Synthesis and biological activity of optically active NCL-1, a lysine-specific demethylase 1 selective inhibitor

Article abstract of DOI:10.1016/j.bmc.2010.12.024

Optically active (1S,2R)-NCL-1 and (1R,2S)-NCL-1 were synthesized and evaluated for their lysine-specific demethylase 1 inhibitory activity and cell growth inhibitory activity. In enzyme assays, the (1S,2R)-isomer was approximately four times more potent than the (1R,2S)-isomer. In cell growth inhibition assays, the two isomers showed similar activity in HEK293 cells and SH-SY5Y cells, whereas the (1S,2R)-isomer showed approximately four times more potent activity than the (1R,2S)-isomer in HeLa cells.

Full text of DOI:10.1016/j.bmc.2010.12.024

Bioorganic & Medicinal Chemistry 19 (2011) 3702–3708
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of optically active NCL-1, a lysine-specific
demethylase 1 selective inhibitor
Daisuke Ogasawara a, Takayoshi Suzuki a,b, , Koshiki Mino c, Rie Ueda a, Mohammed Naseer Ahmed Khan a,
⇑
Takuya Matsubara a, Koichi Koseki c, Makoto Hasegawa c, Ryuzo Sasaki d, Hidehiko Nakagawa a,
a,
Tamio Mizukami c, , Naoki Miyata
⇑
⇑
a Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
b PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
c Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan
d Frontier Pharma Inc., 1281-8 Tamura-cho, Nagahama, Shiga 526-0829, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Optically active (1S,2R)-NCL-1 and (1R,2S)-NCL-1 were synthesized and evaluated for their lysine-specific
demethylase 1 inhibitory activity and cell growth inhibitory activity. In enzyme assays, the (1S,2R)-
isomer was approximately four times more potent than the (1R,2S)-isomer. In cell growth inhibition
assays, the two isomers showed similar activity in HEK293 cells and SH-SY5Y cells, whereas the
(1S,2R)-isomer showed approximately four times more potent activity than the (1R,2S)-isomer in HeLa
cells.
Received 17 September 2010
Revised 2 December 2010
Accepted 8 December 2010
Available online 22 December 2010
Keywords:
Histone demethylase
Inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
Epigenetic gene expression
Lysine-specific demethylase
1. Introduction
cell-active LSD1 selective inhibitor.8 We showed that NCL-1
potently inhibits LSD1 in preference to monoamine oxidase-A
Reversible histone methylation, a process strictly controlled by
histone methyltransferases and histone demethylases, plays a
pivotal role in the regulation of epigenetic gene expression.1
Lysine-specific demethylase 1 (LSD1) removes methyl groups from
mono- and dimethylated Lys4 of histone H3 (H3K4me1/2) through
flavin adenine dinucleotide (FAD)-dependent enzymatic oxidation.2
In prostate cell lines, LSD1 also demethylates H3K9me1/2 and regu-
lates androgen-receptor-mediated transcription.3 It has recently
been reported that H3K9me1/2 is demethylated by LSD1 in cells
infected by herpes virus.4 The targets of LSD1 regulatory demethyl-
ation are not limited to histone H3; LSD1 also demethylates p53,
DNA methyltransferase 1, and E2F1, and regulates their cellular
functions.5
(MAO-A) and MAO-B, which are FAD-dependent oxidases, and
represses cancer cell growth with the accumulation of H3K4me2.
However, the NCL-1 we reported8 is a mixture of (1S,2R)-NCL-1
and (1R,2S)-NCL-1 (Fig. 1). Recently, Mai and co-workers investi-
gated the LSD1 and MAO inhibitory activities of chiral PCPAs and
their derivatives.7k That report prompted us to examine the biolog-
ical activity of optically active NCL-1.
Here we report the synthesis, absolute structure determination,
LSD1 inhibition, and cell growth inhibition of (1S,2R)-NCL-1 and
(1R,2S)-NCL-1.
2. Chemistry
Histone demethylation by LSD1 is suggested to be associated
with certain disease states, such as cancer and herpes simplex.1a,4,6
Therefore, LSD1 inhibitors are of interest not only as tools for eluci-
dating in detail the biological functions of the enzyme, but also as
potential therapeutic agents. Several LSD1 inhibitors, such as
trans-2-phenylcyclopropylamine (PCPA) (Fig. 1), have been identi-
fied so far.7 We have recently reported NCL-1 (Fig. 1) as the first
The routes used for the synthesis of (1S,2R)-NCL-1 are shown in
Schemes 1–3. Scheme 1 shows the preparation of intermediate 4.
N-Boc homoserine 1 was treated with benzylamine in the presence
of PyBOP to give amide 2.
Deprotection of the Boc group of 2 afforded amine 3. Amine 3
was treated with benzoic acid in the presence of PyBOP to give
amide 4.
Scheme 2 shows the preparation of optically active cyclopro-
panecarboxamides (1S,2S)-10 and (1R,2R)-10. Carboxylic acid 5
⇑
Corresponding authors.
E-mail address: miyata-n@phar.nagoya-cu.ac.jp (N. Miyata).
was converted into methyl ester
6 under acidic conditions.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.024

D. Ogasawara et al. / Bioorg. Med. Chem. 19 (2011) 3702–3708
3703
NH2
NH2
PCPA
(1S,2R)-
PCPA
(1R,2S)-
O
O
O
NH2
O
NH2
N
H
N
H
HN
O
HN
O
NCL-1
(1S,2R)-
NCL-1
(1R,2S)-
Figure 1. Structures of PCPA and NCL-1.
O
mixture of (1S,2S)-10 and (1R,2R)-10. The mixture was separated
by column chromatography to give optically active (1S,2S)-10
and (1R,2R)-10.
OH
HOOC
a
b
OH
N
H
NHBoc
NHBoc
Scheme 3 shows the synthesis of (1S,2R)-NCL-1 from (1S,2S)-10.
Amide (1S,2S)-10 was hydrolyzed under alkaline conditions to give
carboxylic acid (1S,2S)-9, which, in turn, was converted into amine
(1S,2R)-11 by the Curtius rearrangement. Deprotection of the Boc
and MOM groups of compound (1S,2R)-11 under acidic conditions
and subsequent Boc protection afforded (1S,2R)-12. Compound
(1S,2R)-12 and compound 4 were subjected to the Mitsunobu reac-
tion to give (1S,2R)-13. Deprotection of the Boc group of (1S,2R)-13
under acidic conditions afforded (1S,2R)-NCL-1. (1R,2S)-NCL-1 was
synthesized from (1R,2R)-10 using a similar procedure to that de-
scribed above. The absolute structure of optically active NCL-1 was
determined as described below.
1
2
O
OH
O
N
H
c
OH
HN
O
N
H
NH2
3
4
Scheme 1. Reagents and conditions: (a) benzylamine, PyBOP, Et3N, DMF, rt; (b) HCl,
1,4-dioxane, CH2Cl2, rt; (c) benzoic acid, PyBOP, Et3N, DMF, rt.
3. Results and discussion
MOM protection of the hydroxyl group of 6 afforded compound
7. Cyclopropanation of olefin
7 was achieved through the
3.1. Absolute structure determination
Corey–Chaykovsky reaction to give cyclopropane compound 8.
Methyl ester 8 was hydrolyzed under alkaline conditions to
give carboxylic acid 9. Carboxylic acid 9 was treated with (S)-
phenylethylamine in the presence of EDCI and HOBt to give a
We initially tried to determine the absolute structure of
prepared NCL-1 by X-ray crystal structure analysis. Although we
O
O
a
b
HO
HO
OH
OMe
5
7
6
8
O
O
c
d
MOMO
MOMO
OMe
OMe
O
MOMO
N
H
(1S,2S)-10
O
e, f
MOMO
OH
O
9
MOMO
N
H
(1R,2R)-10
Scheme 2. Reagents and conditions: (a) concd H2SO4, MeOH, reflux; (b) MOMCl, K2CO3, acetone, rt; (c) Me3S(O)I, NaH, DMSO, rt; (d) KOH, MeOH, rt; (e) (S)-
phenylethylamine, EDCI, HOBt, DMF, rt; (f) column chromatography.

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D. Ogasawara et al. / Bioorg. Med. Chem. 19 (2011) 3702–3708
O
O
a
b
d
MOMO
MOMO
N
H
OH
(1S,2S)-10
9
(1S,2S)-
MOMO
NHBoc
HO
NHBoc
c
11
(1S,2R)-
12
(1S,2R)-
O
O
e
O
NHBoc
O
NH2
BnHN
BnHN
NHBz
(1S,2R)-13
NHBz
(1S,2R)-NCL-1
Scheme 3. Reagents and conditions: (a) KOH, hydrazine, ethylene glycol, 120 °C; (b) DPPA, Et3N, t-BuOH, cyclohexane, reflux; (c) (i) HCl, AcOEt, CH2Cl2, rt; (ii) Boc2O, Et3N,
1,4-dioxane, H2O, rt; (d) 4, PPh3, DIAD, THF, rt; (e) HCl, AcOEt, CH2Cl2, rt.
O
HOOC
HOOC
O
O
O
O
COOH
COOH
O
14
Figure 2. Structure of chiral shift reagent 14.
Figure 4. 1H NMR spectra of NCL-1. (a)
A mixture of (1S,2R)-NCL-1 and
(1R,2S)-NCL-1, (b) a mixture of (1S,2R)-NCL-1 and (1R,2S)-NCL-1 in the presence
of 14, (c) (1S,2R)-NCL-1 (prepared as shown in Scheme 3) in the presence of 14, (d)
(1R,2S)-NCL-1 (the other isomer) in the presence of 14.
structure analysis. We then decided to determine its absolute
structure by 1H NMR analysis using a chiral shift reagent.
As NCL-1 has a PCPA moiety (Fig. 1), the absolute structure of
the prepared NCL-1 can be determined by comparing its 1H NMR
spectrum with that of an enantiopure PCPA in the presence of a
chiral shift reagent. To enantiodiscriminate the PCPA part of NCL-1
by 1H NMR analysis, we used (18-crown-6)-2,3,11,12-tetracarb-
oxylic acid 14 (Fig. 2) as the chiral shift reagent, because it has been
used to discriminate the chirality of various amines.9 We initially
analyzed the 1H NMR spectrum of PCPA (Fig. 3). Peaks assignable
to the C2 methine proton of the cyclopropane of racemic PCPA
were observed at 1.43–1.37 ppm (Fig. 3a). Multiple peaks of race-
mic PCPA were split into two sets of peaks by the addition of chiral
shift reagent 14 (Fig. 3b). One of the two sets of peaks was identical
to that of (1S,2R)-PCPA7k in the presence of compound 14 (Fig. 3c).
Figure 3. 1H NMR spectra of PCPA. (a) PCPA (racemate), (b) PCPA (racemate) in the
presence of 14, (c) (1S,2R)-PCPA in the presence of 14.
tried to crystallize optically active NCL-1 and several intermedi-
ates, we could not obtain crystals suitable for the X-ray crystal

D. Ogasawara et al. / Bioorg. Med. Chem. 19 (2011) 3702–3708
3705
Table 2
Growth inhibition of HeLa cells, HEK 293 cells, and SH-SY5Y cells by PCPA, (1S,2R)-
NCL-1, and (1R,2S)-NCL-1a
Cell
PCPA
(1S,2R)-NCL-1
(1R,2S)-NCL1
GI50
(
lM)
GI50
(
lM)
GI50 (lM)
HeLa
HEK293
SH-SY5Y
>500
>500
500
20
17
9.0
74
25
9.0
a
Values are means of at least three separate experiments.
formation was monitored in the absence and presence of
(1S,2R)-NCL-1 or (1R,2S)-NCL-1. As shown in Figures 5 and 6, both
(1S,2R)-NCL-1 and (1R,2S)-NCL-1 were time-dependent inhibitors
of LSD1, exhibiting nonlinear progress curves and reaching a
plateau. These data suggest that both (1S,2R)-NCL-1 and (1R,2S)-
NCL-1 are irreversible inhibitors.
Figure 5. Steady-state progress curves obtained for the inactivation of LSD1 with 0
(d), 0.8 (j), 2.3 (N), 6.9 (s), and 21 (h) M (1S,2R)-NCL-1.
l
The IC50 values of (1S,2R)-NCL-1 and (1R,2S)-NCL-1 were also
determined. We used the data after the product concentration
reached
a plateau for the determination of the IC50 values
(Figs. 5 and 6). As expected from previous data, both (1S,2R)-NCL-1
and (1R,2S)-NCL-1 showed more potent activity than PCPA.
(1S,2R)-NCL-1 was approximately four times more potent than
(1R,2S)-NCL-1 (IC50 of (1S,2R)-NCL-1 = 1.6
1 = 6.7 M) (Table 1).
lM; IC50 of (1R,2S)-NCL-
l
3.3. Cell growth inhibition assays
We reported that a mixture of (1S,2R)-NCL-1 and (1R,2S)-NCL-1
exerts cell growth inhibitory activity with the accumulation of
H3K4me2.8 Therefore, (1S,2R)-NCL-1 and (1R,2S)-NCL-1 were
evaluated for their ability to inhibit the growth of three cell lines
(Table 2). Interestingly, (1S,2R)-NCL-1 and (1R,2S)-NCL-1 displayed
similar activities against HEK293 cells and SH-SY5Y cells, whereas
(1S,2R)-NCL-1 showed approximately four times more potent
inhibitory activity against HeLa cells than (1R,2S)-NCL-1. The rea-
sons for the differences in results between enzyme assays and
cell-based assays and between HeLa cell line and the other two cell
lines are not clear. Nevertheless, we surmise that the differences
may be due to the sensitivity to LSD1 that complexed with proteins
in the cells. LSD1 has been reported to bind to such proteins as
HDAC1, CoREST, and TLX.10 The activity of NCL-1 may depend on
which protein LSD1 binds to in cells. The binding of LSD1 to the
proteins may alter the structure of the active site and affect the
binding of NCL-1 to LSD1.
Figure 6. Steady-state progress curves obtained for the inactivation of LSD1 with 0
(d), 2.3 (j), 6.9 (N), 20.6 (s), and 62 (h) M (1R,2S)-NCL-1.
l
These results showed that the peaks at higher magnetic fields
(1.47–1.43 ppm, yellow circle) are derived from (1S,2R)-PCPA and
those at lower magnetic fields (1.60–1.55 ppm, blue circle) are de-
rived from (1R,2S)-PCPA. Next, we analyzed the 1H NMR spectrum
of NCL-1 (Fig. 4). As is the case in PCPA (Fig. 3), the addition of chi-
ral shift reagent 14 split the multiple peaks at 1.32–1.29 ppm of
the C2 methine proton of a mixture of (1S,2R)-NCL-1 and (1R,2S)-
NCL-1 (Fig. 4a) into two sets of peaks at 1.58–1.53 and 1.44–1.40
(Fig. 4b). One of the two sets of peaks was identical to that of
one of the two optically active NCL-1s we prepared, as shown in
Scheme 3 (Fig. 4c), and the other set of peaks corresponded to that
of the other optically active NCL-1 (Fig. 4d). Comparing the peak
shift pattern of NCL-1 (Fig. 4) with that of PCPA (Fig. 3), the opti-
cally active NCL-1 prepared as shown in Scheme 3 was determined
to be (1S,2R)-NCL-1, and the other compound was determined to
be (1R,2S)-NCL-1.
4. Conclusion
We have synthesized optically active (1S,2R)-NCL-1 and (1R,2S)-
NCL-1 and evaluated their LSD1 inhibitory activity and cell growth
inhibitory activity. In the enzyme assays, (1S,2R)-NCL-1 was more
potent than (1R,2S)-NCL-1. In the cell growth inhibition assays,
(1S,2R)-NCL-1 and (1R,2S)-NCL-1 showed similar activity in
HEK293 cells and SH-SY5Y cells, whereas (1S,2R)-NCL-1 showed
approximately four times more potent activity in HeLa cells than
(1R,2S)-NCL-1. These findings are expected to contribute to the
development of PCPA-based LSD1 inhibitors.
3.2. Enzyme assays
(1S,2R)-NCL-1 and (1R,2S)-NCL-1 were assayed against LSD1
using a peroxidase-coupled assay as described before.8 Because a
mixture of (1S,2R)-NCL-1 and (1R,2S)-NCL-1 has been reported to
be an irreversible inhibitor that covalently binds to FAD,8 we initially
determined whether the inhibition by (1S,2R)-NCL-1 or (1R,2S)-
NCL-1 was time-dependent or not. The time course of product
5. Experimental section
5.1. Chemistry
Table 1
In vitro LSD1 inhibitory activities (IC50 values) of PCPA, (1S,2R)-NCL-1, and (1R,2S)-
NCL-1a
Melting points were determined with a Yanagimoto micro
melting point apparatus or a Büchi 545 melting point apparatus
and were left uncorrected. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded on a JEOL JNM-LA500 spectrome-
Compound
IC50 M)
PCPA
21
(1S,2R)-NCL-1
(1R,2S)-NCL-1
(l
1.6
6.7
a
Values are means of at least three separate experiments.

3706
D. Ogasawara et al. / Bioorg. Med. Chem. 19 (2011) 3702–3708
ter in solvent as indicated. Chemical shifts (d) were reported in
parts per million relative to the internal standard, tetramethylsil-
the solution was stirred at room temperature for 15 min. Then,
MOMCl (11.2 mL, 0.1476 mmol) was added slowly with a syringe
through a septum. After complete addition, the reaction mixture
was stirred at room temperature for 12 h. Filtration, concentration
in vacuo, and purification by silica gel flash column chromatogra-
phy (AcOEt/n-hexane = 1:10) gave 27.5 g (84%) of 7 as a colorless
oil; 1H NMR (CDCl3, 500 MHz, d; ppm) 7.66 (1H, d, J = 16.2 Hz),
7.30 (1H, t, J = 8.0 Hz), 7.20 (1H, s), 7.17 (1H, d, J = 8.0 Hz), 7.07
(1H, dd, J = 8.0, 2.4 Hz), 6.43 (1H, d, J = 16.0 Hz), 5.19 (2H, s), 3.81
(3H, s), 3.49 (3H, s).
ane. Elemental analysis was performed with
a Yanaco CHN
CORDER NT-5 analyzer, and all values were within 0.4% of the cal-
culated values. Fast atom bombardment (FAB) mass spectra were
recorded on a JEOL JMS-SX102A mass spectrometer. Reagents
and solvents were purchased from Aldrich, Tokyo Kasei Kogyo,
Wako Pure Chemical Industries, and Kanto Kagaku and used with-
out purification. Flash column chromatography was performed
using Silica Gel 60 (particle size 0.046–0.063 mm) supplied by
Merck.
5.1.6. Methyl trans-2-(3-methoxymethoxyphenyl)cyclopropane-
carboxylate (8)
5.1.1. (S)-1-Benzylcarbamoyl-3-hydroxypropyl tert-butylcarba-
mate (2)
Toamixtureof NaH(6.43 g, 0.161 mol, 60 wt %inmineraloil)and
trimethylsulfoxonium iodide (35.4 g, 0.161 mol) was added drop-
wise dry DMSO (140 mL) with stirring at room temperature. The
reaction mixture was stirred at room temperature for 1 h. To the
mixture was added dropwise a solution of 7 (27.5 g, 0.124 mol) ob-
tained above in dry DMSO (140 mL). The mixture was stirred at room
temperature for 6 h, acidified to pH 4 by adding 10% citric acid, and
extracted with AcOEt. The organic layer was washed with brine
and dried over Na2SO4. Filtration, concentration in vacuo, and
purification by silica gel flash column chromatography (AcOEt/n-
hexane = 1:10) gave 4.63 g (16%) of 8 as a colorless oil; 1H NMR
(CDCl3, 500 MHz, d; ppm) 7.19 (1H, t, J = 8.0 Hz), 6.89 (1H, dd,
J = 8.0, 2.5 Hz), 6.78 (1H, t, J = 2.5 Hz), 6.74 (1H, d, J = 8.0 Hz), 5.16
(2H, s), 3.71 (3H, s), 3.47 (3H, s), 2.51–2.49 (1H, m), 1.92–1.89 (1H,
m), 1.61–1.57 (1H, m), 1.34–1.31 (1H, m).
A solution of 1 (1.42 g, 6.50 mmol), PyBOP (3.38 g, 6.50 mmol),
Et3N (1.8 mL, 12.9 mmol), and benzylamine (0.85 mL, 7.77 mmol)
in DMF (13.5 mL) was stirred for 14 h at room temperature. The
reaction mixture was poured into water and extracted with CHCl3.
The organic layer was washed with brine and dried over Na2SO4.
Filtration, concentration in vacuo, and purification by silica gel
flash column chromatography (AcOEt/n-hexane = 2:1) gave 1.24 g
(62%) of 2 as a white solid; 1H NMR (CDCl3, 500 MHz, d; ppm)
7.35–7.32 (2H, m), 7.29–7.25 (3H, m), 6.71 (1H, br s), 5.58 (1H, d,
J = 7.3 Hz), 4.45 (2H, s), 4.35 (1H, s), 3.71 (2H, s), 3.27 (1H, br s),
2.04–1.98 (1H, m), 1.72–1.85 (1H, m), 1.43 (9H, s).
5.1.2. (S)-2-Amino-N-benzyl-4-hydroxybutanamide
hydro- chloride (3ÁHCl)
To a solution of 2 (1.24 g, 3.89 mmol) obtained above in CH2Cl2
(13 mL) was added 4 N HCl in 1,4-dioxane (20 mL) and the mixture
was stirred for 3 h at room temperature. Concentration in vacuo
gave 951 mg (q. y.) of 3 as a white solid; 1H NMR (CD3OD,
500 MHz, d; ppm) 7.44–7.41 (2H, m), 7.34–7.30 (3H, m), 7.27
(1H, br s), 4.43 (2H, s), 3.94 (1H, d, J = 7.0 Hz), 2.03 (2H, s).
5.1.7. 2-(3-Methoxymethoxyphenyl)cyclopropanecarboxylic
acid (9)
To a solution of 8 (4.60 g, 19.5 mmol) obtained above in MeOH
(38 mL) was added a solution of KOH (10.9 g, 194 mmol) in MeOH
(89 mL) at 0 °C. The reaction mixture was stirred for 5 h at room
temperature. The solution was concentrated in vacuo and the
residue was suspended in water and extracted with CH2Cl2. The
organic layer was discarded and the aqueous phase was acidified
with 2 N aqueous HCl to pH 1 and extracted with CH2Cl2. The
organic layer was dried over Na2SO4. Filtration and concentration
in vacuo gave 3.70 g (86%) of 9 as a colorless oil; 1H NMR (CDCl3,
500 MHz, d; ppm) 7.20 (1H, t, J = 8.0 Hz), 6.90 (1H, dd, J = 8.0,
1.9 Hz), 6.79 (1H, t, J = 1.9 Hz), 6.75 (1H, d, J = 8.0 Hz), 5.16 (2H,
s), 3.48 (3H, s), 2.60–2.56 (1H, m), 1.92–1.89 (1H, m), 1.65 (1H,
quintet, J = 5.0 Hz), 1.42–1.38 (1H, m).
5.1.3. (S)-N-(1-Benzylcarbamoyl-3-hydroxypropyl)benzamide
(4)
A solution of 3 (951 mg, 3.89 mmol) obtained above, PyBOP
(3.06 g, 5.88 mmol), benzoic acid (598 mg, 4.90 mmol), and Et3N
(1.3 mL, 9.80 mmol) in DMF (10 mL) was stirred for 12 h at room
temperature. The reaction mixture was poured into water and ex-
tracted with CH2Cl2. The organic layer was washed with water and
brine and dried over Na2SO4. Filtration, concentration in vacuo,
and purification by silica gel flash column chromatography
(AcOEt/n-hexane = 2:1) gave 1.00 g (65%) of 4 as a white solid; 1H
NMR (CDCl3, 500 MHz, d; ppm) 7.83 (2H, d, J = 7.0 Hz), 7.56–7.53
(1H, m), 7.47–7.44 (2H, m), 7.36–7.33 (2H, m), 7.30–7.26 (3H, m),
6.62 (1H, s), 4.86–4.81 (1H, m), 4.54–4.45 (2H, m), 3.78–3.75 (2H,
m), 3.63–3.61 (1H, m), 2.17–2.09 (1H, m), 1.94–1.89 (1H, m).
5.1.8. (1S,2S)-2-(3-(Methoxymethoxy)phenyl)-N-((S)-1-phenylethyl)
cyclopropanecarboxamide ((1S,2S)-10) and (1R,2R)-2-(3-(methoxy-
methoxy)phenyl)-N-((S)-1-phenylethyl) cyclopropanecarboxamide
((1R,2R)-10)
To a solution of 9 (3.6 g, 16.2 mmol) obtained above in DMF
(53 mL) were added EDCI (4.66 g, 24.3 mmol), HOBtÁH2O (3.72 g,
24.3 mmol), and (S)-1-phenylethylamine (3.1 mL, 24.3 mmol).
The reaction mixture was stirred for 13 h at room temperature.
The mixture was poured into water and extracted with CHCl3.
The organic layer was washed with water and brine, and dried
over Na2SO4. Filtration, concentration in vacuo, and purification
by silica gel flash column chromatography (AcOEt/n-hexane = 1:4)
gave 1.35 g (51%) of (1S,2S)-10 as a white solid and 1.33 g (50%)
5.1.4. Methyl trans-3-(3-hydroxylphenyl)propenoate (6)
To a solution of 5 (25.0 g, 0.152 mol) in MeOH (82 mL) was
added concentrated H2SO4 (2.5 mL) at 0 °C and the mixture was
stirred at reflux temperature for 6 h. The reaction mixture was con-
centrated in vacuo and the residue was dissolved in AcOEt. The or-
ganic layer was washed with water, saturated NaHCO3, and brine,
and dried over Na2SO4. Filtration and concentration in vacuo gave
26.3 g (97%) of 6 as a white solid; 1H NMR (CDCl3, 500 MHz, d;
ppm) 7.60 (1H, d, J = 15.8 Hz), 7.21 (1H, t, J = 8.0 Hz), 7.04 (1H, d,
J = 8.0 Hz), 6.98 (1H, t, J = 1.8 Hz), 6.82 (1H, dd, J = 8.0, 1.8 Hz),
6.44 (1H, d, J = 15.8 Hz), 3.77 (3H, s).
of (1R,2R)-10 as
a
white solid. (1S,2S)-10: 1H NMR (CDCl3,
500 MHz, d; ppm) 7.34–7.33 (4H, m), 7.28–7.25 (1H, m), 7.18
(1H, t, J = 8.0 Hz), 6.87 (1H, dd, J = 8.2, 2.4 Hz), 6.76–6.73 (2H,
m), 5.85 (1H, br s), 5.15 (2H, s), 3.47 (3H, s), 2.50–2.45 (1H, m),
1.61–1.57 (1H, m), 1.59 (3H, s), 1.50–1.49 (2H, m), 1.21–1.18
(1H, m). (1R,2R)-10: 1H NMR (CDCl3, 500 MHz, d; ppm) 7.35–
7.31 (4H, m), 7.28–7.24 (1H, m), 7.17 (1H, t, J = 8.0 Hz), 6.86
(1H, dd, J = 8.2, 2.4 Hz), 6.73–6.72 (1H, m), 6.71 (1H, s), 5.84
5.1.5. Methyl trans-3-(3-methoxymethoxyphenyl)propenoate
(7)
To a solution of 6 (26.3 g, 0.148 mmol) obtained above in ace-
tone (220 mL) was added dry K2CO3 (40.8 g, 0.295 mmol), and

D. Ogasawara et al. / Bioorg. Med. Chem. 19 (2011) 3702–3708
3707
(1H, br s), 5.14 (2H, s), 3.47 (3H, s), 2.47–2.44 (1H, m), 1.63–1.57
(1H, m), 1.58 (3H, s), 1.52–1.50 (2H, m), 1.23–1.22 (1H, m).
4.13–4.09 (1H, m), 2.69–2.68 (1H, m), 2.43–2.37 (2H, m),
1.99–1.97 (1H, m), 1.45 (9H, s), 1.14–1.10 (2H, m).
5.1.9. (1S,2S)-2-(3-(Methoxymethoxy)phenyl)cyclopropanecarb-
oxylic acid ((1S,2S)-9)
5.1.13. (1S,2R)-2-(3-((S)-3-Benzamido-4-(benzylamino)-4-oxo-
butoxy)phenyl)cyclopropylamine hydrochloride ((1S,2R)-NCL-
1ÁHCl)
To a suspension of (1S,2S)-10 (1.35 g, 4.15 mmol) obtained
above in ethylene glycol (21 mL) were added hydrazine monohy-
drate (1.0 mL, 16.60 mmol) and KOH (1.40 g, 25.0 mmol), and the
mixture was stirred for 7 days at 120 °C. The reaction mixture
was poured into 2 N aqueous HCl and extracted with CHCl3. The or-
ganic layer was washed with brine and dried over Na2SO4. Filtra-
tion, concentration in vacuo, and purification by silica gel flash
column chromatography (AcOEt/n-hexane = 1:3) gave 531 mg
(58%) of (1S,2S)-9 as a yellow solid; 1H NMR (CDCl3, 500 MHz, d;
ppm) 7.20 (1H, t, J = 8.0 Hz), 6.90 (1H, dd, J = 8.0, 2.0 Hz), 6.79
(1H, s), 6.75 (1H, d, J = 8.0 Hz), 5.16 (2H, s), 3.48 (3H, s), 2.60–
2.55 (1H, m), 1.93–1.90 (1H, m), 1.66–1.63 (1H, m), 1.42–1.38
(1H, m).
To a solution of (1S,2R)-13 (131 mg, 0.241 mmol) obtained
above in CH2Cl2 (1.6 mL) was added 4 N HCl in AcOEt (2.8 mL,
11.2 mmol), and the mixture was stirred for 7 h at room tempera-
ture. The solvent was removed by evaporation and the residue was
recrystallized from CHCl3 and MeOH to give 70 mg (61%) of
(1S,2R)-NCL-1ÁHCl as colorless crystals: >99% ee (1H NMR), mp
208–209 °C; 1H NMR (CD3OD, 500 MHz, d; ppm) 7.84 (2H, d,
J = 8.5 Hz), 7.54 (1H, t, J = 8.5 Hz), 7.46 (2H, t, J = 8.5 Hz), 7.26–
7.17 (6H, m), 6.77 (1H, d, J = 8.0 Hz), 6.72 (1H, d, J = 8.0 Hz), 6.67
(1H, d, J = 2.0 Hz), 4.40 (2H, m), 4.08 (2H, t, J = 6.0 Hz), 2.78–2.76
(1H, m), 2.42–2.40 (1H, m), 2.29–2.24 (2H, m), 1.37–1.31 (1H,
m), 1.28–1.24 (1H, m); 13C NMR (CD3OD, 125 MHz, d; ppm)
173.94, 170.34, 160.52, 141.32, 139.79, 135.21, 132.96, 130.81,
129.57, 129.53, 128.54, 128.45, 128.19, 120.00, 114.14, 113.80,
65.91, 53.13, 44.15, 32.66, 31.95, 22.57, 13.83; MS (FAB) m/z 444
(M+). Anal. Calcd for C27H30ClN3O3Á1/2H2O: C, 66.32; H, 6.39; N,
8.59. Found: C, 66.33; H, 6.16; N, 8.58.
5.1.10. tert-Butyl ((1S,2R)-2-(3-(methoxymethoxy)phenyl)-
cyclopropyl)carbamate ((1S,2R)-11)
To a solution of (1S,2S)-9 (531 mg, 2.39 mmol) obtained above in
dry cyclohexane (22 mL) were added DPPA (0.6 mL, 2.88 mmol),
Et3N (0.4 mL, 2.87 mmol), and dry tert-butanol (9.1 mL, 95.1 mmol)
at 0 °C under nitrogen atmosphere. The reaction mixture was
refluxed for 12 h and then cooled to room temperature. Concentra-
tion in vacuo and purification by silica gel flash column chromatog-
raphy (AcOEt/n-hexane = 1:7) gave 346 mg (49%) of (1S,2R)-11 as a
yellow solid; 1H NMR (CDCl3, 500 MHz, d; ppm) 7.17 (1H, t, J =
8.0 Hz), 6.85 (1H, dd, J = 8.0, 1.5 Hz), 6.77 (1H, s), 6.76 (1H, s), 5.15
(2H, s), 3.47 (3H, s), 2.75–2.72 (1H, m), 2.05–1.99 (1H, m), 1.45
(9H, s), 1.17–1.15 (2H, m).
5.1.14. (1R,2S)-2-(3-((S)-3-Benzamido-4-(benzylamino)-4-oxo-
butoxy)phenyl)cyclopropylamine hydrochloride ((1R,2S)-NCL-
1ÁHCl)
(1R,2S)-NCL-1ÁHCl was synthesized from (1R,2R)-10 using the
procedure described for (1S,2R)-NCL-1ÁHCl as colorless crystals:
>99% ee (1H NMR), mp 138–142 °C; 1H NMR (CD3OD, 500 MHz,
d; ppm) 7.84 (2H, d, J = 8.5 Hz), 7.54 (1H, t, J = 8.5 Hz), 7.46 (2H, t,
J = 8.5 Hz), 7.26–7.18 (6H, m), 6.77 (1H, d, J = 8.0 Hz), 6.72 (1H, d,
J = 8.0 Hz), 6.67 (1H, s), 4.40 (2H, m), 4.08 (2H, t, J = 6.0 Hz), 2.80–
2.76 (1H, m), 2.44–2.39 (1H, m), 2.29–2.24 (1H, m),
1.36–1.35 (1H, m), 1.28–1.25 (1H, m); 13C NMR (CD3OD,
125 MHz, d; ppm) 173.93, 170.34, 160.52, 141.32, 139.79, 135.21,
132.96, 130.80, 129.57, 129.53, 128.54, 128.45, 128.19, 120.01,
114.04, 113.80, 65.87, 53.11, 44.15, 32.67, 31.95, 22.57, 13.85;
MS (FAB) m/z 444 (M+). Anal. Calcd for C27H30ClN3O3Á7/6H2O: C,
64.73; H, 6.50; N, 8.39. Found: C, 64.52; H, 6.13; N, 8.73.
5.1.11. tert-Butyl ((1S,2R)-2-(3-hydroxyphenyl)cyclopropyl)-
carbamate ((1S,2R)-12)
To a solution of (1S,2R)-11 (346 mg, 1.18 mmol) obtained above
in CH2Cl2 (4.0 mL) was added 4 N HCl in AcOEt (4.5 mL,
18.0 mmol), and the solution was stirred for 2.5 h at room temper-
ature. The solvent was removed by evaporation and the residue
was dissolved in 1,4-dioxane (2.2 mL) and H2O (2.2 mL). To the
solution were added Et3N (1.3 mL, 9.35 mmol) and Boc2O
(0.3 mL, 1.31 mmol), and the reaction mixture was stirred at room
temperature. After 9 h, the reaction mixture was poured into aque-
ous 10% citric acid and extracted with AcOEt. The organic layer was
washed with brine and dried over Na2SO4. Filtration, concentration
in vacuo, and purification by silica gel flash column chromatogra-
phy (AcOEt/n-hexane = 1:5) gave 214 mg (73%) of (1S,2R)-12 as a
colorless oil; 1H NMR (CDCl3, 500 MHz, d; ppm) 7.12 (1H, d,
J = 8.0 Hz), 6.70 (1H, d, J = 8.0 Hz), 6.64 (1H, d, J = 8.0 Hz), 6.61
(1H, s), 4.82 (1H, br s), 4.73 (1H, br s), 2.74–2.69 (1H, m),
2.01–1.97 (1H, m), 1.45 (9H, s), 1.16–1.12 (2H, m).
5.2. Biology
5.2.1. Assay for LSD1 inhibitory activity
The assay for LSD1 inhibitory activity was conducted at 25 °C
using the peroxidase-coupled method as described previously.8
The chemically synthesized peptide consisted of the first 21 amino
acid residues of histone H3, incorporating dimethylated lysine at
position 4 (H3K4me2 peptide) (Sigma–Aldrich), and was used as
the substrate of LSD1. The reaction mixture contained 50 mM
HEPES–NaOH, pH 7.5, 0.1 mM 4-aminoantipyrine, 1 mM 3,5-di-
chloro-2-hydroxybenzenesulfonic acid, 5.5 units/mL horseradish
5.1.12. tert-Butyl ((1S,2R)-2-(3-((S)-3-benzamido-4-(benzyl-
amino)-4-oxobutoxy)phenyl)cyclopropyl)carbamate ((1S,2R)-
13)
peroxidase, 20 lM H3K4me2 peptide, and an appropriate amount
of LSD1. To assess the inhibitory effect of the test compounds on
LSD1 activity in comparison with that of PCPA, partially purified
LSD1 obtained at the purification step by HisTrap HP chromatogra-
phy was dissolved in buffer C and used. The test compounds were
dissolved in DMSO. The final concentration of DMSO in the reaction
mixture was adjusted to 5%, and it was confirmed that 5% DMSO
did not affect LSD1 activity. Reaction without the inhibitors was
To a solution of (1S,2R)-12 (214 mg, 0.858 mmol), 4 (536 mg,
1.72 mmol), and PPh3 (675 mg, 2.57 mmol) in dry THF (2.0 mL)
was added DIAD (1.3 mL, 2.57 mmol, 40% in toluene) at 0 °C. The
reaction mixture was stirred at 0 °C for 1 h and then at room tem-
perature for 9 h. Concentration in vacuo and purification by silica
gel flash column chromatography (AcOEt/n-hexane = 1:2) gave
131 mg (28%) of (1S,2R)-13 as a white solid; 1H NMR (CDCl3,
500 MHz, d; ppm) 7.82 (2H, d, J = 8.5 Hz), 7.52 (1H, t, J = 8.5 Hz),
7.46 (2H, t, J = 8.5 Hz), 7.31–7.21 (5H, m), 7.16 (1H, t, J = 8.0 Hz),
6.92 (1H, s), 6.75 (1H, d, J = 8.0 Hz), 6.63 (1H, d, J = 8.0 Hz), 4.88
(1H, q, J = 6.0 Hz), 4.47 (2H, d, J = 6.0 Hz), 4.32–4.29 (1H, m),
also conducted as control. Reaction mixtures (18
all the materials except H3K4me2 peptide were incubated for
5 min. Then, the reactions were started by adding 2 L of 0.2 mM
lL) containing
l
peptide solution into the reaction mixtures. The absorbance at
515 nm was monitored for 30 min in a 384-well plate (Nunc) by
using a microplate reader (SpectraMax M2e; Molecular Devices).

3708
D. Ogasawara et al. / Bioorg. Med. Chem. 19 (2011) 3702–3708
For the determination of IC50 values, enzyme activity was deter-
mined from the linear part of the reaction curve. The ratio of the
enzyme activity measured in the presence of the inhibitor to the
activity of the control was plotted against log[Inh].
References and notes
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5.2.2. Assay for cell growth inhibitory activity
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(50 lL/well) in 96-well plates in RPMI 1640 with 10% fetal bovine
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mL)
bromide (MTT) (Sigma) was added (10
was carried out for 3 h before solubilization buffer (0.04 mol/L
HCl–isopropanol) was added (100 L/well) onto the cultured cells.
of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
lL/well) and incubation
l
The solubilized dye was quantified by colorimetric measurement
at 560 nm using a reference wavelength of 750 nm. The absorbance
values of control wells (C) and test wells (T) were measured. Absor-
bance of the test wells (T0) was also measured at time 0 (addition of
compounds). Using these measurements, cell growth inhibition
(percentage of growth) by a test inhibitor at each concentration
was calculated as follows: % growth = 100 Â [(T À T0)/(C À T0)],
when T > T0 and % growth = 100 Â [(T À T0)/T], when T < T0. Com-
puter analysis of the % growth values afforded the 50% growth inhi-
bition parameter (GI50). GI50 was calculated as 100 Â [(T À T0)/
(C À T0)] = 50.
Acknowledgments
This work was supported in part by JST PRESTO program (T.S.)
and grants from Taisho Pharmaceutical Co., Ltd (T.S.), the Uehara
Memorial Foundation (T.S.), and Ho-ansha Foundation (T.S.). We
thank Mie Tsuchida and Miho Hosoi for their technical support.
8. Ueda, R.; Suzuki, T.; Mino, K.; Tsumoto, H.; Nakagawa, H.; Hasegawa, M.;
Sasaki, R.; Mizukami, T.; Miyata, N. J. Am. Chem. Soc. 2009, 131, 17536.
9. (a) Ann, E.; Lovely, J. W.; Thomas, J. W. J. Org. Chem. 2006, 71, 9178; (b) Thomas,
J. W.; Jolene, E. T. J. Org. Chem. 2000, 65, 1243.
10. Yokoyama, A.; Takezawa, S.; Schüle, R.; Kitagawa, H.; Kato, S. Mol. Cell. Biol.
2008, 28, 3995.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.024.