Synthesis of Carboxamide-Containing Tranylcypromine Analogues as LSD1 (KDM1A) Inhibitors Targeting Acute Myeloid Leukemia

Article abstract of DOI:10.1002/cmdc.202000754

Lysine-specific demethylase 1 (LSD1/KDM1A) oxidatively removes methyl groups from histone proteins, and its aberrant activity has been correlated with cancers including acute myeloid leukemia (AML). We report a novel series of tranylcypromine analogues with a carboxamide at the 4-position of the aryl ring. These compounds, such as 5 a and 5 b with benzyl and phenethylamide substituents, respectively, had potent sub-micromolar IC₅₀ values for the inhibition of LSD1 as well as cell proliferation in a panel of AML cell lines. The dose-dependent increase in cellular expression levels of H3K4me2, CD86, CD11b and CD14 supported a mechanism involving LSD1 inhibition. The tert-butyl and ethyl carbamate derivatives of these tranylcypromines, although inactive in LSD1 inhibition, were of similar potency in cell-based assays with a more rapid onset of action. This suggests that carbamates can act as metabolically labile tranylcypromine prodrugs with superior pharmacokinetics.

Full text of DOI:10.1002/cmdc.202000754

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Synthesis of Carboxamide-Containing Tranylcypromine
Analogues as LSD1 (KDM1A) Inhibitors Targeting Acute
Myeloid Leukemia
Maria Teresa Borrello,[a] Hanae Benelkebir,[a] Adam Lee,[a] Chak Hin Tam,[a] Manar Shafat,[b]
Stuart A. Rushworth,[b] Kristian M. Bowles,[b] Leon Douglas,[c] Patrick J. Duriez,[c] Sarah Bailey,[c]
Simon J. Crabb,[c] Graham Packham,[c] and A. Ganesan*[a]
In memory of the late Professor Maurizio Botta 1950–2019 for his generous friendship and his many outstanding scientific contributions
to medicinal chemistry.
Lysine-specific demethylase 1 (LSD1/KDM1A) oxidatively re-
moves methyl groups from histone proteins, and its aberrant
activity has been correlated with cancers including acute
myeloid leukemia (AML). We report a novel series of tranylcy-
promine analogues with a carboxamide at the 4-position of the
aryl ring. These compounds, such as 5a and 5b with benzyl
and phenethylamide substituents, respectively, had potent sub-
micromolar IC50 values for the inhibition of LSD1 as well as cell
proliferation in a panel of AML cell lines. The dose-dependent
increase in cellular expression levels of H3K4me2, CD86, CD11b
and CD14 supported a mechanism involving LSD1 inhibition.
The tert-butyl and ethyl carbamate derivatives of these
tranylcypromines, although inactive in LSD1 inhibition, were of
similar potency in cell-based assays with a more rapid onset of
action. This suggests that carbamates can act as metabolically
labile tranylcypromine prodrugs with superior pharmacoki-
netics.
Introduction
enzymes has emerged as a successful strategy for the design of
selective inhibitors.[2,3]
Lysine methyltransferases (KMTs) catalyse the addition of one,
two or three methyl groups to the lysine side chains in
proteins.[1] The process is dynamically reversible due to lysine
demethylases (KDMs) which carry out CÀ H oxidation of the
methyl group to a carbinolamine that undergoes hydrolysis to
formaldehyde and the demethylated lysine. KDM1 comprising
the two lysine-specific demethylase (LSD) isoforms LSD1 and
LSD2 employs FAD (flavin adenine dinucleotide) as the oxidant.
In addition, the approximately twenty human isoforms of
KDM2-7 are iron(II)-dependent dioxygenases also known as
Jumonji C (JmjC) demethylases. Targeting the FAD or iron
cofactors respectively within these two families of KDM
LSD1 (KDM1A) contains SWIRM (Swi3, Rsc8 and Moira) and
Tower domains that modulate its incorporation into transcrip-
tionally activating or repressive multiprotein assemblies. As part
of the CoREST (REST corepressor 1) and NuRD (nucleosome
remodelling and histone deacetylase) complexes, LSD1 demeth-
ylates the nucleosome at histone H3K4 residues leading to
gene silencing. Meanwhile, association with androgen or estro-
gen receptors promotes H3K9 demethylation and gene activa-
tion. These events are linked to cell proliferation in leukemia
and hormone-dependent cancers as well as the maintenance of
cancer stem cells resistant to therapy.[4] Further validation of
LSD1 for epigenetic drug discovery comes from its high
expression levels in other cancer types including sarcoma,
neuroblastoma, bladder, gastric and lung.[5,6]
[a] Dr. M. Teresa Borrello, Dr. H. Benelkebir, A. Lee, C. Hin Tam,
Prof. A. Ganesan
One approach to targeting LSD1 involves the repurposing
of monoamine oxidase (MAO) inhibitors that disrupt the FAD
cofactor common to both enzymes.[7,8] The approved antide-
pressant (�)-tranylcypromine (1, Figure 1) for example, is a
substrate mimic that is oxidized to radical cation 2, followed by
strain induced cyclopropyl ring opening to the reactive
intermediate 3 which covalently modifies FAD. Since the
cofactor is tightly bound to LSD1, with one study reporting a Kd
of 180 nM,[9] the formation of tranylcypromine-FAD adducts
effectively results in irreversible enzyme inactivation. While
tranylcypromine is consequently being reinvestigated in clinical
trials as an anticancer agent, it has a modest micromolar
potency against LSD1 and inhibits MAO equally. Both the LSD1
potency and selectivity are improved in second-generation
School of Pharmacy, University of East Anglia
Norwich, NR4 7TJ (UK)
E-mail: a.ganesan@uea.ac.uk
[b] M. Shafat, Dr. S. A. Rushworth, Prof. K. M. Bowles
Norwich Medical School, University of East Anglia
Norwich, NR4 7TJ (UK)
[c] L. Douglas, Dr. P. J. Duriez, Dr. S. Bailey, Prof. S. J. Crabb, Prof. G. Packham
Protein Core Facility and Cancer Sciences
Cancer Research UK Centre and Experimental Cancer Medicines Centre
University of Southampton
Southampton General Hospital
Southampton, SO16 6YD (UK)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000754
This article belongs to the joint Special Collection with ChemBioChem,
“Chemical Epigenetics”.
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acidic removal of the tert-butyl ester afforded the monoacid 11
which was reacted with diphenylphosphoryl azide followed by
Curtius rearrangement and trapping of the resulting isocyanate
with tert-butanol to give Boc protected tranylcypromine 12. The
methyl ester was then hydrolysed to give the common
intermediate 13 which was coupled with a set of primary and
secondary amines. Boc deprotection of the resulting amides
14a–p completed the synthesis of the desired tranylcypromine
analogues (�) 5a–p.
Figure 1. Mechanism of action of (�)-tranylcypromine 1 and examples of
para-substituted second-generation analogues (�)-4–6.
Biology: LSD1 inhibition and antiproliferative activity against
AML cell lines
analogues featuring substitution of the aryl ring or the nitrogen
(or both).[10] One series 4 with a para-anilide substituent was
independently reported by Mai and Takeda.[11–16] We were
interested in the “reverse amide” isosteres 5 and while our
studies were in progress, a Takeda patent disclosed similar
structures.[17] Nevertheless, only compound 5a below was
described and the rest of our analogues are unique to this
work. In parallel, we explored a sulfonamide series 6 which we
have recently published.[18]
Tranylcypromine analogues 5 were investigated for their LSD1
inhibitory activity with a fluorescence-based LSD1 enzymatic
assay according to our previously published procedure. Briefly,
the assay uses a synthetic dimethylated histone H3K4me2
peptide as substrate and AmplexRed® detection of the H2O2 by-
product.[19,20] While tranylcypromine itself had a IC50 of 21.0 μM
in this assay, the analogues were significantly more potent
LSD1 inhibitors with the exception of 5h and 5i (Table 1).
Compounds 5a–c containing an aryl or heteroaryl substituent
linked by a one or two carbon spacer to the carboxamide were
sub-micromolar inhibitors of the enzyme. Switching to the
nonaromatic cyclohexyl isosteres 5d–e led to a loss of activity,
thus suggesting that π–π interactions might be important
within the enzyme active site. In the benzyl compound 5a, para
substitution as in 5f–5i led to reduced activity, most dramati-
cally with the methoxy and biphenyl analogues. We additionally
synthesized the tertiary amides 5j with a dibenzyl group and
5k–5p containing a 4-aminopiperazine moiety. These tertiary
amides, however, did not exhibit a gain in potency compared
to the simpler secondary amides.
Based on the enzyme assay results, selected analogues were
tested for their growth inhibitory potential against acute
myeloid leukemia (AML) cell lines. We selected a panel that
included both the wild type MLL (mixed lineage leukemia) gene
(U937, HL-60 and OCI-AML3) as well as MLL fusions arising from
gene translocation (Kasumi-1: MLL-ETO; THP-1: MLL-AF9; MV4-
11: MLL-AF4), since the latter are particularly sensitive to the
effects of LSD1 inhibition.[21] After 72 hours’ incubation, tranylcy-
promine was found to have low antiproliferative activity, with
IC50 values >50 μM in five of the six cell lines tested (Table 2).
On the other hand, the analogues with a benzylamide (5a, 5f)
or a phenethylamide (5b, 5c) arrested proliferation of at least
one cell line at sub-micromolar concentrations. The cellular
activity of analogues generally tracked with the inhibition
observed in the enzyme assay.
Results and Discussion
Chemistry: Analogue synthesis
Typically, the tranylcypromine scaffold is obtained from a
cyclopropyl carboxylic acid precursor which in turn arises from
the [2+1] cyclization of arylalkenes with carbenoid or
carbanion synthons. In our synthetic route, 4-formylbenzoic
acid (7, Scheme 1) was protected as the methyl ester 8,
followed by a Horner–Wadsworth–Emmons olefination with
tert-butyl diethylphosphonoacetate to provide the differentially
protected cinnamate 9. A Johnson-Corey-Chaykovsky cyclo-
propanation installed the three-membered ring in 10 with the
trans configuration as confirmed by the 1H,1H coupling con-
stants of the cyclopropyl moiety (Jab =4.2–4.5 Hz). Selective
Biology: Mechanism of inhibition and biomarkers
Scheme 1. Synthesis of tranylcypromine analogues 5a–p. a) AcCl, MeOH,
°
0 C to RT, overnight, 91%; b) tert-butyl diethylÀphosphonoacetate, KOtBu,
The nature of cellular growth inhibition was examined with the
analogues through a washout experiment. Cells were exposed
to the compounds for a short period of 6 hours, after which the
culture medium was washed out and replaced by fresh medium
+
°
THF, À 5 C to RT, overnight, 98%; c) Me3S(O) I , KOtBu, DMSO, RT, overnight,
53%; d) TFA, Et3SiH, CH2Cl2, RT, overnight, 72%; e) diphenylphosphoryl azide,
1
2
°
Et3N, tBuOH, reflux, 72 h, 42%; f) LiOH, aq THF, 50 C, 2 h, 82%; g) R R NH,
EDCI, HOBt, iPr2NEt, RT, overnight, 36–67%; h) HCl, THF, 13–71%.
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Table 1. LSD1 enzyme inhibition by tranylcypromine analogues (�)-5a–p.
IC50 values are reported�SEM (n=3).
Table 2. Antiproliferative activity of tranylcypromine (1) and tranylcypro-
mine analogues (5) in AML cancer cell lines. Blank entries indicate the assay
was not performed with that cell line.
Kasumi-1 U937
HL-60
OCI-
AML3
THP-1
MV4-11
IC50 [μM]�SEM (n=5)
R1R2N
IC50 [μM]
0.3�0.08
1
32�1.8
>100
84�15.0 89�13.0 81�3.9 63�8.4
Compound
5a 0.3�0.01 1.6�0.4
5b 0.7�0.02 1.2�0.2
5c 0.4�0.08 0.6�0.1
1.7�0.4
1.8�0.8
0.1�0.1 18.1�7.2
2.7�0.2 19.0�3.6
1.0�0.1 1.7�0.8
12.0�0.5
0.9�0.07 0.6�0.5
6.3�0.1
5a
5d 2.4�0.5
5e 31.0�2.7
5f 0.6�0.08
5g 2.7�0.1
5l 2.3�1.4
5n >100
5o
6.0�0.8
3.4�1.2
1.0�0.2
5b
5c
5d
5e
0.4�0.04
0.6�0.08
2.4�0.99
5.8�0.47
0.4�0.1 2.0�0.7
34.7�3.4 >100
>100
6.1�0.3
>100
>100
>100
>100
20�2.3
>100
2.0�0.7
1.8�0.2
0.3�0.1
2.0�0.1
5p 3.3�0.6
without the inhibitors (Figure 2). Following 72 hours’ incuba-
tion, the antiproliferative effects with or without the washout
were similar, supporting an irreversible mechanism of action as
predicted for tranylcypromines.
Further experiments involving biomarkers supported LSD1
as the cellular target. Analogue 5b was selected for these
studies as this phenethylamide displayed a high level of activity
in the enzyme assay as well as growth inhibition of cell lines
with either wild type MLL (e.g., HL-60) or MLL-fusions (e.g.,
MV4-11 and THP-1). MV4-11 cells were incubated with 5b
(200 nM) for time periods of 2, 4, 6, 48 and 72 hours. After 48
and 72 hours, a time-dependent increase of H3K4me2 was
5f
1.3�0.26
0.9�0.18
32.0�0.25
5g
5h
5i
5j
17.4�3.9
0.7�0.09
5k
5l
1.5�0.2
1.8�0.05
5m
0.5�0.01
5n
5o
0.9�0.01
0.5�0.04
Figure 2. Washout experiment. AML cell lines were treated with 200 nM of
compound 5b or 5c for 6 h followed by washout, or for 72 h continuously.
The percentage viability was measured with Cell-Titer-Glo® and relative
luminescence units (RLU) normalised to DMSO control (NT: not treated).
Statistical analysis using two-way ANOVA and corrected for multiple
comparisons using Bonferroni’s test revealed only insignificant (ns) variations
in cells viability between untreated, washout and continuous treatment. The
results are expressed as percentage of viability compared to controls. Error
bars are mean � SEM (n=5 and three technical replicates).
5p
1.8�0.3
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observed (Figure 3); this is consistent with the inhibition of
LSD1.
FACS sorting analysis revealed a 70–90% increase in the
expression of CD86 compared to control samples (Figure 4). In
addition, we examined the levels of CD11b and CD14, two
glycoproteins on the myeloid cell surface that are exclusively
expressed in mature leucocytes and not in undifferentiated
cells. As anticipated, 5a and 5b triggered cell differentiation as
confirmed by a significant increase in CD11b and CD14
expression (Figure 5).
Further evidence for cellular LSD1 inhibition came from the
increased levels of cell surface proteins associated with myeloid
differentiation. CD86 (cluster of differentiation 86), a linker of
the coinhibitory immune response of the CTL4 and CD86
receptor, is a convenient cellular biomarker as it is among the
most highly upregulated genes following LSD1 inhibition.[22]
Treatment of AML cell lines with 5a, 5b and 5c followed by
Biology: Tranylcypromine versus carbamate derivatives
The mechanism of action of tranylcypromine (Figure 1) requires
the lone pair of electrons in the amine to undergo oxidation by
the FAD cofactor. As expected, the Boc protected precursors 14
of our analogues 5 were inactive in enzyme inhibition assays at
the highest tested concentration of 250 μM even at prolonged
exposure times of 72 and 96 hours. However, to our surprise,
the Boc carbamates displayed significant growth inhibition of
the AML cell lines as illustrated by the representative results for
14b and 14c (Figure 6), the protected versions of 5b and 5c,
respectively (Table 3, additional data are given in the Support-
ing Information). Furthermore, the Boc carbamate often dis-
played a higher cellular activity relative to the corresponding
tranylcypromine at shorter exposure times of 24 or 48 hours
(Figure 7, additional data are given in the Supporting Informa-
tion).
Figure 3. H3K4me2 levels upon treatment with compound 5b in MV4-11
cells. A) Immunoblot of H3K4me2 and H3 (loading control) of lysates
prepared from MV4-11 cells treated with 5b. B) Quantification of (A) by
using ImageJ software. Mean band intensity plotted � SEM (n=3); a one-
way ANOVA was used for statistical analysis (****p<0.0001, n=3). The
figure is representative of three independent biological replicates.
To understand if these effects were specific to the Boc
compounds, we prepared the ethyl carbamates 15b and 15c,
which also exhibited similar levels of antiproliferative activity
(Table 3 and Supporting Information).
The carbamates were indistinguishable from the tranylcy-
promines in their cellular phenotypic effects with a dose-
dependent increase in H3K4me2, CD86, CD11b and CD14 (data
in the Supporting Information). Some of our compounds were
also tested as LSD1 inhibitors against the parasite Schistosoma
mansoni, the causative agent of the neglected tropical disease
schistosomiasis. In this context as well, the Boc tranylcypro-
mines 14 were more active than the free amines 5.[23] Based on
these results, we believe the carbamates are acting as prodrugs
that undergo hydrolysis to liberate the active compound
intracellularly, and we have observed a similar phenomenon in
our sulfonamide series 6.[18] This suggests that derivatization of
tranylcypromines is a promising avenue for the improvement of
pharmacokinetic properties, as indicated by the faster time
course of cancer cell line and parasite inhibition observed with
Figure 4. Evaluation of CD86 expression on MV4-11 cells upon treatment
with 5a, 5b, 5c. Cells were gated based on forward scatter (FSC) and side
scatter (SSC) parameters. The x-axis indicates the mean fluorescence increase
in CD86-FITC stained cells. Percentage of increase is reported compared to
control cells treated with DMSO. The figure is representative of three
biological replicates with three technical replicates.
Table 3. Antiproliferative activity of Boc protected tranylcypromines 14b,c and ethyl carbamates 15b,c in AML cancer cell lines. Blank entries indicate the
assay was not performed with that cell line.
Kasumi-1
IC50 [μM]�SEM (n=5)
U937
HL-60
OCI-AML3
THP-1
MV4-11
5b
0.7�0.02
0.5�0.03
1.2�0.2
0.4�0.04
0.3�0.03
0.6�0.1
0.6�0.02
0.9�0.07
0.4�0.02
0.5�0.08
6.3�0.1
0.6�0.5
0.4�0.02
1.4�0.5
2.7�0.2
19.0�3.6
0.5�0.01
1.3�0.3
1.7�0.8
0.6�0.1
0.5�0.02
14b
15b
5c
14c
15c
0.2�0.04
0.4�0.08
0.7�0.1
1.0�0.1
0.6�0.05
0.3�0.01
0.5�0.04
0.7�0.03
0.5�0.07
0.5�0.04
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Figure 5. Effect of compounds 5a and 5b (200 nM, 48 h) on the expression of CD14 and CD11b. FSC and SSC profiles were applied for the initial gating by
selecting cell size and distribution and removal of cell debris. The isotype controls are shown on the left, followed by the expression of CD11b and CD14 upon
treatment with 5a and 5b, respectively. Statistical significance was determined with a two-way ANOVA and corrected for multiple comparisons with
Bonferroni’s test. Values are expressed as mean % of CD14-CD11b increase � SD (n=3); ****p<0.0001. The reported figure is representative of three
independent experiments.
the carbamates. This probably reflects more efficient cell
permeability of the carbamates relative to the ionizable amine
present in tranylcypromines.
Conclusion
We report a series of tranylcypromines containing a para-
carboxamide substituent in the phenyl ring. Many of the
compounds, including 5a and 5b with the relatively simple
benzyl and phenethyl substituent respectively, robustly inhibit
LSD1 with sub-micromolar IC50 values and have similar levels of
potency in the inhibition of AML cell lines. We demonstrated
LSD1 as the cellular target through measurement of increased
Figure 6. Structures of tranylcypromine carbamates 14b,c and 15b,c.
Figure 7. Cell viability upon 24 hours’ treatment with tranylcypromine 5b and Boc derivative 14b in AML cancer cells at 0.1 and 1 μM. Two-way ANOVA
analysis corrected with Dunnett’s test for multiple comparison revealed a significant decrease in cell viability upon 24 h of treatment with either 5b or 14b
(*p=0.01, **p=0.001, ****p<0.0001) compared to untreated (NT, vehicle) cells. The results are expressed as a percentage viability compared to controls.
Error bars are mean � SEM (n=5 and three technical replicates).
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extracted with EtOAc (100 mL×5). The organic phases were
combined and washed with sat. NaHCO3 (100 mL), H2O (100 mL)
and brine (100 mL) and dried over MgSO4. Evaporation of the
solvent in vacuo provided 9 (14.9 g, 98%) as a white crystalline
histone methylation and the increased expression of cellular
differentiation biomarkers CD86, CD11b and CD14. Interestingly,
tert-butyl and ethyl carbamate derivatives of the tranylcypro-
mines were potent in cellular assays although unable to inhibit
LSD1 in vitro, suggesting they act as metabolically labile
prodrugs.
solid that was used without further purification: Rf =0.64 (petro-
1
°
leum ether/ EtOAc 2:8); m.p. 64 C; H NMR (CDCl3): δ=1.52 (s, 9H),
3.90 (s, 3H), 6.43 (d, J=16.0 Hz, 1H) 7.53–7.59 (m, 3H), 8.04 (d, J=
8.0 Hz, 2H). 13C NMR (CDCl3): δ=28.2, 52.3, 80.9, 122.6, 127.8, 130.1,
131.2, 139.0, 142.1, 165.8, 166.5; HRMS (ESI) m/z calcd for C15H19O4:
263.1278 [M+H]+; found: 263.1278.
Experimental Section
(�)-trans-4-Methyl[2-(tert-butoxycarbonyl) cyclopropyl)]benzoate (10):
A flame dried flask was charged with potassium tert-butoxide
(0.49 g, 4.37 mmol) and trimethylsufoxonium iodide (0.92 g,
4.18 mmol). DMSO (25 mL) was then added. The mixture was stirred
under a nitrogen atmosphere for 30 min, before the dropwise
addition of alkene 9 (1.00 g, 3.81 mmol) dissolved in DMSO (15 mL)
over 30 min. The reaction mixture was stirred at ambient temper-
ature under nitrogen for 16 h. Water (50 mL) and EtOAc (50 mL)
were then added and the organic layer separated. The aqueous
layer was extracted a further 4 times with EtOAc (50 mL) and the
combined organic extracts dried over MgSO4 and concentrated.
Purification by silica gel column chromatography gave 10 as a
Chemistry: General procedures. All chemicals were purchased
from Sigma-Aldrich, Lancaster Synthesis GmbH, Alfa Aesar, and
Novabiochem and used without further purification. TLC was used
to monitor reactions and performed on aluminium-backed silica gel
coated plates (Merck DC, Alufolien Kieselgel 60 F254) with spots
visualized by UV light (λ 254 nm). Product concentration after
reactions and extractions involved the use of a rotary evaporator
operating at reduced pressure of ca. 20 Torr and the term in vacuo
refers to solvent concentration at reduced pressure. When neces-
sary products were purified by flash chromatography using silica
gel (MN Kieselgel 60, 40–63 μm, 230–400 mesh ASTM). NMR spectra
were recorded on a Bruker AC 400 spectrometer and the spectra
were calibrated to the residual deuterated solvent peak (CDCl3,
CD3OD or [D6]DMSO). The chemical shifts are reported in δ (ppm)
units followed by brackets for protons containing spectral details in
this order: multiplicity (s: singlet, d: doublet, t: triplet, q: quartet, m:
multiplet, br: broad), coupling constants (reported in Hz), number
of protons (from integration). HRMS were acquired through the
EPSRC National Mass Spectrometry Service Centre, Swansea.
Melting points were determined with a STUART Melting point
SMP10. IR spectra were determined with Perkin Elmer, Spectrum
GX, FTIR system. RP-HPLC analyses were performed with an Agilent
Technologies 1200 series chromatograph with an Agilent Technol-
ogies ZORBAX Eclipse XDBÀ C18 (5 μm, 4.6×150 mm) column.
Gradient used: 95:5 water: methanol with 0.05% TFA additive to
5:95 water: methanol over 15 min returning to 95:5 water:
methanol over 5 min at a flow rate of 1 mL/min. Purity was
monitored at 220 nm and found to be at least 95% for all
compounds that were tested in biological assays.
white solid (0.56 g, 53%). Rf = 0.66 (petroleum ether/EtOAc 8:2);
1
m.p. 40 C; IR: 1715, 1609 cmÀ 1; H NMR (CDCl3, 400 MHz): δ=1.26
°
(ddd, J=4.8, 6.4, 8.5 Hz, 1H), 1.46 (s, 9H), 1.57 (m, 1H), 1.88 (ddd, J=
4.4, 5.5, 8.6 Hz, 1H), 2.4 (ddd, J=4.1, 5.4, 9.5 Hz, 1H), 3.9 (s, 3H), 7.1
(d, J=8.3 Hz, 2H), 7.9 (d, J=8.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz):
δ=16.5, 24.6, 24.8, 27.1, 50.9, 79.8, 124.9, 127.6, 128.7, 145.07,
165.8, 171.0; HRMS (ESI) m/z calcd. for C16H24O4N: 294.1700 [M+
NH4]+; found: 294.1704.
(�)-trans-[2-(4-(Methoxycarbonyl)phenyl]cyclopropanecarboxylic acid
(11): Trifluoroacetic acid (19.7 g, 13.2 mL, 0.17 mol, 13 equiv.) and
triethylsilane (3.86 g, 5.3 mL, 33.3 mmol, 2.5 equiv.) were added to a
solution of 10 (3.7 g, 13.4 mmol) in dichloromethane (40 mL). The
reaction mixture was stirred at RT and monitored by TLC. After
completion, the reaction mixture was co-evaporated with
acetonitrile (15 mL×3) to give 11 (2.1 g, 72%) as a white crystalline
°
solid that was used without further purification: m.p. 123 C; IR:
3307, 1716, 1608, 1477 cmÀ 1
;
1H NMR (CD3OD, 400 MHz): δ=1.43
(ddd, J=4.6, 6.5, 8.5 Hz, 1H), 1.60 (ddd, J=4.6, 5.3, 9.4 Hz, 1H), 1.92
(ddd, J=4.1, 5.4, 8.5 Hz, 1H), 2.51 (ddd, J=4.0, 6.3, 9.2 Hz, 1H), 3.90
(s, 3H), 7.25 (d, J=8.4 Hz, 2H), 7.93 (d, J=8.44 Hz, 2H); 13C NMR
(CD3OD, 100 MHz,): δ=17.9, 25.6, 26.9, 52.5, 127.1, 129.4, 130.7,
147.6, 168.7, 176.4; HRMS (ESI) m/z calcd. for C12H11O4: 219.0663
[MÀ H]À ; found: 219.0659.
Methyl 4-formylbenzoate (8): 4-Formylbenzoic acid (10.0 g, 66.0 mol,
1.0 equiv.) was dissolved in anhydrous MeOH (100 mL) with cooling
°
(À 5 C) and acetyl chloride (24.1 g, 21.2 mL, 0.33 mol, 5.0 equiv.)
was added dropwise over a period of 10 min. After 30 min, the
reaction mixture was warmed to RT and stirred overnight. The
volatiles were removed in vacuo and the residue dissolved in EtOAc
(70 mL), washed with 1 N NaOH, (100 mL×3) followed by sat.
NaHCO3 (50 mL×3), H2O (50 mL×3) and brine (50 mL×3). The
organic phase was dried over MgSO4, filtered and concentrated to
give 8 (9.8 g, 91%) as a crystalline white solid that was used
(�)-trans-4-Methyl-2-[(tert-butoxycarbonyl)amino]cyclopropyl
ben-
zoate (12): The carboxylic acid 11 (2.1 g, 9.5 mmol, 1.0 equiv.),
diphenylphosphoryl azide (2.86 g, 2.27 mL, 10.5 mmol, 1.1 equiv.)
and triethylamine (1.44 g, 1.98 mL, 14.3 mmol, 1.5 equiv.) were
combined in tert-butanol (11 mL) under argon, heated at reflux and
allowed to react for 72 h. After that time, the reaction mixture was
cooled to RT, diluted with EtOAc (20 mL) and washed with
saturated Na2CO3 solution (20 mL×3). The organic layer was
separated and the aqueous layer further extracted with EtOAc
(20 mL). The organic layers were combined (40 mL), washed with
sat. NaHCO3 (10 mL), H2O (10 mL) and brine (10 mL) and dried over
MgSO4. Concentration in vacuo afforded a yellow oil which was
purified by silica gel column chromatography (Hexane/EtOAc 8:2)
without further purification: Rf =0.4, (petroleum ether/EtOAc 3:7);
1
m.p. 180 C; IR: 1716, 1684, 1428 cmÀ 1; H NMR (CDCl3): δ=3.91 (s,
°
3H), 7.94 (d, J=8.5 Hz, 2H), 8.19 (d, J=8.3 Hz, 2H), 10.00 (s, 1H); 13C
NMR (CDCl3): δ=52.6, 129.6, 130.3, 135.2, 139.3, 166.2, 191.7; HRMS
(ESI) m/z calcd. for C9H9O3 [M+H]+ 165.0546; found: 165.0545.
(E)-Methyl 4-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl) benzoate (9): To a
solution of KOtBu (7.3 g, 64.6 mmol, 1.1 equiv.) in dry THF (100 mL),
tert-butyl diethylphosphonoacetate (16.3 g, 15.4 mL, 64.6 mmol,
°
1.1 equiv.) was slowly added dropwise at À 5 C for a period of
affording 12 (1.6 g, 42%) as a white crystalline solid: Rf=0.57
15 min. The mixture was stirred for 1 h while maintaining the same
temperature. Aldehyde 8 (9.8 g, 58.7 mmol, 1 equiv.), dissolved in
dry THF (40 mL), was then added dropwise to the mixture with
1
°
(hexane/EtOAc 8:2); m.p. 45 C; H NMR (CDCl3, 400 MHz): δ=1.20–
1.24 (m, 2H), 1.43 (s, 9H), 2.07 (td, J=3.1, 7.6 Hz, 1H), 2.70–2.78 (m,
1H) 3.9, (s, 3H), 7.15 (d, J=8.5 Hz, 2H), 7.9 (d, J=8.4 Hz, 2H); 13C
NMR (CDCl3, 100 MHz): δ=14.8, 26.3, 28.0, 28.1, 28.4, 55.3, 113.8,
127.8, 130.6, 132.7, 157.9; 168.7; HRMS (ESI) m/z calcd. for
C15H18N1O4: 276.1241 [MÀ H]À ; found: 276.1243.
°
vigorous stirring at À 5 C for a period of 20 min. The reaction
mixture was warmed to RT and stirred overnight. After that time,
the reaction mixture was poured into iced H2O (100 mL) and
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(�)-trans-4-2-[(tert-Butoxycarbonyl)amino)cyclopropyl]benzoic
acid
1H), 2.42 (ddd, J=3.5, 6.3 Hz, 10.3 1H), 2.90 (t, J=7.7 Hz, 3H), 3.58
(t, J=7.2 Hz, 2H), 7.17–7.20 (m, 1H), 7.24–7.29 (m, 6H) 7.72 (d, J=
7.7 Hz, 2H); 13C NMR (CD3OD): δ=14.3, 22.4, 32.3, 36.3, 42.6,
127.3,127.4, 128.6, 129.5, 129.9, 134.3, 140.6, 143.8, 169.7; HRMS
(ESI) m/z calcd. for C18H21N2O: 281.1648 [M+H]+; found: 281.1648.
(13): To a suspension of 12 (1.6 g, 5.7 mmol, 1 equiv.) in THF:H2O
(3:1 ratio), LiOH (0.4 g, 17.32 mmol, 3 equiv.) was added and the
°
reaction mixture heated to 50 C and stirred for 3 h. The reaction
was monitored by TLC and after completion, the reaction mixture
was diluted with water (10 mL) and acidified to pH 1–2 with sat.
KHSO4. The aqueous layer was extracted with EtOAc (20 mL×3) and
the combined organic layers (60 mL) were washed with sat. NaHCO3
(10 mL), H2O (10 mL) and brine H2O (10 mL) and dried over MgSO4.
Solvent concentration in vacuo afforded 13 (1.3 g, 82%) as a white
solid that was used without further purification. IR: 3314, 2873,
4-(�)-trans-2-Aminocyclopropyl)-N-(2-(thiophen-2-yl)ethyl)benzamide
°
hydrochloride (5c): Yield 47%, yellow solid; m.p. 218 C; IR: 3318,
2775, 2977, 1626, 1505 cmÀ 1
;
1H NMR (CD3OD): δ=1.37–1.42 (m,
1H), 1.48 (ddd, J=4.56, 6.7, 10.2 Hz, 1H), 2.43 (ddd, J=3.6, 6.0,
9.0 Hz, 1H), 2.90–2.94 (m, 1H), 3.13 (t, J=7.0 Hz, 2H), 3.61 (t, J=
7.0 Hz, 2H), 6.88 (dd, J=1.0, 3.4 Hz, 1H), 6.91–6.94 (m, 1H) 7.20 (dd,
1
1681, 1453 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.21–1.25 (m, 1H),
J=1.0, 4.8 Hz, 1H), 7.25 (d, J=8.4 Hz, 2H), 7.75 (d, J=8.4 Hz, 2H); 13
C
1.43 (s, 9H), 2.04 (td, J=3.9, 7.9 Hz, 1H), 2.69–2.65 (m, 1H), 7.19 (d,
J=8.3 Hz, 2H), 7.91 (d, J=8.3 Hz, 2H); 13C NMR (CD3OD, 100 MHz):
δ=17.1, 25.8, 28.7, 35.0, 80.3, 126.9, 129.4, 130.7, 148.5, 158.9,
169.9; HRMS (ESI) m/z calcd. for C17H25NO4: 273.1961 [M+H]+;
found: 273.1962.
NMR (CD3OD): δ=14.3, 22.4, 30.1, 32.2, 42.7, 124.7, 126.3, 127.4,
127.8, 128.6, 134.1, 142.5, 143.7, 169.7.
4-(�)-trans-2-Aminocyclopropyl)-N-(cyclohexylmethyl)benzamide
°
hydrochloride (5d): Yield 58%, yellow solid; m.p. 198 C; IR: 3335,
1712, 1696, 1459 cmÀ 1; 1H NMR (CD3OD, 400 MHz): δ=0.94–1.03 (m,
2H), 1.18–1.31 (m, 4H), 1.33–1.44 (m, 1H), 1.48–1.52 (m, 1H), 1.58–
1.69 (m, 2H), 1.71–1.82 (m, 3H), 2.41–2.49 (m, 1H), 2.88–2.92 (m, 1H)
3.20 (d, J=6.9 Hz, 2H), 7.26 (d, J=8.2 Hz, 2H), 7.76 (d, J=8.2 Hz,
2H); 13C NMR (CD3OD, 100 MHz): δ=14.3, 22.4, 26.9, 27.6, 32.3, 39.2,
39.4, 47.2, 127.4, 128.6, 134.3, 143.6, 169.8; HRMS (ESI) m/z calcd. for
C17H25N2O: 273.1961 [M+H]+; found: 273.1962.
General procedure for 14a–p and 5a–p: To a stirring suspension of
12 in dichloromethane, Hünig’s base (2 equiv.) was added and the
mixture stirred until obtaining a clear solution. Subsequently, HOBt
(0.2 equiv.) and EDCI (1.5 equiv.) were added and the reaction
mixture stirred for 30 min. The desired amine was added to the
mixture and stirring continued at RT overnight. After that time, the
reaction mixture was diluted with dichloromethane and washed
with 2 M HCl and 1 M NaOH. The organic layers were combined,
washed with sat. NaHCO3, H2O and brine and dried over MgSO4.
Concentration in vacuo afforded Boc amides 14a–p. Acid hydrolysis
to remove the Boc protecting group was carried out by stirring the
carbamate in 0.5 mL of THF and 0.5 mL of 4 M HCl in 1,4-dioxane at
RT overnight. After completion, the reaction mixture was diluted
with acetonitrile and concentrated in vacuo afforded to give
tranylcypromines 5a–p as their hydrochloride salt. The salts were
washed with diethyl ether and purified by preparative RP-HPLC.
4-(�)-trans-2-Aminocyclopropyl)-N-(2-cyclohexylethyl)benzamide
°
hydrochloride (5e): Yield 33%, yellow solid; m.p. 79 C; IR: 3318,
1
1632, 1539, 1449 cmÀ 1; H NMR ([D6]DMSO, 400 MHz): δ=0.84–0.96
(m, 2H), 1.08–1.31 (m, 5H), 1.36–1.44 (m, 2H), 1.45–1.51 (m, 1H),
1.64–1.80 (m, 5H), 2.43 (ddd, J=3.5, 5.5, 10.1 Hz, 1H), 2.81–2.89 (m,
1H), 3.22–3.29 (m, 2H), 7.25 (d, J=8.3 Hz, 2H), 7.75 (d, J=8.3 Hz,
2H), 8.39 (t, J=6.0 Hz, 1H), 8.64 (br s, 2H); 13C NMR (CD3OD,
100 MHz): δ=14.3, 22.4, 27.4, 27.6, 32.2, 34.3, 36.8, 37.9, 38.8, 127.4,
128.6, 134.3, 143.6, 169.6; HRMS (ESI) m/z calcd. for C18H27N2O:
287.2118 [M+H]+; found: 287.2119.
tert-Butyl
((�)trans-2-(4-phenethylcarbamoyl)phenylcyclopropyl
carbamate (14b): Yield: 63%, white solid; IR: 3393, 3376, 1701, 1681,
4-((�)-trans-2-Aminocyclopropyl)-N-(4-fluorobenzyl)benzamide
1516 cmÀ 1; H NMR (CDCl3): δ=1.17–1.20 (m, 2H), 1.44 (s, 9H), 2.05
1
°
hydrochloride (5f): Yield 45%, yellow solid; m.p. 98 C; IR: 335, 2486,
1620, 1516, 1458 cmÀ 1; 1H NMR (CD3OD, 400 MHz): δ=1.37–1.42 (m,
1H), 1.49 (ddd, J=4.7, 7.0, 10.2 Hz, 1H), 2.43 (ddd, J=3.6, 6.5,
10.0 Hz, 1H), 2.89–2.96 (m, 1H), 4.53, (s, 2H), 7.0 (t, J=8.7 Hz, 2H),
7.26 (d, J=8.2, 2H), 7.36 (m, 2H), 7.81 (d, J=8.3 Hz, 2H); 13C NMR
(CD3OD, 100 MHz): δ=14.3, 22.4, 32.3, 43.8, 116.2, 127.5, 128.5,
130.4, 133.9, 136.3, 143.9, 163.5 (d, JF-C =220 Hz), 169.6; HRMS (ESI)
m/z calcd. for C17H18F1N2O: 285.1398 [M+H]+; found: 285.1402.
(td, J=3.0, 7.7 Hz, 1H), 2.68–2.77 (m, 1H), 2.92 (t, J=7.0 Hz, 2H),
6.73 (q, J=7.2 Hz, 2H), 4.87 (br s, 1H), 6.08 (br s, 1H), 7.13 (d, J=
8.1 Hz, 2H), 7.21–7.24 (m, 3H), 7.39–7.34 (m, 2H), 7.58 (d, J=8.4 Hz,
2H); 13C NMR (CDCl3): δ=16.8, 22.1, 28.5, 33.1, 35.8, 41.2, 80.8,
125.39, 125.5, 125.8, 127.6, 127,76, 131.21, 137.9, 143.6, 156.7, 166.2;
HRMS m/z calcd for C23H29N2O3: 381.2173 [M+H]+; found: 381.2173.
tert-Butyl
((�)trans-2-(4-(2-thiophenylethylcarbamoyl)
phenylcyclopropyl carbamate (14c): Yield, 51%, white solid; m.p.
4-((�)-trans-2-Aminocyclopropyl)-N-(4-bromobenzyl)benzamide
À 1
;
1H NMR (CDCl3): δ=1.61–
°
94 C; IR: 3358, 3335, 1685, 1449 cm
°
hydrochloride (5g): Yield 65%, white solid; m.p. 97 C; IR: 3320, 3035,
1
2665, 1637, 1533, 14893 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.37–
1.19 (m, 2H), 1.43 (s, 9H), 2.02–2.05 (m, 1H), 2.68–2.76 (m,1H), 3.06
(t, J=6.4 Hz, 2H), 3.62 (q, J=6.4 Hz, 2H), 4.93 (br s, 1H), 6.33 (br s,
1H), 6.83–6.87 (m, 1H), 6.95 (dd, J=3.4, 4.9 Hz, 1H), 7.10–7.16 (m,
3H), 7.61 (d, J=8.3 Hz, 2H); 13C NMR (CDCl3): δ=15.5, 23.9, 27.5,
28.9, 31.8, 40.12, 78.6, 122.9, 124.3, 125.3, 125.8, 125.9, 131.0, 140.2,
143.6, 155.1, 166.13; HRMS m/z calcd. for C21H27N2O3S: 387.1737 [M
+H]+; found: 387.1737.
1.42 (m, 1H), 1.46–1.51 (m, 1H), 2.44 (ddd, J=3.2, 6.3, 9.7 Hz, 1H),
2.90–2.93 (m, 1H), 4.52 (s, 2H), 7.26 (d, J=8.0 Hz, 4H), 7.47 (d, J=
8.3 Hz, 2H), 7.83 (d, J=8.3 Hz, 2H); 13C NMR (CD3OD, 100 MHz): δ=
14.4, 22.4, 32.3, 43.9, 121.8, 127.5, 128.8, 130.5, 132.6, 133.9, 139.6,
144.00, 169.7; HRMS (ESI) m/z calcd. for C17H18BrN2O: 345.0597 [M+
H]+; found: 345.0602.
4-((�)-trans-2- Aminocyclopropyl)-N-benzylbenzamide hydrochloride
4-((�)-trans-2-Aminocyclopropyl)-N-(4-methoxybenzyl)benzamide
°
(5a): Yield 71%, yellow crystalline solid; m.p. 200 C; IR: 3336, 3289,
°
hydrochloride (5h): Yield 52%, yellow solid; m.p. 123 C; IR: 3364,
3075, 1674, 1437 cmÀ 1
;
1H NMR (CD3OD): δ=1.36–1.42 (m, 1H),
1
1672, 1528 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.36–1.41 (m, 1H),
1.46–1.51 (m, 1H), 2.46 (ddd, J=3.3, 6.6, 9.9 Hz, 1H), 2.90–2.92, (m,
1H), 4.6 (s, 2H), 7.11–7.19 (m, 3H), 7.22–7.35 (m, 4H), 7.81 (d, J=
8.2 Hz, 2H); 13C NMR (CD3OD): δ=14.3, 22.4, 32.2, 44.4, 127.5, 128.2,
128.5,129.7, 129.5, 134.1, 140.2, 143.9, 169.6; HRMS (ESI) m/z calcd.
for C17H19N2O: 267.1492 [M+H]+; found: 267.1495.
1.49 (ddd, J=4.6, 6.7, 10.5 Hz, 1H), 2.44 (ddd, J=3.4, 6.3, 9.8 Hz,
1H), 2.91, (ddd, J=4.0, 7.9, 11.7 Hz, 1H), 3.76 (s, 3H), 4.48 (s, 2H),
6.87 (d, J=8.6 Hz, 2H), 7.25 (d, J=8.4 Hz, 2H), 7.26 (d, J=8.4 Hz,
2H), 7.79 (d, J=8.1 Hz, 2H); 13C NMR (CD3OD, 100 MHz): δ=14.3,
22.4, 32.3, 43.9, 55.7, 114.9, 127.5, 128.7, 129.8, 132.2, 134.1, 143.9,
160.4, 169.5; HRMS (ESI) m/z calcd. for C18H21N2O2: 297.1598 [M+
H]+; found: 297.1595.
4-(�)-trans-2-Aminocyclopropyl)-N-phenethylbenzamide hydrochloride
À 1
°
(5b): Yield 67%, yellow solid; m.p. 180 C; IR: 3283, 1637, 1545 cm
;
1H NMR (CD3OD): δ=1.36–1.42 (m, 1H), 1.48 (ddd, J=4.2, 6.7, 10.3
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N-([1,1’-Biphenyl]-4-ylmethyl)-4-(�-trans-2-aminocyclopropyl)
benzamide hydrochloride (5i): Yield 34%, yellow solid; IR: 3402,
3045, 1715, 1632, 1540 cmÀ 1;1H NMR (CD3OD, 400 MHz): δ=1.38–
1.43 (m, 1H), 1.46–1.50 (m, 1H), 2.43 (ddd, J=3.5, 6.3, 10.2 Hz, 1H),
2.92 (ddd, J=3.7, 7.9, 11.6 Hz, 1H), 4.61 (s, 2H), 7.26–7.29 (m, 2H),
7.30–7.34 (m, 1H), 7.39–7.45 (m, 4H), 7.56–7.60 (m, 4H), 7.83 (d, J=
8.4 Hz, 2H); 13C NMR (CD3OD, 100 MHz): δ=14.3, 22.4, 32.2, 44.1,
127.4, 127.8, 128.0,128.2, 128.7, 128.9, 129.7, 134.0, 139.2, 141.3,
142.0, 143.8, 169.6.
4H), 7.28 (d, J=7.6 Hz, 2H), 7.42 (d, J=7.8 Hz, 2H); 13C NMR (CD3OD,
100 MHz): δ=14.5, 22.4, 32.4, 54.9, 68.0, 117.7, 122.3, 126.6, 127.9,
128.4, 128.9, 134.3, 135.9, 142.6, 156.6 (d, JF-C=245.8 Hz), 172.19;
HRMS (ESI) m/z calcd. for C20H23FN3O: 340.1820 [M+H]+; found:
340.1821.
2-(4-(4-(�)-trans-2-Aminocyclopropyl)benzoyl)piperazin-1-yl)
benzonitrile hydrochloride (5p): Yield 39%, white solid; IR: 3353,
1
2856, 2221, 1632, 1562 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.38–
1.42 (m, 1H), 1.44–1.51 (m, 1H), 2.40–2.45 (m, 1H), 2.92 (ddd, J=4.2,
8.0, 11.0 Hz, 1H), 3.18 (br s, 2H), 3.56–3.59 (m, 1H), 3.64–3.68 (m,
3H), 3.71–3.74 (m, 2H), 3.95 (br s, 1H), 7.14 (t, J=7.4 Hz, 1H), 7.19 (d,
J=8.2 Hz, 1H), 7.29 (d, J=8.0 Hz, 2H), 7.43 (d, J=8.0 Hz, 2H), 7.57–
7.61 (m, 1H), 7.65 (dd, J=2.07, 9.06 Hz, 1H); 13C NMR (CD3OD,
100 MHz): δ=14.2, 22.4, 32.2, 43.7, 62.2, 107.7, 119.1, 120.6, 124.0,
127.7, 128.7, 135.1, 135.3, 135.4, 142.3, 156.6, 172.3; HRMS (ESI) m/z
calcd. for C21H23N4O: 347.1866 [M+H]+; found: 347.1869.
4-(�)-trans-2-Aminocyclopropyl)-N,N-dibenzylbenzamide
hydrochloride (5j): Yield 52%, yellow solid; IR: 3214, 1645.7, 1620,
1
1452 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.37–1.38 (m, 1H), 1.49–
1.56 (m, 1H), 2.43 (ddd, J=3.2, 6.4, 9.8 Hz, 1H), 2.96–2.90 (m, 1H),
4.43 (br s, 2H), 4.66 (br s, 2H), 7.12 (br s, 2H), 7.24–7.30 (m, 7H),
7.32–7.39 (m, 5H), 7.43–7.50 (m, 2H); 13C NMR (CD3OD, 100 MHz):
δ=14.1, 22.3, 32.3, 53.7, 127.6, 128.0, 128.2, 128.7, 129.8, 129.9,
135.5, 142.2, 174.2; HRMS (ESI) m/z calcd. for C24H25N2O: 357.1961
[M+H]+; found: 357.1960.
General procedure for 15b and 15c: These carbamates were
prepared from 11 by carrying out the Curtius rearrangement in the
presence of ethanol to give the ethyl carbamate. The methyl ester
was then deprotected and the acid coupled with 2-phenethylamine
or 2-thiophenethylamine to give 15b and 15c respectively.
4-((�)-trans-2-Aminocyclopropyl)phenyl)4-(pyridin-2-yl)piperazin-1-yl)
methanone hydrochloride (5k): Yield 13%, orange oil; IR: 3358, 1603,
1
1545, 1435 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.37–1.43 (m, 1H),
1.51 (ddd, J=4.4, 6.6, 11.1 Hz, 1H), 2.48 (ddd, J=3.5, 6.3, 10.0 Hz,
1H), 2.93 (ddd, J=4.4, 7.6, 11.2 Hz, 1H), 3.8 (br s, 8H), 7.06 (t, J=
6.5 Hz, 1H), 7.32 (d, J=8.0 Hz, 2H), 7.42 (d, J=9.2 Hz, 1H), 7.48 (d,
J=8.0 Hz, 2H), 8.0 (dd, J=1.24, 7.9 Hz, 1H), 8.05 (dt, J=1.6, 8.5 Hz,
1H); 13C NMR (CD3OD, 100 MHz): δ=14.3, 22.4, 32.2, 44.8, 47.2,
114.3, 127.8, 128.8, 131.1, 134.5, 137.5, 142.8, 145.7, 153.7, 172.6;
HRMS (ESI) m/z calcd. for C19H23N4O: 323.1866 [M+H]+; found:
323.1869.
Ethyl ((�)trans-2-(4-Phenethylcarbamoyl)phenylcyclopropyl carbamate
(15b): Yield 54%, white solid; IR: 3312, 3191, 1689, 1637, 1545,
1510 cmÀ 1; 1H NMR (CDCl3): δ=1.20–1.27 (m, 5H), 2.07–2.11 (m, 1H),
2.72–2.76 (m, 1H), 2.92 (t, J=6.9 Hz, 2H), 3.69 (q, J=6.9, Hz, 2H),
4.12 (q, J=6.9 Hz, 2H), 7.14 (d, J=7.9 Hz, 2H), 7.19–7.24 (m, 3H),
7.28–7.34 (m, 2H), 7.58 (d, J=7.8 Hz, 2H); 13C NMR (CDCl3): δ=14.6,
16.4, 25.2, 33.1, 35.7, 41.1, 61.0, 126.6, 126.7, 126.9, 128.7, 128.8,
132.4, 138.8, 144.4, 157.1, 167.2; HRMS m/z calcd. for C21H25N2O3:
353.1860 [M+H]+; found: 353.1860.
4-(�)-trans-2-Aminocyclopropyl-phenyl)-(4-(pyrimidin-2-yl)piperazin-1-
yl)methanone hydrochloride (5l): Yield 22%, red oil; IR: 3353,1678,
Ethyl
((�)trans-2-(4-(2-Thiophenethylcarbamoyl)phenylcyclopropyl
1
1632, 1551 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.39–1.45 (m, 1H),
carbamate (15c): Yield 49%, white solid; IR: 3318, 3214, 1689, 1655,
1.47 (ddd, J=4.5, 6.9, 10.1 Hz, 1H), 2.44 (ddd, J=3.6, 6.4, 10.2 Hz,
1H), 2.92–2.96 (m, 1H), 3.53 (br s, 2H), 3.82 (br s, 4H), 3.94 (br s, 2H),
6.66 (t, J=4.7, Hz, 1H), 7.29 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.3 Hz,
2H), 8.35 (d, J=4.8 Hz, 2H); 13C NMR (CD3OD, 100 MHz): δ=14.2,
22.4, 32.1, 40.4, 41.7, 111.8, 127.7, 128.6, 135.2, 142.3, 159.1, 162.8,
172.45; HRMS (ESI) m/z calcd. for C18H22N5O: 324.1819 [M+H]+;
found: 324.1824.
1551, 1510 cmÀ 1; H NMR (CDCl3): δ=1.20–1.23 (m, 5H), 2.06–2.10
1
(m, 1H), 2.70–2.74 (m, 1H), 3.13 (t, J=6.5 Hz, 2H), 3.69 (q, J=
7.04 Hz, 2H), 4.11 (q, J=6.5 Hz, 2H), 6.85 (dd, J=0.9, 3.3 Hz, 1H),
6.94 (dd, J=3.4, 5.1 Hz, 1H), 7.13–7.17 (m, 3H), 7.61 (d, J=8.1 Hz,
2H); 13C NMR (CDCl3): δ=14.6, 16.4, 25.2, 29.9, 33.1, 41.3, 61.0,
124.1, 125.5, 126.6, 127.0, 127.1, 132.3, 141.3, 141.5, 157.1, 167.2;
HRMS m/z calcd. C19H23N2O3S: 359.1424 [M+H]+; found: 359.1425.
4-((�)-trans-2-Aminocyclopropyl)phenyl)(4-(phenylsulfonyl)piperazin-
1-yl)methanone hydrochloride (5m): Yield 25%, white solid; IR: 3392,
1637, 1652, 1551 cmÀ 1; 1H NMR (CD3OD, 400 MHz): δ=1.33–1.38 (m,
1H), 1.45–1.50 (m, 1H), 2.41–2.43 (m, 1H), 2.45 (s, 3H), 2.87–2.89 (m,
1H), 3.00 (br s, 4H), 3.56 (br s, 2H), 3.77 (br s, 2H), 7.22 (d, J=7.6 Hz,
2H), 7.30, (d, J=7.6 Hz, 2H), 7.44 (d, J=7.8 Hz, 2H), 7.65 (d, J=
7.9 Hz, 2H); 13C NMR (CD3OD, 200 MHz): δ=14.2, 21.5, 22.3, 32.2,
44.2, 47.2, 127.7, 128.6, 128.9, 130.9, 133.8, 134.5, 142.4, 145.6,
172.2; HRMS (ESI) m/z calcd. for C21H26N3O3S: 400.1689 [M+H]+;
found: 400.1687.
Biology
LSD1 enzyme assay: LSD1 residual activity after incubation with the
carboxamide TCP derivatives was measured according to our
previously reported assay.[19]
AML cell line viability assay: To measure the in vitro compound
activity, 100 μL of cell suspension was plated out at a density of
2.5×104 cells/mL in 96 well plates. Tested compounds (stock
250 mM) were dissolved to the appropriate concentration in RPMI
complete medium (10× final concentration) and 10 μL of each
concentration was immediately added to the plated cell suspen-
sion. Each concentration was assessed five times. After treatment,
cells were further cultured for the reported amount of time. Cell
viability was measured with CellTiter-Glo® (Promega, Southampton,
UK) by adding 100 μL of the reagent to each well. After 10 min of
incubation at RT, 100 μL of cell suspension+reagent were trans-
ferred into a white GREINER 96 well plate to eliminate straight light
and bioluminescence was recorded in a BMG Cell star microplate
reader. Collected raw data were normalised to control (vehicle
control) and IC50’s and statistical significance were determined with
GraphPad Prism software (San Diego, CA).
4-(�)-trans-2-Aminocyclopropyl)phenyl(4-(methylsulfonyl)piperazin-1-
yl)methanone hydrochloride (5n): Yield 37%, white solid; IR: 3387,
1
1614, 1562 cmÀ 1; H NMR (CD3OD, 400 MHz): δ=1.37–1.42 (m, 1H),
1.47 (ddd, J=4.5, 6.8, 10.3 Hz, 1H), 2.42 (ddd, J=3.6, 6.6, 10.2 Hz,
1H), 2.87 (s, 3H), 2.89–2.93 (m, 1H), 3.26 (br s, 4H), 3.57 (br s, 2H),
3.8, (br s, 2H), 7.28 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.3 Hz, 2H);
13C NMR (CD3OD, 100 MHz): δ=14.2, 22.4, 32.0, 34.8, 46.7, 68.0,
127.8, 128.6, 134.7, 142.4, 172.3; HRMS (ESI) m/z calcd for
C15H24N3O3S: 324.1380 [M+H]+; found: 324.1380.
4-((�)-trans-2-Aminocyclopropyl)phenyl)4-(2-fluorophenyl)piperazin-1-
yl)methanone hydrochloride (5o): Yield 44%, yellow solid; IR: 3387,
1603, 1505, 1470 cmÀ 1; 1H NMR (CD3OD, 400 MHz): δ=1.37–1.42 (m,
1H), 1.44–1.49 (m, 1H), 2.42 (ddd, J=3.2, 6.3, 9.7 Hz, 1H), 2.89–2.93
(m, 1H), 3.16 (br s, 4H), 3.66 (br s 2H), 3.94 (br s 2H), 7.03–7.13 (m,
ChemMedChem 2021, 16, 1316–1324
www.chemmedchem.org
1323
© 2021 Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000754
ChemMedChem
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°
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concentration determined using BCA assay kit (Thermo Scientific
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: acute myeloid leukemia
· enzyme inhibitors ·
epigenetics · FAD-dependent enzymes · histone demethylases
Manuscript received: September 22, 2020
Revised manuscript received: December 18, 2020
Version of record online: February 3, 2021
ChemMedChem 2021, 16, 1316–1324
www.chemmedchem.org
1324
© 2021 Wiley-VCH GmbH