Piperazinyl-glutamate-pyrimidines as potent P2Y12 antagonists for inhibition of platelet aggregation

Article abstract of DOI:10.1016/j.bmcl.2009.09.017

Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y₁₂ antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compou

Full text of DOI:10.1016/j.bmcl.2009.09.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6148–6156
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Piperazinyl-glutamate-pyrimidines as potent P2Y12 antagonists for inhibition
of platelet aggregation
b
a
b
b
b
John J. Parlow a, , Mary W. Burney , Brenda L. Case , Thomas J. Girard , Kerri A. Hall , Ronald R. Hiebsch ,
Rita M. Huff b, Rhonda M. Lachance b, Deborah A. Mischke a, Stephen R. Rapp b,
Rhonda S. Woerndle a, Michael D. Ennis a
*
a Department of Medicinal Chemistry, Pfizer Global Research and Development, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
b Department of Biology, Pfizer Global Research and Development, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2009
Revised 2 September 2009
Accepted 4 September 2009
Available online 10 September 2009
Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the
4-position of the pyrimidine leading to highly potent P2Y12 antagonists. In particular, 4-substituted
piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physico-
chemical properties were fine-tuned through modifications at the 4-position of the piperidine ring lead-
ing to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
P2Y12 receptor
P2Y12 antagonist
GPCR antagonist
Antiplatelet
Antithrombotic
Cardiovascular disease
Cardiovascular and cerebrovascular diseases are the first and
third most common causes of morbidity and mortality in the wes-
tern world. One current therapy for these diseases includes anti-
platelet agents. There are many antiplatelet agents based on
different mechanisms of action, such as aspirin, which irreversibly
inhibits cyclooxygenase-1; dipyridamole and cilostazol, which are
phosphodiesterase inhibitors; abciximab, eptifibatide, and tirofi-
ban, which are intravenous, potent, glycoprotein IIb/IIIa antago-
nists; and thienopyridines, which are antagonists of the platelet
adenosine diphosphate (ADP) receptor.1 PlavixÒ (clopidogrel),
belonging to the thienopyridine class, acts by means of platelet
ADP inhibition and has been approved for the reduction of throm-
botic events (stroke, myocardial infarction (MI), and death) for pa-
tients with acute coronary syndrome, a recent history of MI, stroke,
or event-established peripheral vascular disease. P2Y12 is an ADP
G-protein coupled receptor (GPCR) found primarily on platelets.2
ADP is an important platelet agonist that can induce a primary
aggregation response and also contributes to secondary aggrega-
tion via release from platelet dense granules. ADP-induced platelet
aggregation is mediated by a dual receptor system involving
activation of P2Y1 (activation of phospholipase C) and P2Y12
(inhibition of adenylyl cyclase). Experimental studies have demon-
strated that selective blockade of either receptor is sufficient to in-
hibit platelet activation. However, P2Y1 has ubiquitous expression
whereas P2Y12 is a platelet-specific receptor and thus represents a
more attractive therapeutic target for selective attenuation of ADP-
induced platelet activation.
The P2Y12 receptor has been identified as the molecular target
of the commercial antiplatelet agent clopidogrel.3 Clopidogrel is
the only oral prescription antiplatelet that is marketed in most
of the world. However, clopidogrel is a prodrug, the active metab-
olite of which irreversibly and selectively inhibits the P2Y12
receptor, requiring a loading dose and several days of treatment
to achieve its full effect.4 Once it is activated, the drug becomes
irreversibly bound to platelets. As a result, clopidogrel not only
has a slow onset, but also a slow offset of pharmacological action.
This makes it less effective in acute settings and difficult to man-
age if a patient bleeds, experiences a trauma, or requires surgery.
In addition, there are subsets of individuals who either do not
metabolize the prodrug adequately or who are resistant to the ef-
fects of clopidogrel (ꢀ12–25% of patients).5 It is anticipated that a
direct-acting, reversible P2Y12 inhibitor will achieve a similar or
significant improvement in efficacy while exhibiting an improved
safety profile. Accordingly, a need still exists for new drug thera-
pies for the treatment of subjects suffering from, or susceptible
to, platelet aggregation-mediated conditions. A new P2Y12 antag-
onist having one or more improved properties, such as safety
* Corresponding author. Tel.: +1 636 247 3494.
E-mail address: john.j.parlow@pfizer.com (J.J. Parlow).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.017

J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
6149
R4
X3 X5
N
S-glutamic acid, a butyl or pentyl carbamate on the piperazine
nitrogen (R1), and the 6-phenylpyridine were optimal for potency
(Fig. 1).7 Thus, these groups were maintained and efforts were fo-
cused on derivatization at the 4-position of the pyrimidine ring.
Assembly of the pyrimidine ring was achieved by condensation
of benzamidine 1 with diethyl oxalacetate sodium salt 2 to provide
the hydroxypyrimidine 3 with the phenyl ring in place. The
hydroxypyrimidine intermediate 3 was reacted with oxalyl chlo-
ride to afford the chloropyrimidine acid chloride 4. Addition of
the piperazinyl-glutamate amine 5 to the pyrimidine acid chloride
4 afforded the versatile 4-chloropyrimidine intermediate 6. The
4-chloropyrimidine intermediate 6 allows for substitution with
nitrogen, oxygen, and sulfur nucleophiles at the 4-position. The
4-chloro was displaced by heating intermediate 6 with an excess
of amine 7 and triethylamine in DMSO at 100 °C followed by
deprotection of the t-butyl ester with TFA to yield the desired
4-aminopyrimidine analogs 9. Similarly, using an excess of thiol
8 with triethylamine in DMSO at 100 °C followed by deprotection
of the t-butyl ester with TFA afforded the desired 4-thiopyrimidine
analogs 10. Displacement of the 4-chloro with oxygen nucleophiles
could be accomplished, but with limited success in a parallel fash-
ion. As a result, the 4-hydroxypyrimidine intermediate 11 was pre-
pared by reacting the piperazinyl-glutamate amine 5 with the
hydroxypyrimidine acid 3 using EDC as the coupling agent with
hydroxybenzotriazole and NMM as the base. Mitsunobu conditions
or direct alkylation of the hydroxypyrimidine 11 provided 4-oxy-
gen analogs 14 as shown in Scheme 1. Direct alkylation of the
4-hydroxypyrimidine 11 was accomplished using 2 equiv of the
electrophile 13 with cesium carbonate as the base and a catalytic
amount of potassium iodide in DMF followed by deprotection of
the t-butyl ester with TFA to provide the desired 4-ether pyrimi-
dine analogs 14. Alternatively, employing Mitsunobu conditions
with 2 equiv of the alcohol 12 using DEAD and triphenylphosphine
O
O
NH
N
I
X3=X5=CH
O
N
II X3=CH, X5=N
R1O
III X3=N, X5=CH
O
OH
Figure 1. Piperazinyl-glutamate-pyridine/pyrimidine core structures.
profile, efficacy, or physical properties, relative to currently avail-
able P2Y12 antagonists could fulfill this unmet need. Several groups
have research efforts aimed toward discovering ADP receptor
antagonists, including AR-C69931MX (Cangrelor) and AZD-6140,
both of which are in clinical studies.6 We also sought to discover
and develop potent, selective P2Y12 antagonists to address the
unmet medical need for safe and effective oral antiplatelet agents.
We and others have previously reported the preparation of pip-
erazinyl-glutamate-pyridines as potent, orally bioavailable, P2Y12
antagonists for inhibition of platelet aggregation.7,8 These pyridyl
compounds are antagonists for the P2Y12 receptor exhibiting sub-
nanomolar P2Y12 binding Ki’s and sub-micromolar platelet rich
plasma (PRP) IC50’s with excellent selectivity over P2Y1 and
P2Y13. In an effort to increase the potency and influence the phar-
macokinetic properties, other core ring systems were evaluated.
Depicted in Figure 1 is the general piperazinyl-glutamate-pyridine
structure I. Focusing on the pyridine ring, one of the studies was to
add an additional nitrogen into the pyridine ring system, resulting
in pyrimidine ring systems II and III.
Synthetic routes to 4-substituted pyrimidine analogs (II) are
shown in Scheme 1. It was found in the pyridine series that an
R4OH
12
OH
OR4
N
N
viii or ix, vi
+
or
O
O
R4X
13
O
O
N
11
N
(II)
NH
NH
X= Br, Cl, I, etc.
O
N
N
14
O
N
N
R1O
R1O
CO2t-Bu
CO2H
vii
N
O
HO
N
CO2H
ii
Cl
COCl
+
NH
NH2
EtOOC
+
i
N
N
N
R1O
N
NH2
COOEt
NaO
5
1
2
Ph
Ph
O
CO2t-Bu
3
4
iii
R4
Cl
N
N
R'R''NH
O
O
O
O
7
N
N
iv or v, vi
+
or
(II)
NH
NH
6
R4SH
N
N
9 R4 = NR'R"
10 R4 = SR4
O
O
N
N
8
R1O
R1O
CO2H
CO2t-Bu
Scheme 1. Synthesis of 4-substituted pyrimidine analogs II. Reagents and conditions: (i) NaOH, H2O; (ii) Cl(CO)2Cl, DCM, DMF, 0 °C; (iii) 1.2 equiv 5, DIEA, DCM; (iv) excess 7,
TEA, DMSO, 100 °C; (v) excess 8, TEA, DMSO, 100 °C; (vi) 10% TFA/DCM; (vii) 1 equiv 5, EDC, NMM, HOBt, DCM/DMF; (viii) 2 equiv 12, DEAD, PPh3, THF; (ix) 2 equiv 13,
Cs2CO3, KI, DMF, rt–100 °C.

6150
J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
in THF followed by deprotection of the t-butyl ester with TFA pro-
vided the 4-ether pyrimidine analogs 14. Using both procedures al-
lowed for a greater diversity of monomer inputs 12 and 13,
ultimately providing a wide array of 4-ether pyrimidine analogs
14. Comparison of 4-ether analogs, prepared by both Mitsunobu
conditions and direct alkylations of intermediate 11, with analogs
prepared by displacement of the 4-chloro intermediate 6 with
alcohols showed the analogs were identical, demonstrating that
alkylation was occurring on the oxygen and not the nitrogen.
The synthesis of 4-substituted pyrimidine analogs (III) is shown
in Scheme 2. The pyrimidine ring was constructed by reacting 5-
amino-3-phenylisoxazole 15 with ethyl oxalyl chloride 16 to afford
intermediate 17, followed by hydrogenation with platinum dioxide
to provide the 4-hydroxypyrimidine intermediate 18.9 Hydrolysis
of the ethyl ester 18 with sodium hydroxide gave the carboxylic
acid, and subsequent treatment of the acid with oxalyl chloride
generated the acid chloride as well as concomitant conversion of
the 4-hydroxy to 4-chloro. Addition of the amine 5 to the acid chlo-
ride provided the versatile 4-chloropyrimidine intermediate 19.
The 4-chloro was displaced by heating intermediate 19 with an ex-
cess of amine 7 followed by deprotection of the t-butyl ester with
TFA to yield the desired 4-aminopyrimidine analogs 20. Displace-
ment of the 4-chloropyrimidine intermediate 19 with oxygen
nucleophiles was met with limited success. As a result, the 4-chlo-
ropyrimidine intermediate 19 was displaced using sodium hydrox-
ide in THF to provide the 4-hydroxypyrimidine intermediate 21,
which could then be directly alkylated or used under Mitsunobu
conditions to yield the desired 4-ether pyrimidine analogs 22.
Several 100 compounds were prepared by the above syntheses
tration, and Ki’s were determined for those compounds with >50%
inhibition.10 These compounds were also tested as antiplatelet
agents by measuring their inhibitory action on the in vitro aggrega-
tion of human platelet rich plasma (PRP) stimulated by 20
using a turbidimetric method.11 The compounds were initially as-
sayed at 10 M and IC50’s were determined for compounds with
lM ADP
l
>50% inhibition of platelet aggregation. Historical analysis of hu-
man PRP potency data for P2Y12 antagonists revealed variations
due to donor-to-donor variability. To reduce the influence of hu-
man donor-to-donor variability, the potency data for two reference
standards (thienopyrimidine12 and AZD-61406) were determined
on each 96-well plate and the data for the test compounds were
normalized to the standards from that same plate. In regards to
the SAR, emphasis was placed on the PRP potency as this was an
indication of the functional activity taking into account the effect
of protein binding.13
In general, the SAR trends for the pyrimidine series were very
similar to the pyridine series.7 Substituting the 4-position of
either pyrimidine ring (II or III) with an amine or oxygen substi-
tuent was preferred for good P2Y12 binding and PRP potency.
Also, aliphatic straight chain carbamates of four- to five-carbon
length (R1) were preferred, and the S-glutamic acid piece was
optimal for potency. Table 1 shows the data for a representative
set of 4-oxygen analogs for both pyrimidine regioisomers 14 and
22. While the SAR trends for both pyrimidine templates were
consistent, the pyrimidine compounds 14 (II) were typically
more potent than the corresponding regioisomers 22 (III).
Extending a heteroatom such as an oxygen or nitrogen two to
four carbon atoms away from the 4-oxygen atom tended to in-
crease the PRP activity. In particular, the 4-oxypiperidine and
and screened in a P2Y12 receptor binding assay at 5–10
l
M concen-
OH
H
N
O
O
O
O
H2N
N
N
i
ii
+
N
Cl
EtO
O
O
EtO
N
18
O
16
15 Ph
17
O
Ph
OEt
iii, iv, v
R'
R"
Cl
N
N
N
O
O
O
N
19
vi, vii
R'R''NH
+
N
(III)
20
NH
7
N
NH
N
O
N
O
N
R1O
CO2t-Bu
R1O
CO2H
viii
OR4
OH
N
N
O
O
O
R4OH
O
N
N
21
(III)
12
NH
NH
ix or x, vii
N
+
N
or
R4X
13
22
O
O
N
N
R1O
R1O
H
CO2
CO2t-Bu
Scheme 2. Synthesis of 4-substituted pyrimidine analogs III. Reagents and conditions: (i) Pyridine, THF, 0 °C–rt; (ii) PtO2, H2, EtOH, 50 °C; (iii) NaOH, H2O, THF; (iv) Cl(CO)2Cl,
DCM, DMF, 0 °C; (v) 1 equiv 5, DIEA, DCM; (vi) excess 7, TEA, DMSO, rt–100 °C; (vii) 10% TFA/DCM; (viii) excess NaOH, H2O, THF, 80 °C; (ix) 2 equiv 12, DEAD, PPh3, THF; (x)
2 equiv 13, Cs2CO3, KI, DMF, rt–100 °C.

J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
6151
Table 1
Binding and PRP activity data for a representative set of 4-ether pyrimidine analogs 14 (II) and 22 (III)13
X3
X5
R1
R4
Kia (nM)
IC50
(
lM)
Norm ratioc
Norm ratiod
b
Cmpd
14A
14a
22a
CH
CH
N
N
N
CH
Bu
Pent
Pent
12
1.2
1.2
2.6
7.9
>10
1.0
1.2
—
3.7
8.8
—
–O(CH
2
)2OMe
O
14B
CH
N
Bu
55
4.4
1.4
3.8
O
14C
14c
22c
CH
CH
N
N
N
CH
Bu
Pent
Pent
72
27
184
3.0
15
>10
1.1
2.4
—
4.3
17
—
–O(CH
2
)2NEt
2
14d
22d
CH
N
N
CH
Pent
Pent
2.8
11
8.7
7.0
1.4
1.2
8.3
6.6
O
N
O
14E
14F
CH
CH
N
N
Bu
Bu
11
19
1.3
1.8
0.3
0.8
1.7
2.8
O
O
NH
N
14G
14g
22g
CH
CH
N
N
N
CH
Bu
Pent
Pent
16
2.2
31
1.2
1.7
9.4
0.3
0.3
1.7
1.0
1.7
8.5
NH
N
O
14H
14h
22h
CH
CH
N
N
N
CH
Bu
Pent
Pent
15
5.7
12
3.0
3.6
3.8
0.9
0.5
0.8
3.7
3.7
4.4
O
O
NEt2
14I
CH
N
Bu
123
49
—
—
O
Membranes from CHO cells expressing recombinant human P2Y12 receptors incubated with 33P ADP and compound. Ki values are corrected from IC50 using Cheng and
a
Prusoff equation and are the geometric mean of n = 2 or greater.
b
IC50 values are from human PRP incubated with 20 lM ADP.
IC50 value of compound/IC50 value of thienopyrimidine control from same plate = normalized IC50 ratio.
IC50 value of compound/IC50 value of AZD-6140 control from same plate = normalized IC50 ratio.
c
d
4-oxymethyl piperidine analogs (E–H) were the most potent
compounds of the series. In general, the butyl carbamates were
slightly more active in the PRP assay than the corresponding
pentyl carbamates for the 4-oxygen substituted pyrimidine
series.
Table 2 shows the data for a representative set of 4-thiopyrim-
idine analogs 10. While the 4-thio substitution provided com-
pounds with binding activity, no PRP activity was observed with
these analogs, presumably due to high protein binding.13
A large number of 4-aminopyrimidine analogs 9 (II) and 20
(III) was synthesized, and the data for a representative set are
shown in Table 3. The SAR trends across the two pyrimidine tem-
plates 9 and 20 were consistent, and, as was observed with the
corresponding 4-oxygen pyrimidines 14 and 22, the pyrimidine
compounds 9 were typically more potent with respect to both
binding and PRP activity than their corresponding regioisomers
20. In general, amino substitution at the 4-position provided
pyrimidine compounds with even greater potency than the 4-
oxygen analogs. The 4-amine analogs substituted with alkyl
groups were less active than compounds with a heteroatom pres-
ent in the side-chain (data not shown). Compounds 9a–d provide
examples of analogs with an oxygen heteroatom, as a hydroxy or
ether, as part of the amine group with good binding and PRP
activity. Extending an amine group, regardless of substitution,
two to three carbon atoms away from the 4-nitrogen atom tended
to increase PRP activity (9e–i). This trend was most notable with
4-substituted piperazines (9h) and piperidines (9i). As a result, ef-
forts focused on the synthesis of additional analogs with various
substitutions on the 4-nitrogen of the piperazine (23, 24) and var-
ious substitutions on the 4-position of the piperidine (25, 26)
(Fig. 2).
Table 2
Binding and PRP activity data for a representative set of 4-thiopyrimidine analogs
1013
b
Cmpd
R1
R4
Kia (nM)
IC50
(
lM)
10a
10b
10c
Pent
Pent
Pent
StBu
S(CH2)2OH
SCH2CF3
42
4.5
22
>10
>10
>10
10d
10e
Pent
Pent
17
70
>10
>10
S
S
A representative set of 4-substituted piperazines (23, 24) is de-
picted in Table 4. Again, the trend was observed where the pyrim-
idine regioisomer II was the preferred isomer in terms of potency,
as the pyrimidine compounds 23 (II) were more potent than the
corresponding regioisomeric analogs 24 (III). These compounds
had good potency with relatively flat SAR across various function-
a
Membranes from CHO cells expressing recombinant human P2Y12 receptors
incubated with 33P ADP and compound. Ki values are corrected from IC50 using
Cheng and Prusoff equation and are the geometric mean of n = 2 or greater.
b
IC50 values are from human PRP incubated with 20 lM ADP.

6152
J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
Table 3
Binding and PRP activity data for a representative set of 4-aminopyrimidine analogs 9 (II) and 20 (III)13
X3
X5
R1
R4
Kia (nM)
IC50
(lM)
Norm ratioc
Norm ratiod
b
Cmpd
9a
20a
H
N
N
H
Pent
Pent
1.2
5.2
2.6
>10
0.5
—
2.2
—
NH(CH2)2OMe
9b
20b
H
N
N
H
Pent
Pent
1.0
11
6.3
>10
2.0
—
7.9
—
N
O
9c
H
H
N
N
Pent
Pent
2.5
1.2
3.5
4.1
0.8
1.0
4.6
3.4
N
N
OH
OEt
9d
9e
20e
H
N
N
H
Pent
Pent
4.8
38
2.6
>10
0.6
—
1.7
—
NH(CH2)3NMe2
9f
20f
H
N
N
H
Pent
Pent
4.1
19
2.6
>10
0.5
—
2.3
—
N
H
NH
N
9g
20g
H
N
N
H
Pent
Pent
9.4
35
3.7
>10
0.9
—
3.5
—
HN
N
9h
20h
H
N
N
H
Pent
Pent
2.7
11
1.4
8.1
0.3
1.8
1.3
8.1
N
9i
20i
H
N
N
H
Pent
Pent
0.69
8.4
0.72
2.7
0.2
0.6
0.5
3.1
N
NH2
a
Membranes from CHO cells expressing recombinant human P2Y12 receptors incubated with 33P ADP and compound. Ki values are corrected from IC50 using Cheng and
Prusoff equation and are the geometric mean of n = 2 or greater.
b
IC50 values are from human PRP incubated with 20 lM ADP.
IC50 value of compound/IC50 value of thienopyrimidine control from same plate = normalized IC50 ratio.
IC50 value of compound/IC50 value of AZD-6140 control from same plate = normalized IC50 ratio.
c
d
R4
X
with excellent PRP activity (25b–c). Extending the amine group
(primary, secondary, or tertiary) by one or two carbon atoms main-
tained both binding and PRP activity (25d–k). Replacing the amine
with an ether link to the piperidine ring (25m–o) resulted in a
three- to fourfold loss in PRP activity. Extending an oxygen atom,
as a hydroxy or ether, from the 4-position of the piperidine ring
also resulted in a loss of PRP activity (25p–r).
N
X3 X5
N
O
O
Compounds with decreased basicity were explored by prepar-
ing the corresponding amide analogs of select amines. For example,
the amide of compound 25h was prepared such that the carbonyl
was at the 2-position of the pyrrolidine ring system to provide
compound 25s, which resulted in a threefold decrease in PRP activ-
ity. However, the presence of a carbonyl on the 4-piperidine meth-
ylene carbon provided compounds with exceptional potency (25y).
In general, the 4-piperidinecarboxamides with small alkyl amides
were the most potent compounds of the series (25t–y). Extending
the amide group by an additional carbon atom away from the
piperidine ring generally resulted in a loss of activity (25aa–ee).
Unlike the 4-ether pyrimidine series (14, 22), there was no consis-
tent trend in PRP activity between the butyl carbamates and the
corresponding pentyl carbamates for the 4-substituted piperi-
dine-4-pyrimidine series (25, 26).
Having identified many compounds with exceptional potency,
we sought to evaluate the in vivo pharmacokinetic properties of
selected analogs. In general, this class of compounds had good
solubility and was chemically stable. These inhibitors also
showed excellent metabolic stability in both the rat and human
microsomal assays.14 The CYP inhibition profiles were satisfac-
tory15 and none showed hERG activity.16 The in vivo pharmacoki-
NH
N
23 X5,X=N, X3=CH
O
N
24 X3,X=N, X5=CH
25 X5=N, X,X3=CH
26 X3=N, X,X5=CH
R1O
O
OH
Figure 2. 4-Substituted piperazine (23, 24) and piperidine (25, 26) 4-pyrimidine
core structures.
alities at the 4-position, including the unsubstituted nitrogen ana-
log 23a, amide 23b, ureas 23c–d, and other various groups 23e–g.
Table 5 shows the data for a representative set of 4-substituted
piperidine-4-pyrimidines (25, 26). Overall, these compounds were
optimal with respect to both binding and PRP potency. The SAR
trends across the two pyrimidine templates (25 and 26) were con-
sistent, and again the pyrimidine compounds 26 (III) were less po-
tent than the corresponding regioisomer pyrimidine compounds
25 (II). Substitution of the piperidine ring system with alkyl groups
(25a) resulted in loss of PRP activity, while substitution with
amines at the 4-position of the piperidine provided compounds

J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
6153
Table 4
Binding and PRP activity data for a representative set of 4-substituted piperazine-4-pyrimidine analogs 23 (II) and 24 (III)13
X3
X5
R1
R4
Kia (nM)
IC50
(lM)
Norm ratioc
Norm ratiod
b
Cmpd
23a
24a
CH
N
N
CH
Pent
Pent
2.0
22
1.3
5.2
0.3
1.1
1.4
6.0
H
23b
24b
CH
N
N
CH
Pent
Pent
3.7
29
1.5
6.5
0.3
1.8
1.0
8.9
O
O
O
23c
24c
CH
N
N
CH
Pent
Pent
1.2
5.8
0.94
3.6
0.2
1.0
0.8
4.7
NH2
23d
24d
CH
N
N
CH
Pent
Pent
2.9
14
1.6
>10
0.3
—
1.5
—
NMe2
O
23e
24e
CH
N
N
CH
Pent
Pent
2.6
6.8
0.53
3.0
0.1
0.8
0.5
3.5
NMe2
O
23f
CH
N
Pent
2.1
1.1
0.2
1.5
N
23g
24g
CH
N
N
CH
Pent
Pent
2.3
12
2.2
5.1
0.3
1.6
1.5
8.3
(CH
2)2OMe
a
Membranes from CHO cells expressing recombinant human P2Y12 receptors incubated with 33P ADP and compound. Ki values are corrected from IC50 using Cheng and
Prusoff equation and are the geometric mean of n = 2 or greater.
b
IC50 values are from human PRP incubated with 20 lM ADP.
IC50 value of compound/IC50 value of thienopyrimidine control from same plate = normalized IC50 ratio.
IC50 value of compound/IC50 value of AZD-6140 control from same plate = normalized IC50 ratio.
c
d
netic characteristics in rat of a representative set of inhibitors are
profiled in Table 6. Most of the initial compounds evaluated had
high clearance values despite good in vitro metabolic stability. As
expected for high molecular weight carboxylic acids, these
compounds generally had low volumes of distribution. The
4-substituted piperidine-4-pyrimidine series (25) provided com-
pounds with exceptional potency and provided a handle to mod-
ulate the pharmacokinetic properties by varying the substituent
at the 4-position of the piperidine ring. It was found that com-
pounds with the 4-piperidine group containing a basic amine
usually had high clearance values and no bioavailability, such as
compound 25b with a pyrrolidine at the 4-position, or 4-amino-
methyl substituted analogs containing a primary 25d, secondary
25e, or tertiary amine 25h. Despite the inferior potency with
the pyrimidine series (III), several were profiled, including 26d.
This compound showed slight improvement with respect to clear-
ance over the corresponding regioisomer (25d) but also lacked
bioavailability.
absorption, as exemplified by 25p (molecular weight 639, Papp
0.4 ꢁ 10ꢂ6).
In vitro receptor binding, signaling, and functional studies have
shown that these pyrimidine analogs are high-affinity, selective,
and competitive antagonists at P2Y12 receptors. All of the com-
pounds tested showed more than 340-fold selectivity for P2Y12
over the other purinergic receptors tested, including the closest
homologue, P2Y13 (48% homology to P2Y12), and a second platelet
purinergic GPCR, P2Y1 (19% homology to P2Y12).
In summary, we have shown that changing the core ring
system of the piperazinyl-glutamate-pyridines from pyridine
(I) to pyrimidine (II and III) provides highly potent P2Y12 antag-
onists. In general, the pyrimidine template (II) was more potent
than the regioisomer pyrimidine template (III). The 4-nitrogen
substituted pyrimidine series proved more potent than the 4-
oxygen and 4-thio substituted pyrimidines. In particular, the
4-substituted piperidine-4-pyrimidine series (25) provided com-
pounds with exceptional potency with sub-nanomolar P2Y12
Making the amine less basic by converting it into a secondary or
tertiary amide had a profound effect on the clearance values. The
primary amide 25t still had a high clearance value, but the second-
ary and tertiary amides had acceptable clearance values as ob-
served with the N-ethylamide 25v, diethylamide 25w, and the
pyrrolidine amide 25y, all with single-digit clearance values. Com-
pound 25w also displayed moderate bioavailability. Another strat-
egy to reduce the basicity was to replace the basic amine on the
piperidine with an alkoxy group (25m–o). This resulted in com-
pounds with a slight loss in potency, but improved clearance val-
ues (relative to the amines) and good oral bioavailability, as seen
with compound 25p. The corresponding regioisomer pyrimidine
26p had a slightly better clearance value, but poor bioavailability.
Despite high molecular weight and, in numerous cases, poor
Caco-2 permeability values, several compounds had good to
acceptable bioavailability and probably have transporter-mediated
binding Ki’s and sub-micromolar PRP IC50’s (vs 20 lM ADP).
With sufficient levels of potency attained, pharmacokinetic
and physicochemical properties were modulated by altering
the substituent at the 4-position of the piperidine ring (25,
26). Compounds with basic groups as the 4-substituent of the
piperidine ring generally had high in vivo clearance. However,
when the amine was replaced with a non-basic group, accept-
able clearance values could be achieved. Non-basic groups, such
as ethers, improved the clearance and provided compounds
with good bioavailability (25p, F = 61%). Other non-basic groups,
such as 4-piperidinecarboxamides with small alkyl amides, pro-
vided low clearance compounds, with compound 25w having a
clearance of 6 mL/min/kg and bioavailability of 38%. The po-
tency, selectivity, safety, and pharmacokinetic profiles for these
piperazinyl-glutamate-pyrimidines P2Y12 antagonists support
further evaluation.

6154
J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
Table 5
Binding and PRP activity data for a representative set of 4-substituted piperidine-4-pyrimidine analogs 25 (II) and 26 (III)13
X3
X5
R1
R4
Kia (nM)
IC50
(l
M)
Norm ratioc
—
Norm ratiod
—
b
Cmpd
25a
CH
N
Pent
Me
7.5
>10
25B
25b
26b
CH
CH
N
N
N
CH
Bu
Pent
Pent
1.7
1.5
7.4
0.66
0.51
2.1
0.2
0.1
0.4
0.8
0.4
2.4
N
25C
25c
26c
CH
CH
N
N
N
CH
Bu
Pent
Pent
1.3
2.9
5.9
0.88
0.78
3.0
0.2
0.2
0.8
1.1
0.7
3.9
N
O
25d
26d
CH
N
N
CH
Pent
Pent
3.0
9.4
0.49
0.77
0.1
0.2
0.6
1.2
NH2
25e
26e
CH
N
N
CH
Pent
Pent
1.2
11
0.74
1.3
0.1
0.4
0.7
1.0
NHMe
NHEt
25f
26f
CH
N
N
CH
Pent
Pent
2.2
11
0.77
1.2
0.1
0.4
0.7
1.6
25g
25h
CH
CH
N
N
Pent
Pent
0.61
0.40
0.86
0.56
0.2
0.1
0.8
0.7
NEt2
N
25i
26i
CH
N
N
CH
Pent
Pent
2.7
3.0
0.62
1.4
0.1
0.4
0.4
1.9
NH2
25j
CH
N
Pent
2.8
0.40
0.1
0.4
NMe2
25k
26k
CH
N
N
CH
Pent
Pent
2.1
4.2
0.81
1.7
0.1
0.4
0.5
1.7
N
25M
25m
26m
CH
CH
N
N
N
CH
Bu
Pent
Pent
1.4
5.0
7.4
1.8
2.7
>10
0.4
0.5
—
2.7
1.6
—
OMe
25n
26n
CH
N
N
CH
Pent
Pent
5.3
7.1
1.4
5.3
0.2
1.2
0.8
5.3
O(CH
O(CH
2
)
2
OMe
OH
25o
26o
CH
N
N
CH
Pent
Pent
0.71
10
1.3
>10
0.4
—
0.9
—
2)
2
25p
26p
CH
N
N
CH
Pent
Pent
8.4
12
2.0
6.8
0.4
1.4
2.8
9.5
OMe
O
25q
26q
CH
N
N
CH
Pent
Pent
4.3
8.1
2.0
>10
0.3
—
1.2
—
OMe
25R
25r
26r
CH
CH
N
N
N
CH
Bu
Pent
Pent
2.0
10.2
8.4
1.2
6.3
>10
0.5
1.3
—
2.1
5.4
—
OMe
O
25s
CH
N
Pent
0.99
1.6
0.4
2.0
N
O
O
25t
26t
CH
N
N
CH
Pent
Pent
2.0
3.5
0.69
2.3
0.1
0.4
0.7
1.9
NH2
25U
25u
CH
CH
N
N
Bu
Pent
1.3
2.5
1.5
0.37
0.2
0.1
1.1
0.3
NMe2

J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
6155
Table 5 (continued)
X3
X5
R1
R4
Kia (nM)
IC50
(l
M)
Norm ratioc
Norm ratiod
b
Cmpd
O
25V
25v
CH
CH
N
N
Bu
Pent
0.38
0.60
0.49
0.93
0.1
0.2
0.6
1.5
NHEt
25W
25w
26w
CH
CH
N
N
N
CH
Bu
Pent
Pent
0.79
1.5
3.6
0.28
0.40
1.3
0.1
0.1
0.4
0.6
0.6
1.5
O
O
NEt2
N
25x
CH
N
Pent
0.83
0.23
0.04
0.3
25Y
25y
26y
CH
CH
CH
N
N
N
Bu
Pent
Pent
0.70
0.46
3.6
0.39
0.27
1.5
0.08
0.06
0.5
0.4
0.4
1.8
O
N
O
25aa
25bb
CH
CH
N
N
Pent
Pent
0.90
0.67
1.1
1.6
0.2
0.3
1.2
1.6
NHMe
O
O
O
O
NHEt
NEt2
N
25cc
CH
CH
CH
N
N
N
Pent
Pent
Pent
3.1
2.6
1.0
1.5
0.6
0.2
0.3
3.3
1.1
2.0
25dd
0.92
1.6
25ee
N
a
Membranes from CHO cells expressing recombinant human P2Y12 receptors incubated with 33P ADP and compound. Ki values are corrected from IC50 using Cheng and
Prusoff equation and are the geometric mean of n = 2 or greater.
b
IC50 values are from human PRP incubated with 20 lM ADP.
IC50 value of compound/IC50 value of thienopyrimidine control from same plate = normalized IC50 ratio.
IC50 value of compound/IC50 value of AZD-6140 control from same plate = normalized IC50 ratio.
c
d
6. (A) For reviews see: (a) Angiolillo, D. J. Am. J. Cardiovasc. Drugs 2007, 7, 423; (b)
Cattaneo, M. Ex. Rev. Cardiovasc. Ther. 2007, 5, 45; (c) Storey, R. F. Curr. Pharm.
Design 2006, 12, 1255; (d) Boeynaems, J. M.; van Giezen, H.; Savi, P.; Herbert, J.
M. Curr. Opin. Invest. Drugs 2005, 6, 275; (B) For papers see: (a) Bryant, J.; Post, J.
M.; Alexander, S.; Wang, Y. X.; Kent, L.; Schirm, S.; Tseng, J. L.; Subramanyam,
B.; Buckman, B.; Islam, I.; Yuan, S.; Sullivan, M. E.; Snider, M.; Morser, J. Thromb.
Res. 2008, 122, 523; (b) Post, J. M.; Alexander, S.; Wang, Y. X.; Vincelette, J.;
Vergona, R.; Kent, L.; Bryant, J.; Sullivan, M. E.; Dole, W. P.; Morser, J.;
Subramanyam, B. Thromb. Res. 2008, 122, 533; (c) Wang, Y. X.; Vincelette, J.; da
Cunha, V.; Martin-McNulty, B.; Mallari, C.; Fitch, R. M.; Alexander, S.; Islam, I.;
Buckman, B. O.; Yuan, S.; Post, J. M.; Subramanyam, B.; Vergona, R.; Sullivan, M.
E.; Dole, W. P.; Morser, J.; Bryant, J. Thromb. Haemostasis 2007, 97, 847; (d)
Springthorpe, B.; Bailey, A.; Barton, P.; Birkinshaw, T. N.; Bonnert, R. V.; Brown,
R. C.; Chapman, D.; Dixon, J.; Guile, S. D.; Humphries, R. G.; Hunt, S. F.; Ince, F.;
Ingall, A. H.; Kirk, I. P.; Leeson, P. D.; Leff, P.; Lewis, R. J.; Martin, B. P.;
McGinnity, D. F.; Mortimore, M. P.; Paine, S. W.; Pairaudeau, G.; Patel, A.; Rigby,
A. J.; Riley, R. J.; Teobald, B. J.; Tomlinson, W.; Webborn, P. J. H.; Willis, P. A.
Bioorg. Med. Chem. Lett. 2007, 17, 6013; (e) Husted, S.; Emanuelsson, H.;
Heptinstall, S.; Sandset, P. M.; Wickens, M.; Peters, G. Eur. Heart J. 2006, 27,
1038; (f) Van Giezen, J. J. J.; Humphries, R. G. Sem. Thromb. Hemost. 2005, 31,
195; (g) De Marco, A.; de Candia, M.; Carotti, A.; Cellamare, S.; De Candia, E.;
Altomare, C. Eur. J. Pharm. Sci. 2004, 22, 153; (h) Yang, J.; Hua, W.; Wang, F.;
Wang, Z.; Wang, X. Bioorg. Med. Chem. 2004, 12, 6547; (i) Bryant, J. A.; Buckman,
B. O.; Islam, I.; Mohan, R.; Morrissey, M. M.; Wei, G. P.; Xu, W.; Yuang, S. WO
052366, 2004; (j) De Candia, M.; Summo, L.; Carrieri, A.; Altomare, C.;
Nardecchia, A.; Cellamare, S.; Carotti, A. Bioorg. Med. Chem. 2003, 11, 1439;
(k) Yang, S. W.; Buivich, A.; Chan, T. M.; Smith, M.; Lachowicz, J.; Pomponi, S. A.;
Wright, A. E.; Mierzwa, R.; Patel, M.; Gullo, V.; Chu, M. Bioorg. Med. Chem. Lett.
2003, 13, 1791; (l) Xu, B.; Stephens, A.; Kirschenheuter, G.; Greslin, A. F.; Cheng,
X.; Sennelo, J.; Cattaneo, M.; Zighetti, M. L.; Chen, A.; Kim, S. A.; Kim, H. S.;
Bischofberger, N.; Cook, G.; Jacobson, K. A. J. Med. Chem. 2002, 45, 5694; (m)
Bryant, J. A.; Buckman, B. O.; Islam, I.; Mohan, R.; Morrissey, M. M.; Wei, G. P.;
Xu, W.; Yuang, S. WO 098856, 2002
Table 6
Rat pharmacokinetic profiles of selected P2Y12 antagonists
Cmpd
CL (mL/min/kg)
Vdss (L/kg)
T1/2
,
(h)
Foral (%)
eff
25b
25d
25e
25h
25o
25p
25t
25V
25v
25w
25y
26d
26p
75
33
31
98
18
14
68
16
8
6
2
16
6
3.9
4.9
2.0
7.7
0.8
1.1
2.4
0.3
0.2
0.21
0.2
0.28
0.15
0.6
1.7
0.7
0.3
0.5
0.9
0.4
0.1
0.3
0.4
0.8
0.2
0.3
—
1
2
1
—
61
—
7
8
38
13
1
6
Male Sprague-Dawley rats (n = 2–4 rats).
Dose: iv infusion at 2 mg/kg; po at 5 mg/kg (n = 2–4 rats).
References and notes
1. Horiuchi, H. Ann. Med. 2006, 38, 162.
2. Gachet, C. Thromb. Haemostasis 2001, 86, 222.
3. Hollopeter, G.; Jantzen, H. M.; Vincent, D.; Li, G.; England, L.; Ramakrishnan, V.;
Yang, R. B.; Nurden, P.; Nurden, A.; Julius, D.; Conley, P. B. Nature 2001, 409,
202.
4. Savi, P.; Pereillo, J. M.; Uzabiaga, M. F.; Combalbert, J.; Picard, C.; Maffrand, J. P.;
Pascal, M.; Herbert, J. M. Thromb. Haemostasis 2000, 84, 891.
5. Gurbel, P. A.; Bliden, K. P.; Hiatt, B. L.; O’Connor, C. M. Circulation 2003, 107,
2908.
7. Parlow, J. J.; Case, B. L.; Girard, T. J.; Hall, K. A.; Hiebsch, R. R.; Huff, R. M.;
Lachance, R. M.; Mischke, D. A.; Rapp, S. R.; Woerndle, R. S.; Ennis, M. D. Bioorg.
Med. Chem. Lett. 2009, 19, 4657.

6156
J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6148–6156
8. (a) Caroff, E.; Hilpert, K.; Meyer, E. WO 050301, 2008; (b) Caroff, E.; Hilpert, K.;
Meyer, E. WO 044217, 2008; (c) Caroff, E.; Fretz, H.; Hilpert, K.; Houille, O.;
Hubler, F.; Meyer, E. WO 114774, 2006.
9. (a) Honma, Y.; Sekine, Y.; Hashiyama, T.; Takeda, M.; Ono, Y.; Tsuzurahara, K.
Chem. Pharm. Bull. 1982, 30, 4314; (b) Shaw, G.; Sugowdz, G. J. Chem. Soc. 1954,
665.
was analyzed using BioAssay Solver to derive IC50 values. The reference
compounds (2S)-4-({4-[4-(1,10-biphenyl-4-ylcarbonyl)piperazine-1-yl]-6-
ethylthieno[2,3-d]pyrimidin-2-yl}oxy)butane-1,2-diol and AZD-6140 were
run on every plate and the test compound IC50 data was normalized against
these two standards to minimize donor-to-donor variability.
12. Ennis, M. D.; Kortum, S. W.; Rahman, H.; Schweitzer, B. A.; Tenbrink, R. E. WO
2006079916, 2006.
13. In most cases, the difference in Ki value from the binding assay to the IC50 value
of the functional PRP assay was attributed to protein binding. These
compounds showed significantly reduced binding affinity when 0.4% human
serum albumin was added to the binding assay.
14. Compounds were incubated in human liver and rat liver microsomes for
30 min and the percent remaining was measured. These compounds can be
categorized as having good metabolic stability with percent remaining values
of >80%.
10. P2Y12 Radioligand Binding Assay: CHO (chinese hamster ovary) cells
transfected with a synthetic human P2Y12-gene were cultured in Alpha-MEM
containing 10% dialyzed FBS. Washed membranes prepared from near
confluent cells were stored at ꢂ80 °C. Binding reactions were conducted in
polypropylene assay plates in a volume of 150 mL of assay buffer (50 mM Tris,
100 mM NaCl, 1 mM EDTA) including 0.3 mg/well membrane protein, 0.2 nM
33P-MeSADP, and serial dilutions of test compounds, vehicle or 300 nM 2-
MeSADP for the definition of non-specific binding. Dry compounds were
prepared as 10 mM DMSO stocks and were diluted in seven-point, threefold
dilution series in assay buffer with 0.02% BSA. Each concentration was run in
triplicate beginning at 10 mM, final concentration in the assay. Binding
reactions were incubated at room temperature for 1 h and stopped by
dilution and transfer/aspiration of the mixture onto GF/B UniFilter 96 Well
Plates (Perkin–Elmer), and washed 3ꢁ with ice-cold 50 mM Tris, pH 7.4. The
filter plates were counted on a Top Count (Perkin–Elmer) and data were
analyzed using GraphPad Prism using a single site binding equation.
15. Compounds were examined at a single concentration (3 lM) and conducted in
duplicate using a minimum of one positive control inhibitor per CYP-specific
assay which included CYP1A2, CYP2C9, CYP2D6, and CYP3A4. Incubation time
for the assays were 30 min at 37 °C and end-point measurements were used to
assess the degree of inhibition. These compounds can be categorized as weak
inhibitors with percent inhibition values of <25%.
16. Compounds were examined at a single concentration (10 lM) in a 384 well
11. Diluted test compounds (10, 5, 2.5, 1.25, 0.625, 0.313, 0.156
concentrations) were pre-incubated with PRP for 5 min at 37 °C and
aggregation was then initiated by addition of 20 M ADP (final concentration).
l
M
final
fluorescence polarization assay using Cy3B tagged N-desmethyl dofetilide to
competitively bind to HEK-hERG membrane homogenates. Incubation time for
the assay was 120 min and end-point measurements were used to assess the
degree of inhibition. These compounds can be categorized as weak inhibitors
with percent inhibition values of <25%.
l
The progress of the platelet aggregation response to ADP was monitored by light
transmission aggregometry at 626 nm using a SpectraMax Plus plate reader for
15 min at 37 °C. Duplicate concentration–response data (15 min end-point read)