Bis-vinyl selenides obtained via iron(iii) catalyzed addition of PhSeSePh to alkynes: Synthesis and antinociceptive activity

Article abstract of DOI:10.1039/c2ob27064a

In the present study the synthesis and antinociceptive activity of bis-vinyl selenides, prepared via FeCl₃ promoted reaction addition of diorganyl dichalcogenides to alkynes, is described. The pharmacological results demonstrated that bis-vinyl selenides 3a, 3d, 3h and 3t elicited antinociceptive effect in the mouse formalin test. The antinociceptive effects of bis-vinyl selenides are not sensitive to electronic effects of the substituents on the aromatic ring directly bonded to the selenium atom. Bis-vinyl selenides 3h and 3t were the most promising molecules for pharmacological purposes since these bis-vinyl selenides were effective in both phases of the formalin test and against edema. A single dose of bis-vinyl selenides 3a, 3d, 3h and 3t did not cause acute toxicity in mice.

Full text of DOI:10.1039/c2ob27064a

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Bis-vinyl Selenides Obtained via Iron(III) Catalyzed Addition of
PhSeSePh to Alkynes: Synthesis and Antinociceptive Activity
Glaubia Sartori,a José S. S. Neto,a Ana Paula Pesarico,a Davi F. Back,b Cristina W. Nogueiraa and Gilson
Zenia,*
5
Received (in XXX, XXX) Xth XXXXXXXXX 201X, Accepted Xth XXXXXXXXX 201X
First published on the web Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
Abstract:
10 In the present study the synthesis and antinociceptive activity of bisꢀvinyl selenides, prepared via FeCl3 promoted reaction
addition of diorganyl dichalcogenides to alkynes, is described. The pharmacological results demonstrated that bisꢀvinyl
selenides 3a, 3d, 3h and 3t elicited antinociceptive effect in the mouse formalin test. The antinociceptive effects of bisꢀvinyl
selenides are not sensitive to electronic effects of the substituents on the aromatic ring directly bonded to the selenium atom.
Bisꢀvinyl selenides 3h and 3t were the most promising molecules for a pharmacological purposes since these bisꢀvinyl selenides
15 were effective in both phases of the formalin test and against the edema.A single dose of bisꢀvinyl selenides 3a, 3d, 3h and 3t
did not cause acute toxicity in mice.
alkynes.10 For example, the nucleophilic addition of
chalcogenolate anions to alkynes affords, preferentially, the Zꢀ
55 vinylic chalcogenides after longer reaction times at room
Introduction
Pain is one of the most prevalent, costly and disabling conditions,
20 which reduces quality of life. The major problem in pain
management is that effective analgesics may have some
unpleasant sideꢀeffects. Based on these considerations, research
leading to the development of new analgesics is of public health
temperature. The reaction is faster at a high temperature;
however, the mixture of Z and E vinylic chalcogenide is obtained
in an almost 1:1 ratio.11 On the other hand, the eletrophilic
addition of organochalcogenyl halides to alkynes gave a mixture
60 of Markownikov and antiꢀMarkownikov adducts, depending on
the nature of the substituents at the triple bond.12 Conversely,
vinylic chalcogenides can be prepared by transitionꢀmetalꢀ
catalyzed cleavage of selenium–selenium bond and addition to
alkynes.13 These wellꢀknown methods, however, suffer from
65 several drawbacks such as the use of extremely strong reducing
agents, expensive transitionꢀmetal reagents, low stereoselectivity,
and mainly the lack of environmental friendliness. Realizing the
importance in the development of environmentally benign
protocols to the preparation of vinyl chalcogenides applying
70 green, mild and relatively cheaper methods, this study aimed at
the preparation of bisꢀvinyl selenides 3 via reaction of alkynes 1
with diorganyl dichalcogenides promoted by iron salts (Scheme
1). The pharmacological potential of bisꢀvinyl selenides was
investigated by screening the antinociceptive activity in mice.
interest.
Under this point of view, organoselenium and
reported as
25 organotellurium compounds have been
pharmacological agents.1 Among organoselenium compounds, a
class of bis and tris selenide alkene derivatives has attracted some
attention. The interest in the chemistry of bisꢀvinyl selenides has
increased remarkably due to their chemical properties and
30 pharmaceutical potential.2 Bis selenide alkene derivatives are
known as effective antioxidants. 2,3ꢀBis(4ꢀchlorophenylselanyl
propꢀ2ꢀenꢀ1ꢀol) is an effective antinoceptive agent in different
chemical3 and thermal4 models of pain in mice.
The
antidepressantꢀlike5 and neuroprotective6 actions of 2,3ꢀbis(4ꢀ
35 chlorophenylselanyl propꢀ2ꢀenꢀ1ꢀol) in rodents has been also
reported. Recently, much attention has been devoted to the
synthesis of vinylic chalcogenides and their derivatives since they
emerged as highly valuable intermediates in a variety of synthetic
applications.7 There are several reasons for this, which include a
40 widely varied synthetic organochemical potential and the fact that
the chalcogen atom exercises a stabilizing effect on neighbouring
positive as well as negative charges. This makes the double bond
in vinylic chalcogenides responsive toward both nucleophilic and
electrophilic attack, an extremely useful feature for organic
45 synthetic purposes. In this sense, metalꢀcatalyzed crossꢀcoupling8
and transmetalation reactions9 have shown to be the most reliable
strategy for the formation of a new carbonꢀcarbon bond since the
method allows the reaction to proceed under very mild conditions
and with very high stereoselectivities. Although various methods
50 are reported for the preparation of vinylic chalcogenides, the
more useful procedure has centred on the nucleophilic or
electrophilic organochalcogen addition to terminal or internal
75
Scheme 1: General scheme.
Chemistry
Our preliminary studies started with the determination of the
influence of reaction atmosphere. Thus, a mixture of diphenyl
80 diselenide (0.25 mmol) and FeCl3 (1 equiv), using MeNO2 as
solvent, was reacted with diphenylacetylene 1a (1 equiv) at room
temperature. A comparison of reaction runs using rigorously
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15
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
DMF
CH3CN
dioxane
THF
ꢀc
57
9
dried systems (Table 1, entry 1) versus those run in an ambient
atmosphere (Table 1, entry 2) showed that the yield was slightly
higher in the latter case. To better understand if the activity of
the iron system is due to the different ligand partner type on the
iron complex or to the effect of dry iron salt, some experiments
were also carried out using other iron sources and a very sluggish
reactivity was observed (Table 1, entries 3ꢀ6). In an effort to
improve on these conversions and subsequent isolated yields and
to reduce the catalyst loading, we turned our attention to the use
10 of 0.5, 0.25 and 0.1 equiv (10 mol%) of FeCl3. We were pleased
to observe that FeCl3 (10 mol%) in MeNO2 efficiently catalyzed
the formation of 3a in an excellent isolated yield (Table 1, entries
7ꢀ9). These results are significant since it suggests that the two
portions of diphenyl diselenide (PhSe) were incorporated in the
15 final product, the reaction was carried out at ambient atmosphere
and required only 10 mol% of FeCl3, which makes our method
more attractive from an economic standpoint, and indicates an
atom economy which is an important concept of green chemistry
philosophy. Next, we evaluated various solvents as reaction
20 media for the ironꢀcatalyzed addition of diphenyl diselenide to
16
17
5
18
ꢀc
19d
20e
21f
CH3NO2
CH3NO2
CH3NO2
20
79
73
[a] Yields for isolated products. [b] Reaction was carried out at
argon atmosphere. [c] 1a was recovered quantitatively. [d] Reaction
carried using reflux temperature of CH3NO2. [e] Reaction was
performed in 2 mL of solvent. [f] Reaction was performed in 5 mL
of solvent.
35
The results described above suggested that the reaction of
diphenyl diselenide 2a with substrate 1a gave the desired bisꢀ
vinyl selenides 3a in better yields when the reaction was carried
alkynes.
dichloroethane,
A
set of solvents including dichloromethane,
toluene, ethanol, dimethyl sulfoxide,
40 out using a 1:1 substrate stoichiometry, FeCl3 (10 mol%), in
nitromethane (1 mL) at room temperature for 12 h, under ambient
atmosphere. On the basis of these results, we undertook a
systematic study applying the conditions of entry 9, table 1, to
several substituted alkynes 1aꢀg in order to test the tolerance of
45 functional groups as well as their effects on the conversion.
These results are collected in table 2. The yields of bisꢀvinyl
selenides 3 varied depending on the nature of diorganoyl
diselenide used. While diaryl diselenides bearing a neutral,
electronꢀdonating or electronꢀwithdrawing group on the aromatic
50 ring provided the best yields of 3 (Table 2, entries 1ꢀ8), the
presence of the alkyl or bulky naphthyl group directly bonded to
the selenium atom, decreased the yield to 58 and 51%
respectively (Table 2, entries 9ꢀ10). The results also indicated
that the reaction seems not to be sensitive to electronic effects of
55 the substituents in the aromatic ring directly bonded to the triple
bond. For example, alkynes having the aromatic ring substituted
by Cl or MeO group gave good yields (Table 2, entries 11ꢀ18).
Furthermore, we found that the reaction with substrate 1f having
an alkyl substituent directly bonded to the triple bond and the
60 terminal alkyne 1g also worked well under the standard reaction
conditions; although the terminal alkyne gave the product in
moderated 60% yield (Table 2, entries 19ꢀ20). Moreover, the
reaction of diorganyl disulfides with alkynes using the standard
reaction conditions yielded the addition product only in
65 moderated yields, while the use of diorganyl ditellurides did not
give any amount of desired bisꢀvinyl tellurides (Table 2, entries
21ꢀ25).
dimethylformamide, acetonitrile, dioxane and tetrahydrofuran
were examined, and nitromethane gave the best results as solvent,
25 although dichloromethane, dichloroethane and toluene also gave
the products in acceptable yields (Table 1, entries 10ꢀ18).
Finally, neither running the reaction at solvent reflux (Table 1,
entry 19) nor changing the concentration (Table 1, entries 20ꢀ21)
gave better yields than those obtained in table 1, entry 9.
30 Table 1: Influence of reaction conditions in the formation of 3a
entry
1
[Fe] (equiv)
FeCl3 (1)
solvent
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH2Cl2
DCE
yield (%)a
73b
89
45
74
ꢀc
2
FeCl3 (1)
3
FeCl2.4H2O (1)
FeCl2.6H2O (1)
Fe° (1.0)
4
5
6
Fe(acac)3 (1.0)
FeCl3 (0.5)
FeCl3 (0.25)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
FeCl3 (0.1)
ꢀc
7
88
89
92
77
79
74
ꢀc
8
Table 2: Synthesis of Eꢀbisꢀvinyl chalcogenides 3a
9
10
11
12
13
14
Entry
R
R1Y
Product
(yield)b
toluene
EtOH
1
C6H5
C6H5Se
1a
2a
3aꢀ92%
DMSO
ꢀc
2
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2
3
1a
1a
pꢀMeOC6H4Se
2b
13
14
15
16
17
18
1b
2f
2a
2a
2d
2f
3bꢀ60%
3mꢀ78%
oꢀMeOC6H4Se
2c
oꢀClC6H4
1c
3nꢀ78%
3oꢀ72%
3p-63%
3qꢀ76%
3rꢀ62%
3cꢀ71%
3dꢀ75%
pꢀMeOC6H4
4
5
1a
1a
pꢀMeC6H4Se
1d
2d
1d
1d
Me
oꢀMeC6H4Se
2e
Se
Ph
Ph
Se
Me
3eꢀ70%
3f-64%
3g-75%
6
7
8
1a
1a
1a
pꢀClC6H4Se
2f
mꢀMeOC6H4
2a
1e
pꢀFC6H4Se
2g
19
20
nꢀC5H11
1f
2a
2a
F3C
mꢀCF3C6H4Se
3sꢀ85%
3tꢀ60%
2h
H
1g
Se
Ph
Ph
Se
CF3
3hꢀ81%
3iꢀ58%
21
22
23
1a
1e
1g
C6H5S
2k
S
Ph
S
9
1a
1a
nꢀBuSe
2i
Ph
3uꢀ62%
10
1ꢀnaphthylSe
2k
2k
2j
3vꢀ42%
S
H
S
3jꢀ51%
3k-91%
3l-64%
Ph
11
12
pꢀClC6H4
2a
2d
3wꢀ40%
1b
24
25
1a
1a
C6H5Te
2l
nꢀBuTe
ꢀc
ꢀc
2m
1b
[a] Reaction condition: the reaction was carried out using ambient
atmosphere, alkyne (0.5 mmol), diorganoyl dichalcogenides (1.0
equiv), FeCl3 (0.1 equiv) in CH3NO2 (1 mL) at room temperature for
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12 hours. [b] Yields for isolated products. [c] 1a was recovered
quantitatively.
phases of test (p ≤ 0. 0467) (Figure 3). 3a and 3d significantly
decreased licking time in the first phase (Figure 2) but had no
45 effect in the second phase of the formalin test (Figure 3).
Based on understanding that in the ironꢀcatalyzed cross coupling
that iron is easily admitted as a complex having an oxidation state
+3 and that this complex could be stabilized by the selenium
atom,14 we proposed a plausible mechanism to support the current
Fe(III)ꢀcatalyzed selenium addition, as illustrated in Scheme 2.
(i) The iron trichloride coordinates with diorganyl diselenides to
form selenoniumꢀlike specie a; (ii) the alkyne coordination to the
5
10 selenium atom provides the seleniranium ion b and selenolate
specie c; (iii) nucleophilic antiꢀattack of the species c on the
activated triple bond leads to Eꢀvinyl selenides 3. In this context,
in view of the high activation of the triple bond by the selenium
group, our methodology showed to be high stereoselective,
15 providing exclusively the desired Eꢀvinyl selenides 3, via an antiꢀ
addition process, as the unique isomer, which was confirmed by
Xꢀray diffraction analysis (Figure 1). The radical pathway was
not discarded; however, when the reaction of diphenylacetylene
1a with diphenyl diselenide was carried out in the presence of
20 TEMPO, a radical inhibitor, bisꢀvinyl selenide 3a was obtained in
Figure 2. Effect of bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t on
licking behavior induced by formalin in the first (0–5 min) phase
of the formalin test. Each column represents the mean ± S.E.M.
50 of 5–7 mice in each group. Asterisks denote significance levels
when compared to the control group (oneꢀway ANOVA followed
by the Duncan’s test) (*) p < 0.05; ** p <0.01 and *** p <0.001.
C means control, a group of mice treated with canola oil.
Standard error (S.E.M.) of all groups represents <10% of means.
87% yield. This result indicates that the pathway does not follow
15
the typical radical addition, where the radical source is PhSe .
•
Scheme 2: Plausible mechanism.
55
Figure 3. Effect of bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t on
licking behavior induced by formalin in the second (15–30 min)
phase of the formalin test. Each column represents the mean ±
S.E.M. of 5–7 mice in each group. Asterisks denote significance
60 levels when compared to the control group (oneꢀway ANOVA
followed by the Duncan’s test) (*) p < 0.05. C means control, a
group of mice treated with canola oil. Standard error (SEM) of all
groups represents <20% of means.
25
Figure 1: ORTEP view of the compound 3h.
Administration of morphine (2.5 mg/kg, i.g.) as a positive control
65 was effective in decreasing the time that mice spent licking the
hind paw in both phases of the formalin test (data not shown).
Studies have demonstrated that the antinociceptive action of other
organoselenium compounds, such as diphenyl diselenide and bisꢀ
selenide derivatives, remain significant up to 2h and 45 min,
70 respectively17 and that morphine, diacetylmorphine and 6ꢀ
acetylmorphine, known analgesic drugs, have approximately
2.5h, 1.8h and 1.7h of the effect duration, respectively.18
The hindpaw edema from animals was assessed to further
confirm the antiꢀinflammatory activity of bisꢀvinyl selenides.
75 Figure 4 shows that only 3h and 3t, administered to mice 2h
before the formalin test, significantly reduced (about 25%) paw
edema induced by intraplantar injection of formalin.
Accordingly, the antiꢀedematogenic effect of 3h and 3t could be
associated to the reduction of nociception by these bisꢀvinyl
80 selenides in the inflammatory phase (second phase) of the
Results and Discussion
In an attempt to investigate the potential antinociceptive and antiꢀ
inflammatory effects of bisꢀvinyl selenides, the formalin test was
30 carried out. The formalin test consists in two phases: the first
phase, also called neurogenic phase, which results basically from
the direct activation of nociceptors, and the second phase, which
involves a period of sensitization during which an inflammatory
phenomena occurs.16
35 The majority of bisꢀvinyl selenides tested exhibited effect on the
first phase of the formalin test (p<0.05), except for one
compound which is pꢀchloro substituted (3f, Figure 2). At 2 h of
preꢀtreatment, bisꢀvinyl selenides 3h at a dose equal to 50 mg/kg
(i.g) decreased the time that mice spent licking the hind paw in
40 the first (p ≤ 0.011837) (Figure 2) and second phases (p ≤0.0029)
(Figure 3) of the formalin test. Bisꢀvinyl selenide 3t elicited
similar effects of 3h in first (p ≤ 0.009658) (Figure 2) and second
4
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formalin test. All the other bisꢀvinyl selenides tested, 3a, 3d and 50 nonspecific locomotor disturbances in mice (see supporting
3f, had no antiꢀedematogenic effect in mice.
information, Table 2). The results obtained with the bisꢀvinyl
selenide substituted with the chlorine group in the aromatic ring
bonded to the selenium atom 3f indicated minor hepatic toxicity,
demonstrated by an inhibition (about 15%) in hepatic δꢀALAꢀD
55 activity and an increase in AST activity, a biochemical marker of
acute hepatic damage (see supporting information, Table 2).
δꢀALAꢀD is a sulfhydrylꢀcontaining enzyme, which is extremely
sensitive to oxidizing agents and to situations associated with
oxidative stress.19 Similar to δꢀALAꢀD, Na+, K+ꢀATPase is a
60 sulfhydryl enzyme sensitive to oxidizing agents.20 However, none
of bisꢀvinyl selenides tested inhibited the Na+, K+ꢀATPase
activity even the compound 3f (see supporting information,
Table 2).
Conclusion
5
Figure 4. Effect of bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t on
edemaꢀinduced by formalin in mice. Each column represents the
mean ± S.E.M. of 5–7 mice in each group. Asterisks denote
significance levels when compared to the control group (oneꢀway
65
In summary, we have explored the addition reaction
behavior of of diorganyl dichalcogenides to alkynes in
presence of catalytic amount of FeCl3 and established a
steroselectivity route to obtain highly substituted bisꢀvinyl
chalcogenides. The reaction works well with a wide range of
ANOVA followed by the
Duncan’s test) *** P <0.001. C
70 substituents in both diorganyl dichalcogenides and alkynes,
generally resulting in good yields, and proceed under
relatively mild conditions. Furthermore, since the reactions
was carried out at room temperature, under ambient
atmosphere and iron salts are easily available commercially,
75 less expensive and relative nonꢀtoxic our method could be
considered an economic and ecoꢀfriendly protocol. The
pharmacological results demonstrated that bisꢀvinyl selenides
3a, 3d, 3h and 3t elicited antinociceptive effect in the mouse
10 means control, a group of mice treated with canola oil. Standard
error (SEM) of all groups represents <10% of means.
Taking these results together it is possible to infer that bisꢀvinyl
selenides 3h and 3t are the most promising molecules for a
pharmacological propose since these bisꢀvinyl selenides were
15 effective in both phases of the formalin test and against the
edema.
Evidence has been found to suggest that the
antinociceptive effects of bisꢀvinyl selenides are not sensitive to
electronic effects of the substituents on the aromatic ring directly
bonded to the selenium atom. A inspection of bisꢀvinyl selenides
20 tested revealed that compounds 3a, 3d, 3h and 3t showed
antinociceptive effects despite the differences of the substituents
on the aromatic ring directly bonded to the selenium atom. While
3h is a bisꢀvinyl selenide with a CF3 group substituted in the
aromatic ring directly bonded to the selenium atom, 3a has a
25 methyl group substituted in the aromatic ring directly bonded to
the selenium atom and 3d has no substituent in the aromatic ring,
ruling out the possibility of a structureꢀactivity relationship. In
support of this idea are the results obtained with 3t, which has the
hydrogen atom in the place of the aromatic ring bonded to the
30 vinyl portion of the molecule.
In addition, antinociceptive effects of bisꢀvinyl selenides (50
mg/kg, i.g.) at other times of preꢀtreatment (30 and 60 min) were
demonstrated (see supporting information, Table 1). Bisꢀvinyl
selenides 3t and 3a administered 30 and 60 min before the test
35 decreased the time that mouse spent licking hind paw in the first
phase. By contrast, only 3t showed the same effect in the second
phase at 60 min of preꢀtreatment (p ≤0.05). Interestingly, 3h and
3d preꢀadministered at 30 and 60 min before the test diminished
the time that mouse spent licking hind paw in the second phase.
40 3h, but not 3d, administered 30 min before the test to mice
elicited the antinociceptive effect in the first phase of the formalin
test (p<0.05) (see supporting information, Table 1). Moreover,
bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t administered to mice at
30 or 60 min before the formalin test had no antiꢀedematogenic
45 (see supporting information, Table 1).
formalin test.
The antinociceptive effects of bisꢀvinyl
80 selenides were not sensitive to electronic effects of the
substituents on the aromatic ring directly bonded to the
selenium atom. Bisꢀvinyl selenides 3h and 3t were the most
promising molecules for a pharmacological propose since
these bisꢀvinyl selenides were effective in both phases of the
85 formalin test and against the edema. Bisꢀvinyl selenide 3f
has no anitinociceptive effect in mice. A single dose of bisꢀ
vinyl selenides 3a, 3d, 3h and 3t did not cause acute toxicity
in mice.
Experimental Section
90
Proton nuclear magnetic resonance spectra (1H NMR) were
obtained at 200 MHz on a DPXꢀ200 NMR spectrometer or at 400
MHz on a DPXꢀ400 NMR spectrometer. Spectra were recorded
in CDCl3 solutions. Chemical shifts are reported in ppm,
referenced to the solvent peak of CDCl3 or tetramethylsilane
95 (TMS) as the external reference. Data are reported as follows:
chemical shift (δ), multiplicity, coupling constant (J) in Hertz and
integrated intensity. Carbonꢀ13 nuclear magnetic resonance
spectra (13C NMR) were obtained either at 50 MHz on a DPXꢀ
200 NMR spectrometer or at 100 MHz on a DPXꢀ400 NMR
100 spectrometer. Spectra were recorded in CDCl3 solutions. All
synthesized and tested compounds were obtained in purity
superior to 98% determined by combustion analysis, HPLC and
gas chromatography.
105 General Procedure for Iron-Promoted Addition of
Diorganoyl Dichalcogenides to Alkynes: To a Schlenk tube,
Concerning the possible toxicity caused by bisꢀvinyl selenides,
the potential acute toxicity and the locomotor activity after a
single oral dose of 3a, 3d, 3f, 3h and 3t were investigated. Most
of the bisꢀvinyl selenides showed neither acute toxicity nor
under ambient atmosphere, containing
a mixture of the
appropriate diorganoyl dichalcogenide (0.55 equiv) in CH3NO2
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(0.5 mL) was added, at room temperature, the FeCl3 (0.1 equiv,
10 mol%). The resulting solution was stirred for 15 min at this
temperature. After that the corresponding alkyne (0.5 mmol) in
CH3NO2 (0.5 mL) was added and resulting solution was stirred
under reflux for 12 hours. The reaction mixture was diluted with
ethyl acetate (20 mL) and washed with a saturated solution of
NH4Cl (3 x 10 mL). The organic phase was separated, dried over
MgSO4, and concentrated under vacuum. The residue was
purified by flash chromatography eluted with hexane/acetate
m/z: 520 (7), 349 (16), 281 (27), 253 (28), 107 (100), 178 (80),
152 (19), 133 (17), 91 (66), 73 (40). Anal. (%) calcd. for
C28H24Se2: C 64.87, H 4.67. Found: C 64.93, H 4.71.
5
55 (E)-1,2-Bis(4-chlorophenylselenyl)-1,2-diphenylethene
(3f):
1
Yield: 0.179 g (65%). Yellow solid. mp = 178ꢀ181 °C. H RMN
(CDCl3, 200 MHz), δ (ppm): 7,23ꢀ7,13 (m, 10H), 7,08 (d, J = 8,3
Hz, 4H), 6,96 (d, J = 8,6 Hz, 4 H). 13C RMN (CDCl3, 100
MHz), δ (ppm):140,2, 136,3, 133,9, 133,1, 129,4, 129,1, 128,1,
60 129,9, 127,7. MS (relative intensity) m/z: 553 (5), 348 (15), 281
927), 253 (21), 214 (12), 207 (100), 176 (28), 151 (10), 96 (3), 91
(54), 73 (40). Anal. HRMS calcd. for C26H18Cl2Se2 (M+Na+):
559.9113. Found: 559.9141.
10 (95:5).
(E)-1,2-Diphenyl-1,2-bis(phenylselenyl)ethene (3a): Yield:
0.226 g (92%). White solid. mp = 155ꢀ158 °C. 1H RMN (CDCl3,
400 MHz), δ (ppm):7.24ꢀ7.21 (m, 4H), 7.19ꢀ7.03 (m, 12H), 7.01ꢀ
6.96 ( m, 4H). RMN 13C (CDCl3, 100 MHz), δ (ppm): 140.5,
15 135.0, 133.1, 130.0, 129.5, 128.3, 127.6, 127.4, 102.3. MS
(relative intensity) m/z: 492 (13), 335 (25), 254 (27), 178 (100),
152 (19), 77 (10). Anal. (%) calcd. for C26H20Se2: C 63.68, H
4.110. Found: C 63.74, H 4.18.
(E)-1,2-Bis(4-fluorophenylselenyl)-1,2-diphenylethene
65 Yield: 0.198 g (75%). White solid. mp = 134ꢀ137 °C. H RMN
(3g):
1
(CDCl3, 400 MHz), δ (ppm): 7.22ꢀ7.01( m, 14H), 6.71ꢀ6.65 (m,
1
4H), 13C RMN (CDCl3, 100 MHz), δ (ppm): 162.5 (d, J = 248
3
Hz), 140.2, 135.5 (d, J = 8.7 Hz), 132.7 ,128.4, 127.8, 127.6,
4
124.5 (d, J = 3.6 Hz), 115.5 (d, 2J = 21.9 Hz). MS (relative
(E)-1,2-Bis(4-methoxyphenylselenyl)-1,2-diphenylethene(3b):
1
20 Yield: 0.165 g (60%). Yellow solid. mp = 164ꢀ167 °C. H RMN
70 intensity) m/z: 528 (8), 353 (20), 281 (4), 253 (28), 207 (100),
178 (80), 152 (19), 133 917), 91 (66), 73 (40). Anal. (%) calcd.
for C26H18F2Se2: C 59.33, H 3.45. Found: C 59.48, H 3.51.
(CDCl3, 400 MHz), δ (ppm): 7.20ꢀ7.11 ( m, 10H), 7.08ꢀ7.05 (m,
4H), 6.54ꢀ6.49 (m, 4H), 3.68 (s, 6H). 13C RMN (CDCl3, 50
MHz), δ (ppm): 159.9, 140.5, 137.2, 132.5, 129.4, 127.7, 127.2,
120.3, 113.9, 55.1. MS (relative intensity) m/z: 552 (9), 373 (7),
25 365 (25), 281 (30), 253 (25), 207 (100), 186 (58), 178 (75), 133
(17), 77 99). Anal. HRMS calcd. for C28H24O2Se2 (M+Na+):
552.0107. Found: 552.0113.
(E)-1,2-Diphenyl-1,2-bis(3-
(trifluoromethyl)phenylselenyl)ethene (3h): Yield: 0.255 g
75 (81%). White solid. mp = 128ꢀ131 °C. 1H RMN (CDCl3, 400
MHz), δ (ppm): 7.39ꢀ7.35 (m, 4H), 7.32ꢀ7. 29 (m, 2H), 7.25ꢀ7.08
(m, 12H). 13C RMN (CDCl3, 100 MHz), δ (ppm): 139.8, 138.1,
133.0, 131.8 (q, J = 4.4 Hz), 131.0, 130.7(q, J = 32.2 Hz), 129.4,
128.6, 128.0, 127.9, 124.2 (q, J =3.6 Hz), 123.5 (q, J = 272.2 Hz).
80 MS (relative intensity) m/z:628 (3), 625 (7), 403 (16), 363 (9),
322 (8), 178 (100), 152 (13), 126 (34). Anal. HRMS calcd. for
C28H18F6Se2 (M+Na+): 697.9643. Found: 697.9758.
(E)-1,2-Bis(2-methoxyphenylselenyl)-1,2-diphenylethene (3c):
1
Yield: 0.196 g (71%). White solid. mp = 156ꢀ159 °C. H RMN
30 (CDCl3, 200 MHz), δ (ppm): 7.37ꢀ7.25 (m, 6H), 7.16ꢀ7.07 (m,
8H), 6.71ꢀ6.63 (m, 2H), 6.58ꢀ6.53 (m, 2H), 3.69 (s, 6H). 13C
RMN (CDCl3, 50 MHz), δ (ppm):158.2, 141.2, 135.5, 133.5,
129.4, 129.0, 128.9, 127.3, 127.2, 120.7, 119.9, 110.2, 55.5. MS
(relative intensity) m/z: 551 (17), 365 (29), 284 (14), 207 (21),
35 178 (100), 159 (17), 107 (36), 77 (33). Anal. HRMS calcd. for
C28H24O2Se2 (M+Na+): 551.0107. Found: 552.0117.
(E)-1,2-Bis(butylselenyl)-1,2-diphenylethene (3i): Yield: 0.132
1
g (58%). Yellow solid. mp = 80ꢀ83 °C. H RMN (CDCl3, 400
85 MHz), δ (ppm): 7.41ꢀ7.35 (m, 8H), 7.32ꢀ7.28 (m, 2H), 2.14 (qui,
J = 7.3 Hz, 4H), 1.33 (qui, J = 7.6 Hz, 4H), 1.12 (sex, J = 7.6 Hz,
4H), 0.71 (t, J = 7.6 Hz, 6H). 13C RMN (CDCl3, 100 MHz), δ
(ppm): 141.1, 130.6, 129.5, 128.1, 127.5, 32.3, 27.0, 22.7, 13.4.
MS (relative intensity) m/z: 352 (10), 304 (23), 228 (21), 207
90 (53), 178 (84), 152 (18), 91 (5), 55 (14). Anal. HRMS calcd. for
C22H28Se2 (M+Na+): 452.0521. Found: 452.0530.
(E)-1,2-Diphenyl-1,2-bis(p-tolylselenyl)ethene (3d): Yield:
0.195 g (75%). Yellow solid. mp = 133ꢀ136 °C. 1H RMN
(CDCl3, 200 MHz), δ (ppm): 7.32ꢀ7.08 (m, 10H), 7.05 (d, J = 8.1
40 Hz, 4H), 6.79 (d, J = 7.8 Hz, 4H), 2.17 (s, 6H). 13C RMN (CDCl3,
100 MHz), δ (ppm): 140.8, 137.3, 135.1, 133.0, 125.5, 129.1,
129.6, 127.3, 126.4, 21.0. MS (relative intensity) m/z: 518 (16),
353 (7), 372 (16), 353 (12), 207 (28), 178 (100), 152 (22), 133
(5), 76 (4). Anal. (%) calcd. for C28H24Se2: C 64.87, H 4.67.
45 Found: C 64.96, H 4.72.
(E)-1,2-Bis(naphthalen-1-ylselenyl)-1,2-diphenylethene (3j):
1
Yield: 0.151 g (51%). White solid. mp = 167ꢀ170 °C. H RMN
(CDCl3, 200 MHz), δ (ppm): 8.19 ꢀ8.12 (m, 2H), 7.71ꢀ7.54 (m,
95 4H), 7.51ꢀ7.38 (m, 6H), 7.12ꢀ6.95 (m, 12H). 13C RMN (CDCl3,
100 MHz), δ (ppm): 150.5, 135.7, 134.9, 133.7, 133.5, 129.5,
129.1, 129.0, 128.4, 128.2, 127.2, 126.3, 125.8, 125.1. MS
(relative intensity) m/z: 392 (5), 304 (23), 228 (21), 207 (53), 178
(84), 152 (18), 128 (22), 126 (19) 115 (100), 77 (7). Anal. (%)
100 calcd. for C34H24Se2: C 69.16, H 4.10. Found: C 69.28, H 4.17.
(E)-1,2-Diphenyl-1,2-bis(o-tolylselenyl)ethene (3e): Yield:
0.182 g (70%). White solid. mp = 190ꢀ193 °C. 1H RMN (CDCl3,
400 MHz), δ (ppm): 7.25ꢀ7.20 (m, 5H), 7.16ꢀ7.09 (m, 5H), 6.97ꢀ
6.95 (m, 2H), 6.84ꢀ6.80 (m, 2H), 2.23 (s, 6H). 13C RMN (CDCl3,
50 50 MHz), δ (ppm):141.3, 140.9, 136.6, 133.5, 131.0, 129.5,
129.0, 128.1, 127.48, 127.3, 125.8, 22.8. MS (relative intensity)
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 9
Organic & Biomolecular Chemistry
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(Z)-(1-(4-Methoxyphenyl)-2-phenylethene-1,2-diyl)bis(p-
(E)-(1-(4-Chlorophenyl)-2-phenylethene-1,2-
diyl)bis(phenylselene) (3k): Yield: 0.255 g (91%). White solid.
tolylselene) (3p): Yield: 0.173 g (63%). Yellow solid. mp = 121ꢀ
123 °C. 1H RMN (CDCl3, 400 MHz), δ (ppm): 7.24ꢀ7.09 (m,
7H), 7.06ꢀ7.03 (m, 4H), 6.82ꢀ7.78 (m, 4H), 6.719ꢀ6.69 (m, 2H),
1
mp = 80ꢀ83 °C. H RMN (CDCl3, 400 MHz), δ (ppm): 7.22ꢀ7.10
(m, 14H), 6.72ꢀ6.65 (m, 4H). 13C RMN (CDCl3, 50 MHz), δ
5
(ppm): 140.5, 139.2, 135.1, 135.6, 134.7, 134.0, 133.2, 131.4, 55 3.74 (s, 3H), 2.19 (s, 6H). 13C RMN (CDCl3, 50 MHz), δ (ppm):
130.9, 129.9, 129.4, 128.8, 128.6, 128.4, 127.9, 127.7, 127.6. MS
(relative intensity) m/z: 525 (9), 368 (14), 333 (27), 331 (15), 252
(22), 211 (100), 176 (80), 156 (22), 150 (26), 77 (14). Anal. (%)
calcd. for C26H19ClSe2: C 59.60, H 3.65. Found: C 59.62, H 3.69.
158.6, 140.8, 137.3,137.1, 135.2, 137.7, 133.2, 132.5, 130.8,
129.5, 129.2, 129.1, 127.5, 127.2, 126.7, 126.4, 113.0, 55.1, 21.1.
MS (relative intensity) m/z: 550 (14), 348 (13), 379 (52), 364 (9),
298 (17), 208 (100), 193 (78), 165 (79), 139 (10), 91 (35). Anal.
60 (%) calcd. for C29H26OSe2: C 63.51, H 4.78. Found: C 63.74, H
4.82.
10 (E)-(1-(4-Chlorophenyl)-2-phenylethene-1,2-diyl)bis(p-
tolylselene) (3l): Yield: 0.177 g (64%). White solid. mp = 135ꢀ
1
138 °C. H RMN (CDCl3, 200 MHz), δ (ppm): 7.23ꢀ6.99 (m,
(E)-(1-(4-Methoxyphenyl)-2-phenylethene-1,2-diyl)bis((4-
chlorophenyl)selene)(3q): Yield: 0.224 g (76%). Yellow solid.
13H), 6.88ꢀ6.77 (m, 4H), 2.22ꢀ1.14 (m, 6H). 13C RMN (CDCl3,
1
100 MHz), δ (ppm): 140.7, 139.4, 137.7, 137.6, 135.2, 134.1,
mp = 80ꢀ86 °C. H RMN (CDCl3, 400 MHz). δ (ppm): 7.25ꢀ7.05
15 133.0, 131.8, 130.9, 130.8, 129.5, 129.4, 129.2, 128.8, 127.8, 65 (m, 10H), 7.03ꢀ6.92 (m, 4H), 6.83ꢀ6.81 (m, 1H), 6.76ꢀ6.68 (m,
127.7, 127.4, 126.6, 126.2, 21.0 (2C). MS (relative intensity) m/z:
556 (5), 553 (10), 383 (19), 383 (19), 348 (31), 281 (16), 268
(10), 212 (86), 176 (59), 170 (20), 151 (21), 83 (10), 91 (100), 73
(25). Anal. HRMS calcd. for C29H26ClSe2 (M+Na+): 553.9819.
20 Found: 553.9829.
2H), 3.73 (s, 3H). 13C RMN (CDCl3, 50 MHz), δ (ppm): 159.0,
141.3, 140.0, 136.3, 136.2, 133.9, 133.8, 129.3, 128.9, 128.5(2C),
128.1, 128.0, 127.8, 127.7, 121.9, 114.8, 113.5, 55.2. MS
(relative intensity) m/z: 591 (4), 401 (13), 399 (29), 364 (19), 208
70 (100), 193 (60), 165 (64), 139 (9), 73 (11). Anal. HRMS calcd.
for C27H20Cl2OSe2 (M+Na+): 589.9222. Found: 589.9230.
(E)-(1-(4-Chlorophenyl)-2-phenylethene-1,2-diyl)bis((4-
chlorophenyl)selene) (3m): Yield: 0.232 g (78%). White solid.
mp = 131ꢀ139 °C. RMN H (CDCl3, 400 MHz), δ (ppm): 7.23ꢀ
7.14 (m, 9H), 7.01ꢀ7.05 (m, 4H), 7.02ꢀ6.95 (m, 4H). RMN 13C
(E)-(1-(3-Methoxyphenyl)-2-phenylethene-1,2-
1
diyl)bis(phenylselene) (3r): Yield: 0.162 g (62%). White solid.
1
mp = 112ꢀ115 °C. RMN H (CDCl3, 400 MHz), δ (ppm): 7.24ꢀ
25 (CDCl3, 100 MHz), δ (ppm):140.2, 138.8, 136.5, 136.27, 134.6, 75 6.97 (m, 16H), 6.87ꢀ6.85 (m, 1H), 6.72 (s, 1H), 6.68ꢀ6.65 (m,
134.3, 134.3, 133.7, 130.8, 129.4, 129.9, 128.7, 128.2, 127.9,
127.8. MS (relative intensity) m/z: 596 (4), 593 (5), 403 (12), 370
(10), 366 (11), 281 (17), 153 (16), 212 (100), 207 (62), 190 (21),
176 (70), 156 (14), 133 (11), 77 (7). Anal. HRMS calcd. for
30 C26H17Cl3Se2 (M+Na+): 593.8726. Found: 593.8738.
1H), 3.69 (s, 3H). RMN 13C (CDCl3, 100 MHz), δ (ppm):158.9,
141.8, 140.5, 135.1, 134.3, 134.0, 128.7, 127.5, 127.4 (2C),122.0,
114.7, 113.6, 55.2. MS (relative intensity) m/z: 522 (23), 520
(21), 350 (13), 313 (10), 285 (19), 253 (17), 208 (100), 178 (73),
80 165 (65), 152 (14), 77 (23). Anal. HRMS calcd. for C27H22OSe2:
C 62.32, H 4.26. Found: C 62.45, H 4.30.
(E)-(1-(4-Chlorophenyl)-2-phenylethene-1,2-
diyl)bis(phenylselene) (3n): Yield: 0.205 g (78%). White solid.
mp = 115ꢀ118 °C. H RMN (CDCl3, 400 MHz), δ (ppm): 7.36ꢀ
7.15 (m, 10H), 7.12ꢀ6.96 (m, 9H). 13C RMN (CDCl3, 100 MHz),
(E)-(1-Phenylhept-1-ene-1,2-diyl)bis(phenylselene) (3s): Yield:
0.206 g (85%). Yellow oil. 1H RMN (CDCl3, 400 MHz), δ (ppm):
7.48ꢀ7.45 (m, 2H), 7.29ꢀ7.23 (m, 5H), 7.13ꢀ7.05 (m, 8H), 2.64 (t,
1
35 δ (ppm): 139.9, 138.8, 136.6, 134.98, 132.8, 132.7, 131.2, 131.1, 85 J = 7.8 Hz, 2H), 1.57 ( qui, J = 7.6 Hz, 2H), 1.27ꢀ1.19 (m, 4H),
129.7, 129.5, 129.3, 128.7, 128.6, 128.4, 128.3, 128.1, 127.8,
127.7, 127.4, 125.9. MS (relative intensity) m/z: 525 (5), 341
(12), 334 (16), 332 (10), 282 (10), 212 (63), 207 (100), 176 (49),
116 (16), 77 (19). Anal. HRMS calcd. for C26H19ClSe2 (M+Na+):
40 525.9506. Found: 525.9533.
0.85 (t, J = 6.6 Hz, 3H). 13C RMN (CDCl3, 100 MHz), δ (ppm):
141.8, 137.3, 134.6, 134.2, 131.3, 130.5, 130.3, 129.5, 129.9,
128.6, 127.6, 127.5, 127.2, 127.1, 37.7, 31.3, 29.0, 22.4, 13.9. MS
(relative intensity) m/z: 488 (4), 486 (13), 258 (35), 207(17), 178
90 (100), 156 (17), 143 (40), 129 (48), 115 (78), 91 (64), 71 (22), 65
(6), 55 (7), 51 (6). Anal. HRMS calcd. for C25H26Se2: C
486.0365. Found: 486.0378.
(E)-(1-(4-Methoxyphenyl)-2-phenylethene-1,2-
diyl)bis(phenylselene) (3o): Yield: 0. 188 g (72%). Yellow solid.
1
mp = 52ꢀ55 °C. H RMN (CDCl3, 400 MHz), δ (ppm): 7.28ꢀ6.94
(E)-(1-Phenylethene-1,2-diyl)bis(phenylselene) (3t): Yield:
0.124 g (60%). Yellow oil. 1H RMN (CDCl3, 400 MHz), δ (ppm):
(m, 16 H), 6.9ꢀ6.84 (m, 1H), 6.76ꢀ6.63 (m, 2H). 13C RMN
45 (CDCl3, 100 MHz), δ (ppm): 158.9, 140.8, 135.1, 134.2, 134.0, 95 7.55ꢀ7. 14 (m, 15H), 7.07 (s, 1H). 13C RMN (CDCl3, 50 MHz), δ
133.2, 131.4, 130.9, 130.5, 130.3, 130.17, 128.5, 128.6, 128.3,
127.6, 127.2, 126.2, 113.1, 55.1. MS (relative intensity) m/z: 522
(11), 365 (42), 284 (12), 208 (100), 193 (62), 165 (66), 139 (9),
77 (9). Anal. HRMS calcd. for C27H22OSe2 (M+Na+): 522.0001.
50 Found: 522.0012.
(ppm): 139.5, 133.0, 132.1, 131.1, 130.6, 130.4, 129.3, 129.2,
128.6, 128.3, 128.4, 127.5, 127.4, 126.0. MS (relative intensity)
m/z: 415 (9), 280 (10), 258 (19), 252 (9), 206 (25), 178 (100), 156
(48), 151 (10), 77 (66). Anal. HRMS calcd. for C20H16Se2: C
100 415.9582. Found: 415.9602.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

Organic & Biomolecular Chemistry
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(E)-1,2-Diphenyl-1,2-bis(phenylthio)ethene) (3u): Yield: 0.123 55 biting the injected paw was recorded during the first 5 min (early
1
phase) and 15–30 min (late phase) postꢀi.pl. Bisꢀvinyl selenides
(50 mg/kg, i.g.) or vehicle (canola oil, p.o., 10 ml/kg) were
administered 30 min, 1 h or 2 h before formalin administration.
The time spent licking the injected paw, indicative of
60 nociception, was timed with a chronometer. In order to assess if
bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t inhibit edema associated
with inflammatory pain, paw edema was measured by comparing
the difference between the weight of the formalinꢀtreated paw and
the weight of the contralateral paw (nonꢀtreated paw). Animals
65 were killed by cervical dislocation 30 min after formalin injection
and both paws were cut at the ankle joint and weighed in an
analytical balance.
g (62%). White solid. mp = 106ꢀ106 °C. H RMN (CDCl3, 400
MHz) , δ (ppm): 7.49ꢀ7.37 (m, 3H), 7.19ꢀ6.99 (m, 16H), 7.00ꢀ
6.89 (m, 1H). 13C RMN (CDCl3, 100 MHz), δ (ppm): 138.9,
134.4, 131.7, 130.6, 129.9, 128.1, 128.3, 127.3, 126.8. MS
(relative intensity) m/z: 396 (49), 287 (30), 271 (13), 253 (41),
209 (51), 206 (56), 178 (100), 165 (43), 152 (42), 108 (29), 77
(18). Anal. HRMS calcd. for C26H20S2 (M+Na+): 396.1006.
Found: 396. 1019.
5
10 (E)-(1-(2-Chlorophenyl)-2-phenylethene-1,2-
diyl)bis(phenylsulfide) (3v): Yield: 0.091 g (42%). White solid.
mp = 43ꢀ46 °C. 1H RMN (CDCl3, 400 MHz), δ (ppm): 7.54ꢀ6.99
(m, 19H). 13C RMN (CDCl3, 100 MHz), δ (ppm):137.6, 137.0,
134.7, 134.2, 133.3, 132.5, 132.0, 131.5, 130.1, 129.6, 129.3,
15 129.0, 128.8, 128.3 (2C), 127.8, 127.7, 127.1, 126.0. MS
(intensidade relativa) m/z: 430 (3), 341 (5), 281 (21), 253 (17),
207 (100), 190 (16), 133 (17), 96 (15), 73 (26). Anal. HRMS
calcd. for C26H19ClS2: 430.0617. Found: 430.0629.
Open-field test: With the purpose of excluding sedative or motor
abnormality, spontaneous locomotor (number of segments
70 crossed with the four paws) and exploratory activity (number of
times reared on the hind limbs) behaviors of mice treated with
bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t were assessed in the
openꢀfield test. The openꢀfield was made of plywood and
surrounded by walls 30 cm in height. The floor of the open field,
75 45 cm in length and 45 cm in width, was divided by masking tape
markers into 9 squares (3 rows of 3). Each animal was placed
individually at the center of the apparatus and the number of
squares crossed with all paws (crossing) was counted in a 4 min
session. The arena floor was cleaned between the trials and the
80 test was carried out in a temperature and light controlled room.22
(E)-(1-Phenylethene-1,2-diyl)bis(phenylsulfide (3w): Yield:
1
20 0.064 g (40%). colorless oil . H RMN (CDCl3, 400 MHz), δ
(ppm): 7.60ꢀ7.56 (m, 2 H), 7.34ꢀ7.17 (m, 15 H), 7.14ꢀ7.09 (m,
1H), 6.89 (s, 1H). 13C RMN (CDCl3, 50 MHz), δ (ppm): 138.9,
137.4, 134.4, 134.2, 131.7, 131.5, 130.6, 129.9, 128.5, 128.3,
127.6 (2C), 127.3, 126.7. MS (relative intensity) m/z: 331 (4),
25 281 (12), 253 (15), 209 (13), 207 (100), 191 (13), 123 (14), 96
(14), 73 (7).
Acute toxicity: The possible acute toxicity of a single dose of
bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t was investigated. Bisꢀ
vinyl selenides at a dose of 50 mg/kg (i.g.) or vehicle were
administered to mice. After 72 h, mice were anesthetized to blood
85 collection by heart puncture; the blood was collected into heparin
containing tubes. Blood samples were centrifuged at 2000×g for
10 min and plasma obtained for biochemical assays. Aspartate
aminotransferase (AST) and alanine aminotransferase (ALT)
activities, biochemical markers of acute hepatic damage, were
90 determined. AST and ALT were expressed as UI/l. The renal
function was investigated by determining plasma urea. Urea was
expressed as mg/dl. All these parameters were determined by
enzymatic colorimetric methods using commercial kits (Labtest
Diagnostica, MG, Brazil).
Animals: The experiments were conducted using male adult
Swiss mice (25ꢀ35 g) from our own breeding colony. The animals
were kept in a separate animal room, on a 12 h light/dark cycle
0
30 with lights on at 7:00 a.m., at room temperature (22 ± 1 C) with
free access to water and food. All manipulations were carried out
between 08:00 a.m. and 04:00 p.m. All experiments were
performed on separate groups of animals. The animals were used
according to the guidelines of the Committee on Care and Use of
35 Experimental Animal Resources, the Federal University of Santa
Maria, Brazil. All efforts were made to minimize animals
suffering and to reduce the number of animals used in the
experiments. Animals were randomly divided into groups of 5ꢀ7
animals each.
95 Tissue preparation: Mice were killed and brains and livers were
quickly removed and placed on ice. For δꢀALAꢀD assay brain and
liver were homogenized in 50 mM Tris/HCl pH 7.5, 1:2 and 1:5
(w/v), respectively. For Na+, K+ꢀATPase assay brain was
40 Drugs: Bisꢀvinyl selenides 3a, 3d, 3f, 3h and 3t were dissolved
in canola oil and administered intragastrically (i.g.). A single dose 100 were centrifuged at 2400 x g and 4000 x g for 10 min at 4 C for
of 50 mg/kg was used for all compounds for comparison
homogenized at 1:10 (w/v) in the same buffer. The homogenates
0
δꢀALAꢀD and Na+, K+ꢀATPase assays, respectively, to yield a
purposes. Morphine, a positive control, was dissolved in saline
and administered at a dose of 2.5 mg/kg by subcutaneous (s.c.)
45 route. Mice received bisꢀvinyl selenides and morphine in a
constant volume of 10 ml/kg of body weight. Appropriate
vehicleꢀtreated groups were also simultaneously assessed. All
compounds were tested at different pretreatment times of 30 min,
1h and 2h.
lowꢀspeed supernatant fraction (S1). S1 freshly prepared was used
for the enzymatic assays. The protein content in homogenates
was quantified by the method of Bradford,23 using bovine serum
105 albumin (1 mg/ml) as a standard.
Na+, K+ꢀ ATPase activity: The reaction mixture for Na+, K+ꢀ
ATPase activity assay contained 3 mM MgCl2, 125 mM NaCl, 20
mM KCl, and 50 mM Tris–HCl, pH 7.4, in a final volume of 500
ꢁl. An aliquot of 50 ꢁl of S1 was added to the reaction mixture
50 Formalin-induced nociceptive behavior and edema: The
formalin test was carried out as previously described.21 Briefly,
formalin was administered by intraplantar (i.pl.) injection (2.5%,
20 ꢁl, v/v) into the mouse dorsal right hind paw. The mice were
returned to the experimental cage and the time spent licking or
0
110 and preꢀincubated at 37 C for 10 min. The reaction was initiated
by the addition of adenosine triphosphate (ATP) to a final
concentration of 3.0 mM and incubated at 37 0C for 30 min.
Controls were carried out under the same conditions with the
8
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addition of 0.1 mM ouabain. Na+, K+ꢀ ATPase activity was
calculated by the difference between the two assays. Released
inorganic phosphate (Pi) was measured by the method of Fiske
and Subbarow.24
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60
65
70
75
80
85
90
95
5
δ-Aminolevulinate dehydratase (δ-ALA-D) activity: δꢀALAꢀD
activity was determined according to the method described by
Sassa25 with some modifications. An aliquot of 200 ꢀl of S1 was
preꢀincubated for 10 min at 37 0C. Enzymatic reaction was
initiated by adding the substrate (δꢀaminolevulinic acid) to a final
10 concentration of 2.2 mM in a medium containing 45 mM
phosphate buffer, pH 6.8 and incubated for 1h (liver) and 3 h
(brain) at 37 0C. The incubation was stopped by adding
trichloroacetic acid solution 10% with 10 mM HgCl2. The
porphobilinogen, which is formed within a fixed time, is mixed
15 with Ehrlich’s reagent, and the color developed is measured
photometrically (555 nm) against a blank.
Statistical Analysis: All experimental results are given as the
mean ± SEM. The statistically significant difference between
groups was calculated by means of oneꢀway followed by the
20 Duncan’s test when appropriate. A value of p ≤ 0.05 was
considered to be significant.
Acknowledgments
The financial support by UFSM, CAPES, FAPERGS
(PRONEX research grant # 10/0005ꢀ1) and CNPq (INCTꢀ
25 catalise) is gratefully acknowledged. We thank CNPq for
fellowships.
Notes and references
a
Laboratório de Síntese, Reatividade, Avaliação Farmacológica
e
Toxicológica de Organocalcogênios, CCNE, UFSM, Santa Maria ꢀ Rio
30 Grande do Sul – Brasil ꢀ 97105ꢀ900.
b Laboratório de Materiais Inorgânicos, CCNE, UFSM, Santa Maria, Rio
Grande do Sul, Brazil, 97105ꢀ900.
23. M. M. Bradford, Anal. Biochem. 1976, 72, 248.
24. C. H. Fiske, Y. J. Subbarow, 1925. Biol. Chem. 1925,
66, 375.
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
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