Enantioselective synthesis of tranylcypromine analogues as lysine demethylase (LSD1) inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.02.017

Asymmetric cyclopropanation of styrenes by tert-butyl diazoacetate followed by ester hydrolysis and Curtius rearrangement gave a series of tranylcypromine analogues as single enantiomers. The o,- m- and p-bromo analogues were all more active than tranylcypromine in a LSD1 enzyme assay. The m- and p-bromo analogues were micromolar growth inhibitors of the LNCaP prostate cancer cell line as were the corresponding biphenyl analogues prepared from the bromide by Suzuki crosscoupling.

Full text of DOI:10.1016/j.bmc.2011.02.017

Bioorganic & Medicinal Chemistry 19 (2011) 3709–3716
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enantioselective synthesis of tranylcypromine analogues as lysine
demethylase (LSD1) inhibitors
Hanae Benelkebir a, Christopher Hodgkinson a, Patrick J. Duriez b, Annette L. Hayden c,
Rosemary A. Bulleid c, Simon J. Crabb c, Graham Packham c, A. Ganesan a,
⇑
a School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom
b Cancer Research UK Protein Core Facility, University of Southampton, Faculty of Medicine, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom
c Cancer Research UK Centre, University of Southampton, Faculty of Medicine, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Asymmetric cyclopropanation of styrenes by tert-butyl diazoacetate followed by ester hydrolysis and
Curtius rearrangement gave a series of tranylcypromine analogues as single enantiomers. The o,- m-
and p-bromo analogues were all more active than tranylcypromine in a LSD1 enzyme assay. The m-
and p-bromo analogues were micromolar growth inhibitors of the LNCaP prostate cancer cell line as were
the corresponding biphenyl analogues prepared from the bromide by Suzuki crosscoupling.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 30 August 2010
Revised 3 February 2011
Accepted 9 February 2011
Available online 13 February 2011
Keywords:
Epigenetics
Prostate cancer
Lysine demethylase
Enzyme inhibitors
1. Introduction
5-methylcytosine in DNA to 5-hydroxymethylcytosine were re-
ported and there is evidence that DNA repair enzymes will replace
Eukaryotic DNA is packaged together with histone proteins to
form chromatin in which genes are usually silenced. The DNA
needs to unwind from the histone complex to be transcriptionally
active and this interaction between DNA and histones is orches-
trated by three sets of players. Firstly, there are the ‘writers’, en-
zymes that introduce post-translational modifications into DNA
and histone proteins and thereby alter their affinity for one an-
other. Next, there are corresponding ‘erasers’ that remove these
modifications and return chromatin to its native state. Finally,
‘readers’ recognize and respond to the pattern of dynamic chroma-
tin post-transcriptional modification, referred to as the ‘epigenetic
code’.1
Among the post-translational modifications, methylation is un-
ique in that it occurs both in DNA (primarily at the C-5 position of
cytosine) and in histone proteins (at lysine and arginine residues).
It is also unique in that it was long considered to be an irreversible
modification although there is no chemical reason why demethyla-
tion would be impossible. In 2004, the first lysine demethylase LSD1
(Lysine Specific Demethylase) was described.2 Soon thereafter in
2006, a second family of lysine and arginine demethylases contain-
the modified base by cytosine thus effecting an indirect
demethylation.4
Currently, nearly 30 human lysine demethylases within the LSD
and JmjC families are known.5 LSDs comprising LSD1 and LSD2 are
homologous to monoamine oxidases (MAOs) and demethylate
mono- and dimethyllysine residues. The reaction involves electron
transfer between the FAD cofactor and the nitrogen lone pair. This
is why trimethyllysine is not a substrate, while mono- or dim-
ethyllysine are oxidized to an iminium ion that is hydrolytically
decomposed to formaldehyde (Fig. 1). The best characterized LSD
substrate is histone H3 on K4 and K9 residues but there are addi-
tional non-histone client proteins of importance. LSD1 mediated
demethylation of the tumor suppressor p53 inhibits its function
by preventing interaction with the co-activator 53BP1,6 while
demethylation of the DNA methyltransferase Dnmt1 is necessary
for maintaining DNA methylation activity.7
Overexpression of LSDs is observed in neuroblastoma, prostate,
breast and bladder cancers where it is believed to repress tran-
scription of repair pathways that would normally lead to apoptosis
instead of proliferation.8 Thus, LSD inhibitors are of interest as
anticancer agents as well as potentially applicable to other human
diseases that exhibit misregulated gene expression. The homology
between LSDs and MAOs, a clinically validated target, suggests that
LSDs are druggable. Indeed, screening known MAO inhibitors has
uncovered micromolar LSD inhibitors among which the best
known is the antidepressant drug tranylcypromine (trans-2-
ing the JmjC domain was identified that are iron-dependent a-keto-
glutarate dioxygenases.3 In 2009, enzymes that convert
⇑
Corresponding author. Tel.: +44 02380 593897; fax: +44 02380 596805.
E-mail address: ganesan@soton.ac.uk (A. Ganesan).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.017

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H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3709–3716
Figure 1. The catalytic mechanism of lysine specific demethylases (LSDs).
phenylcyclopropylamine, Parnate).9 The compound acts as an irre-
versible inhibitor forming a covalent adduct with the FAD cofactor.
X-ray structures of this complex bound within the LSD1 active site
are not completely unambiguous, and it is likely that more than
one adduct is formed (Fig. 2).10
Recently, several groups have reported the evaluation of tranyl-
cypromine analogues as LSD inhibitors. Tranylcypromine itself is
clinically used as a racemate and the same is true of the analogues
described (Fig. 3).10c,11 Here, we report our independent results in
developing a high-throughput LSD1 assay and the synthesis of
tranylcypromine analogues by a route that provides compounds
as single enantiomers.
ing methyllysine and lysine derivatives. Furthermore, substrate
turnover was significantly reduced by the addition of tranylcypro-
mine confirming inhibition of the enzyme.
We next examined colorimetric LSD assays suitable for higher
throughput. Shi has reported2 a coupled assay in which formalde-
hyde, the byproduct of LSD demethylation, is converted by formal-
dehyde dehydrogenase to formic acid with the concomitant
reduction of NAD+ to NADH which is spectrophotometrically mon-
itored at 340 nM. However, no enzyme activity was detected with
this assay. We then turned to the quantitation of hydrogen perox-
ide, the other byproduct of LSD demethylation. Forneris has re-
ported a horseradish peroxidase coupled LSD assay based on the
formation of a chromogenic quinoneimine dye between 3,5-
dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine.12
The results with this assay were inconclusive in our hands. An
alternative method13 based on the peroxidase catalyzed oxidation
of 10-acetyl-3,7-dihydroxyphenoxazine (AmplexRed) to the fluo-
rescent product resorufin however gave reliable and reproducible
results. As a fluorescence-based method, it has intrinsically higher
sensitivity and lower background than the colorimetric readouts.
While this work was in progress, the Cole group has also noted14
the improved sensitivity of the AmplexRed coupled assay and we
recommend it as the preferred LSD assay for screening. As a sec-
ondary assay to eliminate false positives and artefacts, we find
the mass spectrometric method described above to be a useful
and rapid means of directly measuring substrate demethylation.
2. Results and discussion
2.1. LSD1 assays
We expressed His-tagged full length recombinant human LSD1
using a plasmid kindly provided by Shi.2 The purified enzyme was
assayed by Shi’s immunoblot method. Briefly, the enzyme was
incubated with purified histones or a methylated histone peptide
substrate and activity detected with a commercial H3K4(me2) anti-
body. In our hands, this did not give reproducible results. SELDI-
TOF mass spectrometric analysis of the reaction mixture was more
reliable (Fig. 4) and we observed both single and double demethyl-
ation of the dimethyllysine containing peptide to the correspond-
Figure 2. The mechanism of LSD inhibition by tranylcypromine.

H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3709–3716
3711
Figure 3. Examples of tranylcypromine analogues reported as LSD inhibitors. Ki(inact) values are from the literature and not necessarily comparable due to differences in assay
conditions.
Figure 4. Mass spectrometric analysis of LSD1 activity with the synthetic peptide ARTK(me2)QTARKSTGGKAPRKQLA. Due to a calibration error, all masses are M+5. Top: MS
of peptide alone, MW 2283. Middle: MS of peptide after incubation with LSD1 showing monomethyl (MW 2269) and nonmethylated products (MW 2255). Bottom: MS of
peptide incubated with LSD1 and inhibitor tranylcypromine.
2.2. Tranylcypromine stereochemistry
cypromine has a Ki(inact) of 284
l
M compared to 168
l
M for (ꢀ)-
tranylcypromine and the two enantiomers form different adducts
with FAD in the LSD active site. It is possible their assay underes-
timates activity as it uses the less sensitive colorimetric detection
method. Furthermore, their results are based on truncated LSD1
lacking the N-terminal 184 amino acids whereas our assay uses
the full length protein.
Four stereoisomers of 2-phenylcylopropylamine are possible. Of
these, it is the racemate of the trans diastereomer which is used
clinically as a MAO inhibitor. MAO inhibition was found to be rel-
atively insensitive to stereochemistry, with the trans diastereomer
being three fold more potent than the cis. Within the trans series,
the (+) antipode with (1R,2S) stereochemistry was fourfold more
potent than its enantiomer.15 At the time of our studies, the situa-
tion with regards to LSD inhibition was unknown. We took racemic
tranylcypromine and resolved16 it into its two enantiomers which
were individually tested (Fig. 5). The Ki(inact) values observed are
2.3. Enantioselective analogue synthesis
The reported syntheses of tranylcypromine analogues as LSD
inhibitors are in racemic form. Although we found only a small dif-
ference in activity between the two tranylcypromine enantiomers,
it was unclear if this would be further magnified in the case of ana-
logues. For this reason, we designed an enantioselective route
which would exclusively provide the (1R,2S) stereochemistry that
similar to those reported (21 l
M) by Schmidt and McCafferty.9b
Our results indicate a slightly higher activity for the (1R,2S) enan-
tiomer, which is also the more active in MAO inhibition. While this
manuscript was in preparation, Mai has reported10c that (+)-tranyl-

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H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3709–3716
Figure 5. LSD1 inhibition by tranylcypromine as a racemate and single enantiomers.
was the more active of the two enantiomers. Our synthesis began
with the copper-catalyzed cyclopropanation of styrenes by -dia-
electronic properties can be accommodated at ortho, meta and
para positions.
a
zoesters. Pioneering work by Doyle, Pfaltz and Evans among others
has led to chiral catalysts that provide the product with high
enantioselectivity.17 Evans, for example, reports the reaction be-
tween tert-butyl diazoacetate and styrene in the presence of chiral
bisoxazoline ligands to give the cyclopropane in 81:19 trans:cis
diastereoselectivity and 96% ee.18 We repeated the procedure with
substituted styrenes and isolated the pure trans cyclopropane es-
ters by chromatography. Comparison of optical rotation with liter-
ature values indicated high enantioselectivity. These were then
converted to the chiral tranylcypromine analogue in three steps
by conversion of the ester to the acyl azide and Curtius rearrange-
ment (Fig. 6).19
We chose the bromo derivatives for a second reason, namely
the ability to convert these compounds into additional analogues
by palladium catalyzed crosscoupling reactions. This was demon-
strated by Suzuki coupling to give biphenyl analogues 4f–h
(Fig. 7). While substitution of bromide by phenyl in the para posi-
tion was well tolerated, the meta and ortho biphenyl analogues had
low activity in the LSD enzyme assay.
Functional biology studies have indicated potential for inhibi-
tors of LSD1 as anticancer therapeutics. For example, in prostate
cancer cell lines, siRNA induced depletion of LSD1 attenuates its
complex formation with the androgen receptor (AR) and subse-
quent AR mediated transcriptional activation and cell proliferation.
Chemical inhibition of LSD1 in these models (using the MAO inhib-
itor pargyline) was also shown to block demethylation of repres-
sive histone H3 lysine 9 (H3K9) marks and to inhibit androgen
mediated AR dependent gene activation.20 We therefore tested
the in vivo effects of tranylcypromine and our analogues for their
inhibitory effects in cell proliferation assays using the LNCaP pros-
tate adenocarcinoma cell line (Table 1).
2.4. Biological evaluation
Our first three analogues 4a–c probed the electronic effects of
phenyl substitution. In the LSD enzyme assay (Table 1), both the
para-methoxy and para-fluoro compounds were poorer inhibitors
than tranylcypromine suggesting that strongly electron-donating
or electron-withdrawing substituents are detrimental. On the
other hand, the para-bromo analogue 4c was a significantly
better LSD inhibitor than tranylcypromine, a result that was
independently reported by Mai.10c We then prepared the meta-
and ortho- bromo isomer 4d–e and these showed improved
activity as well compared to tranylcypromine. The X-ray struc-
ture of LSD1 indicates a roomy active site compared to MAOs,10
and our results demonstrate that substituents with the right
Tranylcypromine was a very poor inhibitor of the cell line in a
six-day proliferation assay with an IC50 of 174 mM. The
meta- and para-bromo analogues were significantly more potent
micromolar inhibitors with
a 1000 fold increase in activity
compared to tranylcypromine. Surprisingly, all three biphenyl
analogues were micromolar inhibitors of cell growth inhibition
including the meta- and ortho- analogues with weak activity in
the enzyme assay. The trends observed together with the low
Figure 6. Enantioselective synthesis of tranylcypromine analogues.

H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3709–3716
3713
M)
Table 1
Activity of tranylcypromine analogues in LSD enzyme and LNCaP growth inhibition assays
Compound
Substitution R
LSD inhibition Ki(inact)
(lM)
LNCaP inhibition IC50 (l
( )-Tranylcypromine
(+)-Tranylcypromine
(ꢀ)-Tranylcypromine
H
H
H
25.0 9.5
26.6 12.2
28.1 12.9
41.8 1.0
79.6 1.0
3.7 0.5
8.9 3.2
11.7 0.9
6.8 0.3
>100
>100,000
>100,000
>100,000
1706 120
>100,000
111 103
4a
4b
4c
4d
4e
4f
4-OMe
4-F
4-Br
3-Br
2-Br
4-Ph
3-Ph
2-Ph
129
3
5869 470
110 31
226 72
4g
4h
>100
181
6
Figure 7. Synthesis of phenyltranylcypromine by Suzuki crosscoupling.
growth inhibition of tranylcypromine itself suggest that activity in
the cell assay is driven by permeability and lipophilicity. Thus, the
more lipophilic biphenyl analogues retain activity despite being
poor in the enzyme assay. ortho-Bromide 4e is an outlier as its cell
growth inhibition is much lower compared to the isomeric
bromides.
and Q-sepharose columns from GE-Healthcare) were successively
performed and the purified protein was stored in 20 mM
Tris–HCl pH 8.0, 300 mM NaCl and 5% glycerol at ꢀ70 °C.
4.2. Fluorescence based LSD1 enzyme assay
LSD1 enzymatic activity was measured with a synthetic peptide
corresponding to the first 21 amino acids of human histone H3
dimethylated on lysine 4: ARTK(me2)QTARKSTGGKAPRKQLA (BPS
3. Conclusions
Bioscience). Assays were carried out in a final volume of 100
in white 96 microtitre plates (Greiner).
lL
We report the first enantioselective synthesis of tranylcypro-
mine analogues designed as LSD inhibitors. Our results indicate
very little difference between the two enantiomers of tranylcypro-
mine in LSD1 inhibition. The bromo substituted tranylcypromine
analogues 4c–e were superior to tranylcypromine in the enzyme
assay regardless of the position of bromination, with the para-
bromo derivative showing the highest activity. We have prepared
the biphenyl derivatives 4f–h and show that the meta or ortho ana-
logues are relatively inactive while the para analogue is similar in
activity to the bromide. We report the first observations of tranyl-
cypromine analogues as single agents in cancer cell growth inhibi-
tion. In the LNCaP prostate cancer cell line, 4c and 4f are over 1000
fold more potent than tranylcypromine. Work is in progress to pre-
pare further tranylcypromine analogues with a view to improving
cellular potency and selectivity.
An aliquot of 1.25
lg of recombinant LSD1 enzyme was added
to a 50 L reaction containing 35
l
lM of peptide substrate in
50 mM potassium phosphate pH 7.2 and the reaction mixture
was incubated at room temperature for 20 min. Fifty microliter
of a mix containing 0.5
L of 10 U/ml HRP (Sigma) in 50 mM potassium phosphate buffer
were added and the reaction mixture kept at room temperature for
another 30 min before quenching by adding 20 L of AmplexRed
lL of 10 mM AmplexRed (InVitrogen) and
1
l
l
Stop solution (InVitrogen). Fluorescence was measured at 530/
590 nm on a Varioskan Flash microplate reader (Thermo Fisher).
Under these conditions, a Km of 21 lM was observed for the pep-
tide substrate. For assays with inhibitors, the compound was incu-
bated with LSD1 for 10 min prior to the addition of the peptide
substrate. Ki(inact) values are means determined from a minimum
of three dose–response curves for each compound.
4. Experimental
4.1. Expression and purification of full length human LSD1
4.3. Mass spectrometric LSD1 enzyme assay
The plasmid pet15b-His-tagged full length human LSD1 was
kindly provided by Fei Lan in Dr. Yang Shi’s laboratory.2 The plas-
mid was transformed into BL21 RIPL Codon Plus (DE3) bacteria
(Stratagene) and protein expression was induced with 0.1 mM
IPTG for 3 h at room temperature. The bacterial pellet was lysed
(40 mM Tris–HCl pH 8.0, 300 mM NaCl, 0.2 % Triton X100, 5% glyc-
In a total volume of 50
bated alone or in the presence of 2.4
ture for 10 min.
l
L, 50
l
M of tranylcypromine was incu-
g of LSD1 at room tempera-
l
The peptide ARTK(me2)QTARKSTGGKAPRKQLA was added at a
concentration of 35 M and the reaction was carried out for an ex-
l
tra 20 min at room temperature. The samples were kept on ice be-
fore mass spectrometric analysis, carried out with NP20
ProteinChip (BioRad) arrays.
erol, 10 lg/ml DNase I and 10 mM MgCl2 in the presence of prote-
ase inhibitors), sonicated on ice and centrifuged. The supernatant
was used to purify LSD1 recombinant protein using an AKTA Prime
FPLC system. Three chromatography steps (Nickel, gel filtration
An NP20 array was pre-rinsed with 5
lL of ultrapure H2O
and 3 L sample was subsequently applied on each spot. The
l

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H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3709–3716
sample-loaded arrays were then incubated for 15 min in a humid-
ity chamber at room temperature. Then, 1 L of energy absorbing
matrix [EAM; -cyano-4-hydroxycinnamic acid (CHCA) in 200
acetonitrile and 200 L of 1% TFA] was added to each spot, air dried
(m, 1H), 7.57 (dd, J = 7.9, 1.3 Hz, 1H); 13C NMR (75 MHz, CDCl3) d
15.1, 24.3, 26.8, 28.2, 80.6, 126.4, 127.3, 127.4, 128.0, 132.6,
139.4, 172.4; EI MS m/z 240, 242 ([M+-tBu]). Yield 30%.
l
a
lL
l
and reapplied. ProteinChips were read using a PBS II SELDI mass
spectrometer (Bio-Rad). Data acquisition was performed using
the following parameters: Mass range between 0.5 and 20 kDa
with a focus mass of 3 kDa and detector blinding at 0.5 kDa. The la-
ser settings used were: intensity of 3,000nj and 18 shots, 2 warm-
ing shots at an intensity of 3,300nj, acquisition of 1 shot every four
pixels in a randomized fashion.
4.5.2. Ester hydrolysis of 1 to 2
The carboxylate esters 1 (1 equiv), TFA (13 equiv) and triethyl-
silane (2.5 equiv) were taken up in CH2Cl2. The reaction mixture
was stirred for 90 min followed by concentration in vacuo. The
crude material was used with no further purification or purified
by flash chromatography using CH2Cl2 as eluent if required to pro-
vide 2.
4.4. LNCaP cell growth inhibition assay
4.5.2.1. (R,R)-2-(4-Methoxyphenyl)cyclopropane carboxylic acid
(2a)19a
.
1H NMR (300 MHz, CDCl3) d 1.37 (ddd, J = 8.3, 6.7,
Cell proliferation assays were undertaken using the LNCaP pros-
tate adenocarcinoma cell line to produce IC50 values for inhibition
as previously described.21 Experiments are mean values +/ꢀ SEM
4.5 Hz, 1H), 1.63 (dt, J = 9.4, 4.8 Hz, 1H), 1.84 (ddd, J = 8.3, 5.1,
4.2 Hz, 1H), 2.58 (ddd, J = 9.2, 6.6, 4.2 Hz, 1H), 3.80 (s, 3H), 6.81–
6.87 (m, 2H), 7.03–7.09 (m, 2H). Yield 78%.
determined from
compound.
a minimum of two experiments for each
4.5.2.2. (R,R)-2-(4-Fluorophenyl)cyclopropane carboxylic acid
(2b)19a
.
1H NMR (300 MHz, CDCl3) d 1.53 (ddd, J = 8.4, 6.7,
4.5. Compound synthesis
4.7 Hz, 1H), 1.82 (dt, J = 9.4, 4.9 Hz, 1H), 1.96–2.08 (m, 1H), 2.71–
2.82 (m, 1H), 7.08–7.20 (m, 2H), 7.20–7.32 (m, 2H), 10.83 (br s,
1H). Yield 92%.
4.5.1. Styrene asymmetric cyclopropanation to give 1a–1e
(S,S)-2,20-Isopropylidene-bis(4-tert-butyl-2-oxazoline)
(0.01
equiv) and copper(II) trifluoromethanesulfonate (0.01 equiv) were
dissolved in chloroform and stirred for 45 min under argon. Sty-
rene derivatives (5 equiv) and tert-butyl diazoacetate (1 equiv)
were then added and the reaction mixture was stirred for 5 h, fol-
lowed by concentration in vacuo and purification of the crude
material by flash chromatography using CH2Cl2/hexane (2/8) to
provide the pure trans-diastereoisomers.
4.5.2.3. (R,R)-2-(4-Bromophenyl)cyclopropanecarboxylic acid
(2c)25
.
1H NMR (300 MHz, CDCl3) d 1.38 (ddd, J = 8.4, 6.6,
4.8 Hz, 1H), 1.68 (dt, J = 9.4, 5.0 Hz, 1H), 1.88 (ddd, J = 8.5, 5.2,
4.2 Hz, 1H), 2.57 (ddd, J = 9.2, 6.6, 4.0 Hz, 1H), 6.99 (d, J = 8.4 Hz,
2H), 7.42 (d, J = 8.4 Hz, 2H). Yield 98%.
4.5.2.4. (R,R)-2-(3-Bromophenyl)cyclopropanecarboxylic acid
(2d)26
.
1H NMR (300 MHz, CDCl3) d 1.41 (ddd, J = 8.4, 6.8,
4.5.1.1. (R,R)-tert-Butyl 2-(4-methoxyphenyl)cyclopropanecarb-
4.6 Hz, 1H), 1.59 (m, 1H), 1.91 (ddd, J = 8.5, 5.2, 4.2 Hz, 1H), 2.58
(ddd, J = 9.2, 6.6, 4.0 Hz, 1H), 7.05 (dt, J = 7.8, 1.4 Hz, 1H), 7.17 (t,
J = 7.7 Hz, 1H), 7.25–7.28 (m, 1H), 7.33–7.40 (m, 1H). Yield 99%.
oxylate (1a)22
.
½
a 2D3
ꢁ
ꢀ192.2 (c 0.24, CHCl3), [lit.23
½
a 2D4
ꢁ
ꢀ241.4
(c 1.0, CHCl3)]; 1H NMR (300 MHz, CDCl3) d 1.18 (m, 1H), 1.48 (s,
9H), 1.50–1.52 (m, 1H), 1.76 (dt, J = 8.7, 4.6 Hz, 1H), 2.38–2.44
(m, 1H), 3.79 (s, 3H), 6.83 (dd, J = 9.0, 0.4 Hz, 2H), 7.03 (dd,
J = 8.6, 0.3 Hz, 2H); ES+ MS m/z 303 ([M+Na+MeOH]+). Yield 77%.
4.5.2.5. (R,R)-2-(2-Bromophenyl)cyclopropanecarboxylic acid
(2e)27
.
½
a 2D5
ꢁ
ꢀ76.4 (c 0.29, CHCl3), [lit.26 (S,S)-enantiomer
[a]
+109.9 (c 0.77, CHCl3)]; 1H NMR (300 MHz, CDCl3) d 1.44
D
4.5.1.2. (R,R)-tert-Butyl 2-(4-fluorophenyl)cyclopropanecarb-
(ddd, J = 8.4, 7.0, 4.8 Hz, 1H), 1.71 (dt, J = 9.2, 4.7 Hz, 1H), 1.77–
1.90 (m, 1H), 2.80 (ddd, J = 9.2, 7.0, 4.4 Hz, 1H), 7.05 (dd, J = 7.7,
1.5 Hz, 1H), 7.12 (dt, J = 7.6, 1.7 Hz, 1H), 7.20–7.26 (m, 1H), 7.59
(dd, J = 7.9, 1.3 Hz, 1H). Yield 99%.
oxylate (1b)22
.
½
a 2D5
ꢁ
ꢀ213.9 (c 0.44, CHCl3), [lit.22
½
a 2D0
ꢁ
ꢀ182
(c 0.64, CHCl3)]; 1H NMR (300 MHz, CDCl3) d 1.19 (ddd, J = 8.4,
6.4, 4.5 Hz, 1H), 1.48 (s, 9H), 1.52 (dt, J = 4.6, 0.8 Hz, 1H), 1.78
(ddd, J = 8.5, 5.3, 4.2 Hz, 1H), 2.43 (ddd, J = 9.2, 6.5, 4.1 Hz, 1H),
6.93–7.01 (m, 2H), 7.02–7.10 (m, 2H). Yield 46%.
4.5.3. Curtius rearrangement of 2 to 3
The acids 2 (1 equiv), diphenylphosphoryl azide (1.1 equiv) and
triethylamine (1.5 equiv) were combined in tert-butanol under ar-
gon and heated at reflux for 48 h. The mixture was diluted with
EtOAc and saturated Na2CO3 solution. The organic layer was sepa-
rated and the aqueous layer was extracted with EtOAc. The organ-
ics layers were combined, dried over MgSO4 and concentrated in
vacuo. The crude material was purified by flash chromatography
(EtOAc/hexane 2/8) to provide the desired carbamates 3.
4.5.1.3. (R,R)-tert-Butyl 2-(4-bromophenyl)cyclopropanecarb-
oxylate (1c)23
.
½
a 2D5
ꢁ
ꢀ196.1 (c 0.41, CHCl3), [lit.23
½
a 2D4
ꢁ
ꢀ216.7
(c 1.00, CHCl3)]; 1H NMR (300 MHz, CDCl3) d 1.20 (ddd, J = 8.5, 6.4,
4.5 Hz, 1H), 1.47 (s, 9H), 1.54 (td, J = 4.6, 0.8 Hz, 1H), 1.80 (ddd,
J = 8.4, 5.4, 4.2 Hz, 1H), 2.40 (ddd, J = 9.2, 6.3, 4.1 Hz, 1H), 6.70 (d,
J = 8.2 Hz, 2H), 7.02–7.10 (d, J = 8.5 Hz, 2H). Yield 45%.
4.5.1.4. (R,R)-tert-Butyl 2-(3-bromophenyl)cyclopropanecarb-
oxylate (1d)24
.
1H NMR (300 MHz, CDCl3) d 1.22 (ddd, J = 8.5,
4.5.3.1. (1R,2S)-tert-Butyl 2-(4-methoxyphenyl)cyclopropylcar-
6.3, 4.6 Hz, 1H), 1.48 (s, 9H), 1.53 (dd, J = 9.5, 5.1 Hz, 1H), 1.83 (ddd,
J = 8.4, 5.3, 4.2 Hz, 1H), 2.41 (ddd, J = 9.2, 6.2, 4.0 Hz, 1H), 7.03 (dt,
J = 7.7, 1.3 Hz, 1H), 7.14 (t, J = 7.9 Hz, 1H), 7.23 (t, J = 1.8 Hz, 1H),
7.33 (dt, J = 7.7, 1.5 Hz, 1H). Yield 21%.
bamate (3a)19a
.
1H NMR (300 MHz, CDCl3) d 1.03–1.16 (m,
2H), 1.48 (s, 9H), 1.95–2.05 (m, 1H), 2.64–2.70 (m, 1H), 3.78 (s,
3H), 4.91 (br s, 1H), 6.76–6.90 (m, 2H), 7.05–7.15 (m, 2H). Yield 47%.
4.5.3.2. (1R,2S)-tert-Butyl 2-(4-fluorophenyl)cyclopropylcarba-
4.5.1.5. (R,R)-tert-Butyl 2-(2-bromophenyl)cyclopropanecarb-
mate (3b)19a
.
1H NMR (300 MHz, CDCl3) d 1.04–1.19 (m, 2H),
oxylate (1e).
Colorless oil; ½a D25
ꢁ
ꢀ66.3 (c 0.27, CH3OH); IR
1.46 (s, 9H), 1.98–2.12 (m, 1H), 2.60–2.76 (m, 1H), 4.83 (br s, 1H),
6.88–7.01 (m, 2H), 7.11–7.15 (m, 2H). Yield 46%.
3060, 1716, 1442 cmꢀ1 1H NMR (300 MHz, CDCl3) d 1.29 (ddd,
;
J = 8.4, 6.7, 4.6 Hz, 1H), 1.50 (s, 9H), 1.54–1.59 (m, 1H), 1.69 (ddd,
J = 8.4, 5.2, 4.6 Hz, 1H), 2.64 (ddd, J = 9.0, 6.7, 4.5 Hz, 1H), 7.01
(dd, J = 7.7, 1.4 Hz, 1H), 7.09 (dt, J = 7.7, 1.6 Hz, 1H), 7.20–7.26
4.5.3.3. (1R,2S)-tert-Butyl 2-(4-bromophenyl)cyclopropylcarba-
mate (3c)28
.
1H NMR (300 MHz, CDCl3) d 1.08–1.19 (m, 2H),

H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3709–3716
3715
1.46 (s, 9H), 1.94–2.09 (m, 1H), 2.64–2.73 (m, 1H), 4.82 (br s, 1H),
7.03 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H); ES+ MS m/z 334, 336
([M+Na]+). Yield 73%.
1H), 1.32–1.43 (m, 1H), 2.28–2.41 (m, 1H), 2.76 (dt, J = 7.7,
3.7 Hz, 1H), 3.75 (s, 3H), 6.82–6.91 (m, 2H), 7.04–7.16 (m, 2H);
HRMS (ESI) m/z calcd for C10H14NO (M+H)+ 164.1070, found
164.1074. Yield 77%.
4.5.3.4. (1R,2S)-tert-Butyl 2-(3-bromophenyl)cyclopropylcarba-
mate (3d)29
.
1H NMR (300 MHz, CDCl3) d 1.13–1.20 (m, 2H),
4.5.5.2. (1R,2S)-2-(4-Fluorophenyl)cyclopropanamine hydro-
1.46 (s, 9H), 2.02 (td, J = 7.8, 3.0 Hz, 1H), 2.69–2.77 (m, 1H), 4.82
(br s, 1H), 7.04–7.10 (m, 1H), 7.11–7.16 (m, 1H), 7.31 (m, 1H),
7.37–7.43 (m, 1H); ES+ MS m/z 334, 336 ([M+Na]+). Yield 52%.
chloride (4b)19a
.
1H NMR (300 MHz, CD3OD) d 1.30 (dt,
J = 7.8, 6.7 Hz, 1H), 1.42 (ddd, J = 10.2, 6.7, 4.4 Hz, 1H), 2.39 (ddd,
J = 10.1, 6.5, 3.6 Hz, 1H), 2.82 (ddd, J = 7.9, 4.4, 3.6 Hz, 1H), 6.96–
7.10 (m, 2H), 7.14–7.25 (m, 2H); ES+ MS m/z 193 ([M+H+CH3CN]+);
HRMS (ESI) m/z calcd for C9H11FN (M+H)+ 152.0870, found
152.0870. Yield 35%.
4.5.3.5. (1R,2S)-tert-Butyl 2-(2-bromophenyl)cyclopropylcarba-
mate (3e)30
.
1H NMR (300 MHz, CDCl3) d 1.14–1.40 (m, 2H),
1.52 (s, 9H), 2.09–2.34 (m, 1H), 2.65–2.90 (m, 1H), 5.05 (br s,
1H), 7.04–7.18 (m, 2H), 7.22–7.36 (m, 1H), 7.53–7.64 (m, 1H);
ES+ MS m/z 334, 336 ([M+Na]+). Yield 33%.
4.5.5.3. (1R,2S)-2-(4-Bromophenyl)cyclopropanamine hydro-
chloride (4c)29
.
1H NMR (300 MHz, CD3OD) d 1.39 (m, 1H),
1.49 (ddd, J = 10.3, 6.8, 4.5 Hz, 1H), 2.42 (ddd, J = 10.3, 6.5, 3.8 Hz,
1H), 2.90 (dt, J = 7.9, 3.8 Hz, 1H), 7.16 (d, J = 8.5 Hz, 2H), 7.51 (d,
J = 8.5 Hz, 2H); ES+ MS m/z 253, 255 ([M+H+CH3CN]+); HRMS
(ESI) m/z calcd for C9H11BrN (M+H)+ 212.0069, found 212.0073.
Yield 51%.
4.5.4. Suzuki coupling of 3c–e to 3f–h
To a solution of the aryl bromide in a mixture of toluene/meth-
anol/water (80/18/2) were added tetrakis(triphenylphosphine)pal-
ladium (0.2 equiv), phenylboronic acid (4 equiv), and Na2CO3
(2 equiv). The reaction mixture was refluxed overnight. The mix-
ture was then diluted with EtOAc, washed with water and brine
and purified by flash chromatography (EtOAc/hexane 5/95) to pro-
vide the biphenyl product.
4.5.5.4. (1R,2S)-2-(3-Bromophenyl)cyclopropanamine hydro-
chloride (4d)29
.
1H NMR (300 MHz, CD3OD)
d 1.41 (q,
J = 7.0 Hz, 1H), 1.49 (ddd, J = 10.3, 6.3, 4.5 Hz, 1H), 2.42 (ddd,
J = 10.0, 6.5, 3.5 Hz, 1H), 2.93 (dt, J = 8.0, 4.0 Hz, 1H), 7.17–7.23
(m, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.40–7.48 (m, 2H); ES+ MS m/z
253, 255 ([M+H+CH3CN]+); HRMS (ESI) m/z calcd for C9H11BrN
(M+H)+ 212.0069, found 212.0072. Yield 25%.
4.5.4.1.
mate (3f).
(1R,2S)-tert-Butyl-2-(biphenyl-4-yl)cyclopropylcarba-
Mp 98–100 °C; ½a 2D3
ꢀ99.4 (c 0.25, CHCl3); IR
ꢁ
3338, 1687, 1523, 1483 cmꢀ1 1H NMR (300 MHz, CDCl3) d 1.08–
;
1.33 (m, 2H), 1.48 (s, 9H), 2.09 (ddd, J = 9.3, 6.4, 3.1 Hz, 1H), 2.77
(m, J = 15.6 Hz, 1H), 4.88 (br s, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.35
(d, J = 7.4 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 7.48–7.61 (m, 4H); 13C
NMR (75 MHz, CDCl3) d 16.4, 24.8, 28.4, 32.6, 79.7, 126.8, 126.9,
127.0, 128.7, 139.0, 139.9, 141.0, 156.3; ES+ MS m/z 332
([M+Na]+); HRMS (ESI) m/z calcd for C20H23NNaO2 (M+Na)+
332.1621, found 332.1616. Yield 73%.
4.5.5.5. (1R,2S)-2-(2-Bromophenyl)cyclopropanamine hydro-
chloride (4e)30
.
1H NMR (300 MHz, CD3OD) d ppm 1.35–
1.62 (m, 2H), 2.53–2.73 (m, 1H), 2.79–2.96 (m, 1H), 7.09–7.26
(m, 2H), 7.27–7.42 (m, 1H), 7.63 (d, J = 7.9 Hz, 1H). Yield 77%.
4.5.5.6. (1R,2S)-2-(Biphenyl-4-yl)cyclopropanamine hydrochlo-
ride (4f)31
.
1H NMR (300 MHz, CD3OD) d 1.33–1.54 (m, 2H),
4.5.4.2.
(1R,2S)-tert-Butyl-2-(biphenyl-3-yl)cyclopropylcarba-
2.44 (ddd, J = 10.1, 6.7, 3.6 Hz, 1H), 2.91 (dt, J = 7.7, 4.1 Hz, 1H),
mate (3g).
Mp 68–70 °C; ½a 2D5
ꢀ50.8 (c 0.52, CH3OH); IR
ꢁ
7.21–7.39 (m, 3H), 7.44 (t, J = 7.5 Hz, 2H), 7.53–7.70 (m, 4H); ES+
3330, 1687, 1511, 1483 cmꢀ1
;
1H NMR (400 MHz, CDCl3) d 1.13–
MS m/z 251 ([M+H+CH3CN]+); HRMS (ESI) m/z calcd for C15H16
N
1.32 (m, 2H), 1.48 (s, 9H), 2.12 (ddd, J = 9.5, 6.4, 3.3 Hz, 1H),
2.73–2.89 (m, 1H), 4.87 (br s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.31–
7.38 (m, 3H), 7.39–7.50 (m, 3H), 7.52–7.67 (m, 2H); 13C NMR
(100 MHz, CDCl3) d 16.6, 25.1, 28.4, 32.6, 79.7, 125.0, 125.3,
125.4, 127.19, 127.23, 128.69, 128.73, 141.2, 141.3, 156.2; ES+
MS m/z 373 ([M+Na+CH3CN]+); HRMS (ESI) m/z calcd for
(M+H)+ 210.1277, found 210.1282. Yield 94%.
4.5.5.7. (1R,2S)-2-(Biphenyl-3-yl)cyclopropanamine hydrochlo-
ride (4g)31 1H NMR (300 MHz, CD3OD) d 1.38–1.58 (m, 2H),
.
2.50 (ddd, J = 10.1, 6.7, 3.6 Hz, 1H), 2.95 (dt, J = 7.7, 4.1 Hz, 1H),
7.18 (d, J = 7.5 Hz, 1H), 7.32–7.56 (m, 6H), 7.56–7.73 (m, 2H); ES+
MS m/z 210 ([M+H]+), 251 ([M+H+CH3CN]+); HRMS (ESI) m/z calcd
for C15H16N (M+H)+ 210.1277, found 210.1281. Yield 100%.
C
20H23NNaO2 (M+Na)+ 332.1621, found 332.1628. Yield 66%.
4.5.4.3.
mate (3h).
(1R,2S)-tert-Butyl-2-(biphenyl-2-yl)cyclopropylcarba-
Mp 64–66 °C; IR 3338, 1696, 1480 cmꢀ1 1H
;
4.5.5.8. (1R,2S)-2-(Biphenyl-2-yl)cyclopropanamine hydrochlo-
NMR (400 MHz, CDCl3) d 0.98–1.08 (m, 1H), 1.15 (q, J = 6.1 Hz,
1H), 1.46 (s, 9H), 2.00 (ddd, J = 9.9, 6.3, 3.0 Hz, 1H), 2.62–2.87 (m,
1H), 4.56 (br s, 1H), 7.04 (d, J = 7.1 Hz, 1H), 7.20–7.33 (m, 3H),
7.38 (dq, J = 8.8, 4.3 Hz, 1H), 7.45 (d, J = 5.1 Hz, 4H); 13C NMR
(100 MHz, CDCl3) d 17.0, 23.3, 28.3, 33.2, 79.5, 125.0, 125.9,
126.9, 127.5, 128.0, 129.6, 129.7, 137.8, 141.6, 142.5, 156.2; ES+
MS m/z 373 ([M+Na+CH3CN]+); HRMS (ESI) m/z calcd for
ride (4h).
Mp 52–54 °C; ½a 2D5
ꢀ15.3 (c 0.07, CH3OH); IR 1597,
ꢁ
1487, 1454 cmꢀ1; 1H NMR (400 MHz, CD3OD) d 1.19–1.29 (m, 2H),
2.39 (td, J = 8.6, 3.5 Hz, 1H), 2.87–3.03 (m, 1H), 6.98–7.12 (m, 1H),
7.26–7.56 (m, 8H); 13C NMR (100 MHz) d 14.8, 21.0, 32.3, 125.4,
127.7, 128.3, 128.9, 129.5, 130.5, 130.9, 136.7, 142.5, 143.9; ES+
MS m/z 210 ([M+H]+), 251 ([M+H+CH3CN]+); HRMS (ESI) m/z calcd
for C15H16N (M+H)+ 210.1277, found 210.1279. Yield 100%.
C
20H23NNaO2 (M+Na)+ 332.1621, found 332.1626. Yield 18%.
Acknowledgements
4.5.5. Boc deprotection of 3 to 4
Carbamate derivative 3 was taken up in anhydrous EtOAc
cooled at 0 °C and HCl(g) was bubbled into the solution for 40–
90 min. The reaction was monitored by TLC. The resulting HCl salt
was filtered and washed with diethyl ether.
We are grateful to Cancer Research UK for a studentship to H.B.
We thank Cancer Research UK, the Southampton Cancer Research
UK Centre and Experimental Cancer Medicine Centre and COST Ac-
tion TD0905 ‘Epigenetics: Bench to Bedside’ for financial support.
We thank Dr. Bashar Zeidan and Professor Paul Townsend at the
Faculty of Medicine, University of Southampton for assistance with
mass spectrometric studies, and Dr. Wynne Aherne and Dr. Kathy
4.5.5.1. (1R,2S)-2-(4-Methoxyphenyl)cyclopropanamine hydro-
chloride (4a)19a
.
1H NMR (300 MHz, CD3OD) d 1.18–1.31 (m,

3716
H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3709–3716
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Supplementary data
Supplementary data (1H and 13C NMR spectra for novel com-
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