Regioselective synthesis of 3,4-diaryl-5-unsubstituted isoxazoles, analogues of natural cytostatic combretastatin A4

Article abstract of DOI:10.1016/j.ejmech.2018.01.070

4,5-Diarylisoxazoles are potent antiproliferative tubulin-targeting agents. Their isomeric 3,4-diaryl-5-unsubstituted isoxazoles are hardly accessible. The synthesis of 3,4-diaryl-5-unsubstituted isoxazoles 13 was designed based on a condensation of arylb

Full text of DOI:10.1016/j.ejmech.2018.01.070

Accepted Manuscript
Regioselective synthesis of 3,4-diaryl-5-unsubstituted isoxazoles, analogues of
natural cytostatic combretastatin A4
Natalia B. Chernysheva, Anna S. Maksimenko, Fedor A. Andreyanov, Victor P. Kislyi,
Yuri A. Strelenko, Victor N. Khrustalev, Marina N. Semenova, Victor V. Semenov
PII:
S0223-5234(18)30083-7
10.1016/j.ejmech.2018.01.070
EJMECH 10147
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 August 2017
Revised Date: 6 January 2018
Accepted Date: 20 January 2018
Please cite this article as: N.B. Chernysheva, A.S. Maksimenko, F.A. Andreyanov, V.P. Kislyi, Y.A.
Strelenko, V.N. Khrustalev, M.N. Semenova, V.V. Semenov, Regioselective synthesis of 3,4-diaryl-5-
unsubstituted isoxazoles, analogues of natural cytostatic combretastatin A4, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.070.
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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT
Regioselective synthesis of 3,4-diaryl-5-unsubstituted isoxazoles, analogues of natural cytostatic
combretastatin A4
Natalia B. Chernysheva a, Anna S. Maksimenko a,b, Fedor A. Andreyanov a, Victor P. Kislyi a, Yuri A.
Strelenko a, Victor N. Khrustalev c,d, Marina N. Semenova e, Victor V. Semenov a,*
a N. D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russian
Federation
b D. I. Mendeleev University of Chemical Technology of Russia, 9 Miusskaya pl., 125047 Moscow,
Russian Federation
c Faculty of Science, RUDN University, 6 Miklukho-Maklay Street, 117198 Moscow, Russian
Federation
d A. N. Nesmeyanov Institute of Organoelement Compounds RAS, 28 Vavilov Street, 119991
Moscow, Russian Federation
e N. K. Koltzov Institute of Developmental Biology RAS, 26 Vavilov Street, 119334 Moscow, Russian
Federation
Corresponding author: Victor V. Semenov
Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991,
Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966.
E-mail: vs@zelinsky.ru
E-mail addresses:
Natalia B. Chernysheva
Anna S. Maksimenko
Fedor A. Andreyanov
Victor P. Kislyi
info@chemblock.com
chem.annamaks@gmail.com
andreyanovfeodor@gmail.com
vkislyi@yandex.ru
Yuri A. Strelenko
strel@ioc.ac.ru
Victor N. Khrustalev
Marina N. Semenova
Victor V. Semenov
vkh@xray.ineos.ac.ru
ms@chemical-block.com
vs@zelinsky.ru

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ABSTRACT
4,5-Diarylisoxazoles are potent antiproliferative tubulin-targeting agents. Their isomeric 3,4-diaryl-5-
unsubstituted isoxazoles are hardly accessible. The synthesis of 3,4-diaryl-5-unsubstituted isoxazoles
13 was designed based on a condensation of arylbenzaldehydes, arylnitromethanes, and
ethoxycarbonylmethylpyridinium bromide followed by a selective one-step transformation of
intermediate 3,4-diaryl-5-ethoxycarbonyl-4,5-dihydroisoxazole 2-oxides 8. The orientation of aryl
rings in relation to isoxazole heterocycle was confirmed by X-ray crystallography. Targeted
compounds were evaluated for antimitotic microtubule destabilizing activity using a phenotypic sea
urchin embryo assay. 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole 13e and 13h with a
single methoxy substituent were the most potent. Compound 13e showed strong cytotoxicity in NCI60
screen with GI50 for NCI-H522 human lung cancer cell line of 0.023 µM.
Keywords:
3,4-Diarylisoxazoles
Nitrostilbenes
Microtubule destabilization
Sea urchin embryo
Abbreviations:
CA4, combretastatin A-4
SAR, structure-activity relationship

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1. Introduction
o-Diarylazoles can be considered as cis-restricted analogues of combretastatin A-4 (CA4, Fig.
1), a highly potent natural cytostatic originally isolated from the bark of African willow tree
Combretum caffrum [1]. Numerous structure-activity relationship (SAR) studies identified that 3,4,5-
trimethoxyphenyl ring A and 4-methoxyphenyl ring B bound by cis-double bond are essential for
antimitotic microtubule destabilizing effect of CA4, whereas the presence of 3-hydroxy group in the
ring B is irrelevant [2–4]. To avoid cis-trans isomerization that causes activity decrease, diverse N-
containing rings have been introduced to replace the ethene linker, resulting in compounds with
antiproliferative antitubulin effects comparable with those of CA4 [4]. Among them, a number of
diaryl-o-substituted isoxazoles were identified as potent antimitotic microtubule destabilizing agents
[5–10]. Particularly, diaryl-o-isoxazole 1 (Fig. 1) effectively inhibited both cancer cell growth and in
vitro tubulin polymerization [5,7].
Fig. 1. Structures of combretastatin A-4 and their diaryl-o-substituted isoxazole analogues.
The removal of hydroxy group from the ring B also resulted in potent cytotoxic compounds 2,
KRIBB3, and 3 (Fig. 1) that strongly suppressed cancer cell growth both in vitro and in vivo in mouse
xenograft model, disrupted cellular microtubules, induced mitotic arrest followed by apoptosis, and
displayed antiangiogenic effect [6–8]. On the other hand, the position and number of substituents in
benzene rings as well as the orientation of isoxazole heterocycle relating to aryl rings could
significantly affect biological activity [5–8,10].
Up to date, no reliable and universal synthetic strategy for 3,4-diaryl-5-unsubstituted isoxazoles
4b (Fig. 2) has been published, particularly due to the unpredictable regioselectivity. Generally, the
reported reaction routes required expensive catalysts and scarcely available reagents, and could not be
reproduced on a multigram scale [11]. According to literature, a conventional procedure for the
synthesis of polymethoxy-4,5-diarylisoxazoles 4a includes cyclization of easily accessible
diarylketoaldehydes with hydroxylamine (Fig. 2) [5–7,10].

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Fig. 2. General route for the preparation of diarylisoxazoles.
It was reported that the same chemical route afforded both polymethoxy-4,5-diarylisoxazoles
4a and their 3,4-diaryl-substituted analogues 4b, including compound 3 (Fig. 1 and 2) [6]. However,
the identification of the isomers had come into question, since their published NMR spectra were
identical. Similarly, the structure of 3,4-diarylisoxazoles 4b (Fig. 2) obtained using the same reaction
route by another research group [12] required further clarification by proper analytic procedures.
Notably, all other literature data confirmed the formation of isomers 4a by the above cyclization
pathway (Fig. 2) [5,7,10,13]. In the present study a facile synthetic procedure for 5-unsubstituted 3,4-
diarylisoxazoles using available materials was developed. The targeted molecules were evaluated for
their antimitotic microtubule destabilizing activity in a sea urchin embryo model.
2. Results and discussion
2.1. Chemistry
Starting diarylnitrostilbenes 7a–j were easily synthesized by condensation of
phenylnitromethanes 5 with aldehydes 6 [14–16]. At the next step compounds 7 reacted with
ethoxycarbonyl methylpyridinium bromide in the presence of Et3N to afford the respective 3,4-
diarylisoxazoline N-oxide-5-carboxylates 8 with good yields (Scheme 1) [16].

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Scheme 1. Synthesis of diarylnitrostilbenes 7 and 3,4-diaryl-5-ethoxycarbonyl-4,5-dihydroisoxazole 2-
oxides 8.
The reported straightforward rearrangement of isoxazolines to the respective isoxazoles [17]
implied possible application of this reaction to afford 3,4-diaryl-5-ethoxycarbonylisoxazoles A. It was
found, however, that only isoxazolines 8a–j, but not isoxazoles A, were formed at different conditions:
20 °C, 100–200 h, or 60 °C, 4–5 h (Scheme 1) [16]. Nitro derivative 8b was the only exception capable
to convert partially into the respective 3,4-diaryl-5-ethoxycarbonylisoxazole. Therefore, the next step
of the study was to develop a chemical strategy for the transformation of 8a-j to 3,4-diaryl-5-
ethoxycarbonylisoxazoles A. According to literature, the rearrangement of 3,4-diphenylisoxazoline
oxide B proceeded in water solution of 2% NaOH at room temperature during 24 h, yielding
monoxime C (40%) and isoxazole D (30%) (Scheme 2). The plausible mechanism of the reaction
included an opening of 3,4-diphenylisoxazoline N-oxide ring with the cleavage of N–O bond affording
tricarbonylmonoxime C, followed by a cyclization and dehydration resulting in 3,4-
diphenylbenzoylisoxazole D [17a,b].

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Scheme 2. Rearrangement of 3,4-diphenylisoxazoline oxide B.
We intended to use these reaction conditions for the rearrangement of isoxazoline N-oxides 8 to
3,4-diaryl-5-ethoxycarbonylisoxazoles A. However, instead of anticipated compounds A or 5-
carboxyisoxazoles 12a–j, 5-unsubstituted isoxazoles 13a–j with minor impurities of 12a–j were
formed at 20 °C and reaction time of 100–200 h. It was further found that the yields as well as relative
content of minor 12 and major 13 components were similar at different temperatures. Targeted
unsubstituted 3,4-diarylisoxazoles 13 were easily separated in alkaline media from byproduct acids 12.
Finally, optimal reaction conditions were found to be 60 °C (5–6 h) with the ratio of 12:13 = 1:2 – 1:7.
A putative mechanism of this reaction is presented in Scheme 3.
Scheme 3. Proposed mechanism of transformation of 3,4-diaryl-5-ethoxycarbonyl-4,5-
dihydroisoxazole 2-oxides 8 to 5-unsubstituted 3,4-diarylisoxazoles 13.
Starting compounds 8 were not detected by TLC in the reaction mixtures after ~ 2 h. Initially the ester
group of 8 was hydrolyzed to afford isoxazoline acid Na-salts 9. Then isoxazoline ring of 9 was
recyclized with cleavage of N-O bond to afford 2-oxo-carboxylic acid salts 10. It is well known that
such acids are easily decarboxylated in acidic and alkaline media even at room temperature resulting in
oxyimino aldehydes 11 [18]. Then intermediate ketocarboxylic salts 10 and oxyimino aldehydes 11
were cyclized to corresponding 5-carboxyisoxazoles 12 and unsubstituted isoxazoles 13, respectively,
like the cyclization of oxyimino ketone C (Scheme 2) [17a,b].
During the rearrangement of 9a and 9c intermediate aldehydes 11a and 11c were found in the
reaction mixture. Aromatic protons and doublets of CHO and CH-Ar were observed in 1H NMR
spectra of products separated at the reaction half-time (11a:
δ 8.8, d, J = 7.5 Hz; 5.61, d, J = 7.5 Hz;
11c: 8.9, d, J =7.65 Hz; 5.52, d, J =7.6 Hz). Besides, there was proton signal of C=NOH in 11a at
δ

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δ
12.8 ppm. Integral intensities were corresponded to single protons in CHO, CH-Ar, and C=NOH
groups. To confirm this reaction route, isoxazoline oxide acid of 9a was synthesized by acidic
hydrolysis of ester 8a with high yield (98%). The solution of this acid was kept in EtOH–H2O–NaOH
(2%) at room temperature during 100 h, finally yielding 5-carboxyisoxazole 12a (17%) and isoxazole
13a (67%) (Scheme 4). Additionally, 5-carboxyisoxazole 12a failed to transform to 13a under the
same conditions (2% NaOH/EtOH/H2O, 60 °C, 5 h), and 90% of unconverted 12a was separated from
the reaction mixture. This observation was in a good agreement with literature data that
decarboxylation of 3,4-diarylisoxazole-5-carboxylic acids proceeded at high temperature (200 °C, 3 h)
and with low yield (5%) [11b].
Scheme 4. Synthesis and transformation of isoxazoline 2-oxide 9a-acid to corresponding 5-
unsubstituted 3,4-diarylisoxazole 13a.
Finally the study of reaction mechanism resulted in the development of a simple and reliable
procedure for the selective synthesis of hardly accessible 5-unsubstituted 3,4-diarylisoxazoles. The
structures of 12 and 13 were proved by 1H-, 13C-NMR, and MS analyses. To confirm the structure of
regioisomers (4a or 4b, Fig. 2), 5-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole 14 (Fig. 3)
was synthesized according to procedure [7] and compared with the respective 3,4-diarylisoxazole 13e
by 1H NMR.
Fig. 3. Structure of 3,4-diarylisoxazole 13e and its 4,5-diarylisoxazole regioisomer 14.
The difference of 0.27 ppm in the chemical shift of the isoxazole proton in 13e (8.48) and in 14 (8.31)
could be considered as a reliable basis for distinguishing regioisomers 4a and 4b (Fig. 2). In addition,

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the chemical shifts of aromatic protons in 13e and 14 were different as well (Experimental section,
4.1.5.7 and 4.1.5.8).
The structure of isoxazole 13e was characterized by single-crystal X-ray diffraction study (Fig.
4). The geometrical parameters for 13e are available in Supplementary data (Table S1). Unsubstituted
at C-5 position 3,4-diarylisoxazoles were not published previously [19]. Specifically, the isoxazole
ring in 13e was planar with all bond lengths matching conventional O─N, O─C, C─C, N═C and C═C
bonds reported for the related 4,5-diarylisoxazoles unsubstituted at C-3 position [20,21]. The 3- and 4-
aryl substituents were not coplanar to the central isoxazole ring (the dihedral angles are 24.38(6) and
61.56(6)°, respectively). The twist angles of aryl groups at C-4 position were usually larger than those
of aryl groups at C-3 and C-5 positions in the corresponding 3,4- and 4,5-diarylisoxazoles [20,21,22].
Notably, the 4-methoxy group in 3-(4-methoxyphenyl) substituent as well as 3- and 5-methoxy groups
in 4-(3,4,5-trimethoxyphenyl) substituent were found to be within the planes of the corresponding
benzene rings (the C10─C9─O2─C12, C14─C15─O3─C19 and C18─C17─O5─C21 torsion angles
are -5.9(2), -6.9(3) and -8.4(3)°, respectively), whereas the 4-methoxy group in 4-(3,4,5-
trimethoxyphenyl) substituent was rotated relative to the benzene plane (the C15─C16─O4─C20
torsion angle is -100.5(2)°).

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Fig. 4. Molecular structure of 3-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole 13e (50%
ellipsoids).
2.2. Biological evaluation
Compounds 7, 8, 12 and 13 were screened for antimitotic microtubule destabilizing activity
using a phenotypic sea urchin embryo assay [23]. The two-step assay includes the treatment of
fertilized eggs to assess cell division (cleavage) alteration/arrest. Specific swimming changes of
hatched blastulae exposed to a compound, namely, rapid spinning at the bottom of the culture well
instead of forward swimming near the surface, indicate microtubule destabilizing mechanism of action.
The results are presented in Table 1.

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Table 1.
Effects of diarylethylenes 7 and diarylisoxazoles 8, 12, and 13 on sea urchin embryos
Sea urchin embryo effects, EC (µM)a
Compd
Cleavage alteration
Cleavage arrest
Embryo spinning
1
0.2
2
4
2
>4
2
7e
7f
>4
>4
4
7g
2
4
7h
2
4
1
7j
>4
>4
4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>40
>5
>4
>4
>4
>10
>10
1
8a
>4
8b
>4
8c
>4
>4
>4
>4
>4
20
>4
8d
>4
8e
>4
8j
>4
12a
12c
12d
12e
12j
13a
13b
13c
13d
13e
14
>4
>40
2 (TE)b
>4
0.2
>4
>4
>4
0.52
0.2
0.01
0.005
0.5
>4
0.005
4
>4
>4
4 (TE)b
2 (TE)b
0.1
0.1
>4
0.5
>4
>4
0.1
>4
>4
0.05
13f
13g
13h
13i
13j
CA4
>4
0.05
>4
2
>4
0.002
0.01

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a The sea urchin embryo assay was conducted as described previously [23]. 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 TE: tuberculate eggs typical of microtubule destabilizing agents.
It was reported previously that compound 7e inhibited growth of chick embryonic cardiac
fibroblasts [14]. Our data confirmed moderate antiproliferative effects of diarylnitroethylenes 7
associated with both microtubule destabilization and tubulin unrelated toxicity. Specifically,
compounds 7e, 7f, and 7j caused embryo destruction and death at 2–4 µM concentration. According to
literature, introduction of NO2 group into the double bond of benzyl CA4 analogues dramatically
decreased cytotoxicity, although did not affect antitubulin activity [24]. Similar to our results for series
7, 1-(3′,4′,5′-trimethoxyphenyl)-2-nitroethylene, together with inhibition of tubulin polymerization,
was found to exhibit tubulin-unrelated cytotoxicity [25]. Transformation of 7 to dihydroisoxazole 2-
oxides 8 resulted in activity loss. Further transformation to diarylisoxazole-5-carbonic acids 12 also
afforded inactive compounds with the only one exception for 3,4,5-trimethoxyphenyl derivative 12e
that produced weak microtubule destabilizing effect. Final 3,4-diarylisoxazoles 13c–e, h with
polymethoxy-substituted rings A and B displayed antimitotic antitubulin activity. Among them,
surprisingly, 13h with only one methoxy group was the most potent in the sea urchin embryo assay,
whereas trimethoxy-substituted 13i showed negligible activity. A close analogue of CA4, 3-(4-
methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole 13e, inhibited cleavage at low nanomolar
concentration. This compound exhibited pronounced cytotoxicity in NCI60 screen with average GI50
of 0.17 µM for 60 human cancer cell lines. NCI-H522 non-small cell lung cancer cell line was the
most sensitive with GI50 = 0.023 µM and TGI (total growth inhibition concentration) = 0.075 µM.
4,5-Diarylisoxazole 14, the corresponding regioisomer of 13e, showed similar but slightly higher
antimitotic antitubulin activity. Thiophene derivative 13f could be considered as antiproliferative agent
with non-tubulin mode of action.
3. Conclusions
The synthesis of 3,4-diaryl-5-unsubstituted isoxazoles 13 was developed using readily available
starting phenylnitromethanes 5 and arylbenzaldehydes 6 that condensed easily with formation of 1,2-
diaryl-1-nitroethylenes 7. Further reaction with ethoxycarbonylmethylpyridinium bromide at 60 °C for
4–6 h yielded intermediate 3,4-diaryl-5-ethoxycarbonyl-4,5-dihydroisoxazole 2-oxides 8. Subsequent
one-step isomerization with decarboxylation of 8 afforded the targeted 3,4-diaryl-5-unsubstituted
isoxazoles 13. The synthesized compounds were evaluated for antiproliferative activity in a sea urchin
embryo model. 4-(4-Methoxyphenyl)-3-phenylisoxazole 13h and 3-(4-methoxyphenyl)-4-(3,4,5-

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trimethoxyphenyl)isoxazole 13e were identified as the most potent antimitotic microtubule
destabilizing agents. 13e exhibited high cytotoxicity against a panel of human cancer cell lines in
NCI60 screen.
4. Experimental section
4.1. Chemistry. Materials and methods
Melting points were measured on a Boetius melting point apparatus and were uncorrected. 1H
NMR and 13C NMR spectra were recorded on a Bruker DRX-500 instrument [working frequencies of
500.13 MHz (1H) and 125.76 MHz (13C)]. Chemical shifts were stated in parts per million (ppm) and
referenced to the appropriate NMR solvent peaks. Spin-spin coupling constants (J) were reported in
Hertz (Hz). NMR spectra (Supplementary data) were prepared using an original software designed at
N. D. Zelinsky Institute of Organic Chemistry RAS (Moscow, Russian Federation)
(http://nmr.ioc.ac.ru:8080/SDF2PDF.kl1). Low resolution mass spectra (m/z) were recorded on a
Finnigan MAT/INCOS 50 mass spectrometer at 70 eV using direct probe injection. High resolution
mass spectra (HRMS) were measured on a Bruker maXis and micrOTOF II instruments using
electrospray ionization (ESI). Elemental analysis was performed on the automated Perkin-Elmer 2400
CHN microanalyzer. Flash chromatography was carried out on silica gel (Acros, 0.035–0.070 mm, 60
Å). TLC was performed on Merck 60 F254 plates. Solvents, benzaldehydes, and ethyl bromoacetate
were purchased from Acros Organics (Belgium) at the highest commercial quality and used as
received. 4,7-Dimethoxy-2H-1,3-benzodioxole-5-carbaldehyde was synthesized from apiol, isolated
from parsley seed extract [26]. Phenylnitromethanes 5 were synthesized by modified procedure
[16,27].
4.1.1. General procedure for the preparation of 1,2-diaryl-1-nitroethylenes 7 [14,15,16]
A mixture of phenylnitromethane 5 (12 mmol), aldehyde 6 (13 mmol), MeOH (4 mL),
MeNH2 HCl (0.1 g, 1.46 mmol), and NaHCO3 (0.04 g, 0.48 mmol) was stirred at room temperature for
120–200 h (TLC control) or at 60 °C for 4–6 h. Methanol was evaporated and methylene chloride (70
mL) was added to the mixture. The solution was washed with water (3 × 20 mL), dried by filtration
through cotton wool, then the solvent was removed and the residue was triturated with minimal
amount of MeOH. Nitroethylene crystals were filtered, washed with methanol and dried in vacuo.
4.1.2. General procedure for the preparation of 3,4-diaryl-5-ethoxycarbonyl-4,5-dihydroisoxazole 2-
oxides 8 [16].

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A mixture of the corresponding nitroethylene (5 mmol), ethoxycarbonylmethylpyridinium
bromide [28] (7.5 mmol), and triethylamine (15 mmol) in dry acetonitrile (40 mL) was stirred at 60 °C
for 4–5 h (TLC control). Acetonitrile was removed in vacuo, and then methylene chloride (150 mL)
was added. The methylene chloride extract was washed with water, 5% aqueous HCl, water until pH 7,
and dried by filtration through cotton wool. Methylene chloride was removed in vacuo and the residue
was triturated in ethanol. The collected crystals were washed with ethanol and dried in air to obtain a
4,5-dihydroisoxazole 2-oxides 8. When crystallization was unavailable, 4,5-dihydroisoxazole 2-oxides
8 were isolated by column chromatography. For each compound, the appropriate conditions are
specified.
4.1.3. Synthesis of 5-carboxy-4-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydroisoxazole 2-oxide
(9a).
A solution of compound 8a (0.546 g, 1.5 mmol) in dioxane (15 mL) and aqueous HCl (9%, 3
mL) was heated at ~ 60 °C for 5 h, then kept overnight at room temperature. Dioxane was evaporated
in vacuo and the residue was extracted with ethyl acetate (3 × 25 mL). The extracts were washed with
water, brine, dried with anhydrous sodium sulfate. The solvent was removed in vacuo to afford 9a as
white solid (0.512 g, 98% yield); mp 116–119 °C; 1H NMR (500 MHz, DMSO-d6)
δ 13.75 (1H, br s,
COOH), 7.85 (2H, d, J = 9.0 Hz, H-2′,6′), 7.45 (2H, d, J = 8.4 Hz, H-3′′,5′′), 7.39 (2H, d, J = 8.4 Hz,
H-2′′,6′′), 6.97 (2H, d, J = 9.0 Hz, H-3′,5′), 5.39 (1H, d, J = 2,4 Hz, H-4), 4.99 (1H, d, J = 2.4 Hz, H-
5), 3.75 (3H, s, OMe); 13C NMR (500 MHz, DMSO-d6)
δ 173.3, 1159.9, 138.0, 132.7, 129.2 (2C),
129.1 (2C), 128.2 (2C), 117.4, 114.3 (2C), 114.0, 77.4, 55.2, 51.9; EIMS m/z 347 [M]+(17), 316 (17),
303 (16), 135 (100). Anal. Calcd for C17H14ClNO5: C, 58.72; H, 4.06; N, 4.03. Found: C, 58.65; H,
4.16; N, 4.25.
4.1.4. Synthesis of 4-(4-chlorophenyl)-3-(4-methoxyphenyl)isoxazole-5-carboxylic acid (12a) and 4-(4-
chlorophenyl)-3-(4-methoxyphenyl)isoxazole (13a).
A mixture of acid 9a (1 mmol, 0.348 g), 2% ethanolic NaOH solution (7 mL), and water (3
mL) was stirred at 60 °C for 6 h, then ethanol was removed in vacuo. The residue was suspended in
methylene chloride (100 mL), aqueous alkaline layer was separated, and the methylene chloride
extract was washed with water (2 × 20 mL). The methylene chloride solution was dried by filtration
through cotton wool and evaporated in vacuo to afford 0.19 g (67%) of 4-(4-chlorophenyl)-3-(4-
methoxyphenyl)isoxazole 13a as white crystals; mp 89–91 °C. 1H NMR (500 MHz, CDCl3)
(1H, s, H-5), 7.41 (2H, d, J = 8.8 Hz, H-2′,6′), 7.33 (2H, d, J = 8.5 Hz, H-2′′,6′′), 7.19 (2H, d, J = 8.5
Hz, H-3′′,5′′), 6.90 (2H, d, J = 8.8 Hz, H-3′,5′), 3.83 (3H, s, OMe); 13C NMR (500 MHz, CDCl3)
δ 8.48
δ

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160.8, 159.7, 156.2, 134.0, 130.1 (2C), 130.0 (2C), 129.0 (2C), 127.7, 120.4, 119.6, 114.2 (2C), 55.3;
EIMS m/z 285 [M]+(100), 257 (29), 242 (46). Anal. Calcd for C16H12ClNO2: C, 67.26; H, 4.23; N,
4.90. Found: C, 67.42; H, 4.31; N, 4.72.
The washings were combined with the aqueous alkaline layer. The combined alkaline solution
was acidified using 10% aqueous HCl (pH 1-2) and extracted with ethyl acetate (2 × 35 mL). The
combined extracts were washed with water (2 × 20 mL), brine, and dried with anhydrous Na2SO4. The
solvent was removed in vacuo, and the residue was triturated in benzene. Separated crystals were
filtered, washed with benzene, and dried to afford 0.056 g (17%) of acid 12a as a white solid; mp 183–
185 °C; 1H NMR (500 MHz, DMSO-d6)
δ 14.25 (1H, br s, COOH), 7.47 (2H, d, J = 8.5 Hz, H-3′′,5′′),
7.34 (2H, d, J = 8.5 Hz, H-2′′,6′′), 7.26 (2H, d, J = 8.8 Hz, H-2′,6′), 6.96 (2H, d, J = 8.9 Hz, H-3′,5′),
3.76 (3H, s, OMe); EIMS m/z 329 [M]+(3), 284 (4), 256 (13), 123 (100). Anal. Calcd for
C17H12ClNO4: C, 61.92; H, 3.67; N, 4.25. Found: C, 61.80; H, 3.55; N, 4.33.
4.1.5. General procedure for the preparation of 3,4-diarylisoxazoles 13 and 3,4-diarylisoxazole-5-
carboxylic acids 12.
A mixture of the corresponding 5-(ethoxycarbonyl)-4,5-dihydroisoxazole N-oxides- 8 (1.5
mmol), 2% ethanolic NaOH solution (10 mL) and water (2 mL) was stirred at 60 °C for 5–6 h, then
ethanol was removed in vacuo. The residue was suspended in methylene chloride (100 mL) and the
aqueous alkaline layer was separated. The methylene chloride extract was washed with water (2 × 20
mL). The methylene chloride solution was dried by filtration through cotton wool and evaporated in
vacuo. The residue was crystallized from methanol to afford corresponding 3,4-diarylisoxazole 13. 13f
was purified by column chromatography (silica gel, benzene/ethyl acetate 19:1). Water washings were
combined with the aqueous alkaline layer, acidified with 10% aqueous HCl (pH 1–2), and extracted
with ethyl acetate (100 mL). The extract was washed with water (2 × 20 mL) and brine and then dried
with anhydrous Na2SO4. The solvent was removed, the residue was mixed with benzene, and the
crystals of the corresponding 3,4-diarylisoxazole-5-carboxylic acid 12 were filtered and dried in vacuo.
Acids 12b,f–i were not specifically separated.
4.1.5.1. 4-(4-Chlorophenyl)-3-(4-methoxyphenyl)isoxazole (13a). 20 °C, 200 h; 0.15 g (53%).
4.1.5.2. 3-(4-Methoxyphenyl)-4-(4-nitrophenyl)isoxazole (13b). 20 °C, 190 h; cream crystals; 0.05 g
(15%); mp 111–113 °C; 1H NMR (500 MHz, CDCl3)
3′′,5′′), 7.44 (2H, d, J = 8.6 Hz, H-2′,6′), 7.38 (2H, d, J = 8.6 Hz, H-2′′,6′′), 6.92 (2H, d, J = 8.6 Hz, H-
3′,5′), 3.85 (3H, s, OMe); 13C NMR (500 MHz, CDCl3)
161.0, 159.7, 157.0, 147.3, 136.0, 130.0
δ 8.64 (1H, s, H-5), 8.21 (2H, d, J = 8.6 Hz, H-
δ

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(2C), 129.3 (2C), 124.0 (2C), 119.8, 118.4, 114.3 (2C), 55.3; EIMS m/z 296 [M]+(100), 268 (23), 253
(20), 207 (18). Anal. Calcd for C16H12N2O4: C, 64.86; H, 4.08; N, 9.45. Found: C, 64.72; H, 4.18; N,
9.36.
4.1.7.3. 4-(3-Methoxyphenyl)-3-(4-methoxyphenyl)isoxazole (13c). Yellowish oil; 0.16 g (57%); 1H
NMR (500 MHz, DMSO-d6) δ 9.21 (1H, s, H-5), 7.39 (2H, d, J = 8.7 Hz, H-2′,6′), 7.29 (1H, t, J = 7.9
Hz, H-5′′), 7.01 (2H, d, J = 8.7 Hz, H-3′,5′), 6.93 (1H, d, J = 7.3 Hz, H-6′′), 6.87 (1H, br s, H-2′′), 6.84
(1H, d, J = 7.7 Hz, H-4′′), 3.79 (3H, s, O′Me), 3.70 (3H, s, O′′Me); 13C NMR (500 MHz, DMSO-d6)
δ
160.3, 159.3, 159.2, 158.0, 130.0, 129.8, 129.7 (2C), 120.7, 120.3, 119.1, 114.12 (2C), 114.08,
113.3, 55.1, 55.0; EIMS m/z 281 [M]+(100), 253 (53), 238 (41), 210 (38). Anal. Calcd for C17H15NO3:
C, 72.58; H, 5.37; N, 4.98. Found: C, 72.40; H, 5.48; N, 4.70.
4.1.5.4. 4-(3-Methoxyphenyl)-3-(4-methoxyphenyl)isoxazole-5-carboxylic acid (12c). White solid;
0.044 g (14%); mp 145–147 °C; 1H NMR (500 MHz, DMSO-d6)
δ 7.27–7.32 (3H, m, H-2′,6′, H-6′′),
6.91 (4H, m, H-3′,5′, H-2′′,5′′), 6.82 (1H, br d, J = 7.5 Hz, H-4′′), 3.75 (3H, s, O′Me), 3.71 (3H, s,
O′′Me); EIMS m/z 325 [M]+ (33), 298 (13), 280 (33), 252 (100). Anal. Calcd for C18H15NO5: C,
66.46; H, 4.65; N, 4.31. Found: C, 66.70; H, 4.60; N, 4.43.
4.1.5.5. 4-(3,5-Dimethoxyphenyl)-3-(4-methoxyphenyl)isoxazole (13d). White solid; 0.296 g (64%);
mp 82–84 °C; 1H NMR (500 MHz, CDCl3)
(2H, d, J = 8.8 Hz, H-3′,5′), 6.44 (1H, t, J = 2.3 Hz, H-4′′), 6.41 (2H, d, J = 2.3 Hz, H-2′′,6′′), 3.82
(3H, s, OMe′), 3.71 (6H, s, OMe′′); 13C NMR (500 MHz, CDCl3)
161.3, 161.1, 160.2, 156.7 (2C),
δ 8.49 (1H, s, H-5), 7.48 (2H, d, J = 8.8 Hz, H-2′,6′), 6.89
δ
131.4, 130.5 (2C), 121.2, 120.5, 114.4 (2C), 107.4 (2C), 100.5, 55.8 (2C), 55.7; EIMS m/z 311 [M]+
(20), 283 (29), 268 (16), 92 (100). Anal. Calcd for C18H17NO4: C, 69.44; H, 5.50; N, 4.50. Found: C,
69.51; H, 5.61; N, 4.65.
4.1.5.6. 4-(3,5-Dimethoxyphenyl)-3-(4-methoxyphenyl)isoxazole-5-carboxylic acid (12d). White solid;
0.05 g (9%); mp 187–189 °C; 1H NMR (500 MHz, D2O, Na-salt)
s, H-3′,5′), 6.29–6.21 (3H, m, H-4′′,2′′,6′′), 3.39 (6H, s, OMe′′), 3.35 (3H, s, OMe′); 13C NMR (500
MHz, D2O, Na-salt) 162.4, 161.4, 161.1, 159.7, 159.5 (2C), 131.3, 129.1 (2C), 119.8, 119.1, 113.3
δ 7.00 (2H, br s, H-2′,6′), 6.4 (2H, br
δ
(2C), 108.2 (2C), 99.6, 54.6 (2C), 54.4; EIMS m/z 355 [M]+(100), 327 (38), 310 (28), 282 (25). Anal.
Calcd for C19H17NO6: C, 64.22; H, 4.82; N, 3.94. Found: C, 64.42; H, 4.70; N, 3.73.

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4.1.5.7. 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole (13e). White solid; 0.235 g (46%);
mp 143–145 °C; 1H NMR (500 MHz, CDCl3)
6.90 (2H, d, J = 8.9 Hz, H-3′,5′), 6.46 (2H, s, H-2′′,6′′), 3.88 (3H, s, OMe′), 3.83 (3H, s, OMe′′), 3.74
(6H, s, OMe′′); 13C NMR (500 MHz, CDCl3)
160.7, 159.7, 155.9 (2C), 153.4, 137.8, 130.1 (2C),
δ 8.48 (1H, s, H-5), 7.49 (2H, d, J = 8.9 Hz, H-2′,6′),
δ
124.6, 120.8, 120.0, 114.0 (2C), 106.1 (2C), 61.0, 56.1 (2C), 55.3; EIMS m/z 341 [M]+(38), 326 (4),
92 (71), 77 (100). Anal. Calcd for C19H19NO5: C, 66.85; H, 5.61; N, 4.10. Found: C, 66.77; H, 5.52; N,
4.38.
4.1.5.8. 5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole (14). White solid; 0.32 g (69%); mp
148–150 °C; 1H NMR (500 MHz, CDCl3)
(2H, d, J = 8.7 Hz, H-3′,5′), 6.58 (2H, s, H-2′′,6′′), 3.90 (3H, s, OMe′), 3.84 (3H, s, OMe′′), 3.79 (6H,
s, OMe′′); 13C NMR (500 MHz, CDCl3)
163.9, 160.9, 153.6 (2C), 151.7, 137.8, 128.8 (2C), 125.8,
δ 8.30 (1H, s, H-3), 7.62 (2H, d, J = 8.7 Hz, H-2′,6′), 6.91
δ
120.1, 114.9, 114.1 (2C), 105.9 (2C), 61.0, 56.2 (2C), 55.3; EIMS m/z 341 [M]+(60), 326 (14), 136
(21), 135 (100), 107 (11), 92 (15), 77 (26). Anal. Calcd for C19H19NO5: C, 66.85; H, 5.61; N, 4.10.
Found: C, 66.80; H, 5.65; N, 4.22.
4.1.5.9. 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole-5-carboxylic acid (12e). White
solid; 0.15 g (25%); mp 203–205 °C; 1H NMR (500 MHz, DMSO-d6)
δ 14.1 (1H, br s, COOH), 8.33
(2H, d, J = 8.8 Hz, H-2′,6′), 7.97 (2H, d, J = 8.9 Hz, H-3′,5′), 6.64 (2H, s, H-2′′,6′′), 3.76 (3H, s,
OMe′), 3.70 (3H, s, OMe′′), 3.64 (6H, s, OMe′′); EIMS m/z 385 [M]+(33), 342 (38), 312 (34), 298
(63), 77 (100). Anal. Calcd for C20H19NO7: C, 62.33; H, 4.97; N, 3.63. Found: C, 62.52; H, 4.80; N,
3.81.
4.1.5.10. 3-(4-Methoxyphenyl)-4-(2-thienyl)isoxazole (13f). Gray solid; 0.169 g (33%); mp 75–77 °C;
1H NMR (500 MHz, CDCl3)
5.2 Hz, J = 1.1 Hz, H-5′′), 7.01 (1H, dd, J = 5.2 Hz, J = 3.6 Hz, H-4′′), 6.93 (1H, dd, J = 3.6 Hz, J =
1.1 Hz, H-3′′), 6.91 (2H, d, J = 8.8 Hz, H-3′,5′), 3.83 (3H, s, OMe); 13C NMR (500 MHz, CDCl3)
δ 8.51 (1H, s, H-5), 7.50 (2H, d, J = 8.8 Hz, H-2′,6′), 7.29 (1H, dd, J =
δ
160.8, 159.8, 156.3 (2C), 130.1, 129.4 (2C), 128.3, 127.3, 125.9, 120.4, 114.3 (2C), 55.2; EIMS m/z
257 [M]+(100), 229 (18), 214 (35), 186 (13). Anal. Calcd for C14H11NO2S: C, 65.35; H, 4.31; N, 5.44.
Found: C, 65.58; H, 4.42; N, 5.64.
4.1.5.11. 3-(4-Methoxyphenyl)-4-(1-methylpyrazol-4-yl)isoxazole (13g). White solid; 0.339 g (67%);
mp 86–88 °C; 1H NMR (500 MHz, CDCl3)
δ 8.47 (1H, s, H-5), 7.52 (2H, d, J = 8.6 Hz, H-2′,6′), 7.43
(1H, s, H-3′′), 7.26 (1H, s, H-5′′), 6.93 (2H, d, J = 8.6 Hz, H-3′,5′), 3.88 (3H, s, OMe), 3.85 (3H, s,

17
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NMe); 13C NMR (500 MHz, CDCl3)
δ 160.7, 159.9, 155.3, 138.6, 129.8 (2C), 128.7, 120.9, 114.0
(2C), 111.3, 109.4, 55.2, 39.1; EIMS m/z 255 [M]+(100), 227 (10), 212 (21), 184 (8). Anal. Calcd for
C14H13N3O2: C, 65.87; H, 5.13; N, 16.46. Found: C, 65.68; H, 5.23; N, 16.64.
4.1.5.12. 4-(4-Methoxyphenyl)-3-phenylisoxazole (13h). Yellowish oil; 0.05 g (25%); 1H NMR (500
MHz, CDCl3)
(2H, d, J = 8.2 Hz, H-2′′,6′′), 6.86 (2H, d, J = 8.2 Hz, H-3′′,5′′), 3.80 (3H, s, OMe); 13C NMR (500
MHz, CDCl3) 160.1, 159.4, 155.8, 130.0 (2C), 129.5, 128.7, 128.6 (2C), 128.6 (2C), 121.0, 119.9,
δ 8.43 (1H, s, H-5), 7.50 (2H, d, J = 7.3 Hz, H-2′,6′), 7.40–7.33(3H, m, H-3′,4′,5′), 7.15
δ
114.2 (2C), 55.2; HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for C16H14NO2 252.1019; Found
252.1023. Anal. Calcd for C16H13NO2: C, 76.48; H, 5.21; N, 5.57. Found: C, 76.65; H, 5.35; N, 5.77.
4.1.5.13. 3-Phenyl-4-(3,4,5-trimethoxyphenyl)isoxazole (13i). White solid; 0.13 g (54%); mp 72–77
°C; 1H NMR (500 MHz, DMSO-d6)
(3H, m, H-3′,4′,5′), 6.44 (2H, s, H-2′′,6′′), 3.87 (3H, s, OMe), 3.71 (6H, s, OMe); 13C NMR (500 MHz,
DMSO-d6) 160.2, 155.9, 153.4 (2C), 137.8, 129.7, 128.8 (2C), 128.64, 128.56, 124.3, 120.3, 106.0
δ 8.55 (1H, s, H-5), 7.55 (2H, d, J = 7.5 Hz, H-2′,6′), 7.43–7.37
δ
(2C), 60.9, 56.0 (2C); HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for C18H18NO4 312.1230; Found
312.1231; [M + Na]+ Calcd for C18H17NO4Na 334.1050; Found 334.1046. Anal. Calcd for
C18H17NO4: C, 69.44; H, 5.50; N, 4.50. Found: C, 69.64; H, 5.55; N, 4.80.
4.1.5.14. 4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-3-phenylisoxazole (13j). White solid; 0.22 g
(51%); mp 166–168 °C; 1H NMR (500 MHz, DMSO-d6)
6.51(1H, s, H-6′′), 6.06 (2H, s, OCH2O), 3.69 (3H, s, OMe), 3.40 (3H, s, OMe); 13C NMR (500 MHz,
DMSO-d6) (+ or – is a sign in the APT procedure) 160.7 (–), 158.9 (+), 139.1 (–), 138.0 (–), 137.2 (–
δ 9.04 (1H, s, H-5), 7.46–7.40 (5H, m, Ph),
δ
), 136.0 (–), 130.0 (+), 129.5 (+2C), 129.2 (–), 128.0 (+2C), 115.9 (–), 114.6 (–), 110.2 (+), 102.3 (–),
59.5 (+), 56.9 (+); HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for C18H16NO5 326.1023; Found
326.1019; [M + K]+ Calcd for C18H15NO5K 364.0582; Found 364.0582. Anal. Calcd for C18H15NO5:
C, 66.46; H, 4.65; N, 4.31. Found: C, 66.61; H, 4.54; N, 4.52.
4.1.5.15. 4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-3-phenylisoxazole-5-carboxylic acid (12j).
White solid; 89 mg (18%); mp 198–200 °C; 1H NMR (500 MHz, DMSO-d6)
6.62 (1H, s, H-6′′), 6.09 (2H, s, OCH2O), 3.72 (3H, s, OMe), 3.42 (3H, s, OMe); 13C NMR (500 MHz,
DMSO-d6) 62.6, 157.9, 157.5, 138.6, 138.0, 137.2, 135.6, 130.1, 128.8 (2C), 128.2, 127.6 (2C),
119.2, 114.0, 110.4, 102.0, 59.1, 56.6; HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for C19H16NO7
δ 7.48–7.40 (5H, m, Ph),
δ

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370.0921; Found 370.0913; [M + K]+ Calcd for C19H15NO7K 408.0480; Found 408.0484. Anal. Calcd
for C19H15NO7: C, 61.79; H, 4.09; N, 3.79. Found: C, 61.63; H, 4.22; N, 3.54.
4.1.6. X-ray crystal structure determination.
Data were collected using a Bruker APEX-II CCD diffractometer (λ(MoKα)-radiation, graphite
monochromator, ω and φ scanning mode) and corrected for absorption using the SADABS program
[29] (For the details, see Table S1, Supplementary data). The crystal structures of 13e were determined
by direct methods and refined by a full-matrix least squares technique on F2 with anisotropic
displacement parameters for non-hydrogen atoms. The hydrogen atoms in both compounds were
placed in calculated positions and refined within the riding model with fixed isotropic displacement
parameters (Uiso(H) = 1.5Ueq(C) for the CH3-groups and 1.2Ueq(C) for the other groups). All
calculations were carried out using the SHELXTL program [30]. Crystallographic data for 13e have
been deposited with the Cambridge Crystallographic Data Center. CCDC 1535084 (13e) contain
supplementary crystallographic data for this paper. These data can be obtained free of charge from the
Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk or www.ccdc.cam.ac.uk).
4.2. Biology. Phenotypic sea urchin embryo assay [23].
Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean
Sea on 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
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 Biolam light
microscope (LOMO, St. Petersburg, Russia). For treatment with the test compounds, 5 mL aliquots of
embryo suspension were transferred to six-well plates and incubated as a monolayer at a concentration
up to 2000 embryos/mL. Stock solution of 12d was prepared in distilled water at 10 mM
concentration. Stock solutions of other compounds were prepared in DMSO at 10–20 mM
concentration followed by a 10-fold dilution with 96% EtOH. This procedure enhanced the solubility
of the test compounds in the salt-containing medium (seawater), as evidenced by microscopic
examination 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 nonspecific alteration and retardation of the sea urchin embryo
development independent of the treatment stage. Combretastatin A-4 (synthesized as reported
previously [26] served as a positive control. The antiproliferative activity was assessed by exposing
fertilized eggs (8–15 min after fertilization, 45–55 min before the first mitotic cycle completion) to 2-

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fold decreasing concentrations of the compound. Cleavage alteration and arrest were clearly detected
at 2.5 h and 5.5 h after fertilization, when control embryos reached 8-cell and early blastula stages,
respectively. The effects were estimated quantitatively as an effective threshold concentration,
resulting in cleavage alteration and embryo death before hatching 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 concentrations the compounds failed to produce any effect.
For microtubule-destabilizing activity, the compounds were tested on free-swimming blastulae just
after hatching (8–10 h after fertilization), which originated from the same embryo culture. Embryo
spinning 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 illustrations are available at
http://www.chemblock.com. Sea urchin embryo assay data are available at http://www.zelinsky.ru.
Experiments with the sea urchin embryos fulfill the requirements of biological ethics. The artificial
spawning does not cause animal death, embryos develop outside the female organism, and both post
spawned adult sea urchins and the excess of intact embryos are returned to the sea, their natural
habitat.

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Acknowledgments
This work was carried out with the financial support of the Ministry of Education and Science
of the Russian Federation (Agreement number 02.a03.21.0008) and the Russian Foundation for Basic
Research (Grant No. 16-03-00648 dated of February 15, 2016). We thank the National Cancer Institute
(NCI) (Bethesda, MD, USA) for screening compound 13e by the Developmental Therapeutics
Program at NCI (Anti-cancer Screening Program; http://dtp.cancer.gov).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version, at
........................ These include X-ray data for 13e, analytical data, 1H and 13C NMR spectra of
compounds 5a,b, 7a–j, 8a–j, 9a, 12a,c–e,j, and 13a–j (PDF).
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Highlights
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A facile synthesis of 3,4-diaryl-5-unsubstituted isoxazoles is developed.
Rearrangement of diaryl-5-ethoxycarbonylisoxazoline 2-oxides to diarylisoxazoles.
High antitubulin activity of 4-(4-methoxyphenyl)-3-phenyl isoxazole.