3-(5-)-Amino-o-diarylisoxazoles: Regioselective synthesis and antitubulin activity

Article abstract of DOI:10.1016/j.ejmech.2013.12.006

A regioselective synthesis of both 5-amino- and 3-aminodiarylisoxazoles substituted with polyalkoxyaryl pharmacophores has been validated. Starting materials for the synthetic scheme were easily available from plant extracts. The targeted molecules were f

Full text of DOI:10.1016/j.ejmech.2013.12.006

European Journal of Medicinal Chemistry 73 (2014) 112e125
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
3-(5-)-Amino-o-diarylisoxazoles: Regioselective synthesis and
antitubulin activity
Dmitry V. Tsyganova, Victor N. Khrustalev b, Leonid D. Konyushkin a,
,
Mikhail M. Raihstat a c, Sergei I. Firgang a, Roman V. Semenov a, Alex S. Kiselyov d,
,
,
*
Marina N. Semenova c e, Victor V. Semenov a
a N. D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation
b A. N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilov Street, 119991 Moscow, Russian Federation
c Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus
d Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology, Institutsky Per. 9, Dolgoprudny, 141700 Moscow Region,
Russian Federation
e N. K. Kol’tsov Institute of Developmental Biology RAS, 26 Vavilov Street, 119334 Moscow, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
A regioselective synthesis of both 5-amino- and 3-aminodiarylisoxazoles substituted with polyalkoxyaryl
pharmacophores has been validated. Starting materials for the synthetic scheme were easily available
from plant extracts. The targeted molecules were further tested in the phenotypic sea urchin embryo
assay to identify compounds with antimitotic microtubule destabilizing activity. Structureeactivity
relationship studies suggested that the structural features essential for potent antiproliferative activity
include: 1) 5-aminoisoxazole bridge linking biaryl substituents (rings A and B); 2) unsubstituted 5-amino
group; 3) 3,4,5-methoxy substituted benzene and 4-methoxy benzene pharmacophores as rings A and B,
respectively. The most potent compounds also showed strong in vitro cytotoxicity in NCI60 anticancer
drug screen against a panel of 60 human cancer cell lines, including multi-drug resistant cells.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 19 May 2013
Received in revised form
3 December 2013
Accepted 5 December 2013
Available online 16 December 2013
Keywords:
Diarylaminoisoxazoles
Combretastatin
Plant polyalkoxybenzenes
Microtubule destabilizing agents
Sea urchin embryo
Cytotoxicity
1. Introduction
Natural products of combretastatin family, namely com-
bretastatins A-2 and A-4 (Fig. 1; CA2, CA4) are reported antimitotic
Many anticancer drugs selectively target microtubules respon-
sible for the formation of the mitotic spindle and required for
proper chromosomal separation during cell division. It is generally
agreed that agents affecting tubulin polymerization impair micro-
tubule dynamics and structure, and consequently arrest cell cycle in
mitosis [1e4].
compounds found in the bark of Combretum caffrum Kuntze
(Combretaceae) [5]. It is generally recognized that these agents
bind to the colchicine binding site of tubulin and affect its assembly
into microtubules [6,7]. Several phosphorylated prodrugs including
CA4 disodium phosphate (CA4P, Zybrestat) and combretastatin A-1
phosphate (Oxi4503) are being evaluated in the late stages of
clinical trials. In addition to affecting cancer cell division, they have
been found to disrupt tumor microvasculature [3,8e10].
However, despite of their therapeutic promise these agents
display systemic toxicity. A significant effort has been dedicated to
the discovery of synthetic combretastatins featuring potencies
similar to the parent molecules but with better safetyeefficacy
profiles [11,12]. Examples of synthetic modifications of the ring B
with diverse pharmacophores (e.g., OH, NH2, F) are quite abundant
in the literature [11,12]. At the same time, structureeactivity rela-
tionship (SAR) studies of substituents in the ring A have been
conducted to a lesser extent. This is presumably due to the lack of
Abbreviations: CA2, combretastatin A-2; CA4, combretastatin A-4; SAR, struc-
tureeactivity relationship.
* Corresponding author. Tel.: þ7 916 620 9584; fax: þ7 499 137 2966.
E-mail addresses: tsyg@ioc.ac.ru (D.V. Tsyganov), vkh@xray.ineos.ac.ru (V.
N. Khrustalev), LeonidK@chemical-block.com (L.D. Konyushkin), mr@chemical-
block.com (M.M. Raihstat), cbi@chemical-block.com (S.I. Firgang), rs@chemical-
block.com (R.V. Semenov), akiselyov@chemdiv.com (A.S. Kiselyov), ms@chemical-
block.com (M.N. Semenova), vs@zelinsky.ru, vs@chemical-block.com (V.
V. Semenov).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.006

D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
113
synthesized
regioselectively
when
4,4-dimethyl-3-
oxopentanenitrile reacted with hydroxylamine in a weakly basic
aqueous media [54]. It was confirmed that the initial step of this
conversion involved hydroxylamine reaction with carbonyl- or
nitrile-group to afford corresponding 5-amino- or 3-
aminoisoxazoles depending on either nature of substituents or
pH of the reaction medium [54].
Alternative regioselective routes to 5-aminoisoxazoles include
addition of acetonitrile anion to a-chloroximes, condensation of a-
bromoketoximines with cyanide or LiAlH4-mediated rearrange-
ment of 5-alkyl-4-cyanoisoxazoles [51,52,55,56]. To the best of our
knowledge, there is only one reference to the synthesis of 3-amino-
4,5-diarylisoxazoles via the reaction of 4-chlorophenylacetic acid
amidoxime with benzoic acid chlorides [57].
Fig. 1. Structure of combretastatins and their aminoisoxazole derivatives.
appropriate building blocks substituted with more than three
alkoxy groups. It was assumed that similar modification of ring A
with an additional methoxy functionality could yield potent anti-
mitotic agents. Tetraalkoxybenzene moiety is well-represented in
natural products exhibiting antimitotic properties [13e16].
A linker unit in the combretastatin derivatives is essential to
lock cis-configuration of the biaryl pharmacophore confirmed to be
necessary for the efficient interaction of a molecule with the
colchicine binding site of tubulin [17,18]. As a further elaboration,
five-membered heterocycles were previously reviewed to provide a
nonisomerizable and metabolically stable isosteric replacement for
cis-styrene [11,12]. Subsequently a number of novel ortho-diary-
lsubstituted four- and five-membered heterocyclic combretastatin
analogs with cytotoxicity comparable to CA4 have been synthe-
sized: 2-azetidinones [19,20], 4,5-diaryloxazoles [21], isoxasoles
[22,23], diarylthiazoles [24], thiazol-2(3H)-ones [25], imidazol-2-
Considering the potent anticancer activity of diary-
laminoisoxazoles, we developed a synthesis of both 5-amino- and
3-aminodiarylisoxazoles (Fig. 1, I, II) substituted with poly-
alkoxyaryl pharmacophores. The targeted molecules were further
tested in our phenotypic sea urchin embryo assay to identify
antimitotic compounds with microtubule destabilizing activity
[58]. The key phenotypic parameters that were monitored included
(a) cleavage alteration and/or arrest of a fertilized egg suggestive of
the antimitotic activity and (b) blastulae behavior treated with test
articles immediately after hatching. Rapid embryo spinning around
the animalevegetal axis near the bottom of the vessel is an evi-
dence of a microtubule destabilizing activity of a compound (video
illustrations are available at http://www.chemblock.com). The
acquisition of a specific tuberculate morphology of arrested eggs
could be considered as additional indirect proof of the ability of a
molecule to destabilize microtubules [58]. Several compounds with
strong antimitotic antitubulin effects in the sea urchin embryo
assay were further selected for cytotoxicity assessment in the
NCI60 human tumor cell line anticancer drug screen.
ones [26], pyrazolones [27],
b-lactams [28], tetrazoles [29], fu-
ranes [30], 1,2,4- [31,32], and 1,2,3-triazoles [33e38].
Recently, a series of 4,5-(polymethoxy)diaryl-3-aminopyrazole
analogs of CA4 have been prepared via short synthetic sequences
from polyalkoxybenzoic acid esters and respective aromatic aceto-
nitriles [39,40]. Most of the key polyalkoxybenzene intermediates
(benzaldehydes, benzoic acids, benzyl alcohols, aromatic acetoni-
triles) were easily synthesized using polyalkoxyallylbenzenes from
dill and parsley seed extracts [40e42].
2. Results and discussion
2.1. Chemistry
o-Diarylsubstituted isoxazoles were found to be the most potent
antiproliferative agents [22,23,43,44]. Within this class of com-
pounds aminoisoxazoles and their unsubstituted congeners dis-
played similar cytotoxic properties. For instance, cytotoxicity of
some 5-amino-3,4-diaryl derivatives (Fig. 1, I) featuring 3,4,5-
trimethoxyphenyl fragment was comparable with that of the
parent CA4, when tested against five cancer cell lines [45]. These
compounds inhibited purified tubulin polymerization, caused cell
cycle arrest in G2/M phase, and induced apoptosis. Docking studies
showed that 5-amino-3,4-diarylisoxazole overlapped well with
CA4 in the colchicine binding site of tubulin [45].
As published before, 5-amino-3,4-diarylisoxazoles were synthe-
sized by condensation of diarylcyanoketones with hydroxylamine
hydrochlorides in refluxing alcohol [45]. Our attempts to follow the
reported protocol yielded a mixture of regioisomers, specifically 5-
amino-3,4-diaryl- (I) and 3-amino-4,5-diarylisoxazoles (II) (Fig. 1).
Because of a high potency of 5-amino-3,4-diarylisoxazoles (I), we
decided to reproduce the study by Liu et al. [45] with the aim
to compare the activity of regioisomers (I) and (II), and to devise
an approach to the selective synthesis of 3-amino-3,4-diarylisoxa
zoles (II).
The original route based on chlorooximes was not suitable for
the synthesis of polyalkoxyaryl 5-amino-3,4-diarylisoxazoles pri-
marily due to the chlorination of the electron rich benzene ring. In
order to address this issue, we investigated condensation of cya-
noketones with hydroxylamines. In our hands, the reaction of
diarylcyanoketones with hydroxylamine hydrochlorides in reflux-
ing alcohol resulted in two regioisomers, namely 5-amino-3,4-
diarylisoxazoles 1aee, 1hek, 1m, and 1n and 3-amino-4,5-
diarylisoxazoles 2c, 2hej, and 2k (Scheme 1). In most cases, the
ratio of regioisomers 5-amino/3-amino was in the 1:1e1:1.5 range.
Individual compounds were isolated by column chromatography.
Notably, reaction of 5-aminoisoxazoles with Ac2O in pyridine
resulted in diacylisoxazoles 100a, 100b, and 100d. The respective in-
termediate mono-acyl derivatives 10a, 10b, and 10d were not
separated.
Signals of the NH2-group corresponding to regioisomers 1 and 2
were well separated
(d in DMSO was 5.2e5.3 ppm for 3-
aminoisoxazoles and 6.4e6.9 ppm for 5-aminoisoxazoles, respec-
tively). In addition, 3-aminoisomers usually had melting point
values up to 15e20 ꢀC higher than respective 5-amino derivatives.
Structure of 3-aminoisoxazoles were further confirmed by an in-
dependent crystallography data.
It has been reported earlier that the cyclization of b-ketonitriles
with hydroxylamine results in corresponding 5-aminoisoxazoles
[46e53]. In a representative example, 5-amino-3-tert-butylisox-
azole was prepared by reacting 4,4-dimethyl-3-oxopentanenitrile
and hydroxylamine hydrochloride [46]. Notably, an alternative
The molecular structure of 2c was unambiguously determined
by a single crystal X-ray diffraction study. Compound 2c was a 3-
amino-4,5-diarylisoxazole derivative, which crystallized in the
triclinic space group P-1 featuring two independent molecules per
crystallographic unit (Fig. 2). The geometry of these two
regioisomer,
namely
3-amino-5-tert-butylisoxazole,
was

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D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
Scheme 1. Synthesis of mixtures of 5-amino- and 3-aminodiarylisoxazoles by condensation of cyanoketones with hydroxylamines. Reagents and conditions: (a) Refs. [40e42]; (b)
Ref. [40]; (c) NH2OH$2HCleNaOAc, EtOH, reflux, 5 h; (d) PyeAc2O, r.t., 24 h.
independent molecules was very similar. Specifically, the isoxazole
ring was planar with all bond lengths matching conventional OeN,
OeC, CeC, N]C and C]C bonds reported for this heterocyclic
system (Table S1, Supplementary data). Hydrogen bonds and
crystal packing for 2c and other details are presented in Table S2
and Fig. S1 (Supplementary data). Aryl substituents (methox-
yphenyl and 5-methoxy-1,4-benzodioxane) were not coplanar to
the central isoxazole ring. Nitrogen atoms of the amino groups
adopted a trigonal-planar configuration. Notably, the amino groups
were found to be within the planes of the isoxazole ring, and the
methoxy groups were aligned with the benzene planes. Crystallo-
graphic data for 2c have been deposited with the Cambridge
Crystallographic Data Center (CCDC 915647).
We attempted to prepare 3-aminoisoxazoles via the reaction of
phenylacetic acid amidoximes with benzoyl chlorides in pyridine
(Scheme 2). In a representative example, condensation of 4-
chlorophenylacetic acid amidoxime with tolyl chloride according
to the published procedure [57] did afford the anticipated 3-
aminoisoxazole derivative 4l albeit in a moderate yield (43%).
Moreover, similar protocol conducted with amidoxime derivatives
of electron-rich arylacetic acid (ex., CH3O-substituted) 3pes fur-
nished benzyloxadiazoles 5aef, h, and t. Formation of benzylox-
adiazoles 5 from phenylacetic acid aldoximes 3 was unequivocally
confirmed by the high-resolution NMR experiments including
heteronuclear coupling observation: 13C (SF ¼ 125.76), (1He13C)
HSQC and (1He13C)HMBC for 5c and h. Traces of 3-aminoisoxazole

D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
115
Fig. 2. Molecular structure of 3-aminoisoxazole 2c. Two crystallographically independent molecules are presented. The alternative positions of the disordered eCH2eCH2e
fragments are shown by thick dashed lines. Thin dashed lines indicate the intermolecular NeH/O hydrogen bonds.
4h (ca. 10%) were detected in a single instance, namely upon
condensation of 3q with p-methoxybenzoylchloride (Scheme 2).
As a next step, we studied the reaction of equimolar 4-
methoxyphenylacetic acid amidoxime 3p with m-methox-
ybenzoyl chloride to yield the intermediate O-benzoyl derivative 6f
(Scheme 2). Optimization of reaction conditions allowed for the
development of a regioselective synthesis of 3-aminoisoxazoles.
Depending on the reaction time and temperature (20e60 ꢀC),
addition of a 3e4 fold excess of Ac2O afforded both mono- (20f) and
di-N-acetyl (200f) derivatives of 3-aminoisoxazoles in modest yields.
Similar reaction conducted in the refluxed pyridine furnished
oxadiazole 5f (ca 25%) in addition to the products listed above.
Treatment with a two-fold excess of AcCl at room temperature for
1 h improved yields of monoacetyl 3-aminoisoxazoles 20aeh, 20n,
and 20o (50e60%), respective oxadiazoles were not detected in the
reaction mixtures. Structures of N-acyl-3-aminoisoxazoles were
confirmed by high resolution NMR-experiments: (1He1H) NOESY
Aminoisoxazole 1i could be considered as an antitubulin agent as
well, because at 2 M it caused formation of tuberculate eggs,
although it did not induce embryo spinning. Benzyloxadiazoles 5ae
f, h, and t were inactive up to 4 M concentration (data not shown).
m
m
2.2.1. Structureeactivity studies in the sea urchin embryo assay.
Modifications of aminoisoxazole bridge
Comparing the activity of regioisomers showed that 5-
aminoisoxazoles were consistently more potent antimitotic
agents than respective 3-aminoisoxazoles (by ca. 2-fold, compare
1c and 2c, 1h and 2h, 1i and 2i). For 3-aminoisoxazoles, N-acety-
lation yielded inactive molecules (compare 2c and 20c; 2h and 20h).
Other N-monoacetyl-3-aminoisoxazoles 20a, 20b, 20deg, 20n, and
20o had no effect on the sea urchin embryo development as well up
to 4 mM concentration. Similarly, N-benzoyl compound 4h did not
cause any developmental abnormalities. Moreover, N,N-diacetyl-5-
aminoisoxazoles were consistently less active than the respective
amino derivatives (compare 1a and 100a, 1b and 100b). For tetra-
substituted 5-aminoisoxazoles, diacetylation increased the anti-
mitotic effect (compare 1d and 100d). It is worth noting that iso-
xazoles 1a and 1b were considerably more active than their
corresponding pyrazole analogs (compounds 12a and 12b, [40]).
13
for 20a and (1He13C)HMBC and C (SF ¼ 125.76) for 20h. Longer
reaction time (5e15 h) led to the extensive formation of side
products and lower yields of the targeted 3-aminoisoxazoles. For
the derivative 6d, we isolated intermediate product, namely N-
acyl-O-benzoylamidoxime 7d, which was successfully converted to
the targeted 3-acylaminoisoxazole 20d upon heating in pyridine in a
60% yield.
2.2.2. Substitutions in the rings A and B
In the 5-aminoisoxazoles series with p-methoxy ring B, the ef-
fect on sea urchin embryos in relation to the structure of ring A
decreased in the following order: 3,4-methylenedioxy-5-methoxy
phenyl (1b) > 3,4,5-trimetoxyphenyl (1a) z 3,4-ethylenedioxy-5-
methoxyphenyl (1c) > 2,3-dimethoxy-4,5-methylenedioxyphenyl
(1e) > 2,5-dimethoxy-3,4-methylenedioxyphenyl (1d). These data
suggested that tetraalkoxy substitution in the ring A reduced the
antimitotic effect of a molecule. Similarly, activity of compounds
endowed with tetraalkoxysubstituted ring B and p-methoxy ring A
(1j, 1k, 2j, and 2k) was markedly reduced.
Pharmacophore ‘swapping’ (exchange of rings A and B) in the
regioisomers of 5-aminoisoxazoles yielded mixed results. For
example, the antimitotic effects of compounds 1a vs 1h and 1d vs 1j
were comparable. In other cases, molecules with monomethoxy
phenyl substituent for the ring A were considerably less active
(compare 1b and 1i, 1e and 1k).
2.2. Antimitotic microtubule destabilizing activity in the phenotypic
sea urchin embryo assay
The antimitotic and microtubule destabilizing effects of syn-
thesized aminoisoxazoles were investigated using the phenotypic
sea urchin embryo assay [58]. The results are presented in Table 1.
CA2 and CA4 served as reference compounds.
Table 1 illustrates that 5-aminoisoxazoles 1aec, 1e, 1h, 1m, 100a,
100b, and 100d and 3-aminoisoxazoles 2c and 2h exhibited well-
defined cleavage alteration, cleavage arrest, and embryo spinning
with EC values ranging from 0.0005 to 0.5 mM, providing evidence
for their antimitotic microtubule destabilizing activity. The data
correlated well with the literature, namely compounds 1a and 100a
inhibited purified tubulin polymerization, and 100a also caused G2/
M arrest typical for tubulin targeting agents [45]. Among 5-amino
derivatives, molecules 1a, 1c, and 1h exhibited activity similar to
that of CA2 and CA4 in the sea urchin embryo assay, whereas
compound 1b was determined to be even more potent. 5-
In summary, structureeactivity relationship studies further
suggested that the structural features essential for yielding potent
antimitotic compounds with microtubule destabilizing properties

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D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
Scheme 2. Regioselective synthesis of 3-aminoisoxazoles. Reagents and conditions: a) EtOHeH2O, reflux, 5 h; b) Py, reflux, 1 h; c) CH3CN, CDI, r.t., 8 h; d) AcCl, Py, r.t., 1.5 h; e) Ac2O,
Py, 60 ꢀC, 24 h.
include: 1) 5-aminoisoxazole bridge linking biaryl substituents
(rings A and B); 2) unsubstituted 5-amino group; 3) 3,4,5-methoxy
substituted benzene and 4-methoxy benzene pharmacophores as
rings A and B, respectively.
number of cancer cell lines with GI50 < 10 nM. In addition, mole-
cules 1a and 1c inhibited cell growth of COLO 205, HT-29 (both
colon cancer), and 786-0 (renal cancer) significantly more than CA4
(Table S3, Supplementary data).
Generally, the most potent compounds identified in the sea
urchin embryo assay (1aec) showed strong cytotoxicity against
human cancer cells with GI50 in a low nanomolar concentration
range. Interestingly, all molecules tested in NCI60 screen were more
potent against P-glycoprotein-overexpressing multi-drug resistant
cell line NCI/ADR-RES than against the parent ovarian cancer cell
line OVCAR-8 (Table 2).
2.3. In vitro cancer cell growth inhibition
Compounds 1aec, 1e, 1i, and 2i were further selected for eval-
uation in the NCI-60 human tumor cell line anticancer drug screen.
The results are presented in Table 2 and Table S3 (Supplementary
data).
Non-small cell lung carcinoma (NCI-H522), leukemia (SR),
melanoma (MDA-MB-435), and colon carcinomas were the most
sensitive cell lines towards 5-aminoisoxazoles 1aec (Table S3,
Supplementary data). These compounds affected growth of a
It is worth noting that methylenedioxy-methoxy derivative 1b
showed higher activity than the respective thrimethoxyphenyl
analog 1a (ca. 4-fold) in the sea urchin embryo assay, whereas the
cytotoxicity data were reversed, namely, 1a was more active than

D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
117
Table 1
Effects of 3- and 5-aminoisoxazoles on sea urchin embryo.
Compd
Bridge
R1
R2
R3
R4
R5
R6
R7
R8
R9
Sea urchin embryo effects, EC (
Cleavage alteration Cleavage arrest Embryo spinning
m
M)a
CA2
CA4
0.002
0.002
0.01
0.01
0.05
0.05
1a
H
H
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
H
H
H
H
OCH3
OCH3
H
H
H
H
0.002
0.005
0.5
100a
0.05
0.2
1
20a
200a
1b
H
H
H
H
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
>4
>4
>4
NDb
NDb
0.002
0.1
NDb
0.05
0.5
eOCH2Oe
eOCH2Oe
eOCH2Oe
OCH3
OCH3
0.0005
0.02
100b
20b
1c
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
>4
>4
>4
0.1
0.5
>4
>5
eOCH2CH2O
e
0.002
0.005
>4
0.01
0.02
>4
2c
eOCH2CH2O
e
20c
1d
eOCH2CH2O
e
OCH3 eOCH2Oe
OCH3 eOCH2Oe
OCH3 eOCH2Oe
2
>5
100d
OCH3
OCH3
H
H
OCH3
H
H
0.5
2
5
20d
1e
H
H
H
H
OCH3
OCH3
H
H
H
H
>4
>4
>4
OCH3 OCH3 eOCH2Oe
OCH3 OCH3 eOCH2Oe
0.02
0.2
2
20e
20f
H
H
H
H
OCH3
OCH3
H
H
H
H
>4
>4
>4
>4
>4
>4
H
H
OCH3
OCH3
H
H
H
H
200f
H
H
H
OCH3
OCH3
H
H
H
NDb
NDb
NDb
20g
1h
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
>4
>4
>4
OCH3 OCH3 OCH3
0.002
0.01
0.1
(continued on next page)

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D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
Table 1 (continued )
Compd
Bridge
R1
R2
R3
R4
R5
R6
R7
R8
R9
Sea urchin embryo effects, EC (
Cleavage alteration Cleavage arrest Embryo spinning
m
M)a
2h
H
H
H
H
OCH3
OCH3
H
H
H
H
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
H
H
0.004
0.02
0.2
20h
>4
>4
>4
4h
1i
H
H
H
H
OCH3
OCH3
H
H
H
H
OCH3 OCH3 OCH3
H
H
>4
>4
>4
>5
OCH3 eOCH2Oe
0.2
2
2i
1j
H
H
H
H
OCH3
OCH3
H
H
H
H
OCH3 eOCH2Oe
OCH3 eOCH2Oe
H
0.2
4
>4
>4
>10
>4
OCH3
2j
H
H
H
H
OCH3
OCH3
H
H
H
OCH3 eOCH2Oe
OCH3 >4
>4
>4
>4
>4
1k
OCH3 OCH3 eOCH2Oe
OCH3 OCH3 eOCH2Oe
H
H
>4
2k
4l
H
H
H
H
OCH3
CH3
H
H
4
>4
>4
H
H
Cl
H
H
NDb
NDb
NDb
1m
1n
H
H
H
H
H
H
H
H
H
OCH3 OCH3
H
H
H
H
0.5
10
10
OCH3
H
Cl
>4
>4
>4
20n
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
Cl
H
H
H
H
>4
>4
>4
>4
>4
>4
20o
NO2
a
The sea urchin embryo assay was conducted as described previously [58]; fertilized eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of
compounds; duplicate measurements showed no differences in effective threshold concentration (EC) values.
b
ND: not determined.
1b. This relationship was also observed for combretastatins (CA4
and CA2) [42] and 4-aza-podophyllotoxins [59]. Whereas this
discrepancy deserves a detailed investigation, it could be attributed
to the fact that cultured cancer cells usually divide every 20e24 h as
compared to a much shorter mitotic cycle (35e45 min) of the sea
urchin blastomeres. As a result, microtubule destabilizing agents
are likely to affect predominantly interphase microtubules in can-
cer cells. Their main target in the sea urchin embryo could be the
mitotic spindle. These two types of microtubules may exhibit
different sensitivity when treated with an antitubulin agent.
It was reported that 5-aminoisoxazoles 1a and 1h [45] and
respective 3-aminopyrazoles [39] displayed comparable cell
growth inhibition against five human cancer cell lines. In the pre-
sent study isoxazoles 1a and 1b were considerably more active than
their pyrazole analogs (12a and 12b, [40]) in the sea urchin embryo
assay (ca. 50-fold). Similarly, in NCI60 screen isoxazole 1a was ca.
15-fold more cytotoxic than pyrazole 12a [40]. In this context it
should be noted that GI50 values of 5-aminoisoxazole 1a against
non-small cell lung cancer cell line A549 and prostate cancer cell
line PC-3 in NCI60 screen were <10 nM (Table S3, Supplementary
data), whereas GI50 values reported previously were 4.49
3.03 M, respectively [45].
mM and
m
3. Conclusions
series of both 5-amino- and 3-aminodiarylisoxazoles
A
substituted with polyalkoxyaryl pharmacophores has been syn-
thesized. Starting materials for our synthetic scheme were easily

D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
119
Table 2
EtOAc/petroleum ether, 1:3e1:5). The yield of targeted mixtures
was 25e60%, with the ratio of the isolated isomers 1:1e1:1.5.
Cell growth inhibition of aminoisoxazoles 1aec, 1e, 1i, and 2i (GI50, nM) in NCI60
screen (NCI Anti-cancer Screening Program; http://dtp.cancer.gov) with CA4 as a
reference compound.
4.1.1.1. 3-(3,4,5-Trimethoxyphenyl)-4-(4-methoxyphenyl)-5-
Compound
NSC
NCI60 screen, cell growth inhibition (GI50
nM)a
,
isoxazolamine (1a). White solid; 13% yield; mp 165e167 ꢀC (lit. [45]
157e159 ꢀC); 1H NMR (CDCl3):
d
3.67 (s, 6H, 2ꢂ OCH3-30,50), 3.82 (s,
Meanb
OVCAR-8c
NCI/ADR-RESd
3H, OCH3-40), 3.85 (s, 3H, OCH3-400), 4.48 (s, 2H, NH2-5), 6.71 (s, 2H,
H-20,60), 6.93 (d, J ¼ 8.7 Hz, 2H, H-300,500), 7.17 (d, J ¼ 8.7 Hz, 2H, H-
CA4
1a
1b
1c
1e
1i
613729
758029
758031
760955
758030
767978
763186
7.68
19.0
55.0
1.00
13.9
58.5
1.00
<10
19.1
200,600); 13C NMR (DMSO-d6):
d
55.52 (CH3, OCH3-400), 55.95 (2CH3,
OCH3-30,50), 60.43 (CH3, OCH3-40), 91.12 (C, C-4), 105.63 (2CH, C-
20,60), 114.44 (2CH, C-300,500), 123.30 (C, C-100), 125.66 (C, C-10), 131.57
(2CH, C-200,600), 138.43 (C, C-40), 153.00 (2C, C-30,50), 158.38 (C, C-400),
160.93 (C, C-3), 167.63 (C, C-5); EIMS m/z 356 [M]þ (27), 341 (13),
195 (100),194 (39),135 (67),134 (50), 92 (15), 77 (34), 44 (37); Anal.
Calcd for C19H20N2O5: C 64.04; H 5.66; N 7.86. Found: C 64.23; H
5.76; N 7.67.
33.0
21.5
<10
389.0
2570
2951
448.0
3620
4260
126.0
563.0
684.0
2i
a
GI50: Concentration required for 50% cell growth inhibition.
Mean GI50 values for 60 human cancer cell lines.
OVCAR-8: ovarian cancer cell line 8.
b
c
d
NCI/ADR-RES: P-glycoprotein-overexpressing multi-drug resistant cell line
derived from OVCAR-8.
4.1.1.2. 3-(7-Methoxy-1,3-benzodioxol-5-yl)-4-(4-methoxyphenyl)-
5-isoxazolamine (1b). White solid; 32% yield; mp 128e130 ꢀC; 1H
NMR (CDCl3): d
3.73 (s, 3H, OCH3-70), 3.82 (s, 3H, OCH3-400), 4.48 (s,
available from plant extracts. It was found that the condensation of
diarylcyanoketones with hydroxylamine hydrochlorides described
earlier [45] yielded a mixture of regioisomers. We explored a se-
lective synthesis of 3-amino-4,5-diarylisoxazoles based on the N,O-
acylation of amidoximes followed by their intramolecular cycliza-
tion. The targeted molecules were further tested in our phenotypic
sea urchin embryo assay to identify compounds with antimitotic
microtubule destabilizing activity. It was shown that 5-amino
isoxazoles were ca. 2-fold more potent than the respective 3-
aminoisoxazoles. Compounds exhibiting 5-aminoisoxazole bridge
were found to be considerably more active than their corre-
sponding pyrazole analogs both in the sea urchin embryo assay and
in the NCI60 anticancer drug screen. The most potent 5-amino-3,4-
diarylisoxazoles identified in the sea urchin embryo assay (1a, b,
and c) featured 3,4,5-trimethoxyphenyl, 3,4-methylenedioxy-5-
methoxyphenyl, or 3,4-ethylenedioxy-5-methoxyphenyl pharma-
cophores as ring A and p-methoxyphenyl substituent as ring B.
These compounds also showed strong in vitro cytotoxicity against
human cancer cell lines, including multi-drug resistant cells.
2H, NH2-5), 5.96 (s, 2H, OCH2O), 6.62 (d, J ¼ 2.4 Hz, 1H, H-60), 6.72
(d, J ¼ 2.4 Hz, 1H, H-40), 6.92 (d, J ¼ 8.8 Hz, 2H, H-300,500), 7.14 (d,
J ¼ 8.8 Hz, 2H, H-200,600); 13C NMR (DMSO-d6):
d 55.00 (CH3, OCH3-
400), 55.90 (CH3, OCH3-70), 90.61 (C, C-4), 101.46 (CH2, OCH2O),
101.63 (CH, C-40), 107.86 (CH, C-60), 114.02 (2CH, C-300,500), 122.74 (C,
C-100), 123.98 (C, C-50), 130.85 (2CH, C-200,600), 135.40 (C, C-10), 142.90
(C, C-70), 148.21 (C, C-30), 157.81 (C, C-400), 160.41 (C, C-3), 167.14 (C,
C-5); EIMS m/z 340 [M]þ (43), 312 (15), 179 (100), 178 (74), 135 (89),
134 (79), 119 (31), 77 (54), 76 (47), 44 (58); Anal. Calcd for
C18H16N2O5: C 63.52; H 4.74; N 8.23. Found: C 63.64; H 4.76; N 8.12.
4.1.1.3. 3-(2,3-Dihydro-8-methoxy-1,4-benzodioxin-6-yl)-4-(4-
methoxyphenyl)-5-isoxazolamine (1c). White solid; 20% yield; mp
164e167 ꢀC (HPLC); 1H NMR (DMSO-d6):
d
3.56 (s, 3H, OCH3-80),
3.75 (s, 3H, OCH3-400), 4.20 (m, 4H, OCH2CH2O), 6.42 (d, J ¼ 1.9 Hz,
1H, H-70), 6.51 (d, J ¼ 1.9 Hz, 1H, H-50), 6.60 (s, 2H, NH2-5), 6.93 (d,
J ¼ 8.7 Hz, 2H, H-300,500), 7.08 (d, J ¼ 8.7 Hz, 2H, H-200,600); 13C NMR
(DMSO-d6):
d
55.30 (CH3, OCH3-400), 55.77 (CH3, OCH3-80), 64.19
(2CH2, OCH2CH2O), 90.31 (C, C-4), 104.12 (CH, C-70), 108.70 (CH, C-
50), 114.66 (2CH, C-300,500), 120.56 (C, C-100), 121.88 (CH, C-60),
131.80(CH, C-40) (2CH, C-200,600), 134.10 (C, C-10), 141.92 (CH, C-40),
148.77 (CH, C-80), 159.91 (C, C-400), 161.73 (C, C-3), 167.54(C, C-5);
EIMS m/z 354 [M]þ (5), 193 (11), 192 (8), 149 (15), 135 (9), 134 (6),
119 (7), 111 (17), 109 (14), 107 (6), 97 (30), 57 (100), 43 (84); Anal.
Calcd for C19H18N2O5: C 64.40; H 5.12; N 7.91. Found: C 64.34; H
5.07; N 7.81.
4. Experimental protocols
4.1. Chemistry and chemical methods
NMR spectra were collected on a Bruker DR-500 instrument
[working frequencies of 500.13 MHz (1H) and 125.76 MHz (13C)].
Mass spectra were obtained on a Finnigan MAT/INCOS 50 instru-
ment (70 eV) using direct probe injection. Elemental analysis was
accomplished with the automated PerkineElmer 2400 CHN
microanalyzer. The compound purity has been determined by
NMR, HPLC, and elemental analyses. Purity of compounds 1aee,
1hek, 1m, 1n, 100a, 100b, 100d, 2c, 2hek, 20aeh, 20n, 20o, 200a, 200f, 4h,
4l, 5aef, 5h, 5t was determined to be ꢁ 95%.
4.1.1.4. 3-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-4-(4-
methoxyphenyl)-5-isoxazolamine (1d). White solid; 25% yield; mp
162e164 ꢀC; 1H NMR (CDCl3):
d
3.41 (s, 3H, OCH3-40), 3.78 (s, 3H,
OCH3-70), 3.82 (s, 3H, OCH3-400), 4.53 (s, 2H, NH2-5), 5.98 (s, 2H,
OCH2O), 6.59 (s, 1H, H-60), 6.86 (d, J ¼ 8.7 Hz, 2H, H-300,500), 7.07 (d,
J ¼ 8.7 Hz, 2H, H-200,600); 13C NMR (DMSO-d6):
d 54.92 (CH3, OCH3-
Polymethoxyphenyl aldehydes, acids, acetonitriles [42], and
polymethoxydiarylcyanketones [40] were synthesized from plant
polyalkoxyallylbenzenes extracted from dill and parsley as
described earlier [41,42].
400), 56.42 (CH3, OCH3-70), 59.15 (CH3, OCH3-40), 92.42 (C, C-4),
101.84 (CH2, OCH2O), 109.13 (CH, C-60), 113.80 (2CH, C-300,500), 116.23
(C, C-50), 123.56 (C, C-100), 128.70 (2CH, C-200,600), 135.71 (CH, C-40),
137.19 (C, C-10), 138.38 (C, C-30), 138.59 (C, C-70), 157.21 (C, C-400),
159.72 (C, C-3), 165.83 (C, C-5); EIMS m/z 370 [M]þ (15), 209 (11),
208 (9), 163 (11), 162 (16), 135 (99), 134 (100), 119 (41), 107 (24), 106
(31), 93 (42), 92 (31), 91 (33), 77 (75); Anal. Calcd for C19H18N2O6: C
61.62; H 4.90; N 7.56. Found: C 61.74; H 4.96; N 7.42.
4.1.1. General procedure for synthesis of mixtures of 5-aminoisoxa
zoles 1aee, h, iek, m, n and 3-aminoisoxazoles 2c, h, iek
A mixture of ketonitrile (1.2 mmol), NH2OH$2HCl (2.4 mmol)
and NaOAc$3H2O (2.4 mmol) in 5 mL of EtOH was refluxed for 5 h,
concentrated in vacuo, extracted with 50 mL of CH2Cl2. The
resulting extract washed with 2 ꢂ 50 mL of water, dried over
anhydrous Na2SO4 and separated by column chromatography (SiO2,
4.1.1.5. 3-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)-4-(4-
methoxyphenyl)-5-isoxazolamine (1e). Resin; 12% yield; 1H NMR
(DMSO-d6):
d
3.36 (s, 3H, OCH3-60), 3.70 (s, 3H, OCH3-70), 3.89 (s,

120
D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
3H, OCH3-400), 6.03 (s, 2H, OCH2O), 6.46 (s, 1H, H-40), 6.68 (s, 2H,
(s, 3H, OCH3-300), 3.74 (s, 3H, OCH3-400), 6.62 (dd, J ¼ 8.2, 2.0 Hz, 1H,
H-600), 6.65 (d, J ¼ 2.0 Hz, 1H, H-200), 6.66 (s, 2H, NH2-5), 6.89 (d,
J ¼ 8.2 Hz, 1H, H-500), 7.37 (m, 5H, C6H5); EIMS m/z 296 [M]þ (21),
281 (4), 268 (15), 253 (8), 250 (1), 178 (8), 165 (22), 164 (29), 163
(21), 148 (9), 135 (5), 119 (25), 105 (100), 104 (86), 89 (10), 77 (55);
Anal. Calcd for C17H16N2O3: C 68.91; H 5.44. Found: C 68.87; H 5.33.
NH2-5), 6.83 (d, J ¼ 8.8 Hz, 2H, H-300,500), 6.99 (d, J ¼ 8.8 Hz, 2H, H-
200,600); 1H NMR (CDCl3):
d
3.53 (s, 3H, OCH3-60), 3.77 (s, 3H, OCH3-
70), 3.97 (s, 3H, OCH3-400), 4.53 (s, 2H, NH2-5), 5.95 (s, 2H, OCH2O),
6.47 (s, 1H, H-40), 6.83 (d, J ¼ 8.8 Hz, 2H, H-300,500), 7.07 (d, J ¼ 8.8 Hz,
2H, H-200,600); 13C NMR (DMSO-d6):
d
54.88 (CH3, OCH3-400), 59.75
(CH3, OCH3-70), 60.94 (CH3, OCH3-60), 92.28 (C, C-4), 101.71 (CH2,
OCH2O), 103.23 (CH, C-40), 113.80 (2CH, C-300,500), 116.79 (C, C-50),
123.30 (C, C-100), 128.74 (2CH, C-200,600), 137.08 (C, C-70), 138.37 (C, C-
10), 144.04(C, C-30), 144.88 (CH, C-60), 157.16 (C, C-400), 159.62 (C, C-
3), 165.87 (C, C-5); EIMS m/z 370 [M]þ (100), 339 (6), 327 (18), 312
(21), 311 (60), 209 (38), 208 (29), 193 (29), 192 (30), 179 (11), 135
(94), 134 (77), 107 (22), 91 (70), 77 (64), 44 (75); Anal. Calcd for
4.1.1.11. 4-(4-Chlorophenyl)-3-(4-methoxyphenyl)-5-isoxazolamine
(1n). Yield 65% (mixture of 3-amino- and 5-aminoizoxazoles ¼ 6:1
according to 1H NMR analysis). 1n: white solid; mp 148e150 ꢀC;
Rf ¼ 0.40; 1H NMR (DMSO-d6):
d
3.76 (s, 3H, OCH3-40), 6.87 (s, 2H,
NH2-5), 6.94 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.11 (d, J ¼ 8.5 Hz, 2H, H-
300,500), 7.23 (d, J ¼ 8.8 Hz, 2H, H-20,60), 7.36 (d, J ¼ 8.5 Hz, 2H, H-
200,600); EIMS m/z 302 [Mþ2]þ (17), 300 [M]þ (50), 287 (5), 285 (18),
272 (5), 257 (10), 242 (16), 165 (10), 153 (11), 152 (21), 151 (35), 150
(17), 149 (87), 148 (41), 139 (19), 138 (21), 135 (100), 134 (82), 125
(11), 123 (38), 113 (7), 111 (19), 108 (21), 92 (24), 89 (17), 77 (34);
Anal. Calcd for C16H13ClN2O2: C 63.90; H 4.36; N 9.31. Found: C
63.82; H 4.29; N 9.39. Corresponding 3-aminoizoxazole was not
separated as pure compound.
C
19H18N2O6: C 61.62; H 4.90; N 7.56. Found: C 61.79; H 4.98; N 7.39.
4.1.1.6. 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-5-
isoxazolamine (1h). White solid; 18% yield; mp 156e158 ꢀC (lit. [45]
146e148 ꢀC); 1H NMR (DMSO-d6):
d
3.63 (s, 6H, 2ꢂ OCH3-300,500),
3.65 (s, 3H, OCH3-400), 3.76 (s, 3H, OCH3-40), 6.35 (s, 2H, H-200,600),
6.78 (s, 2H, NH2-5), 6.95 (d, J ¼ 8.7 Hz, 2H, H-30,50), 7.30 (d,
J ¼ 8.7 Hz, 2H, H-20,60); 13C NMR (DMSO-d6):
d
55.21 (CH3, OCH3-40),
55.59 (2CH3, OCH3-300,500), 60.00 (CH3, OCH3-400), 91.12 (C, C-4),
106.46 (2CH, C-200,600), 113.84 (2CH, C-30,50), 122.19 (C, C-10), 126.42
(C, C-100), 129.56 (2CH, C-20,60), 135.78 (C, C-400), 152.82 (2C, C-300,500),
159.92 (C, C-40), 160.81 (C, C-3), 166,99 (C, C-5); EIMS m/z 356 [M]þ
(13), 341 (2), 328 (2), 313 (2), 297 (2), 208 (2), 195 (5), 194 (6), 193
(7), 149 (8),135 (100), 134 (48),121 (7), 92 (5), 77 (9); Anal. Calcd for
4.1.1.12. 3-Amino-4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
3-isoxazolamin (2a). This was found in evaporated reaction
mixture by 1H NMR analysis but was not separated.
4.1.1.13. 5-(2,3-Dihydro-8-methoxy-1,4-benzodioxin-6-yl)-4-(4-
methoxyphenyl)-3-isoxazolamine (2c). White solid; 15% yield; mp
C
19H20N2O5: C 64.04; H 5.66; N 7.86. Found: C 64.19; H 5.71; N 7.74.
189e191 ꢀC (HPLC); 1H NMR (DMSO-d6):
d
3.60 (s, 3H, OCH3-800),
3.80 (s, 3H, OCH3-40), 4.20 (m, 4H, OCH2CH2O), 5.27 (s, 2H, NH2-3),
6.49 (d, J ¼ 1.9 Hz, 1H, H-700), 6.63 (d, J ¼ 1.9 Hz, 1H, H-500), 7.04 (d,
J ¼ 8.7 Hz, 2H, H-30,50), 7.25 (d, J ¼ 8.7 Hz, 2H, H-20,60); EIMS m/z 354
[M]þ (84), 311 (5), 283 (15), 255 (12), 193 (66), 192 (49), 161 (80),
149 (14), 135 (14), 134 (76), 119 (21), 111 (14), 109 (17), 107 (16), 97
(23), 57 (100), 43 (99); Anal. Calcd for C19H18N2O5: C 64.40; H 5.12;
N 7.91. Found: C 64.26; H 5.04; N 7.98.
4.1.1.7. 4-(7-Methoxy-1,3-benzodioxol-5-yl)-3-(4-methoxyphenyl)-
5-isoxazolamine (1i). White solid; 12% yield; mp 162e164 ꢀC; 1H
NMR (DMSO-d6): d
3.71 (s, 3H, OCH3-700), 3.76 (s, 3H, OCH3-40), 5.97
(s, 2H, OCH2O), 6.28 (d, J ¼ 1.5 Hz, 1H, H-600), 6.35 (d, J ¼ 1.5 Hz, 1H,
H-400), 6.71 (s, 2H, NH2-5), 6.94 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.28 (d,
J ¼ 8.8 Hz, 2H, H-20,60); 13C NMR (DMSO-d6):
d
55.18 (CH3, OCH3-40),
56.00 (CH3, OCH3-700), 90.94 (C, C-4), 101.15 (CH2, OCH2O), 103.39
(CH, C-400), 109.03 (CH, C-600), 113.91 (2CH, C-30,50), 122.14 (C, C-10),
125.09 (C, C-500), 129.38 (2CH, C-20,60), 133.37 (C, C-100), 143.25 (C, C-
700), 148.41 (C, C-300), 159.91 (C, C-40), 160.70 (C, C-3), 167.12 (C, C-5);
EIMS m/z 340 [M]þ (57), 339 (25), 325 (8), 312 (10), 309 (1), 192
(32), 179 (16), 178 (21), 135 (100), 134 (61), 92 (10), 77 (18), 43 (43);
Anal. Calcd for C18H16N2O5: C 63.52; H 4.74; N 8.23. Found: C 63.58;
H 4.77; N 8.14.
4.1.1.14. 5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3-
isoxazolamine (2h). White solid; 11% yield; mp 156e158 ꢀC; 1H
NMR (DMSO-d6):
d
3.71 (s, 3H, OCH3-40), 3.72 (s, 6H, 2ꢂ OCH3-30,50),
3.76 (s, 3H, OCH3-400), 5.38 (s, 2H, NH2-3), 6.59 (s, 2H, H-20,60), 6.97
(d, J ¼ 8.7 Hz, 2H, H-300,500), 7.43 (d, J ¼ 8.7 Hz, 2H, H-200,600); 13C NMR
(DMSO-d6): d
55.08 (CH3, OCH3-400), 55.76 (2CH3, OCH3-30,50), 59.87
(CH3, OCH3-40), 106.57 (C, C-4), 106.91 (2CH, C-20,60), 114.12 (2CH, C-
300,500), 120.31 (C, C-100), 124.97 (C, C-10), 127.80 (2CH, C-200,600),
136.98 (C, C-40), 153.21 (2C, C-30,50), 160.12 (C, C-400), 162.04 (C, C-5),
162.94 (C, C-3); EIMS m/z 356 [M]þ (49), 341 (14), 313 (6), 270 (2),
255 (3), 254 (4), 253 (2), 238 (4), 221 (19), 194 (20), 193 (11), 162 (6),
149 (15), 135 (100), 134 (49), 119 (5), 107 (8), 92 (13), 77 (23); Anal.
Calcd for C19H20N2O5: C 64.04; H 5.66; N 7.86. Found: C 64.12; H
5.69; N 7.79.
4.1.1.8. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-3-(4-
methoxyphenyl)-5-isoxazolamine (1j). White solid; 8% yield; mp
145e147 ꢀC; 1H NMR (DMSO-d6):
d
3.46 (s, 3H, OCH3-400), 3.70 (s,
3H, OCH3-700), 3.74 (s, 3H, OCH3-40), 6.02 (s, 2H, OCH2O), 6.33 (s, 1H,
H-600), 6.46 (s, 2H, NH2-5), 6.91 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.28 (d,
J ¼ 8.8 Hz, 2H, H-20,60); EIMS m/z 370 [M]þ (51), 339 (36), 327 (5),
222 (19), 207 (22), 193 (7), 170 (12), 163 (17), 149 (23),135 (100),134
(41), 107 (15), 92 (28), 77 (44), 44 (49); Anal. Calcd for C19H18N2O6:
C 61.62; H 4.90; N 7.56. Found: C 61.74; H 4.95; N 7.48.
4.1.1.15. 4-(7-Methoxy-1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl)-
3-isoxazolamine (2i). White solid; 22% yield; mp 178e181 ꢀC; 1H
4.1.1.9. 4-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)-3-(4-
NMR (DMSO-d6): d
3.77 (s, 3H, OCH3-70), 3.79 (s, 3H, OCH3-400), 5.35
methoxyphenyl)-5-isoxazolamine (1k). White solid; 12% yield; mp
(s, 2H, NH2-3), 6.05 (s, 2H, OCH2O), 6.50 (d, J ¼ 1.4 Hz,1H, H-40), 6.57
158e161 ꢀC; 1H NMR (DMSO-d6):
d
3.42 (s, 3H, OCH3-600), 3.74 (s,
(d, J ¼ 1.4 Hz, 1H, H-60), 6.97 (d, J ¼ 8.9 Hz, 2H, H-300,500), 7.41 (d,
3H, OCH3-700), 3.92 (s, 3H, OCH3-40), 5.99 (s, 2H, OCH2O), 6.28 (s, 1H,
H-400), 6.42 (s, 2H, NH2-5), 6.91 (d, J ¼ 8.9 Hz, 2H, H-30,50), 7.30 (d,
J ¼ 8.9 Hz, 2H, H-20,60); EIMS m/z 370 [M]þ (100), 355 (29), 339 (88),
327 (11), 235 (6), 207 (13), 135 (19), 43 (29); Anal. Calcd for
J ¼ 8.9 Hz, 2H, H-200,600); 13C NMR (DMSO-d6):
d 55.15 (CH3, OCH3-
400), 56.12 (CH3, OCH3-70), 101.33 (CH2, OCH2O), 103.66 (CH, C-40),
106.37 (C, C-4), 109.41 (CH, C-60), 114.16 (2CH, C-300,500), 120.28 (C, C-
100), 123.47 (C, C-50), 127.72 (2CH, C-200,600), 134.53 (C, C-10), 143.57
(C, C-70), 148.72 (C, C-30), 160.08 (C, C-400), 162.03 (C, C-5), 162.99 (C,
C-3); EIMS m/z 340 [M]þ (45), 325 (6), 312 (10), 205 (17), 178 (21),
135 (100), 134 (49), 92 (20), 77 (32), 43 (37); Anal. Calcd for
C
19H18N2O6: C 61.62; H 4.90; N 7.56. Found: C 61.77; H 4.96; N 7.45.
4.1.1.10. 4-(3,4-Dimethoxyphenyl)-3-phenyl-5-isoxazolamine (1m).
White solid; 55% yield; mp 147e149 ꢀC; 1H NMR (DMSO-d6):
3.61
d
C18H16N2O5: C 63.52; H 4.74; N 8.23. Found: C 63.60; H 4.75; N 8.17.

D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
121
4.1.1.16. 4-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(4-
vacuo, and the residue was washed by water (3 ꢂ 30 mL) and dried.
Yield 75e80%.
methoxyphenyl)-3-isoxazolamine (2j). White solid; 17% yield; mp
118e120 ꢀC; 1H NMR (DMSO-d6): 3.61 (s, 3H, OCH3-40), 3.75 (s, 3H,
d
OCH3-70), 3.76 (s, 3H, OCH3-400), 5.21 (s, 2H, NH2-3), 6.08 (s, 2H,
OCH2O), 6.47 (s, 1H, H-60), 6.96 (d, J ¼ 8.9 Hz, 2H, H-300,500), 7.38 (d,
J ¼ 8.9 Hz, 2H, H-200,600); EIMS m/z 370 [M]þ (69), 355 (3), 236 (6),
235 (41), 208 (14),193 (20),136 (11),135 (100),134 (39),107 (17), 92
(31), 77 (48), 43 (26); Anal. Calcd for C19H18N2O6: C 61.62; H 4.90; N
7.56. Found: C 61.68; H 4.93; N 7.51.
4.1.3.1. N0-Hydroxy-2-(4-methoxyphenyl)ethanimidamide
White solid; 85% yield; mp 88e91 ꢀC (lit. [60] 108e109 ꢀC); 1H NMR
(DMSO-d6): 3.18 (s, 2H, CH2), 3.71 (s, 3H, OCH3-4), 5.33 (s, 2H,
(3p).
d
NH2), 6.84 (d, J ¼ 8.7 Hz, 2H, H-3,5), 7.18 (d, J ¼ 8.7 Hz, 2H, H-2,6),
8.85 (s,1H, OH); EIMS m/z 180 [M]þ (54),163 (51),147 (71),132 (35),
121 (100), 107 (18), 104 (15), 91 (23), 77 (35); Anal. Calcd for
C9H12N2O2: C 59.99; H 6.71; N 15.55. Found: C 59.84; H 6.67; N
15.73.
4.1.1.17. 4-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)-5-(4-
methoxyphenyl)-3-isoxazolamine (2k). White solid; 55% yield; mp
172e175 ꢀC; 1H NMR (DMSO-d6):
d
3.48 (s, 3H, OCH3-60), 3.76 (s, 3H,
4.1.3.2. N0-Hydroxy-2-(3,4,5-trimethoxyphenyl)ethanimidamide
OCH3-70), 3.96 (s, 3H, OCH3-400), 5.29 (s, 2H, NH2-3), 6.06 (s, 2H,
OCH2O), 6.46 (s, 1H, H-60), 6.96 (d, J ¼ 8.9 Hz, 2H, H-300,500), 7.38 (d,
J ¼ 8.9 Hz, 2H, H-200,600); EIMS m/z 370 [M]þ (100), 355 (3), 339 (4),
338 (6), 327 (2), 312 (11), 235 (35), 193 (17), 135 (67), 134 (20), 107
(11), 92 (19), 77 (33), 44 (18); Anal. Calcd for C19H18N2O6: C 61.62; H
4.90; N 7.56. Found: C 61.70; H 4.93; N 7.45.
(3q). White solid; 75% yield; mp 126e128 ꢀC; 1H NMR (DMSO-d6):
d
3.18 (s, 2H, CH2), 3.62 (s, 3H, OCH3-4), 3.75 (s, 6H, OCH3-3,5), 5.35
(s, 2H, NH2), 6.59 (s, 2H, H-2,6), 8.86 (s, 1H, OH); EIMS m/z 240 [M]þ
(37), 223 (17), 209 (25), 192 (13), 181 (100), 167 (20), 150 (43), 107
(21), 79 (33), 77 (37); Anal. Calcd for C11H16N2O4: C 54.99; H 6.71; N
11.66. Found: C 55.11; H 6.74; N 11.57.
4.1.2. General procedure for synthesis of diacyl derivatives 100a,b,d
A suspension of 5-aminoisoxazole 1a,b,d (1.46 mmol) in 2 mL
pyridine was treated with 0.37 g (3.62 mmol) of Ac2O. The reaction
mixture was stirred for 24 h, poured into EtOAc (30 mL), washed
with 5% aqueous HCl (2 ꢂ 15 mL), water (2 ꢂ 20 mL), concentrated,
and purified by column chromatography (EtOAc/petroleum ether,
1:6, Rf ¼ 0.5). Intermediate monoacyl derivatives 10a,b,d were not
separated.
4.1.3.3. N0-Hydroxy-2-(4-nitrophenyl)ethanimidamide
White solid; 88% yield; mp 168e170 ꢀC (lit. [61] 170 ꢀC); 1H NMR
(DMSO-d6):
2H, H-2,6), 8.17 (d, J ¼ 8.7 Hz, 2H, H-3,5), 9.01 (s, 1H, OH); EIMS m/z
195 [M]þ (50), 178 (12), 161 (6), 156 (7), 154 (7), 136 (11), 132 (100),
121 (100), 104 (20), 90 (30), 89 (49), 78 (30), 77 (21); Anal. Calcd for
C8H9N3O3: C 49.23; H 4.65; N 21.53. Found: C 49.08; H 4.59; N 21.73.
(3s).
d
3.42 (s, 2H, CH2), 5.54 (s, 2H, NH2), 7.55 (d, J ¼ 8.7 Hz,
4.1.4. General procedure for synthesis of oxadiazoles 5aef, h, t
4 .1. 2 .1. N - A c e t yl - N - [ 4 - ( 4 - m e t h o x y p h e nyl ) - 3 - ( 3 , 4 , 5 -
A solution of amidoxime (5 mmol) and benzoylchloride
trimethoxyphenyl)-5-isoxazolyl]acetamide (100a). Yellow solid; 45%
(7.5 mmol) in dry pyridine (10 mL) was refluxed for 1 h, brought to
room temperature, poured into ice cold water (100 mL). The
resulting precipitate of 5 was collected, washed with 2ꢂ 10 mL of
cold water and purified by column chromatography (SiO2, EtOAc/
petroleum ether). Yield 12e56%.
yield; mp 125e127 ꢀC (lit. [45].119e121 ꢀC); 1H NMR (CDCl3):
d 2.32
(s, 6H, 2ꢂ NC(O)CH3), 3.67 (s, 6H, 2ꢂ OCH3-30,50), 3.82 (s, 3H, OCH3-
40), 3.87 (s, 3H, OCH3-400), 6.74 (s, 2H, H-20,60), 6.92 (d, J ¼ 8.8 Hz, 2H,
H-300,500), 7.12 (d, J ¼ 8.8 Hz, 2H, H-200,600); EIMS m/z 440 [M]þ (13),
398 (17), 357 (10), 356 (13), 210 (23), 195 (10), 194 (11), 168 (39), 147
(51), 135 (18), 43 (100); Anal. Calcd for C23H24N2O7: C 62.72; H 5.49;
N 6.36. Found: C 62.84; H 5.54; N 6.29.
In our hands the same procedure [57] using N0-hydroxy-2-
(3,4,5-trimethoxyphenyl)ethanimidamide
and
p-methox-
ybenzoylchloride furnished 3-aminoisoxazole 4h, whereas the re-
action between N0-hydroxy-2-(4-chlorophenyl)ethanimidamide
and p-methoxybenzoylchloride afforded only 3-aminoisoxazole 4l.
4.1.2.2. N-Acetyl-N-[3-(7-methoxy-1,3-benzodioxol-5-yl)-4-(4-
methoxyphenyl)-5-isoxazolyl]acetamide (100b). White solid; 65%
yield; mp 150e152 ꢀC; 1H NMR (CDCl3):
d
2.31 (s, 6H, 2ꢂ NC(O)
4.1. 4.1. 4-Methoxy-N-[5-(4-methoxyphenyl)-4-(3, 4, 5-
CH3), 3.73 (s, 3H, OCH3-70), 3.83 (s, 3H, OCH3-400), 6.01 (s, 2H,
OCH2O), 6.67 (d, J ¼ 1.5 Hz, 1H, H-60), 6.73 (d, J ¼ 1.5 Hz, 1H, H-40),
6.91 (d, J ¼ 8.8 Hz, 2H, H-300,500), 7.08 (d, J ¼ 8.8 Hz, 2H, H-200,600);
EIMS m/z 424 [M]þ (4), 382 (7), 167 (12), 147 (16), 43 (100); Anal.
Calcd for C22H20N2O7: C 62.26; H 4.75; N 6.60. Found: C 62.34; H
4.78; N 6.46.
trimethoxyphenyl)-3-isoxazolyl]benzamide (4h). White solid; 10%
yield; mp 205e207 ꢀC; 1H NMR (DMSO-d6): 3.68 (s, 3H, OCH3-400),
d
3.72 (s, 6H, 2ꢂ OCH3-300,500), 3.85 (s, 3H, OCH3-4000), 3.86 (s, 3H,
OCH3-40), 7.02 (s, 2H, H-200,600), 7.09 (d, J ¼ 8.9 Hz, 2H, H-3000,5000), 7.14
(d, J ¼ 8.9 Hz, 2H, H-30,50), 8.03 (d, J ¼ 8.9 Hz, 2H, H-2000,6000), 8þ.07 (d,
J ¼ 8.9 Hz, 2H, H-20,60), 10.38 (s, 1H, NH); EIMS m/z 490 [M] (17),
195 (16), 135 (97), 107 (5), 92 (7), 77 (12), 43 (100); Anal. Calcd for
4.1.2.3. N-Acetyl-N-[3-(4,7-dimethoxy-1,3-benzodioxol-5-yl)-4-(4-
C27H26N2O7: C 66.11; H 5.34; N 5.71. Found: C 66.22; H 5.38; N 5.62.
methoxyphenyl)-5-isoxazolyl]acetamide (100d). White solid; 14%
yield; mp 156e162 ꢀC; 1H NMR (CDCl3):
d
2.32 (s, 6H, 2ꢂ NC(O)
4.1.4.2. N-[4-(4-Chlorophenyl)-5-(4-methylphenyl)-3-isoxazolyl]-4-
CH3), 3.36 (s, 3H, OCH3-40), 3.80 (s, 3H, OCH3-70), 3.85 (s, 3H, OCH3-
400), 5.99 (s, 2H, OCH2O), 6.64 (c, 1H, H-60), 6.87 (d, J ¼ 8.8 Hz, 2H, H-
300,500), 7.08 (d, J ¼ 8.8 Hz, 2H, H-200,600); EIMS m/z 454 [M]þ (1),
412(16), 371 (6), 209 (10), 197 (34), 182 (11), 162 (10), 147 (10), 135
(18), 91 (19), 43 (100); Anal. Calcd for C23H22N2O8: C 60.79; H 4.88;
N 6.16. Found: C 60.84; H 4.92; N 6.10.
methylbenzamide (4l). White solid; 43% yield; mp 236e237 ꢀC (lit.
[57] 233e234 ꢀC); 1H NMR (DMSO-d6): 2.40 (s, 3H, CH3-4000), 2.41
d
(s, 3H, CH3-40), 7.37 (d, J ¼ 8.1 Hz, 2H, H-3000,5000), 7.40 (d, J ¼ 8.1 Hz,
2H, H-30,50), 7.55 (d, J ¼ 8.7 Hz, 2H, H-300,500), 7.73 (d, J ¼ 8.7 Hz, 2H,
H-200,600), 7.95 (d, J ¼ 8.1 Hz, 2H, H-2000,6000), 8.00 (d, J ¼ 8.1 Hz, 2H, H-
20,60), 10.56 (s, 1H, NH); EIMS m/z 404 [M þ 2]þ (24), 402 [M]þ (68),
141 (13), 139 (41), 119 (100), 113 (7), 111 (29), 91 (80); Anal. Calcd for
4.1.3. General procedure for synthesis of amidoximes 3p,q,s
C24H19ClN2O2: C 71.55; H 4.75; N 6.95; Cl 8.80. Found: C 71.69; H
The solution of NaOH (2.71 g, 67.93 mmol) in water (20 mL) was
added at room temperature to the solution of poly-
methoxyphenylacetonitrile [40] (5.0 g, 33.96 mmol) and hydrox-
ylamine hydrochloride (4.72 g, 67.93 mmol) in EtOH (100 mL). The
reaction mixture was refluxed for 5 h, the solvent was evaporated in
4.81; N 6.87; Cl 8.67.
4.1.4.3. 3-(4-Methoxybenzyl)-5-(3,4,5-trimethoxyphenyl)-1,2,4-
oxadiazole (5a). White solid; 32% yield; mp 116e118 ꢀC; 1H NMR
(DMSO-d6):
d
3.73 (s, 3H, OCH3-40), 3.76 (s, 3H, OCH3-400), 3.88 (s,

122
D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
6H, 2ꢂ OCH3-300,500), 4.09 (s, 2H, CH2), 6.90 (d, J ¼ 8.7 Hz, 2H, H-
30,50), 7.26 (d, J ¼ 8.7 Hz, 2H, H-20,60), 7.32 (s, 2H, H-200,600); EIMS m/z
356 [M]þ (34), 341 (2), 196 (13), 195 (55), 122 (12), 121 (100), 91 (9),
78 (16), 77 (20); Anal. Calcd for C19H20N2O5: C 64.04; H 5.66; N 7.86.
Found: C 64.17; H 5.70; N 7.76.
(C-20,60), 114.98 (C-300,500), 115.84 (C-600), 129.83 (C-200,600), 131.39 (C-
10), 136.46 (C-40), 152.93 (C-30,50), 163.03 (C-40), 169.76 (C-3), 174.92
(C-5); EIMS m/z 356 [M]þ (62), 341 (24), 181 (22), 165 (7), 149 (7),
148 (8), 147 (7), 137 (14), 136 (27), 135 (100), 133 (15), 121 (7), 120
(8), 107 (28), 92 (34), 78 (20), 77 (57); Anal. Calcd for C19H20N2O5: C
64.04; H 5.66; N 7.86. Found: C 64.16; H 5.70; N 7.77.
4.1.4.4. 5-(7-Methoxy-1,3-benzodioxol-5-yl)-3-(4-methoxybenzyl)-
1,2,4-oxadiazole (5b). White solid; 22% yield; mp 96e98 ꢀC; 1H
4.1.4.10. 5-(4-Nitrophenyl)-3-(4-methoxybenzyl)-1,2,4-oxadiazole
(5t). White solid; 56% yield; mp 122e124 ꢀC; 1H NMR (DMSO-d6):
d03.73 (s, 3H, OCH3-40), 4.14 (s, 2H, CH2), 6.91 (d, J ¼ 8.7 Hz, 2H, H-
3 ,50), 7.29 (d, J ¼ 8.7 Hz, 2H, H-20,60), 8.33 (d, J ¼ 8.9 Hz, 2H, H-
200,600), 8.42 (d, J ¼ 8.9 Hz, 2H, H-300, 500); EIMS m/z 311 [M]þ (88), 161
(100), 150 (17), 146 (9), 137 (10), 134 (30), 121 (83), 118 (7), 116 (6),
104 (32), 92 (17), 91 (20), 90 (21), 78 (35), 77 (43), 76 (42); Anal.
Calcd for C16H13N3O4: C 61.73; H 4.21; N 13.50. Found: C 61.84; H
4.30; N 13.36.
NMR (DMSO-d6): d
3.73 (s, 3H, OCH3-40), 3.91 (s, 3H, OCH3-700), 4.06
(s, 2H, CH2), 6.16 (s, 2H, OCH2O), 6.90 (d, J ¼ 8.7 Hz, 2H, H-30,50), 7.25
(d, J ¼ 1.5 Hz, 1H, H-600), 7.26 (d, J ¼ 8.7 Hz, 2H, H-20,60), 7.34 (d,
J ¼ 1.5 Hz, 1H, H-400); EIMS m/z 340 [M]þ (37), 180 (10), 179 (10), 161
(14), 151 (16), 121 (92), 95 (21), 91 (12), 78 (30), 77 (27); Anal. Calcd
for C18H16N2O5: C 63.52; H 4.74; N 8.23. Found: C 63.39; H 4.68; N
8.37.
4.1.4.5. 5-(8-Methoxy-2,3-dihydro-1,4-benzodioxin-6-yl)-3-[(4-
methoxybenzyl)methyl]-1,2,4-oxadiazole (5c). White solid; 18%
4.1.5. General procedure for synthesis of mixture of monoacetyl-3-
aminoisoxazoles 20a,f and diacetyl 3-aminoisoxazoles 200a,f
A solution of benzoylamidoxime (1.0 g, 3.18 mmol) and Ac2O
(0.86 g, 10.5 mmol) in pyridine (10 mL) was stirred for 24 h at 60 ꢀC,
poured into water (50 mL), extracted with CH2Cl2 (2ꢂ 20 mL), dried
over anhydrous Na2SO4, concentrated in vacuo, and separated by
column chromatography (EtOAc/petroleum ether, 1:4e1:1). First
fraction (Rf ¼ 0.8) yielded 30 mg of the diacyl derivative 200a,f,
second fraction (Rf ¼ 0.2) yielded monoacyl derivative 20a,f (25 mg).
Attempts to remove the acyl group under a variety of experimental
conditions were unsuccessful.
yield; mp 125e127 ꢀC; 1H NMR (DMSO-d6): 3.73 (s, 3H, OCH3-40),
d
3.85 (s, 3H, OCH3-800), 4.06 (s, 2H, CH2), 4.30 (m, 2H, H-200), 4.33 (m,
2H, H-300), 6.90 (d, J ¼ 8.7 Hz, 2H, H-30,50), 7.18 (s, 2H, H-500,700), 7.26
(d, J ¼ 8.7 Hz, 2H, H-20,60); 13C: 30.61 (CH2), 55.08 (OCH3-40), 56.05
(OCH3-800), 63.95 (C-200), 64.27 (C-300), 103.70 (C-700), 109.63 (C-500),
114.04 (C-30,50), 115.03 (C-600), 127.67 (C-10), 129.99 (C-20,60), 137.45
(C-400a), 144.10 (C-800a), 149.31 (C-800), 158.25 (C-40), 170.16 (C-3),
174.84 (C-5); EIMS m/z 354 [M]þ (57), 194 (11), 193 (100), 165 (10),
121 (98), 107 (11), 91 (10), 78 (21), 77 (21); Anal. Calcd for
C
19H18N2O5: C 64.40; H 5.12; N 7.91. Found: C 64.56; H 5.19; N 7.78.
4.1.4.6. 5-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-3-(4-
4.1.6. General procedure for synthesis of benzoylamidoximes 6
A suspension of benzoic acid (8.8 mmol) in dry MeCN (20 mL)
was treated with carbonyl diimidazole (11.5 mmol) at 20 ꢀC. The
resulting mixture was stirred for 30 min, treated with amidoxime
3pes (8.8 mmol), stirred for additional 8 h, and poured into water
(100 mL). The solid residue was filtered and recrystallized from 50%
EtOH/water.
methoxybenzyl)-1,2,4-oxadiazole (5d). White solid; 16% yield; mp
95e97 ꢀC; 1H NMR (DMSO-d6):
d
3.73 (s, 3H, OCH3-40), 3.84 (s, 3H,
OCH3-400), 3.91 (s, 3H, OCH3-700), 4.07 (s, 2H, CH2), 6.18 (s, 2H,
OCH2O), 6.90 (d, J ¼ 8.7 Hz, 2H, H-30,50), 7.18 (s, 1H, H-600), 7.26 (d,
J ¼ 8.7 Hz, 2H, H-20,60); EIMS m/z 370 [M]þ (24), 209 (40), 207 (48),
195 (6), 194 (10), 179 (17), 163 (6), 147 (13), 135 (15), 121 (100), 91
(13), 78 (27), 77 (29); Anal. Calcd for C19H18N2O6: C 61.62; H 4.90; N
7.56. Found: C 61.80; H 4.97; N 7.41.
4.1.6.1. 2-(4-Methoxyphenyl)-N0-[(3,4,5-trimethoxybenzoyl)oxy]
ethanimidamide (6a). White solid; 82% yield; mp 161e163 ꢀC; 1H
4.1.4.7. 5-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)-3-(4-
NMR (DMSO-d6): d 3.36 (s, 2H, CH2), 3.73 (s, 3H, OCH3-4), 3.74 (s,
methoxybenzyl)-1,2,4-oxadiazole (5e). White solid; 21% yield; mp
3H, OCH3-40), 3.85 (s, 6H, 2ꢂ OCH3-30,50), 6.51 (s, 2H, NH2), 6.89 (d,
J ¼ 8.6 Hz, 2H, H-3,5), 7.30 (d, J ¼ 8.6 Hz, 2H, H-2,6), 7.31 (s, 2H, H-
20,60); EIMS m/z 374 [M]þ (0.2), 196 (9), 195 (100), 122 (6), 121 (28),
78 (7), 77 (10); Anal. Calcd for C19H22N2O6: C 60.95; H 5.92; N 7.48.
Found: C 61.07; H 5.96; N 7.42.
102e104 ꢀC; 1H NMR (DMSO-d6):
d
3.73 (s, 3H, OCH3-40), 3.79 (s,
3H, OCH3-700), 3.98 (s, 3H, OCH3-600), 4.06 (s, 2H, CH2), 6.14 (s, 2H,
OCH2O), 6.90 (d, J ¼ 8.7 Hz, 2H, H-30,50), 7.10 (s, 1H, H-400), 7.27 (d,
J ¼ 8.7 Hz, 2H, H-20,60); EIMS m/z 370 [M]þ (18), 353 (3), 341 (2), 209
(27), 207 (33), 206 (24),193 (5),179 (8),166 (6),161 (5),147 (10),135
(16), 121 (100), 91 (13), 78 (24), 77 (27); Anal. Calcd for C19H18N2O6:
C 61.62; H 4.90; N 7.56. Found: C 61.78; H 4.93; N 7.48.
4.1.6.2. N0-{[(7-Methoxy-1,3-benzodioxol-5-yl)carbonyl]oxy}-2-(4-
methoxyphenyl)ethanimidamide (6b). White solid; 70% yield; mp
116e118 ꢀC; 1H NMR (DMSO-d6):
d 3.34 (s, 2H, CH2), 3.73 (s, 3H,
4.1.4.8. 5-(3-Methoxyphenyl)-3-(4-methoxybenzyl)-1,2,4-oxadiazole
OCH3-4), 3.89 (s, 3H, OCH3-70), 6.11 (s, 2H, OCH2O), 6.52 (s, 2H,
NH2), 6.89 (d, J ¼ 8.7 Hz, 2H, H-3,5), 7.29 (d, J ¼ 8.7 Hz, 2H, H-2,6),
7.33 (d, J ¼ 1.5 Hz, 1H, H-60), 7.39 (d, J ¼ 1.5 Hz, 1H, H-40); EIMS m/z
358 [M]þ (8), 341 (3), 340 (12), 196 (10), 195 (6), 180 (20), 179 (100),
162 (14), 151 (19), 147 (9), 121 (50), 95 (15), 78 (17), 77 (12); Anal.
Calcd for C18H18N2O6: C 60.33; H 5.06; N 7.82. Found: C 60.39; H
5.08; N 7.69.
(5f). White solid; 22% yield; mp 52e54 ꢀC; 1H NMR (DMSO-d6):
d
3.73 (s, 3H, OCH3-40), 3.85 (s, 3H, OCH3-300), 4.10 (s, 2H, CH2), 6.90
(d, J ¼ 8.7 Hz, 2H, H-30,50), 7.26 (dd, J ¼ 7.8, 2.7 Hz, 1H, H-400), 7.27 (d,
J ¼ 8.7 Hz, 2H, H-20,60), 7.53 (t, J ¼ 7.8 Hz, 1H, H-500), 7.54 (dd, J ¼ 2.7,
2.5 Hz, 1H, H-200), 7.66 (dd, J ¼ 7.8, 2.5 Hz, 1H, H-600); EIMS m/z 296
[M]þ (63), 281 (1), 162 (13), 161 (79), 146 (7), 137 (11), 135 (100), 133
(29), 121 (65), 107 (47), 92 (37), 78 (39), 77 (82); Anal. Calcd for
C
17H16N2O3: C 68.91; H 5.44; N 9.45. Found: C 68.84; H 5.40; N 9.53.
4.1.6.3. N0-{[(8-Methoxy-2,3-dihydro-1,4-benzodioxin-6-yl)carbonyl]
oxy}-2-(4-methoxyphenyl)ethanimidamide (6c). White solid; 85%
4.1.4.9. 5-(4-Methoxyphenyl)-3-[(3,4,5-trimethoxybenzyl)methyl]-
yield; mp 165e167 ꢀC; 1H NMR (DMSO-d6):
d 3.33 (s, 2H, CH2), 3.73
1,2,4-oxadiazole (5h). White solid; 12% yield; mp 86e88 ꢀC; 1H
(s, 3H, OCH3-4), 3.82 (s, 3H, OCH3-80), 4.26 (m, 2H, H-20), 4.29 (m,
2H, H-30), 6.49 (s, 2H, NH2), 6.88 (d, J ¼ 8.6 Hz, 2H, H-3,5), 7.18 (d,
J ¼ 1.8 Hz,1H, H-70), 7.29 (d, J ¼ 8.6 Hz, 2H, H-2,6), 7.30 (d, J ¼ 1.8 Hz,
1H, H-50); EIMS m/z 372 [M]þ (2), 355 (3), 354 (15), 210 (83), 195
(17), 193 (100), 165 (10), 164 (21), 163 (16), 154 (15), 139 (17), 122
NMR (DMSO-d6):
d
3.63 (s, 3H, OCH3-40), 3.76 (s, 6H, 2ꢂ OCH3-30,50),
3.86 (s, 3H, OCH3-400), 4.06 (s, 2H, CH2), 6.67 (s, 2H, H-20,60), 7.15 (d,
J ¼ 8.9 Hz, 2H, H-300,500), 8.03 (d, J ¼ 8.9 Hz, 2H, H-200,600); 13C: 31.67
(CH2), 55.64 (OCH3-400), 65.89 (OCH3-30,50), 60.00 (OCH3-40), 106.36

D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
123
(39),121 (60),111 (14),107 (10), 91 (10), 78 (20), 77 (25); Anal. Calcd
for C19H20N2O6: C 61.28; H 5.41; N 7.52. Found: C 61.37; H 5.43; N
7.48.
4.1.7.1. N-[N0-{[(4,7-Dimethoxy-1,3-benzodioxol-5-yl)carbonyl]oxy}-
2-(4-methoxyphenyl)ethanimidoyl]acetamide (7d). White solid; 16%
yield; mp 168e170 ꢀC; 1H NMR (DMSO-d6):
d 2.24 (s, 3H, COCH3),
3.71 (s, 3H, OCH3-4), 3.81 (s, 3H, OCH3-70), 4.06 (s, 3H, OCH3-50),
4.11 (s, 2H, CH2), 6.17 (s, 2H, OCH2O), 6.87 (d, J ¼ 8.7 Hz, 2H, H-3,5),
7.17 (s, 1H, H-60), 7.22 (d, J ¼ 8.7 Hz, 2H, H-2,6), 10.88 (s, 1H, NH);
EIMS m/z 430 [M]þ (2), 210 (14), 209 (100), 207 (5),194 (8),121 (30),
78 (7), 77 (7), 65 (5); Anal. Calcd for C21H22N2O8: C 58.60; H 5.15; N
6.51. Found: C 58.78; H 5.21; N 6.46.
4.1.6.4. N0-{[(4,7-Dimethoxy-1,3-benzodioxol-5-yl)carbonyl]oxy}-2-
(4-methoxyphenyl)ethanimidamide (6d). White solid; 55% yield;
mp 124e126 ꢀC; 1H NMR (DMSO-d6):
d 3.32 (s, 2H, CH2), 3.73 (s, 3H,
OCH3-4), 3.82 (s, 3H, OCH3-40), 3.83 (s, 3H, OCH3-70), 6.11 (s, 2H,
OCH2O), 6.30 (s, 2H, NH2), 6.89 (d, J ¼ 8.7 Hz, 2H, H-3,5), 7.00 (s, 1H,
H-60), 7.29 (d, J ¼ 8.7 Hz, 2H, H-2,6); EIMS m/z 388 [M]þ (5), 371 (1),
370 (2), 357 (6), 211 (8), 210 (37), 209 (100), 194 (17), 166 (9), 147
(7),134 (9),121 (72),106 (12), 93 (9), 78 (21), 77 (20); Anal. Calcd for
4.1.7.2. N-[4-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-3-
isoxazolyl]acetamide (20a). White solid; 38% yield; mp 205e207 ꢀC;
C
19H20N2O7: C 58.76; H 5.19; N 7.21. Found: C 58.83; H 5.23; N 7.16.
1H NMR (DMSO-d6): 2.10 (s, 3H, NC(O)CH3), 3.74 (s, 3H, OCH3-400),
d
3.81 (s, 3H, OCH3-40), 3.90 (s, 6H, 2ꢂ OCH3-300,500), 7.06 (d, J ¼ 8.8 Hz,
2H, H-30,50), 7.29 (s, 2H, H-200,600), 7.68 (d, J ¼ 8.8 Hz, 2H, H-20,60),
9.94 (s, 1H, NH-3); EIMS m/z 398 [M]þ (100), 383 (5), 367 (1), 356
(23), 195 (13), 194 (18), 135 (41), 43 (17); Anal. Calcd for
4.1.6.5. N0-{[(6,7-Dimethoxy-1,3-benzodioxol-5-yl)carbonyl]oxy}-2-
(4-methoxyphenyl)ethanimidamide (6e). White solid; 75% yield; mp
125e127 ꢀC; 1H NMR (DMSO-d6):
d
3.34 (s, 2H, CH2), 3.73 (s, 6H, 2ꢂ
OCH3-4,70), 3.93 (s, 3H, OCH3-60), 6.08 (s, 2H, OCH2O), 6.33 (s, 2H,
NH2), 6.89 (d, J ¼ 8.6 Hz, 2H, H-3,5), 7.02 (s, 1H, H-40), 7.28 (d,
J ¼ 8.6 Hz, 2H, H-2,6); EIMS m/z 388 [M]þ (3), 371 (1), 370 (2), 357
(7), 226 (6), 211 (6), 210 (18), 209 (100), 194 (5), 166 (8), 164 (7), 147
(6), 134 (8), 121 (50), 91 (6), 78 (13), 77 (13); Anal. Calcd for
C21H22N2O6: C 63.31; H 5.57; N 7.03. Found: C 63.47; H 5.65; N 6.91.
4 .1. 7 . 3 . N - A c e t yl - N - [ 4 - ( 4 - m e t h o x y p h e nyl ) - 5 - ( 3 , 4 , 5 -
trimethoxyphenyl)-3-isoxazolyl]acetamide (200a). White solid; 15%
yield; mp 205e207 ꢀC; 1H NMR (CDCl3):
d
2.35 (s, 6H, 2ꢂ NC(O)
C
19H20N2O7: C 58.76; H 5.19; N 7.21. Found: C 58.88; H 5.24; N 7.11.
CH3), 3.83 (s, 3H, OCH3-400), 3.93 (s, 6H, 2ꢂ OCH3-300,500), 3.97 (s, 3H,
OCH3-40), 6.99 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.30 (s, 2H, H-200,600), 7.53
(d, J ¼ 8.8 Hz, 2H, H-20,60); EIMS m/z 440 [M]þ (2), 398 (19), 383 (3),
357 (7), 195 (30), 180 (10), 168 (5), 135 (22), 92 (17), 43 (100); Anal.
Calcd for C23H24N2O7: C 62.72; H 5.49; N 6.36. Found: C 62.79; H
5.52; N 6.31.
4.1.6.6. N0-[(3-Methoxybenzoyl)oxy]-2-(4-methoxyphenyl)ethanimi-
damide (6f). White solid; 72% yield; mp 107e109 ꢀC; 1H NMR
(DMSO-d6):
d 3.35 (s, 2H, CH2), 3.73 (s, 3H, OCH3-4), 3.82 (s, 3H,
OCH3-30), 6.53 (s, 2H, NH2), 6.89 (d, J ¼ 8.7 Hz, 2H, H-3,5), 7.20 (dd,
J ¼ 8.0, 2.6 Hz, 1H, H-40), 7.30 (d, J ¼ 8.7 Hz, 2H, H-2,6), 7.41 (t,
J ¼ 8.0 Hz, 1H, H-50), 7.56 (dd, J ¼ 2.6, 1.5 Hz, 1H, H-20), 7.68 (dd,
J ¼ 8.0, 1.5 Hz, 1H, H-60); EIMS m/z 314 [M]þ (6), 297 (1), 296 (2), 162
(23), 147 (20), 135 (100), 121 (36), 107 (26), 92 (20), 78 (16), 77 (33);
Anal. Calcd for C17H18N2O4: C 64.96; H 5.77; N 8.91. Found: C 65.06;
H 5.79; N 8.85.
4.1.7.4. N-[5-(7-Methoxy-1,3-benzodioxol-5-yl)-4-(4-
methoxyphenyl)-3-isoxazolyl]acetamide (20b). White solid; 24%
yield; mp 195e197 ꢀC; 1H NMR (DMSO-d6):
d 2.09 (s, 3H, NC(O)
CH3), 3.81 (s, 3H, OCH3-700), 3.94 (s, 3H, OCH3-40), 6.12 (s, 2H,
OCH2O), 7.05 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.25 (d, J ¼ 1.1 Hz,1H, H-600),
7.29 (d, J ¼ 1.1 Hz, 1H, H-400), 7.67 (d, J ¼ 8.8 Hz, 2H, H-20,60), 9.93 (s,
1H, NH-3); EIMS m/z 382 [M]þ (24), 340 (27), 179 (31), 178 (31), 163
(15), 135 (100), 107 (11), 77 (31), 43 (70); Anal. Calcd for
4.1.6.7. N0-[(4-Methoxybenzoyl)oxy]-2-(4-methoxyphenyl)ethanimi-
damide (6g). White solid; 85% yield; mp 142e145 ꢀC; 1H NMR
(DMSO-d6):
d
3.32 (s, 2H, CH2), 3.73 (s, 3H, OCH3-4), 3.83 (s, 3H,
C20H18N2O6: C 62.82; H 4.74; N 7.33. Found: C 62.91; H 4.78; N 7.19.
OCH3-40), 6.47 (s, 2H, NH2), 6.89 (d, J ¼ 8.7 Hz, 2H, H-3,5), 7.01 (d,
J ¼ 8.9 Hz, 2H, H-30,50), 7.29 (d, J ¼ 8.7 Hz, 2H, H-2,6), 8.05 (d,
J ¼ 8.9 Hz, 2H, H-20, 60); EIMS m/z 314 [M]þ (1), 296 (1), 162 (9), 136
(10),135 (100),121 (22),107 (7), 92 (11), 78 (10), 77 (22); Anal. Calcd
for C17H18N2O4: C 64.96; H 5.77; N 8.91. Found: C 65.02; H 5.80; N
8.84.
4.1.7.5. N-[5-(2,3-Dihydro-8-methoxy-1,4-benzodioxin-6-yl)-4-(4-
methoxyphenyl)-3-isoxazolyl]acetamide (20c). White solid; 35%
yield; mp 221e223 ꢀC; 1H NMR (DMSO-d6):
d 2.08 (s, 3H, NC(O)
CH3), 3.81 (s, 3H, OCH3-800), 3.87 (s, 3H, OCH3-40), 4.30 (m, 4H,
OCH2CH2O), 6.63 (d, J ¼ 1.9 Hz, 1H, H-500), 7.05 (d, J ¼ 8.8 Hz, 2H, H-
30,50), 7.16 (s, 2H, H-500,700), 7.65 (d, J ¼ 8.8 Hz, 2H, H-20,60), 9.92 (s,
1H, NH); EIMS m/z 396 [M]þ (18), 354 (20), 193 (34), 192 (47), 136
(16), 135 (100), 107 (14), 92 (19), 77 (30), 43 (70); Anal. Calcd for
4.1.6.8. N0-[(4-Methoxybenzoyl)oxy]-2-(3,4,5-trimethoxyphenyl)
ethanimidamide (6h). White solid; 75% yield; mp 178e180 ꢀC; 1H
NMR (DMSO-d6):
d
3.35 (s, 2H, CH2), 3.64 (s, 3H, OCH3-4), 3.78 (s,
C21H20N2O6: C 63.63; H 5.09; N 7.07. Found: C 63.74; H 5.14; N 6.94.
6H, 2ꢂ OCH3-3,5), 3.83 (s, 3H, OCH3-40), 6.50 (s, 2H, NH2), 6.73 (s,
2H, H-2,6), 7.02 (d, J ¼ 8.9 Hz, 2H, H-30,50), 8.05 (d, J ¼ 8.9 Hz, 2H, H-
20,60); EIMS m/z 374 [M]þ (4), 356 (1), 207 (6), 181 (8), 152 (6), 136
(10),135 (100), 92 (5), 77 (9); Anal. Calcd for C19H22N2O6: C 60.95; H
5.92; N 7.48. Found: C 61.10; H 5.98; N 7.35.
4.1.7.6. N-[5-(4,7-Dimethoxy-1,3-benzodioxol-5-yl)-4-(4-
methoxyphenyl)-3-isoxazolyl]acetamide (20d). White solid; 12%
yield; mp 218e222 ꢀC; 1H NMR (DMSO-d6):
d 2.10 (s, 3H, NC(O)
CH3), 3.80 (s, 3H, OCH3-400), 3.87 (s, 3H, OCH3-700), 3.95 (s, 3H, OCH3-
40), 6.17 (s, 2H, OCH2O), 7.06 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.12 (s,1H, H-
600), 7.59 (d, J ¼ 8.8 Hz, 2H, H-20,60), 9.94 (s, 1H, NH-3); EIMS m/z 412
[M]þ (45), 370 (27), 209 (48), 208 (61), 135 (100), 107 (91), 77 (16),
43 (34); Anal. Calcd for C21H20N2O7: C 61.16; H 4.89; N 6.79. Found:
C 61.21; H 4.88; N 6.69.
4.1.7. General procedure for selective synthesis of 3-
acetylaminoisoxazoles 20aeh, n, o and 3-diacetylaminoisoxazoles
200a,f
A solution of benzoylamidoxime (5.3 mmol) in dry pyridine
(10 mL) was treated dropwise with acetylchloride (11 mmol) at
10 ꢀC (ice bath), the reaction mixture was stirred at room temper-
ature for 1.5 h, and poured into water (100 mL). The precipitate was
collected and purified by column chromatography (SiO2, EtOAc/
petroleum ether ¼ 1:5). During this procedure in case of derivative
6d besides isoxazol 2d the intermediate acetylated benzoylami-
doxime 7d was isolated.
4.1.7.7. N-[5-(6,7-Dimethoxy-1,3-benzodioxol-5-yl)-4-(4-
methoxyphenyl)-3-isoxazolyl]acetamide (20e). White solid; 18%
yield; mp 140e143 ꢀC; 1H NMR (DMSO-d6):
d 2.09 (s, 3H, NC(O)
CH3), 3.70 (s, 3H, OCH3-600), 3.80 (s, 3H, OCH3-700), 4.00 (s, 3H, OCH3-
40), 6.11 (s, 2H, OCH2O), 7.06 (d, J ¼ 8.7 Hz, 2H, H-30,50), 7.09 (s, 1H,
H-400), 7.59 (d, J ¼ 8.7 Hz, 2H, H-20,60), 9.93 (s, 1H, NH-3); EIMS m/z

124
D.V. Tsyganov et al. / European Journal of Medicinal Chemistry 73 (2014) 112e125
412 [M]þ (26), 370 (13), 209 (23), 208 (36), 193 (11), 163 (12), 135
(100), 107 (11), 92 (14), 77 (24), 43 (46); Anal. Calcd for C21H20N2O7:
C 61.16; H 4.89; N 6.79. Found: C 61.29; H 4.97; N 6.64.
4.2. Biology. Sea urchin embryo assay [58]
Adult sea urchins Paracentrotus lividus were collected from the
Mediterranean Sea at the Cyprus coast and kept in an aerated
seawater tank. Gametes were obtained by intracoelomic injection
of 0.5 M KCl. Eggs were washed with filtered seawater and fertilized
by adding drops of a diluted sperm. Embryos were cultured at room
temperature under gentle agitation with a motor-driven plastic
paddle (60 rpm) in filtered seawater. The embryos were observed
with a light microscope Biolam (LOMO, S.-Petersburg, Russia). For
treatment with the test compounds, 5 mL aliquots of embryo sus-
pension were transferred to 6-well plates and incubated as a
monolayer at a concentration up to 2000 embryos/mL. Stock so-
lutions of compounds were prepared in DMSO at 10 mM concen-
tration, followed by a 10-fold dilution with 96% EtOH. This
procedure enhanced solubility of the test compounds in the salt-
containing medium (seawater), as evidenced by microscopic ex-
amination of the samples. The maximal tolerated concentrations of
DMSO and EtOH in the in vivo assay were determined to be 0.05%
and 1%, respectively. Higher concentrations of either DMSO (ꢁ0.1%)
or EtOH (>1%) caused non-specific alteration and retardation of the
sea urchin embryo development independent of the treatment
stage. Combretastatins A-4 and A-2 served as reference
compounds.
4.1.7.8. N-[5-(3-Methoxyphenyl)-4-(4-methoxyphenyl)-3-isoxazolyl]
acetamide (20f). White solid; 8% yield; mp 185e187 ꢀC; 1H NMR
(DMSO-d6): d
2.09 (s, 3H, NC(O)CH3), 3.81 (s, 3H, OCH3-300), 3.86 (s,
3H, OCH3-40), 7.07 (d, J ¼ 8.8 Hz, 2H, H-30,50), 7.12 (dd, J ¼ 8.0, 2.4 Hz,
1H, H-400), 7.48 (t, J ¼ 8.0 Hz, 1H, H-500), 7.52 (t, J ¼ 2.4 Hz, 1H, H-200),
7.63 (d, J ¼ 8.0 Hz, 1H, H-600), 7.67 (d, J ¼ 8.0 Hz, 2H, H-20,60), 9.99 (s,
1H, NH-3); EIMS m/z 338 [M]þ (99), 297 (18), 296 (100), 163 (17),
136 (17), 135 (94), 107 (9), 92 (11), 77 (15), 43 (26); Anal. Calcd for
C
19H18N2O4: C 67.45; H 5.36; N 8.28. Found: C 67.59; H 5.42; N 8.17.
4.1.7.9. N-Acetyl-N-[5-(3-methoxyphenyl)-4-(4-methoxyphenyl)-3-
isoxazolyl]acetamide (200f). Oil; 8% yield; 1H NMR (CDCl3):
2.40 (s,
d
6H, 2ꢂ NC(O)CH3), 3.85 (s, 3H, OCH3-40), 3.89 (s, 3H, OCH3-300), 6.98
(d, J ¼ 8.9 Hz, 2H, H-30,50), 7.04 (dd, J ¼ 8.0, 2.6 Hz, 1H, H-400), 7.40 (t,
J ¼ 8.0 Hz, 1H, H-500), 7.54 (d, J ¼ 8.9 Hz, 2H, H-20,60), 7.59 (t,
J ¼ 2.6 Hz, 1H, H-200), 7.67 (dd, J ¼ 8.0, 2.6 Hz, 1H, H-600); EIMS m/z
380 [M]þ (8), 398 (19), 338 (28), 297 (10), 296 (50), 204 (30), 161
(47), 135 (100), 121 (41), 107 (60), 92 (71), 43 (100); Anal. Calcd for
C
21H20N2O5: C 66.31; H 5.30; N 7.36. Found: C 62.37; H 5.32; N 7.30.
The antiproliferative activity was assessed by exposing fertilized
eggs (8e20 min after fertilization, 43e55 min before the first
mitotic cycle completion) to 2-fold decreasing concentrations of
the compound. Cleavage alteration and arrest were clearly detected
at 2.5e5.5 h after fertilization. The effects were quantitatively
estimated as effective threshold concentrations (EC) resulting in
cleavage alteration or full mitotic arrest. At these concentrations, all
tested microtubule destabilizers caused 100% cleavage alteration
and embryo death before hatching, whereas at 2-fold lower con-
centrations, the compounds failed to produce any effect. For
microtubule destabilizing activity, the compounds were tested on
free-swimming blastulae just after hatching (9e10 h after fertil-
ization), originated from the same embryo culture. Embryo spin-
ning was observed after 15 min to 20 h of treatment, depending on
the structure and concentration of the compound. Both spinning
and lack of forward movement were interpreted to be the result of
the microtubule destabilizing activity of a molecule. Video illus-
trations are available at http://www.chemblock.com.
4.1.7.10. N-[4,5-Bis(4-methoxyphenyl)-3-isoxazolyl]acetamide (20g).
White solid; 16% yield; mp 201e203 ꢀC; 1H NMR (DMSO-d6):
2.09
d
(s, 3H, NC(O)CH3), 3.81 (s, 3H, OCH3-40), 3.85 (s, 3H, OCH3-400), 7.06
(d, J ¼ 8.8 Hz, 2H, H-30,50), 7.11 (d, J ¼ 8.8 Hz, 2H, H-300,500), 7.63 (d,
J ¼ 8.8 Hz, 2H, H-20,60), 7.97 (d, J ¼ 8.8 Hz, 2H, H-200,600), 9.92 (s, 1H,
NH-3); EIMS m/z 338 [M]þ (45), 297 (18), 296 (50), 163 (10), 162 (6),
136 (12), 135 (100), 134 (34), 107 (6), 92 (12), 77 (14), 43 (28); Anal.
Calcd for C19H18N2O4: C 67.45; H 5.36; N 8.28. Found: C 67.49; H
5.39; N 8.21.
4.1.7.11. N-[5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3-
isoxazolyl]acetamide (20h). White solid; 26% yield; mp 196e199 ꢀC;
1H NMR (DMSO-d6): 2.11 (s, 3H, NC(O)CH3), 3.72 (s, 3H, OCH3-40),
d
3.86 (s, 9H, 3ꢂ OCH3-30,50,400), 6.99 (s, 2H, H-20,60), 7.12 (d, J ¼ 8.8 Hz,
2H, H-300,500), 8.02 (d, J ¼ 8.8 Hz, 2H, H-200,600), 10.03 (s, 1H, NH);
EIMS m/z 398 [M]þ (68), 356 (16), 342 (13), 341 (64), 222 (16), 195
(60), 135 (100), 134 (40), 92 (9), 77 (18), 43 (74); Anal. Calcd for
Both sea urchin embryo assay and DTP NCI60 cell line activity
data are available free of charge via the Internet at http://www.
zelinsky.ru.
C
21H22N2O6: C 63.31; H 5.57; N 7.03. Found: C 63.42; H 5.60; N 6.94.
4.1.7.12. N-[5-(4-Methoxyphenyl)-4-(4-chlorophenyl)-3-isoxazolyl]
acetamide (20n). White solid; 85% yield; mp 252e254 ꢀC (MeOH);
Acknowledgments
1H NMR (DMSO-d6):
d
2.11 (s, 3H, COCH3), 3.85 (s, 3H, OCH3-400),
7.12 (d, J ¼ 8.5 Hz, 2H, H-300,500), 7.54 (d, J ¼ 8.0 Hz, 2H, H-30,50), 7.68
(d, J ¼ 8.0 Hz, 2H, H-20,60), 8.00 (d, J ¼ 8.5 Hz, 2H, H-200,600), 10.11 (s,
1H, NH); EIMS m/z 344 [Mþ2]þ (4), 342 [M]þ (12), 302 (7), 300 (22),
167 (6), 141 (13), 139 (40), 135 (31), 134 (100), 113 (9), 111 (29), 90
(5), 77 (5), 43 (58); Anal. Calcd for C18H15ClN2O3: C 63.07; H 4.41; N
8.17. Found: C 63.15; H 4.37; N 8.28.
We thank the National Cancer Institute (NCI) (Bethesda, MD,
USA) for screening compounds 1a, 1b, 1c, 1e, and 2i by the
Developmental Therapeutics Program at NCI (Anti-cancer
Screening Program; http://dtp.cancer.gov). This work was sup-
ported by the grant # 13-03-90455 from Russian Foundation for
Basic Research (RFBR), grant # F53.4/056 from State Fund for
Fundamental Research (DFFD), Ukraine, and a grant from Chemical
Block Ltd.
4.1.7.13. N-[5-(4-Methoxyphenyl)-4-(4-nitrophenyl)-3-isoxazolyl]
acetamide (20o). White solid; 25% yield; mp 243e247 ꢀC (MeOH);
1H NMR (DMSO-d6):
d
2.14 (s, 3H, COCH3), 3.86 (s, 3H, OCH3-400),
7.14 (d, J ¼ 8.8 Hz, 2H, H-300,500), 7.87 (d, J ¼ 8.9 Hz, 2H, H-20,60), 8.05
(d, J ¼ 8.8 Hz, 2H, H-200,600), 8.31 (d, J ¼ 8.9 Hz, 2H, H-30,50), 10.40 (s,
1H, NH); EIMS m/z 353 [M]þ (14), 311 (32), 178 (3), 150 (13), 135
(38), 134 (100), 119 (5), 117 (4), 104 (22), 92 (10), 90 (6), 77 (7), 76
(18), 43 (69); Anal. Calcd for C18H15N3O5: C 61.19; H 4.28; N 11.89.
Found: C 61.25; H 4.36; N 11.81.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.12.
006. These data include MOL files and InChiKeys of the most
important compounds described in this article.

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