Steric structure–activity relationship of cyproheptadine derivatives as inhibitors of histone methyltransferase Set7/9

Article abstract of DOI:10.1016/j.bmc.2016.07.024

Set7/9 is a histone lysine methyltransferase, but it is also thought to be involved in a wide variety of pathophysiological functions. We previously identified cyproheptadine, which has a characteristic butterfly-like molecular conformation with bent tricyclic dibenzosuberene and chair-form N-methylpiperidine moieties, as a Set7/9 inhibitor. In this work, we synthesized several derivatives in order to examine the steric structure–inhibitory activity relationship. We found that even a small change of molecular shape due to reduction or replacement of the 10,11-olefinic bond of the tricyclic ring generally resulted in a drastic decrease of the inhibitory activity. Our results should be useful not only for development of more potent and selective inhibitors, but also for the construction of novel inhibitor scaffolds.

Full text of DOI:10.1016/j.bmc.2016.07.024

Bioorganic & Medicinal Chemistry 24 (2016) 4318–4323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Steric structure–activity relationship of cyproheptadine derivatives
as inhibitors of histone methyltransferase Set7/9
Takashi Fujiwara a, Kasumi Ohira a, Ko Urushibara b, Akihiro Ito c,d, Minoru Yoshida c,d,e, Misae Kanai b,
Aya Tanatani b, Hiroyuki Kagechika a, Tomoya Hirano a,
⇑
a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
b Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
c Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
d Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
e Drug Discovery Platforms Cooperation Division, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Set7/9 is a histone lysine methyltransferase, but it is also thought to be involved in a wide variety of
pathophysiological functions. We previously identified cyproheptadine, which has a characteristic but-
terfly-like molecular conformation with bent tricyclic dibenzosuberene and chair-form N-methylpiperi-
dine moieties, as a Set7/9 inhibitor. In this work, we synthesized several derivatives in order to examine
the steric structure–inhibitory activity relationship. We found that even a small change of molecular
shape due to reduction or replacement of the 10,11-olefinic bond of the tricyclic ring generally resulted
in a drastic decrease of the inhibitory activity. Our results should be useful not only for development of
more potent and selective inhibitors, but also for the construction of novel inhibitor scaffolds.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 17 June 2016
Revised 11 July 2016
Accepted 12 July 2016
Available online 12 July 2016
Keywords:
Set7/9
Inhibitor
Histone methyltransferase
Cyproheptadine
Epigenetics
1. Introduction
Dot1L.26–33 We have reported
a bisubstrate-type inhibitor,
DAAM-3, based on an aza-derivative of coenzyme adenosylhomo-
cysteine (AzaAdoMet) bearing an alkylamino group (Fig. 1a).34
Recently, the first practically selective and potent inhibitor, (R)-
PFI-2, was reported,35 and subsequently DC-S239 was also devel-
oped.36 In addition, we recently identified cyproheptadine (1a)
based on fluorescence-based assay system,37 which has been used
clinically as a serotonin receptor antagonist or histamine receptor
(H1) antagonist, as a Set7/9 inhibitor.38 Cyproheptadine is reported
to inhibit methylation-mediated stabilization of estrogen receptor
Histone lysine methyltransferases (HKMTs) are ‘writers’ of
histone protein methylation, transferring the methyl group of
S-adenosylmethionine (SAM) to specific lysine residues.1–4 The
HKMTs include SET domain-containing lysine methyltransferase
7/9 (Set7/9, also known as SETD7 or KMT7), which was reported
to methylate lysine 4 on histone H3 (H3K4), leading to activation
of gene transcription.5 Since then, it has also been found to regulate
other biomolecules,6 including TAF10,7 p53,8 estrogen receptor
(ER
),9 androgen receptor (AR),10 retinoic acid receptor (RAR),11
a
a
a (ERa). It also inhibits estrogen-dependent growth of breast can-
STAT3,12 FoxO3,13 Tat,14 HIF-1a15 and DNA methyltransferase
(DNMT1).16 Due to this diversity of substrates, Set7/9 is considered
to influence a wide range of physiological functions, including dif-
ferentiation, cell cycle, metabolism and inflammation, and altered
function of Set7/9 is associated with various diseases.17–20 How-
ever, its exact function is still unclear, as Set7/9 knockdown or
depletion appears not to induce expected function.21–25 Inhibitors
of Set7/9 are needed for studies of its physiological function and
for potential therapeutic use, but only a few have been reported,
in contrast to the situation for other KHMTs, such as G9a and
cer MCF-7 cells, and has been proposed as a potential therapeutic
agent for breast cancer.
DAAM-3 was shown to bind to the coenzyme-binding site by
analysis of the crystal structure of the complex of DAAM-3 with
Set7/9,39 and DC-S239 is expected to bind to the same site.36 On
the other hand, (R)-PFI-2 binds to the substrate-binding site of
Set7/9.35 Cyproheptadine (1a) may also bind to the substrate-bind-
ing site, as indicated by its co-crystal structure and kinetic studies.
Crystal structure and NMR analyses showed that cyproheptadine
(1a) exists in a butterfly-like conformation with a bent tricyclic
dibenzosuberene moiety and chair-form piperidine ring.40,41 This
characteristic structure is retained in the co-crystal of cyprohep-
tadine (1a) with Set7/9, but interestingly, the only interaction
⇑
Corresponding author. Tel.: +81 3 5280 8128; fax: +81 3 5280 8127.
E-mail address: hira.chem@tmd.ac.jp (T. Hirano).
http://dx.doi.org/10.1016/j.bmc.2016.07.024
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

T. Fujiwara et al. / Bioorg. Med. Chem. 24 (2016) 4318–4323
4319
with a saturated bond (2a), an oxygen atom (3a), a sulfur atom
(4a), a direct linkage between the rings (5a) or no linkage (6a).
Ab initio calculations indicated that these conversions would
change the ring geometry.42 In addition, the olefinic bond linking
the tricyclic ring to piperidine was changed to a carbon–nitrogen
bond (compounds 1b, 3b and 4b) or an ether bond (compounds
1c, 3c and 4c) (Fig. 2).
Compounds 1a and 4a are commercially available. Compounds
2a, 3a, 5a and 6a were prepared via Grignard reaction of 4-chloro-
1-methylpiperidine with the corresponding arylketones (2d, 3d, 5d
and 6d), followed by dehydration, according to the reported
method (Scheme 1).42,43
Compounds 1f and 3f were converted to the corresponding
chlorinated compounds (1g and 3g), and then treated with 1-
methylpiperazine to yield 1b and 3b (Scheme 2). Compound 4b
was similarly prepared from 4f, which was derived from thioxan-
thone by reduction. Compounds 1c and 3c were synthesized by
condensation of 4-hydroxy-1-methylpiperidine with the corre-
sponding alcohols (1f and 3f) under acidic conditions. Compound
4c was prepared from thioxanthone via 4f.
2.2. Inhibitory activities towards Set7/9
The Set7/9-inhibitory activities of the synthesized compounds
were evaluated by means of the AlphaLISA method, which is one
of reported methods for evaluating the activity of Set7/9,44 and
consists of biotylated substrate peptide, two beads conjugated
with streptavidin and antibody against methylated substrate,
respectively (Table 1). In methylated state of substrate peptide
by Set7/9, both beads could be attached, resulting in emission of
luminescence. In this assay, cyproheptadine (1a) inhibited Set7/9
with an IC50 value of 3.4
ified tricyclic ring, compound 4a (X = S) showed similar inhibitory
activity (IC50: 4.3 M for 4a) to cyproheptadine. The compound
lM. Among test compounds with a mod-
l
Figure 1. (a) Structures of reported Set7/9 inhibitors. (b) Schematic representation
of the three-dimensional structure of cyproheptadine (1a).
with the oxygen atom (3a) showed a small decrease of inhibitory
activity, and those with the saturated bond (2a), direct linkage
(5a) or no linkage (6a) showed greatly decreased activities. Com-
pounds in which the double bond linking the tricyclic ring and
piperidine was converted to a carbon–nitrogen bond (1b, 3b, 4b)
or an ether bond (1c, 3c, 4c) showed a dramatic loss of inhibitory
activity, which indicated that this double bond is important for
the inhibitory activity.
2.3. Structural study for the inhibition of Set7/9
Although calculated optimized structures of 1a–6a have been
reported,42 we performed X-ray crystallographical analysis of
Figure 2. Derivatives of cyproheptadine (1a).
between the inhibitor and the receptor appeared to occur at the
N-methylpiperidine ring, especially the nitrogen atom, of cypro-
heptadine (1a). Thus, the importance of the overall molecular
shape remains unclear. So in this study, we synthesized several
derivatives of cyproheptadine (1a) with different tricyclic ring
systems or different linking groups between the tricyclic ring and
monocyclic amine, in order to investigate the structural require-
ments for inhibition of Set7/9.
2. Results and discussion
2.1. Design and synthesis of derivatives of cyproheptadine
We designed three types of cyproheptadine derivatives. The
10,11-olefinic bond in the tricyclic ring (X in Fig. 2) was replaced
Scheme 1. Synthesis of 2a, 3a, 5a and 6a.

4320
T. Fujiwara et al. / Bioorg. Med. Chem. 24 (2016) 4318–4323
selected compounds, cyproheptadine (1a), 2a, 4a and 5a (Table 2),
for precise evaluation of the steric structure–activity relationship.
As shown in Figure 3, the crystal structure of cyproheptadine
(1a) was similar to that bound to Set7/9.38 Since there are no obvi-
ous direct interactions such as hydrogen bonds between its tri-
cyclic dibenzosuberene moiety and Set7/9 in the crystal
structure, we assumed that the molecular shape would be impor-
tant for the activity. Compound 2a (X = –CH2CH2–) showed rather
similar molecular shapes with cyproheptadine (1a), though signif-
icant distortion of the benzene rings was observed in 2a. Com-
pound 4a also showed a similar structure of the tricyclic part to
cyproheptadine (1a), while the conformation of the piperidine ring
was different. In the front view, it can be seen that the dihedral
angles of the two benzene rings of 1a, 2a and 4a were similar,
about 120–130°. Compound 5a formed a flatter structure (see side
view). Previously reported structural study by ab initio calculations
indicated that 4a would have similar butterfly-like conformation to
cyproheptadine (1a), 2a would be also similar, whereas 5a would
have similar flat structure.42 Our crystallographic study showed
similar insights of 4a and 5a, however, structural characteristic
only for 2a, which would be distortion of benzene ring due to flex-
ible saturated bond, was also observed. Cyproheptadine (1a) and
its derivatives are reported to be serotonin 5-HT2A receptor ligands,
and the binding affinities of 1a, 2a, 4a and 5a have also been
reported. Compound 5a lacked affinity for both Set7/9 and 5-
HT2A, probably due to its flatter structure, whereas 4a showed sim-
ilar activity to cyproheptadine (1a) for both targets, and 2a showed
5-fold less activity for 5-HT2A and 18-fold less activity for Set7/9.
Thus, rigidity of the tricyclic structure due to the presence of the
unsaturated central double bond may be important for Set7/9-
inhibitory activity.
Scheme 2. Synthesis of 1b, 3b, 4b, 1c, 3c and 4c.
Table 1
In our previous report, the N-methyl group of cyproheptadine
(1a) was shown to be important for Set7/9 inhibition: even the
change to an ethyl group decreased the activity. In accordance with
this, the crystal structure of cyproheptadine (1a) bound to Set7/9
indicates that the N-methylpiperidine moiety is held within the
lysine access channel by hydrogen bond formation and hydropho-
bic van der Waals interactions, while the importance of other
structural components is harder to predict. In this work, we have
shown that the folded molecular shape of cyproheptadine (1a) is
important, because loss of the rigid olefinic bond linking the diben-
zosuberene and piperidine rings drastically decreased the inhibi-
tory activity. Interestingly, reduction of the 10,11-olefinic bond of
the tricyclic ring had a greater effect on the inhibitory activity
towards Set7/9 than on that towards 5-HT2A, the original target
molecule of cyproheptadine. The present findings may provide
Inhibitory activity towards Set7/9
IC50 (lM)
1a
2a
3a
4a
5a
6a
3.4
59.1
16.3
4.3
41.5
94.6
1b
3b
4b
>100
>100
>100
1c
3c
4c
>100
>100
>100
Table 2
X-ray structural analysis of cyproheptadine (1a), 2a, 4a and 5a
Cyproheptadine (1a)
2a
4a
5a
Recryn. solvent
Formula
Crystal system
Space group
a (Å)
EtOH, H2O
21H21
Monoclinic
P21/c
5.4909(8)
10.5745(16)
27.313(3)
90
92.410(2)
90
CH2Cl2, hexane
Hexane
C19H19NS
Triclinic
Hexane
C
N
C21H23
N
C19H19
Orthorhombic
Pnma
20.141(3)
12.653(2)
5.3569(9)
90
N
Monoclinic
P21/n
10.0058(12)
5.6890(7)
27.915(3)
90
93.533(2)
90
4
ꢀ
P1
10.5730(9)
17.3009(15)
17.9732(15)
105.9721(14)
101.3888(14)
96.0237(14)
8
b (Å)
c (Å)
a
(°)
b (°)
90
90
4
c
(°)
Z
4
T (K)
93
93
93
93
GOF
0.987
1.004
1.043
1.151
R1 [I > 2
wR2 [I > 2
r
(I)]
(I)]
0.0494
0.1173
0.0358
0.0876
0.0369
0.0995
0.0420
0.0963
r

T. Fujiwara et al. / Bioorg. Med. Chem. 24 (2016) 4318–4323
4321
Figure 3. Steric structure and biological activities of cyproheptadine (1a), and its derivatives 2a, 4a and 5a. In the crystal of 4a, four molecules exist independently in the
asymmetric unit. All of them have similar structures, and only one of them is shown. aData from Ref. 42.
clues for the development of more selective and potent inhibitors
of Set7/9, to avoid any supposed adverse effects in its clinical appli-
cation due to the activity against those receptors.
5a: 1H NMR (CDCl3, 400 MHz) d 7.89 (br d, J = 7.6 Hz, 2H), 7.77
(m, 2H), 7.34–7.25 (m, 4H), 3.28 (t, J = 6.0 Hz, 4H), 2.65 (t,
J = 6.0 Hz, 4H), 2.36 (s, 3H); HRMS (ESI+) Calcd for C19H20N [M
+H]+ : 262.1590. Found 262.1586.
6a: 1H NMR (CDCl3, 400 MHz) d 7.30–7.26 (m, 4H), 7.22–7.18
(m, 2H), 7.14–7.12 (m, 4H), 2.46–2.39 (m, 8H), 2.30 (s, 3H); HRMS
(ESI+) Calcd for C19H22N [M+H]+ : 264.1747. Found 264.1748.
3. Conclusion
In this work, we synthesized several derivatives of cyprohep-
tadine in order to examine the steric structure–activity relation-
ship for Set7/9-inhibitory activity. We found that the overall
folded molecular shape of cyproheptadine is important. Our results
should be helpful for the development of more potent and selective
Set7/9 inhibitors, and for finding novel inhibitor scaffolds.
4.2.2. Preparation of 1b and 3b
Under an argon atmosphere, thionyl chloride (0.30 ml,
4.1 mmol) was added to a solution of 1f (0.30 g, 1.4 mmol) and pyr-
idine (0.20 ml, 2.5 ml) in dichloromethane (3 ml) at 0 °C. The
mixture was stirred overnight at room temperature. The solvent
was removed under reduced pressure to yield 1g, which was used
without further purification. Compound 1g was dissolved in THF
(8 ml), and N-methylpiperadine (0.32 ml, 2.9 mmol) was added
slowly to the resulting solution at À70 °C. The reaction mixture
was stirred for 3 h at room temperature, then diluted with diethy-
lether, and the organic layer was washed with brine. The extract
was dried over anhydrous sodium sulfate, and the solvent was
removed under reduced pressure. The residue was purified by sil-
ica gel column chromatography (ethyl acetate–methanol = 10:1) to
afford 1b (0.14 g, 0.48 mmol, y. 33%) as a white powder. 1H NMR
(CDCl3, 400 MHz) d 7.40–7.34 (m, 4H), 7.33–7.27 (m, 4H), 6.58 (s,
2H), 4.28 (s, 1H), 2.17 (br, 4H); 13C NMR (CDCl3, 125 MHz) d
138.2, 134.4, 130.4, 130.1, 129.6, 128.0, 127.0, 78.0, 54.9, 51.4,
45.7; Anal. Calcd for C20H22N2: C, 82.72; H, 7.64, N, 9.65. Found
C, 82.46; H, 7.59; N, 9.57.; mp (hexane): 144.5–146.0 °C.
4. Experimental
4.1. 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 Bruker 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.
4.2. Preparation of compounds
3b was similarly prepared from 3f and N-methylpiperadine. 1H
NMR (CDCl3, 400 MHz) d 7.34 (q, J = 1.5, 7.8 Hz, 2H), 7.31–7.28 (m,
4.2.1. Preparation of 1a–6a
Cyproheptadine (1a) and 4a were obtained commercially. Com-
pounds 2a, 3a, 5a and 6a were prepared according to reported
methods from 4-chloro-1-methylpiperidine and corresponding
arylketones (2d, 3d, 5d and 6d) via Grignard reaction and acidic
dehydration.42,43 Those analytical data were shown below.
2a: 1H NMR (CDCl3, 400 MHz) d 7.14–7.07 (m, 8H), 3.46–3.37
(m, 2H), 2.86–2.78 (m, 2H), 2.65–2.60 (m, 2H), 2.47–2.38 (m,
4H), 2.28 (s, 3H), 2.17–2.11 (m, 2H); HRMS (ESI+) Calcd for
2H), 7.15–7.10 (m, 4H), 4.81 (s, 1H), 2.39 (br, 8H), 1.61 (s, 3H); 13
C
NMR (CDCl3, 125 MHz) d 153.4, 130.4, 128.6, 122.9, 120.1, 116.1,
60.5, 55.2, 47.7, 45.7; Anal. Calcd for C18H20N2O: C, 77.11; H,
7.19, N, 9.99; O, 5.71. Found C, 77.29; H, 7.18; N, 9.85.; mp (hex-
ane): 115.0–117.0 °C.
4.2.3. Preparation of 4b
21H24N [M+H]+ : 290.1903. Found 290.1906.
Sodium borohydride (0.70 g, 19 mmol) was added to a solution
of thioxanthone (1.0 g, 4.7 mmol) in methanol (30 ml) at 0 °C. The
mixture was heated at reflux for 20 min, then cooled to room tem-
perature, and quenched with water. The aqueous layer was
extracted with ethyl acetate. The organic layer was dried over
C
3a: 1H NMR (CDCl3, 400 MHz) d 7.34 (dd, J = 1.6, 7.6 Hz, 2H),
7.26–7.18 (m, 4H), 7.13–7.09 (m, 2H), 2.85 (t, J = 6.0 Hz, 4H), 2.37
(t, J = 6.0 Hz, 4H), 2.26 (s, 3H); HRMS (ESI+) Calcd for C19H20NO
[M+H]+: 278.1539. Found 278.1538.

4322
T. Fujiwara et al. / Bioorg. Med. Chem. 24 (2016) 4318–4323
anhydrous sodium sulfate. The solvent was removed under
reduced pressure to yield 4f. This product was dissolved in dichlor-
omethane under an argon atmosphere, and thionyl chloride
(1.0 ml, 14 mmol) and pyridine (0.60 ml, 7.4 mmol) were added
to the solution at 0 °C. The mixture was stirred overnight at room
temperature. The solvent was removed under reduced pressure to
yield 4g, which was dissolved in THF (25 ml). N-Methylpiperadine
(1.0 ml, 9.0 mmol) was added slowly to the resulting solution at
À70 °C. The reaction mixture was stirred for 5 h at room tempera-
ture, and then diluted with diethylether. The organic layer was
washed with brine and dried over anhydrous sodium sulfate, and
the solvent was removed under reduced pressure. The residue
was purified by silica gel column chromatography (ethyl acetate–
methanol = 10:1) to afford 4b (89 mg, 0.30 mmol, y. 6.0%) as a
brown powder. 1H-NMR (CDCl3, 400 MHz) d 7.45–7.43 (m, 2H),
7.36–7.34 (m, 2H), 7.25–7.23 (m, 4H), 4.52 (s, 1H), 2.33 (br, 8H)
2.18 (s, 3H); 13C-NMR (CDCl3, 125 MHz) d 134.1, 132.9 131.4,
127.2, 126.8, 125.7, 70.3, 55.1, 50.0, 45.7; Anal. Calcd for
125 MHz) d 136.8, 132.7, 127.2, 127.2, 126.6, 126.2, 45.9, 31.3;
HRMS (ESI+) Calcd for C19H21NOS [M+H]+ : 312.1417. Found
312.1408.
4.3. AlphaLISA assay
An AlphaLISA enzymatic assay was performed as described
elsewhere.44 Briefly, recombinant Set7/9 protein was incubated
with a biotinylated histone H3-derived peptide (final concentra-
tion 50 nM) and SAM (final concentration 400 nM) in 10 lL of
assay buffer (50 mM Tris–HCl [pH 8.8], 0.01% Tween-20, 5 mM
MgCl2, 1 mM DTT). After 60 min at room temperature, anti-
H3K4me1-2 acceptor beads (final concentration 20
streptavidin donor beads (final concentration 20
added and incubated for an additional 30 min at room tempera-
ture. Then, the signal was detected with an EnSpire Alpha plate
l
g/mL) and
l
g/mL) were
a
reader (PerkinElmer, Waltham, MA, USA).
C
18H20N2S: C, 72.93; H, 6.80, N, 9.45; S, 10.82. Found C, 73.02; H,
4.4. X-ray crystallographic analysis
6.68; N, 9.24.; mp (hexane): 126.0–147.5 °C.
Crystallographic data were collected on a Bruker SMART APEX II
ULTRA diffractometer attached with a CCD detector and graphite-
4.2.4. Preparation of 1c and 3c
4-Hydroxy-1-methylpiperidine (0.31 g, 2.7 mmol) was added to
a solution of 1f (0.50 g, 2.4 mmol) and p-toluenesulfonic acid
(0.20 g, 1.2 mmol) in toluene (40 ml). The mixture was heated at
reflux overnight, then cooled to room temperature, and 2 M aque-
ous sodium hydroxide was added to it. The aqueous layer was
extracted with dichloromethane. The organic layer was dried over
anhydrous sodium sulfate. The solvent was removed under
reduced pressure, and the residue was purified by silica gel column
chromatography (ethyl acetate–methanol = 10:1) to afford 1c
(0.53 g, 1.7 mmol, y. 73%) as a yellow oil. 1H NMR (CDCl3,
400 MHz) d7.74 (d, J = 7.6 Hz, 2H), 7.39 (dt, J = 1.2, 7.6 Hz, 2H),
7.29 (dd, J = 1.2, 7.6 Hz, 2H), 7.21 (dt, J = 1.2, 7.4 Hz, 2H), 4.86 (s,
1H), 3.55 (m, 1H), 2.73 (m, 2H), 2.29 (s, 3H), 2.19 (br, 2H), 1.93
monochromated MoKa (k = 0.71073 Å) radiation. Data were cor-
rected for absorption by the multiscan semiempirical method
implemented in SADABS45 and their crystal structures were solved
by direct methods SHELXS-9746 end refined by SHELXTL-2014.47
Full-matrix least-squares refinement was performed on F2 for all
unique reflections with anisotropic displacement parameters for
non-hydrogen atoms. All hydrogen atoms were included as their
calculated positions. The crystallographic data have been depos-
ited at the Cambridge Crystallographic Data Centre as CCDC
1484063 for 1a, CCDC 1484064 for 2a, CCDC 1484065 for 4a and
CCDC 1484066 for 5a.
(br, 2H), 1.85 (m, 2H); HRMS (ESI+) Calcd for C21H23NO [M + H]+
306.1852. Found 306.1848.
:
Acknowledgements
We thank Akiko Nakata for AlphaLISA assay. 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 T.H.,
26670052 to H.K., 26221204 to M.Y., 16K08318 to A.T.). T.H. and
A.T. gratefully acknowledges funding from Terumo Life Science
Foundation. A.T. also acknowledges funding from the Naito Foun-
dation. This work was also partially supported by Project for Devel-
opment 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.
3c was similarly prepared from 3f and 4-hydroxy-1-
methylpiperidine. 1H NMR (CDCl3, 400 MHz) d 7.52 (dd, J = 1.8,
7.8 Hz, 2H), 7.35 (dt, J = 1.6, 7.6 Hz, 2H), 7.16 (m, 4H), 5.81 (s,
1H), 3.39 (m, 1H), 2.58 (m, 2H), 2.12 (s, 3H), 1.98 (br, 2H), 1.67
(br, 4H); 13C NMR (CDCl3, 125 MHz) d 152.3, 130.2, 129.6, 123.2,
121.1, 116.8, 68.7, 53.5, 46.1, 32.5; HRMS (ESI+) Calcd for
C
19H21NO2 [M+H]+ : 296.1645. Found 296.1640.
4.2.5. Preparation of 4c
Sodium borohydride (0.56 mg, 15 mmol) was added to a solu-
tion of thioxanthone (0.80 g, 3.8 mmol) in methanol (25 ml) at
0 °C. The mixture was heated at reflux for 1 h, then cooled to room
temperature, and quenched with water. The aqueous layer was
extracted with ethyl acetate. The extract was dried over anhydrous
sodium sulfate. The solvent was removed under reduced pressure
to yield 4f, which was dissolved in toluene (55 ml). 4-Hydroxy-1-
methylpiperidine (0.31 g, 2.7 mmol) and p-toluenesulfonic acid
(0.27 mg, 1.5 mmol) were added to the resulting solution. The mix-
ture was heated at reflux overnight, then cooled to room tempera-
ture, and 2 M aqueous sodium hydroxide was added to it. The
aqueous layer was extracted with dichloromethane. The organic
layer was dried over anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and the residue was purified
by silica gel column chromatography (ethyl acetate–methanol =
10:1) to afford 4c (0.19 mg, 0.59 mmol, y. 16%) as a yellow oil. 1H
NMR (CDCl3, 400 MHz) d 7.60 (dd, J = 0.8, 7.2 Hz, 2H), 7.49 (dd,
J = 1.2, 7.6 Hz, 2H), 7.31 (dt, J = 1.6, 7.4 Hz, 2H), 7.24 (dt, J = 1.6,
7.8 Hz, 2H), 5.15 (s, 1H), 3.62 (m, 1H), 2.79 (m, 2H), 2.32 (s, 3H),
2.24 (br, 1H), 1.98 (m, 2H), 1.85 (m, 2H); 13C NMR (CDCl3,
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