A greener protocol for the synthesis of phosphorochalcogenoates: Antioxidant and free radical scavenging activities

Article abstract of DOI:10.1016/j.ejmech.2020.113052

In this contribution, a metal- and base-free protocol has been developed for the synthesis of phosphorochalcogenoates (Se and Te) by using DMSO as solvent at 50 °C. A variety of phosphorochalcogenoates were prepared from diorganyl dichalcogenides and H-phosphonates, leading to the formation of a Chal-P(O) bond, in a rapid procedure with good to excellent yields. A full structural elucidation of products was accessed by 1D and 2D NMR, IR, CGMS, and HRMS analyses, and a stability evaluation of the phosphorochalcogenoates was performed for an effective operational description of this simple and feasible method. Typical ⁷⁷Se{¹H} (δ_Se = 866.0 ppm), ¹²⁵Te{¹H} (δ_Te = 422.0 ppm) and ³¹P{¹H} (δ_P = ?1.0, ?13.0 and ?15.0 ppm) NMR chemical shifts were imperative to confirm the byproducts, in which this stability study was also important to select some products for pharmacological screening. The phosphorochalcogenoates were screened in vitro and ex vivo tests for the antioxidant potential and free radical scavenging activity, as well as to investigation toxicity in mice through of the plasma levels of markers of renal and hepatic damage. The pharmacological screening of phosphorochalcogenoates indicated that compounds have antioxidant propriety in different assays and not changes plasma levels of markers of renal and hepatic damage, with excision of 3g compound that increased plasma creatinine levels and decreased plasma urea levels when compared to control group in the blood mice. Thus, these compounds can be promising synthetic antioxidants that provide protection against oxidative diseases.

Full text of DOI:10.1016/j.ejmech.2020.113052

European Journal of Medicinal Chemistry 213 (2021) 113052
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
A greener protocol for the synthesis of phosphorochalcogenoates:
Antioxidant and free radical scavenging activities
Daniela H. Mailahn a, Lucas E.B. Iarocz a, Patrick C. Nobre a, Gelson Perin a, Airton Sinott b,
b
b
b
a, *
ꢀ
Ana Paula Pesarico , Paloma T. Birmann , Lucielli Savegnago , Marcio S. Silva
a LASOL - CCQFA, Universidade Federal de Pelotas - UFPel, P.O. Box 354, 96010-900, Pelotas, RS, Brazil
Programa de Pos-Graduaçao Em Biotecnologia, Grupo de Pesquisa Em Neurobiotecnologia, Centro de Biotecnologia, Universidade Federal de Pelotas, RS,
b
ꢀ
~
Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this contribution, a metal- and base-free protocol has been developed for the synthesis of phos-
phorochalcogenoates (Se and Te) by using DMSO as solvent at 50 ꢀC. A variety of phosphor-
ochalcogenoates were prepared from diorganyl dichalcogenides and H-phosphonates, leading to the
formation of a Chal-P(O) bond, in a rapid procedure with good to excellent yields. A full structural
elucidation of products was accessed by 1D and 2D NMR, IR, CGMS, and HRMS analyses, and a stability
evaluation of the phosphorochalcogenoates was performed for an effective operational description of
Received 3 September 2020
Received in revised form
17 November 2020
Accepted 22 November 2020
Available online 27 November 2020
this simple and feasible method. Typical 77Se{1H} (dSe ¼ 866.0 ppm), 125Te{1H} (dTe ¼ 422.0 ppm) and 31
P
{1H} (dP ¼ ꢁ1.0, ꢁ13.0 and ꢁ15.0 ppm) NMR chemical shifts were imperative to confirm the byproducts,
in which this stability study was also important to select some products for pharmacological screening.
The phosphorochalcogenoates were screened in vitro and ex vivo tests for the antioxidant potential and
free radical scavenging activity, as well as to investigation toxicity in mice through of the plasma levels of
markers of renal and hepatic damage. The pharmacological screening of phosphorochalcogenoates
indicated that compounds have antioxidant propriety in different assays and not changes plasma levels
of markers of renal and hepatic damage, with excision of 3g compound that increased plasma creatinine
levels and decreased plasma urea levels when compared to control group in the blood mice. Thus, these
compounds can be promising synthetic antioxidants that provide protection against oxidative diseases.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
groups could improve applications in agrochemical and pharma-
ceutical fields.
Organic chalcogen compounds have become versatile building
blocks for organic synthesis [1e7], particularly under mild condi-
tions [8e15]. Among the organic chalcogen compounds, phos-
phorochalcogenoates are important intermediates in organic
synthesis [16e21] and can be an alternative route to access het-
erocyclic compounds [22]. In addition, there is an interest in
phosphorochalcogenoate derivatives because of their numerous
biological properties, especially those containing sulfur elements
[23e25], and because of their similarities with the phosphate
functional group [26e28]. Thus, a combination of the biological
activities of chalcogen compounds [29e31] with phosphonate
Distinctive methodologies have been developed to form
SeeP(O) bond (Scheme 1) [32e39]. According to the literature,
various synthetic protocols employ harsh conditions, as metals
[32,35e37], bases [34,35,37], oxidant [38], radical specie, [33] and
harmful organic solvents [32e34,38]. Additionally, the more recent
method developed to form SeP(O) bond, the authors have reported
the obtaining phosphoroselenoates using H-phosphonate and N-
(phenylselene)phthalimide as starting materials under solvent-free
conditions, however, only two compounds are obtained [39].
Regarding the biological activity evaluation, Hajra and co-workers
prepared phosphoroselenoates from diselenides and H-phospho-
nates using zinc (dust) in EtOH and evaluated their biological ac-
tivity [36]. In vitro antimicrobial and antifungal evaluations
demonstrated that these compounds possess relevant biological
applications. This is the unique biological study carried out for
* Corresponding author.
E-mail addresses: luciellisavegnago@yahoo.com.br (L. Savegnago), silva.ms@
ufpel.edu.br (M.S. Silva).
https://doi.org/10.1016/j.ejmech.2020.113052
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
Scheme 1. Examples of the synthesis of phosphorochalcogenoates (Se and Te).
these phosphorochalcogenoates (Se), emphasizing the needs to
improve the discernments about this organic class of compounds.
Although there is an ample number of methods for forming
SeP(O) bond [40e47], a simple and rapid methodology for the
synthesis of phosphorochalcogenoate (Se and Te) frameworks re-
mains necessary. Moreover, the methodologies to access SeeP(O)
and TeeP(O) bond formation involve metals, bases, harsh condi-
tions and time-consuming methods, which decompose the phos-
phorochalcogenoates containing tellurium element due to their
instability compared to selenium element. According to the litera-
ture only one synthetic procedure was able to prepare phosphor-
ochacogenoates using both elements (Se and Te), employing CuI
and Et2NH (Scheme 1) [35].
Based on the development of a greener and practical protocol
with a greater substrate scope (Se and Te) to obtain phosphor-
ochalcogenoates, and in terms of antioxidant activities of chalcogen
compounds [29e31] and phosphorylated derivatives [48e53], we
report a simple and rapid method for the synthesis of some novel
Se- and Te-aryl and alkyl phosphorochalcogenoates using only
DMSO as solvent at 50 ꢀC, where neither catalyst nor additive is
required. Moreover, the major limitation of these prepared com-
pounds is the sensitivity to air, moisture, and harsh experimental
conditions, which was accessed by the stability study, especially
those containing tellurium atom. Furthermore, the results were
extremely important to design the biological study, as antioxidant
and radical scavenging activity, since byproducts from phosphor-
ochalcogenoate compounds could interfere in biological assays,
hampering the interpretation.
diselenide 1a was not effective in improving the reaction conditions,
an excess of H-phosphonate 2a increased the yield to 91% (Table 1,
entry 3 vs 4). When the temperature was increased to 50 ꢀC, yield
increased to 97% in 2.0 h of reaction time (Table 1, entry 6). Reaction
at 90 ꢀC showed a significant decrease inyield when compared tothe
reaction at 50 ꢀC (Table 1, entry 7 vs 6). Almost quantitative forma-
tion of the desired product 3a in DMSO at 50 ꢀC with no catalyst, base
or oxidant was achieved after 2.0 h. Subsequently, other solvents
were also evaluated, including H2O, glycerol, THF, EtOH, MeCN, DMF
and PEG-400 (Table 1, entries 8e14). No reaction occurred in H2O,
glycerol and THF as solvent (Table 1, entries 8e11). On the other
hand, the use of MeCN, EtOH, DMF and PEG-400 proved to be effi-
cient in obtaining product 3a, but only after a long reaction time
(Table 1, entries 11e14 vs 6). In solvent-free conditions the formation
of product 3a was not efficient (Table 1, entry 15). Then, aiming to
decrease the time, the reaction was performed under ultrasound
irradiation (US), and the expected product 3a was obtained in 84%
yield after 0.5 h (Table 1, entry 16).
Thus, the best reaction condition to prepare compound 3a
involve the stirring of a mixture of diphenyl diselenide 1a
(0.125 mmol) and dimethyl H-phosphonate 2a (0.300 mmol) in
DMSO (0.5 mL) as a solvent at 50 ꢀC for 2.0 h in an open flask
(Table 1, entry 6).
Next, we turned our attention to the reactional substrate scope
(Scheme 2), evaluating different diselenides 1 and H-phosphonates
2 under optimized reaction conditions. The reaction provided
phosphoroselenoates 3a-d in excellent yield (93e98%), regardless
of the steric effect of the H-phosphonate. Only in the case of a
phenyl group (Scheme 2, product 3e) was the reaction not effective,
probably due to the lower basicity of the diphenyl H-phosphonate
[54,55]. After evaluating the influence of the H-phosphonate sub-
stituents, the effect of the structure of the other starting material
(diphenyl diselenide 1) on the yield and reaction time was inves-
tigated. Several diaryl and dialkyl diselenides 3f-m were employed,
all with good to excellent yields (82e98%). The electronic nature of
the substituents on the diaryl diselenide did not change the profile
of the SeeP(O) bond formation (Scheme 2). However, when
dinaphthyl diselenide was used only traces of the product were
observed (Scheme 2, product 3l).
2. Results and discussion
2.1. Chemistry
We commenced optimization of the reaction conditions using
diphenyl diselenide 1a with dimethyl H-phosphonate 2a. The re-
action conditions were investigated as outlined in Table 1. Initially,
the reaction was carried out using 0.25 mmol of 1a with 0.125 mmol
of 2a in DMSO at 25 ꢀC in the presence of KOH (0.05 mmol). After
7.5 h, the yield was just 69% (Table 1, entry 1). To our delight, when
the reaction was performed without catalyst, product 3a was ob-
tained in 83% yield (Table 1, entry 2 vs 1). Although an excess of
To exemplify the full structural elucidation, 1H, 13C{1H}, 31P{1H},
HSQC and HMBC NMR experiments were performed with the
2

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
Table 1
Optimization of the reaction conditions for the synthesis of phosphorochalcogenoate 3aa..
#
1 (mmol)
2 (mmol)
Solvent
Temperature (ꢀC)
Time (h)
Yield (%)b
1c
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16d
0.125
0.125
0.150
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.250
0.250
0.250
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
H2O
Glycerol
THF
MeCN
EtOH
25
25
25
25
30
50
90
50
50
50
50
50
50
50
50
e
7.5
7.5
7.5
7.5
7.5
2.0
2.0
24.0
24.0
24.0
24.0
24.0
24.0
24.0
24.0
0.5
69
83
70
91
91
97
67
NR
NR
NR
61
95
89
85
28
84
DMF
PEG-400
e
DMSO
a
Reactions performed using dimethyl H-phosphonate 1a, diphenyl diselenide 2a and solvent (0.5 mL) in an open flask.
Yields of isolated product.
Reaction performed using KOH (0.05 mmol).
Reaction performed with ultrasound energy; NR ¼ no reaction.
b
c
d
Scheme 2. Substrate scope for the synthesis of phosphoroselenoates 3a-m.
product 3j. As can be seen in the support information, 1H NMR
bond formation was explored (Scheme 3). On evaluating the H-
phosphonates to obtain phosphorotelluroates 3n-u, the result was
similar to the diselenides (Scheme 3, 57e94%). Moreover, the
reactivity of diphenyl H-phosphonate was also ineffective in
obtaining TeeP(O) bond formation under optimized reaction con-
ditions. The variation with ditelluride was less effective than with
diselenide. As can be seen in Scheme 3, the yields decreased, and
the substrate scope was limited. In addition, when diphenyl ditel-
luride with an electron withdrawing group in the phenyl group was
used, only traces of the product were observed even after a long
reaction time (Scheme 3, product 3t).
signals appear at dH ¼ 3.82 ppm (methyl groups), presenting a
coupling constant with phosphorus atom (JH-P ¼ 13.2 Hz), and at
aromatic region with a typical meta-substitution multiplicity. The
13C{1H} NMR experiment showed eight signals with fluoride and
phosphorus scalar couplings, which according to the HSQC NMR
experiment was possible to designate the carbons attached to the
protons. Based on the carbon-13 chemical shifts and HMBC NMR
experiment the quaternary carbons were confirmed. Additionally,
coupling constants with phosphorus atom were imperative to aid
the structural characterization in all compounds prepared, such as
the quaternary carbon bonded to the selenium atom in the product
3j that demonstrates a coupling constant with phosphorus atom
(JP-C ¼ 2.5 Hz).
The full structural characterization was also carried with phos-
phorotelluorates. Similar to selenium compounds elucidation, the
1H NMR spectrum of product 3q (SI) demonstrates a signal at
dH ¼ 5.08 ppm with a scalar coupling with phosphorus atom (JH-
After determining the substrate scope regarding substituted
diorganyl diselenides, the effect of ditelluride 1j-m on TeeP(O)
¼ 13.2 Hz), and six signals at aromatic region. Considering the
P
3

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
Scheme 3. Substrate scope for the synthesis of phosphorotelluroates 3n-u.
integral values, it was possible to discriminate the aromatic ring
attached to the tellurium atom with benzyl aromatic rings in the 1H
NMR spectrum. The 13C{1H} NMR spectrum also shows coupling
constants with phosphorus atom, which supports the carbons
discrimination. HSQC and HMBC NMR experiments have confirmed
the quaternary carbons. It is important to mention that the 2D NMR
experiments were not performed for all compounds, since the
scalar couplings with phosphorus atom provide useful data for
structures elucidation.
NMR, as given in the literature [56,57] and starting material 1j was
identified by 125Te-{1H} NMR spectroscopy. In DCM solvent, prod-
uct 3o was less reactive but still observed in small amounts (~20%).
In DMSO and EtOH solvents, product 3o degrades completely and
only by-product 4 was observed by 31P-{1H} NMR spectroscopy.
Diphenyl ditelluride 1j was not detected, and we believe that it was
transformed to tellurium oxide, as seen by the formation of a white
solid. When acid or base media in DMSO at 50 ꢀC were tested, only
by-product 4 was observed, together with this white solid.
Based on the explored substrate scope, the experimental fea-
tures of the reactions and the distinctive results obtained from the
diselenide and ditelluride starting materials, we decided to verify
the stability of these products. For this investigation phosphor-
oselenoate 3b and phosphotelluroate 3o were chosen as standard
compounds. Product 3b was stable in the presence of light (Blue
LED) for 24.0 h in different solvents (DMSO, DCM and EtOH) and
when submitted to acid (TsOH, 1.0 equiv) or base (Na2CO3, 1.0
equiv) in DMSO solvent at 50 ꢀC for 24.0 h it remained intact. These
results emphasize the high stability of the SeeP(O) bonding and
explain the greater number of methods for obtaining phosphor-
oselenoates (Scheme 1).
On the other hand, when phosphorotelluroate 3o was investi-
gated in the stability study, different by-products were identified
(Scheme 4). Product 3o is more reactive in the outlined conditions
and by-products were formed in the presence of light in 2.0 h of
reaction time. By-products 4 and 5 were identified by 31P-{1H}
After demonstrating the stability of the products against light
and acidebase media, we evaluated the SeeP(O) bond reactivity
against molecular bromine. It is well-known that bromine reacts
with diselenides to form reactive electrophiles (Scheme 5, BreSe-
Aryl6) [58]. When a THF solution ofmolecularbrominewas added to
a THF solution of phosphoroselenoate 3b at 0 ꢀC, the starting ma-
terials were obtained. The reactive electrophile phenylselanyl bro-
mide 4 was formed in around of 10% yield (Scheme 5). The products
were confirmed by 31P-{1H} and 77Se-{1H} NMR spectroscopy
(Figures 96S and 97S). Based on this result, it is possible to observe
that SeeP(O) bonding is also labile but stronger than TeeP(O)
bonding. On evaluating the stability of phosphorotelluroate 3o
against bromine to obtain the electrophile phenyltellanyl bromide,
the result was the same as with phosphoroselenoate 3b (SI).
Based on the stability studies we can conclude that the higher
instability of phosphorotelluroates is because of the weak TeeP(O)
bonding, which justified the small substrate scope obtained from
Scheme 4. Stability evaluation of phosphorotelluroate 3o under different reaction conditions.
4

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
Scheme 5. Stability evaluation of phosphoroselenoate 3b against molecular bromine.
diorganyl ditellurides in our procedure. In addition, the studies also
suggest possible reasons why some of the protocols demonstrated
in the literature (Scheme 1) to form phosphorotelluroates did not
work. After evaluating the stability of the products, we decide to
scale-up this protocol in an attempt to estimate the stability in a
gram-scale reaction. The procedure was performed using 5 mmol of
diethyl H-phosphonate 2b with 2.1 mmol of diphenyl dichalcoge-
nide 1a or 1j in DMSO at 50 ꢀC. The yields obtained were compa-
rable to the small-scale reactions, affording the products in 3b and
3o in 87% and 90%, respectively.
Scheme 7. A plausible mechanistic pathway.
We have carried out some control experiments to investigate
the mechanistic pathway to obtain phosphorochalcogenoates.
Thus, when the reaction between diphenyl diselenide 1a and H-
phosphonate 2b was performed in the presence of radical scaven-
gers (2.0 equiv), including 2,2,6,6-tetramethyl-1-piperidinyloxy
(TEMPO) and 1,4-benzoquinone (BQ), the yields stayed the same
in 3.0 h of reaction time (Scheme 6). Therefore, these results clearly
supported that the free radical mechanism was not involved in
phosphorochalcogeonate formation. Then we performed a reaction
under argon and observed that the yield was not affected by an
inert atmosphere (Scheme 6).
On the basis of the control experiments and the scope profile of
this procedure, a plausible mechanism was proposed (Scheme 7).
Initially, tautomerization of the H-phosphonate occurs to form the
intermediateA. The basicityof tautomerA is well-known[47,54] and
reaction with dichalcogenides provides phosphorochalcogenoates.
Furthermore, intermediate B is also formed, which is oxidized in situ
to dichalcogenides for subsequent runs. This oxidation procedure
reutilizes intermediate B and increases the atomic efficiency of the
reaction. For phosphorotelluroate, instability hampers the reaction
scope and some of these products degrade to by-products 4 and 5,
together with tellurium oxide.
The greener synthetic procedure developed is desirable for
pharmacological studies, since no catalyst and metal were
employed, which avoids contaminations. Additionally, DMSO is a
useful solvent because the application in chemical processes, and
due the lower toxicity compared to hydrocarbons, and chlorinated
solvents [59]. Overall, the substrate scope evaluation has provided
the stability profile of the distinct phosphorochalcogenoates syn-
thesized. In addition to the stability parameters accessed (light,
solvent, and acid/base media), the substrate scope demonstrated
that heating and long reaction-time were imperative to the
decreasing of the yields. The phosphorotelluroates are not stable
enough for biological evaluation, because the standard experi-
mental conditions. The use of tellurium products could generate
non-confident results. In this sense, the phosphoroselenoates were
selected as the proper substrates for biological analyses. As can be
seen in Scheme 2, the compounds 3a, 3c 3d, 3f, 3g, and were
designated for the biological study due to their higher stability
during the storage, and the structural distinction.
a
Scheme 6. Control experiments. Yield of isolated product.
5

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
Table 3
2.2. In vitro biological results
Effect phosphoroselenoates on reactive species production assays in the cerebral
cortex of mice.
2.2.1. Thiobarbituric acid reactive species assay in brain total of
mice
The levels of TBARS in the lipid peroxidation induced by SNP in
cerebral tissue were reduced for compounds at different concen-
trations. As shown in Table 2, 3f and 3d compound were effective at
Concentrations (
Compounds 10
m
M)
50
e
100
500
3a
3c
3d
3f
71.50 13.00 *
98.30 7.15
61.70 15.00 *
70.30 18.90
94.00 11.10
e
e
76.10 7.90 * 61.40 2.76 **
e
e
concentration of 10
mM, while the compounds 3c and 3g showed a
e
e
84.90 13.40 70.10 17.50
94.10 3.92 67.20 15.80 *
62.7 12.3
e
significant inhibition of the lipid peroxidation at the concentration
3g
of 500
levels.
mM. The 3a compound was not effective in lowering TBARS
Data are expressed as mean
82.10 0.05; Induced: 139.00 26.80; Trolox (10
SEM (n ¼ 3) of units of fluorescence. Control:
M, positive control): 99 15.53.
m
The letters represent significant difference (*p < 0.05 and **p < 0.01) when
compared to induced. One-way ANOVA followed by the Tukey.
2.2.2. Reactive species assay in brain total of mice
In this study was evaluated the capacity of the compounds in
neutralizing the RS in cerebral tissue of mice (Table 3). The results
from the RS assay demonstrate that compounds 3a and 3d
decreased the formation of RS induced by azide in the cerebral
Table 4
Effect phosphoroselenoates on the FRAP assay.
Concentration (
10
mM)
tissue of mice at concentrations equal to or higher than 10
while 3c and 3g compounds were effective only at the concentra-
tion of 50 M and 100 M, respectively. The 3f compound was not
mM,
Compounds
50
100
500
m
m
3a
3c
3d
3f
0.11 0.02
0.11 0.02
0.16 0.01
0.14 0.01
0.16 0.01
0.12 0.01
0.15 0.01
0.23 0.04
0.17 0.016
0.16 0.01
0.12 0.02
0.15 0.01
0.40 0.03
0.17 0.04
0.19 0.03
0.16 0.01
effective in neutralizing the RS in cerebral tissue of mice.
0.3 0.01 ***
1.02 0.18 ***
0.29 0.04 *
0.26 0.02 **
2.2.3. Ferric ion reducing antioxidant power
3g
The FRAP assay was used to determine the reducing power of
compounds to elucidate the relationship between the antioxidant
effect and the reducing power (Table 4). Compounds 3c, 3d, 3f and
Data expressed mean SEM (n ¼ 3) of absorbance; Vehicle: 0.11 0.02; Trolox
(10 M, positive control): 0.39 0.132 The letters represent significant (*p < 0.05,
**p < 0.01and ***p < 0.001) when compared to vehicle. One-way ANOVA followed
m
by the Tukey.
3g exhibited ferric-reducing ability at concentration of 500
3a compound did not present this power.
mM and
Table 5
Effect phosphoroselenoates on the DPPH radical scavenging.
2.2.4. Radical scavenging activity
Table 5 demonstrates that the compounds 3a and 3g present a
significant DPPH radical scavenging activity at concentrations
500 mM, while the other compounds did not demonstrate radical
Concentration
50
(mM)
Compounds
10
100
500
3a
3c
3d
3f
5.53 3.67
6.14 6.14
2.19 2.19
12.20 6.48
3.72 3.72
7.83 4.09
18.38 7.49
3.94 3.94
6.83 2.93
6.95 3.81
4.68 2.39
12.21 6.20
3.72 3.72
9.87 9.87
11.92 4.69
21.93 0.88 *
7.38 4.18
5.04 5.04
3.62 3.62
19.06 3.60 *
scavenging efficiency.
Table 6 shows the antioxidant ability of 3a, 3f and 3g com-
pounds in ABTS þ assay. The compound 3a and 3f were effective at
3g
concentrations equal to and higher than 50 mM, while the 3g pre-
sented radical scavenging efficiency at concentration equal to
500 M.
Data expressed mean SEM (n ¼ 3) percentage of scavenging activity in relation to
vehicle. Vehicle: 0.00 0.00; Trolox (10 M, positive control): 93.5 0.28.The letters
m
m
represent significant difference (*p < 0.05) when compared to vehicle group. One-
way ANOVA followed by the Tukey.
2.3. Ex vivo biological results
2.3.1. Thiobarbituric acid reactive species assay in brain, liver and
kidney of mice
Table 6
Effect phosphoroselenoates on the ABTSþ radical scavenging.
Table 7 demonstrates that the compounds 3f and 3g present
antioxidant activity in the brains of mice at the dose of 10 mg/kg,
while that in the liver and kidney none compounds presented ac-
tivity. Interestingly, the 3a compound presents pro-oxidant activity.
Concentration
50
(mM)
Compounds 10
100
500
3a
3c
3d
3f
5.02 3.19
1.92 1.92
3.85 3.85
10.35 1.09 *
8.76 0.79
3.06 3.06
e
e
3.84 3.14
1.86 0.93
29.90 1.92 ***
6.91 3.29
2.34 2.34
12.40 6.53
e
Table 2
11.50 1.22 12.50 2.64
6.76 2.59 8.60 2.43
Effect phosphoroselenoates on the TBARS assays in cerebral tissue of mice.
3g
19.80 2.57 **
Concentrations (
Compounds 10
m
M)
50
Data expressed mean SEM (n ¼ 3) percentage of scavenging activity in relation to
vehicle. Vehicle: 0.00 0.00; Trolox (10 M, positive control): 94.33 2.60. The
letters represent significant difference (*p < 0.05 **p < 0.01 ***p < 0.001) when
m
100
500
compared with vehicle group. One-way ANOVA followed by the Tukey.
3a
3c
3d
3f
84.69 8.41
79.35 4.97
59.51 14.15 *
73.00 0.69 *
87.64 2.42
80.59 11.5 85.89 7.34 79.77 4.97
83.79 5.83 90.36 3.05 57.02 13.05 **
e
e
e
e
e
e
3g
78.64 2.68 78.11 0.72 57.96 8.95 ***
2.3.2. SOD and GPx activity in brain of mice
Data are expressed as mean SEM (n ¼ 3) of % of lipid peroxidation in cerebral
tissue; Control: 33.50 5.65; Induced: 100; Trolox (10 M, positive control):
29.75 0.85. (*p < 0.05, **p < 0.01 and ***p < 0.001) when compared to induced
As seen in Table 8, the compounds 3a, 3c and 3g, at the dose of
10 mg/kg, decreased the activity of GPx, and 3f and 3g decreased
the activity of SOD. 3d did not demonstrate any effect.
m
(100% of oxidation). One-way ANOVA followed by the Tukey.
6

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
Table 7
In vitro, phosphoroselenoates protected against lipid peroxida-
tion induced by SNP and reactive species induced by SNP in mice
brain homogenate. The mechanism of action by which phosphor-
oselenoates shows antioxidant activity is related to its DPPH and
ABTS þ -scavenging activity as well as reducing power of iron.
Based on the in vitro results, we expanded the study to investigate
the effect of oral administration of compounds. It was demon-
strated that 3f and 3g compounds decreased the oxidative stress
parameters in the brains of mice and the 3f reduced the lipid per-
oxidation levels and decreased the SOD and GPx activities. The
results indicate that a reduction of reactive species, could decrease
the SOD activity and consequently it causes decrease of GPx ac-
tivity. The compounds 3a, 3c and 3g altered the antioxidant en-
zymes, that can be a mechanism to neutralize oxidative stress,
because the SOD catalyzes the dismutation of superoxide into ox-
ygen and hydrogen peroxide. The compounds affected only the
brain, probably because this organ contains a high degree of
polyunsaturated fatty acids, substrates particularly vulnerable to
oxidation, and is rich in non-haem iron, which is catalytically
involved in the generation of free radicals [60].
Recently studies proved that phosphorus-containing com-
pounds increase the pharmacokinetics propriety of drugs [61].
Thus, organoselenium compounds could have their proprieties
improved by adding a phosphorus molecule in their structures.
Furthermore, a new study on phosphorus supply and selenium
uptake on plants [62] shows that increase supply of P enhances Se
transportation across the cell membrane raising the availability of
selenium in the cell, as a result the new selenium availability inside
the cell could be used to the formation of enzymes and seleno-
proteins used in the antioxidant system [63].
Basal levels of reactive oxygen species and reactive nitrogen
species are physiologically produced by many mechanisms,
including oxidative deamination of biogenic amines and reduction
of O2 via mitochondrial electron transport chain [64,65]. However,
when there is an imbalance between the production of reactive
species and the natural antioxidant defenses result in oxidative
stress [66]. There is some evidence that reactive species, including
superoxide anion, hydrogen peroxide and singlet oxygen, play an
important role in the pathogenesis of cancer, neurodegeneration,
and psychiatric disorders as well as aging [67,68]. For this reason,
these compounds may be used as synthetic antioxidants that pro-
vide protection in pathologies related to oxidative stress including
diabetes, cardiovascular diseases, cancer, and several neurodegen-
erative diseases. In addition, we can highlight the compound 3 g,
which in addition to presenting good to excellent yields (98%), also
showed promising antioxidant effect, both in vitro and ex vivo assay,
this may be due to its chemical structure presenting methox-
yphenyl group, because the methoxyphenyl derived compounds
usually exhibit a series of pharmacological properties such as
anticancer [69], antioxidant [69], antibacterial [70], and analgesic
[71]. However, 3 g compound (10 mg/kg) increased plasma levels of
creatinine in the blood of mice, therefore, in future studies should
be investigated the toxicity of this compound and check if at lower
doses will have a promising antioxidant effect without causing
changes in the markers of kidney damage, we point out that this
was a limitation of this study.
Effect of 3a, 3c, 3d, 3f and 3g (10 mg/kg) on lipid peroxidation in the brain, liver and
kidney mice.
Compounds TBARS in the brain TBARS in the liver TBARS in the kidney
Control
3a
3c
3d
3f
91.91 9.38
87.34 13.19
87.71 18.57
83.00 11.89
54.79 7.41*
64.64 7.63*
271.5 43.88
326.0 11.72
249.40 44.63
263.80 24.2
199.90 39.95
280.80 65.93
438.20 27.59
676.60 80.76*
371.00 27.89
580.5 66.58
455.4 32.70
422.5 81.08
3g
Data are reported as mean SEM of 6 animals per group. (*) denotes p < 0.05 as
compared to the control group (canola oil); (the Student’s t-test).a TBARS levels/g
tissue.
Table 8
Effect of 3a, 3c, 3d, 3f and 3g (10 mg/kg) on SOD and GPx activity in the brain of
mice.
Compounds
GPXa
SODb
Control
3a
3c
3d
3f
12.8 2.28
7.18 2.86*
5.50 1.35*
6.39 1.55
7.48 2.78
4.98 0.61**
25.44 4.12
25.94 5.22
23.85 1.84
17.54 0.91
7.37 0.46**
13.47 2.89*
3g
Data are reported as mean SEM of 6 animals per group. (*) denotes p < 0.05 and
(**) denotes p < 0.01 as compared to the control group (canola oil); (the Student’s t-
a
test). nmolNADPH/g tissue; b units/min.
2.3.3. Toxicological parameters
Table 9 demonstrates that, at the dose of 10 mg/kg, 3g decreased
plasma urea and increased plasma creatinine. None of the com-
pounds at the dose of 10 mg/kg altered alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) plasma levels.
Data are reported as mean SEM of 5/6 animals per group. (*)
denotes p < 0.05 and (***) denotes p < 0.001 as compared to the
control group; (the Student’s t-test). a mg/dL; b IU/L.
2.4. Biological discussion
The antioxidant potential of phosphoroselenoates compounds
was investigated because the involvement of oxidative stress has
been attributed in a variety of human diseases. Thus, the 3a, 3c, 3d,
3f and 3g compounds were tested in vitro and ex vivo for the
antioxidant potential, as well as to investigation toxicity in mice
through of the plasma levels of markers of renal and hepatic
damage. The results of the present study demonstrate that the
phosphoroselenoates compounds had an in vitro antioxidant ac-
tivity, in addition, the 3a, 3c, 3f and 3g compounds, at dose 10 mg/
kg, presented ex vivo antioxidant activity. It is noteworthy that the
compounds not caused changes in plasma markers of renal and
hepatic damage in mice, only the compound 3 g increased the
creatinine levels, but more studies are necessary to investigate this
change.
Table 9
Plasma toxicological parameters of hepatic and renal damage of 3a, 3c, 3d, 3f and 3g
(10 mg/kg) on mice.
3. Conclusions
Compounds Ureaa
Creatininea
ASTb
ALTb
In summary, a simple and efficient protocol for the formation of
SeeP(O) and TeeP(O) bonds from dichalcogenides and H-phos-
phonate was developed under catalyst-, and base-free conditions.
The methodology provided a greener alternative to generate valu-
able chalcogen phosphonates via a rapid procedure. Full structural
assignment and a stability evaluation of products were performed
Control
3a
3c
3d
3f
90.31 0.74
1.77 0.05
1.78 0.15
1.75 0.27
1.52 0.18
1.96 0.22
148.00 8.08 110.90 12.4
151.20 4.50 117.40 4.94
159.30 4.07 140.00 7.86
155.30 2.66 138.10 8.55
147.40 9.75 119.00 4.49
80.30 6.54
105.00 3.94
98.59 5.91
80.24 9.80
3g
74.39 1.78*** 2.36 0.18* 151.00 5.07 119.50 4.26
7

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
for an effective operational description of this simple and feasible
method. The stability study was vital to design the substrate scope
for pharmacological activity. According to the biological studies, we
could conclude that the phosphoroselenoates present antioxidant
effect in different assays and these compounds are effectives to act
against oxidative stress in biological systems.
respectively, with a desolvation temperature of 180 ꢀC. For data
acquisition, processing and isotopes simulations, Compass 1.3 for
micrOTOF-Q II software (Bruker daltonics, USA) was used. Melting
point (mp) values were measured in a Marte PFD III instrument
with a 0.1 ꢀC precision.
4.1.2. General procedure for chalcogen (Se and Te)-phosphorus
bond formation
4. Experimental section
The corresponding H-phosphonate (0.3 mmol), dichalcogenide
(0.125 mmol) and DMSO (0.5 mL) were put into an open glass tube.
The mixture was stirred at 50 ꢀC for 1e4 h. After the reaction was
complete, 10.0 mL of water was added. The mixture was extracted
with ethyl acetate (3 ꢂ 10.0 mL). The combined organic layer was
dried over MgSO4 and evaporated in vacuo. The residue was puri-
fied by silica gel column chromatography to give the corresponding
phosphorochalcogenoates 3.
4.1. Chemistry
4.1.1. General information
The reactions were monitored by TLC carried out on Merck silica
gel (60 F254) by using UV light as visualizing agent and 5% vanillin in
10% H2SO4 and heat as developing agents. Baker silica gel (particle
size 0.040e0.063 mm) was used for flash chromatography.
Hydrogen nuclear magnetic resonance spectra (1H NMR) were
obtained on Bruker Avance III HD 400 MHz employing a direct
broadband probe. Spectra were recorded in CDCl3 solutions.
Chemical shifts are reported in ppm, referenced to tetramethylsi-
lane (TMS) as the external reference. Coupling constant (J) are re-
ported in Hertz. Abbreviations to denote the multiplicity of a
particular signal are s (singlet), d (doublet), dd (doublet of doublet),
dt (doublet of triplet), t (triplet), td (triplet of doublet), quint
(quintet), sex (sextet) and m (multiplet). Carbon-13 nuclear mag-
netic resonance spectra (13C NMR) were obtained on Bruker Avance
III HD 400 MHz employing a direct broadband probe. Chemical
shifts are reported in ppm, referenced to the solvent peak of CDCl3.
Coupling constant (J) are reported in Hertz. Abbreviations to denote
the multiplicity of a particular signal are d (doublet), dd (doublet of
doublet), q (quartet), qd (quartet of doublet) and m (multiplet).
Phosphorus-31 nuclear magnetic resonance spectra (31P NMR)
were obtained on Bruker Avance III HD 400 MHz employing a direct
broadband probe. Spectra were recorded in CDCl3 solutions.
Chemical shifts are reported in ppm, referenced to triphenylphos-
phate (TPP). Coupling constant (J) are reported in Hertz. Abbrevi-
ations to denote the multiplicity of a particular signal are quint
(quintet), sept (septet) and sept d (septet of doublet). Selenium-77
nuclear magnetic resonance spectra (77Se NMR) were obtained on
Bruker Avance III HD 400 MHz employing a direct broadband
probe. Spectra were recorded in CDCl3 solutions. Chemical shifts
are reported in ppm, referenced to the diphenyl diselenide
(462 ppm). Coupling constant (J) are reported in Hertz. Abbrevia-
tions to denote the multiplicity of a particular signal are d (doublet)
and d quint (doublet of quintet). Tellurium-125 nuclear magnetic
resonance spectra (125Te NMR) were obtained on Bruker Avance III
HD 400 MHz employing a direct broadband probe. Spectra were
recorded in CDCl3 solutions. Chemical shifts are reported in ppm,
referenced to diphenyl ditelluride (422 ppm). Coupling constant (J)
are reported in Hertz. Abbreviations to denote the multiplicity of a
particular signal are d (doublet) and d quint (doublet of quintet).
Fluorine-19 nuclear magnetic resonance spectra (19F NMR) were
obtained on Bruker Avance III HD 400 MHz employing a direct
broadband probe. Spectra were recorded in CDCl3 solutions.
4.1.2.1. O,O-dimethyl Se-phenyl phosphoroselenoate (3a)[38].
Yield: 0.0645 g (97%); Yellowish oil. 1H NMR (400 MHz, CDCl3)
d
(ppm) ¼ 7.58e7.56 (m, 2H); 7.32e7.20 (m, 3H); 3.72 (d, JH-
¼ 13.1 Hz, 6H). 13C-{1H} NMR (100 MHz, CDCl3)
(ppm) ¼ 135.6 (d,
d
P
JC-P ¼ 4.6 Hz), 129.5 (d, JC-P ¼ 2.1 Hz), 128.9 (d, JC-P ¼ 2.6 Hz), 123.2
(d, JC-P ¼ 8.5 Hz), 53.9 (d, JC-P ¼ 5.6 Hz). 31P NMR (162 MHz, CDCl3)
d
(ppm) ¼ 21.9 (sept, JP-H ¼ 13.1 Hz). 77Se-{1H} NMR (76 MHz,
CDCl3)
d
(ppm) ¼ 244.1 (d, JSe-P ¼ 490.5 Hz). IR (cmꢁ1): 2994, 2865,
1484, 1456, 1238, 1012, 854, 736. MS: m/z (rel. int., %) 266 (Mþ,
27.7), 157 (10.2), 109 (100.0), 79 (16.8), 77 (17.5), 51 (7.5). HRMS
(APCI-QTOF) calculated mass for C8H11O3PSe [MþH]þ: 266.9689,
found: 266.9686.
4.1.2.2. O,O-diethyl Se-phenyl phosphoroselenoate (3 b). [37] Yield:
0.0684 g (93%); Yellow oil. 1H NMR (400 MHz, CDCl3)
d
(ppm) ¼ 7.57
(d, J ¼ 7.6 Hz, 2H); 7.30e7.22 (m, 3H); 4.18e4.03 (m, 4H); 1.23 (t,
J ¼ 7.1 Hz, 6H). 13C-{1H} NMR (100 MHz, CDCl3)
(ppm) ¼ 135.4 (d,
d
JC-P ¼ 4.6 Hz), 129.4 (d, JC-P ¼ 1.9 Hz), 128.7 (d, JC-P ¼ 2.4 Hz), 123.7
(d, JC-P ¼ 8.4 Hz), 63.7 (d, JC-P ¼ 6.0 Hz), 15.8 (d, JC-P ¼ 7.3 Hz). 31P
NMR (162 MHz, CDCl3)
d
(ppm) ¼ 18.0 (quint, JP-H ¼ 9.0 Hz). 77Se-
{1H} NMR (76 MHz, CDCl3)
d
(ppm) ¼ 263.8 (d, JSe-P ¼ 480.6 Hz). IR
(cmꢁ1): 2988, 2821, 1484, 1479, 1271, 986, 854, 744, 713. MS: m/z
(rel. int., %) 294 (Mþ, 32.8), 238 (6.5), 158 (32.0), 137 (25.9), 109
(100.0), 81 (39.7), 77 (28.7), 51 (10.2). HRMS (APCI-QTOF) calcu-
lated mass for C10H15O3PSe [MþH]þ: 295.0002, found: 295.010.
4.1.3. O,O-dibutyl Se-phenyl phosphoroselenoate (3c)
[38] Yield: 0.0858 g (98%); Yellowish oil. 1H NMR (400 MHz,
CDCl3)
d
(ppm) ¼ 7.59e7.56 (m, 2H); 7.29e7.21 (m, 3H); 4.08e3.97
(m, 4H); 1.55 (quint, J ¼ 7.4 Hz, 4H); 1.27 (sex, J ¼ 7.4 Hz, 4H); 0.82 (t,
J ¼ 7.4 Hz, 6H). 13C-{1H} NMR (100 MHz, CDCl3)
(ppm) ¼ 135.4 (d,
d
JC-P ¼ 4.7 Hz), 129.3 (d, JC-P ¼ 1.9 Hz), 128.6 (d, JC-P ¼ 2.3 Hz), 123.7
(d, JC-P ¼ 8.4 Hz), 67.4 (d, JC-P ¼ 6.4 Hz), 31.9 (d, JC-P ¼ 7.2 Hz), 18.5,
13.4.31P NMR (162 MHz, CDCl3)
d
(ppm) ¼ 18.0 (quint, JP-H ¼ 8.0 Hz).
77Se-{1H} NMR (76 MHz, CDCl3)
d
(ppm)
¼
263.4 (d, JSe-
¼ 478.5 Hz). IR (cmꢁ1): 2967, 2869, 1576, 1434, 1258, 1019, 740,
P
534. MS: m/z (rel. int., %) 350 (Mþ, 23.9), 294 (17.4), 238 (88.6), 158
(45.6), 137 (50.7), 77 (27.5), 57 (100.0), 55 (20.8), 51 (9.2), 41 (52.8).
HRMS (APCI-QTOF) calculated mass for C14H23O3PSe [MþH]þ:
351.0628, found: 351.0643.
Chemical shifts are reported in ppm, referenced to
a,a,a-tri-
fluorotoluene (ꢁ63.72 ppm). Coupling constant (J) are reported in
Hertz. Abbreviations to denote the multiplicity of a particular signal
is m (multiplet). Low-resolution mass spectra (MS) were obtained
with a Shimadzu GC-MS-QP2010 mass spectrometer. The high-
resolution mass spectrometry (QTOF) analysis was performed on
a Bruker Daltonics micrOTOF-Q II instrument in operating positive
mode. The samples were solubilized in HPLC-grade acetonitrile and
injected into APCI source by means of a syringe pump at a flow rate
4.1.4. O,O-dibenzyl Se-phenyl phosphoroselenoate (3d)
[38] Yield: 0.1024 g (98%); Orange solid; m.p. ¼ 75.4e77.8 ꢀC. 1H
NMR (400 MHz, CDCl3)
d
(ppm) ¼ 7.48 (d, J ¼ 7.7 Hz, 2H); 7.28e7.14
(m, 13H); 5.08e4.99 (m, 4H). 13C-{1H} NMR (100 MHz, CDCl3)
d
(ppm) ¼ 135.8 (d, JC-P ¼ 4.5 Hz), 135.2 (d, JC-P ¼ 7.8 Hz),129.4 (d, JC-
of 5.0
capillary and cone voltages were set to þ3500 V and ꢁ500 V,
m
L minꢁ1. The follow instrument parameters were applied:
¼ 1.9 Hz), 128.8 (d, JC-P ¼ 2.5 Hz), 128.5 (d, JC-P ¼ 2.3 Hz), 128.0,
P
123.2 (d, JC-P ¼ 8.5 Hz), 69.1 (d, JC-P ¼ 6.1 Hz). 31P NMR (162 MHz,
8

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
CDCl3)
(76 MHz, CDCl3)
d
(ppm) ¼ 18.6 (quint, JP-H ¼ 8.0 Hz). 77Se-{1H} NMR
d
(ppm) ¼ 7.90 (s, 1H); 7.85 (d, J ¼ 7.7 Hz, 1H); 7.63 (d, J ¼ 7.8 Hz,
d
(ppm) ¼ 275.0 (d, JSe-P ¼ 495.1 Hz). IR (cmꢁ1):
1H); 7.46 (t, J ¼ 7.8 Hz, 1H), 3.82 (d, JH-P ¼ 13.2 Hz, 6H). 13C-{1H}
2984, 2892, 2823, 1465, 1291, 986, 747, 702. MS: m/z (rel. int., %) 418
(Mþ, 2.3), 248 (5.4), 91 (100.0), 77 (7.2), 65 (14.3), 51 (4.8). HRMS
(APCI-QTOF) calculated mass for C20H19O3PSe [MþH]þ: 419.0315,
found: 419.0324.
NMR (100 MHz, CDCl3)
d
(ppm) ¼ 138.8 (d, JC-P ¼ 4.1 Hz),
132.3e132.0 (m), 131.8 (qd, JC-F/CeP ¼ 32.3 and 2.5 Hz), 129.9 (d, JC-
¼ 1.6 Hz), 125.8e125.7 (m), 124,4 (d, JC-P ¼ 8.6 Hz), 123.4 (q, JC-
P
¼ 271.2 Hz), 54.1 (d, JC-P ¼ 5.8 Hz). 31P NMR (162 MHz, CDCl3)
F
d
(ppm) ¼ 20.7 (sept, JP-H ¼ 13.2 Hz). 77Se-{1H} NMR (76 MHz,
4.1.5. O,O-dimethyl Se-(p-tolyl) phosphoroselenoate (3f)
CDCl3)
d
(ppm) ¼ 255.9 (d, JSe-P ¼ 475.1 Hz). IR (cmꢁ1): 3031, 2945,
[33] Yield: 0.0623 g (89%); Yellow oil. 1H NMR (400 MHz, CDCl3)
1534, 1476, 1242, 1050, 901, 793.
d
(ppm) ¼ 7.51 (dd, J ¼ 8.0 and 1.6 Hz, 2H); 7.12 (d, J ¼ 7.9 Hz, 2H);
3.78 (d, JH-P ¼ 13.1 Hz, 6H); 2.34 (s, 3H). 13C-{1H} NMR (100 MHz,
4.1.10. Se-mesityl O,O-dimethyl phosphoroselenoate (3k)
CDCl3)
d
(ppm) ¼ 139.1 (d, JC-P ¼ 2.9 Hz), 135.6 (d, JC-P ¼ 4.3 Hz),
Yield: 0.0755 g (98%); Yellow oil. 1H NMR (400 MHz, CDCl3)
130.3 (d, JC-P ¼ 2.1 Hz), 119.3 (d, JC-P ¼ 8.7 Hz), 53.8 (d, JC-P ¼ 5.6 Hz),
d
(ppm) ¼ 6.95 (s, 2H); 3.72 (d, JH-P ¼ 12.7 Hz, 6H); 2.56 (s, 6H); 2.26
21.1.31P NMR (162 MHz, CDCl3)
d
(ppm) ¼ 22.3 (sept, JP-H ¼ 13.1 Hz).
(s, 3H). 13C-{1H} NMR (100 MHz, CDCl3)
d
(ppm) ¼ 143.7 (d, JC-
77Se-{1H} NMR (76 MHz, CDCl3)
d
(ppm)
¼
234.4 (d, JSe-
¼ 3.9 Hz), 139.4 (d, JC-P ¼ 3.7 Hz), 128.9 (d, JC-P ¼ 3.1 Hz), 121.2 (d,
P
¼ 495.6 Hz). IR (cmꢁ1): 2990, 2859, 1495, 1449, 1210, 1029, 802,
JC-P ¼ 9.2 Hz), 53.9 (d, JC-P ¼ 7.0 Hz), 24.7, 20.8.31P NMR (162 MHz,
P
731. MS: m/z (rel. int., %) 279 (Mþ, 37.1), 171 (11.3), 109 (100.0), 91
(40.0), 79 (17.3), 65 (8.9). HRMS (APCI-QTOF) calculated mass for
C9H13O3PSe [MþH]þ: 280.9846, found: 280.9840.
CDCl3)
d
(ppm) ¼ 20.6 (sept, JP-H ¼ 12.7 Hz). 77Se-{1H} NMR
(76 MHz, CDCl3)
d
(ppm) ¼ 150.1 (d, JSe-P ¼ 513.9 Hz). IR (cmꢁ1):
2949, 2823,1462,1398,1222, 987, 889, 802. MS: m/z (rel. int., %) 307
(Mþ, 15.2), 198 (15.8), 119 (100.0), 109 (6.0), 91 (13.2), 77 (5.6), 41
(2.8). HRMS (APCI-QTOF) calculated mass for C11H17O3PSe [MþH]þ:
309.0159, found: 309.0155.
4.1.6. Se-(4-methoxyphenyl) O,O-dimethyl phosphoroselenoate
(3 g)
Yield: 0.0725 g (98%); Yellow oil. 1H NMR (400 MHz, CDCl3)
d
(ppm) ¼ 7.52 (dd, J ¼ 8.7 and 1.9 Hz, 2H); 6.85 (d, J ¼ 8.7 Hz, 2H);
4.1.11. Se-butyl O,O-dimethyl phosphoroselenoate (3 m)
3.80 (s, 6H); 3.77 (s, 3H). 13C-{1H} NMR (100 MHz, CDCl3)
[38] Yield: 0.0589 g (86%); Yellowish oil. 1H NMR (400 MHz,
d
(ppm) ¼ 160.4 (d, JC-P ¼ 2.6 Hz), 137.3 (d, JC-P ¼ 4.1 Hz), 115.3 (d, JC-
CDCl3)
d
(ppm) ¼ 3.78 (d, JH-P ¼ 13.2 Hz, 6H); 2.92e2.84 (m, 2H);
¼ 2.3 Hz), 112.9 (d, JC-P ¼ 8.7 Hz), 55.2, 53.9 (d, JC-P ¼ 5.8 Hz). 31
P
1.75 (quint, J ¼ 7.4 Hz, 2H); 1.43 (sex, J ¼ 7.4 Hz, 2H); 0.93 (t,
P
NMR (162 MHz, CDCl3)
d
(ppm) ¼ 22.3 (sept, JP-H ¼ 13.0 Hz). 77Se-
J ¼ 7.4 Hz, 3H). 13C-{1H} NMR (100 MHz, CDCl3)
d
(ppm) ¼ 53.5 (d,
{1H} NMR (76 MHz, CDCl3)
d
(ppm) ¼ 230.6 (d, JSe-P ¼ 500.7 Hz). IR
JC-P ¼ 5.6 Hz), 33.2 (d, JC-P ¼ 4.6 Hz), 26.1 (d, JC-P ¼ 4.7 Hz), 22.6,
(cmꢁ1): 2995, 2866, 1492, 1428, 1256, 1008, 880, 719. MS: m/z (rel.
int., %) 295 (Mþ, 57.1), 263 (10.4), 187 (26.7), 121 (9.1), 109 (100.0),
79 (16.0), 63 (10.8), 43 (2.8). HRMS (APCI-QTOF) calculated mass for
C9H13O4PSe [MþH]þ: 296.9795, found: 296.9796.
13.3.31P NMR (162 MHz, CDCl3)
77Se NMR (76 MHz, CDCl3)
d
(ppm) ¼ 25.3 (sept, JP-H ¼ 13.2 Hz).
d
(ppm) ¼ 95.0 (d quint, JSe-P/
SeeH ¼ 497.2 and 9.8 Hz). IR (cmꢁ1): 2961, 2844, 1465, 1412, 1210,
989, 812, 723. MS: m/z (rel. int., %) 246 (Mþ, 14.7), 190 (88.4), 109
(100.0), 79 (70.0), 55 (24.8), 41 (23.2). HRMS (APCI-QTOF) calcu-
lated mass for C6H15O3PSe [MþH]þ: 247.0002, found: 246.9998.
4.1.7. Se-(4-chlorophenyl) O,O-dimethyl phosphoroselenoate (3h)
[38] Yield: 0.0652 g (87%); Yellow oil. 1H NMR (400 MHz, CDCl3)
d
(ppm) ¼ 7.57 (dd, J ¼ 8.5 and 1.9 Hz, 2H); 7.29 (d, J ¼ 8.5 Hz, 2H);
4.1.12. O,O-dimethyl Te-phenyl phosphorotelluroate (3n)
3.80 (d, JH-P ¼ 13.2 Hz, 6H). 13C-{1H} NMR (100 MHz, CDCl3)
[33] Yield: 0.0553 g (70%); Orange oil. 1H NMR (400 MHz, CDCl3)
d
(ppm) ¼ 136.8 (d, JC-F ¼ 4.5 Hz), 135.5 (d, JC-F ¼ 3.0 Hz), 129.7 (d, JC-
d
(ppm) ¼ 7.84 (dt, J ¼ 7.7 and 1.4 Hz, 2H); 7.37 (td, J ¼ 7.7 and 1.4 Hz,
¼ 2.1 Hz), 121.3 (d, JC-F ¼ 8.7 Hz), 54.0 (d, JC-F ¼ 5.9 Hz). 31P NMR
1H); 7.26 (t, J ¼ 7.7 Hz, 2H); 3.73 (d, JH-P ¼ 13.6 Hz, 6H). 13C-{1H}
F
(162 MHz, CDCl3)
NMR (76 MHz, CDCl3)
d
(ppm) ¼ 21.2 (sept, JP-H ¼ 13.2 Hz). 77Se-{1H}
NMR (100 MHz, CDCl3)
d
(ppm) ¼ 139.9 (d, JC-P ¼ 3.9 Hz), 129.7 (d,
d
(ppm) ¼ 243.3 (d, JSe-P ¼ 483.0 Hz). IR
JC-P ¼ 1.8 Hz), 129.0 (d, JC-P ¼ 2.2 Hz), 108.3 (d, JC-P ¼ 8.1 Hz), 53.6 (d,
(cmꢁ1): 2987, 2891, 1502, 1471, 1227, 1018, 851, 730. MS: m/z (rel.
int., %) 299 (Mþ, 17.1), 190 (5.4), 156 (7.2), 109 (100.0), 79 (13.8), 47
(2.2). HRMS (APCI-QTOF) calculated mass for C8H10ClO3PSe
[MþH]þ: 300.9300, found: 300.9299.
JC-P ¼ 5.4 Hz). 31P NMR (162 MHz, CDCl3)
d
(ppm) ¼ 3.7 (sept, JP-
¼ 13.6 Hz). 125Te-{1H} NMR (126 MHz, CDCl3)
(ppm) ¼ 349.9 (d,
d
H
JTe-P ¼ 1374.6 Hz). IR (cmꢁ1): 3011, 2892, 1491, 1483, 1259, 1001, 891,
787. MS: m/z (rel. int., %) 316 (Mþ, 20.7), 207 (12.2), 155 (14.0), 109
(100.0), 79 (20.7), 77 (68.0), 51 (30.3).
4.1.8. Se-(4-fluorophenyl) O,O-dimethyl phosphoroselenoate (3i)
Yield: 0.0682 g (96%); Yellow oil. 1H NMR (400 MHz, CDCl3)
4.1.13. O,O-diethyl Te-phenyl phosphorotelluroate (3ꢀ)
d
(ppm) ¼ 7.64e7.60 (m, 2H); 7.03 (t, J ¼ 8.6 Hz, H); 3.80 (d, JH-
[33] Yield: 0.0808 g (94%); Orange oil. 1H NMR (400 MHz, CDCl3)
¼ 13.2 Hz, 6H). 13C-{1H} NMR (100 MHz, CDCl3)
d
(ppm) ¼ 163.3
d
(ppm) ¼ 7.84 (d, J ¼ 7.5 Hz, 2H); 7.36 (t, J ¼ 7.5 Hz, 1H); 7.25 (t,
P
(dd, JC-F/CeP ¼ 248.2 and 2.9 Hz), 137.7 (dd, JC-F/CeP ¼ 8.1 and
4.3 Hz),117.7 (dd, JC-P/CeF ¼ 8.7 and 3.5 Hz),116.8 (dd, JC-F/CeP ¼ 21.8
and 2.2 Hz), 54.0 (d, JC-P ¼ 5.8 Hz). 31P NMR (162 MHz, CDCl3)
J ¼ 7.5 Hz, 2H); 4.21e4.06 (m, 4H); 1.31 (t, J ¼ 7.0 Hz, 6H). 13C-{1H}
NMR (100 MHz, CDCl3)
d
(ppm) ¼ 139.8 (d, JC-P ¼ 3.9 Hz), 129.6 (d,
JC-P ¼ 1.6 Hz), 128.8 (d, JC-P ¼ 2.1 Hz), 108.7 (d, JC-P ¼ 8.0 Hz), 63.4 (d,
d
(ppm) ¼ 21.5 (sept d, JP-H/PeF ¼ 13.1 and 4.2 Hz); 77Se-{1H} NMR
JC-P ¼ 5.5 Hz), 15.7 (d, JC-P ¼ 7.3 Hz). 31P NMR (162 MHz, CDCl3)
(76 MHz, CDCl3)
(376 MHz, CDCl3)
d
(ppm) ¼ 239.8 (d, JSe-P ¼ 486.7 Hz). 19F NMR
d
(ppm) ¼ ꢁ1.1 (quint, JP-H ¼ 9.3 Hz). 125Te-{1H} NMR (126 MHz,
d
(ppm) ¼ ꢁ111.66 to ꢁ111.75 (m). IR (cmꢁ1):
CDCl3)
d
(ppm) ¼ 382.7 (d, JTe-P ¼ 1341.8 Hz). IR (cmꢁ1): 2972, 2861,
3007, 2979, 1515, 1489, 1455, 1276, 1041, 899, 764. MS: m/z (rel. int.,
%) 284 (Mþ, 18.7), 175 (10.4), 109 (100.0), 95 (5.8), 79 (16.5), 57 (3.0).
HRMS (APCI-QTOF) calculated mass for C8H10FO3PSe [MþH]þ:
284.9595, found: 284.9597.
1453, 1429, 1220, 983, 880, 717.MS: m/z (rel. int., %) 344 (Mþ, 24.0),
205 (11.5), 137 (18.8), 109 (100.0), 81 (45.1), 77 (65.7), 51 (25.5).
HRMS (APCI-QTOF) calculated mass for C10H15O3PTe [MþH]þ:
344.9899, found: 344.9908.
4.1.9. O,O-dimethyl Se-(3-(trifluoromethyl)phenyl)
phosphoroselenoate (3j)
4.1.14. O,O-dibutyl Te-phenyl phosphorotelluroate (3p)
[32] Yield: 0.0810 g (81%); Orange oil. 1H NMR (400 MHz, CDCl3)
[38] Yield: 0.0685 g (82%); Yellow oil. 1H NMR (400 MHz, CDCl3)
d
(ppm) ¼ 7.84 (dt, J ¼ 8.0 and 1.3 Hz, 2H); 7.36 (td, J ¼ 7.5 and
9

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
1.3 Hz, 1H); 7.27e7.22 (m, 2H); 4.13e3.99 (m, 4H); 1.63 (quint,
J ¼ 7.4 Hz, 6H); 1.34 (sex, J ¼ 7.4 Hz, 4H); 0.89 (t, J ¼ 7.4 Hz, 6H). 13C-
4.2.2. In vitro assays
Phosphorochalcogenoates 3a, 3c, 3d, 3f and 3g and positive
control, trolox (10 M), were dissolved in dimethyl sulfoxide
{1H} NMR (100 MHz, CDCl3)
d
(ppm) ¼ 139.9 (d, JC-P ¼ 4.1 Hz), 129.6
m
(d, JC-P ¼ 1.7 Hz), 128.8 (d, JC-P ¼ 2.2 Hz), 108.8 (d, JC-P ¼ 8.1 Hz), 67.2
(DMSO) for all in vitro assays. Mice were euthanized and samples of
the whole brain were rapidly removed, placed on ice and weighed.
Tissues were immediately homogenized in cold 50 mM TriseHCl,
pH 7.4 (1/5, weight/volume, w/v). The homogenate was centri-
fuged for 10 min at 2400 g for 10 min at 4 ꢀC to yield a pellet that
was discarded, and a low-speed supernatant (S1) was obtained. S1
was used to carry out the effect of compounds 3a, 3c, 3d, 3f and 3g
on lipid peroxidation and reactive species levels.
(d, JC-P ¼ 6.2 Hz), 31.9 (d, JC-P ¼ 7.3 Hz), 18.7, 13.5.31P NMR (162 MHz,
CDCl3)
(126 MHz, CDCl3)
d
(ppm) ¼ ꢁ0.9 (quint, JP-H ¼ 8.1 Hz) 125Te-{1H} NMR
d
(ppm) ¼ 381.4 (d, JTe-P ¼ 1337.1 Hz). IR (cmꢁ1):
2947, 2841, 1478, 1437, 1230, 996, 850, 741.MS: m/z (rel. int., %) 400
(Mþ, 17.8), 342 (6.1), 288 (22.3), 206 (12.5), 137 (60.9), 77 (51.6), 57
(100.0). HRMS (APCI-QTOF) calculated mass for
C14H23O3PTe
[MþH]þ: 401.0525, found: 401.0525.
4.2.3. Lipid peroxidation assay
4.1.15. O,O-dibenzyl Te-phenyl phosphorotelluroate (3q)
[32] Yield: 0.0936 g (80%); Yellow oil. 1H NMR (400 MHz, CDCl3)
Lipid peroxidation in the cerebral tissue was performed by the
formation of thiobarbituric acid reactive species (TBARS) during an
acid-heating reaction as previously described by Ohkawa, Ohishi
d
(ppm) ¼ 7.75 (d, J ¼ 7.8 Hz, 2H); 7.37e7.30 (m, 7H); 7.27e0.7.24
(m, 4H); 7.18 (t, J ¼ 7.8 Hz, 2H); 5.08 (dd, J ¼ 8.6 and 3.2 Hz, 4H). 13C-
{1H} NMR (100 MHz, CDCl3)
d
(ppm) ¼ 140.2 (d, JC-P ¼ 3.9 Hz), 135.2
and Yagi [72]. The 20
in the presence of compounds (10e500
m
L aliquot of the brain total was pre-incubated
M) with sodium nitro-
m
(d, JC-P ¼ 7.8 Hz), 129.6 (d, JC-P ¼ 2.0 Hz), 128.9 (d, JC-P ¼ 2.1 Hz),
prusside (30 mM) at 37 ꢀC for 1 h. The sodium nitroprusside was
used as classical inductors of lipid peroxidation. The mixture was
then incubated with sodium dodecyl sulfate (SDS, 8.1%), TBA (0.8%),
and acetic acid/HCl (pH 3.4) at 95 ꢀC for 2 h. Malondialdehyde
(MDA), an end product of the peroxidation of lipids, was used as a
biomarker of fatty acid peroxidation. The absorbance of the sample
was measured at 532 nm. Results are expressed as percentage of
lipid peroxidation.
128.5, 128.5, 128.1, 108.5 (d, JC-P ¼ 8.0 Hz), 68.7 (d, JC-P ¼ 5.8 Hz). 31
P
NMR (162 MHz, CDCl3)
d
(ppm) ¼ ꢁ1.8 (quint, JP-H ¼ 8.6 Hz). 125Te-
{1H} NMR (126 MHz, CDCl3)
d
(ppm) ¼ 424.7 (d, JTe-P ¼ 1387.4 Hz).
IR (cmꢁ1): 3007, 2866, 1476, 1419, 1279, 1021, 895, 756. MS: m/z (rel.
int., %) 468 (Mþ, 0.6), 207 (3.0), 91 (100.0), 78 (28.5), 65 (14.4), 51
(20.7). HRMS (APCI-QTOF) calculated mass for
C20H19O3PTe
[MþH]þ: 469.0212, found: 469.0208.
4.1.16. O,O-dimethyl Te-(p-tolyl) phosphorotelluroate (3s)
4.2.4. Reactive species assay
[33] Yield: 0.0742 g (90%); Brown oil. 1H NMR (400 MHz, CDCl3)
To estimate the level of reactive species production, samples of
d
(ppm) ¼ 7.72 (d, J ¼ 7.3 Hz, 2H); 7.07 (d, J ¼ 7.3 Hz, 2H); 3.72 (d, JH-
¼ 13.5 Hz, 6H); 2.34 (s, 3H). 13C-{1H} NMR (100 MHz, CDCl3)
(ppm) ¼ 140.1 (d, JC-P ¼ 3.7 Hz), 139.3 (d, JC-P ¼ 2.7 Hz), 130.7 (d, JC-
brain total were incubated with 10
1 mM) in the presence or the absence of a pro-oxidant (sodium
azide; 1 mM) and compound (1e500 M). The reactive species
ml of dichlorofluorescein (DCF;
P
d
m
¼ 2.1 Hz), 104.1 (d, JC-P ¼ 8.1 Hz), 53.5 (d, JC-P ¼ 5.3 Hz), 21.2.31
P
P
levels were determined by a spectrofluorimetric method, using
20,70-dichlorofluorescein diacetate (DCHF-DA) assay. The oxidation
of DCHF-DA to fluorescent dichlorofluorescein (DCF) is measured
for the detection of intracellular reactive species. The DCF fluores-
cence intensity emission was recorded at 520 nm (with 488 nm
excitation) 30 min after the addition of DCHF-DA to the medium
[73]. The reactive species levels were expressed as arbitrary units of
fluorescence.
NMR (162 MHz, CDCl3)
d
(ppm) ¼ 3.8 (sept, JP-H ¼ 13.5 Hz). 125Te-
{1H} NMR (126 MHz, CDCl3)
d
(ppm) ¼ 346.7 (d, JTe-P ¼ 1390.3 Hz).
IR (cmꢁ1): 2957, 2830, 1478, 1461, 1227, 985, 877, 747. MS: m/z (rel.
int., %) 330 (Mþ, 23.4), 298 (11.1), 221 (15.3), 169 (17.1), 109 (78.5),
91 (100.0), 79 (16.9), 65 (37.3), 44 (6.34). HRMS (APCI-QTOF)
calculated mass for C9H13O3PTe [MþH]þ: 330.9743, found:
330.9748.
4.1.17. Te-butyl O,O-dimethyl phosphorotelluroate (3u)
4.2.5. Ferric ion reducing antioxidant power
The ferric ion reducing antioxidant power (FRAP) assay evaluate
the ability of the compounds to reduce the ferric ions [74]. Different
[32]Yield: 0.0422 g (57%); Orange oil. 1H NMR (400 MHz, CDCl3)
d
(ppm) ¼ 3.66 (d, JH-P ¼ 13.6 Hz, 6H); 2.81 (dt, J ¼ 12.8 Hz and
J ¼ 6.4, 2H); 1.77 (quint, J ¼ 7.4 Hz, 2H); 1.33 (sex, J ¼ 7.4 Hz, 2H);
0.86 (d,
7.4 Hz, 3H). 13C-{1H} NMR (100 MHz, CDCl3)
concentrations of compounds (10e500
mM) and FRAP reagent
J
¼
(FeCl3, triazine and sodium acetate buffer pH 3.2; 10:1:1) were
mixed and the mixture was incubated at 37 ꢀC for 40 min in the
dark. The absorbance of the resulting solution was measured at
593 nm.
d
(ppm) ¼ 53.2 (d, JC-P ¼ 5.3 Hz), 34.3 (d, JC-P ¼ 3.5 Hz), 24.8,13.2, 8.6
(d, JC-P ¼ 3.9 Hz). 31P NMR (162 MHz, CDCl3)
(ppm) ¼ 1.6 (sept, JP-
d
¼ 13.5 Hz). 125Te NMR (126 MHz, CDCl3)
d
(ppm) ¼ 91.4 (d quint,
H
J
Te-P/TeeH. ¼ 1391.7 and 22.0 Hz). IR (cmꢁ1): 2961, 2839, 1477, 1434,
1263, 1002, 840, 717. MS: m/z (rel. int., %) 296 (Mþ, 14.7), 240 (54.0),
4.2.6. 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity
The scavenger activity of the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical was evaluated according to the methodology of
the literature with some modifications [75]. For this assay different
238 (50.8), 109 (100.0), 93 (17.6), 79 (33.1), 57 (31.9), 41 (28.1).
4.2. Biological materials and methods
concentrations (10e500
mixed with 990 L of an ethanolic solution containing the DPPH
(50
mM) of the phosphoroselenoates were
4.2.1. Animals
m
The experiments were conducted using male adult Swiss mice
(25e35 g) from our own breeding colony. The animals were kept in
a separate animal room, on a 12 h light/dark cycle with lights on at
7:00 a.m., at room temperature (22 1 ꢀC) with free access to water
and food. The animals were used according to the guidelines of the
Committee on Care and Use of Experimental Animal Resources, the
Federal University of Pelotas, Brazil (13,008e2020). All efforts were
made to minimize animals suffering and to reduce the number of
animals used in the experiments.
mM) radicals. Subsequently, this mixture was stirred and incu-
bated for 30 min at 30 ꢀC in the dark. Thereafter, the absorbance
was read at a wavelength of 517 nm, which the decrease in absor-
bance indicates the neutralization of the DPPH radicals.
4.2.7. Azinobis-3-ethyl-benzothiazoline-6-sulfonic acid radical
scavenging activity
The neutralization evaluation of the radiation of azinobis-3-
ethyl-benzothiazoline-6-sulfonic
acid
(ABTSþ.)
by
10

D.H. Mailahn, L.E.B. Iarocz, P.C. Nobre et al.
European Journal of Medicinal Chemistry 213 (2021) 113052
phosphoroselenoate was carried out according to a description
4.2.13. Statistical analysis
methodology [76] with some modifications. Different concentra-
The results as means standard error of the mean (SEM) for the
experiments data were performed using one-way analysis of vari-
ance followed by Tukey test when appropriated and Student-t test.
All tests were performed at least three times in duplicate. The p
values less than 0.05 (p < 0.05) were considered as indicative of
significance.
tions of compounds (10e500
mM) were mixed with 990 mL the
ABTSþ. solution. The decrease in absorbance was measured at
734 nm, which depicted the scavenging activity of compounds
against ABTS radical.
4.2.8. Ex vivo assays
Declaration of competing interest
Considering that the in vitro experiments pointed out the anti-
oxidant properties of compounds, the 3a, 3c, 3d, 3f and 3g were
used to investigate its ex vivo antioxidant effect in the brain, kidney,
and liver of Swiss mice.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Mice were divided into 6 groups of 5e6 animals each. Animals
belonging to group I received oral application of canola oil (10 mL/
kg of body weight). Mice of groups II, III, IV, V and VI received oral
administration of 3a, 3c, 3d, 3f and 3g (10 mg/kg, intragastric
route), respectively. Thirty minutes after the treatment, mice were
slightly anesthetized with isoflurane for blood collection by heart
puncture in tubes containing heparin. After that the mice were
killed and the samples of liver, kidneys and brain were quickly
removed. The organs were homogenized as described in Section
4.2.2. The low-speed supernatants (S1) were separated and used for
ex vivo assays.
Acknowledgements
~
This study was financed in part by the Coordenaçao de Aper-
feiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance
Code 001. FAPERGS (PqG 17/2551-0000987-8) and FAPERGS/CAPES
April 2018 - DOCFIX 18/2551-0000511-8, CNPq and FINEP are
acknowledged for financial support.
Appendix A. Supplementary data
It is important to mention that the dose (10 mg/kg) of the
compounds and the time (30 min) were determined based on
previous studies by our research group, which tested other organic
selenium compounds showed antioxidant effect in male Swiss mice
in these same dose and time [77,78].
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113052.
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