Development of Dihydrodibenzooxepine Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Ligands of a Novel Binding Mode as Anticancer Agents: Effective Mimicry of Chiral Structures by Olefinic E/Z-Isomers

Article abstract of DOI:10.1021/acs.jmedchem.8b01200

A novel class of PPARγ ligand 1 (EC₅₀ = 197 nM) with a dibenzoazepin scaffold was identified through high-throughput screening campaign. To avoid the synthetically troublesome chiral center of 1, its conformational analysis using the MacroModel was conducted, focusing on conformational flip of the tricyclic ring and the conformational restriction by the methyl group at the chiral center. On the basis of this analysis, scaffold hopping of dibenzoazepine into dibenzo[b,e]oxepine by replacing the chiral structures with the corresponding olefinic E/Z isomers was performed. Consequently, dibenzo[b,e]oxepine scaffold 9 was developed showing extremely potent PPARγ reporter activity (EC₅₀ = 2.4 nM, efficacy = 9.5%) as well as differentiation-inducing activity against a gastric cancer cell line MKN-45 that was more potent than any other well-known PPARγ agonists in vitro (94% at 30 nM). The X-ray crystal structure analysis of 9 complexed with PPARγ showed that it had a unique binding mode to PPARγ ligand-binding domain that differed from that of any other PPARγ agonists identified thus far.

Full text of DOI:10.1021/acs.jmedchem.8b01200

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Article
Development of Dihydrodibenzooxepine Peroxisome Proliferator-Activated
Receptor (PPAR) gamma Ligands of a Novel Binding Mode as Anticancer
Agents: Effective Mimicry of Chiral Structures by Olefinic E/Z-Isomers
Keisuke Yamamoto, Tomohiro Tamura, Kazuki Henmi, Takeshi Kuboyama, Arata Yanagisawa, Masahiro
Matsubara, Yuichi Takahashi, Michihiko Suzuki, Jun-ichi Saito, Kimihisa Ueno, and Satoshi Shuto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01200 • Publication Date (Web): 23 Oct 2018
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Journal of Medicinal Chemistry
Development of Dihydrodibenzooxepine Peroxisome
Proliferator-Activated Receptor (PPAR) gamma Ligands
of a Novel Binding Mode as Anticancer Agents:
Effective Mimicry of Chiral Structures by Olefinic E/Z-
Isomers
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Keisuke Yamamoto*,†,‡, Tomohiro Tamura†, Kazuki Henmi†, Takeshi Kuboyama†, Arata Yanagisawa†, Masahiro
Matsubara†, Yuichi Takahashi†, Michihiko Suzuki†, Jun-ichi Saito†, Kimihisa Ueno† and Satoshi Shuto*,‡,§
†Fuji Research Park, RD Division, Kyowa Hakko Kirin, 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shiuoka,
Japan
‡Faculty of Pharmaceutical Sciences and §Center for Research and Education on Drug Discovery, Hokkaido
University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
ABSTRACT : A novel class of PPARγ ligand 1 (EC50 = 197 nM) with a dibenzoazepin scaffold was identified
through high-throughput screening campaign. To avoid the synthetically troublesome chiral center of 1, its
conformational analysis using the MacroModel was conducted, focusing on conformational flip of the tricyclic
ring and the conformational restriction by the methyl group at the chiral center. Based on this analysis, scaffold
hopping of dibenzoazepine into dibenzo[b,e]oxepine by replacing the chiral structures with the corresponding
olefinic E/Z isomers was performed. Consequently, dibenzo[b,e]oxepine scaffold 9 was developed showing
extremely potent PPARγ reporter activity (EC50 = 2.4 nM, efficacy = 9.5%) as well as differentiation-inducing
activity against a gastric cancer cell line MKN-45 that was more potent than any other well-known
PPARγ agonists in vitro (94% at 30 nM). The X-ray crystal structure analysis of 9 complexed with
PPARγ showed that it had a unique binding mode to PPARγ ligand-binding domain that differed from that of
any other PPARγ agonists identified thus far.
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INTRODUCTION
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Peroxisome proliferator-activated receptor gamma (PPARγ), a member of the nuclear receptor of the
superfamily of ligand-inducible transcription factors, is known as a master regulator of adipose cells.1,2 PPARγ
is predominantly expressed in adipose tissue, while it is lower expressed in liver, muscle, and other tissue.3
Owing to its outstanding insulin resistance-restoring activity, the thiozolidinedione (TZD) class of PPARγ full
agonists, e.g., pioglitazone and rosiglitazone, have been used as antidiabetic drugs for the treatment of type 2
diabetes mellitus.4,5,6
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There are three protein members of the PPAR family, PPARα PPARγ and PPARβ/δ each of which
regulates different genes.7 Many selective PPARγ agonists have been reported, and the structures of well-
known PPARγ full agonists and ligands are shown in Figure 1.8,9,10,11,12,13 PPARγ forms a heterodimer with
retinoid X receptor (RXR) and the dimer binds to PPAR response element (PPRE).14 Consequently regulating
the expression of adipose-related genes that code adipocyte-related proteins, represented by adipocyte fatty
acid-binding protein 2 (aP2),15 adipose differentiation-related protein (ADFP) and adiponectin.16 In this way,
PPARγ agonists promote the differentiation of adipose tissues,17 and there are many reports demonstrating that
PPARγ agonists strongly promote the differentiation of fibroblast-like cells such as 3T3-L1 cells to
adipocytes.18,19,20,21
Given the correlation between PPARγ activation and the differentiation of pre-adipocytes, PPARγ activation
potentially induces the differentiation of cancer cells. Indeed, efatutazone (CS-7017), a potent PPARγ full
agonist, showed differentiation-inducing activity in anaplastic thyroid carcinoma,6,22 non-small cell lung
cancer,23 and pancreatic cancer6 under low concentrations in vitro, while such differentiation effects have not
been reported for any other PPARγ agonists. In addition, a recent phase 1 clinical study of efatutazone
demonstrated that its treatment prolonged the overall survival of anaplastic carcinoma patients. However,
several patients have reported adverse effects, such as localized edema, likely due to the chemical structure of
the TZD moiety.22,24
Based on the abovementioned results, we suspected that it might be feasible to develop the novel therapeutic
drugs for treating undifferentiated carcinoma with reduced side effects compared with efatutazone by creating
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a novel PPARγ agonist with a scaffold other than TZD. We herein report our findings regarding a new class of
PPARγ ligands of dihydrodibenzo[b,e]oxepine scaffold with very potent in vitro differentiation-inducing
activity and a unique binding mode to the PPARγ ligand-binding domain (LBD), different from those of any
known PPARγ agonists.
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RESULT
Identification of Dihydrodibenzocycloheptene Derivative 5. PPARγ activation by efatutazone as
observed in PPRE3-tk-Luc transfected DRO reporter gene assay is reportedly well correlated with the
differentiation activity of cancer cell lines; for example, reporter gene assay found that the EC50 was
approximately 1 nM, compared with 63% inhibition of cologenic growth at 1 nM.9 We explored
PPARγ agonists with a novel core structure for cancer therapy through a high-throughput screening campaign
with PPARγ reporter gene assay using our chemical library. We ultimately identified dibenzoazepine
derivative 1 as a hit, showing 24% efficacy of reporter gene expression at 1 μM compared with that of
pioglitazone at 1 μM (100%); furthermore, this molecule had a scaffold that differed from any PPARγ agonists
reported to date (Figure 2). The hit compound 1 was one of R/S enantiomers,25 and one of the enantiomers was
the eutomer. We believed that efficient lead optimization using a chiral compound as a lead might be
troublesome, as the enantioselective construction of the asymmetric center adjacent to the nitrogen atom on the
azepine ring was likely to require great effort. Therefore, to discover an alternative scaffold that was easy to
prepare while mimicking the active form of 1, a conformation analysis of each enantiomer of 1 was conducted.
The interaction between Tyr473 on PPARγ LBD and an acidic moiety of PPARγ full agonist is known to be
essential for agonistic activity due to the stabilization of Helix12 that composes a section of the transcriptional
coactivator binding region of the LBD.26,27 This suggests that the acidic tetrazole in 1 may also be essential for
the such activity. Then, we searched the dominant conformations focusing on the relative arrangement between
the tetrazole group and the dibenzoazepine/dihydrodibenzocycloheptene moiety, using the molecular modeling
tool MacroModel.
The conformations of 6/7/6-tricyclic ring systems like dibenzoazepine, which are often included in
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pharmacologically active compounds, have been well studied (Figure 3).28,29,30 These studies suggested that, in
this kind of 6/7/6-tricyclic ring system, significant conformational change occurs with the reversal motion of
the central seven-membered ring (conformational flip), as shown in Figure 3.
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Figures 4 shows the structures of the tricyclic compounds (R)-1’ and (S)-1’ as model compounds for the
conformational studies; these compounds had a simplified methyl substituent instead of imidazopyridine side
chain at the 2-position of the dibenzoazepine moiety. The stable conformations of (R)-1’ and its enantiomer
(S)-1’ calculated by MacroModel are shown in Figures 4A and 4B, respectively. The conformations were
mainly changed depending on the flip of the central azepine ring (indicated with up and down based on the
direction of 2-methyl group) and also the orientation of the tetrazole ring to the dibenzoazepine ring (indicated
with syn and anti to the 2-methyl group). Thus, two significantly stable conformations down-syn and up-anti
for (R)-1’ and up-syn and down-anti for (S)-1’ were obtained,31 where the relationships between down-syn of
(R)-1’ and up-syn of (S)-1’ and between up-anti of (R)-1’and down-anti of (S)-1’ were enantiomeric of each
other. These results show that the configurational difference, i.e. R or S, of the methyl-substituted asymmetric
carbon center adjacent to the central 7-membered ring restricts the orientation of the tetrazole ring, probably
due to the steric repulsion for the tricyclic ring structure, and accordingly one of the four conformationally
restricted structures, down-syn and up-anti for (R)-1’ and up-syn and down-anti for (S)-1’, might be a bioactive
form binding to the LBD of PPARγ. This sort of effective conformational restriction depending on the
configuration (R or S) of an asymmetric center due to the steric effect of a methyl or ethyl substituents attached
to the asymmetric center has previously been reported by our laboratory.32,33,34,35,36,37,38,39,40
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Given the results of the conformation analysis, we suspected that the syn/anti-orientation of the tetrazole
moiety might be mimicked by the corresponding E/Z-olefinic structures without the asymmetric center. Stable
conformations of designed olefinic model compounds 2’ and 3’ were calculated using MacroModel. As shown
in Figures 4C and 4D, two stable conformations down-Z and up-Z for 2’ and up-E and down-E for 3’ were
obtained depending on the flip of the dihydrocycloheptene ring, which likely mimicked the four structures:
down-syn and up-anti for (R)-1’ and up-syn and down-anti for (S)-1’, as we expected. These results showed
that scaffold hopping of the asymmetric carbon substituent dibenzoazepine into dihydrodibenzocycloheptene
might be feasible.
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Figure 5 shows the superimposition of the most stable conformations of (R)-1’ and (S)-1’, E/Z isomers 2’
and 3’, respectively, in which the tricyclic ring and the tetrazole in four pairs (down-syn of (R)-1’ vs. down-Z
of 2’ (5A), up-anti of (R)-1’ vs. up-E of 3’ (5B), up-syn of (S)-1’ vs. up-Z of 2’ (5C), down-anti of (S)-1’ vs.
dowm-E of 3’ (5D)) were overlaid very closely. These results suggest that one of the stable conformer of
dihydrodibenzocycloheptene derivatives 2 or 3 with the imidazopyridine side chain effectively mimic the
bioactive conformer of the eutomer of 1. We then synthesized E/Z isomers 2 and 3, and another E/Z isomers 4
and 5, which had an oxadiazolone instead of a tetrazole as an acidic moiety.
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A reporter gene assay of the dihydrodibenzocycloheptene E/Z-isomers 2-5 at 1 μM was carried out, and the
efficacies of the reporter gene expression relative to that of pioglitazone at 1 μM and EC50 values are
summarized in Table 1. The E-isomers 2 and 4 were almost inactive; however, not only the efficacies of Z-
isomers 3 and 5 were more than 20% at 1 μM, which was comparable to that of representative PPARγ ligands
like INT-131 and metaglidasen (Table 3), but also their potencies were almost equal to that of hit compound 1
(197 nM vs. 251, 177 nM). This result suggested that the three-dimensional structure of the
dihydrodibenzocycloheptene Z-isomers was likely to be analogous to that of the eutomer of 1.
PPARγ ligands, such as INT-131 and metaglidasen, often show low efficacy (<20% of control) in HEK293
reporter gene assays; however, potent antidiabetic activities of these PPARγ ligands have been reported in
preclinical and clinical studies.10,41,42,43 In a preclinical study, the glucose level was significantly reduced by
approximately 60% in INT-131 treated db/db diabetic model mice compared with vehicle mice, roughly equal
to the effects of pioglitazone. Although the in vitro efficacies of 3 and 5 were analogous, we selected 544 as the
lead for further study due to its lower hydrophobicity than 3 (ClogP of 3 and 5:5.76 vs. 6.03, calculated by
ChemBioDraw Ultra 14.0).
Optimization of Lead Compound 5. To explore compounds having more potent differentiation activity
than lead compound 5, we performed the structural modification of 5. The EC50 value in a PPARγ reporter
gene assay was employed as an indicator of the activity, as the evaluation system was convenient and have
been used effectively in previous developments of PPARγ ligands showing an excellent in vitro and in vivo
antidiabetic activity.45 Lead compound 5 had rather high lipophilicity (CLogP 6.03) and needed multistep
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reactions for its synthesis due to the dihydrodibenzocycloheptene scaffold. To solve these issues, we designed
and synthesized compound 6 (CLogP 5.00), in which the dihydrodibenzocycloheptene scaffold of 5 was
replaced by an analogue tricyclic dibenzo[b,e]oxepine scaffold.
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As a result, compound 6 with its novel dibenzo[b,e]oxepine scaffold had a good EC50 value of 84 nM,
which was 2-fold more potent than the lead compound 5 and thereby encouraged us to prepare additional
dibenzo[b,e]oxepine analogues. We further designed and synthesized benzoimidazole analogue 7, removing
the nitrogen atom on the imidazo[4,5-b]pyridine ring. Compound 7 showed promising activity (EC50 = 17 nM),
that was approximately 10 times more potent than lead compound 5.
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Regarding modifications at the 2-position on the benzoimidazole moiety, we succeeded to find 2-propyl
analogue 8, which possessed the best in vitro activity in this chemical series, with an EC50 of 2.7 nM.
Compound 9, a 4-methyl analogue of 8, was further synthesized and showed extremely potent in vitro activity
with an EC50 of 2.4 nM comparable to that of 8. This result suggested that modification at the 4-position of
benzoimidazole was tolerated. To reduce the lipophilicity, analogues 10-13, which had polar substituents at the
4-position, such as hydroxy and carbamoyl groups, were synthesized. Unfortunately, these compounds had
lower efficacy and/or potency than 9. Therefore, polar substituents at the 4-position were deemed unlikely to
be tolerated. In addition, the imidazole analogues 14 and 15, in which the benzoimidazole ring was replaced by
a 4-aryllimidazole ring, also showed a significantly decreased activity compared with 9.
Differentiation Effects of Dibenzo[b,e]oxepine Analogue 9 in Cancer Cells. We estimated the
differentiation activity of dibenzo[b,e]oxepine derivative 9 as well as well-known PPARγ agonists using
MKN-45 cells, a poorly differentiated gastric cancer cell line.46 The differentiation activity was denoted by the
aggregation effect, which was quantified by analyzing the morphological change using an IN Cell Analyzer.47
As expected, 9 effectively induced the differentiation of MKN-45 cells at significantly lower concentrations
(94% at 30 nM) than the other PPARγ full agonist and ligands, pioglitazone, INT-131, metaglidasen and FK-
614, which showed EC50 values of >500 nM (Figure 6).
We next examined the effects of 9 on the gene expression (Figure 7). Quantitative polymerase chain reaction
(qPCR) results of 9-treated MKN-45 cells showed that it promoted the expressions of angiopoietin-like 4
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(angptl4),48 a PPARγ-regulated downstream gene and adipose differentiation-related protein (adfp)49 at 100
nM, which was the concentration at which the effective induction of the MKN-45 cell aggregation by 9 was
observed. In contrast, the expression of vimentin, a mesenchymal cell marker,50 was effectively impaired at the
same 100 nM concentration. Since the expression of these differentiation makers was changed at the same
concentration range (10-100 nM), these data suggested that the differentiation activity of 9 was likely
associated with transactivation activity on PPARγ.
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As mentioned above, the differentiation-inducing activity of cancer cells was indicated by the aggregation
activity of MKN-45 cells. We therefore plotted the EC50 values of compounds in a reporter gene assay with
HEK293 versus those in the aggregation assay with MKN-45. As shown in Figure 8, the correlation between
the EC50 values in the two different assay systems was extremely high (correlation coefficient r2 = 0.924).
These data suggest that 9 promotes the differentiation of MKN-45 cells due to its PPARγ agonistic activity.
Novel Binding Mode of 9 to PPARγ. A number of different crystal structures of the PPARγ LBD
complexed with its ligands in various binding modes have been reported. The characteristic biological activity,
particularly the remarkable differentiation against cancer cells, probably due to the unique scaffold of 9 as a
PPARγ ligand prompted us to investigate its binding mode to PPARγ LBD. We successfully obtained the X-
ray crystallographic structure of 9 binding to the PPARγ LBD (Figure 9A). The acidic proton of the
oxadiazolone of 9 formed a hydrogen bond with the phenolic oxygen of Tyr473 on Helix12 (H12), which
plays an important role in its agonistic activity.51 The benzoimidazole side chain of 9 filled the region between
Helix3 (H3) and Helix 5 (H5), known as the canonical ligand-binding pocket, with which interactions of a
number of PPARγ ligands have been reported.52 In contrast to the binding mode of rosiglitazone (Figure 9B),
in the binding mode of 9, Phe282 on H3, was moved toward H5 to form a small hydrophobic pocket where the
rigid tricyclic structure was accommodated. In addition, one benzene ring of dibenzo[b,e]oxepine moiety
of 9 occupied the hydrophobic region surrounded by H3 and helix 11 (H11), which was absent when
complexed with rosiglitazone (Figure 9B). Furthermore, an edge-to-face interaction between the benzene ring
of the benzoimidazole side chain and Phe282 was also observed.
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Next, the binding mode of 9 was compared with those of other PPARγ ligands, including the recently
reported MRL20 (Figure 1). Figure 9C shows the superimposed binding structures of PPARγ full
agonist; rosiglitazone and PPARγ ligands; INT131, metaglidasen, MRL20,52 and 9. Although the acidic groups
of rosiglitazone, MRL20 and 9 were closely overlaid and the benzoimidazole side chain of 9 filled the
canonical ligand binding pocket like other ligands, the tricyclic dibenzo[b,e]oxepine moiety was
accommodated in a hydrophobic pocket surrounded by H3 and H11, in contrast to all other ligands.
Finally, we confirmed the bioactive form. Among the four stable calculated conformations of (R)-1 and (S)-
1 and the two stable conformations of 3, down-syn of (R)-1 and down-Z of 3 were well superimposable into the
structure of 9 in the X-ray-analyzed binding mode (Figure 10). The acidic groups and the core structures of all
compounds showed excellent overlap. These results demonstrated that the compound design based on a
comparison of the global minimum energy conformations between each enantiomer of 1c and 2c, 3c was
effective. Given this good conformation overlap of 1 and 9, we concluded that (R)-1 is the eutomer and down-
syn of (R)-1 (aquamarine) and down-Z of 3 (bluepurple) are the bioactive form. The binding mode of 9 differed
from those of any other PPARγ agonists reported to date. This characteristic binding mode of 9 might lead to a
unique activity to PPARγ related to the differentiation effects.
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Synthetic Chemistry. Compounds 2-11, 14 and 15 were prepared from E/Z congeners 2-(2-
(hydroxymethyl)-10,11-dihydro-5H-dibenzocycloheptene-5-ylidene)propanenitrile 16Z and 16E, and (E)-2-(8-
(hydroxymethyl)dibenzo[b,e]oxepin-11(6H)-ylidene)propanenitrile 17E (supporting information) respectively
as summarized in Scheme 1. Mitsunobu reaction of 16E and 16Z with an imidazo[4,5-b]pyridine ring provided
the corresponding coupling products 20E, 20Z and 21E respectively. Bromination or chlorination reaction of
17E furnished halides 18 and 19, respectively. The halides were reacted with azole rings to afford
dibenzo[b,e]oxepine analogues 22E-29E.
The cyano groups of 20E and 20Z were cyclized by treatment with trimethylsilyl azide and then dibutyltin
oxide gave tetrazoles 2 and 3, respectively. Furthermore, the addition of hydroxyamine to the cyano group of
22E-24E and 26E-29E, and subsequent acylation using ethyl chlorofomate, followed by treatment with tert-
BuOK afforded the target oxadiazolone ring-closure compounds 4-11, 14, and 15.
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Conversion of the substituents at the 4-position of bennzo[d]imidazole ring of 25E, was conducted as shown
in Scheme 2. Hydrolysis of ester of 25E, followed by condensation with 2-aminoethanol gave 31. Curtius
rearrangement of 4-carboxylic acid in 30 and removal of the Boc group provided 4-sulfonamide 32. Finally,
the cyclization step was conducted as described above to give the target compounds 12 and 13.
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DISCUSSION and CONCLUSION
The conformation studies suggested that the syn/anti-orientation of the tetrazole moiety in the racemic
dibenzoazepine of hit 1 would be mimicked by the corresponding E/Z-olefinic structures that did not contain
an asymmetric center. Based on these findings, we designed a novel dihydrodibenzocycloheptene scaffold, and
subsequent examination successfully led to the discovery of lead compound 5, which possessed
PPARγ reporter activity comparable to that of hit compound 1. As a result of our lead optimization,
dibenzo[b,e]oxepine derivative 9 with potent reporter and differentiation-inducing activities was obtained. The
X-ray crystal structure of 9 complexed with PPARγ showed its unique binding mode to PPARγ LBD, which
differed from those of any other PPARγ agonists identified to date. Interestingly, the calculated stable
conformations of (R)-1 (down-syn) overlapped well with the X-ray-analyzed LBD-binding structure
of 9, suggesting that (R)-1 was the eutomer in which its bioactive form was the down-syn conformation.
In conclusion, we achieved scaffold hopping of hit compound 1 through a conformation study and
successfully discovered a novel class of PPARγ ligand with potent cancer cell differentiation-inducing activity.
EXPERIMENTAL SECTION
General Methods. All reagents and solvents were procured from commercial sources and used as received.
Thin layer chromatography (TLC) was carried out using Merck GmbH Precoated silica gel 60 F254.
Chromatography on silica gel was carried out using prepacked silica gel cartridges (Yamazen Hi-Flash
Column Silicagel or Wako Presep® Silicagel). Chemical shifts in 1H NMR spectra were reported in δ values
(ppm) relative to trimethylsilane. HPLC analyses were performed following conditions: Waters Xbrige® C18
column (3.5 μm, 4.6 mm × 50 mm), 30 °C column temperature, 1.0 mL/min flow rate, photodiode array
detection (254 nm), and linear mobile phase gradient of 20%−90% B over 5 min, holding for 3.5 min at 20% B
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(mobile phase A, 0.05% trifluoroacetic acid in water; mobile phase B, acetonitrile), by which the purities of
final compounds were confirmed as >95%. Mass spectra were recorded on a Waters 2695 (ESI-MS).
(E)-3-((5-(1-(1H-tetrazol-5-yl)ethylidene)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-2-yl)methyl)-2-
ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (2). To a solution of 20E (193 mg, 0.447 mmol) and
dibutyltin oxide (44 mg, 0.18 mmol) in toluene (5.5 mL) was added trimethylsilylazide (0.474 mL, 3.57 mmol)
and the solution was stirred at 80 °C for 48 h. To the reaction mixture was added methanol and the solution
was concentrated to dryness. The obtained residue was then purified by flash column chromatography on silica
gel (100:0 to 96:4 CHCl3/methanol) to afford 2 (190 mg, 90%) as an amorphous. 1H NMR (300 MHz, CDCl3):
δ 1.28 (t, J = 7.5 Hz, 3H), 2.31 (s, 3H), 2.53 (s, 3H), 2.58 (s, 3H), 2.66–2.78 (m, 4H), 3.09–3.25 (m, 2H), 5.41
(s, 2H), 6.88–6.91 (m, 2H), 6.99–7.02 (m, 2H), 7.13–7.32 (m, 4H). The proton of tetrazole was not observed.
LC/MS (ESI, [M + H]+, m/z) 476. HPLC: purity 99%, RT 3.25 min.
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(Z)-3-((5-(1-(1H-tetrazol-5-yl)ethylidene)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-2-yl)methyl)-2-
ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (3). A solution of 20Z (212 mg, 0.49 mmol) in toluene (6.0
mL) at room temperature was treated with dibutyltin oxide (48 mg, 0.19 mmol) and trimethylsilylazide (0.516
mL, 3.88 mmol), and then the mixture was stirred at 80 °C for 48 h. To the reaction mixture was added
methanol and the solution was concentrated to dryness. The obtained residue was then purified by flash
column chromatography on silica gel (100:0 to 96:4 CHCl3/methanol) to afford 3 (171 mg, 74%) as an
amorphous. 1H NMR (300 MHz, CDCl3): δ 1.31 (t, J = 7.6 Hz, 3H), 2.33 (s, 3H), 2.54 (s, 3H), 2.61 (s, 3H),
2.68–2.82 (m, 2H), 2.75 (q, J = 7.6 Hz, 2H), 3.15–3.34 (m, 2H), 5.38 (s, 2H), 6.83–6.94 (m, 4H), 7.14–7.24
(m, 4H). The proton of tetrazole was not observed. LC/MS (ESI, [M + H]+, m/z) 476. HPLC: purity 99%, RT
3.57 min.
(E)-3-(1-(2-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)-10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5-ylidene)ethyl)-1,2,4-oxadiazol-5(4H)-one (4). A solution of 20E (192 mg, 0.44
mmol) in ethanol (4.4 mL) was treated with NH2OH solution 50wt.% in water (0.54 mL, 8.9 mmol), and the
resulting mixture was refluxed for 3 days. After the consumption of the starting material, the reaction mixture
was poured into water and extracted twice with ethyl acetate. The combined organic layers were washed with
brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to dryness and the obtained
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residue was dissolved in DMF (4.4 mL). To a stirred solution was added pyridine (44 μL, 0.53 mmol) and
ethyl chloroformate (52 μL, 0.53 mmol) and the solution was stirred for 1h at room temperature. The reaction
mixture was poured into saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate.
The combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic
layer was concentrated to give the residue. To a solution of the residue in toluene (4.4 mL) was added tert-
BuOK (100 mg, 0.888 mmol) and the solution was stirred for 30 minutes at room temperature. The reaction
mixture was poured into 5% aqueous citric acid solution and extracted twice with ethyl acetate. The combined
organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer was
concentrated to give the residue. The obtained residue was then purified by flash column chromatography on
silica gel (100:0 to 98:2 CHCl3/methanol) to give 4 (166 mg, 76%) as an amorphous. 1H NMR (300 MHz,
CDCl3): δ 1.31 (t, J = 7.5 Hz, 3H), 2.09 (s, 3H), 2.57 (s, 3H), 2.62 (s, 3H), 2.72–2.87 (m, 4H), 3.21–3.37 (m,
2H), 5.40 (s, 2H), 6.91–6.94 (m, 3H), 7.06–7.11 (m, 2H), 7.19–7.35 (m, 3H). The proton of oxadiazolone was
not observed. LC/MS (ESI, [M + H]+, m/z) 492. HPLC: purity 97%, RT 3.63 min.
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(Z)-3-(1-(2-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)-10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5-ylidene)ethyl)-1,2,4-oxadiazol-5(4H)-one (5). To a stirred solution of 20Z (197
mg, 0.456 mmol) in ethanol (4.5 mL) was added NH2OH solution 50wt.% in water (0.56 mL, 9.24 mmol), and
the solution was refluxed for 3 days. After the consumption of the starting material, the reaction mixture was
poured into water and extracted twice with ethyl acetate. The combined organic layers were washed with
brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to dryness and the obtained
residue was dissolved in DMF (4.5 mL). To a stirred solution was added pyridine (0.046 mL, 0.55 mmol) and
ethyl chloroformate (0.054 mL, 0.55 mmol) and the solution was stirred for 1 h at room temperature. The
reaction mixture was poured into saturated aqueous sodium bicarbonate solution and extracted twice with ethyl
acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The
organic layer was concentrated to give the residue. To a solution of the residue in toluene (4.5 mL) was added
tert-BuOK (104 mg, 0.924 mmol) and the solution was stirred for 30 minutes at room temperature. The
reaction mixture was poured into 5% aqueous citric acid solution and extracted twice with ethyl acetate. The
combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer
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was concentrated to give the residue. The obtained residue was then purified by flash column chromatography
on silica gel (100:0 to 98:2 CHCl3/methanol) to give 5 (126 mg, 56%) as an amorphous. 1H NMR (300 MHz,
CDCl3): δ 1.33 (t, J = 7.5 Hz, 3H), 2.11 (s, 3H), 2.55 (s, 3H), 2.63 (s, 3H), 2.75–2.83 (m, 2H), 2.79 (q, J = 7.5
Hz, 2H), 3.24–3.34 (m, 2H), 5.42 (s, 2H), 6.89–6.94 (m, 3H), 7.05–7.24 (m, 5H). The proton of oxadiazolone
was not observed. LC/MS (ESI, [M + H]+, m/z) 492. HPLC: purity 95%, RT 3.85 min.
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(E)-3-(1-(8-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
ylidene)ethyl)-1,2,4-oxadiazol-5(4H)-one (6). To a stirred solution of 21E (145 mg, 0.334 mmol) in ethanol
(1.5 mL) was added NH2OH solution 50wt.% in water (0.41 mL, 6.7 mmol), and the solution was refluxed for
3 days. After the consumption of the starting material, the reaction mixture was poured into water and
extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium
sulfate, and filtered. The organic layer was concentrated to dryness and the obtained residue was dissolved in
DMF (1.0 mL). To a stirred solution was added pyridine (0.053 mL, 0.66 mmol) and ethyl chloroformate
(0.090 mL, 0.7 mmol) and the solution was stirred for 1h at room temperature. The reaction mixture was
poured into saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate. The
combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer
was concentrated to give the residue. To a solution of the residue in toluene (3.0 mL) was added tert-BuOK
(70 mg, 0.66 mmol) and the solution was stirred for 30 minutes at room temperature. The reaction mixture was
poured into 5% aqueous citric acid solution and extracted twice with ethyl acetate. The combined organic
layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to
give the residue. The obtained residue was then purified by flash column chromatography on silica gel (100:0
to 98:2 CHCl3/methanol) to give 6 (60 mg, 36%) as an amorphous. 1H-NMR (300 MHz, CDCl3):δ 1.23 (t, J =
7.5 Hz, 3H), 2.06 (s, 3H), 2.54 (s, 3H), 2.58 (s, 3H), 2.68 (q, J = 7.5 Hz, 2H), 4.51 (d, J = 12.6 Hz, 1H), 5.28
(d, J = 12.6 Hz, 1H), 5.44 (s, 2H), 6.65 (dd, J = 8.3, 1.1 Hz, 1H), 6.80 (td, J = 7.5, 1.1 Hz, 1H), 6.90 (s, 1H),
7.01 (dd, J = 7.8, 1.7 Hz, 1H), 7.06 (s, 1H), 7.11–7.15 (m, 1H), 7.17 (br s, 2H). The proton of oxadiazolone
was not observed. LC/MS (ESI, [M + H]+, m/z) 494. HPLC: purity 99%, RT 3.6 min.
(E)-3-(1-(8-((2-ethyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-ylidene)ethyl)-1,2,4-
oxadiazol-5(4H)-one (7). To a stirred solution of 22E (157 mg, 0.387 mmol) in ethanol (3.0 mL) was added
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NH2OH solution 50wt.% in water (0.712 mL, 11.6 mmol), and the solution was refluxed for 3 days. After the
consumption of the starting material, the reaction mixture was poured into water and extracted twice with ethyl
acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The
organic layer was concentrated to dryness and the obtained residue was dissolved in THF (2.2 mL). To a
stirred solution was added Et3N (81 μL, 0.58 mmol) and ethyl chloroformate (56 μL, 0.58 mmol) and the
solution was stirred for 1h at room temperature. The reaction mixture was poured into saturated aqueous
sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic layers were washed
with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. To a
solution of the residue in toluene (3.9 mL) was added tert-BuOK (65 mg, 0.58 mmol) and the solution was
stirred for 30 minutes at room temperature. The reaction mixture was poured into 5% aqueous citric acid
solution and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried
over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The obtained residue
was then purified by flash column chromatography on silica gel (99:1 to 95:5 CHCl3/methanol) to to give 7
(8.7 mg, 4.8%) as an amorphous. 1H-NMR (300 MHz, CDCl3):δ 1.30 (t, J = 7.4 Hz, 3H), 2.31 (s, 3H), 2.90 (q,
J = 7.4 Hz, 2H), 4.67 (d, J = 12.6 Hz, 1H), 5.29–5.44 (m, 2H), 5.53 (d, J = 12.6 Hz, 1H), 6.77–6.86 (m, 1H),
6.87–7.07 (m, 3H), 7.11–7.37 (m, 6H), 7.70–7.84 (m, 1H). The proton of oxadiazolone was not observed.
LC/MS (ESI, [M + H]+, m/z) 465. HPLC: purity 98%, RT 3.45 min.
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(E)-3-(1-(8-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-ylidene)ethyl)-1,2,4-
oxadiazol-5(4H)-one (8). To a stirred solution of 23E (344 mg, 0.821 mmol) in ethanol (8.2 mL) was added
NH2OH solution 50wt.% in water (0.754 mL, 34.6 mmol), and the solution was refluxed for 3 days. After the
consumption of the starting material, the reaction mixture was poured into water and extracted twice with ethyl
acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The
organic layer was concentrated to dryness and the obtained residue was dissolved in DMF (4.1 mL). To a
stirred solution was added pyridine (80 μL, 0.98 mmol) and ethyl chloroformate (94 μL, 0.98 mmol) and the
solution was stirred for 1h at room temperature. The reaction mixture was poured into saturated aqueous
sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic layers were washed
with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. To a
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solution of the residue in toluene (8.2 mL) was added tert-BuOK (138 mg, 1.23 mmol) and the solution was
stirred for 30 minutes at room temperature. The reaction mixture was poured into 5% aqueous citric acid
solution and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried
over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The obtained residue
was then purified by flash column chromatography on silica gel (100:0 to 99:1 CHCl3/methanol) to give 8 (46
mg, 12%) as an amorphous. 1H-NMR (300 MHz, CDCl3):δ 0.94–1.06 (m, 3H), 1.80–1.96 (m, 2H), 2.28 (s,
3H), 2.74–2.84 (m, 2H), 4.71 (d, J = 12.6 Hz, 1H), 5.35 (s, 2H), 5.48 (d, J = 12.6 Hz, 1H), 6.74–6.86 (m, 1H),
6.86–6.97 (m, 1H), 6.98–7.04 (m, 1H), 7.04–7.32 (m, 7H), 7.64–7.78 (m, 1H). The proton of oxadiazolone was
not observed. LC/MS (ESI, [M + H]+, m/z) 479. HPLC: purity 99%, RT 3.6 min.
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(E)-3-(1-(8-((4-methyl-2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
ylidene)ethyl)-1,2,4-oxadiazol-5(4H)-one (9). To a stirred solution of 24E (449 mg, 1.04 mmol) in ethanol
(10 mL) was added NH2OH solution 50wt.% in water (1.9 mL, 31 mmol), and the solution was refluxed for 3
days. After the consumption of the starting material, the reaction mixture was poured into water and extracted
twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and
filtered. The organic layer was concentrated to dryness and the obtained residue was dissolved in DMF (5.2
mL). To a stirred solution was added pyridine (0.10 mL, 1.2 mmol) and ethyl chloroformate (0.12 mL, 1.2
mmol) the solution was stirred for 1h at room temperature. The reaction mixture was poured into saturated
aqueous sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic layers were
washed with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to give the
residue. To a solution of the residue in toluene (10 mL) was added tert-BuOK (190 mg, 1.55 mmol) and the
solution was stirred for 30 minutes at room temperature. The reaction mixture was poured into 5% aqueous
citric acid solution and extracted twice with ethyl acetate. The combined organic layers were washed with
brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The
obtained residue was then purified by flash column chromatography on silica gel (100:0 to 99:1
CHCl3/methanol) to give 9 (109 mg, 22%) as an amorphous. 1H-NMR (300 MHz, DMSO-d6):δ 0.80–0.93 (m,
3H), 1.59–1.72 (m, 2H), 2.14 (s, 3H), 2.44–2.54 (m, 3H), 2.70–2.83 (m, 2H), 4.74–4.94 (m, 1H), 5.35–5.52
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(m, 3H), 6.74–6.80 (m, 1H), 6.85–7.06 (m, 5H), 7.10–7.32 (m, 4H). The proton of oxadiazolone was not
observed. LC/MS (ESI, [M + H]+, m/z) 493. HPLC: purity 99%, RT 3.7 min.
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(E)-3-(1-(8-((4-(hydroxymethyl)-2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
ylidene)ethyl)-1,2,4-oxadiazol-5(4H)-one (10). To a stirred solution of 26E (90 mg, 0.20 mmol) in ethanol (2
mL) was added NH2OH solution 50wt.% in water (0.368 mL, 6.00 mmol), and the solution was refluxed for 3
days. After the consumption of the starting material, the reaction mixture was poured into water and extracted
twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and
filtered. The organic layer was concentrated to dryness and the obtained residue was dissolved in THF (1 mL).
To a stirred solution was added Et3N (0.042 mL, 0.30 mmol) and ethyl chloroformate (0.029 mL, 0.30 mmol)
the solution was stirred for 1h at room temperature. The reaction mixture was poured into saturated aqueous
sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic layers were washed
with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. To a
solution of the residue in toluene (2 mL) was added tert-BuOK (34 mg, 0.30 mmol) and the solution was
stirred for 30 minutes at room temperature. The reaction mixture was poured into 5% aqueous citric acid
solution and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried
over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The obtained residue
was then purified by flash column chromatography on silica gel (100:0 to 95:5 CHCl3/methanol) to give 10 (10
mg, 10%) as an amorphous. 1H-NMR (300 MHz, DMSO-d6):δ 0.85-1.00 (m, 3H), 1.67–1.84 (m, 2H), 2.27 (s,
3H), 2.93–3.04 (m, 2H), 4.76 (d, J = 12.6 Hz, 1H), 5.06 (s, 2H), 5.41 (s, 2H), 5.56 (d, J = 12.6 Hz, 1H), 6.78–
6.87 (m, 1H), 6.86–7.01 (m, 2H), 7.04–7.24 (m, 7H). The protons of oxadiazolone and OH were not observed.
LC/MS (ESI, [M + H]+, m/z) 509. HPLC: purity 98%, RT 3.4 min.
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(E)-3-(1-(8-((4-(2-hydroxypropan-2-yl)-2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-
11(6H)-ylidene)ethyl)-1,2,4-oxadiazol-5(4H)-one (11). To a stirred solution of 27E (147 mg, 0.308 mmol) in
ethanol (2 mL) was added NH2OH solution 50wt.% in water (0.566 mL, 9.24 mmol), and the solution was
refluxed for 3 days. After the consumption of the starting material, the reaction mixture was poured into water
and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over
sodium sulfate, and filtered. The organic layer was concentrated to dryness and the obtained residue was
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dissolved in THF (2 mL). To a stirred solution was added Et3N (0.064 mL, 0.46 mmol) and ethyl
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chloroformate (0.044 mL, 0.46 mmol) the solution was stirred for 1 h at room temperature. The reaction
mixture was poured into saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate.
The combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic
layer was concentrated to give the residue. To a solution of the residue in toluene (2 mL) was added tert-BuOK
(52 mg, 0.46 mmol) and the solution was stirred for 30 minutes at room temperature. The reaction mixture was
poured into 5% aqueous citric acid solution and extracted twice with ethyl acetate. The combined organic
layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to
give the residue. The obtained residue was then purified by flash column chromatography on silica gel (100:0
to 95:5 CHCl3/methanol) to give 11 (61 mg, 37%) as an amorphous. 1H-NMR (300 MHz, DMSO-d6):δ 0.92–
1.06 (m, 3H), 1.71 (s, 6H), 1.78–1.98 (m, 2H), 2.31 (s, 3H), 2.70–2.82 (m, 2H), 4.77 (d, J = 12.6 Hz, 1H), 5.33
(s, 2H), 5.51 (d, J = 12.6 Hz, 1H), 6.70–6.87 (m, 1H), 6.87–6.98 (m, 1H), 6.98–7.24 (m, 8H). The protons of
oxadiazolone and OH were not observed. LC/MS (ESI, [M + H]+, m/z) 537. HPLC: purity 96%, RT 3.7 min.
(E)-N-(2-hydroxyethyl)-1-((11-(1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)ethylidene)-6,11-
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dihydrodibenzo[b,e]oxepin-8-yl)methyl)-2-propyl-1H-benzo[d]imidazole-4-carboxamide (12). To a stirred
solution of 31 (240 mg, 0.474 mmol) in ethanol (2.4 mL) was added NH2OH solution 50wt.% in water (0.73
mL, 24 mmol), and the solution was refluxed for 3 days. After the consumption of the starting material, the
reaction mixture was poured into water and extracted twice with ethyl acetate. The combined organic layers
were washed with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to dryness
and the obtained residue was dissolved in CH2Cl2 (2.4 mL). To a stirred solution was added Et3N (99 μL, 0.71
mmol) and ethyl chloroformate (68 μL, 0.71 mmol) the solution was stirred for 1 h at room temperature. The
reaction mixture was poured into saturated aqueous sodium bicarbonate solution and extracted twice with
ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and filtered.
The organic layer was concentrated to give the residue. To a solution of the residue in toluene (1.2 mL) was
added tert-BuOK (106 mg, 0.948 mmol) and the solution was stirred for 30 minutes at room temperature. The
reaction mixture was poured into 5% aqueous citric acid solution and extracted twice with ethyl acetate. The
combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer
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was concentrated to give the residue. The obtained residue was then purified by flash column chromatography
on silica gel (100:0 to 97:3 CHCl3/methanol) to give 12 (70 mg, 26%) as an amorphous. 1H-NMR (300 MHz,
DMSO-d6):δ 0.85–1.01 (m, 3H), 1.72–1.90 (m, 2H), 2.18 (s, 3H), 2.77–3.01 (m, 2H), 3.43–3.65 (m, 4H),
4.72–5.05 (m, 2H), 5.38–5.52 (m, 1H), 5.59 (s, 2H), 6.73–6.86 (m, 1H), 6.86–6.99 (m, 1H), 7.02–7.13 (m,
2H), 7.13–7.36 (m, 4H), 7.61–7.75 (m, 1H), 7.75–7.93 (m, 1H), 9.93–10.13 (m, 1H), 12.19 (br s, 1H). LC/MS
(ESI, [M + H]+, m/z) 566. HPLC: purity 99%, RT 3.3 min.
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(E)-N-(1-((11-(1-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)ethylidene)-6,11-dihydrodibenzo[b,e]oxepin-8-
yl)methyl)-2-propyl-1H-benzo[d]imidazol-4-yl)methanesulfonamide (13). To a stirred solution of 32 (45
mg, 0.088 mmol) in ethanol (1.0 mL) was added NH2OH solution 50wt.% in water (0.27 mL, 4.4 mmol), and
the solution was refluxed for 3 days. After the consumption of the starting material, the reaction mixture was
poured into water and extracted twice with ethyl acetate. The combined organic layers were washed with brine,
dried over sodium sulfate, and filtered. The organic layer was concentrated to dryness and the obtained residue
was dissolved in CH2Cl2 (1.0 mL). To a stirred solution was added Et3N (18 μL, 0.13 mmol) and ethyl
chloroformate (13 μL, 0.13 mmol) the solution was stirred for 1 h at room temperature. The reaction mixture
was poured into saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate. The
combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer
was concentrated to give the residue. To a solution of the residue in toluene (1.0 mL) was added tert-BuOK
(20 mg, 0.18 mmol) and the solution was stirred for 30 minutes at room temperature. The reaction mixture was
poured into 5% aqueous citric acid solution and extracted twice with ethyl acetate. The combined organic
layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to
give the residue. The obtained residue was then purified by flash column chromatography on silica gel (100:0
to 97:3 CHCl3/methanol) to give 13 (14 mg, 28%) as an amorphous. 1H-NMR (300 MHz, CDCl3):δ 0.89–1.07
(m, 3H), 1.74–1.90 (m, 2H), 2.32 (s, 3H), 2.78–2.92 (m, 2H), 3.11 (s, 3H), 4.79 (d, J = 12.6 Hz, 1H), 5.38 (s,
2H), 5.55 (d, J = 12.6 Hz, 1H), 6.80–6.88 (m, 1H), 6.91–7.01 (m, 2H), 7.01–7.09 (m, 2H), 7.09–7.26 (m, 4H),
7.41–7.51 (m, 1H). The protons of oxadiazolone and sulfonamide were not observed. LC/MS (ESI, [M + H]+,
m/z) 572. HPLC: purity 99%, RT 3.52 min.
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(E)-3-(1-(8-((4-phenyl-2-propyl-1H-imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-ylidene)ethyl)-1,2,4-
oxadiazol-5(4H)-one (14). To a stirred solution of 28E (182 mg, 0.409 mmol) in ethanol (2.0 mL) was added
NH2OH solution 50wt.% in water (1.35 mL, 20.5 mmol), and the solution was refluxed for 3 days. After the
consumption of the starting material, the reaction mixture was poured into water and extracted twice with ethyl
acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The
organic layer was concentrated to dryness and the obtained residue was dissolved in CH2Cl2 (2.0 mL). To a
stirred solution was added Et3N (86 μL, 0.62 mmol) and ethyl chloroformate (59 μL, 0.62 mmol), and the
solution was stirred for 1 h at room temperature. The reaction mixture was poured into saturated aqueous
sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic layers were washed
with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. To a
solution of the residue in toluene (2.0 mL) was added tert-BuOK (91 mg, 0.82 mmol) and the solution was
stirred for 30 minutes at room temperature. The reaction mixture was poured into 5% aqueous citric acid
solution and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried
over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The obtained residue
was then purified by flash column chromatography on silica gel (100:0 to 90:10 CHCl3/methanol) to give 14
(75 mg, 36%) as an amorphous. 1H-NMR (300 MHz, CDCl3):δ 0.92–1.02 (m, 3H), 1.67–1.87 (m, 2H), 2.26 (s,
3H), 2.58–2.69 (m, 2H), 4.78 (d, J = 12.6 Hz, 1H), 5.11 (s, 2H), 5.52 (d, J = 12.6 Hz, 1H), 6.81–6.87 (m, 1H),
6.87–6.95 (m, 1H), 6.98–7.11 (m, 4H), 7.11–7.27 (m, 3H), 7.27–7.39 (m, 2H), 7.68–7.79 (m, 2H). The proton
of oxadiazolone was not observed. LC/MS (ESI, [M + H]+, m/z) 505. HPLC: purity 95%, RT 3.9 min.
(E)-3-(1-(8-((2-propyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
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ylidene)ethyl)-1,2,4-oxadiazol-5(4H)-one (15). To a stirred solution of 29E (43 mg, 0.097 mmol) in ethanol
(1.0 mL) was added NH2OH solution 50wt.% in water (0.32 mL, 4.8 mmol), and the solution was refluxed for
3 days. After the consumption of the starting material, the reaction mixture was poured into water and
extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium
sulfate, and filtered. The organic layer was concentrated to dryness and the obtained residue was dissolved in
CH2Cl2 (1.0 mL). To a stirred solution was added Et3N (20 μL, 0.15 mmol) and ethyl chloroformate (14 μL,
0.15 mmol), and the solution was stirred for 1 h at room temperature. The reaction mixture was poured into
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saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic
layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to
give the residue. To a solution of the residue in toluene (1.0 mL) was added tert-BuOK (22 mg, 0.19 mmol)
and the solution was stirred for 30 minutes at room temperature. The reaction mixture was poured into 5%
aqueous citric acid solution and extracted twice with ethyl acetate. The combined organic layers were washed
with brine, dried over sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The
obtained residue was then purified by flash column chromatography on silica gel (100:0 to 90:10
CHCl3/methanol) to give 15 (3.6 mg, 8.3%) as an amorphous. 1H-NMR (300 MHz, CDCl3):δ 0.93–1.04 (m,
3H), 1.67–1.85 (m, 2H), 2.32 (s, 3H), 2.55–2.71 (m, 2H), 4.67 (d, J = 12.6 Hz, 1H), 5.12 (s, 2H), 5.45 (d, J =
12.6 Hz, 1H), 6.79–6.89 (m, 2H), 6.89–7.02 (m, 1H), 7.07–7.32 (m, 5H), 7.55–7.68 (m, 2H), 8.32–8.45 (m,
2H). The proton of oxadiazolone was not observed. LC/MS (ESI, [M + H]+, m/z) 506. HPLC: purity 96%, RT
3.2 min.
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(E)-2-(2-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)-10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5-ylidene)propanenitrile (20E). To a stirred solution of 16E (367 mg, 1.33 mmol),
2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (367 mg, 2.09 mmol) and PPh3 (1.4 g, 2.8 mmol, polymer-
bound, ~approximately 3 mmol/g triphenylphosphine loading, Sigma-Aldrich) in THF (13 mL) was added di-
tert-butyl azodicarboxylate (642 mg, 2.79 mmol), and the solution was stirred for 2 h at room temperature. The
reaction mixture was filtered, and the filtrate was concentrated. The obtained residue was then purified by flash
column chromatography on silica gel (80:20 to 65:35 hexane/ethyl acetate) to give 20E (399 mg, 69%) as an
amorphous. 1H NMR (300 MHz, CDCl3): δ 1.31 (t, J = 7.6 Hz, 3H), 1.99 (s, 3H), 2.58 (s, 3H), 2.63 (s, 3H),
2.73–2.88 (m, 2H), 2.76 (q, J = 7.6 Hz, 2H), 3.18–3.34 (m, 2H), 5.41 (s, 2H), 6.90–7.01 (m, 4H), 7.13 (dd, J =
7.1, 1.6 Hz, 1H), 7.18–7.28 (m, 2H), 7.39 (dd, J = 7.1, 1.6 Hz, 1H). LC/MS (ESI, [M + H]+, m/z) 433.
(Z)-2-(2-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)-10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5-ylidene)propanenitrile (20Z). To a stirred solution of 16Z (399 mg, 1.45 mmol),
2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (367 mg, 2.09 mmol) and PPh3 (1.4 g, 2.8 mmol, polymer-
bound, ~3 mmol/g triphenylphosphine loading, Sigma-Aldrich) in THF (13 mL) was added di-tert-butyl
azodicarboxylate (642 mg, 2.79 mmol), and the solution was stirred for 2 h at room temperature. The reaction
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mixture was filtered, and the filtrate was concentrated. The obtained residue was then purified by flash column
chromatography on silica gel (80:20 to 65:35 hexane/ethyl acetate) to give 20Z (376 mg, 68%) as an
amorphous. 1H NMR (300 MHz, CDCl3): δ 1.31 (t, J = 7.4 Hz, 3H), 2.00 (s, 3H), 2.56 (s, 3H), 2.63 (s, 3H),
2.74–2.84 (m, 2H), 2.75 (q, J = 7.6 Hz, 2H), 3.20–3.30 (m, 2H), 5.40 (s, 2H), 6.83 (s, 1H), 6.88 (s, 1H), 6.97
(dd, J = 8.0, 1.6 Hz, 1H), 7.03–7.06 (m, 1H), 7.15–7.26 (m, 3H), 7.36 (d, J = 7.9 Hz, 1H). LC/MS (ESI, [M +
H]+, m/z) 433.
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(E)-2-(8-((2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
ylidene)propanenitrile (21E). 2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (218 mg, 1.25 mmol) was
added to a solution of 17E (231 mg, 0.834 mmol), PPh3 (1.4 g, 2.8 mmol, polymer-bound, ~approximately 3
mmol/g triphenylphosphine loading) and di-tert-butyl azodicarboxylate (382 mg, 1.66 mmol) in THF (4 mL),
and the solution was stirred for 2 h at room temperature. The reaction mixture was filtered, and the filtrate was
concentrated. The obtained residue was then purified by flash column chromatography on silica gel (70:30 to
20:80 hexane/ethyl acetate) to give 21E (228 mg, 63%) as an amorphous. 1H NMR (300 MHz, CDCl3): δ 1.31
(t, J = 7.6 Hz, 3H), 2.24 (s, 3H), 2.58 (s, 3H), 2.65 (s, 3H), 2.77 (q, J = 7.6 Hz, 2H), 4.74 (d, J = 12.6 Hz, 1H),
5.40 (d, J = 12.6 Hz, 1H), 5.44–5.51 (m, 2H), 6.79–6.86 (m, 1H), 6.86–6.95 (m, 2H), 7.00–7.10 (m, 2H), 7.12–
7.25 (m, 2H), 7.37–7.46 (m, 1H). LC/MS (ESI, [M + H]+, m/z) 435.
(E)-2-(8-((2-ethyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-ylidene)propanenitrile
(22E). 18 (300 mg, 1.01 mmol) was added to a solution of 2-ethyl-1H-benzo[d]imidazole (163 mg, 1.12
mmol) and K2CO3 (701 mg, 5.07 mmol) in DMF (5 mL), and the solution was stirred overnight at room
temperature. The reaction mixture was poured into water and extracted twice with ethyl acetate. The
combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer
was concentrated to give the residue. The obtained residue was then purified by flash column chromatography
on silica gel (90:10 to 50:50 hexane/ethyl acetate) to give 22E (318 mg, 77%) as an amorphous. 1H NMR (300
MHz, CDCl3): δ 1.43 (t, J = 7.6 Hz, 3H), 2.24 (s, 3H), 2.83 (q, J = 7.6 Hz, 2H), 4.72 (d, J = 12.6 Hz, 1H), 5.35
(s, 2H), 5.41 (d, J = 12.6 Hz, 1H), 6.80–6.99 (m, 3H), 7.02–7.15 (m, 2H), 7.16–7.33 (m, 4H), 7.43–7.50 (m,
1H), 7.72–7.83 (m, 1H). LC/MS (ESI, [M + H]+, m/z) 406.
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(E)-2-(8-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-ylidene)propanenitrile
(23E). 18 (300 mg, 1.01 mmol) was added to a solution of 2-propyl-1H-benzo[d]imidazole (179 mg, 1.12
mmol) and K2CO3 (701 mg, 5.07 mmol) in DMF (5 mL), and the solution was stirred overnight at room
temperature. The reaction mixture was poured into water and extracted twice with ethyl acetate. The
combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer
was concentrated to give the residue. The obtained residue was then purified by flash column chromatography
on silica gel (90:10 to 70:30 hexane/ethyl acetate) to give 23E (448 mg, 100%) as an amorphous. 1H NMR
(300 MHz, CDCl3): δ 0.95–1.12 (m, 3H), 1.82–1.98 (m, 2H), 2.26 (s, 3H), 2.72–2.89 (m, 2H), 4.73 (d, J = 12.6
Hz, 1H), 5.35 (s, 2H), 5.41 (d, J = 12.6 Hz, 1H), 6.81–6.99 (m, 3H), 7.01–7.15 (m, 2H), 7.15–7.30 (m, 4H),
7.40–7.48 (m, 1H), 7.74–7.84 (m, 1H). LC/MS (ESI, [M + H]+, m/z) 420.
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(E)-2-(8-((4-methyl-2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
ylidene)propanenitrile (24E). 19 (1.15 g, 3.61 mmol) was added to a solution of 4-methyl-2-propyl-1H-
benzo[d]imidazole (691 mg, 3.97 mmol) and K2CO3 (2.49 g, 18.0 mmol) in DMF (20 mL), and the solution
was stirred overnight at room temperature. The reaction mixture was poured into water and extracted twice
with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and
filtered. The organic layer was concentrated to give the residue. The obtained residue was then purified by
flash column chromatography on silica gel (90:10 to 70:30 hexane/ethyl acetate) to give 24E (891 mg, 57%) as
an amorphous. 1H NMR (300 MHz, CDCl3): δ 0.95–1.05 (m, 3H), 1.74–1.91 (m, 2H), 2.25 (s, 3H), 2.69 (s,
3H), 2.78–2.88 (m, 2H), 4.73 (d, J = 12.6 Hz, 1H), 5.34 (s, 2H), 5.41 (d, J = 12.6 Hz, 1H), 6.80–6.98 (m, 3H),
6.98–7.18 (m, 5H), 7.17–7.28 (m, 1H), 7.38–7.48 (m, 1H). LC/MS (ESI, [M + H]+, m/z) 434.
Methyl (E)-1-((11-(1-cyanoethylidene)-6,11-dihydrodibenzo[b,e]oxepin-8-yl)methyl)-2-propyl-1H-
benzo[d]imidazole-4-carboxylate (25E). 19 (853 mg, 2.89 mmol) was added to a solution of methyl 2-
propyl-1H-benzo[d]imidazole-4-carboxylate (600 mg, 2.75 mmol) and K2CO3 (1.9 g, 14 mmol) in DMF (16
mL), and the solution was stirred overnight at room temperature. The reaction mixture was poured into water
and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over
sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The obtained residue was
then purified by flash column chromatography on silica gel (80:20 to 20:80 hexane/ethyl acetate) to give 25E
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(970 mg, 74%) as an amorphous. 1H NMR (300 MHz, CDCl3): δ 0.97–1.10 (m, 3H), 1.83–1.97 (m, 2H), 2.24
(s, 3H), 2.84–2.99 (m, 2H), 4.04 (s, 3H), 4.64–4.75 (m, 1H), 5.28–5.49 (m, 3H), 6.82–6.96 (m, 3H), 7.02–7.14
(m, 2H), 7.18–7.30 (m, 2H), 7.31–7.39 (m, 1H), 7.39–7.46 (m, 1H), 7.89–8.00 (m, 1H). LC/MS (ESI, [M +
H]+, m/z) 478.
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(E)-2-(8-((4-(hydroxymethyl)-2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
ylidene)propanenitrile (26E). 19 (137 mg, 0.465 mmol) was added to a solution of (2-propyl-1H-
benzo[d]imidazole-4-yl)methanol (97 mg, 0.51 mmol) and K2CO3 (321 mg, 2.33 mmol) in DMF (2.3 mL), and
the solution was stirred overnight at room temperature. The reaction mixture was poured into water and
extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium
sulfate, and filtered. The organic layer was concentrated to give the residue. The obtained residue was then
purified by flash column chromatography on silica gel (50:50 to 20:80 hexane/ethyl acetate) to give 26E (185
mg, 89%) as an amorphous. 1H NMR (300 MHz, CDCl3): δ 0.91–1.06 (m, 3H), 1.77–1.92 (m, 2H), 2.25 (s,
3H), 2.72–2.84 (m, 2H), 4.22–4.33 (m, 1H), 4.73 (d, J = 12.6 Hz, 1H), 5.09–5.19 (m, 2H), 5.35 (s, 2H), 5.41
(d, J = 12.6 Hz, 1H), 6.83–6.99 (m, 3H), 7.02–7.17 (m, 5H), 7.17–7.29 (m, 1H), 7.40–7.50 (m, 1H). LC/MS
(ESI, [M + H]+, m/z) 450.
(E)-2-(8-((4-(2-hydroxypropan-2-yl)-2-propyl-1H-benzo[d]imidazol-1-yl)methyl)dibenzo[b,e]oxepin-
11(6H)-ylidene)propanenitrile (27E). A solution of K2CO3 (404 mg, 2.92 mmol) and 2-(2-propyl-1H-
benzo[d]imidazol-4-yl)propan-2-ol (190 mg, 0.642 mmol) in DMF (3 mL) was treated with 19 (173 mg, 0.584
mmol), and the solution was stirred overnight at room temperature. The reaction mixture was poured into
water and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over
sodium sulfate, and filtered. The organic layer was concentrated to give the residue. The obtained residue was
then purified by flash column chromatography on silica gel (90:10 to 60:40 hexane/ethyl acetate) to give 27E
(295 mg, 100%) as an amorphous. 1H NMR (300 MHz, CDCl3): δ 0.89–1.07 (m, 3H), 1.73 (s, 6H), 1.78–1.98
(m, 2H), 2.25 (s, 3H), 2.70–2.85 (m, 2H), 4.75 (d, J = 12.6 Hz, 1H), 5.32 (s, 2H), 5.43 (d, J = 12.6 Hz, 1H),
6.76–7.01 (m, 4H), 7.01–7.34 (m, 5H), 7.39–7.51 (m, 1H). The proton of OH was not observed. LC/MS (ESI,
[M + H]+, m/z) 478.
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(E)-2-(8-((4-phenyl-2-propyl-1H-imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-ylidene)propanenitrile
(28E). To a stirred solution of 19 (131 mg, 0.445 mmol) and 4-phenyl-2-propyl-1H-imidazole (91 mg, 0.49
mmol) in DMF (2 mL) was added K2CO3 (307 mg, 2.22 mmol), and the solution was stirred overnight at room
temperature. The reaction mixture was poured into water and extracted twice with ethyl acetate. The combined
organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer was
concentrated to give the residue. The obtained residue was then purified by flash column chromatography on
silica gel (100:0 to 70:30 hexane/ethyl acetate) to give 28E (182 mg, 91%) as an amorphous. 1H NMR (300
MHz, CDCl3): δ 0.92–1.04 (m, 3H), 1.65–1.84 (m, 2H), 2.26 (s, 3H), 2.57–2.70 (m, 2H), 4.79 (d, J = 12.6 Hz,
1H), 5.11 (s, 2H), 5.43 (d, J = 12.6 Hz, 1H), 6.80–6.97 (m, 2H), 7.00–7.12 (m, 3H), 7.13–7.30 (m, 3H), 7.30–
7.40 (m, 2H), 7.43–7.52 (m, 1H), 7.72–7.83 (m, 2H). LC/MS (ESI, [M + H]+, m/z) 446.
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(E)-2-(8-((2-propyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)methyl)dibenzo[b,e]oxepin-11(6H)-
ylidene)propanenitrile (29E). To a stirred solution of 19 (69 mg, 0.24 mmol) and 2-propyl-4-(pyridine-4-yl)-
1H-imidazole (44 mg, 0.235 mmol) in DMF (1.5 mL) was added K2CO3 (160 mg, 1.18 mmol), and the
solution was stirred overnight at room temperature. The reaction mixture was poured into water and extracted
twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and
filtered. The organic layer was concentrated to give the residue. The obtained residue was then purified by
flash column chromatography on silica gel (100:0 to 90:10 CHCl3/methanol) to give 29E (43 mg, 40%) as an
amorphous. 1H NMR (300 MHz, CDCl3): δ 0.93–1.06 (m, 3H), 1.68–1.85 (m, 2H), 2.25 (s, 3H), 2.61–2.73 (m,
2H), 4.81 (d, J = 12.6 Hz, 1H), 5.13 (s, 2H), 5.43 (d, J = 12.6 Hz, 1H), 6.79–6.97 (m, 2H), 7.03–7.32 (m, 4H),
7.35–7.55 (m, 2H), 7.57–7.67 (m, 2H), 8.47–8.60 (m, 2H). LC/MS (ESI, [M + H]+, m/z) 447.
(E)-1-((11-(1-cyanoethylidene)-6,11-dihydrodibenzo[b,e]oxepin-8-yl)methyl)-2-propyl-1H-
benzo[d]imidazole-4-carboxylic acid (30). To a stirred solution of 25E (100 mg, 0.209 mmol) in ethanol (1
mL) was added 4M NaOH aqueous solution (1.0 mL, 4.0 mmol) and the solution was stirred for 2 h at 70 °C.
The reaction mixture was acidified with 4 M HCl and the resultant solid was filtered, washed with water, and
dried under reduced pressure to give 30 (86 mg, 89%) as a white solid.1H NMR (300 MHz, DMSO-d6): δ
0.87–1.01 (m, 3H), 1.60–1.82 (m, 2H), 2.18 (s, 3H), 2.96–3.12 (m, 2H), 4.93 (d, J = 12.6 Hz, 1H), 5.44 (d, J =
12.6 Hz, 1H), 5.76 (s, 2H), 6.80–6.87 (m, 1H), 6.90– 7.00 (m, 1H), 7.14–7.35 (m, 3H), 7.35–7.41 (m, 1H),
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7.41–7.51 (m, 2H), 7.85–8.01 (m, 2H). The proton of CO2H was not observed. LC/MS (ESI, [M - H]-, m/z)
462.
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(E)-N-(1-((11-(1-cyanoethylidene)-6,11-dihydrodibenzo[b,e]oxepin-8-yl)methyl)-2-propyl-1H-
benzo[d]imidazol-4-yl)-3-hydroxypropanamide (31). To a stirred solution of 30 (220 mg, 0.475 mmol),
EDCI·HCl (109 mg, 0.571 mmol) and HOBt·H2O (87 mg, 0.57 mmol) in DMF (4 mL) was added 2-
aminoethanol (57 μL, 0.95 mmol) and the solution was stirred overnight at room temparature. The reaction
mixture was poured into saturated sodium hydrogen carbonae solution and the resultant solid was filtered,
washed with water, and dried under reduced pressure. The obtained residue was then purified by flash column
chromatography on silica gel (100:0 to 95:5 hexane/CHCl3) to give 31 (241 mg, 99%) as an amorphous. 1H
NMR (300 MHz, CDCl3): δ 0.88–1.13 (m, 3H), 1.79–1.97 (m, 2H), 2.26 (s, 3H), 2.72–2.88 (m, 2H), 3.38–3.59
(m, 1H), 3.71–3.86 (m, 2H), 3.86–3.97 (m, 2H), 4.68–4.78 (m, 1H), 5.33–5.49 (m, 3H), 6.81–6.96 (m, 3H),
7.03–7.15 (m, 2H), 7.20–7.37 (m, 3H), 7.41–7.50 (m, 1H), 8.03–8.17 (m, 1H), 10.22–10.35 (m, 1H). The
protons of OH and amide were not observed. LC/MS (ESI, [M + H]+, m/z) 507.
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(E)-N-(1-((11-(1-cyanoethylidene)-6,11-dihydrodibenzo[b,e]oxepin-8-yl)methyl)-2-propyl-1H-
benzo[d]imidazol-4-yl)methanesulfonamide (32). To a stirred solution of 30 (810 mg, 1.86 mmol) and Et3N
(1.3 ml, 9.3 mmol) in CHCl3 (9 mL) was added DPPA (2.1 mL, 9.3 mmol) and the solution was stirred for 5 h
at room temparature. tert-BuOH (9 ml) was added and the reaction mixture was stirred overnight at 100 °C.
The reaction mixture was poured into saturated sodium hydrogen carbonate solution and extracted twice with
CHCl3. The combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The
organic layer was concentrated to give the residue. To a stirred solution of the obtained residue in CH2Cl2 (1
mL) was added TFA (0.31 mL) and the solution was stirred for 2 h at room temparature. The reaction mixture
was poured into saturated sodium hydrogen carbonate solution and extracted twice with CHCl3. The combined
organic layers were washed with brine, dried over sodium sulfate, and filtered. The organic layer was
concentrated to give the residue. The obtained residue was purified by flash column chromatography on silica
gel (70:30 to 20:80 hexane/ethyl acetate). The resulting residue was dissolved in CH2Cl2 (2 mL) and DMAP (2
mg, 0.02 mmol) , MeSO2Cl (7.8 μL, 0.10 mmol) were added. The reaction mixture was stirred 5 h at room
temparature. After the consumption of starting material, the mixture was poured into 2 mol/L HCl and
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extracted twice with CHCl3. The combined organic layers were washed with brine, dried over sodium sulfate,
and filtered. The organic layer was concentrated to give the residue. The obtained residue was then purified by
flash column chromatography on silica gel (80:20 to 20:80 hexane/ethyl acetate) to afford 32 (47 mg, 49%) as
an amorphous. 1H NMR (300 MHz, CDCl3): δ 0.93–1.03 (m, 3H), 1.73–1.88 (m, 2H), 2.25 (s, 3H), 2.72–2.84
(m, 2H), 3.09 (s, 3H), 4.76 (d, J = 12.6 Hz, 1H), 5.33 (s, 2H), 5.43 (d, J = 12.6 Hz, 1H), 6.79–6.88 (m, 1H),
6.88–7.01 (m, 3H), 7.03–7.13 (m, 2H), 7.13–7.20 (m, 1H), 7.20–7.25 (m, 1H), 7.36–7.42 (m, 1H), 7.42–7.47
(m, 1H), 7.67–8.26 (m, 1H). LC/MS (ESI, [M + H]+, m/z) 513.
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Biological Methods.
Chimeric GAL4-PPARγ transactivation reporter assay. Test compounds were screened for agonist activity
on PPARγ-GAL4 chimeric receptors in transiently transfected HEK293EBNA cells. The cells were maintained
in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) containing 10% fetal bovine serum
(Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific) and
incubated at 37 oC in 5% CO2. To transfect the reporter constract into HEK293EBNA cells, cells were seeded
at 1x 105 cells/ml in tissue culture dish (Iwaki, Chiba, Japan). After 24h incubation, transfections were
performed with Superfect transfection reagent (QIAGEN) according to the instructions of the manufacturer. In
brief, pM-human PPARγ/GAL4 expression vector and pZAC19-Luc vector were premixed and transfected into
the cells followed by 2.5 h incubation. After a further 24 h incubation with growth medium, the transfected
cells were seeded into 96-well assay plates, and test compounds were added (1-3000 nM, N = 3 per
concentration). The test compounds were initially dissolved in DMSO, and then diluted in DMEM without any
supplement. Steady-Glo luciferase assay reagent (Promega) was used as a substrate, and the luciferase activity
was measured using the Microplate Scintillation and luminescence counter TopCount NXT (Packard,
Groningen, Netherlands). The luciferase activity was normalized to that of pioglitazone at 1000 nM. The
maximum activation (efficacy) of pioglitazone was taken as 100%. The efficacy of each compound was
calculated as the percentage of the maximum activation obtained with pioglitazone. EC50 values were
determined by the concentration that was 50% of its maximum activity using the XLFit.
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MKN45 cell aggregation assay. MKN45 cells were aggregated when cells were incubated with PPARγ
agonists. The cells were maintained in RPMI1640 medium (Thermo Fisher Scientific) containing 10% fetal
bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/ml streptomycin(Thermo Fisher
Scientific) and incubated at 37 oC in 5% CO2. Cells were seeded at 2500 cells/well in 96-well assay plates and
incubated with test compounds, (1-1000 nM, N = 3 per concentration), for 5 days. Cell images were captured
using an IN Cell Analyzer 1000 (GE healthcare). Nuclei were stained with the Hoechst 33342 (SIGMA) dyes,
and the nuclei area was calculated. An area exceeding 1,015.58 μm2 was defined as an aggregated cell cluster.
The area ratio of aggregated cell clusters to the total cell area was calculated and taken as the formation rate of
aggregation. The cell aggregation-inducing activity was normalized to that of 9, with the maximum activity of
9 taken as 100%. The maximum activity of each compound was then calculated as the percentage of the
maximum activity of 9. EC50 values was determined as the concentration achieving 50% of its maximum
activity using the XLFit.
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MKN-45 cell gene expression analyses. MKN45 cells were seeded in assay plate and incubated with test
compounds, (N = 3 per concentration), for 72 h. Total RNA was isolated from MKN45 cells using a RNeasy
Mini Kit (QIAGEN). The cells were washed with cold PBS and lysed with buffer according to the instructions
of the manufacturer. The RNA was reverse-transcribed using a SuperScript VILO cDNA Synthesis kit
(Thermo Fisher Scientific) and synthesized to cDNA. Quantitative PCR was performed with Taqman
fluorescent dye using an ABI PCR system. For PCR primers and probes, we used the Taqman® Gene
Expression Assays system (Thermo Fisher Scientific) for ANGPTL4 (Hs_01101127_m1), VIM
(Hs00958116_m1), and ADFP (Hs_00765634_m1). The PCR primer/probe sequences for GAPDH were as
follows. Forward: ACAGTCAGCCGCATCTTCTTT, Reverse: CCCAATACGACCAAATCCGT, Probe:
6FAM-CGAGCCACATCGCTCAGACACCAT-Tamra (Operon). The gene expression value was corrected
based on the value of GAPDH. The fold-induction ratio to the control was calculated.
Molecular modelling methods. The three-dimensional molecule structures of 1-3 were built using
Schrödinger MacroModel10.9. The conformational search of each compound was performed using mixed
torsional/low-mode sampling as implemented in MacroModel 10.9 with OPLS_2005 force field. The
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conformational analysis was carried out without solvent. The default values were used for all other settings.
The obtained global minimum energy conformations of the ligands were superimposed.
■ ASSOCIATED CONTENT
9
Supporting Information
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General methods (S2)
Experimental details of tricyclic intermediates 16E/Z,18,19 (S2)
NMR chart of compound 4 and 5 (S11)
Crystallographic data table of compound 9 (S13)
Electron density map of compound 9 (S14)
Molecular formula strings
Accession Codes
The X-ray structure of 9 with PPARγ LBD has been deposited with the Protein Data Bank. The code is 6AD9.
We will release the atomic coordinates and experimental data upon article publication.
■ AUTHOR INFORMATION
Corresponding Authors
*For K.Y.: phone, +81-55-989-3989; fax, +81-55-986-7430; Email, keisuke.yamamoto@kyowa-kirin.co.jp;
address, Small Molecule Drug Research Laboratories, Fuji Research Park, Shimotogari, Nagaizumi-cho,
Sunto-gun, Shizuoka 411-8731, Japan.
*For S.S.: phone/fax, +81-11-706-3769; E-mail, shu@pharm.hokudai.ac.jp; address, Faculty of
Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank Hiromi Nagai for supporting the synthesis of the tricyclic compounds. We thank Takahiro Kabeya
for his assistance for the preparation of Figure 8. We thank Yusuke Yamada, Atsuko Sato and Mizuki
Watanabe for the conformation study. We thank Kenta Nakajima for supporting our validation of the in vitro
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assay data. We thank Hideyuki Onodera, Seiji Aratake and Kuniyuki Kishikawa for supporting the edit of this
manuscript. We thank Daisuke Nakashima for screening the compounds for PAINS.
■ ABBREVIATIONS USED
9
DPPA, diphenylphosphoryl azide; DTBAD, di-tert-butyl azodicarboxylate; EDCI; 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide, HOBt, 1-hydroxybenzotriazole; PPRE, proliferator hormone response
elements
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ANCILLARY INFORMATION
Present author address
Kazuki Henmi, Arata Yanagisawa: Otemachi Financial City Grand Cube, 1-9-2 Otemachi, Chiyoda-ku,
Tokyo, Japan
Masahiro Matsubara: Tokyo Research Park, 3-6-6 Asahi-machi, Machida-shi, Tokyo, Japan
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