Synthesis and biological evaluation of novel 5-hydroxylaminoisoxazole derivatives as lipoxygenase inhibitors and metabolism enhancing agents

Article abstract of DOI:10.1016/j.bmc.2015.12.040

A versatile synthesis of novel 5-hydroxylaminoisoxazoles bearing adamantane moieties has been accomplished using the heterocyclization reactions of readily available unsaturated esters by the treatment with tetranitromethane in the presence of triethylami

Full text of DOI:10.1016/j.bmc.2015.12.040

Accepted Manuscript
Synthesis and biological evaluation of novel 5-hydroxylaminoisoxazole deriv-
atives as lipoxygenase inhibitors and metabolism enhancing agents
Elena B. Averina, Dmitry A. Vasilenko, Yulia A. Gracheva, Yuri K. Grishin,
Eugene V. Radchenko, Vladimir V. Burmistrov, Gennady M. Butov, Margarita
E. Neganova, Tatyana P. Serkova, Olga M. Redkozubova, Elena F. Shevtsova,
Elena R. Milaeva, Tamara S. Kuznetsova, Nikolay S. Zefirov
PII:
S0968-0896(15)30206-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.12.040
BMC 12733
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 October 2015
18 December 2015
23 December 2015
Please cite this article as: Averina, E.B., Vasilenko, D.A., Gracheva, Y.A., Grishin, Y.K., Radchenko, E.V.,
Burmistrov, V.V., Butov, G.M., Neganova, M.E., Serkova, T.P., Redkozubova, O.M., Shevtsova, E.F., Milaeva,
E.R., Kuznetsova, T.S., Zefirov, N.S., Synthesis and biological evaluation of novel 5-hydroxylaminoisoxazole
derivatives as lipoxygenase inhibitors and metabolism enhancing agents, Bioorganic & Medicinal Chemistry (2015),
doi: http://dx.doi.org/10.1016/j.bmc.2015.12.040
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Synthesis and biological evaluation of novel 5-
hydroxylaminoisoxazole derivatives as lipoxygenase
inhibitors and metabolism enhancing agents
E.B. Averina, D.A. Vasilenko, Y.A. Gracheva, Y.K. Grishin, E.V. Radchenko, V.V. Burmistrov, G.M. Butov,
M.E. Neganova, T.P. Serkova, O.M. Redkozubova, E.F. Shevtsova, E.R. Milaeva, T.S. Kuznetsova, N.S.
Zefirov

Bioorganic & Medicinal Chemistry
Synthesis and biological evaluation of novel 5-hydroxylaminoisoxazole derivatives as
lipoxygenase inhibitors and metabolism enhancing agents
a,b
a
a
a
Elena B. Averina,  Dmitry A. Vasilenko, Yulia A. Gracheva, Yuri K. Grishin, Eugene V.
a,b
c
c
b
Radchenko, Vladimir V. Burmistrov, Gennady M. Butov, Margarita E. Neganova, Tatyana P.
b
b
a,b
a,b
a
Serkova, Olga M. Redkozubova, Elena F. Shevtsova, Elena R. Milaeva, * Tamara S. Kuznetsova,
a,b
Nikolay S. Zefirov
a
Lomonosov Moscow State University, Department of Chemistry, Leninskie Gory, 1-3, Moscow 119991, Russia
IPAC RAS, Severnyi Proezd, 1, Chernogolovka, Moscow Region, 142432, Russia
b
c
Volgograd State Technical University, VSTU, Lenina avenue, 28, Volgograd, 400005, Russia
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A versatile synthesis of novel 5-hydroxylaminoisoxazoles bearing adamantane moieties has been
accomplished using the heterocyclization reactions of readily available unsaturated esters by the
treatment with tetranitromethane in the presence of triethylamine and subsequent reduction of
2
resulting 5-nitroisoxazoles by SnCl with the participation of THF. A number of obtained
Available online
isoxazole derivatives were evaluated for their antioxidative activity, inhibition of lipoxygenases
and impact on the rat liver mitochondria. The majority of tested compounds demonstrated
moderate antiradical activity in DPPH test (up to EC50 16 μM). The same compounds strongly
inhibited soybean lipoxygenase (up to IC50 0.4 μM) and Fe - and Fe -induced lipid
peroxidation (LP) of rat brain cortex homogenate (up to IC50 0.3 μM). All tested isoxazole
derivatives promoted the phosphorylating respiratory activity simultaneously with maximal
stimulated respiratory activity of mitochondria and do not reveal any toxicity towards the
primary culture of rat cortex neurons.
2+
3+
Keywords:
Isoxazole
Heterocyclization
Reduction
Lipoxygenase
Mitochondria
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
biosynthesis, but also due to their involvement in induction of
lipid peroxidation. Therefore, a search for selective lipoxygenase
inhibitors and multitarget antioxidants with LOX-inhibiting and
antioxidant activities is of a considerable interest. Although many
attempts to develop selective LOX inhibitors have been made,
Zileuton, which is an iron-binding 5-LOX inhibitor with
antiasthmatic action, remains the only approved drug product.
Oxidative stress induced by reactive oxygen species has been
implicated in the pathogenesis of various diseases and disorders.
One of the main features and biomarkers for oxidative stress is
the lipid peroxidation, which may be induced through non-
enzymatic or enzymatic pathways involving lipoxygenase,
cyclooxygenase or cytochrome P450. Lipoxygenases (LOXs) are
non-heme iron-containing dioxygenases which catalyze the
conversion of unsaturated fatty acids containing one or more
cis,cis-pentadiene fragments into hydroperoxy fatty acids. This
process is the first step in a series of reactions producing
inflammatory-mediating
hydroxyeicosatetraenoic acids (HETEs) which are associated
with a variety of disease states including asthma, rheumatoid
5-10
Recently we described the first synthesis of previously
unknown 5-[hydroxy(tetrahydrofuran-2-yl)amino]isoxazoles
1
1
-4
via unusual reduction of 5-nitroisoxazoles with the participation
1
1
of THF used as solvent, as well as the novel approach to hardly
accessible functionalized 5-nitroisoxazoles based on the
heterocyclization of electrophilic alkenes under the action of
leukotrienes
(LTs)
and
1
2,13
tetranitromethane (TNM) – triethylamine complex
(Scheme
1
1).
arthritis, inflammatory bowel disease, psoriasis, and allergy. The
human lipoxygenases are indisputably involved in human
pathology not only due to their essential role in LTs and HETEs
In the present work we proposed a new scaffold for structure
design of LOXs inhibitors – novel N-hydroxylaminoisoxazoles 1
—
——

Corresponding authors. Tel.: +7-495-939-3969; fax: +7-495-939-3969 (E.B.A.); Tel.: +7-495-939-3864; fax: +7-495-939-0290 (E.R.M.);
e-mail: elaver@org.chem.msu.ru (E.B. Averina), e-mail: milaeva@med.chem.msu.ru (E.R. Milaeva)

11-13
Scheme 1. A described procedure
for the synthesis of N-hydroxylaminoisoxazoles 1.
possessing the fragment of THF which appear to be certain
structural analogue of Zileuton.
2.2. Biological evaluation
2
.2.1. Lipoxygenase inhibitory assessment
Since peroxyl radical is formed in all LOX-catalyzed
reactions, LOX activation evokes lipid peroxidation. During
oxidative stress, LOX can exhibit uncontrolled activity and may
cause cell membrane destruction by phospholipid oxidation.
Thus, lipoxygenase inhibition under pathologic conditions helps
All lipoxygenases are homologues and contain the same two
domains: N-terminal “C2-like” domain and a larger C-termina1l
catalytic domain containing a single atom of non-heme iron.
Indeed, it was shown in various investigations that the molecules
with bulky lipophilic fragments generally exhibit an efficient
14-19
inhibition of enzymes, including LOXs.
Particularly
22
to decrease the level of total oxidative stress. The LOX
inhibition activity of the studied compounds was evaluated in
order to determine capacity of compounds to decrease oxidative
stress level. Lipoxygenase-1 from soybeans (LOX-1) has been
used in the present study since it shares many important
characteristics with LOXs from other sources and represents a
adamantane-based compounds, due to their specific
conformation, frequently demonstrated the enhanced interaction
20
with the target enzyme’s active site. Moreover, the introduction
of adamantane moieties increases the membrane permeability of
20
the modified compound.
adamantane framework in target heterocycles 1 employing
synthetically available α,β-unsaturated esters for
Therefore we introduced the
2
3-25
routine model for studies on animal system.
heterocyclization. The versatility of our synthetic methodology
allowed to prepare a library of compounds 1 by varying the
substituents in the 3-position of isoxazole core.
Сompounds 1a-c, g, h (Table 2, entries 1-3, 7, 8) demonstrate
IC50 values lower than 50 μM and can be considered as
promising lipоxygenase inhibitors. The most active compounds
were 1b, c, g (Table 2, entries 2, 3, 7), the IC50 values for these
compounds were in the range from 0.4 to 4μM. In the presence of
Herein, we report the design and synthesis of novel
adamantane-containing
5-[hydroxy(tetrahydrofuran-2-
1g – one of the most active compounds – the activity of LOX
yl)amino]isoxazoles 1 and evaluation of their antioxidative
potential, as well as LOX inhibition and mitochondria functional
characteristics.
was inhibited almost completely at 50 µM of 1g (Figure 1).
26
According to literature data and our experimental results (IC50
100 μM) the reference compound Zileuton does not inhibit the
soybean LOX, but its inhibitory activity towards human LOX-5
>
2
. Results and discussion
.1. Chemistry
For the synthesis of novel hydroxylamines 1a-h, we proposed
7,26
(
IC50 = 1.1 µM) is similar to that of isoxazoles 1b, c, g towards
2
soybean
a three-step scheme including the acylation of adamantane-
containing alcohols 2a-h by acryloyl chloride followed by the
heterocyclization of unsaturated esters 3a-h employing our
1
2,13
previously published procedure
(Table 1). Subsequent
reduction of 5-nitroisoxazoles 4a-h by SnCl
resulted in desired heterocycles 1a-h.
2
-HCl in THF
The preparation of the starting alkenes 3a-h from the alcohols
bearing the adamantane moieties proceeds smoothly under
21
reported conditions using equimolar amounts of acryloyl
chloride and Et N. Somewhat poorer yield was obtained for
alkene 3g (Table 1, entry 7) due to low solubility of
corresponding alcohol in CH Cl . Dilution of the reaction
mixture or the use of ether solvents (Et O, THF, 1,4-dioxane) did
3
2
2
2
not provide any substantial increase in the alkene yield. The
electrophilic alkenes 3a-h were involved in the heterocyclization
Figure 1. Kinetic curves of linolic acid hydroperoxide formation in
upon the treatment by in situ generated C(NO
2 4 3
) -Et N complex
the presence of 50µM 1g
resulting in 5-nitroisoxazoles 4a-h with satisfactory yields. In
some cases the decrease in yields of the heterocycles 4e, f may
result from the partial oxidation of alkene 3f (Table 1, entry 6)
under the reaction conditions or from the formation of small
amount of 3,3,3-trinitropropanoic acid esters (Table 1, entry 5).
For the successful transformation of 5-nitroisoxazoles 4a-h into
desired 5-[hydroxy(tetrahydrofuran-2-yl)amino]isoxazoles 1a-h,
THF, previously distilled from NaOH, must be bubbled by air for
1
.1.1. DPPH radical scavenging activity
The radical scavenging activity of compounds 1a-h has been
1
2
studied in the process of hydrogen atom transfer from the
molecule to the stable free radical 2,2-diphenyl-1-picrylhydrazyl
(DPPH) producing diphenylpicrylhydrazine and radicals which
can undergo further reactions such as coupling, fragmentation
and addition. The reaction is accompanied by a color change
from violet to yellow, which can be monitored
spectrophotometrically by measuring the decrease in absorbance
2
0 hours. All synthesized compounds were characterized and
1 13
identified using H and C NMR and HRMS.

Table 1. Synthesis of 5-[hydroxy(tetrahydrofuran-2-yl)amino]isoxazoles 1a-h from adamantane-containing alcohols.
a
Yield, %
Entry
1
Compound
R
3
4
1
a
84
57
46
b
c
d
e
f
90
78
72
61
82
54
78
58
84
55
33
36
63
68
50
47
52
54
62
60
75
2
3
4
5
6
7
g
h
8
a
Isolated yields
Table 2. Redox activity and LOX-inhibition (soybean lipoxygenase LOX-1) of 5-[hydroxy(tetrahydrofuran-2-
yl)amino]isoxazoles 1a-h.
3
+
2+
Entry
Compound
LP, Fe
LP, Fe
DPPH
(EC50 μM)
16±2
LOX-1 inhibition
IC50, μM
25±3
IC50 μM
1.37 ± 0.32
1.21± 0.20
5.25 ± 0.8
35 ± 2
IC50 μM
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
0.33 ± 0.19
10.54± 1.34
3.15 ± 0.93
71±8
4±0.6
21±3
0.4±0.1
na
a
na
>100
1.58 ± 0.07
5.45 ± 1.52
1.48 ± 0.23
22 ± 3
2.74 ± 1.79
3.41 ± 1.04
5.16 ± 2.4
6.58 ± 1.44
>100
77±6
>100
na
1g
1h
38±5
1±0.4
70±5
16±2

at 517 nm.27 Antiradical activity was evaluated as the amount of
antioxidant necessary to decrease the initial concentration of
DPPH by 50% (Efficient Concentration = EC50). The EC50 values
reflect the reactivity of a compound toward DPPH but give
limited information on the mechanistic outcome of the reaction.
The results of the DPPH test at 20 °C show that the most active
compounds were 1a-c, g (Table 2, entries 1-3, 8). The antiradical
activity was the highest for compound 1a (Table 2, entry 1)
which completed the reaction with DPPH after several seconds at
equimolar ratio of reagents. The compounds 1a-c, g, h were
found to demonstrate high antiradical activity. To compare the
antioxidative properties of the tested compounds with the
standard, the antioxidative potency of Zileuton was estimated in
our experimental conditions. The obtained data clearly indicate
that the antiradical activity of Zileuton (this work: EC50=14 μM;
28
for comparison, literature data: IC50=43 μM ) is also similar to
that of isoxazoles 1a-c, g, h.
Figure 2. Influence of isoxazoles 1a-h on the viability of rat
cerebellar granule cells (CGC) and neuroblastoma SH-SY5Y. Control
samples contain DMSO (≤ 1%). *indicates p<0.05, # - p<0.01 compared
to the control cultures (T-test).
1
.1.2. Influence on lipid peroxidation
Not only lipoxygenases (LOX), but also the mitochondrial
respiratory chain, nitric oxide synthase, NADPH oxidase and
cyclooxygenases (COX) are the main sources of reactive oxygen
species in the cells. It was shown that noncompetitive LOX
inhibitors may act as reducing agents or iron ligands which affect
not only the LOX activity, but also the LOX-independent
1.1.4. Mitochondrial function measurements
Oxygen consumption rate (OCR) by mitochondria was
determined using the Seahorse extracellular flux (XF-96)
analyzer (Seahorse Bioscience, Chicopee, MA, USA).
Respiration of the mitochondria (0.5 mg protein/well) with or
without 5-hydroxylaminoisoxazoles 1a, c-h was sequentially
measured in a coupled state with substrate of complex II
succinate and rotenone to inhibit complex I of respiratory chain
present (basal respiration), followed by phosphorylating
respiration, in the presence of ADP and substrate,
nonphosphorylating or resting respiration induced with the
addition of oligomycin and then maximal uncoupler-stimulated
respiration after addition of uncoupler FCCP, followed by
nonrespiratory oxygen consumption in the presence of inhibitor
of complex II antimycin A (Figure 3a).
28
oxidative stress. Inhibitors of LOX also possess the antioxidant
activity, not only by inhibiting the enzymatic activity of LOXs,
but also directly by scavenging free radicals. Zileuton, an iron-
chelating inhibitor of 5-LOX, was a rather weak free radical
2+
scavenger and antioxidant with IC50>50µM against Fe -induced
28
lipid peroxidation in rat brain homogenate . However, the new
-hydroxylaminoisoxazoles 1a-h reveal significant antioxidant
5
properties. We have studied the influence of these compounds on
oxidative degradation of lipids in rat brain homogenate induced
by ferrous and ferric ions. All synthesized compounds inhibit
2+
3+
Fe - and Fe -induced lipid peroxidation (LP) of rat brain cortex
homogenate. The most active was compound 1a, but a
structurally similar compound 1h reveals lower activity and the
compound 1d is the weakest inhibitor of LP. The inhibition of
ferric and ferrous ion-induced lipid peroxidation by 5-
hydroxylaminoisoxazoles for all compounds except 1e and 1f has
some correlation with their free radical scavenging capacity
Treatment of mitochondria with isoxazoles 1a, c-h led to an
increase in phosphorylating respiration and moreover, in
maximum stimulated respiration after uncoupling of oxygen
consumption (or electron transport) and ATP synthesis with
FCCP treatment (Figure 3b). But there was no noted increase in
OCR in the presence of olygomycin and we cannot propose the
uncoupling mode of action of the compounds on mitochondrial
respiratory chain.
(Table 2).
1.1.3. Cell viability
The electron transport along the mitochondrial respiratory
chain is accompanied by the formation of electrochemical
gradient of hydrogen ions at the inner mitochondrial membrane.
The electrical part of electrochemical potential, i.e. ΔΨ, can be
measured in isolated mitochondria by changing the absorbance of
the cationic dye safranine. We have shown that compounds 1a-h
do not influence ΔΨ and calcium-induced depolarization of
mitochondria regardless of the used substrates of mitochondrial
respiration: succinate and rotenone or glutamate/malate (data not
shown). These results are in agreement with the above mentioned
absence of any change in oligomycin-dependent oxygen
consumption. The increase of the phosphorylating respiration
At the next step of our work we have studied the influence of
the compounds 1a-h on the viability of the cultured tumor cells
neuroblastoma SH-SY5Y) and primary neuron culture from rat
brain cortex (Figure 2). Cell viability was measured by standard
MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium
bromide) assay after 24 h incubation with 30 µM of isoxazoles
a-h. Cell viability significantly decreased in neuroblastoma cells
to 22±1% at 30 µM of 1h in comparison with the control). In
primary cortex neurons only compound 1c decreased the viability
of cells (to 77±5%). Moreover, for compounds 1d-h a
statistically significant increase in the MTT-connected activity
was observed. MTT is a tetrazolium salt reduced to purple
formazan crystals mainly by mitochondrial succinate
dehydrogenase. Thus the use of this assay as an indicator of
metabolically active mitochondria may overestimate the number
of viable cells due to increasing activity of succinate
dehydrogenase or other cellular dehydrogenases in response to
some compounds. Thus we have studied the direct influence of
our compounds on mitochondrial respiration and other
mitochondrial functions.
(
1
(
(with succinate as substrate) and of the maximal stimulated
respiration caused by the studied compounds may be the reason
of the overestimated MTT-dependent index of cell viability.
However, more significantly, these results allow one to suggest
that 5-[hydroxy(tetrahydrofuran-2-yl)amino]isoxazoles 1a-h may
be useful as a scaffold for the development of novel metabolic
enhancers.

about 50% brain penetration, intestinal absorption is about 80–
00%, and hERG pK is about 4.5. In fact, the properties of the
1
i
studied compounds are comparable or better than those of the
reference compounds (Zileuton, BW-B 70C and Phenidone). In
particular, the predicted blood-brain barrier permeability of the
adamantane derivatives is significantly higher than that of
Zileuton and BW-B 70C, which may be important in the therapy
of the brain oxidative stress.
3. Conclusions
In conclusion, we have developed a universal synthetic
methodology for the synthesis of isoxazole derivatives with a
wide range of substituents based on the heterocyclization of
readily available unsaturated esters by the treatment of TNM-
TEA complex and subsequent reduction of the resulting
5
-nitroisoxazoles by SnCl
2
with the participation of THF
(
solvent). number of 5-[hydroxy(tetrahydrofuran-2-
A
yl)amino]isoxazoles containing a bulky adamantane framework
were synthesized using this method. The obtained compounds
were evaluated for their antioxidative activity, inhibition of
lipoxygenase and impact on rat liver mitochondrial function. The
compounds 1a-c, g, h were found to demonstrate high
antioxidative activity, as well as high inhibitory activity towards
lipoxygenase. All tested compounds promote the phosphorylating
respiratory activity simultaneously with maximal stimulated
respiratory activity of mitochondria and do not reveal any
toxicity towards the primary culture of rat cortex neurons. The
attempt to correlate the in vitro biological results with some
estimated physicochemical and pharmacokinetic properties was
not quite successful. In particular, lipophilicity does not have a
significant influence on the biological activity while the steric
requirements are more important. However, the predicted
properties are comparable or better than those of the reference
compounds and seem quite acceptable for a lead candidate. Thus,
novel 5-[hydroxy(tetrahydrofuran-2-yl)amino]isoxazoles may be
considered as promising and useful scaffold for the creation of
novel drugs with antioxidant and metabolic enhancing properties.
Figure
3.
Influence
of
5-[hydroxy(tetrahydrofuran-2-
yl)amino]isoxazoles on representative respirometric profile of
mitochondria isolated from rat liver in the presence of 10 μM of
compounds 1a, c-h (a) and FCCP-stimulated OCR (b). Final
concentrations of additives after port injections were 4 mM ADP; 2,5
μg/ml oligomycin; 4 μM FCCP; and 4 μM antimycin A. Calculated RCR
was 7.95±0.8.
4. Experimental
4.1. Chemistry
4.1.1. General experimental details
NMR spectra were recorded on spectrometer Agilent 400-MR
2
.3. Structure-activity relationships and physico-chemical
1
13
(
400.0 MHz for H; 100.6 MHz for C) at room temperature;
chemical shifts were measured with reference to the solvent
CDCl ,  = 7.24 ppm,  = 77.0 ppm). Chemical shifts () are
properties
(
3
H
C
Analysis of this limited series of compounds suggests that
their biological activity is defined by a common structural
scaffold. The elongation of the linker between adamantane and
isoxazole moieties (1d,f) as well as the enlarging of the
adamantane core (1e) lead to a decrease in antiradical activities
and inhibition of lipoxygenase.
given in ppm; J values are given in Hz. When necessary,
assignments of signals in NMR spectra were made using 2D
techniques. Accurate mass measurements (HRMS) were
measured on Jeol GCMate II mass spectrometer (70 eV).
Analytical thin layer chromatography was carried out with
Silufol silica gel plates (supported on aluminum); the detection
was done by UV lamp (254 and 365 nm). Column
chromatography was performed on silica gel (230–400 mesh,
We have performed a preliminary computational estimation of
some physicochemical and pharmacokinetic properties of the
studied compounds that may be relevant in the drug development
context. In particular, the lipophilicity (logP) and aqueous
solubility values were predicted as well as the blood-brain barrier
permeability (LogBB), human intestinal absorption and
cardiotoxicity potential (hERG affinity). The predicted values are
presented in the Supplementary Material (Table S1). No direct
correlations between the physicochemical parameters and in vitro
biological results can be discerned. However, for most of the
compounds (except 1f) the properties seem quite acceptable for a
lead candidate. Their LogP values are about 2–2.5 and the
2
9
30
31
32
33,34
35
36
37
Merck). Compounds: 2a, 2b, 2c, 2d, 2e,
2f, 2g, 2h,
2
1
11
11
3
h, 4h, 1h, were synthesized by known procedures. All other
starting materials were commercially available. All reagents
except commercial products of satisfactory quality were purified
by literature procedures prior to use.
4
.1.2. General procedure for the preparation of compounds 3a-h
To the solution of starting alcohol 2 (6 mmol) in
dichloromethane (8 mL) was added Et N (0.84 mL, 606 mg, 6
3
mmol) in atmosphere of Ar. The mixture was cooled to 0…–5 °C
and the acryloyl chloride (0.49 mL, 543 mg, 6 mmol) in
dichloromethane (2 mL) was added dropwise. The resulting
–
4
solubility is about 10 M that is significantly higher than IC50
LogBB for all compounds is close to zero that corresponds to
.

mixture was stirred at this temperature for 3 h and then at room
temperature for 24 h. The reaction mixture was poured into water
(Ad)), 36.8 (3CH
(2CH (Ar)), 128.2 (2CH (Ar)), 128.4 (=CH), 130.9 (=CH
132.9 (C), 151.6 (C), 166.1 (C=O); HRMS (ESI, m/z): calcd for
2
(Ad)), 43.1 (3CH
2
(Ad)), 66.2 (CH
2
O), 125.1
2
),
(
15 mL) and extracted with dichloromethane (4×20 mL). The
combined organic layer was washed with brine solution (3×20
mL) and was dried over anhydrous Mg SO . The solvent was
+
+
C
20 24
H O
2
Na [М+Na] 319.1669; found 319.1674.
2
4
3,5-Dimethyl-1-adamantyl acrylate (3g). Obtained as colorless
evaporated in vacuo; the residue was purified by preparative
column chromatography on silica gel.
oil (0.76 g, 54 %); Rf 0.71 (petroleum ether:EtOAc 50:1); 
H
(
400 MHz, CDCl
1.15-1.21 (m, 1H, CH
2H, 2CH ), 1.72-1.83 (m, 4H, 2CH
2
3
): 0.85 (s, 6H, 2CH
), 1.23-1.29 (m, 2H, 2CH
), 1.96-1.98 (m, 2H, CH
3
), 1.09-1.14 (m, 1H, CH
2
),
2-Adamantyl acrylate (3b). Obtained as colorless solid (1.11
2
2
), 1.34-1.40 (m,
g, 90 %); mp 64-65°С; Rf 0.49 (petroleum ether:EtOAc 50:1); 
H
2
2
),
3
2
3
(
(
(
4
1
400 MHz, CDCl
m, 2H, 2CH (Ad)), 1.74-1.80 (m, 2H, 2CH
m, 4H, 2CH+CH (Ad)), 1.98 – 2.07 (m, 4H, 2CH
3
): 1.52-1.59 (m, 2H, 2CH
2
(Ad)), 1.71– 1.74
(Ad)), 1.81-1.88
+2CH (Ad)),
2.20 (sept, J = 3.1 Hz, 1H, CH), 5.68 (dd, J = 1.6 Hz, J = 10.4
3 3
2
2
2
Hz, 1H, =CH ), 5.99 (dd, J = 10.4 Hz, J = 17.3 Hz, 1H, =CH),
2 3
2
2
6.26 (dd, J = 1.6 Hz, J = 17.3 Hz, 1H, =CH
CDCl ): 29.9 (2CH ), 31.1 (CH (Ad)), 33.8 (2C (Ad)), 39.7 (CH
(Ad)), 42.5 (2CH (Ad)), 47.1 (2CH (Ad)), 50.5 (CH (Ad)),
81.9 (C-O), 129.2 (=CH ), 130.4 (=CH), 165.3 (C=O); HRMS
2 C
);  (101 MHz,
2
3
.96-5.01 (m, 1H, CHO), 5.79 (dd, J = 1.6 Hz, J = 10.4 Hz,
3
3
2
3
3
H, =CH
2
), 6.13 (dd, J = 10.4 Hz, J = 17.3 Hz, 1H, =CH), 6.39
2
2
2
2
3
(dd, J = 1.6 Hz, J = 17.3 Hz, 1H, =CH
); 
2 C
(101 MHz, CDCl
(Ad)) 31.9 (2CH
(Ad)), 77.1 (CHO), 129.3
2
), 165.5 (C=O); HRMS (ESI, m/z): calcd for
3
):
2
+
+
2
7.0 (CH (Ad)), 27.2 (CH (Ad)), 31.8 (2CH
2
(ESI, m/z): calcd for C15
257.1514
22 2
H O Na [М+Na] 257.1512; found
(Ad)), 36.3 (2CH
2
(Ad)), 37.4 (CH
2
(=CH), 129.9 (=CH
+
+
4.1.3. General procedure for the preparation of compounds 4a-h
To the solution of C(NO (0.75 mL, 1.225 g, 6.25 mmol) in
C
13 18
H O
2
Na [М+Na] 229.1199; found 229.1200.
2 4
)
2
-(1-Adamantyl)ethyl acrylate (3c). Obtained as colorless oil
1.10 g, 78 %); Rf 0.56 (petroleum ether:EtOAc 50:1);  (400
) 1.46-1.52 (m, 6H,
(Ad)), 1.63-1.70 (m, 3H,
1,4-dioxane (7 mL) was added Et N (0.70 mL, 505 mg, 5 mmol)
3
(
H
in atmosphere of Ar for 30 min at –10…–5 °C. The resulting
3
MHz, CDCl
3
): 1.42 (t, J = 7.4 Hz , 2H, CH
(Ad)), 1.56-1.62 (m, 3H, 3CH
(Ad)), 1.88-1.94 (m, 3H, 3CH (Ad)), 4.17 (t, J = 7.4 Hz ,
2
mixture was stirred at this temperature for 10 min and then the
alkene 3 (2.5 mmol) in 1,4-dioxane (3 mL) was added dropwise.
The reaction mixture was stirred at 80°C for 2 h and then the
solvent was evaporated in vacuo; the residue was purified by
preparative column chromatography on silica gel.
3
3
2
CH
CH
H, OCH
2
2
3
2
2
3
2
), 5.75 (dd, J = 1.5 Hz, J = 10.4 Hz, 1H, =CH ), 6.06
2
3
3
2
(
dd, J = 10.4 Hz, J = 17.3 Hz, 1H, =CH), 6.33 (dd, J = 1.5 Hz,
3
J = 17.3 Hz, 1H, =CH
Ad)), 31.7 (C), 37.0 (3CH
0.9 (CH O), 128.7 (=CH), 130.2 (=CH
ESI, m/z): calcd for C15
57.1524.
2
); 
(Ad)), 42.4 (CH
), 166.2 (C=O); HRMS
C
(101 MHz, CDCl
3
): 28.5 (3CH
1
-Adamantyl 5-nitroisoxazole-3-carboxylate (4a). Obtained as
(
6
(
2
2
2
); 42.5 (3CH
2
(Ad)),
colorless solid (0.42 g, 57 %); mp 127-128°С; Rf 0.33 (petroleum
2
2
+
+
ether:EtOAc 40:1); H (400 MHz, CDCl ): 1.65-1.75 (m, 6H,
3
22 2
H O Na [М+Na] 257.1512; found
3
CH
CH); 
41.2 (3CH (Ad)), 85.7 (C (Ad)), 102.2 (CH), 156.1 (C=O),
2
(Ad)), 2.24 (br.s, 9H, 3CH
2
(Ad) + 3CH (Ad)), 7.30 (s, 1H,
C
(101 MHz, CDCl ): 31.0 (3CH (Ad)), 35.9 (3CH
3
2
(Ad)),
2
-(1-Adamantyloxy)ethyl acrylate (3d). Obtained as colorless
oil (1.08 g, 72%); Rf 0.60 (petroleum ether:EtOAc 10:1);  (400
MHz, CDCl ): 1.53-1.65 (m, 6H, CH (Ad)), 1.69 – 1.74 (m, 6H,
CH (Ad)), 2.09-2.15 (m, 3H, 3CH (Ad)), 3.63 (m, 2H, CH ),
), 5.79 (dd, J = 1.5 Hz, J = 10.4 Hz, 1H,
2
H
1
C
59.4 (C), 165.4 (br.s., CNO
); HRMS (ESI, m/z): calcd for
14 16 2 5
H N O Na [М+Na] 315.0951; found 315.0955.
2
+ +
3
2
2
2
2
3
2-Adamantyl 5-nitroisoxazole-3-carboxylate (4b). Obtained as
4
.23 (m, 2H, CH
CH ), 6.12 (dd, J = 10.4 Hz, J = 17.3 Hz, 1H, =CH), 6.38 (dd,
J = 1.5 Hz, J = 17.3 Hz, 1H, =CH
0.5 (3CH (Ad)), 36.4 (3CH (Ad)), 41.4 (3CH
CH ), 64.5 (CH ), 72.5 (C-O), 128.5 (=CH), 130.7 (=CH
2
2
3
3
colorless solid 0.43 g, 59 %); mp 117-118°С; Rf 0.27 (petroleum
ether:EtOAc 40:1);  (400 MHz, CDCl ): 1.60 – 1.67 (m, 2H,
=
2
2
3
H
3
2
).; 
C
(101 MHz, CDCl
(Ad)), 58.0
),
3
):
2
2
CH
CH
2
(Ad)), 1.75 – 1.78 (m, 2H, 2CH
(Ad)), 1.86-1.95 (m, 4H, 2CH+CH (Ad)), 2.05-2.12 (m,
2
2
(Ad)), 1.78 – 1.85 (m, 2H,
3
(
1
2
2
2
2
2
2
+
+
2
2H, 2CH (Ad)), 2.13 – 2.18 (m, 2H, 2CH (Ad)), 5.24 – 5.27 (m,
66.2 (C=O); HRMS (ESI, m/z): calcd for C15
73.1461; found 273.1458.
22 3
H O Na [М+Na]
1
(
H, CHO), 7.37 (с, 1H, CH); 
CH (Ad)), 27.0 (CH (Ad)), 31.7(2CH
36.2 (2CH (Ad)), 37.1 (CH (Ad)), 80.8 (CH-O), 102.3 (CH),
C
(101 MHz, CDCl
3
): 26.7
2
(Ad)), 31.8 (2CH (Ad)),
i-Propyl 3-(acryloyloxy)adamantane-1-carboxylate (3e).
Obtained as colorless solid (1.07 g, 61 %); mp 66-67°С; Rf 0.42
2
2
156.9 (C=O), 158.6 (C) 165.5 (br.s., CNO ); HRMS (ESI, m/z):
2
3
+
+
(
=
2
2
=
petroleum ether:EtOAc 20:1); 
6.3 Hz, 6H, 2CH ), 1.58-1.71 (m, 2H, CH
CH ), 2.08 - 2.14 (m, 2H, 2CH ), 2.16-2.22 (m, 2H, 2CH
.24-2.26 (m, 2H, CH ), 2.27 – 2.32 (m, 2H, 2CH), 4.98 (sept, J
6.3 Hz, 1H, OCH), 5.74 (dd, J = 1.6 Hz, J = 10.4 Hz, 1H,
H
(400 MHz, CDCl
), 1.78-1.88 (m, 4H,
),
3
): 1.21 (d, J
calcd for C14
16 2 5
H N O Na [М+Na] 315.0951; found 315.0959.
3
2
2
-(1-Adamantyl)ethyl 5-nitroisoxazole-3-carboxylate (4c).
2
2
2
3
Obtained as colorless solid (0.67 g, 84 %); mp 101-102°С; Rf
2
2
3
0.33 (petroleum ether:EtOAc 50:1);  (400 MHz, CDCl ): 1.52-
H
3
3
3
3
2 2
1.56 (m, 6H, 3CH (Ad)), 1.58 (t, J = 7.5 Hz, 2H, CH ), 1.59-
CH ), 6.03 (dd, J = 10.4 Hz, J = 17.3 Hz, 1H, CHCO), 6.30 (dd,
2
2
3
1.65 (m, 3H, 3CH (Ad)), 1.67-1.74 (m, 3H, 3CH (Ad)), 1.93-
2
2
J = 1.6 Hz, J = 17.3 Hz, 1H, CH
2CH ), 30.3 (2CH), 35.2 (CH ), 37.8 (2CH
CH ), 43.9 (C), 67.4 (CHO), 80.2 (C), 129.5 (=CH
=CH), 165.1 (C=O), 175.5 (C=O); HRMS (ESI, m/z): calcd for
); 
2 C
(101 MHz, CDCl
), 40.4 (2CH
), 130.2
3
): 21.7
3
1
1
3
.98 (m, 3H, 3CH (Ad)), 4.47 (t, J = 7.5 Hz, 2H, OCH
H, CH);  (101 MHz, CDCl ): 28.5 (3CH(Ad)), 31.8 (C (Ad)),
6.9 (3CH (Ad)), 42.0 (CH ), 42.4 (3CH (Ad)), 63.9 (CH O),
2
), 7.36 (s,
(
(
(
C
3
2
2
2
), 42.3
C
3
2
2
2
2
2
2
+
+
102.2 (CH), 157.7 (C=O), 158.3 (C), 165.6 (br.s., CNO ); HRMS
2
17
H
24
O
4
Na [М+Na] 315.1567; found 315.1564.
+ +
(ESI, m/z): calcd for C16
20 2 5
H N O Na [М+Na] 343.1264; found
4-(1-Adamantyl)benzyl acrylate (3f). Obtained as colorless
343.1253.
solid (1.46 g, 82 %); mp 57-58°С; Rf 0.81 (petroleum
ether:EtOAc 10:1);  (400 MHz, CDCl ): 1.72-1.83 (m, 6H,
CH (Ad)), 1.86-1.97 (m, 6H, CH (Ad)), 2.07-2.13 (m, 3H, 3CH
Ad)), 5.18 (c, 2H,CH O), 5.83 (dd, J = 1.5 Hz, J = 10.4 Hz,
2
-(1-Adamantyloxy)ethyl 5-nitroisoxazole-3-carboxylate (4d).
H
3
Obtained as colorless solid (0.46 g, 55%); mp 45-46°С; Rf 0.11
(petroleum ether:EtOAc 20:1);  (400 MHz, CDCl ): 1.52-1.64
(m, 6H, 3CH (Ad)), 1.68-1.72 (br.s, m ,6H, 3CH (Ad)), 2.10-
2 2
2.14 (m, 3H, 3CH (Ad)), 3.72-3.76 (m, 2H, CH ), 4.47-4.51 (m,
2
H, CH
2
2
2
3
H
3
(
2
3
3
1
H, CH
2
), 6.16 (dd, J = 10.4 Hz, J = 17.3 Hz, 1H, CHCO), 6.44
2
3
(dd, J = 1.5 Hz, J = 17.3 Hz, 1H, CH
2
), 7.31-7.39 (m, 4H,
2
(
2
), 7.37 (с, 1H, CH); 
C
(101 MHz, CDCl
3
): 30.4 (3CH
4
CH(Ar)); 
C
(101 MHz, CDCl
3
): 28.9 (3CH (Ad)), 36.1 (C
Ad)), 36.3 (3CH
2
(Ad)), 41.4 (3CH
2
(Ad)), 57.5 (C (Ad)), 66.9

+
+
(
1
[
CH
2
O), 72.8 (CH
); HRMS (ESI, m/z): calcd for C16
М+Na] 359.1214; found 359.1212.
2
O), 102.3 (CH), 157.6 (C=O), 158.1 (C),
m/z): calcd for C18
371.1576.
H
24
N
2
O
5
Na [М+Na] 371.1577; found
+
65.6 (br.s.,CNO
2
20 2 6
H N O Na
+
2
-Adamantyl
5-[hydroxy(tetrahydrofuran-2-
3
-(i-Propoxycarbonyl)-1-adamantyl
carboxylate (4e). Obtained as colorless solid (0.31 g, 33%); mp
5-56°С; Rf 0.29 (petroleum ether:EtOAc 15:1);  (400 MHz,
5-nitroisoxazole-3-
yl)amino]isoxazole-3-carboxylate (1b). Obtained as yellowish oil
(70 mg, 50 %); Rf 0.42 (petroleum ether:EtOAc 3:1);  (400
MHz, CDCl ): 1.54-1.62 (m, 2H, 2CH (Ad)), 1.72-1.76 (m, 2H,
2CH (Ad)), 1.76-1.83 (m, 2H, 2CH (Ad)), 1.83-1.90 (m, 4H,
2CH+CH (Ad)), 1.88-1.96 (m, 1H, CH ), 2.06-2.17 (m, 6H,
2CH (Ad)+2CH (Ad)+CH ), 2.20-2.29 (m, 1H, CH ), 3.83-3.89
(m, 1H, CH O), 4.03 – 4.09 (m, 1H, CH O), 5.16-5.18 (m, 1H,
CHO (Ad)), 5.58 (dd, J = 4.0 Hz, J = 6.8 Hz, 1H, CHO), 6.00
(s, 1H, CH), 6.48 (br. s, 1H, OH);  (101 MHz, CDCl ): 25.3
(CH ), 26.9 (CH (Ad)), 27.1 (CH (Ad)), 27.9 (CH ), 31.7 (2CH
(Ad)), 31.8 (2CH (Ad)), 36.2 (2CH (Ad)), 37.2 (CH (Ad)), 69.9
(CH O), 79.1 (CHO (Ad)), 87.0 (CH), 92.4 (CHO), 157.4 (C) ,
159.3 (C=O), 172.7 (C-NOH); HRMS (ESI, m/z): calcd for
H
5
H
3
2
3
CDCl
3
): 1.20 (d, J = 6.2 Hz, 6H, 2CH
Ad)), 1.83-1.85 (m, 4H, 2CH (Ad)), 2.16 - 2.21 (m, 2H, 2CH
Ad)), 2.24-2.30 (m, 2H, 2CH (Ad)), 2.32-2.36 (m, 4H, CH
3
), 1.62-1.72 (m, 2H, CH
2
2
2
2
2
(
(
(
2
2
2
2
2
2
2
3
Ad)+2CH (Ad)), 4.96 (sept, J = 6.2 Hz, 1H, OCH), 7.30 (s, 1H,
(101 MHz, CDCl ): 21.7 (2CH ), 30.4 (2CH (Ad)), 34.8
(Ad)), 37.5 (2CH (Ad)), 40.2 (2CH (Ad)), 42.1 (CH
Ad)), 44.1 (C(Ad)), 67.7 (CHO), 85.1 (C-O), 102.2 (CH), 156.1
C=O), 159.2 (C), 165.4 (br.s, .CNO ), 175.0 (C=O); HRMS
2
2
3
3
CH); 
C
3
3
(
(
(
(
CH
2
2
2
2
C
3
2
2
2
2
2
2
+
+
ESI, m/z): calcd for C18
01.1312.
H
22
N
2
O
7
Na [М+Na] 401.1319; found
2
4
+
+
C
18 24
H N
2
O
5
Na [М+Na] 371.1577; found 371.1578.
4-(1-Adamantyl)benzyl 5-nitroisoxazole-3-carboxylate (4f).
Obtained as colorless solid (0.34 g, 36 %); mp 155-156°С; Rf
2-(1-Adamantyl)ethyl 5-[hydroxy(tetrahydrofuran-2-
0
1
.47 (petroleum ether:EtOAc 10:1); 
.82 (m, 6H, CH (Ad)), 1.88-1.91 (m, 6H, CH
O), 7.37 (c, 1H, CH), 7.39
(101 MHz, CDCl ): 28.9 (3CH (Ad)),
6.2 (C (Ad)), 36.7 (3CH (Ad)), 43.1 (3CH (Ad)), 68.7 (CH
O), 102.3 (CH), 125.4 (2CH (Ar)), 128.8 (2CH(Ar)), 131.0 (C
H
(400 MHz, CDCl
3
): 1.70-
yl)amino]isoxazole-3-carboxylate (1c). Obtained as yellowish
solid (71 mg, 47 %); mp 105-107°С; Rf 0.14 (petroleum
2
2
(Ad)), 2.06-2.12
(
(
3
m, 3H, 3CH (Ad)) 5.41 (c, 2H, CH
br.s, 4H, 4CH (Ar)); 
2
ether:EtOAc 5:1); 
3CH (Ad)+CH ), 1.58-1.65 (m, 3H, 3CH
3H, 3CH (Ad)), 1.87 – 1.97 (m, 4H, 3CH (Ad) + CH
2.28 (m, 3H, 2CH
1H, CH O), 4.38 (t, J = 7.4 Hz, 2H, CH
H
(400 MHz, CDCl
3
): 1.51-1.57 (m, 8H,
(Ad)), 1.66-1.73 (m,
), 2.05-
C
3
2
2
2
2
2
2
-
2
2
2
), 3.83-3.90 (m, 1H, CH
2
O), 4.03 – 4.10 (m,
O), 5.58 (dd, J = 4.0
3
3
(Ar)), 152.5 (C (Ar)), 157.5 (C=O), 158.2 (C), 165.6 (br.s.,
2
2
+
+
3
CNO
2
); HRMS (ESI, m/z): calcd for C21
22
H N
2
O
5
Na [М+Na]
Hz, J = 6.8 Hz, 1H, CHO), 5.98 (s, 1H, CH), 6.37 (s, 1H, OH);
(101 MHz, CDCl ): 25.3 (CH ), 27.9 (CH ), 28.5 (3CH (Ad)),
(Ad)), 42.1 (CH ), 42.4 (3CH (Ad)),
O), 87.0 (CH), 92.3 (CHO), 157.0 (C),
405.1421; found 405.1414.

C
3
2
2
3
6
1
C
1.8 (C (Ad)), 36.9 (3CH
9.9 (CH
60.1 (C=O), 172.7 (C); HRMS (ESI, m/z): calcd for
20
2
2
2
3,5-Dimethyl-1-adamantyl
5-nitroisoxazole-3-carboxylate
2
O), 85.3 (CH
2
(
4g). Obtained as colorless solid (0.50 g, 63 %); mp 103-104°С;
Rf 0.37 (petroleum ether:EtOAc 40:1);  (400 MHz, CDCl ):
(Ad)), 1.21-1.26 (m,
(Ad)), 1.39-1.45 (m, 2H,
(Ad)) 1.90-1.95 (m, 2H,
(Ad)), 2.29 (sept, J = 3.2
(101 MHz, CDCl ): 29.7
), 31.2 (CH (Ad)), 34.3 (2C (Ad)), 39.6 (CH (Ad)), 42.2
(Ad)), 47.0 (2CH (Ad)), 50.2 (CH (Ad)), 86.9 (C (Ad)),
02.2 (CH), 156.2 (C=O), 159.4 (C), 165.4 (br.s., CNO ); HRMS
Na [М+Na] 343.1264; found
H
3
+
+
28
H N
2
O
5
Na [М+Na] 399.1890; found 399.1887.
0
1
2
2
.91 (s, 6H, 2CH
H, CH (Ad)), 1.29-1.35 (m, 2H, 2CH
CH (Ad)), 1.83-1.88 (m, 2H, 2CH
CH (Ad)), 2.08 – 2.11 (m, 2H, CH
3 2
), 1.15-1.20 (m, 1H, CH
2
2
2-(1-Adamantyloxy)ethyl 5-[hydroxy(tetrahydrofuran-2-
2
2
yl)amino]isoxazole-3-carboxylate (1d). Obtained as brownish
solid (82 mg, 52 %); mp 98-101°С; Rf 0.24 (petroleum
ether:EtOAc 3:1);  (400 MHz, CDCl ): 1.51-1.63 (m, 6H,
3CH (Ad)), 1.69-1.72 (m, 6H, 3CH (Ad)), 1.82-1.94 (m, 1H,
2CH ), 2.04-2.14 (m, 5H, 3CH (Ad) + 2CH ), 2.16-2.27 (m, 1H,
2CH ), 3.69-3.72 (m, 2H, CH ), 3.78-3.85 (m, 1H, CH O), 3.99 –
4.06 (m, 1H, CH O), 4.36-4.39 (m, 2H, CH ), 5.54 (dd, J = 4.0
Hz, J = 6.7 Hz, 1H, CHO), 5.94 (s, 1H, CH), 7.46 (br.s, 1H,
OH);  (101 MHz, CDCl ): 25.3 (CH ), 27.9 (CH ), 30.4 (3CH
Ad)), 36.3 (3CH (Ad)), 41.3 (3CH (Ad)), 57.8 (CH O), 65.6
CH O), 69.9 (CH O), 73.1 (C (Ad)), 86.6 (CH), 92.1 (CHO),
56.6 (C), 160.0 (C=O), 173.0 (C-NOH); HRMS (ESI, m/z):
3
2
2
Hz, 1H, CH (Ad)), 7.29 (s, 1H, CH); 
C
3
H
3
(
(
2CH
2CH
3
2
2
2
2
2
2
2
2
1
2
2
2
2
+
+
3
(ESI, m/z): calcd for C16
H
20
N
2
O
5
2
2
3
343.1260.
C
3
2
2
4.1.4. General procedure for the preparation of compounds 1a-h
(
(
2
2
2
THF was distilled over NaOH. Air was bubbled through the
2
2
THF during 20 h prior its use in the reaction. To the solution of
nitroisoxazole 4 (0.4 mmol) in a mixture (1:1) H O-THF (14 mL)
was added SnCl (265 mg, 1.4 mmol) in one portion, and conc.
HCl (0.3 mL) dropwise at 0 C. The resulting mixture was stirred
at this temperature for 3 h, then THF was evaporated in vacuo
and saturated aq. NaHCO
water phase was extracted with dichloromethane (4×20 mL). The
combined organic layer was dried over anhydrous Mg SO . The
solvent was evaporated in vacuo; the residue was purified by
preparative column chromatography on silica gel.
1
+
+
2
calcd for C20
28 2 6
H N O Na [М+Na] 415.1840; found 415.1852.
2
о
3
-(i-Propoxycarbonyl)-1-adamantyl
5-
[hydroxy(tetrahydrofuran-2-yl)amino]isoxazole-3-carboxylate
(1e). Obtained as yellowish oil (94 mg, 54 %); Rf 0.10
3
(15 mL) was added to the residue. The
3
(petroleum ether:EtOAc 4:1); 
6.3 Hz, 6H, 2CH ), 1.57-1.71 (m, 2H, CH
H, 2CH (Ad)), 1.87-1.96 (m, 1H, CH ), 2.04-2.19 (m, 4H, CH
Ad)+CH ), 2.20-2.34 (m, 7H, 2CH (Ad)+2CH (Ad) +CH ),
.82-3.89 (m, 1H, OCH ), 4.02-4.09 (m, 1H, OCH ), 4.95 (sept,
J = 6.3 Hz, 1H, CHO), 5.55 (dd, J = 4.0 Hz, J = 6.9 Hz, 1H,
CHO), 5.93 (s, 1H, CH), 6.57 (br.s., 1H, OH);  (101 MHz,
CDCl ): 21.7 (2CH ), 25.3 (CH ), 27.9 (CH ), 30.4 (2CH (Ad)),
5.0 (CH (Ad)), 37.6 (2CH (Ad)), 40.2 (2CH (Ad)), 42.2 (CH
Ad)), 44.0 (C (Ad)), 67.6 (CHO), 69.9 (CH O), 83.0 (C (Ad)),
H
(400 MHz, CDCl
3
): 1.18 (d, J =
3
2
(Ad)), 1.78-1.86 (m,
2
4
4
2
2
2
(
2
2
2
3
2
2
1
-Adamantyl
yl)amino]isoxazole-3-carboxylate (1a). Obtained as yellowish oil
64 mg, 46 %); Rf 0.45 (petroleum ether:EtOAc 3:1);  (400
MHz, CDCl ): 1.62-1.73 (m, 6H, 3CH (Ad)), 1.86-1.96 (m, 1H,
CH ), 2.05-2.17 (m, 2H, CH ), 2.17-2.29 (m, 10H, 3CH (Ad) +
CH (Ad) + CH ), 3.82-3.89 (m, 1H, CH O), 4.02-4.09 (m, 1H,
CH O), 5.56 (dd, J = 4.0 Hz, J = 6.7 Hz, 1H, CHO), 5.93 (s,
H, CH), 6.44 (br. s, 1H, OH);  (101 MHz, CDCl ): 25.3
CH ), 27.9 (CH ), 30.9 (3CH (Ad)), 36.0 (3CH (Ad)), 41.2
3CH (Ad)), 69.9 (CH O), 83.6 (C (Ad)), 87.1 (CH), 92.4
CHO), 158.2 (C), 158.6 (C=O), 172.5 (C-NOH); HRMS (ESI,
5-[hydroxy(tetrahydrofuran-2-
3
3
3
C
(
H
3
3
2
2
3
2
3
2
2
2
2
2
2
(
2
3
2
2
2
8
1
7.0 (CH), 92.4 (CHO), 157.9 (C), 158.6 (C=O), 172.6 (C),
3
3
+
2
75.4 (C=O); HRMS (ESI, m/z): calcd for C22
30
H N
2
O
7
Na
1
C
3
+
[М+Na] 457.1945; found 457.1939.
(
(
(
2
2
2
4-(1-Adamantyl)benzyl 5-[hydroxy(tetrahydrofuran-2-
yl)amino]isoxazole-3-carboxylate (1f). Obtained as yellowish oil
2
2

(
109 mg, 62%); Rf 0.09 (petroleum ether:EtOAc 5:1); 
MHz, CDCl ): 1.69-1.81 (m, 6H, 3CH (Ad)), 1.84-1.94 (m, 7H,
(Ad)+CH ), 2.02-2.13 (m, 5H, 3CH (Ad) + 2CH ), 2.14-
.25 (m, 1H, CH ), 3.80-3.87 (m, 1H, CH O), 4.00-4.07 (m, 1H,
O), 5.32 (s, 2H, CH
CHO), 5.99 (s, 1H, CH), 6.55 (br.s), 7.34-7.35 (m, 4H, 4CH
H
(400
cuvettes was 0.1 mM. The samples were incubated for 30 min at
20°C in methanol and the decrease in the absorbance values of
DPPH solution was measured at λmax 517 nm on the Thermo
Scientific Multiscan Go microplate spectrophotometer. Results
were expressed as scavenging activity, calculated as follows:
3
2
3
2
CH
CH
2
2
2
2
2
3
3
2
2
), 5.55 (dd, J = 4.0 Hz, J = 6.8 Hz, 1H,
0 1 0
Scavenging activity, % = [(A −A )/A ] × 100
(
(
(
1
1
Ar)); 
Ad)), 36.1 (C (Ad)), 36.7 (3CH
CH O), 69.9 (CH O), 87.0 (CH), 92.2 (CHO), 125.1 (2CH (Ar),
28.5 (2CH (Ar)), 131.9 (C (Ar)), 151.9 (C (Ar)), 156.8 (C),
59.9 (C=O), 172.8 (C); HRMS (ESI, m/z): calcd for
C
(101 MHz, CDCl
3 2 2
): 25.3 (CH ), 27.8 (CH ), 28.9 (3CH
2
(Ad)), 43.1 (3CH
2
(Ad)), 67.5
The concentration of the compound needed to decrease 50%
of the initial substrate concentration (EC ) is a parameter widely
2
2
5
0
2
7
used to measure the antioxidant effect. For determination of
EC , the values of DPPH decrease after 30 min were used. The
50
+
+
C
25 30
H N
2
O
5
Na [М+Na] 461.2047; found 461.2036.
EC50 values were calculated graphically by plotting scavenging
activity against compound concentration. Different sample
concentrations (0.01, 0.02, 0.05 and 0.1 mM) were used in order
to obtain kinetic curves and to calculate the EC50 values.
3,5-Dimethyl-1-adamantyl 5-[hydroxy(tetrahydrofuran-2-
yl)amino]isoxazole-3-carboxylate (1g). Obtained as yellowish oil
90 mg, 60 %); Rf 0.14 (petroleum ether:EtOAc 5:1);  (400
MHz, CDCl ): 0.88 (6H, 2CH ), 1.12-1.24 (m, 2H, CH
.26-1.32 (m, 2H, 2CH (Ad)), 1.37-1.43 (m, 2H, 2CH
.81-1.97 (m, 5H, 2CH (Ad)+CH
+CH (Ad)), 3.83-3.90 (m, 1H,
(
H
3
3
2
(Ad)),
2
(Ad)),
4.2.5. Lipoxygenase activity
1
1
2
Lipoxidase (LOX) type 1-B from Glycine max (soybean),
boric acid, linoleic acid, NaOH, ethanol (96%) were used with no
further purification. LOX inhibition activity was determined
spectrophotometrically by measuring the increase in absorbance
2
2 2
), 2.05-2.09 ((m, 2H, CH
(
Ad)), 2.09-2.28 (m, 4H, 2CH
CH O), 4.03-4.10 (m, 1H, CH O), 5.57 (dd, J = 4.1 Hz, J = 6.9
Hz, 1H, CHO), 5.91 (s, 1H, CH), 5.94 (s, 1H, OH);  (101
2
3
3
2
2
43
C
at 234 nm for the oxidation of linoleic acid The reaction
mixture contained (final concentration) the test compounds,
dissolved in DMSO at concentrations of 1-100 μM, or the solvent
MHz, CDCl
3
): 25.3 (CH
Ad)), 34.1 (2C (Ad)), 39.6 (CH
2CH (Ad)), 50.4 (CH (Ad)), 69.9 (CH
CH), 92.3 (CHO), 158.1 (C), 158.7 (C=O), 172.6 (C); HRMS
2 2 3
), 27.9 (CH ), 29.8 (2CH ), 31.2 (CH
(
(
(
(
2
(Ad)), 42.4 (2CH
2
(Ad)), 47.1
2
2
2
O), 84.9 (C (Ad)), 86.9
(
control) and linoleic acid 100 μM, in borate buffer (pH=9.0).
The reaction was started by adding a lipoxygenase in amount of
00 units. Six different concentrations of each complex were
+
+
28 2 5
ESI, m/z): calcd for C20H N O Na [М+Na] 399.1890; found
5
3
99.1889.
used for the inhibition activity experiments. The increase in
absorbance was recorded every 10 s during 10 min under
controlled temperature 25 ˚C.
4
.2. Biological studies
4
.2.1. Animals
The degree of LOX activity (A%) in the presence of the
complexes was calculated according to the following.
The experiments were carried out on outbreed albino male rats
44
weighing 200-300 g. The animals had free access to food and
water.
A,% = (ν
inhibitor) * 100, where ν
0
in the presence of inhibitor/ν
is the initial rate.
0
in the absence of
0
4.2.2. Brain homogenate
-1
The value of the initial rate (v
0
, μM • min ) was calculated
The animals were narcotized with carbon dioxide and
according to the formulas:
decapitated using a guillotine. The procedure is in compliance
with the Guidelines for Animal Experiments at IPAC RAS. Their
brains were removed and homogenized in 120 mM KCl/20 mM
HEPES on ice. For isolation of the subcellular fraction, the brain
homogenate was centrifuged at 4000 rpm and supernatant was
used in further experiments on the same day. The protein content
in brain homogenate and mitochondria extracts was measured
0
v = ΔC/Δt = ΔA/Δtε = tgα/Δtε,
where C is the product concentration (hydroperoxylinoleic
acid), t is the reaction time, ε is molar absorbance coefficient of
hydroperoxylinoleic acid, tgα is the slope of the kinetic curve
plotted as absorbance vs time.
38
using a microbiuret method.
.2.3. Mitochondria
Mitochondria were isolated by conventional differential
IC50 values were obtained graphically by plotting logistic
curve in coordinates inhibition activity (A, %) vs inhibitor
concentration ([I], μM). The general curve equation is the
following:
4
centrifugation from the livers of adult Wistar strain rats fasted
overnight as in this work. The standard mitochondrial fraction
contained 130-150 mg of protein per liver. The mitochondria
39
A, % = 100 x (1/(1+[I]/IC50))
о
were stored at 4 С. Functional activity of the rat liver
4.2.6. Mitochondrial membrane potential
mitochondria remained constant for 2.5…4 h.
Mitochondrial membrane potential of isolated rat liver
mitochondria was measured using potential-dependent indicator
4.2.4. Redox activity
о
45
Safranine A at 25 С as in our previous work.
.2.7. Mitochondrial O consumption rate (OCR) measurements
Mitochondrial O consumption measurements were made
Lipid peroxidation intensity in brain homogenate was assayed
4
0
4
2
using a modified TBA test.
2
Fe(II)-chelating activity was evaluated using a modified
41
using an XF96 Extracellular Flux Analyzer (Seahorse
method described elsewhere.
46
Bioscience) as described. Mitochondria were added to XF96
The free radical scavenging activity was evaluated using the
Analyzer plate with 30 µM of compounds in the presence of
succinate (5mM) and rotenone (1µM) and then the measurement
of the oxygen consumption was started. Final concentrations of
the additives after port injections were 4 mM ADP; 2,5 μg/ml
oligomycin; 4 μM FCCP; and 4 μM antimycin A. Calculated
RCR was 7.95±0.8. OCR was automatically calculated and
recorded by the Seahorse XF96 software.
stable radical DPPH, according to the method described by
42
Brand-Williams et al.
with a slight modification. All
compounds were tested in 1:1 ratio expressed as moles of
compound per mole of DPPH radical. A 1 mL sample of
compound solution in methanol was added to 1 mL of DPPH
solution in methanol so that the initial DPPH concentration in the

4
.2.8. Viability of cultured neuroblastoma SH-SY5Ycells and rat
18. Milaeva, E. R.; Orlova, S. I.; Osolodkin, D. I.; Palyulin, V. A.;
Fursova, E. Yu.; Ovcharenko, V. I.; Zefirov, N. S. Russ. Chem.
Bull., Int. Ed. 2011, 60, 2564-2571.
cerebellar granule cells (CGC)
Cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-
1
9. Milaeva, E. R.; Meleshonkova, N. N.; Shpakovsky, D. B.;
Paulavets, V. V.; Orlova, S. I.; Osolodkin, D. I.; Palyulin, V. A.;
Loginov, S. V.; Storozhenko, P. A.; Zefirov, N. S. Doklady
Chemistry 2012, 443, 77–79.
4
7
yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the
SH-SY5Y cells or primary culture of rat cerebellar granule cells
were incubated with 30 µM of compounds or equal volume of
vehicle (less than 1% of the total volume of medium under the
cell layer) during 24 h. Then 20ꢀμL of MTT (2ꢀmg/mL in PBS)
were added to each well and the cells were incubated at 37°C for
2
0. Wanka, L.; Iqbal, K.; Schreiner, P. R. Chem. Rev. 2013, 113,
3
516−3604.
2
1. Wang, W.; Li, Y.; Sun, M.; Zhou, C.; Zhang, Y.; Lia, Y.; Yang,
Q. Chem. Commun. 2012, 48, 6040–6042.
2
ꢀh. The supernatants were aspirated carefully, 200ꢀμl of DMSO
22. Novo, E.; Parola, M. Fibrogenesis Tissue Repair, 2008, 1:5. 58pp.
2
3. Ribeiro, D.; Freitas, M.; Tomé, S. M.; Silva, A. M. S.; Porto, G.;
Cabrita, E. J.; Marques, M. M. B.; Fernandes, E. Eur. J. Med.
Chem. 2014, 72, 137-145.
were added to each well to dissolve the precipitate, and
absorbance was measured at 570ꢀnm using a microplate reader
Victor (Perkin Elmer).
2
4. Kouatly, O.; Geronikaki, A.; Kamoutsis, C.; Hadjipavlou-Litina
D.; Eleftheriou P. Eur. J. Med. Chem. 2009, 44, 1198-1204.
5. Lomnitski, L.; Bar-Natan, R.; Sklan, D.; Grossman, S. Biochim.
Biophys. Acta 1993, 1167, 331–338.
4.2.9. Statistics
2
Data are presented as the mean ± SD. Statistical comparisons
were made by Student’s t-test. Values of <0.05 and <0.01
were considered significant.
26. Chan, K. Y.; Mohamad, K.; Ooi, A. J. A.; Imiyabir, Z.; Chung, L.
Y. Fitoterapia 2012, 83, 961–967.
2
2
7. Molyneux, P. Songklanakarin J. Sci. Technol., 2004, 26, 211–219.
8. Czapski, G. A; Czubowicz, K.; Strosznajder, R. P. Pharmacol
Rep. 2012 64, 1179-1188.
Acknowledgments
2
3
9. Baklan, V. F.; Khil'chevskii, A. N.; D'yakovskaya, V. M.;
Sologub, L. S.; Kukhar, V. P.; Russ. J. Org. Chem. 1993, 29, 454–
This work was partially supported by the Russian Foundation
for Basic Research (grant no. 14-03-00469, 13-03-01151, 15-03-
4
56.
0. Schraml, J.; Vcelak, J.; Chvalovsky, V.; Engelhardt, G.; Jancke,
03057) and the Presidium of RAS (Program N 8). The research
H.; Vodicka, L.; Hlavaty, J. Collect. Czech. Chern.Commun. 1978,
work was carried out using NMR spectrometer Agilent 400-MR
purchased under the program of MSU development. The
equipment of Center for Collective Use IPAC RAS (agreement
N14.621.21.0008, ID RFMEFI62114X0008) was used in the
biological experiments.
4
3, 3179.
3
3
3
1. Takahashi, N.; Mochizuki, D. US Patent; 5658923, 1997; Chem.
Abstr. 1997, 127, 248269.
2. Reznikov, A. N.; Skomorokhov, M. Y.; Klimochkin, Y. N. Russ.
J. Org. Chem. 2010, 46, 1741–1742.
3. Anderson, G.; Burks, W.; Harruna, I. Synth. Commun. 1988, 18,
1
967-1974.
References and notes
34. Kadaba, P. Synthesis, 1971, 316–317.
3
3
3
5. Stepanov, F. N.; Dikolereko, E. J.; Danilenko, G. J. Russ J. Org.
Chem. 1966, 2, 640.
6. Avdeev, M. V.; Bagrii, E. I.; Maravin, G. B.; Korolev, Y. M.;
Borisov, R. S. Petroleum Chem. 2000, 40, 391–398.
7. Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.;
Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk,
P.; Huang, Q.; Han, S.-P.; et al.; J. Med. Chem. 2005, 48, 5025–
1
2
.
.
Haeggstrӧm, J. Z.; Funk, C. D. Chem. Rev. 2011, 111, 5866–5898.
Gilbert, N. C.; Bartlett, S. G.; Waight, M. T.; Neau, D. B.;
Boeglin, W. E.; Brash, A. R.; Newcomer, M. E. Science 2011,
3
31, 217-219.
3
4
5
6
.
.
.
.
Charlier, C.; Michaux, C. Eur. J. Med. Chem. 2003, 38, 645-659.
Brash, A. R. J. Biol. Chem. 1999, 274, 23679-23682.
Pergola, C.; Werz, O. Expert Opin. Ther. Pat. 2010, 20, 355-375.
Elkady, M.; Nieß, R.; Schaible, A. M.; Bauer, J.; Luderer, S.;
Ambrosi, G.; Werz, O.; Laufer, S. A. J. Med. Chem. 2012, 55,
5
037.
3
8. Dawson, R. M. C.; Elliott, D. C., Elliott, W. H., Jones, K. M. Data
for Biochemical Research. Third Edition, Clarendon Press; 1991.
9. Whipps, D. E.; Halestrap, A. P. Biochem. J. 1984, 221, 147–152.
0. Armstrong, D.; Browne, R. Adv. Exp. Med. Biol. 1994, 366, 43–
3
4
8
958−8962.
Wu, Y.; He, C.; Gao, Y.; He, S.; Liu, Y.; Lai L. J. Med. Chem.
012, 55, 2597−2605.
7
8
9
1
.
.
.
5
8.
1. Perez, C. A.; Wei, Y.; Guo, M. J. Inorg. Biochem. 2009, 103, 326–
2.
2
4
4
4
Liedtke, A. J.; Keck, P. R. W. E. F.; Lehmann, F.; Koeberle, A.;
Werz, O.; Laufer, S. A. J. Med. Chem. 2009, 52, 4968–4972.
Chowdhury, M. A.; Abdellatif, K. R. A.; Dong, Y.; Das, D.;
Suresh, M. R.; Knaus E. E. J. Med. Chem. 2009, 52, 1525–1529.
3
2. Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Food Sci.
Technol. 1995, 28, 25–30.
3. Ozturk, I.; Filimonova, S.; Hadjikakou, S. K.; Kourkoumelis, N.;
Dokorou, V.; Manos, M. J.; Tasiopoulos, A. J.; Barsan, M. M.;
Butler, I. S.; Milaeva, E. R.; Balzarini, J.; Hadjiliadis, N. Inorg.
Chem. 2010, 49, 488–501.
4. Xanthopoulou, M. N.; Hadjikakou, S. K.; Hadjiliadis, N.; Milaeva,
E. R.; Gracheva, J. A.; Tyurin, V. Y.; Kourkoumelis, N.;
Christoforidis, K. C.; Metsios, A. K.; Karkabounas, S.;
Charalabopoulos, K. Eur. J. Med. Chem. 2008, 43, 327–335.
5. Milaeva, E. R.; Shpakovsky, D. B.; Gracheva, Y. A.; Antonenko,
T. A.; Osolodkin, D. I.; Palyulin, V. A.; Shevtsov, P. N.;
Neganova, M. E.; Vinogradova, D. V.; Shevtsova, E. F. J.
Organomet. Chem. 2015, 782, 96–102.
0. Peduto, A.; Bruno F.; Dehm, F.; Krauth V.; de Caprariis, P.;
Weinigel C.; Barz, D.; Massa A.; De Rosa M.; Werz, O.; Filosa R.
Eur. J. Med. Chem. 2014, 81, 492-498.
1. Averina, E. B.; Vasilenko, D. A.; Samoilichenko, Y. V.; Grishin,
Y. K.; Rybakov, V. B.; Kuznetsova, T. S.; Zefirov, N. S.
Synthesis, 2014, 46, 1107-1113.
1
1
1
4
4
2. Volkova, Y. A.; Averina, E. B.; Grishin, Y. K.; Bruheim, P.;
Kuznetsova, T. S.; Zefirov, N. S. J. Org. Chem., 2010, 75, 3047-
3
052.
3. Regiochemistry reported in ref. [12] has been revised in ref [13]:
Averina, E. B.; Volkova, Y. A.; Samoilichenko, Y. V.; Grishin, Y.
K.; Rybakov, V. B.; Kutateladze, A. G.; Elyashberg, M. E.;
Kuznetsova, T. S.; Zefirov, N. S. Tetrahedron Lett., 2012, 53,
472–1475.
4. Ulbrich, H.; Fiebich, B.; Dannhardt, G. Eur. J. Med. Chem. 2002,
7, 953-959.
5. Pontiki, E.; Hadjipavlou-Litina, D.; Litinas, K.; Nicolotti, O.;
Carotti, A. Eur. J. Med. Chem. 2011, 46, 191-200.
6. Pavi, K.; Perkovi, I.; Cindri, M.; Pranji, M.; Martin-Kleiner, I.;
Kralj, M.; Schols, D.; Hadjipavlou-Litina, D.; Katsori, A.-M.;
Zorc, B. Eur. J. Med. Chem. 2014, 86, 502-514.
4
4
6. Rogers, G. W.; Brand, M. D.; Petrosyan, S.; Ashok, D.; Elorza, A.
A.; Ferrick, D. A.; Murphy, A. N. PLoS ONE 2011, 6(7): e21746,
doi:10.1371/journal.pone.0021746
1
1
1
1
7. . Niks, M.; Otto, M. J. Immunol. Methods 1990, 130, 149–151.
3
Supplementary Material
Supplementary data (some details of biological studies,
computational estimation of some physicochemical and
pharmacokinetic properties of compounds 1a–h and reference
1
7. Pontiki, E.; Hadjipavlou-Litina, D Med. Res. Rev. 2008, 28, 39-
1
17.

compounds, and NMR spectra for compounds 3a–g, 4a-g, 1a–g)
associated with this article can be found in the online version.