Synthesis of Isoxazolines by the Electrophilic Chalcogenation of β,γ-Unsaturated Oximes: Fishing Novel Anti-Inflammatory Agents

Article abstract of DOI:10.1021/acs.joc.9b01754

Herein, we describe a new strategy to prepare chalcogen-functionalized isoxazolines. The strategy involves the reaction of β,γ-unsaturated oximes with electrophilic selenium and tellurium species, affording 19 new selenium- and tellurium-containing isoxaz

Full text of DOI:10.1021/acs.joc.9b01754

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Article
Synthesis of isoxazolines by the electrophilic chalcogenation of
#,#-unsaturated oximes: fishing novel anti-inflammatory agents
Eric F. Lopes, Filipe Penteado, Samuel Thurow, Mikaela Pinz, Angelica Reis, Ethel A. Wilhelm, Cristiane
Luchese, Thiago Barcellos, Bianca Dalberto, Diego Alves, Marcio S. da Silva, and Eder J. Lenardao
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01754 • Publication Date (Web): 11 Sep 2019
Downloaded from pubs.acs.org on September 12, 2019
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Synthesis of isoxazolines by the electrophilic
chalcogenation of β,γ-unsaturated oximes: fishing novel
anti-inflammatory agents
9
Eric F. Lopes,a Filipe Penteado,a Samuel Thurow,a Mikaela Pinz,b Angelica Reis,b Ethel
Wilhelm,b Cristiane Luchese,b Thiago Barcellos,c Bianca Dalbertoa, Diego Alves,a
Marcio S. da Silvaa and Eder J. Lenardãoa,*
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a Laboratório de Síntese Orgânica Limpa - LASOL - CCQFA - Universidade Federal de
Pelotas - UFPel - P.O. Box 354 - 96010-900, Pelotas, RS, Brazil.
b Lab. de Pesquisa em Farmacologia Bioquímica - LaFarBio - CCQFA - Universidade
Federal de Pelotas - UFPel - P.O. Box 354 - 96010-900, Pelotas, RS, Brazil
c Laboratory of Biotechnology of Natural and Synthetic Products - Universidade de
Caxias do Sul - UCS, 95070-560, Caxias do Sul, RS, Brazil.
* Email: lenardao@ufpel.edu.br
ABSTRACT
We describe herein a new strategy to prepare chalcogen-functionalized
isoxazolines. The strategy involves the reaction of β,γ-unsaturated oximes with
electrophilic selenium and tellurium species, affording 19 new selenium- and
tellurium-containing isoxazolines in good yields after 1 hour at room temperature.
The method was efficiently extended to the synthesis of 5 new (bis)isoxazoline
ditellurides.
One
of
the
prepared
compounds,
3-phenyl-5-
((phenylselanyl)methyl)-isoxazoline, demonstrated better anti-inflammatory and
anti-edematogenic effects than the reference drug Celecoxib.
INTRODUCTION
Heterocyclic compounds play an important role in several areas of
chemical industry, including the development of agrochemicals and dyes.1
Besides, 70% of marketed drugs, and 8 in 10 of the most consumed ones present
at least one heterocyclic unity.2 In this context, isoxazole and its derivatives are
a valuable class of heterocyclic compounds, which are ranked among the top 25
nitrogen-containing frequently found motifs in drugs approved by the U. S. FDA.3
Among them, Valdecoxib and Parecoxib are alluring examples of selective COX-
2 inhibitors, used against inflammatory processes. In the mid-2000s, Valdecoxib
was removed from the market due to adverse effects.4 The close-related
isoxazoline derivatives, in turn, are quite interesting compounds, showing a
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variety of activities, including COX-2 inhibitory effects (like compound ISO-1),5
cytotoxic activity towards HT-29, HeLa, MCF-7 and PC-3 cancer cells (like
compound I),6 and are present in a new class of the highly-active oomycete
fungicide Zorvec® (Figure 1).7
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O
O
N
O
N
HO
OMe
ISO-1
I
HO
HO
F
S
N
N
N
N
N
O
O
F3C
Zorvec®
F
Figure 1. Biologically active isoxazolines.
The first general method for the selective synthesis of isoxazolines was
developed by Quilico in 19508 and it involves the 1,3-cycloaddition of nitrile oxides
to alkenes. During many years this was the method of choice to prepare
differently substituted isoxazoline derivatives.9 More recently, new methods do
access isoxazolines have emerged, ranging from metal-catalyzed10 to metal-
promoted cyclization of oximes, using overstoichiometric amounts of transition
metals.11 The radical cyclization of alkenyl oximes using a diversity of radical
initiators was also explored12 and the fluoro- and iodo-promoted cyclizations of
alkenyl oximes to prepare fluoro-13 and iodo-isoxazolines14 were recently
developed.
Organochalcogen compounds are widely known to be versatile
intermediates in organic synthesis.15 For instance, vinyl selenides were employed
in 1,3-cycloaddition reactions to construct selenium-containing isoxazolines,
which were further converted to isoxazole derivatives through selenoxide
elimination pathways.16 Recently, Yu and Cai17 have disclosed a radical
cyclization/sulfanylation and selenylation of β,γ-unsaturated oximes using iodine
hypervalent species (PIDA) in the presence of base to access chalcogen-
containing isoxazolines in moderate to good yields (Scheme 1).
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Yu & Cai's work: radical cyclization
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Se
)2 (1.5 equiv)
PhI(OAc)2 (1.5 equiv)
DBU (1.5 equiv)
R
OH
Se
R
O
N
N
MeCN, r.t.
Ar, 6h
Ph
Ph
2 examples
R = Ph, Bu
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This work: electrophilic chalcogenation
OH
Y Y
R/ )2
O
N
N
Y
Y
R X/ X4
MeCN, r.t.
1 h
R
R
Y = Se, Te
25 examples
Scheme 1. Synthetic methods to chalcogen-containing isoxazolines.
Despite the biological importance of isoxazolines and organochalcogen
compounds,5,18 there are no general protocols to prepare selenium and tellurium-
functionalized isoxazolines. This causes a limitation in the study of the biological
activities of such molecular hybrids. Thus, based on the efficiency of chalcogen
electrophilic species to construct new C-Y bonds (Y = S, Se or Te) by the reaction
with electron-rich substrates,19 we present herein a general electrophilic
cyclization of β,γ-unsaturated oximes to access chalcogen-containing
isoxazolines, through a simple, fast and open-air protocol. The methodology was
extended to unprecedented tellurium-containing isoxazolines.
RESULTS AND DISCUSSION
(E)-1-Phenylbut-3-en-1-one oxime 1a and PhSeCl 2a were chosen as the
standard substrates to determine the best reaction conditions to prepare the
respective isoxazoline 3a (Table 1). We started by determining the best solvent
for the reaction at room temperature under argon atmosphere (Table 1, entries
1-6). The desired product 3a was accessed in 82% yield using MeCN as the
solvent (Table 1, entry 1). Solvents like hexane, DMSO, γ-valerolactone and
EtOAc did not present better results and the expected product 3a was obtained
in a yield range of 71-80% (Table 1, entries 2 to 5). In addition, based on previous
works in the use of deep eutectic solvents (DES) in the synthesis of
organochalcogen compounds,20 the choline chloride (ChCl):urea-based DES
was tested as the reaction medium; however, the isoxazoline 3a was obtained in
only 49% yield (Table 1, entry 6).
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Table 1: Optimization of the reaction conditions.a
6
7
8
9
OH
O
Se
Ph
N
N
Conditions
r.t., 1 h
Se
Ph Cl
+
Ph
Ph
1
2a
3a
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Entry
Solvent
Yield (%)b
1
2
MeCN
hexanes
DMSO
82
74
72
80
71
49
74
81
82
3c
4
γ-valerolactone
EtOAc
5
6d
7c
8d
9d,e
1 ChCl: 2 Urea
MeCN
MeCN
MeCN
a
In a test tube saturated with argon was added β,γ-
unsaturated oxime 1a (0.5 mmol), solvent (0.5 mL) and
PhSeCl 2a (0.6 mmol). The reaction was carried out at
room temperature for 1 h. The yields refer to the
isolated product obtained by column chromatography,
except when noted.
PhSeCl 2a. d The reaction was carried out in an open-
air flask. The crude reaction was quenched with a
saturated K2CO3 and extracted with EtOAc. After
evaporation of the solvent, the resulting solid was
rinsed with hexanes and dried under vacuum, to yield
pure 3a.
b
c
PhSeBr was used instead
e
Following, the reaction was performed using PhSeBr as the selenium
electrophilic source instead PhSeCl, giving the product 3a in 74% yield (Table 1,
entry 7). When the reaction was carried out in an open-air flask, 3a was obtained
in 81% yield, showing that the reaction is not sensible to air atmosphere (Table
1, entry 8).
It is worth to mention that using hexane or acetonitrile as solvent caused
the precipitation of the product 3a that, after a simple filtration, could be obtained
in 40% yield. However, the yield of 3a was increased to 82% after the crude
reaction was quenched with a saturated solution of K2CO3 and extracted with
EtOAc. After evaporation of the solvent, the resulting solid was rinsed with
hexanes and dried under vacuum (Table 1, entry 9).
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Based on these results, the best reaction condition was defined as stirring
a mixture of unsaturated oxime 1a and PhSeCl 2a (1.2 equiv) in MeCN in an
open-air vessel for 1 h at room temperature. Additionally, purification of the
product was performed just by rinsing it with hexanes after extraction.
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With the best reaction conditions in hand, a study on the reaction scope
was performed by varying the substituents in the aromatic ring of the β,γ-
unsaturated oxime 1 in the reaction with PhSeCl 2a (Table 2, 1b-i). As shown in
Table 2, β,γ-unsaturated oximes bearing halogen groups in the para-position
were good substrates for the reaction, affording the respective products 3b (R1 =
4-Br), 3c (R1 = 4-Cl) and 3d (R1 = 4-F) in 76%, 92% and 78% yield. On the other
hand, the electron-rich β,γ-unsaturated oximes 1e (R1 = 4-Me) and 1f (R1 = 4-
OMe) were less reactive substrates, affording the expected products 3e and 3f in
58% and 46% yield, respectively. Some limitations were found by employing
meta- and ortho-substituted β,γ-unsaturated oximes, accessing the products 3g
(R1 = 3-Cl) and 3i (R1 = 2-Me) in 17% and 15% yield, respectively. Unfortunately,
the meta-NO2 substituted oxime 1h was not a suitable substrate to our protocol,
and only trace amounts of product 3h were detected (Table 2).
Table 2. Scope for selenium-containing isoxazolines 3.a
OH
O
Se
Ar
N
N
MeCN
r.t., 1 h
+
Se
Ar Cl
1a-i
2a-e
"open-air"
3a-m
R1
O
R1
O
O
O
Se
Ph
Se
Se
Se
Ph
Ph
Ph
N
N
N
N
N
3a
3b
3c
3d
, 82%
, 76%
, 92%
, 78%
H
Br
Cl
F
O
O
O
O
Se
Ph
Se
Ph
Se
Ph
Se
Ph
N
N
N
Cl
O2N
[b]
3e
3g
3h
, traces
, 58%
O
, 17%
3f
, 46%
Me
MeO
Se
Ph
N
O
O
Me
Se
Se
N
N
Me
O
OMe
Ph
Ph
[c]
3k
, traces
3i
, 15%
3j
, 30%
CF3
O
Se
Se
N
N
Cl
Ph
Ph
3m
, 60%
3l
, 80%
a In a test tube was added β,γ-unsaturated oxime 1 (0.5 mmol), MeCN (0.5 mL) and ArSeCl 2 (0.6
mmol). The reaction was carried out at room temperature for 1 h. Isolated yields were obtained
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by rinsing the crude reaction mixture with hexanes solvent. b The product was purified by column
chromatography (1:9 mixture of AcOEt/hexanes as eluent).c Obtained in a mixture 2:1 with the
respective oxime.
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Following, the reactivity of differently substituted arylselenyl chlorides 2
was evaluated in the reaction with (E)-1-phenylbut-3-en-1-one oxime 1a under
the optimal conditions. It was observed that the presence of electron-donor
groups in the para-position of the benzene ring, like in 2b (Ar = 4-Me-C6H4) and
2c (Ar = 4-MeO-C6H4) reduced the reactivity, and the respective products 3j and
3k were obtained in 30% yield and trace amounts, respectively. These results
can be explained in part by the lower electrophilicity of the selenium atom in these
species and by the stabilization of the in situ formed seleniranium intermediate,
once in both cases the total consumption of oxime 1a was observed (see Scheme
2 for a plausible mechanism). On the other hand, the presence of electron-
withdrawing groups positively affects the reaction and products 3l (Ar = 4-Cl-
C6H4) and 3m (Ar = 3-CF3-C6H4) were obtained in 80% and in 60% yield (Table
2). These results demonstrate that the electron-withdrawing effect enhances the
electrophilicity of the selenium atom, facilitating the reaction.
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Based on the positive results obtained in the synthesis of the selenium-
containing isoxazolines 3 (Table 2), we were encouraged to expand the reaction
scope to tellurium-containing isoxazolines 5 (Table 3). For this purpose, since
tellanyl halides are not bench stable, aryltellanyl iodides 4 were prepared in situ
in a solution of THF, and the reaction was subsequently performed in the
presence of β,γ-unsaturated oximes 1.21
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Table 3. Scope for tellurium-containing isoxazolines 5.a
OH
O
Te
N
Ar
N
MeCN
r.t., 1 h
+
Te
Ar
I
"open-air"
1a-f
4
5a-i
R1
R1
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O
O
O
N
Te
Te
Te
Ph
Ph
Ph
N
N
N
5a
5b
5c
, 66%
, 71%
, 70%
O
H
F
Br
Cl
O
O
Te
Te
Te
Ph
Ph
Ph
N
N
[b]
5d
5e
5f
, 69%
, 68%
, 45%
Me
MeO
O
O
O
Te
5g
Te
5h
Te
5i
N
N
N
Me
OMe
Ph
Cl
Ph
Ph
, 57%
, traces
, 76%
a The ArTeI 4 (1.0 mL, 1M in THF) was prepared immediately before the use, in a separate vessel,
from a mixture of ArTeTeAr (0.5 mmol) and I2 (0.5 mmol) in THF (1.0 mL). This solution was
added to a solution of β,γ-unsaturated oxime 1 (0.5 mmol) in MeCN (0.5 mL), and the resulting
mixture was stirred at room temperature for 1 h. The crude reaction was quenched (sat. K2CO3)
and extracted with EtOAc. After evaporation of the solvent, the resulting solid was rinsed with
hexanes. b Yield after rinsing the crude with a 1:9 mixture of AcOEt/hexanes.
The yields of the tellurium-containing isoxazolines were slightly lower than
those observed for the selenium-analogues 3. Thus, neutral (E)-1-phenylbut-3-
en-1-one oxime 1a reacted smoothly with phenyltellanyl iodide 4a (Ar = C6H5) to
give the product 5a in 71% yield (Table 3). Unlike the observed in the synthesis
of selenium- isoxazolines 3b-d (Table 2), halogen-substituted β,γ-unsaturated
oximes 1b-d (R1 = Br, Cl and F) presented a similar reactivity in the reaction with
PhTeI 4a, giving the respective products 5b-d in 70%, 66% and 69% yield, similar
to the unsubstituted analogue 4a (Table 3). The presence of electron-donor
groups negatively influences the reaction, as observed for the selenium
analogues, and para-methyl-substituted oxime 1e (R1 = Me) reacted with 4a to
give 5e in 68% yield, while oxime 1e (R1 = OMe) afforded the respective product
5f in only 45% yield (Table 3).
Regarding the ArTeI 4 counterpart, the reactivity was less sensible to
electronic effects, except in the case when the strong electron-donor methoxy
group was present (4h, Ar = 4-OMeC6H4), that completely inhibited the reaction
with oxime 1a, with only trace amounts of 5h being detected (Table 3). The
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presence of para-chlorine substituent (4i, Ar = 4-Cl-C6H4) increased the reactivity,
and product 5i was obtained in 76% yield. In an attempt to extend the
methodology, PhSCl and ArS-succinimides (Ar = Ph, p-MeOC6H4 and p-ClC6H4)
were used as electrophiles in the reaction with oxime 1a. However, the expected
sulfur-containing isoxazolines were not formed under the optimal conditions.
Based on the literature reports,23 HRMS data and in our observations, a
plausible mechanism for the formation of the chalcogen-containing isoxasoline
3d is proposed in Scheme 2. Initially, the β,γ-unsaturated oxime 1d reacts with
the electrophilic selenium species 2a, giving the seleniranium cation intermediate
A. From the intermediate A, the reaction can follow three different pathways to
access the product 3d. I the path A, the seleniranium species A is directly
converted to 3d through the intramolecular nucleophilic attack by the oxygen and
elimination of HCl. Alternatively, the seleniranium A undergoes a ring-opening
reaction by the intermolecular attack by oxime 1d, giving the oxonium
intermediate B (detected by HRMS; Figures S1 and S2). Finally, an
intramolecular annulation occurs, releasing oxime 1d as leaving group, and
giving the desired product 3d (Scheme 2, path B). A third pathway involves the
attack of the seleniranium specie A to a second PhSeCl 2a to give the
intermediate C, which is easily converted to D (detected by HRMS; Figures S1
and S3) by an intramolecular cyclization. Finally, another unit of β,γ-unsaturated
oxime 1d reacts with the intermediate D to give the desired product 3d and
releasing the key intermediate A for a new reaction (Scheme 2, path C).
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OH
N
4
5
6
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8
1d
F
Se
Ph Cl
2a
OH
9
Ph
Se
N
Cl-
path B
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A
path C
F
HCl
path A
2a
1d
3d
F
HCl
Se
Ph
O
Ph
B
Intermediate
N
Se
OH
detected by HRMS-APCI
C26H22F2N2O2Se+ [M]+
m/z 515.1043 u
N
H
N
O
Se
C
Ph
F
B
C
D
Intermediates and
F
1d
detected by HRMS-APCI
C22H19FNOSe2+ [M]+
m/z 491.9770 u
Se
Ph
1d
O
N
O
N
SePh
Se
Ph
3d
A
D
F
F
Scheme 2. Mechanism proposal for the electrophilic chalcogenation.
Inspired by the results above, we envisioned to accomplish the synthesis
of diisoxazoline ditellurides 7, by using TeCl4 6a as electrophile in the annulation
of the β,γ-unsaturated oximes 1, followed by a sequential reduction-oxidation
step, as previously described by Comasseto et al.22 To our delight, the reaction
between (E)-1-phenylbut-3-en-1-one oxime 1a and TeCl4 6a under our optimal
conditions (r.t., 1 h), followed by the Comasseto’s reaction, gave the expected
diisoxazoline ditelluride 7a in an overall 67% yield (Table 4).
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Table 4. Scope for diisoxazoline dichalcogenides 7.a
5
6
7
8
O
OH
Y
)
N
2
N
1) MeCN, r.t., 1 h
Y
Cl4
+
2) NaBH4, H2O/THF
3) sat. NH4Cl, 0 oC, air
1a-e
7a-f
6a:
Y = Te
Y = Se
9
R1
6b:
R1
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O
O
O
Te
Te
Te
Se
)
)
)
N
N
N
2
2
2
b
7a
7b
7c
, 67%
, 40%
, 30%
O
H
Me
MeO
O
O
Te
Te
)
2
)
)
2
N
N
N
2
7d
7e
7f
, 48%
, 44%
, 51%
Cl
F
H
a
In a test tube was added β,γ-unsaturated oxime 1a (0.5 mmol), 0.5 mL of MeCN
and TeCl4 6a (0.6 mmol). The reaction mixture was stirred at room temperature for
1 h. Then, 3 mL of THF was added, followed by the dropwise addition of an aqueous
solution of NaBH4 (0.75 M) at 0 °C. The resulting mixture was stirred for additional
20 min and quenched (sat. NH4Cl) under air atmosphere.22 The solvent was removed
under reduced pressure and the resulting solid was rinsed with hexanes. b Yield after
rinsing the crude with a 3:7 mixture ratio of AcOEt/hexanes.
The reaction was extended to para-substituted β,γ-unsaturated oximes 1
bearing electron-releasing and electron-withdrawing groups. In all cases, the
expected ditellurides were obtained in slightly lower yields compared to the
unsubstituted oxime 1a. There is no a clear effect caused by the substituents and
products 7b (R1 = 4-Me), 7d (R1 = 4-Cl) and 7e (R1 = 4-F) were obtained in 40%,
44% and 51% yield, respectively. The strong electron-donating methoxy group
caused an even more decreasing in the reaction yield, affording the respective
product 7c (R1 = 4-MeO) in only 30% yield. In order to expand the scope of this
approach to the diselenide analogue, SeCl4 6b was satisfactorily employed as an
electrophile under the optimal conditions, affording the expected diisoxazoline
diselenide 7f in 48% yield (Table 4).
Based in the literature22 and in our own findings, a plausible mechanism
for the formation of ditellurides 7a-e (and by extension to diselenide 7f) involves
the initial formation of the tellurium trichloride isoxazoline 8, after attack by the
oxime 1 to TeCl4 6. Following, the Te(IV) trichloride 8 is reduced to the unstable
tellurol 9, that rapidly is oxidized by air to the respective ditelluride 7 (Scheme 3).
The formation of the tellurium trichloride isoxazoline 8 as an intermediate was
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confirmed by 125Te NMR and HRMS analysis (Figure S4). The registered 125Te
NMR of tellurium tetrachloride 6 (in CD3CN) showed one signal at  1707 ppm.
After the addition of the unsaturated oxime 1, almost instantaneous consumption
of TeCl4 and the appearance of a new signal at  1248 ppm are observed. Using
HRMS analysis it was possible to confirm that the signal observed in the 125Te
NMR is in fact the tellurium trichloride 8.
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OH
N
O
Te
Cl3
N
Te
Cl4
+
Ar
Ar
1
6
8
HCl
1) aq. NaBH4
2) H3O+
O
O
Te)2
Te
N
H
N
air
[O]
Ar
Ar
7
9
not isolable
Scheme 3. Mechanism proposal for the formation of ditellurides 7.
As mentioned before, in the introduction, a number of biological activities
are attributed to the isoxazoline core and to organoselenium compounds. The
similarity of the chalcogen-containing isoxazolines 3 and 5 with ISO-1, a potent
anti-inflammatory agent,5 we wonder if the new prepared compounds could
present
such
bioactivity.
To
test
our
hypothesis,
3-phenyl-5-
[(phenylselanyl)methyl]-isoxazoline 3a (PPSEI45) was chosen and evaluated for
its antiedematogenic and anti-inflammatory effects in mice (Figs. 2 and 3). Here,
ear edema was induced by croton-oil application, as described elsewhere.24-25
Importantly, treatment with PPSEI45, at dose of 50 mg/kg, or Celecoxib,
only at dose of 100 mg/kg, reduced the ear edema induced by croton oil,
presenting anti-edematogenic effect (ANOVA: F (4, 25) = 93.17, p< 0.0001). The
inhibition of ear edema by PPSEI45 (50 mg/kg) was 51%, while by Celecoxib
(100 mg/kg) was 36% (Figure 2).
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Figure 2. Ear swelling treatment with PPSEI45 (3a) and Celecoxib.
In order to extend our knowledge about the anti-inflammatory potential of
PPSEI45, the MPO activity was determined. An elevated level of MPO is
considered as an indicator of neutrophil activation under the influence of pro-
oxidative and pro-inflammatory conditions.26-30 As demonstrated in Figure 3, the
data analysis revealed that croton oil significantly increased the MPO activity in
ears of mice, when compared to the control group. Here, pre-treatment with
PPSEI45 (3a, 50 mg/kg) or Celecoxib (100 mg/kg) protected against the increase
on the MPO activity in mouse ear induced by croton oil (ANOVA: F (4, 25) =
7.313, p< 0.001). PPSEI45 (50 mg/kg) and Celecoxib (100 mg/kg) reduced
around 74% and 79% of the MPO activity, respectively. This result suggests that
PPSEI45 suppressed the severity of croton oil via inhibition of MPO activity.
Figure 3. MPO activity for PEPSEI45 (3a) and Celecoxib.
Of particular importance is that the single oral administration of PPSEI45
(300 mg/kg) did not cause death of animals. The alanine aminotransferase ALT
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(df = 0.4070; t = 2, p > 0.05) and aspartate aminotransferase AST (df = 2; t =
1.016, p > 0.05) activities, as well as the levels of urea (df = 2; t = 1.110, p > 0.05)
remained unaltered after treatment with PPSEI45, when compared to those mice
of the control group (Table S1). An increasing of plasmatic AST activity is a
marker of cardiac damage,31 which is one of the adverse effects of coxibs.32
Additionally, the ability of organoselenium compounds to oxidize sulfhydryl
groups from biological molecules can be involved both in their pharmacological
and/or toxicological effects. δ-ALA-D is a sulfhydryl-containing enzyme that is
extremely sensitive to oxidizing agents.33 A single oral administration of
compound PPSEI45, at dose 300mg/kg, did not alter the enzyme activity in
samples of brain, kidney or liver (Table S2).
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The findings of the present study indicated, for the first time, that PPSEI45,
an organoselenium compound possessing structural elements of Valdecoxib,
exerts anti-inflammatory action in mice. Importantly, PPSEI45 (3a) reduced ear
edema formation and protected against the increase on the myeloperoxidase
(MPO) activity in mouse ear. Indeed, our results are consistent with the fact that
PPSEI45 was more effective than Celecoxib, a reference drug, in this model of
acute inflammation.
CONCLUSIONS
In conclusion, we have developed a new and general method to prepare
chalcogen-containing (Se and Te) isoxazolines. A simple and mild electrophilic
cyclization of β,γ-unsaturated oximes allowed the preparation of 25 isoxazolines,
including unprecedented tellurium-functionalized ones and diisoxazoline
ditellurides. HRMS and NMR analysis afforded information on the reaction
mechanism, showing the involvement of cationic species. One of the prepared
compounds, 3-phenyl-5-((phenylselanyl)methyl)-isoxazoline, exerts anti-
inflammatory effect in an applicable experimental model and at lower doses than
the reference drug (Celecoxib).
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EXPERIMENTAL SECTION
5
6
General Information
7
8
9
The reactions were monitored by TLC carried out on pre-coated TLC sheets
ALUGRAM® Xtra SIL G/UV254 by using UV light as visualization agent and the
mixture of 5% vanillin in 10% H2SO4 under heating conditions as developing
agent. Merck silica gel (particle size 63-200 m) was used to flash
chromatography. Hydrogen nuclear magnetic resonance spectra (1H NMR) were
obtained at 400 MHz on Bruker Nuclear Ascend 400 spectrometer. The spectra
were recorded in CDCl3 and DMSO-d6 solutions. The chemical shifts are reported
in ppm, referenced to tetramethysilane (TMS) as the external reference.
Hydrogen coupling patterns are described as singlet (s), doublet (d), triplet (t),
doublet of doublets (dd), doublet of doublet of doublet of doublets (dddd) and
multiplet (m). Coupling constants (J) are reported in Hertz. Carbon-13 nuclear
magnetic resonance spectra (13C NMR) were obtained at 100 MHz on Bruker
Nuclear Ascend 400 spectrometer. The chemical shifts are reported in ppm,
referenced to the solvent peak of CDCl3 or DMSO-d6. Electrospray ionization
(ESI-QTOF) high resolution mass spectrometry (EMAR) analyzes were
performed on a Bruker Daltonics micrQTOF-Q II instrument in positive or negative
modes. The samples were solubilized in HPLC grade acetonitrile and injected
into the APCI source via a syringe at a flow rate of 5.0 μL min-1. The following
instrument parameters were applied: the capillary and cone voltages were
adjusted to +3500 V and -500 V, respectively, with a desolvation temperature of
180 °C. Compass 1.3 software for micrOTOF-Q II (Bruker Daltonics, USA) was
used for data acquisition and processing. Data were collected in the m/z range of
50-1200 at the rate of two sweeps per second. The starting oximes 1a-I were
prepared as previously described in the literature.10a,35-37
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General Procedure for the synthesis aryl selanyl chlorides 2: Reactions
using diphenyl diselenide were performed in a 5 mmol scale; the other reactions
were carried out in a 2.5 mmol scale. In a 50 mL round-bottomed flask saturated
with argon, containing a solution of the diaryl diselenide (5 mmol) in DCM (10.0
mL) at 0 ºC, it was added dropwise 50% of a solution of sulfuryl chloride (0.67 g,
5.0 mmol) in DCM (10.0 mL) under stirring. Following, the remaining solution of
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SO2Cl2 in the addition funnel was diluted with 10.0 mL of DCM and the resulting
solution was added dropwise under vigorous stirring at 0 ºC. The system was
stirred for additional 1 h at room temperature. Finally, the obtained mixture was
concentrated under reduced pressure to give an orange solid, which was used
as substrate without further purification.38
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General Procedure for the synthesis of selenium-containing isoxazolines 3:
In a round-bottomed flask were added the β,γ-unsaturated oxime 1 (0.5 mmol),
MeCN (0.5 mL) and ArSeCl 2 (0.6 mmol). The resulting mixture was stirred at
room temperature for 1 h. After reaction completion, a saturated solution of K2CO3
(2.0 mL) was added carefully and the product was extracted with ethyl acetate
(4x 2.0 mL). The solvent was removed under reduced pressure and the resulting
solid was rinsed with hexanes (8x 1.0 mL). Following, the solid was dried in a
vacuum pump to access the product 3 in the tabulated yields (Table 2). Product
3f was isolated by column chromatography (a 9:1 mixture of hexanes/AcOEt was
used as eluent).
3-Phenyl-5-[(phenylselanyl)methyl]-4,5-dihydroisoxazole (3a). Yield: 130 mg
(82%); white solid; mp 94-96 ºC. 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.6 - 7.62
(m, 2H); 7.57 - 7.55 (m, 2H); 7.41 - 7.39 (m, 3H); 7.29 - 7.26 (m, 3H); 4.91 (dddd,
J = 10.3, 9.1, 6.9, 4.5 Hz, 1H); 3.44 (dd, J = 16.8, 10.3 Hz, 1H); 3.31 (dd, J = 12.6,
4.5 Hz, 1H); 3.20 (dd, J = 16.8, 6.9 Hz, 1H); 3.00 (dd, J = 12.6, 9.0 Hz, 1H).
13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 156.3, 133.4, 130.3, 129.6, 129.5, 128.8,
127.7, 126.8, 80.5, 40.2, 31.5 (d, 1JC-Se = 68.5 Hz). HRMS (ESI-TOF) m/z: [M +
H]+ Calcd for C16H16NOSe 318.0392; Found 318.0391.
3-(4-Bromophenyl)-5-[(phenylselanyl)methyl]-4,5-dihydroisoxazole
(3b).
1
Yield: 150 mg (76%); white solid; mp 94-96 ºC. H NMR (CDCl3, 400 MHz) δ
(ppm) 7.56 - 7.54 (m, 2H); 7.53 - 7.47 (m, 4H); 7.29 - 7.28 (m, 3H); 4.95 - 4.87
(m, 1H); 3.40 (dd, J = 16.8, 10.3 Hz, 1H); 3.30 (dd, J = 12.6, 4.5 Hz, 1H); 3.16
(dd, J = 16.8, 7.0 Hz, 1H); 2.99 (dd, J = 12.6, 9.0 Hz, 1H). 13C{1H} NMR (CDCl3,
100 MHz) δ (ppm) 155.5, 133.4, 132.0, 129.4, 128.7, 128.5, 128.2, 127.7, 124.5,
80.8, 39.9, 31.4 (d, 1JC-Se = 68.6 Hz). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C16H15BrNOSe 394.9494; Found 394.9500.
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3-(4-Chlorophenyl)-5-[(phenylselanyl)methyl]-4,5-dihydroisoxazole
(3c).
1
Yield: 161 mg (92%); white solid; mp 58-60 ºC. H NMR (CDCl3, 300 MHz) δ
(ppm) 7.79 - 7.76 (m, 2H); 7.55 - 7.51 (m, 2H); 7.38 - 7.30 (m, 3H); 7.26 - 7.20
(m, 2H); 4.99 (dddd, J = 10.2, 9.1, 7.2, 4.4 Hz, 1H); 3.41 (dd, J = 16.8, 10.2 Hz,
1H); 3.29 (dd, J = 12.1, 4.5 Hz, 1H); 3.09 - 3.01 (m, 2H). 13C{1H} NMR (CDCl3, 75
MHz) δ (ppm) 155.3, 136.1, 133.3, 129.4, 129.0, 128.6, 127.9, 127.9, 127.6, 80.7,
39.9, 31.3. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H15ClNOSe 351.9999;
Found 351.9998.
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3-(4-Fluorophenyl)-5-[(phenylselanyl)methyl]-4,5-dihydroisoxazole
(3d).
1
Yield: 130 mg (78%); yellow solid; mp 72-74 ºC. H NMR (CDCl3, 400 MHz) δ
(ppm) 7.63 - 7.59 (m, 2H); 7.56 - 7.54 (m, 2H); 7.29 - 7.27 (m, 3H); 7.10 - 7.05
(m, 2H); 4.90 (dddd, J = 10.3, 9.0, 6.9, 4.5 Hz, 1H); 3.41 (dd, J = 16.8, 10.3 Hz,
1H); 3.30 (dd, J = 12.6, 4.5 Hz, 1H); 3.17 (dd, J = 16.8, 6.9 Hz, 1H); 2.99 (dd, J =
12.6, 9.0 Hz, 1H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 163.9 (d, 1J = 250.7
Hz), 155.3, 133.3, 129.4, 128.7 (d, 3J = 8.4 Hz), 127.8, 127.7, 125.8 (d, 4J = 3.4
2
1
19
Hz), 116.0 (d, J = 22.0 Hz), 80.6, 40.2, 31.5 (d, JC-Se = 68.3 Hz). F NMR
(CDCl3, 376 MHz) δ (ppm) 112.38 (tt, J = 8.3, 5.2 Hz). 77Se NMR (CDCl3, 76 MHz)
δ (ppm) 259.0. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H15FNOSe 336.0298;
Found 336.0298.
5-[(Phenylselanyl)methyl]-3-(p-tolyl)-4,5-dihydroisoxazole (3e). Yield: 96 mg
(58%); light red oil. 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.56 - 7.54 (m, 1H); 7.52
(d, J = 8.3 Hz, 2H); 7.28 - 7.25 (m, 3H); 7.19 (d, J = 8.0 Hz, 2H); 4.87 (dddd, J =
10.3, 9.1, 6.8, 4.6 Hz, 1H); 3.41 (dd, J = 16.8, 10.2 Hz, 1H); 3.30 (dd, J = 12.5,
4.5 Hz, 1H); 3.17 (dd, J = 16.8, 6.8 Hz, 1H); 2.98 (dd, J = 12.5, 9.0 Hz, 1H); 2.37
(s, 3H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 156.3, 140.5, 133.3, 129.5,
1
129.4, 128.9, 127.6, 126.8, 126.7, 80.3, 40.3, 31.5 (d, JC-Se = 68.5 Hz), 21.6.
77Se NMR (CDCl3, 76 MHz) δ (ppm) 258.9. HRMS (ESI-TOF) m/z: [M + H]+ Calcd
for C17H18NOSe 332.0549; Found 332.0550.
3-(4-Methoxyphenyl)-5-[(phenylselanyl)methyl]-4,5-dihydroisoxazole (3f).
1
Yield: 79 mg (46%); yellow solid; mp 74-76 °C. H NMR (CDCl3, 400 MHz) δ
(ppm) 7.57 - 7.53 (m, 4H); 7.28 - 7.26 (m, 3H); 6.91 - 6.88 (m, 2H); 4.89 - 4.82
(m, 1H); 3.82 (s, 3H); 3.40 (dd, J = 16.7, 10.2 Hz, 1H); 3.29 (dd, J = 12.6, 4.5 Hz,
1H); 3.16 (dd, J = 16.7, 6.8 Hz, 1H); 2.97 (dd, J = 12.6, 9.0 Hz, 1H). 13C{1H} NMR
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(CDCl3, 100 MHz) δ (ppm) 161.1, 155.9, 133.2, 129.4, 128.9, 128.3, 127.6, 122.0,
114.2, 80.1, 55.4, 40.4, 31.5 (d, 1JC-Se = 67.9 Hz). 77Se NMR (CDCl3, 76 MHz) δ
(ppm) 259.3. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H18NO2Se 348.0497;
Found 348.0498.
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3-(3-Chlorophenyl)-5-[(phenylselanyl)methyl]-4,5-dihydroisoxazole
(3g).
Yield: 25.0 mg (15%), yellow oil. 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.65 - 7.45
(m, 2H), 7.39 - 7.26 (m, 7H), 4.92 (dddd, J = 10.3, 9.0, 6.9, 4.5 Hz, 1H), 3.41 (dd,
J = 16.9, 10.3 Hz, 1H), 3.31 (dd, J = 12.6, 4.5 Hz, 1H), 3.17 (dd, J = 16.8, 6.9 Hz,
13
1H), 2.99 (dd, J = 12.6, 9.0 Hz, 1H). C{1H} NMR (CDCl3, 100 MHz) δ (ppm)
155.1, 137.4, 134.7, 133.2, 130.1, 129.9, 129.3, 127.6, 126.7, 124.7, 80.7, 39.7,
31.3. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H15ClNOSe 351.9827; Found
351.9825.
5-[(Phenylselanyl)methyl]-3-(o-tolyl)-4,5-dihydroisoxazole (3i). Yield: 28 mg
(17%), yellowish oil. 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.57 - 7.55 (m, 2H), 7.28
- 7.19 (m, 3H), 4.85 (dddd, J = 10.2, 8.8, 6.8, 4.6 Hz, 1H), 3.48 (dd, J = 16.7, 10.2
Hz, 1H), 3.30 (dd, J = 12.5, 4.6 Hz, 1H), 3.23 (dd, J = 16.7, 6.8 Hz, 1H), 3.01 (dd,
13
J = 12.6, 8.8 Hz, 1H), 2.54 (s, 3H). C{1H} NMR (CDCl3 , 100 MHz) δ (ppm)
157.2, 138.1, 133.3, 131.7, 129.5, 129.4, 129.0, 128.6, 127.6, 125.9, 79.4, 42.7,
31.6 (d, 1J C-Se = 68.1 Hz), 23.1. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C17H18NOSe 332.0371; Found 332.0374.
3-Phenyl-5-[(p-tolylselanyl)methyl]-4,5-dihydroisoxazole (3j). Yield: 49 mg
(30%); white solid; mp 83-85 °C. 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.65 - 7.63
(m, 2H); 7.48 - 7.45 (m, 2H); 7.41 - 7.38 (m, 3H); 7.11 - 7.09 (m, 2H); 4.92 - 4.84
(m, 1H); 3.43 (dd, J = 16.8, 10.2 Hz, 1H); 3.27 (dd, J = 12.5, 4.5 Hz, 1H); 3.20
(dd, J = 16.9, 6.9 Hz, 1H); 2.94 (dd, J = 12.5, 9.2 Hz, 1H); 2.34 (s, 3H). 13C{1H}
NMR (CDCl3, 100 MHz) δ (ppm) 156.3, 137.8, 133.8, 130.2, 130.2, 129.5, 128.8,
1
1
126.8, 124.9 (d, JC-Se = 101.2 Hz), 80.5, 40.1, 31.7 (d, JC-Se = 68.3 Hz), 21.2.
HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H18NOSe 332.0549; Found
332.0546.
5-{[(4-Chlorophenyl)selanyl]methyl}-3-phenyl-4,5-dihydroisoxazole
(3l).
1
Yield: 140 mg (80%); brown solid; mp 65-67 ºC. H NMR (CDCl3, 300 MHz) δ
(ppm) 7.65 - 7.62 (m, 2H); 7.49 (d, J = 8.5 Hz, 2H); 7.42 - 7.38 (m, 3H); 7.25 (d,
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J = 8.6 Hz, 2H); 4.90 (dddd, J = 10.3, 8.6, 6.8, 4.7 Hz, 1H); 3.45 (dd, J = 16.8,
10.3 Hz, 1H); 3.27 (dd, J = 12.7, 4.7 Hz, 1H); 3.19 (dd, J = 16.8, 6.9 Hz, 1H); 3.01
(dd, J = 12.6, 8.5 Hz, 1H). 13C{1H} NMR (CDCl3, 75 MHz) δ (ppm) 156.3, 134.7,
134.0, 133.4, 130.4, 129.4, 128.9, 127.1, 126.8, 80.2, 40.3, 32.0. HRMS (ESI-
TOF) m/z: [M + H]+ Calcd for C16H15ClNOSe 351.9999; Found 352.0001.
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3-Phenyl-5-{[(3-(trifluoromethyl)phenyl)selanyl]methyl}-4,5-
dihydroisoxazole (3m). Yield: 115 mg (60%); white solid; mp 89-91 ºC. 1H NMR
(CDCl3, 400 MHz) δ (ppm) 7.80 (s, 1H); 7.74 (d, J = 7.8 Hz, 1H); 7.66 - 7.63 (m,
1H); 7.53 (d, J = 7.8 Hz, 1H); 7.42 - 7.39 (m, 4H); 4.94 (dddd, J = 10.3, 8.2, 6.8,
4.8 Hz, 1H); 3.48 (dd, J = 16.8, 10.3 Hz, 1H); 3.34 (dd, J = 12.7, 4.8 Hz, 1H); 3.21
(dd, J = 16.8, 6.8 Hz, 1H); 3.10 (dd, J = 12.6, 8.2 Hz, 1H). 13C{1H} NMR (CDCl3,
100 MHz) δ (ppm) 156.3, 136.2 (q, 4J = 1.5 Hz), 131.7 (q, 2J = 32.4 Hz), 130.4,
130.3, 129.75, 129.65 (q, 3J = 3.8 Hz), 129.4, 128.9, 126.8, 124.4 (q, 3J = 3.8 Hz),
1
19
123.7 (d, J = 272.9 Hz), 80.1, 40.4, 31.9. F NMR (CDCl3, 376 MHz) δ (ppm)
62.8. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H15F3NOSe 386.0266; Found
386.0281.
General Procedure for the synthesis of tellurium-containing isoxazolines 5:
The electrophilic species ArTeI was prepared immediately before the use by
stirring a mixture of ArTeTeAr (0.5 mmol) and I2 (0.127 g, 0.5 mmol) in THF (1.0
mL) for 2 h at room temperature.21 The solution of ArTeI (1.0 mL, 1M in THF) was
added to the β,γ-unsaturated oxime 1 (0.5 mmol), in MeCN (0.5 mL) and the
resulting mixture was stirred for 1 h at room temperature. The extraction and
purification were performed as described for the products 3, above to afford
products 5 in the tabled yields (Table 3). Product 5f was isolated by rinsing the
solid with a 9:1 mixture of hexanes/AcOEt.
3-Phenyl-5-[(phenyltellanyl)methyl]-4,5-dihydroisoxazole (5a). Yield: 130 mg
1
(71%); red solid; mp 38-40 °C. H NMR (CDCl3, 400 MHz) δ (ppm) 7.80 - 7.78
(m, 2H); 7.62 - 7.60 (m, 2H); 7.40 - 7.37 (m, 3H); 7.34 - 7.30 (m, 1H); 7.25 - 7.21
(m, 2H); 5.03 - 4.95 (m, 1H); 3.45 (dd, J = 16.8, 10.2 Hz, 1H); 3.30 (dd, J = 12.1,
4.4 Hz, 1H); 3.12 - 3.03 (m, 2H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 156.2,
139.0, 130.2, 129.6, 129.6, 128.8, 128.3, 126.8, 110.6, 82.1, 41.3, 13.3 (d, 1JC-Te
= 173.3 Hz). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H16NOTe 368.0289;
Found 368.0287.
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3-(4-Bromophenyl)-5-[(phenyltellanyl)methyl]-4,5-dihydroisoxazole
(5b).
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Yield: 155 mg (70%); light orange solid; mp 89 - 91 °C. H NMR (CDCl3, 400
MHz) δ (ppm) 7.78 - 7.76 (m, 2H); 7.50 (d, J = 8.7 Hz, 2H); 7.44 (d, J = 8.7 Hz,
2H); 7.33 - 7.29 (m, 1H); 7.23 – 7.19 (m, 2H); 4.98 (dddd, J = 10.3, 9.0, 7.3, 4.5
Hz, 1H); 3.39 (dd, J = 16.7, 10.2 Hz, 1H); 3.28 (dd, J = 12.2, 4.5 Hz, 1H); 3.03 -
3.00 (m, 1H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 155.4, 139.0, 132.0, 129.5,
128.5, 128.3, 128.2, 124.4, 110.6, 82.4, 41.0, 13.1 (d, 1JC-Te = 174.1 Hz). HRMS
(ESI-TOF) m/z: [M + H]+ Calcd for C16H15BrNOTe 445.9376; Found 445.9375.
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3-(4-Chlorophenyl)-5-[(phenyltellanyl)methyl]-4,5-dihydroisoxazole
(5c).
Yield: 132 mg (66%); light orange solid; m. p. 102-103 °C. 1H NMR (CDCl3, 400
MHz) δ (ppm) 7.79 - 7.77 (m, 2H); 7.53 - 7.51 (m, 2H); 7.36 - 7.30 (m, 3H); 7.24
- 7.20 (m, 2H); 5.03 - 4.95 (m, 1H); 3.40 (dd, J = 16.8, 10.2 Hz, 1H); 3.29 (dd, J
= 12.1, 4.5 Hz, 1H); 3.08 - 3.01 (m, 2H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm)
155.3, 139.0, 136.1, 129.6, 129.0, 128.3, 128.1, 128.0, 110.6, 82.4, 41.1, 13.2.
125Te NMR (CDCl3, 126 MHz) δ (ppm) 433.0. HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C16H15ClNOTe 401.9889; Found 401.9889.
3-(4-Fluorophenyl)-5-[(phenyltellanyl)methyl]-4,5-dihydroisoxazole
(5d).
Yield: 132 mg (69%); light orange solid; mp 95-97 °C. 1H NMR (CDCl3, 400 MHz)
δ (ppm) 7.79 - 7.77 (m, 2H); 7.60 - 7.57 (m, 2H); 7.34 - 7.30 (m, 1H); 7.24 - 7.20
(m, 2H); 7.09 - 7.05 (m, 2H); 4.98 (dddd, J = 10.2, 9.0, 7.2, 4.5 Hz, 1H); 3.41 (dd,
J = 16.7, 10.2 Hz, 1H); 3.29 (dd, J = 12.1, 4.5 Hz, 1H); 3.09 - 3.03 (m, 2H). 13C{1H}
1
NMR (CDCl3, 100 MHz) δ (ppm) 163.8 (d, J = 250.8 Hz), 155.2, 139.0, 129.5,
128.7 (d, 3J = 8.4 Hz), 128.3, 125.9 (d, 4J = 3.4 Hz), 115.9 (d, 2J = 21.9 Hz), 110.6,
82.2, 41.3, 13.2 (d, 1JC-Te = 173.5 Hz). 19F NMR (CDCl3, 376 MHz) δ (ppm) 109.90
(tt, J = 8.7, 5.4 Hz). 125Te NMR (CDCl3, 126 MHz) δ (ppm) 432.0. HRMS (ESI-
TOF) m/z: [M + H]+ Calcd for C16H15FNOTe 386.0195; Found 386.0189.
5-[(Phenyltellanyl)methyl]-3-(p-tolyl)-4,5-dihydroisoxazole (5e). Yield: 129
mg (68%); red oil. 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.80 - 7.77 (m, 2H); 7.51 -
7.49 (m, 2H); 7.34 - 7.30 (m, 1H); 7.24 - 7.18 (m, 4H); 4.97 (dddd, J = 10.1, 9.1,
7.1, 4.5 Hz, 1H); 3.43 (dd, J = 16.7, 10.1 Hz, 1H); 3.30 (dd, J = 12.1, 4.5 Hz, 1H);
3.10 - 3.03 (m, 2H); 2.37 (s, 3H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 56.2,
140.5, 139.1, 129.6, 129.5, 128.3, 126.8, 126.7, 110.7, 81.9, 41.4, 21.6, 13.34.
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HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H18NOTe 382.0446; Found
5
6
382.0440.
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3-(4-Methoxyphenyl)-5-[(phenyltellanyl)methyl]-4,5-dihydroisoxazole (5f).
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Yield: 89 mg (68%); light brown solid; mp 85-87. H NMR (CDCl3, 400 MHz) δ
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(ppm) 7.79 - 7.77 (m, 2H); 7.55 - 7.53 (m, 2H); 7.33 - 7.29 (m, 1H); 7.24 - 7.20
(m, 2H); 6.91 - 6.89 (m, 2H); 4.95 (dddd, J = 10.1, 9.1, 7.1, 4.5 Hz, 1H); 3.83 (s,
3H); 3.41 (dd, J = 16.7, 10.1 Hz, 1H); 3.29 (dd, J = 12.1, 4.5 Hz, 1H); 3.09 - 3.03
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(m, 2H). C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 161.1, 155.8, 139.0, 129.5,
128.3, 128.2, 122.2, 114.2, 110.7, 81.8, 55.5, 41.5, 13.4. 125Te NMR (CDCl3, 126
MHz) δ (ppm) 431.5. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H18NO2Te
398.0395; Found 398.0395.
3-Phenyl-5-[(p-tolyltellanyl)methyl]-4,5-dihydroisoxazole (5g). Yield: 108 mg
(57%); light orange solid; mp 55-56°C. 1H RMN (CDCl3, 400 MHz) δ (ppm) 7.69
(d, J = 7.7 Hz, 2H); 7.62 - 7.60 (m, 2H); 7.40 - 7.37 (m, 3H); 7.05 (d, J = 7.7 Hz,
2H); 5.00 - 4.92 (m, 1H); 3.43 (dd, J = 16.7, 10.2 Hz, 1H); 3.27 (dd, J = 12.0, 4.5
Hz, 1H); 3.12 - 2.98 (m, 2H); 2.35 (s, 3H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm)
156.2, 139.4, 138.5, 130.5, 130.1, 129.7, 128.8, 126.7, 106.4, 82.1, 41.2, 21.3,
13.2 (d, 1JC-Te = 173.4 Hz). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H18NOTe
382.0445; Found 382.0445.
5-{[(4-Chlorophenyl)tellanyl]methyl}-3-phenyl-4,5-dihydroisoxazole
(5i):
Yield: 152 mg (76%); dark orange solid; mp 90-92 °C. 1H NMR (CDCl3, 400 MHz)
δ (ppm) 7.72 - 7.68 (m, 2H); 7.62 - 7.60 (m, 2H); 7.41 - 7.38 (m, 3H); 7.20 - 7.186
(m, 2H); 5.01 - 4.94 (m, 1H); 3.45 (dd, J = 16.7, 10.2 Hz, 1H); 3.27 (dd, J = 12.2,
4.8 Hz, 1H); 3.11 - 3.05 (m, 2H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 156.3,
140.4, 130.3, 129.8, 129.5, 128.8, 126.8, 108.2, 81.7, 41.4, 13.9 (d, 1JC-Te = 173.0
Hz). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H15ClNOTe 401.9888; Found
401.9892.
General Procedure for the synthesis of diisoxazoline dichalcogenides 7: In
a round-bottomed flask were added the β,γ-unsaturated oxime 1 (0.5 mmol),
MeCN (0.5 mL) and TeCl4 (0.161 g, 0.6 mmol) or SeCl4 (0.132 g, 0.6 mmol). The
resulting mixture was stirred for 1 h at room temperature. Then, the solution was
diluted with THF (3.0 mL) and an aqueous solution of NaBH4 (2.0 mL, 0.75M) was
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added dropwise at 0 ºC.22 The resulting dark red (for tellurium) or yellowish (for
selenium) solution was treated with saturated NH4Cl (15 mL) and extracted with
AcOEt (3x 15 mL). The organic layer was washed with brine (10 mL) and dried
over MgSO4. The solvent was removed under reduced pressure and the resulting
solid was rinsed with hexanes (5x 2.0 mL). Following, the solid was dried in a
vacuum pump to access the product 7 in the tabulated yields (Table 4). For
product 7c, a 7:3 mixture of hexanes/AcOEt was used in the washing step.
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2-Bis[(3-phenyl-4,5-dihydroisoxazol-5-yl)methyl]ditellane (7a). Yield: 97 mg
1
(67%); red solid; mp 35-36 °C. H NMR (CDCl3, 300 MHz) δ (ppm) 7.68 - 7.64
(m, 2H); 7.41 - 7.39 (m, 3H); 5.02 - 4.91 (m, 1H); 3.66 - 3.45 (m, 3H); 3.20 - 3.12
(m, 1H). 13C{1H} NMR (CDCl3, 75 MHz) δ (ppm) 156.4, 156.4, 130.3, 129.5,
128.9, 126.8, 83.0, 83.0, 41.1, 10.0, 9.9. HRMS (ESI-TOF) m/z: [M + H]+ Calcd
for C20H21N2O2Te2 576.9693; Found 576.9693.
1,2-Bis{[3-(p-tolyl)-4,5-dihydroisoxazol-5-yl]methyl}ditellane (7b). Yield: 60
mg (40%); orange solid; mp 125-127 °C. 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.54
(d, J = 7.8 Hz, 2H); 7.20 (d, J = 7.8 Hz, 2H); 4.98 - 4.89 (m, 1H); 3.63 - 7.57 (m,
1H); 3.50 - 3.44 (m, 2H); 3.17 - 3.10 (m, 1H); 2.37 (s, 3H). 13C{1H} NMR (CDCl3,
100 MHz) δ (ppm) 156.3, 156.3, 140.5, 129.5, 126.7, 126.7, 82.8, 82.8, 41.2,
21.6, 10.1, 9.9. 125Te NMR decoupled (CDCl3, 126 MHz) δ (ppm) 99.4 (t, 2JTe-H
=
2
33.0 Hz), 97.3 (t, J = 33.6 Hz). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C22H25N2O2Te2 605.0007; Found 604.9981.
1,2-Bis((3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)ditellane
(7c). Yield: 47 mg (30%); dark brown solid; mp 140-142 °C. 1H RMN (CDCl3, 400
MHz) δ (ppm) 7.59 - 7.57 (m, 2H); 6.91 - 6.88 (m, 2H); 4.95 - 4.87 (m, 1H); 3.82
(s, 3H); 3.62 - 3.56 (m, 1H); 3.48 - 3.42 (m, 2H); 3.15 - 3.09 (m, 1H). 13C{1H} NMR
(CDCl3, 100 MHz) δ (ppm) 161.2, 156.0, 155.9, 128.3, 122.0, 114.2, 82.7, 82.6,
55.5, 55.4, 41.3, 10.0, 9.9. 125Te NMR decoupled (CDCl3, 126 MHz) δ (ppm) 99.2
(t, 2JTe-H = 32.9 Hz), 96.9 (t, 2J = 32.5 Hz). HRMS (ESI-TOF) m/z: [M + H]+ Calcd
for C22H25N2O4Te2 636.9905; Found 636.9905.
1,2-Bis{[3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl]methyl}ditellane (7d).
1
Yield: 97 mg (44%); orange solid; mp 99-101 °C. H NMR (CDCl3, 400 MHz) δ
(ppm) 7.61 - 7.56 (m, 2H); 7.39 - 7.35 (m, 2H); 5.03 - 4.92 (m, 1H); 3.65 - 3.58
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(m, 1H); 3.51 - 3.42 (m, 2H); 3.16 - 3.08 (m, 1H). 13C{1H} NMR (CDCl3, 100 MHz)
δ (ppm) 155.9, 136.7, 129.6, 128.5, 118.7, 83.7, 83.7, 41.4, 10.3, 10.1. HRMS
(ESI-TOF) m/z: [M + H]+ Calcd for C20H19Cl2N2O2Te2 646.8906; Found 646.8895.
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1,2-Bis{[3-(4-fluorophenyl)-4,5-dihydroisoxazol-5-yl]methyl}ditellane (7e).
Yield: 78 mg (51%); light yellow solid; mp 120-122 °C. 1H NMR (CDCl3, 400 MHz)
δ (ppm) 7.65 - 7.62 (m, 2H); 7.10 - 7.05 (m, 2H); 5.00 - 4.91 (m, 1H); 3.64 - 3.57
(m, 1H); 3.50 - 3.43 (m, 2H); 3.16 - 3.10 (m, 1H). 13C{1H} NMR (CDCl3, 100 MHz)
δ (ppm) 163.8 (d, 1J = 251.0 Hz), 155.4, 128.6 (d, 2J = 8.5 Hz), 125.7 (d, 4J = 3.4
Hz), 115.9 (d, 3J = 22.0 Hz), 83.0, 83.0, 41.1, 9.8, 9.7. 19F NMR (CDCl3, 376 MHz)
δ (ppm) -109.60 -109.67 (m). 125Te NMR decoupled (CDCl3, 126 MHz) δ (ppm)
101.6 (t, J = 32.4 Hz), 100.0 (t, J = 32.4 Hz). HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C20H19F2N2O2Te2 612.9505; Found 612.9509.
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1,2-Bis[(3-phenyl-4,5-dihydroisoxazol-5-yl)methyl]diselane (7f). Yield: 57 mg
1
(48%), white solid; mp 122 - 124°C. H NMR (CDCl3, 400 MHz) δ (ppm) 7.66 -
7.63 (m, 2H), 7.41-7.37 (m, 3H), 5.06 - 4.97 (m, 1H), 3.51 - 3.42 (m, 1H), 3.25 -
3.19 (m, 1H), 3.04 - 2.92 (m, 2H). 13C{1H} NMR (CDCl3, 100 MHz) δ (ppm) 156.5,
130.2, 130.2, 129.4, 128.8, 126.8, 126.7, 81.0, 80.7, 40.4, 40.4, 28.7, 28.6.
HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C20H21N2O2Se2 478.9942; Found
478.9948.
Bioassays: Animals experimental and drugs
The experiments were conducted using male adult Swiss mice (25-35 g). The
animals were kept in a separate animal room, in a 12 h light/dark cycle (with lights
on at 7:00 a.m.), at a room temperature of 22 ± 2 °C, with free access to food and
water. All procedures were approved by the Committee on Care and Use of
Experimental Animal of Federal University of Pelotas, Brazil (CEEA1987). All
efforts were made to minimize animal suffering and to reduce the number of
animals used in the experiments. PPSEI45 (3a) was dissolved in canola oil, while
Celecoxib was dissolved in 0.9 % saline solution. The mice received oral
administration (p.o., by intragastric gavage) in a constant volume of 10 mL/kg
body weight. Celecoxib was obtained commercially, and it was used as reference
drug. All other chemicals were of analytical grade and obtained from standard
commercial suppliers.
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Ear edema induced by croton oil.
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Ear edema was induced by croton oil based on previous studies with some
modifications.39,40 Mice were treated with compound 3a (50 mg/kg, p.o.),
Celecoxib (50 mg/kg and 100mg/kg, p.o.) or vehicle, before the topical application
of 2.5 % croton oil in acetone (20 µL/ear) in the right ear of each mouse. The
identical volume (20 µL) of acetone was applied to the left ear. Four hours after
the application, animals were euthanized and both ears were cut at the base and
the weight difference between the samples of the control ear (left) and the croton
oil-treated ear (right) was established on an analytical balance.41 The results
obtained were represented in weight (mg). Subsequently, ears were used to
determine the myeloperoxidase (MPO) activity.
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MPO assay.
The MPO activity was evaluated according to the method previously proposed
with some modifications.31 The samples of ears were homogenized in potassium
phosphate
buffer
(20
mmol/L,
pH
7.4;
1:5,
w/v)
containing
ethylenediaminetetraacetic acid (0.1 mmol/L). After this process, samples were
subjected to ultrasonic bath and then frozen and thawed three times and
centrifuged at 2.500 rotations per minute (rpm) at 4 °C for 10 min to yield a low
speed supernatant fraction. Then, the supernatant fractions were centrifuged
again at 14.000 rpm at 4 °C for 15 min to yield a final pellet that was resuspended
in medium containing potassium phosphate buffer (50 mmol/L, pH 6.0) and
hexadecyltrimethyl ammonium bromide (0.5%). For the enzyme assay, an aliquot
of final pellet resuspended (100 μL) was added to a medium containing the
medium of resuspension and N,N,N′,N′-tetramethylbenzidine (1.5 mmol/L). The
kinetic analysis of MPO was started after H2O2 (0.01%) addition and results were
expressed as optic density (OD)/mg protein/min.
Acute toxicity
To examine the potential acute toxicity caused by 3a, mice received a single oral
dose of the compound (300 mg/kg) or vehicle (canola oil). After drug
administration, animals were observed for up to 72 h to determine the lethal
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potential of this compound. After this time of exposure, mice were anesthetized
for a heart puncture through which blood was collected in tubes containing
heparin. Plasma was obtained by centrifugation at 900×g for 10 min and used for
biochemical assays, which were performed with commercial test kits. Plasma
aspartate (AST) and alanine (ALT) aminotransferase activities, used as the
biochemical markers for early acute hepatic damage were determined by
colorimetric method.42 Here, AST activity was also used as marker of cardiac
damage. The values were expressed as U/L. Renal function was analyzed by
determining plasma urea levels.33 The values were expressed as mg/dL.
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Samples of liver, kidney and brain were also collected to determine the δ-
aminolevulinate dehydratase (δ-ALA-D) activity, a marker of oxidative damage.43
Samples were homogenized in 50mM Tris/HCl pH 7.5, (1:10, w/v) and
centrifuged at 900×g for 10 min at 4 °C to yield a supernatant (S1). δ-ALA-D
activity was assayed by the method of Sassa.44 An aliquot of S1 was pre-
incubated for 10 min at 37 °C. The enzymatic reaction was initiated by adding the
substrate (δ-ALA) to a final concentration of 2.2mM in a medium containing 45
mM phosphate buffer, pH 6.8 and incubated for 1 h at 37 °C. The incubation was
stopped by adding 10% trichloroacetic acid (TCA) solution with 10mM HgCl2. The
reaction product porphobilinogen (PBG) was measured at 555 nm using modified
Ehrlich's reagent. The values were expressed as nmol PBG/mg protein/h. The
protein concentration was measured by the method of Bradford,45 using bovine
serum albumin as the standard.
Conflicts of Interest
There are no conflicts to declare.
Associated Content
13
Support Information: Contains the full spectroscopy (1H, C{1H} NMR) data for
all synthesized compounds.
Acknowledgements
The authors are grateful to CNPq, FAPERGS and FINEP for the financial support.
This study was financed in part by the Coordenação de Aperfeiçoamento de
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Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. CNPq is also
acknowledged for Fellowships to E.W., C.L., D.A. and E.J.L..
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Notes and References
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