Effective Synthesis of 3,4-Diaryl-isoxazole-5-carboxamides and their Antiproliferative Properties

Article abstract of DOI:10.1002/ejoc.201900643

A simple scalable procedure for the synthesis of 3,4-diaryl-isoxazole-5-carboxamides 6 under mild conditions from readily available material was developed. The targeted compounds 6, structural analogues of heat shock protein inhibitors, were obtained by the rearrangement of intermediate 3,4-diaryl-5-carboxamido-isoxazoline N-oxides 5. In contrast to carboxamido-isoxazoline oxides 5, base-catalyzed recyclization of 3,4-diaryl-5-(ethoxycarbonyl)isoxazoline N-oxides 9c unexpectedly yielded 5-hydroxy-1,2-oxazin-6-ones 17c instead of ethyl 3,4-diaryl-isoxazole-5-carboxylates 10. Crystal and molecular structure of 4-(2,5-dimethoxy-3,4-methylenedioxyphenyl)-5-hydroxy-3-phenyl-6H-1,2-oxazin-6-one 17c was established by single-crystal X-ray diffraction study. In a phenotypic sea urchin embryo assay, carboxamide 6f showed moderate antimitotic antitubulin activity compared to 5-unsubstituted 3,4-diarylisoxazoles 15, which featured strong microtubule destabilizing effect.

Full text of DOI:10.1002/ejoc.201900643

DOI: 10.1002/ejoc.201900643
Full Paper
Isoxazoles and Isoxazoline-N-Oxides
Effective Synthesis of 3,4-Diaryl-isoxazole-5-carboxamides and
their Antiproliferative Properties
Anna S. Maksimenko,[a] Victor P. Kislyi,*[a] Natalia B. Chernysheva,[a] Yuri A. Strelenko,[a]
Yan V. Zubavichus,[b] Victor N. Khrustalev,[c] Marina N. Semenova,[d] and
Victor V. Semenov[a]
Abstract: A simple scalable procedure for the synthesis of 3,4- diaryl-isoxazole-5-carboxylates 10. Crystal and molecular struc-
diaryl-isoxazole-5-carboxamides 6 under mild conditions from ture of 4-(2,5-dimethoxy-3,4-methylenedioxyphenyl)-5-hydroxy-
readily available material was developed. The targeted com- 3-phenyl-6H-1,2-oxazin-6-one 17c was established by single-
pounds 6, structural analogues of heat shock protein inhibitors, crystal X-ray diffraction study. In a phenotypic sea urchin em-
were obtained by the rearrangement of intermediate 3,4-diaryl- bryo assay, carboxamide 6f showed moderate antimitotic anti-
5-carboxamido-isoxazoline N-oxides 5. In contrast to carbox- tubulin activity compared to 5-unsubstituted 3,4-diarylisox-
amido-isoxazoline oxides 5, base-catalyzed recyclization of 3,4- azoles 15, which featured strong microtubule destabilizing ef-
diaryl-5-(ethoxycarbonyl)isoxazoline N-oxides 9c unexpectedly fect.
yielded 5-hydroxy-1,2-oxazin-6-ones 17c instead of ethyl 3,4-
Introduction
4,5-Diarylisoxazole scaffold can be found in a wide variety of
non steroidal anti-inflammatory drugs (NSAIDs),[1,2] inhibitors of
platelet aggregation,[3] tubulin polymerization inhibitors,[4,5] es-
trogen receptor modulators, antiviral, anti-diabetic, anti-convul-
sant, and other agents.[6] Polyoxygenated 4,5-diarylisoxazoles
Figure 1. Structures of 4,5-diarylisoxazole-3-carboxamides (Hsp90 inhibitors,
A) and isomeric 3,4-diarylisoxazole-5-carboxamides (B).
with 3-carboxamide groups (Figure 1, A) were reported to in-
hibit heat shock protein Hsp90, a chaperone that assists folding
of various proteins including those implicated in malignancy.
Namely, Hsp90 inhibitors suppressed growth of human cancer
cell lines.[2,7] Some of these compounds are currently undergo-
ing clinical evaluation as anticancer agents.[8–11] However, bio-
logical effects of isomeric 3,4-diarylisoxazole-5-carboxamides
(Figure 1, B) were less investigated due to the challenges in
regioselective synthesis and the lack of efficient synthetic
routes for their intermediates, 3,4-diarylisoxazoline N-oxides.
Published protocols[2,12–17] usually included a variety of draw-
backs, such as toxic and expensive reagents, using of anhydrous
conditions, and cumbersome reaction steps. Therefore, an effi-
cient and simple synthetic scheme to afford 3,4-diarylisoxazole-
5-carboxamides is of considerable interest.
In our previous work, starting 3,4-diaryl-isoxazoline N-oxide-
5-carboxylic esters were synthesized in high yields.[18] Unfortu-
nately, their subsequent rearrangement was not selective, giv-
ing decarboxylated 3,4-diaryl-5H-isoxazoles as the main prod-
ucts with only negligible amount of the required 3,4-diarylisox-
azole-5-carboxylic acids.[4] Considering the literature evidence,
the present study was aimed to develop an efficient, inexpen-
sive, and scalable protocol for regioselective synthesis of 3,4-
diaryl-isoxazole- 5-carboxamides from available materials under
mild conditions.
Results and Discussion
Chemistry
[a] N. D. Zelinsky Institute of Organic Chemistry RAS,
47 Leninsky Prospect, 119991, Moscow, Russian Federation
E-mail: vkislyi@yandex.ru
A simple synthetic protocol was developed based on an easily
available starting material. Specifically, arylnitromethanes 1
were readily transformed to nitrostilbenes 2 in yields of 44–
73 %, and chloroacetamides 3 were treated with pyridine to
afford quaternized pyridinium salts 4 in almost quantitative
yields. At the next step, compounds 2 reacted with pyridinium
salts 4 in the presence of Et3N to afford the respective 3,4-
diaryl-5-carboxamido-isoxazoline N-oxides 5 with good and
[b] Russian Research Centre “Kurchatov Institute”,
Kurchatov sq. 1, 123182 Moscow, Russian Federation
[c] Peoples' Friendship University of Russia (RUDN University),
6 Miklukho-Maklay Street, 117198, Moscow, Russian Federation
[d] N. K. Koltzov Institute of Developmental Biology RAS,
26 Vavilov Street, 119334, Moscow, Russian Federation
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201900643.
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moderate yields (Scheme 1). Then, intermediates 5 were sepa- the desired 3,4-diarylisoxazole-5-carboxamides 6. Cyclization of
rated from the reaction mixtures and further rearranged into nitrostilbenes with pyridinium salts without separation of inter-
Anna Maksimenko was born in 1993 in Moscow, Russia. Graduated from the Faculty of Technology of Organic Substances and Chemical
Pharmaceutical Compounds at the Mendeleev University of Chemical Technology of Russia in 2018. In 2015, she started working at Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences, and became a PhD student at the same organization in 2018. Area of interest
includes synthetic method developments and study of structure-activity relationships for further applications in cancer therapy.
Dr. Victor Kislyi graduated from The Chemical Department of Moscow State University in 1988. He joined the High Energy Materials Research
Laboratory at N. D. Zelinsky Institute of Organic Chemistry in 1988. Received PhD degree in 1995. Since 2006 and up to date he is working in
the Medicinal Chemistry Laboratory at N. D. Zelinsky Institute of Organic Chemistry. His research area includes chemistry of nitro compounds
and nitrogen heterocycles, synthesis of anticancer molecules and NSAIDs, prediction of NMR spectra and receptor binding at the DFT level of
theory.
Dr. Natalia Chernysheva graduated from The Chemical Department of Moscow State University in 1962. During 1965–1977 hold different
positions in USSR Research Vitamin Institute and then Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences. Received
PhD degree at The Chemical Department of Moscow State University in 1972. Since 1977 and up to date she is working at N. D. Zelinsky
Institute of Organic Chemistry. Fields of research interests: nitro compounds, nitrogen heterocycles, anticancer molecules.
Dr. Yuri Strelenko was born on 1949 in Moscow, USSR. He received his Ph.D. “NMR of 199Hg” from Moscow State University under the supervision
of Prof. Yu.A. Ustynyuk in 1980. After that he worked in GNIIHTEOS, Moscow in NMR of Silicone olygomers and polymers. He studies nitro-
compounds by NMR at Zelinsky Institute of Organic Chemistry since 1985 up today.
Prof. Dr. Yan Zubavichus was born in Krasnoyarsk in 1974. Graduated from the Higher Chemical College of RAS in Moscow in 1997. Received
his PhD degree in physical chemistry in 2001 from the Institute of Organoelement Compounds of RAS in Moscow. After that he spent two years
in Germany as a postdoc in the University of Heidelberg in the group of Prof. Michael Grunze. During 2005–2018 hold different positions in the
National Research Center “Kurchatov Institute”. Received DSc degree in physics and mathematics in 2013 from the NRC “Kurchatov Institute”.
Moved to the Institute of Catalysis in Novosibirsk in November, 2018. Fields of research interests: various applications of synchrotron radiation
techniques to structural chemistry and materials science, combination of X-ray spectroscopic and scattering techniques, methodological develop-
ment.
Prof. Dr. Victor N. Khrustalev graduated with honours (cum laude) from M.V. Lomonosov Moscow Institute of Fine Chemical Technology (MIFCT)
in February 1993 getting a diploma in Physics and Chemistry of Solid State. In March 1993 he joined the Laboratory of X-ray diffraction studies
at the A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences (INEOS RAS), in which he worked for 25 years,
having gone from an engineer to a leading researcher. He obtained his Ph.D. in Inorganic chemistry at the MIFCT in January 2000 and D.Sc. in
Physical Chemistry and Organoelement Chemistry at the INEOS RAS in February 2008. Starting from 2006 he is a visiting professor of New
Mexico Highlands University (USA), and from 2007 – Baku State University (Azerbaijan). In 2015 Victor N. Khrustalev became the Head of
Inorganic Chemistry Department at the Peoples' Friendship University of Russia (RUDN University), and since 2017 is also the Director of Research
Institute of Chemistry of RUDN University. His main research area includes: (a) structural studies of Main Group compounds with unusual valent
and coordination states; (b) investigations of the nature of chemical bond and specific non-valent interactions; (c) supramolecular chemistry; (d) stereochemistry
and catalysis; (e) polymorphism and phase transitions.
Dr. Marina Semenova graduated from the Biomedical Department, Moscow Medical Institute, Russia, getting a diploma in biochemistry. In 1977
she joined the Laboratory of General Physiology at the Institute of Developmental Biology, Russian Academy of Sciences, where she has got
PhD degree and is working up to date. She has been involved in several scientific projects concerning the regulation of embryonic development,
mostly in marine invertebrates, and took part in several marine expeditions. In 2004 she with co-workers focused attention on the development
of new antitumoral molecules and devised an assay for the evaluation of tubulin modulators using the sea urchin embryo model. The phenotypic
sea urchin embryo assay is used successfully for the identification and SAR studies of antimitotic molecules that affect tubulin/microtubule
dynamics.
Prof. Dr. Victor Semenov graduated from The Chemical Department of Moscow State University in 1973. After that he joined the High Energy
Materials Research Laboratory at N. D. Zelinsky Institute of Organic Chemistry, where he received PhD degree (1977) and DSc degree (1987) in
organic chemistry. During 1988–1996 he was Deputy Director of N. D. Zelinsky Institute of Organic Chemistry. In 1989 Victor Semenov became
the Head of Technological Laboratory. Since 2006 up to date he is the Head of Medicinal Chemistry Laboratory in N. D. Zelinsky Institute of
Organic Chemistry. His research area includes chemistry of nitro compounds and nitrogen heterocycles, synthesis of anticancer molecules,
chemistry of natural compounds, application of acetylene and carbon dioxide in organic chemistry
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Scheme 1. Synthesis of isoxazoline N-oxides 5 and isoxazole-5-carboxamides 6.
mediate isoxazoline oxides was reported, and a mechanism for obtained from isoxazoles 6 in such reaction conditions. These
isomerization of 4-aryl-isoxazolineoxide-3,5-dicarboxylates to 4- observations provided additional evidence for the ring-opening
arylisoxazole-3,5-dicarboxylates without ring-opening was pro- and cyclization mechanism.
posed, however, without experimental confirmation.[19] In our
hands, one-pot synthesis of 6 without separation of 5 failed
due to numerous by-products formed.
Dehydration of 5-carboxamido-isoxazoline N-oxides 5 to af-
ford isoxazole-5-carboxamides 6 proceeded via a complex reac-
tion mechanism including deprotonation, subsequent electro-
cyclic ring opening, and cyclization of ketoamide 7, as de-
scribed previously for isoxazoline-5-carboxylates (Scheme 2).[4]
The intermediate ketoamides similar to 7 (Scheme 2) were iden-
tified in the reaction mixtures.[4] In the present study it was
found that oximes 8 could be formed only by hydrolysis of
Unlike isoxazoline N-oxides with two ester groups in 3 and
5 positions,[19–23] isoxazolines 5 cannot be deprotonated by
simple amines (Et3N or BuNH2). As evidence, amide 5c was re-
turned unchanged after 15 h refluxing in acetonitrile with Et3N.
In this case recyclization could not proceed probably due to
failure of anion C formation. Isoxazoline N-oxide 5c in ethanolic
NaOH afforded mainly isoxazole-5-carboxamide 6c and unex-
pected oxime 8, along with other by-products. Intermediate
alpha-keto amides 7 likely disappeared from the reaction mix-
ture in the presence of water or alcohols by various ways.
Isoxazole-5-carboxamides 6 were obtained with the highest
intermediate ketoamide 7 with malonic acid monoamide for- yields in anhydrous CH3CN and DBU (Scheme 1). Alternative
mation. Particularly, we showed that oximes 8 could not be quicker synthesis of amides 6 was assumed through the corre-
Scheme 2. Mechanism of formation of isoxazole-5-carboxamides 6.
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Scheme 3. Alternative pathway to isoxazole-5-carboxamides 6.
sponding 5-ethoxycarbonyl-isoxazoline N-oxides 9, followed by acetonitrile. Instead, 3,4-diaryl-4-hydroxy-1,2-oxazin-6-one 17c
their rearrangement into ethyl isoxazole-5-carboxylates 10 or was separated unexpectedly from the reaction mixture.
isoxazole-5-carboxylic acids 11 (Scheme 3).
To confirm this synthetic scheme the reactivity of esters 9 in
X-ray Crystallography
different conditions was studied. Ester 9a in acidic media (aque-
ous HCl in dioxane) was hydrolyzed almost quantitatively into
3,4-diaryl-5-carboxy-isoxazoline N-oxides 12a in 98 % yield
(Scheme 4).[18] In contrast, in ethanolic NaOH the rearrange-
ment of both esters 9 and acids 12 afforded mainly 3,4-diaryl-
5H-isoxazoles 15 with small amounts of isoxazole-5-carboxylic
acids 11 (0–9 %). Refluxing of esters 9 with Et3N in CH3CN failed
to afford desired ethyl isoxazole-5-carboxylates 10 or isoxazole-
5-carboxylic acids 11 as well. Similarly, no traces of isoxazole-5-
carboxylic esters 10 were identified in the presence of DBU in
The structure of the product 17c was unambiguously estab-
lished by X-ray diffraction study and is shown in Figure 2 along
with the atomic numbering scheme The full geometrical pa-
rameters for 17c are available in the Supporting Information.
Figure 2. Molecular structure of 4-(2,5-dimethoxy-3,4-methylenedioxyphenyl)-
5-hydroxy-3-phenyl-6H-1,2-oxazin-6-one 17c (50 % ellipsoids).
The central 1,2-oxazine ring in 17c is almost planar (rms de-
viation is 0.006 Å). It is interesting to note that the O1–N2
(1.423(3) Å) and N2=C3 (1.325(3) Å) bond lengths are longer,
but the O1–C6 (1.345(3) Å) is shorter than those in the oxazine
rings of the compounds studied previously.[24–26]
The 3- and 4-aryl substituents are twisted by 28.76(8) and
68.10(11)°, respectively, relative to the oxazine ring plane. The
5-methoxy group of the apiolyl substituent is coplanar to the
plane of the parent benzene ring (the C18–C19–O19–C21 tor-
sion angle is –6.9(5)°), whereas the 2-methoxy group is rotated
relative to this plane (the C15A–C16–O16–C20 torsion angle is
33.1(5)°). In the crystal, the molecules of 17c form centro-
symmetrical H-bonded dimers by the two intermolecular O5–
H5···O6 (–x + 2, –y + 1, –z + 1) hydrogen bonds [O···O 2.668(3)
Å, H···O 1.87(4) Å, ∠O–H···O 146(4)°] (Figure 3). The crystal
packing of the H-bonded dimers is stacked along the crystallo-
Scheme 4. Mechanism of formation of 4-hydroxy-1,2-oxazin-6-ones 17.
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Table 1. Effect of 3,4-diarylisoxazole-5-carboxamides 6c–f, h, i and reference diarylisoxazoles 11 and 15 on sea urchin embryos.
[a]
Cmpd ID
Ar1
Ar2
R
EC, μM
Cleavage alteration Cleavage arrest
Embryo spinning
6c
6d
6e
6f
6h
6i
Ph
Ph
Ph
2,5-(MeO)2-3,4-(OCH2O)-C6H
2,5-(MeO)2-3,4-(OCH2O)-C6H
2,5-(MeO)2-3,4-(OCH2O)-C6H
3,4,5-(MeO)3-C6H2
2,5-(MeO)2-3,4-(OCH2O)-C6H
2,5-(MeO)2-3,4-(OCH2O)-C6H
2,5-(MeO)2-3,4-(OCH2O)-C6H
3,4,5-(MeO)3-C6H2
H
Et
>4
>4
>4
1
>4
>4
>4
0.2
2
>4
>4
>4
>4
>4
>4
>4
>4
>4
>4
>5
>4
1
3,4-(MeO)2 -C6H3CH2CH2
H
4-Br-C6H4
4-F2ClCO-C6H4
–
–
–
–
4-MeO-C6H4
Ph
Ph
Ph
4-MeO-C6H4
Ph
4-MeO-C6H4
4 TE[b]
>4
>4
>4
11c[4]
11b[4]
15c[4]
15b[4]
2 TE[b]
>4
2,5-(MeO)2-3,4-OCH2O-C6H
3,4,5-(MeO)3-C6H2
0.01
0.1
[a] The sea urchin embryo assay was conducted as described previously.[6,27] Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing
concentrations of compounds. Duplicate measurements showed no differences in effective concentration (EC) values. [b] TE: tuberculate eggs typical of
microtubule destabilizing agents.
graphic b axis. Within the stacks, the dimers are bound to each
Compound 6f with 4-methoxyphenyl and 3,4,5-trimethoxy-
other by the weak π···π stacking interactions between the oxaz- phenyl fragments was the only one exhibiting moderate anti-
ine and phenyl rings at the C···C distances of 3.379(4)-3.508(4) mitotic antitubulin effect, because this molecule induced for-
Å, the O···C distances of 3.329(3)-3.360(3) Å, and the N···C dis- mation of tuberculate arrested eggs typical of microtubule de-
tances of 3.390(3)-3.453(3) Å (Figure 3).
stabilizing agents.[27] As shown previously,[4] introduction of
-COOH group in isoxazole ring reduced antimitotic activity
(compare 15b and 11b; 15c and 11c (Table 1). The respective
C-5 substitution in 15b with amide group yielded even less
active molecule 6f. Isoxazoles 6 featuring 2,5-dimethoxy-3,4-
methylenedioxy moiety (6c–e, h, i) failed to produce any effect
at concentration up to 4 μM.
Conclusions
In summary, a novel and facile synthetic procedure has been
developed for the synthesis of 3,4-diaryl-isoxazole-5-carbox-
amides 6, structural analogues of heat shock protein inhibitors.
The compounds were obtained from readily available starting
materials (chloroacetamides, aldehydes, and arylnitromethanes)
by the rearrangement of intermediate 3,4-diaryl-5-carbox-
amido-isoxazoline N-oxides 5. In contrast to 5-carboxamido-is-
oxazoline N-oxides 5, base-catalyzed recyclization of 5-ethoxy-
Figure 3. The centrosymmetrical H-bonded dimer of 17c. The intermolecular
O–H···O hydrogen bonds are depicted by dashed lines.
Previously 4-hydroxy-1,2-oxazin-6-ones were obtained as sin-
gle products in the reaction of aldehydes with nitroacetic esters
in presence of bimolar excess of amines.[26] The approach to 4-
hydroxy-1,2-oxazin-6-ones 17 described in the present study
could be considered as a novel efficient method for synthesis of
this class of compounds with still unknown biological activities.
carbonyl-isoxazoline N-oxides
9 unexpectedly yielded 5-
hydroxy-1,2-oxazin-6-ones 17 instead of ethyl 3,4-diaryl-isox-
azole-5-carboxylates 10. Evaluation of carboxamides 6 for anti-
proliferative activity in a phenotypic sea urchin embryo assay
identified 3-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isox-
azole-5-carboxamide 6f as antimitotic antitubulin agent, al-
though less active than the corresponding 5-unsubstituted 3,4-
diarylisoxazole 15f with strong microtubule destabilizing effect.
Biological Evaluation
In our previous work 3,4-diaryl isoxazoles were identified as
antimitotics with microtubule destabilizing mode of action.[4]
Therefore, the targeted 3,4-diaryl-isoxazole-5-carboxamides
6c–f,h,i were evaluated for antimitotic microtubule destabiliz-
ing activity using a phenotypic sea urchin embryo assay.[6,27]
Antimitotic properties of a tested molecule were estimated by
the ability to disrupt cleavage – quick successive embryonic cell
divisions, whereas specific alteration of embryo motility, spin-
ning on the bottom of the culture vessel instead of forward
swimming, indicated microtubule destabilizing mechanism of
action. The results are presented in Table 1.
Experimental Section
Chemistry
Materials and Methods: Melting points were measured using a
Boetius melting point apparatus and were uncorrected. 1H NMR
and 13C NMR spectra were recorded on a Bruker DRX-500 instru-
ment [working frequencies of 500.13 MHz (1H) and of 125.76 MHz
(
13C), respectively] and Bruker AVANCE-II [working frequencies of
300.13 MHz (1H), of 282.4 MHz (19F) and of 75.47 MHz (13C), respec-
tively]. Chemical shift values were reports in parts per million (ppm)
and referenced to the appropriate NMR solvent peaks. Spin–spin
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coupling constants (J) were reported in Hertz (Hz). In isoxazoline N-
131.6 (C-1-Ar), 127.5 (2C, C-3,5-Py), 120.5 (C-6-Ar), 112.7 (C-2-Ar),
oxides and isoxazoles, atoms of isoxazole ring were marked by “iso” 112.0 (C-5-Ar), 61.6 (CH2CO), 55.6 (OCH3), 55.5 (OCH3), 41.0 (CH2NH),
(H-4-iso); numbers of atoms of the aryl ring in 3 position have no
apostrophe (H-1); numbers of atoms of the aryl ring in 4 position
were marked one apostrophe (H-1′); atoms of substituent at amide
nitrogen atom were marked double apostrophe (H-1′′). Symbols +
and – in the JMOD procedure signal assignment denote carbons
with even or odd number of hydrogens. IR spectra nave been meas-
ured by Bruker AlphaT instrument. Low resolution mass spectra
(m/z) were recorded on a Finnigan MAT/INCOS 50 mass spectrome-
ter at 70 eV using direct probe injection. High resolution mass spec-
tra (HRMS) were measured on a Bruker maXis and micrOTOF II in-
struments using electrospray ionization (ESI). Thin layer chromatog-
raphy (TLC) analysis was performed using Merck 60 F254 plates.
Flash chromatography was accomplished using Silica (Acros, 0.035–
0.070 mm, 60 Å). All solvents and reagents were purified according
to standard procedures. Arylnitromethanes 1 and nitrostilbenes 2
were prepared accordingly published procedures.[4]
34.5 (CH2Ar) ppm; HRMS (ESI/QTOF) m/z calcd. for C17H20N2O3 + H+:
301.1547 [M + H]+, found 301.1552.
1-(2-(3,4-Methelenedioxybenzylamino)-2-oxoethyl)pyridin-1-
ium Chloride (4g): Beige crystals (2.6 g, yield 85 %), m.p. 198–
202 °C; 1H NMR (500.13 MHz, [D6]DMSO) δ=9.62 (s, 1H, NH), 9.12 (d,
3J = 5.7 Hz, 2H, H-2,6-Py), 8.67 (t, 3J = 7.8 Hz, 1H, H-4-Py), 8.20 (t,
3
3J = 6.6 Hz, 2H, H-3,5-Py), 6.93 (s, 1H, H-2-Ar), 6.85 (d, J = 7.8 Hz,
H-6-Ar), 6.80 (d, 3J = 7.8 Hz, H-5-Ar), 6.00 (s, 2H, OCH2O), 5.57 (s, 2H,
CH2Ar), 4.27 (d, 3J = 5.4 Hz, 2H, CH2NH) ppm; 13C NMR (125.76 MHz,
[D6]DMSO) δ=164.3 (CO), 147.2 (C-4-Ar), 146.2 (2C, C-2,6-Py), 146.1
(C-3-Ar), 145.9 (C-4-Py), 132.3 (C-1-Ar), 127.4 (2C, C-3,5-Py), 120.6 (C-
6-Ar), 108.0 (C-2-Ar), 107.9 (C-5-Ar), 100.8 (OCH2O), 61.3 (CH2CO),
42.2 (NHCH2) ppm; HRMS (ESI/QTOF) m/z calcd. for C15H14N2O3
+
H+: 271.1077 [M + H]+, found 271.1071.
1-(2-(4-Bromophenylamino)-2-oxoethyl)pyridin-1-ium Chloride
(4h): Beige crystals (2.9 g, yield 89 %), m.p. 235–236 °C (lit. m.p.
235–237 °C[30]); 1H NMR (300.13 MHz, [D6]DMSO) δ=11.45 (s, 1H,
NHCO), 9.10 (d, 3J = 8.1 Hz, 2H, H-2,6-Py), 8.69 (t, 3J = 7.8 Hz, 1H,
General Procedure for the Synthesis of Chloroacetamides (3):
Chloroacetyl chloride (11.5 mmol) was added dropwise to stirred
solution of the corresponding amine (10 mmol) dissolved in aceto-
nitrile (10 mL), and then triethylamine (13 mmol) was added. A
reaction mixture was stirred during 1 h at room temperature, then
solvent was removed in vacuo, water(5 mL) and ethyl acetate (2 ×
10 mL) were added. The resulting product was dried by magnesium
sulfate, evaporated in vacuo, and crystallized from hexane.
3
3
H-4-Py), 8.23 (t, J = 6.9 Hz, 2H, H-3,5-Py), 7.63 (d, J = 8.7 Hz, 2H,
3
H-2,6-Ar), 7.54 (d, J = 8.7 Hz, 2H, H-3,5-Ar), 5.75 (s, 2H, CH2) ppm.
1-(2-(4-Chlorodifluoromethoxyphenylamino)-2-oxoethyl)pyr-
idin-1-ium Chloride (4i): Beige crystals (2.7 g, yield 77.5 %), m.p.
215–217 °C; 1H NMR (500.13 MHz, [D6]DMSO) δ=11.84 (s, 1H, NHCO),
9.17 (d, 3J = 6.1 Hz, 2H, H-2,6-Py), 8.71 (t, 3J = 7.8 Hz, 1H, H-4-Py),
N-(4-Chlorodifluoromethoxy)phenyl 2-Chloroacetamide (3i):
3
3
8.23 (t, J = 6.9 Hz, 2H, H-3,5-Py), 7.81 (d, J = 9.1 Hz, 2H, H-2,6-Ar),
7.35 (d, 3J = 8.8 Hz, 2H, H-3,5-Ar), 5.84 (s, 2H, CH2) ppm; 13C NMR
(125.76 MHz, [D6]DMSO) δ=163.5 (CO), 146.4 (2C, C-2,6-Py), 146.2
(C-4-Py), 145.0 (C-4-Ar), 137.6 (C-1-Ar), 127.5 (2C, C-3,5-Py), 124.9 (t,
3J = 286.7 Hz, OCF2Cl), 122.0 (2C, C-2,6-Ar), 120.5 (2C, C-3,5-Ar), 62.1
(CH2) ppm; HRMS (ESI/QTOF) m/z calcd. for C14H11ClF2N2O2 + H+:
313.0550 [M + H]+, found 313.0545.
Off-white crystals (2.04 g, yield 75.5 %), m.p. 104–108 °C (hexane);
3
1H NMR (300.13 MHz, [D6]DMSO) δ=10.52 (s, 1H, NH), 7.72 (d, J =
3
8.7 Hz, 2H, H-2,4-Ar), 7.35 (d, J = 8.7 Hz, 2H, H-3,5-Ar), 4.29 (s, 2H,
CH2) ppm; 13C NMR (75.47 MHz, [D6]DMSO) δ=164.8 (CONH), 145.1
(C-4-Ar), 137.6 (C-1-Ar), 124.9 (t, 3J = 378.1 Hz, OCF2Cl), 121.9 (2C, C-
2,6-Ar), 120.7 (2C, C-3,5-Ar), 43.4 (CH2Cl) ppm; 19F NMR (282.4 MHz,
[D6]DMSO) δ= –25.63 (OCF2Cl) ppm; EIMS m/z 273 (11), 272 (6), 271
(67), 270 (14), 269 [M]+ (100), 234 (13), 220 (15), 193 (47).
General Procedure for the Synthesis of Isoxazolines (5): Nitro-
stilbene 2 (1.5 mmol) was dissolved in methanol (10 mL) with heat-
ing, if necessary. Pyridinium salt 4 (3 mmol) was added and then
NEt3 (4.35 mmol) was added whilst stirring at 50 °C. The reaction
mixture was stirred for 6 h (TLC control). After completion of reac-
tion the solvent was evaporated in vacuo, the crude product was
washed with water and extracted with dichloromethane. Combined
extracts were dried with magnesium sulfate, and the solvent was
evaporated in vacuo to give yellowish solids 5.
General Procedure for the Synthesis of 2-Amino-2-oxoethylpyr-
idinium Chlorides (4): Pyridine (20–40 mmol) and corresponding
chloroacetamide (10 mmol) were stirred at 80 °C for 40 min, and
then precipitate was filtered and washed with CH3CN. The crystals
were air dried.
1-(2-Amino-2-oxoethyl)pyridin-1-ium Chloride (4a): Off-white
crystals (1.59 g, yield 92.2 %), m.p. 205–207 °C (lit. m.p. 207–
1
3
209 °C[28]); H NMR (300.13 MHz, [D6]DMSO) δ=9.05 (d, J = 6.0 Hz,
5-Carbamoyl-4-(4-methoxyphenyl)-3-phenyl-4,5-dihydroisoxaz-
ole 2-Oxide (5a): White crystals (290 mg, yield 61 %), m.p. 99–
101 °C (purified by column chromatography, silica gel, gradient
benzene, benzene/ethyl acetate, 1:1); Rf = 0.6 (benzene/ethyl acet-
ate, 1:1); 1H NMR (500.13 MHz, [D6]DMSO) δ=7.91 (d, 3J = 7.6 Hz,
3
2H, H-2,6-Py), 8.68 (t, J = 7.8 Hz, 1H, H-4-Py), 8.32 (s, 1H, CONH2),
8.18 (t, 3J = 6.8 Hz, 2H, H-3,5-Py), 7.70 (s, 1H, CONH2), 5.52 (s, 2H,
CH2) ppm.
1-(2-Ethylamino-2-oxoethyl)pyridin-1-ium Chloride (4d): Purple
3
crystals (1.72 g, yield 86 %), m.p. 180–185 °C (lit. m.p. 177–
2H, H-2,6), 7.86 (s, 1H, NH), 7.65 (s, 1H, NH), 7.40 (t, J = 7.1 Hz, 2H,
1
3
179 °C[29]); H NMR (300.13 MHz, [D6]DMSO) δ=9.03 (d, J = 6.2 Hz,
H-3,5), 7.35 (t, 3J = 7.0, 1H, H-4), 7.31 (d, 3J = 8.6 Hz, 2H, H-2′,6′),
6.93 (d, 3J = 8.6 Hz, 2H, H-3′,5′), 5.27 (d, 3J = 2.6 Hz, 1H, H-4-iso),
3
2H, H-2,6-Py), 8.95 (1H, br.s, CONH), 8.65 (t, J = 7.8 Hz, 1H, H-4-Py),
8.17 (t, J = 6.9 Hz, 2H, H-3,5-Py), 5.49 (s, 2H, CH2CO), 3.15 (m, J =
6.6 Hz, 2H, CH2CH3), 1.07 (t, J = 7.2 Hz, 3H, CH2CH3) ppm.
3
3
3
4.82 (d, J = 2.6 Hz, 1H, H-5-iso), 3.71 (s, 3H, OCH3) ppm; 13C NMR
3
(125.76 MHz, [D6]DMSO) δ=170.3 (CONH2), 159.0 (C-4′), 131.2
(C-1′), 129.5 (C-4), 128.8 (2C, C-2,6), 128.6 (2C, C-2′,6′), 126.6 (2C, C-
3,5), 125.6 (C-1), 115.7 (C-3-iso), 114.6 (2C, C-3′,5′), 79.4 (C-5-iso),
1-(2-(2-(3,4-Dimethoxyphenyl)-ethylamino)-2-oxoethyl)pyridin-
1-ium Chloride (4e): Dark crystals (3.33 g, yield 99 %), m.p. 95–
98 °C; 1H NMR (500.13 MHz, [D6]DMSO) δ=9.23 (1H, br.s, CONH),
9.05 (d, 3J = 5.7 Hz, 2H, H-2,6-Py), 8.67 (t, 3J = 5.4 Hz, 1H, H-4-Py),
55.2 (C-4-iso), 52.1 (OCH ) ppm; IR (KBr) ν= 3458, 3144, 1697, 1614,
˜
3
1512, 1252, 1178 cm–1; HRMS (ESI/QTOF) m/z calcd. for C17H16N2O4
+ H+: 313.1183 [M + H]+, found 313.1184; m/z calcd. for C17H16N2O4
+ Na+: 335.1002 [M + Na]+, found 335.0998.
3
8.19 (t, J = 7.0 Hz, 2H, H-3,5-Py), 6.87–6.85 (m, 2H, H-2,6-Ar), 6.75
3
(d, J = 8.0 Hz, 1H, H-5-Ar), 5.55 (s, 2H, CH2-Py), 3.75 (s, 3H, OCH3),
3
3.72 (s, 3H, OCH3), 3.34 (q, 3J = 6.7 Hz, 2H, CH2), 2.71 (t, J = 7.4 Hz,
5-Carbamoyl-4-(3,4-dimethoxyphenyl)-3-phenyl-4,5-dihydro-
2H, CH2) ppm; 13C NMR (125.76 MHz, [D6]DMSO) δ=164.2 (CO), isoxazole 2-Oxide (5b): White crystals (280 mg, yield 55 %), m.p.
148.7 (C-4-Ar), 147.3 (C-3-Ar), 146.2 (2C, C-2,6-Py), 146.1 (C-4-Py),
82–84 °C; Rf = 0.3 (benzene/ethyl acetate, 1:1); 1H NMR (500.13 MHz,
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3
[D6]DMSO) δ=7.92 (d, J = 7.4 Hz, 2H, H-2,6), 7.85 (s, 1H, NH), 7.64
55.9(OCH3), 55.8(OCH3), 49.0(C-4-iso), 40.8 (CH2NH), 34.8 (CH2Ar)
(s, 1H, NH), 7.41 (t, 3J = 7.9 Hz, 2H, H-3,5), 7.36 (t, 3J = 7.4 Hz, 1H,
H-4), 7.03 (d, 4J = 2.1 Hz, 1H, H-2′), 6.92 (d, 3J = 8.3 Hz, 1H, H-5′),
ppm; IR (KBr) ν= 3406, 2936, 1683, 1616, 1515, 1451, 1236, 1140,
˜
1064 cm–1; HRMS (ESI/QTOF) m/z calcd. for C29H30N2O9 + H+:
551.2024 [M + H]+, found 551.2015; [M + Na]+ calcd. for C29H30N2O9
+ Na+: 573.1841, found 573.1835.
4
3
3
6.84 (dd, J = 2.1 Hz, J = 8.3 Hz, 1H, H-6′), 5.24 (d, J = 2.7 Hz, 1H,
H-4-iso), 4.85 (d, 3J = 2.7 Hz, 1H, H-5-iso), 3.75 (s, 3H, OCH3), 3.71 (s,
3H, OCH3) ppm; 13C NMR (125.76 MHz, [D6]DMSO) δ=170.1 (CONH),
149.0 (C-4′), 148.4 (C-3′), 131.4 (C-1′), 129.4 (C-4), 128.6 (2C, C-2,6),
126.4 (2C, C-3,5), 125.6 (C-1), 119.2 (C-5′), 115.5 (C-3-iso), 112.1
(C-2′), 111.1 (C-6′), 79.2 (C-5-iso), 55.5 (OCH3), 55.4 (OCH3), 52.3
5-Carbamoyl-3-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
4,5-dihydroisoxazole 2-Oxide (5f): Off-white crystals (360 mg,
yield 60 %), m.p. 81–85 °C (benzene); Rf = 0.75 (ethyl acetate); 1H
NMR (500.13 MHz, [D6]DMSO) δ=7.90 (d, 3J = 8.5 Hz, 2H, H-2,6), 7.82
(C-4-iso) ppm; IR (KBr) ν= 3430, 2937, 1685, 1615, 1517, 1261,
˜
3
(s, 1H, NH), 7.63 (s, 1H, NH), 6.99 (d, J = 8.4 Hz, 2H, H-3,5), 6.68 (s,
1143 cm–1; HRMS (ESI/QTOF) m/z: [M + H]+ calcd. for C18H18N2O5 +
H+: 343.1288, found 343.1291; calcd. for C18H18N2O5 + K+: 381.0847
[M + K]+, found 381.0844.
2H, H-2′,3′), 5.20 (s, 1H, H-4-iso), 4.86 (s, 1H, H-5-iso), 3.74 (s, 9H,
3 × OCH3) 3.63 (s, 3H, OCH3) ppm; 13C NMR (125.76 MHz, [D6]DMSO)
δ=170.2 (CONH), 159.8 (C-4), 153.2 (2C, C-3′,5′), 137.0 (C-1′), 135.0
(C-4′), 128.2 (2C, C-2,6), 117.8 (C-1), 115.1 (C-3-iso), 114.2 (2C, C-3,5),
104.6 (2C, C-2′,6′), 78.8 (C-5-iso), 59.8 (OCH3), 55.8 (2C, OCH3), 55.2
5-Carbamoyl-4-(2,5-dimethoxy-3,4-methylenedioxyphenyl)-3-
phenyl-4,5-dihydroisoxazole 2-Oxide (5c): Off-white crystals
(430 mg, yield 75 %), m.p. 98–101 °C; Rf = 0.25 (hexane/ethyl acet-
ate, 1:1); 1H NMR (500.13 MHz, [D6]DMSO) δ=7.86 (d, 3J = 7.5 Hz,
2H, H-2,6), 7.83 (s, 1H, NH), 7.62 (s, 1H, NH), 7.42 (t, J = 7.2 Hz, 2H,
H-3,5), 7.36 (t, J = 7.3 Hz, 1H, H-4), 6.57 (s, 1H, H-6′), 6.03 (br.s, 2H,
(OCH ), 52.9 (C-4-iso) ppm; IR (KBr) ν= 3396, 2960, 2939, 2839, 1676,
˜
3
1609, 1510, 1460, 1260, 1127 cm–1; HRMS (ESI/QTOF) m/z calcd. for
C
20H22N2O7 + H+: 403.1500 [M + H]+, found 403.1488; m/z calcd. for
3
C20H22N2O7 + Na+: 425.1319 [M + Na]+, found 425.1304.
3
3
3
OCH2O), 5.36 (d, J = 4.0 Hz, 1H, H-4 iso), 4.82 (d, J = 4.0, 1H, H-5
iso), 3.81 (s, 3H, OCH3), 3.73 (s, 3H, OCH3) ppm; 13C NMR
(125.76 MHz, [D6]DMSO) δ=170.6 (CONH), 139.4 (C-3′), 139.3 (C-5′),
137.0 (C-4′), 136.2 (C-2′), 129.8 (C-4), 129.2 (2C, C-2,6), 126.7 (2C, C-
3,5), 126.2 (C-1), 124.5 (C-1′), 115.2 (C-3-iso), 108.4 (C-6′), 102.4
(OCH2O), 79.0 (C-5-iso), 60.59 (OCH3), 57.2 (OCH3), 48.9 (C-4-iso)
5-(N-(3,4-Methelenedioxybenzyl)carbamoyl)-3-(4-methoxy-
phenyl)-4-(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole 2-Ox-
ide (5g): Off-white crystals (410 mg, yield 51 %), m.p. 76–78 °C
(benzene); Rf = 0.8 (benzene/ethyl acetate, 1:1); 1H NMR
(500.13 MHz, [D6]DMSO) δ=8.93 (t, 3J = 5.9 Hz, 1H, NH), 7.90 (d, 3J =
3
8.7 Hz, 2H, H-2,6), 6.99 (d, J = 8.7 Hz, 2H, H-3,5), 6.86 (s, 1H, H-2′′),
ppm; IR (KBr) ν= 3336, 3195, 1678, 1614, 1501, 1450, 1064 cm–1
;
3
6.82 (d, J = 7.9 Hz, 1H, H-6′′), 6.75 (d, 3J = 7.9 Hz, 1H, H-5′′), 6.68
˜
HRMS (ESI/QTOF) m/z: [M + H]+ calcd. for C19H18N2O7 + H+:
(s, 2H, H-2′,6′), 5.95 (s, 2H, OCH2O), 5.25 (d, 3J = 3.3 Hz, 1H, H-4-iso),
4.96 (d, 3J = 3.3 Hz, 1H, H-5-iso), 4.27 (d, 3J = 6.0 Hz, 2H, NHCH2),
+
387.1187, found 387.1193; m/z calcd. for C19H18N2O7 + NH4
:
404.1452 [M + NH4]+, found 404.1457.
3.76 (s, 3H, OCH3), 3.73(s, 6H, 2 × OCH3), 3.62(s, 3H, OCH3) ppm; 13
C
NMR (125.76 MHz, [D6]DMSO) δ=168.1 (CONH), 159.9 (C-4), 153.3
(2C, C-3′,5′), 147.3 (C-3′′), 146.2 (C-4′′), 137.1 (C-1′), 135.0 (C-4′), 132.7
(C-1′′), 128.4 (2C, C-2,6), 120.6 (C-2′′), 117.9 (C-1), 115.2 (C-3-iso),
114.3 (2C, C-3,5), 108.1 (C-5′′), 108.0 (C-6′′), 104.7 (2C, C-2′,6′), 100.9
(OCH2O), 79.2 (C-5 iso), 60.0 (OCH3), 56.0 (2C, 2 × OCH3), 55.3
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-5-(N-ethylcarb-
amoyl)-3-phenyl-4,5-dihydroisoxazole 2-Oxide (5d): Off-white
crystals (220 mg, yield 35 %), m.p. 129–131 °C (heptane); Rf = 0.64
(benzene/ethyl acetate, 1:1); 1H NMR (500.13 MHz, [D6]DMSO) δ=
3
3
8.45 (t, J = 5.4 Hz, 1H, NH), 7.85 (d, J = 7.8 Hz, 2H, H-2,6), 7.42 (t,
3
3J = 7.6 Hz, 2H, H-3,5), 7.36 (t, J = 7.3 Hz, 1H, H-4), 6.56 (s, 1H, H-
(OCH ), 53.2 (C-4-iso), 42.2 (NHCH ) ppm; IR (KBr) ν= 3436, 1686,
˜
3
2
1618, 1508, 1459, 1259, 1128 cm–1 HRMS (ESI/QTOF) m/z calcd. for
C28H28N2O9 + H+: 537.1868 [M + H]+, found 537.1856; m/z calcd. for
C28H28N2O9 + K+: 575.1426 [M + K]+, found 575.1420.
3
6′), 6.02 (br.s, 2H, OCH2O), 5.36 (d, J = 4.2 Hz, 1H, H-4-iso), 4.82 (d,
3J = 4.2 Hz, 1H, H-5-iso), 3.80 (s, 3H, OCH3), 3.73 (s, 3H, OCH3) ppm;
13C NMR (125.76 MHz, [D6]DMSO) δ=167.7 (CONH), 139.0 (C-3′),
138.9 (C-5′), 136.6 (C-4′), 135.8 (C-2′), 129.4 (C-4), 128.8 (2C, C-2,6),
126.3 (2C, C-3,5), 125.8 (C-1), 124.0 (C-1′), 114.8 (C-3-iso), 108.0
(C-6′), 102.1 (OCH2O), 78.8 (C-5-iso), 60.2 (OCH3), 56.8 (OCH3), 48.7
5-(N-(4-Bromophenyl)carbamoyl)-4-(2,5-dimethoxy-3,4-methyl-
enedioxyphenyl)-3-phenyl-4,5-dihydroisoxazole 2-Oxide (5h):
White crystals (480 mg, yield 59 %), m.p. 105–109 °C; Rf = 0.8 (benz-
ene/ethyl acetate, 10:1); 1H NMR (500.13 MHz, [D6]DMSO) δ=10.45
(C-4-iso) ppm; IR (KBr) ν= 3373, 2941, 1679, 1613, 1507, 1457, 1217,
˜
1139, 1068 cm–1; HRMS (ESI/QTOF) m/z calcd. for C21H22N2O7 + H+:
3
3
(s, 1H, NH), 7.86 (d, J = 7.4 Hz, 2H, H-2,6), 7.64 (d, J = 8.9 Hz, 2H,
415.1500 [M + H]+, found 415.1494; m/z calcd. for C21H22N2O7
+
3
3
H-3′′,5′′), 7.52 (d, J = 8.9 Hz, 2H, H-2′′,6′′), 7.42 (t, J = 7.9 Hz, 2H,
H-3,5), 7.36 (t, J = 7.3 Hz, 1H, H-4), 6.66 (s, 1H, H-6′), 6.01 (br.s, 2H,
NH4+: 432.1765 [M + NH4]+, found 432.1760.
3
OCH2O), 5.50 (d, 3J = 4.3 Hz, 1H, H-4-iso), 5.03 (d, 3J = 4.3 Hz, 1H,
H-5-iso), 3.82 (s, 3H, OCH3) 3.74 (3H, s, OCH3) ppm; 13C NMR
(125.76 MHz, [D6]DMSO) δ=167.2 (CONH), 139.4 (C-3′), 139.2 (C-5′),
137.9 (C-1′′), 137.2 (C-4′), 136.3 (C-2′), 132.0 (2C, C-3′′,5′′), 129.8 (C-
4), 129.1 (2C, C-2,6), 126.7 (2C, C-3,5), 126.2 (C-1), 124.0 (C-1′), 122.3
(2C, C-2′′,6′′), 116.4 (C-4′′), 115.0 (C-3-iso), 108.8 (C-6′), 102.4
(OCH2O), 79.4 (C-5-iso), 60.5 (OCH3), 57.3 (OCH3), 49.1 (C-4-iso) ppm;
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-5-(N-(3,4-di-
methoxyphenylethyl)carbamoyl)-3-phenyl-4,5-dihydroisoxaz-
ole 2-Oxide (5e): Reaction condition: 6 equiv. NEt3, 60 °C, 5.5 h.
Beige crystals (520 mg, yield 64 %), m.p. 129–31 °C; Rf = 0.64 (benz-
1
ene/ethyl acetate, 1:1); H NMR (500.13 MHz, [D6]DMSO) δ=8.45 (t,
3J = 5.7 Hz, 1H, NH), 7.85 (d, 3J = 7.5 Hz, 2H, H-2,6), 7.43 (t, 3J =
3
4
7.3 Hz, 2H, H-3,5), 7.37 (t, J = 7.3 Hz, 1H, H-4), 6.80 (d, J = 1.9 Hz,
IR (KBr) ν= 3394, 2934, 1687, 1618, 1525, 1450, 1397, 1225, 1140,
˜
3
4
3
1H, H-2′′), 6.72 (d, J = 8.2 Hz, 1H, H-5′′), 6.67 (dd, J = 1.8 Hz, J =
8.1 Hz, 1H, H-6′′), 6.53 (s, 1H, H-6′), 6.01 (br.s, 2H, OCH2O), 5.30 (d,
3J = 4.0 Hz, 1H, H-4-iso), 4.84 (d, 3J = 4.0 Hz, 1H, H-5-iso), 3.78 (s,
3H, OCH3), 3.73 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.65 (s, 3H, OCH3),
3.37 (m, 2H, CH2N), 2.70 (t, 3J = 7.1 Hz, 2H, CH2Ar) ppm; 13C NMR
1064 cm–1; HRMS (ESI/QTOF) m/z calcd. for C25H21N2O7Br + H+:
541.0605 [M + H]+, found 541.0596; m/z calcd. for C25H21N2O7Br +
NH4+: 558.0870 [M + NH4]+, found 558.0861.
5-(N-(4-Chlorodifluoromethoxyphenyl)carbamoyl)-4-(2,5-di-
(125.76 MHz, [D6]DMSO) δ=168.3 (CONH), 149.1 (C-4′′), 147.7 methoxy-3,4-methylenedioxyphenyl)-3-phenyl-4,5-dihydroisox-
(C-3′′), 139.4 (C-3′), 139.3 (C-5′), 137.0 (C-4′), 136.2 (C-2′), 132.0
(C-1′′), 129.8 (C-4), 129.2 (2C, C-2,6), 126.7 (2C, C-3,5), 126.2 (C-1),
azole 2-Oxide (5i): Beige crystals (315 mg, yield 37 %), m.p. 81–
83 °C (benzene); Rf = 0.55 (benzene/ethyl acetate, 10:1); 1H NMR
124.5 (C-1′), 120.9 (C-6′′), 115.2 (C-3-iso), 113.0 (C-5′′), 112.2 (C-2′′), (500.13 MHz, [D6]DMSO) δ=10.66 (s, 1H, NH), 7.88 (d, 3J = 7.8 Hz,
3
3
108.4 (C-6′), 102.5 (OCH2O), 79.1 (C-5-iso), 60.6 (OCH3), 57.3 (OCH3),
2H, H-2,6), 7.80 (d, J = 9.0 Hz, 2H, H-2′′,6′′), 7.44 (t, J = 7.8 Hz, 2H,
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H-3,5), 7.37 (m, 3H, H-4,3′′,5′′), 6.65 (s, 1H, H-6′), 6.03 (br.s, 2H, 4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-N-ethyl-3-phen-
OCH2O), 5.52 (d, 3J = 4.3 Hz, 1H, H-4-iso), 5.07 (d, 3J = 4.3 Hz, 1H, H-
5-iso), 3.81 (s, 3H, OCH3) 3.74 (s, 3H, OCH3) ppm; 13C NMR (500 MHz,
[D6]DMSO) 167.4 (+CONH), 145.8 (+C-4′′), 139.4 (+C-3′), 139.2
(+C-5′), 137.8 (+C-4′), 137.1 (+C-1′′), 136.3 (+C-2′), 129.8 (-C-4), 129.2
(–2C, C-2,6), 126.8 (–2C, C-3,5), 126.2 (+C-1), 125.4 (+t, 3J = 286.7 Hz,
ylisoxazole-5-carboxamide (6d): This was purified by column
chromatography (silica gel, gradient benzene, benzene/ethyl acet-
ate, 3:1). White crystals (67 mg, yield 42 %), m.p. 154–156 °C; Rf =
0.8 (benzene/ethyl acetate, 1:1); 1H NMR (500.13 MHz, [D6]DMSO)
δ=8.85 (t, 3J = 5.5 Hz, 1H, NH), 7.47–7.40 (m, 5H, Ph), 6.55 (s, 1H,
OCF2Cl), 124.1 (+C-1′), 122.5 (–2C, C-3′′,5′′), 121.8 (–2C, C-2′′,6′′), H-6′), 6.05 (s, 2H, OCH2O), 3.70 (s, 3H, OCH3), 3.40 (s, 3H, OCH3), 3.24
115.0 (+C-3-iso), 108.8 (-C-6′), 102.5 (+OCH2O), 79.3 (-C-5-iso), 60.5
(-OCH ), 57.3 (-OCH ), 49.3 (-C-4-iso); IR (KBr) ν= 3392, 2939, 1695,
(p, 3J = 6.5 Hz, 2H, OCH2), 1.09 (t, 3J = 7.0 Hz, 3H, CH3) ppm; 13C NMR
(125.76 MHz, [D6]DMSO) δ=162.4 (CONH), 160.0 (C-3-iso), 155.8 (C-
5-iso), 138.6 (C-3′), 138.1 (C-5′), 137.1 (C-4′), 135.8 (C-2′), 130.0 (C-4),
128.8 (2C, C-2,6), 128.3 (C-1), 127.6 (2C, C-3,5), 116.2 (C-4-iso), 114.1
(C-1′), 110.6 (C-6′), 101.9 (OCH2O), 59.0 (OCH3), 56.7 (OCH3), 30.7
˜
3
3
1618, 1533, 1508, 1200 cm–1; HRMS (ESI/QTOF) m/z calcd. for
C
26H21ClF2N2O8 + H+: 563.1027 [M + H]+, found 563.1013.
General Procedure for the Synthesis of Ioxazole-5-carbox-
amides (6): DBU (0.44 mmol) was added to the solution of corre-
sponding isoxazoline N-oxide 5 (0.4 mmol) in dry acetonitrile
(3.5 mL), and reaction mixture was stirred for 4 h at 40 °C (or until
the full conversion monitored by TLC). Then the solvent was evapo-
rated in vacuo, the crude product was washed with water (4 mL)
and extracted with ethyl acetate (2 × 5 mL). The resulting mixture
was dried with magnesium sulfate and the solvents evaporated in
vacuo to yield corresponding isoxazole 6.
(CH NH), 14.5 (CH ) ppm; IR (KBr) ν= 3308, 1670, 1520, 1439, 1238,
˜
2
3
1060 cm–1; HRMS (ESI/QTOF) m/z calcd. for C21H20N2O6 + H+:
397.1394 [M + H]+, found 397.1390; [M + K]+ calcd. for C21H20N2O6
+ K+: 435.0953, found 435.0952.
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-N-(3,4-dimeth-
oxyphenethyl)-3-phenylisoxazole-5-carboxamide 6e: The reac-
tion proceeded at 30 °C for 5.5 h. The product was purified by
column chromatography (silica gel, gradient benzene, benzene/
ethyl acetate, 3:1).White crystals (79 mg, yield 37 %), m.p. 157–
158 °C; Rf = 0.45 (benzene/ethyl acetate, 10:1); 1H NMR (500.13 MHz,
[D6]DMSO) δ=8.88 (t, 3J = 5.3 Hz, 1H, NH), 7.47–7.39 (m, 5H, Ph),
6.85(d, 3J = 8.1 Hz, 1H, H-5′′), 6.81 (s, 1H, H-2′′), 6.71 (d, 3J = 8.1, 1H,
H-6′′), 6.52 (s, 1H, H-6′), 6.05 (s, 2H, OCH2O), 3.72 (s, 3H, OCH3), 3.71
(s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.37 (s, 3H, OCH3), 3.44 (q, 3J =
6.8 Hz, 2H, CH2NH), 2.75 (t, 3J = 7.3, 2H, CH2Ar) ppm; 13C NMR
(125.76 MHz, [D6]DMSO) δ=162.3 (CONH), 159.7 (C-3-iso), 155.8
(C-5-iso), 148.5 (C-4′′), 147.2 (C-3′′), 138.5 (C-3′), 138.0 (C-5′), 136.9
(C-4′), 135.6 (C-2′), 131.4 (C-1′′), 129.9 (C-4), 128.7 (2C, C-2,6), 128.2
(C-1), 127.5 (2C, C-3,5), 120.3 (C-6′′), 116.2 (C-4-iso), 113.9 (C-1′),
112.3 (C-5′′), 111.7 (C-2′′), 110.3 (C-6′), 101.8 (OCH2O), 58.9 (OCH3),
56.5 (OCH3), 55.4 (OCH3), 55.2 (OCH3), 40.3 (CH2NH), 34.2 (CH2Ar)
4-(4-Methoxyphenyl)-3-phenylisoxazole-5-carboxamide (6a):
White crystals (63 mg, yield 54 %), m.p. 204–207 °C (EtOH); Rf = 0.7
(benzene/ethyl acetate, 1:1); 1H NMR (500.13 MHz, [D6]DMSO) δ=
8.22 (s, 1H, NH2), 7.96 (s, 1H, NH2), 7.47–7.35 (m, 5H, Ph), 7.20 (d,
3
3J = 8.2 Hz, 2H, H-2′,6′), 6.93 (d, J = 8.2 Hz, 2H, H-3′,5′), 3.78 (s, 3H,
OCH3) ppm; 13CNMR (125.76 MHz, [D6]DMSO) δ=162.0 (CONH),
159.8 (C-3-iso), 158.9 (C-4′), 157.8 (C-5-iso), 131.4 (2C, C-2,6), 129.8
(C-4), 128.6 (2C, C-2′,6′), 128.3 (2C, C-3,5), 128.0 (C-1), 120.0 (C-1′),
119.9 (C-4-iso), 113.5 (2C, C-3′,5′), 55.0 (OCH ) ppm; IR (KBr) ν= 3359,
˜
3
3177, 1695, 1634, 1603, 1515, 1353, 1248 cm–1; HRMS (ESI/QTOF)
m/z calcd. for C17H14N2O4 + H+: 295.1077 [M + H]+, found 295.1081;
m/z calcd. for C17H14N2O3 + NH4+: 312.1343 [M + NH4]+, found
312.1345; m/z calcd. for C17H14N2O3 + K+: 333.0636 [M + K]+, found
333.0636.
ppm; IR (KBr) ν= 3390, 2947, 1681, 1514, 1236, 1160 cm–1; HRMS
˜
(ESI/QTOF) m/z calcd. for C29H28N2O8 + H+: 533.1918 [M + H]+,
found 533.1912; m/z calcd. for C29H28N2O8 + K+: 571.1477 [M + K]+,
found 571.1472.
4-(3,4-Dimethoxyphenyl)-3-phenylisoxazole-5-carboxamide
(6b): White crystals (74 mg, yield 57 %), m.p. 203–205 °C (EtOH);
Rf = 0.55 (benzene/ethyl acetate, 1:1); 1H NMR (500.13 MHz,
[D6]DMSO) δ=8.19 (s, 1H, NH2), 7.97 (s, 1H, NH2), 7.48–7.38 (m, 5H,
Ph), 6.95 (d, J = 8.3 Hz, 1H, H-5′), 6.89 (d, 4J = 2.0 Hz, 1H, H-2′), 6.82
(dd, 4J = 2.0 Hz, 3J = 8.2 Hz, 1H, H-6′), 3.78 (s, 3H, OCH3), 3.60 (s,
3H, OCH3) ppm; 13C NMR (125.76 MHz, [D6]DMSO) δ=162.5 (CONH),
159.4 (C-3-iso), 158.4 (C-5-iso), 149.2 (C-4′), 148.5 (C-3′), 130.2 (C-4),
129.1 (2C, C-2,6), 128.8 (2C, C-3,5), 128.4 (C-1), 123.2 (C-5′), 120.6
(C-1′), 120.4 (C-4-iso), 114.5 (C-2′), 111.7 (C-6′), 55.8 (OCH3), 55.7
3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole-5-
carboxamide (6f): The reaction proceeded at 50 °C for 5 h. The
white crystals (86 mg, yield 56 %), m.p. 187–189 °C (ethyl acetate);
Rf = 0.5 (benzene/ethyl acetate, 1:1); 1H NMR (500.13 MHz,
[D6]DMSO) δ=8.06 (s, 1H, NH2), 7.88 (s, 1H, NH2), 7.34 (d, 3J = 8.0 Hz,
3
3
2H, H-3,5), 6.97 (d, J = 8.0 Hz, 2H, H-2,6), 6.62 (d, J = 8.0 Hz, 2H,
H-2′,6′), 3.77 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.65 (s, 6H, 2 × OCH3)
ppm; 13C NMR(125.76 MHz, [D6]DMSO) δ=161.5 (CONH2), 160.4 (C-
3-iso), 159.0 (C-4), 157.9 (C-5-iso), 152.4 (2C, C-3′,5′), 137.2 (C-4′),
129.7 (2C, C-2,6), 123.6 (C-1′), 120.0 (C-1), 119.8 (C-4-iso), 114.0 (2C,
C-3,5), 107.9 (2C, C-2′,6′), 60.0 (OCH3), 55.8 (2C, 2 × OCH3), 55.2
(OCH ); IR (KBr) ν= 3447, 1702, 1459, 1255, 1133 cm–1; HRMS (ESI/
(OCH ) ppm; IR (KBr) ν= 3435, 3225, 1690, 1601, 1586, 1518,
˜
3
1250 cm–1; HRMS (ESI/QTOF) m/z calcd. for C18H16N2O4 + H+:
325.1183 [M + H]+, found 325.1189; m/z calcd. for C18H16N2O4
+
Na+: 347.1002 [M + Na]+, found 347.1005; m/z calcd. for C18H16N2O4
+ K+: 363.0742 [M + K]+, found 363.0745.
3
˜
QTOF) m/z calcd. for C20H20N2O6 + H+: 385.1394 [M + H]+, found
385.1390; m/z calcd. for C20H20N2O6 + NH4+: 402.1660 [M + NH4]+,
found 402.1656.
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-3-phenylisox-
azole-5-carboxamide (6c): White crystals (94 mg, yield 64 %), m.p.
234–237 °C (EtOH); Rf = 0.75 (benzene/ethyl acetate, 1:1); 1H NMR
(500.13 MHz, [D6]DMSO) δ=8.14 (s, 1H, NH2), 7.91 (s, 1H, NH2), 7.47–
7.39 (m, 5H, Ph), 6.55 (s, 1H, H-6′), 6.05 (s, 2H, OCH2O), 3.70 (s, 3H,
N-(3,4-Methylenedioxybenzyl)-3-(4-methoxyphenyl)-4-(3,4,5-tri-
methoxyphenyl)isoxazole-5-carboxamide (6g): White crystals
OCH3), 3.41 (s, 3H, OCH3) ppm; 13C NMR (125.76 MHz, [D6]DMSO) (151 mg, yield 73 %), m.p. 178–180 °C; Rf = 0.2 (benzene/ethyl acet-
δ=162.3 (CONH), 159.8 (C-3-iso), 157.5 (C-5-iso), 138.5 (C-3′), 138.0
(C-5′), 136.9 (C-4′), 135.6 (C-2′), 129.8 (C-4), 128.7 (2C, C-2,6), 128.2
(C-1), 127.5 (2C, C-3,5), 116.4 (C-4-iso), 114.1 (C-1′), 110.3 (C-6′), 101.8
ate, 10:1); 1H NMR (500.13 MHz, [D6]DMSO) δ=9.31 (t, 3J = 6.1Hz,
3
3
1H, NH), 7.35 (d, J = 8.8 Hz, 2H, H-3,5), 6.98 (d, J = 8.8 Hz, 2H, H-
2,6), 6.85 (d, 4J = 1.6 Hz, 1H, H-2′′), 6.83 (d, 3J = 8.0 Hz, 1H, H-5′′),
(OCH O), 58.9 (OCH ), 56.5 (OCH ) ppm; IR (KBr) ν= 3161, 1687, 6.74 (dd, 4J = 1.5, 3J = 8.0, 1H, H-6′′), 6.59 (s, 2H, H-2′,6′), 5.98 (s, 2H,
˜
2
3
3
1602, 1500, 1432, 1344 cm–1; HRMS (ESI/QTOF) m/z calcd. for
OCH2O), 4.33 (d, 3J = 6.1 Hz, 2H, CH2), 3.77 (s, 3H, OCH3), 3.69 (s,
C19H16N2O6 + H+: 369.1081 [M + H]+, found 369.1073; m/z calcd. for 3H, OCH3), 3.60 (s, 6H, 2 × OCH3) ppm; 13C NMR(125.76 MHz,
C19H16N2O6 + Na+: 391.0901 [M + Na]+, found 391.0887.
[D6]DMSO) δ=161.9 (+CONH2), 161.0 (+C-3-iso), 159.5 (+C-4), 156.9
Eur. J. Org. Chem. 0000, 0–0
www.eurjoc.org
8
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
(+C-5-iso), 153.0 (+2C, C-3′,5′), 147.7 (+C-4′′), 146.6 (+C-3′′), 138.0
(+C-4′), 132.9 (+C-1′′), 130.2 (–2C, C-2,6), 123.9 (+C-1′), 121.1 (-C-2′′),
pyridinium bromide (7.5 mmol), and triethylamine (15 mmol) in dry
acetonitrile (40 mL) was stirred at 60 °C for 5 h (TLC control). Aceto-
120.4 (+C-1), 120.2 (+C-4-iso), 114.6 (–2C, C-3,5), 108.4 (–2C, C-2′,6′), nitrile was removed in vacuo, and then dichloromethane (150 mL)
108.3 (–2C, C-5′′,6′′), 101.3 (+OCH2O), 60.5 (-OCH3), 56.3 (–2C, OCH3),
55.7 (-OCH ), 42.5 (+CH ) ppm; IR (KBr) ν= 3351, 2939, 1681, 1584,
was added. The dichloromethane extract was washed with water,
5 % aqueous HCl, water until pH 7.0, and dried by magnesium sulf-
˜
3
2
1243, 1128 cm–1; HRMS (ESI/QTOF) m/z calcd. for C28H26N2O8 + H+: ate. Methylene chloride was removed in vacuo and the residue was
519.1762 [M + H]+, found 519.1751; m/z calcd. for C28H26N2O8
+
triturated in ethanol. The collected crystals were washed with eth-
anol and dried in air to obtain 9c as white solid (1.16 g, yield 56 %),
m.p. 129–132 °C; Rf = 0.46 (benzene/ethyl acetate, 9:1); 1H NMR
(500.13 MHz, [D6]DMSO) δ=7.84 (d, 3J = 7.5 Hz, 2H, H-2,6), 7.43
Na+: 541.1581 [M + Na]+, found 541.1571.
N-(4-Bromophenyl) 4-(2,5-dimethoxy-3,4-methylenedioxy-
phenyl)-3-phenylisoxazole-5-carboxamide (6h): White crystals
(115 mg, yield 55 %), m.p. 240–243 °C (acetonitrile); Rf = 0.9 (benz-
ene/ethyl acetate, 10:1); 1H NMR (500.13 MHz, [D6]DMSO) δ=10.95
3
3
(t, J = 7.5 Hz, 2H, H-3,5), 7.37 (t, J = 7.5 Hz, 1H, H-4), 6.50 (s, 1H,
3
H-6′), 6.03 (s, 2H, OCH2O), 5.44 (d, J = 3.5 Hz, 1H, H-4-iso), 5.08 (d,
3J = 3.5 Hz, 1H, H-5-iso), 4.25 (m, 2H, OCH2), 3.83 (s, 3H, OCH3), 3.73
3
3
(br.s, 1H, NH), 7.72 (d, J = 8.8 Hz, 2H, H-3′′,5′′), 7.54 (d, J = 8.8 Hz,
2H, H-2′′,6′′), 7.49–7.43 (m, 5H, Ph), 6.59 (s, 1H, H-6′), 6.05 (s, 2H,
OCH2O), 3.69 (s, 3H, OCH3), 3.42 (s, 3H, OCH3) ppm; 13CNMR
(500.13 MHz, [D6]DMSO) δ=162.5 (CONH), 159.4 (C-3-iso), 154.5
(C-5-iso), 138.5 (C-3′), 138.0 (C-5′), 137.3 (C-1′′), 137.0 (C-4′), 135.6
(C-2′), 131.5 (2C, C-3′′,5′′), 130.0 (C-4), 128.7 (2C, C-2,6), 128.0 (C-1),
127.6 (2C, C-3,5), 122.4 (2C, C-2′′,6′′), 117.3 (C-4′′), 116.2 (C-4-iso),
113.6 (C-1′), 110.3 (C-6′), 101.8 (OCH2O), 58.9 (OCH3), 56.5 (OCH3)
(s, 3H, OCH3), 1.27 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (125.76 MHz,
3
[D6]DMSO) δ=169.1, 139.3, 139.3, 137.1, 136.0, 129.9, 129.2 (2C),
126.7 (2C), 125.9, 123.9, 114.7, 108.0, 102.5, 77.5, 62.1, 60.6, 57.2,
48.8, 14.3; IR (KBr) ν= 1763, 1618, 1509, 1214, 1066 cm–1; HRMS (ESI/
˜
QTOF) m/z calcd. for C21H22NO8 + K+: 454.0899 [M + K]+, found
454.0893.
General Procedure for the Preparation of 3,4-Diarylisoxazoles
(15) and 3,4-Diarylisoxazole-5-carboxylic Acids (11). A mixture
of the corresponding 5-(ethoxycarbonyl)-4,5-dihydroisoxazole N-ox-
ppm; IR (KBr) ν= 3293, 1684, 1594, 1527, 1492, 1232, 1057,
˜
1020 cm–1; HRMS (ESI/QTOF) m/z calcd. for C25H19N2O6Br + H+:
523.0499 [M + H]+, found 523.0491; m/z calcd. for C25H19N2O6Br + ides 9[18] (1.5 mmol), 2 % ethanolic NaOH solution (10 mL) and
Na+: 545.0319 [M + Na]+, found 545.0313.
water (2 mL) was stirred at 60 °C for 5–6 h, then ethanol was re-
moved in vacuo. The residue was suspended in dichloromethane
(100 mL) and the aqueous alkaline layer was separated. The di-
chloromethane extract was washed with water (2 × 10 mL), filtered
through silica pad, and the solvents evaporated in vacuo to afford
corresponding 3,4- diarylisoxazole 15. Water washings were com-
bined with the aqueous alkaline layer, evaporated to 1/3 initial vol-
ume, acidified with 10 % aqueous HCl (pH 1.0–2.0), and extracted
with ethyl acetate (100 mL). The extract was washed with brine and
then dried with anhydrous Na2SO4. The solvent was removed, the
residue was mixed with benzene, and the crystals of the corre-
sponding 3,4-diarylisoxazole-5-carboxylic acid 11 were filtered and
dried in vacuo.
N-(4-(Chlorodifluoromethoxy)phenyl)-4-(2,5-dimethoxy-3,4-
methylenedioxyphenyl)-3-phenylisoxazole-5-carboxamide (6i):
White crystals (105 mg, yield 48 %), m.p. 232–233 °C (acetonitrile);
Rf = 0.85 (benzene/ethyl acetate, 10:1); 1H NMR (500.13 MHz,
[D6]DMSO) δ=11.06 (s, 1H, NH), 7.88 (d, 3J = 8.9 Hz, 1H, H-3′′,5′′),
3
7.50–7.45 (m, 5H, Ph), 7.36 (d, J = 8.8 Hz, 2H, H-2′′,6′′), 6.61 (s, 1H,
H-6′), 6.06 (s, 2H, OCH2O), 3.69 (s, 3H, OCH3), 3.42 (s, 3H, OCH3) ppm;
13C NMR (125.76 MHz, [D6]DMSO) δ=162.5 (CONH), 159.2 (C-3-iso),
154.5 (C-5-iso), 145.5 (C-4′′), 138.5 (C-3′), 138.0 (C-5′), 137.1 (C-4′),
137.0 (C-1′′), 135.6 (C-2′), 130.0 (C-4), 128.8 (2C, C-2,6) 128.0 (C-1),
127.6 (2C, C-3,5), 124.9 (OCF2Cl), 121.9 (4C, C-2′′,3′′,5′′,6′′), 117.5 (C-
4-iso), 113.6 (C-1′), 110.3 (C-6′), 101.9 (OCH2O), 59.0 (OCH3), 56.5
(OCH ) ppm; IR (KBr) ν= 3315, 1693, 1606, 1542, 1199, 1021 cm–1
;
3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole-5-
˜
3
HRMS (ESI/QTOF) m/z calcd. for C26H19ClF2N2O7 + H+: 545.0922 [M
+ H]+, found 545.0919; m/z calcd. for C26H19ClF2N2O7 + Na+:
567.0741 [M + Na]+, found 567.0734.
carboxylic Acid (11b): White solid (0.15 g, yield 27 %), m.p. 203–
1
205 °C; H NMR (125.76 MHz, [D6]DMSO) δ = 14.1 (br s, 1H, COOH),
8.33 (d, J = 8.8 Hz, 2H, H-2,6), 7.97 (d, J = 8.9 Hz, 2H, H-3,5), 6.64 (s,
2H, H-2′,6′), 3.76 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.64 (s, 6H, OCH3);
MS (70eV): m/z (%): 385 (33) [M]+, 342 (38), 312 (34), 298 (63), 77
(100); elemental analysis calcd. (%) for C20H19NO7: C 62.33; H 4.97;
N 3.63. found: C 62.52; H 4.80; N 3.81.
2-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-1-phenylethan-
one oxime 8c: A mixture of the corresponding isoxazoline N-oxide
5 (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 acidified up to pH 5.0 and dichloromethane 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)isoxazole
(50 mL) was added. The dichloromethane extract evaporated in
vacuo, and residue was purified by column chromatography (silica
gel, gradient benzene, benzene/ethyl acetate, 1:1). Off-white crys-
tals (52 mg, yield 11 %), m.p. 86–88 °C; Rf = 0.4 (benzene/ethyl
(15b): White solid (0.235 g, yield 48 %), m.p. 143–145 °C; 1H NMR
(500 MHz, CDCl3) δ = 8.48 (s, 1H, H-5-iso), 7.49 (d, J = 8.9 Hz, 2H,
H-2,6), 6.90 (d, J = 8.9 Hz, 2H, H-3,5), 6.46 (s, 2H, H-2′,6′), 3.88 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3), 3.74 (s, 6H, OCH3); 13C NMR (125.76 MHz,
acetate, 10:1); 1H NMR (500.13 MHz, CDCl3) δ=8.40–8.10 (br.s, 1H, CDCl3) δ = 160.7, 159.7, 155.9 (2C), 153.4, 137.8, 130.1 (2C), 124.6,
NOH), 7.61 (m, 2H, H-2,6), 7.32 (m, 3H, H-3,4,5), 6.34 (s, 1H, H-6′),
5.93 (s, 2H, OCH2O), 4.13 (s, 2H, CH2), 3.86 (s, 3H, OCH3), 3.74 (s, 3H,
OCH3) ppm; 13C NMR (125.76 MHz, [D6]DMSO) δ=158.1 (C=N), 139.1
120.8, 120.0, 114.0 (2C), 106.1 (2C), 61.0, 56.1 (2C), 55.3; MS (70eV):
m/z (%): 341 (38) [M]+, 326 (4), 92 (71), 77 (100); elemental analysis
calcd. (%) for C19H19NO5: C 66.85; H 5.61; N 4.10. found: C 66.77;
(C-3′), 138.5 (C-4′), 136.2 (C-5′), 135.5 (C-1), 135.4 (C-2′), 129.2 (C-4), H 5.52; N 4.38.
128.4 (2C, C-3,5), 126.5 (2C, C-2,6), 121.9 (C-6′), 108.0 (OCH2O), 59.9
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-3-phenylisox-
(CH O), 56.8 (CH O), 25.4 (CH ) ppm; IR (CCl ) ν= 3594 (NOH), 1643
˜
3
3
2
4
azole-5-carboxylic Acid (11c): White solid (89 mg, yield 21 %), m.p.
198–200 °C; 1H NMR (500 MHz, [D6]DMSO) δ = 7.48–7.40 (m, 5H,
Ph), 6.62 (s, 1H, H-6′), 6.09 (s, 2H, OCH2O), 3.72 (s, 3H, OCH3), 3.42
(s, 3H, OCH3); 13C NMR (125.76 MHz, [D6]DMSO) δ = 162.6, 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 calcd. for
(C=N), 1505, 1454, 1432, 1272 (NOH), 1272, 1140, 1054 cm–1; HRMS
(ESI/QTOF) m/z calcd. for C27H17N1O5 + H+: 316.1179 [M + H]+,
found 316.1166.
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-5-(ethoxycarb-
onyl)-3-phenyl-4,5-dihydroisoxazole N-oxide 9c: A mixture of the
corresponding nitroethylene (5 mmol), ethoxycarbonylmethyl- C19H15NO7 + H+: 370.0921 [M + H]+, found 370.0913; m/z calcd. for
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C19H15NO7 + K+: 408.0480 [M + K]+, found 408.0484; elemental anal-
ysis calcd. (%) for C19H15NO7: C 61.79; H 4.09; N 3.79. found: C 61.63;
H 4.22; N 3.54.
of 1.0° and ꢁ scan mode. The data were indexed and integrated
using the utility iMOSFLM from the CCP4 program suite[31] and then
scaled and corrected for absorption using the Scala program (Tmin
=
0.920; Tmax = 0.940).[32] The structure was determined by direct
methods and refined by full-matrix least square technique on F2 in
anisotropic approximation for non-hydrogen atoms. The hydrogen
atom of the OH-group was objectively localized in the difference-
Fourier map and refined isotropically with fixed displacement pa-
rameters [Uiso(H) = 1.5Ueq(O)]. The other hydrogen atoms were
placed in calculated positions and refined within the riding model
with fixed isotropic displacement parameters [Uiso(H) = 1.5Ueq(C)
for the methyl groups and 1.2Ueq(C) for the other groups]. The final
divergence factors were R1 = 0.074 for 2074 independent reflections
with I > 2σ(I) and wR2 = 0.206 for all independent reflections, S =
1.077. All calculations were carried out using the SHELXTL program
suite.[33]
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-3-phenylisoxaz-
ole (15c): White solid (0.22 g, yield 53 %), m.p. 166–168 °C; 1H NMR
(500 MHz, [D6]DMSO) δ = 9.04 (s, 1H, H-5), 7.46–7.40 (m, 5H, Ph),
6.51(s, 1H, H-6′), 6.06 (s, 2H, OCH2O), 3.69 (s, 3H, OCH3), 3.40 (s, 3H,
OCH3); 13C NMR (125.76 MHz, [D6]DMSO) δ = 160.7, 158.9, 139.1,
138.0, 137.2, 136.0, 130.0, 129.5, 129.2, 128.0, 115.9, 114.6, 110.2,
102.3, 59.5, 56.9; HRMS (ESI/QTOF) m/z calcd. for C18H15NO5 + H+:
326.1023 [M + H]+, found 326.1019; m/z calcd. for C18H15NO5 + K+:
364.0582 [M + K]+, found 364.0582; elemental analysis calcd. (%) for
C18H15NO5: C 66.46; H 4.65; N 4.31. found: C 66.61; H 4.54; N 4.52.
5-Carboxy-4-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-di-
hydroisoxazole N-Oxide (12a): A solution of compound 9a[4]
(0.546 g, 1.5 mmol) in dioxane (15 mL) and aqueous HCl (9 %, 3 mL)
was heated at ca. 60 °C for 5 h, then kept overnight at room temper-
ature. Dioxane was evaporated in vacuo and the residue was ex-
tracted 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 12a as white solid (0.512 g, yield
98 %), m.p. 116–119 °C; Rf = 0.31 (benzene/methanol/acetic acid,
45:8:4); 1H NMR (500 MHz, [D6]DMSO) δ=13.75 (br s, 1H, COOH),
7.85 (d, 3J = 9.0 Hz, 2H, H-2′,6′), 7.45 (d, 3J = 8.5 Hz, 2H, H-3′′,5′′),
7.39 (d, 3J = 8.5 Hz, 2H, H-2′′,6′′), 6.97 (d, 3J = 9.0 Hz, 2H, H-3′,5′),
5.39 (d, 3J = 2.5 Hz, 1H, H-4-iso), 4.99 (d, 3J = 2.5 Hz, 1H, H-5-iso),
3.75 (s, 3H, OCH3) ppm; 13C NMR (500 MHz, [D6]DMSO) δ = 173.3,
159.9, 138.0, 132.7, 129.2 (2C), 129.1 (2C), 128.2 (2C), 117.4, 114.3
CCDC 1912623 (for 17c) contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre.
Phenotypic Sea Urchin Embryo Asay:[27] Adult sea urchins, Para-
centrotus lividus, 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 micro-
scope (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 solutions of compounds were pre-
(2C), 114.0, 77.4, 55.2, 51.9; IR (KBr) ν= 1752, 1609, 1510, 1260,
˜
1170 cm–1; MS (70eV): m/z (%): 347 (17) [M]+, 316 (17), 303 (16),
135 (100); elemental analysis calcd. (%) for C17H14ClNO5: C 58.72; H
4.06; N 4.03. found: C 58.65; H 4.16; N 4.25.
pared in DMSO at 10 mM concentration followed by a 10-fold dilu-
tion with 96 % EtOH. This procedure enhanced the solubility of the
test compounds in the salt-containing medium (seawater), as evi-
denced 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 con-
centrations of either DMSO (>0.1 %) or EtOH (>1 %) caused nonspe-
cific alteration and retardation of the sea urchin embryo develop-
ment independent of the treatment stage. The antiproliferative ac-
tivity was assessed by exposing fertilized eggs (8–15 min after fertil-
ization, 45–55 min before the first mitotic cycle completion) to 2-
fold decreasing concentrations of the compound. Cleavage altera-
tion and arrest were clearly detected at 2.5 h and 5.5 h after fertiliza-
tion, when control embryos reached 8-cell and early blastula stages,
respectively. The effects were estimated quantitatively as an effec-
tive threshold concentration, resulting in cleavage alteration and
embryo death before hatching or full mitotic arrest. At these con-
centrations all tested microtubule destabilizers caused 100 % cleav-
age 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. 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. Experi-
ments with the sea urchin embryos fulfil the requirements of bio-
logical 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.
4-(2,5-Dimethoxy-3,4-methylenedioxyphenyl)-5-hydroxy-3-
phenyl-6H-1,2-oxazin-6-one (17c): DBU (0.52 mmol, 0.08 g) was
added to solution of 4,5-dihydroisoxazole N-oxide 9c (0.2 g,
0.48 mmol) in acetonitrile (4.4 mL) whilst stirring and mixture was
kept for a night. Solvent was evaporated in vacuo, water (2 mL) and
then 1 N HCl was added dropwise to achieve pH 1.0–2.0. A product
was extracted with ethyl acetate, organic layer was dried with mag-
nesium sulfate and ethyl acetate was evaporated. Crude oxazinone
17c was recrystallized from EtOH. Beige crystals (60 mg, yield 33 %),
m.p. 176–182 °C; Rf = 0.2 (benzene/ethyl acetate, 1:1); 1H NMR
(500.13 MHz [D6]DMSO) δ=11.90 (s, 1H, OH), 7.38–7.30 (m, 3H, H-
3,4,5-Ph), 7.23 (d, 3J = 7.5 Hz, 2H, H-2,6-Ph), 6.48 (s, 1H, CH), 5.96
(br.s, 2H, OCH2O), 3.65 (s, 3H, OCH3), 3.42 (s, 3H, OCH3); 13C NMR
(125.76 MHz, [D6]DMSO) δ=163.1, 159.2, 146.3, 138.3, 137.4, 136.9,
135.2, 132.7, 129.3, 128.4 (2C), 127.8 (2C), 115.9, 115.5, 110.0, 101.6,
58.9, 56.48; HRMS (ESI/QTOF) m/z calcd. for C19H15NO7 + H+:
370.0920 [M + H]+, found 370.0909; m/z calcd. for C19H15NO7 + K+:
408.0480 [M + K]+, found 408.0478.
X-ray Crystal Structure Determination
The colorless prismatic crystal of 17c (C19H15NO7, M = 369.32) is
monoclinic, space group P21/c, at T = 100 K: a = 11.959(2) Å,
b = 7.0189(14) Å, c = 20.934(4) Å, ꢀ = 105.28(3)°, V = 1695.1(6) Å3,
Z = 4, dcalc = 1.447 g/cm3, F(000) = 768, μ = 0.236 mm–1. The intensi-
ties of 13800 reflections (3464 independent reflections, Rint = 0.113)
were measured on the “Belok” beamline of the Kurchatov Synchro-
tron Radiation Source (National Research Center “Kurchatov Insti-
tute”, Moscow, Russian Federation) using a Rayonix SX165 CCD de-
tector at λ = 0.96260 Å. A total of 360 images for two different
orientations of the crystal were collected using an oscillation range
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Acknowledgments
This work was carried out with the financial support of the
Russian Scientific Foundation (grant no. 18–13–00044). X-ray
structure determination was supported by the RUDN University
Program “5–100”.
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Received: April 30, 2019
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Isoxazoles and Isoxazoline-N-Oxides
A. S. Maksimenko, V. P. Kislyi,*
N. B. Chernysheva, Yu. A. Strelenko,
Y. V. Zubavichus, V. N. Khrustalev,
M. N. Semenova,
V. V. Semenov ................................... 1–12
Effective Synthesis of 3,4-Diaryl-
isoxazole-5-carboxamides and their
Antiproliferative Properties
3,4-Diaryl-isoxazole-5-carboxamides 6, isoxazoline N-oxides 5. In contrast,
structural analogues of heat shock base-catalyzed recyclization of 3,4-di-
protein inhibitors, were obtained by a aryl-5-(ethoxycarbonyl)isoxazoline N-
simple scalable rearrangement of in- oxides
9 unexpectedly yielded 5-
termediate 3,4-diaryl-5-carboxamido- hydroxy-1,2-oxazin-6-ones 17.
DOI: 10.1002/ejoc.201900643
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