Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B

Article abstract of DOI:10.1016/j.bmcl.2008.01.003

A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members such as monoamine oxidases A and B.

Full text of DOI:10.1016/j.bmcl.2008.01.003

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3047–3051
Facile synthesis of substituted
trans-2-arylcyclopropylamine inhibitors of the human
histone demethylase LSD1 and monoamine oxidases A and B
David M. Gooden, Dawn M. Z. Schmidt, Julie A. Pollock,
Ami M. Kabadi and Dewey G. McCafferty*
Department of Chemistry, Duke University, Durham, NC 27708, USA
Received 27 October 2007; revised 22 December 2007; accepted 2 January 2008
Available online 8 January 2008
Abstract—A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-
based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members
such as monoamine oxidases A and B.
Ó 2008 Elsevier Ltd. All rights reserved.
The functional capacity of genetically encoded proteins
is powerfully expanded by reversible posttranslational
modification. Within eukaryotic cells, the regulation of
gene expression is intimately linked with posttransla-
tional modification of histone proteins. Reversible his-
amine oxidase domain-containing enzyme lysine-specific
demethylase 1 (LSD1).7 Catalysis by this enzyme is a fla-
vin-dependent process in which formaldehyde and per-
oxide are produced as by-products of histone
demethylation (Scheme 1).7,8 The requirement for for-
mation of positive charge on the e-nitrogen atom of ly-
sine in the imine intermediate limits LSD1 to
demethylation of dimethylated and monomethylated ly-
sine. The amine oxidase domain of LSD1 is homologous
to equivalent domains found in polyamine oxidase
(PAO, 22.4% identity) and monoamine oxidases A and
B (MAO A, 17.6% identity; MAO B, 17.6% identity).
tone
posttranslational
modifications
include
acetylation of lysine, phosphorylation of serine and
threonine, and methylation of lysine and arginine.1
Additionally, lysine residues may be mono-, di-, or
trimethylated, while arginine residues may be mono-
or dimethylated. The resulting complexity of modifica-
tions has been postulated to act as a ‘histone code’, by
which these patterns of modifications are ‘read’ by cellu-
lar machinery to produce a specific gene regulatory
outcome.1
Monoamine oxidase inhibitors (MAOIs) are well-
known drugs that have been used clinically for the treat-
ment of depression, anxiety, and Parkinson’s disease.
Because of the similarities between the catalytic mecha-
nisms and architecture of the enzyme active sites of
MAO A and B and LSD1, we hypothesized that fla-
vin-targeted irreversible inhibitors of monoamine oxi-
dases might simultaneously inhibit the function of
LSD1, perhaps leading to pleiotropic effects beneficial
to the biological action of these therapeutics.
Two classes of human enzymes capable of demethylat-
ing methylated lysine residues within histones have been
recently identified. JmjC domain-containing demethy-
lases including JHDM3A/JMJD2A, GASC1/JMJD2C,
JHDM2A, and JHDM1 comprise one class2–6; these en-
zymes appear to utilize the cofactors Fe(II) and a-keto-
glutarate to demethylate via hydroxylation of the methyl
group with subsequent elimination of formaldehyde.
The second class of histone demethylases includes the
Indeed, upon evaluation of this hypothesis, we discov-
ered that clinically relevant concentrations of trans-2-
phenylcyclopropylamine9 (2-PCPA, brand name
Parnate), an irreversible inhibitor of monoamine and
polyamine oxidases, proved effective at inhibiting
LSD1-mediated histone demethylation in vivo and
Keywords: Lysine-specific histone demethylase; LSD1; trans-2-Cyclop-
roylamine; Parnate; Tranylcypromine; Monoamine oxidase; Inhibitor.
*
Corresponding author. Tel.: +1 919 660 1516; fax: +1 919 668
5483; e-mail: dewey.mccafferty@duke.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.003

3048
D. M. Gooden et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3047–3051
H2O2
O2
Histone H3K4
Histone H3K4
Histone H3K4
FAD FADH2 H2O
Repeat
Cycle
(CH2)4
(CH2)4
(CH2)4
LSD1
HCHO
H3C
N
CH3
H3C
NH
NH2
Scheme 1. The reaction catalyzed by the histone demethylase LSD1.
O
in vitro. Our group subsequently reported the first
mechanistic studies of LSD1 inactivation by 2-PCPA.10
As depicted in Scheme 2, LSD1 inhibition by 2-PCPA
occurs via formation of a covalent adduct joined at
the N5 and C4a positions of the flavin ring (Scheme
2).11 To pave the way for ongoing LSD1-selective inhib-
itor discovery, we report here a facile route to substi-
tuted trans-2-arylcyclopropylamines and the kinetic
evaluation of a representative subset of 2-PCPA deriva-
tives as mechanism-based inactivators of the human fla-
voenzyme oxidase lysine-specific histone demethylase
LSD1 and related monoamine oxidases A and B.
Ar
OH
a
O
O
NH2. HCl
e, f or g
c or d
Ar
O
Ar
Ar
O
4
2
3
b
O
+
Ar-I
O
Scheme 3. Reagents and condition: (a) TMSCHN2, C6H6, MeOH; (b)
Pd(OAc)2, K2CO3, NBu4Cl, DMF; (c) 1.6 mol% Pd(OAc)2, CH2N2,
THF; or (d) Me3S(O)I/NaH, DMSO; (e) i—aq NaOH, MeOH; ii—aq
HCl; (f) i—diphenylphosphoryl azide, Et3N, t-BuOH; ii—aq HCl,
THF; or (g) i—ethyl chloroformate, Et3N, acetone; ii—aq NaN3; iii—
2-(trimethylsilyl)ethanol; iv—NBu4F, THF.
Results: The general synthetic route to trans-2-arylcyclo-
propylamines commenced with the synthesis of esters
2a–k either by esterification of the commercially avail-
able carboxylic acids or by palladium catalyzed cross-
coupling between the appropriate aryl iodide and methyl
acrylate (Scheme 3). Two cyclopropanation methods
were employed for the preparation of compounds 3a–k
(Table 1). Reaction of a,b-unsaturated esters 2a–j with
the Corey–Chaykovsky reagent in DMSO (Method A)
generally gave poor yields of the desired cyclopropanat-
ed products.12–14 Detosylation of the indole nitrogen in
2k was anticipated under the strongly basic conditions
of Method A, thus cyclopropanation by this method
was not attempted. On the other hand, use of diazo-
methane with palladium (II) acetate15 (Method B) affor-
ded the corresponding cyclopropanated products in
excellent yields with only two exceptions (entries 9 and
10, Table 1).
For these two substrates, cyclopropanation by Method
B gave complex reaction mixtures whereas all other sub-
strates reacted under these conditions yielding the cyclo-
propyl adduct as the sole reaction product. However,
cyclopropanation with Method A provided 3i and 3j
in acceptable yield as the sole reaction products without
the need for further purification.
The trans stereochemistry of the 2-arylcyclopropylcarb-
oxylate esters, obtained by either method, was con-
firmed by the vicinal H–1H coupling constants of the
1
cyclopropyl moiety (Jab = 4.2–4.5 Hz). Alkaline hydro-
lysis gave the desired trans-arylcyclopropanecarboxylic
acids in high yields. These acids were transformed to
the corresponding primary amines by Curtius rearrange-
ments in yields ranging from 41% to 56% over 4 steps
(Scheme 3).16,17
FAD
FADox
NH2
NH2
NH2
FAD
Additionally, as shown in Scheme 4, the synthesis of 2,2-
diphenylcyclopropylamine (7) was accomplished in four
steps.18 Cyclopropanation of 5 was achieved via cop-
per(II) sulfate catalyzed addition of ethyl diazoacetate
to 1,1-diphenylethene. The resulting ester was hydro-
lyzed to give 6. Curtius rearrangement afforded the de-
sired amine 7.
R
N
R
N
N
O
N
O
O
H2O
NH
NH
N
H
N
H
O
O
NH2
R
N
R
N
We next examined inhibition kinetics for a representa-
tive subset of the 2-PCPA derivatives against human
LSD1 and monoamine oxidase A and B enzymes (Table
2). Intriguingly, biphenyl (7) and naphthyl (8) 2-PCPA
analogues showed only minor inhibitory potential with
respect to LSD1 at concentrations up to 1 mM (Table
3). However, the remaining derivatives displayed
approximately 4- to 5-fold increased values of kinact/KI
N
O
O
N
O
-H2O
NH
NH
N
N
O
HO
Scheme 2. The proposed mechanism of inhibition of LSD1 by 2-PCPA
via flavin adduct formation.

D. M. Gooden et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3047–3051
Table 1. Cyclopropanation methods for the preparation of esters 3a–k
3049
O
O
method A: ((CH3)3S(O)I/NaH)
method B: (CH2N2/Pd(OAc)2)
Ar
O
Ar
O
3a-k
2a-k
Entry
Compound
Ar
% Yielda (Method A)4
% Yielda (Method B)5
1
3a
3b
3c
3d
3e
3f
4-CF3C6H4
4-BrC6H4
4-CH3OC6H4
4-NO2C6H4
2-Thienyl
27
50
50
5
96
94
98
65
97
99
94
99
—
—
97
2
3
4
5
40
59
27
21
34
65
—
6
3-Thienyl
2-Furyl
7
3g
3h
3i
8
3-Furyl
3-Pyridyl
9b
10b
11c
3j
3k
1-Naphthyl
N(Ts)-5-indoyl
a Yields are for isolated product using 1.4–1.8 mmol of alkene substrate.
b Cyclopropanation by Method B gave inseparable and/or complex mixtures.
c Cyclopropanation by Method A not attempted on this substrate.
night, then diluting 100-fold into assay solution. Only
LSD1 incubated with the biphenyl derivative (7) showed
any residual activity, approximately 2% (data not
shown). This demonstrates that both compounds 7
and 8 are able to inhibit LSD1 over a longer period of
time. Absorbance scans of LSD1 inactivated by the
compounds showed the formation of the reduced flavin
species previously reported with 2-PCPA (data not
shown).10
O
NH2
a, b
c, d
OH
5
6
7
Scheme 4. Reagents and conditions: (a) N2CHCO2Et, 3 mol% CuSO4,
C6H6 reflux; (b) NaOH, EtOH (87% after 2 steps); (c) diphenylphos-
phoryl azide, Et3N, t-BuOH; (d) 1 M HCl, MeOH (52% overall).
Table 2. Representative 2-PCPA derivatives synthesized and kineti-
cally evaluated for LSD1, MAO A, and MAO B inhibition
The structure of the 2-PCPA–flavin adduct was recently
determined11 and was found to differ from that seen in
the crystal structure of MAO B inactivated by 2-PCPA19
(Figs. 1 and 2). In LSD1, cyclopropyl ring opening ap-
pears to lead to formation of a benzylic radical which at-
tacks the flavin at C4(a) to form an initial adduct, which
then undergoes cyclization to form a five-membered ring
R2
NH2
R1
Compound
R1
R2
7
8
Ph
Ph
H
H
H
H
H
1-Naphthyl
4-CF3C6H4
4-BrC6H4
4-CH3OC6H4
3-Thienyl
9
R
N
R
N
10
11
12
N
O
N
O
O
NH
NH
N
H
N
O
O
for inactivation of LSD1. Each of the inhibitors was
tested for their ability to irreversibly inhibit LSD1 by
incubating 50 lM LSD1 with 250 lM inhibitor over-
Figure 1. Structures of the 2-PCPA–flavin adducts identified in the
crystal structures of MAO B (left) and LSD1 (right).
Table 3. Kinetic parameters for inactivation of LSD1, MAO B, and MAO A by trans-2-phenylcyclopropylamine derivatives
Inhibitor
LSD1
MAO B
MAO A
kinact (sÀ1
)
KI (lM)
kinact/KI
(MÀ1 sÀ1
kinact (sÀ1
)
KI (lM)
kinact/KI
(MÀ1 sÀ1
kinact (sÀ1
)
KI (lM)
kinact/KI
(MÀ1 sÀ1
)
)
)
2-PCPA
7
0.026 0.001
NDa
ND
477 79
ND
ND
55
<1
0.024 0.002
ND
13.6 3.0
ND
1779
<1.5
4837
7507
2356
802
0.018 0.001
ND
37.3 8.4
ND
469
<14
362
8
<28
190
184
262
<10
0.047 0.001
0.058 0.002
0.078 0.005
0.070 0.002
0.035 0.005
9.8 0.6
7.7 1.4
32.9 9.1
86.6 7.8
1548 312
0.014 0.001
0.085 0.004
0.070 0.005
0.023 0.001
ND
38.7 8.3
29.8 3.3
36.5 6.2
20.5 3.0
ND
9
0.067 0.008
0.104 0.007
0.049 0.004
ND
352 95
566 73
188 52
ND
2859
1918
1112
<18
10
11
12
22.3
a ND, not determined.

3050
D. M. Gooden et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3047–3051
Figure 2. Active sites of (A) LSD1, (B) MAO B, and (C) MAO A.
joining the N5 and C4(a) atoms of the isoalloxazine
ring.
thyl derivative was equal in potency to 2-PCPA and less
potent than the para-bromo and para-methoxy deriva-
tives, opposite of the trend in MAO B. For both mono-
amine oxidases compound 9 was the most potent.
Although the exact structure of the 2-PCPA–flavin ad-
duct formed in MAO A is unknown, superposition of
the LSD1 and MAO B adducts in the active site of
MAO A suggests that the MAO B adduct will be formed
(data not shown). Ile335 in MAO A replaces Tyr326 in
MAO B, potentially creating additional inhibitor access.
Cys323 serves as an upper limit on the space above the
para-carbon of the phenyl group of 2-PCPA; the sulfur
Formation of the adduct structure seen in MAO B is
apparently prohibited in LSD1 by a steric clash between
the phenyl ring of 2-PCPA and F538 and Y761.11 There
appears to be ample room at the para-position of the
phenyl ring of the 2-PCPA–flavin adduct in LSD1 to
accommodate substituents of varying sizes at this posi-
tion. However, formation of an adduct with naphthyl
derivative 8 is predicted to be limited in either possible
orientation by Val333 on one side of the active site
and Ala809 on the other. The active site cavity in the
vicinity of the adduct carbon adjacent to flavin C4(a)
is not large enough to efficiently accommodate an addi-
tional phenyl moiety in the biphenyl derivative 7.
˚
is located 10.2 A away.
Presently we are investigating whether analysis of cells
exposed to 2-PCPA or other MAOIs reveals some of
the contributing factors responsible for the poorly
understood therapeutic effects and side effects of these
antidepressants.
Three of the six tested 2-PCPA derivatives showed in-
creased inhibitory potential toward MAO B relative to
the starting compound; these included the naphthyl
(8), para-trifluoromethyl (9), and para-bromo (10) deriv-
atives (Tables 2 and 3). It is somewhat unclear how the
active site of MAO B is able to accept the naphthyl
derivative, but rotation of the carbon–carbon bond be-
tween the naphthyl group and the rest of the adduct
likely prevents a steric clash with Gln206 (Fig. 2). As
with LSD1, the biphenyl derivative showed little inhibi-
tory potential at concentrations up to 1 mM. Interest-
ingly, the para-methoxy substituted derivative showed
a decrease in kinact/KI of approximately 50% relative to
2-PCPA, with a nearly 7-fold increase in KI. In the active
site of MAO B, Leu171, Tyr326, and Gln206 form a
‘cap’ above the para-position of the phenyl ring of 2-
PCPA, likely limiting the length of substituents permis-
sible at that position, and suggesting a means to develop
inhibitors selective for LSD1 over MAO B. We are cur-
rently conducting enantioselective syntheses of the most
potent and selective inhibitors of LSD1 to determine the
degree to which absolute stereochemistry affects inhibi-
tory potency.
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