Novel terpene based 1,4,2-dioxazoles: Synthesis, characterization, molecular properties and screening against Entamoeba histolytica

Article abstract of DOI:10.1016/j.ejmech.2011.06.005

In present investigation a series of 20 dioxazole analogues (1-20) were synthesized, characterized and subjected to molecular properties prediction, anti-amoebic screening and cytotoxicity evaluation. Out of the twenty compounds viz. 3,5-substituted-1,4,2-dioxazoles, six compounds have shown IC₅₀ values in the range (1.00-1.10 μM) lower than the standard drug metronidazole (IC₅₀ = 1.45 μM). The toxicological studies of the active compounds on H9c2 rat cardiac myoblasts showed that all compounds were nontoxic. The pKa, and log P values have also been predicted. Compound 8 showed the most promising results based on anti-amoebic evaluation, cytotoxicity studies and physico-chemical properties prediction.

Full text of DOI:10.1016/j.ejmech.2011.06.005

European Journal of Medicinal Chemistry 46 (2011) 4742e4752
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Novel terpene based 1,4,2-dioxazoles: Synthesis, characterization, molecular
properties and screening against Entamoeba histolytica
,
b
c
b
Mohmmad Younus Wani a, Fareeda Athar a , Attar Salauddin , Subhash Mohan Agarwal , Amir Azam ,
**
***
,
d,
*
Inho Choi d , Abdul Roouf Bhat
a Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India
b Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India
c Bioinformatics Division, Institute of Cytology and Preventive Oncology, Noida 201301, India
d School of Biotechnology, Yeungnam University, 241-1, Dae-Dong, Gyeongsan 712749, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In present investigation a series of 20 dioxazole analogues (1e20) were synthesized, characterized and
subjected to molecular properties prediction, anti-amoebic screening and cytotoxicity evaluation. Out of
the twenty compounds viz. 3,5-substituted-1,4,2-dioxazoles, six compounds have shown IC50 values in
Received 26 April 2011
Received in revised form
29 May 2011
Accepted 7 June 2011
Available online 14 June 2011
the range (1.00e1.10
m
M) lower than the standard drug metronidazole (IC50 ¼ 1.45
mM). The toxicological
studies of the active compounds on H9c2 rat cardiac myoblasts showed that all compounds were
nontoxic. The pKa, and log P values have also been predicted. Compound 8 showed the most promising
results based on anti-amoebic evaluation, cytotoxicity studies and physico-chemical properties
prediction.
Keywords:
Dioxazole
Terpene
Ó 2011 Elsevier Masson SAS. All rights reserved.
Anti-amoebic activity
Cytotoxicity studies
1. Introduction
antiviral, antiallergenic, antispasmodic, antihyperglycemic, antiin-
flammatory, chemotherapeutic and immunomodulatory properties
Amoebiasis, an infectious disease caused by Entamoeba histo-
lytica, results in severe liver and brain abscess and causes high rate
of morbidity and mortality in humans. Metronidazole, the
5-nitroimidazole drug has been the drug of choice for several
decades in the treatment of amoebiasis [1]. However, there are
concerns regarding its carcinogenicity [2] and recent studies have
reported several toxic effects such as genotoxicity, gastric mucus
irritation, and spermatozoid damage [3,4]. Furthermore, there are
occasional reports of failure with metronidazole suggesting that
this could probably be heralding the development of drug resis-
tance clinically [5]. Recurrence of amoebic liver abscess even after
treatment with metronidazole has been reported and parasites
may survive in spite of adequate treatment [6]. Terpenoids are
natural molecules with antimicrobial, antifungal, antiparasitic,
[7e18]. The versatile biological applications of azoles made it
a targeted investigatory class of compound [19]. Counting the
antiparasitic properties of terpenes [20e22] and anti-amoebic
properties of 1,4,2-dioxazoles [23e25], we proposed the synthesis
of terpene based 1,4,2-dioxazoles.
In the development of drugs intended for oral use, good drug
absorption and appropriate drug delivery are very important [26].
About 30% of oral drugs fail in development due to poor phar-
macokinetics [27]. Among the pharmacokinetic properties, a low
and highly variable bioavailability is indeed the main reason for
stopping further development of the drug [28]. Moreover, the
knowledge of ionization constant is an important challenge for
understanding various phenomena like biological uptake, phar-
macological activity or in vivo studies. Hence, the discovery of new
molecules require accurate determination of pKa and log P values.
In the present research work some new terpene-based dioxazole
derivatives 1e20 have been synthesized, screened for anti-
amoebic activity and cell cytotoxicity. Their pKa and log P values
have been determined in order to understand the chemical
interactions between the compound of interest and its pharma-
cological target.
* Corresponding author. Tel.: þ82 53 810 3589; fax: þ82 53 810 4769.
** Corresponding author. Tel.: þ82 53 810 3024; fax: þ82 53 810 4769.
*** Corresponding author. Tel.: þ91 11 26981717x4492; fax: þ91 11 26980164.
E-mail addresses: fareeda.cirbs@jmi.ac.in (F. Athar), inhochoi@ynu.ac.kr (I. Choi),
abroouf@gmail.com (A. Roouf Bhat).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.005

M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
4743
2. Results and discussion
9.43e11.13 ppm and 6.90e7.80 due to (NeOH) and (N]CH)
respectively in all the compounds 1e20. The singlet at
5.65e6.96 ppm, which arose due to (CeH) group present at C-3 of
the dioxazole ring, confirmed the condensation of oximes IeII with
different hetero aromatic aldehydes/ketones and terpene moieties
and oximes IIIeV with different terpene moieties. Methyl signals
were obtained for compounds which contain methyl protons at C-3
of the dioxazole ring. For methyl group, a singlet at 1.90e2.54 ppm
appeared in compounds 1, 3, 5, 8, 10 and 12. In addition, the signals
for the different ring protons appeared in their respective range in
all the compounds. 13C NMR spectra further supported the confir-
matory structures. The (C]N) signal around 155.1e169.6 ppm and
(eOCOe) signal around 85.9e96.8 ppm clearly favoured the
formation of dioxazole rings. The signals due to the different hetero
aromatic and aliphatic carbons resonate at their usual positions and
the values are given in the experimental section.
2.1. Chemistry
The outline synthesis of dioxazole derivatives (1e20) is given in
Scheme 1. The reaction of corresponding oximes (IeV) with
different aromatic aldehydes or ketones in ethylacetate using
sodium hypochlorite and triethylamine yielded the respective
dioxazole derivatives. The compounds in solid state showed sharp
melting points and the elemental analysis was found in accordance
with ꢀ0.3%. The compounds were stable and were soluble in
DMSO, methanol, and chloroform. These terpene based dioxazole
derivatives 1e20 were recrystallized from appropriate solvents,
and final yield of 25e60% was obtained. The dioxazole derivatives
were characterized by electronic, IR, 1H and 13C NMR, and mass
spectroscopy.
Characteristic IR bands provide significant indications for the
formation of the oximes IeV and dioxazoles 1e20. The absence of
a band at/or around 2665 cmꢁ1 due to aldehydic proton and the
appearance of characteristic bands at 3245e3260 cmꢁ1 and
2.2. Pharmacology
1622e1635 cmꢁ1 due to
n(NOeH) and n(C]N) respectively,
All the dioxazole derivatives (1e20) were screened in vitro
against HM1:IMSS strain of E. histolytica using microdilution
method. All the experiments were carried out in triplicate at each
concentration level and repeated thrice. Toxicity of active
compounds was studied by MTT assay on H9c2 cardiac myoblasts.
The results of biological activity and toxicity are summarized in
Table 1 and Fig. 1. Besides the in vitro cytotoxic studies, their pKa
and log P values have been determined, for understanding the
chemical interactions, between the compound of interest and its
pharmacological target.
confirmed the formation of the oximes IeV. The absence of a band at
3245e3260 cmꢁ1 in all the dioxazoles 1e20 confirmed the
conversion of the corresponding oximes into their respective diox-
azoles. The
n(C]N) band in dioxazoles was in the range of
1642e1660 cmꢁ1 and a new band in the range of 1120e1185 cmꢁ1
arose due to (CeOeC) group, however, in some cases this band split.
In addition to these the representative bands due to carbonecarbon
stretching of aliphatic and aromatic groups were also present.
The electronic spectra of the compounds 1e20 studied in the UV
region, exhibited three absorption bands at 314e354 nm,
2.2.1. Anti-amoebic activity
295e314 nm and 250e295 nm assigned to n / p* p* and
, p /
Preliminary experiments were carried out to determine the
in vitro anti-amoebic activity of all the compounds 1e20 by
microdilution method using HM1:IMSS strain of E. histolytica. The
results are summarized in Table 1. The data are presented in terms
of percent growth inhibition relative to untreated controls, and
plotted as probit values as a function of drug concentration. The
n / s* transitions respectively. The band at 314e354 nm was
assigned to the transition involving the azomethine group (C]N).
The other two absorption bands at 295e314 nm and 250e295 nm
were due to
p /
p* transition of dioxazole ring and n / s* tran-
sition of azomethine nitrogen respectively.
The structure of the oximes IeV was further confirmed by 1H
NMR spectra. A singlet at 6.90e7.90 ppm due to (CeH]N) proton
showed condensation between carbonyl group of aldehydes and
amino group of hydroxylamine hydrochloride. The signal at
9.43e11.13 ppm due to (NeOH) proton further confirmed the
formation of the oximes. The structure of oximes IeV was further
supported by 13C NMR spectra. The absence of aldehydic carbon
signal at 192 ppm and the presence of a signal at 146.0e149.80 ppm
due to (C]N) confirmed the formation of oximes. The formation of
Table 1
In vitro anti-amoebic activity of compounds (1e20) against HM1:IMSS strain of
E. histolytica and toxicity profile. The compounds with bold font IC50 values are more
active than metronidazole.
Compound
Anti-amoebic activity
Toxicity profile
IC50
(
m
M)
S.D.a (ꢀ)
IC50
(m
M)
Safety index
(SI)
1
2
3
4
5
6
7
8
2.25
2.98
2.02
2.42
3.01
2.90
1.48
1.00
1.03
1.10
1.09
2.68
3.50
1.39
2.22
1.06
2.78
1.05
3.05
2.45
1.45
0.62
0.13
0.46
0.27
0.29
0.16
0.18
0.25
0.28
0.14
0.13
0.20
0.36
0.39
0.22
0.43
0.29
0.31
0.19
0.26
0.33
N.D
N.D
N.D
N.D
N.D
N.D
N.D
>200
40
>200
>200
N.D
N.D
N.D
N.D
85
N.D
200
N.D
N.D
>200
N.C
N.C
N.C
N.C
N.C
N.C
N.C
>200
38.83
>181.81
>183.48
N.C
N.C
N.C
N.C
>80.18
N.C
dioxazoles was supported by the absence of
a
signal at
Method 1/
Method II
H
O
a
O
N
R1
R2
NH2OH.HCl
+
OH
R
N
O
R
H
R
I-V
1-20
9
Where
R =
10
11
12
13
14
15
16
17
18
19
20
MNZ
S
=
III
V
=
I
=
=
N
O
O
O
O
=
II
IV
190.47
N.C
N.C
For
R1 and R 2 see Table 2 structures
Scheme 1. General Method for the synthesis of Dioxazole derivatives via Oximes.
Reagents and Conditions: Method I: Pyridine, C2H5OH, reflux 24 h. Method II: H2O,
NaHCO3, Stirring, r.t. (a) aq. NaOCl, Et3N, EtOAc, (R1eCOeR2), where (R1eCOeR2)
represents the different aldehydes, ketones and terpene moieties as given in Table 2.
>137.90
N.D. Not done, N.C. Not Calculated, S.D. (ꢀ) Standard deviation.
a
The value obtained in at least three separate assay done in triplicate.

4744
M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
Fig. 1. Percentage of viable cells after 48 h pre-treatment of H9c2 myoblasts with Metronidazole, compounds 8, 9, 10, 11, 16 and 18, evaluated by MTT assay.
anti-amoebic effect was compared with the most widely used
anti-amoebic medication metronidazole with 50% inhibitory
against the H9c2 cardiac myoblasts. Compound 8 showed least
cytotoxicity among all the compounds screened, with a remarkable
concentration (IC50) of 1.45
mM in our experiments. Since the target
viability of 88% at a concentration of 200 mM. The cytotoxicity IC50
compounds were deigned in three different ways based on the
substituted molecules at position-3 and 5 of the dioxazole ring. The
compounds (1e7), (8e14) and (15e20) showed activity in the range
values along with the standard deviation values of the compounds
and metronidazole are given in Table 1. To further investigate the
selectivity of the compounds, the “safety index” (SI), defined as the
toxicity IC50/protozoal IC50, was calculated. This allows estimating
the efficacy of compounds. The results are summarized in Table 1.
Compound 8 showed higher safety index value. Compounds 10, 11
and 18 show safety index values better than metronidazole. These
results showed that further studies of these compounds can give
a better lead in the drug field. The results of physico-chemical
properties are also in good support with the above results.
of IC50
¼
1.40e3.01
M respectively. The results showed that the
(IC50 1.00 M), (IC50 1.03 M), 10
M), 11 (IC50 ¼ 1.09 M), 16 (IC50 ¼ 1.06 M) and 18
M) exhibited better anti-amoebic activity than the
m
M, IC50
¼
1.00e3.50
m
M
and
IC50 ¼ 1.05e3.05
m
compounds
(IC50 ¼ 1.10
(IC50 ¼ 1.05
8
¼
m
m
9
¼
m
m
m
m
standard drug metronidazole while compounds 7 & 14 were
moderately active. Based on IC50 values, six compounds 8, 9, 10, 11,
16 & 18 were more active than the standard drug metronidazole.
From the above results it can be inferred that most of the new
compounds having piperonal ring skeleton in conjugation with
pyridine ring or furan ring in the same compound showed signifi-
cant anti-amoebic activity and the modification of functionality
produced significant change of activity. Thus, in the dioxazole
series, anti-amoebic activity can be positively modulated through
the introduction of pyridine, furan and piperonal ring residue on
the dioxazole ring. It is worthy to mention that the position of the
CH3 group at position 3 of the dioxazole ring has also a marked
effect on the activity and toxicity of the synthesized compounds as
depicted by compounds 8 and 9 besides the presence and position
of pyridine ring has a noticeable effect on the anti-amoebic activity
and cytotoxicity of the compounds. The results were also statisti-
cally evaluated by analysis of variance. The null hypothesis was
tested using t-test. The significativity of the difference between the
IC50 values of metronidazole and the compounds 8, 9, 10, 11, 16 and
18 was evaluated by t-test. The values of the calculated T were
found higher than the Table value of T at 5% level, thus concluding
that the character under study is said to be significantly influenced
by the treatment.
2.3. Molecular properties prediction
A molecular property is a complex balance of various structural
features which determine whether a particular molecule is similar
to the known drugs. It generally means “molecules which contain
functional groups and/or have physical properties consistent with
most of the known drugs”. These properties, mainly molecular size,
flexibility, hydrophobicity and presence of various pharmacophoric
features influence the behaviour of molecules in a living organism,
including bioavailability. A good bioavailabilitycan be achieved with
an appropriate balance between solubility and partitioning prop-
erties. Thus in order to achieve good oral drugs we have subjected
a series of 1,4,2-dioxazole derivatives (1e20) for the prediction of
lipophilicity, solubility and Lipinski’s “Rule of Five” [29].
The knowledge of acidity constant and lipophilicity is a key
parameter for understanding the chemical interactions, between
the compound of interest and its pharmacological target. Relation-
ship between acidity constant, pKa and structure may prove useful
in drug design studies and in explaining the biopharmaceutical
properties of substances like benzoxa-, benzothia- or benzoselena-
zolinone [30,31]. Many biologically active molecules are fully or
partially ionized at physiological pH, and it has often been shown
that the presence of ionizable groups is necessary for biological
activity and/or solubility. Moreover, the acidebase property of
a drug molecule is the key parameter for drug development because
it governs solubility, absorption, distribution, metabolism and
elimination. Particularly for developing new API’s, the pKa has
become of great importance because the transport of drugs into cells
and across other membranes is a function of physico-chemical
properties, and of the pKa and log P of the drugs [32].
2.2.2. MTT assay
To ensure the toxicity of the most active compounds 8, 9, 10, 11,
16 and 18, they were tested against H9c2 cardiac myoblasts. A sub-
confluent population of H9c2 cells was treated with increasing
concentrations of these compounds and the number of viable cells
was measured after 48 h by MTT cell viability assay. The concen-
tration range of all the compounds was 3.13e200 mM. Fig. 1 depicts
that all the compounds including the reference compound metro-
nidazole showed a viability of 100% at the concentration range of
3.13
metronidazole exhibited ꢂ82% viability at the concentration range
of 3.13e25 M. On increasing the concentration range up to 50, 100
and 200 M the compounds showed moderate to high cytotoxicity
mM. Fig. 1 depicts that the compounds 8, 9, 10, 11, 16 and 18 and
2.3.1. pKa and log P
Poor solubility and poor permeability are amongst the main
causes for failure during drug development [33e35]. It is therefore,
m
m

M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
4745
important to determine the physico-chemical properties associated
with a drug. In order to calculate pKa values of the compounds,
Marvin was used that has been described as a method of choice in
a published paper where comparison of predictive performance of
five different pKa prediction tools was undertaken [36].
In cases where multiple tautomeric forms exist the one which is
expected to be the most stable was considered for calculating pKa
values. As shown in Fig. 2a, the pKa values can be correlated with
the activity of the compounds under study. In particular, the most
active compounds have pKa values of w2 (Table 2), and at physi-
ological pH values, their rings will be uncharged while those of the
less active species will be charged (to a lesser or greater extent)
[32]. The presence of certain substituents which result in resonance
stabilization/charge delocalization have also a very major effect on
the pKa of the compounds [37].
(Lipinski’s Rule of Five) mean that they can readily get past ester/
phosphate groups in skin membranes. The log P values of the
compounds are shown in Table 2. There also exists a good rela-
tionship between log P and activity and in this study we have found
that compounds having lower log P and IC50 values are the most
active. However the compounds having lower IC50 values and
higher log P values are the least active and similarly the compounds
having higher IC50 values and lower log P values are also least
active. As shown in Fig. 2, the compounds with log P values close to
the linear line and have lower IC50 values are the most active.
Hansch et al. [38] have also shown the relationship of the type.
Activity ¼ m log P þ K0ðLinearÞ
The octanolewater partition coefficient (P) (also referred to as
Kow) is a measure of the propensity of a neutral compound to
differentially dissolve in these immiscible phases. It is usually
referred to as the logarithmic ratio, log P, and serves as a quantita-
tive descriptor of lipophilicity. Compounds with log P values ꢃ5
2.3.2. “Rule of Five” properties
High oral bioavailability is an important factor for the devel-
opment of bioactive molecules as therapeutic agents. Good intes-
tinal absorption, reduced molecular flexibility (measured by the
number of rotatable bonds), low polar surface area or total
hydrogen bond count (sum of donors and acceptors), are important
predictors of good oral bioavailability [39,40]. Molecular properties
such as membrane permeability and bioavailability are always
associated with some basic molecular descriptors such as log P
(partition coefficient), molecular weight (MW), or hydrogen bond
acceptors and donors count in a molecule [41]. Lipinski [29] used
these molecular properties in formulating his “Rule of Five”. The
rule states that most molecules with good membrane permeability
have log P ꢃ 5, molecular weight ꢃ500, number of hydrogen bond
acceptors ꢃ10, and number of hydrogen bond donors ꢃ5. This rule
is widely used as a filter for drug-like properties. A compound that
fulfils at least three out of the four criteria is said to adhere to
Lipinski’s “Rule of Five”. A poor permeation or absorption is more
likely when there are more than 5 H-bond donors, 10 H-bond
acceptors. The series (1e20) under investigation has most of the
compounds possessing less number of hydrogen bond donors (<5)
but do possess considerable number of acceptors (<10) as shown
in Table 3. Lipinski’s rule was used as a filter to choose the
reasonable scaffolds as potential lead scaffolds. Tables 2 and 3 list
the values of these properties for the new scaffolds and suggest
that the active compounds are reasonable starting points for a drug
discovery effort.
3. Conclusion
The combination of extended synthetic analogues of natural
molecules leads to discovery of chemical entities which might be
excellent anti-amoebic compounds as depicted in our results.
3,5-substituted 1,4,2-dioxazole derivatives 1e20, bearing
terpene moieties were synthesized and screened against
HM1:IMSS strain of E. histolytica. Compound 8 (2-(3-(benzo[d][1,
3]dioxol-5-yl)-5-methyl-1,4,2-dioxazol-5-yl)pyridine) showed
the most promising results based on anti-amoebic screening,
cytotoxicity studies and molecular properties prediction. Being
highly anti-amoebic this compound can be explored in future as
an option for decreasing pathogenic potential of infecting
E. histolytica species. The compounds showing lower IC50 values
than metronidazole also follow Lipinski’s “Rule of Five”.
4. Experimental protocol
Fig. 2. a. Graph showing correlation between the PIC50 for E. histolytica growth inhi-
bition by dioxazoles and the computed pka values of the parent bases. The line is to
guide the eye. b. Graph showing correlation between log P and IC50 values. The
compounds whose log P values are close to the linear line and have lower IC50 values
are the most active. The most active compounds are encircled.
Solvents and organic reagents were purchased from Sigma
Aldrich, Merck (Germany) and Loba Chemie (India) and were used
without further purification. Melting points (mp) were performed

4746
M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
Table 2
pKa and log P values of compounds 1e20.
C. No.
% Yield
Neutral
Tautomer
LogP
pKa
N+
O
CH3
H3C
O
H
CH3
H3C
N
N
O
O
N
N
N
N
1a
25
4.50 ꢀ 0.66
Not Predicted
CH3
CH3
H3C
CH3
H3C
H3C
N+
O
H
O
H
H
O
O
2a
30
25
50
3.96 ꢀ 0.65
5.15 ꢀ 0.66
4.61 ꢀ 0.66
Not Predicted
CH3
CH3
CH3
H3C
H3C
CH3
H3C
O
CH3
O
N
CH3
O
CH3
O
N
O
O
3
1.2e1.3
H3C
H3C
CH3
H3C
H3C
+
O
H
O
H
N
O
H
O
N
N
N
O
O
4
1.2e1.3
H3C
H3C
CH3
H3C
H3C
+
O
CH3
S
O
CH3
H
N
O
O
5
40
5.67 ꢀ 0.66
1.6
S
CH3
H3C
H3C
CH3
H3C
H3C
HN+
O
H
O
H
S
O
O
6
30
5.13 ꢀ 0.66
1.6
S
CH3
H3C
H3C
CH3
H3C
N+
O
H
O
H
H
H3C
N
O
O
O
O
7
40
5.31 ꢀ 0.67
1.3e1.4
O
O
CH3
H3C
CH3
H3C

M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
4747
Table 2 (continued)
C. No.
% Yield
Neutral
Tautomer
LogP
pKa
O
N
CH3
O
CH3
N
N
O
O
O
8a
60
27
29
40
50
52
58
2.34 ꢀ 0.91
0.30e0.40
O
O
+
H
N
O
O
O
N
H
N
N
H
O
O
9a
1.80 ꢀ 0.90
2.99 ꢀ 0.91
2.45 ꢀ 0.90
3.51 ꢀ 0.92
2.97 ꢀ 0.91
5.44 ꢀ 0.92
0.20e0.30
0.10e0.20
0.10
O
O
O
O
N+
H
+
O
O
H
N
CH3
N
CH3
O
O
O
O
10
11
12
13
14
O
O
O
O
H
HN+
O
O
N
N
H
O
O
O
O
O
O
O
O
+
O
O
H
N
CH3
CH3
O
O
O
0.40e0.50
0.40e0.50
1.2e1.3
O
O
S
S
O
+
O
O
H
H
H
N
H
N
N
O
O
O
O
O
O
S
S
N+
CH3
O
H
CH3
O
H
O
O
O
O
O
O
H3C
CH3
H3C
CH3
CH3
N+
CH3
O
H
N
O
H
H
O
O
15a
60
4.08 ꢀ 0.89
1.00
N
N
H3C
CH3
H3C
CH3
(continued on next page)

4748
M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
Table 2 (continued)
C. No.
% Yield
Neutral
Tautomer
LogP
pKa
+
O
H
O
H
H
N
N
16a
50
34
32
60
1.80 ꢀ 0.90
Not Predicted
O
O
O
O
N
N
O
O
N+
O
O
H
H
H
N
CH3
CH3
O
O
17
18
19
5.25 ꢀ 0.90
2.97 ꢀ 0.91
4.74 ꢀ 0.90
0.50e0.60
O
O
CH3
H3C
CH3
H3C
+
O
H
O
H
H
N
N
Not Predicted
O
O
O
O
O
O
O
O
+
O
O
H
H
H
N
N
CH3
CH3
O
O
Not Predicted
S
S
CH3
H3C
CH3
H3C
N+
O
O
H
H
H
N
20
50
2.45 ꢀ 0.90
Not Predicted
O
O
O
O
O
S
S
O
a
Have multiple tautomeric forms. The one which is the most stable has been mentioned.
using a Mel-temp instrument, and the results are uncorrected.
Precoated aluminium sheets (silica gel 60 F254, Merck Germany)
were used for thin-layer chromatography (TLC) and spots were
visualized under UV light. Elemental analyses were performed on
Heraeus Vario EL III analyzer at Central Drug Research Institute,
Lucknow, India. Electronic spectra were recorded on a Shimadzu UV
1601 PC UVeVisible spectrophotometer. IR spectra were recorded
on PerkineElmer model 1600 FT-IR RX1 spectrophotometer as KBr
discs. 1H NMR and 13C NMR spectra were recorded on Bruker
AVANCE 300 spectrometer using DMSO-d6 as solvent with TMS as
internal standard. Splitting patterns are designated as follows; s,
singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet.
Chemical shift values are given in ppm. ESI-MS was recorded on
a MICROMASS QUATTRO II triple quadrupole mass spectrometer.
4.1.1. Method I
An aldehyde (1 mmol) and hydroxylamine hydrochloride
(1.25 mmol) 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 solid mass was
collected, washed with water, and crystallized from methanol to
give the corresponding oxime. However in case of citral oxime an
oil layer was formed which was extracted with ether and separated.
The organic layer was dried with NaSO4 and concentrated under
reduced pressure.
4.1.2. Method II
To a solution of aldehyde (1 eq) and hydroxylamine hydrochlo-
ride (1.25 eq) in water (5 ml), a solution of sodium bicarbonate
(1.25 eq) in water (10 ml) was added gradually with stirring, and the
mixture was stirred for further 2e5 h with the formation of a solid
precipitate, which was filtered and dried. However in case of citral
oxime (I), an organic layer was formed, which was separated and the
aqueous layer was extracted with ether and added to the organic
layer. The organic layer was dried with NaSO4 and evaporated.
4.1. General procedure for the synthesis of oximes (IeV)
The oximes were synthesized by two methods (I & II). Although
both methods resulted in good yields, however the method II is
favoured, because of higher yield and less reaction time (Table 4).

M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
4749
found: C 47.05, H 4.06, N 11.09%; IR nmax cmꢁ1: 3254 (NOeH), 3132
(CeH), 1628 (C]N), 935 (NeO stretch); 1H NMR (DMSO-d6)
Table 3
The pharmacokinetic properties of compounds 1e20 according to Lipinski’s rule.
d
(ppm): 11.13 (broad s, 1H, NeOH), 6.90 (s, 1H, CH]NeOH), 7.20 (d,
Compound
Molecular
weight
Log P
H-bond
donors
H-bond
acceptors
Rule of 5
criteria
met
1H, furan ring-H, J ¼ 7.6 Hz) 6.01 (dd 2H furan ring-H, J ¼ 8.5,
8.9 Hz); 13C NMR (DMSO-d6)
d(ppm): 146.0 (C]N), 142.8, 134.7,
110.8, 109.5; ESI-MS m/z: [Mþþ1] 111.03.
1
2
3
4
5
6
7
8
286.17
272.15
275.15
261.14
291.13
277.11
315.15
284.08
270.06
273.06
259.05
289.04
275.03
315.15
272.15
270.06
261.14
259.05
275.03
289.04
4.50 ꢀ 0.66
3.96 ꢀ 0.65
5.15 ꢀ 0.66
4.61 ꢀ 0.66
5.67 ꢀ 0.66
5.13 ꢀ 0.66
5.31 ꢀ 0.67
2.34 ꢀ 0.91
1.80 ꢀ 0.90
2.99 ꢀ 0.91
2.45 ꢀ 0.90
3.51 ꢀ 0.92
2.97 ꢀ 0.91
5.44 ꢀ 0.92
4.08 ꢀ 0.89
1.80 ꢀ 0.90
5.25 ꢀ 0.90
2.97 ꢀ 0.91
4.74 ꢀ 0.90
2.45 ꢀ 0.90
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
4
4
4
4
4
5
6
6
6
6
6
6
5
4
6
4
6
6
6
Y
Y
N
Y
N
N
N
Y
Y
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
4.1.2.5. (E)-N-hydroxy-1-(thiophen-2-yl)methanimine (V). White;
mp 98e101 ꢄC; Anal. Calc. For C5H5NOS: C 47.23, H 3.96, N 11.01%.
found: C 47.12, H 4.15, N 10.89%; IR nmax cmꢁ1: 3256 (NOeH), 3064
(CeH), 1622 (C]N), 942 (NeO stretch); 1H NMR (DMSO-d6)
d(ppm): 11.05 (broad s, 1H, NeOH), 7.80 (s, 1H, CH]NeOH),
9
7.10e7.00 (dd, 2H, Thp-H, J ¼ 7.8, 8.0 Hz), 6.80 (d, 1H, Thp-H,
10
11
12
13
14
15
16
17
18
19
20
J ¼ 5.4 Hz); 13C NMR (DMSO-d6)
d(ppm): 151.8 (C]N), 145.8,
129.5, 126.0, 124.9; ESI-MS m/z: [Mþþ1] 128.03.
4.2. General procedure for the synthesis of dioxazoles (1e20)
A 13% aqueous solution of NaOCl (1.6 equiv.) was added to
a solution of aldehydes (1 equiv.) and triethylamine (0.1 equiv.) in
ethylacetate under argon atmosphere. The oxime (1 equiv.) in
ethylacetate was added dropwise (over a period of 1 h) at 0 ꢄC to the
above solution and stirred at room temperature for 12e15 h and
refluxed for additional 10e12 h, depending upon the reaction time
(monitored by TLC). The reaction mixture was cooled to room
temperature and water was added to it. The organic layer was
separated and the aqueous layer was extracted with ethylacetate.
The combined organic layers were washed with water, brine, dried
over NaSO4, filtered and concentrated under vacuo. The compounds
were crystallized using dichloromethaneehexane solution.
Y. Yes, N. No (Rule of 5 criteria met or not).
4.1.2.1. (1E,2E)-N-hydroxy-3,7-dimethylocta-2,6-dien-1-imine (I). Oil;
Anal. Calc. For C10H17NO: C 71.81, H 10.25, N 8.37%; found: C 71.79, H
10.26, N 8.39%; IR nmax cmꢁ1: 3250 (NOeH), 2864 (CeH), 1632 (C]
N), 936 (NeO stretch); 1H NMR (DMSO-d6)
d(ppm): 9.43 (broad s,
1H, NeOH), 7.32 (s, 1H, CH]NeOH), 5.90 (d, 1H,]CHeCH]N,
J ¼ 11.7 Hz), 5.08 (s, 1H,]CHeCH2), 2.30e2.03 (m, 4H, CH2eCH2),
1.83 (s, 6H, CH3), 1.68 (s, 3H, CH3); 13C NMR (DMSO-d6)
d(ppm):
154.58 (C]N), 151.2, 132.2, 125.6, 122.4, 43.5, 28.0, 26.2, 20.5, 18.9;
4.2.1. 2-(5-methyl-3-((E)-2,6-dimethylhepta-1,5-dienyl)-1,4,2-dio-
xazole-5-yl) pyridine (1)
ESI-MS m/z: [Mþþ1] 167.13.
Cream; mp 183e185 ꢄC; Anal. Calc. For C17H22N2O2: C 71.30, H
7.74, N 9.78%; found: C 71.27, H 7.70, N 9.79%; UV lmax nm: 352, 311,
250; IR nmax cmꢁ1: 3054 (CeH stretch py), 1652 (C]N), 1645 (C]C
4.1.2.2. (E)-1-(1,3-benzodioxol-5-yl)-N-hydroxymethanimine (II). -
White; mp 100e105 ꢄC; Anal. Calc. For C8H7NO3: C 58.18, H 4.27, N
8.48%. found: C 58.34, H 4.36, N 8.49%; IR nmax cmꢁ1: 3245 (NOeH),
2921 (CeH), 1635 (C]N), 937 (NeO stretch); 1H NMR (DMSO-d6)
stretch), 1615 (C]N, py); 1H NMR (DMSO-d6)
d(ppm): 8.60 (d, 1H,
py-H, J ¼ 7.4 Hz), 8.40 (d, 1H, py-H, J ¼ 7.2 Hz), 8.12 (dd, 1H, py-H,
J ¼ 8.4, 8.5 Hz), 7.95 (dd, 1H, py-H, J ¼ 7.8, 8.2 Hz), 5.86 (s,1H,]
CHeC), 5.01 (s, 1H,]CHeCH2), 2.57e2.61 (m, 4H, CH2eCH2), 2.17 (s,
3H, CH3 dioxazole ring), 1.99 (s, 3H, CH3), 1.68 (s, 6H, CH3), 13C NMR
d
(ppm): 10.85 (broad s, 1H, NeOH), 7.90 (s, 1H, CH]NeOH),
7.30e6.50 (m, 3H, AreH), 5.90 (s, 2H, eOeCH2eOe); 13C NMR
(DMSO-d6) d(ppm): 148.10 (C]N), 147.3, 145.0, 126.6, 122.5, 115.0,
112.8, 101.6 (eOeCH2eO); ESI-MS m/z: [Mþþ1] 166.04.
(DMSO-d6) d(ppm):162.4 (C]N), 160.1 (C]N), 154.2, 133.5, 123.7,
121.5, 40.5, 26.7, 25.1, 19.4, 18.1, (Aliphatic chain-C), 147.6, 134.4,
122.8, 120.6, (AreC), 95.1(OeCeO) 26.2 (CeCH3 dioxazole ring);
ESI-MS m/z: [Mþþ1] 287.17.
4.1.2.3. (E)-N-hydroxy-1-(pyridin-2-yl)methanimine (III). Pink; mp
118e120 ꢄC; Anal. Calc. For C6H6N2O: C 59.01, H 4.95, N 22.94. found:
C 58.95, H 4.96, N 23.09%; IR nmax cmꢁ1: 3260 (NOeH), 2920 (CeH),
1634 (C]N), 1630 (C]N), 937 (NeO stretch); 1H NMR (DMSO-d6)
4.2.2. 2-(3-((E)-2,6-dimethylhepta-1,5-dienyl)-1,4,2-dioxazole-5-yl)
pyridine (2)
d
(ppm): 10.76 (broad s,1H, NeOH), 8.60 (d,1H, py-H, J ¼ 7.4 Hz), 8.45
(d, 1H, py-H, J ¼ 7.2 Hz), 8.03e8.11 (dd, 1H, py-H, J ¼ 8.7 Hz),
Oil; Anal. Calc. For C16H20N2O2: C 70.56, H7.40, N 10.29%; found:C
7.90e8.02 (dd, 1H, py-H, J ¼ 8.4 Hz), 6.97 (s, 1H, CH]N-OH); 13C
70.51, H 7.48, N 10.35%; UV lmax nm: 321, 299, 267; IR nmax cmꢁ1
:
NMR (DMSO-d6) d(ppm): 151.60 (C]N), 146.10 (C]N), 145.0, 136.9,
3064 (CeH stretch py),1645 (C]N),1660 (C¼N)1642 (C]C stretch);
125.5, 101.6 (eOeCH2eO); ESI-MS m/z: [Mþþ1] 122.04.
1H NMR (DMSO-d6)
d(ppm): 7.80 (dd, 1H, py-H), 7.71 (d, 1H, py-H,
J ¼ 7.4 Hz), 7.62 (d, 1H, py-H, J ¼ 7.2 Hz),7.26 (dd, 1H, py-H), 6.91 (s,
1H, CeH (dioxazole ring)) 5.08 (s, 2H, eCH]C), 2.33e2.10 (m, 4H,
CH2eCH2), 1.68 (s, 6H, CH3), 1.55 (s, 3H, CH3); 13C NMR (DMSO-d6)
4.1.2.4. (E)-1-(furan-2-yl)-N-hydroxymethanimine (IV). White; mp
112e115 ꢄC; Anal. Calc. For C5H5NO2: C 47.23, H 3.96, N 11.01%.
d(ppm): 155.1 (C]N), 158.5 (C]N py), 154.8, 134.8, 98.2, 38.5, 25.7,
Table 4
24.3, 20.4, 18.7, 18.6, (Aliphatic-C) 149.3, 137.4, 123.9, 121.0, (AreC),
87.5(OeCeO); ESI-MS m/z: [Mþþ1] 273.15.
Synthesis of oximes IeV by methods I & II.
Oximes
Method I
(Yield, %)
Reaction
time (h)
Method II
(Yield, %)
Reaction
time (h)
4.2.3. 5-(furan-2-yl)-5-methyl-3-((E)-2,6-dimethylhepta-1,5-
dienyl)-1,4,2-dioxazole (3)
Oil; Anal. Calc. For C16H21NO3: C 69.79, H 7.69, N 5.09%; found: C
69.76, H 7.70, N 5.11%; UV lmax nm: 332, 298, 276; IR nmax cmꢁ1: 3130
(CeH stretch furan ring), 1632 (C]N) 1641 (C]C stretch); 1H NMR
I
II
III
IV
V
76
80
70
70
72
24
24
24
24
24
92
90
83
80
87
2
3e5
2e3
3
3e4
(DMSO-d6)
d
(ppm): 8.02 (d, 1H furan ring-H, J ¼ 5.6 Hz), 6.52e6.45

4750
M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
(m 2H furan ring-H), 5.86 (s,1H,]CHeC), 5.07 (s, 1H,]CHeCH2),
2.61e2.57 (m, 4H, CH2eCH2), 2.54 (s, 3H, CH3 dioxazole ring),1.68 (s,
1650 (C]N py); 1H NMR (DMSO-d6)
d(ppm): 7.83 (d, 1H, py-H,
J ¼ 7.2 Hz), 7.71 (d, 1H, py-H, J ¼ 7.2 Hz), 7.60 (dd, 1H, py-H,
J ¼ 7.8, 8.2 Hz), 7.34 (dd, 1H, py-H, J ¼ 8.2, 8.6 Hz), 6.92e6.85 (m,
3H, AreH), 6.07 (s, 2H, eOeCH2eO), 1.90 (s, 3H, CH3 dioxazole
3H, CH3),1.53 (s, 6H, CH3),13C NMR (DMSO-d6)
d(ppm): 159.6(C]N),
152.2, 134.8, 124.3, 114.5, 41.2, 28.7, 26.6, 19.5, 18.3 (Aliphatic chain-
C) 155.6, 140.2, 110.5, 106.4 (AreC) 96.8 (OeCeO), 25.5 (CeCH3
dioxazole ring); ESI-MS m/z: [Mþþ1] 276.15.
ring); 13C NMR (DMSO-d6)
d(ppm): 167.5 (C]N), 162.5 (C]N py),
150.6, 148.7, 146.2, 145.5, 123.4, 122.4, 121.2, 120.0, 115.7, 113.0,
(AreC), 101.6 (eOeCH2eO), 94.8 (OeCeO), 27.8 (CeCH3 dioxazole
ring); ESI-MS m/z: [Mþþ1] 285.10.
4.2.4. 5-(furan-2-yl)-3-((E)-2, 6-dimethylhepta-1,5-dienyl)-1,4,2-
dioxazole (4)
Oil; Anal. Calc. For C15H19NO3: C 68.94, H 7.33, N 5.36%; found: C
69.14, H 7.28, N 5.37%; UV lmax nm: 336, 312, 286; IR nmax cmꢁ1: 3030
(CeH stretch furan ring), 1648 (C]N), 1643 (C]C stretch); 1H NMR
4.2.9. 2-(3-(benzo[d] [1,3]dioxol-5-yl)-1,4,2-dioxazol-5-yl)
pyridine (9)
Cream; mp 130e134 ꢄC; Anal. calc. for C14H10N2O4: C 62.22, H
3.73, N 10.37%; found: C 63.01, H 3.83, N 10.26%; UV lmax nm: 346,
307, 255; IR nmax cmꢁ1: 3065 (CeH), 3025 (AreH), 1656 (C]N),
(DMSO-d6)
d
(ppm): 8.08 (d,1H furan ring-H, J ¼ 5.2 Hz), 6.52 (dd 1H
furan ring-H, J ¼ 5.6, 5.6 Hz), 6.16 (dd 1H furan ring-H, J ¼ 5.8, 5.4 Hz),
5.90 (s,1 H, CeH (dioxazole ring), 5.08 (s, 2H,]CHeCH2), 2.25e2.05
(m, 4H, CH2-CH2), 1.83 (s, 3H, CH3) 1.50 (s, 6H, CH3); 13C NMR
1643 (C]N py); 1H NMR (DMSO-d6)
d(ppm):): 8.81 (d, 1H, py-H,
J ¼ 7.6 Hz), 8.33 (d, 1H, py-H, J ¼ 7.5 Hz), 8.02 (dd, 1H, py-H,
J ¼ 8.6, 8.4 Hz), 7.71 (dd, 1H, py-H, J ¼ 8.2, 8.4 Hz), 6.91e7.59 (m,
3H, AreH), 6.52 (s, 1H, CeH (dioxazole ring), 5.94 (s, 2H,
(DMSO-d6) d(ppm): 168.4 (C]N), 162.4 (C]N) 122.6, 154.3, 137.8,
40.7, 28.1, 25.3, 20.5,17.8 (Aliphatic chain-C) 151.6,138.4,111.7,106.7,
(AreC)), 90.7(OeCeO); ESI-MS m/z: [Mþþ1] 262.14.
eOeCH2eOe); 13C NMR (DMSO-d6)
d(ppm): 162.4 (C]N), 159.8
(C]N py), 152.5, 150.2, 148.4, 146.5, 136.4, 124.5, 123.0, 122.1, 114.7,
112.5 (AreC), 103.5 (eOeCH2eO), 87.5 (OeCeO); ESI-MS m/z:
[Mþþ1] 271.08.
4.2.5. 5-methyl-3-((E)-2, 6-dimethylhepta-1, 5-dienyl)-5-(thioph-
enyl-2-yl)-1,4,2-dioxazole (5)
Oil; Anal. Calc. For C16H21NO2S: C 65.95, H 7.26, N 4.81%; found:
C 65.84, H 7.28, N 4.87%; UV lmax nm: 344, 304, 281; IR nmax cmꢁ1
:
4.2.10. 3-(benzo[d][1,3]dioxol-5-yl)-5-(furan-2-yl)-5-methyl-1,4,2-
dioxazole (10)
3125 (CeH stretch thiophene ring), 1652 (C]N),1640 (C]C
stretch); 1H NMR (DMSO-d6)
d
(ppm): 7.83 (dd 1H Thp-H, J ¼ 8.6,
Cream; mp 150e155 ꢄC; Anal. calc. for C14H11NO5: C 61.54, H
4.06, N 5.13%; found: C 61.63, H 3.93, N 5.23%; UV lmax nm: 338,
306, 275; IR nmax cmꢁ1: 3072 (AreH), 3045 (CeH), 1650 (C]N); 1H
8.4 Hz), 7.74 (d, 1H Thp-H, J ¼ 7.6 Hz), 7.13 (d, 1H Thp-H, J ¼ 7.6 Hz),
6.08 (s, 1H,]CHeC), 5.08 (s, 1H,]CHeCH2), 2.54 (s, 3H, CH3 diox-
azole ring), 2.27e2.13 (m, 4H, CH2eCH2), 1.38 (s, 6H, CH3), 1.35 (s,
NMR (DMSO-d6)
d
(ppm): 7.83 (d, 1H, furan ring-H, J ¼ 5.6 Hz)
3H, CH3), 13C NMR (DMSO-d6)
d(ppm): 160.9 (C]N), 149.0, 133.0,
7.51e7.31 (m, 3H Ar-H), 7.25 (d, 1H, furan ringeH, J ¼ 5.4 Hz), 7.13
(dd,1H, furan ring-H, J ¼ 7.6, 7.6 Hz), 5.86 (s, 2H, eOeCH2eOe),1.90
123.5, 36.5, 30.1, 21.6, 21.6, 16.9, (Aliphatic chain-C) 153.2, 135.5,
112.0, 105.6, 102.5, (AreC), 95.0 (OeCeO), 26.4 (CeCH3 dioxazole
ring); ESI-MS m/z: [Mþþ1] 292.13.
(s, 3H, dioxazole ring); 13C NMR (DMSO-d6)
d(ppm): 162.4 (C]N),
157.2, 150.4, 148.1, 141.4, 128.5, 123.7, 121.6, 114.5, 110.5, 104.0, 101.5
(AreC), 91.5 (OeCeO), 25.6 (CeCH3 dioxazole ring); ESI-MS m/z:
[Mþþ1] 274.06.
4.2.6. 3-((E)-2,6-dimethylhepta- 1,5-dienyl)- 5-(thiophen-2-yl)-
1,4,2-dioxazole (6)
Oil; Anal. Calc. For C15H19NO2S: C 64.95, H 6.90, N 5.05%; found:
4.2.11. 3-(benzo[d][1,3]dioxol-5-yl)-5-(furan-2-yl)-1,4,2-dioxazole (11)
Brown; mp 153e155 ꢄC; Anal. calc. for C13H9NO5: C 60.24, H
3.50, N 5.40%; found: C 60.58, H 3.42, N 5.33%; UV lmax nm: 354,
312, 265; IR nmax cmꢁ1: 3061 (AreH), 3027 (CeH), 1638 (C]N); 1H
C 65.04, H 6.98, N 5.05%; UV lmax nm: 319, 299, 261; IR nmax cmꢁ1
:
3095 (CeH stretch thiophene ring), 1660 (C]N), 1644 (C]C
stretch), 1H NMR (DMSO-d6)
d
(ppm): 7.74 (dd 1H Thp-H) 7.53 (d, 1H
Thp-H, J ¼ 5.6 Hz), 7.43 (d, 1H Thp-H, J ¼ 5.6 Hz), 6.30 (s, 1H, CeH
(dioxazole ring), 5.90 (s, 1H,]CHeC), 5.10 (s, 1H,]CHeCH2),
NMR (DMSO-d6)
d
(ppm): 7.80 (d, 1H furan ring-H, J ¼ 5.7 Hz),
7.74e7.60 (m, 3H, AreH), 7.26 (dd 1H furan ring-H, J ¼ 5.6, 5.6 Hz),
7.13 (d, 1H furan ring-H, J ¼ 5.6 Hz), 6.14 (s, 2H, eOeCH2eOe) 6.08
2.25e2.13 (m, 4H, CH2eCH2), 1.38 (s, 6H, CH3), 1.35 (s, 3H, CH3), 13
C
NMR (DMSO-d6)
d(ppm): 166.5 (C]N), 153.0, 135.0, 121.2, 114.2,
(s, 1H, CeH (dioxazole ring)); 13C NMR (DMSO-d6)
d(ppm): 162.6
34.5, 32.5, 25.6, 20.5, 17.0; (Aliphatic chain-C) 153.2, 135.5, 105.6,
(C]N), 156.9, 150.4, 148.1, 141.4, 128.5, 123.7, 114.5, 110.5, 108.2,
102.5, (AreC), 89.5 (OeCeO); ESI-MS m/z: [Mþþ1] 278.17.
104.0, 100.2 (AreC) 87.5 (OeCeO); ESI-MS m/z: [Mþþ1] 260.04.
4.2.7. 5-(benzo[d][1,3]dioxol-5-yl)-3-((E)-2,6-dimethylhepta-1,5-
dienyl)-1,4,2-dioxazole (7)
4.2.12. 3-(benzo[d][1,3]dioxol-5-yl)-5-methyl-5-(thiophen-2-yl)-
1,4,2-dioxazole (12)
White; mp 123e125 ꢄC; Anal. Calc. For C18H21NO4: C 68.55, H
6.71, N 4.44%; found: C 68.54, H 6.78, N 4.40%; UV lmax nm: 354,
314, 271; IR nmax cmꢁ1: 1647 (C]N), 1645 (C]C stretch), 1H NMR
Yellowish; mp 120e123 ꢄC; Anal. calc. for C14H11NO4S: C 58.12, H
3.83, N 4.84%; found: C 58.34, H 3.73, N 4.84%; UV lmax nm: 330, 311,
291; IR nmax cmꢁ1: 3100 (AreH), 3065 (CeH), 1656 (C]N); 1H NMR
(DMSO-d6)
d
(ppm): 7.80e7.56 (m, 3H, AreH), 6.96 (s, 1H, CeH
(DMSO-d6)
d
(ppm): 7.80 (dd, 1H, Thp-H, J ¼ 5.8, 5.8 Hz), 7.71e7.56
(dioxazole ring), 6.16 (s, 2H, eOeCH2eOe), 5.43 (s, 1H,]CHeCH2),
4.75 (s, 1H,]CHeC), 2.14e2.03 (m, 4H, CH2eCH2), 1.90 (s, 6H, CH3),
(m, 3H, Ar-H), 7.26 (d, 1H Thp-H, J ¼ 7.8 Hz), 6.91 (d, 1H Thp-H,
J ¼ 7.4 Hz), 6.09 (s, 2H, eOeCH2eOe), 2.19 (s, 3H, dioxazole ring);
1.38 (s, 3H, CH3); 13C NMR (DMSO-d6)
d
(ppm): 162.4 (C]N), 150.5,
13C NMR (DMSO-d6)
d(ppm): 164.4 (C]N),154.6,148.3,147.5,128.4,
134.3, 120.5, 116.0, 34.9, 30.5, 23.5, 22.6, 17.5, (Aliphatic chain-C)
147.5, 145.6, 136.0, 120.5, 114.6, 110.7, 100.5, (AreC) 89.7(OeCeO);
ESI-MS m/z: [Mþþ1] 316.18.
126.8, 125.5, 123. 0, 115.4, 112.2, 108.5, 102.5 (AreC), 92.5 (OeCeO),
28.5 (CeCH3 dioxazole ring); ESI-MS m/z: [Mþþ1] 290.04.
4.2.13. 3-(benzo[d][1,3]dioxol-5-yl)-5-(thiophen-2-yl)-1,4,2-
dioxazole (13)
4.2.8. 2-(3-(benzo[d][1, 3]dioxol-5-yl)-5-methyl-1,4,2-dioxazol-5-yl)
pyridine (8)
Oil; Anal. calc. for C13H9NO4S: C 56.72, H 3.30, N 5.09%; found: C
Cream; mp 140e145 ꢄC; Anal. calc. for C15H12N2O4: C 63.38, H
4.25, N 9.85%; found: C 63.45, H 4.30, N 9.96%; UV lmax nm: 340,
301, 265; IR nmax cmꢁ1: 3054 (AreH), 3020 (CeH), 1660 (C]N),
56.80, H 3.40, N 5.23%; UV lmax nm: 325, 297, 255; IR nmax cmꢁ1
:
3055 (CeH), 3034 (AreH), 1640 (C]N); 1H NMR (DMSO-d6)
(ppm): 7.83 (dd, 1H, Thp-H, J ¼ 5.6, 5.6 Hz), 7.26e7.13 (m, 3H, Ar-
d

M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
4751
H), 6.92 (d, 1H, Thp-H, J ¼ 7.2 Hz), 6.80 (d, 1H, Thp-H, J ¼ 7.4 Hz),
6.04 (s, 1H, CeH (dioxazole ring)), 5.70 (s, 2H, eOeCH2eOe); 13C
2H, eOeCH2eOe), 5.90 (s, 1H, CeH (dioxazole ring)); 13C NMR
(DMSO-d6) (ppm): 167.0 (C]N), 152.0, 147.9, 142.6, 140.4, 135.6,
d
NMR (DMSO-d6)
d
(ppm): 168.1 (C]N), 158.5, 152.6, 147.5, 128.2,
120.8, 117.4, 112.6, 109.5, 108.2, 98.5 (AreC), 96.3 (OeCeO); ESI-
MS m/z: [Mþþ1] 260.05.
126.5, 124.5, 123.6, 121.5, 115.4, 113.5, 109.2, (AreC) 89.0 (OeCeO);
ESI-MS m/z: [Mþþ1] 276.03.
4.2.19. 5-((E)-2, 6-dimethylhepta-1,5-dienyl)-3-(thiophen-2-yl)-1,4,2-
dioxazole (19)
4.2.14. 3-(benzo[d][1,3]dioxol-6-yl)-5-((E)-2,6-dimethylhepta-1,5-
dienyl)-1,4,2-dioxazole (14)
Oil; Anal. calc. for C15H19NO2S: C 64.95, H 6.90, N 5.05%; found: C
Grey; mp 145e147 ꢄC; Anal. calc. for C18H21NO4: C 68.55, H 6.71,
N 4.44%; found: C 68.32, H 6.73, N 4.33%; UV lmax nm: 335, 298,
275; IR nmax cmꢁ1: 3075 (CeH), 3065 (AreH), 1650 (C]N); 1H NMR
64.84, H 7.01, N 5.14%; UV lmax nm: 348, 310, 291; IR nmax cmꢁ1
:
1642 (C]C stretch), 3135 (CeH stretch thiophene ring), 3054
(AreH), 1639 (C]N); 1H NMR (DMSO-d6)
d(ppm): 8.04 (dd, 2H,
(DMSO-d6)
d
(ppm): 7.53e7.13 (m, 3H, Ar-H), 6.04 (s, 2H,
Thp-H, J ¼ 5.6, 5.6 Hz) 7.73 (d,1H Thp-H, J ¼ 7.2 Hz), 6.04 (s,1H, CeH
(dioxazole ring)), 5.86 (s, 1H,]CHeC), 5.07 (s, 1H,]CHeCH2),
eOeCH2eOe) 5.88 (s, 1H,]CHeC), 5.65 (s, 1H, CeH (dioxazole
ring)), 5.10 (s, 1H,]CHeCH2), 2.26e2.13 (m, 4H, CH2eCH2), 1.38 (s,
2.14e2.03 (m, 4H, CH2eCH2), 1.90 (s, 3H, CH3), 1.60 (s, 6H, CH3); 13
C
3H, CH3), 1.35 (s, 6H, CH3); 13C NMR (DMSO-d6)
d(ppm): 162.4 (C]
NMR (DMSO-d6) d(ppm): 164.5 (C]N), 134.9, 131.5, 126.8, 120.4,
N), 135.0, 133.6, 124.5, 121.2, 32.5, 34.5, 25.6, 20.5, 17.0 (Aliphatic
chain-C) 151.5, 146.2, 127.5, 123.5, 113.5, 109.2, 103.2 (AreC) 86.7
(OeCeO); ESI-MS m/z: [Mþþ1] 316.15.
40.5, 36.5, 27.4, 19.5, 17.8 (Aliphatic-C), 130.6, 127.5, 125.4, 123.2,
(AreC), 87.3 (OeCeO); ESI-MS m/z: [Mþþ1] 278.11.
4.2.20. 5-(benzo[d][1,3]dioxol-5-yl)-3-(thiophen-2-yl)-1,4,2-
dioxazole (20)
Oil; Anal. calc. for C13H9NO4S: C 56.70, H 3.30, N 5.09%; found: C
56.84, H 3.47, N 5.14%; UV lmax nm: 318, 295, 250; IR nmax cmꢁ1: 3054
(Ar CeH), 3130 (CeH stretch thiophene ring) 1660 (C]N); 1H NMR
4.2.15. 2-(5-((E)-2, 6-dimethylhepta-1,5-dienyl)-1,4,2-dioxazol-3-yl)
pyridine (15)
Grey; mp 140e143 ꢄC; Anal. calc. for C16H20N2O2: C 70.56, H 7.40,
N 10.29%; found: C 70.64, H 7.33, N 10.24%; UV lmax nm: 321, 303,
263; IR nmax cmꢁ1: 1642 (C]C stretch), 3054 (CeH stretch py), 1650
(DMSO-d6)
d
(ppm): 7.83 (dd, 2H, Thp-H, J ¼ 5.6, 5.8 Hz), 7.13 (d, 1H.
(C]N); 1635 (C]N py); 1H NMR (DMSO-d6)
d
(ppm): 8.08 (d,1H, py-
Thp-H, J ¼ 7.2 Hz), 6.92e6.85 (m, 3H, AreH), 6.02 (s, 2H,
H, J ¼ 5.2 Hz), 7.73 (d,1H, py-H, J ¼ 5.6 Hz), 7.56 (dd,1H, py-H, J ¼ 5.4,
5.4 Hz), 7.26 (dd,1H, py-H, J ¼ 5.6, 5.6 Hz), 6.07 (s,1H, CeH (dioxazole
ring), 5.90 (s,1H,]CHeC), 5.08 (s,1H,]CHeCH2), 2.61e2.29 (m, 4H,
CH2eCH2), 1.99 (s, 3H, CH3), 1.68 (s, 6H, CH3); 13C NMR (DMSO-d6)
eOeCH2eOe), 5.86 (s, 1H, CeH (dioxazole ring)); 13C NMR (DMSO-
d6) d(ppm): 156.7 (C]N),152.6,149.9,134.6,130.6,127.5,125.6,124.0,
122.0, 118.3, 114.6, 99.7, (AreC), 85.9 (OeCeO); ESI-MS m/z: [Mþþ1]
276.03.
d(ppm): 159.8 (C]N),154.5 (C]N py),138.8,134.7,122.8,120.5, 36.9,
34.5, 24.6, 22.8, 17.5, (Aliphatic chain-C), 146.5, 136.9, 127.5, 123.5
4.3. In vitro anti-amoebic assay
(Ar-C) 87.5 (OeCeO); ESI-MS m/z: [Mþþ1] 273.15.
All the compounds 1e20 were screened in vitro for anti-amoebic
activity against HM1:IMSS strain of E. histolytica by microdilution
method [42]. E. histolytica trophozoites were cultured in wells of
96-well microtiter plate by using Diamond TYIS-33 growth
medium [43]. The test compounds (1 mg) were dissolved in DMSO
4.2.16. 2-(5-(benzo[d][1, 3] dioxol-5-yl)-1,4,2-dioxazol-3-yl)
pyridine (16)
Yellowish; mp 135e137 ꢄC; Anal. calc. for C14H10N2O4: C 62.22, H
3.73, N 10.37%; found: C 62.34, H 3.64, N 10.24%; UV lmax nm: 350,
313, 265; IR nmax cmꢁ1: 3075 (CeH), 3054 (AreH), 1659 (C]N);
(40 ml, level at which no inhibition of amoeba occurs) [44,45]. The
1630 (C]N py); 1H NMR (DMSO-d6)
d(ppm): 8.50 (d, 1H, py-H,
stock solutions of the compounds were prepared freshly before use
at a concentration of 1 mg/ml. Two-fold serial dilutions were made
in the wells of 96-well microtiter plate (costar). Each test includes
metronidazole as a standard amoebicidal drug, control wells
(culture medium plus amoebae) and a blank (culture medium
only). All the experiments were carried out in triplicate at each
concentration level and repeated thrice. The amoeba suspension
was prepared from a confluent culture by pouring off the medium
at 37 ꢄC and adding 5 ml of fresh medium, chilling the culture tube
on ice to detach the organisms from the side of the flask. The
number of amoeba/ml was estimated with a haemocytometer,
using trypan blue exclusion to confirm the viability. The suspension
J ¼ 7.6 Hz), 7.83 (d, 1H, py-H, J ¼ 7.4 Hz), 7.74 (dd, 1H, py-H,
J ¼ 8.9, 8.6 Hz), 7.26 (dd, 1H, py-H, J ¼ 8.4, 8.6 Hz), 6.92e6.85 (m,
3H, AreH), 6.04 (s, 1H, CeH (dioxazole ring)), 5.88 (s, 2H,
eOeCH2eOe); 13C NMR (DMSO-d6)
d(ppm): 160.5 (C]N), 156.9
(C]N), 150.4, 148.5, 147.2, 134.6, 128.5, 116.8, 126.6, 122.5, 112.9,
108.7, 102.5, (AreC), 93.7 (OeCeO); ESI-MS m/z: [Mþþ1] 271.06.
4.2.17. 3-(furan-2-yl)-5-((E)-2, 6-dimethylhepta-1, 5-dienyl)-1,4,2-
dioxazole (17)
Oil; Anal. calc. for C15H19NO3: C 68.94, H 7.33, N 5.36%; found: C
69.08, H 7.31, N 5.34%; UV lmax nm: 322, 296, 273; IR nmax cmꢁ1
:
1642 (C]C stretch), 3125 (CeH), 1643 (C]N); 1H NMR (DMSO-d6)
was diluted to 105 organism/ml by adding fresh medium and 170
of this suspension was added to the test and control wells in the
plate so that the wells were completely filled (total volume, 340 l).
ml
d
(ppm): 8.81 (d, 1H furan ring-H, J ¼ 5.6 Hz), 6.96 (dd, 2H furan ring
eH, J ¼ 5.8, 5.6 Hz) 5.86 (s, 1H, CeH (dioxazole ring)); 5.45 (s, 1H,]
CHeC), 5.10 (s, 1H,]CHeCH2), 2.61e2.54 (m, 4H, CH2eCH2), 1.99 (s,
m
An inoculum of 1.7 ꢅ 104 organisms/well was chosen so that
confluent, but not excessive growth, took place in control wells.
Plates were sealed and gassed for 10 min with nitrogen before
incubation at 37 ꢄC for 72 h. After incubation, the growth of amoeba
in the plate was checked with a low power microscope. The culture
medium was removed by inverting the plate and shaking gently.
Plate was then immediately washed with sodium chloride solution
(0.9%) at 37 ꢄC. This procedure was completed quickly and the plate
was not allowed to cool in order to prevent the detachment of
amoebae. The plate was allowed to dry at room temperature and
the amoebae were fixed with methanol and when dried, stained
with (0.5%) aqueous eosin for 15 min. The stained plate was washed
3H, CH3), 1.60 (s, 6H, CH3), 13C NMR (DMSO-d6)
d(ppm): 162.4 (C]
N), 138.4, 134.7, 125.5, 120.5, 36.9, 34.5, 24.6, 22.8, 17.5 (Aliphatic
chain-C), 140.5, 138.4, 109.1, 105.6, (AreC), 96.3 (OeCeO); ESI-
MS m/z: [Mþþ1] 262.14.
4.2.18. 5-(benzo[d][1,3]dioxol-5-yl)-3-(furan-2-yl)-1,4,2-dioxazole(18)
Brown; mp 145e147 ꢄC; Anal. calc. for C13H9NO5: C 60.24, H
3.50, N 5.40%; found: C 60.14, H 3.51, N 5.44%; UV lmax nm: 327, 303,
278; IR nmax cmꢁ1: 3065 (AreH), 3030 (C-H), 1660 (C]N); 1H NMR
(DMSO-d6)
d
(ppm): 7.99 (d, 1H furan ring-H, J ¼ 7.2 Hz), 7.26e6.98
(m, 3H, AreH), 6.55 (dd, 2H, furan ring-H, J ¼ 7.8, 7.8 Hz), 5.94 (s,

4752
M. Younus Wani et al. / European Journal of Medicinal Chemistry 46 (2011) 4742e4752
[5] M. Gomez, D.G. Perez, P. Ayala, J. Samuleson, E. Orozco, Arch. Med. Res. 27
(1996) 421e425.
[6] F.E. Pittman, J.C. Pittman, Am. J. Trop. Med. Hyg. 23 (1974) 146e150.
[7] R. Paduch, M. Kandefer-Szerszen, M. Trytek, J. Fiedurek, Arch. Immunol. Ther.
Exp. 55 (2007) 315e327.
once with tap water, then twice with distilled water and allowed to
dry. A 200 l portion of 0.1 N sodium hydroxide solution was added
m
to each well to dissolve the protein and release the dye. The optical
density of the resulting solution in each well was determined at
490 nm with a microplate reader. The % inhibition 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 line from which the IC50 value was found. The IC50 values in
[8] E. Breitmaier, Terpenes: Flavors, Fragrances, Pharmaca, Pheromones.
Wiley-VCH, Weinheim, Germany, 2006.
[9] K.A. Hammer, C.F. Carson, T.V. Riley, J. Appl. Microbiol. 95 (2003) 853e860.
[10] M. Friedman, P.R. Henika, R.E. Mandrell, J. Food Prot. 65 (2002) 1545e1560.
[11] N. Theis, M. Lerdau, Int. J. Plant Sci. 164 (2003) S93eS103.
[12] P.L. Crowell, R.R. Chang, Z.B. Ren, C.E. Elson, M.N. Gould, Biol. Chem. 266
(1991) 17679e17685.
mM are reported in Table 1.
[13] D.A. Wiseman, S.R. Werner, P.L. Crowell, J. Pharmacol. Exp. Ther. 320 (2007)
1163e1170.
[14] Y.D. Burke, A.S. Ayoubi, S.R. Werner, B.C. McFarland, D.K. Heilman, A. Ruggeri,
P.L. Crowell, Anticancer. Res. 22 (2002) 3127e3134.
4.4. Cytotoxicity studies (MTT assay)
[15] T.J. Raphael, G. Kuttan, Immunopharmacol. Immunotoxicol. 25 (2003) 285e294.
[16] S.A. Look, W. Fenical, G.K. Matsumoto, J. Clardy, J. Org. Chem. 51 (1986)
5140e5145.
[17] A.T. Peana, P.S. D’Aquila, M.L. Chessa, M.D. Moretti, G. Serra, Eur. J. Pharmacol.
460 (2003) 37e41.
H9c2 rat cardiac myoblasts were cultured and maintained as
monolayer in Dulbecco’s modified Eagle’s medium (DMEM), high
glucose, supplemented with 10% fetal bovine serum (heat inacti-
[18] A.T. Peana, P.S. D’Aquila, F. Panin, G. Serra, P. Pippia, M.D. Moretti, Phytome-
dicine 9 (2002) 721e726.
[19] N.H. Georgopapadakou, J.W. Thomas, Antimicrob. Agents Chemother. 40
(1996) 279e291.
[20] A.C.U. Uys, S.F. Malan, S. Dyk, R.L. Zyl, ChemInform 33 (2002) 180.
[21] H. Miyaoka, M. Shimomura, H. Kimura, Y. Yamada, H. Kim, Y. Wataya,
Tetrahedron 54 (1998) 13467e13474.
[22] K.S. Moore, S. Wehrli, H. Roder, M. Rogers, J.N. Forrest, D. McCrimmon,
M. Zasloff, Proc. Nat. Acad. Sci. U.S.A. 90 (1993) 1354e1358.
[23] A.R. Bhat, F. Athar, A. Azam, Eur. J. Med. Chem. 44 (2009) 926e936.
[24] P.F. Iqbal, H. Parveen, A.R. Bhat, F. Hayat, A. Azam, Eur. J. Med. Chem. 44 (2009)
4747e4751.
vated), 100 units/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/
ml amphotericin B, at 37 ꢄC in humidified incubator with 5% CO2
[46]. Only viable cells were used in the assay. Exponentially
growing cells were plated at 1.2 ꢅ 104 cells per well into 96-well
plates and incubated for 48 h before the addition of drugs to ach-
ieve the maximum confluency of the cells. Stock solutions were
prepared by dissolving the compounds in 20% (v/v) DMSO and
further diluted with fresh complete medium to achieve 1 M
concentration. The growth-inhibitory effects of the compounds
were measured using standard tetrazolium MTT assay. Cells were
incubated with different concentrations of metronidazole and
compounds 8, 9, 10, 11, 16 & 18 for 48 h at 37 ꢄC in 5% CO2
humidified incubator together with untreated control sample. At
appropriate time points, cells were washed in PBS, treated with
[25] I. Irfan, N. Sawangjaroen, A.R. Bhat, A. Azam, Eur. J. Med. Chem. 45 (2010)
1648e1653.
[26] T.J. Hou, X.J. Xu, Curr. Pharm. Des. 10 (2004) 1011e1033.
[27] H. De Waterbeemd, E. Gifford, Nat. Rev. Drug Discov. 2 (2003) 192e204.
[28] T. Hou, J. Wang, W. Zhang, X. Xu, J. Chem. Inf. Model. 47 (2007) 460e463.
[29] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 23
(1997) 3e25.
[30] J.H. Poupaert, P. Carato, E. Colacino, Curr. Med. Chem. 12 (2005) 877e885.
[31] C. Foulon, C. Danel, C. Vaccher, S. Yous, J.P. Bonte, J.F. Goossens, J. Chromatogr.
A 1035 (2004) 131e136.
50
4 h at 37 ꢄC. At the end of the incubation period, the medium was
removed and pure DMSO 100 L was added to each well. The
mL MTT solution (5 mg/ml, tetrazolium salt) and incubated for
m
[32] M. Andrasi, P. Buglyo, L. Zekany, A. Gaspar, Pharm. Biomed. Anal. 44 (2007)
1040e1047..
metabolized MTT product dissolved in DMSO was quantified by
measuring the absorbance at 570 nm on an ELISA plate reader
(Labsystems Multiskan RC, Helsinki, Finland) with a reference
wavelength of 655 nm. All assays were performed in triplicate and
repeated thrice. Percent viability was defined as the relative
absorbance of treated versus untreated control cells.
[33] A. Avdeef, Curr. Top. Med. Chem. 1 (2001) 277e351.
[34] T.I. Oprea, Comput. J. Aided Mol. Des 16 (2002) 325e334.
[35] U. Norinder, A.S. Bergstr, Chem. Med. Chem. 1 (2006) 920e934.
[36] G.T. Balogh, B. Gyarmati, B. Nagy, L. Molnar, G.M. Keseru, Qsar Comb. Sci. 28
(2009) 1148e1155.
[37] G. Subhash, M.W. Julian, R.L. Christopher, A.M. Gary, C.L. Jared, S.T. Zev,
M.F. Ryan, C.L. Timothy, B. Iris, K. Howard, L.C. Simon, G.K. Robert, K. Seiki,
N. Tomoyoshi, O. Eric, J. Med. Chem. 47 (2004) 175e187.
[38] C. Hansch, T. Fujita, J. Am. Chem. Soc. 86 (1964) 1616e1625.
[39] D.F. Veber, S.R. Johnson, H.Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kapple,
J. Med. Chem. 45 (2002) 2615e2623.
[40] H.H.F. Refsgaard, B.F. Jensen, P.B. Brockhoff, S.B. Padkjaer, M. Guldbrandt,
M.S. Christensen, J. Med. Chem. 48 (2005) 805e811.
[41] I. Muegge, Med. Res. Rev. 23 (2003) 302e321.
[42] C.W. Wright, M.J. O’Neill, J.D. Phillipson, D.C. Warhurst, Antimicrob. Agents
Chemother. 32 (1988) 1725e1729.
Acknowledgements
This work was supported by Department of Science and Tech-
nology (Grant no. SR/FT/LS-069/2007). The authors are thankful to
Higher Education Department, Jammu and Kashmir Government
for their co-operation.
[43] L.S. Diamond, D.R. Harlow, C. Cunnick, Trans. Soc. Trop. Med. Hyg. 72 (1978)
431e432.
[44] F.D. Gillin, D.S. Reiner, M. Suffness, Antimicrob. Agents Chemother. 22 (1982)
342e345.
[45] A.T. Keene, A. Harris, J.D. Phillipson, D.C. Warhurst, Planta. Med. 52 (1986)
278e285.
[46] M.K. Gupta, T.V. Neelakantan, M. Sanghamitra, R.K. Tyagi, A. Dinda, S. Mualik,
References
[1] A. Lichtenstein, A.T. Kondo, G.S. Visvesvara, A. Fernandez, E.F. Paiva, T. Mauad,
M. Dolhnikoff, M.A. Martins, Thorax 60 (2005) 350e352.
[2] P. Upcroft, J.A. Upcroft, Clin. Microbiol. Rev. 14 (2001) 150e164.
[3] F.A. el-Nahas, M.I. el-Ashmawy, Basic Clin. Pharmacol. Toxicol. 94 (2004)
226e231.
C.K. Mukhopadhyay, S.K. Goswami, Antioxi. Redox Signal.
8 (2006)
1081e1093.
[4] V. Purohit, K.A. Basu, Chem. Res. Toxicol. 13 (2000) 673e692.