Discovery of CC-90011: A Potent and Selective Reversible Inhibitor
of Lysine Specific Demethylase 1 (LSD1)
Toufike Kanouni, Christophe Severin, Robert W. Cho, Natalie Y.-Y. Yuen, Jiangchun Xu, Lihong Shi,
Chon Lai, Joselyn R. Del Rosario, Ryan K. Stansfield, Lee N. Lawton, David Hosfield, Shawn O’Connell,
Matt M. Kreilein, Paula Tavares-Greco, Zhe Nie, Stephen W. Kaldor, James M. Veal, Jeffrey A. Stafford,
and Young K. Chen*
ABSTRACT: Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of
monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a
number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of
LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors
derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors.
Exploration of structure−activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical
candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and
small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is
currently in phase 2 trials in patients with first line, extensive stage SCLC ( identifier: NCT03850067).
Differentiation Agents. Tumors that are characterized as
poorly differentiated are among the most lethal and difficult to
treat as a result of uncontrolled proliferation of immature cells.
Efforts to combat poorly differentiated tumors with targeted
cytotoxic agents have had limited success, as resistance often
develops. While cytotoxic agents are generally effective in
debulking tumors, they often leave quiescent population of
tumor cells to evolve and recruit auxiliary pathways for survival
and evasion, hence, rendering cytotoxic therapies ineffective
with prolonged treatment. An alternative approach to targeting
poorly differentiated tumor is by inducing progression to
terminal differentiation, as terminally differentiated cells have a
limited capacity to proliferate and a finite lifespan.1 An early
example of a differentiation inducing agent is all-trans retinoic
acid (ATRA). ATRA, a metabolite of retinol-A, functions by
inducing immature leukemic blast cells to undergo terminal
differentiation and apoptosis. This discovery has been
transformative for patients suffering from acute-promyelocytic
leukemia (APL), a subset of AML with a chromosomal
translocation of retinoic acid receptor. Prior to the discovery of
ATRA, the prognosis for patients with APL was extremely
poor, with patients succumbing to rapid onset of hematological
toxicity. The cure rates for ATRA in combination with arsenic
trioxide treatment are as high as 80% for APL patients.2
Isocitrate dehydrogenase inhibitors (IDH1 and IDH2) have
also been shown to successfully induce differentiation in
another subset of AML patients that harbor IDH mutations.
The IDH mutants produce the oncometabolite D-2-hydrox￾yglutarate that inhibits downstream 2-oxoglutarate dependent
epigenetic modifiers, resulting in differentiation arrest of blast
cells.3 More recently, the discovery of inhibitors of
dihydroorotate dehydrogenase (DHODH), an enzyme in￾volved in the de novo pyrimidine synthesis pathway, is
Received: June 8, 2020 Drug Annotation
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anticipated to further broaden the repertoire of differentiation
inducing agents for combating AML.4 While advances in
differentiation agents are showing great promise for hemato￾logical malignancy, the same is not true for patients with solid
tumors.5 Herein, we report the discovery of clinical candidate
CC-90011, a potent reversible inhibitor for lysine demethylase-
1 LSD1 (KDM1A) that induces cellular differentiation of
poorly differentiated and highly metastatic small cell lung
carcinoma (SCLC).
LSD1 is a member of a broad family of monoamine oxidases
that requires FAD as a cofactor to catalyze demethylase
activity.6 LSD1 catalyzes demethylation at histone 3 lysine 4
methyl 1/2 (H3K4me1/2) resulting in a transcriptional
repressive state. However, LSD1 can also demethylate histone
3 lysine 9 methyl 1/2 (H3K9me1/2) to act as a transcriptional
activator in certain cellular context. LSD1 is also known to
demethylate nonhistone protein p53 and DNMT1.7 The LSD1
protein structure is comprised of three domains. The SWIRM
and Tower domains function as scaffolding for multiprotein
complex formation. The amine oxidase domain (AO) contains
the active site, wherein a catalytic lysine residue (K661) acts to
deprotonate a methylated histone lysine (e.g., H3K4me2),
prompting hydride transfer to FAD.8 It is noteworthy that
catalytic activity of the protein is lost upon mutations to K661.
However, recent reports have demonstrated that the AO
pocket also functions as scaffolding for transcription regulators
of cellular differentiation and proliferation, such as GFI1 and
INSM1.9 These proteins bind to the AO pocket via their
SNAG domains.
Under normal physiological conditions, epigenetic machi￾nery tightly regulates stem cell fate to self-renew, differentiate,
and proliferate. When functioning properly, this process is
orderly and self-limiting. However, dysregulation of epigenetic
machinery has been implicated in pathological conditions
found in various cancers. Dysregulation and overexpression of
LSD1 have been implicated in multiple cancer types that are
trapped in differentiation arrest. LSD1 is enriched in 60% of
leukemic blasts with oncogenic fusion protein MLL-AF9.10 In
this context, genetic knock down experiment of LSD1 in a
THP-1 cell line resulted in induction of cellular differentiation,
as measured by CD11b and CD86 expression. LSD1 also
regulates cellular differentiation and proliferation of AML with
RUNX1-RUNX1T1 translocation (AML1-ETO).11 In solid
tumors, LSD1 regulates differentiation and proliferation of
SCLC, a neuroendocrine tumor that is morphologically
characterized as immature, small, round, blue cells with
granular chromatin.12 SCLC is known to be highly proliferative
and to have high metastatic potential. The expression level of
LSD1 in primary SCLC samples is elevated in 98% of the
samples that have been tested.13 High expression levels of
LSD1 have also been found in breast cancer, neuroblastoma,
colorectal cancer, and prostate cancer. Aberrant dysregulation
of LSD1 causes differentiation arrest in both hematological and
solid tumors, making it an attractive target for cancer therapy.7
Clinical Stage LSD1 Inhibitors. Tranylcypromine (TCP),
a MAO inhibitor marketed for the treatment of severe
depression, and structurally related analogs of TCP have
been developed to selectively and irreversibly inhibit LSD1
(Figure 1). These compounds belong to a class of inhibitors
that covalently modify FAD in the AO pocket. ORY1001, the
most clinically advanced inhibitor in this class, is currently in
phase 2 trials for AML and SCLC.14 GSK2879552 is another
irreversible inhibitor; however, clinical trials of GSK2879552 in
AML and SCLC patients have recently been terminated
( identifier NCT02034123).13 Additional
clinical stage irreversible inhibitors IMG7289 (ClinicalTrials.-
gov identifier NCT02842827) and INCB059872 (Clinical￾ identifier NCT02712905) are in clinical trials for
AML and solid tumors. A reversible N-acylhydrazone LSD1
inhibitor was reported and is currently being evaluated in a
phase 1 trial for AML.15
Rationale for Reversible Inhibitors. Given our under￾standing of LSD1 biology, we hypothesized that reversible
inhibition of LSD1 could provide improved therapeutic
outcomes compared to irreversible inhibition. LSD1 plays
critical roles in maintaining cellular homeostasis in both
embryonic stem cells (ESCs) and adult hematopoiesis. Genetic
deletion of LSD1 in murine ESCs resulted in early death, while
knock down of LSD1 in hematopoietic in adults leads to
pancytopenia, albeit clinically manageable.16 Given these
findings, we surmised that irreversibly disabling LSD1 could
lead to increase of on-target toxicity. Furthermore, reversible
differentiation agents, such as ATRA, have been successfully
employed for treating APL patients with minimal toxicity. As
such, a reversible inhibitor of LSD1 could provide a
therapeutic window to induce differentiation of tumor cells
while limiting the potential on-target toxicity to normal cells.
Identification of Lead Reversible Inhibitors. A high
throughput screen of a 300 000 compound diversity library and
a smaller structure-based design library resulted in 75
compounds of interest from several distinct chemotypes.
Within this subset, we identified compound 1 (1 μM) as a
highly attractive starting point for further SAR exploration. A
binding model of compound 1 was created from a crystal
structure of the LSD1 AO pocket (PDB code 2HKO) (Figure
2) and used to guide SAR optimization.6b In the model,
Figure 1. Clinical stage LSD1 inhibitors.
Figure 2. Model of compound 1 in the AO pocket of LSD1 (PDB
code 2HKO).
compound 1 forms an important H-bonding interaction
between the nitrogen of the benzonitrile and catalytic lysine
(K661) in the FAD groove. A salt-bridge interaction between
the positively charged pyrrolidine side chain and D555 further
enhances binding in the AO pocket. The Cl substituent
projects into a hydrophobic pocket (W695, I356, L677, L692,
and F358) adjacent to the narrow groove of FAD.
Introduction of lipophilic groups such as p-tolyl resulted in
pyrimidine 2 (LSD1 IC50 = 2 nM, CD11b EC50 = 28 nM;
Table 1), which represents a 500-fold improvement in
biochemical potency and potent induction of CD11b in a
THP1 cell line, as assessed by fluorescence-activated cell
sorting (FACS) (Supporting Information). CD11b is a
myeloid differentiation surface marker that is directly regulated
by LSD1, and it was employed as a biomarker,10 while we were
pleased that pyrimidine 2 could induce CD11b expression in a
pharmacologically useful range, supporting the hypothesis that
reversible inhibition is a viable strategy. However, 2 was
limited by poor oral bioavailability (AUC0−6h = 1 μM·h) and
potent hERG inhibition (>95% at 10 μM). Replacement of the
pyrrolidine of 2 with a fused bicyclic ring system served to
rigidify and lock 2 in a low energy conformation for optimal
engagement of D555 and simultaneously eliminated an
unproductive H-bond donor. The resulting pyrimidine 3 had
good biochemical and cellular potency (LSD1 IC50 = 1.4 nM,
CD11b IC50 = 73 nM) with improved oral exposure (AUC0−6h
= 15 μM·h). The propensity of lipophilic cationic amines to be
potent hERG channel blockers is well-known and can be a
major challenge to overcome.17 In order to address this
liability, we devised a strategy of incremental structural changes
and judicious placement of heteroatoms to lower the overall
lipophilicity, all while maintaining potent cellular activity and
oral exposure. To this end, modification of the fused bicyclic
ring in 3 to a structurally simple aminopiperidine 4 maintained
good potency (LSD1 IC50 = 3.0 nM, CD11b IC50 = 28 nM)
and lowered overall lipophilicity, as measured by cLogD (2.6).
Replacement of the central pyrimidine core with pyrazinone 5
retained potency (LSD1 IC50 = 1.4 nM, CD11b IC50 = 47 nM)
while maintaining good oral exposure (AUC0−6h = 7.3 μM·h).
Opportunities for modification of the benzonitrile were limited
due to lack of space in the narrow FAD groove. However,
addition of a fluorine at the 3-position of the benzonitrile 6 was
accommodated and provided a further boost in potency (LSD1
IC50 = 0.5 nM, CD11b IC50 = 18 nM). The positional isomer
with a fluorine substituent in the 2-position (7) was disfavored,
resulting in a significant loss in biochemical and cellular
potency (LSD1 IC50 = 11 nM, CD11b IC50 = 120 nM).
Pyrimidinone 8 compared favorably to pyrazinone 5 with
respect to potency (LSD1 IC50 = 0.5 nM, CD11b IC50 = 18
nM), and replacement of p-Me with p-OMe (9) resulted in
meaningful lowering of hERG channel inhibition (67%).
Addition of fluorine in the 3-position of methoxyphenyl
group (10) further reduced hERG channel inhibition (48%)
while maintaining good overall properties. Addition of a
fluorine substitution in the 3-position of the benzonitrile
resulted in compound 11 (CC-90011). Compound 11
demonstrated potent inhibition of LSD1 at 0.3 nM and
induction of on-target cellular differentiation marker CD11b in
THP-1 cell line with EC50 = 7 nM, antiproliferative activity in
AML kasumi-1 cells with EC50 = 2 nM, and no effect in normal
human fibroblasts (IMR-90). The selectivity profile of
compound 11 was tested against FAD dependent amine￾oxidases LSD2, MOA-A, and MAO-B, and no enzymatic
inhibition was observed below 10 μM (Supporting Informa￾tion).
Mouse pharmacokinetic properties were evaluated in CD-1
mice by administration of a single 5 mg/kg dose of compound
11 intravenously (iv) and by oral gavage (Table 2). After iv
administration, compound 11 had systemic clearance of 32.4
mL min−1 kg−1
, elimination half-life of 2 h, and a high volume
of distribution of 7.5 L/kg. Compound 11 was readily
absorbed after oral administration with an AUC0−24h of 1.8
μM·h, Cmax of 0.36 μM, and oral bioavailability of 32%. The
Table 1. Summary of SAR of LSD1 Inhibitors
LSD1 IC50 was determined using a TR-Fret assay. b
CD11b assay was
assessed by FACS in THP1 cell line. c
10 mg/kg dose. d
Percent hERG
channel inhibition at 10 μM. e
Calculated log D.
Table 2. Pharmacokinetics and hERG Profile of 11
parameter 11
iv/po (mg/kg) 5/5
Cl (mL min−1 kg−1
) 32.4
Vdss (L/kg) 7.5
T1/2 (h) 2
AUC0−24h (μM·h) 1.8
Cmax (μM) 0.36
F (%) 32
hERG (μM) 3.4
mouse PPB (%) 92
effect of compound 11 on hERG potassium channels
(HEK293 cells stably transfected with hERG cDNA) was
examined by patch clamp. The IC50 value for compound 11
was determined to be 3.4 μM which provided sufficient
cardiotoxicity safety margins to advance the compound for in
vivo studies.
Cocrystal Structure of CC-90011/LSD1. An X-ray crystal
structure of compound 11 bound to the LSD1/CoREST
complex was determined at 2.92 Å (PDB code 6W4K) (Figure
3). As expected, the inhibitor forms a rigid Y-shaped
configuration with the central pyrimidinone core projecting
the aminopiperidine moiety to form a salt-bridge with D555.
The benzonitrile projects into a hydrophobic pocket formed by
FAD and several amino acid side chains. An H-bond
interaction of catalytic K661 with the nitrile appears to have
displaced the catalytic water molecule. The 2-F-anisole ring
forms an additional hydrophobic interaction within a shallow
pocket adjacent to the FAD pocket.
Synthesis of 11 or CC-90011. The synthesis of 11 was
achieved in six steps starting from readily available
perchloropyrimidine 12, as shown in Scheme 1. The
chemoselective displacement of the chlorine in the 4-position
of perchloropyrimidine was achieved using aqueous base (1.3
equiv of NaOH) at ambient temperature, resulting in 69%
yield of pyrimidinone 13. Treatment of 13 with iodomethane
resulted in the thermodynamically favorable N-methylpyrimi￾dinone adduct 14 in good yield (62%). Treatment of N￾methylpyrimidinone 14 with N-Boc-aminopiperidine led to the
displacement of the 2-position chlorine to give 15 in excellent
yield (80%). Selective Suzuki cross coupling reaction with (4-
cyano-3-fluorophenyl)boronic acid in ACN/water at 85 °C
furnished intermediate 16 (57% yield). A second Suzuki cross
coupling with intermediate 16 and (3-fluoro-4-
methoxyphenyl)boronic acid in dioxane/water at 80 °C
afforded 17 in excellent yield (92%). Finally, deprotection
with BSA in the presence of formic acid furnished compound
11 (CC-90011) as the benzenesulfonate salt in 95% yield.
Notably, each chlorine atom in perchloropyrimidine 12 was
chemoselectively functionalized to furnish CC-90011 in six
linear steps and 17% overall yield.
Pharmacology of Compound 11. LSD1 inhibitors have
been shown to suppress GRP expression in neuroendocrine
tumors such as SCLC. ChIP-seq analysis in SCLC cell lines
(H69 and H209) showed LSD1 co-occupies sites with the
enhancer mark H3K4me1 surrounding the GRP locus
(Supporting Information) linking LSD1 to regulation of
gastrin-releasing peptide (GRP) expression, which was used
as a pharmacodynamic (PD) biomarker. Consistent with the
ChIP-seq results, suppression of GRP was observed with
treatment of compound 11 (4 days) in a dose-dependent
manner and at pharmacologically useful concentrations (EC50
= 3 nM, H209 and 4 nM, H1417) (Figure 4A). In addition,
treatment of SCLC cells with compound 11 for an extended
incubation period (12 days) in vitro resulted in potent
antiproliferative activity (EC50 = 6 nM, H1417) that correlated
with GRP suppression (Figure 4B). It has been suggested that
loss of GRP expression is a result of SCLC cells undergoing
differentiation and losing their neuroendocrine status.13
Given the excellent on-target in vitro activity and PK
properties, compound 11 was advanced into a SCLC human
tumor xenograft (H1417) study in mice. Mice were inoculated
with H1417 tumor cells and treated QD at 2.5, 5, and 10 mg/
kg of compound 11 for 4 consecutive days. GRP levels were
assessed by qPCR 24 h after the last dose (Figure 4C).
Treatment with compound 11 resulted in robust down￾regulation of GRP mRNA levels at 2.5 mg/kg and maximum
suppression of GRP at 5 mg/kg. In a separate study, mice were
implanted with H1417 SCLC tumors and treated with
compound 11 daily at 2.5 and 5 mg/kg, which resulted in
tumor growth regressions of 159% and 178%, respectively (P￾value of 0.001 and 0.0001) (Figure 4D). Compound 11 was
well tolerated for the duration of this study (65 days) with an
average maximal body weight gain of up to 7% (Supporting
Information). Maximum efficacy was observed at 5 mg/kg QD
in both xenograft studies. These results established 5 mg/kg
QD of 11 as the efficacious dose for the in vivo studies.
To assess efficacy of compound 11 in clinically relevant
patient-derived xenograft models (PDX), mice were inoculated
with human LXFS 615 SCLC cells and treated with compound
11 at 5 mg/kg daily for 30 consecutive days. Compound
treatment resulted in 78% TGI (P-value of 0.001) (Figure 5).
Compound 11 was well tolerated with mean body weight loss
of <1% (Supporting Information). The efficacy observed in
Figure 3. Cocrystal structure of compound 11 in the AO pocket of
LSD1 (PDB code 6W4K).
Scheme 1. Synthesis of 11 or CC-90011a
Reagent and conditions: (a) 1.3 equiv of NaOH, THF, rt, overnight,
69% yield; (b) MeI, K2CO3, DMF, rt, overnight, 62% yield; (c) tert￾butyl piperidin-4-ylcarbamate, DIEA, DMF, rt, overnight, 80% yield;
(d) 4-cyano-3-fluorophenyl)boronic acid, Pd(PPh3)4, K2CO3,
MeCN/H2O, 85 °C, 57% yield; (e) (3-fluoro-4-methoxyphenyl)-
boronic acid, Pd(PPh3)4, K2CO3, dioxane/H2O, 80 °C, 92% yield; (f)
benzenesulfonic acid, formic acid.
this PDX model is in line with other SCLC PDX models that
had been treated with compound 11.
Compound 11 was evaluated in a separate SCLC PDX
model (LU2514) wherein treatment with compound 11 at 5
and 10 mg/kg achieved tumor growth inhibition of 39% and
56%, respectively (Figure 6A). Additionally, marked cellular
morphology changes (Figure 6B) were observed upon H&E
staining of tumor cells collected 15 days after the final dose (10
mg/kg group). Vehicle-treated tumor cells maintained a classic
poorly differentiated round cell morphology with granular
chromatin. On the other hand, compound 11 treated tumor
cells was shown to have a looser cellular structure, decreased
nucleus to cytoplasm ratio, and higher number of apoptotic
bodies (indicated by yellow arrows). These results clearly
demonstrate a change in differentiation status of LU2514 cells
consistent with the mechanistic hypothesis. The phenotypic
effect upon inhibition LSD1 is generally cytostatic, but the
underlining differentiation biology is pronounced and warrants
further investigation.
Conventional targeted agents for combating cancers have
focused on promoting apoptosis in rapidly proliferating cancer
cells. These agents have proven effective in reducing tumor
bulk, but prolonged treatment has often resulted in tumors
developing resistance. An alternative strategy is to suppress
proliferation by inducing terminal differentiation of the cancer
cells. This strategy is employed less frequently but has been
highly effective in subsets of AML. The discovery of CC-
90011, a highly potent and reversible inhibitor of LSD1,
provides a novel differentiation strategy for the treatment of
neuroendocrine tumors and AML. Phase 1 study of CC-90011
in patients with advanced solid tumors has been completed,
and the safety, tolerability, and preliminary efficacy have been
reported.18 CC-90011 is currently in phase 2 trials in patients
with first line, extensive stage SCLC (
identifier NCT03850067).
Figure 4. (A) GRP mRNA expression in H209 and H1417 cell lines with treatment of compound 11. (B) Cell-Titer Glo viability assay in H1417
cell line with treatment using compound 11. (C) In vivo effect of compound 11 treatment on human GRP mRNA expression in SCLC H1417
model at 2.5 mg/kg, 5 mg/kg, and 10 mg/kg QD dosing for 4 days, n = 3 mice/group. (D) In vivo efficacy of compound 11 in mice bearing SCLC
xenograft model H1417 at 2.5 mg/kg and 5 mg/kg QD dosing for 65 days, n = 7 or 8 mice/group.
Figure 5. In vivo efficacy of compound 11 in mice bearing PDX
model LXFS615 at 5 mg/kg QD dosing for 30 days, n = 10 mice/
General Chemical Synthesis Methods. All solvents were
purchased commercially and utilized without further purification.
All reagents were obtained from commercial suppliers and used
without further purification. All reactions were conducted under an
inert atmosphere at room temperature unless otherwise stated. 1
NMR spectra were recorded on a Bruker Advance 400. Chemical
shifts are presented in parts per million (ppm). Coupling constants
are in units of hertz (Hz). Splitting patterns describe apparent
multiplicities and are designated as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), and br (broad). Low-resolution mass
spectrometry (MS) and compound purity data were acquired on a
Shimazu LC/MS single quadrupole system equipped with electro￾spray ionization (ESI) source and UV detector (220 and 254 nm).
The purity of final tested compounds was ≥95% as determined by 1
2,5,6-Trichloropyrimidin-4-ol (13). To a solution of perchlor￾opyrimidine 12 (1 kg, 4.63 mol) in THF (6 L) was added 1 N NaOH
(6 L, 6.0 mol) dropwise, and the mixture was stirred overnight at
room temperature. Upon completion, the solution was acidified with
1 N HCl and extracted with DCM (3×). The combined organic layers
were dried (Na2SO4) and concentrated in vacuo. The solids were
suspended and stirred in Et2O for 30 min. The heterogeneous mixture
was successively filtered, washed with Et2O, and dried to give 635 g
(69%) of the title compound 13. LCMS (C18 column, column size
4.6 mm × 50 mm; mobile phase 20−40%, acetonitrile−water−0.02%
NH4OAc): tR = 2.785 min; [M + H] calcd for C4HCl3N2O, 199;
found, 199.
2,5,6-Trichloro-3-methyl-3-hydropyrimidin-4-one (14). A
solution of 2,5,6-trichloropyrimidin-4-ol 13 (670 g, 3.38 mol) and
K2CO3 (560 g, 4.06 mol) in DMF (5 L) was stirred at room
temperature for 15 min and then cooled to 0 °C. Iodomethane (528
g, 3.72 mol) was added dropwise, and the mixture was stirred at room
temperature for 17 h. The reaction mixture was taken up in ethyl
acetate and the combined organic layers were successively washed
with brine, dried (Na2SO4), and concentrated in vacuo. The residue
was purified by silica gel chromatography (PE:EA, 10:1) to give 447 g
(62%) of the title compound 14. 1
H NMR (400 MHz, CDCl3): δ
3.74 (s, 3 H). LCMS (C18 column; column size 4.6 mm × 50 mm;
mobile phase 20−95%, acetonitrile−water−0.02% NH4OAc): tR =
2.986 min; [M + H] calcd for C5H3Cl3N2O, 213; found, 213.
yl))(4-piperidyl)](tert-butoxy)carboxamide (15). A solution of
2,5,6-trichloro-3-methyl-3-hydropyrimidin-4-one 14 (532 g, 2.51
mol), DIEA (648 g, 5.02 mol), and (tert-butoxy)-N-(4-piperidyl)-
carboxamide (502 g, 2.51 mol) in DMF (800 mL) was heated to 120
°C for 1 h. The solvent was removed in vacuo and the residue was
purified by silica gel chromatography (PE:EA, 1:1) to give 751 g
(80%) of the title compound 15. 1
H NMR (400 MHz, CDCl3): δ
1.45 (s, 9H), 1.50−1.58 (m, 2H), 2.06−2.10 (m, 2H), 2.98−3.05 (m,
2H), 3.48 (s, 3 H), 3.53−3.56 (m, 2H), 3.70 (s, 1H), 4.52 (s, 1H).
LCMS (C18 column; column size 4.6 mm × 50 mm; mobile phase
20−95%, acetonitrile−water−0.02% NH4OAc): tR = 4.006 min; [M +
H] calcd for C15H22Cl2N4O3, 377; found, 321 (MW-tBu).
tert-Butyl 1-(5-Chloro-4-(3-fluoro-4-cyanophenyl)-1-meth￾yl-6-oxo-1,6-dihydropyrimidin-2-yl)piperidin-4-ylcarbamate
(16). To a solution of N-[1-(5,6-dichloro-3-methyl-4-oxo(3-hydro￾pyrimidin-2-yl))(4-piperidyl)](tert-butoxy)carboxamide 15 (150 g,
0.40 mol) in ACN (4 L), under N2 atmosphere, was added 3-fluoro-4-
cyanophenylboronic acid (65.8 g, 0.40 mol), Pd(Ph3P)4 (9.3 g, 8
mmol), and Na2CO3 (0.4 N, 2 L, 0.80 mol). The mixture was stirred
at 85 °C for 2 h. Upon completion, the reaction was allowed to cool
to room temperature. Water (2 L) was added, and the aqueous
mixture was extracted with ethyl acetate (3×). The combined organic
layers were successively washed with water (3×), brine, dried
(Na2SO4), and concentrated in vacuo. The residue was purified by
silica gel chromatography (PE:EA, 3:1) to give 95 g (57%) of the title
compound 16. 1
H NMR (400 MHz, CDCl3): δ 1.45 (s, 9 H), 1.54−
1.61 (m, 2H), 2.05−2.13 (m, 2H), 2.99−3.08 (m, 2H), 3.53−3.58 (s,
5H), 3.70 (s, 1H), 4.54 (d, J = 6.0 Hz, 1H), 7.68−7.80 (m, 3 H).
LCMS (C18 column; column size 4.6 mm × 50 mm; mobile phase
5%−95%, acetonitrile−water−0.1% TFA): tR = 4.443 min; [M + H]
calcd for C22H25ClFN5O3, 462; found, 462.
tert-Butyl N-[1-[4-(4-Cyano-3-fluorophenyl)-5-(3-fluoro-4-
yl]carbamate (17). A mixture of (tert-butoxy)-N-{1-[5-chloro-6-(4-
piperidyl)}carboxamide 16 (295.8 g, 640 mmol), 3-fluoro-4-
methoxybenzeneboronic acid (217.7 g, 1281 mmol), Pd(Ph3P)4
(18.5 g, 16.0 mmol), and K2CO3 (265.5 g, 1921 mmol) in degassed
dioxane:H2O (3:1, 3550 mL:887 mL) was stirred at 80 °C under N2
atmosphere for 2 h. Upon completion, the reaction mixture was
allowed to cool to room temperature and water (11 L) was added.
The slurry was stirred for 1 h and filtered. The solids were successively
washed with water (4 L) followed by MeOH:water (1:1, 4 L) mixture.
The filter cake was taken up and stirred in MeOH (4 L) for 10 min.
The slurry was filtered, and the solids were rinsed with MeOH (4 L)
followed by washing with MTBE (4 L). The solids were taken in
DCM (16 L), and the reaction mixture was stirred for 15 min. 2-
Mercaptoethyl ethyl sulfide silica (400 g) was then added, and the
reaction mixture was stirred at room temperature for at least 1 h
under N2 atmosphere. The reaction mixture was filtered through
Celite in a fritted filter, and the solids were rinsed with DCM (1 L).
The volume of DCM filtrate was reduced to near dryness, and MeOH
(4 L) was added and concentrated in vacuo. The solids were taken up
in MeOH (4 L), and the slurry was cooled to 15 °C and filtered. The
filter cake was washed further with MeOH (0.7 L) followed by MTBE
(0.7 L) and dried in a vacuum oven (45 °C) to a constant weight of
324.6 g (91.9%) of the title compound (17). 1
H NMR (400 MHz,
CDCl3): δ 1.46 (s, 9 H), 1.60 (d, J = 10.11 Hz, 2 H), 2.11 (d, J =
11.62 Hz, 2 H), 3.06 (t, J = 12.00 Hz, 2 H), 3.54 (s, 3 H), 3.60 (d, J =
13.64 Hz, 2 H), 3.72 (br s, 1 H), 3.88 (s, 3 H), 4.52 (br s, 1 H),
6.79−6.89 (m, 2 H), 6.97 (d, J = 12.38 Hz, 1 H), 7.13 (d, J = 8.34 Hz,
1 H), 7.31 (d, J = 9.85 Hz, 1 H), 7.42 (br s, 1 H). LCMS (C18
Figure 6. (A) In vivo efficacy of compound 11 in mice bearing PDX model LU2514 at 5 and 10 mg/kg QD dosing for 28 days, n = 8 or 10 mice/
group. (b) H&E staining of vehicle and compound 11 treated (10 mg/kg) tumor (LU2514) 14 days after last dose.
column; column size 4.6 mm × 50 mm; mobile phase: 5%−95%,
acetonitrile−water−0.1% TFA): tR = 6.979; [M + H] calcd for
C29H31F2N5O4, 552; found, 552.
4-[2-(4-Aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphen￾yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzo￾nitrile, Besylate Salt (11 or CC90011). A solution of tert-butyl N-
methyl-6-oxopyrimidin-2-yl]piperidin-4-yl]carbamate (17) (5.0 g,
9.06 mmol) and benzenesulfonic acid monohydrate (1.9 g, 10.9
mmol) in formic acid (41.5 mL) was stirred at room temperature
until reaction completion. The solution was filtered through a 0.45
μm filter. Water (25 mL) was added to the formic acid solution. Seeds
of 4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-
methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile, besy￾late salt (0.05g) were introduced, and the solution was aged for 30
min. Water (up to 50 mL) was added to the mixture over 6 h. The
batch was then allowed to age for at least 12 h. The batch was filtered,
and the cake was washed with 80/20 water/formic acid (v/v) and
dried at 40−50 °C in a vacuum oven with a nitrogen bleed to give the
(5.25 g, 95%) of title compound (11 or CC-90011). 1
H NMR (400
MHz, CD3OD): δ 1.69 (q, 2H, J = 11.4 Hz), 2.00 (d, 2H, J = 10.2
Hz), 2.99 (t, 2H, J = 12.3 Hz), 3.31 (bs, 1H), 3.42 (s, 3H), 3.72 (d,
2H, J = 13.2 Hz), 3.81 (s, 3H), 6.78 (d, 1H, J= 8.4 Hz), 7.01−7.06
(m, 1H), 7.04−7.06 (m, 1H), 7.19 (dd, 1H, 1.2 Hz), 7.32 (m, 2H),
7.32 (m, 1H), 7.46 (dd, 1H, J = 10.5, 1.2 Hz), 7.61 (m, 2H), 7.82 (dd,
1H, J = 8.1, 7.2 Hz), 7.92 (bs, 1H), 7.92 (bs, 2H). LCMS (column,
Agilent Zorbax SB-C8, 4.6 mm × 50 mm, 3.5 μm particle size; mobile
phase 5%−95%, acetonitrile−water−0.1% TFA): tR = 3.854; [M + H]
calcd for C24H23F2N5O2, 452; found, 452. A second recrystallization
from 80/20 water/formic acid was performed as described above to
provide material greater than 99% pure as determined by LC analysis.
*sı Supporting Information
The Supporting Information is available free of charge at
Biology and pharmacology of 11, crystallization
methods, and purity information (PDF)
Molecular formula strings and standard error of
measurement for all biological data present (CSV)
Accession Codes
Authors will release the atomic coordinates and experimental
data upon article publication (PDB code 6W4K for compound
11 bound to LSD1/CoREST).
Corresponding Author
Young K. Chen − Bristol Myers Squibb, San Diego, California
92121, United States;;
Email: [email protected]
Toufike Kanouni − Fount Therapeutics, LLC, San Diego,
California 92130, United States
Christophe Severin − Bristol Myers Squibb, San Diego,
California 92121, United States
Robert W. Cho − Quanticel Pharmaceuticels, San Francisco,
California 94158, United States
Natalie Y.-Y. Yuen − Oric Pharmaceuticals, South San
Francisco, California 94080, United States
Jiangchun Xu − Bristol Myers Squibb, San Diego, California
92121, United States
Lihong Shi − Bristol Myers Squibb, San Diego, California
92121, United States
Chon Lai − Bristol Myers Squibb, San Diego, California 92121,
United States
Joselyn R. Del Rosario − Bristol Myers Squibb, San Diego,
California 92121, United States
Ryan K. Stansfield − 858 Therapeutics, Inc., San Diego,
California 92121, United States
Lee N. Lawton − Dana-Farber Cancer Institute, Boston,
Massachusetts 02215, United States
David Hosfield − University of Chicago, Chicago, Illinois
60637, United States
Shawn O’Connell − Pfizer Inc., San Diego, California 92121,
United States
Matt M. Kreilein − Bristol Myers Squibb, Summit, New Jersey
07901, United States
Paula Tavares-Greco − Bristol Myers Squibb, Summit, New
Jersey 07901, United States
Zhe Nie − Schrödinger, Inc., San Diego, California 92121,
United States
Stephen W. Kaldor − Fount Therapeutics, LLC, San Diego,
California 92130, United States
James M. Veal − 858 Therapeutics, Inc., San Diego, California
92121, United States
Jeffrey A. Stafford − 858 Therapeutics, Inc., San Diego,
California 92121, United States
Complete contact information is available at:
The authors declare no competing financial interest.
Y.K.C. is grateful to Laura D’Agostino and Robert Borzilleri for
valuable discussions, proofreading, and unwavering support
during the drafting of this manuscript.
This manuscript is dedicated to the memory of our dear friend
and colleague Christophe Severin. His astute observations and
perseverance were crucial to the success of the discovery of
LSD1, lysine demethylase 1; ATRA, all-trans retinoic acid;
IDH, isocitrate dehydrogenase; DHODH, dihydroorotate
dehydrogenase; H3K4me1/2, histone 3 lysine 4 methyl 1/2;
H3K9 me1/2, histone 3 lysine 9 methyl 1/2; GFI1, growth
factor independent 1; INSM1, insulinoma associated protein 1;
SNAG, Snail1; ESC, embryonic stem cell; H&E, hemotoxylin
and eosin
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