Development of novel silicon-containing inverse agonists of retinoic acid receptor-related orphan receptors

Article abstract of DOI:10.1016/j.bmc.2014.01.023

Retinoic acid receptor (RAR)-related orphan receptors (RORs) regulate a variety of physiological processes, including hepatic gluconeogenesis, lipid metabolism, circadian rhythm and immune function. The RAR agonist: all-trans retinoic acid was reported to be an RORβ inverse agonist, but no information is available regarding ROR activity of its synthetic analogue Am580. Therefore, we screened Am580 and some related tetramethyltetrahydronaphthalene derivatives and carried out structural development studies, including substitution of carbon atoms with silicon, with the aim of creating a potent ROR transcriptional inhibitor. The phenyl amide disila compound 22 showed the most potent ROR-inhibitory activity among the compounds examined. Its activity towards RORα, RORβ and RORγ was increased compared to that of Am580. The IC₅₀ values for RORα, RORβ and RORγ are 1.3, >10 and 4.5 μM, respectively.

Full text of DOI:10.1016/j.bmc.2014.01.023

Bioorganic & Medicinal Chemistry 22 (2014) 1948–1959
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel silicon-containing inverse agonists of retinoic
acid receptor-related orphan receptors
a
a
b
Hirozumi Toyama a, , Masaharu Nakamura , Masahiko Nakamura , Yotaro Matsumoto ,
⇑
Madoka Nakagomi c, Yuichi Hashimoto a
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Faculty of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan
c Research Foundation Itsuu Laboratory, 2-28-10 Tamagawa, Setagaya-ku, Tokyo 158-0094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Retinoic acid receptor (RAR)-related orphan receptors (RORs) regulate a variety of physiological pro-
cesses, including hepatic gluconeogenesis, lipid metabolism, circadian rhythm and immune function.
The RAR agonist: all-trans retinoic acid was reported to be an RORb inverse agonist, but no information
is available regarding ROR activity of its synthetic analogue Am580. Therefore, we screened Am580 and
some related tetramethyltetrahydronaphthalene derivatives and carried out structural development
studies, including substitution of carbon atoms with silicon, with the aim of creating a potent ROR tran-
scriptional inhibitor. The phenyl amide disila compound 22 showed the most potent ROR-inhibitory
Received 25 December 2013
Revised 14 January 2014
Accepted 18 January 2014
Available online 8 February 2014
Keywords:
Retinoic acid receptor-related orphan
receptor
Silicon
activity among the compounds examined. Its activity towards ROR
pared to that of Am580. The IC50 values for ROR , RORb and ROR
Ó 2014 Elsevier Ltd. All rights reserved.
a
, RORb and ROR
c
was increased com-
a
c
are 1.3, >10 and 4.5
lM, respectively.
Inverse agonist
1. Introduction
which have the consensus RGGTCA core motif, in the regulatory
region of target genes.5 Most other nuclear receptors, such as RARs,
ligand X receptors (LXRs) and so on, form heterodimers with
retinoid-X receptors (RXRs) and these dimers bind to the corre-
sponding promoter region. In contrast, RORs bind to ROREs in mono-
mer form and do not form heterodimers with retinoid-X receptors.6
Nuclear receptors, including RORs, have two transcriptional
activation sites. One is activation function 1 (AF1), which perma-
nently activates transcription without ligand binding. The other is
Nuclear receptors are a highly conserved group of transcription
factors that regulate a range of metabolic, endocrine and immuno-
logic disorders, including cancer, inflammation, diabetes and
atherosclerosis. Forty-eight NRs have been identified in humans
and approximately half of them have been characterized as ligand-
activated transcription factors regulating the expression of target
genes.
A large number of these receptors are still classified as orphan
receptors due to the lack of a characterized natural ligand. Retinoic
acid receptor (RAR)-related orphan receptor (ROR) is example of
an orphan nuclear receptor that plays critical roles in immunity, cel-
lular metabolism and circadian rhythm.1 There are three subtypes of
ROR, which are differentiated by alternative promoter usage and
activation function 2 (AF2), which activates transcription in
response to ligand binding. AF2 is in helix12 and consists of the motif
PLYKELF, which is 100% conserved among RORs.7 Deletion of helix12
or point mutation within helix12 causes loss of ROR transactiva-
tion.7 It is believed that helix10 plays a critical role in homodimer-
ization and heterodimerization of nuclear receptors.7 Structural
analyses revealed that the presence of a kink in helix10 of the LBD
exon splicing.2 In humans, ROR
RORb and ROR each have two isoforms, RORb1, b2 and ROR
t), respectively.3 RORs have a typical nuclear receptor domain
a
has four isoforms, ROR
a1–a
4.
c
c1,
c2
of ROR
is consistent with the fact that RORs do not form homodimers or het-
erodimers with other RORs or RXRs. ROR is expressed in various
a
and RORb impairs the dimerization ability of RORs.8 This
(c
structure consisting of four major functional domains: a N-terminal
(A/B) domain, a DNA-binding domain (DBD), a hinge domain and a
C-terminal ligand-binding domain (LBD).4 RORs regulate gene tran-
scription by binding to specific DNA response elements (ROREs),
a
tissues, including kidney and liver, but is most highly expressed in
the brain, particularly cerebellum and thalamus.9 In contrast, RORb
is expressed only in certain regions of the brain and retina.5 ROR
c1
and ROR
expression: ROR
liver, skeletal muscle, and kidney, whereas ROR
expressed in cell types associated with the immune system.10
c
t (ROR
c
2) exhibit distinct patterns of tissue-specific
1 is expressed in a variety of tissues, including
t is exclusively
c
c
⇑
Corresponding author. Tel.: +81 3 5841 7847; fax: +81 3 5841 8495.
E-mail address: hashimot@iam.u-tokyo.ac.jp (H. Toyama).
http://dx.doi.org/10.1016/j.bmc.2014.01.023
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
1949
O
Recent studies have demonstrated that RORs function as ligand-
dependent transcriptional factors.11 Additionally, ROR
and ROR
O
a
c
O
OH
have essential role for the development of TH17 cells and are
OH
N
H
relevant to autoimmune disease.12 Among synthetic ligands, LXR
agonist T0901317 was reported as a ROR
and T0901317 derivative SR1001 was reported as ROR
a/c
dual inverse agonist,12
a/
ATRA (all-trans retinoic acid)
Am580 (1)
c
-selective inverse agonist without LXR agonist activity.12–16
Among natural ligands, 7a-hydroxycholesterol was reported as a
Figure 2. The chemical structures of Am580 and ATRA.
ROR
ated by ROR
an ROR
agonist.18 Likewise, digoxin19,20 and ursolic acid21 were
reported to be ROR inverse agonists. Moreover, all-trans retinoic
a
/
c
inverse agonist that diminished basal transcription medi-
a/ .
c 17
Cholesterol sulfate was reported to function as
a
which was extracted from the known structure of the ROR
c
LBD–
c
digoxin complex (PDB ID: 3B0W), using AutoDock 4.2 software. Di-
acid (ATRA), which is a natural RAR agonist, was also reported to
goxin is an ROR
c
inverse agonist.19,20 The result is illustrated in
act as a RORb inverse agonist (Fig. 1).22
Figure 3, as visualized with PyMOL software.31
ATRA is a first-generation topical retinoid that is widely used to
treat acne vulgaris and keratosis pilaris. It is also used to treat
acute promyelocytic leukemia (APL) under the brand name Vesa-
noidÒ (Roche). ATRA is the best studied retinoid for treatment of
photoaging,23 and is widely used for treatment of acne.24 It is also
used to treat hair loss25 and as a component of commercial prod-
ucts intended to ameliorate skin aging or to remove wrinkles.26,27
Topical ATRA is used to improve the appearance of stretch marks
via induction of increased collagen production in the dermis.28
As Am580 (1) was successfully docked to the ROR
c LBD, we
anticipated that Am580 would bind to the RORc LBD and show
ROR-inhibitory activity. Therefore, we next screened Am580
and compounds 2, 3 and 4 containing TMN structure32 for ROR-
inhibitory activity, using luciferase reporter gene assay with
nuclear receptor expression plasmids hRORa1, hRORb1 and hRORc1
and a luciferase reporter, RORE-TK-Luc. T0901317 and SR1001
were used as positive controls.
As shown in Table 1, Am580 showed weak ROR-inhibitory
activity. Surprisingly, compound 2, which has a benzoyl group,
and compound 3, which has an acetyl group, were more potent
inhibitors than Am580. Further, compound 2 exhibited slight RORb
Am580 (1), an analog of ATRA that acts as a selective RARa ago-
nist, was developed by Kagechika in 1988, and contains an aro-
matic carboxylic acid moiety instead of the polyenecarboxylic
acid of ATRA (Fig. 2).29 Its IC50 values for RAR
a
, RARb and RAR
are 0.3, 8.6 and 13 nM, respectively. Notably, Am580 induces
IL-4, IL-5 and IL-13, inhibits IL-12 and IFN synthesis, and induces
c
selectivity and compound 3 exhibited slight RORa/c-selectivity.
Compound 4 (TMN) showed little activity.
c
As mentioned earlier, ROR
mune disease and ROR inverse agonist has potential utility in
the treatment of autoimmune diseases. Therefore, we focused on
structural development of compound 3 to create a novel ROR
preferential transcriptional inhibitor possessing TMN structure.
a and RORc are relevant to autoim-
cell differentiation with over 7 times the potency of ATRA in vitro.
Am580 has powerful and selective cyto-differentiating effects on
APL,30 but there has been no report on its ROR ligand activity.
We considered that Am580 and its derivatives might be novel
ROR inverse agonists. Based on this hypothesis, we designed
screening, computational docking and structural development
studies to create novel ROR ligands. Here, we describe synthesis
of several tetramethyltetrahydronaphthalene (TMN) derivatives
and evaluation of their ROR inverse agonistic activities in luciferase
reporter gene assays.
a/c
a/c-
Moreover, considering the characteristic differences between
carbon compounds and their silicon analogs, we also synthesized
silicon analogs. The strategy of the structural development study
is illustrated in Figure 4.
(A) Conversion of TMN structure to 5-membered ring, Si-ring,
TMS group or other group to examine whether ring size
and C/Si exchange influence ROR-inhibitory activity.
2. Results and discussion
(B) Conversion of acetyl group to alcohol, aldehyde, amide or
other group to examine whether ketone and amide struc-
tures are important for ROR-inhibitory activity.
2.1. Docking study and screening for ROR-inhibitory activity
First, we performed a computational docking study of Am580
(1) with the structure of the ROR
c
ligand binding domain (LBD),
F3C
H
N
O
O
N
S
S
S
F3C
HO
NH
O
O
F3C
HO
O
N
CF3
CF3
T0901317
RORα/γ
inverse agonist
SR1001
RORα/γ
inverse agonist
O
O
OH
H
H
O
H
HO
O
HO
HO
O
O
H
OH
O
O
HO
H
H
Digoxin (natural RORγ inverse agonist)
Figure 1. Chemical structures of natural and synthetic ROR ligands.
Figure 3. Binding model of Am580 (1) at the RORc LBD.

1950
H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
Table 1
RORa/b/c-inhibitory activity (percent) by Am580 and TMN derivatives at 1 and 10 lM
ROR
a
inhibition
10
RORb inhibition
M (%) 10
ROR
c
inhibition
10
1
l
M (%)
l
M (%)
1
l
l
M (%)
1
l
M (%)
l
M (%)
O
O
OH
N
H
<5.0
12.2
<5.0
11.6
5.8
9.1
1
(Am580)
O
12.8
22.7
<5.0
10.8
27.6
<5.0
6.6
20.2
17.8
<5.0
<5.0
7.0
18.4
24.4
<5.0
2
O
<5.0
<5.0
3
4
<5.0
O
Si
Si
Si
A) 5-membered ring, Si-ring, TMS group
a
b
etc..
B) alcohol,
aldehyde,
amide
OH
O
O
4
3
Ph
etc..
O
H
N
H
Cl
Si
Si Cl
Cl
c
HO
HO
OH
+
Si
Si
Si
Si Cl
Cl
etc..
Ph
C) silanediol
TMNAc
Cl
24
25
26
Figure 4. Strategy for structural development of compound 3.
TMS
O
OTMS
Si
Si
Si
Si
Si Cl
Si Cl
d
30
X
X
X
e
(C) Conversion of acetyl group to silanol and silanediol, which is
an isostere of ketone, to examine the effect of silicon
substitution.33
X = CH2: 26
X = (CH2)2: 27
X = CH2: 28
X = (CH2)2:
31
X = CH2:
X = (CH2)2: 32
29
OH
O
Si
g
Si
Si
f
X
X
Si
2.2. Synthesis of derivatives of compound 3
14
: X = CH2
7
: X = CH2
5: X = (CH2)2
Compound 3, 5, 6, and 7 were synthesized as shown in
Scheme 1. Compound 3 was synthesized from benzene and 2,5-di-
chloro-2,5-dimethylhexane with aluminum(III) chloride by means
of Friedel–Crafts reaction. Dialkyne 28 and 29 were synthesized
from 26 and 27 by reaction with ethynylmagnesium chloride,
and then cyclized with 3-trimethylsilyloxy-1-butyne (30) in the
presence of cobalt catalyst to give the disila compounds 31 and
32.34 Compounds 31 and 32 were deprotected with acetic acid
and the resulting alcohols 14 and 11 were oxidized with MnO2 to
give the desired ketones 7 and 5. Compound 34 was synthesized
from 1,3-indanedione (33) with trimethylaluminium. The crude
mixture was reacted with acetyl chloride and aluminum(III) chlo-
ride by means of Friedel–Crafts reaction to afford compound 6.
Compounds 9 and 10 were synthesized as shown in Scheme 2.
First, 3- or 4-bromoacetophenone (35 or 36) was ketalized with
ethylene glycol and then reacted with Mg and trimethylsilyl
chloride to give intermediate 39 or 40. Deprotection afforded
3- or 4-(trimethylsilyl)acetophenone 9 or 10.
11: X = (CH2)2
O
O
O
i
h
33
34
6
(crude)
Scheme 1. Synthesis of compound 3 and ring-modified derivatives 5–7. Reagents
and conditions: (a) 2,5-dichloro-2,5-dimethylhexane, AlCl3, CH2Cl2, reflux, 12 h,
67%; (b) acetyl chloride, AlCl3, CH2Cl2, reflux, 2 h, 78%; (c) Mg, tetrahydrofuran,
reflux, 60 °C, 20 h, 29%; (d) ethynylmagnesium chloride, tetrahydrofuran, rt to
reflux, 2 h, 21–87%; (e) 3-trimethylsilyloxy-1-butyne (30), CpCo(CO)2, xylene,
reflux, 9 h, crude; (f) acetic acid, methanol, reflux, 28 h, 1–14% (2 steps); (g)
MnO2, 0 °C–rt, CH2Cl2, 1 h, 33%.; (h) trimethylaluminium, toluene, reflux, 24 h,
crude; (i) acetyl chloride, AlCl3, CH2Cl2, reflux, 2 h, 4% (2 steps).
Compounds 12 and 13 were synthesized as shown in Scheme 3.
Compound 29 was cyclized with propiolaldehyde diethyl acetal

H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
1951
O
O
O
O
O
O
o
o
o
o
a
b
m
p
m
p
m
p
m
p
TMS
Br
Br
TMS
m-Br: 35
36
m-Br: 37
38
m-Br: 39
40
m-TMS: 9
10
p-TMS:
p-Br:
p-Br:
p-Br:
Scheme 2. Synthesis of TMS compounds 9 and 10. Reagents and conditions: (a) ethylene glycol, p-toluenesulfonic acid, benzene, reflux, 39 h, 31–40%; (b) trimethylsilyl
chloride, Mg, tetrahydrofuran, reflux, 1.5 h, 6–23% (2 steps).
(41) in the presence of cobalt catalyst, followed by hydrolysis with
hydrochloric acid to give the disila compound 12. Compound 12
was oxidized with OxoneÒ to give the desired carboxylic acid 13.
The chemical structures of the silanediol and silanol derivatives
are shown in Figure 5. These compounds were synthesized
according to the reported method.34
Disila-amide compounds 18, 20 and 22 were synthesized as
shown in Scheme 4. Compound 13 was amidated with primary
amine, EDC hydrochloride (1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride), DMAP or HOBt (1-hydroxyben-
zotriazole) to give the corresponding amide compounds 18, 20
and 22.
HO OH
HO OH
Si
HO
Si
Si
Ph
15
16
17
Figure 5. Chemical structures of silanediol and silanol derivatives.
O
O
Si
Si
Si
Si
R
a
OH
N
H
Amide compounds 19, 21 and 23 were synthesized as shown in
Scheme 5. Compound 42 was amidated according to method (a) or
(b) to give the corresponding products 19, 21 and 23.
13
18: R=Me
20
: R=t-Bu
22: R=Ph
Scheme 4. Synthesis of disila-amide compounds 18, 20 and 22. Reagents and
conditions: (a) amine reagent, EDC hydrochloride, DMAP or HOBt, N,N-dimethyl-
formamide, room temperature, overnight, 11–94%.
2.3. Evaluation of ROR-inhibitory activity of carbon compounds
and silicon analogs
As shown in Figure 6 and Table 2, the ROR-inhibitory activity of
the six-membered-ring silicon compound 5 was greater than that
of the corresponding carbon analog 3. Similarly, the activity of
the five-membered ring silicon compound 7 was greater than that
of the corresponding carbon analog 6. These results indicate that
changing quaternary carbon to silicon is effective for increasing
O
O
R
a or b
OH
N
H
42
19: R=Me
21: R=t-Bu
23
the inhibitory activity. In addition, the RORa/c-inhibitory activities
: R=Ph
of the six-membered-ring compounds 5 and 3 were higher than
those of the five-membered-ring compounds 6 and 7. However,
the differences between the six-membered and five-membered
compounds were smaller than those between the silicon com-
pounds and the corresponding carbon analogs. The order of ROR-
inhibitory activities was as follows.
Scheme 5. Synthesis of amide compounds 19, 21 and 23. Reagents and conditions:
(a) (1) NHS (N-hydroxysuccinimide), EDC hydrochloride (1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride) CH2Cl2, room temperature; (2) amine
reagent; (3) triethylamine, CH2Cl2, room temperature, 55–63% (3 steps); (b) (1)
(COCl)2, N,N-dimethylformamide (cat.), CH2Cl2, 0 °C to room temperature; (2)
concentration in vacuo; (3) amine reagent, triethylamine, pyridine, CH2Cl2, room
temperature, 59% (3 steps).
5 (Si6) > 7 (Si5) > 3 (C6) > 6 (C5)
To examine the reason for the differences of inhibition activity
between silicon compounds and their carbon analogs, and between
five-membered and six-membered compounds, we calculated the
molecular sizes of the compounds as described below.
(A) The ring size of six-membered-ring carbon compound 3 is
similar that of five-membered-ring silicon compound 7, sug-
gesting that the Me2CCH2CH2CMe2 group of 3 is equivalent
in size to Me2SiCH2SiMe2 of 7.
(B) The ring size of silicon compound 5 is larger than that of the
carbon analogue 3, as expected, because the atomic size of
silicon is larger than that of carbon.
2.4. Calculation of molecular size and charge distribution of
carbon compounds and silicon analogs
The structures of model compounds were obtained by geometry
optimizations at the B3LYP/6-31+G⁄ level with the Gaussian07 pro-
gram package, and superpositions of the calculated structures are
shown in Figure 7.
(C) The ring size of 3 is clearly larger than that of 6.
(D) In common with (B), the ring size of 6 is larger than that of 7.
As mentioned above, the order of ROR-inhibitory activity is as
follows: 5 (Si6) > 7 (Si5) > 3 (C6) > 6 (C5). However, this order is
not to the same as that of ring size. Therefore, some other factor
should also be important for ROR-inhibitory activity.
Electrostatic potential maps of the model compounds are
shown in Figure 8. The silicon compounds have electronic varia-
tions in the ring structure due to the difference of electronegativity
between silicon and carbon. In contrast, the corresponding carbon
analogs have no electronic variation in the ring moiety. As noted
above, the ROR-inhibitory activities of silicon compounds are high-
er than those of the corresponding carbon analogs, and this may be
OEt
OEt
41
a
O
O
Si
Si
Si
Si
b
OH
H
29
12
13
Scheme 3. Synthesis of aldehyde 12 and carboxylic acid 13. Reagents and
conditions: (a) (1) propargylaldehyde diethylacetal (41), CoBr2/Zn, acetonitrile, rt,
16 h; (2) HCl/H2O, rt, 30 min, 28% (2 steps); (b) OxoneÒ, N,N-dimethylformamide, rt,
overnight, 32%.

1952
H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
O
O
O
O
Si
Si
Si
Si
3
5
6
7
F3C
H
O
S
N
O
N
S
S
F3C
HO
NH
O
O
F3C
HO
O
N
CF3
CF3
T0901317 (positive control)
SR1001 (positive control)
Figure 6. RORc-inhibitory activity (percent of control) of compound 3 and the derivatives.
Table 2
RORa/b/c-inhibitory activity (percentage) of 3 and the derivatives at 1 and 10 lM
Compound
ROR
a
inhibition
10
RORb inhibition
10
ROR
c
inhibition
10 lM (%)
1
lM (%)
lM (%)
1
l
M (%)
lM (%)
1
lM (%)
3
5
6
7
22.7
28.4
11.3
24.5
27.6
37.4
26.7
28.1
<5.0
10.4
10.4
22.7
17.8
22.4
14.5
23.6
7.0
24.4
46.2
<5.0
36.8
20.2
<5.0
26.4
related to the difference charge distribution arising from replace-
ment of carbon with silicon.
2.6. Evaluation of ROR-inhibitory activity of compounds 5, 7
and their alcohol, aldehyde and carboxylic acid derivatives
2.5. Evaluation of ROR-inhibitory activity of compounds
containing a TMS moiety
As shown in Table 4, the ROR-inhibitory activity of alcohol
11 is weaker than that of the corresponding ketone derivative
5. Similarly, the inhibitory activity of alcohol 14 is weaker
than that of the corresponding ketone 7. Aldehyde 12 showed
cytotoxicity at 1 and 10 lM. The carboxylic acid 13 showed
weak ROR-inhibitory activity compared to 5. Those results
As shown in Table 3, compound 8 showed little ROR-inhibitory
activity. In contrast, TMS compounds 9 and 10 showed weak
activity, being less potent than 5. This result indicates that a
silicon-containing ring is an important functional group.
indicate that the acetyl group is an important functional

H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
1953
(A)
(B)
(C)
(D)
3 (grey)
3 (grey)
7 (green)
5 (purple)
3 (grey)
6 (orange)
6 (orange)
7 (green)
Figure 7. Superpositions of calculated structures of the model compounds.
+251.0
kcal/mol
Compound 3
Compound 5
–25.1
kcal/mol
Compound 6
Compound 7
Figure 8. Electrostatic potential maps of calculated structures of the model compounds.
group. Further, the inhibition activity of 11 is similar to that
of 14. This result indicates that, among the five-membered
compounds, the difference between alcohol and ketone deriv-
atives is smaller than among the six-membered-ring
compounds.
2.7. Evaluation of ROR-inhibitory activity of 3 and its silanediol
and silanol derivatives
As shown in Table 5, compounds 15 and 16 both showed weak-
er ROR-inhibitory activity than 3. On the other hand, the

1954
H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
Table 3
RORa/b/c-inhibitory activity (percentage) of derivatives of 5 containing TMS structure at 1 and 10 lM
RORa
inhibition
10
RORb inhibition
10
ROR
c
inhibition
10 lM (%)
1
l
M (%)
lM (%)
1
l
M (%)
l
M (%)
1
l
M (%)
Toxica
Toxica
<5.0
8.7
<5.0
<5.0
<5.0
<5.0
5.1
7.9
8
<5.0
<5.0
9
<5.0
28.4
<5.0
37.4
<5.0
10.4
17.9
22.4
7.6
14.5
46.2
10
20.2
5
a
The compound showed weak cytotoxicity.
Table 4
ROR /b/c-inhibitory activity (percentage) of compound 5 and 7 and their derivatives at 1 and 10 lM
a
R
Si
Si
X
Compound
ROR
M (%)
a
inhibition
RORb inhibition
ROR
c
inhibition
X
R
1
l
10
l
M (%)
1
l
M (%)
10
l
M (%)
1
l
M (%)
10 lM (%)
O
5
CH2CH2
28.4
<5.0
Toxic
37.4
10.4
5.3
22.4
20.2
<5.0
Toxic
46.2
23.5
OH
11
12
CH2CH2
CH2CH2
21.3
21.9
b
O
Toxic
Toxic
Toxic
Toxic
H
O
O
13
7
CH2CH2
CH2
<5.0a
24.5
23.4
<5.0a
28.1
26.8
13.3
22.7
18.6
10.8
23.6
22.2
<5.0
26.4
15.8
<5.0
36.8
19.3
OH
OH
14
CH2
b
a
The compound showed weak cytotoxicity.
Racemic mixture.
b
ROR
a
/
c
-inhibitory activity of 17 was equivalent to that of 3, while
its RORb-inhibitory activity is higher than that of 5. In contrast, the
corresponding carbon analog 19 showed little activity. The RORb/
the RORb-inhibitory activity of 17 was greater than that of 3. These
results indicate that 17 showed slight b-selectivity. Among the
silanediol and silanol derivatives, 17 showed the most potent ROR-
inhibitory activity, suggesting that introduction of a bulky phenyl
group at the silicon atoms is beneficial for ROR-inhibitory activity.
c-inhibitory activity of the t-butyl amide disila compound 20 is
weaker than that of 5, and the corresponding carbon analog 21
showed a similar tendency. Among all the carbon and silicon com-
pounds, the phenyl amide disila compound 22 showed the most
potent ROR-inhibitory activity, being more potent than 5. Com-
pound 22 was a much greater inverse agonist of RORs as compared
2.8. Evaluation of ROR-inhibitory activity of 5 and its amide
derivatives
with Am580, with IC50 values for ROR
and 4.5 M, respectively (Fig. 9). In contrast, the corresponding
carbon analog 23 showed IC50 values of >10 M for each of ROR
RORb and ROR . It is possible that the phenyl group is involved in
a, RORb and RORc of 1.3, >10,
l
As shown in Table 6, the ROR
methyl amide disila compound 18 is equivalent to that of 5, while
a
/c-inhibitory activity of the
l
a,
c

H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
1955
Table 5
ROR /b/c-inhibitory activity (percentage) of 3 and its silanediol and silanol derivatives at 1 and 10 lM
a
Compound
ROR
M (%)
a
inhibition
10
RORb inhibition
M (%) 10
ROR
M (%)
c
inhibition
10 lM (%)
R
1
l
lM (%)
1
l
l
M (%)
1
l
O
3
22.7
27.6
<5.0
17.8
7.0
24.4
HO
HO
HO
OH
OH
15
16
<5.0a
<5.0
<5.0a
<5.0
<5.0
<5.0
14.4
7.9
<5.0
<5.0
17.5
<5.0
Si
Si
Si
17
13.1
22.5
19.8
40.9
14.4
27.9
Ph
a
The compound showed weak cytotoxicity.
p
–
p
interaction with an adjacent amino acid in the ROR LBD.
13C NMR spectra were recorded on a JEOL JNM-GX500 (500 MHz)
spectrometer. Chemical shifts are expressed in d (ppm) values with
tetramethylsilane (TMS) as an internal reference. The following
abbreviations are used: s = singlet, d = doublet, t = triplet, q = quar-
tet, qn = quintet, sext = sextet, sep = septet, o = octet, n = nonet,
m = multiplet, br = broad. Fast atom bombardment mass spectra
(FAB-MS) and high resolution mass spectra (HRMS) were recorded
on a JEOL JMS-DX303 spectrometer with m-nitrobenzyl alcohol.
Flash column chromatography was performed on silica gel 60 Kan-
Among these amide compounds, the silicon compounds tend to
be more potent than their carbon analogs. This further supports
the idea that replacing quaternary carbon with silicon is favorable
for ROR-inhibitory activity.
3. Conclusion
Am580 is a synthetic analog of ATRA, which is a natural RORb in-
verse agonist. Therefore, we speculated that Am580 and its deriva-
tives might be novel ROR inverse agonists. Screening assay
showed that Am580 and several derivatives exhibited weak ROR-
inhibitory activity. So, based on computational docking studies, we
designed candidate ROR inverse agonists containing TMN structure
and investigated the effect of silicon substitution. We found that the
ROR-inhibitory activities of silicon compounds tended to be higher
than those of the corresponding carbon analogs, possibly as a result
of differences of molecular size and charge distribution. The phenyl
amide disila compound 22 was the most potent ROR inhibitor
among all the silicon and carbon compounds examined. The IC50 val-
to Kagaku (40–100 lm).
4.2. Synthesis of TMN compounds
4.2.1. Bis(chlorodimethylsilyl)methane (26)
A mixture of Me2SiCl2 (24) (55.3 mL, 0.460 mol) and dimethyl-
chloromethylchlorosilane (25) (20.1 mL, 0.153 mol) in tetrahydro-
furan (32.5 mL) was added dropwise to a solution of Mg (chips,
4.06 g, 0.167 mol) in tetrahydrofuran (10 mL) at 60 °C. The reaction
mixture was refluxed with stirring for 20 h and the precipitate was
removed by filtration. The solvent and unconsumed Me2SiCl2 were
distilled off. Then the reaction mixture was distilled and the frac-
tion with bp 75–80 °C (10 mmHg) was collected (8.98 g, 29%). This
material was used in the next step without further purification. 1H
NMR (500 MHz, CDCl3) d 0.51 (s, 12H), 0.58 (s, 2H).
ues for RORa, RORb and RORc of 1.3, >10, and 4.5 lM, respectively.
4. Experimental
4.1. General
4.2.2. Bis(ethynyldimethylsilyl)methane (28)
Melting points were determined by using a Yanagimoto hot-
stage melting point apparatus and are uncorrected. 1H NMR and
To a solution of bis(chlorodimethylsilyl)methane (26) (5.79 g)
in tetrahydrofuran was added 130 mL of ethylmagnesium chloride
Table 6
RORa/b/c-inhibitory percentage of 5 and its amide derivatives at 1 and 10 lM
ROR
M (%)
a
inhibition
10
RORb inhibition
M (%) 10
ROR
M (%)
c
inhibition
10 lM (%)
Compound
X
R
1
l
lM (%)
1
l
lM (%)
1
l
5
—
Si
C
Si
C
—
28.4
<5.0
6.6
37.4
37.6
<5.0
10.4
<5.0
<5.0
14.0
<5.0
21.2
<5.0
22.4
41.2
<5.0
22.0
11.2
26.9
19.1
20.2
6.3
46.2
41.0
6.7
20.6
15.5
64.1
34.6
18
19
20
21
22
23
Me
Me
t-Bu
t-Bu
Ph
<5.0
15.5
20.4
27.1
<5.0
35.2a
23.7a
49.4
<5.0
38.6a
27.9a
67.6
20.7
Si
C
Ph
a
RORa showed weak cytotoxicity.

1956
H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
O
Si
N
H
Si
Compound 22
Figure 9. RORs-inhibitory activity (percent of control) of compound 22.
solution in tetrahydrofuran (0.5 M). The mixture was stirred for 1 h
at room temperature, refluxed at 95 °C for 1 h, then poured into
satd aq NH4Cl and extracted with ethyl acetate. The organic layer
was washed with water, satd aq NaHCO3 and brine. The organic
layer was dried over MgSO4, concentrated in vacuo and distilled.
The fraction with bp 40–45 °C (5 mmHg) was collected. Bis(ethy-
nyldimethylsilyl)methane (28) was isolated in a yield of 1.09 g
(21%). 1H NMR (500 MHz, CDCl3) d 2.40 (s, 2H), 0.26 (s, 12H),
0.04 (s, 2H). MS (FAB) not found.
4.2.5. 1-(1,1,3,3-Tetramethyl-2,3-dihydrobenzo[1,3]disilol-5-
yl)ethanone (7)
To a solution of 14 (35.0 mg, 0.699 mmol) in anhydrous CH2Cl2
(10 mL), MnO2 (250.4 mg, 1.44 mmol) was added at 0 °C. The reac-
tion mixture was stirred for 1 h at room temperature, then filtered
and the solvents were removed under vacuum. The residue was
purified by silica gel chromatography, affording1-(1,1,3,3-tetra-
methyl-2,3-dihydrobenzo[1,3]disilol-5-yl)ethanone (7) in a yield
of 10.4 mg (33%) as a yellow solid. 1H NMR (500 MHz, CDCl3) d
8.10 (d, 1H, J = 1.0 Hz), 7.91 (dd, 1H, J = 7.3, 2.0 Hz), 7.65 (d, 1H,
J = 7.0 Hz), 2.62 (s, 3H), 0.33 (s, 6H), 0.31 (s, 6H), 0.01 (s, 2H). 13C
NMR (500 MHz, CDCl3) d 199.16, 157.21, 151.16, 136.94, 131.99,
131.08, 128.24. 26.87, 0.56, 0.37, À2.35. MS (FAB, [M+H]+) m/z
249. Mp 34–36 °C.
4.2.3. 1,1,3,3-Tetramethyl-5-[1-(trimethylsilyloxy)ethyl]-2,3-
dihydrobenzo[d][1,3]disiline (31)
To a solution of 28 (0.516 g, 2.86 mmol) and 3-trimethylsilyl-
oxy-1-butyne (0.249 mL, 1.43 mmol) in xylene (2 mL) was added
dicarbonylcyclopentadienyl cobalt(I) (38.1 lL, 0.286 mmol). The
reaction mixture was refluxed at 170 °C for 9 h, and then concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (n-hexane/ethyl acetate = 20:1) and the product was
used in the next step without further purification. 1H NMR
(500 MHz, CDCl3) d 7.51 (s, 2H), 7.33 (s, 1H), 7.28 (dd, 1H, J = 7.0,
2.0 Hz), 4.87 (s, 1H), 2.30 (d, 3H, J = 25 Hz), 0.29 (s, 12H), 0.09 (s,
9H), À0.05 (s, 2H). 13C NMR (500 MHz, CDCl3) d 136.68, 133.48,
131.65, 128.67, 126.06, 125.87, 70.96, 26.87, 0.72, 0.60, 0.27,
À0.08, À2.20, À2.38. MS (FAB) not found.
4.2.6. 1,1,3,3-Tetramethyl-2,3-dihydro-1H-indene (34)
To a solution of trimethylaluminium in toluene (23.3 mL,
42 mmol, 1.8 M) was added dropwise a solution of 1,3-indanedi-
one (33) (3.07 g, 21.0 mmol) in toluene (80 mL). The reaction mix-
ture was refluxed at 130 °C for 24 h. The reaction was quenched
with ice–water and the whole was extracted with hexane. The or-
ganic layer was washed with water and brine, and dried over
MgSO4. The organic layer was concentrated in vacuo and purified
by silica gel column chromatography using hexane. The product
was used in the next step without further purification.
4.2.4. 1-(1,1,3,3-Tetramethyl-2,3-dihydrobenzo[1,3]disilol-5-
yl)ethanol (14)
4.2.7. 1-(1,1,3,3-Tetramethyl-2,3-dihydro-1H-inden-5-
yl)ethanone (6)
The oil 31 was diluted with methanol (2 mL) and acetic acid
(0.5 mL) was added to the solution. The mixture was refluxed for
28 h at 95 °C, poured into satd aq NaHCO3 and extracted with ethyl
acetate. The organic layer was washed with water and brine, dried
over MgSO4 and concentrated in vacuo to afford a colorless oil. This
oil was purified with silica gel column chromatography (n-hexane/
ethyl acetate = 4:1) to afford a colorless oil (32.3 mg, 14% (2 steps)).
1H NMR (500 MHz, CDCl3) d 7.56 (d, 2H, J = 7.0 Hz), 7.39 (dd, 1H,
J = 8.0, 2.0 Hz), 4.91 (m, 1H), 1.52 (d 3H, J = 6.5 Hz), 0.29 (m,
12H), À0.04 (s, 2H). 13C NMR (500 MHz, CDCl3) d 155.14, 149.90,
145.97, 132.08, 128.71, 126.03, 70.81, 25.14, 0.65, À2.22. MS
(FAB, [MÀOH]+) m/z 233.
To a solution of 34 (393.0 mg) in dehydrated CH2Cl2 (4 mL) was
added AlCl3 (451.0 mg, 3.38 mmol) and acetyl chloride (0.240 mL,
3.38 mmol) at room temperature. The mixture was stirred at
80 °C for 2 h, then poured into water and extracted with ethyl ace-
tate. The organic layer was washed with water, satd aq NaHCO3
and brine, dried over MgSO4 and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography (n-hex-
ane/ethyl acetate = 50:1) to afford a colorless oil (198.3 mg, 4.4%
(2 steps)). 1H NMR (500 MHz, CDCl3) d 7.82 (dd, 1H, J = 8.0,
2.0 Hz), 7.73 (d, 1H, J = 1.5 Hz), 7.19 (d, 1H, J = 8.0 Hz), 2.60 (s,
3H), 1.95 (s, 2H), 1.34 (s, 6H), 1.32 (s, 6H). 13C NMR (500 MHz,

H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
1957
CDCl3) d 198.26, 157.27, 151.99, 136.52, 127.83, 122.69, 56.54,
42.81, 42.57, 31.55, 31.32, 26.84. MS (FAB, [M+H]+) m/z 217.
solution in tetrahydrofuran (0.5 M). The reaction mixture was stir-
red for 1 h at room temperature, refluxed at 95 °C for 1 h, then
poured into satd aq NH4Cl and extracted with ethyl acetate. The or-
ganic layer was washed with water, satd aq NaHCO3 and brine,
dried over MgSO4, and concentrated in vacuo. Bis(ethynyldimeth-
ylsilyl)ethane (29) was isolated in quantitative yield. 1H NMR
(500 MHz, CDCl3) d 2.38 (s, 2H), 0.62 (s, 4H), 0.18 (s, 12H). MS
(FAB) not found.
4.2.8. 2-(3-Bromopheny1)-2-methyl-1,3-dioxolane (37)
A mixture of 3-bromoacetophenone 35 (3.30 mL, 25.0 mmol),
ethylene glycol (2.33 g, 37.5 mmol), and p-toluenesulfonic acid
(238 mg, 1.25 mmol) in 180 mL of dry benzene was heated at re-
flux in a flask equipped with a Dean–Stark apparatus. After 8 h,
ethylene glycol (1.55 g, 25 mmol) was added. The reaction mixture
was refluxed for 31 h, then washed with 1 N NaHCO3, H2O, and
brine, and dried over Na2SO4. The residue was purified by silica
gel column chromatography (n-hexane/ethyl acetate = 20:1) to af-
ford 2-(3-bromopheny1)-2-methyl-1,3-dioxolane 37 as a colorless
liquid (2.42 g, 40%). 1H NMR (500 MHz, CDCl3) d 7.64 (m, 1H), 7.41
(m, 2H), 7.21 (t, 1H, J = 7.5 Hz), 4.04 (m, 2H), 3.77 (m, 2H), 1.63 (s,
3H). 13C NMR (500 MHz, CDCl3) d 145.93, 131.01, 129.99, 128.62,
124.08, 122.49, 108.24, 64.63, 27.65. MS (FAB) not found.
4.2.13. 1,1,4,4-Tetramethyl-1,2,3,4-tetrahydrobenzo[1,4]
disiline-6-carbaldehyde (12)
A mixture of iodine (96.8 mg, 381 lmol of I2), zinc (249 mg,
3.80 mmol) and acetonitrile (18 mL) was stirred at 20 °C until the
brown coloration disappeared and a grey suspension was obtained.
Compound 29 (3.70 g, 19.0 mmol), 41 (3.80 mL, 26.6 mmol) and
cobalt(II) bromide in 9 mL of acetonitrile (415.6 mg, 1.90 mmol)
were then added sequentially in single portions to the stirred mix-
ture. The resulting dark green solution was stirred at 20 °C for 16 h.
Then, hydrochloric acid (2 M, 18.5 mL) was added and the reaction
mixture was stirred at 20 °C for 30 min. Ethyl acetate was added
and the organic phase was separated and washed with brine. The
aqueous phases were combined, extracted with a further portion
of ethyl acetate and discarded. The organic phases were combined,
dried over MgSO4, filtered and concentrated. The resulting brown
oil was purified by flash column chromatography (n-hexane/CH2-
Cl2 = 50:1) to yield 1.32 g of a yellow oil. This material was used
in the next step without further purification. 1H NMR (500 MHz,
CDCl3) d 10.01 (s, 1H), 7.95 (d, 1H, J = 1.0 Hz), 7.79 (dd, 1H,
J = 7.5, 2.0 Hz), 7.66 (d, 1H, J = 7.0 Hz), 1.04 (s, 4H), 0.27 (s, 6H),
0.26 (s, 6H) 13C NMR (500 MHz, CDCl3) d 193.21, 154.78, 147.11,
135.44, 134.44, 134.06, 128.63, 7.41, 7.31, À1.54, À1.66. HRMS
(FAB) calcd for C13H20OSi2: 248.1053; found: 249.1045 (M+H)+.
4.2.9. 2-(4-Bromopheny1)-2-methyl-1,3-dioxolane (38)
A mixture of 4-bromoacetophenone 36 (4.98 g, 25.0 mmol),
ethylene glycol (2.33 g, 37.5 mmol), and p-toluenesulfonic acid
(238 mg, 1.25 mmol) in 180 mL of dry benzene was heated at re-
flux in a flask equipped with a Dean–Stark apparatus. After 8 h,
ethylene glycol (1.55 g, 25 mmol) was added and the mixture
was refluxed for 31 h. The mixture was washed with 1 N NaHCO3,
H2O, and brine and dried over Na2SO4. The residue was purified by
silica gel column chromatography (n-hexane/ethyl acetate = 20:1)
to afford 2-(4-bromopheny1)-2-methyl-1,3-dioxolane (38) as col-
orless prisms (1.86 g, 31%). 1H NMR (500 MHz, CDCl3) d 7.46 (d,
2H, J = 8.5 Hz), 7.36 (d, 2H, J = 8.5 Hz), 4.03 (m, 2H), 3.75 (m, 2H),
1.63 (s, 3H). 13C NMR (500 MHz, CDCl3) d 142.54, 131.41, 127.26,
121.98, 108.56, 64.57, 27.62. Mp 34–36 °C. MS (FAB) not found.
4.2.10. 3-(Trimethylsily1)acetophenone (9)
4.2.14. 1,1,4,4-Tetramethyl-1,2,3,4-tetrahydrobenzo[1,4]
disiline-6-carboxylic acid (13)
To a solution of Mg powder (55.7 mg, 2.29 mmol) and trimethyl-
silyl chloride (0.33 mL, 2.58 mmol) was added a solution of 37
(500 mg, 2.07 mmol) in 6 mL of tetrahydrofuran. The mixture was
heated at reflux for 1.5 h, and stirred overnight at room temperature,
then diluted with ether and washed with H2O and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuo. The residue
was purified by silica gel column chromatography (n-hexane/ethyl
A mixture of OxoneÒ (6.53 g, 10.6 mmol), 12 (1.32 g, 5.31 mmol)
in 20 mL of N,N-dimethylformamide was stirred overnight at room
temperature, then diluted with ethyl acetate, and washed with
H2O and brine. The organic layer was dried over Na2SO4 and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane/ethyl acetate = 5:1) to afford a white
solid (448.1 mg, 9%). 1H NMR (500 MHz, CDCl3) d 8.20 (d, 1H,
J = 1.5 Hz), 8.02 (dd, 1H, J = 8.0, 1.5 Hz), 7.61 (d, 1H, J = 7.5 Hz),
1.04 (s, 4H), 0.27 (s, 6H), 0.25 (s, 6H) 13C NMR (500 MHz, CDCl3)
d 172.70, 153.58, 146.53, 134.65, 133.63, 129.12, 128.47, 7.47,
7.36, À1.55, À1.64. MS (FAB, [M+H]+) m/z 264. Mp 134–136 °C.
acetate = 50:1) to afford 3-(trimethylsily1)acetophenone
9
(24.5 mg, 6%) as colorless crystals. 1H NMR (500 MHz, CDCl3) d
8.10 (s, 1H), 7.93 (dt, 1H, J = 8.0, 1.5 Hz), 7.72 (dt, 1H, J = 7.5,
1.5 Hz), 7.45 (t, 1H, J = 7.5 Hz), 2.62 (s, 3H), 0.30 (s, 9H). 13C NMR
(500 MHz, CDCl3) d 198.76, 141.37, 138.12, 136.40, 133.00, 128.89,
128.00, 26.76, À1.12. MS (FAB, [M+H]+) m/z 193. Mp 194–200 °C.
4.2.15. 1,1,4,4-Pentamethyl-1,2,3,4-tetrahydrobenzo-1,4-
disiline-6-carboxamide (18)
4.2.11. 4-(Trimethylsily1)acetophenone (10)
To a solution of Mg powder (55.7 mg, 2.29 mmol) and trimeth-
ylsilyl chloride (0.33 mL, 2.58 mmol) was added a solution of 38
(500 mg, 2.07 mmol) in 6 mL of tetrahydrofuran. The mixture
was heated at reflux for 1.5 h, stirred overnight at room tempera-
ture, then diluted with ether, and washed with H2O and brine.
The organic layer was dried over Na2SO4 and concentrated in va-
cuo. The residue was purified by silica gel column chromatography
(n-hexane/ethyl acetate = 50:1) to afford 4-(trimethylsily1)aceto-
Methylamine solution in 40% methanol (0.341 mmol, 34.8 lL)
was added to a solution of 13 (0.227 mmol, 60 mg), EDC hydro-
chloride (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride) (0.341 mmol, 65.4 mg), HOBt (1-hydroxybenzotriazole)
(0.341 mmol, 52.2 mg) in N,N-dimethylformamide (5 mL) and stir-
red at room temperature overnight, then diluted with ethyl ace-
tate, and washed with H2O and brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (n-hexane/ethyl ace-
tate = 5:1) to afford a white solid (8.2 mg, 11%) 1H NMR (500 MHz,
CDCl3) d 7.86 (d, 1H, J = 1.5 Hz), 7.66 (dd, 1H, J = 7.8, 2.0 Hz), 7.54
(d, 1H, J = 7.5 Hz), 6.24 (s, 1H), 3.01 (d, 3H, J = 4.0 Hz), 1.02 (s,
4H), 0.24 (s, 6H), 0.23 (s, 6H). 13C NMR (500 MHz, CDCl3) d
168.78, 150.20, 146.62, 133.88, 133.67, 131.49, 125.97, 26.92,
phenone 10 (91.7 mg, 23%) as
a
colorless liquid. 1H NMR
(500 MHz, CDCl3) d 7.93 (dd, 2H, J = 6.5, 1.5 Hz), 7.62 (dd, 2H,
J = 6.5, 1.5 Hz), 2.60 (s, 3H), 0.29 (s, 9H), 13C NMR (500 MHz, CDCl3)
d 198.46, 147.30, 137.19, 133.60, 133.55, 127.30, 127.24, 26.73,
26.67, À1.25, À1.32. MS (FAB, [M+H]+) m/z 193.
4.2.12. Bis(ethynyldimethylsilyl)ethane (29)
To a solution of bis(chlorodimethylsilyl)ethane (27) (5.0 g) in
tetrahydrofuran was added 104 mL of ethylmagnesium chloride
7.53, 7.40, À1.49, À1.55. HRMS (FAB) calcd for
C14H23OSi2:
277.1318; found: 278.1337 (M+H)+. Mp 132–134 °C.

1958
H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
4.2.16. N-tert-Butyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydro
benzo-1,4-disiline-6-carboxamide (20)
4.2.21. Methyl-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphtha
len-2-yl)silanediol (15)
tert-Butylamine (0.341 mmol, 35.9
l
L) was added to a solution
The title compound was prepared according to the reported
method.33 White paste. 1H NMR (500 MHz, CDCl3) d 7.60 (1H, s),
7.40 (1H, s), 7.32 (1H, s), 2.98 (2H, s), 1.68 (4H, s), 1.29 (6H, s),
1.28 (6H, s), 0.39 (3H, s). 13C NMR (500 MHz, CDCl3) d: 147.31,
144.36, 132.66, 131.85, 130.51, 126.16, 35.11, 34.93, 34.34, 34.18,
31.84, 31.72. HRMS (FAB) m/z: calcd for C15H24O2SiLi 271.1706,
found 271.1693 (M+Li+).
of 13 (0.227 mmol, 60 mg), EDC hydrochloride (1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride) (0.341 mmol,
65.4 mg) and DMAP (0.341 mmol, 41.7 mg) in N,N-dimethylform-
amide (5 mL). The mixture was stirred at room temperature
overnight, diluted with, ethyl acetate and washed with H2O and
brine. The organic layer was dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (n-hexane/ethyl acetate = 5:1) to afford
a
white solid
4.2.22. Dimethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronapht
halen-2-yl)silanol (16)
(12.2 mg, 17%) 1H NMR (500 MHz, CDCl3)
d
7.87 (d, 1H,
J = 1.5 Hz), 7.56 (dd, 1H, J = 7.8, 2.0 Hz), 7.52 (d, 1H, J = 8.0 Hz),
5.92 (s, 1H), 1.47 (s, 9H), 1.02 (s, 4H), 0.25 (s, 6H), 0.22 (s, 6H).
13C NMR (500 MHz, CDCl3) d 167.49, 149.87, 146.68, 135.08,
133.49, 131.68, 125.52, 51.68, 28.97, 7.56, 7.41, À1.52, À1.55.
HRMS (FAB) calcd for C17H29OSi2: 319.1788; found: 320.1808
(M+H)+. Mp 154–157 °C.
The title compound was prepared according to the reported
method.33 Pale yellow solid. Mp 89–90 °C. 1H NMR (500 MHz,
CDCl3) d 7.44 (1H, d, J = 1.8 Hz), 7.27 (1H, dd, J = 1.8, 7.9 Hz), 7.21
(1H, d, J = 7.9 Hz), 1.68 (4H, s), 1.57 (6H, s), 1.29 (6H, s), 1.28 (6H,
s). 13C NMR (500 MHz, CDCl3) d: 145.97, 144.57, 143.23, 126.26,
122.27, 121.82, 72.46, 35.23, 35.08, 34.40, 33.98, 31.90, 31.84,
31.6. HRMS (FAB) m/z: calcd for
C17H26O 246.1984, found
4.2.17. 1,1,4,4-Tetramethyl-N-phenyl-1,2,3,4-tetrahydrobenzo-
1,4-disiline-6-carboxamide (22)
246.1972 (M+Li+).
Aniline (0.341 mmol, 31.0
l
L) was added to a solution of 13
4.2.23. Phenyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphth
alen-2-yl)silanediol (17)
(0.227 mmol, 60 mg), EDC hydrochloride (1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride) (0.341 mmol,
65.4 mg), DMAP (0.341 mmol, 41.7 mg) in N,N-dimethylformamide
(5 mL). The mixture was stirred at room temperature overnight,
then diluted with, ethyl acetate and washed with H2O and brine.
The organic layer was dried over Na2SO4 and concentrated in va-
cuo. The residue was purified by silica gel column chromatography
(n-hexane/ethyl acetate = 5:1) to afford a white solid (72.3 mg,
94%) 1H NMR (500 MHz, CDCl3) d 7.99 (d, 1H, J = 1.5 Hz), 7.76
(dd, 1H, J = 8.0, 2.5 Hz), 7.71 (d, 2H, J = 7.5 Hz), 7.58 (d, 1H,
J = 8.0 Hz), 7.36 (t, 2H, J = 7.5, 7.0 Hz) 7.15 (t, 1H, J = 7.5, 7.3 Hz)
1.05 (s, 4H), 0.27 (s, 6H), 0.26 (s, 6H). 13C NMR (500 MHz, CDCl3)
d 166.45, 150.92, 147.02, 138.12, 134.27, 133.79, 131.83, 129.15,
125.96, 124.61, 120.36, 7.53, 7.39, À1.50, À1.53. HRMS (FAB) calcd
for C19H25OSi2: 339.1475; found: 340.1496 (M+H)+. Mp 149–
152 °C.
The title compound was prepared according to the reported
method.35 White solid. Mp 98–100 °C. 1H NMR (500 MHz, CDCl3)
d: 7.70 (2H, d, J = 6.7 Hz), 7.66 (1H, s), 7.44–7.31 (4H, m), 7.30
(1H, d, J = 8.0 Hz), 1.68 (4H, s), 1.27 (6H, s), 1.26 (6H, s). 13C NMR
(500 MHz, CDCl3) d: 147.50, 144.35, 134.54, 134.45, 134.35,
132.75, 131.34, 130.66, 130.36, 127.90, 126.16, 35.10, 34.93,
34.36, 34.18, 31.83, 31.70. HRMS (FAB) m/z: calcd for C20H26O2SiLi
333.1862, found 333.1872 (M+Li+).
4.2.24. 5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalene-2-
carboxamide (19)
The title compound was prepared according to the procedure
described for Section 2.2, starting from compound 42, and method
(a). Mp: 136.5–138 °C. 1H NMR (500 MHz, CDCl3) d 7.74(d, 1H,
J = 2.0 Hz), 7.44 (dd, 1H, J = 8.0, 2.0 Hz), 7.34 (d, 1H, J = 8.0 Hz),
6.04 (br s, NH), 1.69 (s, 4H), 1.30 (s, 6H), 1.28 (s, 6H). HRMS
(FAB) calcd for C16H23NO: 246.1858; found: 246.1822 (M+H)+.
4.2.18. 5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl-
ethanone (3)
The title compound was prepared according to the procedure
described for Section 2.2, starting from benzene. 1H NMR
(500 MHz, CDCl3) d 7.92 (d, 1H, J = 1.9 Hz), 7.70 (dd, 1H, J = 8.0,
1.9 Hz), 7.38 (d, 1H, J = 8.0 Hz), 2.55 (s, 3H), 1.69 (s, 4H), 1.30 (s,
6H), 1.28 (s, 6H). HRMS (FAB) calcd for C16H23O: 231.1749; found:
231.1780 (M+H)+.
4.2.25. N-tert-Butyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydrona
phthalene-2-carboxamide (21)
The title compound was prepared according to the procedure
described for Section 2.2, starting from compound 42, and method
(b). Mp: 154–155.5 °C. 1H NMR (500 MHz, CDCl3) d 7.77 (d, 1H,
J = 2.0 Hz), 7.36 (dd, 1H, J = 8.0, 2.0 Hz), 7.32 (d, 1H, J = 8.0 Hz),
5.89 (br s, NH), 1.69 (s, 4H), 1.46 (s, 9H), 1.30 (s, 6H), 1.27 (s,
6H). MS (FAB, [M+H]+) m/z 288. Calcd for C19H29NO: C, 79.39; H,
10.17; N, 4.87; O, 5.57. found: C, 79.23; H, 10.12; N, 4.91.
4.2.19. 1,1,4,4-Tetramethyl-1,2,3,4-tetrahydrobenzo-1,4-disilin-
6-yl-thanone (5)
The title compound was prepared according to the procedure
described for Section 2.2, starting from compound 27. 1H NMR
(500 MHz, CDCl3) d 8.05 (d, 1H, J = 2.0 Hz), 7.86 (dd, 1H, J = 7.0,
2.0 Hz), 7.59 (d, 1H, J = 7.0 Hz), 2.60 (s, 3H), 1.03 (s, 4H), 0.26 (s,
6H), 0.24 (s, 6H). HRMS (FAB) calcd for C14H23OSi2: 263.1287;
found: 263.1284 (M+H)+.
4.2.26. 5,5,8,8-Tetramethyl-N-phenyl-5,6,7,8-tetrahydronaphth
alene-2-carboxamide (23)
The title compound was prepared according to the procedure
described for Section 2.2, starting from compound 42, and method
(a). Mp: 124–127.5 °C. 1H NMR (500 MHz, CDCl3) d 7.86 (d, 1H,
J = 2.0 Hz), 7.72 (br s, NH), 7.64 (d, 1H, J = 7.0 Hz), 7.55 (dd, 1H,
J = 8.0, 2.0 Hz), 7.41 (d, 1H, J = 8.0 Hz), 7.37 (t, 2H, J = 7.0 Hz), 7.15
(t, 1H, J = 7.0 Hz), 1.72 (s, 4H), 1.33 (s, 6H), 1.30 (s, 6H). HRMS
(FAB) calcd for C21H25NO: 308.2014; found: 308.1980 (M+H)+.
4.2.20. 1,1,4,4-Tetramethyl-1,2,3,4-tetrahydrobenzo-1,4-disilin-
6-yl-ethan-1-ol (11)
The title compound was prepared according to the procedure
described for Section 2.2, starting from compound 27. Yellow oil.
1H NMR (500 MHz, CDCl3) d 7.49 (m, 2H), 7.36 (dd, 1H, J = 7.5,
2.0 Hz), 4.88 (q, 1H, J = 6.0 Hz), 1.51 (d, 3H, J = 6.5), 1.01 (s, 4H),
0.23 (s, 6H), 0.22 (s, 6H). 13C NMR (500 MHz, CDCl3) d 146.34,
145.27, 145.15, 133.86, 130.48, 125.29, 70.74, 25.06, 7.65, 7.58,
À1.38, À2.39. MS (FAB, [MÀOH]+) m/z 247.
4.3. Computational methods
All calculations were carried with the Gaussian 09 program
package. The molecular structures and harmonic vibrational fre-
quencies were obtained using the hybrid density functional

H. Toyama et al. / Bioorg. Med. Chem. 22 (2014) 1948–1959
3. He, Y. W.; Deftos, M. L.; Ojala, E. W.; Bevan, M. J. Immunity 1998, 9, 797.
1959
method based on Becke’s three-parameter exchange function and
the Lee–Yang–Parr nonlocal correlation functional (B3LYP). The
6-31+G⁄ basis set was used for the all atoms. Geometry optimiza-
tion and vibrational analysis were performed at the same level. All
stationary points were optimized without any symmetry assump-
tions, and characterized by normal coordinate analysis at the same
level of theory (number of imaginary frequencies, NIMAG, 0 for
minima).
4. Evans, R. M. Science 1988, 240, 889.
5. Andre, E.; Gawlas, K.; Becker-Andre, M. Gene 1998, 216, 277.
6. Schräder, M.; Danielsson, C.; Wiesenberg, I.; Carlberg, C. J. Biol. Chem. 1996, 271,
19732.
7. Kurebayashi, S.; Ueda, E.; Sakaue, M.; Patel, D. D.; Medvedev, A.; Zhang, F.;
Jetten, A. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10132.
8. Kallen, J. A.; Schlaeppi, J. M.; Bitsch, F.; Geisse, S.; Geiser, M.; Delhon, I.;
Fournier, B. Structure 2002, 10, 1697.
9. Becker-Andre, M.; Andre, E.; DeLamarter, J. F. Biochem. Biophys. Res. Commun.
1993, 194, 1371.
10. Eberl, G.; Littman, D. R. Immunol. Rev. 2003, 195, 81.
11. Solt, L.; Burris, T. Trends Endocrinol. Metab. 2012, 23, 619.
12. Solt, L.; Kumar, N.; Nuhant, P.; Wang, Y.; Lauer, J.; Liu, J.; Istrate, M.;
Kamenecka, T.; Roush, W.; Vidovic´, D.; Schürer, S.; Xu, J.; Wagoner, G.; Drew,
P.; Griffin, P.; Burris, T. Nature 2011, 472, 491.
13. Wang, Y.; Kumar, N.; Nuhant, P.; Cameron, M.; Istrate, M.; Roush, W.; Griffin,
P.; Burris, T. ACS Chem. Biol. 2010, 5, 1029.
14. Kumar, N.; Kojetin, D.; Solt, L.; Kumar, K.; Nuhant, P.; Duckett, D.; Cameron, M.;
Butler, A.; Roush, W.; Griffin, P.; Burris, T. ACS Chem. Biol. 2011, 7, 218.
15. Kumar, N.; Lyda, B.; Chang, M.; Lauer, J.; Solt, L.; Burris, T.; Kamenecka, T.;
Griffin, P. ACS Chem. Biol. 2012, 7, 672.
16. Kumar, N.; Solt, L. A.; Conkright, J. J.; Wang, Y.; Istrate, M. A.; Busby, S. A.;
Garcia-Ordonez, R. D.; Burris, T. P.; Griffin, P. R. Mol. Pharmacol. 2010, 77,
228.
17. Wang, Y.; Kumar, N.; Solt, L.; Richardson, T.; Helvering, L.; Crumbley, C.;
Garcia-Ordonez, R.; Stayrook, K.; Zhang, X.; Novick, S.; Chalmers, M.; Griffin, P.;
Burris, T. J. Biol. Chem. 2010, 285, 5013.
4.4. Reporter gene assay of nuclear receptors
4.4.1. Cell culture conditions
Human embryonic kidney (HEK) 293 cells were cultured in
DMEM containing 5% FBS, penicillin and streptomycin mixture at
37 °C in a humidified atmosphere of 5% CO2 in air.
4.4.2. Plasmids
CMX-GAL4N-hRAR
CMX-GAL4N-hLXR
CMX-b-GAL were provided by Professor Dr. Makishima. pcDNA
1 and
a, CMX-GAL4N-hRARb, CMX-GAL4N-hRAR
c,
a,
CMX-GAL4N-hLXRb, TK-MH100Â4-Luc and
3.1(À)-hROR
a
1, pcDNA3.1(À)-hRORb1, pcDNA3.1(À)-hROR
c
RORE-TK-Luc were provided by Itsuu Laboratory.
18. Kallen, J.; Schlaeppi, J.-M.; Bitsch, F.; Geisse, S.; Geiser, M.; Delhon, I.; Fournier,
B. Structure 2002, 10, 1697.
19. Fujita-Sato, S.; Ito, S.; Isobe, T.; Ohyama, T.; Wakabayashi, K.; Morishita, K.;
Ando, O.; Isono, F. J. Biol. Chem. 2011, 286, 31409.
20. Huh, J.; Leung, M.; Huang, P.; Ryan, D.; Krout, M.; Malapaka, R.; Chow, J.; Manel,
N.; Ciofani, M.; Kim, S.; Cuesta, A.; Santori, F.; Lafaille, J.; Xu, H.; Gin, D.;
Rastinejad, F.; Littman, D. Nature 2011, 472, 486.
21. Xu, T.; Wang, X.; Zhong, B.; Nurieva, R.; Ding, S.; Dong, C. J. Biol. Chem. 2011,
286, 22707.
22. Stehlin-Gaon, C.; Willmann, D.; Zeyer, D.; Sanglier, S.; Van Dorsselaer, A.;
Renaud, J.-P.; Moras, D.; Schüle, R. Nat. Struct. Biol. 2003, 10, 820.
23. Stefanaki, C.; Stratigos, A.; Katsambas, A. J. Cosmet. Dermatol. 2005, 4, 130.
24. MedlinePlus Drug Information: Tretinoin Topical.
25. Rogers, . N.; Avram, M. J. Am. Acad. Dermatol. 2008, 59, 547.
26. Babamiri, K.; Nassab, R. Aesthet. Surg. J. 2010, 30, 74.
27. Serri, R.; Iorizzo, M. Clin. Dermatol. 2008, 26, 633.
28. Perry, A. W. Straight Talk about Cosmetic Surgery; Yale University Press, 2007. p.
63, ISBN 978-0-300-12104-9.
4.4.3. Transfection
HEK293 cells were seeded at a density of 20% confluence/96-
well plate 24 h prior to transfection. One well of cells was cotrans-
fected with 150 ng of a nuclear receptor expression plasmid,
500 ng of a luciferase reporter and 10 ng of CMX-b-GAL expression
vector. Transfections were performed by the calcium phosphate
co-precipitation method. After 8 h, transfected cells were treated
with test compounds or dimethyl sulfoxide (DMSO) for 16 h. Trea-
ted cells were assayed for luciferase activity in a luminometer. The
luciferase activity of each sample was normalized by the level of b-
galactosidase activity. Each transfection was carried out in
triplicate.
29. Charton, J.; Deprez-Poulain, R.; Hennuyer, N.; Tailleux, A.; Staels, B.; Deprez, B.
Bioorg. Med. Chem. Lett. 2009, 19, 489.
30. Kagechika, H.; Kawachi, E.; Hashimoto, Y.; Shudo, K.; Himi, T. J. Med. Chem.
1988, 31, 2182.
Supplementary data
31. DeLano, W. The PyMol Molecular Graphics System. DeLano Scientific LLC: San
Carlos, CA, USA available from http://www.pymol.org.
32. Nakamura, M.; Hamasaki, T.; Tokitou, M.; Baba, M.; Hashimoto, Y.; Aoyama, H.
Bioorg. Med. Chem. 2009, 17, 4740.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.023.
33. Nakamura, M.; Matsumoto, Y.; Toyama, M.; Baba, M.; Hashimoto, Y. Chem.
Pharm. Bull. 2013, 61, 237.
References and notes
34. Büttner, M.; Penka, M.; Doszczak, L.; Kraft, P.; Tacke, R. Organometallics 2007,
26, 1295.
1. Jetten, A. Nucl. Recept. Signal. 2009, 7, e003.
2. Andre, E.; Conquet, F.; Steinmayr, M.; Stratton, S. C.; Porciatti, V.; Becker-Andre,
M. EMBO J. 1998, 17, 3867.