Synthesis and antioxidant activity of O-alkyl selenocarbamates, selenoureas and selenohydantoins

Article abstract of DOI:10.1016/j.ejps.2012.12.016

The preparation of three different families of lipophilic organoselenium compounds (aryl- and sugarderived selenoureas, O-alkyl selenocarbamates and selenohydantoins) has been carried out in order to evaluate their in vitro antioxidant profile, analyzing the influence of the selenium-containing functional group, and the substituents on the activity. Title compounds have therefore been studied for the first time as free radical, hydrogen peroxide, alkyl peroxides and nitric oxide scavengers using colorimetric methods; furthermore, their glutathione peroxidase-like activity has also been analyzed by NMR spectroscopy. Free radical scavenging activity has been evaluated using the DPPH method; the strongest free radical scavengers were found to be both, aryl- and sugar-derived selenoureas, with EC50 values ranging 19-46 lM. Concerning anti-H₂O₂ activity, measured by the horseradish peroxidase-mediated oxidation of phenol red, the best results were achieved for aryl selenohydantoins, showing a 61-76% inhibition at 0.5 mM concentration. Organoselenium compounds were also found to be capable of inhibiting the chain reaction involving lipid peroxidation (ferric thiocyanate method); thus, when tested at 0.74 mM, sugar selenocarbamates exhibited 49-71% inhibition of alkyl peroxides-mediated degradation of linoleic acid. Nitric oxide scavenging was studied by transforming sodium nitroprusside into nitrite ion, which in turn was transformed into an easily UV-detectable azocompound; aryl selenocarbamates exhibited 64-80% inhibition at 0.71 mM concentration. It has also been demonstrated that selenoxo compounds can behave as excellent glutathione peroxidase mimics; thus a 0.05 molar equiv. of the title compounds catalyzed efficiently the H₂O₂-mediated oxidation of dithiothreitol into the corresponding cyclic disulfide, mimicking removal of H ₂O₂ exerted by glutathione peroxidase; t1/2 values were found to be quite low for aryl- and sugar-derived selenoureas (2.0-12.7 min).

Full text of DOI:10.1016/j.ejps.2012.12.016

European Journal of Pharmaceutical Sciences 48 (2013) 582–592
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Synthesis and antioxidant activity of O-alkyl selenocarbamates, selenoureas and
selenohydantoins
⇑
Penélope Merino-Montiel, Susana Maza, Sergio Martos, Óscar López , Inés Maya,
José G. Fernández-Bolaños
⇑
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Seville, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation of three different families of lipophilic organoselenium compounds (aryl- and sugar-
derived selenoureas, O-alkyl selenocarbamates and selenohydantoins) has been carried out in order to
evaluate their in vitro antioxidant profile, analyzing the influence of the selenium-containing functional
group, and the substituents on the activity. Title compounds have therefore been studied for the first time
as free radical, hydrogen peroxide, alkyl peroxides and nitric oxide scavengers using colorimetric meth-
ods; furthermore, their glutathione peroxidase-like activity has also been analyzed by NMR spectroscopy.
Free radical scavenging activity has been evaluated using the DPPH method; the strongest free radical
scavengers were found to be both, aryl- and sugar-derived selenoureas, with EC50 values ranging
Received 1 September 2012
Received in revised form 19 December 2012
Accepted 20 December 2012
Available online 31 December 2012
Keywords:
Organoselenium compounds
Antioxidant
Free radical scavenger
Oxidative stress
Glutathione peroxidase mimics
19–46 lM. Concerning anti-H2O2 activity, measured by the horseradish peroxidase-mediated oxidation
of phenol red, the best results were achieved for aryl selenohydantoins, showing a 61–76% inhibition
at 0.5 mM concentration. Organoselenium compounds were also found to be capable of inhibiting the
chain reaction involving lipid peroxidation (ferric thiocyanate method); thus, when tested at 0.74 mM,
sugar selenocarbamates exhibited 49–71% inhibition of alkyl peroxides-mediated degradation of linoleic
acid. Nitric oxide scavenging was studied by transforming sodium nitroprusside into nitrite ion, which in
turn was transformed into an easily UV-detectable azocompound; aryl selenocarbamates exhibited
64–80% inhibition at 0.71 mM concentration. It has also been demonstrated that selenoxo compounds
can behave as excellent glutathione peroxidase mimics; thus a 0.05 molar equiv. of the title compounds
catalyzed efficiently the H2O2-mediated oxidation of dithiothreitol into the corresponding cyclic
disulfide, mimicking removal of H2O2 exerted by glutathione peroxidase; t1/2 values were found to be
quite low for aryl- and sugar-derived selenoureas (2.0–12.7 min).
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
and Fanburg, 2000) or gene transcription (Fang et al., 2002); some
others, like NO, behave as a neurotransmitter, or are involved in
Reactive Oxygen Species (ROS) and Reactive Nitrogen Species
(RNS), such as free radicals, superoxide anion, hydroperoxides,
organic peroxides, nitric oxide (NO), or peroxynitrite, are by-
products in the endogenous cellular metabolism. Generation of
free radicals and related compounds takes place in aerobic cells,
and especially in mitochondria (Turrens, 2003), through enzyme
catalyzed-electron transfer reactions to ground state molecular
oxygen in a sequence of steps (Tiwari et al., 2012); on the contrary,
production of NO is obtained under hypoxic conditions in the mito-
chondrial respiratory chain (Reuter et al., 2010).
immune response, when produced by neuronal cells or by macro-
phages, respectively (Fang et al., 2002).
Nevertheless, a disruption of the balance between prooxidant
ROS/RNS and natural antioxidant defences (a complex machinery
comprised of both, low molecular weight compounds, and antiox-
idant enzymes) leads to the accumulation of the former species,
and to a cellular state known as oxidative stress (Kohen and Nyska,
2002). The excessive formation of ROS and RNS provokes a
deleterious effect in cells, and has been related to the degradation
of crucial biomolecules (e.g. lipid peroxidation, alteration of
proteins or DNA) (Reuter et al., 2010). Oxidative stress has been
associated with the development of chronic inflammation (Reuter
et al., 2010), and as a result, to diseases like cancer (Grek and Tew,
2010), or neurodegenerative disorders (e.g. Alzheimer’s disease)
(Butterfield et al., 2006).
Some of such species, like free radicals, are involved in critical
biological processes when present in low concentrations (Dröge,
2002), such as intra- and inter-cellular redox signaling (Thannickal
⇑
Corresponding authors.
E-mail addresses: osc-lopez@us.es (Ó. López), bolanos@us.es (J.G. Fernández-
Therefore, the development of new potent antioxidant agents is
a major goal of Pharmaceutical and Medicinal Chemistry, as a way
Bolaños).
0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.12.016

P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
583
of removing the excess of ROS and RNS, and thus, to ameliorate
their hazardous effects on human beings. In this context, many
organoselenium compounds have shown unique redox properties
in connection with scavenging of prooxidant species (Battin and
Brumaghim, 2009); very frequently they behave as glutathione
peroxidase (GPx) mimics (Merino-Montiel et al., 2012), the enzyme
being one of the most prominent natural antioxidant defences.
Nevertheless, readily-accessible selenoxo (C@Se) derivatives, like
selenoureas (Takahashi et al., 2005a), selenocarbamates (Takahashi
et al., 2005b), or selenoamides (Takahashi et al., 2004), have only
been assayed as superoxide anion scavengers.
2.1.2.2. O-Ethyl-N-(p-methylphenyl)selenocarbamate (3b). Isoselen-
ocyanate 2b (59 mg, 0.30 mmol) was used, and the reaction pro-
ceeded for 7 h. Yield: 63 mg, 86%; RF 0.41 (10:1 hexane–EtOAc);
IR mmax 3202, 2989, 1596, 1534, 1417, 1326, 1220, 1180, 1028,
810 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.23 (brs, 1H, NH), 7.15
;
(m, 4H, ArAH), 4.74 (q, 2H, JH,H = 7.1 Hz, CH2CH3), 2.31 (s, 3H, CH3-
Ar), 1.43 (t, 3H, CH2CH3); 13C NMR (75.5 MHz, CDCl3) d 190.8
(C@Se), 135.9, 134.6 (ArAC), 129.8, 121.8 (ArACH), 72.8 (CH2CH3),
21.1 (CH3Ar), 14.3 (CH2CH3); CIMS m/z 244 ([M+H]+, 14%); HRCI-
MS m/z calcd. for C10H14NO80Se: ([M+H]+): 244.0241, found:
244.0258.
Herein we report the synthesis and the unprecedented in vitro
assay of a panel of related selenoxo compounds (selenoureas,
selenocarbamates and selenohydantoins, having in common a sel-
enocarbamoyl group) as potent RNS and ROS scavengers. Our main
target was to find a relationship between the electronic structure
of the selenoxo group and the antioxidant activity.
2.1.2.3. O-Ethyl-N-(o-methylphenyl)selenocarbamate (3c). Isoselen-
ocyanate 2c (59 mg, 0.30 mmol) was used, and the reaction pro-
ceeded for 9 h. Yield: 67 mg, 92%; RF 0.36 (10:1 hexane–EtOAc);
IR mmax 3168, 2971, 1515, 1339, 1183, 1038, 748 cmꢀ1 1H NMR
;
(300 MHz, CDCl3) d 8.74 (brs, 1H, NH), 7.26–7.20 (m, 4H, ArAH),
4.69 (q, 2H, JH,H = 7.1 Hz, CH2CH3), 2.28 (s, 3H, CH3Ar), 1.35 (t, 3H,
CH2CH3); 13C NMR (75.5 MHz, CDCl3) d 192.1 (C@Se), 135.4,
132.1, 131.0, 127.4, 126.8, 125.3 (ArAC), 72.7 (CH2CH3), 18.1 (CH3-
Ar), 14.3 (CH2CH3); LSIMS m/z 244 ([M+H]+, 100%); HRLSI-MS m/z
calcd. for C10H14NO80Se ([M+H]+): 244.0241, found: 244.0230.
2. Materials and methods
2.1. Chemistry
2.1.2.4.
O-Ethyl-N-(2-isopropyl-6-methylphenyl)selenocarbamate
2.1.1. General procedures
(3d). Isoselenocyanate 2d (71 mg, 0.30 mmol) was used, and the
reaction proceeded for 4 days. 1H NMR data indicated the existence
of E/Z isomers in a 5:1 ratio in CDCl3, and 3:2 in DMSO-d6. Yield:
72 mg, 85%; RF 0.38 (10:1 hexane–EtOAc); IR mmax 3150, 2959,
CH2Cl2, EtOAc, hexane, MeOH, n-PrOH and n-BuOH were dis-
tilled before use; the rest of the chemicals were used without prior
purification.
Optical rotations were measured with a Jasco P-2000 polarime-
ter. 1H (300 MHz) and 13C (75.5 MHz) NMR spectra were recorded
on Bruker Avance-300 spectrometer using CDCl3 as solvent. Chem-
ical shifts are reported in d units (ppm) relative to the solvent peak
(7.26 and 77.16 ppm for 1H and 13C NMR, respectively).
The assignments of 1H and 13C signals were confirmed by
homonuclear COSY, and heteronuclear HSQC spectra, respectively.
Mass spectra (CI and LSI) were recorded on Micromass AutoSpec-Q
mass spectrometer with a resolution of 1000 or 10,000 (10% valley
definition). For LSI spectra, ions were produced by a beam of xenon
atoms and Cs+ ions, using thioglycerol as matrix and NaI as addi-
tive. TLCs were performed on aluminum pre-coated sheets (E.
Merck Silica Gel 60 F254); spots were visualized by UV light, by
charring with 10% H2SO4 in EtOH or with vanillin (1.5% in EtOH
in the presence of 1% H2SO4). Column chromatography was per-
1516, 1338, 1193, 1118, 1045, 779 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) E isomer: d 8.53 (brs, 1H, NH), 7.27–7.02 (m, 3H, ArAH),
4.60 (q, 2H, JH,H = 7.1 Hz, CH2CH3), 3.04 (sept, 1H, JH,H = 6.9 Hz,
CH(CH3)2), 2.20 (s, 3H, CH3Ar), 1.21 (t, 3H, CH2CH3), 1.14 (d, 6H,
CH(CH3)2); Z isomer: d 7.80 (brs, 1H, NH), 7.27–7.02 (m, 3H, ArAH),
4.65 (m, 2H, CH2CH3), 3.04 (sept, 1H, JH,H = 6.9 Hz, CH(CH3)2), 2.24
(s, 3H, CH3Ar), 1.40 (t, 3H, CH2CH3), 1.26, 1.12 (2d, 3H each,
CH(CH3)2); 13C NMR (75.5 MHz, CDCl3) E isomer: d 192.8 (C@Se),
145.8, 135.7, 133.3 (ArAC), 128.9, 128.1, 123.9 (ArACH), 72.3
(CH2CH3), 28.9 (CH(CH3)2), 23.6 (CH(CH3)2), 18.6 (CH3Ar), 14.4
(CH2CH3); Z isomer: d 195.9 (C@Se), 146.7, 136.6, 133.9 (ArAC),
129.1, 128.4, 124.4 (ArACH), 70.5 (CH2CH3), 28.8 (CH(CH3)2),
24.3, 23.4 (CH(CH3)2), 18.8 (CH3Ar), 14.4 (CH2CH3); LSIMS m/z
286 ([M+H]+, 35%); HRLSI-MS m/z calcd. for C13H20NO80Se
([M+H]+): 286.0710, found: 286.0706.
formed using E. Merck Silica Gel 60 (40–63 lm). Microanalyses
were performed at the Instituto de Investigaciones Químicas
CSIC-US, using an elemental analyzer LECO Truspec CHN/CHNS.
2.1.2.5. O-Ethyl-N-(1-naphthyl)selenocarbamate (3e). Isoselenocya-
nate 2e (70 mg, 0.30 mmol) was used, and the reaction proceeded
for 7 h. Yield: 79 mg, 95%; mp: 124–126 °C (EtOH); RF 0.29 (10:1
hexane–EtOAc); IR mmax 3150, 2986, 1527, 1342, 1189, 1127,
2.1.2. General procedure for the synthesis of O-ethyl-N-
arylselenocarbamates (3a-e)
1043, 772 cmꢀ1 1H NMR (300 MHz, CDCl3) d 9.05 (brs, 1H, NH),
;
7.91 (dd, 1H, J6,8 = 1.6 Hz, J7,8 = 7.5 Hz, H-8), 7.89 (dd, 1H,
J5,6 = 7.5 Hz, J5,7 = 1.9 Hz, H-5), 7.83 (brt, 1H, H-3), 7.57 (m, 1H,
J6,7 = 6.8 Hz, H-7), 7.54 (m, 1H, H-6), 7.47 (m, 2H, H-2, H-4), 4.69
(q, 2H, JH,H = 7.0 Hz, CH2CH3), 1.26 (t, 3H, CH2CH3); 13C NMR
(75.5 MHz, CDCl3) d 193.1 (C@Se), 134.3, 132.5, 128.4 (C-1, C-4a,
C-8a), 128.6 (C-5), 128.1 (C-3), 127.1 (C-7), 126.7 (C-6), 125.4,
123.2 (C-2, C-4), 122.2 (C-8), 72.7 (CH2CH3), 14.2 (CH2CH3); LSIMS
m/z 280 ([M+H]+, 65%); HRLSI-MS m/z calcd. for C13H14NO80Se
([M+H]+): 280.0241, found: 280.0249; Anal. calcd. for C13H13NOSe:
C, 56.12; H, 4.71; N, 5.03, found: C, 56.19; H, 4.68; N, 5.12.
A solution of aryl isoselenocyanates 2a–e (0.3 mmol) in EtOH
(6 mL) was refluxed in the darkness during the time indicated in
each case. Then, the crude reaction was concentrated to dryness
and the residue was purified by column chromatography (hex-
ane ? 20:1 hexane–EtOAc) to give selenocarbamates 3a–e.
2.1.2.1. O-Ethyl-N-phenylselenocarbamate (3a). Isoselenocyanate 2a
(55 mg, 0.30 mmol) was used, and the reaction proceeded for 7 h.
Yield: 64 mg, 94%; RF 0.32 (10: 1 hexane–EtOAc); IR mmax 3171,
2962, 1547, 1331, 1180, 1098, 1031, 726 cmꢀ1
;
1H NMR
(300 MHz, CDCl3) d 8.95 (brs, 1H, NH), 7.37–7.18 (m, 5H, ArAH),
4.76 (q, 2H, JH,H = 7.1 Hz, CH2CH3), 1.46 (t, 3H, CH2CH3); 13C NMR
(75.5 MHz, CDCl3) d 191.5 (C@Se), 137.2, 129.4, 126.0, 121.7
(ArAC), 73.1 (CH2CH3), 14.3 (CH2CH3); LSIMS m/z 230 ([M+H]+,
100%); HRLSI-MS m/z calcd. for C9H12NO80Se ([M+H]+): 230.0084,
found: 230.0083.
2.1.3. (S)-5-Isopropyl-3-phenyl-2-selenoxoimidazolidin-4-one (5)
To a solution of phenyl isoselenocyanate (242 mg, 1.33 mmol)
in anhydrous THF (10 mL) were added L-valine ethyl ester hydro-
chloride (362 mg, 1.99 mmol, 1.5 equiv.) and Et3N (0.61 mL,
4.39 mmol, 3.3 equiv.). The mixture was kept stirring at rt under
Ar and in the darkness for 2 h. Then, it was concentrated to dryness

584
P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
and the residue was purified by column chromatography
(t, 1H, J1,2 = 8.7 Hz, H-2), 3.76 (m, 1H, H-5); 13C NMR (75.5 MHz,
CDCl3) major isomer: d 199.4 (C@Se), 171.5, 170.8, 170.0, 169.7
(4CH3CO), 84.8 (C-1), 73.9 (C-5), 72.7 (C-3), 70.6 (C-2), 68.4 (C-4),
61.7 (C-6), 61.0 (OCH3), 20.9, 20.8, 20.7 (x2) (4CH3CO); minor iso-
mer: d 196.5 (C@Se), 80.0 (C-1), 73.7 (C-5), 72.6 (C-3), 70.4 (C-2),
68.1 (C-4); CIMS m/z 470 ([M+H]+, 19%), 409 ([MꢀAcOH]+, 24%);
HRCI-MS m/z calcd. for C16H24NO1080Se ([M+H]+): 470.0565, found:
470.0541.
(CH2Cl2 ? 10:1 CH2Cl2ꢀMeOH) to give
5 (374 mg, quant.).
½
a 2D5
ꢁ
þ 3 (c 1.05, CH2Cl2); 1H NMR (300 MHz, CDCl3): d 8.85 (brs,
1H, NH), 7.54–7.47 (m, 3H, ArAH), 7.31–7.24 (m, 2H, ArAH), 4.00
(d, 1H, JH,H = 3.9 Hz, CHAN), 2.38 (m, 1H, CH(CH3)2), 1.15 (d, 3H,
JH,H = 7.0 Hz, CH(CH3)2), 1.05 (d, 3H, JH,H = 6.8 Hz, CH(CH3)2);
13C NMR (75.5 MHz, CDCl3): d 185.4 (C@Se), 172.6 (C@O), 133.5,
129.7, 129.3, 128.6 (ArAC), 66.3 (CHAN), 31.1 (CH(CH3)2), 18.9,
16.4 (CH(CH3)2; CIMS m/z 282 (M+, 100%); HRCI-MS m/z calcd.
for C12H14N2O280Se (M+): 282.0271, found: 282.0280.
2.1.5.2.
O-Ethyl-N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)sele-
nocarbamate (10b). Ethanol was used. Yield: 53 mg (quant.).
2.1.4. (1S)-N-(1-Ethoxycarbonyl-2-methylpropyl)-N0-(1-
naphthyl)selenourea (6) and (S)-5-isopropyl-3-(1-naphthyl)-2-
selenoxoimidazolidin-4-one (7)
½
a 2D8
ꢁ
þ 16 (c 0.87, CH2Cl2); 1H NMR (300 MHz, CDCl3) major
isomer: d 7.32 (d, 1H, JNH,1 = 9.0 Hz, NH), 5.74 (t, 1H, J1,2 = 9.3 Hz,
H-1), 5.36 (t, 1H, J2,3 = 9.6 Hz, J3,4 = 9.3 Hz, H-3), 5.06 (t, 1H,
J4,5 = 9.9 Hz, H-4), 5.02 (t, 1H, H-2), 4.71–4.49 (m, 2H, OCH2CH3),
4.30 (dd, 1H, J5,6a = 4.8 Hz, J6a,6b = 12.6 Hz, H-6a), 4.11 (dd, 1H,
J5,6b = 1.6 Hz, H-6b), 3.86 (ddd, 1H, H-5), 2.06, 2.05, 2.02, 2.00 (4s,
3H each, 4OAc), 1.33 (t, 3H, JH,H = 7.2 Hz, OCH2CH3); minor isomer:
d 7.81 (d, 1H, JNH,1 = 7.8 Hz, NH), 5.26 (t, 1H, J2,3 = J3,4 = 9.6 Hz, H-3),
5.10–5.04 (m, 2H, H-1, H-4), 4.93 (t, 1H, J1,2 = 9.6 Hz, H-2), 4.21 (dd,
1H, J5,6a = 4.5 Hz, J6a,6b = 12.3 Hz, H-6a), 4.08 (dd, 1H, J5,6b = 1.8 Hz,
H-6b), 3.76 (m, 1H, H-5); 13C NMR (75.5 MHz, CDCl3) major
isomer: d 198.1 (C@Se), 171.3, 170.7, 169.9, 169.7 (4CH3CO), 84.5
(C-1), 73.8 (C-5), 72.7 (C-3), 70.9 (OCH2CH3), 70.5 (C-2), 68.3
(C-4), 61.7 (C-6), 20.8 (x2), 20.6 (x2) (4CH3CO), 14.1 (OCH2CH3);
minor isomer: d 195.1 (C@Se), 170.6, 170.5, 170.1, 169.4 (4CH3CO),
79.9 (C-1), 73.8 (C-5), 72.6 (C-3), 70.3 (C-2), 68.2 (C-4), 61.8 (C-6);
CIMS m/z 484 ([M+H]+, 10%); HRCI-MS m/z calcd. for C17H26NO1080Se
([M+H]+): 484.0722, found: 484.0729.
To
a
solution of 1-naphthyl isoselenocyanate (188 mg,
-valine ethyl
0.81 mmol) in anhydrous THF (10 mL) were added
L
ester hydrochloride (221 mg, 1.21 mmol, 1.5 equiv.) and Et3N
(0.37 mL, 2.68 mmol, 3.3 equiv.). The mixture was kept stirring at
rt under Ar and in the darkness for 2 h. Then, it was concentrated
to dryness and the residue was purified by column chromatogra-
phy (hexane ? 2:1 hexane–EtOAc) to give 6 and 7.
Eluated first was 6: 163 mg, 53%. ½a D25
ꢁ
+59 (c 0.97, CH2Cl2);
1H NMR (300 MHz, CDCl3): d 8.62 (brs, 1H, NHAAr), 8.02 (m, 1H,
ArAH), 7.96–7.86 (m, 2H, ArAH), 7.61–7.45 (m, 4 H, ArAH), 6.49
(d, 1H, JNH,CH = 8.3 Hz, NHACH), 5.14 (dd, 1H, JH,H = 4.5 Hz, CHAN),
4.11–4.02 (m, 2H, CH2ACH3), 2.30–2.23 (m, 1H, CH(CH3)2), 1.18 (t,
3H, JH,H = 7.1 Hz, CH2ACH3), 0.78, 0.75 (2d, 3H each, JH,H = 7.0 Hz,
(CH3)2CH); 13C NMR (75.5 MHz, CDCl3): d 180.3 (C@Se), 171.2
(C@O), 134.8, 131.5, 129.6, 128.7, 127.7, 127.4, 125.8, 125.1,
122.6 (ArAC), 65.3 (CHN), 61.5 (CH2CH3), 31.7 (CH(CH3)2), 18.4,
18.3 (CH(CH3)2), 14.2 (CH2CH3); CIMS m/z 378 (M+, 47%); HRCI-
MS m/z calcd. for C18H22N2O280Se (M+): 378.0846, found: 378.0855.
2.1.5.3. O-n-Propyl-N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)sel-
enocarbamate (10c). Propan-1-ol was used. Yield: 54 mg (quant.).
Eluated second was 7: 68 mg, 25%. ½a D25
ꢁ
ꢀ 20 (c 1.00, CH2Cl2); 1H
½
a 2D7
ꢁ
þ 12 (c 0.78, CH2Cl2); 1H NMR (300 MHz, CDCl3) major
NMR (300 MHz, CDCl3): 1.2:1 atropisomeric mixture d 9.02 (m, 2H,
NH, NH), 8.02–7.93 (m, 4H, ArAH), 7.63–7.52 (m, 8H, ArAH), 7.45
(dd, 1H, JH,H = 1.1 Hz, JH,H = 7.3 Hz, ArAH), 7.37 (dd, 1H, JH,H = 1.1
Hz, JH,H = 7.3 Hz, ArAH), 4.16 (m, 1H, CHAN), 4.08 (m, 1H CHAN),
2.47–2.39 (m, 2H, CH(CH3)2), 1.20 (d, 3H, JH,H = 7.0 Hz, CH(CH3)2),
1.18 (d, 3H, JH,H = 7.1 Hz, CH(CH3)2), 1.16 (d, 3H, JH,H = 7.0 Hz,
CH(CH3)2), 1.10 (d, 3H, JH,H = 6.8 Hz, CH(CH3)2); 13C NMR
(75.5 MHz, CDCl3): d 185.9 (C@Se), 173.0 (C@O), 134.5, 130.6,
130.3, 130.2, 128.9, 128.8, 127.9, 127.7, 127.5, 126.9, 126.8,
125.6, 126.8, 125.6, 125.5, 122.4, 122.3 (ArAC), 66.9, 66.5 (CHAN),
31.2, 30.6 (CH(CH3)2), 19.2, 18.9, 17.1, 16.5 (CH(CH3)2); CIMS m/z
332 (M+, 100%); HRCI-MS m/z calcd. for C16H16N2O280Se (M+):
332.0428, found: 332.0434.
isomer: d 7.27 (d, 1H, JNH,1 = 8.7 Hz, NH), 5.75 (t, 1H, J1,2 = 9.3 Hz,
H-1), 5.37 (t, 1H, J2,3 = J3,4 = 9.6 Hz, H-3), 5.07 (t, 1H, J4,5 = 9.9 Hz,
H-4), 5.03 (t, 1H, H-2), 4.49 (m, 2H, OCH2CH2CH3), 4.32 (dd, 1H,
J5,6a = 4.5 Hz, J6a,6b = 12.3 Hz, H-6a), 4.12 (dd, 1H, J5,6b = 2.1 Hz,
H-6b), 3.87 (ddd, 1H, H-5), 2.08, 2.06, 2.03, 2.02 (4s, 3H each,
4OAc), 1.75 (m, 2H, OCH2CH2CH3), 0.95 (t, 3H, JH,H = 7.5 Hz, OCH2-
CH2CH3); minor isomer: d 7.76 (d, 1H, JNH,1 = 8.7 Hz, NH), 5.26
(t, 1H, J2,3 = 9.3 Hz, J3,4 = 9.6 Hz, H-3), 5.06 (m, 1H, H-4), 5.04 (m,
1H, H-1), 4.94 (t, 1H, J1,2 = 10.2 Hz, H-2), 4.65–4.52 (m, 2H, OCH2-
CH2CH3), 4.22 (dd, 1H, J5,6a = 4.8 Hz, J6a,6b = 12.9 Hz, H-6a), 4.08
(dd, 1H, J5,6b = 2.4 Hz, H-6b), 3.74 (m, 1H, H-5), 1.82 (m, 2H, OCH2-
CH2CH3), 1.00 (t, 3H, JH,H = 7.2 Hz, OCH2CH2CH3); 13C NMR
(75.5 MHz, CDCl3) major isomer: d 198.3 (C@Se), 171.4, 170.8,
169.9, 169.7 (4CH3CO), 84.6 (C-1), 76.5 (OCH2CH2CH3), 73.9 (C-5),
72.7 (C-3), 70.5 (C-2), 68.3 (C-4), 61.7 (C-6), 21.9 (OCH2CH2CH3),
20.9, 20.8, 20.7 (x2) (4CH3CO), 10.3 (OCH2CH2CH3); minor isomer:
d 195.3 (C@Se), 170.7, 170.1 (x2), 169.5 (4CH3CO), 80.0 (C-1), 77.9
(OCH2CH2CH3), 73.8 (C-5), 72.6 (C-3), 70.3 (C-2), 68.2 (C-4), 61.8
(C-6), 22.0 (OCH2CH2CH3), 10.5 (OCH2CH2CH3); CIMS m/z 497
(M+, 12%) 437 ([MꢀAcOH]+, 9%); HRCI-MS m/z calcd. for C18H27
NO1080Se (M+): 497.0800, found: 497.0796.
2.1.5. General procedure for the synthesis of N-
glucopyranosylselenocarbamates 10a–d
A solution of O-protected glucopyranosyl isoselenocyanate 9
(50 mg, 0.11 mmol) in the corresponding alcohol (HPLC grade or
distilled, 8 mL) was kept stirring at 70 °C (or refluxing for metha-
nol) under N2 in the darkness for 2 h. Then, the solvent was re-
moved under reduced pressure to afford selenocarbamates 10a–d
as pure compounds.
2.1.5.4. O-n-Butyl-N-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)sele-
2.1.5.1. O-Methyl-N-(2,3,4,6-tetra-O-acetyl-b-
D
-glucopyranosyl)sele-
nocarbamate (10d). Butan-1-ol was used. Yield: 57 mg (quant.).
nocarbamate (10a). Methanol was used. Yield: 51 mg (quant.).
½
a 3D0
ꢁ
þ 12 (c 1.00, CH2Cl2); 1H NMR (300 MHz, CDCl3) major isomer:
½
a 2D8
ꢁ
þ 12 (c 0.82, CH2Cl2); 1H NMR (300 MHz, CDCl3) major
d 7.24 (d, 1H, JNH,1 = 12.0 Hz, NH), 5.76 (t, 1H, J1,2 = 9.6 Hz, H-1),
5.37 (t, 1H, J2,3 = J3,4 = 9.6 Hz, H-3), 5.07 (t, 1H, J4,5 = 9.6 Hz, H-4),
5.02 (t, 1H, H-2), 4.66–4.44 (m, 2H, OCH2(CH2)2CH3), 4.32 (dd,
1H, J5,6a = 4.5 Hz, J6a,6b = 12.6 Hz, H-6a), 4.12 (dd, 1H, J5,6b = 2.1 Hz,
H-6b), 3.87 (ddd, 1H, H-5), 2.08, 2.06, 2.03, 2.02 (4s, 3H each,
4OAc), 1.69 (m, 2H, OCH2CH2CH2CH3), 1.42–1.32 (m, 2H, O(CH2)2-
CH2CH3), 0.93 (t, 3H, JH,H = 7.1 Hz, O(CH2)3CH3); minor isomer:
d 7.73 (d, 1H, JNH,1 = 9.3 Hz, NH), 5.28 (t, 1H, J2,3 = J3,4 = 9.3 Hz, H-3),
isomer: d 7.33 (d, 1H, JNH,1 = 9.0 Hz, NH), 5.72 (t, 1H, J1,2 = 9.3 Hz,
H-1), 5.37 (t, 1H, J2,3 = 9.3 Hz, J3,4 = 9.6 Hz, H-3), 5.07 (t, 1H,
J4,5 = 10.0 Hz, H-4), 5.02 (t, 1H, H-2), 4.31 (dd, 1H, J5,6a = 4.8 Hz,
J6a,6b = 12.6 Hz, H-6a), 4.12 (dd, 1H, J5,6b = 2.1 Hz, H-6b), 4.11 (s,
3H, OCH3), 3.87 (ddd, 1H, H-5), 2.07, 2.06, 2.03, 2.02 (4s, 3H each,
4OAc); minor isomer: d 7.76 (d, 1H, JNH,1 = 7.2 Hz, NH), 5.27 (t, 1H,
J2,3 = 10.3 Hz, J3,4 = 9.6 Hz, H-3), 5.10–5.02 (m, 2H, H-1, H-4), 4.93

P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
585
5.10–5.04 (m, 2H, H-1, H-4), 4.93 (t, 1H, J1,2 = 9.3 Hz, H-2), 4.65–
4.54 (m, 2H, OCH2(CH2)2CH3), 4.23 (dd, 1H, J5,6a = 4.8 Hz,
J6a,6b = 13.2 Hz, H-6a), 4.23 (dd, 1H, J5,6b = 1.5 Hz, H-6b), 3.73 (m,
1H, H-5), 1.85–1.75 (m, 2H, OCH2CH2CH2CH3), 1.59–1.45 (m, 2H,
O(CH2)2CH2CH3, 0.99–0.90 (m, 3H, O(CH2)3CH3); 13C NMR
(75.5 MHz, CDCl3): d 198.2 (C@Se), 171.4, 170.7, 170.0, 169.7
(4CH3CO), 84.6 (C-1), 74.9 (OCH2(CH2)2CH3), 73.9 (C-5), 72.2 (C-
3), 70.5 (C-2), 68.3 (C-4), 61.8 (C-6), 30.5 (OCH2CH2CH2CH3), 20.9,
20.8, 20.7 (x2) (4CH3CO), 19.1 (O(CH2)2CH2CH3), 13.8 (O(CH2)3
CH3); minor isomer: d 195.3 (C@Se), 170.6, 170.1 (x2), 169.4 (4CH3
CO), 79.9 (C-1), 76.3 (OCH2(CH2)2CH3), 73.8 (C-5), 72.6 (C-3), 70.3
(C-2), 68.1 (C-4), 61.8 (C-6), 29.8 (OCH2CH2CH2CH3), 19.2 (O(CH2)2
CH2CH3), 14.0 (O(CH2)3CH3); CIMS m/z 512 ([M+H]+, 21%), 451
([MꢀAcOH]+, 9%); HRCI-MS m/z calcd. for C19H30NO1080Se
([M+H]+): 512.1035, found: 512.1019.
hydrochloride (55 mg, 0.34 mmol) was used. Column chromatog-
raphy (7:3 CH2Cl2ꢀEtOAc) afforded 11c (94 mg, 72%). ½a D24
þ 31
ꢁ
(c 1.00, CH2Cl2); 1H NMR (300 MHz, CDCl3): d 7.85 (d, 1H,
JNH,H1 = 6.9 Hz, NH), 7.64 (d, 1H, JNH ,H1 = 8.1 Hz, NH0), 5.90 (brs,
0
0
1H, H-10), 5.37–5.27 (m, 2H, H-30, H-1), 5.10 (t, 1H, J1 ,3
=
0
0
J2 ,3 = 9.3 Hz, H-20), 5.09 (t, 1H, J3 ,4 = J4 ,5 = 9.3 Hz, H-40), 4.34 (dd,
0
0
0
0
0
0
1H, J5 ,6 a = 4.2 Hz, J6 a,6 b = 12.3 Hz, H-60a), 4.33–4.20 (m, 2H, OCH2
0
0
0
0
CH3), 4.15 (dd, 1H, J5 ,6 b = 2.1 Hz H-60b), 4.11–4.00 (m, 2H, H-2),
3.88 (ddd, 1H, H-50), 3.33 (brs, 1H, OH), 2.08, 2.06, 2.03, 2.01 (4s,
3H each, 4OAc), 1.30 (t, 3H, JH,H = 6.0 Hz, OCH2CH3); 13C NMR
(75.5 MHz, CDCl3): d 185.3 (C@Se), 171.2 (x2), 170.7, 170.1, 170.0
(5C@O), 84.6 (C-10), 73.8 (C-50), 73.1 (C-30), 70.6 (C-20), 68.3
(C-40), 62.8 (C-2), 62.3 (OCH2CH3), 61.9 (C-1), 61.7 (C-60), 20.9
(x2), 20.7 (3CH3CO), 14.2 (OCH2CH3); CIMS m/z 571 ([M+H]+,
0
0
56%); HRCI-MS m/z calcd. for
C
20H30N2O1280Se ([M+H]+):
571.1042, found: 571.1028.
2.1.6. General procedure for the synthesis of selenoureas 11a–e
To a solution of O-protected glucopyranosyl isoselenocyanate 9
(100 mg, 0.23 mmol) in anhydrous THF (6 mL) was added the cor-
2.1.6.4.
tetra-O-acetyl-b-
(1S)-N-(1-Ethoxycarbonyl-2-methylpropyl)-N0-(20,30,40,60-
-glucopyranosyl)selenourea (11d). -Valine ethyl
D
L
responding aminoacid ester (0.34 mmol) and Et3N (106
lL,
ester hydrochloride (62 mg, 0.34 mmol) was used. Column chro-
0.76 mmol). The mixture was kept stirring at rt under N2 in the
darkness during 20 h. After that, the solvent was removed, and
the residue was purified by column chromatography, using the
eluant indicated in each case.
matography (9:1 CH2Cl2ꢀAcOEt) afforded 11d (94 mg, 72%).
½
a 2D30 (c 0.79, CH2Cl2); 1H NMR (300 MHz, CDCl3): d 7.57 (d, 1H,
ꢁ
JNH,1 = 7.5 Hz, NH), 7.37 (d, 1H, JNH ,H1 = 6.6 Hz, NH0), 5.76 (brs,
0
0
1H, H-10), 5.31 (t, 1H, J2 ,3 = J3 ,4 = 9.3 Hz, H-30), 5.19 (brs, 1H, H-
0
0
0
0
1), 5.04 (t, 1H, J4 ,5 = 9.3 Hz, H-40), 4.99 (t, 1H, J1 ,2 = 9.3 Hz, H-20),
0
0
0
0
2.1.6.1. N-(Ethoxycarbonylmethyl)-N0-(20,30,40,60-tetra-O-acetyl-b-
D
-
4.33 (dd, 1H, J5 ,6 a = 4.8 Hz, J6 a,6 b = 12.6 Hz, H-60a), 4.25–4.13 (m,
0
0
0
0
2H, OCH2CH3), 4.09 (dd, 1H, J5 ,6 b = 1.2 Hz H-60b), 3.87 (ddd, 1H,
H-50), 2.32–2.25 (m, 1H, H-2), 2.04, 2.01, 1.99, 1.97 (4s, 3H each,
4OAc), 1.26 (t, 3H, JH,H = 7.2 Hz, OCH2CH3), 0.97 (d, 3H, JH,H = 6.9 Hz,
CH3), 0.89 (d, 3H, CH3); 13C NMR (75.5 MHz, CDCl3): d 185.6
(C@Se), 171.9, 171.1, 170.8, 170.0, 169.8 (5C@O), 84.2 (C-10), 73.5
(C-50), 72.9 (C-30), 70.5 (C-20), 68.3 (C-40), 64.7 (C-1), 61.8 (C-60),
61.7 (OCH2CH3), 31.6 (C-2), 20.8, 20.7, 20.6 (x2) (4CH3CO), 18.8,
18.2 (CH(CH3)2), 14.2 (OCH2CH3); CIMS m/z 583 ([M+H]+, 35%);
HRCI-MS m/z calcd. for C22H35N2O1180Se ([M+H]+): 583.1406,
found: 583.1396; Anal. calcd. for C22H34N2O1180Se: C, 45.44; H,
5.89; N, 4.82, found: C, 45.37; H, 5.60; N, 4.86.
0
0
glucopyranosyl)selenourea (11a). Glycine ethyl ester hydrochloride
(47 mg, 0.34 mmol) was used. Column chromatography (7:3 CH2-
Cl2ꢀEtOAc) afforded 11a (94 mg, 76%). ½a D28
ꢀ 4 (c 0.97, CH2Cl2);
ꢁ
1H NMR (300 MHz, CDCl3): d 7.32 (t, 1H, JNH,CH2 = 3.8 Hz, NH),
7.26 (m, 1H, NH0), 5.70 (brs, 1H, H-1), 5.36 (t, 1H, J2 ,3 = J3 ,4 = 9.6 -
0
0
0
0
Hz, H-30), 5.08 (t, 1H, J4 ,5 = 10.2 Hz, H-40), 5.03 (t, 1H, J1 ,2 = 9.6 Hz,
0
0
0
0
H-20), 4.31 (dd, 1H, J5 ,6a = 4.8 Hz, J6a ,6b = 12.3 Hz, H-6a0), 4.44 (brs,
2H, NACH2), 4.25 (q, 2H, JH,H = 7.1 Hz, OCH2CH3), 4.16 (dd, 1H,
0
0
0
0
J5 ,6b = 2.1 Hz, H-6b0), 3.89 (ddd, 1H, H-50), 2.07 (x2), 2.04, 2.02
(4s, 3H each, 4OAc), 1.30 (t, 3H, OCH2CH3); 13C NMR (75.5 MHz,
CDCl3): d 185.0 (C@Se), 171.6, 170.9, 170.0, 169.8, 169.6 (5C@O),
84.3 (C-10), 73.7 (C-50), 72.9 (C-30), 70.8 (C-20), 68.4 (C-40), 62.2
(OCH2CH3), 61.9 (C-60), 49.4 (NACH2), 20.9 (x2), 20.8 (x2) (4CH3-
CO), 14.2 (OCH2CH3); CIMS m/z 541 ([M+H]+, 7%); HRCI-MS m/z
calcd. for C19H29N2O1180Se ([M+H]+): 541.0937, found: 541.0917;
Anal. calcd. for C19H28N2O1180Se: C, 42.31; H, 5.23; N, 5.19, found:
C, 42.16; H, 5.01; N, 5.11.
0
0
2.1.6.5.
(200,300,400,600-tetra-O-acetyl-b-
-Tryptophan methyl ester hydrochloride (91 mg, 0.34 mmol) was
(1S)-N-[2-(1H-Indol-30-yl)-1-methoxycarbonylethyl]-N0-
-glucopyranosyl)selenourea (11e).
D
L
used. Column chromatography (9:1 CH2Cl2ꢀEtOAc) afforded 11e
(134 mg, 89%). ½a D22
ꢁ
þ 17 (c 1.08, CH2Cl2); 1H NMR (300 MHz,
CDCl3): d 8.49 (brs, 1H, H-10), 7.47 (d, 1H, J4 ,5 = 7.8 Hz, H-40),
0
0
0
0
2.1.6.2.
tetra-O-acetyl-b-
(1S)-N-(1-Ethoxycarbonyl-2-phenylethyl)-N0-(20,30,40,60-
-glucopyranosyl)selenourea (11b). -Phenylalanine
7.34 (d, 1H, J6 ,7 = 8.0 Hz, H-70), 7.28 (brs, 1H, NH), 7.16 (brt, 1H,
0
0
D
L
J5 ,6 = 7.3 Hz, H-60), 7.08 (brt, 1H, H-50), 6.87 (brs, 1H, H-20), 6.13
ethyl ester hydrochloride (79 mg, 0.34 mmol) was used. Column
(brs, 1H, H-100), 5.55 (m, 1H, H-1), 5.21 (m, 1H, H-300), 4.98 (t, 1H,
00 00
00 00
00 00
chromatography (9:1 CH2Cl2ꢀEtOAc) afforded 11b (119 mg, 82%).
J3 ,4 = 9.6 Hz, J4 ,5 = 9.9 Hz, H-400), 4.91 (brt, 1H, J1 ,2 = 9.3 Hz,
00 00
½
a 2D3
ꢁ
þ 20 (c 1.13, CH2Cl2); 1H NMR (300 MHz, CDCl3): d 7.28–7.27
J2 ,3 = 9.6 Hz, H-200), 4.15 (m, 1H, H-600a), 3.88 (m, 1H, H-600b),
(m, 5H, ArAH, NH), 7.10 (m, 2H, ArAH, NH0), 5.70 (brs, 1H, H-10),
3.88–3.69 (m, 1H, H-500), 3.69 (s, 3H, OMe), 3.64 (dd, 1H,
J1,2a = 4.8 Hz, J2a,2b = 15.0 Hz, H-2a), 3.33 (dd, 1H, J1,2b = 3.4 Hz, H-
2b), 2.02, 1.98, 1.97, 1.96 (4s, 3H each, 4OAc); 13C NMR
(75.5 MHz, CDCl3): d 184.5 (C@Se), 172.2, 171.2, 171.1, 170.0,
169.9 (5C@O), 136.1 (C-30a), 127.9 (C-70a), 123.3 (C-20), 122.3
(C-60), 119.9 (C-50), 119.0 (C-40), 111.5 (C-50), 109.4 (C-30), 83.9
(C-100), 73.2 (C-500), 72.8 (C-300), 70.4 (C-200), 68.3 (C-400), 61.8
(C-600), 60.5 (C-1), 52.7 (OMe), 27.1 (C-2), 20.8 (x2), 20.6 (x2)
(4CH3CO); CIMS m/z 655 (M+, 2%); HRCI-MS m/z calcd. for C27H33
N3O1180Se (M+): 655.1280, found: 655.1268.
5.44 (brs, 1H, H-1), 5.32 (t, 1H, J2 ,3 = J3 ,4 = 9.3 Hz, H-30), 5.06 (t,
0
0
0
0
1H, J4,5 = 9.3 Hz, H-40), 5.00 (t, 1H, J1 ,2 = 9.6 Hz, H-20), 4.32 (dd, 1H,
0
0
J5 ,6 a = 4.2 Hz, J6 a,6 b = 12.3 Hz, H-60a), 4.20–4.12 (m, 2H, OCH2CH3),
4.10–4.04 (m, 1H, H-60b), 3.79 (m, 1H, H-50), 3.39–3.21 (m, 2H, CH2-
Ph), 2.06, 2.03, 2.02, 2.01 (4s, 3H each, 4OAc), 1.28–1.25 (m, 3H,
OCH2CH3); 13C NMR (75.5 MHz, CDCl3): d 184.4 (C@Se), 171.6,
171.3, 170.8, 170.0, 169.7 (5C@O), 135.8, 129.6, 128.6, 127.3 (ArAC),
83.9 (C-10), 73.5 (C-50), 72.8 (C-30), 70.6 (C-20), 68.2 (C-40), 62.0
(OCH2CH3), 61.7 (C-60), 60.5 (C-1), 37.7 (C-2), 20.8 (x2), 20.6 (x2)
(CH3CO), 14.2 (OCH2CH3); CIMS m/z 631 ([M+H]+, 45%); HRCIMS
0
0
0
0
m/z calcd. for
C
26H35N2O1180Se ([M+H]+): 631.1406, found:
2.2. Antioxidant activity
631.1436; Anal. calcd. for C26H34N2O1180Se: C, 49.61; H, 5.44; N,
4.45, found: C, 49.76; H, 5.70; N, 4.09.
2.2.1. Free radical scavenging (DPPH assay)
The free radical scavenging activity was measured using the
DPPH (1,1-diphenyl-2-picrylhydrazyl radical) assay (Prior et al.,
2005) in a Hitachi U-2900 spectrophotometer, using PS cuvettes.
2.1.6.3. (1S)-N-(1-Ethoxycarbonyl-2-hydroxyethyl)-N0-(20,30,40,60-tetra-
O-acetyl-b- -glucopyranosyl)selenourea (11c). L-Serine ethyl ester
D

586
P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
Acontrol ꢀ Asample
The methanolic solution of DPPH exhibits a strong purple color,
% Inhibition ¼
ꢂ 100
with a strong absorption at 515 nm, which is reduced in the pres-
ence of an antioxidant agent, giving a yellowish solution. There-
fore, there is an inverse relationship between the remaining
DPPH concentration and the anti-radical activity of the antioxidant
(Cefarelli et al., 2006).
Acontrol
where Acontrol refers to the solution containing MeOH instead of the
methanolic organoselenium solution, and Asample refers to the
absorbance of solutions containing the organoselenium compounds.
To a 60
was added 30
l
M methanolic solution of DPPH (HPLC-grade, 1.17 mL)
2.2.4. Nitric oxide scavenging activity
Nitric oxide scavenging activity was measured using the Griess
Illosvoy reaction (Garratt, 1964).
To a solution of 40 mM phosphate buffer saline (pH 7.4,
0.15 mL) and MeOH (0.10 mL) were added a 10 mM methanolic
l
L of the methanolic solution of the antioxidant (at 5
different concentrations), or pure methanol (for the control). The
corresponding mixtures were kept in the darkness for 30 min
and then, the absorbance at 515 nm was measured against a MeOH
blank in order to calculate the EC50 values, that is, the concentra-
solution of the antioxidant (50
side in buffer (0.4 mL). The control solution was prepared using
MeOH (50 L) instead of the sample. After incubation at rt for
lL) and a 10 mM sodium nitroprus-
tion of the antioxidant (expressed in
lM) required to reduce the
concentration of the DPPH to 50% of its initial value. All the
measurements were carried out in triplicate. The remaining DPPH
concentration was calculated using the expression:
l
125 min, a 2% (w/v) solution of sulfanilamide in 4% (v/v) H3PO4
(0.3 mL) was added, and the mixture was incubated at rt for
5 min. After that, a 0.2% (w/v) solution of N-(1-naphthy)lethylen-
ediamine dihydrochloride in water (0.3 mL) was added, and the
mixture was incubated at rt for further 30 min. Then, the absor-
bance was measured at 540 nm against a blank (11.5% buffer,
11.5% MeOH, 53.8% H2O, 23.1% H3PO4 (4%)). Methanolic 10 mM
and 100 mM ascorbic acid solutions were used as the reference
compound.
Asample
Acontrol
% DPPH remaining ¼
ꢂ 100
where Asample and Acontrol refer to the absorbances at 515 nm of the
free radical in the sample and control solutions, respectively.
2.2.2. H2O2 scavenging
The results are expressed as the percentage of nitric oxide
scavenging:
The procedure reported by Bahorun et al. was used (Bahorun
et al., 1996). To a solution of 0.003% H2O2 (100
(100 L) and 0.1 M phosphate buffer (pH 7.4, 700
a 10 mM methanolic solution of the organoselenium compound
(100 L), or methanol (for the control). The corresponding mixture
lL), 0.1 M NaCl
Acontrol ꢀ Asample
l
lL) was added
% NO scavenging ¼
ꢂ 100
Acontrol
l
where Acontrol refers to the solution containing pure MeOH instead
of the methanolic organoselenium solution, and Asample refers to
the absorbance of solutions containing the organoselenium
compounds.
was incubated in the darkness at 37 °C for 20 min. Then, a solution
containing phenol red (0.2 mg/mL) and horseradish peroxidase
(0.1 mg/mL) in 0.1 M phosphate buffer (pH 7.4) (1.0 mL) was
added. The corresponding solution was kept at 37 °C during
15 min. Then, 1 M NaOH (100 lL) was added, and the mixture
2.2.5. Glutathione peroxidase (GPx)-like activity
was kept at rt for 10 min, and finally, the absorbance was read at
610 nm against a 18.7:3:1 phosphate buffer:H2O:methanol blank.
All the measurements were carried out in triplicate. The results
are expressed as percentages of reduction of H2O2:
The organoselenium derivatives were evaluated as glutathione
peroxidase mimics by NMR spectroscopy using Iwaoka’s method-
ology (Iwaoka and Kumakura, 2008), based on the reduction of
H2O2 in the presence of dithiothreitol (DTTred) as a thiol cofactor.
In a typical experiment, DTTred (25 mg, 0.161 mmol) and the
A
ꢀ Asample
H2O2
% Reduction H2O2 ¼
ꢂ 100
organoselenium compound (8.0
dissolved in CD3OD (0.6 mL). The reaction was initiated by the
addition of 30% H2O2 (18 L, 0.176 mmol), and monitored follow-
ing the disappearance of the CH protons of DTTred (3.68 ppm),
and the appearance of the CH protons of the cyclic disulfide (DTTox
3.51 ppm) at different time intervals. The ratio between DDTred and
DTTox was estimated by integration of such signals, and the t1/2
lmol, 0.05 molar equiv.) were
A
H2O2
l
where AH2O2 refers to the control solution with MeOH instead of the
methanolic organoselenium solution, and Asample refers to the
absorbance of solutions containing the organoselenium compounds.
,
,
2.2.3. Lipid peroxidation assay (Ferric thiocyanate method, FTC)
The inhibition of the 2,20-azobis(2-amidinopropane) dihydro-
chloride (APPH)-induced linoleic acid peroxidation was assayed
using the modification reported by Olszewska et al. (2012) on
the original method developed by Azuma et al. (1999).
that is, the time required for the disappearance of 50% of the initial
DTTred was estimated. A control experiment (without the organo-
selenium catalyst) was also conducted, and the ratio of DDTox
was substrated from the selenium-containing experiment.
To a solution of the seleno compound (10.0 mM in MeOH,
2.2.6. Statistical analysis
37
(88
cap vial was added 55.3 mM AAPH in buffer (25
solution was prepared using MeOH (37 L) instead of the sample.
The vial was incubated at 50 0.1 °C for 72 h in the darkness. After
that, to a solution of 30 L of the crude reaction in H2O (2.18 mL)
and MeOH (0.73 mL) were added a 10% aqueous solution of NH4-
SCN (30 L) and 20 mM FeCl2 in 3.5% HCl (30 L); after 3 min of
l
l
L) and 1.3% (w/v) methanolic linoleic acid (175
L) and 0.2 M phosphate buffer (pH 7.0, 175 L) in a screw-
L). The control
lL) in H2O
All tests were run in triplicate for each experimental condition.
Values are expressed as the confidence interval, which was calcu-
lated for n = 3, P = 0.95 using the Student’s t-distribution.
l
l
l
l
3. Results and discussion
l
l
3.1. Synthesis of N-aryl selenocarbamates, N-(b-D-glucopyranosyl)
incubation at rt, the absorbance was measured at 546 nm against
a blank (24.8% of MeOH, 0.4% of buffer, 73.8% of H2O and 1% of
3.5% HCl). BHT (butylated hydroxytoluene, 10 mM in MeOH) was
used as the reference compound.
The results are expressed as the percentage of lipid peroxida-
tion inhibition:
selenocarbamates, N-aryl selenohydantoins, and amino-acid derived
N-glucopyranosyl selenoureas
We envisioned the possibility of preparing lipophilic organose-
lenium compounds with relevant antioxidant properties, as a po-
tential way of combating oxidative stress in lipophilic media. In

P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
587
this context, we have accomplished the synthesis of a series of O-
alkyl selenocarbamates derived from N-aryl isoselenocyanates
(Scheme 1). The latter compounds were prepared starting from
their corresponding formamides, following a procedure previously
developed in our group (López et al., 2009), consisting of dehydra-
tion promoted by triphosgene, followed by coupling of the tran-
sient isocyanide with elemental selenium black. Derivatization of
isoselenocyanates 2a–e into O-ethyl selenocarbamates 3a–e was
carried out by refluxing the heterocumulenes in ethanol; such
derivatives were obtained in excellent yields (85–95%).
Spectroscopic data confirm the structure of the selenocarba-
mates; the quaternary carbon resonance in 13C NMR for the ANCSe
group changed from roughly 130 ppm to 190.8–193.1 ppm.
The 1H NMR spectrum for O-ethyl selenocarbamate 3d, the one
with the most prominent steric hindrance, showed the presence of
two rotational E/Z isomers in a 5:1 ratio in CDCl3; the ratio of the Z
isomer was found to increase when DMSO-d6 was used as solvent
(E/Z 3:2).
The presence of two rotational isomers is due to the fact that
the NACSe bond has a partial double character, due to the delocal-
ization of the nitrogen lone pair to the selenoxo group; further-
more, the presence of bulky substituents on ortho and ortho’
positions on the aryl moiety in 3d makes the rotational barrier to
be high enough to enable the detection of E/Z isomers by NMR
spectroscopy. Due to steric hindrance associated with the bulky
selenium atom, we suggest the E isomer to be the major one. A sig-
nificant deshielding of the NH proton was found for the E isomer
(8.53 ppm vs. 7.80 ppm); a similar situation can be found in litera-
ture for N-arylselenoacetamides (Murai et al., 1998).
located on the same plane as the ANAC@Se moiety; this arrange-
ment was also found for N-(4-methoxyphenyl)selenoacetamide
(Murai et al., 1998). Moreover, for the minor rotamer of selenocar-
bamate 3d, it was found that both methyl groups of the isopropyl
moiety are not equivalent, and thus resonate at different
frequencies (1.26 and 1.12 ppm), whereas for the major rotamer,
both methyl groups are equivalent, (d = 1.14 ppm). Such behavior
suggests that for the Z isomer, there is a chirality axis in the
NAC(Ar) bond, coming from the restricted rotation, provoked by
the bulky aryl substituents. The change from the aR to the aS con-
formation is slow enough in the NMR time scale for both methyl
groups being diastereotopic and thus, distinguishable by NMR
spectroscopy.
From the above selenocarbamates, derivatives 3a and 3e were
selected for the antioxidant assays, as the prominent steric hin-
drance exerted by bulky alkyl groups on the rest of examples pre-
sumably would reduce their activity.
Using some of the above mentioned aryl isoselenocyanates, or
glucopyranosyl isoselenocyanate 9 we have also accomplished
the preparation of peptidomimetics by reaction with commer-
cially-available
bond is mimicked by a selenoureido moiety (Schemes 2 and 3).
Thus, reaction of phenyl isoselenocyanate with -valine ethyl ester
L-configured a-aminoesters, where the peptidic
L
hydrochloride, in the presence of an excess of triethylamine affor-
ded the corresponding selenourea, which spontaneously under-
went an intramolecular cyclization involving the ArANH and the
ethoxycarbonyl groups, evolving to N-aryl selenohydantoin 5 in a
quantitative yield (Scheme 2). The formation of compound 5 is evi-
denced by the lack of the ethoxy moiety in NMR spectroscopy.
Hemantha and Sureshbabu (2010) recently reported the prepara-
tion of 5 from the isoselenocyanate derived from the aminoester
and aniline.
Furthermore, for compound 3d, a NOESY experiment showed a
cross-peak between the NH proton and one of the methyl groups of
the isopropyl moiety, suggesting that the aromatic ring is not
Scheme 1. Synthesis of N-aryl O-alkylselenocarbamates 3a–e.

588
P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
HN
Se
Se
O
N
NCSe
HN
N
H
COOEt
H3N
COOEt
Cl
Et3N
2a
4 (not isolated)
5 (quant)
HN
Se
Se
O
NCSe
HN
N
H
COOEt
N
H3N
COOEt
Cl
+
Et3N
2e
6 (53%)
7 (25%)
Scheme 2. Synthesis of N-arylselenoureas and selenohydantoins 5–7.
OAc
OAc
1) AFA, NaHCO3
O
O
NH2·HBr
NCSe
R3
AcO
AcO
AcO
AcO
2) Triphosgene, Et3N
3) Se black
OAc
OAc
9
8
R1OH
OAc
COOR2
H3N
Cl
Δ
O
H
N
Et3N
O
AcO
AcO
R1
OAc
OAc
O
Se
O
H
H
N
R1 = Me (quant)
N
10a
AcO
AcO
OR2
10b R1 = Et (quant)
10c R1 = Pr (91%)
OAc
Se
R3
R
1 = Bu (92%)
10d
R2= Et, R3= H (76%)
11a
11b R2= Et, R3= CH2Ph (82%)
11c R2= Et, R3= CH2OH (72%)
R2= Et, R3= iPr (72%)
11d
11e R2= Me, R3=
(89%)
NH
Scheme 3. Synthesis of N-glycosylselenocarbamates 10a–d and N-glycosylselenoureas 11a–e.
When
a
-napthyl isoselenocyanate 2e was coupled with
L
-valine
heating of 9 with a series of aliphatic alcohols afforded O-alkyl sel-
enocarbamates 10a–d in almost quantitative yields as the first
examples of N-glycopyranosyl selenocarbamates reported so far
in the literature. A total conversion into compounds 10a–d was
observed, without the formation of side-products, and just an
elimination of the corresponding alcohol is required, without any
further purification. In a similar way as found for N-(o,o0-disubsti-
tuted phenyl) selenocarbamate 3d, N-glycopyranosyl selenocarba-
mates were found to exist as a non-resolvable mixture of E/Z
isomers (3:2 for 10a, and 2.7:1 for 10b–d).
ethyl ester hydrochloride, a separable mixture of N-naphthyl sele-
nourea 6 (53%) and N-naphthyl selenohydantoin 7 (25%) was ob-
tained. The different behavior observed for 2e, in comparison
with 2a, which only led to the selenohydantoin, must be due to
the more prominent steric hindrance exerted by the naphthyl res-
idue, provoking an impairment for the intramolecular cyclization.
We have carried out the preparation of the first examples of
glyconjugates comprised of sugar-derived selenoureas containing
natural aminoacid residues (Scheme 3), starting from glucosyl
isoselenocyanate 9, readily available (López et al., 2009) upon
Alternatively, coupling of sugar-derived isoselenocyanate 9
with commercially-available aminoesters derived from natural
N-formilation
of
per-O-acetylated-b-D-glucopyranosylamine
hydrobromide 8 with acetic–formic acid (AFA), followed by dehy-
dration with triphosgene and coupling of the transient glycopyr-
anosyl isocyanide with elemental selenium black.
Isoselenocyanate 9 was found to be a versatile intermediate in
the preparation of selenium-containing glycoconjugates; thus,
L-amino acids (Scheme 3) gave access to protected selenoureas
11a–e in excellent yields, which can be considered as a novel fam-
ily of glycoconjugates. Attempts to convert such selenoureas into
selenohydantoins were unsuccessful, probably due to the steric
hindrance exerted by the saccharidic residue, thus, preventing

P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
589
the intramolecular cyclization involving the NH and the ester
moieties. 1H NMR spectroscopy of compounds 11a–e showed a sig-
nificant deshielding of H-1 proton when compared with parent
isoselenocyanate 9, probably indicating that the major conforma-
tion for these compounds must be the one indicated in Scheme 3,
where the anomeric proton is located on the deshielding region of
the selone group.
Organoselenium compounds were found to be quite stable in
solution when kept at ꢀ20 °C in the darkness. When solutions
are kept at rt in a physiological pH, stability is increased in the
darkness. For example, selenohydantoin 7, apparently the least sta-
ble compound, releases elemental red selenium when exposed to
indirect sunlight for 48 h.
activity. Remarkably, naphthyl selenourea 6, with an EC50 value
of 19 M, behaved as the best free radical scavenger.
It is noteworthy that compounds 3e, 6, and 11a–d showed sim-
ilar, or even better scavenging activity than very well-known anti-
3
l
oxidants like ascorbic acid (13
3
lM), trolox (14
5 lM), or BHT
(70 M), thus demonstrating an excellent antioxidant profile.
9
l
From the data depicted in Fig. 1, it is clear that the increasing
order of activity could be: selenohydantoins < selenocarba-
mates < selenoureas. This sequence of activity was established
considering selenohydantoin 7, selenocarbamate 3e and selenou-
rea 6, where all of them keep in common an
a-naphthyl residue,
whereas the selenium functionality is changed. It is also clear that
organoselenium compounds behaved as more potent free radical
scavengers when bearing aromatic residues, and quite noticeable,
an
a-naphthyl moiety, as can be observed when comparing sele-
nocarbamates 3a, 3e, and 10a–d.
3.2. Organoselenium compounds as free radical scavengers
In order to explain the above results, we postulate that the orga-
noselenium compounds 12, bearing a selenoxo peptide linkage, can
act as free radical scavengers by donating a H atom from an NH
group to DPPH (Scheme 4); as a result, the resonance-stabilized
free-radical 14 is obtained. The existence of an aromatic residue
at the nitrogen atom, specially a naphthyl ring, can better stabilize
the resulting organoselenium radical, and thus, lead to an in-
creased activity when compared with alkyl groups (e.g.
carbohydrates).
The different families of organoselenium compounds reported
herein were evaluated as ROS scavengers, in particular against free
radicals and hydrogen peroxide; it is remarkable that no similar
study has been accomplished previously on this series of organose-
lenium derivatives.
The anti-free radical activity was monitored using commer-
cially-available DPPH, a stable free radical, widely used for study-
ing the anti-radical activity of numerous antioxidants, like
natural and synthetic polyphenols (Sharma and Bhat, 2009).
We have evaluated the influence of the selenium-containing
functional groups (selenocarbamate, selenourea, selenohydantoin),
together with the nature of the substituents on the activity of the
organoselenium compounds prepared herein. The results, in terms
of EC50 (effective concentration for scavenging 50% of the initial
DPPH) are depicted in Fig. 1, together with the reported activity
of some known antioxidant agents (ascorbic acid, trolox, BHT) used
herein as positive controls.
3.3. Organoselenium compounds as H2O2 scavengers
The organoselenium compounds were also evaluated as H2O2
scavengers; the assay considers the modification described by
Bahorun et al. (1996) on the original procedure developed by Pick
and Keisari (1980). The method is based on the oxidation reaction
by phenol red indicator in the presence of H2O2 and a peroxidase,
leading to a compound exhibiting an intense absorption at 610 nm;
a reduction of the absorbance in the presence of an antioxidant
agent is directly correlated with the scavenging activity of such
antioxidant.
The scavenging activity for compounds 3a,e, 5–7, 10a,c,d and
11a–d is depicted in Fig. 2 (0.5 mM final concentration). Interest-
ingly, the order of potency as H2O2 scavengers is reversed when
compared with the anti-free radical activity for those compounds
bearing N-aryl substituents 3a,e, 5–7. Thus, selenohydantoins 5,
7 and selenocarbamates 3a,e exhibit the strongest activity, consid-
erably higher than related selenourea 6 (76 2% for 7, 61 3% for
3a, 51 3% for 5, 44 1% for 3e, 38 1% for 6). Therefore, N-phenyl
selenohydantoin 7, the poorest free radical scavenger, was found to
be the most potent peroxide scavenger; the complete opposite sit-
uation was found for N-naphthyl selenourea 6.
As deduced from the EC50 data, the compounds with the lowest
anti-radical capacity were found to be N-naphthyl selenohydantoin
7
(379 11
10a,c,d (383 32, 325 59, 360 70
ate activities were achieved for O-ethyl-N-phenyl selenocarbamate
3a (148 18 M) and N-phenyl selenohydantoin 5 (147 18 M).
-Naphthylselenocarbamate 3e (43 M) and sugar-derived sel-
enoureas 11a–d (25 5, 40 10, 39 11, 46 16 M) turned out to
l
M) and sugar-derived O-alkyl selenocarbamates
lM, respectively). Intermedi-
l
l
a
7 l
l
be excellent scavengers of free radicals; glycine-based selenourea
11a was found to be slightly more active than their counterparts
11b–d, probably due to steric factors. The latter compounds, bear-
ing either polar or non-polar substituents on the
a-carbon of the
aminoacid residue presented almost no different in activity, thus
indicating no influence of such substituents on the anti-radical
Considering the carbohydrate-derived selenocarbamates
10a,c,d, and the selenoureas 11a–d, activity is quite similar,
although slightly higher for selenoureas (63 1% for 11a, 73 1%
for 11b, 74 1% for 11c, 69 1% for 11d) when compared with car-
bohydrate-derived selenocarbamates (57 2% for 10a, 60 1% for
10c, 56 1% for 10d).
These results were compared with that of hydroxytyrosol,
2-(30,40-dihydroxyphenyl)ethanol, a natural polyphenol with po-
tent antioxidant properties (Fernández-Bolaños et al., 2008);
hydroxytyrosol (HT) behaved as
a stronger H2O2 scavenger
(69 5% at 60 M concentration) than organoselenium derivatives.
l
3.4. Organoselenium compounds as lipid peroxidation inhibitors
Compounds synthesized herein were tested as inhibitors of ini-
tial steps of lipid peroxidation; for that purpose, the free radical-in-
duced peroxidation of linoleic acid was initiated by the thermal
decomposition of 2,20-azobis(2-amidinopropane) hydrochloride
Fig. 1. DPPH scavenging activity of selected selenocarbamoyl derivatives. Values
are expressed as the confidence interval, which was calculated for n = 3, P = 0.95
using the Student’s t-distribution.

590
P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
Se
Se
NO2
R1
R1
Se
2
2
N
R
N
R
R1
O2N
N
N
14
2
N
H
R
+
+
NO2
NO2
12
13
O2N
NH
N
1
R = alkyl, aryl
2
R
= O-alkyl, NH-Ar
NO2
15
Scheme 4. Mechanism for the scavenging of free radicals by selenocarbamoyl derivatives.
Fig. 2. Percentage of scavenging of H2O2 by selected selenocarbamoyl derivatives
using a 0.50 mM concentration, except for HT, tested at 0.06 mM. Values are
expressed as the confidence interval, which was calculated for n = 3, P = 0.95 using
the Student’s t-distribution.
Fig. 3. Percentage of inhibition of lipid peroxidation by selected selenocarbamoyl
derivatives using a 0.74 mM concentration. Values are expressed as the confidence
interval, which was calculated for n = 3, P = 0.95 using the Student’s t-distribution.
(APPH), which upon a chain reaction furnishes alkyl peroxides.
Such lipid peroxides react with Fe2+ to give Fe3+, which in turn,
upon interchange with ammonium thiocyanate, produces ferric
thiocyanate, a brick-red complex which is determined spectropho-
tometrically at 540 nm (Azuma et al., 1999). The presence of an
efficient ROS scavenger reduces the concentration of the alkyl per-
oxides, and therefore, decreases the observed absorbance. This
methodology is therefore, suitable for estimating the incipient ran-
cidity of lipids induced by free radicals.
Herein we have determined the percentage of inhibition of lipid
peroxidation of the new organoselenium derivatives (final concen-
tration 0.74 mM) upon incubation for 72 h, and compared with
BHT, as a positive control; results are summarized in Fig. 3. Lipid
peroxidation was inhibited by all families of the organoselenium
compounds, in a range 26–71%. Antioxidant activity was especially
remarkable for sugar-derived O-alkyl selenocarbamates 10a
(49 15%), 10c (69 4%), and 10d (71 16%), which is similar or
even better than BHT (57 10%); aryl selenocarbamates 3a
(57 3%), and 3e (46 17%) were found to be less active than su-
gar-derived counterparts.
Compounds
Fig. 4. Percentage of scavenging of nitric oxide by selected selenocarbamoyl
derivatives, using a 0.71 mM concentration, except for ascorbic acid, tested at
7.1 mM. Values are expressed as the confidence interval, which was calculated for
n = 3, P = 0.95 using the Student’s t-distribution.
3.5. Organoselenium compounds as nitric oxide (NO) scavengers
Similar activities were found for sugar-derived selenoureas 11b
(47 12%) and 11c (41 5%), naphthyl thiourea 6 (41 11%), and
selenohydantoin 7 (46 17%).
Therefore, all these results further demonstrate the efficient
anti-ROS activity of selenoxo derivatives, which might be suitable
agents for reducing the rancidity of lipidic media.
Nitric oxide can be generated from sodium nitroprusside at
physiological pH, and, upon interaction with atmospheric oxygen
is transformed into nitrite ion, which is determined spectrophoto-
metrically by transformation into a purple azo-compound (Garratt,
1964). Although NO is involved in numerous relevant biological
processes (Fang et al., 2002), abnormally high production of NO

P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
591
3.6. Organoselenium compounds as glutathione peroxidase mimics
Glutathione peroxidases (GPxs) comprise a group of selenium-
containing enzymes which act as a natural defense against several
ROS, like H2O2, via a redox-based catalytic cycle in which selenium
and thiol cofactors are involved (Arthur, 2000).
Numerous organoselenium derivatives have been reported to
exhibit GPx-like activity, and therefore, to act as enzyme mimics
(Battin and Brumaghim, 2009), but to the best of our knowledge,
such study has not been previously conducted on selenoxo deriva-
tives, like selenocarbamates, selenoureas, or selenohydantoins.
We have evaluated such activity using NMR spectroscopy, in
which the organoselenium compound was used in a catalytic
amount (5%), in the presence of H2O2 and dithiothreitol as the thiol
source. The efficiency as glutathione peroxidase mimics was eval-
uated by estimating t1/2, that is, the time required for converting
50% of initial dithiothreitol (DTTred) into the corresponding cyclic
disulfide (DTTox), mediated by H2O2.
t (s)
Fig. 5. Plot of decrease of DTT (dithiothreitol) vs. time in the glutathione
peroxidase-like activity, measured by 1H NMR.
Compound 6
Such reaction is quite slow in the absence of catalyst (Fig. 5),
and the rate was efficiently increased when a selenoxo derivative
is present. Nevertheless, an important difference in rate was found
among the different families of the organoselenium compounds
tested (Fig. 5).
y = 0,1194x + 35,405
R² = 0,9697
The least efficient compounds were found to be aryl and sugar-
derived selenocarbamates (e.g. 3e, 10c), together with selenohyd-
antoin 7, with t1/2 values ranging from 100 to 108 min.
t (s
An spectacular increase in the reaction rate was achieved by
using a selenoureido motif, as the t1/2 was reduced up to 40 times
(2.0–12.9 min). Especially relevant are naphthyl selenourea 6 (t1/
2 = 2.0 min), and sugar-derived selenourea 11a (t1/2 = 2.9 min),
which represent a roughly 700-fold increase in rate when com-
pared with non-catalyzed background; in fact, the reaction is com-
plete after just 12 min. Glycine-derived selenourea 11a behaved as
a better mimic than compounds 11b–d (derived from phenyl ala-
nine, serine and valine, respectively), suggesting that substitution
Fig. 6. Linear regression plot for the estimation of t1/2 value for compound 6 in the
glutathione peroxidase-like activity test.
has been associated to the development of certain diseases (Shah
et al., 2004), including sepsis and hepatic failure; therefore, NO
scavengers are valuable targets from a pharmacological point of
view.
Although most of the tested new compounds did not show sig-
nificant anti-NO activity, it is remarkable to mention the scaveng-
ing activity exerted by aryl selenocarbamates 3a (64 5%) and 3e
(80 7%), when tested at a final concentration of 0.71 mM (Fig. 4).
For reaching a similar activity (63 8%), ascorbic acid required a
concentration of 7.1 mM, thus indicating a 10-fold potency of
the above mentioned selenocarbamates compared with ascorbic
acid.
on the
a-position of the aminoacid framework slightly reduces
the catalytic activity, presumably due to steric factors.
Fig. 6 depicts the data for selenourea 6, adjusted to a linear
regression for estimating the value of t1/2
.
Monitoring of the conversion of DTTred into DTTox by NMR for
selenourea 6 as catalyst is shown in Fig. 7a; for comparison, the
non-catalyzed background reaction is also included (Fig. 7b).
Fig. 7. Monitoring by 1H NMR of the oxidation of DTTred into DTTox in the presence of compound 6 (a), and for the background reaction (b).

592
P. Merino-Montiel et al. / European Journal of Pharmaceutical Sciences 48 (2013) 582–592
CONACYT of Mexico and the Fundación Carolina of Spain, and
S.M. the MICINN for the award of fellowships.
O
O
H2O
H2O2
Se
Se
Se
R
R
R
N
H
R'
References
N
H
R'
N
H
R'
17
16
Arthur, J.R., 2000. The glutathione peroxidases. Cell. Mol. Life Sci. 57, 1825–1835.
Azuma, K., Nakayama, M., Koshioka, M., Ippoushi, K., Yamaguchi, Y., Kohata, K.,
Yamauchi, Y., Ito, H., Higashio, H., 1999. Phenolic antioxidants from the leaves
of Corchorus olitorius L. J. Agric. Food Chem. 47, 3963–3966.
Bahorun, T., Gressier, B., Trotin, F., Brunet, C., Dine, T., Luyckx, M., Vasseur, J., Cazin,
J.C., Pinkas, M., 1996. Oxygen species scavenging activity of phenolic extracts
from hawthorn fresh plant organs and pharmaceutical preparations. Arzneim.
Forsch. 46, 1086–1089.
R
S
S
2 O
+
H
R
HSRRSH
HO
SH
SH
R
(DTTred
)
HSRRSH=
HO
R
S
S
H
OH
Battin, E.E., Brumaghim, J.L., 2009. Antioxidant activity of sulfur and selenium: a
review of reactive oxygen species scavenging, glutathione peroxidase, and
metal-binding antioxidant mechanisms. Cell. Biochem. Biophys. 55, 1–23.
Butterfield, D.A., Perluigi, M., Sultana, R., 2006. Oxidative stress in Alzheimer’s
disease brain: new insights from redox proteomics. Eur. J. Pharmacol. 545, 39–
50.
HO
HO
Se
S
S
(DTTox
)
R
RSSR=
N
R'
H
18
Cefarelli, G., D’Abrosca, B., Fiorentino, A., Izzo, A., Mastellone, C., Pacifico, S., Piscopo,
V., 2006. Free-radical-scavenging and antioxidant activities of secondary
metabolites from reddened cv. Annurca apple fruits. J. Agric. Food Chem. 54,
803–809.
Scheme 5. Mechanism for the glutathione peroxidase-like activity of selenocarba-
moyl derivatives.
Dröge, W., 2002. Free radicals in the physiological control of cell function. Physiol.
Rev. 82, 47–95.
Fang, Y.-Z., Yang, S., Wu, G., 2002. Free radicals, antioxidants, and nutrition.
Nutrition 18, 872–879.
Fernández-Bolaños, J.G., López, Ó., Fernández-Bolaños, J., Rodríguez-Gutiérrez, G.,
2008. Hydroxytyrosol and derivatives: isolation, synthesis and biological
properties. Curr. Org. Chem. 12, 442–463.
Garratt, D.C., 1964. The Quantitative Analysis of Drugs, vol. 3. Chapman and Hall
Ltd., Japan, pp. 456–458.
Grek, C.L., Tew, K.D., 2010. Redox metabolism and malignancy. Curr. Opin.
Pharmacol. 10, 362–368.
Hemantha, H.P., Sureshbabu, V.V., 2010. Isoselenocyanates derived from amino acid
esters: an expedient synthesis and application to the assembly of
The proposed catalytic cycle for the organoselenium derivatives
(selenocarbamates, selenohydantoins and selenoureas) acting as
glutathione peroxidase mimics is depicted in Scheme 5. The
organoselenium compound is firstly oxidized by H2O2 to give selen-
oxide-type derivative 17, stabilized by resonance. Next, intermedi-
ate 17 reacts with one thiol group of dithiothreitol (DTTred) to give a
selenyl-sulfide adduct (18); the latter, upon interaction with the
second thiol group, evolves to cyclic disulfide DTTox, regenerating
the selenoxo compound which can take part again in the catalytic
cycle.
selenoureidopeptidomimetics,
unsymmetrical
selenoureas
and
selenohydantoins. J. Pept. Sci. 16, 644–651.
The major stabilization of 17 in the case of selenoureas might
explain the higher reactivity of such compounds in the glutathione
peroxidase assay.
Iwaoka, M., Kumakura, F., 2008. Applications of water-soluble selenides and
selenoxides to protein chemistry. Phosphorous, Sulphur Silicon Relat. Elem.
183, 1009–1017.
Kohen, R., Nyska, A., 2002. Oxidation of biological systems: oxidative stress
phenomena, antioxidants, redox reactions, and methods for their
quantification. Toxicol. Pathol. 30, 620–650.
López, Ó., Maza, S., Ulgar, V., Maya, I., Fernández-Bolaños, J.G., 2009. Synthesis of
sugar-derived isoselenocyanates, selenoureas and selenazoles. Tetrahedron 65,
2556–2566.
The lipid peroxidation activity, that might be related to free rad-
ical scavenging, and to GPx-like activity, was found to be slightly
better for sugar-derived selenocarbamates 10a,c,d; nevertheless,
such compounds were shown to be remarkably worse than su-
gar-derived selenoureas 11a–d when tested as free radical scav-
engers (DPPH assay) and as glutathione peroxidase mimics. The
explanation could be the better stability of selenocarbamates un-
der the lipid peroxidase assay conditions, which involve prolonged
heating.
In conclusion, we have carried out the preparation of three dif-
ferent families of selenium-containing derivatives: selenocarba-
mates, selenoureas and selenohydantoins. Some of the new
families prepared herein can be considered as peptidomimetics,
combining a carbohydrate residue and a natural aminoacid frame-
work connected by a selenoureido-type linker.
Merino-Montiel,
P.,
López,
Ó.,
Fernández-Bolaños,
J.G.,
2012.
L-
Isofucoselenofagomine and derivatives: dual activities as antioxidants and as
glycosidase inhibitors. Tetrahedron 68, 3591–3595.
Murai, T., Niwa, N., Ezaka, T., Kato, S., 1998. Structure of N-aryl selenoacetamides in
solution and in the solid state. J. Org. Chem. 63, 374–376.
Olszewska, M.A., Presler, A., Michel, P., 2012. Profiling of phenolic compounds and
antioxidant activity of dry extracts from the selected Sorbus species. Molecules
17, 3093–3113.
Pick, E., Keisari, Y., 1980.
A simple colorimetric method for the treatment of
hydrogen peroxide produced by cells in culture. J. Immunol. Methods 38, 161–
170.
Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements. J. Agric.
Food Chem. 53, 4290–4302.
These compounds have been subjected to the first in vitro study
as ROS and RNS scavengers, in particular against free radical, hydro-
gen peroxide, alkyl peroxides and nitric oxide; their glutathione
peroxidase-like activity has also been evaluated. We have carried
out a qualitative structure–activity study, analyzing the influence
of both, the functional group in which selenium is incorporated,
as well as the nature of the substituents. Antioxidant activity was
found to be strongly dependent on the electronic structure of the
selenoxo group. Remarkably, in such tests, some of the analyzed
compounds turned out to be sensibly more active than some
well-known antioxidants, like ascorbic acid, trolox or BHT.
Reuter, S., Gupta, S.C., Chaturvedi, M.M., Aggarwal, B.B., 2010. Oxidative stress,
inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49,
1603–1616.
Shah, V., Lyford, G., Gores, G., Farrugia, G., 2004. Nitric oxide in gastrointestinal
health and disease. Gastroenterology 126, 903–913.
Sharma, O.P., Bhat, T.K., 2009. DPPH antioxidant assay revisited. Food Chem. 113,
1202–1205.
Takahashi, H., Nishina, A., Fukumoto, R.-H., Kimura, H., Koketsu, M., Ishihara, H.,
2005a. Selenoureas and thioureas are effective superoxide radical scavengers
in vitro. Life Sci. 76, 2185–2192.
Takahashi, H., Nishina, A., Fukumoto, R.-H., Kimura, H., Koketsu, M., Ishihara, H.,
2005b. Selenocarbamates are effective superoxide anion scavengers in vitro.
Eur. J. Pharm. Sci. 24, 291–295.
Takahashi, H., Nishina, A., Kimura, H., Motoki, K., Koketsu, M., Ishihara, H., 2004.
Tertiary selenoamide compounds are useful superoxide radical scavengers
in vitro. Eur. J. Pharm. Sci. 23, 207–211.
Acknowledgments
Thannickal, V.J., Fanburg, B.L., 2000. Reactive oxygen species in cell signaling. Am. J.
Physiol. Lung Cell. Mol. Physiol. 279, L1005–L1028.
Tiwari, S.K., Rohilla, A., Rohilla, S., Kushnoor, A., 2012. Reactive oxygen species: a
comprehensive review. Int. J. Curr. Pharm. Res. 4, 19–23.
Turrens, J.F., 2003. Mitochondrial formation of reactive oxygen species. J. Physiol.
552, 335–344.
We thank the Dirección General de Investigación of Spain and
the Junta de Andalucía (Grants Numbers CTQ2008-02813, co-
financed with FEDER funds and FQM134). P.M.-M. thanks the