Identification of 1-{2-[4-chloro-1′-(2,2-dimethylpropyl)-7-hydroxy-1, 2-dihydrospiro[indole-3,4′-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3] thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and orally bioavailable P2Y1 antagonist as an antiplatelet agent

Article abstract of DOI:10.1016/j.bmcl.2014.01.066

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y₁ receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y₁ antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y₁₂ antagonist clopidogrel in rat efficacy/bleeding models.

Full text of DOI:10.1016/j.bmcl.2014.01.066

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1294–1298
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 1-{2-[4-chloro-10-(2,2-dimethylpropyl)-7-hydroxy-
1,2-dihydrospiro[indole-3,40-piperidine]-1-yl]phenyl}-3-{5-chloro-
[1,3]thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and
orally bioavailable P2Y1 antagonist as an antiplatelet agent
a,
a
a
a
a
Yoon T. Jeon a, Wu Yang a, , Jennifer X. Qiao , Ling Li , Rejean Ruel , Carl Thibeault , Sheldon Hiebert ,
Tammy C. Wang a, Yufeng Wang a, Yajun Liu a, Charles G. Clark a, Henry S. Wong a, Juliang Zhu a,
Dauh-Rurng Wu a, Dawn Sun a, Bang-Chi Chen a, Arvind Mathur a, Silvi A. Chacko c, Mary Malley c,
Xue-Qing Chen c, Hong Shen c, Christine S. Huang c, William A. Schumacher b, Jeffrey S. Bostwick b,
Anne B. Stewart b, Laura A. Price b, Ji Hua b, Danshi Li c, Paul C. Levesque c, Dietmar A. Seiffert b,
Robert Rehfuss b, Ruth R. Wexler a, Patrick Y. S. Lam a
⇑
⇑
a Discovery Chemistry, Bristol-Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA
b Discovery Biology, Cardiovascular, Bristol-Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA
c Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor.
Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spi-
ropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in
the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile
in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved
bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 12 December 2013
Revised 13 January 2014
Accepted 21 January 2014
Available online 31 January 2014
Keywords:
P2Y1 antagonist
Spiropiperidinylindolines
Antiplatelet agent
Atherothrombosis is the number one cause of mortality and
morbidity worldwide. Platelet activation plays a key role in the
development of arterial thrombosis. The two G-protein coupled
receptors P2Y1 and P2Y12 are important in regulating haemostasis
and thrombosis. Synergistic activation of both P2Y1 and P2Y12
receptors is essential for a complete platelet response. Blockade
of either receptor significantly decreases both in vitro and
ex vivo ADP-induced platelet aggregation and reduces thrombosis
formation.1 Inhibitors of P2Y12 have resulted in several marketed
drugs such as the widely used clopidogrel, and recently approved
prasugrel and ticagrelor. An attractive alternative therapeutic ap-
proach would be the discovery of antagonists of the P2Y1 receptor.
Preclinical models with P2Y1 knock-out mice or with selective P2Y1
antagonists such as the adenosine nucleotide analog MRS25002
and the recently disclosed diaryl ureas3,4 showed complete blockade
of ADP-induced platelet aggregation and effective reduction of
arterial thrombosis with only moderate prolongation of bleeding
time. With the potential of providing a reduced bleeding liability
and equivalent efficacy compared to P2Y12 antagonism, P2Y1
antagonism is a promising target for antithrombotic therapy.5
Recently, we reported on the discovery of 1-(2-(10-neopentyl-
spiro[indoline-3,40-piperidine]-1-yl)phenyl)-3-(4-(trifluorometh-
oxy)phenyl)urea (compound 1)4 as a P2Y1 receptor antagonist.
Compound 1 inhibited platelet aggregation (PA) in human plate-
let-rich plasma with an IC50 of 4.9 lM at 10 lM ADP, which is
five-to ten-fold less potent than the desired potency range to
achieve a reasonable human dose projection. Additionally, the
modestly improved aqueous solubility of compound 1 (4 lg/ml,
amorphous) was a desired property. Described in this article is
the discovery of 7-hydroxyl substitution on the indoline that re-
sulted in improvements in both potency and solubility. Optimiza-
tion of this series resulted in the identification of compound 3e,
which showed more than 20-fold improvement in in vitro potency
in FLIPR (fluorescent imaging plate reader) and PA assays and had
⇑
Corresponding authors. Tel.: +1 609 818 6493 (W.Y.); +1 609 818 5298 (J.X.Q.).
E-mail addresses: wu.yang@bms.com (W. Yang), jennifer.qiao@bms.com
(J.X. Qiao).
http://dx.doi.org/10.1016/j.bmcl.2014.01.066
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Y. T. Jeon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1294–1298
1295
OCF3
OCF3
Table 1
O
O
B
N
SAR of 4-substituted 7-hydroxyl indolines
N
H
N
H
N
H
N
H
HO
7
6
OCF3
N
O
4 or 5-OH, FLIPR: >150 nM
6-OH, FLIPR IC50 = 4.6 nM
PA IC50 = 5.9 µM
A
N
H
N
H
HO
7
6
5
4
N
N
N
2
1
2
5
FLIPR IC50 = 2.6 nM
PA IC50 = 4.9 µM
Sol: 4 µg/ml
FLIPR IC50 = 0.23 nM
PA IC50 = 1.6 µM
Sol: 30 µg/ml
4
R
N
(TFA salt, pH = 6.5, amorphous)
(TFA salt, pH = 6.5, amorphous)
Compds
R
FLIPRa (nM) PAb IC50
(
l
M) LM (h, r)c % remaining
Cl
O
N
80, 87d
78, 70d
79, 88
59, 51
21, 75
100, 98
48, 57
N
2
H
F
Cl
CN
0.27 0.11
0.37 0.04
0.04 0.02
0.04 0.00
1.6 0.45
S
N
H
N
H
HO
10a
10b
10c
10d
10e
10f
0.92 0.32
0.45(n = 1)
1.1 0.11
N
3e
FLIPR IC50 = 0.12 nM
PA IC50 = 0.13 µM
Sol: 680 µg/ml
SO2NMe2 0.13 0.01
CF3
CO2Me
0.42(n = 1)
0.13 0.04
0.70 0.26
Cl
N
0.14 0.02
0.15 0.01
(MSA salt, pH = 3.9, crystalline)
a
Figure 1. Identification of 3e from compound 1.
FLIPR was tested with 1
l
M 2-methylthio-ADP, and IC50 values are reported as
the mean SD; n P2.
b
PA was tested with 10 lM of ADP, and IC50 values are reported as the
mean SD; n P2 unless otherwise stated. See Ref. 4 and Supporting information for
enhanced solubility compared with 1 (Fig. 1). Compound 3e also
demonstrated robust antithrombotic effect and improved
bleeding risk profile compared to P2Y12 antagonist clopidogrel in
rat efficacy/bleeding models.
detailed assay conditions.
a
c
Liver microsome stability: percentage of compound remaining after incubation
with 0.5
l
M of human (h) or rat (r) liver microsomes.
d
3
lM liver microsomes were used.
Shown in Scheme 1 is a general synthesis of 7-hydroxy
spiropiperidinylindoline analogs listed in Table 1. Methyl or benzyl
protected anilino phenols 4 were converted to their corresponding
hydrazines 5 by treatment with sodium nitrate followed by tin(II)
chloride in the presence of concentrated HCl. The requisite spiro-
piperidinylindolines 7 were prepared via Fischer indoline synthesis
by reacting hydrazines 5 with aldehyde 6 in the presence of acids,
followed by reduction of the imines formed in situ with NaBH4,
NaBH(OAc)3 or NaCNBH3. N-Arylation of compound 7 with 1-bro-
mo-2-nitrobenzene provided compound 8 which were sequen-
tially reduced, first by Zn/NH4Cl, then by Red-Al to anilines 9.
Treatment of 9 with 4-trifluoromethoxyphenylisocyanate afforded
the diaryl ureas, which were deprotected with either BCl3/Bu4NI
(when methyl was the protecting group) or Pd/C/H2 (when benzyl
was the protecting group) to provide the final compounds 10a–e.
Compound 10f (R = CO2Me) can be obtained by hydrolysis of com-
pound 10e (R = CF3) with aqueous NaOH in methanol.6
Table 2
Arylurea modifications
O
Ar
N
H
N
H
HO
N
Cl
N
Compds Ar
FLIPR
(nM)
PA IC50
(
l
M) LM (h, r)
%remaining
OCF3
CF3
10b
3a
0.04 0.02 0.45(n = 1)
0.08 0.03 0.40(n = 1)
79, 88
93, 93
Cl
N
N
3b
3c
0.03 0.01 0.17 0.06
87, 66
OP
S
H
N
CHO
OP
OP
NH2
NHNH2
c, d
e
a, b
0.06 0.02 0.23(n = 1)
100, 89
S
N
+
F
N
R
N
O
R
R
3d
3e
0.17 0.05 0.32 0.09
96, 93
94, 93
S
N
O
Cl
Cl
4
5
6
7
OCF3
0.12 0.05 0.13(n = 1)
O
S
OP
R
OP
N
P = Me
h, i
f, g
N
H
N
NO2
NH2
N
N
N
HO
H
N
P = Bn
j, h
R
The compounds in Table 2 with various heteroaryl ureas were
prepared in a similar way as described in Scheme 2 for the synthe-
sis of compound 3e. 5-Chlorothiazolo[5,4-b]pyridin-2-amine 11
was obtained in one step by treatment of 2,6-dichloropyridin-3-
amine with KSCN. The aniline 9 was first activated with p-nitro-
phenylchloroformate, and then reacted with 11 in the presence
of DMAP to give 12. Demethylation with AlCl3 under microwave
irradiation at 100 °C or with BCl3–Me2S under conventional heat-
ing at 50 °C afforded compound 3e.
N
R
N
O
10a-e
8
9
10e (R = 4-CF3)
P = Me, when R = H, 4-Cl, 4-CF3, 4-SO2NEt2
P = Bn, when 4-F, 4-CN
k
10f (R = 4-CO2Me)
Scheme 1. Synthesis of 7-OH spiroindolines. Reagents and conditions: (a) NaNO2, 6
N HCl, 0 °C; (b) SnCl2, concd HCl, 64–98% for 2 steps; (c) concd H2SO4/EtOH, or
HOAc, or TFA/toluene/CH3CN, or HCl/dioxane, rt ꢀ80 °C; (d) NaBH4 or NaBH(OAc)3,
or NaCNBH3, MeOH, ꢀ20 °C to rt, 24–88% for 2 steps; (e) o-Br-NO2Ph, Pd2(dba)3, rac-
BINAP, Cs2CO3, toluene, 110–120 °C, 65–96%; (f) Zn/NH4Cl/MeOH, 82–98%; (g) Red-
Al, CH2Cl2, 45–90%; (h) OCN-p-OCF3-Ph, CH2Cl2, 90–98%; (i) BCl3, tetrabutylammo-
nium iodide, CH2Cl2, ꢀ50 °C to rt, 23–70%; (j) Pd/C, H2, ammonium acetate, MeOH,
40–99%; (k) 1 N NaOH, MeOH, 70%.
As previously reported, a nitrogen atom ‘walk’ around the A and
B rings of compound 1 was performed in order to increase aqueous
solubility, however, these aza-analogs resulted in weaker P2Y1

1296
Y. T. Jeon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1294–1298
Cl
N
N
Cl
a
N
S
the spiropiperidine indoline template to study the SAR of the ter-
minal urea substitutions.
+
H2N
N
S
K
N
Cl
H2N
The SAR of aryl and heteroaryl ureas were investigated and rep-
resentative compounds are shown in Table 2. A variety of substi-
tuted phenyl and heteroaryl urea analogs were found to be
highly potent in both the FLIPR and the PA assays. Among the ureas
surveyed, the small chlorothiazole (3b) and 5-chloro-2-methyl-
thiazolo[5,4-b]pyridine urea (3e) were the most potent in the PA
assay. These compounds were subsequently differentiated in rat
cassette PK studies with compound 3e showing the best overall
rat PK profile. Compound 3e was further characterized in vitro
and in vivo as a potential candidate for advancement.
11
Cl
O
N
OMe
N
b
c
NH2
S
N
H
N
MeO
H
N
N
H2N
S
Cl
N
Cl
N
11
Cl
N
9
12
Cl
O
N
N
S
N
H
N
HO
H
Compound 3e was selective against P2Y12 and P2Y2 receptors
N
with IC50s greater than 10 lM, and with over a thousand fold selec-
tivity against the P2X1 receptor in the FLIPR assay (IC50 = 180 nM).
It generated less than 1% GSH adduct formation in the reactive
intermediate assay. However, in vitro patch clamp assay indicated
Cl
N
3e
3e inhibited the cardiac hERG current by 96% at 1 lM. To follow up
Scheme 2. Synthesis of compound 3e. Reagents and conditions: (a) concd HCl/
EtOH, 100 °C, 24 h, 46–89%; (b) ClCOO-p-NO2Ph, K2CO3, CH2Cl2, then compound 11,
DMAP, 80 °C, 5 h, 84–96%; (c) AlCl3, CH2Cl2, microwave irradiation, 100 °C, 10 min,
63–97% or BCl3–Me2S, CH2Cl2, 50 °C to rt, 80–95%.
on this observation, an anesthetized rabbit electrophysiology (EP)
study was performed to evaluate effects of 3e on cardiac electro-
physiology in vivo. At increasing IV doses of 3, 10 and 30 mg/kg
which generated plasma concentrations of 24, 62, 158 lM respec-
tively, compound 3e had no effect on PR and QRS intervals on sur-
face electrocardiography (ECG), but with a trend toward QTc
increase at 10 and 30 mg/kg doses. However, the increase was
small and signified minimal proarrhythmic risk. The lack of a sig-
nificant QTc increase in vivo despite potent hERG inhibition in
the patch clamp assay was probably due to the high protein bind-
ing of 3e (rabbit: 99.3%; human: 99.3%; and rat: 99.8% bound) as
hERG activity is free fraction driven.
activity.4 Another approach to improve solubility was to add a hy-
droxyl group onto compound 1. Toward this goal, a hydroxyl group
was attached to various positions of the A ring. Of the four posi-
tions surveyed on the A ring, the 4- and 5-OH analogs showed sig-
nificantly decreased potency while the 6-OH substitution was
about two fold weaker compared with compound 1. The 7-OH ana-
log (compound 2) in contrast resulted in a 10-fold improvement in
potency as measured in the FLIPR assay and was three fold more
potent in the human PA inhibition assay. The FLIPR assay measured
the activation of the calcium signaltransduction pathway by P2Y1
in washed human platelets in a more physiologically relevant set-
ting using the endogenously expressed receptor and a reduced
amount of protein (ꢁ0.1% BSA) and was more sensitive than the
binding assay (Ki). The Ki of compound 2 was determined to be
8 nM, which was similar to compound 1 (Ki = 4 nM), due to the
sensitivity of the binding assay (the assay bottomed out at single
digit nanomolar). Compound 2 also showed modestly enhanced
Since the free base aqueous solubility of compound 3e was poor
(<1
a mono-methanesulfonic acid (MSA) salt was identified as the pre-
ferred salt form with good aqueous solubility (680 g/ml, crystal-
lg/ml, crystalline, at pH 6.5), salt screening was performed and
l
line, at pH 3.9). The MSA salt was used in subsequent PK studies.
The PK profile of compound 3e in different preclinical species is
listed in Table 3. Compound 3e demonstrated low clearance, good
T1/2 and moderate oral bioavailability across species and was cho-
sen to proceed to rat efficacy and bleeding models.
Compound 3e was evaluated in established efficacy models of
FeCl2-induced carotid thrombosis (AT), cuticle bleeding time
(CBT) and mesenteric bleeding time (MBT) conducted in pentobar-
bital-anesthetized rats.9 Vehicle (1 ml/kg of 10% N,N-dimethylacet-
amide/90% dextrose 5% water) or compound 3e (0.3, 1, 3, 10 mg/
kg) was administered as an oral gavage 1 h before induction of
AT or BT (n per group indicated in figures). Blood samples were ob-
tained at the end of the AT protocol to assess platelet aggregation
aqueous solubility (30
lg/ml) compared with compound 1 (4 lg/
ml) both as amorphous form.
When compound 2 was tested for in vitro bioactivation in hu-
man liver microsomes using GSH,7 significant amounts (ca. 38%)
of glutathione (GSH) addition to hydroxylindoline was observed
(M+GSH).8 The reactivity was probably due to the electron-rich
nature of the hydroxyindoline ring system, which could readily
undergo oxidation to form a quinone and subsequently serve as a
Michael accepter for GSH. To decrease the electron density of the
7-hydroxyl indoline ring, various electron-withdrawing groups were
installed at the 4-position (Table 1). Analogs containing 4-fluoro
and 4-chloro substitutions (10a and 10b) showed improved PA
IC50 by approximately two and four fold respectively and main-
tained good metabolic stability when tested for oxidative metabo-
lism in liver microsomes. Both compounds significantly reduced
GSH adduct formation (M + GSH <1%) compared with compound
2 as measured in the assays for reactive intermediate formation.8
Analogs with a 4-CN or 4-CO2Me substitution (10c and 10f),
though still potent, had reduced liver microsomal stability. The
analog with a 4-dimethylsulfonamide substitution (10d) showed
poor liver microsomal stability and also generated 5% of the GSH
adduct. The 4-CF3 analog (10e) was potent and metabolically
stable, but was found to be chemically unstable under basic
conditions. Overall, the 4-chloro substitution demonstrated the
optimal profile with the best potency, liver microsomal stability,
much reduced GSH adduct formation and was chosen to serve as
responses to 10 lM ADP and obtain exposure levels (7 1, 39 14,
81 11, 240 49 nM at 0.3, 1, 3, 10 mg/kg, respectively). Com-
pound 3e produced a dose-dependent reduction in thrombus
weight and maintenance of blood flow (Fig. 2). Dose-dependent
inhibition of ex vivo platelet aggregation and prolongation of BTs
were also observed (Fig. 3). The 10 mg/kg dose produced maxi-
mum efficacy with complete preservation of blood flow in all
Table 3
PK data for compound 3e
Rata
Dogb
Cynob
Dose (mpk) (IV/PO)
Clearance (ml/min/kg)
Vdss (l/kg)
T1/2 (h)
F%
0.4/15
7.3
3.8
8.4
23%
0.2/5
4
2.5
9.4
27%
0.2/5
7.7
3.3
8.2
22%
a
Rat IV/oral PK formulation: DMAC/cremophor/ethanol/water (1/1/1/7).
Dog and cyno IV/oral PK formulation: PEG400/ethanol/water (50/5/45).
b

Y. T. Jeon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1294–1298
1297
7
Vehicle
0.3
6/6
5/5
5/5
3/6
0/6
6
Proportion of
total number
that occluded
5
1
3
Compound3e
(mg/kg, p.o.)
4
3
2
1
0
*
*
*
10
*
p<0.05 compared to
vehicle
*
*
6
5
4
3
2
1
= SEM
120
100
80
60
40
20
0
50% FeCl2
10
0
0
20
30
40
50
60
Time (min)
Figure 2. Effects of compound 3e on arterial thrombosis in rats.
Vehicle
0.3
*
p<0.05 compared
1
3
Compound 3e
(mg/kg, p.0.)
to vehicle
= SEM
10
600
500
400
300
200
100
0
100
80
160
140
120
100
80
*
*
*
*
60
*
*
40
20
0
60
*
40
20
9
9
9
7
7
6
5
5
6
6
10
7
7
5
7
0
Figure 3. Effects of compound 3e on platelet aggregation and bleeding times in rats. Number of rats per group indicated at bottom of each bar.
drug-treated rats, a 66 6% reduction in thrombus weight, and a
74 4% decrease in platelet aggregation. The ID50s were similar
for inhibition of thrombosis (7.3 mg/kg) and ex vivo platelet aggre-
gation (6.6 mg/kg). The IC50 of compound 3e for thrombus weight
issue of reactive intermediate formation. Further SAR studies of
the urea group resulted in discovery of compound 3e as a potent,
orally bioavailable P2Y1 antagonist with a suitable PK profile in dif-
ferent preclinical species for further advancement. Compound 3e
demonstrated a robust antithrombotic effect with an improved
bleeding profile compared to P2Y12 antagonist clopidogrel in rat
efficacy/bleeding models.
reduction was 0.13 lM, which compared to 0.47 lM obtained pre-
viously for oral administration of compound 1 in the same model.
Relative to vehicle, compound 3e at 10 mg/kg prolonged CBT by
2.5 0.4 fold and MBT by 2.2 0.3 fold. In comparison, a 10 mg/
kg oral dose of clopidogrel tested in these same models reduced
thrombus weight by an equivalent 67 5%, but prolonged CBT by
6.2 1.1 fold and MBT by 8.2 0.2 fold.9
In conclusion, SAR studies on the spiropiperidinylindoline
chemotype identified 7-hydroxyl substitution which both im-
proved P2Y1 potency and also increased solubility. Electron-with-
drawing groups were introduced at the 4-position to address the
Acknowledgments
We would like to thank Dr. William R. Ewing for his help during
the preparation of this manuscript and BBRC Chemical Synthesis
Group for large scale synthesis of intermediates 9 and 11. We are
also thankful for Drs. Mary F. Grubb and Jonathan L. Josephs for
reviewing the data for RI formation.

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Y. T. Jeon et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1294–1298
S.; Atherton, J. Bioorg. Med. Chem. Lett. 2008, 18, 3338; (b) Morales-Ramos, A. I.;
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7.
A
fluorescent agent, dansylglutathione(5-(dimethylamino)naphthalene-1-
sulfonyl glutathione) (dGSH) was utilized to give a quantitative estimate of
GSH adduct formed in human liver microsomes. For fluorescent compounds or
for those which form fluorescent metabolites, underivatized glutathione (GSH)
was utilized as an in vitro trapping agent, in which case the reactive metabolites
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8. Several 4-substituted 7-hydroxyl indolines with p-trifluoromethoxyphenyl urea
also formed glutathione adduct (m/z = 511) with the cleaved right-hand
a
portion of the molecule (p-CF3OPhNHCOGSH) in amounts of ꢁ5–10%.
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5. For recent publications on non-nucleotide small molecule P2Y1 antagonists, see:
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