N-methylthioureas as new agonists of retinoic acid receptor-related orphan receptor

Article abstract of DOI:10.1007/s12272-012-0809-0

Thirty two thiourea derivatives were prepared and their agonistic activities on the retinoic acid receptor-related orphan receptor α (RORα) were evaluated. The replacement of the 3-allyl-2-imino-thiazolidin- 4-one moiety of the lead compound CGP52608 (1) with various functional group substituted aromatic rings, improved the agonistic activity of RORα. Among the prepared derivatives, 1-methyl-3-(4-phenoxy-benzyl)-thiourea (32) showed 2.6-fold higher agonistic activity than CGP52608 in the RORα-activation assay.

Full text of DOI:10.1007/s12272-012-0809-0

Arch Pharm Res Vol 35, No 8, 1393-1401, 2012
DOI 10.1007/s12272-012-0809-0
N-Methylthioureas as New Agonists of Retinoic Acid Receptor-Related
Orphan Receptor
Yohan Park1, Suckchang Hong2, Myungmo Lee2, Hyojun Jung2, Won-Jea Cho3, Eun-Jin Kim4, Ho-Young Son4,
Mi-Ock Lee4, and Hyeung-geun Park 2
2
1College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, Korea, Research Institute of
3
Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, Korea, College of Phar-
4
macy Chonnam National University, Kwangju 500-757, Korea, and Bio-MAX Institute and College of Pharmacy, Seoul
National University, Seoul 151-742, Korea
(Received March 15, 2012//Accepted April 12, 2012)
Thirty two thiourea derivatives were prepared and their agonistic activities on the retinoic
acid receptor-related orphan receptor
α
(ROR
α
) were evaluated. The replacement of the 3-
) with various func-
tional group substituted aromatic rings, improved the agonistic activity of ROR . Among the
prepared derivatives, 1-methyl-3-(4-phenoxy-benzyl)-thiourea (32) showed 2.6-fold higher ago-
nistic activity than CGP52608 in the ROR -activation assay.
allyl-2-imino-thiazolidin-4-one moiety of the lead compound CGP52608 (
1
α
α
Key words: Thiourea derivatives, Agonists, Retinoid acid receptor-related orphan receptor α,
CGP52608
homeostasis of cholesterol (Wada et al., 2008). In
addition, ROR participates in the maintenance of
Retinoic acid related-receptor orphan receptors, bone tissue (Meyer et al., 2000). Since those physiolo-
ROR (Becker-Andre et al., 1993; Giguere et al., 1994, gical functions of ROR can be possibly modulated by
1995), ROR (Carlberg et al., 1994), and ROR (Hirose exogenous ligands, the discovery of new non-natural
et al., 1994; Ortiz et al., 1995; Medvedev et al., 1996; ligands may lead to the development of novel thera-
He et al., 1998; Jetten et al., 2001; He, 2002; Jetten peutics for human diseases that involve ROR . In
and Ueda, 2002; Eberl and Littman, 2003), are trans- 1995 and 1996, the pineal gland hormone melatonin
cription factors belonging to the steroid hormone re- and thiazolidinone type CGP52608 ( ), respectively,
ceptor superfamily. These receptors have been regarded were identified as efficient agonists of ROR and showed
INTRODUCTION
α
α
α
β
γ
α
1
α
as critical factors in the regulation of a number of phy- antiarthritic activity in vivo, indicating that they may
siological processes (Jetten, 2004). Among the receptors, be used as novel therapeutics for the treatment of
ROR
α plays an important role in the development of rheumatoid arthritis and other autoimmune diseases
the cerebellum by regulating Purkinje cell differen- (Steinhilber et al., 1995; Wiesenberg et al., 1995; Missbach
tiation and proliferation of granule cell progenitors et al., 1996). Recent studies on the crystal structure
(Gold et al., 2003). ROR
steroid metabolism including lipogenesis and fatty that cholesterol sulfate is an efficient agonist of ROR
acid oxidation (Vu-Dac et al., 1997; Lau et al., 2008), (Kallen et al., 2002, 2004). The activated transcription
hepatic lipid metabolism (Wang et al., 2010), and of ROR by cholesterol itself increased apoA-I expres-
α also plays a role in lipid and and transcriptional activity of RORα also revealed
α
α
sion, which carries cholesterol from peripheral cells to
the liver to protect against cholesterol accumulation in
arterial walls, preventing coronary arteriosclerosis (Vu-
Dac et al., 1997; Raspe et al., 2001).
Correspondence to: Hyeung-geun Park, Research Institute of
Pharmaceutical Sciences and College of Pharmacy, Seoul
National University, Seoul 151-742, Korea
Tel: 82-2-880-7871, Fax: 82-2-872-9129
In this paper, we report the synthesis and ROR
α ac-
E-mail: hgpk@snu.ac.kr
tivity of thiourea derivatives, based on thiazolidinone
1393

1394
Y. Park et al.
(MS) were measured on a JEOL JMS-AX 505wA,
JEOL JMS-HX/HX 110A spectrometer. Melting points
were measured on a Buchi B-540 melting point appar-
atus and were not corrected.
General procedure
The coupling of methylisocynate with various ben-
Fig. 1. Structure of CGP52608 (1) and N-methyl thiourea
analogues (2).
zylic amines
ureas ( 29). N-Methyl isothiocyanate (0.15 mL, 2.20
mmol) was added to a stirred solution of substituted
). As part of our program to develop benzylamine (1.00 mmol) in diethyl ether or dichloro-
novel drug-like ROR agonists for the treatment of methane (4 mL). The reaction mixture was stirred suf-
metabolic disorders such as fatty liver diseases, we ficiently at ambient temperature until benzylamines
chose the first non-natural ligand, CGP52608 ( ) as a were disappeared by TLC analysis. Then the reaction
lead compound and attempted to replace the thiazolidin- mixture was evaporated and diluted with diethyl ether
4-one moiety of with the phenyl rings, substituted with (100 mL) and filtered to afford N-methylthioureas.
3 gave the corresponding N-methylthio-
6-
type CGP52608 (
1
α
1
1
various functional groups. Based on the similarity of The residue was purified by column chromatography
the two moieties in size, we assumed that the N-methyl- (silica gel, hexanes-EtOAc = 3:1 or 1:1) to afford N-
thiourea analogues (2) could retain the agonistic ac- methylthioureas (Scheme 1).
tivity against ROR (Fig. 1).
α
The formation of imine from aldehydes
5 and N-
methylthiourea in the presence of Ti(OiPr)4 under
THF, followed by reduction with NaBH4 provided the
MATERIALS AND METHODS
Chemistry
corresponding N-methylthioureas (30-36) (Armstrong
et al., 1997). Magnetic bar was added to two-neck round
Organic solvents were concentrated under reduced bottom flask and vaccumed. After Ar gas substituted,
pressure using a Büchi rotary evaporator. Syringes, N-Methyl thiourea (450.8 mg, 5.00 mmol) was added
needles and cannulae were oven dried at 100oC. TLC to a stirred solution of substituted benzaldehyde (1.00
analyses were performed using Merck precoated TLC mmol) in anhydrous THF (4 mL). Then titanium(IV)
plate (silica gel 60 GF254, 0.25 mm). Flash column chro- isopropoxide (0.50 mL, 1.70 mmol) was added to a reac-
matography was carried out using E. Merck Kieselgel tion mixture. The reaction mixture was refluxed until
60 (230~400 mesh). Infrared (IR) spectra were recorded benzaldehyde were disappeared by TLC analysis. Then
on a JASCO FT/IR-300E and Perkin-Elmer 1710 FT the reaction mixture was cooled down to room tem-
spectrometer. Nuclear magnetic resonance (1H-NMR perature and NaBH4 (37.8 mg, 1.00 mmol) was added.
and 13C-NMR) spectra were measured on JEOL JNM- After stirring for 1 h, neutralized reaction mixture
LA 300 [300 MHz (1H), 75 MHz (13C)] spectrometer, using 1 M HCl. The reaction mixtures was diluted
JEOL JNM-GSX 400 [400 MHz (1H), 100 MHz (13C)] with dichloromethane (50 mL), washed with brine (20
spectrometer, and Bruker AMX 500 [500 MHz (1H), mL), dried over anhydrous MgSO4, filtered, and concen-
125 MHz (13C)] spectrometer, using CHCl3-d, CH3OH- trated in vacuo. The residue was purified by column
d
, DMSO-d as solvents. Nuclear magnetic resonance chromatography (silica gel, hexanes-EtOAc = 2:1) to
(1H-NMR and 13C-NMR) spectra using CHCl3-d were afford N-methylthioureas (Scheme 1).
reported in ppm relative to CHCl3 (
δ
δ
7.24) for 1H-NMR
77.23) resonance 1-Benzyl-3-methyl-thiourea (6)
and relative to the central CDCl3 (
for 13C-NMR. Nuclear magnetic resonance (1H-NMR White solid; yield 99%; m.p. 89oC; IR (KBr) 3258,
and 13C-NMR) spectra using CH3OH-
in ppm relative to CH3OH (
relative to the central CH3OH (
d were reported 3062, 3029, 2931, 2854, 1954, 1870, 1810, 1657, 1557,
1
δ
4.87) for H-NMR and 1496, 1453, 1372, 1346, 1293, 1227, 1202, 1173, 1090,
δ
49.15) resonance for 1070, 1028, 959, 742, 697 cm−1; FAB-MS (m/z): 181
13C-NMR. Nuclear magnetic resonance (1H-NMR and [M+H]+; H-NMR (300 MHz, CDCl3)
δ 7.29-7.20 (m,
1
13C-NMR) spectra using DMSO-
ppm relative to DMSO (
2.50) for 1H-NMR and NMR (100 MHz, CDCl3)
relative to the central DMSO (
13C-NMR. Coupling constants (
d
were reported in 5H), 6.69 (s, 2H), 4.58 (s, 2H), 2.81 (s, 3H) ppm; 13C-
δ
δ
181.8, 137.0, 128.2, 127.1,
δ 39.51) resonance for 126.9, 47.7, 30.5 ppm.
J) in 1H-NMR are in
Hz. Low-resolution mass spectra (MS) were recorded 1-(4-Fluoro-benzyl)-3-methyl-thiourea (7)
on a VG Trio-2 GC-MS spectrometer and mass spectra White solid; yield 81%; m.p. 92oC; IR (KBr) 3254, 1604,

N-Methylthioureas as Novel ROR
α
Agonists
1395
1557, 1509, 1345, 1296, 1223, 1157, 1081, 1016, 964, 822 3242, 2923, 1571, 1511, 1458, 1348, 1293, 1219, 1174,
cm−1; FAB-MS (m/z): 199 [M+H]+; 1H-NMR (300 MHz, 1108, 948, 848, 736 cm−1; FAB-MS (m/z): 226 [M+H]+;
CD3OD)
δ
7.35-7.30 (m, 2H), 7.05-6.99 (m, 2H), 4.68 (s, 1H-NMR (300 MHz, DMSO)
δ
8.18 (d,
8.07 (s, 1H), 7.67 (s, 1H), 7.51 (d, = 8.4 Hz, 2H), 4.79
(s, 2H), 2.83 (s, 3H) ppm; 13C-NMR (100 MHz, DMSO)
183.7, 148.0, 146.3, 128.6, 1223.8, 46.4, 30.9 ppm.
J = 8.4 Hz, 2H),
2H), 2.93 (s, 3H) ppm; 13C-NMR (75 MHz, CD3OD)
165.1, 161.8, 136.1, 130.4, 116.2, 72.0, 31.0 ppm.
δ
J
δ
1-(3,4-Difluoro-benzyl)-3-methyl-thiourea (8)
White solid; yield 55%; m.p. 122oC; IR (KBr) 3255, 1-(4-Dimethylamino-benzyl)-3-methyl-thiourea (14)
1611, 1558, 1519, 1437, 1344, 1286, 1210, 1115, 966, Yellow solid; yield 47%; m.p. 89oC; IR (KBr) 3261,
816, 778 cm−1; FAB-MS (m/z): 217 [M+H]+; H-NMR 2936, 1615, 1555, 1523, 1345, 1287, 1228, 1190, 1166,
1
(400 MHz, DMSO)
δ
7.97 (s, 1H), 7.52 (s, 2H), 7.40- 1064, 1018, 947, 807, 752 cm−1; FAB-MS (m/z): 223
7.29 (m, 2H), 7.13 (s, 1H), 4.63 (s, 2H), 2.83 (s, 3H) [M+H]+; H-NMR (400 MHz, DMSO)
δ
7.73 (s, 1H),
= 6.3 Hz, 2H), 6.68 (d, = 6.45
Hz, 2H), 4.49 (s, 2H), 2.87-2.83 (m, 9H) ppm; 13C-NMR
(100 MHz, DMSO) 149.7, 128.4, 126.8, 112.4, 46.8,
40.3, 31.2, 30.9 ppm.
1
ppm; 13C-NMR (100 MHz, DMSO)
δ
183.7, 150.4, 7.33 (s, 1H), 7.13 (d,
J
J
148.0, 137.6, 123.9, 117.2, 45.8, 30.7 ppm.
δ
1-(2,4-Difluoro-benzyl)-3-methyl-thiourea (9)
Yellow solid; yield 99%; m.p. 119oC; IR (KBr) 3225,
1569, 1505, 1428, 1375, 1353, 1272, 1231, 1137, 1103, 1-Methyl-3-(4-methyl-benzyl)-thiourea (15)
1078, 966, 859, 746 cm−1; FAB-MS (m/z): 217 [M+H]+; White solid; yield 69%; m.p. 116oC; IR (KBr) 3245,
1H-NMR (400 MHz, CD3OD)
δ 7.40 (dd, J1 = 14.84 Hz, 3021, 2924, 2103, 1557, 1516, 1454, 1344, 1295, 1229,
J2 = 8.00 Hz, 1H), 6.93-6.88 (m, 2H), 4.72 (s, 2H), 2.94 1083, 1020, 963, 930, 802, 747 cm−1; FAB-MS (m/z):
(s, 3H) ppm; 13C-NMR (100 MHz, CD3OD)
164.1, 163.3, 161.7, 132.5, 124.0, 112.9, 42.9, 31.5 ppm. (m, 4H), 6.29 (s, 2H), 4.67 (s, 2H), 3.02 (d,
δ
165.7, 195 [M+H]+; H-NMR (300 MHz, CDCl3)
δ
J
7.36-7.21
= 4.2 Hz,
1
3H), 2.42 (s, 3H) ppm; 13C-NMR (100 MHz, CDCl3)
δ
1-(4-Chloro-benzyl)-3-methyl-thiourea (10)
182.3, 137.6, 133.8, 129.5, 127.5, 48.2, 30.8, 21.0 ppm.
White solid; yield 71%; m.p. 110oC; IR (KBr) 3255,
3062, 1557, 1492, 1343, 1292, 1091, 1014, 964, 931, 1-Methyl-3-(3-methyl-benzyl)-thiourea (16)
1
802 cm−1; FAB-MS (m/z): 215 [M+H]+; H-NMR (300 White solid; yield 99%; m.p. 77oC; IR (KBr) 3254, 3060,
MHz, CDCl3)
2H), 2.96 (d,
MHz, CDCl3)
30.75 ppm.
δ
7.32-7.24 (m , 4H), 5.96 (s, 2H), 4.68 (s, 2920, 1607, 1557, 1463, 1341, 1295, 1223, 1081, 1024,
= 4.59 Hz, 3H) ppm; 13C-NMR (100 959, 776, 742, 692 cm−1; FAB-MS (m/z): 195 [M+H]+;
182.55, 135.71, 133.51, 128.87, 47.69, 1H-NMR (300 MHz, CDCl3)
7.40-7.33 (m, 2H), 7.24-
7.21 (m, 2H), 6.34 (s, 2H), 4.73 (s, 2H), 3.07 (d,
4.59 Hz, 3H), 2.46 (s, 3H) ppm; 13C-NMR (100 MHz,
CDCl3) 182.50, 138.69, 136.91, 128.80, 128.67, 128.33,
J
δ
δ
J
=
1-(3,4-Dichloro-benzyl)-3-methyl-thiourea (11)
δ
White solid; yield 39%; m.p. 136oC; IR (KBr) 3230, 124.64, 48.51, 30.88, 21.40 ppm.
1557, 1515, 1471, 1299, 1205, 1130, 1030, 961, 824
cm−1; FAB-MS (m/z): 249 [M+H]+; 1H-NMR (300 MHz, 1-Methyl-3-(2-methyl-benzyl)-thiourea (17)
CD3OD)
= 2.01 Hz, 1H), 4.70 (s, 2H), 2.81 (s, 3H) ppm; 13C- 1556, 1461, 1344, 1298, 1078, 1020, 962, 746 cm−1;
NMR (100 MHz, CD3OD)
183.2, 142.2, 132.8, 130.0, FAB-MS (m/z): 195 [M+H]+; 1H-NMR (400 MHz,
δ
7.47-7.42 (m, 2H), 7.23 (dd, J1 = 9.15 Hz, J2 White solid; yield 99%; m.p. 90oC; IR (KBr) 3245, 3064,
δ
126.8, 126.5, 125.8, 46.3, 30.6 ppm.
CD3OD)
2.27 (s, 3H) ppm; 13C-NMR (100 MHz, CD3OD)
183.25, 137.06, 135.51, 129.90, 126.82, 125.72, 45.28,
δ 7.18-7.14 (m, 4H), 4.60 (s, 2H), 2.86 (s, 3H),
δ
1-(2,4-Dichloro-benzyl)-3-methyl-thiourea (12)
White solid; yield 94%; m.p. 110oC; IR (KBr) 3256, 30.92, 18.71 ppm.
3062, 1556, 1297, 1102, 1049, 965, 938, 865, 830, 755
cm−1; FAB-MS (m/z): 250 [M+H]+; 1H-NMR (300 MHz, 1-(4-tert-Butyl-benzyl)-3-methyl-thiourea (18)
CDCl3)
δ 7.42 (d, J
= 2.01 Hz, 1H), 7.35-7.25 (m, 2H), White solid; yield 99%; m.p. 78oC; IR (KBr) 3255, 2961,
4.76 (s, 2H), 2.95 (s, 3H) ppm; 13C-NMR (75 MHz, 1558, 1515, 1465, 1343, 1295, 1081, 1018, 964, 935, 818,
1
CDCl3)
46.9, 32.0 ppm.
δ
173.8, 137.2, 135.6, 135.2, 131.9, 130.8, 128.9, 755 cm−1; FAB-MS (m/z): 237 [M+H]+; H-NMR (300
MHz, CDCl3)
Hz, 2H), 5.86 (s, 2H), 4.61 (s, 2H), 2.96 (d,
1.3 (s, 9H) ppm; 13C-NMR (100 MHz, CDCl3)
δ
7.37 (d,
J
= 8.2 Hz, 2H), 7.24 (d,
= 4.6 Hz, 3H),
182.7,
J = 8.1
J
1-Methyl-3-(4-nitro-benzyl)-thiourea (13)
δ
Yellow solid; yield 89%; m.p. 155oC; IR (KBr) 3378, 151.1, 133.7, 127.4, 126.1, 48.3, 34.5, 31.8, 30.8 ppm.

1396
Y. Park et al.
1-(4-Methoxy-benzyl)-3-methyl-thiourea (19)
1512, 1439, 1352, 1274, 1128, 1025, 805, 762 cm−1; FAB-
White solid; yield 57%; m.p. 98oC; IR (KBr) 3256, MS (m/z): 227 [M+H]+; 1H-NMR (300 MHz, CD3OD)
δ
2935, 2835, 1612, 1556, 1512, 1463, 1345, 1301, 1248, 6.82-6.68 (m, 3H), 4.50 (s, 2H), 3.77 (s, 3H), 2.89 (s,
1176, 1080, 1032, 963, 819 cm−1; FAB-MS (m/z): 211 3H) ppm; 13C-NMR (100 MHz, CD3OD)
δ 149.1, 148.4,
1
[M+H]+; H-NMR (300 MHz, CDCl3)
δ 7.24-7.20 (m , 120.7, 116.5, 113.5, 57.3 ppm.
2H), 6.88-6.83 (m, 2H), 5.98 (s, 2H), 4.55 (s, 2H), 3.77
(s, 3H), 2.94 (d,
= 4.59 Hz, 3H) ppm; 13C-NMR (100 1-(3,4-Dihydroxy-benzyl)-3-methyl-thiourea (25)
MHz, CDCl3)
182.18, 159.13, 128.90, 114.12, 55.21, Yellow solid; yield 58%; m.p. 137oC; IR (KBr) 3430,
J
δ
47.89, 30.76 ppm.
1615, 1572, 1512, 1443, 1342, 1288, 1112, 1016, 863,
1
780, 596 cm−1; FAB-MS (m/z): 213 [M+H]+; H-NMR
1-Benzo[1,3]dioxol-5-ylmethyl-3-methyl-thiourea
(20)
(300 MHz, CD3OD)
δ
6.76-6.61 (m, 3H), 4.50 (s, 2H),
183.4,
2.93 (s, 3H) ppm; 13C-NMR (125 MHz, CD3OD)
δ
Yellow solid; yield 89%; m.p. 148oC; IR (KBr) 3361, 146.4, 145.7, 131.1, 120.1, 116.2, 115.8, 59.8, 25.8 ppm.
3198, 3023, 1576, 1525, 1498, 1447, 1365, 1252, 1212,
1038, 936, 822, 636 cm−1; FAB-MS (m/z): 225 [M+H]+; 1-Methyl-3-(4-trifluoromethyl-benzyl)-thiourea (26)
1H-NMR (300 MHz, DMSO)
δ
7.84 (s, 1H), 7.46 (s, Yellow solid; yield 75%; m.p. 90oC; IR (KBr) 3261,
1H), 6.88-6.75 (m, 3H), 5.97 (s, 2H), 4.53 (s, 2H), 2.82 3065, 2925, 1620, 1559, 1420, 1327, 1231, 1165, 1122,
(s, 3H) ppm; 13C-NMR (100 MHz, DMSO) 183.25, 1066, 1018, 968, 941, 820, 751 cm−1; FAB-MS (m/z):
147.10, 146.01, 133.32, 120.46, 107.92, 100.75, 48.58, 249 [M+H]+; 1H-NMR (300 MHz, CDCl3)
7.59 (d,
= 8.04 Hz, 2H), 5.98 (s, 2H),
= 4.95 Hz, 2H), 2.98 (d, = 4.95 Hz, 3H)
182.9, 141.5, 130.1,
δ
δ
J =
46.68, 30.59 ppm.
8.25 Hz, 2H), 7.44 (d,
4.81 (d,
ppm; 13C-NMR (75 MHz, CDCl3)
J
J
J
1-(2,4-Dimethoxy-benzyl)-3-methyl-thiourea (21)
δ
White solid; yield 85%; m.p. 90oC; IR (KBr) 3264, 127.6, 125.6, 122.1, 47.9, 30.7 ppm.
2938, 1614, 1589, 1555, 1508, 1463, 1346, 1286, 1208,
1157, 1129, 1081, 10435, 919, 833, 753 cm−1; FAB-MS
(S
)-1-[1-(4-Methoxy-phenyl)-ethyl]-3-methyl-thio-
1
(
m/z): 241 [M+H]+; H-NMR (300 MHz, CD3OD)
δ
urea (27)
7.18-7.15 (m, 1H), 6.43-6.40 (m, 2H), 4.46 (s, 2H), 3.78 Yellow solid; yield 99%; m.p. 119oC; IR (KBr) 3255,
(d,
= 12.09 Hz, 6H), 2.93 (s, 3H) ppm; 13C-NMR (100 2967, 1611, 1552, 1513, 1456, 1344, 1248, 1178, 1107,
MHz, CD3OD)
181.7, 160.7, 158.0, 130.6, 117.2, 104.3, 1034, 832, 755 cm−1; FAB-MS (m/z): 225 [M+H]+; 1H-
J
δ
99.4, 55.4, 55.3, 43.3, 31.5 ppm.
NMR (300 MHz, CD3OD)
δ
7.24 (d,
= 8.61 Hz, 2H), 5.33 (s, 1H), 3.71 (s, 3H),
= 4.23 Hz, 3H), 1.37 (d, = 6.78 Hz, 3H)
181.9, 158.0,
J = 8.61 Hz, 2H),
6.87 (d,
2.82 (d,
J
J
1-Methyl-3-(3,4,5-trimethoxy-benzyl)-thiourea (22)
J
White solid; yield 92%; m.p. 149oC; IR (KBr) 3344, ppm; 13C-NMR (75 MHz, CD3OD)
δ
2939, 2837, 1594, 1556, 1506, 1460, 1423, 1331, 1235, 136.3, 136.3, 127.2, 113.5, 55.0, 51.7, 30.5, 22.2 ppm.
1181, 1126, 1004, 831, 753 cm−1; FAB-MS (m/z): 271
1
[M+H]+; H-NMR (300 MHz, DMSO)
δ
7.83 (s, 1H),
(R)-1-[1-(4-Methoxy-phenyl)-ethyl]-3-methyl-thio-
7.46 (s, 1H), 6.64 (s, 2H), 4.55 (s, 2H), 3.74 (s, 6H), 3.62 urea (28)
(s, 3H), 2.83 (s, 3H) ppm; 13C-NMR (100 MHz, DMSO) Yellow oil; yield 99%; IR (KBr) 3257, 2967, 2835, 1612,
δ
182.8, 152.7, 136.4, 134.9, 104.8, 60.0, 55.8, 47.3, 1553, 1513, 1461, 1345, 1248, 1179, 1108, 1034, 833, 755
30.6 ppm.
cm−1; FAB-MS (m/z): 225 [M+H]+; 1H-NMR (300 MHz,
DMSO) 7.17-7.14 (m, 2H), 6.79-6.74 (m, 2H), 5.21 (s,
1H), 3.66 (s, 3H), 2.82 (s, 3H), 1.35 (d, = 6.8 Hz, 3H)
160.2, 136.9, 128.3,
δ
1-(3,5-Dimethoxy-benzyl)-3-methyl-thiourea (23)
J
White solid; yield 70%; m.p. 128oC; IR (KBr) 3256, ppm; 13C-NMR (75 MHz, DMSO)
2938, 2837, 1462, 1430, 1345, 1296, 1204, 1156, 1064, 114.9, 55.7, 53.8, 22.5 ppm.
959, 1833, 751 cm−1; FAB-MS (m/z): 241 [M+H]+; 1H-
δ
NMR (300 MHz, CD3OD)
δ 6.48 (d, J = 2.22 Hz, 2H), 1-Biphenyl-4-ylmethyl-3-methyl-thiourea (29)
6.35 (t,
J
= 2.22 Hz, 1H), 4.62 (s, 2H), 3.74 (s, 6H), 2.94 White solid; yield 87%; m.p. 139oC; IR (KBr) 3242, 3054,
(s, 3H) ppm; 13C-NMR (75 MHz, CD3OD)
106.3, 100.1, 55.7 ppm.
δ
162.5, 1556, 1488, 1343, 1299, 1078, 1009, 940, 846, 762, 696
cm−1; FAB-MS (m/z): 257 [M+H]+; 1H-NMR (300 MHz,
CDCl3)
6.02 (s, 2H), 4.40 (s, 2H), 5.65 (d,
13C-NMR (100 MHz, CDCl3)
182.5, 140.7, 140.3, 136.0,
δ
7.25 (d,
J
= 7.8 Hz, 4H), 7.15-7.02 (m, 5H),
1-(3-Hydroxy-4-methoxy-benzyl)-3-methyl-thiourea
(24)
J
= 4.23 Hz, 4H) ppm;
δ
White solid; yield 64%; m.p. 150oC; IR (KBr) 3334, 1558, 128.7, 127.9, 127.4, 127.3, 126.9, 48.1, 30.8 ppm.

N-Methylthioureas as Novel ROR
α
Agonists
1397
1-Methyl-3-(4-pyridin-2-yl-benzyl)-thiourea (30)
1-[4-(4-Fluoro-phenoxy)-benzyl]-3-methyl-thiourea
Yellow solid; yield 79%; m.p. 109oC; IR (KBr) 3253, (35)
2058, 1557, 1469, 1435, 1343, 1292, 1085, 1015, 965, Yellow oil; yield 67%; IR (KBr) 3254, 3062, 1557, 1496,
1
940, 777, 751 cm−1; FAB-MS (m/z): 258 [M+H]+; H- 1334, 1250, 1213, 1192, 1167, 1089, 1013, 964, 876, 831
NMR (300 MHz, CDCl3)
7.94 (d, = 8.22 Hz, 2H), 7.78-7.68 (m, 2H), 7.40 (d,
= 8.04 Hz, 2H), 7.21 (s, 1H), 6.17 (s, 1H), 5.99 (s, 1H), 2H), 4.63 (d,
4.71 (s, 2H), 2.98 (d,
= 4.56 Hz, 3H) ppm; 13C-NMR ppm; 13C-NMR (100 MHz, CD3OD)
(100 MHz, CDCl3) 182.8, 156.9, 149.3, 138.5, 138.4, 155.4, 130.9, 122.3, 120.1, 118.1, 118.0, 62.3, 21.7 ppm.
δ 8.66 (d, J
= 4.95 Hz, 1H), cm−1; FAB-MS (m/z): 291 [M+H]+; 1H-NMR (300 MHz,
J
J
CDCl3)
δ
7.28-7.26 (m, 2H), 7.05-6.91 (m, 6H), 5.91 (s,
= 4.74 Hz, 2H), 2.97 (d, = 4.95 Hz, 3H)
161.9, 160.0, 159.0,
J
J
J
δ
δ
137.0, 127.8, 127.2, 122.2, 120.7, 48.0, 30.9 ppm.
1-[4-(4-Methoxy-phenoxy)-benzyl]-3-methyl-thio-
urea (36)
1-(4-Benzyloxy-benzyl)-3-methyl-thiourea (31)
Yellow solid; yield 30%; m.p. 127oC; IR (KBr) 3317, Yellow solid; yield 84%; m.p. 102oC; IR (KBr) 3255,
1610, 1549, 1509, 1453, 1173, 1014, 948, 843, 816, 741, 2939, 1556, 1499, 1464, 1344, 1295, 1227, 1101, 1033,
696 cm−1; FAB-MS (m/z): 287 [M+H]+; H-NMR (300 964, 875, 842, 753 cm−1; FAB-MS (m/z): 303 [M+H]+;
1
MHz, CD3OD)
Hz, 2H), 6.97-6.94 (m, 2H), 5.07 (s, 2H), 4.55 (s, 2H), 6.84 (m, 6H), 6.15 (s, 2H), 4.58 (s, 2H), 2.93 (s, 3H) ppm;
2.82 (s, 3H) ppm; 13C-NMR (75 MHz, CD3OD) 182.8, 13C-NMR (100 MHz, CDCl3)
182.4, 158.3, 15630, 149.6,
δ 7.44-7.28 (m, 5H), 7.21 (d, J δ 7.24-7.20 (m, 2H), 6.93-
= 8.43 1H-NMR (400 MHz, CDCl3)
δ
δ
168.9, 157.3, 145.5, 137.2, 131.6, 128.6, 127.7, 114.5, 128.8, 121.1, 117.8, 117.2, 114.9, 55.6, 48.0 ppm.
69.1, 46.4, 31.3 ppm.
1-Methyl-3-naphthalen-2-ylmethyl-thiourea (37)
1-Methyl-3-(4-phenoxy-benzyl)-thiourea (32)
N-Methyl thiourea (180.3 mg, 2.00 mmol) was added
Yellow solid; yield 65%; m.p. 104oC; IR (KBr) 3254, to a stirred solution of 2-bromomethyl naphthalene
1589, 1556, 1505, 1488, 1343, 1237, 1166, 1073, 1017, (221.1 mg, 1.00 mmol) in MeOH (4 mL). After stirring
871, 753, 692 cm−1; FAB-MS (m/z): 273 [M+H]+; H- for 2 h, the reaction mixtures was evaporated and
1
NMR (300 MHz, CDCl3)
(m, 1H), 6.98-6.92 (m, 4H), 6.04 (s, 2H), 4.62 (s, 2H), mL), dried over anhydrous MgSO4, filtered, and con-
2.95 (d,
= 4.56 Hz, 3H) ppm; 13C-NMR (100 MHz, centrated in vacuo. The residue was purified by column
CDCl3) 182.5, 157.1, 156.8, 131.6, 129.8, 129.2, 123.5, chromatography (silica gel, EtOAc-MeOH = 10:1) to
δ 7.33-7.23 (m, 4H), 7.11-7.06 diluted with EtOAc (50 mL), washed with brine (20
J
δ
119.0, 48.0, 30.8 ppm.
afford 37 (131.3 mg) as white solid; yield 57%; m.p.
97oC; IR (KBr) 3052, 1650, 1603, 1508, 1413, 1165, 1019,
1
1-Methyl-3-(3-phenoxy-benzyl)-thiourea (33)
860, 820, 754 cm−1; FAB-MS (m/z): 231 [M+H]+; H-
Yellow solid; yield 84%; m.p. 93oC; IR (KBr) 3254, NMR (400 MHz, DMSO)
δ 9.27 (s, 1H), 7.91-7.86 (m,
3062, 1574, 1556, 1487, 1444, 1341, 1252, 1213, 1163, 5H), 7.55-7.51 (m, 3H), 4.71 (s, 2H), 2.83 (s, 3H) ppm;
1073, 1023, 965, 758, 691 cm−1; FAB-MS (m/z): 273 13C-NMR (100 MHz, DMSO)
δ 163.2, 133.7, 132.7,
δ 7.33-7.25 (m, 132.2, 128.3, 128.1, 128.0, 127.6, 126.9, 126.2, 34.71,
[M+H]+; H-NMR (400 MHz, CD3OD)
1
3H), 7.09-7.05 (m, 2H), 6.97-6.95 (m, 3H), 6.83 (dd, J1 30.5 ppm.
= 6.44 Hz, J2 = 1.48 Hz, 1H), 4.68 (s, 2H), 2.92 (s, 3H)
ppm; 13C-NMR (100 MHz, CD3OD)
δ 184.8, 159.6,
Biological assay
159.4, 143.2, 131.6, 125.2, 124.0, 120.7, 120.0, 119.5,
119.2, 42.3, 34.3 ppm.
HepG2 cells obtained from the American Type Cul-
ture Collection were maintained at 37oC in 5% CO2 in
Dulbecco’s Modification of Eagle’s Medium with 10%
fetal bovine serum. HepG2 cells were plated at a density
1-Methyl-3-(4-
o-tolyloxy-benzyl)-thiourea (34)
Yellow solid; yield 24%; m.p. 156oC; IR (KBr) 3230, of 1
×
105 cells per well of a 12-well plate and were
3063, 1557, 1486, 1453, 1343, 1233, 1075, 875, 752, 691 transfected with RORE-tk-Luc plasmid using Welfect-
cm−1; FAB-MS (m/z): 273 [M+H]+; 1H-NMR (300 MHz, EXTM PLUS (WelGENE Inc.). After transfection, cells
CD3OD)
δ 7.40-7.31 (m, 3H), 7.26-7.21 (m, 1H), 7.13- were treated with increasing concentration of compound
7.06 (m, 2H), 6.98-6.95 (m, 2H), 6.85-6.82 (m, 1H), 4.72
1
(lead compound) or 32 (a derivative); from 1 to 100
(s, 2H), 2.90 (s, 3H) ppm; 13C-NMR (75 MHz, CD3OD)
(
µ
M). Reporter gene analysis was as previously describ-
δ
158.9, 156.0, 130.9, 130.4, 129.7, 125.2, 124.8, 124.2, ed (Kim et al., 2008).
119.9, 119.2, 54.8, 44.1 ppm.
Molecular modeling
For the docking study of 32 into the binding site of

1398
Y. Park et al.
RORα, Surflex-Dock was used as the docking algori- using HepG2 cells transfected with RORE-Luc, as
thm in Sybyl version 8.1.1 (Tripos Associates), which described previously (Kim et al., 2008). The fold activ-
was operated under Red Hat Linux 4.0 on an IBM com- ation in the presence of test compounds was compared
puter (Intel Pentium 4, 2.8 GHz CPU, 1GB memory). with that of no treatment. As shown in Table I, most
As the binding site of ROR
cholesterol sulfate complex structure (PDB# 1S0X) ity of ROR
α
from the given ROR
α
-
of thiourea derivatives induced transcriptional activ-
. The agonistic activity was not sensitive
α
was prepared to give a protomol, the chemical struc- to the nature of the functional groups; however, the
ture of 32 was also prepared using the Sybyl package substituted position was important to their biological
with standard bond lengths and angles. The structure activity. Generally, para-substitutents showed higher
was minimized through the conjugate gradient method agonistic activity than the corresponding meta- or
by applying Gasteiger-Huckel charge with a distance- ortho-substitutents. Further substitution at the meta
-
dependent dielectric function. The Surflex-Dock per- and ortho-position in addition to the para-position led
formed the docking to the protomol, which represents to a significant decrease of activity. Some para-sub-
the ligand-binding pocket of ROR
is critical and essential for docking since it elucidates nistic activity (15, 4-CH3, 3.9; 19, 4-CH3O, 3.4) except
potential interactions with the site allowing for rapid for the bulky groups (14, (CH3)2N, 1.3; 18 tert-butyl,
docking of ligands into simplistic representations of 0.9). A large loss of activity was observed for the
the site. As a result of the Surflex-Dock of 32, twenty methylated analogues of 19 27, 0.7; 28, 0.6) at
α, and the protomol stituted electron donating groups exhibited high ago-
,
(
R
-
S-
conformers were aligned in a high scoring sequence, benzylic position, which implies that the agonistic
and the best scoring conformer was selected to further activity is quite sensitive to the conformation. The
investigate the binding mode of the ligand to the biphenyl derivative (29, 0.2) had the lowest agonistic
active site.
activity, but high activity was observed for the 2’-pyri-
dyl analogue (30, 1.9). The ether linkage analogue
showed the highest agonistic activity (32, 4.6). A similar
decreases in activity were observed for the meta- and
RESULTS AND DISCUSSIONS
Thirty two N-methylthiourea derivatives were easily ortho-analogues (33, 1.0; 34, 1.3). The cumulative
prepared in one step from the corresponding amines results indicate that the binding pocket of ROR is
α
or aldehydes (Scheme 1). All compounds gave satisfac- not spacious but rather linear. Unfortunately, a further
tory spectroscopic data consistent with the proposed increase in activity by substitution of functional group
structures. The coupling of methylisocynate with vari- at 32 was not observed (35, 1.5; 36, 2.1).
ous benzylic amines
methylthioureas ( 28). The formation of imine from and 32 in activating RORE-Luc and examined the
aldehydes and N-methylthiourea in the presence of transcriptional activity depending on the concentra-
Ti(OiPr)4 under THF, followed by reduction with NaBH4 tion along with CGP52608 (
provided the corresponding N-methylthioureas (29 tional activity of CGP52608 (
37) (Armstrong et al., 1997).
The ROR agonistic activities of the prepared deri- might be due to the less sensitivity of our assay system.
3
gave the corresponding N-
We chose two of the highest effective agonists 31
6-
4
1
). The ROR
α transcrip-
-
1) was lower than that of
the previous report (Wiesenberg et al., 1995), which
α
vatives were evaluated based on transcription activity The transcriptional activity was clearly proportional
to the concentration of 31 and 32 (Fig. 2).
To examine the docking of the most effective agonist
32 into the ligand-binding pocket of ROR
crystal structure of ROR complex with cholesterol
sulfate (PDB# 1S0X) was applied (Kallen et al., 2002).
The binding site of ROR contains two characteristic
α, the X-ray
α
α
regions: hydrophilic and hydrophobic areas in which
some amino residues interact with the functional groups
of 32 (Fig. 3). In terms of hydrophilic interactions, two
hydrogen bonds between each NH of thiourea in 32
and the carbonyl oxygen of Gln 289 were observed. In
terms of hydrophobic interactions, the phenoxy benzene
ring of 32 was thoroughly enclosed with residues Cys
323, Ile 327, Ala 330, Phe 365 and Phe 381 of the lipo-
philic pocket, suggesting that compound 32 fit well
Scheme 1. General procedure for preparation of N-methyl-
thioureas.

N-Methylthioureas as Novel ROR
α
Agonists
1399
Table I. In vitro biological activity of the N-methylthioureas in the RORα transcriptional assay
Compound No.
Fold activationa
1.5
Compound No.
Fold activation
6
22
2.2
1.0
7
8
2.5
0.8
23
24
1.9
9
0.9
2.6
1.0
25
26
27
0.6
2.3
0.7
10
11
12
13
14
0.6
1.6
1.3
28
29
30
0.6
0.2
1.9
15
16
3.9
1.1
31
32
3.6
4.8
17
18
0.7
0.9
33
34
1.0
1.3
19
20
3.4
1.7
35
36
1.5
2.1
21
1.1
37
0.7
aFold activation of the derivative was the ratio of activating activity of RORE-Luc in the presence of derivative (100 mM)
compared to that of no treatment15 and fold activation values were estimated from 3 times replicates [
(n = 3)].
p < 0.01 vs vehicle

1400
Y. Park et al.
ACKNOWLEDGEMENTS
This work was supported by the National Research
Foundation (NRF) grant funded by the Korea govern-
ment (MEST) (No. 201000017).
REFERENCES
Armstrong, J. D., III, Wolfe, C. N., Keller, J. L., Lynch, J.,
Bhupathy, M., Volante, R. P., and De Vita, R. J., A novel
synthesis of disubstituted ureas using titanium (IV) iso-
propoxide and sodium borohydride. Tetrahedron Letts.,
38, 1531-1532 (1997).
Becker-Andre, M., Andre, E., and DeLamarter, J. F., Identi-
fication of nuclear receptor mRNAs by RT-PCR amplifica-
tion of conserved zinc-finger motif sequences. Biochem.
Biophys. Res. Commun., 194, 1371-1379 (1984).
Carlberg, C., Hooft van Huijsduijnen, R., Staple, J. K., De-
Lamarter, J. F., and Becker-Andre, M., RZRs, a new family
of retinoid-related orphan receptors that function as both
monomers and homodimers. Mol. Endocrinol., 8, 757-770
(1994).
Fig. 2. RORα transcriptional activity of 31 and 32. HepG2
cells were transfected with the RORE-Luc and treated with
compounds (31 and 32). ** < 0.01 and *** < 0.001 vs
vehicle; ##p < 0.01 and ###p < 0.001 vs
(100 M) (n = 3).
p
p
1
µ
Eberl, G. and Littman, D. R., The role of the nuclear hormone
receptor RORgammat in the development of lymph nodes
and Peyer's patches. Immunol. Rev., 195, 81-90 (2003).
Giguere, V., Tini, M., Flock, G., Ong, E., Evans, R. M., and
Otulakowski, G., Isoform-specific amino-terminal domains
dictate DNA-binding properties of ROR alpha, a novel
family of orphan hormone nuclear receptors. Genes Dev.
,
8, 538-553 (1994).
Giguere, V., Beatty, B., Squire, J., Copeland, N. G., and
Jenkins, N. A., The orphan nuclear receptor ROR alpha
(RORA) maps to a conserved region of homology on
human chromosome 15q21-q22 and mouse chromosome 9.
Genomics, 28, 596-598 (1995).
Gold, D. A., Baek, S. H., Schork, N. J., Rose, D. W., Larsen,
D. D., Sachs, B. D., Rosenfeld, M. G., and Hamilton, B. A.,
RORalpha coordinates reciprocal signaling in cerebellar
development through sonic hedgehog and calcium-de-
pendent pathways. Neuron, 40, 1119-1131 (2003).
He, Y. W., Deftos, M. L., Ojala, E. W., and Bevan, M. J.,
RORgamma t, a novel isoform of an orphan receptor,
negatively regulates Fas ligand expression and IL-2 pro-
duction in T cells. Immunity, 9, 797-806 (1998).
Fig. 3. The H-bond mode of 32 docked with amino acids in
the binding site of ROR
α.
into the binding site of ROR
In conclusion, thirty two thiourea derivatives were
prepared and their agonistic activities against ROR
α.
α
were evaluated. A significant increase in agonistic
activities were observed by replacing the 3-allyl-2-
imino-thiazolidin-4-one moiety of the lead compound
He, Y. W., Orphan nuclear receptors in T lymphocyte devel-
opment. J. Leukoc. Biol., 72, 440-452 (2002).
CGP52608 (1) with substituted phenyl rings. Among
Hirose, T., Smith, R. J., and Jetten, A. M., ROR gamma: the
third member of ROR/RZR orphan receptor subfamily
that is highly expressed in skeletal muscle. Biochem. Bio-
phys. Res. Commun., 205, 1976-1983 (1994).
Jetten, A. M., Kurebayashi, S., and Ueda, E., The ROR
nuclear orphan receptor subfamily: critical regulators of
multiple biological processes. Prog. Nucl. Acid Res., 69,
205-247 (2001).
them, 1-methyl-3-(4-phenoxy-benzyl)-thiourea (32) showed
the best agonistic activity. We believe this pharmaco-
phore information would be very useful in the design
of more potent agonistic scaffolds for the treatment of
metabolic disorders such as fatty liver diseases. The
further focused structure-activity relationship study is
now under investigation.
Jetten, A. M. and Ueda, E., Retinoid-related orphan receptors

N-Methylthioureas as Novel ROR
α
Agonists
1401
(RORs): roles in cell survival, differentiation and disease.
Cell Death Differ., 9, 1167-1177 (2002).
Jetten, A. M., Recent advances in the mechanisms of action
and physiological functions of the retinoid-related orphan
receptors (RORs). Curr. Drug Targets Inflamm. Allergy, 3,
395-412 (2004).
Kallen, J. A., Schlaeppi, J. M., Bitsch, F., Geisse, S., Geiser,
M., Delhon, I., and Fournier, B., X-ray structure of the
hRORalpha LBD at 1.63 A: structural and functional data
that cholesterol or a cholesterol derivative is the natural
ligand of RORalpha. Structure, 10, 1697-1707 (2002).
Kallen, J., Schlaeppi, J. M., Bitsch, F., Delhon, I., and Fournier,
B., Crystal structure of the human RORalpha Ligand
binding domain in complex with cholesterol sulfate at 2.2
A. J. Biol. Chem., 279, 14033-14038 (2004).
Kim, E. J., Yoo, Y. G., Yang, W. K., Lim, Y. S., Na, T. Y., Lee,
I. K., and Lee, M.-O., Transcriptional activation of HIF-1 by
RORalpha and its role in hypoxia signaling. Arterioscler.
Thromb. Vasc. Biol., 28, 1796-1802 (2008).
Lau, P., Fitzsimmons, R. L., Raichur, S., Wang, S. C., and
Lechtken, A., The orphan nuclear receptor, RORalpha, regu-
lates gene expression that controls lipid metabolism:
staggerer (SG/SG) mice are resistant to diet-induced
obesity. J. Biol. Chem., 283, 18411-18421 (2008).
Medvedev, A., Yan, Z. H., Hirose, T., Giguere, V., and Jetten,
A. M., Cloning of a cDNA encoding the murine orphan re-
ceptor RZR/ROR gamma and characterization of its re-
sponse element. Gene, 181, 199-206 (1996).
Meyer, T., Kneissel, M., Mariani, J., and Fournier, B., In
vitro and in vivo evidence for orphan nuclear receptor
RORalpha function in bone metabolism. Proc. Natl. Acad.
Sci. U. S. A., 97, 9197-9202 (2000).
Missbach, M., Jagher, B., Sigg, I., Nayeri, S., Carlberg, C.,
and Wiesenberg. I., Thiazolidine diones, specific ligands of
the nuclear receptor retinoid Z receptor/retinoid acid re-
ceptor-related orphan receptor alpha with potent antiar-
thritic activity. J. Biol. Chem., 271, 13515-13522 (1996).
Ortiz, M. A., Piedrafita, F. J., Pfahl, M., and Maki, R., TOR:
a new orphan receptor expressed in the thymus that can
modulate retinoid and thyroid hormone signals. Mol.
Endocrinol., 9, 1679-1691 (1995).
Raspe, E., Duez, H., Gervois, P., Fievet, C., Fruchart, J. C.,
Besnard, S., Mariani, J., Tedgui, A., and Staels, B., Tran-
scriptional regulation of apolipoprotein C-III gene expres-
sion by the orphan nuclear receptor RORalpha. J. Biol.
Chem., 276, 2865-2871 (2001).
Steinhilber, D., Brungs, M., Werz, O., Wiesenberg, I.,
Danielsson, C., Kahlen, J.-P., Nayeri, S., Schrader, M., and
Carlberg, C., The nuclear receptor for melatonin represses
5-lipoxygenase gene expression in human B lymphocytes.
J. Biol. Chem., 270, 7037-7040 (1995).
Vu-Dac, N., Gervois, P., Grötzinger, T., De Vos, P., Schoonjans,
K., and Fruchart, J. C., Transcriptional regulation of apo-
lipoprotein A-I gene expression by the nuclear receptor
RORalpha. J. Biol. Chem., 272, 22401-22404 (1997).
Wada, T., Kang, H. S., Jetten, A. M., and Xie, W., The emerg-
ing role of nuclear receptor RORalpha and its crosstalk
with LXR in xeno- and endobiotic gene regulation. Exp.
Biol. Med., 233, 1191-1201 (2008).
Wang. Y., Solt, L. A., and Burris, T. P., Regulation of FGF21
expression and secretion by retinoic acid receptor-related
orphan receptor alpha. J. Biol. Chem., 285, 15668-15673
(2010).
Wiesenberg, I., Missbach, M., Kahlen, J.-P., Schraeder, M.,
and Carlberg, C., Transcriptional activation of the nuclear
receptor RZR alpha by the pineal gland hormone melato-
nin and identification of CGP 52608 as a synthetic ligand.
Nucleic Acids Res., 23, 327-333 (1995).