Discovery of clinical candidate BMS-906024: A potent pan-notch inhibitor for the treatment of leukemia and solid tumors

Article abstract of DOI:10.1021/acsmedchemlett.5b00001

Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in

Full text of DOI:10.1021/acsmedchemlett.5b00001

Letter
pubs.acs.org/acsmedchemlett
Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch
Inhibitor for the Treatment of Leukemia and Solid Tumors
Ashvinikumar V. Gavai,*,† Claude Quesnelle,† Derek Norris,† Wen-Ching Han,† Patrice Gill,†
Weifang Shan,† Aaron Balog,† Ke Chen,§ Andrew Tebben,† Richard Rampulla,† Dauh-Rurng Wu,†
Yingru Zhang,† Arvind Mathur,† Ronald White,† Anne Rose,† Haiqing Wang,† Zheng Yang,†
Asoka Ranasinghe,† Celia D’Arienzo,† Victor Guarino,† Lan Xiao,† Ching Su,† Gerry Everlof,†
Vinod Arora,‡ Ding Ren Shen,† Mary Ellen Cvijic,† Krista Menard,† Mei-Li Wen,† Jere Meredith,‡
George Trainor,† Louis J. Lombardo,† Richard Olson,‡ Phil S. Baran,§ John T. Hunt,† Gregory D. Vite,†
Bruce S. Fischer,† Richard A. Westhouse,† and Francis Y. Lee†
†Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, United States
‡Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States
§Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: Structure−activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides
identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the
basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft
models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.
KEYWORDS: Notch inhibitor, γ-secretase inhibitor, SAR, triple-negative breast cancer, T-acute lymphoblastic leukemia
otch signaling is an evolutionally conserved cell signaling
pathway involved in cell fate control and multiple cell
The involvement of each of the four Notch receptors in cancer
development has been well established.2 Notch1 is the most
commonly activated oncogene in T-acute lymphoblastic
leukemia (TALL). Notch1 and Notch4 are coexpressed in
breast cancer, but not in normal breast tissue. There is
increasing evidence supporting the role of Notch3-mediated
signaling in solid tumors. These data support our central
hypothesis that pan-Notch inhibition will provide the broadest
spectrum of antitumor efficacy in humans.
γ-Secretase is a membrane bound protease with presenilin-1
or presenilin-2 as the catalytic core and three accessory
proteins, Aph-1, Pen-2, and nicastrin.5 It is also involved in the
sequential cleavage of amyloid precursor proteins to release β-
amyloid (Aβ) peptides that are thought to play a causative role
in Alzheimer’s disease (AD). Several APP-selective γ-secretase
inhibitors (GSIs) were developed for the treatment of AD but
demonstrated side effects attributed to the inhibition of
Notch1. Some of these GSIs that were developed as Aβ
inhibitors are now being repositioned in the clinic as Notch
inhibitors for the treatment of cancer (Figure 1).5
N
differentiation processes during embryonic and adult life.
Recent literature reports provide a strong rationale for pursuing
Notch signaling inhibition as a cancer treatment strategy.1−4
Deregulation of the Notch pathway has been shown to be
oncogenic in many human solid tumors and hematologic
malignancies. Inappropriate activation of the Notch pathway
produces many of the hallmarks of cancer, including
uncontrolled proliferation, restricted differentiation leading to
increased self-renewal capacity, evasion of apoptosis, and
enhancement of angiogenesis and metastasis. In addition,
there is strong evidence that Notch signaling plays a role in the
maintenance and survival of cancer stem cells.4 Drug resistance
during chemotherapy has also been attributed to activation of
the Notch pathway.2 Finally, targeting the Notch−ligand
interaction in endothelial cells can produce antitumor effects
through inhibition of tumor angiogenesis that is distinct from
the VEGF pathway.
The mammalian Notch receptor family has four homologues
(Notch1−4). Signaling is normally activated by contact of a
Notch receptor-bearing cell with a cell carrying one of the five
transmembrane ligands (Jagged 1 and 2, and Delta-like ligand 1,
3, and 4). Binding of ligand promotes two successive
proteolytic processing events, the second of which is catalyzed
by γ-secretase. As a result of proteolysis, the Notch intracellular
domain (NICD) is liberated and can enter the nucleus to
engage DNA-binding proteins and regulate gene expression.
Chronic inhibition of Notch1 signaling has been associated
with development of severe gastrointestinal (GI) toxicity due to
conversion of proliferative cells in intestinal crypts to goblet
Received: January 2, 2015
Accepted: March 11, 2015
© XXXX American Chemical Society
A
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TALL-1 cell proliferation assay (Table 1).9 TALL-1 is a T-cell
acute lymphoblastic leukemia cell line sensitive to Notch
inhibition,10 which was recently reported to have an activating
mutation in Notch3.11
Table 1. Effects of Succinamide Modifications on Notch1/3
Inhibitory Potency and in Vitro Metabolic Stability
Figure 1. Examples of Notch inhibitors undergoing clinical evaluation
in cancer.
cells.6 However, intermittent dosing with GSIs or combination
with glucocorticoids have been shown to mitigate GI toxicity
without affecting antitumor effects.7 With these strategies,
Notch1 inhibition can be tolerated, but inhibition of this
receptor subtype is still likely to be the source of dose-limiting
toxicity. Therefore, a balanced inhibitor that provides roughly
equally potent inhibition of the important Notch2/3/4
receptors as well as the critical but dose-limiting Notch1
receptor should produce the optimal efficacy/tolerability
spectrum. In addition, the relatively short half-life of small
molecule Notch inhibitors should offer an advantage over
antibody-based therapies by enabling regimens that allow for
target recovery in key cell types which mediate toxicity. This
Letter describes structure−activity relationships (SARs) in a
series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides as inhib-
itors of γ-secretase mediated signaling of Notch1/2/3/4
receptors leading up to the identification of 12 (BMS-
906024), which has advanced into clinical development.8
At Bristol-Myers Squibb (BMS), the Notch program was
initiated with the development of high-throughput cell-based
transcriptional reporter assays for Notch1−4 receptors.9 Initial
screening of the collection of GSIs was carried out in Notch1
and -3 assays and selected hits were subsequently evaluated in
all the four Notch signaling assays. The caprolactam 1 screening
hit was preferred since it provided a similar level of inhibitory
potency for Notch1 and -3 receptors (Figure 2). Early SAR
Figure 2. Notch inhibitors: Hit to Lead.
a
Data reported as average of three test results. See ref 9 for description
b
of assay conditions. Percentage of the parent compound (0.5 μM)
investigation suggested that the caprolactam can be replaced
with a 1,4-benzodiazepin-2-one moiety without any loss of
potency (compound 2). This change was preferred since it
improved pharmacokinetic properties in the lead series (data
not shown). Further SAR exploration established that the
primary amide was essential for potency, lipophilic groups were
preferred on the succinamide moiety, and the S,R,S
configuration was optimal for this series of pan-Notch
inhibitors. These efforts led to the discovery of compound 3
remaining after a 10 min incubation with 1 mg/mL of HLM or MsLM.
c
Not determined.
Compound 3 exhibited very poor metabolic stability in
isolated human or mouse liver microsomes (Table 1).
Therefore, biotransformation studies were conducted to
identify the metabolic soft spots (Figure 3). Abundance of
the metabolites (Met1−5) was estimated based on their liquid
chromatography−ultraviolet (LC−UV) peak heights compared
to that of 3 at the start of incubation. Structural analysis of the
as a highly potent pan-Notch inhibitor (Notch1/2/3/4 IC50
2/1/3/2 nM) with a commensurate level of potency in the
=
B
DOI: 10.1021/acsmedchemlett.5b00001
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a
Scheme 1. Synthesis of 12
Figure 3. Oxidative metabolites of 3 in human liver microsomes
a
(HLM) and mouse liver microsomes (MsLM). Yield of microsomal
transformation.
1
metabolites by mass spectroscopy and H NMR indicated that
alkyl groups on the succinamide moiety were the preferred sites
for oxidative metabolism. Met1, Met2, and Met4 incorporated
two additional oxygen atoms in the succinamide side chain,
whereas the structure of Met3 represented a net gain of three
oxygen atoms followed by the loss of a water molecule. The
methine functionality on the isobutyl group appeared to be the
most susceptible to enzymatic oxidation since the correspond-
ing hydroxylated compound (Met5) was isolated as the most
abundant metabolite. This information was utilized to initiate
succinamide SAR with the objective to improve metabolic
stability (Table 1). Incorporation of an additional methyl group
(compound 4) in place of the metabolically labile methine
proton in the isobutyl moiety in 3 maintained potency but did
not improve oxidative stability, suggesting that oxidation of the
3-pentyl group was also likely. Indeed, successive replacement
of the 3-pentyl moiety with propyl (compound 5) and
trifluoropropyl (compound 6) groups provided dramatic
improvement in metabolic stability. However, these changes
were accompanied by reduction in potency in the Notch1/3
signaling and the TALL-1 cell proliferation assays. Further
optimization was attempted by varying the R1 alkyl group on
the succinamide. A comparison of 6 with 4 and 7 indicates that
both alkyl groups on the succinamide contribute to the overall
metabolic stability in this series. Surprisingly, even small
structural variations at the R1 position led to significant
changes in potency in the Notch1/3 signaling assays (e.g.,
compare 7 to 8). The profile of succinamide 10 represented an
encouraging combination of Notch1/3 and TALL-1 potency
along with oxidative stability in the liver microsomes. The
corresponding regioisomeric compound 11 exhibited an
improvement in TALL-1 potency while retaining a similar
level of metabolic stability. When these two changes were
incorporated together, the resulting bis(trifluoropropyl)
succinamide 12 provided a very high level of in vitro potency
together with complete oxidative stability when incubated with
human or mouse liver microsomes.
a
Reagents and conditions: (a) sodium hexamethyldisilazane, THF,
BrCH2CO2t-Bu, −78 °C, 68%; (b) lithium hydroxide, water, hydrogen
peroxide, 0 °C, 83%; (c) LDA, THF, CF3CH2CH2OTf, −78 to 0 °C,
80%; 5:1 ratio of diastereomers at *stereocenter; (d) LDA, THF,
Et2AlCl, hexane, −78 to 0 °C, 90%; 9:1 ratio of diastereomers at
*stereocenter; (e) (1) LDA, toluene, THF, −78 °C; (2) LDA, toluene,
THF, t-Bu 5,5,5-trifluoropentanoate, −78 °C; (3) Cu(2-ethyl-
hexanoate)2, −78 to 40 °C, 66%; 1.6:1 ratio of diastereomers at
*stereocenter; (f) lithium hydroxide, water, hydrogen peroxide, 0 °C,
86%; (g) LDA, THF, Et2AlCl, hexane, −78 to 0 °C, 99%; 9:1 ratio of
diastereomers at *stereocenter; (h) 19, TBTU, Et3N, DMF, 89%; (i)
TFA, CH2Cl2, 0 °C, 75%; (j) EDC, HOBt, iPr2NEt, NH4Cl, DMF,
79%.
diethylaluminum chloride to give 15 as a 9:1 mixture in favor of
the desired diastereomer. An alternate synthetic route was
developed for the large scale synthesis of 15. Alkylation of 16
with t-butyl 2-bromoacetate afforded 17 in 68% yield.14
Removal of the chiral auxiliary and subsequent alkylation of
the resulting succinate with trifluoropropyl trifluoromethane-
sulfonate provided 18, which was epimerized with LDA and
diethylaluminum chloride to give 15 as a 9:1 mixture in favor of
the desired diastereomer. Coupling of acid 15 with (3S)-amino-
1,4-benzodiazepin-2-one 1915 in the presence of TBTU and
triethylamine produced the amide 20 in 89% yield. Trifluoro-
acetic acid-mediated ester hydrolysis and subsequent coupling
with ammonium chloride under standard conditions afforded
12 in 59% yield from intermediate 20. The absolute
configuration of 12 was further verified by X-ray diffraction
studies.
New compounds were prepared according to the sequence
illustrated in Scheme 1 for the synthesis of 12. An oxidative
intermolecular enolate heterocoupling reaction was employed
to prepare the 2,3-disubstituted succinate 15.12 Thus, reaction
of the lithium enolate of t-butyl trifluoropentanoate with the
lithium enolate of trifluoropentanoyl oxazolidinone 13 in the
presence of copper(II) 2-ethylhexanoate afforded the adduct 14
in 66% yield as a 1.6:1 mixture in favor of the depicted
diastereomer. Chemoselective removal of the chiral auxiliary
with lithium peroxide provided the corresponding carboxylic
acid, which was subjected to epimerization13 with LDA and
Compound 12 inhibited Notch processing with IC50 values
of 1.6, 0.7, 3.4, and 2.9 nM for Notch1, -2, -3, and -4 receptors,
respectively, and exhibited greater than 200-fold selectivity
against a panel of diverse protein targets (receptors, ion
channels, and enzymes). In cancer cell proliferation assays, 12
inhibited both leukemia (TALL-1) and triple-negative breast
C
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ACS Medicinal Chemistry Letters
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cancer (MDA-MB-468) cells with IC50 of ∼4 nM. In MDA-
MB-468 cells, 12 elicited dose-dependent inhibition of Notch3
intracellular domain formation, and the potency of this
inhibition was consistent with the potency observed for the
inhibition of cell proliferation.16 Extensive in vitro and in vivo
analysis identified several tumor cell lines that were sensitive
(e.g., HCC70 and MDA-MB-157) and resistant (e.g.,
HCC1419 and SKBR3) to treatment with compound 12.16
The pharmacokinetic parameters obtained for 12 in four
species are summarized in Table 2. The compound has good
Table 2. Pharmacokinetic Parameters for 12
a
a
a
b
parameter
mouse
rat
dog
cyno
c
d
e
f
g
f
h
d
dose (mg kg−1) iv/po
1 /1
1 /1
0.05 / 0.05
0.4/0.01
0.2 / 0.2
0.3/0.01
0.5/0.1
Cmax (μM) iv/po
1.0/0.3
3.4/1.9
1.7/0.1
2.0/0.8
i
j
AUC(0−24h) (μM·h)
iv/po
4.8 / 0.6
tmax (h) iv/po
t1/2 (h) iv/po
MRT (h) iv
Fpo (%)
0.4/0.8
4.6/5.3
3.8
0.2/1.2
6.6/4.2
3.0
0.2/5.3
0.2/2.0
6.7/13.0
8.2
51.1/32.1
62.3
Figure 4. In vivo antitumor activity of 12 against TALL-1 leukemia
xenografts (schedule: qd × 10; 2 days off; qd × 5).
57
46
29
b
30
a
Data reported as average of three animals. Data reported as average
c
d
of two animals. Vehicle: PEG400/water (70:30). PEG300/TPGS/
e
f
EtOH (80:10:10). PEG400/water (75:25). PEG300/TPGS/EtOH
(Log Cell Kill >1). Nearly complete tumor stasis was observed
throughout the dosing regimen at this dose, but tumor growth
resumed following the cessation of treatment. At higher doses,
a significant delay in tumor growth was observed even after the
dosing was completed. Dose-dependent antitumor activity was
also demonstrated in the MDA-MB-468 triple negative breast
cancer xenograft model. No overt toxicity as measured by
morbidity or significant body weight loss was observed at the
indicated doses in these experiments. Subsequently, 12
demonstrated broad-spectrum antineoplastic activity against a
wide array of human cancer xenografts, including colon
carcinoma, ovarian carcinoma, nonsmall cell lung carcinoma,
HER2-positive breast carcinoma, pancreatic carcinoma, glio-
blastoma, and neuroblastoma.17
In conclusion, optimization of the in vitro potency and
oxidative metabolic stability in a series of (2-oxo-1,4-
benzodiazepin-3-yl)-succinamides led to the identification of
12, a pan-Notch inhibitor that was orally efficacious in human
tumor xenografts dependent on the Notch signaling pathway.
On the basis of its favorable in vitro pharmacology, broad
spectrum in vivo efficacy in multiple tumor models, and a
satisfactory pharmacokinetic profile, 12 was advanced into
clinical trials.18
g
(60:20:20). Saline/hydroxypropyl β-cyclodextrin/PEG300 (87:12:1).
h
i
j
PEG400 (100). AUC(0−168h). AUC(0−72h)
.
intrinsic permeability in Caco-2 cells (104 nm/s) and was well
absorbed following oral administration from solution for-
mulations at the doses denoted in Table 2. Plasma half-life and
mean residence time (MRT) were longer in dogs as compared
to other species. Systemic clearance was less than 10% of the
hepatic blood flow, and volume of distribution (Vss) was larger
than total body water, indicating extensive extravascular
distribution. The measured oral bioavailability (Fpo) ranged
from 29% in dogs to 57% in mice. Compound 12 (10 μM) was
88.7% bound to human serum proteins and 95.9%, 94.3%,
95.4%, and 87.6% bound to the serum proteins of mouse, rat,
dog, and cynomolgus monkey, respectively.
Results of the in vivo activity of 12 in leukemia and solid
tumor xenograft models are summarized in Table 3. As
illustrated in Figure 4, once daily (10 days on; 2 days off; 5 days
on) oral administration of 12 in mice bearing the TALL-1
tumors resulted in a dose-dependent inhibition of tumor
growth with 1 mg/kg/day as the minimum efficacious dose
Table 3. In Vivo Antitumor Activity of 12 against MDA-MB-
468 Triple-Negative Breast Cancer and TALL-1 Leukemia
Xenografts (Schedule: qd × 10; 2 days off; qd × 5)
ASSOCIATED CONTENT
* Supporting Information
Experimental details for synthetic procedures and associated
chemical data for compounds 3−20. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
a
b
c
tumor model
TALL-1
dose (mg kg−1
)
LCK
%TGI
0.5
1
0.4
1.1
3.1
6.1
73
100
103
107
84
2
AUTHOR INFORMATION
Corresponding Author
*Phone: 609-252-5091. E-mail: ashvinikumar.gavai@bms.com.
Notes
■
5
d
MDA-MB-468
1
ND
ND
ND
d
4
92
d
6
94
The authors declare no competing financial interest.
a
b
Vehicle: PEG300/TPGS/EtOH (80:10:10). Log Cell Kill (LCK) at
ACKNOWLEDGMENTS
We thank William Frietze, Nenghui Wang, David Williams, and
the Department of Discovery Synthesis for resynthesis support.
c
■
the predetermined median tumor target size. Percent tumor growth
inhibition (TGI) at the end of the dosing period, P < 0.05. Not
d
determined.
D
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ABBREVIATIONS
■
EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide;
HLM, human liver microsomes; HOBt, 1-hydroxybenzotria-
zole; LDA, lithium diisopropylamide; MsLM, mouse liver
microsomes; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetrame-
thyluronium tetrafluoroborate; TFA, trifluoroacetic acid;
TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate;
VEGF, vascular endothelial growth factor
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