Selenoureas and thioureas are effective superoxide radical scavengers in vitro

Article abstract of DOI:10.1016/j.lfs.2004.08.037

Oxygen radicals, such as superoxide radicals, embellishing DNA, protein, lipids, etc., and carrying out the obstacle of the function of a cell is known. It depends for the oxidant level in the living body on the balance of a generation system and an elimination system of oxygen radicals, and research which controls an oxidant level in the living body is briskly done by taking in the substance which eliminates an oxygen radical. We investigated scavenging effects of superoxide radicals by selenoureas and thioureas using a highly sensitive and quantitative chemiluminescence method. At 330 nM, five selenoureas and five thioureas scavenged fractions of superoxide radicals (O ₂^-) ranging from 8.4% to 87.6%. Among five N,N-unsubstituted selenoureas and N,N-unsubstituted thioureas 1-selenocarbamoylpiperidine and 1-thiocarbamoylpyrrolidine were the most effective scavengers. A possibility that selenoureas could use it as a new superoxide anion-scavenging substance from the result of this research became clear.

Full text of DOI:10.1016/j.lfs.2004.08.037

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Life Sciences 76 (2005) 2185–2192
www.elsevier.com/locate/lifescie
Selenoureas and thioureas are effective superoxide radical
scavengers in vitro
a
a,
a
b
*
Hitoe Takahashi , Atsuyoshi Nishina , Ryo-hei Fukumoto , Hirokazu Kimura ,
Mamoru Koketsuc, Hideharu Ishiharad
aGunma Industrial Technology Center, 884-1 Kamesato, Maebashi, Gunma, 379-2147, Japan
bGunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki, Maebashi, Gunma, 371-0052, Japan
cDivision of Instrumental Analysis, Life Science Research Center, Gifu University, Gifu, 501-1193, Japan
dDepartment of Chemistry, Faculty of Engineering, Gifu University, Gifu, 501-1193, Japan
Received 19 May 2004; accepted 18 August 2004
Abstract
Oxygen radicals, such as superoxide radicals, embellishing DNA, protein, lipids, etc., and carrying out the
obstacle of the function of a cell is known. It depends for the oxidant level in the living body on the balance of a
generation system and an elimination system of oxygen radicals, and research which controls an oxidant level in
the living body is briskly done by taking in the substance which eliminates an oxygen radical. We investigated
scavenging effects of superoxide radicals by selenoureas and thioureas using a highly sensitive and quantitative
chemiluminescence method. At 330 nM, five selenoureas and five thioureas scavenged fractions of superoxide
radicals (O2À) ranging from 8.4% to 87.6%. Among five N,N-unsubstituted selenoureas and N,N-unsubstituted
thioureas 1-selenocarbamoylpiperidine and 1-thiocarbamoylpyrrolidine were the most effective scavengers. A
possibility that selenoureas could use it as a new superoxide anion-scavenging substance from the result of this
research became clear.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Selenourea; Thiourea; Superoxide radicals; Scavenging effect; Superoxide anion-scavenging activity (SOSA)
* Corresponding author. Tel.: +81 27 290 3030; fax: +81 27 290 3040.
E-mail address: nishina@tec-lab.pref.gunma.jp (A. Nishina).
0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2004.08.037

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H. Takahashi et al. / Life Sciences 76 (2005) 2185–2192
Introduction
The cells of an organism generate large amounts of reactive oxygen species (ROS) as oxygen
metabolites, inevitably resulting in exposure to these injurious chemical species. ROS including
superoxide radicals (OÀ2 ), hydrogen peroxide (H2O2), and hydroxyl radicals (.OH), cause degeneration of
biological macromolecules such as DNA, representing oxidative and sometimes genotoxic stress in vivo
(Ramirez et al., 2003; Long et al., 1997). OÀ2 is considered to be generated primarily by mitochondria in
various cells, and by phagocytes such as granulocytes and monocytes/macrophages (Ricci et al., 2003).
Under physiologic conditions, O2À is converted to H2O2 in hydrophilic solvents such as water by a
disproportion reaction (Ueda et al., 1994). In addition, OÀ2 can react with nitric oxide (NO) and generate
highly toxic ROS including ONOOÀ and nitrogen oxides (NOx) (Hu et al., 2002). Thus, elimination of
O2À is an important biologic need.
Various antioxidant enzymes including the superoxide dismutases (SODs), catalase, and glutathione
peroxidase, as well as antioxidant vitamins (C and E) directly scavenge and eliminate ROS. An important
antioxidant enzyme, glutathione peroxidase (GPX), contains a selenium molecule in its active domain.
GPX effectively scavenges and eliminates H2O2 both in vitro and in vivo. In addition, previous studies
have shown that selenium compounds such as selenoproteins, protect cells against oxidative stress (Jeong
et al., 2002; Taino et al., 2000). Accordingly, a variety of selenium compounds may effectively scavenge
and eliminate ROS. On the other hand, certain thioureas that effect the twitch tension of the rat
diaphragm upon stimulation of the phrenic nerve-diaphragm also scavenge O2À (Crosland, 1995).
However, details concerning of the superoxide anion-scavenging activity of thioureas are not known.
This study was performed to determine how effectively selenoureas and thioureas scavenge O2À in vitro.
Materials and methods
Materials
A cypridina luciferin analogue, (2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazin-
3-one (MCLA), was obtained from Tokyo Kasei (Tokyo, Japan) for use as a chemiluminescent probe for
superoxide radicals. MCLAwas dissolved in doubly distilled water, and stored at À808C until needed. The
concentration of MCLA solution was determined by absorbance at 430 nm using an absorbance coefficient
value of q = 9600 MÀ1cmÀ1, as previously described (Kimura and Nakano, 1988). Horse heart cytochrome
c (type IV), SOD (from bovine erythrocytes, 3000 units/mg protein), xanthine oxidase (XOD grade III),
and bovine serum albumin (BSA, acid-and globulin-free) were purchased from Sigma Chemical (St.
Louis, MO). Hypoxanthine was purchased from Wako Chemicals (Tokyo, Japan) and used without further
purification. All other chemicals and solvents were analytical grade and used without further purification.
General
Synthetic methods for the production of selenoureas and thioureas
N,N-Dimethyselenourea (4a). HCl (1N; 2 mL, 2 equivalent) in anhydrous diethyl ether was added to
N,N-dimethylcyanamide (0.08 mL, 1 equivalent) of tetrahidroflane (THF) solution (10 mL). The initially

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H. Takahashi et al. / Life Sciences 76 (2005) 2185–2192
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colorless and clear reaction mixture became a milky white suspension in less than 30 s, and then was
stirred at 08C for 2 h. Subsequently, LiAlHSeH (2) (1 equivalent) (Ishihara et al., 2001) was added to the
reaction mixture. The reaction mixture was stirred further at 08C for 3 h, and then was extracted with
dichloromethane and washed with water. The organic layer was dried over sodium sulfate and
evaporated to dryness. The residue was purified by flash chromatography on silica gel with
dichloromethane : ether (1:1) to give 4a (0.105 g; yield, 70%); Mp. 172.2 to 172.88C; IR (KBr)
1
3366, 3162, 1551 cmÀ1; H NMR (DMSO-d6) y 3.18 (6 H, br s, N-CH3), 7.60 (2 H, br s, NH); 13C
NMR (DMSO-d6) y 37.9, 45.3, 177.7; 77Se NMR (DMSO-d6) y 230.4; MS (CI): m/z = 153 [M+1];
HRMS (EI) calculated for C3H8N2Se 151.98522; found, 151.98346.
1
N,N-Diethyselenourea (4b). Mp. 121.8 to 122.78C; IR (KBr) 3340, 3176, 1534 cmÀ1; H NMR
(CDCl3) y 1.27 (6 H, t, J = 6.8 Hz, CH3), 3.49 (2 H, br s, CH2), 3.97 (2 H, br s, CH2), 6.48 (2 H, br s,
NH); 13C NMR (CDCl3) y 12.2, 42.9, 51.1, 176.6; 77Se NMR (CDCl3) y 209.8; MS (CI): m/z = 181
[M+1]; HRMS (EI) calculated for C5H12N2Se 180.0165; found, 180.0147.
1
1-Selenocarbamoylpyrrolidine (4c). Mp. 215.1 to 215.98C; IR (KBr) 3292, 3159, 1523 cmÀ1; H
NMR (DMSO-d6) y 1.80 (2 H, m, CH2), 1.98 (2 H, m, CH2), 3.26 (2 H, t, J = 6.8 Hz, CH2), 3.62 (2 H, t,
J = 6.8 Hz, CH2), 7.53 (2 H, br s, NH); 13C NMR (DMSO-d6) y 24.4, 25.9, 47.5, 54.2, 173.6; 77Se NMR
(DMSO-d6) y 245.1; MS (CI): m/z = 179 [M++1].
1-Selenocarbamoylpiperidine (4d). Mp. 145.0 to 146.28C; IR (KBr) 3303, 3169, 1522 cmÀ1; 1H NMR
(DMSO-d6) y 1.47 (4 H, m, CH2), 1.59 (2 H, m, CH2), 3.76 (4 H, br s, CH2), 7.75 (2 H, br s, NH); 13
C
NMR (DMSO-d6) y 23.6, 25.3, 176.0; 77Se NMR (DMSO-d6) y 218.5; MS (CI): m/z = 193 [M+1];
HRMS (EI) calculated for C6H12N2Se, 192.0165; found 192.01499.
1
4-Selenocarbamoylmorpholine (4e). Mp. 207.8 to 209.08C; IR (KBr) 3316, 3214, 1523 cmÀ1; H
NMR (DMSO-d6) y 3.57 (4 H, t, J = 4.4 Hz, CH2), 3.79 (4 H, br s, CH2), 7.95 (2 H, br s, NH); 13C
NMR (DMSO-d6) y 65.6, 178.0; 77Se NMR (DMSO-d6) y 225.0; MS (CI): m/z = 195 [M++1].
1
N,N-Dimethythiourea (5a). Mp. 163.0 to 164.28C; IR (KBr) 3385, 3183, 1542 cmÀ1; H NMR
(DMSO-d6) y 3.11 (6 H, br s, N-CH3), 7.15 (2 H, br s, NH); 13C NMR (DMSO-d6) y 181.6; MS (CI): m/
z = 105 [M++1].
N,N-Diethythiourea (5b). Mp. 99.2 to 100.68C; IR (KBr) 3376, 3192, 1522 cmÀ1; 1H NMR (DMSO-
d6) y 1.06 (6 H, t, J = 6.8 Hz, CH3), 3.53 (4 H, br s, CH2), 7.10 (2 H, br s, NH); 13C NMR (DMSO-d6) y
12.6, 44.5, 179.8; MS (CI): m/z = 133 [M++1].
1
1-Thiocarbamoylpyrrolidine (5c). Mp. 197.3 to 199.08C; IR (KBr) 3316, 3171, 1511 cmÀ1; H NMR
(DMSO-d6) y 1.80 (2 H, m, CH2), 1.98 (2 H, m, CH2), 3.26 (2 H, t, J = 6.8 Hz, CH2), 3.62 (2 H, t, J = 6.8 Hz,
CH2),7.53(2H,brs,NH);13CNMR(DMSO-d6)y24.6,25.9,47.5,51.4,178.3;MS(CI):m/z =131[M++1].
1
1-Thiocarbamoylpiperidine (5d). Mp. 127.4 to 129.18C; IR (KBr) 3334, 3188, 1510 cmÀ1; H NMR
(DMSO-d6) y 1.45 (4 H, m, CH2), 1.56 (2 H, m, CH2), 3.69 (4 H, br s, CH2), 7.25 (2 H, br s, NH); 13
NMR (DMSO-d6) y 23.7, 25.3, 48.2, 180.2; MS (CI): m/z=145 [M++1].
C

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H. Takahashi et al. / Life Sciences 76 (2005) 2185–2192
4-Thiocarbamoylmorpholine (5e). Mp. 178.9 to 179.78C; IR (KBr) 3326, 3216, 1509 cmÀ1; 1H NMR
(DMSO-d6) y 3.56 (4 H, t, J = 4.8 Hz, CH2), 3.71 (4 H, t, J = 4.4 Hz, CH2), 7.49 (2 H, br s, NH); 13
NMR (DMSO-d6) y 47.4, 65.7, 181.4; MS (CI): m/z = 147 [M++1].
C
Assay of superoxide anion-scavenging activity (SOSA)
The SOSA of selenoureas and thioureas was measured by a previously reported method
(Kimura and Nakano, 1988; Kato et al., 2002; Kato et al., 2003). In brief, the standard reaction
mixture contained 10À7 M MCLA, 5
10À5 M hypoxanthine, XOD (6.5 U), SOD (0.2 to 20
Â
ng / mL) and 50 mM Tris-HCl buffer containing 0.1 mM EDTA (pH 7.8), in a total volume of
3.0 ml, in the presence or absence of various concentrations of the selenoureas or thiourea being
tested. Chemiluminescence measurement using a luminometer (Aloka, BLR102) at 258C was
initiated upon the addition of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-
one hydrochloride (MCLA) to the standard incubation mixture excluding XOD. Measurement
continued for 2 min without XOD and then for an additional 2 min after addition of XOD. A
representative example of a measurement of the effect of 5c on MCLA-dependent luminescence is
shown in Fig. 1.
The inhibition (%) of compounds had strong SOSA at 333 nM was also measured at 33.3 and
3.33 nM. The luminescence intensity (count / min) of the solution which does not contain a
substance at all, and the solution containing 4c, 4d, 4e, or 5c was measured, and the inhibition (%)
was computed. The inhibition (%) and the concentration of 4c, 4d, 4e, or 5c were plotted, and
concentration equivalent to 50% of rates of inhibition was set to IC50.
This study considered the elimination of superoxide anion generated by XOD by selenamides.
Exact SOSA cannot be measured if selenamides inhibit the activity of XOD. We checked that
measured electron spin resonance of super oxide anion and selenamides did not inhibit the activity
of XOD (Tanigawa, 1990) (data not shown).
Fig. 1. Dose-dependent effect of 1-thiocarbamoylpyrrolidine (5c) on 2-methyl-6-( p-methoxyphenyl)-3,7-dihydroimidazo-[1,2-
a] pyrazin-3-one (MCLA)-dependent luminescence. Incubation conditions are given in the text. Arrows indicate the time at
which MCLA or xanthine oxidase (XOD) was added.