingly, as the antiprotozoal potency of
5j is much lower than that of 5h and 5i. In summary,
although the observed bioactivity of the synthesized
molecules is low in comparison to the reference com-
pounds, this study still indicates the antiprotozoal potential
of 4,5-dihydroisoxazoles, particularly when their toxic
potential is reduced after further medicinal chemistry
efforts.
Nitrolic acid 3a–3p (1.0 mmol) was added to phenyl (or
methyl) vinyl sulfone (1 mmol) in 5 cm3 dry toluene and
the mixture was heated to reflux for 30 min. The reaction
mixture was concentrated in vacuo, and the crude residue
was purified by flash column chromatography (eluent
petroleum ether/ethyl acetate 2:1) to give the title com-
pounds as yellow solids.
(R)-4,5-Dihydro-5-(phenylsulfonyl)-3-(pyridin-2-yl)-
isoxazole (5a, C14H12N2O3S)
Experimental
Recrystallized from ethylacetate/n-hexane to afford crys-
tals suitable for single-crystal X-ray diffraction. Yield
158 mg (55 %); m.p.: 127–129 °C (Ref. [54] 126–127 °C).
Melting points were determined on an Electro Thermal
melting point apparatus. Infrared spectra were recorded on
Shimadzu FTIR spectrophotometer. Mass spectra (low and
high resolution) were recorded on a Fisons VG Platform II
spectrometer and on a Micromass Q-TOF Micro spec-
trometer (ESI-interface). NMR spectra were recorded on a
Bruker DPX 400 spectrometer operating at 400 MHz for
1H and at 100 MHz for 13C at 25 °C. All chemical shifts
are reported in ppm downfield from TMS. Coupling con-
Crystal data: C14H12N2O3S, Mr = 288.32; monoclinic,
˚
˚
P21/n; a = 5.3810(10) A, b = 8.2117(7) A, c = 29.564(7)
3
˚
˚
A, b = 92.742(9)°, V = 1304.9(5) A ; Z = 4, Dx = 1.468
Mg m-3, Dm not measured; Mo Ka radiation, k =
˚
0.71073 A; cell parameters from 3,214 reflections, h = 2.5–
28.3°, l = 0.257 mm-1; T = 90 K; colorless needle, 0.37 9
0.07 9 0.03 mm.
1
stants (J) are reported in Hz. Multiplicity in H NMR is
reported as singlet (s), doublet (d), doublet doublet (dd),
double triplet (dt), double triple doublet (dtd), double
quartet (dq), triplet (t), and multiplet (m).
(R)-4,5-Dihydro-5-(phenylsulfonyl)-3-(pyridin-3-yl)-
isoxazole (5b, C14H12N2O3S)
Obtained as a colorless solid which was recrystallized from
ethylacetate/n-hexane to afford suitable crystals for single-
crystal X-ray diffraction. Yield 205 mg (71 %); m.p.: 154–
155 °C; Rf = 0.59 (ethyl acetate/n-hexane 1:2); IR (KBr):
Pyridyl- (3a–3g) and other aryl-substituted (3h–3p)
nitrolic acids were prepared according to the procedures
described previously and used without further purification.
Their structural characteristics were checked and compared
with the data in the literature [47, 58, 59].
ꢀm = 1,580, 1,451, 1,311, 1,306, 1,155, 851, 770, 735 cm-1
;
1H NMR (400 MHz, CDCl3): d = 8.75 (s, 1H), 8.60
123

Synthesis, crystal structure, and in vitro antiprotozoal activity
713
(s, 1H), 7.91 (t, 3H), 7.61 (t, 1H), 7.50 (t, 2H), 7.30 (m, 1H),
5.56 (dd, J = 10.9, 4.6 Hz, 1H), 4.00 (dd, J = 8.3, 4.5 Hz,
1H), 3.75 (q, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d = 154.7 (C=N, isoxazole), 151.8 (C=N, pyridine), 147.8,
135.1, 134.7, 134.2, 129.7, 129.3, 128.3, 123.7, 93.5,
36.3 ppm; MS: m/z (%) = 146 (100, [M-PhSO2]?), 78
(46), 51 (25); HRMS-TOF–MS (ES?): found 289.0633,
calculated for C14H12N2O3S ? H 289.0647.
J = 4.0 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.65 (t, 1H),
7.30 (t, 1H), 5.50 (dd, J = 11.0, 4.8 Hz, 1H), 4.15 (dd,
J = 19.2, 4.9 Hz, 1H), 3.85 (q, 1H), 2.90 (s, 3H) ppm; 13C
NMR (100 MHz, CDCl3): d = 156.9 (C=N, isoxazole),
150.0 (C=N, pyridine), 135.2, 134.5, 132.8, 131.7, 130.8,
130.6, 129.9, 129.2, 128.9, 128.7, 127.0, 93.9, 39.1,
33.9 ppm; HRMS-TOF–MS (ES?): found 227.0480, cal-
culated for C9H11N2O3S ? H 227.0490.
Crystal data: C14H12N2O3S, Mr = 288.32; orthorhombic,
˚
(R)-4,5-Dihydro-5-(methylsulfonyl)-3-(pyridin-3-yl)-
isoxazole (5f, C9H11N2O3S)
˚
P212121; a = 5.2052(10) A, b = 9.894(2) A, c = 25.166(7)
3
A, V = 1296.1(5) A ; Z = 4, Dx = 1.478 Mg m-3, Dm not
Orange solid; yield 182 mg (80 %); m.p.: 144–146 °C;
Rf = 0.33 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
˚
˚
˚
ꢀ
measured; Mo Ka radiation, k = 0.71073 A; cell parame-
ters from 1,779 reflections, h = 2.5–28.7°, l = 0.258
1,411, 1,365, 1,305, 1,263, 1,130, 1,028, 979, 939, 858,
804, 773 cm-1; 1H NMR (400 MHz, CDCl3): d = 8.90 (d,
J = 1.7 Hz, 1H), 8.70 (dd, J = 4.8, 1.4 Hz, 1H), 8.05 (d,
J = 8.1 Hz, 1H), 7.40 (t, 1H), 5.60 (dd, J = 10.8, 4.7 Hz,
1H), 4.05 (dd, J = 18.2, 4.7 Hz, 1H), 3.80 (dd, J = 18.2,
7.3 Hz, 1H), 3.09 (s, 3H) ppm; 13C NMR (100 MHz,
CDCl3): d = 156.9 (C=N, isoxazole), 152.0 (C=N, pyri-
dine), 148.1, 135.2, 134.5, 132.8, 131.7, 130.8, 130.6,
129.9, 129.2, 128.9, 128.7, 127.0, 93.9 (CHSO2Ph), 37.0
(CH3SO2Ph), 35.4 (CH2) ppm; HRMS-TOF–MS (ES?):
found 227.0484, calculated for C9H11N2O3S 227.0490.
mm-1; T = 90 K; colorless plate, 0.37 9 0.22 9 0.03 mm.
(R)-4,5-Dihydro-5-(phenylsulfonyl)-3-(pyridin-4-yl)-
isoxazole (5c, C14H12N2O3S)
Yellow solid; yield 170 mg (49 %); m.p.: 151–152 °C;
ꢀ
Rf = 0.50 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
1,595, 1,444, 1,408, 1,363, 1,309, 1,146, 862, 824, 735
cm-1
;
1H NMR (400 MHz, CDCl3): d = 8.72 (d,
J = 5.8 Hz, 2H), 8.02 (d, J = 7.6 Hz, 2H), 7.73 (t, 1H),
7.61 (t, 1H), 7.50 (d, J = 5.9 Hz, 2H), 5.56 (dd, J = 10.9,
4.6 Hz, 1H), 4.10 (dd, J = 8.3, 4.6 Hz, 1H), 3.80 (q, 1H)
ppm; 13C NMR (100 MHz, CDCl3): d = 155.5 (C=N,
isoxazole), 150.3 (C=N, pyridine), 137.2, 135.0, 134.8,
129.7, 129.4, 120.9, 93.6, 35.9 ppm; MS: m/z (%) = 146
(100, [M-PhSO2]?), 78 (44), 51 (28); HRMS-TOF–MS
(ES?): found 289.0633, calculated for C14H12N2O3S ? H
289.0647.
(R)-3-(6-Chloropyridin-3-yl)-4,5-dihydro-5-
(methylsulfonyl)isoxazole (5g, C9H10ClN2O3S)
White solid; yield 239 mg (92 %); m.p.: 159–160 °C;
ꢀ
Rf = 0.39 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
1,602, 1,552, 1,433, 1,379, 1,305, 1,139, 1,109, 1,010, 933,
864, 765 cm-1; 1H NMR (400 MHz, CDCl3): d = 8.56 (d,
J = 2.4 Hz, 1H), 7.95 (dd, J = 8.4, 2.4 Hz, 1H), 7.35 (d,
J = 8.3 Hz, 1H), 5.50 (dd, J = 10.8, 4.7 Hz, 1H), 3.90
(dd, J = 18.2, 4.9 Hz, 1H), 3.75 (dd, J = 18.1, 10.8 Hz,
1H) ppm; 13C NMR (100 MHz, CDCl3): d = 156.6 (C=N,
isoxazole), 155.6 (Cl–C, pyridine), 152.1 (C=N, pyridine),
148.1, 135.2, 134.5, 132.8, 131.7, 130.8, 130.6, 129.9,
129.2, 128.9, 128.7, 127.0, 94.7 (CHSO2Ph), 37.2
(CH3SO2Ph), 35.8 (CH2, isoxazole) ppm; HRMS-TOF–
MS (ES?): found 261.0095, calculated for C9H10ClN2O3S
261.0101.
(R)-3-(6-Chloropyridin-3-yl)-4,5-dihydro-5-
(phenylsulfonyl)isoxazole (5d, C14H11ClN2O3S)
Yellow solid; yield 268 mg (72 %); m.p.: 142–144 °C;
ꢀ
Rf = 0.47 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
1,584, 1,480, 1,408, 1,369, 1,308, 1,151, 1,110, 859, 765,
729 cm-1 1H NMR (400 MHz, CDCl3): d = 8.56 (d,
;
J = 2.0 Hz, 1H), 7.98 (m, 3H), 7.70 (m, 1H), 7.58 (t, 3H),
7.37 (d, J = 5.9 Hz, 2H), 5.60 (dd, J = 10.9, 4.5 Hz, 1H),
4.10 (dd, J = 18.3, 4.5 Hz, 1H), 3.80 (q, 1H) ppm; 13C
NMR (100 MHz, CDCl3): d = 153.9 (C=N, isoxazole),
148.5 (C=N, pyridine), 136.6, 135.1, 129.7, 129.4, 124.7,
122.7, 93.3, 36.1 ppm; MS: m/z (%) = 180 (100,
[M-PhSO2]?), 152 (19), 112 (28), 76 (44), 50 (28);
HRMS-TOF–MS (ES?): found 323.0267, calculated for
C14H11ClN2O3S ? H 323.0257.
(R)-4,5-Dihydro-3-(4-nitrophenyl)-5-(phenylsulfonyl)-
isoxazole (5-regioisomer, 5h, C15H12N2O5S)
and (R)-4,5-dihydro-3-(4-nitrophenyl)-4-
(phenylsulfonyl)isoxazole (4-regioisomer, C15H12N2O5S)
It was deduced from 1H NMR spectra that the ratio
between 5-regioisomer and 4-regioisomer was 3.46:1. The
mixed compound was obtained as a yellow solid. Yield
232 mg (70 %); m.p.: 143–145 °C (Ref. [53] 187–189 °C
for the 5-regioisomer); Rf = 0.35 (ethyl acetate/n-hexane
(R)-4,5-Dihydro-5-(methylsulfonyl)-3-(pyridin-2-yl)-
isoxazole (5e, C9H11N2O3S)
Yellow solid; yield 199 mg (88 %); m.p.: 140 °C (dec.);
ꢀ
ꢀ
1:2); IR (KBr): m = 1,600, 1,585, 1,521, 1,446, 1,348,
Rf = 0.45 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
1,581, 1,469, 1,444, 1,367, 1,305, 1,126, 945, 862,
1,151, 848, 732 cm-1 1H NMR (400 MHz, CDCl3):
;
786 cm-1 1H NMR (400 MHz, CDCl3): d = 8.55 (d,
;
d = 8.20 (m, 4H), 7.98–7.40 (m, 5H), 5.55 (dd,
123

714
Y. Du¨ru¨st et al.
J = 10.9, 4.6 Hz, 1H), 5.20 (dd, J = 11.2, 2.9 Hz, 1H,
4-regioisomer), 5.10 (dd, J = 10.1, 2.9 Hz, 1H, 4-regio-
isomer), 4.60 (t, 1H, 4-regioisomer), 4.05 (dd, J = 18.3,
4.6 Hz, 1H, 5-regioisomer), 3.75 (dd, J = 18.3, 10.9 Hz,
1H, 5-regioisomer) ppm; 13C NMR (100 MHz, CDCl3):
d = 155.5 (C=N), 149.1, 135.1, 135.0, 134.8, 133.3, 129.7,
129.4, 129.2, 129.1, 128.5, 127.9, 124.1, 123.9, 93.6 (C-5
of 5-regioisomer), 73.0 (C-4 of 4-regioisomer), 71.2 (C-5
of 4-regioisomer), 36.2 (C-4 of 5-regioisomer) ppm; MS:
m/z (%) = 180 (100, [M-PhSO2]?), 152 (19), 112 (28), 76
(44), 50 (28); HRMS-TOF–MS (ES?): found 333.0531,
calculated for C15H12N2O5S ? H 333.0545.
148.2 (C=N, pyridine), 135.2, 134.5, 132.8, 131.7, 130.8,
130.6, 129.9, 129.2, 128.9, 128.7, 127.0, 93.9, 39.1 ppm;
MS: m/z (%) = 178 (100, [M-PhSO2]?), 150 (19), 111
(28), 89 (32), 75 (44), 50 (28); HRMS-TOF–MS (ES?):
found 322.0291, calculated for C15H12ClNO3S ? H
322.0305.
(R)-4,5-Dihydro-3-(4-methoxyphenyl)-5-
(phenylsulfonyl)isoxazole (5l)
Yellow solid; yield 91 mg (69 %); m.p.: 125.5–127 °C
(Ref. [57] 128–130 °C, Ref. [54] 129–130 °C).
(R)-4,5-Dihydro-3-(4-methoxyphenyl)-5-
(methylsulfonyl)isoxazole (5m)
(R)-4,5-Dihydro-3-(2-nitrophenyl)-5-(phenylsulfonyl)-
isoxazole (5i, C15H12N2O5S)
Yellow solid; yield 96 mg (75 %); m.p.: 167.5–169 °C
(Ref. [56] 167–168 °C).
Yellow solid; yield 179 mg (69 %); m.p.: 159–160 °C;
ꢀ
Rf = 0.41 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
1,599, 1,579, 1,512, 1,348, 1,317, 1,143, 1,085, 846,
(R)-4,5-Dihydro-3-(4-fluorophenyl)-5-(phenylsulfonyl)-
isoxazole (5n, C15H12FNO3S)
729 cm-1
;
1H NMR (400 MHz, CDCl3): d = 8.30 (d,
Yellow solid; yield 88 mg (58 %); m.p.: 135–136 °C;
ꢀ
J = 2.0 Hz, 2H), 8.00 (d, J = 2.0 Hz, 2H), 7.85 (d,
J = 2.0 Hz, 2H), 7.70 (t, 1H), 7.60 (t, 2H), 5.60 (dd,
J = 10.9, 4.5 Hz, 1H), 4.15 (dd, J = 18.3, 4.5 Hz, 1H),
3.85 (dd, J = 18.3, 10.9 Hz, 1H) ppm; 13C NMR
(100 MHz, CDCl3): d = 153.9 (C=N, isoxazole), 148.5
(C=N, pyridine), 136.6, 135.1, 129.7, 129.4, 124.7, 122.7,
93.3, 36.1 ppm; MS: m/z (%) = 190 (100, [M-PhSO2]?),
143 (29), 89 (80); HRMS-TOF–MS (ES?): found
333.0539, calculated for C15H12N2O5S ? H 333.0545.
Rf = 0.43 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
1,596, 1,514, 1,307, 1,236, 1,142, 856 cm-1 1H NMR
;
(400 MHz, CDCl3) : d = 8.03 (d, 2H), 7.74–7.70 (m, 1H),
7.66–7.59 (m, 4H), 7.13 (dd, J = 11.5, 6.6 Hz, 2H), 5.58
(dd, J = 15.3, 6.3 Hz, 1H), 4.48 (dd, J = 18.2, 13.7 Hz,
1H), 3.81 (dd, J = 18.2, 10.8 Hz, 1H) ppm; 13C NMR
(100 MHz, CDCl3): d = 163.0, 155.9, 134.6, 129.7, 129.2,
129.1, 123.6, 116.3, 116.0, 93.3, 36.8 ppm; HRMS-TOF–
MS (ES?): found 328.0430, calculated for C15H12FNO3S ?
Na 328.0420.
(R)-4,5-Dihydro-3-(3-nitrophenyl)-5-(phenylsulfonyl)-
isoxazole (5j, C15H12N2O5S)
(R)-4,5-Dihydro-3-(4-fluorophenyl)-5-(methylsulfonyl)-
isoxazole (5o, C10H10FNO3S)
Yellow solid; yield 216 mg (65 %); m.p.: 162–163 °C;
ꢀ
Rf = 0.31 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
Yellow solid; yield 95 mg (78 %); m.p.: 155–157 °C;
1,531, 1,352, 1,311, 1,149, 720 cm-1; 1H NMR (400 MHz,
CDCl3): d = 8.48 (t, 1H), 8.35 (dd, J = 8.2, 1.2 Hz, 1H),
8.03 (t, 2H),7.75 (t, 1H), 7.70–7.60 (m, 5H), 5.65 (dd,
J = 11.5, 4.4 Hz, 1H), 4.15 (dd, J = 18.3, 4.5 Hz, 1H),
3.80 (dd, 18.3, 7.4 Hz, 1H) ppm; 13C NMR (100 MHz,
CDCl3): d = 156.9 (C=N, isoxazole), 148.4 (C=N, pyri-
dine), 135.5, 135.2, 133.7, 131.1, 129.9, 129.8, 129.2,
125.9, 121.9, 93.7, 36.8 ppm; MS: m/z (%) = 189 (100,
[M-PhSO2]?), 143 (20), 116 (21), 89 (74), 76 (44), 50
(29); HRMS-TOF–MS (ES?): found 333.0541, calculated
for C15H12N2O5S ? H 333.0545.
Rf = 0.29 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
ꢀ
1
1,601, 1,559, 1,516, 1,306, 1,227, 1,134, 842 cm-1; H
NMR (400 MHz, CDCl3) : d = 7.77–7.63 (m, 2H), 7.15 (t,
J = 8.5 Hz, 2H), 5.55 (dd, J = 10.8, 4.7 Hz, 1H), 4.02
(dd, J = 18.1, 4.6 Hz, 1H), 3.79 (dd, J = 18.1, 10.8 Hz,
1H), 3.06 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3):
d = 165.7 (F–C), 156.2 (C=N, isoxazole), 129.4, 123.4,
116.3, 91.9, 37.0 (CH3SO2), 36.1 (CH2, isoxazole) ppm;
HRMS-TOF–MS (ES?): found 266.0261, calculated for
C10H10FNO3S ? Na 266.0263.
(R)-3-(Furan-2-yl)-4,5-dihydro-5-(phenylsulfonyl)-
isoxazole (5p, C13H11NO4S)
Yellow oil; yield 161 mg (58 %); Rf = 0.36 (ethyl acetate/
(R)-3-(4-Chlorophenyl)-4,5-dihydro-5-(phenylsulfonyl)-
isoxazole (5k, C15H12ClNO3S)
ꢀ
Yellow solid; yield 247 mg (60 %); m.p.: 136–138 °C;
n-hexane 1:2); IR (neat): m = 1,583, 1,483, 1,446, 1,319,
1
Rf = 0.44 (ethyl acetate/n-hexane 1:2); IR (KBr): m =
1,309, 1,151, 1,085, 840, 783 cm-1; H NMR (400 MHz,
ꢀ
1,591, 1,477, 1,446, 1,321, 1,151, 1,084, 852, 759,
CDCl3): d = 7.90 (t, 2H), 7.60 (t, 1H), 7.45 (m, 4H), 6.70
(d, J = 4.0 Hz, 1H), 6.40 (dd, J = 3.4, 1.7 Hz, 1H), 5.45
(dd, J = 10.8, 4.4 Hz, 1H), 4.00 (dd, J = 18.3, 4.4 Hz,
1H), 3.85 (dd, J = 18.2, 10.8 Hz, 1H) ppm; 13C NMR
(100 MHz, CDCl3): d = 159.0 (C=N, isoxazole), 149.4,
732 cm-1 1H NMR (400 MHz, CDCl3): d = 8.05 (d,
;
J = 7.4 Hz, 1H), 7.72 (t, 1H), 7.60 (m, 2H), 7.46–7.25 (m,
5H), 5.63 (t, 1H), 4.10 (dd, J = 11.3, 7.4 Hz, 2H) ppm; 13
C
NMR (100 MHz, CDCl3): d = 156.9 (C=N, isoxazole),
123

Synthesis, crystal structure, and in vitro antiprotozoal activity
715
21. Lattmann E, Kinchington D, Singh H, Merino I, Begum A,
Ayuko WO, Tisdale MJ (2001) Pharm Pharmacol Lett 11:5
22. Ma H-J, Li Y-H, Zhao Q-F, Zhang T, Xie R-L, Mei X-D, Ning J
(2010) J Agric Food Chem 58:4356
147.0, 136.6, 135.4, 134.5, 129.7, 125.1, 122.1, 93.7,
36.8 ppm; HRMS-TOF–MS (ES?): found 278.0489, cal-
culated for C13H11NO4S 278.0442.
23. Hwang IT, Kim HR, Choi JS, Jeon DJ, Hong KS, Song JH, Cho
KY (2006) Weed Biol Manage 6:102
In vitro antiprotozoal and cytotoxic activity studies
24. Choi K, Siegel M, Piper JL, Yuan L, Cho E, Strnad P, Omary B,
Rich KM, Khosla C (2005) Chem Biol 12:469
25. Conti P, De Amici M, Grazioso G, Roda G, Pinto A, Bø Hansen
K, Nielsen B, Madsen U, Brauner-Osborne H, Egebjerg J, Vestri
V, Pellegrini-Giampietro DE, Sibille P, Acher FC, De Micheli C
(2005) J Med Chem 48:6315
The bioassays were carried out as described elsewhere
[43].
˙
Acknowledgments Abant Izzet Baysal University, Directorate
of Research Projects Commission (BAP grant no. 2007.03.03.260)
26. Zambach W, Renold
P (2010) Preparation of insecticidal
¨
˙
N-hydroxy- or N-oxy-benzamidine isoxazoles. PCT Int Appl WO
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27. Zambach W, Renold P (2009) Chem Abstr 150:472699
28. Milinkevich KA, Yoo CL, Sparks TC, Lorsbach BA, Kurth MJ
(2009) Bioorg Med Chem Lett 19:5796
and TUBITAK (Turkish Scientific and Technological Research
Council, Grant no. 106T645) are gratefully acknowledged for finan-
cial support.
29. Alam A, Pal C, Goyal M, Kundu MK, Kumar R, Iqbal MS, Dey
S, Bindu S, Sarkar S, Pal U, Maiti NC, Adhikari S, Bandyo-
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