New derivatives of 3,5-substituted-1,4,2-dioxazoles: Synthesis and activity against Entamoeba histolytica

Article abstract of DOI:10.1016/j.ejmech.2008.02.001

Dioxazole derivatives (1-33) were synthesized in two steps via their corresponding oximes (I-III). All the compounds were characterized using various spectroscopic techniques. A comparative study of in vitro antiamoebic activity of these heterocyclic compounds, viz. 3-o-chloro (1-11), 3-m-chloro (12-22) and 3-p-chloro (23-33) dioxazoles having same substituents at 5-position of dioxazole ring, was performed against HM1:IMSS strain of Entamoeba histolytica. The results showed a regular pattern in the activity and out of 33 compounds assayed 15 compounds showed better antiamoebic activity than metronidazole with IC₅₀ values in the range 0.41-1.73 μM and 1.80 μM. Dioxazoles having o-chloro, m-chloro, p-chloro, dichloro and pyridine substituents at 5-position were more active than the standard drug metronidazole. The toxicity studies against human kidney epithelial cell line showed that all the compounds were non-toxic. 3,5-Bis-[2-chlorophenyl]-1,4,2-dioxazole (10) was most active and least toxic among all the compounds.

Full text of DOI:10.1016/j.ejmech.2008.02.001

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 926e936
http://www.elsevier.com/locate/ejmech
Short communication
New derivatives of 3,5-substituted-1,4,2-dioxazoles: Synthesis and
activity against Entamoeba histolytica
1
Abdul R. Bhat, Fareeda Athar , Amir Azam
*
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 15 January 2008; received in revised form 5 February 2008; accepted 7 February 2008
Available online 4 March 2008
Abstract
Dioxazole derivatives (1e33) were synthesized in two steps via their corresponding oximes (IeIII). All the compounds were characterized
using various spectroscopic techniques. A comparative study of in vitro antiamoebic activity of these heterocyclic compounds, viz. 3-o-chloro
(1e11), 3-m-chloro (12e22) and 3-p-chloro (23e33) dioxazoles having same substituents at 5-position of dioxazole ring, was performed against
HM1:IMSS strain of Entamoeba histolytica. The results showed a regular pattern in the activity and out of 33 compounds assayed 15 compounds
showed better antiamoebic activity than metronidazole with IC50 values in the range 0.41e1.73 mM and 1.80 mM. Dioxazoles having o-chloro,
m-chloro, p-chloro, dichloro and pyridine substituents at 5-position were more active than the standard drug metronidazole. The toxicity studies
against human kidney epithelial cell line showed that all the compounds were non-toxic. 3,5-Bis-[2-chlorophenyl]-1,4,2-dioxazole (10) was most
active and least toxic among all the compounds.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Dioxazole; Antiamoebic activity; Entamoeba histolytica; MTT assay
1. Introduction
metallic taste, nausea, vomiting, diarrhea, sensory neuropathies
and toxicity with ataxia, vertigo, seizures and encephalopathy
[6,7]. Despite these side effects, there is lack of ideal drug, and
immunity acquired to already available drugs and the side effects
is a major hurdle in eradicating these diseases [8,9].
Amoebiasis, causative protozoan parasite Entamoeba histoly-
tica, is the second leading cause of death from parasitic disease
worldwide [1]. E. histolytica causes approximately 50 million
cases and approximately 100,000 deaths annually [2,3]. Liver
abscesses and brain abscesses are dreadful complications of
this disease [4]. The treatment for amoebiasis remains the
nitroimidazole derivatives (metronidazole, tinidazole, ornida-
zole, emitine). However, there are reports of failure of treatment
with metronidazole [1,5]. Although most antiamoebic drugs
have shown to be relatively efficient for the treatment of clinical
cases, the long-term use of medications produces undesirable
side effects in patients. The side effects of metronidazole are
Azoles have long been targeted for synthetic investigation
because of their known biological properties like cognitive-
enhancing and anxiolytic-like activities [10e12]. The antifun-
gal agents of azoles are useful drugs and are widely used for
the treatment of topic or inner mycoses, in particular AIDS-
related mycotic pathologies [13e16]. Oxazoles, an important
member of the azole family also contains a number of biolog-
ically active molecules, which play an important role in the
drug chemistry. A number of compounds had been screened
for antituberculosis agents including dihydrophinazines [17],
indoles and ureas [18]. Oxybenzylglycine, possessing an
oxazoline group is currently in clinical development for the
treatment of type 2 diabetes and dyslipidemia [19]. Consider-
ing the facts that nearly all the classes of the azole family are
biologically active in one way or the another, we tried to
* Corresponding author. Tel.: þ91 11 26981717/3250; fax: þ91 11
26981232.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
1
Present address: Centre for Interdisciplinary Research in Basic Sciences,
Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.001

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
927
develop a new series of dioxazoles and screened it against the
HM1:IMSS strain of E. histolytica to develop an effective drug
that can be better than metronidazole, a member of azole
family and the commercially available drug for amoebiasis.
assigned to n / p*, p / p* and n / s* transition,
respectively. The band at 356e317 nm was assigned to the
transition involving the azomethine group (C]N). Two other
absorption bands at 318e299 nm and 290e250 nm were due
to p / p* transition of dioxazole ring and n / p* transition
of azomethine nitrogen, respectively.
2. Results and discussion
1
N
2
O
The dioxazoles were prepared by treating aldo-oximes with
different aldehydes and ketones as described elsewhere [20]
(Scheme 1). The oximes (IeIII) and dioxazoles (1e33)
were characterized by different spectroscopic techniques and
the purity was confirmed by the elemental analysis and melt-
ing points. All the compounds showed sharp melting points
and the elemental analysis was found in accordance with
ꢀ0.3%. The oximes (IeIII) and dioxazoles (1e33) were con-
firmed by IR spectroscopy. The characteristic bands of n(NOeH)
and n(C]N) in oximes showed at 3267e3250 cmꢁ1 and
1634e1623 cmꢁ1, respectively. Besides the common aromatic
bands, the band n(C]C) at 1611e1434 cmꢁ1 and the bands
for ortho-substituted benzene in oxime I at 773 cmꢁ1, meta-
3
5
R'
O
4
R''
R
A
The formation of dioxazoles was supported by the absence
of a signal at 11.16e11.37 ppm due to NeOH in all the
compounds (1e33). The singlet at 5.86e6.28 ppm arises due
to CH group present at C-3 of the dioxazole ring (A) in
compounds 1e4, 10e15, 21e26, 32 and 33. Methyl and ethyl
signals were found in compounds where methyl and ethyl
groups are attached at C-3 of the dioxazole ring. For methyl
group, a singlet at 2.10e2.53 ppm was found in compounds
5, 7, 9, 16, 18, 20, 27, 29 and 31, while for ethyl group,
a multiplet at 1.87e2.34 ppm for CH2 protons and a triplet
at 1.11e1.54 ppm for CH3 protons were found in compounds
6, 9, 17, 19, 28 and 30. Additional signals for aromatic region
were found in 6.9e7.8 ppm with least chemical shifting in all
the compounds. The compounds 9, 20 and 31 containing
pyridine showed two double doublets at 7.89e8.03 ppm and
8.06e8.17 ppm and two doublets at 8.21e8.51 ppm and
8.34e8.68 ppm. The structure of the compounds 1, 2, 9, 12,
13, 20, 23, 24 and 31 were further supported by 13C NMR.
The C]N signal was found in the range of 157.1e
164.2 ppm and the characteristic signal for eOCOe was
found in the range 111.1e115.8 ppm which clearly favors
the formation of dioxazole ring. The signals due to the
aromatic aliphatic carbons resonate at their usual positions
and are shown in the data given in Section 3.
substituted benzene in oxime II at 748 cmꢁ1 and 805 cmꢁ1
,
and for para-substituted benzene in oxime III at 811 cmꢁ1
were also found. The n(C]N) band in dioxazoles was in the
range 1611e1673 cmꢁ1 and a new band in the range from
1193 cmꢁ1 to 1125 cmꢁ1 arises due to CeOeC group, which
was splitted in some cases. In addition to C]C of aromatic
region, the respective bands of substituted benzene were also
1
present. The structure was further confirmed by H NMR. In
the oximes (IeIII), a singlet at 6.74e6.94 ppm represents
the CH]N proton which shows the occurrence of condensa-
tion between the CH]O and H2eNeOH groups. The signal
at 11.16e11.37 ppm due to NeOH proton further supports
the formation of the oximes (IeIII).
The electronic spectra of the cyclised dioxazole analogues
studied in the UV region in chloroform exhibited three absorp-
tion bands at 356e317 nm, 318e299 nm and 290e250 nm
O
H
NOH
a
NH2OH.HCl
+
R
R
i)
ii)
iii)
R= 2-Cl
R= 3-Cl
R= 4-Cl
b
1
N
2
O
3
5
R'
O
4
R''
R
Scheme 1. General method for the synthesis of dioxazole derivatives. (a) Pyridine, C2H5OH, reflux 24 h; (b) aq. NaOCl, Et3N, CH2Cl2, (R0eCOeR00), where
(R0eCOeR00) represents the different aldehydes and ketones as given in Table 1.

928
A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
2.1. Antiamoebic activities
(200e400 mesh). Elemental analyses were performed by
Central Drug Research Institute, Lucknow, using Heraeus
Vario EL III analyzer and the results were within ꢀ0.3 of
the theoretical values. Electronic spectra were recorded in
methanol on a Shimadzu UV-1601 PC UVevisible spectro-
photometer. IR spectra on KBr disks were recorded on a Perkin
Elmer model 1620 FT-IR spectrophotometer. All the 1H NMR
and 13C NMR spectra were recorded in CDCl3 at ambient tem-
perature using a Bruker Spectrospin DPX-300 MHz spectro-
photometer with TMS as internal standard. Splitting patterns
are designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. Chemical shift values are given in parts per million.
The FAB mass spectra of all the compounds were recorded on
a JEOL SX 102/DA-6000 Mass Spectrometer/Data System
using argon/xenon (6 kV, 10 mA) as the FAB gas and m-nitro
benzyl alcohol (NBA) was used as the matrix.
Preliminary experiments were carried out to determine the
in vitro antiamoebic activities by using HM1:IMSS strain of E.
histolytica cultured in TYIS-33 growth medium. The results
are summarized in Tables 1e3. Out of 33 compounds, nine
showed best activity with 2.09e4.39 times more active than
metronidazole. The IC50 values for these compounds were
0.41 mM (10), 0.48 mM (32), 0.51 mM (13), 0.53 mM (24),
0.62 mM (21), 0.71 mM (2), 0.72 mM (11), 0.81 mM (1), and
0.86 mM (33) in comparison to metronidazole (IC50 1.8 mM).
Six compounds were found to be moderately active with
1.04e1.97 times better than metronidazole. The IC50 values
of these compounds were 0.91 mM (22), 1.13 mM (23),
1.21 mM (12), 1.58 mM (31), 1.62 mM (9), and 1.73 mM (20).
The results were statistically evaluated by analyses of
variance. The null hypothesis was tested using t-test and the
significance of the differences between the IC50 value(s) of
metronidazole vs. 1, 2, 10, 11, 13, 21, 24, 32 and 33 was
evaluated. The calculated t-values were higher than the table
values at the 4% level. Hence, the character under study was
significantly influenced by the treatment [21]. The activity of
the dioxazole derivatives was found structure dependent. It
was found that 4-chloro and 2-chloro phenyl groups present
at 5-position of the dioxazole ring were more active than the
compounds with 3-chloro phenyl groups. The activity further
depends upon the nature of the substituents at carbon-3 of
the dioxazole ring. Compounds with only phenyl groups
attached at carbon-3 were found more active than those with
additional methyl or ethyl groups present at the same carbon.
SAR further reveals that the phenyl rings with electron-
withdrawing groups present at carbon-3 of the dioxazole
ring were more active than those with electron-donating
groups. The phenyl rings substituted with Br atom were not
found active that may be due to the presence of the methyl
and ethyl groups at carbon-3 atom of the dioxazole ring.
3.1. Preparation of oxime I
A solution of 2-chlorobenzaldehyde (1 equiv) and hydrox-
ylamine hydrochloride (1.08 equiv) in a solution of ethanol
and pyridine (2:1) was refluxed with stirring for 24 h. After
cooling the mixture was concentrated and then poured into
600 ml of ice-cold water. The precipitated solid was collected
and recrystallized from methanol to give the corresponding
oxime.
Yield 70%, m.p. 73 ꢂC; IR nmax (cmꢁ1): 3250 (NeOH),
1
3011 (ArCeH), 2885 (CH), 1634 (CeN), 773 (benzene); H
NMR (CDCl3) d/ppm: 7.20e7.34 (m, 4H, AreH), 6.94 (s,
1H, CH]N), 11.37 (s, 1H, NeOH); FAB MS: m/z
(C7H6NOCl þ H, Mþ þ 1) 156.7, calcd 155.5.
3.2. General procedure for the preparation of
3,5-substituted 1,4,2-dioxazoles (1e11)
The 4% aqueous solution of NaOCl (1.6 equiv) was added
to a solution of dipolarophile (respective aldehyde and ketone,
1 equiv) and triethylamine (0.1 equiv) in dichloromethane
under argon atmosphere. The oxime I (1 equiv) in dichlorome-
thane was added dropwise (over a period of 1 h) at 0 ꢂC to the
above solution. After being stirred at room temperature for
24 h and refluxed for additional 24 h, water was added to
the reaction mixture and the aqueous layer was extracted
with dichloromethane. The combined organic layers were
washed with water and brine, dried (MgSO4), filtered and
concentrated under vacuum. The compounds were recrystal-
lized using dichloromethane hexane solution.
2.2. Toxicity profile
Compounds 1e33 were tested to find the toxic effects on
human kidney epithelial cell line. No one inhibited cell growth
at a concentration of 100 mM. To investigate the selectivity of
the compounds, the ‘‘safety index’’ (SI) was calculated and
defined as toxicity IC50/protozoal IC50, where toxicity IC50
is defined as the concentration of compound that kills 50%
of the human (kidney epithelial) cell line and protozoal IC50
is the concentration that kills 50% of amoeba protozoa. This
allows to estimate which compound might be efficacious or
toxic against human cells and potentially in vivo. The numer-
ical results for each compound are given in Tables 1e3.
3.2.1. 3-[3,4-Dichlorophenyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (1)
Yield 34%, m.p. 130 ꢂC. Anal. calcd for C14H8NO2Cl3: C
51.13, H 2.45, N 4.26%; found: C 51.19, H 2.41, N 4.30%;
UV/vis l (nm): 317, 311, 256, 221; IR nmax (cmꢁ1): 3036
3. Experimental
1
All the chemicals were purchased from Aldrich Chemical
Company (USA). Analytical thin-layer chromatography was
performed on precoated silica gel 60 F254 plates and flash col-
(ArCeH), 2851 (CH), 1621 (C]N), 773, 825 (benzene); H
NMR (CDCl3) d/ppm: 5.93 (s, 1H, CH), 7.05 (d, 1H,
7.3 Hz, ArCeH), 7.12 (s, 1H, ArCeH), 7.14 (d, 1H, 7.3 Hz,
ArCeH), 7.31e7.37 (m, 4H, AreH); 13C NMR (CDCl3)
˚
umn chromatography was accomplished using silica gel, 60 A

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
929
Table 1
Dioxazoles (1e11), their antiamoebic activity against HM1:IMSS strain of E. histolytica and toxicity profile
N
O
R'
O
R''
Cl
(I)
Compound
R0
H
R00
Antiamoebic activity
IC50 (mM)
Toxicity profile
IC50 (mM)
>100
S.D.a (ꢀ)
0.55
Safety index (SI)
1
0.81
>123.5
Cl
Cl
2
H
0.51
>1.80
>1.80
>1.80
>1.80
>1.80
>1.80
1.62
0.38
1.32
1.26
1.09
0.71
0.51
0.76
0.61
0.32
0.26
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>196.1
>55.55
>55.55
>55.55
>55.55
>55.55
>55.55
>61.72
>243.9
>138.8
Cl
3
H
CH3
C2H5
4
H
5
CH3
C2H5
CH3
C2H5
CH3
H
Cl
6
Cl
7
Br
Br
8
9
N
Cl
10
0.41
11
H
0.72
Cl
a
Standard deviation. The compounds with bold font IC50 values are more active than metronidazole.

930
A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
Table 2
Dioxazoles (12e22), their antiamoebic activity against HM1:IMSS strain of E. histolytica and toxicity profile
N
O
R'
O
R''
Cl
(II)
Compound
R0
H
R00
Antiamoebic activity
IC50 (mM)
Toxicity profile
IC50 (mM)
>100
S.D.a (ꢀ)
0.65
Safety index (SI)
12
1.21
>82.6
Cl
Cl
13
14
15
16
17
18
19
20
21
H
0.71
>1.80
>1.80
>1.80
>1.80
>1.80
>1.80
1.73
0.53
1.03
0.98
1.14
0.96
0.91
0.53
0.39
0.23
0.34
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>140.8
>55.55
>55.55
>55.55
>55.55
>55.55
>55.55
>57.8
Cl
H
CH3
C2H5
H
CH3
C2H5
CH3
C2H5
CH3
H
Cl
Cl
Br
Br
N
Cl
0.62
>161.3
>109.89
22
H
0.91
Cl
a
Standard deviation. The compounds with bold font IC50 values are more active than metronidazole.

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
931
Table 3
Dioxazoles (23e33), their antiamoebic activity against HM1:IMSS strain of E. histolytica and toxicity profile
N
O
R'
O
R''
Cl
(III)
Compound
R0
H
R00
Antiamoebic activity
IC50 (mM)
Toxicity profile
IC50 (mM)
>100
S.D.a (ꢀ)
0.86
Safety index (SI)
23
1.13
>88.5
Cl
Cl
24
25
26
27
28
29
30
31
32
33
H
0.53
>1.80
>1.80
>1.80
>1.80
>1.80
>1.80
1.58
0.29
0.71
0.36
0.71
0.45
0.61
0.53
0.68
0.28
0.54
0.32
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>188.7
>55.55
>55.55
>55.55
>55.55
>55.55
>55.55
>63.3
Cl
H
CH3
H
C2H5
CH3
C2H5
CH3
C2H5
CH3
H
Cl
Cl
Br
Br
N
Cl
0.48
>208.3
>116.27
>55.55
H
0.86
Cl
Metronidazole
1.80
a
Standard deviation. The compounds with bold font IC50 values are more active than metronidazole.

932
A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
d/ppm: 157.1 (C]N), 139.0, 137.1, 135.8, 134.6, 130.9,
130.2, 128.2, 121.9 (AreC), 111.1 (eOeCeOe); FAB MS:
m/z (Mþ þ 1) 329.73, calcd 328.56.
(d, 1H, 7.2 Hz, ArCeH), 7.23 (dd, 1H, AreH), 7.32e7.39 (m,
4H, AreH); FAB MS: m/z (Mþ þ 1) 322.37, calcd 322.10.
3.2.7. 3-[3-Bromophenyl],3-[methyl]-5-
3.2.2. 3-[4-Chlorophenyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (2)
[2-chlorophenyl]-1,4,2-dioxazole (7)
Yield 37%, m.p. 147 ꢂC. Anal. calcd for C15H11NO2BrCl:
C 51.07, H 3.14, N 3.97%; found: C 51.16, H 3.19, N
3.92%; UV/vis l (nm): 290, 305, 334; IR nmax (cmꢁ1): 3041
(ArCeH), 2963 (CH3), 1637 (C]N), 753, 813 (benzene);
1H NMR (CDCl3) d/ppm: 2.23 (s, 3H, CH3), 7.15 (s, 1H,
AreH), 7.26 (dd, 1H, ArCeH), 7.34e7.41 (m, 5H, AreH),
7.77e7.80 (d, 1H, 7.6 Hz, AreH); FAB MS: m/z (Mþ þ 1)
353.76, calcd 352.40.
Yield 34%, m.p. 235 ꢂC. Anal. calcd for C14H8NO2Cl3: C
57.12, H 3.08, N 4.76%; found: C 57.15, H 3.12, N 4.72%;
UV/vis l (nm): 250, 299, 318; IR nmax (cmꢁ1): 3045 (ArCeH),
2872 (CH), 1611 (C]N), 757, 813 (benzene); 1H NMR
(CDCl3) d/ppm: 6.2 (s, 1H, CH), 7.13 (d, 2H, 7.5 Hz, Ar-
CeH), 7.16 (d, 2H, 7.5 Hz, ArCeH), 7.21e7.28 (m, 4H,
AreH); 13C NMR (CDCl3) d/ppm: 158.2 (C]N), 140.3,
137.8, 136.6, 135.1, 131.4, 128.6, 122.3 (AreC), 112.8
(eOeCeOe); FAB MS: m/z (Mþ þ 1) 294.36, calcd 294.07.
3.2.8. 3-[3-Bromophenyl],3-[ethyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (8)
3.2.3. 3-[4-Methylphenyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (3)
Yield 34%, m.p. 163 ꢂC. Anal. calcd for C16H13NO2BrCl:
C 52.38, H 3.57, N 3.87%; found: C 52.41, H 3.82, N
3.92%; UV/vis l (nm): 290, 304, 342; IR nmax (cmꢁ1): 3058
(ArCeH), 2925 (CH3), 2871 (CH2), 1664 (C]N), 758, 829
Yield 29%, m.p. 138 ꢂC. Anal. calcd for C15H12NO2Cl2: C
58.23, H 3.91, N 4.51%; found: C 58.28, H 3.87, N 4.53%;
UV/vis l (nm): 255, 318, 331; IR nmax (cmꢁ1): 3026 (ArCeH),
2857 (CH), 1614 (C]N), 776, 819 (benzene); 1H NMR
(CDCl3) d/ppm: 2.31 (s, 3H, CH3), 5.86 (s, 1H, CH), 6.97
(d, 1H, 7.6 Hz, ArCeH), 7.02 (d, 2H, 7.6 Hz, ArCeH),
7.11e7.16 (m, 4H, AreH); FAB MS: m/z (Mþ þ 1) 310.29,
calcd 309.09.
1
(benzene); H NMR (CDCl3) d/ppm: 1.19 (t, 3H, CH3), 1.94
(m, 2H, CH2), 7.17 (s, 1H, AreH), 7.24 (d, 1H, 7.8 Hz,
AreH), 7.31e7.39 (m, 5H, AreH), 7.41e7.44 (d, 1H,
7.8 Hz, AreH); FAB MS: m/z (Mþ þ 1) 367.68, calcd 366.50.
3.2.9. 3-[2-Pyridyl],3-[methyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (9)
3.2.4. 3-[4-Ethylphenyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (4)
Yield 34%, m.p. 134 ꢂC. Anal. calcd for C14H11N2O2Cl: C
61.20, H 4.03, N 5.08%; found: C 61.25, H 4.09, N 5.13%; UV/
vis l (nm): 290, 317, 341; IR nmax (cmꢁ1): 3035 (ArCeH), 2948
(CH3), 1664 (C]N), 758 (benzene); 1H NMR (CDCl3) d/ppm:
2.32 (s, 3H, CH3), 7.53e7.77 (m, 4H, AreH), 7.93e8.01
(dd, 1H, pyeH), 8.09e8.16 (dd, 1H, pyeH), 8.47e8.50 (d,
1H, pyeH), 8.62e8.65 (d, 1H, pyeH); 13C NMR (CDCl3)
d/ppm: 158.4 (C]N), 154.4 (C]N), 140.2, 139.8, 139.1,
135.4, 134.1, 130.8, 130.2, 128.2 (AreC), 115.1 (eOeCeOe),
26.2 (CH3); FAB MS: m/z (Mþ þ 1) 275.76, calcd 274.50.
Yield 37%, m.p. 125 ꢂC. Anal. calcd for C16H14NO2Cl: C
67.07, H 4.95, N 4.91%; found: C 67.13, H 5.01, N 5.89%;
UV/vis l (nm): 271, 309, 334; IR nmax (cmꢁ1): 3043 (ArCeH),
2915 (CH3), 2895 (CH2), 1645 (C]N), 743, 756 (benzene);
1H NMR (CDCl3) d/ppm: 1.53 (t, 3H, CH3), 2.14 (m, 2H,
CH2), 6.13 (s, 1H, CH), 6.93 (d, 2H, 7.7 Hz, ArCeH), 6.97
(d, 2H, 7.7 Hz, ArCeH), 7.16e7.23 (m, 4H, AreH); FAB
MS: m/z (Mþ þ 1) 285.46, calcd 284.61.
3.2.5. 3-[4-Chlorophenyl],3-[methyl]-5-
[2-chlorophenyl]-1,4,2-dioxazole (5)
3.2.10. 3,5-Bis-[2-chlorophenyl]-1,4,2-dioxazole (10)
Yield 34%, m.p. 142 ꢂC. Anal. calcd for C14H9NO2Cl2:
C 57.12, H 3.08, N 4.76%; found: C 57.19, H 3.14, N
4.71%; UV/vis l (nm): 256, 305, 346; IR nmax (cmꢁ1): 3023
(ArCeH), 2856 (CH), 1625 (C]N), 758 (benzene); 1H
NMR (CDCl3) d/ppm: 6.12 (s, 1H, CH), 7.53e7.64 (m, 8H,
AreH); FAB MS: m/z (Mþ þ 1) 295.38, calcd 294.07.
Yield 19%, m.p. 129 ꢂC. Anal. calcd for C15H11NO2Cl: C
58.42, H 3.59, N 4.54%; found: C 58.47, H 3.53, N 4.58%;
UV/vis l (nm): 255, 317, 334; IR nmax (cmꢁ1): 3053 (ArCeH),
2946 (CH3), 1657 (C]N), 746, 793 (benzene); 1H NMR
(CDCl3) d/ppm: 2.53 (s, 3H, CH3), 7.16 (d, 2H, 7.2 Hz,
ArCeH), 7.19 (d, 2H, 7.2 Hz, ArCeH), 7.32e7.39 (m, 4H,
AreH); FAB MS: m/z (Mþ þ 1) 309.35, calcd 308.08.
3.2.11. 3-[3-Chlorophenyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (11)
3.2.6. 3-[3-Chlorophenyl],3-[ethyl]-5-[2-chlorophenyl]-1,
4,2-dioxazole (6)
Yield 34%, m.p. 156 ꢂC. Anal. calcd for C14H9NO2Cl2:
C 57.12, H 3.08, N 4.76%; found: C 57.15, H 2.98, N
4.71%; UV/vis l (nm): 275, 321, 342; IR nmax (cmꢁ1): 3029
Yield 21%, m.p. 137 ꢂC. Anal. calcd for C16H13NO2Cl2: C
59.60, H 4.06, N 4.34%; found: C 59.65, H 4.13, N 4.38%;
UV/vis l (nm): 290, 318, 331; IR nmax (cmꢁ1): 3051 (ArCeH),
2927 (CH3), 2892 (CH2), 1661 (C]N), 720, 748 (benzene);
1H NMR (CDCl3) d/ppm: 1.34 (t, 3H, CH3), 2.34 (m, 2H,
CH2), 7.14 (s, 1H, AreH), 7.16 (d, 1H, 7.2 Hz, ArCeH), 7.19
1
(ArCeH), 2869 (CH), 1639 (C]N), 751, 816 (benzene); H
NMR (CDCl3) d/ppm: 6.09 (s, 1H, CH), 7.16 (s, 1H, AreH),
7.18 (d, 1H, 7.2 Hz, ArCeH), 7.22 (d, 1H, 7.2 Hz, ArCeH),
7.26 (dd, 1H, AreH), 7.29e7.36 (m, 4H, AreH); FAB MS:
m/z (Mþ þ 1) 295.28, calcd 294.07.

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
933
3.3. Preparation of oxime II
130.1, 128.9, 128.6, 126.2 (AreC), 110.9 (eOeCeOe);
FAB MS: m/z (Mþ þ 1) 295.07, calcd 294.07.
A solution of 3-chlorobenzaldehyde (1 equiv) and hydrox-
ylamine hydrochloride (1.08 equiv) in a solution of ethanol
and pyridine (2:1) was refluxed with stirring for 24 h. After
cooling the mixture was concentrated and then poured into
600 ml of ice water. The precipitated solid was collected and
then recrystallized from methanol to give the corresponding
oxime.
3.4.3. 3-[4-Methylphenyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (14)
Yield 29%, m.p. 135 ꢂC. Anal. calcd for C15H12NO2Cl2: C
58.23, H 3.91, N 4.51%; found: C 58.28, H 4.03, N 4.56%;
UV/vis l (nm): 251, 322, 346; IR nmax (cmꢁ1): 3078 (ArCe
1
H), 2864 (CH), 1658 (C]N), 790, 889 (benzene); H NMR
Yield 57%, m.p. 84 ꢂC; IR nmax (cmꢁ1): 3267 (NeOH),
3035 (ArCeH), 2849 (CH), 1630 (CeN), 805, 748 (m-
(CDCl3) d/ppm: 2.43 (s, 3H, CH3), 6.08 (s, 1H, CH), 6.95
(d, 2H, 7.2 Hz, AreH), 6.98 (d, 2H, 7.2 Hz, AreH), 7.04 (d,
1H, 7.3 Hz, AreH), 7.09 (s, 1H, AreH), 7.12 (d, 1H,
7.3 Hz, AreH), 7.16 (dd, 1H, AreH); FAB MS: m/z
(Mþ þ 1) 310.41, calcd 309.09.
1
substituted benzene); H NMR (CDCl3) d/ppm: 6.74 (s, 1H,
CH]N), 7.13 (s, 1H, AreH), 7.17 (d, 1H, 7.4 Hz, ArCeH),
7.23 (d, 1H, 7.4 Hz, ArCeH), 7.27 (dd, 1H, AreH), 11.56
(s, 1H, NeOH); FAB MS: m/z (Mþ þ 1) 156.43, calcd 155.5.
3.4.4. 3-[4-Ethylphenyl]-5-[3-chlorophenyl]-1,
3.4. General procedure for the preparation of
3,5-substituted 1,4,2-dioxazoles (12e22)
4,2-dioxazole (15)
Yield 29%, m.p. 145 ꢂC. Anal. calcd for C16H14NO2Cl: C
67.07, H 4.95, N 4.91%; found: C 67.16, H 5.03, N 4.86%;
UV/vis l (nm): 253, 317, 346; IR nmax (cmꢁ1): 3062 (Ar-
CeH), 2918 (CH3), 2891 (CH2), 1629 (C]N), 791, 869
The 4% aqueous solution of NaOCl (1.6 equiv) was added
to a solution of dipolarophile (respective aldehyde and ketone,
1 equiv) and triethylamine (0.1 equiv) in dichloromethane
under argon atmosphere. The oxime II (1 equiv) in dichloro-
methane was added dropwise (over a period of 1 h) at 0 ꢂC
to the above solution. After being stirred at room temperature
for 24 h and refluxed for additional 24 h, water was added to
the reaction mixture and the aqueous layer was extracted
with dichloromethane. The combined organic layers were
washed with water and brine, dried (MgSO4), filtered and
concentrated under reduced pressure. The compounds were
recrystallized using dichloromethane hexane solution.
1
(benzene); H NMR (CDCl3) d/ppm: 1.58 (t, 3H, CH3), 2.19
(m, 2H, CH2), 5.96 (s, 1H, CH), 6.95 (d, 2H, 7.8 Hz, ArCeH),
6.99 (d, 2H, 7.8 Hz, ArCeH), 7.11 (d, 1H, 7.4 Hz, AreH),
7.14 (s, 1H, AreH), 7.16 (dd, 1H, AreH), 7.20 (d, 1H,
7.3 Hz, AreH); FAB MS: m/z (Mþ þ 1) 288.39, calcd 287.61.
3.4.5. 3-[4-Chlorophenyl],3-[methyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (16)
Yield 22%, m.p. 149 ꢂC. Anal. calcd for C15H11NO2Cl: C
58.42, H 3.59, N 4.54%; found: C 58.47, H 3.65, N 4.59%;
UV/vis l (nm): 283, 313, 354; IR nmax (cmꢁ1): 3048 (ArCeH),
2925 (CH3), 1637 (C]N), 789, 886 (benzene); 1H NMR
(CDCl3) d/ppm: 2.52 (s, 3H, CH3), 7.01 (d, 2H, 7.6 Hz,
AreH), 7.21 (d, 1H, 7.3 Hz, AreH), 7.24 (s, 1H, AreH),
7.26 (d, 2H, 7.3 Hz, AreH), 7.29 (d, 2H, 7.3 Hz, AreH),
7.31 (dd, 1H, AreH); FAB MS: m/z (Mþ þ 1) 309.42, calcd
308.08.
3.4.1. 3-[3,4-Dichlorophenyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (12)
Yield 32%, m.p. 152 ꢂC. Anal. calcd for C14H8NO2Cl3: C
51.13, H 2.45, N 4.26%; found: C 51.07, H 2.39, N 4.21%;
UV/vis l (nm): 252, 308, 354; IR nmax (cmꢁ1): 3074 (Ar-
CeH), 2843 (CH), 1616 (C]N), 790, 885 (benzene); 1H
NMR (CDCl3) d/ppm: 6.17 (s, 1H, CH), 7.08 (d, 1H,
7.6 Hz, AreH), 7.14 (s, 1H, AreH), 7.16 (d, 1H, 7.6 Hz,
ArCeH), 7.21 (dd, 1H, AreH), 7.25 (s, 1H, AreH), 7.28 (d,
1H, 7.3 Hz, ArCeH), 7.31 (d, 1H, 7.3 Hz, AreH); 13C
NMR (CDCl3) d/ppm: 157.2 (C]N), 138.9, 138.1, 137.6,
136.8, 135.1, 134.3, 130.4, 130.1, 128.5 (AreC), 114.1
(eOeCeOe); FAB MS: m/z (Mþ þ 1) 329.7, calcd 328.56.
3.4.6. 3,5-Bis[3-chlorophenyl]-3-[ethyl]-1,
4,2-dioxazole (17)
Yield 31%, m.p. 129 ꢂC. Anal. calcd for C16H13NO2Cl2:
C 59.60, H 4.06, N 4.34%; found: C 59.64, H 4.05, N
4.39%; UV/vis l (nm): 285, 321, 334; IR nmax (cmꢁ1): 3075
(ArCeH), 2852 (CH2), 2935 (CH3), 1673 (C]N), 783, 846
1
(benzene); H NMR (CDCl3) d/ppm: 1.13 (t, 3H, CH3), 2.19
3.4.2. 3-[4-Chlorophenyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (13)
Yield 31%, m.p. 163 ꢂC. Anal. calcd for C14H8NO2Cl3: C
57.12, H 3.08, N 4.76%; found: C 57.17, H 3.16, N 4.78;
UV/vis l (nm): 257, 300, 329; IR nmax (cmꢁ1): 3074
(m, 2H, CH2), 7.10 (s, 1H, AreH), 7.11 (d, 1H, 7.3 Hz,
AreH), 7.13 (d, 1H, 7.3 Hz, AreH), 7.17 (dd, 1H, AreH),
7.21 (s, 1H, AreH), 7.24 (d, 2H, 7.2 Hz, AreH), 7.26
(d, 2H, 7.2 Hz, AreH), 7.28 (dd, 1H, AreH); FAB MS: m/z
(Mþ þ 1) 323.05, calcd 322.04.
1
(AreH), 2846 (CH), 1617 (C]N), 787, 868 (benzene); H
NMR (CDCl3) d/ppm: 6.21 (s, 1H, CH), 7.07 (d, 1H,
7.3 Hz, AreH), 7.11 (s, 1H, AreH), 7.14 (d, 1H, 7.3 Hz,
AreH), 7.18 (dd, 1H, AreH), 7.25 (d, 2H, 7.6 Hz, AreH),
7.29 (d, 2H, 7.6 Hz, AreH); 13C NMR (CDCl3) d/ppm:
160.8 (C]N), 136.4, 135.9, 135.2, 134.8, 132.9, 131.6,
3.4.7. 3-[3-Bromophenyl],3-[methyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (18)
Yield 43%, m.p. 175 ꢂC. Anal. calcd for C15H11NO2BrCl:
C 51.07, H 3.14, N 3.97%; found: C 50.95, H 3.18, N
4.04%; UV/vis l (nm): 290, 318, 346; IR nmax (cmꢁ1): 3035

934
A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
1
(ArCeH), 2981 (CH3), 1652 (C]N), 790, 864 (benzene); H
NMR (CDCl3) d/ppm: 2.29 (s, 3H, CH3), 7.11 (s, 1H, AreH),
7.13 (d, 2H, 7.2 Hz, AreH), 7.15 (dd, 1H, AreH), 7.26 (s, 1H,
AreH), 7.28 (d, 2H, 7.5 Hz, AreH), 7.33 (d, 2H, 7.5 Hz,
AreH), 7.42 (dd, 1H, AreH), 7.81e7.89 (d, 1H, 7.4 Hz,
AreH); FAB MS: m/z (Mþ þ 1) 353.6, calcd 352.4.
3.5. Preparation of oxime III
A solution of 4-chlorobenzaldehyde (1 equiv) and hydrox-
ylamine hydrochloride (1.08 equiv) in a solution of ethanol
and pyridine (2:1) was refluxed with stirring for 24 h. After
cooling the mixture was concentrated and then poured into
600 ml of ice water. The precipitated solid was collected and
then recrystallized from methanol to give the corresponding
oxime.
Yield 79%, m.p. 67 ꢂC; IR nmax (cmꢁ1): 3261 (NeOH),
3034 (ArCeH), 2879 (CH), 1623 (C]N), 781 (benzene);
1H NMR (CDCl3) d/ppm: 6.89 (s, 1H, CH]N), 7.29 (d, 2H,
7.6 Hz, AreH), 7.32 (d, 2H, 7.6 Hz, AreH), 11.16 (s, 1H,
NeOH); FAB MS: m/z 156.6 (Mþ þ 1), calcd 155.5.
3.4.8. 3-[3-Bromophenyl],3-[ethyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (19)
Yield 39%, m.p. 165 ꢂC. Anal. calcd for C16H13NO2BrCl:
C 52.38, H 3.57, N 3.87%; found: C 52.39, H 3.52, N
3.91%; UV/vis l (nm): 256, 300, 346; IR nmax (cmꢁ1): 3065
(ArCeH), 2913 (CH3), 2865 (CH2), 1651 (C]N), 732, 861
1
(benzene); H NMR (CDCl3) d/ppm: 1.23 (t, 3H, CH3), 1.87
(m, 2H, CH2), 7.18 (s, 1H, AreH), 7.21 (d, 1H, 7.8 Hz,
AreH), 7.24 (d, 1H, 7.8 Hz, AreH), 7.28 (dd, 1H, AreH),
7.33 (s, 1H, AreH), 7.34 (d, 1H, 7.4 Hz, AreH), 7.37 (d,
1H, 7.4 Hz, AreH), 7.43 (dd, 1H, AreH); FAB MS: m/z
(Mþ þ 1) 367.23, calcd 366.50.
3.6. General procedure for the preparation of 3,
5-substituted 1,4,2-dioxazoles (23e33)
The 4% aqueous solution of NaOCl (1.6 equiv) was added
to a solution of dipolarophile (respective aldehyde and ketone,
1 equiv) and triethylamine (0.1 equiv) in dichloromethane
under argon atmosphere. The oxime III (1 equiv) in dichloro-
methane was added dropwise (over a period of 1 h) at 0 ꢂC to
the above solution. After being stirred at room temperature for
24 h and refluxed for additional 24 h, water was added to the
reaction mixture and the aqueous layer was extracted with
dichloromethane. The combined organic layers were washed
with water and brine, dried (MgSO4), filtered and concentrated
under reduced pressure. The compounds were recrystallized
using dichloromethane hexane solution.
3.4.9. 3-[2-Pyridyl],3-[methyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (20)
Yield 44%, m.p. 154 ꢂC. Anal. calcd for C14H11N2O2Cl: C
61.20, H 4.03, N 5.08%; found: C 61.24, H 4.08, N 5.13%;
UV/vis l (nm): 278, 321, 354; IR nmax (cmꢁ1): 3021 (ArCe
1
H), 2976 (CH3), 1658 (C]N), 790, 893 (benzene); H NMR
(CDCl3) d/ppm: 2.48 (s, 3H, CH3), 7.23 (s, 1H, AreH), 7.25
(d, 1H, 7.4 Hz, AreH), 7.28 (d, 1H, 7.4 Hz, AreH), 7.31
(dd, 1H, AreH), 8.03e8.15 (dd, 1H, pyeH), 8.17e8.25 (dd,
1H, pyeH), 8.51e8.53 (d, 1H, pyeH), 8.68e8.71 (d, 1H,
pyeH); 13C NMR (CDCl3) d/ppm: 164.2 (C]N), 160.2
(C]N), 139.2, 138.4, 136.2, 132.9, 130.6, 130.1, 128.9,
128.2 (AreC), 113.8 (eOeCeOe), 26.5 (CH3); FAB MS:
m/z (Mþ þ 1) 275.41, calcd 274.50.
3.6.1. 3-[3,4-Dichlorophenyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (23)
Yield 38%, m.p. 172 ꢂC. Anal. calcd for C14H8NO2Cl3: C
51.13, H 2.45, N 4.26%; found: C 51.19, H 2.43, N 4.31%;
UV/vis l (nm): 259, 302, 334; IR nmax (cmꢁ1): 3024 (ArCeH),
2813 (CH), 1611 (C]N), 728, 830 (benzene); 1H NMR
(CDCl3) d/ppm: 6.26 (s, 1H, CH), 7.21 (s, 1H, ArCeH),
7.23 (d, 1H, 7.5 Hz, ArCeH), 7.28 (d, 1H, 7.5 Hz, AreH),
7.35 (d, 2H, 7.4 Hz, AreH), 7.39 (d, 2H, 7.4 Hz, AreH);
13C NMR (CDCl3) d/ppm: 160.5 (C]N), 142.0, 139.8,
136.1, 135.6, 131.2, 128.6, 121.8 (AreC), 112.2
(eOeCeOe); FAB MS: m/z (Mþ þ 1) 329.65, calcd 328.61.
3.4.10. 3-[2-Chlorophenyl]-5-[3-chlorophenyl]-1,
4,2-dioxazole (21)
Yield 26%, m.p. 134 ꢂC. Anal. calcd for C14H9NO2Cl2: C
57.12, H 3.08, N 4.76%; found: C 57.08, H 3.13, N 4.69%;
UV/vis l (nm): 281, 309, 348; IR nmax (cmꢁ1): 3041 (ArCeH),
2823 (CH), 1648 (C]N), 754, 887 (benzene); 1H NMR
(CDCl3) d/ppm: 6.28 (s, 1H, CH), 7.15 (s, 1H, AreH), 7.22
(d, 1H, 7.5 Hz, AreH), 7.25 (d, 1H, 7.5 Hz, AreH), 7.28 (dd,
1H, AreH), 7.48e7.59 (m, 8H, AreH); FAB MS: m/z
(Mþ þ 1) 295.09, calcd 294.00.
3.6.2. 3,5-Bis-[4-chlorophenyl]-1,4,2-dioxazole (24)
Yield 31%, m.p. 162 ꢂC. Anal. calcd for C14H8NO2Cl3: C
57.12, H 3.08, N 4.76%; found: C 57.19, H 3.16, N 4.79%;
UV/vis l (nm): 254, 300, 347; IR nmax (cmꢁ1): 2998 (AreH),
2821 (CH), 1659 (C]N), 756, 838 (benzene); 1H NMR
(CDCl3) d/ppm: 6.11 (s, 1H, CH), 7.19 (d, 2H, 7.6 Hz,
AreH), 7.24 (d, 2H, 7.7 Hz, AreH), 7.33 (d, 2H, 7.6 Hz,
AreH), 7.38 (d, 2H, 7.7 Hz, AreH); 13C NMR (CDCl3) d/
ppm: 158.5 (C]N), 141.2, 138.6, 138.2, 136.2, 135.8, 130.9,
129.1, 122.5 (AreC), 115.5 (eOeCeOe); FAB MS: m/z
(Mþ þ 1) 295.31, calcd 294.07.
3.4.11. 3,5-Bis-[3-chlorophenyl]-1,4,2-dioxazole (22)
Yield 29%, m.p. 152 ꢂC. Anal. calcd for C14H9NO2Cl2: C
57.12, H 3.08, N 4.76%; found: C 57.18, H 3.14, N 4.79%;
UV/vis l (nm): 251, 300, 353; IR nmax (cmꢁ1): 3030
1
(ArCeH), 2861 (CH), 1655 (C]N), 762, 873 (benzene); H
NMR (CDCl3) d/ppm: 5.99 (s, 1H, CH), 7.17 (d, 1H, 7.4
Hz, AreH), 7.21 (d, 1H, 7.6 Hz, AreH), 7.24 (d, 1H,
7.6 Hz, AreH), 7.26e7.41 (m, 4H, AreH), 7.43 (d, 1H,
7.4 Hz, AreH); FAB MS: m/z (Mþ þ 1) 295.01, calcd 294.00.

A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
935
3.6.3. 3-[4-Methylphenyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (25)
3.6.8. 3-[3-Bromophenyl],3-[ethyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (30)
Yield 29%, m.p. 139 ꢂC. Anal. calcd for C15H12NO2Cl2: C
58.23, H 3.91, N 4.51%; found: C 58.27, H 3.95, N 4.56%;
UV/vis l (nm): 255, 309, 354; IR nmax (cmꢁ1): 3068 (ArCeH),
2852 (CH), 1658 (C]N), 775, 834 (benzene); 1H NMR
(CDCl3) d/ppm: 2.51 (s, 3H, CH3), 5.99 (s, 1H, CH), 6.98
(d, 2H, 7.3 Hz, AreH), 7.04 (d, 2H, 7.3 Hz, AreH), 7.26 (d,
2H, 7.5 Hz, AreH), 7.31 (d, 2H, 7.5 Hz, AreH); FAB MS:
m/z (Mþ þ 1) 310.29, calcd 309.09.
Yield 37%, m.p. 136 ꢂC. Anal. calcd for C16H13NO2BrCl:
C 52.38, H 3.57, N 3.87%; found: C 52.43, H 3.62, N
3.79%; UV/vis l (nm): 259, 306, 356; IR nmax (cmꢁ1): 3065
(ArCeH), 2937 (CH3), 2897 (CH2), 1657 (C]N), 754, 830
1
(benzene); H NMR (CDCl3) d/ppm: 1.11 (t, 3H, CH3), 1.98
(m, 2H, CH2), 7.04 (d, 2H, 7.2 Hz, AreH), 7.07 (d, 2H,
7.2 Hz, AreH), 7.17e7.31 (m, 4H, AreH); FAB MS: m/z
(Mþ þ 1) 367.63, calcd 366.50.
3.6.9. 3-[2-Pyridyl],3-[methyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (31)
3.6.4. 3-[4-Ethylphenyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (26)
Yield 40%, m.p. 129 ꢂC. Anal. calcd for C14H11N2O2Cl: C
61.20, H 4.03, N 5.08%; found: C 61.23, H 4.12, N 5.12%;
UV/vis l (nm): 254, 313, 346; IR nmax (cmꢁ1): 3045 (ArCeH),
2948 (CH3), 1658 (C]N), 755, 830 (benzene); 1H NMR
(CDCl3) d/ppm: 2.39 (s, 3H, CH3), 7.11 (d, 2H, 7.6 Hz,
AreH), 7.15 (d, 2H, 7.6 Hz, AreH), 7.89e7.95 (dd, 1H,
pyeH), 8.06e8.17 (dd, 1H, pyeH), 8.21e8.24 (d, 1H, pyeH),
8.34e8.38 (d, 1H, pyeH); 13C NMR (CDCl3) d/ppm: 160.4
(C]N), 153.2 (C]N), 141.3, 138.8, 136.2, 134.1, 130.6,
130.2, 128.9, 128.3 (AreC), 116.8 (eOeCeOe), 25.9
(CH3); FAB MS: m/z (Mþ þ 1) 275.32, calcd 274.50.
Yield 41%, m.p. 158 ꢂC. Anal. calcd for C16H14NO2Cl: C
67.07, H 4.95, N 4.91%; found: C 67.13, H 5.01, N 4.96%;
UV/vis l (nm): 257, 313, 346; IR nmax (cmꢁ1): 3075 (ArCeH),
2947 (CH3), 2876 (CH2), 1656 (C]N), 791, 834 (benzene);
1H NMR (CDCl3) d/ppm: 1.42 (t, 3H, CH3), 2.24 (m, 2H,
CH2), 6.02 (s, 1H, CH), 6.92 (d, 2H, 7.5 Hz, AreH), 6.97
(d, 2H, 7.5 Hz, AreH), 7.22 (d, 2H, 7.6 Hz, AreH), 7.26 (d,
2H, 7.6 Hz, AreH); FAB MS: m/z (Mþ þ 1) 288.84, calcd
287.61.
3.6.5. 3,5-Bis-[4-chlorophenyl]-3-[methyl]1,
4,2-dioxazole (27)
3.6.10. 3-[2-Chlorophenyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (32)
Yield 27%, m.p. 152 ꢂC. Anal. calcd for C15H11NO2Cl: C
58.42, H 3.59, N 4.54%; found: C 58.47, H 3.56, N 4.59%;
UV/vis l (nm): 282, 311, 356; IR nmax (cmꢁ1): 3034 (ArCeH),
2941 (CH3), 1657 (C]N), 753, 828 (benzene); 1H NMR
(CDCl3) d/ppm: 2.10 (3H, CH3), 7.12 (d, 2H, 7.4 Hz,
AreH), 7.17 (d, 1H, 7.4 Hz, AreH), 7.29 (d, 2H, 7.6 Hz,
AreH), 7.34 (d, 2H, 7.6 Hz, AreH); FAB MS: m/z
(Mþ þ 1) 309.2, calcd 308.08.
Yield 35%, m.p. 154 ꢂC. Anal. calcd for C14H9NO2Cl2: C
57.12, H 3.08, N 4.76%; found: C 57.16, H 3.11, N 4.81%;
UV/vis l (nm): 256, 309, 346; IR nmax (cmꢁ1): 3051
(ArCeH), 1654 (C]N), 753, 830 (benzene); 1H NMR
(CDCl3) d/ppm: 6.16 (s, 1H, CH), 7.09e7.24 (m, 4H, AreH),
7.32 (d, 2H, 7.7 Hz, AreH), 7.38 (d, 2H, 7.7 Hz, AreH);
FAB MS: m/z (Mþ þ 1) 295.24, calcd 294.00.
3.6.11. 3-[3-Chlorophenyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (33)
3.6.6. 3-[3-Chlorophenyl],3-[ethyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (28)
Yield 29%, m.p. 165 ꢂC. Anal. calcd for C14H9NO2Cl2: C
57.12, H 3.08, N 4.76%; found: C 57.16, H 3.13, N 4.72%;
UV/vis l (nm): 260, 316, 336; IR nmax (cmꢁ1): 3038 (ArCeH),
2861 (CH), 1659 (C]N), 754, 830 (benzene); 1H NMR
(CDCl3) d/ppm: 6.18 (s, 1H, CH), 7.08 (d, 1H, 7.4 Hz,
AreH), 7.12 (d, 1H, 7.4 Hz, AreH), 7.16 (s, 1H, AreH),
7.18 (dd, 1H, AreH), 7.27 (d, 2H, 7.6 Hz, AreH), 7.32 (d,
2H, 7.6 Hz, AreH); FAB MS: m/z (Mþ þ 1) 295.32, calcd
294.0.
Yield 31%, m.p. 146 ꢂC. Anal. calcd for C16H13NO2Cl2: C
59.60, H 4.06, N 4.34%; found: C 59.64, H 4.03, N 4.39%;
UV/vis l (nm): 256, 300, 336; IR nmax (cmꢁ1): 3033
(ArCeH), 2941 (CH3), 2896 (CH2), 1656 (C]N), 756, 837
1
(benzene); H NMR (CDCl3) d/ppm: 1.54 (t, 3H, CH3), 2.14
(m, 2H, CH2), 7.09 (d, 1H, 7.4 Hz, AreH), 7.13 (s, 1H,
AreH), 7.15 (d, 1H, 7.4 Hz, AreH), 7.19 (dd, 1H, AreH),
7.25 (d, 2H, 7.7 Hz, AreH), 7.29 (d, 2H, 7.7 Hz, AreH);
FAB MS: m/z (Mþ þ 1) 323.14, calcd 322.10.
3.7. Antiamoebic activity
3.6.7. 3-[3-Bromophenyl],3-[methyl]-5-[4-chlorophenyl]-1,
4,2-dioxazole (29)
Compounds 1e33 were screened in vitro for antiamoebic
activity against the HM1:IMSS strain of E. histolytica by the
microdilution method [22]. E. histolytica trophozoites were
cultured in TYIS-33 growth medium as described previously
in 96-well microtiter plates (Costar) [23]. The test compounds
were dissolved in DMSO (40 ml), at which level no inhibition
of the amoeba occurs [24,25]. Then culture medium was
added to obtain a concentration of 1 mg/ml. Twofold serial
Yield 46%, m.p. 151 ꢂC. Anal. calcd for C15H11NO2BrCl:
C 51.07, H 3.14, N 3.97%; found: C 51.13, H 3.16, N
3.93%; UV/vis l (nm): 253, 317, 343; IR nmax (cmꢁ1): 3046
(ArCeH), 2972 (CH3), 1657 (C]N), 789, 843 (benzene);
1H NMR (CDCl3) d/ppm: 2.46 (s, 3H, CH3), 7.06e7.18 (m,
4H, AreH), 7.23 (d, 2H, 7.3 Hz, AreH), 7.29 (d, 2H,
7.3 Hz, AreH); FAB MS: m/z (Mþ þ 1) 353.32, calcd 352.4.

936
A.R. Bhat et al. / European Journal of Medicinal Chemistry 44 (2009) 926e936
dilutions were then made. Each test includes metronidazole as
a standard amoebic drug, control wells (culture medium plus
amoeba), and a blank (culture medium only). The number of
amoeba per millimeter was estimated with a haemocytometer
and trypan blue exclusion was used to confirm viability. The
cell suspension used was diluted to 105 organisms/ml by
adding fresh medium, and 170 ml of this suspension was added
to the test and control wells in the plate. An inoculum of
1.7 ꢃ 104 organisms/well was chosen, so that confluent, but
not excessive, growth took place. The plates were sealed and
gassed for 10 min with N2, and then incubated at 37 ꢂC for
72 h. After incubation the growth of the amoeba was checked
with a low power microscope. The culture medium was
removed by inverting the plate and shaking gently. The plates
were immediately washed with 0.9% aq. NaCl solution at
37 ꢂC. This procedure was performed quickly, and the plate
was not allowed to cool to prevent the detachment of amoebae.
The plate was allowed to dry at room temperature, and the
amoebae were fixed with chilled methanol by keeping it in
an ice bath for 15 min, dried, and stained with 0.5% aq. eosin
for 15 min. The stained plate was washed once with tap H2O
and then twice with distilled H2O, and allowed to dry. Then
0.1 N aq. NaOH solution (200 ml) was added to each well to
dissolve the protein and to release the dye (eosin). The optical
density of the resulting solution in each well was determined
at 490 nm with a microplate reader. The inhibition (in %) of
amoebal growth was calculated from the optical densities of
the control and test wells, and plotted against the logarithm
of the dose of the drug tested. Linear-regression analysis
was used to determine the best-fitting straight line from which
IC50 values were determined.
of the 33 compounds prepared, nine were found with 2e4
times better activity than the reference drug metronidazole.
The MTT assay revealed that all the compounds are non-
toxic to the human kidney epithelial (Graham) cells. The
in vivo studies of these nine compounds are currently in
progress.
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This work examined the biological activities of the 3,5-
substituted-1,4,2-dioxazoles prepared from the oximes. Out