Design and Synthesis of Styrenylcyclopropylamine LSD1 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.0c00060

Leveraging the catalytic machinery of LSD1 (KDM1A), a series of covalent styrenylcyclopropane LSD1 inhibitors were identified. These inhibitors represent a new class of mechanism-based inhibitors that target and covalently label the FAD cofactor of LSD1.

Full text of DOI:10.1021/acsmedchemlett.0c00060

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Letter
Design and synthesis of styrenylcyclopropylamine LSD1 inhibitors
Victor S Gehling, John P. McGrath, Martin Duplessis, Avinash Khanna, Francois Brucelle, Rishi
G. Vaswani, Alexandre Cote, Jacob Stuckey, Venita Watson, Richard T. Cummings, Srividya
Balasubramanian, Priyadarshini Iyer, Priyanka Sawant, Andrew C Good, Brian K. Albrecht, Jean-
Christophe Harmange, James E Audia, Steven F. Bellon, Patrick Trojer, and Julian R. Levell
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00060 • Publication Date (Web): 06 May 2020
Downloaded from pubs.acs.org on May 12, 2020
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Table of Contents Only
Title: Design and synthesis of styrenylcyclopropylamine LSD1 inhibitors.
*
†
‡
Victor S. Gehling , John P. McGrath, Martin Duplessis , Avinash Khanna, Francois Brucelle , Rishi G.
Δ
±
Vaswani, Alexandre Cote , Jacob Stuckey, Venita Watson , Richard T. Cummings, Srividya
†
@
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Balasubramanianˆ, Priyadarshini Iyerˇ, Priyanka Sawant*, Andrew C. Good , Brian K. Albrecht , Jean-
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Christophe Harmange , James E. Audia , Steven F. Bellon , Patrick Trojer, Julian R. Levell
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Title: Design and synthesis of styrenylcyclopropylamine LSD1 inhibitors.
*
†
‡
Victor S. Gehling , John P. McGrath, Martin Duplessis , Avinash Khanna, Francois Brucelle , Rishi G.
Δ
±
Vaswani, Alexandre Cote , Jacob Stuckey, Venita Watson , Richard T. Cummings, Srividya
†
@
Balasubramanianˆ, Priyadarshini Iyerˇ, Priyanka Sawant*, Andrew C. Good , Brian K. Albrecht , Jean-
%
#
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Christophe Harmange , James E. Audia , Steven F. Bellon , Patrick Trojer, Julian R. Levell
Constellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, MA 02142
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*Email: Victor.Gehling@constellationpharma.com
†Current address: C4 Therapeutics, 490 Arsenal Way, Suite 200, Watertown, MA 02472
‡Current address: Foghorn Therapeutics, 100 Binney Street, Suite 610, Cambridge, MA 02142
Δ
th
th
Current address: Schrodinger, 120 West 45 Street, 17 Floor, New York, NY 10036
±
th
Current address: KSQ Therapeutics, 610 Main Street North, 4 Floor, Cambridge, MA 02142
ˆ Current address: AstraZeneca, 35 Gatehouse Drive, Waltham, MA 02451
ˇ Current address: Agenus, 3 Forbes Road, Lexington, MA 02421
*Current address: Syros Pharmaceuticals, 620 Memorial Drive, Suite 300, Cambridge, MA 02139
@Current address: Rheos Medicines, 38 Sidney Street, Cambridge, MA 02139
%
th
Current address: Goldfinch Bio, 215 First Street, 4 Floor, Cambridge, MA 02142
#Current address: Northwestern University, 633 Clark St, Evanston, IL 60208
Abstract:
Leveraging the catalytic machinery of LSD1 (KDM1A), a series of covalent styrenylcyclopropane LSD1
inhibitors were identified. These inhibitors represent a new class of mechanism-based inhibitors that
target and covalently label the FAD cofactor of LSD1. The series was rapidly progressed to potent
biochemical and cellular LSD1 inhibitors with good physical properties. This effort resulted in the
identification of 34, a highly potent (<4 nM biochemical, 2 nM cell, and 1 nM GI ), and selective LSD1
5
0
inhibitor. In-depth kinetic profiling of 34 confirms its covalent mechanism of action, validates the
styrenylcylopropane as an FAD-directed warhead, and demonstrates that the potency of this inhibitor is
driven by improved non-covalent binding (K ). 34 demonstrates robust cell-killing activity in a panel of AML
I
cell lines and robust anti-tumor activity in a KASUMI-1 xenograft model of AML when dosed orally at 1.5
mg/kg once daily.
Keywords:
LSD1, KDM1A, histone demethylase, irreversible inhibition, styrenylcyclopropane, AML
Introduction:
The dynamic organization of the genome in eukaryotic cells is regulated in large part by diverse post-
translational modifications of histones, which control access to DNA and ultimately alter gene
1
,2
expression.
The identification of recurrent genetic alterations in genes encoding histone-modifying
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enzymes in cancer cells of all types suggests that manipulation of chromatin structure is a strategy
commonly employed by cancer cells to invoke transcriptional programs that prevent differentiation and
promote proliferation. Lysine-specific demethylase LSD1 (also known as KDM1A) acts on histone H3 both
as a transcriptional co-repressor through demethylation of lysine-4 (H3K4) and as a co-activator by
4
,5
targeting the methylated lysine-9 residue (H3K9) . These activities have been shown to play an essential
role in a variety of normal physiological processes, including cell proliferation , hematopoietic
differentiation , chromosome segregation , spermatogenesis , stem cell pluripotency , and embryonic
development . LSD1 is highly expressed in many cancer types , where it plays a key role in oncogenic
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processes, including compromised differentiation, proliferation, migration and invasion, as well as
13,14
metabolic reprogramming
.
LSD1 has drawn considerable and increasing attention as a therapeutic target in human malignancies due
to its involvement in a variety of disease states, including acute myelogenous leukemia (AML) , small-cell
15
1
6
17
lung cancer , and myelofibrosis . This interest has resulted in the advancement of multiple LSD1
inhibitors into clinical trials (See figure 1). Many of these inhibitors are derived from tranylcypromine (1),
a mechanism-based, covalent inhibitor of monoamine oxidase A and B (MAO A and B), which
demonstrates weak activity against LSD1. These clinical-stage inhibitors support LSD1 inactivation via
selective reduction of the LSD1-bound flavin adenine dinucleotide (FAD) cofactor.
Figure 1. Tranylcypromine and related LSD1 inhibitors.
O
H
O
NH2
O
N
H
N
O
NH
N
N
N
OH
F
H
N
H
N
OH
NH2
H
H
N
O
N
(
+/-) tranylcypromine
ORY1001
GSK2879552
INCB059872
IMG7289
5
N
1
2
3
4
N
Given our interest in targeting disease-relevant, chromatin-regulatory mechanisms for therapeutic
applications we began a hit finding campaign with the goal of identifying novel covalent inhibitors of LSD1.
LSD1 has a catalytic domain that is structurally homologous with those of monoamine oxidases, which
utilize a non-covalently bound FAD as a cofactor . The similarity in the catalytic and structural properties
of LSD1 and MAO A and B prompted the investigation of anti-MAO chemotypes as potential LSD1
1
8
19
inhibitors . A survey of the MAO literature identified propargylamines and cyclopropylamines as
functional groups capable of labeling the FAD cofactor of the MAOs. Using these cyclopropylamines and
propargylamines to inform our design, we generated an initial hit-finding collection that included MAO
inhibitors, commercially available cyclopropylamine and propargylamine compounds, and de-novo
designs. Screening of this collection using a LSD1 enzymatic assay (see supporting information) identified
several interesting cyclopropylamine derivatives as starting points, including spirocyclopropylamines
and styrenylcyclopropylamine 6. Herein we report our efforts on optimizing the styrenylcycloproplyamine
20,21
6
due to its impressive potency compared to tranylcypromine (1).
Table 1: Exploration of the styrene.
LSD1 TR-FRET
ClogDa
CPI#
1b
Structure
IC ± SD μM (n)
5
0
NH2
22.9 ± 6.9 (42)
-0.5
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6b
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0.444 ± 0.165 (3)
20.4 ± 2.2 (2)
0.1
0.1
0.3
NH2
NH2
8b
4.28 ± 0.83 (4)
NH2
NH2
NH2
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0.898 ± 0.199 (3)
0.412 ± 0.146 (2)
0.0
0.4
(
±)-10
1b
NH2
0.788 ± 0.201 (2)
9.40 ± 0.488 (2)
0.1
1
2b
NH2
-0.4
1
aCalculated using Stardrop 6.6.
bMixtures of diastereomers.
The initial medicinal chemistry efforts explored substitution and modification of the styrenyl group. The
Z-styrene analog (7) lost significant activity (>40 fold) against LSD1 demonstrating that the geometry of
the styrene is critical for productive binding. Subsequently, substitution of the styrene by incorporating a
methyl group at the 1- or 2-position, afforded 8 and 9. There was clear preference for substitution at the
2
-position over the 1 position (see, compounds 8 and 9). The 2-substituted compound 9 demonstrated
similar inhibition of LSD1 relative to the unsubstituted starting point 6 while being ClogD neutral.
Extension from the methyl group to an ethyl group resulted in 10, a compound with comparable activity
to the methyl substituted analog 9 but with a modest impact on the physical properties (ΔClogD = 0.4)
relative to 6. In addition to substitution more dramatic modifications to the styrenyl group were
investigated, including cyclization to form dihydronaphthyl derivative 11, and removal of the phenyl ring
to afford cyclohexene 12. The dihydronaphthyl derivative retained similar activity to the ethyl substituted
compound, however removal of the aryl ring, as in 12, resulted in a >10-fold loss in activity. Further
optimization of this series focused upon exploration of the amine substitution on
styrenylcyclopropylamines 6 and 9.
Table 2. N-substitution of early styrenylcyclopropylamine analogs.
LSD1 TR-FRET
LY96 mRNA
50
a
b
CPI#
Structure
ClogD / LLE
IC ± SD μM (n)
EC ± SD μM (n)
5
0
n.t.c
n.t. c
n.t. c
0.5 / NCd
0.5 / NCd
0.4 / NCd
N
H
1
3
0.0072 ± 0.003 (3)
0.0042 ± 0.001 (7)
NH
NH
N
H
(±)-14
±)-15
NH
(
N
H
<0.0028 ± NA (3)
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H
NH2
(
±)-16e
0.0029 ± 0.001 (5)
0.028 ± 0.016 (4)
0.056 ± 0.016 (5)
0.0088 ± 0.002 (4)
0.025±0.006 (5)
n.t. c
-0.1 / NCd
0.3 / 6.2
0.7 / 5.6
0.5 / 5.9
0.3 / 6.2
N
H
H
NH
(
±)-17
N
0.312 (1)
H
H H
N
1
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(
±)-18 e
O
0.54 ± 0.11 (3)
0.362 (1)
N
H
H
H
N
N
H
(
±)-19
N
H
OH
(
±)-20 e
0.327 ± 0.057 (3)
N
H
H
NH
(±)-21f
0.020 ± 0.004 (4)
0.173 (1)
0.5 / 6.3
N
O
H
a
b
c
Calculated using Stardrop 6.6.
LLE = - log (LY96 EC ) – ClogD.
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n.t. = not tested
d
NC = not calculated.
eSingle unknown diastereomer.
fMixture of diastereomers.
Our initial N-substituted analogs appended a piperidinyl group to the unsubstituted styrenyl, as seen in
compound 13 (Table 2). These initial compounds demonstrated good potency (< 10 nM) in our
biochemical assay. Further profiling of 13 identified poor plasma stability as a potential liability. This
observation was made when attempting to collect plasma protein binding information (PPB), as it was
observed that ≤ 5% of parent compound remained after incubation in plasma. Efforts to address this issue
focused upon substitution of the styrene as we hypothesized this group was contributing to the poor
stability. The methyl substituted analog 14 was synthesized to test this hypothesis and encouragingly, 14
had comparable biochemical potency to 13 and demonstrated increased plasma stability across species.
With promising activity demonstrated by 14, we began a systematic investigation of amine substituents
on the methyl substituted scaffold (Table 2). Our goal in the optimization process was to improve the
biochemical and cellular potency while ensuring that these compounds remained in drug-like chemical
space. A variety of saturated heterocycles were investigated including piperidine 15, trans-
cyclohexylamine 16, and azetidine 17. These initial analogs were highly potent biochemical LSD1
inhibitors. Interestingly, analogs containing a linker consisting of 4 to 5 bond lengths between the two
amine atoms (such as 14, 15, 16) demonstrated superior potency in the biochemical assay compared to
analogs with shorter linkers between the two amine atoms (such as 17).
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Azetidine 17 was subjected to a cell target engagement assay. Compound-induced changes in
expression of the LSD1 target gene lymphocyte antigen 96 (LY96) were measured in human MV4-11 AML
2
2
cells (see supporting information) . In the LY96 target engagement assay compound 17 demonstrated
good activity (< 1 µM), simultaneously validating our triage scheme and confirming the activity of the
styrenylcycloprane scaffold in cellular settings. While the LSD1 TR-FRET assay was initially useful in SAR
analysis, many of the N-substituted analogs could not be differentiated with this assay. Thus, we
transitioned to driving the program based on the LY96 assay data including using this data to calculate the
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lipophilic ligand efficiency (LLE) and track optimization of this series.
Next, non-basic substituents were investigated, including amide 18, imidazole 19, alcohol 20, and lactam
2
1. These non-basic substituents were designed to probe whether an explicit hydrogen bond donor could
replace the basic amine heterocycles present in earlier analogs. While the mono-basic compounds
afforded promising activity in the biochemical assay, these analogs did not significantly improve upon the
initial cellular activity seen with azetidine 17 and were net neutral with respect to LLE.
Table 3: Comparison of methyl and ethyl styrenylcyclopropylamines
LSD1 TRFRET
LY96 mRNA
a
b
CPI#
Structure
ClogD / LLE
IC ± SD μM (n)
EC ± SD μM (n)
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0
50
H H
N
OH
0
.0026 ± 0.0001
6)
(
(
(
(
±)-22c
0.052 ± 0.60 (4)
0.55 ± NA (1)
-0.1 / 7.4
-1.4 / 7.6
0.2 / 8.0
-1.1 / 7.5
N
(
H H
H H
N
OH
±)-23c
±)-24c
±)-25c
O
0.017 ± 0.009 (4)
<0.0018 ± NA (2)
0.020 ± 0.007 (3)
N
H H
H H
N
OH
0.006 ± NA (1)
0.43 ± 0.091 (2)
N
H H
H H
N
OH
O
N
H H
a
b
Calculated using Stardrop 6.6.
LLE = - log (LY96 EC ) – ClogD.
5
0
cSingle unknown diastereomer.
In search of improved activity in the LY96 assay, we focused additional effort around substitution
of the distal amine. Initially, we introduced polar groups, such as the ethanol group in 22 or the acid as in
2
3. The ethanol group in 22 increased potency in both our biochemical and LY96 gene induction assays
relative to earlier compounds. This change also resulted in an impressive lipophilic ligand efficiency (LLE)
of 7.4 for compound 22. Introduction of an acid, as in 23, resulted in a large potency loss in the LY96 assay
(10-fold) but the dramatic decrease in the ClogD resulted in a net improvement in the LLE (see, 22 and
2
3).
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Further exploration of the scaffold focused on ethyl substituted styrene analogs of 22 and 23 in
an effort to modestly increase their ClogD. Synthesis of the ethyl analogs of these compounds afforded
ethanol 24 and acid 25. Introduction of the ethyl group afforded improved activity in the cellular assay for
ethanol analog 24 (~9 fold, 22 vs 24). The improvement in cellular activity offset the increased lipophilicity,
as demonstrated by an increase in LLE from 22 to 24. Interestingly, introduction of the ethyl group did not
have the same effect on the carboxylic acid analog, as compound 25 and compound 23 demonstrate
similar activity in the LY96 assay.
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With the impressive LY96 activity and LLE observed with compound 24, we focused our efforts on
the ethyl cyclopropylamine scaffold. To enable this effort, we developed a scalable synthesis of the
enantioenriched cyclopropylamine building block. The synthesis begins with the condensation of
butanaldehyde (26) and benzaldehyde (27) to afford enal 28. Reaction of the Horner-Wadsworth-Emmons
reagent with enal 28 afforded the diene 29 in excellent yield. This diene was then treated with the Corey-
Chaykovsky reagent to afford the cyclopropane 30 in modest yield. Hydrolysis of the ester, followed by a
Curtius rearrangement and workup afforded the racemic, trans-cyclopropane 10. Resolution of 10 with
(S)-mandelic acid afforded the enantioenriched (1R,2S)-ethyl cyclopropylamine 31. The absolute
stereochemistry of 31 was confirmed by small molecule x-ray crystallography.
Scheme 1: Synthesis of (1R, 2S)-31
Continuing our SAR efforts on the ethylcyclopropylamine scaffold, we re-examined the impact
of the linker between the two amines on activity and ADME properties. We kept the ethanolamine
fragment in place and surveyed a variety of saturated heterocyclic linkers, including piperidine 32, 4,6-
spiro 33, and 4,4-spiro 34. These heterocyclic linkers (compounds 32 to 34) afforded highly active LSD1
inhibitors with TR-FRET IC ’s less than 3 nM with LY96 EC ’s less than 10 nM and LLE’s ≥ 7.
5
0
50
With the impressive potency of the ethylstyrenyl cyclopropane compounds in the LY96 target
engagement assay we began triaging compounds in our phenotypic assay (see supporting information).
In the phenotypic assay we assessed for growth inhibition in the KASUMI-1 cell line upon 12-days of
compound treatment. For these inhibitors there was very good correlation between all three assays,
the TR-FRET IC , the LY96 EC and the phenotypic GI . While multiple inhibitors afforded potent target
5
0
50
50
engagement and phenotypic activity, we chose to focus on the 4,4-spiro system for further exploration
due to the promising liver microsome (LM) data and high LLE of compound 34.
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4: Structure activity relationships on (1R, 2S)-ethylcyclopropylamine 31.
Liver microsome
LSD1 TR-FRET
IC50 ± SD μM
LY96 mRNA
EC50 ± SD μM
(n)
KASUMI-1
GI50 (μM)
clearance
(m / r / d / h)
a
b
CPI#
Structure
ClogD / LLE
c
(
n)
(
μl/min/mg protein)
OH
N
<
0.0026 ± NA
0.004 ± 0.001
(2)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
2
3
0.003
0.001
0.7/7.7
1.1/7.3
46/27/106/41
N
H
(3)
OH
N
<0.0030 ± NA
(4)
0.005 ± 0.003
(3)
28/19/39/10
N
H
OH
N
<
0.0030 ± NA
0.002 ± 0.001
(9)
3
3
4
5
0.001
0.011
0.8/7.9
0.8/7.5
25/15/41/9
N
H
(4)
NH
0.0053 ±
0.0009 (4)
0.005 ± 0.002
(3)
17/19/14/11
N
H
O
N
0
.0030 ±
0.004 ± 0.002
(3)
36
0.001
0.011
0.041
1.6/6.8
1.9/6.3
0.9/7.0
85/43/283/49
883/175/480/133
76/48/87/18
N
H
0.0011 (5)
F
N
<
0.0030 ± NA
0.007 ± 0.003
(4)
F
3
3
7
8
N
H
(2)
O
N
N
0.0077 ±
0.0014 (3)
0.014 ± 0.001
(2)
N
H
O
O
S
0.0040 ±
0.006 ± 0.001
(2)
3
9
0.017
0.8/7.4
0.2/7.0
812/80/105/67
n.t.
N
H
0.0019 (4)
0.212 ±
.130 (34)
0.054 ± 0.031
(48)
3
-
~0.020
0
a
b
Calculated using Stardrop 6.6.
LLE = - log (LY96 EC ) – ClogD.
5
0
cm / r / d / h = mouse / rat / dog / human
d
Calculated using Stardrop 6.6.
A subset of the 4,4-spiro analogs synthesized is presented in table 4. Unsubstituted spiro-analog
35 afforded a potent LSD1 inhibitor in the TR-FRET, LY96, and phenotypic assay. Additional substitutions
aimed at modulating the basicity of the amine, as with oxetane 36 and difluoroethyl 37 afforded two
highly potent LSD1 inhibitors with good activity in thephenotypic assay. These two compounds attenuate
2
4
the basicity of the distal amine by >3 pKa units and demonstrate that modulated bases can afford potent
LSD1 inhibitors. Next, we tested the requirement for a second basic site in this spiro-scaffold by
synthesizing acetamide 38 and sulfonamide 39. Both mono-basic compounds afforded potent activity in
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the LY96 assay with good activity in the phenotypic assay. Additional profiling of the attenuated bases, 36
and 37, and the mono-basic compounds, 38 and 39, demonstrated a significant decrease in microsomal
stability compared to compound 34. Compound 34 was selected for further characterization due to its
impressive combination of potency, LLE, and low molecular weight.
Scheme 2. Synthesis of 34
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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9
0
O
O
N
O
NH
OH
(
R)
O
O
N
O
1. DIPEA, TFAA
2. TFA
(S)
NH2
(S)
N
NaBH(OAc)3
8% yield
HO
N
H
O
CF3
85% yield
-steps
4
3
1
40
41
2
O
O
OH
OH
N
OH
N
HO
NaOH
N
N
H
F
F
3
2
4% yield
-steps
NaBH3CN
used crude
O
F
42
34
2
5
The synthesis of 34 began from styrenyl cyclopropylamine 31 via a reductive amination with tert-butyl
-oxo-2-azaspiro[3.3]heptane-2-carboxylate. This afforded the spirocyclic cyclopropylamine 40 in good
6
yield. Protection of the cyclopropylamine as the trifluoroacetamide followed by Boc deprotection
afforded amine 41. This material was then subjected to a reductive amination with glycoaldehyde dimer
to afford the penultimate intermediate 42. Removal of the trifluoroacetamide group under basic
conditions, followed by purification afforded 34 in good yield.
Figure 2: Further characterization of 34
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Further characterization of 34 in a panel of FAD-utilizing enzymes confirmed that this compound
was highly selective for inhibition of LSD1, with no activity detected against the homologous enzyme LSD2
and related monoamine oxidases MAO-A and MAO-B (Figure 2A). Additional biochemical characterization
of 34 demonstrated that this molecule displays time-dependent inhibition of LSD1 biochemical activity
corroborating its hypothesized, covalent mechanism of inhibition (Figure 2C). This covalent inhibition
occurs via a two-step mechanism consisting first of a reversible, non-covalent binding event followed by
covalent inactivation of the FAD co-factor, in accordance with previous characterization of
1
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tranylcypromine-based LSD1 inhibitors.
Interestingly, ~1,500-fold improved inactivation efficiency (
푘푖푛푎푐푡
)
of 34 over 3 is driven through the reversible, non-covalent binding event, while the observed rate of
퐾퐼
inactivation, kinact, is comparable to that of the tranylcypromine-based 3.14,27
Profiling compound 34 in a panel of human AML cell lines resulted in dose-dependent effects on
cell growth with 5/8 cell lines as highly sensitive to inhibitor treatment (GI50 < 100 nM). In vivo
characterization of 34 began with a single dose PK experiment in mice (Figure 2D). This PK experiment
revealed that 34 is a high clearance, high volume compound with acceptable oral bioavailability to enable
in vivo efficacy studies.
Figure 3. Kasumi-1 xenograft data for compound 34.
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In a Kasumi-1 xenograft model, 34 demonstrated significant and dose-dependent tumor growth
inhibition (TGI) when administrated at 0.5 mg/kg and 1.5 mg/kg PO QD (Figure 3) for 14-days. Both doses
were well-tolerated, with ≤5% body weight loss observed at 1.5 mg/kg PO and no significant body weight
changes at the 0.5 mg/kg PO (see supporting information). Compound exposure and target engagement
were measured in the plasma and tumor samples at 1 and 24 hours post last dose. Target engagement
was monitored via induction of LY96 in the tumor and a good correlation between induction of LY96,
compound concentration in the tumor, and TGI was observed.
Herein we’ve described our efforts directed towards the discovery of novel covalent inhibitors of LSD1.
This research program identified the styrenylcyclopropane structure as a previously unreported
mechanism-based inhibitor of LSD1 which covalently labels the FAD cofactor. This novel chemotype
diversifies upon the clinically relevant tranylcypromine-based scaffolds and potentially offers access to
different physical properties, FAD-adducts, and biological effects. Optimization of this series culminated
in the identification of compound 34, a highly potent biochemical, cellular, and efficient (LLE) inhibitor.
Further, we characterized the kinetics of its covalent inhibition and demonstrate that 34 has an improved
inactivation efficiency compared to tranylcypromine-derived 3. This covalent inhibitor is differentiated
from the current tranylcypromine-based inhibitors by its highly potent non-covalent binding, KI, which
results in an impressive inactivation efficiency roughly 3 orders of magnitude greater than
tranylcypromine-based 3. Additional characterization of 34 across a panel of AML cell lines demonstrate
the broad potential utility of this mechanism in AML. Finally, we show that 34 provides dose-dependent
tumor growth inhibition in an AML xenograft model at tolerated doses.
Supporting information
The supporting information is available free of charge via the internet at http://pubs.acs.org.
Methods and materials, experimental procedures, LSD1 biochemical assay, LSD1 Kinact / K studies, LSD1
i
LY96 assay, cell proliferation assays, in vivo Kasumi-1 xenograft experiment
Corresponding Author
*E-mail: victor.gehling@constellationpharma.com. Phone:1-617-714-0540
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