Development of Novel Inhibitors for Histone Methyltransferase SET7/9 based on Cyproheptadine

Article abstract of DOI:10.1002/cmdc.201800233

The histone methyltransferase SET7/9 methylates not only histone but also non-histone proteins as substrates, and therefore, SET7/9 inhibitors are considered candidates for the treatment of diseases. Previously, our group identified cyproheptadine, used clinically as a serotonin receptor antagonist and histamine receptor (H1) antagonist, as a novel scaffold of the SET7/9 inhibitor. In this work, we focused on dibenzosuberene as a substructure of cyproheptadine and synthesized derivatives with various functional groups. Among them, the compound bearing a 2-hydroxy group showed the most potent activity. On the other hand, a 3-hydroxy group or another hydrophilic functional group such as acetamide decreased the activity. Structural analysis clarified a rationale for the improved potency only by tightly restricted location and type of the hydrophilic group. In addition, a SET7/9 loop, which was only partially visible in the complex with cyproheptadine, became more clearly visible in the complex with 2-hydroxycyproheptadine. These results are expected to be helpful for further structure-based development of SET7/9 inhibitors.

Full text of DOI:10.1002/cmdc.201800233

Accepted Article
Title: Development of novel inhibitors for histone methyltransferase
SET7/9 based on cyproheptadine
Authors: Tomoya Hirano, Takashi Fujiwara, Hideaki Niwa, Michitake
Hirano, Kasumi Ohira, Yusuke Okazaki, Shin Sato, Takashi
Umehara, Yuki Maemoto, Akihiro Ito, Minoru Yoshida, and
Hiroyuki Kagechika
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10.1002/cmdc.201800233
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Development of novel inhibitors for histone methyltransferase
SET7/9 based on cyproheptadine
Tomoya Hirano,*[a] Takashi Fujiwara,[a] Hideaki Niwa,[b] Michitake Hirano,[a] Kasumi Ohira,[a] Yusuke
Okazaki,[a] Shin Sato,[b] Takashi Umehara,[b] Yuki Maemoto,[c] Akihiro Ito,[c],[d] Minoru Yoshida,[d],[e],[f] and
Hiroyuki Kagechika[a]
Abstract: The histone methyltransferase SET7/9 methylates not
only histone but also non-histone proteins as substrates, and
therefore SET7/9 inhibitors are considered candidates for treatment
of diseases. Previously, our group identified cyproheptadine, which
is used clinically as a serotonin receptor antagonist or histamine
receptor (H1) antagonist, as a novel scaffold of SET7/9 inhibitor. In
this work, we focused on dibenzosuberene as a substructure of
cyproheptadine, and synthesized derivatives with various functional
groups. Among them, compound bearing a 2-hydroxy group showed
the most potent activity. On the other hand, a 3-hydroxy group or
other hydrophilic functional groups such as acetamide decreased the
activity. Structural analysis clarified a rationale for the improved
potency only by tightly restricted location and type of hydrophilic
group. In addition, a SET7/9 loop, which was only partially visible in
the complex with cyproheptadine, became more clearly visible in that
with 2-hydroxycyproheptadine. These results are expected to be
helpful for further structure-based development of SET7/9 inhibitors.
family, and methylates lysine 4 of histone H3 (H3K4). However,
its substrate scope is not limited to histone protein, as it also
methylates TAF10,[7] p53,[8] estrogen receptor
α
(ERα),[9]
androgen receptor (AR),[10] retinoic acid receptor (RAR),[11]
STAT3,[12] FoxO3,[13] Tat,[14] HIF-1,[15]
and DNA
methyltransferase (DNMT1).[16] Thus, SET7/9 plays important
roles in various physiological functions and related diseases,
and SET7/9 inhibitors are expected to be useful as therapeutic
agents.[17-20] Recently, (R)-PFI-2 was reported as the first
practical SET7/9 inhibitor,[21] followed by a few inhibitors such as
DC-S239.[22] We have reported a bisubstrate-type inhibitor,
DAAM-3, that consists of the coenzyme derivative AzaAdoMet,
in which the bridging sulfur atom of the coenzyme S-
adenosylmethionine (SAM) is replaced with nitrogen, mimicking
the side chain of lysine (a SET7/9 substrate).[23,24] However,
structure-activity studies of these inhibitors are quite limited, and
no compound has yet been approved for clinical use. We also
identified cyproheptadine (Figure 1, 1),
a clinically used
serotonin receptor antagonist or histamine receptor (H1)
antagonist, as a SET7/9 inhibitor.[25,26] Cyproheptadine (1) blocks
the ubiquitination of ERα by the methylation of lysine (Lys302),
and induces the digestion of ERα, so it could be a candidate
therapeutic agent for diseases associated with upregulation of
ERα, such as breast cancer. As cyproheptadine is already
approved for clinical use, we considered that it would be a
promising lead compound for optimization as a SET7/9 inhibitor.
Cyproheptadine (1) consists of N-methylpiperidine and tricyclic
dibenzosuberene moieties. Our previous study indicated the
importance of the N-methyl group on the piperidine ring, and a
key role of the characteristic butterfly-like conformation.[27]
Replacement of the N-methyl group with any other substituent
examined resulted in a reduction of the inhibitory activity. X-ray
structural analysis confirmed that the space around the N-methyl
group is quite restricted in the cyproheptadine-bound SET7/9
structure. Nevertheless, the structural analysis revealed that a
flexible loop prior to the C-terminal helix interacted with the
dibenzosuberene moiety, suggesting potential for optimization of
the compound. In the present study, we thus focused on the
dibenzosuberene moiety, and introduced various functional
groups on the phenyl ring that might potentially interact with
proximal amino-acid residues, including those in the associated
loop. We present here cyproheptadine derivatives with improved
potency, and also describe a structural analysis of one of the
derivatives, which provided a rationale for the improved potency.
Introduction
Posttranscriptional modifications of histone proteins are well-
known regulatory mechanisms of gene transcription. [1-2] Among
the enzyme families involved, histone lysine methyltransferases
(HKMTs) serve as “writers” of histone protein methylation.[3-6]
SET domain-containing lysine methyltransferase 7/9 (SET7/9,
also known as SETD7 or KMT7) is a member of the HKMT
[a]
Dr. T. Hirano, Dr. T. Fujiwara, M. Hirano, K. Ohira, Y. Okazaki, Prof. H.
Kagechika
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental
University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-
0062, Japan
E-mail: hira.chem@tmd.ac.jp
[b]
Dr. H. Niwa, S. Sato, Dr. T. Umehara
Epigenetics Drug Discovery Unit, RIKEN Center for Life Science
Technologies (CLST), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-
0045, Japan
[c]
[d]
Dr. Y. Maemoto, Prof. A. Ito
School of Life Sciences, Tokyo University of Pharmacy and Life
Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Prof. A. Ito, Prof. M. Yoshida
Chemical Genomics Research Group, RIKEN Center for Sustainable
Resource Science (CSRS), 2-1 Hirosawa, Wako, Saitama 351-0198,
Japan
[e]
[f]
Prof. M. Yoshida
Drug Discovery Platforms Cooperation Division, RIKEN Center for
Sustainable Resource Science (CSRS), 2-1 Hirosawa, Wako, Saitama
351-0198, Japan
Prof. M. Yoshida
Results and Discussion
Graduate School of Agricultural and Life Sciences, The University of
Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
In the co-crystal structure of the SET7/9 complex with
cyproheptadine (1), amino acid residues around the
dibenzosuberene ring were Asn263, Gly264, Tyr335 and Gly336
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under : https://doi.org/xxx
1
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10.1002/cmdc.201800233
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[26], so the introduction of suitable functional groups that could
form favorable interactions with these amino acid residues might
be good strategy. In addition, as described above, the loop
(residues 337 to 349) located prior to the C-terminal helix
seemed to be associated at least in part with the
dibenzosuberene moiety. Hence, in order to develop more
potent inhibitors, we introduced various types of functional
groups including a hydrophilic hydroxy group, hydrophobic
halogen atom and methoxy group into the dibenzosuberene
moiety (Figure 1, 2a – 2p).
Figure 1. Design of cyproheptadine derivatives as candidate SET7/9 inhibitors.
Cyproheptadine derivatives were synthesized via Grignard
reaction
between
4-chloro-1-methylpiperidine
and
dibenzosuberenone derivatives according to the reported
method.[28,29] 2-Chloro-7-methoxydibenzosuberenone (6a) was
prepared by the reported procedure[30] as shown in Scheme 1a.
Wittig reaction of 2-formyl-5-methoxybenzoic acid (3) with the
phosphonium salt of m-chlorobenzyl chloride (4a) afforded a
mixture of cis- and trans 5a. Transformation of 5a into the acid
chloride, followed by ring-closing Friedel-Crafts acylation, gave
compound 6a. Compound 6b bearing a bromo atom was
similarly prepared from 3, though the yield of compound 5
without a halogen atom was relatively low. So, another synthetic
scheme was employed, as shown in Scheme 1b. Bromination of
2-methyl-4-methoxybenzoic acid ester (7a) with NBS afforded
compound 8a, which reacted with benzaldehyde (9a) to yield
10a. After ester hydrolysis, 11a was cyclized to 12a. Other
dibenzosuberenone moieties (12b
– 12g) were prepared
similarly.
Reaction of the Grignard reagent prepared from 4-chloro-N-
methylpiperidine with or 12 afforded cyproheptadine
derivatives (2a – 2i). Demethylation of compounds 2a – 2i
6
yielded the corresponding hydroxy compounds (2j
–
2p)
Scheme 1. Synthesis of dibenzosuberenones (6 and 12)
(Scheme 2).
2
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The SET7/9-inhibitory activities of the synthesized compounds
were evaluated by the AlphaLISA method (Table 1).[31]Most
compounds showed decreased inhibitory activities, including
compounds with a hydrophobic bromo group (2h and 2i). Only
the compound with a hydrophilic hydroxy group at the 2-position
(2n) showed more potent activity: its IC50 was 0.41 µM. The
enhancement of the inhibitory activity of compound 2n
compared with cyproheptadine was also observed in the
immunoblotting for the detection of methylation level of H3K4
(Figure S1). Interestingly, the isomeric compound 2j with the
hydroxy group at the 3-position (IC50: 7.1 µM), and compounds
2o and 2p with an additional bromo group on the other phenyl
ring showed decreased activities (IC50: 2.1 and 6.8 µM). Most
compounds showed only weak inhibitory activity against another
HKMT, G9a (Table S1).
To further explore the effects of hydrophilic groups, we
synthesized compounds 13a - d with cyano, aminocarbonyl,
amino, acetamido and carboxylate groups, respectively. These
compounds were prepared from 2-bromocyproheptadine (2i), as
shown in Scheme 3. The bromo group of 2i could be
transformed to a cyano group by reaction with potassium
ferrocyanide and palladium catalyst[32] to yield 13a, which was
subsequently transformed to aminocarbonyl compound (13b).
Compound 13c bearing an amino group was prepared from 2i
by reaction with sodium azide and cuprous oxide,[33] and its
acetylated derivative 13d was also synthesized. In addition, 2i
was shown to be easily transformed to those with various
functional groups, which would be useful for further work.
Scheme 2. Synthesis of cyproheptadine derivatives 2
Table 1. Inhibitory activity of 2 towards SET7/9.
aIC50 determination experiments were performed in triplicate, and each
standard deviation is also shown.
Scheme 3. Synthesis of cyproheptadine derivatives (13) with various
hydrophilic functional groups.
3
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The inhibitory activities of compounds 13 are summarized in
Table 2. Compounds 13b and 13c with a 2-aminocarbonyl or 2-
amino group showed similar enhancement of inhibitory activity to
compound 2n bearing a 2-hydroxy group, while 13d with a 2-
acetamido group showed weaker activity. So, for the
improvement of SET7/9-inhibitory activity, introduction of a
suitable hydrophilic group at an appropriate position is
necessary
electron density map. Addition of bromine to the other side of the
benzene ring of 2n (i.e. 2o and 2p) decreased the inhibitory
activity (Table 1). The bromine atom would be close to the
carbonyl group of Trp260 in 2o or to the side chain of Trp260 in
2p. In either case, the interaction seems to be unfavorable for
inhibitor binding.
Table 3. Crystallographic data collection and refinement statistics
SET7/9–Sinefungin–2n
Table 2. Inhibitory activity towards SET7/9.
Data Collection
Space group
Unit-cell dimensions (Å)
Resolution range (Å)
Rsym
P21212
a = 110.18, b = 34.96, c = 67.87
50 – 1.69 (1.72 – 1.69) [a]
0.107 (1.284)
0.117 (1.425)
0.046 (0.604)
0.997 (0.467)
22.6 (1.9)
Rmeas
Rpim
aIC50 determination experiments were performed in triplicate, and each
standard deviation is also shown.
CC1/2
I/σ(I)
In order to clarify the reason for the favorable effect of the 2-
hydroxy group of 2n, we conducted X-ray crystallographic
analysis of SET7/9 complexed with a cyproheptadine derivative.
We crystallized the ternary complex of SET7/9, a coenzyme
analogue Sinefungin, and 2n by the soaking method, and
determined its structure at the resolution of 1.69 Å (Table 3 and
Figure 2A). Compound 2n was clearly observed in the electron
density map, and showed a bulge corresponding to the 2-
hydroxy group on the benzene ring near the substrate-binding
site (Figure 2B). Residues from Tyr337 to Ser345 were visible in
the electron density map, although residues 346–348 just before
the C-terminal helix could not be refined because the electron
density was too low.
Completeness (%)
Redundancy
99.9 (99.3)
6.3 (5.4)
Refinement
Resolution range (Å)
No. of reflections (total)
No. of reflections (test set)
Rwork/Rfree
42.77 – 1.69
30193
1762
0.168/0.200
Compound 2n binds to SET7/9 at the same position and with the
same orientation as cyproheptadine (Figure 3). As in the
No. of atoms, Average B-factors (Å2)
Total
SET7/9–SAM–cyproheptadine
complex,[26]
the
N-
2217, 30.7
1952, 30.1
methylpiperidine nitrogen atom of 2n forms a 2.6 Å hydrogen
bond with the carbonyl oxygen atom of Thr266, and the
methylpiperidine ring makes hydrophobic contacts with the side
chain of Tyr335. Importantly, the hydroxy group of 2n forms 2.6
Å and 3.0 Å hydrogen bonds with the side-chain oxygen atom
and main-chain nitrogen atom of Asp338, respectively, which
contributes to the increased affinity for SET7/9 (Figures 2B and
3). In the case of the 3-hydroxy compound 2j, such an
interaction presumably does not exist, because the hydroxy
group lies further from the protein and is more exposed to the
solvent. The dibenzosuberene moiety is surrounded by
hydrophobic residues Pro341, Pro342, Pro350, Trp352 and
Tyr353. Interestingly, the side chain of Tyr337, which occupies
the cyproheptadine-binding site to form the lysine access
channel when bound with a substrate peptide, is completely
flipped outwards (Figures 2B and 3). In the SET7/9–SAM–
cyproheptadine complex, the electron density of Tyr337 is
absent, and only weak electron density of a short stretch in the
loop region is visible (Figure 3).[26] On the other hand, the
interactions between compound 2n and Asp338 are likely to
restrain the conformation of the flexible loop, thereby making
Tyr337 and the subsequent residues clearly visible in the
Protein
Coenzyme (Sinefungin)
Inhibitor (2n)
27,
23,
18,
17.9
24.3
55.8
Glycerol
Water
197, 37.3
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
Ramachandran plot (%)
Favored
0.006
0.89
95.5
4.5
0
Allowed
Outliers
[a] Values in parentheses are for the highest resolution shell.
4
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Figure 2. X-ray crystallographic analysis of the compound 2n-bound SET7/9 SET domain. A) Ribbon representation of the overall structure. α-Helices, β-strands
and the loop regions are shown in red, cyan and pale orange, respectively. Compound 2n (magenta) and Sinfungin (pink) are shown in stick models. B)
Compound 2n (magenta) and surrounding residues represented by a stick model are shown in stereo. Hydrogen bonds are indicated by dashed lines. A
simulated annealing 2mFo-DFc omit map for the compound contoured at 1.2 σ is shown in gray mesh.
We also tried co-crystallization of 13b and 13c with SET7/9–
Sinefungin, as well as the soaking method, but without success.
Simple substitution of the hydroxy group of 2n with an
aminocarbonyl group would cause a steric clash with the side
chain of Asp338. Therefore, the flexible loop might form a
different conformation to accommodate these compounds (i.e.
13b and 13c) for submicromolar inhibitory activity (IC50)
comparable to 2n, which might alter the crystal packing.
Superimposition of the structures of SET7/9–Sinefungin bound
with compound 2n and SET7/9–SAM bound with the reported
inhibitor (R)-PFI-2[21] is shown in Figure 4. The both inhibitors
block the binding tunnel for the side chain of the methylation
target lysine of a substrate protein between the peptide-binding
site and the coenzyme. The piperidine ring of compound 2n and
the pyrrolidine ring of (R)-PFI-2 occupy a similar position, and
the side chain of Tyr335 has hydrophobic contacts with both
rings. However, their binding modes are different in several
points. First, compound 2n occupies only the binding site of the
substrate lysine side chain, whereas (R)-PFI-2 additionally
blocks the site of the residues at the (-1) and (-2) positions from
the target lysine. Second, although both inhibitors interact with
and stabilize the flexible C-terminal loop, its conformations in the
two complexes largely differ. In particular, Tyr337 in compound
2n-bound SET7/9 is flipped out as described above, whereas
Tyr337 in (R)-PFI-2-bound SET7/9 does not change its
conformation. It should be noted that although the compound 2n
itself does not occupy the main chain-binding site of the
substrate protein, residues at Pro342, Gly343 and Lys344 in the
C-terminal loop occupy the binding groove for the side chain of
the residue at the (-2) position from the target lysine. Third,
hydrophilic interactions formed by the two inhibitors are distinct
from each other and there are no common hydrophilic
interactions between the two complexes (Figure 4). Thus, the
binding mode of compound 2n toward SET7/9 is different from
that of (R)-PFI-2, confirming its unique scaffold as a SET7/9
inhibitor.
Our results suggest that introduction of a hydrophilic functional
group of an appropriate type at an appropriate position in
cyproheptadine (1) is necessary to increase the inhibitory
potency. Similar tight restriction was also observed in our
previous report,[27] where a change of molecular shape due to
change of the linking group between the tricyclic
dibenzosuberene moiety and N-methylpiperidine or replacement
of the 10,11-olefinic bond of dibenzosuberene, or even a small
change due to reduction of 10,11-olefinic bond, caused a drastic
decrease of the inhibitory activity.
5
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Figure 3. Stereoview of the superimposition of SET7/9–Sinefungin–compound 2n and SET7/9–SAM–cyproheptadine. SET7/9 in the complex with compound 2n
is shown in pale orange, and the residues from Tyr337 to Ser345, which became clearly visible in this structure, are colored orange. Compound 2n is shown in
magenta. SET7/9 in the complex with cyproheptadine is shown in pale blue color, and cyproheptadine is shown in cyan.
Figure 4. Stereoview of the superimposition of SET7/9–Sinefungin–compound 2n and SET7/9-SAM-(R)-PFI-2. The former complex is depicted as follows:
SET7/9 (orange), Sinefungin (light pink), compound 2n (magenta), key residue labels (black), and hydrogen bonds (dashed black lines). The latter complex is
depicted as follows: SET7/9 (light green), SAM (gray), (R)-PFI-2 (dark green), key residues labels (green), and hydrogen bonds (dashed green lines).
Conclusions
Experimental Section
We have developed a potent SET7/9 inhibitor by structural
Chemistry
development of our previously obtained inhibitor, cyproheptadine,
with the aid of a fine co-crystal structure. The presence of a
hydrophilic group on the dibenzosuberene moiety is important,
but its permissible size and location are tightly restricted. To
date, only a few inhibtors such as (R)-PFI-2 have been repored,
and the study of structure and activity relationship seems still
immature. Our newly developed inhibitor 2n and the structural
insights should be helpful for developing superior SET7/9
inhibitors, which might be candidate agents for treatment of
diseases such as breast cancer.
General: All reagents were purchased from Sigma-Aldrich Chemical,
Tokyo Kasei Kogyo, Wako Pure Chemical Industries, and Kanto Kagaku.
Silica gel for column chromatography was purchased from Kanto Kagaku.
NMR spectra were recorded on a Bruker Avance 400 or Bruker Avance
500 spectrometer. Mass spectral data was obtained on a Brucker
Daltonics microTOF-2focus in the positive and negative ion detection
modes. Melting points were taken on a Yanagimoto micro melting point
apparatus and are uncorrected.
6
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Preparation of 6: m-Chlorobenzyl chloride (4a, 1.3 g, 8.4 mmol) was
added to a solution of triphenylphosphine (2.2 g, 8.4 mmol) in methanol
(10 ml), and the reaction mixture was heated under reflux for 2 h under
argon. The reaction mixture was cooled to 0°C, then 3 (1.0 g, 5.6 mmol)
was added, followed by a solution of sodium methoxide (0.75 g, 14
mmol) in methanol (15 ml). The reaction mixture was stirred overnight,
and poured into ice water. The suspension was washed with
dichloromethane, acidified with aqueous hydrochloric acid, and then
extracted with dichloromethane. The organic phase was dried over
sodium sulfate, and the solvent was removed in vacuo. The residue was
purified by silica gel column chromatography (ethyl acetate : n-hexane =
1 : 5) to afford a mixture of cis- and trans-5a (4 : 1, 0.40 g, 1.4 mmol, y.
25%) as white powder, which was used in the next reaction without
further purification. The obtained mixture was dissolved in
dichloromethane (7 ml), and a solution of thionyl chloride (1.1 ml) in
dichloromethane (3 ml) was slowly added to the solution. The reaction
mixture was heated under reflux for 1.5 h. Then, a suspension of
aluminium chloride (0.22 g) in dichloromethane (10 ml) was added, and
the reaction mixture was stirred overnight at room temperature. The
reaction mixture was poured into ice water, and the aqueous phase was
extracted with dichloromethane. The organic phase was dried over
sodium sulfate, and the solvent was removed in vacuo. The residue was
purified by silica gel column chromatography (ethyl acetate : n-hexane =
1 : 20), and 6a (0.29 g, 1.0 mmol, y. 77%) was obtained as a brown
7.42 (d, J = 8.8 Hz, trans-2H), 7.27 (d, J = 8.4 Hz, cis-2H), 7.15 (d, J =
2.4 Hz, trans-1H), 7.10 (d, J = 12.0 Hz, cis-1H), 6.94 (d, J = 8.4 Hz, cis-
2H), 6.85 (d, J = 16.4 Hz, trans-1H), 6.83 (dd, J = 2.4, 8.8 Hz, trans-1H),
6.81 (dd, J = 2.4, 8.8 Hz, cis-1H), 6.64 (d, J = 2.4 Hz, cis-1H), 6.56 (d, J =
12.0 Hz, cis-1H), 3.90 (s, trans-3H), 3.89 (s, trans-3H), 3.86 (s, cis-3H),
3.63 (s, cis-3H).
10c (obtained as a mixture (2 : 1) of cis- and trans-compounds): 1H NMR
(CDCl3, 500 MHz) δ 8.11 (d, J = 16.5 Hz, trans-1H), 8.03 (d, J = 8.5 Hz,
cis-1H), 8.00 (d, J = 9.0 Hz, trans-1H), 7.71-7.70 (m, trans-1H), 7.50 (brd,
J = 7.5 Hz, trans-1H), 7.43-7.40 (m, trans-1H), 7.27-7.23 (m, cis-2H +
trans-1H), 7.18-7.16 (m, trans-1H), 7.15 (d, J = 12.5 Hz, cis-1H), 7.04-
6.98 (m, cis-2H), 6.92-6.83 (m, trans-2H), 6.84 (dd, J = 2.5, 9.0 Hz, cis-
1H), 6.68 (d, J = 2.5 Hz, cis-1H), 6.59 (d, J = 12.5 Hz, cis-1H), 3.93 (s,
trans-3H), 3.92 (s, trans-3H), 3.89 (s, cis-3H), 3.65 (s, cis-3H).
10e (obtained as a cis-compound): 1H NMR (CDCl3, 400 MHz) δ 8.01 (m,
1H), 7.34-7.32 (m, 2H), 7.25-7.13 (m, 3H), 7.11 (d, J = 12.0 Hz, 1H),
6.99-6.92 (m, 2H), 6.57 (d, J =12.0 Hz, 1H), 3.90 (s, 3H).
10f (obtained as a cis-compound): 1H NMR (CDCl3, 500 MHz) δ 7.50 (d, J
= 2.5 Hz, 1H), 7.17-7.07 (m, 6H), 6.98 (d, J = 12.0 Hz, 1H), 6.85 (dd, J =
2.5, 8.5 Hz, 1H), 6.59 (d, J = 12.0 Hz, 1H), 3.89 (s, 3H), 3.84 (s, 3H).
1
powder. H-NMR (CDCl3, 400 MHz) δ 8.21 (d, J = 8.4 Hz, 1H), 7.77 (d, J
10g (obtained as a mixture (2 : 1) of cis- and trans-compounds): 1H NMR
(CDCl3, 500 MHz) δ 7.91 (d, J = 16.5 Hz, trans-1H), 7.64 (d, J = 8.5 Hz,
trans-1H), 7.51 (d, J = 3.0 Hz, cis-1H), 7.46 (d, J = 8.5 Hz, trans-2H),
7.44 (d, J = 2.5 Hz, trans-1H), 7.38 (d, J = 8.5 Hz, trans-2H), 7.26 (d, J =
8.5 Hz, cis-2H), 7.08 (d, J = 8.5 Hz, cis-1H), 7.08-7.05 (m, trans-1H),
7.02 (d, J = 12.0 Hz, cis-1H), 6.94 (d, J = 8.5 Hz, cis-2H), 6.84 (dd, J =
3.0, 8.5 Hz, cis-1H), 6.84 (d, J = 16.5 Hz, trans-1H), 6.51 (d, J = 12.0 Hz,
cis-1H), 3.93 (s, trans-3H), 3.89 (s, cis-3H), 3.86 (s, trans-3H), 3.84 (s,
cis-3H).
= 3.2 Hz, 1H), 7.52-7.46 (m, 3H), 7.22 (dd, J = 2.8, 8.4 Hz, 1H), 7.06 (d, J
= 12.0 Hz, 1H), 6.95 (d, J = 12.0 Hz, 1H), 3.94 (s, 3H).
6b was similarly prepared from m-bromobenzyl chloride (4b) and
compound 3. 1H-NMR (CDCl3, 400 MHz) δ 8.13 (d, J = 8.8 Hz, 1H), 7.77
(d, J = 2.8 Hz, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.63 (dd, J = 2.0, 8.8 Hz, 1H),
7.50 (d, J = 8.4 Hz,1H), 7.23 (dd, J = 2.8, 8.4 Hz, 1H), 7.07 (d, J = 12.0
Hz, 1H), 6.85 (d, J = 12.0 Hz, 1H), 3.95 (s, 3H).
Preparation of 10: The reaction mixture of 7a (3.8 g, 21 mmol) and N-
bromosuccinimide (4.1 g, 23 mmol) in chlorobenzene (30 ml) was heated
to 70°C, and a solution of 2,2'-azobis(isobutyronitrile) (0.13 g, 0.80 mmol)
in chlorobenzene (10 ml) was added. This mixture was stirred for 1 h at
70°C, then cooled to room temperature, and the solvent was removed in
vacuo. The residue containing compound 8a was used for the next step
without purification. It was added to a solution of triphenylphosphine (5.5
g, 21 mmol) in acetone (25 ml), and the reaction mixture was refluxed for
1 h. It was cooled to room temperature, and the precipitate was collected.
This precipitate and sodium methoxide (1.3 g, 25 mmol) were dissolved
in methanol (30 ml), and the solution was stirred for 30 min at room
temperature, and then heated under reflux. Benzaldehyde (9a, 1.1 g, 11
mmol) was added, and stirring was continued for 6 h under reflux. The
reaction mixture was poured into ice water, and the suspension was
washed with dichloromethane, acidified with aqueous hydrochloric acid,
and extracted with dichloromethane. The organic phase was dried over
sodium sulfate, and the solvent was removed in vacuo. The residue was
purified by silica gel column chromatography (ethyl acetate : n-hexane =
1 : 15), affording a mixture (2 : 1) of cis- and trans-10a (1.6 g, 5.8 mmol,
54%) as colorless oil. 1H NMR (CDCl3, 400 MHz) δ 8.09 (d, J = 16.0 Hz,
trans-1H), 7.98 (d, J = 9.0 Hz, cis-1H), 7.96 (d, J = 9.0 Hz, trans-1H),
7.56 (d, J = 7.5 Hz, trans-2H), 7.36 (t, J = 7.5 Hz, trans-2H), 7.27 (t, J =
7.5 Hz, trans-1H), 7.18-7.07 (m, cis-6H + trans-1H), 6.98 (d, J = 16.0 Hz,
trans-1H), 6.82 (dd, J = 2.5, 8.5 Hz, trans-1H), 6.79 (dd, J = 2.5, 8.5 Hz,
cis-1H), 6.68 (d, J = 2.5 Hz, cis-1H), 6.65 (d, J = 12 Hz, cis-1H), 3.91 (s,
trans-3H), 3.89 (s, trans-3H), 3.57 (s, cis-3H).
Preparation of 11: A mixture of 10a (1.26 g, 4.7 mmol) and potassium
hydroxide (0.53 g, 9.4 mmol) in methanol (20 ml) and water (0.8 ml) was
heated under reflux for 4h, and then cooled to room temperature. The
reaction mixture was poured into ice water, acidified with aqueous
hydrochloric acid, and extracted with ethyl acetate. The organic phase
was dried over sodium sulfate, and the solvent was removed in vacuo to
afford a mixture (2 : 1) of cis- and trans-11a (1.1 g, 4.4 mmol, 93%) as a
white solid. 1H NMR (CDCl3, 400 MHz) δ 8.14-8.08 (m, cis-1H + trans-2H),
7.56 (d, J = 7.2 Hz, trans-2H), 7.38 (t, J = 7.2 Hz, trans-2H), 7.28 (t, J =
7.2 Hz, trans-1H), 7.19-7.09 (m, cis-6H + trans-1H), 7.00 (d, J = 16.0 Hz,
trans-1H), 6.87 (dd, J = 2.4, 8.8 Hz, trans-1H), 6.82 (dd, J = 2.4, 8.4 Hz,
cis-1H), 6.70-6.67 (m, cis-2H), 3.93 (s, trans-3H), 3.57 (s, cis-3H).
11b – 11g were similarly prepared from corresponding compounds 10.
Those analytical data were given below.
11b (obtained as a mixture (5 : 2) of cis- and trans-compounds): 1H NMR
(CDCl3, 400 MHz) δ 8.13-8.08 (m, cis-1H + trans-2H), 7.49 (d, J = 8.8 Hz,
trans-2H), 7.42 (d, J = 8.8 Hz, trans-2H), 7.28 (d, J = 8.4 Hz, cis-2H),
7.16 (d, J = 2.4 Hz, trans-1H), 7.13 (d, J = 12.0 Hz, cis-1H), 6.95 (d, J =
8.4 Hz, cis-2H), 6.91 (d, J = 15.6 Hz, trans-1H), 6.88 (dd, J = 2.4, 8.8 Hz,
trans-1H), 6.84 (dd, J = 2.8, 8.8 Hz, cis-1H), 6.66 (d, J = 2.8 Hz, cis-1H),
6.58 (d, J = 12.0 Hz, cis-1H), 3.91 (s, trans-3H), 3.64 (s, cis-3H).
11c (obtained as a mixture (2 : 1) of cis- and trans-compounds): 1H NMR
(CDCl3, 500 MHz) δ 8.13-8.08 (m, cis-1H + trans-2H), 7.71-7.70 (m,
trans-1H), 7.57-7.52 (m, trans-1H), 7.42-7.39 (m, trans-1H), 7.27-7.22 (m,
cis-2H + trans-1H), 7.16 (d, J = 12.0 Hz, cis-1H), 7.16-7.14 (m, trans-1H),
7.02-6.97 (m, cis-2H), 6.92-6.87 (m, trans-2H), 6.85 (dd, J = 3.0, 8.5 Hz,
cis-1H), 6.67 (d, J = 3.0 Hz, cis-1H), 6.58 (d, J = 12.0 Hz, cis-1H), 3.92 (s,
trans-3H), 3.63 (s, cis-3H).
10b – 10g were similarly prepared from corresponding compounds 7, 8
and 9. Those analytical data were given below.
10b (obtained as a mixture (5 : 2) of cis- and trans-compounds): 1H NMR
(CDCl3, 400 MHz) δ 8.08 (d, J = 16.4 Hz, trans-1H), 8.00 (d, J = 8.8 Hz,
cis-1H), 7.97 (d, J = 8.4 Hz, trans-1H), 7.48 (d, J = 8.8 Hz, trans-2H),
7
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10.1002/cmdc.201800233
ChemMedChem
FULL PAPER
11e (obtained as a cis-compound): 1H NMR (CDCl3, 400 MHz) δ 8.13-
8.11 (m, 1H), 7.38-7.34 (m, 2H), 7.24-7.18 (m, 3H), 7.14 (d, J = 12.0 Hz,
1H).7.01-6.94 (m, 2H), 6.59 (d, J = 12.0 Hz, 1H).
temperature, and
a
solution of 12f (0.15 g, 0.63 mmol) in dry
tetrahydrofuran (8 ml) was added to it. The reaction mixture was stirred
overnight at room temperature. After the addition of saturated aqueous
ammonium chloride, the aqueous phase was extracted with ethyl acetate.
The organic phase was dried over sodium sulfate, and the solvent was
removed in vacuo. The residue was dissolved in 6 M hydrochloric acid
(20 ml), and the mixture was stirred under reflux for 3 h. After the addition
of aqueous sodium hydroxide, the aqueous phase was extracted with
ethyl acetate. The organic phase was dried over sodium sulfate, and the
solvent was removed in vacuo. The residue was purified by silica gel
column chromatography (dichloromethane : methanol = 8 : 1) to afford 2a
(0.14 mg, 0.44 mmol, y. 69%) as a white solid. 1H NMR (CDCl3, 400
MHz) δ 7.33-7.29 (m, 2H), 7.25-7.23 (m, 2H), 7.18 (brd, J = 7.5 Hz, 1H),
6.86 (d, J = 12.0 Hz, 1H), 6.81 (d, J = 12.0 Hz, 1H), 6.80 (dd, J = 2.5, 8.5
Hz, 1H), 6.74 (d, J = 2.5 Hz, 1H), 3.81 (s, 3H), 2.60-2.49 (m, 2H), 2.41-
2.31 (m, 2H), 2.24 (s, 3H), 2.25-2.05 (m, 4H); 13CNMR (CDCl3, 125 MHz)
δ 159.7, 140.4, 138.4, 135.1, 135.0, 133.5, 130.5, 129.5, 129.1, 128.5,
128.1, 128.0, 127.5, 126.3, 113.2, 112.4, 57.2, 55.4, 46.0, 30.2, 30.0;
HRMS (ESI+) Calcd. for C22H24NO [M+H]+: 318.1852. Found 318.1853.
11f (obtained as a cis-compound): 1H NMR (CDCl3, 500 MHz) δ 7.64 (d, J
= 2.5 Hz, 1H), 7.19-7.08 (m, 6H), 7.04 (d, J = 12.0 Hz, 1H), 6.90 (dd, J =
2.5, 9.0 Hz, 1H), 6.62 (d, J = 12.0 Hz, 1H), 3.85 (s, 3H).
11g (obtained as a mixture (2 : 1) of cis- and trans-compounds): 1H NMR
(CDCl3, 500 MHz) δ 7.98 (d, J = 16.5 Hz, trans-1H), 7.66 (d, J = 8.5 Hz,
trans-1H), 7.64 (d, J = 3.0 Hz, cis-1H), 7.59 (d, J = 2.5 Hz, trans-1H),
7.47 (d, J = 8.5 Hz, trans-2H), 7.39 (d, J = 8.5 Hz, trans-2H), 7.27 (d, J =
8.5 Hz, cis-2H), 7.14-7.11 (m, trans-1H), 7.12 (d, J = 8.5 Hz, cis-1H),
7.07 (d, J = 12.0 Hz, cis-1H), 6.95 (d, J = 8.5 Hz, cis-2H), 6.92 (dd, J =
3.0, 8.5 Hz, cis-1H), 6.86 (d, J = 16.5 Hz, trans-1H), 6.54 (d, J = 12.0 Hz,
cis-1H), 3.88 (s, trans-3H), 3.86 (s, cis-3H).
Preparation of 12: 11a (0.25 g, 0.98 mmol) was dissolved in
dichloromethane (20 ml), and thionyl chloride (0.13 g, 1.1 mmol) was
slowly added to the solution. The reaction mixture was heated under
reflux for 1.5 h, then aluminium chloride (0.17 g) was added to it, and
stirring was continued overnight at room temperature. The reaction
mixture was poured into ice water, and the aqueous phase was extracted
with dichloromethane. The organic phase was dried over sodium sulfate,
and the solvent was removed in vacuo. The residue was purified by silica
gel column chromatography (ethyl acetate : n-hexane = 1 : 3) to afford
12a (91 mg, 0.38 mmol, y. 39%) as a brown powder. 1H NMR (CDCl3,
500 MHz) δ 8.32-8.28 (m, 2H), 7.67-7.63 (m, 1H), 7.59-7.55 (m, 2H),
7.12 (dd, J = 2.0, 8.0 Hz, 1H), 7.07 (d, J = 12.5 Hz, 1H), 7.01 (d, J = 12.5
Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 3.95 (s, 3H).
2b – 2i were similarly prepared from corresponding compounds 6 and 12.
Those analytical data are given below.
2b: 1 H NMR (CDCl3, 500 MHz) δ 7.35-7.33 (m, 2H), 7.23 (d, J = 8.5 Hz,
1H), 7.15 (d, J = 8.5 Hz, 1H), 6.87 (d, J = 11.5 Hz, 1H), 6.81 (dd, J = 3.0,
8.5 Hz, 1H), 6.72 (d, J = 11.5 Hz, 1H), 6.71 (d, J = 3.0 Hz, 1H), 3.82 (s,
3H), 2.62-2.55 (m, 2H), 2.42-2.34 (m, 2H), 2.26 (s, 3H), 2.26-2.17 (m,
2H), 2.11-2.06 (m, 2H); 13C NMR (CDCl3, 125 MHz) 160.0, 140.04,
140.01, 136.0, 134.2, 132.5, 131.3, 131.1, 129.8, 129.7, 129.5, 128.0,
121.7, 113.3, 112.9, 57.1, 55.6, 46.0, 30.2, 30.1; HRMS (ESI+) Calcd. for
C22H23BrNO [M + H]+: 396.0958. Found 396.0958.
12b – 12g were similarly prepared from corresponding compounds 11.
Those analytical data are given below.
1
2c: H-NMR (CDCl3, 400 MHz) δ 7.44-7.40 (m, 2H), 7.23 (d, J = 8.4 Hz,
1H), 7.04 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 11.6 Hz, 1H), 6.81 (dd, J = 2.4,
8.4 Hz, 1H), 6.71 (d, J = 2.4 Hz, 1H), 6.70 (d, J = 11.6 Hz, 1H), 3.81 (s,
3H), 2.54-2.51 (m, 2H), 2.40-2.28 (m, 2H), 2.25 (s, 3H), 2.25-2.12 (m,
2H), 2.10-2.03 (m, 2H); 13C-NMR (CDCl3, 125 MHz) δ159.9, 140.2, 137.1,
137.0, 135.7, 132.5, 131.9, 130.6, 130.3, 130.1, 129.8, 127.7, 127.6,
120.0, 113.3, 112.5, 57.0, 55.4, 45.9, 30.1, 30.0; HRMS (ESI+) Calcd. for
C22H23BrNO [M + H]+: 396.0958. Found 396.0956.
12b: 1 H NMR (CDCl3, 400 MHz) δ 8.43 (d, J = 2.0 Hz, 1H), 8.25 (d, J =
8.8 Hz, 1H), 7.68 (dd, J = 2.0, 8.4 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.11
(dd, J = 2.8, 8.8 Hz, 1H), 7.02-6.95 (m, 3H), 3.93 (s, 3H).
12c: 1 H NMR (CDCl3, 500 MHz) δ 8.26 (d, J = 8.5 Hz, 1H), 8.16 (d, J =
8.5 Hz, 1H), 7.68 (d, J = 2.0 Hz, 1H), 7.64 (dd, J = 2.0, 8.5 Hz, 1H), 7.11
(dd, J = 2.5, 8.5 Hz, 1H), 7.02 (d, J = 12.0 Hz, 1H), 6.96 (d, J = 2.5 Hz,
1H), 6.92 (d, J = 12.0 Hz, 1H), 3.92 (s, 3H).
1
2d: H-NMR (CDCl3, 400 MHz) δ 7.25-7.21 (m, 3H), 7.08 (d, J = 8.8 Hz,
1H), 6.87 (d, J = 11.6 Hz, 1H), 6.80 (dd, J = 2.4, 8.8 Hz, 1H), 6.70 (d, J =
2.4 Hz, 1H), 6.68 (d, J = 11.6 Hz, 1H), 3.80 (s, 3H), 2.55-2.52 (m, 2H),
2.39-2.31 (m, 2H), 2.24-2.15 (m, 2H), 2.24 (s, 3H), 2.13-2.04 (m, 2H);
13C-NMR (CDCl3, 125 MHz) δ159.9, 140.3, 136.7, 136.6, 135.9, 132.4,
132.0, 131.8, 129.8, 129.8, 127.8, 127.7, 127.7, 127.4, 113.3, 112.5,
57.0, 55.4, 45.9, 30.2, 30.0; HRMS (ESI+) Calcd. for C22H23ClNO [M +
H]+ : 352.1463. Found 352.1454.
12d (obtained from : 1 H NMR (CDCl3, 400 MHz) δ 8.37 (d, J = 2.4 Hz,
1H), 8.21 (d, J = 8.0 Hz, 1H), 7.72 (dd, J = 2.4, 8.4 Hz, 1H), 7.68-7.63 (m,
1H), 7.59-7.54 (m, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 12.0 Hz, 1H),
7.00 (d, J = 12.0 Hz, 1H).
12e: 1 H NMR (CDCl3, 400 MHz) δ 8.23-8.20 (m, 1H), 8.08 (d, J = 8.8 Hz,
1H), 7.70 (d, J = 2.0 Hz, 1H), 7.68-7.63 (m, 2H), 7.59-7.53 (m, 2H), 7.10
(d, J = 12.0 Hz, 1H), 6.95 (d, J = 12.0 Hz, 1H).
2e: 1 H NMR (CDCl3, 400 MHz) δ 7.33-7.29 (m, 2H), 7.24-7.17 (m, 2H),
7.10 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 12.0 Hz, 1H), 6.89 (dd, J = 2.4, 8.4
Hz, 1H), 6.85 (d, J = 12.0 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 2.53-2.49 (m,
2H), 2.38-2.32 (m, 2H), 2.23 (s, 3H), 2.22-2.15 (m, 2H), 2.11-2.05 (m,
2H); 13C NMR (CDCl3, 125 MHz) 158.1, 139.7, 136.0, 135.2, 134.8,
133.1, 132.5, 131.6, 131.0, 129.8, 128.6, 128.4, 127.9, 126.3, 114.4,
112.5, 57.4, 55.5, 46.2, 30.32, 30.28; HRMS (ESI+) Calcd for C22H24NO
[M + H]+: 318.1852. Found 318.1849.
12f: 1H NMR (CDCl3, 400 MHz) δ 8.27-8.25 (m, 1H), 7.77 (d, J = 2.8 Hz,
1H), 7.65-7.61 (m, 1H), 7.55-7.50 (m, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.21
(dd, J = 2.8, 8.8 Hz, 1H), 7.04 (d, J = 12.0 Hz, 1H), 6.97 (d, J = 12.0 Hz,
1H), 3.94 (s, 3H).
12g: 1H NMR (CDCl3, 500 MHz) δ 8.40 (d, J = 2.0 Hz, 1H), 7.75 (d, J =
2.5 Hz, 1H), 7.72 (dd, J = 2.0, 8.5 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.41
(d, J = 8.0 Hz, 1H), 7.23 (dd, J = 2.5, 8.5 Hz, 1H), 7.06 (d, J = 12.0 Hz,
1H), 6.91 (d, J = 12.0 Hz, 1H), 3.95 (s, 3H).
2f: 1 H NMR (CDCl3, 500 MHz) δ 7.35-7.32 (m, 2H), 7.16 (d, J =8.5 Hz,
1H), 7.09 (d, J = 8.0 Hz, 1H), 6.91 (dd, J = 2.5, 8.5 Hz, 1H), 6.87 (d, J =
11.5 Hz, 1H), 6.83 (d, J = 11.5 Hz, 1H), 6.81 (d, J = 2.5 Hz, 1H), 3.80 (s,
3H), 2.57-2.50 (m, 2H), 2.39-2.30 (m, 2H), 2.24 (s, 3H), 2.21-2.16 (m,
13
Preparation of 2: Magnesium (46 mg, 1.9 mmol) and iodine (2 pieces)
were added to a two-necked flask. Under an argon atmosphere, dry
tetrahydrofuran (8 ml), dibromoethane (2 drops) and 4-chloro-1-
methylpiperidine (0.25 g, 1.9 mmol) were added to the flask. The mixture
was heated under reflux with stirring for 1.5 h, then cooled to room
2H), 2.10-2.06 (m, 2H);
C NMR (CDCl3, 125 MHz) δ 158.1, 141.0,
136.1, 135.7, 133.7, 131.7, 131.6, 131.5, 130.9, 130.2, 129.7, 129.1,
121.9, 114.4, 112.5, 57.11, 57.08, 55.3, 46.0, 30.2, 30.1; HRMS (ESI+)
Calcd. for C22H23BrNO [M + H]+: 396.0958. Found 396.0957.
8
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10.1002/cmdc.201800233
ChemMedChem
FULL PAPER
2g: 1H NMR (CDCl3, 400 MHz) δ 7.44 (d, J = 2.0 Hz, 1H), 7.42 (dd, J =
2.0, 8.4 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.91
(dd, J = 2.8, 8.4 Hz, 1H), 6.89 (d, J = 11.6 Hz, 1H), 6.82 (d, J = 2.8 Hz,
1H), 6.81 (d, J = 11.6 Hz, 1H), 3.80 (s, 3H), 2.61-2.56 (m, 2H), 2.41-2.34
(m, 2H), 2.26 (s, 3H), 2.28-2.08 (m, 4H); 13C NMR (CDCl3, 125 MHz)
158.1, 138.2, 136.6, 135.5, 132.4, 132.3, 131.9, 131.0, 130.8, 130.6,
130.0, 129.9, 129.6, 119.9, 114.6, 112.6, 56.9, 55.3, 45.8, 29.8, 29.7;
HRMS (ESI+) Calcd. for C22H23BrNO [M + H]+: 396.0958. Found
396.0948
8.4 Hz, 1H), 6.76 (br, 1H), 6.66 (d, J = 12.0 Hz, 1H), 3.08-2.99 (m, 2H),
2.77-2.47 (m, 4H), 2.50 (s, 3H), 2.44-2.31 (m, 2H); 13C NMR (CD3OD,
125 MHz) δ 159.7, 140.4, 138.3, 137.8, 136.9, 133.9, 133.4, 131.6, 130.7,
130.1, 129.0, 128.8, 127.9, 115.8, 115.3, 56.36, 56.34, 43.9, 28.6, 28.4;
HRMS (ESI+) Calcd. for C21H21ClNO [M + H]+: 338.1306. Found
338.1297.
2n: 1 H NMR (CD3OD, 500 MHz) δ 7.37-7.34 (m, 2H), 7.27 (td, J = 1.0,
7.5 Hz, 1H), 7.19 (dd, J = 1.5, 7.0 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H), 6.90
(d, J = 12.0 Hz, 1H), 6.82 (dd, J = 2.0, 8.5 Hz, 1H), 6.82 (d, J = 12.0 Hz,
1H), 6.77 (d, J = 2.0 Hz, 1H), 2.57-2.50 (m, 2H), 3.11-3.09 (m, 2H), 2.70-
2.64 (m, 2H), 2.65 (s, 3H), 2.57-2.50 (m, 2H), 2.39-2.30 (m, 2H); 13C
NMR (CD3OD, 125 MHz) δ 158.3, 140.6, 138.7, 138.1, 136.8, 132.9,
132.1, 131.2, 131.0, 130.4, 129.9, 129.7, 128.6, 117.4, 116.2, 57.62,
57.59, 45.2, 29.80, 29.76; HRMS (ESI+) Calcd. for C21H22NO [M + H]+:
304.1696. Found 304.1702.
1
2h: H NMR (CDCl3, 400 MHz) δ 7.36-7.30 (m, 4H), 7.26 (d, J = 1.6, 7.2
Hz, 1H), 7.18 (dd, J = 1.6, 7.2 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 6.94 (d, J
= 11.6 Hz, 1H), 6.84 (d, J = 11.6 Hz, 1H), 2.56-2.50 (m, 2H), 2.41-2.30
(m, 2H), 2.24 (s, 3H), 2.22-2.05 (m, 4H); 13C NMR (CDCl3, 125 MHz)
140.6, 138.6, 134.6, 133.7, 132.1, 131.6, 130.9, 129.8, 129.6, 129.2,
128.5, 128.2, 128.0, 126.5, 121.8, 57.00, 56.98, 45.9, 30.0; HRMS (ESI+)
Calcd. for C21H21BrN [M + H]+: 366.0852. Found 366.0859.
2o: 1 H NMR (CD3OD, 400 MHz) δ 7.40 (dd, J = 2.0, 8.4 Hz, 1H), 7.34 (d,
J = 2.0 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.85 (s,
2H), 6.83 (dd, J = 2.4, 8.0 Hz, 1H) , 6.75 (d, J = 2.4 Hz, 1H), 2.81-2.77 (m,
2H), 2.45-2.19 (m, 6H), 2.38 (s, 3H); 13C NMR (CD3OD, 125 MHz) δ
156.1, 140.5, 135.7, 133.8, 133.6, 132.7, 131.4, 130.2, 129.9, 129.7,
129.3, 129.1, 129.0, 121.2, 115.3, 113.9, 55.84, 55.80, 43.9, 28.52,
28.47; HRMS (ESI+) calcd. for C21H21BrNO [M + H]+: 382.0801. Found
382.0803.
2i: 1 H NMR (CDCl3, 400 MHz) δ 7.45 (br, 1H), 7.42 (d, J = 2.0, 8.4 Hz,
1H), 7.35-7.31 (m, 2H), 7.26-7.24 (m, 1H), 7.18 (brd, J = 7.6 Hz, 1H),
7.06 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 12.0 Hz, 1H), 6.81 (d, J = 12.0 Hz,
1H), 2.54-2.50 (m, 2H), 2.37-2.30 (m, 2H), 2.23 (s, 3H), 2.19-2.14 (m,
2H), 2.10-2.05 (m, 2H); 13C NMR (CDCl3, 125 MHz)
139.0, 137.9,
136.7, 136.0, 134.5, 132.4, 132.3, 130.7, 130.6, 130.1, 129.5, 128.5,
128.4, 128.1, 126.5, 119.9, 57.13, 57.11, 46.0, 30.2; HRMS (ESI+) calcd.
for C21H21BrN [M + H]+: 366.0852. Found 366.0845.
2p: 1 H NMR (DMSO-d6, 500 MHz) δ 9.52 (s, 1H), 7.63 (d, J = 2.0 Hz,
1H), 7.55 (d, J = 2.0, 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.01 (d, J =
8.5 Hz, 1H), 6.93 (d, J = 12.0 Hz, 1H) , 6.91 (d, J = 12.0 Hz, 1H), 6.84
(dd, J = 2.5, 8.5 Hz, 1H), 6.78 (d, J = 2.5 Hz, 2H), 2.48-2.40 (m, 2H),
2.30-2.23 (m, 2H), 2.14 (s, 3H), 2.06-1.97 (m, 4H); 13C NMR (DMSO-d6,
125 MHz) δ 156.2, 137.3, 136.7, 135.5, 134.9, 132.1, 130.6, 130.4, 129.7,
129.4, 129.0, 128.9, 119.8, 115.5, 114.1, 55.4, 55.3, 43.1, 27.7; HRMS
(ESI+) Calcd. for C21H21BrNO [M + H]+: 382.0801. Found 382.0791.
Preparation of compound 2j: A solution of compound 2a (50 mg, 0.16
mmol) in dichloromethane (2 ml) was cooled to -78°C under an argon
atmosphere, and a 1.0 M solution of boron tribromide in dichloromethane
(0.78 ml, 0.78 mmol) was added to it. The reaction mixture was stirred for
2 h at room temperature, then poured into ice-water, and extracted with
dichloromethane. The organic phase was dried over sodium sulfate, and
the solvent was removed in vacuo. The residue was purified by silica gel
column chromatography (dichloromethane : methanol = 8 : 1) to afford 2j
1
(45 mg, 0.15 mmol, y. 92%) as a white solid. H NMR (CD3OD, 500 MHz)
Preparation of 13a: Sodium carbonate (20 mg, 0.19 mmol) and
potassium hexacyanoferrate(II) trihydrate (18 mg, 0.041 mmol) were
ground to a fine powder, placed in pressure tube, and dried in vacuo at
δ 7.41-7.31 (m, 3H), 7.25 (d, J = 8.5 Hz, 1H), 7.24 (brd, J = 7.5 Hz, 1H),
6.89 (d, J = 11.5 Hz, 1H), 6.82 (d, J = 11.5 Hz, 1H), 6.79 (dd, J = 2.0, 8.5
Hz, 1H), 6.70 (d, J = 2.0 Hz, 1H), 3.31-3.25 (m, 2H), 2.88 (br, 2H), 2.80 (s,
3H), 2.70-2.56 (m, 2H), 2.51-2.37 (m, 2H); 13C NMR (CD3OD, 125 MHz)
δ 160.0, 141.3, 139.6, 139.2, 137.2, 132.6, 132.0, 130.3, 130.2, 130.1,
129.71, 129.68, 128.9, 128.94, 128.88, 116.2, 115.9, 57.31, 57.28, 44.7,
29.4, 29.3; HRMS (ESI+) Calcd. for C21H22NO [M + H]+: 304.1696. Found
304.1685.
80°C overnight.
methylpyrrolidone (1.5 ml),
A
solution of 2i (70 mg, 0.19 mmol) in N-
3.8 mM solution of 1,1'-
a
bis(diphenylphosphino)ferrocene in N-methylpyrrolidone (0.1 ml, 0.38
μmol) and a 1.9 mM solution of palladium acetate in N-methylpyrrolidone
(0.1 ml, 0.19 μmol) were then added to it. The atmosphere in the
pressure tube was replaced with argon, and the tube was sealed. The
contents were heated with stirring at 120°C for 19 h, then cooled to room
temperature, poured into water, and extracted with dichloromethane. The
organic phase was dried over sodium sulfate, and the solvent was
removed in vacuo. The residue was purified by silica gel column
chromatography (dichloromethane : methanol = 10 : 1) to afford 13a (34
mg, 0.11 mmol, y. 57%) as a brown solid. 1H NMR (CDCl3, 500 MHz) δ
7.60 (d, J = 1.5 Hz, 1H), 7.57 (dd, J = 1.5, 8.0 Hz, 1H), 7.39-7.26 (m, 4H),
7.19 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 11.5 Hz, 1H), 6.86 (d, J = 11.5 Hz,
1H), 2.54-2.52 (m, 2H), 2.39-2.30 (m, 2H), 2.24 (s, 3H), 2.20-2.04 (m,
4H); 13C NMR (CDCl3, 125 MHz) 143.7, 138.7, 137.4, 136.1, 134.7,
133.6, 132.5, 132.3, 131.0, 129.8, 129.1, 129.0, 128.9, 127.2, 119.2,
110.5, 57.3, 46.3, 30.5, 30.4; HRMS (ESI+) Calcd. for C22H21N2 [M + H]+:
313.1699. Found 313.1708.
2k – 2p were similarly prepared from corresponding compounds 2.
Those analytical data were given below.
2k: 1 H NMR (CD3OD, 400 MHz) δ 7.37 (dd, J = 2.0, 8.0 Hz, 1H), 7.28 (d,
J = 2.0 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.86 (d,
J = 11.6 Hz, 1H), 6.80 (d, J = 2.8, 8.4 Hz 1H), 6.71 (d, J = 11.6 Hz, 1H),
6.68 (d, J = 2.8 Hz, 1H), 3.01 (br, 2H), 2.70 (br, 2H), 2.51 (s, 3H), 2.51-
2.38 (m, 4H); 13C NMR (CD3OD, 125 MHz) δ 158.0, 138.9, 134.8, 134.2,
131.2, 130.1, 129.8, 129.5, 129.4, 126.61, 126.57, 121.0, 114.1, 113.9,
55.4, 55.3, 43.2, 27.9, 27.7; HRMS (ESI+) Calcd. for C21H21BrNO [M +
H]+: 382.0801. Found 382.0806.
2l: 1H-NMR (CDCl3, 500 MHz) δ 7.44 (d, J = 1.5 Hz, 1H), 7.38 (brd, J =
8.5 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.97 (brd, J = 8.5 Hz, 1H), 6.88 (d,
J = 12.0 Hz, 1H), 6.75 (brd, J = 8.5 Hz, 1H), 6.67 (d, J = 12.0 Hz, 1H),
6.62 (d, J = 2.0 Hz, 1H), 2.79-2.71 (m, 2H), 2.57-2.25 (m, 4H), 2.24 (s,
3H), 2.01-1.93 (m, 2H); 13C NMR (CDCl3, 125 MHz) δ 157.8, 140.0,
137.3, 136.8, 133.9, 133.6, 132.2, 130.9, 130.4, 130.3, 130.1, 127.2,
126.9, 120.3, 115.1, 115.0, 56.4, 45.8, 30.1, 29.3; HRMS (ESI+) Calcd.
for C21H21BrNO [M+H]+: 382.0801. Found 382.0790.
Preparation of compound 13b: A flame-dried flask fitted with a
magnetic stirring bar was charged with 13a (30 mg, 0.10 mmol),
potassium tert-butoxide (32 mg, 0.30 mmol) and tert-butanol (3 ml). The
reaction mixture was stirred at room temperature for 21 h under an argon
atmosphere. After addition of water, the aqueous phase was extracted
with dichloromethane. The organic phase was dried over sodium sulfate,
and the solvent was removed in in vacuo. The residue was purified by
silica gel column chromatography (dichloromethane : methanol = 5 : 1) to
afford 13b (15 mg, 0.045 mmol, y. 46%) as a white powder. 1H NMR
(CD3OD, 500 MHz) δ 7.91 (d, J = 2.0 Hz, 1H), 7.88 (dd, J = 2.0, 8.0 Hz,
2m: 1H-NMR (CDCl3, 400 MHz) δ 7.21-7.17 (m, 2H), 7.14, (dd, J = 2.0,
8.0 Hz), 6.91-6.88 (m, 1H), 6.86 (d, J = 12.0 Hz, 1H), 6.82 (dd, J = 2.4,
9
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10.1002/cmdc.201800233
ChemMedChem
FULL PAPER
1H), 7.42-7.24 (m, 5H), 7.04 (dd, J = 11.5 Hz, 1H), 7.01 (d, J = 11.5 Hz,
1H), 2.65-2.62 (m, 2H), 2.43-2.37 (m, 2H), 2.27 (s, 3H), 2.22-2.11 (m,
4H) ; 13C NMR (CD3OD, 125 MHz) 172.8, 144.3, 140.6, 137.2, 136.89,
136.88, 135.8, 134.1, 133.9, 132.1, 130.5, 130.4, 130.3, 130.2, 129.6,
128.8, 128.7, 58.6, 46.8, 31.44, 31.40; HRMS (ESI+) Calcd. for
C22H23N2O [M + H]+: 331.1805. Found 331.1801.
nitrogen. Data were collected at the BL17A beamline at the Photon
Factory (KEK, Tsukuba, Japan), and processed using HKL-2000 and the
CCP4 suite to 1.69 Å resolution. Molecular replacement was performed
with Phaser using the protein portion of the SET7/9–SAM–
cyproheptadine complex (PDB code 5AYF). The topology and parameter
files for the inhibitor were generated with the eLBOW module of PHENIX.
Model building and refinement were carried out with COOT and PHENIX,
respectively. The crystallographic statistics are summarized in Table 3.
The figures were prepared using PyMOL (Schrödinger, LLC). The
coordinates and structure factors have been deposited in the Protein
Data Bank (PDB) with the accession code 5YLT.
Preparation of compound 13c: 2i (0.10 g, 0.27 mmol), proline (40 mg,
0.35 mmol), sodium azide (35 mg, 0.54 mmol), and copper(I) oxide (39
mg, 0.27 mmol) were added to a round-bottomed flask. Under an argon
atmosphere, dimethyl sulfoxide (2.5 mL) was added to the flask, and the
reaction mixture was stirred at 100°C for 7 h. Saturated aqueous
ammonium chloride was added, and the aqueous phase was extracted
with dichloromethane. The organic phase was dried over sodium sulfate,
and the solvent was removed in vacuo. The residue was purified by silica
gel column chromatography (dichloromethane : methanol = 10 : 1) to
afford 13c (69 mg, 0.23 mmol, y. 84%) as a brown solid. 1H NMR (CDCl3,
500 MHz) δ 7.31-7.15 (m, 4H), 6.98 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 11.5
Hz, 1H), 6.78 (d, J = 11.5 Hz, 1H), 6.68 (dd, J = 2.5, 8.0 Hz, 1H), 6.20 (d,
J = 2.5 Hz, 1H), 3.61 (br, 2H), 2.40-2.33 (m, 2H), 2.27 (s, 3H); 13C NMR
(CD3OD, 125 MHz) 148.1, 141.5, 137.7, 137.0, 136.9, 134.7, 133.2,
132.6, 131.9, 130.9, 130.2, 130.0, 129.5, 128.1, 117.6, 116.5, 58.64,
58.61, 46.7, 31.32, 31.27; HRMS (ESI+) Calcd. for C21H23N2 [M + H]+:
303.1856. Found 303.1852.
Acknowledgements
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of
Education, Science, Sports and Culture, Japan (Grants No
15K08019 to TH, 26670052 to HK, 24613007 to TU, 26221204
to MY). We thank the BL17A beamline staff at the Photon
Factory (KEK, Tsukuba, Japan) for their help in data collection.
TH gratefully acknowledges funding from Terumo Life Science
Foundation. This work was also partially supported by Project for
Development of Innovative Research on Cancer Therapeutics,
the Platform for Drug Discovery, Informatics, and Structural Life
Science (MEXT, Japan), and JSPS Core-to-Core Program A,
Advanced Research Networks. A part of this research is based
on the Cooperative Research Project of Research Center for
Biomedical Engineering.
Preparation of compound 13d: A solution of 13c (20 mg, 0.066 mmol)
and acetic anhydride (2 drops) in pyridine (1 ml) was stirred at room
temperature for 5 h, then poured into ice-water, and extracted with ethyl
acetate. The organic phase was dried over sodium sulfate, and the
solvent was removed in vacuo. The residue was purified by silica gel
column chromatography (dichloromethane : methanol = 10 : 1) to afford
1
13d (11 mg, 0.029 mmol, y. 44%) as a white solid. H NMR (CD3OD, 400
MHz) δ 7.90 (s, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.51 (dd, J = 2.0, 8.0 Hz,
1H), 7.36-7.17 (m, 4H), 7.13 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 12 Hz, 1H),
6.86 (d, J = 12 Hz, 1H), 2.62-2.56 (m, 2H), 2.40-2.32 (m, 2H), 2.23 (s,
3H), 2.12 (s, 3H), 2.21-2.06 (m, 4H); 13C NMR (CD3OD, 125 MHz) δ
170.2, 138.8, 137.0, 135.2, 134.8, 134.6, 134.0, 133.5, 131.0, 130.3,
128.3, 128.0, 127.9, 127.6, 126.1, 119.4, 119.1, 56.3, 44.4, 29.0, 22.4;
HRMS (ESI+) Calcd. for C23H25N2O [M + H]+: 345.1961. Found 345.1954.
Keywords: SET7/9 • inhibitor • histone methyltransferase •
cyproheptadine • epigenetics
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Entry for the Table of Contents
New series of SET7/9 inhibitors: SET7/9 methylates not only histone but also non-histone proteins as substrates, so its inhibitors
are considered candidates for treatment of diseases. Here, we have developed a new family of inhibitors based on our previously
identified scaffold, cyproheptadine.
12
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