Design, synthesis, and biological evaluation of novel transrepression- selective liver X receptor (LXR) ligands with 5,11-dihydro-5-methyl-11- methylene-6 H-dibenz[ b, e ]azepin-6-one skeleton

Article abstract of DOI:10.1021/jm3002394

To obtain novel transrepression-selective liver X receptor (LXR) ligands, we adopted a strategy of reducing the transactivational agonistic activity of the 5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-one derivative 10, which exhibits LXR-mediated transrepressional and transactivational activity. Structural modification of 10 based on the reported X-ray crystal structure of the LXR ligand-binding domain led to a series of compounds, of which almost all exhibited transrepressional activity at 1 or 10 μM but showed no transactivational activity even at 30 μM. Among the compounds obtained, 18 and 22 were confirmed to have LXR-dependent transrepressional activity by using peritoneal macrophages from wild-type and LXR-null mice. A newly developed fluorescence polarization assay indicated that they bind directly to LXRα. Next, further structural modification was performed with the guidance of docking simulations with LXRα, focusing on enhancing the binding of the ligands with LXRα through the introduction of substituents or heteroatom(s). Among the compounds synthesized, compound 48, bearing a hydroxyl group, showed potent, selective, and dose-dependent transrepressional activity.

Full text of DOI:10.1021/jm3002394

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Novel
Transrepression-Selective Liver X Receptor (LXR) Ligands with 5,11-
Dihydro-5-methyl-11-methylene‑6H‑dibenz[b,e]azepin-6-one
Skeleton
Atsushi Aoyama,† Kaori Endo-Umeda,‡ Kenji Kishida,§ Kenji Ohgane,† Tomomi Noguchi-Yachide,†
Hiroshi Aoyama,∥ Minoru Ishikawa,† Hiroyuki Miyachi,§ Makoto Makishima,‡ and Yuichi Hashimoto*,†
†Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
‡Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
§Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-Naka, Kita-ku, Okayama
700-8530, Japan
∥Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-City, Tokyo 192-0392, Japan
S
* Supporting Information
ABSTRACT: To obtain novel transrepression-selective liver X receptor (LXR) ligands, we adopted a strategy of reducing the
transactivational agonistic activity of the 5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-one derivative 10, which
exhibits LXR-mediated transrepressional and transactivational activity. Structural modification of 10 based on the reported X-ray
crystal structure of the LXR ligand-binding domain led to a series of compounds, of which almost all exhibited transrepressional
activity at 1 or 10 μM but showed no transactivational activity even at 30 μM. Among the compounds obtained, 18 and 22 were
confirmed to have LXR-dependent transrepressional activity by using peritoneal macrophages from wild-type and LXR-null mice.
A newly developed fluorescence polarization assay indicated that they bind directly to LXRα. Next, further structural
modification was performed with the guidance of docking simulations with LXRα, focusing on enhancing the binding of the
ligands with LXRα through the introduction of substituents or heteroatom(s). Among the compounds synthesized, compound
48, bearing a hydroxyl group, showed potent, selective, and dose-dependent transrepressional activity.
ligand-binding pockets are almost identical.4 LXRα is highly
expressed in liver, intestine, and macrophages, while LXRβ
seems to be ubiquitous in organs and tissues. LXRα and LXRβ
1. INTRODUCTION
Nuclear receptors (NRs) belong to the structurally conserved
nuclear receptor superfamily of ligand-dependent transcription
are known to function coordinately. Oxysterols act on both
factors, which control diverse biological functions including
LXRα and LXRβ and activate the transcription of LXR target
reproduction, differentiation, homeostasis, and the immune
system.1 Forty-eight kinds of NRs have been identified. Among
genes, such as abca1, abcg1, apoe, and glut4, which have the
LXR response element (LXRE) on their promoter region.5 As
them, liver X receptors (LXRs) were originally orphan
receptors, and their endogenous ligands were not known.2
the products of these genes are involved in lipid metabolism,
reverse cholesterol transport, and glucose transport, it is
However, in 1996, Janowsky et al. indicated that several
oxysterols, including 22(R)-hydroxycholesterol (1, Figure 1)
and 24(S),25-epoxycholesterol (2), were endogenous ligands of
thought that LXRs are promising drug targets for treatment
of atherosclerosis, hyperlipidemia, or metabolic syndrome. This
LXR-mediated activation of gene transcription by certain
LXRα, and they proposed that LXRα functions as an internal
cholesterol sensor.3 LXRs consist of two subtypes, LXRα and
LXRβ, which have about 78% amino acid homology of the
ligand binding domain (LBD); in particular, the surfaces of the
Received: February 23, 2012
Published: August 8, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of endogenous LXR ligands (1 and 2), synthetic ligands (3, 4, and 6), and a transrepression-selective ligand (5).
oxysterols is called transactivational action. Currently known
LXR ligands can be classified into two categories with respect
to transactivational action, i.e., those that activate expression of
the target genes and those that repress expression of the target
genes. We define these as transactivational agonists and
transactivational antagonists, respectively. It has been shown
that the activities of LXR ligands depend upon their effect on
recruitment of cofactors to helix 12 of the LXRs;6 many
transactivational agonists induce dissociation of corepressors
(e.g., nuclear receptor corepressor (NCoR) and silencing
mediator for retinoid and tyroid receptors (SMRT)) and
association of coactivators (e.g., steroid receptor coactivator 1
(SRC1) and vitamin D receptor interacting protein (DRIP)),
whereas many transactivational antagonists stabilize the binding
of corepressors. Most of the reported LXR ligands, including N-
2,2,2-trifluoroethyl-N-[4-(2,2,2-trifluoro-1-hydroxy-1-
trifluoromethylethyl)phenyl]benzenesulfonamide (T1317, 3)7a
and 3-[3-[[[2-chloro-3-(trifluoromethyl)phenyl]methyl](2,2-
diphenylethyl)amino]propoxy]benzeneacetic acid (GW3965,
4)7b are transactivational agonists for both LXRα and LXRβ.
However, transactivational agonists may induce serious hyper-
triglyceridemia because genes involved in lipid synthesis, such
as srebp-1c and fas, have LXRE in their promoter region and are
activated by LXR transactivational agonists.8 This is a major
barrier to medical application of LXR ligands.
Recent studies have revealed that LXRs also function in the
regulation of inflammation in macrophages.9 In vitro, several
LXR ligands inhibit the expression of proinflammatory
mediators such as interleukin-6 (IL-6), interleukin-1β (IL-1β),
and inducible nitric oxide synthase (iNOS) in response to
bacterial infection or lipopolysaccaride (LPS) stimulation.
Studies in LXR knockout mice indicated that these actions
are mediated by both LXRα and LXRβ. Therefore, it is
speculated that LXRs are potential targets for treatment of not
only atherosclerosis or hyperlipidemia but also inflammatory
diseases such as rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE), and Castleman’s disease.10 This function
of LXR is called transrepressional action. The promoter regions
of il-6, il-1β, inos, and other proinflammatory genes do not
contain LXRE, which is present in all of the genes showing
LXR transactivation. Thus, the mechanisms of transactivational
and transrepressional action are clearly different. Transrepres-
sional activities of other NRs have also been reported. For
example, in the case of peroxisome proliferator-activated
receptor γ (PPARγ), an NR that is expressed at high levels in
macrophages, SUMOylation of PPARγ by rosiglitazone (a
PPARγ ligand) leads to stabilization of the transrepressional
complex, which includes NCoR, on the promoter region of
each proinflammatory gene, resulting in inhibition of gene
expression under conditions of LPS stimulation.11 In 2007,
Ghisletti et al. proposed that an LXR agonist (4) showed
transrepressional activity via a similar mechanism, i.e., through
SUMOylation of LXR and prevention of NCoR clearance.12
Compounds 3 and 4 exhibit both transactivational and
transrepressional actions, and many reported LXR ligands are
thought to have both activities. However, 25-hydroxycholester-
ol (an endogenous LXR ligand) and 27-hydroxycholesterol
showed only transactivational activity, and this suggested that
transactivational and transrepressional activities might be
separable.12 So far, only one series of transrepression-selective
LXR modulators (a representative compound 5 is shown in
Figure 1) has been reported, by GlaxoSmithKline in 2008.13
Compound 5 has interesting properties (it has similar
transrepressional activity to compound 3 without causing any
increase of triglyceride (TG) accumulation), but it showed
weak transactivational agonistic activity in a cell-based reporter
assay (EC50 = 3−10 μM in our assay system).
In this paper, we describe the structure-based design,
synthesis, and biological evaluation of a novel series of 2-
substituted 5,11-dihydro-5-methyl-11-methylene-6H-dibenz-
[b,e]azepin-6-one LXR ligands that exhibit potent and dose-
dependent transrepression-selective activity.
2. RESULTS AND DISCUSSION
2.1. Identification of a Lead Compound Possessing
Both Transrepressional and Transactivational Activity.
In our previous studies of LXR transactivational agonists and
antagonists,14 we measured transactivational activity using a
Gal4-human LXR reporter system (transactivational agonistic
activity was measured at a concentration of 0.1−30 μM test
compound and antagonistic activity was measured under the
same conditions in the presence of 0.1 μM compound 3).15
Percent efficacy of agonists was given relative to the positive
control 6.14a,16 In the present work, we focused on the
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Figure 2. General structure and chemical structures of LXR transactivational antagonist (7) and transactivational agonists (8−10).
a
Table 1. Transactivational and Transrepressional Activities of Compounds 7−10 in Cell-Based Assays
LXR transcriptional assay
agonistic activity (EC50 (μM))
antagonistic activity (IC50 (μM))
compd
% inhibition of IL-6 production at 10 μM
LXRα (% efficacy)
LXRβ (% efficacy)
LXRα
LXRβ
3
49
NT
NA
54
0.34 0.17 (124)
0.63 0.029 (100)
NA
0.09 0.0059 (118)
0.18 0.014 (100)
NA
NT
NA
NT
NA
NA
NA
NA
NA
6
7
4.1 1.7
NA
8
4.2 0.97 (12)
0.91 0.023 (117)
1.7 0.53 (45)
2.7 0.36 (49)
0.26 0.0153 (108)
0.77 0.102 (73)
9
20
NA
10
71
NA
a
Transactivational activity was measured on LXR-Gal4 chimeric receptors in transiently transfected HEK293 cells. The EC50 value is the molar
concentration of test compound that affords 50% of the maximal reporter activity. Percent efficacy is given relative to the positive control 6. The IC50
value is the molar concentration of test compound that affords a 50% decrease in the maximal reporter activity of 100 nM compound 3.
Transrepressional activity was measured in TPA-treated THP-1 cells. NT means not tested. NA means no activity at 30 μM.
transrepressional activity of phenanthridin-6-one, dibenz[b,f]-
[1,4]oxazepin-11-one, 11,12-dihydrodibenz[b,f ]azocin-6-one,
and 5,11-dihydro-11-methylene-6H-dibenz[b,e]azepin-6-one
derivatives (Figure 2). Transrepressional activity was evaluated
by measuring the reduction in the amount of IL-6 induced by
LPS in the presence of test compounds. Percent inhibition of
IL-6 production of compounds (10 μM) in each table (vide
infra) was measured at the same experiment side-by-side, and
the dose-dependency of compounds was determined before-
hand by separate experiments (Supporting Information).
Compounds possessing strong inhibition of IL-6 production
were selected, and their IC50 values were then determined (vide
infra). Transactivational and transrepressional activities of
representative compounds 7−10 (Figure 2) are shown in
Figure 3. Dose-dependent transrepressional activity of compound 3
Table 1. Novel compound 10, which has a 5,11-dihydro-5-
and 10, evaluated in terms of IL-6 production by means of ELISA.
Normalization was done according to the live cell count.
methyl-11-methylene-6H-dibenz[b,e]azepin-6-one skeleton,
showed the most potent transrepressional activity; it inhibited
LPS-induced IL-6 production in a dose-dependent manner
(Figure 3), showing 71% inhibition at 10 μM. It also exhibited
important for transactivational agonistic activity.17 According
transactivational agonistic activity, with EC50 values for LXRα
and LXRβ of 1.6 μM and 0.77 μM, respectively. In our assay
system, compound 10 showed a similar level of transrepres-
sional activity to compound 5 (data not shown). Therefore, we
selected compound 10 as a new lead compound.
2.2. Design of Transrepression-Selective Compounds.
Our strategy to obtain transrepression-selective LXR ligands
was to reduce the transactivational agonistic activity of
compound 10. Proper folding of C-terminal helix 12 is
to the reported X-ray crystal structures of compound 3 and
hLXRs LBD,18 the hydroxyl group of 3 forms a hydrogen bond
with a histidine residue (His421 for LXRα and His435 for
LXRβ) in helix 11, adjacent to helix 12, and this histidine
residue interacts with a tryptophan residue (Trp443 for LXRα
and Trp457 for LXRβ) in helix 12; these interactions induce
proper folding of helix 12, affording transcriptionally active
LXRs (Figure 4a). Compound 10 contains a hexafluoropropa-
nol moiety, like compound 3. Therefore, we initially focused on
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Figure 4. X-ray crystal structure of compound 3 and hLXRα LBD (a) and design strategy for our compounds (b).
a
Scheme 1. Synthesis of 10, 16−22
a
Reagents and conditions: (a) NBS, DCM, rt, 38−77%; (b) tributylvinyltin, Pd(PPh3)4, toluene, 100 °C or 2,4,6-trivinylcyclotriboroxane−pyridine
complex, Pd(PPh3)4, K2CO3, DME/H2O, reflux, 29−65%; (c) 2-iodobenzoyl chloride, Et3N, DCM, rt, 46%−quant; (d) MeI, NaH, DMF, 0 °C to rt,
46−99%; (e) Pd(OAc)2, PPh3, NaOAc, DMF, 100 °C, 34−88%.
replacement of the hexafluoropropanol moiety with alkyl
groups (Figure 4b), which are expected to be unable to form
the hydrogen bond with the histidine residue in LXRs so that
the histidine residue may not be appropriately located to allow
proper folding of helix 12 for transactivational agonistic activity.
2.3. Synthesis of compounds 16−22. 2-Substituted 5,11-
dihydro-5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-ones
16−22 were prepared via intramolecular Heck reaction as the
key step, as shown in Scheme 1. 4-Substituted anilines 11 were
monobrominated (12) and vinylated to give compounds 13.
Compounds 13 were amidated with 2-iodobenzoyl chloride in
the presence of triethylamine to afford compounds 14 in 46%−
quantitative yield. N-Methylation afforded 15. The intra-
molecular Heck reaction proceeded smoothly with the use of
Pd(OAc)2 and PPh3 in the presence of NaOAc as the base in
DMF to afford compounds 16−22 (Scheme 1). Structure
determination of representative compound 18 was performed
by 1H NMR, 13C NMR, heteronuclear multiple quantum
coherence (HMQC), and mass spectrometry.
2.4. Structure−Activity Relationship (SAR) of Alkyl
Substituent. The synthesized compounds 16−22 all lacked
transactivational agonistic activity at 30 μM, as expected, but
showed weak transactivational antagonistic activity (Table 2).
Next, we investigated the transrepressional activity of these
compounds. Among compounds with various lengths of the
alkyl substituent (16−20), the ethyl compound (18) showed
the best transrepressional activity, while a shorter (16) or
longer (20) alkyl substituent resulted in weaker activity. Among
16−18, 21, and 22, we found that bulkier substituents tended
to be associated with more potent transrepressional activity. On
the basis of these results, we selected 18 and 22 as second-
generation lead compounds.
2.5. Verification of Transrepression/Transactivation
Selectivity. First, we examined in detail the transrepression/
transactivation selectivity of the second-generation leads. To
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a
Table 2. Transactivational and Transrepressional Activities of Compounds 16−22
LXR transcriptional assay
antagonistic activity (IC50
agonistic activity (EC50 (μM))
(μM))
compd
R1
% inhibition of IL-6 production at 10 μM
LXRα (% efficacy)
LXRβ (% efficacy)
LXRα
LXRβ
3
49
73
NA
38
51
38
NA
44
61
0.34 0.17 (124)
0.09 0.0059 (118)
NT
NA
NT
NA
>10
>10
>30
>10
>30
>10
>10
10
16
17
18
19
20
21
22
HO(F3C)2C
1.7 0.53 (45)
0.77 0.102 (73)
H
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
>10
Me
>10
Et
>30
n-hex
n-dodecyl
i-Pr
>10
>30
1.7 0.0
5.4 1.7
t-Bu
a
Transactivational activity was measured on LXR-Gal4 chimeric receptors in transiently transfected HEK293 cells. The EC50 value is the molar
concentration of test compound that affords 50% of the maximal reporter activity. % Efficacy is given relative to the positive control 6. The IC50 value
is the molar concentration of test compound that affords a 50% decrease in the maximal reporter activity of 100 nM compound 3. Transrepressional
activity was measured in TPA-treated THP-1 cells. NT means not tested. NA means no activity at 30 μM.
Figure 5. Dose dependence of transactivational antagonistic activity of compounds 3, 18, and 22 in cellular mammalian two-hybrid assays using
pCMX-Gal4-NCoR and -SMRT.
investigate transactivational antagonistic activity, we performed
mammalian two-hybrid (M2H) assay using pCMX-Gal4-SRC1
and -DRIP205 expression vectors to examine coactivator
recruitment and -NCoR and -SMRT expression vectors to
examine corepressor recruitment.19 While compound 3
strongly recruited coactivators SRC1 and DRIP205 (data not
shown), compound 18 did not recruit them at 10 μM and
compound 22 did so only slightly. However, compounds 18
and 22 both dose-dependently stabilized corepressors NCoR
and SMRT, whereas compound 3 did not (Figure 5). These
results indicate that compounds 18 and 22 exerted transactiva-
tional antagonistic activity by stabilizing the interaction
between LXRα and corepressors. Taking into account the
experimental result that complex formation of LXRs and NCoR
is important for transrepressional activity, compounds 18 and
22 might be possibly to suppress LPS-induced IL-6 production
via LXRs-mediated transrepression. We next examined the
transrepression/transactivation selectivity of the second-gen-
eration leads by means of mRNA expression analysis using
wild-type mouse peritoneal macrophages and human hepato-
cellular liver carcinoma (Huh-7) cells. In mouse peritoneal
macrophages, we examined the mRNA expression of il-6, il-1β
(transrepressional genes) and abca1 (transactivational gene).
Compound 18 inhibited LPS-induced il-6 and il-1β mRNA
expression, as did compounds 3 and 10 (Figure 6a,b), but did
not induce upregulation of abca1 mRNA expression, unlike
compounds 3 and 10 (Figure 6c). These results indicate that
compound 18 is transrepression-selective. Moreover, we
examined mRNA expression of srebp-1c and fas, which are
associated with increased blood triglyceride and may be
transactivated by LXR ligands. As shown in parts d and e of
Figure 6, unlike compound 3 or the first-generation lead
compound 10, compound 18 did not increase the mRNA
expression of srebp-1c or fas in Huh-7 cells. Therefore,
compound 18 should not increase blood triglyceride and may
be a better candidate as an anti-inflammatory medicine than
ligands that have transactivational agonist activity.
2.6. Synthesized Compounds Showed Transrepres-
sional Activity in an LXR-Dependent Manner. LPS
transmits inflammatory signaling intracellularly through Toll-
like receptor 4 (TLR4) in the plasma membrane, and this
induces degradation of the transrepressional complex on the
promoter region of proinflammatory genes (e.g., IL-6 and IL-
1β), thereby activating gene transcription.20 So, inhibitory
activity toward LPS-induced IL-6 production is also exhibited
by TLR4 antagonists21 and TLR4-signaling inhibitors.22
Therefore, we examined whether the IL-6 production−
inhibitory activity of our second-generation lead compounds
was LXR-dependent. To check this point, we used peritoneal
macrophages from wild-type and LXR-null mice (LXRα−/−
β
/
−/− (Nr1h3−/−Nr1h2−/−) or LXRα−/− (Nr1h3−/−)). As shown
in Figure 7a, inhibition of LPS-induced IL-6 production by the
LXR agonist 3 (3 μM), 10, and 18 was entirely abolished in
LXRα−/−/β−/− macrophages, as determined by ELISA (mouse
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Figure 6. Effects of compounds 10 and 18 on mRNA expression of il-6 (a), il-1β (b), and abca1 (c) in wild-type mice peritoneal macrophages and
on mRNA expression of srebp-1c (d) and fas (e) in human hepatocellular liver carcinoma (Huh-7) cells.
IL-6 protein). LXR-independent induction of IL-6 in LXRα−/−
/
LPS-induced IL-6 production by compounds 3 and 10 was
partially abolished in LXRα−/− macrophages as determined at
the mRNA level by means of real-time quantitative PCR
analysis (Figure 7b). When these results were compared with
those in wild-type mice, shown in Figure 6a, we surprisingly
found that the inhibition by compound 18 in particular was
almost entirely abrogated, which suggests that compound 18
might show transrepressional activity predominantly in an
LXRα-dependent manner. The inhibition of LPS-induced IL-1β
production by compounds 3, 10, and 18 was partially abolished
at the mRNA level in LXRα−/− macrophages (Supporting
Information).
β−/− macrophages was observed in response to compounds 3
and 10. The reason for this result is unclear at present, but the
result for compound 3 is in agreement with reported data,9a so
there may be another site of action. Compound 22 lacked
transrepressional activity in LXRα−/−/β−/− macrophages. This
result also indicates that LXRα/β contribute inhibition of IL-6
production by compounds 10, 18, and 22, at least in part,
although we cannot exclude the possibility that our compounds
may inhibit LPS induced IL-6 production through other
mechanism. We next focused on LXRα, which is highly
expressed in immune cells such as macrophages. Inhibition of
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Figure 7. Inhibition of LPS-induced IL-6 production by compounds 10 and 18 (and also 22) was abolished in LXRα−/−β−/− macrophages as
determined at the protein level by ELISA (a) and in LXRα−/− macrophages as determined at the mRNA level by real-time quantitative PCR analysis
(b). Values represent means SE from at least three independent measurements. ***, p < 0.001 vs LPS+, n.s., not significant, ANOVA.
Figure 8. Structure of a reported fluorescent LXR ligand, 2-(4-(4-methoxyphenethyl)phenyl)-5- (dimethylamino)isoindoline-1,3-dione (23) (a).
Compound 23 specifically binds to hLXRα (b). Compounds 18 and 22 specifically bind to GST-hLXRα (c).
2.7. Compounds 18 and 22 Bind Directly to LXRα.
Experiments using the knockout mice confirmed that the
synthesized compounds exhibited transrepressional activity in
an LXR-dependent manner. Because 18 and 22 showed
transactivational antagonistic action, we considered that they
might bind to the same binding site as compound 3. Therefore,
we next investigated whether compounds 18 and 22 bind
directly to LXR. Although scintillation proximity assay (SPA)23
has been used to screen LXR ligands, there are no commercial
available radioactive ligands at present. Because fluorescent
ligands have been widely used for binding assay of target
proteins,24 we decided to employ fluorescence polarization
(FP) assay using the reported fluorescent compound 23
(Figure 8a25). First of all, we investigated whether the
fluorescent compound specifically binds to LXRα. A fixed
concentration of the fluorescent compound (1 μM) was mixed
with different concentrations of GST-hLXRα or GST protein
(X-axis), and the FP value (Y-axis) was measured. As shown in
Figure 8b, fluorescence polarization increased in a dose-
dependent manner with GST-hLXRα but not GST. This result
indicated the fluorescent compound binds specifically to
hLXRα. Furthermore, compound 3, which binds to LXRs
LBD, dose-dependently decreased the FP value in the presence
of fluorescent compound 23 (1 μM) and GST-hLXRα (1 μM)
(Figure 8c). This result demonstrated that fluorescent
compound and compound 3 competitively bind to hLXRα
LBD. Under the conditions used, 18 (20 μM) and 22 (10 μM)
both caused a dose-dependent decrease of the FP value,
indicating that they also both bind directly to GST-hLXRα.
These effective concentrations of compounds were predeter-
mined by a separate experiment. The results also demonstrate
that our fluorescence-based LXR LBD binding assay is effective.
In summary, our second-generation leads 18 and 22 were
confirmed to be transrepression-selective by means of reporter
gene assay, IL-6 ELISA, M2H assay, and mRNA expression
analysis, and we also demonstrated that inhibition of IL-6
production by these lead compounds is mediated by LXR via
direct binding of the compounds to the LXR LBD.
2.8. Selectivity over Other Nuclear Receptors. We next
confirmed the selectivity of the second-generation leads toward
other NRs, that is, farnesoid X receptor (FXR), PPARγ, and
retinoid X receptor α (RXRα). We selected three NRs based on
the following knowledge. (1) LXR and FXR are categorized
into the same subgroup of NRs based on their sequence
homology, and compound 3 is known to bind to FXR.26 (2)
Disorders of lipid consumption and processing influence gene
expression programs are orchestrated by PPARγ and LXR.27
(3) LXRα and LXRβ form heterodimers with the obligate
partner RXR. As far as we examined under experimental
conditions used, the second-generation lead 18 did not show
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Figure 9. Design of candidate ligands interacting with Arg316 (a) and with heteroatom(s) in place of the alkyl group (b).
any FXR agonistic/antagonistic activities, PPARγ antagonistic
activity, or RXRα agonistic/antaogonistic activities (Supporting
Information). Compound 18 only showed very weak PPARγ
agonistic activity (ca. 20% activation at 10 μM). These results
indicate that compound 18 showed sufficient selectivity over
other NRs, FXR, PPAR, and RXR.
with boron tribromide and triflation with trifluoromethane-
sulfonic anhydride were performed to obtain compound 32.
Conversion of triflate 32 to borate ester 33 and subsequent
phenylation with haloaryls 35a−d were performed in moderate
yield. Hydrolysis of 36a−d proceeded to afford compounds
37−40. Starting from commercially available 41a, 41c, and 41d,
and compound 41b synthesized by the method reported in the
literature,30 we synthesized compounds 46, 48, 49, and 52 in
the same manner as illustrated in Scheme 1 (Scheme 3).
Compound 46 was reduced with tin(II) chloride to obtain 47.
Compound 49 was reduced with lithium borohydride to obtain
50. Compound 49 was also hydrolyzed to obtain 51.
2.11. Transrepressional and Transactivational Activ-
ities of the New Compounds. The transrepressional and
transactivational activities of the newly synthesized compounds
were evaluated. Compounds 37−40 broadly showed increased
transactivational antagonistic activity (especially when a 4-
substituent was present) (Table 3). However, the transrepres-
sional activity of compounds 39 and 40 was greatly decreased
relative to compound 18, and compounds 37 and 38 lacked
transrepressional activity. These results indicated that inter-
action with the arginine residue was disadvantageous for
transrepressional activity. Further, because there was no clear
correlation between transactivational antagonistic activity and
transrepressional activity, the key interaction sites with LXR for
the two activities might be different. Another possible
explanation of weak transrepressional activity of this carboxylic
acid series might be due to low penetration into THP-1 cells.
As for compounds 46−52, which were expected to be able to
form a hydrogen bond with the histidine residue in LXR
because of their heteroatom(s), they showed no transactiva-
tional agonistic activity at 30 μM (Table 4). Thus, a substituent
2.9. Further Structural Development to Obtain More
Potent Transrepression-Selective Ligands. Because we
thought the low binding affinity for LXR of compounds 18 and
22 might account for their low transrepressional activity relative
to compound 10, we considered further structural modifica-
tions. One approach was to introduce a group that could
interact with another amino acid residue in LXR based on the
X-ray structure of compound 4 and LXRα LBD (PDB;
3IPQ),28 and the other was to substitute the alkyl group with
heteroatom(s). As shown in Figure 9a, compound 4 binds with
LXRα by interacting with an arginine residue (Arg316). This
position is rather far away from helix 12, and we guessed that
this interaction with arginine, unlike that with histidine in helix
11, may not play a critical role in exerting transactivational
agonistic activity. Therefore, we modified our compounds by
introducing a substituent (R = 3-CH2COOH), based on the
results of docking simulation with AUTODOCK 4.2,29 using
the LXRα LBD (Figure 9).
2.10. Synthesis of Compounds 37−40 and 46−52.
Intermediate 13c was amidated with 2-iodo-5-methoxybenzoic
acid (27), which was prepared from 5-hydroxyanthranilic acid
(24) in three steps in the presence of Vilsmeier reagent to
afford compound 28 (Scheme 2). N-Methylation was
performed to obtain 29. The intramolecular Heck reaction of
29 proceeded smoothly with the use of Pd(OAc)2 and PPh3 in
the presence of NaOAc to afford compound 30. Demethylation
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a
Scheme 2. Synthesis of 37−40
a
Reagents and conditions: (a) NaNO2, conc HCl, 0 °C; KI, H2O, 0 °C to reflux; (b) (MeO)2SO2, K2CO3, acetone, reflux; (c) 2 N NaOH aq,
MeOH, reflux, 47% (3 steps); (d) 27, chloromethylenedimethyliminium chloride, DCM, 0 °C to rt; Et3N, DCM, 83%; (e) MeI, NaH, DMF, 0 °C to
rt, 78%; (f) Pd(OAc)2, PPh3, NaOAc, DMF, 100 °C, 95%; (g) BBr3, DCM, 0 °C to rt, 90%; (h) Tf2O, py, DCM, 0 °C to rt, 47%; (i)
bis(pinacolato)diboron, (PPh3)2PdCl2, KOAc, 1,4-dioxane, 100−120 °C, 57%; (j) conc H2SO4, MeOH, reflux, 91−99%; (k) 35a−d, Pd(PPh3)4,
K3PO4, DMF, 25−83%; (l) 2 N NaOH aq, EtOH, 14−98%.
a
Scheme 3. Synthesis of 46−52
a
Reagents and conditions: (a) NBS, DCM, rt, 76−87%; (b) tributylvinyltin, Pd(PPh3)4, toluene, 100 °C or 2,4,6-trivinylcyclotriboroxane−pyridine
complex, Pd(PPh3)4, K2CO3, DME/H2O, reflux, 47−85%; (c) 2-iodobenzoyl chloride, Et3N, DCM, rt, 88%−quant; (d) MeI, NaH, DMF, 0 °C to rt,
from 44b to 45e (15%), 45a, 45c, 45d (32−91%); (e) Pd(OAc)2, PPh3, NaOAc, DMF, 100 °C, 42−95%; (f) SnCl2·2H2O, AcOEt, 70 °C, 75%; (g)
LiBH4, THF, rt, 26%; (h) 1 N NaOH aq, EtOH, rt, quant.
with appropriate acidity or hydrogen bond-forming ability is
important for transactivational agonistic activity. We found that
compound 48, with a hydroxyl group, exhibited the most
potent transrepressional activity, exceeding that of lead
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a
Table 3. Transactivational and Transrepressional Activities of Compounds 37−40
LXR transcriptional assay
agonistic activity (EC50
(μM))
antagonistic activity (IC50 (μM))
compd
R1
position
% inhibition of IL-6 production at 10 μM
LXRα
LXRβ
LXRα
LXRβ
18
37
38
39
40
78
NA
NA
27
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
>30
>30
COOH
3
4
3
4
>30
>30
COOH
1.3 0.58
21 4.2
2.2 0.72
9.5 2.7
27 0.12
15 0.61
CH2COOH
CH2COOH
9
a
Transactivational activity was measured on LXR-Gal4 chimeric receptors in transiently transfected HEK293 cells. The EC50 value is the molar
concentration of test compound that affords 50% of the maximal reporter activity. The IC50 value is the molar concentration of test compound that
affords a 50% decrease in the maximal reporter activity of 100 nM compound 3. Transrepressional activity was measured in TPA-treated THP-1
cells. NA means no activity at 30 μM.
a
Table 4. Transactivational and Transrepressional Activities of Compounds 46−52
LXR transcriptional assay
agonistic activity (EC50 (μM))
antagonistic activity (IC50 (μM))
compd
R2
% inhibition of IL-6 production at 10 μM
LXRα (% efficacy)
LXRβ (% efficacy)
LXRα
LXRβ
3
49
73
49
58
78
29
27
53
0.34 0.17 (124)
0.09 0.0059 (118)
NT
NA
NT
10
46
47
48
50
51
52
HO(F3C)2C
O2N
1.7 0.53 (45)
0.77 0.102 (73)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
6.1 0.050
19 1.0
4.1 0.045
>10
13 1.9
27 5.2
11 0.43
>10
H2N
HO
HOH2C
HO2C
Ac
14 3.2
4.9 0.95
26 1.8
7.5 1.0
a
Transactivational activity was measured on LXR-Gal4 chimeric receptors in transiently transfected HEK293 cells. The EC50 value is the molar
concentration of test compound that affords 50% of the maximal reporter activity. Percent efficacy is given relative to the positive control 6. The IC50
value is the molar concentration of test compound that affords a 50% decrease in the maximal reporter activity of 100 nM compound 3.
Transrepressional activity was measured in TPA-treated THP-1 cells. NT means not tested. NA means no activity at 30 μM.
Figure 10. Dose dependence of transrepressional activity of compounds 10, 18, 22, and 48 evaluated in terms of IL-6 production by means of
ELISA. Normalization was done according to the live cell count.
compound 10. Finally, we investigated the dose-dependency of
transrepressional activity of compounds 18, 22, and 48. As
shown in Figure 10, all the compounds showed dose-dependent
transrepressional activity and compound 48 was found to have
the most potent transrepressional activity, as expected (its IC50
value was estimated to be 3.5 μM). Transactivational and
transrepressional activities of representative compounds 3, 5,
10, and 48 are summarized in Table 5. Relative efficacy (RE) of
transrepressional activity and reported potency of 3 and 5 are
also indicated.
2.12. Early in Vitro ADME of the Representative
Compound. Compound 48 was further assessed for early in
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Table 5. Summary of Transrepressional and Transactivational Activities of Representative LXR Modulators
transrepression (inhibition of IL-6 production)
transactivation (LXR agonistic activity EC50 (μM))
a
ab
,
c
cd
,
a
a
c
compd
IC50 (μM)
RE
IC50 (nM)
RE
LXRα
LXRβ
LXRα/β
3
>10
>10
5.8
<0.6
<1
1
100
15
1
0.34 0.17
7.1 0.60
1.7 0.53
0.09 0.0059
3.1 1.72
5
1
3−5
10
48
0.77 0.102
NA at 30 μM
3.5
1.3
NA at 30 μM
a
b
c
d
Data in our assay condition. Relative efficacy compared with 10. Data taken from ref 13. Relative efficacy with compound 3.
with m-nitrobenzyl alcohol. Flash column chromatography was
performed on silica gel 60 Kanto Kagaku (40−100 μm). The purity
of each test compound was determined by HPLC (>95% purity; see
Supporting Information).
7-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)-10-methyl-
dibenz[b,f ][1,4]oxazepin-11(10H)-one (8). Melting point 187.5−
188.0 °C. 1H NMR (500 MHz, DMSO-d6) δ 8.95 (s, 1H), 7.76
(dd, J = 7.8, 1.5 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.62−7.58 (m, 2H),
7.54 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.32 (dd, J = 8.3, 7.3
Hz, 1H), 3.50 (s, 3H). HRMS (FAB) calcd for C17H12F6NO3
392.0721; found 392.0705 (M + H)+.
2-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)-5-methyl-11,12-
dihydrodibenz[b,f ]azocin-6-one (9). Melting point 195.7−196.1 °C.
1H NMR (500 MHz, CDCl3) δ 7.44−7.40 (m, 2H), 7.18 (dd, J = 7.3,
1.8 Hz, 1H), 7.14 (d, J = 7.3 Hz, 1H), 7.11−7.04 (m, 2H), 6.89 (d, J =
7.3 Hz, 1H), 3.60 (s, 1H), 3.47−3.40 (m, 1H), 3.41 (s, 3H), 3.30−3.23
(m, 1H), 3.00−2.90 (m, 2H). HRMS (FAB) calcd for C19H16F6NO2
404.1085; found 404.1046 (M + H)+.
vitro absorption−distribution−metabolism−excretion (ADME)
properties. The permeability was evaluated in the Caco-2
intestinal epithelial cell line. In this assay, 48 was found to
exhibit excellent permeability (mean A−B permeability = 84.3
× 10−6 cm/s), and it was classified as high permeability (Papp
≥
20 × 10−6 cm/s). Next, metabolic stability was evaluated in
human liver microsomes. The metabolic stability was measured
as the percentage of the unmodified parent compound
remaining in the mixture. Compound 48 was very stable and
was almost recovered after incubation (mean compound
remaining = 87.8%). Overall, compound 48 possessed both
reasonable in vitro ADME properties and potent transrepres-
sional activity and could be considered as a chemical tool for
advanced studies.
3. CONCLUSION
General Procedure 1 (GP1). 2-Bromo-4-methylaniline (12b). To a
solution of p-toluidine (11b) (1.2 g, 11.2 mmol) and 10 mL of DCM
was added N-bromosuccinimide (2.2 g, 12.4 mmol). The mixture was
stirred for 20 h at room temperature, washed with water and brine,
dried over anhydrous magnesium sulfate, and concentrated. The
residue was purified by silica gel column chromatography (eluent: n-
hexane/ethyl acetate = 9:1, v/v) to afford 924 mg (44%) of the title
compound as a brown oil. 1H NMR (500 MHz, CDCl3) δ 7.23 (d, J =
1.2 Hz, 1H), 6.91 (dd, J = 7.9, 1.2 Hz, 1H), 6.68 (d, J = 7.9 Hz, 1H),
3.92 (br s, 2H), 2.22 (s, 3H).
2-Bromo-4-ethylaniline (12c). This compound was prepared from
4-ethylaniline (11c) by means of GP1 as a brown oil (68%). 1H NMR
(500 MHz, CDCl3) δ 7.25 (d, J = 1.8 Hz, 1H), 6.94 (dd, J = 7.9, 1.8
Hz, 1H), 6.70 (d, J = 7.9 Hz, 1H), 3.94 (br s, 2H), 2.52 (q, J = 7.9 Hz,
2H), 1.18 (t, J = 7.9 Hz, 3H).
We focused on novel transrepression-selective LXR ligands
because of their potential value as tools for functional analysis
of LXRs. On the basis of screening of transrepressional activity
(inhibitory activity toward LPS-induced IL-6 production) of
our LXR ligands, we selected tricyclic compound 10 as a lead
compound. Because it possessed both transrepressional activity
and transactivational agonistic activity, we next aimed to
decrease the transactivational agonistic activity of 10. We
designed novel compounds that should be unable to form the
hydrogen bond with a histidine residue on helix 11 in the LXR
LBD, which is important for agonistic activity. All the
synthesized compounds showed no transactivational agonistic
activity at 30 μM, but showed weak transactivational
antagonistic activity, and most of them had transrepressional
activity. Compounds 18 and 22 were confirmed to be selective
for transrepression over transactivation by means of M2H assay
and mRNA expression analysis. We also showed that the
transrepressional activity of these second-generation lead
compounds was LXR-dependent, and we used a newly
developed fluorescence polarization assay with fluorescent
compound 23 as a nonradiolabeled LXR binding assay system
to establish that the compounds bind directly to LXRα. Finally,
further structural development directed at improving the
binding affinity was performed. Among the compounds
obtained, compound 48, containing a hydroxyl group, showed
potent, selective, and dose-dependent transrepressional activity.
2-Bromo-4-hexylaniline (12d). This compound was prepared from
1
11d by means of GP1 as a brown oil (77%). H NMR (500 MHz,
CDCl3) δ 7.22 (s, 1H), 6.91 (d, J = 7.9 Hz, 1H), 6.69 (d, J = 7.9 Hz,
1H), 3.93 (br s, 2H), 2.46 (t, J = 7.9 Hz, 2H), 1.50 (br s, 2H), 1.28 (br
s, 6H), 0.88 (t, J = 6.7 Hz, 3H). MS (FAB) 255, 257 (M + H)+.
2-Bromo-4-dodecylaniline (12e). This compound was prepared
1
from 11e by means of GP1 as a brown solid (66%). H NMR (500
MHz, CDCl3) δ 7.23 (d, J = 1.8 Hz, 1H), 6.91 (dd, J = 7.9, 1.8 Hz,
1H), 6.69 (d, J = 7.9 Hz, 1H), 3.94 (br s, 2H), 2.46 (t, J = 7.9 Hz, 2H),
1.55−1.50 (m, 2H), 1.30−1.23 (m, 18H), 0.88 (t, J = 7.3 Hz, 3H). MS
(FAB) 339, 341 (M + H)+.
2-Bromo-4-isopropylaniline (12f). This compound was prepared
from 11f by means of GP1 as a yellow oil (41%). 1H NMR (500 MHz,
CDCl3) δ 7.33 (d, J = 1.8 Hz, 1H), 7.04 (dd, J = 7.9, 1.8 Hz, 1H), 6.99
(d, J = 7.9 Hz, 1H), 2.86−2.77 (m, 1H), 1.20 (d, J = 6.7 Hz, 6H).
4-tert-Butyl-2-bromoaniline (12g). This compound was prepared
4. EXPERIMENTAL SECTION
1
from 11g by means of GP1 as a brown oil (38%). H NMR (500
Chemistry. General Methods. 1H NMR spectra were recorded on
a JEOL ALPHA500 (500 MHz) spectrometer, and 13C NMR spectra
were recorded on a JEOL ALPHA500 (125 MHz) spectrometer.
Chemical shifts (δ) are expressed in parts per million (ppm) relative to
deuteriochloroform as an internal reference, with coupling constants in
Hz. The abbreviations s, d, t, q, br, and m signify singlet, doublet,
triplet, quartet, broad, and multiplet, respectively. Fast atom
bombardment mass spectra (FAB-MS) and high-resolution mass
spectra (HRMS) were recorded on a JEOL JMS-HX110 spectrometer
MHz, CDCl3) δ 7.40 (d, J = 1.8 Hz, 1H), 7.13 (dd, J = 7.9, 1.8 Hz,
1H), 6.72 (d, J = 7.9 Hz, 1H), 3.95 (br s, 2H), 1.26 (s, 3H).
General Procedure 2 (GP2). 2-Vinylaniline (13a). To a solution of
2-bromoaniline (12a) (182 mg, 1.06 mmol) and Pd(PPh3)4 (108 mg,
10 mol %) in 5 mL of toluene was added tributylvinyltin (350 μL, 1.20
mmol). The mixture was stirred at 100 °C under an argon atmosphere
for 6 h and extracted with ethyl acetate. The extract was washed with
water and brine, dried over anhydrous magnesium sulfate, and
concentrated. The residue was purified by silica gel column
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chromatography (eluent: n-hexane/ethyl acetate = 9:1 to 5:1, v/v,
silica gel/KF = 10:1, w/w) to afford 51.7 mg (41%) of the title
compound as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 7.29 (d, J =
7.9 Hz, 1H), 7.09 (dd, J = 7.9, 7.9 Hz, 1H), 6.81−6.74 (m, 2H), 6.69
(d, J = 7.9 Hz, 1H), 5.63 (dd, J = 17, 1.2 Hz, 1H), 5.32 (dd, J = 11, 1.2
Hz, 1H), 3.75 (br s, 2H).
Hz, 1H), 5.40 (d, J = 11 Hz, 1H), 2.66 (q, J = 7.9 Hz, 2H), 1.25 (t, J =
7.9 Hz, 3H). MS (FAB) 378 (M + H)+.
N-(4-Hexyl-2-vinylphenyl)-2-iodobenzamide (14d). This com-
pound was prepared from 13d by means of GP4 as a brown solid
1
(quant). H NMR (500 MHz, CDCl3) δ 7.92 (dd, J = 7.9, 7.9 Hz,
2H), 7.53 (d, J = 7.9 Hz, 1H), 7.45 (dd, J = 7.9, 7.9 Hz, 1H), 7.33−
7.27 (m, 2H), 7.20−7.14 (m, 2H), 6.90 (dd, J = 17, 11 Hz, 1H), 5.70
(d, J = 17 Hz, 1H), 5.40 (d, J = 11 Hz, 1H), 2.61 (t, J = 7.9 Hz, 2H),
1.65−1.57 (m, 2H), 1.38−1.27 (m, 6H), 0.89 (t, J = 7.3 Hz, 3H). MS
(FAB) 434 (M + H)+.
4-Methyl-2-vinylaniline (13b). This compound was prepared from
1
12b by means of GP2 as a brown oil (34%). H NMR (500 MHz,
CDCl3) δ 7.11 (d, J = 1.8 Hz, 1H), 6.90 (dd, J = 7.9, 1.8 Hz, 1H), 6.76
(dd, J = 17, 11 Hz, 1H), 6.60 (d, J = 7.9 Hz, 1H), 5.62 (dd, J = 17, 1.8
Hz, 1H), 5.29 (d, J = 11 Hz, 1.8H), 3.63 (br s, 2H), 2.25 (s, 3H).
4-Ethyl-2-vinylaniline (13c). This compound was prepared from
N-(4-Dodecyl-2-vinylphenyl)-2-iodobenzamide (14e). This com-
1
pound was prepared from 13e by means of GP4 (95%). H NMR
1
(500 MHz, CDCl3) δ 7.92 (dd, J = 7.9, 7.9 Hz, 2H), 7.53 (d, J = 7.9
Hz, 1H), 7.45 (dd, J = 7.9, 7.9 Hz, 1H), 7.33−7.27 (m, 2H), 7.20−
7.14 (m, 2H), 6.90 (dd, J = 17, 11 Hz, 1H), 5.70 (d, J = 17 Hz, 1H),
5.40 (d, J = 11 Hz, 1H), 2.60 (t, J = 7.9 Hz, 2H), 1.66−1.56 (m, 2H),
1.40−1.24 (m, 18H), 0.88 (t, J = 7.3 Hz, 3H). MS (FAB) 518 (M +
H)+.
2-Iodo-N-(4-isopropyl-2-vinylphenyl)benzamide (14f). This com-
pound was prepared from 13f by means of GP4 as a white solid
(99%). MS (FAB) 392 (M + H)+.
N-(4-tert-Butyl-2-vinylphenyl)-2-iodobenzamide (14g). This com-
pound was prepared from 13g by means of GP4 as a white solid
(88%). 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 7.9 Hz, 1H), 7.92
(d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.49−7.43 (m, 2H), 7.40
(d, J = 7.9 Hz, 1H), 7.34 (s, 1H), 7.16 (dd, J = 7.9, 7.9 Hz, 1H), 6.92
(dd, J = 17, 11 Hz, 1H), 5.71 (d, J = 17 Hz, 1H), 5.41 (d, J = 11 Hz,
1H), 1.34 (s, 9H). MS (FAB) 406 (M + H)+.
12c by means of GP2 as a brown oil (37%). H NMR (500 MHz,
CDCl3) δ 7.12 (d, J = 1.8 Hz, 1H), 6.94 (dd, J = 7.9, 1.8 Hz, 1H), 6.77
(dd, J = 17, 11 Hz, 1H), 6.63 (d, J = 7.9 Hz, 1H), 5.63 (dd, J = 17, 1.8
Hz, 1H), 5.30 (dd, J = 11, 1.8 Hz, 1H), 3.64 (br s, 2H), 2.55 (q, J = 7.9
Hz, 3H), 1.20 (t, J = 7.9 Hz, 3H).
4-Hexyl-2-vinylaniline (13d). This compound was prepared from
12d by means of GP2 as a yellow oil (57%). The resulting crude 13d
was used directly in the next step.
4-Dodecyl-2-vinylaniline (13e). This compound was prepared from
12e by means of GP2 as a yellow solid (65%). The resulting crude 13e
was used directly in the next step.
General Procedure 3 (GP3). 4-Isopropyl-2-vinylaniline (13f). To a
solution of 12f (429 mg, 2.00 mmol) in 10 mL of DME was added
Pd(PPh3)4 (115 mg, 5 mol %), and the mixture was stirred at room
temperature under an argon atmosphere for 15 min. Potassium
carbonate (330 mg, 2.39 mmol), 3 mL of water, and 2,4,6-
trivinylcyclotriboroxane−pyridine complex (496 mg, 2.06 mmol)
were added, and the reaction mixture was heated under reflux for
4.5 h. The mixture was extracted with ethyl acetate, and the extract was
washed with water and brine, dried over anhydrous magnesium sulfate,
and concentrated. The residue was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate = 9:1, v/v) to afford
192 mg (59%) of the title compound as a brown oil. The resulting
crude 13f was used directly in the next step.
General Procedure 5 (GP5). 2-Iodo-N-methyl-N-(2-vinylphenyl)-
benzamide (15a). To a solution of 14a (107 mg, 0.308 mmol) in 2
mL of DMF were added sodium hydride (38.8 mg, 1.62 mmol) and
iodomethane (40 μL, 0.643 mmol) at 0 °C. The mixture was stirred
for 6.5 h at room temperature and extracted with ethyl acetate. The
organic layer was washed with water and brine, dried over anhydrous
magnesium sulfate, and concentrated. The residue was purified by
silica gel column chromatography (eluent: n-hexane/ethyl acetate =
3:1, v/v) to afford 103 mg (92%) of the title compound as a colorless
oil. 1H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 7.9 Hz, 1H), 7.49 (d, J
= 7.9 Hz, 1H), 7.30 (d, J = 7.9 Hz, 1H), 7.16 (dd, J = 7.9, 7.9 Hz, 1H),
7.08 (dd, J = 7.9, 7.9 Hz, 1H), 7.01 (dd, J = 7.9, 7.9 Hz, 1H), 6.97−
6.90 (m, 2H), 6.82 (dd, J = 7.9, 7.9 Hz, 1H), 5.80 (d, J = 17 Hz, 1H),
5.48 (d, J = 11 Hz, 1H), 3.41 (s, 3H). MS (FAB) 364 (M + H)+.
2-Iodo-N-methyl-N-(4-methyl-2-vinylphenyl)benzamide (15b).
This compound was prepared from 14b by means of GP5 as a
4-tert-Butyl-2-vinylaniline (13g). This compound was prepared
1
from 12g by means of GP2 as a brown oil (29%). H NMR (500
MHz, CDCl3) δ 7.29 (d, J = 1.8 Hz, 1H), 7.13 (dd, J = 7.9, 1.8 Hz,
1H), 6.79 (dd, J = 17, 11 Hz, 1H), 6.64 (d, J = 7.9 Hz, 1H), 5.63 (dd, J
= 17, 1.8 Hz, 1H), 5.31 (dd, J = 11, 1.8 Hz, 1H), 3.66 (br s, 2H), 1.29
(s, 3H).
General Procedure 4 (GP4). 2-Iodo-N-(2-vinylphenyl)benzamide
(14a). To a mixture of 13a (51.7 mg, 0.434 mmol) and Et3N (70 μL,
0.505 mmol) in 2 mL of DCM was added 2-iodobenzoyl chloride (177
mg, 0.664 mmol). The resulting mixture was stirred for 20 h at room
temperature under argon atmosphere. The organic layer was washed
with water and brine, dried over anhydrous magnesium sulfate, and
concentrated. The residue was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate = 5:1, v/v) to afford
1
colorless oil (99%). H NMR (500 MHz, CDCl3) δ 7.69 (dd, J = 7.9,
1.8 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 7.18 (d, J = 7.9 Hz, 1H), 7.03
(dd, J = 7.9, 7.9 Hz, 1H), 6.97−6.87 (m, 3H), 6.83 (dd, J = 7.9, 7.9 Hz,
1H), 5.78 (d, J = 17 Hz, 1H), 5.44 (d, J = 11 Hz, 1H), 3.38 (s, 3H),
2.24 (s, 3H). MS (FAB) 378 (M + H)+.
N-(4-Ethyl-2-vinylphenyl)-2-iodo-N-methylbenzamide (15c). This
compound was prepared from 14c by means of GP5 as a colorless oil
(94%). 1H NMR (500 MHz, CDCl3) δ 7.69 (dd, J = 7.9, 1.8 Hz, 1H),
7.29 (d, J = 1.8 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.02 (dd, J = 7.9, 7.9
Hz, 1H), 6.96−6.88 (m, 3H), 6.82 (dd, J = 7.9, 7.9 Hz, 1H), 5.79 (d, J
= 17 Hz, 1H), 5.44 (d, J = 11 Hz, 1H), 3.39 (s, 3H), 2.54 (q, J = 7.9
Hz, 2H), 1.16 (t, J = 7.9 Hz, 3H). MS (FAB) 392 (M + H)+.
N-(4-Hexyl-2-vinylphenyl)-2-iodo-N-methylbenzamide (15d).
This compound was prepared from 14d by means of GP5 as a
brown oil (87%). The resulting crude 15d was used directly in the next
step.
1
107 mg (71%) of the title compound as a white solid. H NMR (500
MHz, CDCl3) δ 8.07 (d, J = 7.9 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H),
7.54 (d, J = 7.9 Hz, 1H), 7.56−7.52 (m, 2H), 7.49−7.43 (m, 2H), 7.22
(dd, J = 7.9, 7.9 Hz, 1H), 7.17 (dd, J = 7.9, 7.9 Hz, 1H), 6.92 (dd, J =
17, 11 Hz, 1H), 5.71 (d, J = 17 Hz, 1H), 5.43 (d, J = 11 Hz, 1H). MS
(FAB) 350 (M + H)+.
2-Iodo-N-(4-methyl-2-vinylphenyl)benzamide (14b). This com-
pound was prepared from 13b by means of GP4 as a white solid
(46%). 1H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 7.9 Hz, 1H), 7.89
(d, J = 7.9 Hz, 1H), 7.54 (dd, J = 7.9, 1.8 Hz, 1H), 7.45 (dd, J = 7.9,
7.9 Hz, 1H), 7.32−7.28 (m, 2H), 7.19−7.14 (m, 2H), 6.90 (dd, J = 17,
11 Hz, 1H), 5.70 (d, J = 17 Hz, 1H), 5.40 (d, J = 11 Hz, 1H), 2.36 (s,
3H). MS (FAB) 364 (M + H)+.
N-(4-Dodecyl-2-vinylphenyl)-2-iodo-N-methylbenzamide (15e).
This compound was prepared from 14e by means of GP5 as a
colorless oil (97%). The resulting crude 15e was used directly in the
next step.
N-(4-Ethyl-2-vinylphenyl)-2-iodobenzamide (14c). This com-
pound was prepared from 13c by means of GP4 as a white solid
(58%). 1H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 7.9 Hz, 1H), 7.92
(d, J = 7.9 Hz, 1H), 7.53 (dd, J = 7.9, 1.8 Hz, 1H), 7.45 (dd, J = 7.9,
7.9 Hz, 1H), 7.33−7.29 (m, 2H), 7.20 (dd, J = 7.9, 1.8 Hz, 1H), 7.16
(dd, J = 7.9, 7.9 Hz, 1H), 6.91 (dd, J = 17, 11 Hz, 1H), 5.71 (d, J = 17
2-Iodo-N-(4-isopropyl-2-vinylphenyl)-N-methylbenzamide (15f).
This compound was prepared from 14f by means of GP5 as a
colorless oil (46%). MS (FAB) 406 (M + H)+.
N-(4-tert-Butyl-2-vinylphenyl)-2-iodo-N-methylbenzamide (15g).
This compound was prepared from 14g by means of GP5 as a white
solid (85%). 1H NMR (500 MHz, CDCl3) δ 7.69 (dd, J = 7.9, 1.8 Hz,
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1H), 7.46 (d, J = 1.8 Hz, 1H), 7.19 (d, J = 7.9 Hz, 1H), 7.09 (dd, J =
7.9, 1.8 Hz, 1H), 7.01 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 6.93 (dd, J = 7.9,
1.8 Hz, 1H), 6.92 (dd, J = 17, 11 Hz, 1H), 6.81 (ddd, J = 7.9, 7.9, 1.8
Hz, 1H), 5.78 (dd, J = 17, 1.2 Hz, 1H), 5.45 (dd, J = 11, 1.2 Hz, 1H),
3.39 (s, 3H), 1.23 (s, 9H). MS (FAB) 420 (M + H)+.
(s, 1H), 3.57 (s, 3H), 1.31 (s, 9H). HRMS (FAB) calcd for C20H22NO
292.1701; found 292.1748 (M + H)+.
2-(1,1,1,3,3,3-Hexafluoro-2-hydroxypropan-2-yl)-5-methyl-11-
methylene-6H-dibenz[b,e]azepin-6-one (10). This compound was
prepared from 11h by means of a series of procedures similar to those
used for 16 as a white solid (16% in 4 steps); Mp 205.0−206.2 °C. 1H
NMR (500 MHz, CDCl3) δ 7.96 (dd, J = 7.9, 1.8 Hz, 1H), 7.65−7.60
(m, 2H), 7.48 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 7.39 (ddd, J = 7.9, 7.9,
1.8 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.30−7.27 (m, 1H), 5.55 (s,
1H), 5.48 (s, 1H), 3.61 (s, 1H), 3.59 (s, 3H). HRMS (FAB) calcd for
C19H14F6NO2 402.0929; found 402.0924 (M + H)+.
General Procedure 6 (GP6). 5,11-Dihydro-5-methyl-11-methyl-
ene-6H-dibenz[b,e]azepin-6-one (16). A mixture of 15a (103 mg,
0.282 mmol), Pd(OAc)2 (16.8 mg, 26 mol %), PPh3 (34.7 mg, 50 mol
%), and NaOAc (44.0 mg, 0.369 mmol) in 2 mL of DMF was stirred
for 8 h at 100 °C under an argon atmosphere. After filtration through
Celite, the mixture was extracted with ethyl acetate. The extract was
washed with water and brine, dried over anhydrous magnesium sulfate,
and concentrated. The residue was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate = 5:1, v/v) to afford
2-Iodo-5-methoxybenzoic Acid (27). A solution of NaNO2 (0.71 g,
10.3 mmol) in 4 mL of water was added slowly to a cold (0 °C) stirred
solution of 5-hydroxyanthranilic acid (1.5 g, 9.8 mmol) and 8 mL of
concentrated HCl in 20 mL of water. After the addition was
completed, the solution was stirred for an additional 30 min at 0 °C. A
solution of KI (2.5 g, 14.8 mmol) in 4 mL of water was added slowly.
The resulting mixture was stirred for 20 min at 0 °C and then heated
to 90 °C for 30 min to remove N2. The mixture was cooled to room
temperature and extracted with ethyl acetate. The organic extract was
washed with satd NaHSO3 aq and water and dried over anhydrous
magnesium sulfate. The solvent was removed in vacuo to give 25 as a
yellowish−red solid, which was used directly for the next step. To a
solution of 25 and K2CO3 (2.78 g, 20.1 mmol) in 50 mL of anhydrous
acetone was added dimethyl sulfate (1.9 mL, 20.0 mmol). The reaction
mixture was heated under reflux for 5 h, cooled to room temperature,
and filtered. The filtrate was removed in vacuo to give 26 as a yellow
oil, which was used directly for the next step. To a solution of 26 in 30
mL of MeOH was added 10 mL of 2 N NaOH aq, and the mixture was
heated under reflux for 2 h. The solvent was removed in vacuo, and the
residue was dissolved in water and extracted with ethyl acetate. The
aqueous layer was acidified with 2 N HCl and extracted with ethyl
acetate. The combined extract was dried over anhydrous magnesium
sulfate and concentrated in vacuo. The residue was purified by silica
gel column chromatography (eluent: n-hexane/ethyl acetate = 1:1, v/
1
48.3 mg (73%) of the title compound as a yellow oil. H NMR (500
MHz, CDCl3) δ 7.94 (dd, J = 7.9, 1.8 Hz, 1H), 7.45 (ddd, J = 7.9, 7.9,
1.8 Hz, 1H), 7.36 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 7.32−7.21 (m, 4H),
7.15 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 5.49 (d, J = 1.2 Hz, 1H), 5.44 (d, J
= 1.2 Hz, 1H), 3.58 (s, 3H). HRMS (FAB) calcd for C16H14NO
236.1075; found 236.1034 (M + H)+.
5,11-Dihydro-2,5-dimethyl-11-methylene-6H-dibenz[b,e]azepin-
6-one (17). This compound was prepared from 15b by means of GP6
as a colorless oil (45%). 1H NMR (500 MHz, CDCl3) δ 7.93 (dd, J =
7.9, 1.8 Hz, 1H), 7.42 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 7.35 (ddd, J =
7.9, 7.9, 1.8 Hz, 1H), 7.23 (dd, J = 7.9, 1.8 Hz, 1H), 7.11−7.10 (m,
2H), 7.07 (s, 1H), 5.46 (d, J = 1.2 Hz, 1H), 5.42 (d, J = 1.2 Hz, 1H),
3.56 (s, 3H), 2.32 (s, 3H). HRMS (FAB) calcd for C17H16NO
250.1232; found 250.1233 (M + H)+.
5,11-Dihydro-2-ethyl-5-methyl-11-methylene-6H-dibenz[b,e]-
azepin-6-one (18). This compound was prepared from 15c by means
1
of GP6 as a colorless oil (53%). H NMR (500 MHz, CDCl3) δ 7.94
(dd, J = 7.9, 1.8 Hz, 1H), 7.43 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 7.35
(ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 7.26−7.23 (m, 1H), 7.14−7.12 (m,
2H), 7.08 (s, 1H), 5.47 (d, J = 1.2 Hz, 1H), 5.43 (d, J = 1.2 Hz, 1H),
3.56 (s, 3H), 2.62 (q, J = 7.9 Hz, 2H), 1.23 (t, J = 7.9 Hz, 3H). 13C
NMR (125 MHz, CDCl3) δ 168.28, 148.04, 143.49, 141.73, 138.39,
137.66, 131.68, 131.24, 131.17, 127.92, 127.68, 127.15, 126.24, 122.11,
116.35, 38.32, 28.05, 15.36. HRMS (FAB) calcd for C18H18NO
264.1388; found 264.1357 (M + H)+.
5,11-Dihydro-2-hexyl-5-methyl-11-methylene-6H-dibenz[b,e]-
azepin-6-one (19). This compound was prepared from 15d by means
of GP6 as a yellow oil (88%). 1H NMR (500 MHz, CDCl3) δ 7.94 (d,
J = 7.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.25
(d, J = 7.9 Hz, 1H), 7.14−7.08 (m, 2H), 7.06 (s, 1H), 5.46 (s, 1H),
5.42 (s, 1H), 3.56 (s, 3H), 2.56 (t, J = 7.9 Hz, 2H), 1.62−1.52 (m,
2H), 1.37−1.27 (m, 6H), 0.88 (t, J = 6.7 Hz, 3H). HRMS (FAB) calcd
for C22H26NO 320.2014; found 320.2006 (M + H)+.
5,11-Dihydro-2-(1-dodecyl)-5-methyl-11-methylene-6H-dibenz-
[b,e]azepin-6-one (20). This compound was prepared from 15e by
means of GP6 as a colorless oil (76%). 1H NMR (500 MHz, CDCl3) δ
7.94 (d, J = 7.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.35 (t, J = 7.9 Hz,
1H), 7.25 (d, J = 7.9 Hz, 1H), 7.13−7.08 (m, 2H), 7.06 (s, 1H), 5.46
(s, 1H), 5.42 (s, 1H), 3.56 (s, 3H), 2.56 (t, J = 7.9 Hz, 2H), 1.62−1.55
(m, 2H), 1.34−1.22 (m, 18H), 0.88 (t, J = 7.3 Hz, 3H). HRMS (FAB)
calcd for C28H38NO 404.2953; found 404.2919 (M + H)+.
5,11-Dihydro-2-isopropyl-5-methyl-11-methylene-6H-dibenz-
[b,e]azepin-6-one (21). This compound was prepared from 15f by
means of GP6 as a colorless oil (34%). 1H NMR (500 MHz, CDCl3) δ
7.94 (dd, J = 7.9, 1.2 Hz, 1H), 7.43 (ddd, J = 7.9, 7.3, 1.2 Hz, 1H), 7.36
(ddd, J = 7.9, 7.3, 1.2 Hz, 1H), 7.26 (dd, J = 7.3, 1.2 Hz, 1H), 7.16−
7.13 (m, 2H), 7.09 (d, J = 1.2 Hz, 1H), 5.47 (d, J = 1.2 Hz, 1H), 5.43
(d, J = 1.2 Hz, 1H), 3.57 (s, 3H), 2.92−2.85 (m, 1H), 1.24 (dd, J = 6.6,
1.8 Hz, 6H). HRMS (FAB) calcd for C19H20NO 278.1545; found
278.1536 (M + H)+.
1
v) to afford 1.27 g (47%) of the title compound as a white solid. H
NMR (500 MHz, CDCl3) δ 7.90 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 3.1
Hz, 1H), 6.81 (dd, J = 8.5, 3.1 Hz, 1H), 3.84 (s, 3H).
N-(4-Ethyl-2-vinylphenyl)-2-iodo-5-methoxybenzamide (28). To
a solution of 27 (280 mg, 1.01 mmol) in 2 mL of DCM was added
chloromethylenedimethyliminium chloride (130 mg, 1.02 mmol) at 0
°C, and the mixture was stirred for 30 min at room temperature. To a
solution of 13c (117 mg, 0.795 mmol) in 2 mL of DCM were added
130 μL of Et3N and the above solution at 0 °C, and stirring was
continued for 12 h at room temperature. The reaction mixture was
washed with water and brine, dried over anhydrous magnesium sulfate,
and concentrated. The residue was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate = 5:1 to 4:1, v/v) to
afford 258 mg (80%) of the title compound as a white solid. 1H NMR
(500 MHz, CDCl3) δ 7.94−7.89 (m, 1H), 7.76 (d, J = 7.9 Hz, 1H),
7.36−7.30 (m, 2H), 7.20 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 3.1 Hz, 1H),
6.91 (dd, J = 17, 11 Hz, 1H), 6.75 (dd, J = 7.9, 3.1 Hz, 1H), 5.71 (d, J
= 17 Hz, 1H), 5.41 (d, J = 11 Hz, 1H), 3.83 (br s, 3H), 2.66 (q, J = 7.9
Hz, 2H), 1.25 (t, J = 7.9 Hz, 3H).
N-(4-Ethyl-2-vinylphenyl)-2-iodo-5-methoxy-N-methylbenza-
mide (29). This compound was prepared from 28 by means of GP5 as
a yellow oil (78%). 1H NMR (500 MHz, CDCl3) δ 7.51 (d, J = 7.9 Hz,
1H), 7.31 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 6.95−6.88 (m,
2H), 6.53 (d, J = 3.1 Hz, 1H), 6.42 (dd, J = 7.9, 3.1 Hz, 1H), 5.80 (d, J
= 17 Hz, 1H), 5.45 (d, J = 11 Hz, 1H), 3.56 (s, 3H), 3.39 (s, 3H), 2.56
(q, J = 7.9 Hz, 2H), 1.17 (t, J = 7.9 Hz, 3H).
2-Ethyl-5,11-dihydro-8-methoxy-5-methyl-11-methylene-6H-
dibenz[b,e]azepin-6-one (30). This compound was prepared from 29
by means of GP6 as a yellow oil (95%). 1H NMR (500 MHz, CDCl3)
δ 7.46 (d, J = 2.4 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 7.12 (s, 2H), 7.07
(s, 1H), 6.98 (dd, J = 7.9, 2.4 Hz, 1H), 5.41 (s, 1H), 5.38 (s, 1H), 3.82
(s, 3H), 3.56 (s, 3H), 2.62 (q, J = 7.9 Hz, 2H), 1.23 (t, J = 7.9 Hz,
3H).
5,11-Dihydro-2-tert-butyl-5-methyl-11-methylene-6H-dibenz-
[b,e]azepin-6-one (22). This compound was prepared from 15g by
means of GP6 as a colorless oil (63%). 1H NMR (500 MHz, CDCl3) δ
7.95 (d, J = 7.9 Hz, 1H), 7.44 (dd, J = 7.9, 7.9 Hz, 1H), 7.36 (dd, J =
7.9, 7.9 Hz, 1H), 7.31 (dd, J = 7.9, 1.8 Hz, 1H), 7.28−7.25 (m, 1H),
7.23 (d, J = 1.8 Hz, 1H), 7.15 (d, J = 7.9 Hz, 1H), 5.47 (s, 1H), 5.43
2-Ethyl-5,11-dihydro-8-hydroxy-5-methyl-11-methylene-6H-
dibenz[b,e]azepin-6-one (31). To a solution of 30 (120 mg, 0.410
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mmol) in 2 mL of dehydrated DCM was added dropwise 1.6 mL of
boron tribromide (1.0 M solution in dichloromethane) at 0 °C under
an argon atmosphere. The mixture was stirred for 3 h and poured into
ice−water. The organic layer was washed with water and brine, dried
over anhydrous magnesium sulfate, and concentrated. The residue was
purified by silica gel column chromatography (eluent: n-hexane/ethyl
acetate = 3:1 to 1:1, v/v) to afford 103 mg (90%) of the title
Methyl 4-(5,11-Dihydro-2-ethyl-5-methyl-11-methylene-6-
oxodibenz[b,e]azepin-8-yl)benzoate (36b). This compound was
prepared from 35b by means of GP8 as a yellow oil (79%). 1H
NMR (500 MHz, CDCl3) δ 8.21 (d, J = 1.8 Hz, 1H), 8.09 (d, J = 7.9
Hz, 2H), 7.69 (dd, J = 7.9, 1.8 Hz, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.35
(d, J = 7.9 Hz, 1H), 7.18−7.13 (m, 2H), 7.11 (s, 1H), 5.53 (s, 1H),
5.48 (s, 1H), 3.93 (s, 3H), 3.59 (s, 3H), 2.63 (q, J = 7.9 Hz, 2H), 1.24
(t, J = 7.9 Hz, 3H).
1
compound as a yellow oil. H NMR (500 MHz, CDCl3) δ 7.80−7.78
Methyl 2-(3-(5,11-Dihydro-2-ethyl-5-methyl-11-methylene-6-
(m, 2H), 7.16−7.12 (m, 3H), 7.08 (s, 1H), 6.98 (dd, J = 8.5, 2.4 Hz,
1H), 5.41 (s, 1H), 5.38 (s, 1H), 3.57 (s, 3H), 2.63 (q, J = 7.9 Hz, 2H),
1.23 (t, J = 7.9 Hz, 3H). MS (FAB) 279 (M)+.
oxodibenz[b,e]azepin-8-yl)phenyl)acetate (36c). This compound
1
was prepared from 35c by means of GP8 as a brown oil (83%). H
NMR (500 MHz, CDCl3) δ 8.17 (d, J = 1.8 Hz, 1H), 7.65 (dd, J = 7.9,
1.8 Hz, 1H), 7.52−7.46 (m, 2H), 7.39 (t, J = 7.9 Hz, 1H), 7.32 (d, J =
7.9 Hz, 1H), 7.27 (d, J = 7.9 Hz, 1H), 7.17−7.12 (m, 2H), 7.11 (s,
1H), 5.52 (s, 1H), 5.46 (s, 1H), 3.69 (s, 3H), 3.67 (s, 2H), 3.59 (s,
3H), 2.63 (q, J = 7.9 Hz, 2H), 1.24 (t, J = 7.9 Hz, 3H). MS (FAB) 412
(M + H)+.
Methyl 2-(4-(5,11-Dihydro-2-ethyl-5-methyl-11-methylene-6-
oxodibenz[b,e]azepin-8-yl)phenyl)acetate (36d). This compound
was prepared from 35d by means of GP8 as a colorless oil (25%).
1H NMR (500 MHz, CDCl3) δ 8.16 (d, J = 1.8 Hz, 1H), 7.64 (dd, J =
7.9, 1.8 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.36−7.30 (m, 3H), 7.17−
7.10 (m, 3H), 5.51 (s, 1H), 5.46 (s, 1H), 3.71 (s, 3H), 3.66 (s, 2H),
3.59 (s, 3H), 2.63 (q, J = 7.3 Hz, 2H), 1.23 (t, J = 7.3 Hz, 3H). MS
(FAB) 412 (M + H)+.
General Procedure 9 (GP9). 3-(5,11-Dihydro-2-ethyl-5-methyl-11-
methylene-6-oxodibenz[b,e]azepin-8-yl)benzoic Acid (37). To a
solution of 36a (8.2 mg, 0.020 mmol) in 1 mL of ethanol was
added 0.5 mL of 2 N aqueous NaOH. The mixture was stirred for 3 h
at 80 °C and extracted with ethyl acetate. The extract was washed with
2 N HCl, water, and brine, dried over anhydrous magnesium sulfate,
and concentrated to afford 4.9 mg (64%) of the title compound as a
colorless oil. 1H NMR (500 MHz, CDCl3) δ 8.35 (s, 1H), 8.24 (d, J =
1.8 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.72
(dd, J = 7.9, 1.8 Hz, 1H), 7.53 (dd, J = 7.9, 7.9 Hz, 1H), 7.37 (d, J =
7.9 Hz, 1H), 7.17−7.15 (m, 2H), 7.12 (s, 1H), 5.54 (s, 1H), 5.49 (s,
1H), 3.61 (s, 3H), 2.64 (q, J = 7.3 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H).
HRMS (FAB) calcd for C25H22NO3 384.1600; found 384.1582 (M +
H)+.
2-Ethyl-5,11-dihydro-5-methyl-11-methylene-6-oxodibenz[b,e]-
azepin-8-yl trifluoromethanesulfonate (32). To a solution of 31
(10.0 mg, 0.0358 mmol) and 8 μL of pyridine in 1 mL of DCM was
added dropwise trifluoromethanesulfonic anhydride (8 μL, 0.0474
mmol) at 0 °C. The mixture was stirred for 2 h at room temperature
and poured into ice−water. The organic layer was washed with satd
NaHCO3 aq, water, and brine, dried over anhydrous magnesium
sulfate, and concentrated. The residue was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate = 3:1 to 1:1, v/v) to
1
afford 7.0 mg (47%) of the title compound as a yellow oil. H NMR
(500 MHz, CDCl3) δ 7.85 (s, 1H), 7.34 (s, 2H), 7.20−7.14 (m, 2H),
7.08 (s, 1H), 5.51 (s, 1H), 5.51 (s, 1H), 3.56 (s, 3H), 2.64 (q, J = 7.9
Hz, 2H), 1.24 (t, J = 7.9 Hz, 3H). MS (FAB) 412 (M + H)+.
2-Ethyl-5,11-dihydro-5-methyl-11-methylene-8-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)-6H-dibenz[b,e]azepin-6-one (33).
A mixture of 32 (20.1 mg, 0.0489 mmol), bis(pinacolato)diboron
(26.4 mg, 0.104 mmol), PdCl2(PPh3)2 (16.4 mg, 48 mol %), and
KOAc (19.5 mg, 0.199 mmol) in 1 mL of dioxane was stirred for 3 h at
120 °C under an argon atmosphere. The mixture was filtered through
Celite, and the filtrate was extracted with ethyl acetate. The extract was
washed with water and brine, dried over anhydrous magnesium sulfate,
and concentrated. The residue was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate = 5:1, v/v) to afford
1
10.9 mg (57%) of the title compound as a yellow oil. H NMR (500
MHz, CDCl3) δ 8.38 (s, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 7.9
Hz, 1H), 7.11 (s, 2H), 7.06 (s, 1H), 5.48 (s, 1H), 5.44 (s, 1H), 3.55 (s,
3H), 2.60 (q, J = 7.9 Hz, 2H), 1.31 (s, 6H), 1.30 (s, 6H), 1.21 (t, J =
7.9 Hz, 3H). MS (FAB) 390 (M + H)+.
General Procedure 7 (GP7). Methyl 3-(3-Iodophenyl)propanoate
(35c). To a solution of 34c (50.0 mg, 0.191 mmol) in 1 mL of MeOH
was added 0.1 mL of concentrated H2SO4, and the mixture was
refluxed for 4 h. The organic solution was extracted with DCM, and
the extract was washed with water and brine, dried over anhydrous
magnesium sulfate, and concentrated to obtain 48.1 mg (91%) of the
title compound as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.64
(s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.25 (d, J = 7.9 Hz, 1H), 7.06 (t, J =
7.9 Hz, 1H), 3.70 (s, 3H), 3.57 (s, 2H).
4-(5,11-Dihydro-2-ethyl-5-methyl-11-methylene-6-oxodibenz-
[b,e]azepin-8-yl)benzoic Acid (38). This compound was prepared
1
from 36b by means of GP9 as a white solid (14%). H NMR (500
MHz, CDCl3) δ 8.22 (d, J = 2.4 Hz, 1H), 8.14 (d, J = 7.9 Hz, 2H),
7.72−7.67 (m, 3H), 7.37 (d, J = 7.9 Hz, 1H), 7.17−7.15 (m, 2H), 7.11
(s, 1H), 5.54 (s, 1H), 5.49 (s, 1H), 3.60 (s, 3H), 2.64 (q, J = 7.3 Hz,
2H), 1.24 (t, J = 7.3 Hz, 3H). HRMS (FAB) calcd for C25H22NO3
384.1600; found 384.1643 (M + H)+.
2-(3-(5,11-Dihydro-2-ethyl-5-methyl-11-methylene-6-oxodibenz-
[b,e]azepin-8-yl)phenyl)acetic Acid (39). This compound was
prepared from 36c by means of GP9 as a white solid (98%). 1H
NMR (500 MHz, CDCl3) δ 8.16 (d, J = 1.8 Hz, 1H), 7.65 (dd, J = 7.9,
1.8 Hz, 1H), 7.52−7.49 (m, 2H), 7.39 (t, J = 7.9 Hz, 1H), 7.32 (d, J =
7.9 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.17−7.12 (m, 2H), 7.10 (s,
1H), 5.51 (s, 1H), 5.46 (s, 1H), 3.70 (s, 2H), 3.59 (s, 3H), 2.63 (q, J =
7.9 Hz, 2H), 1.23 (t, J = 7.9 Hz, 3H). HRMS (FAB) calcd for
C26H24NO3 398.1756; found 398.1774 (M + H)+.
Methyl 3-(4-Bromophenyl)propanoate (35d). This compound was
1
prepared from 34d by means of GP7 as a colorless oil (99%). H
NMR (500 MHz, CDCl3) δ 7.45 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9
Hz, 2H), 3.70 (s, 3H), 3.58 (s, 2H).
General Procedure 8 (GP8). Ethyl 3-(5,11-Dihydro-2-ethyl-5-
methyl-11-methylene-6-oxodibenz[b,e]azepin-8-yl)benzoate (36a).
A mixture of 33 (12.7 mg, 0.0326 mmol), ethyl 3-iodobenzoate (35a)
(6 μL, 0.0356 mmol), Pd(PPh3)4 (30.6 mg, 80 mol %), and K3PO4
(15.0 mg, 0.0710 mmol) in 1 mL of DMF was stirred for 3 h at 120 °C
under an argon atmosphere. After filtration through Celite, the mixture
was extracted with ethyl acetate and the extract was washed with water
and brine, dried over anhydrous magnesium sulfate, and concentrated.
The residue was purified by silica gel column chromatography (eluent:
n-hexane/ethyl acetate = 5:1, v/v) to afford 8.2 mg (61%) of the title
2-(4-(5,11-Dihydro-2-ethyl-5-methyl-11-methylene-6-oxodibenz-
[b,e]azepin-8-yl)phenyl)acetic Acid (40). This compound was
1
prepared from 36d by means of GP9 as a colorless oil. H NMR
(500 MHz, CDCl3) δ 8.17 (d, J = 1.8 Hz, 1H), 7.64 (dd, J = 7.9, 1.8
Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.36−7.30 (m, 3H), 7.17−7.12 (m,
2H), 7.10 (s, 1H), 5.51 (s, 1H), 5.46 (s, 1H), 3.69 (s, 2H), 3.59 (s,
3H), 2.63 (q, J = 7.9 Hz, 2H), 1.23 (t, J = 7.9 Hz, 3H). HRMS (FAB)
calcd for C26H24NO3 398.1756; found 398.1709 (M + H)+.
1
compound as a yellow oil. H NMR (500 MHz, CDCl3) δ 8.26 (s,
1H), 8.21 (d, J = 1.8 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 7.9
Hz, 1H), 7.70 (dd, J = 7.9, 1.8 Hz, 1H), 7.50 (dd, J = 7.9, 7.9 Hz, 1H),
7.36 (d, J = 7.9 Hz, 1H), 7.17−7.14 (m, 2H), 7.11 (s, 1H), 5.53 (s,
1H), 5.48 (s, 1H), 4.40 (q, J = 7.3 Hz, 2H), 3.60 (s, 3H), 2.64 (q, J =
7.3 Hz, 2H), 1.40 (t, J = 7.3 Hz, 3H), 1.24 (t, J = 7.3 Hz, 3H). MS
(FAB) 412 (M + H)+.
2-Bromo-4-nitroaniline (42a). This compound was prepared from
41a by means of GP1 as a yellow solid (81%). H NMR (500 MHz,
CDCl3) δ 8.38 (d, J = 1.8 Hz, 1H), 8.04 (dd, J = 7.9, 1.8 Hz, 1H), 6.74
(d, J = 7.9 Hz, 1H), 4.82 (br s, 2H).
4-Amino-3-bromophenyl Acetate (42b). This compound was
prepared from 41b by means of GP1 as a white solid (83%). 1H NMR
1
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(500 MHz, CDCl3) δ 7.19 (d, J = 1.8 Hz, 1H), 6.86 (dd, J = 7.9, 1.8
Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 2.26 (s, 3H).
white solid (62%). 1H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 7.9 Hz,
1H), 7.20 (d, J = 7.9 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H), 6.95−6.81 (m,
4H), 6.54 (dd, J = 7.9, 2.4 Hz, 1H), 5.75 (d, J = 17 Hz, 1H), 5.46 (d, J
= 11 Hz, 1H), 3.37 (s, 3H). MS (FAB) 380 (M + H)+.
Ethyl 4-Amino-3-bromobenzoate (42c). This compound was
1
prepared from 41c by means of GP1 as a yellow solid (87%). H
NMR (500 MHz, CDCl3) δ 8.12 (d, J = 1.8 Hz, 1H), 7.80 (dd, J = 7.9,
1.8 Hz, 1H), 6.74 (d, J = 7.9 Hz, 1H), 4.32 (q, J = 6.7 Hz, 2H), 1.37 (t,
J = 6.7 Hz, 3H).
1-(4-Amino-3-bromophenyl)ethanone (42d). This compound was
prepared from 41d by means of GP1 as a brown solid (76%) and used
for the next step without purification.
5,11-Dihydro-5-methyl-11-methylene-2-nitro-6H-dibenz[b,e]-
azepin-6-one (46). This compound was prepared from 45a by means
1
of GP6 as a brown oil (88%). H NMR (500 MHz, CDCl3) δ 8.19−
8.16 (m, 2H), 7.96 (d, J = 7.9 Hz, 1H), 7.51 (dd, J = 7.9, 7.9 Hz, 1H),
7.42 (dd, J = 7.9, 7.9 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.29 (d, J = 7.9
Hz, 1H), 5.64 (s, 1H), 5.56 (s, 1H), 3.62 (s, 3H). HRMS (FAB) calcd
for C16H13N2O3 281.0926; found 281.0925 (M + H) +. Anal. Calcd for
C16H12N2O3: C, 68.56; H, 4.32; N, 9.99. Found: C, 68.75; H, 4.51; N,
9.73.
4-Nitro-2-vinylaniline (43a). This compound was prepared from
1
42a by means of GP2 as a yellow solid (85%). H NMR (500 MHz,
CDCl3) δ 8.19 (d, J = 1.8 Hz, 1H), 8.00 (dd, J = 7.9, 1.8 Hz, 1H),
6.70−6.63 (m, 2H), 5.76 (d, J = 18 Hz, 1H), 5.50 (d, J = 11 Hz, 1H),
4.44 (br s, 2H).
4-Amino-3-vinylphenyl Acetate (43b). This compound was
prepared from 42b by means of GP2 as a yellow oil (69%). 1H
NMR (500 MHz, CDCl3) δ 7.00 (d, J = 2.4 Hz, 1H), 6.81 (dd, J = 7.9,
2.4 Hz, 1H), 6.71 (dd, J = 17, 11 Hz, 1H), 6.66 (d, J = 7.9 Hz, 1H),
5.62 (d, J = 17 Hz, 1H), 5.34 (d, J = 11 Hz, 1H), 3.75 (br s, 2H), 2.27
(s, 3H).
5,11-Dihydro-2-hydroxy-5-methyl-11-methylene-6H-dibenz[b,e]-
azepin-6-one (48). This compound was prepared from 45e by means
1
of GP6 (95%). H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 7.9 Hz,
1H), 7.43 (dd, J = 7.9, 7.9 Hz, 1H), 7.36 (dd, J = 7.9, 7.9 Hz, 1H), 7.22
(d, J = 7.9 Hz, 1H), 7.09 (d, J = 7.9 Hz, 1H), 6.77 (dd, J = 7.9, 2.4 Hz,
1H), 6.74 (d, J = 2.4 Hz, 1H), 5.46 (d, J = 1.2 Hz, 1H), 5.43 (d, J = 1.2
Hz, 1H), 5.24 (br s, 1H), 3.54 (s, 3H). HRMS (FAB) calcd for
+
C16H14NO2 252.1025; found 252.1003 (M + H) . Anal. Calcd for
Ethyl 4-Amino-3-vinylbenzoate (43c). This compound was
prepared from 42c by means of GP3 as a white solid (47%). 1H
NMR (500 MHz, CDCl3) δ 8.00 (d, J = 1.8 Hz, 1H), 7.80 (dd, J = 7.9,
1.8 Hz, 1H), 6.79−6.72 (m, 2H), 5.73 (d, J = 18 Hz, 1H), 5.42 (d, J =
11 Hz, 1H), 4.34 (q, J = 6.7 Hz, 2H), 1.38 (t, J = 6.7 Hz, 3H).
1-(4-Amino-3-vinylphenyl)ethanone (43d). This compound was
prepared from 42d by means of GP2 as a yellow solid. The resulting
crude 43d was used directly in the next step.
2-Iodo-N-(4-nitro-2-vinylphenyl)benzamide (44a). This com-
pound was prepared from 43a by means of GP4 as a yellow oil
(quant). The resulting crude 44a was used directly in the next step.
4-(2-Iodobenzamido)-3-vinylphenyl Acetate (44b). This com-
pound was prepared from 43b by means of GP4 as a white solid
(94%). 1H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 7.9 Hz, 1H), 7.93
(d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.46 (dd, J = 7.9, 7.9 Hz,
1H), 7.34 (s, 1H), 7.20 (d, J = 1.8 Hz, 1H), 7.17 (dd, J = 7.9, 7.9 Hz,
1H), 7.09 (dd, J = 7.9, 1.8 Hz, 1H), 6.87 (dd, J = 17, 11 Hz, 1H), 5.71
(d, J = 17 Hz, 1H), 5.46 (d, J = 11 Hz, 1H), 2.32 (s, 3H). MS (FAB)
408 (M + H)+.
C16H13NO2·1/4H2O: C, 75.13; H, 5.32; N, 5.48. Found: C, 75.43; H,
5.30; N, 5.45.
Ethyl 5,11-Dihydro-5-methyl-11-methylene-6-oxodibenz[b,e]-
azepine-2-carboxylate (49). This compound was prepared from
1
45c by means of GP6 as a colorless oil (42%). H NMR (500 MHz,
CDCl3) δ 7.99−7.93 (m, 3H), 7.47 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H),
7.38 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 7.30−7.26 (m, 2H), 5.55 (s, 1H),
5.51 (s, 1H), 4.42−4.35 (m, 2H), 3.60 (s, 3H), 1.39 (t, J = 7.3 Hz,
3H). MS (FAB) 308 (M + H)+.
2-Acetyl-5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]-
azepin-6-one (52). This compound was prepared from 45d by means
1
of GP6 as a brown viscous oil (22% in 2 steps). H NMR (500 MHz,
CDCl3) δ 7.95 (dd, J = 7.9, 1.2 Hz, 1H), 7.90 (dd, J = 7.9, 1.2 Hz, 1H),
7.87 (d, J = 1.2 Hz, 1H), 7.48 (ddd, J = 7.9, 7.9, 1.2 Hz, 1H), 7.39
(ddd, J = 7.9, 7.9, 1.2 Hz, 1H), 7.31−7.27 (m, 2H), 5.56 (s, 1H), 5.51
(s, 1H), 3.60 (s, 3H), 2.59 (s, 3H). HRMS (FAB) calcd for
C18H16NO2 278.1181; found 278.1198 (M + H)+. Anal. Calcd for
C18H15NO2·8/9H2O: C, 73.70; H, 5.77; N, 4.78. Found: C, 73.51; H,
5.38; N, 4.45.
Ethyl 4-(2-Iodobenzamido)-3-vinylbenzoate (44c). This com-
pound was prepared from 43c by means of GP4 as a white solid
(88%). 1H NMR (500 MHz, CDCl3) δ 8.35 (d, J = 8.5 Hz, 1H), 8.11
(d, J = 2.4 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H),
7.60−7.51 (m, 2H), 7.47 (dd, J = 8.5, 7.3 Hz, 1H), 7.19 (dd, J = 7.3,
6.7 Hz, 1H), 6.88 (dd, J = 17, 11 Hz, 1H), 5.79 (d, J = 17 Hz, 1H),
5.53 (d, J = 11 Hz, 1H), 4.39 (q, J = 7.3 Hz, 2H), 1.41 (t, J = 7.3 Hz,
1H). MS (FAB) 422 (M + H)+.
N-(4-Acetyl-2-vinylphenyl)-2-iodobenzamide (44d). This com-
pound was prepared from 43d by means of GP4 as a white solid
(50% in 2 steps). 1H NMR (500 MHz, CDCl3) δ 8.40 (d, J = 7.9 Hz,
1H), 8.03 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.62 (s, 1H), 7.54 (d, J =
7.9 Hz, 1H), 7.47 (dd, J = 7.9, 7.9 Hz, 1H), 7.19 (dd, J = 7.9, 7.9 Hz,
1H), 6.88 (dd, J = 17, 11 Hz, 1H), 5.79 (d, J = 17 Hz, 1H), 5.55 (d, J =
11 Hz, 1H), 2.62 (s, 3H). MS (FAB) 392 (M + H)+.
2-Iodo-N-methyl-N-(4-nitro-2-vinylphenyl)benzamide (45a). This
compound was prepared from 44a by means of GP5 as a yellow
viscous oil (53%). MS (FAB) 409 (M + H)+.
Ethyl 4-(2-Iodo-N-methylbenzamido)-3-vinylbenzoate (45c). This
compound was prepared from 44c by means of GP5 as a colorless oil
(91%). MS (FAB) 436 (M + H)+.
N-(4-Acetyl-2-vinylphenyl)-2-iodo-N-methylbenzamide (45d).
This compound was prepared from 44d by means of GP5 (32%).
1H NMR (500 MHz, CDCl3) δ 8.08 (d, J = 1.8 Hz, 1H), 7.71 (dd, J =
7.9, 1.8 Hz, 1H), 7.65 (dd, J = 7.9, 1.8 Hz, 1H), 7.37 (d, J = 7.9 Hz,
1H), 7.03 (ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 6.98−6.91 (m, 2H), 6.85
(ddd, J = 7.9, 7.9, 1.8 Hz, 1H), 5.93 (d, J = 18 Hz, 1H), 5.58 (d, J = 12
Hz, 1H), 3.41 (s, 3H), 2.54 (s, 3H). MS (FAB) 406 (M + H)+.
N-(4-Hydroxy-2-vinylphenyl)-2-iodo-N-methylbenzamide (45e).
This compound was prepared from 44b by means of GP5 as a
2-Amino-5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]-
azepin-6-one (47). To a solution of 46 (207 mg, 0.737 mmol) in 5
mL of ethyl acetate was added tin(II) chloride dihydrate (860 mg, 3.81
mmol). The mixture was heated to reflux for 3.5 h. After cooling, the
mixture was poured into ice water, then satd NaHCO3 aq was added.
The organic layer was washed with water and brine, dried over
anhydrous magnesium sulfate, and concentrated. The residue was
purified by silica gel column chromatography (eluent: n-hexane/ethyl
acetate = 1:1, v/v, triethylamine 0.1%, v/v) to afford 138 mg (75%) of
1
the title compound as a yellow−white solid. H NMR (500 MHz,
CDCl3) δ 7.92 (d, J = 7.9 Hz, 1H), 7.42 (dd, J = 7.9, 7.9 Hz, 1H), 7.35
(dd, J = 7.9, 7.9 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.03 (d, J = 7.9 Hz,
1H), 6.70−6.62 (m, 2H), 5.43 (s, 1H), 5.41 (s, 1H), 3.52 (s, 3H).
HRMS (FAB) calcd for C16H15N2O 251.1184; found 251.1196 (M +
H)+. Anal. Calcd for C16H14N2O·1/4H2O: C, 75.42; H, 5.74; N, 10.99.
Found: C, 75.20; H, 5.61; N, 10.63.
5,11-Dihydro-2-hydroxymethyl-5-methyl-11-methylene-6H-
dibenz[b,e]azepin-6-one (50). To a solution of 49 (35.7 mg, 0.116
mmol) in 1.5 mL of dehydrated THF was added LiBH4 (13.0 mg,
0.597 mmol) at 0 °C. The mixture was stirred for 10.5 h at room
temperature and extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous magnesium sulfate,
and concentrated. The residue was purified by silica gel column
chromatography (eluent: n-hexane/ethyl acetate = 1:1, v/v) to afford
1
8.0 mg (26%) of the title compound as a colorless oil. H NMR (500
MHz, CDCl3) δ 7.93 (dd, J = 7.9, 1.2 Hz, 1H), 7.43 (ddd, J = 7.9, 7.3,
1.2 Hz, 1H), 7.35 (ddd, J = 7.9, 7.3, 1.2 Hz, 1H), 7.31−7.27 (m, 2H),
7.24 (dd, J = 7.3, 1.2 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 5.49 (s, 1H),
5.45 (s, 1H), 4.67 (s, 2H), 3.56 (s, 3H). HRMS (FAB) calcd for
C17H16NO2 266.1181; found 266.1173 (M + H)+.
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5,11-Dihydro-5-methyl-11-methylene-6-oxodibenz[b,e]azepine-
2-carboxylic Acid (51). To a solution of 49 (33.8 mg, 0.110 mmol) in
2 mL of ethanol was added 1 mL of 1 N aqueous NaOH. The mixture
was stirred for 2 h at room temperature, then extracted with diethyl
ether. The extract was washed with 2 N HCl, water, and brine, dried
over anhydrous magnesium sulfate, and concentrated to afford 30.7 mg
incubated for 24 h. Cells were stimulated with or without test sample 6
h prior to treatment with or without LPS (100 ng/mL for mRNA
measurement and 10 ng/mL for ELISA). After 18 h, cells were
harvested or the concentration of mIL-6 in the culture supernatant was
determined with an ELISA kit according to the manufacturer’s
protocol (R&D Systems). HuH-7 cells were plated at a density of 2 ×
105 cells/well (6-well plate) 24 h prior to treatment with test
compounds. Then, cells were treated with test compounds or with
ethanol for 24 h.
Real-Time Quantitative RT-PCR Analysis. Total RNAs from
samples were prepared by the acid guanidine thiocyanate-phenol/
chloroform method. cDNAs were synthesized using the ImProm-II
reverse transcription system (Promega, Madison, WI). Real-time PCR
was performed on the ABI PRISM 7000 sequence detection system
(Applied Biosystems, Foster City, CA) using Power SYBR Green PCR
Master Mix (Applied Biosystems). Some primer sequences are listed in
Supporting Information. The mRNA values were normalized to the
expression level of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA.
Expression and Purification of GST Fusion Proteins. Glutathione
S-transferase (GST) fusion proteins were expressed in transformed
Rosetta (DE3) competent cells derived from Escherichia coli BL21
grown in LB by induction with 0.4 mM IPTG (isopropyl-β-D-
thiogalactopyranoside) and 2% ethanol at 15 °C for 24 h. Cell
suspensions were centrifuged at 8000 rpm for 5 min. Bacterial pellets
were resuspended in 50 mM Tris-HCl buffer containing 500 mM
NaCl, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl
fluoride (PMSF). Cell suspensions were sonicated and incubated for
20 min after adding 1% Triton-X 100 (v/v) and centrifuged at 12000g
for 30 min. Supernatants were purified by GSTrap FF according to the
manufacturer’s protocol (GE Healthcare). To ensure that equal
amounts of proteins were used for fluorescence polarization assay,
eluted proteins were quantified by Coomassie Blue staining (96-well
scale) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) with SYPRO Orange staining.
LXRα Binding Assay (Fluorescence Polarization Assay). Recombi-
nant GST-hLXRα (full-length) and GST proteins were produced and
purified as described above. Serial dilutions of GST-hLXRα (full-
length) or GST were incubated in the presence of compound 23 (1
μM) at 0 °C for 1 h in 50 mM Tris-HCl buffer (50 mM Tris-HCl, 10
mM reduced glutathione, pH 8.0). After incubation, the fluorescence
polarization of compound 23 was measured on fluorescence
spectrometer (Jasco FP-6500) with excitation at 400 nm and emission
detection at 500 nm. In competitive displacement studies, compounds
3, 18, or 22 was incubated with 1 μM GST-hLXRα (full-length) or
GST for 1 h in the presence of compound 23.
1
(quant) of the title compound as a brown solid. H NMR (500 MHz,
CDCl3) δ 8.04 (dd, J = 7.9, 1.8 Hz, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.96
(dd, J = 7.9, 1.2 Hz, 1H), 7.48 (ddd, J = 7.9, 7.9, 1.2 Hz, 1H), 7.39
(ddd, J = 7.9, 7.9, 1.2 Hz, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.29 (dd, J =
7.9, 1.2 Hz, 1H), 5.57 (s, 1H), 5.52 (s, 1H), 3.62 (s, 3H). HRMS
(FAB) calcd for C17H14NO3 280.0974; found 280.0951 (M + H)+.
Anal. Calcd for C17H13NO3·1/2H2O: C, 70.82; H, 4.89; N, 4.86.
Found: C, 70.54; H, 4.91; N, 4.51.
Biology. Cell Culture Conditions. Human acute monocytic
leukemia THP-1 cells were cultured in RPMI 1640 containing 10%
fetal bovine serum (FBS), penicillin, and streptomycin mixture at 37
°C in a humidified atmosphere of 5% CO2 in air. Human embryonic
kidney (HEK) 293 cells were cultured in D-MEM containing 5% FBS,
penicillin, and streptomycin mixture at 37 °C in a humidified
atmosphere of 5% CO2 in air. Human hepatocellular liver carcinoma
HuH-7 cells were cultured in D-MEM containing 5% FBS, penicillin,
and streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in
air.
Transient Transfection Assays. HEK293 cells were plated at a
density corresponding to 20% confluence in a 96-well plate 24 h prior
to transfection. Cells were cotransfected with 15 ng of CMX-Gal4N-
hLXRα, CMX-Gal4N-hLXRβ, CMX-VP16N-hLXRα, CMX-Gal4N-
SRC1, CMX-Gal4N-DRIP205, CMX-Gal4N-NCoR, or CMX-Gal4N-
SMRT expression plasmid, 50 ng of a tk-MH100 × 4-luc reporter, and
10 ng of CMX-β-galactosidase expression vector. Transfections were
performed by the calcium phosphate coprecipitation method. After 8
h, transfected cells were treated with test compound or dimethyl
sulfoxide (DMSO) for 16 h. Treated cells were assayed for luciferase
activity in a luminometer. The luciferase activity of each sample was
normalized by the level of β-galactosidase activity. Each transfection
was carried out in triplicate.
Measurement of hIL-6. THP-1 cells were plated (2 × 106 cells per
10 cm dish) and stimulated with 10 nM TPA for 48 h. The
differentiated cells were plated at a density of 3 × 104 cells/well (96-
well plate) and stimulated with or without samples 6 h prior to
treatment with or without LPS (100 ng/mL). After 18 h, the
concentration of IL-6 in the culture supernatant was determined with
an ELISA kit according to the manufacturer’s protocol (GE
Healthcare).
Mice and Isolation of Murine Peritoneal Macrophages. C57BL/
6J mice were obtained from Nihon CLEA, LXR-null mice were kindly
provided by Dr. Mangelsdorf, University of Texas Southwestern
Medical Center at Dallas, TX,31 and these mice were maintained under
controlled temperature (23 1 °C) and humidity (45−65%) with free
access to water and chow (Lab. Animal Diet MF; Oriental Yeast Co.,
Ltd., Tokyo, Japan). Eight-to-10-week-old Nr1h3(LXRα)−/− and
Nr1h3(LXRα)−/−Nr1h2(LXRβ)−/− mice on a C57BL/6J background
were maintained on standard chow and injected intraperitoneally with
1 mL/20 g weight of 3% thioglycollate solution (thioglycollate
medium; GIBCO) 4 days prior to harvesting of the macrophages.
Briefly, the mice were sacrificed, and ice-cold phosphate-buffered
saline (PBS) was injected into the peritoneal cavity of each mouse.
This fluid was carefully withdrawn and centrifuged, and the cell pellet
was resuspended in RPMI 1640 containing 10% fetal bovine serum
(FBS), penicillin, and streptomycin mixture. The cells were pooled
and plated at 2.0 million cells/mL, and the macrophages were allowed
to adhere for 2−6 h. The medium was then replaced, and the cells
were incubated for 24 h. The experimental protocol adhered to the
Guidelines for Animal Experiments of the Nihon University School of
Medicine and was approved by the Ethics Review Committee for
Animal Experimentation of Nihon University School of Medicine.
Compound Treatment. Murine peritoneal macrophages were
plated at a density of 4 × 106 cells/well (6-well plate) for mRNA
measurement and 2 × 105 cells/well (96-well plate) for ELISA and
ADME Study. Caco-2 A-B permeability (pH6.5/7.4) analysis was
carried out at Cerep (www.cerep.com). Test compounds were applied
at standard concentrations (10 μM). Data are expressed in Papp
(apparent permeability) in 10−6 cm/s. Colchicine (mean A−B = 0.1
× 10−6 cm/s) and ranitidine (mean A−B = 0.4 × 10−6 cm/s) were
used as low permeability controls, propranolol (mean A−B = 35.7 ×
10−6 cm/s) was used as a high permeability control, and labetalol
(mean A−B = 3.9 × 10−6 cm/s) was used as a P-gp substrate control.
Metabolic stability assay with human liver microsomes was also carried
out at Cerep. Test compounds were applied at standard concen-
trations (0.1 μM). LC/MS/MS was utilized to quantitate remaining
test compounds. Imipramine (mean compound remaining = 92.2%),
propranolol (mean compound remaining = 88.6%), terfenadine (mean
compound remaining = 3.7%), and verapamil (mean compound
remaining = 36.7%) were used as positive controls.
ASSOCIATED CONTENT
* Supporting Information
■
S
The dose-dependency of compounds, the inhibition of LPS-
induced IL-1β production by compounds 3, 10, and 18,
selectivity of 18 over other NRs, primer sequences, and purity
of the synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Journal of Medicinal Chemistry
Article
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81 3 5841 7847. Fax: +81 3 5841 8495. E-mail:
hashimot@iam.u-tokyo.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work described in this paper was partially supported by
Grants-in Aid for Scientific Research from The Ministry of
Education, Culture, Sports, Science and Technology, Japan, and
the Japan Society for the Promotion of Science. We are grateful
to Dr. Shigeyuki Uno and Dr. Michiyasu Ishizawa for their help
with mouse experiments and Dr. David J. Mangelsdorf of the
Howard Hughes Medical Institute and University of Texas
Southwestern Medical Center at Dallas for providing LXR-null
mice. A.A. is a research fellow of the Japan Society for the
Promotion of Science (JSPS).
ABBREVIATIONS USED
■
LXR, liver X receptor; NR, nuclear receptor; LBD, ligand
binding domain; LXRE, LXR response element; NCoR, nuclear
receptor corepressor; SMRT, silencing mediator for retinoid
and tyroid receptors; SRC1, steroid receptor coactivator 1;
DRIP, vitamin D receptor interacting protein; IL-6, interleukin-
6; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase;
LPS, lipopolysaccaride; RA, rheumatoid arthritis; SLE, systemic
lupus erythematosus; PPARγ, peroxisome proliferator-activated
receptor γ; TG, triglyceride; M2H, mammalian two-hybrid; FP,
fluorescence polarization
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