Part II: Piperazinyl-glutamate-pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation

Article abstract of DOI:10.1016/j.bmcl.2009.12.110

Efforts to refine the SAR of the piperazinyl-glutamate-pyridines for more potent analogs with improved pharmacokinetic profiles are described. Exploring substituted piperidines and other ring systems at the 4-pyridyl position led to compounds with improved potency and pharmacokinetic properties over candidate I. In particular, compounds 4t and 5t were discovered with a 10-fold improvement over potency and improved pharmacokinetic profiles in both the rat and dog.

Full text of DOI:10.1016/j.bmcl.2009.12.110

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1388–1394
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Part II: Piperazinyl-glutamate-pyridines as potent orally bioavailable 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 , Peter K. Harris ,
Ronald R. Hiebsch b, 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 & Development, 700 Chesterfield Parkway West, Chesterfield, MO 63017, United States
b Department of Biology, Pfizer Global Research & Development, 700 Chesterfield Parkway West, Chesterfield, MO 63017, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Efforts to refine the SAR of the piperazinyl-glutamate-pyridines for more potent analogs with improved
pharmacokinetic profiles are described. Exploring substituted piperidines and other ring systems at the
4-pyridyl position led to compounds with improved potency and pharmacokinetic properties over can-
didate I. In particular, compounds 4t and 5t were discovered with a 10-fold improvement over potency
and improved pharmacokinetic profiles in both the rat and dog.
Received 23 November 2009
Accepted 30 December 2009
Available online 4 January 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
P2Y12 receptor
GPCR antagonist
Antiplatelet
Antithrombotic
Cardiovascular disease
The P2Y12 receptor is a G-protein coupled receptor, primarily a
platelet specific receptor, which is an attractive therapeutic target
for selective modulation of adenosine diphosphate (ADP)-induced
platelet activation.1 PlavixÒ (clopidogrel) is an irreversible P2Y12
antagonist that acts by means of platelet ADP inhibition. Clopido-
grel is an orally prescribed antiplatelet agent used for the treat-
ment of peripheral artery disease and acute coronary syndrome.
Clopidogrel is a pro-drug, the active metabolite of which irrevers-
ibly and selectively inhibits the P2Y12 receptor. Once it is activated,
the drug becomes irreversibly bound to platelets. As a result, clop-
idogrel has a slow onset and slow offset of pharmacological ac-
tion.2 This makes it less effective in acute settings and difficult to
manage if a patient bleeds, experiences a trauma, or requires emer-
gency surgery. It is anticipated that a direct acting P2Y12 inhibitor
will not be associated with such difficulties and will therefore ex-
hibit a significant improvement in efficacy while maintaining a
better safety profile.
selectivity over P2Y1 and P2Y13. Candidate I (Fig. 1) was discovered
from this series and showed good potency in a human platelet rich
plasma assay with a good pharmacokinetic profile demonstrating
efficacy in the rat ferric chloride thrombosis model. Efforts from
our backup program were focused on refining the SAR for more po-
tent analogs with improved pharmacokinetic properties. It was pre-
viously found that substitution at the 4-position of the pyridine ring
greatly influenced the SAR. In particular, substituted piperidines at
the 4-pyridyl position provided compounds with good potency. It
was established that modulation of the potency and pharmacoki-
netic properties could be achieved through various substitutions at
OMe
N
As part of our ongoing effort to identify reversible, potent, orally
bioavailable P2Y12 antagonists for inhibition of platelet aggregation,
we recently described the synthesis and evaluation of piperazinyl-
glutamate-pyridines.3 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
O
O
N
NH
N
I
O
N
C5H11
O
O
OH
* Corresponding author. Tel.: +1 636 247 3494.
E-mail address: john.j.parlow@pfizer.com (J.J. Parlow).
Figure 1. Piperazinyl-glutamate-pyridine candidate I.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.110

J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1388–1394
1389
the 4-position of the piperidine ring. Described in this report are ef-
forts at exploring substituted piperidines and other ring systems at
the 4-pyridyl position to identify compounds with improved po-
tency and pharmacokinetic properties over candidate I.
lated by 20 lM ADP using a turbidity method. The compounds
were assayed with IC50’s reported. Historical analysis of human
PRP potency data for P2Y12 antagonists revealed significant do-
nor-to-donor variability. To reduce this effect, the potency data
for a reference standard (AZD-61404) was determined on each
96-well plate and the data for the test compounds were normal-
ized to the standard from the same plate. Emphasis, in regard to
the SAR, was placed on the PRP potency as this was an indication
of the functional activity taking into account the effect of protein
binding.5
The synthesis of 4-substituted pyridines with cyclic amines of
varying ring sizes is shown in Scheme 1. The 4-chloropyridine inter-
mediates 1a–d were reacted with substituted piperidines, pyrrol-
idines, and azetidines in DMSO at 100 °C to afford the cyclic
amino-4-pyridine analogs. Deprotection of the t-butyl ester using
TFA afforded the desired final products 3–9. Cyclic amines, substi-
tuted with a free carboxylic acid, reacted cleanly with the 4-chloro-
pyridine intermediates to afford the carboxylic acid intermediates 2.
The carboxylic acid intermediates 2 were coupled with amines using
polymer-bound carbodiimide as the coupling agent with hydroxy-
benzotriazole to afford the amides, followed by deprotection of the
t-butyl ester to afford the desired final amide products.
The synthesis of 4-substituted piperazine-4-pyridine analogs 12
is depicted in Scheme 2. The 4-chloropyridine intermediate 1c was
reacted with commercially available mono-substituted piperazines
in DMSO at 100 °C to afford the substituted piperazine analogs.
Deprotection of the t-butyl ester using TFA afforded the desired fi-
nal 4-substituted piperazine-4-pyridine analogs 12. Likewise, Cbz
protected piperazine 10 was reacted with 4-chloropyridine inter-
mediate 1c using the same conditions followed by Cbz deprotec-
tion with hydrogen and palladium on carbon to afford the
unsubstituted piperazine intermediate 11. The unsubstituted
piperazine 11 was reacted with various electrophiles including ali-
phatic halides, acid chlorides, and carbamoyl chlorides, followed
by deprotection of the t-butyl ester to afford the desired final
substituted piperazine-4-pyridine analogs 12.
A representative set of 4-substituted piperidine-4-pyridine ana-
logs is depicted in Table 1. Replacing the methoxy of candidate I
with amines provided compounds with improved binding and
PRP activity (4b–f, 5a–f). However, these amino compounds had
high in vivo rat clearance values (5a, Table 6). Extending the meth-
oxy of compound I by an additional methylene (5g) resulted in
similar potency. Extending the amino compounds by an additional
methylene (5h–j) provided compounds that were slightly more
potent in the PRP assay than their corresponding aminomethyl
analogs. Not unexpectedly, these compounds were highly cleared
as well (Table 6, 5i). Compounds with decreased basicity were
explored by preparing the corresponding amide analogs of select
potent amines. For example, acylating the primary amine of 5a
provided compound 5k, which resulted in a significant loss of
PRP activity. The amide of 4f was prepared such that the carbonyl
was at the 2-position of the pyrrolidine ring system to provide
compound 4l, which resulted in a slight decrease in activity. How-
ever, the presence of a carbonyl on the methylene linking the
piperidine and R group provided compounds with exceptional
potency (4o–t, 5m–t) with the tertiary amides being slightly more
potent in the PRP assay. As has been the trend, the butyl and pentyl
carbamates of the piperazine tail (R1) were superior to the propyl
and hexyl carbamates in terms of both binding and PRP potency
as illustrated by the representative examples 3r and 6r.
The compounds were screened in a human P2Y12 receptor bind-
ing assay with Ki’s determined. These compounds were also tested
as antiplatelet agents by measuring their inhibitory action on the
in vitro aggregation of human platelet rich plasma (PRP) stimu-
COOH
N
O
O
N
2
NH
N
O
N
R1O
O
OtBu
i
iii, ii
O
R4
N
Cl
O
i, ii
O
N
1
O
NH
NH
N
N
O
N
O
N
R1O
R1O
O
Ot
Bu
O
OH
R
R
R
N
R
a R1=Pr
R4 =
b R1=Bu
c R1=Pent
d R1=Hex
N
N
N
3-6
7
8
9
Scheme 1. Synthesis of substituted cyclic amino-4-pyridine analogs 3–9. Reagents and conditions: (i) excess cyclic amine, TEA, DMSO, 100 °C; (ii) 10% TFA/DCM; (iii) 2 equiv
PS-carbodiimide resin, 3 equiv amine, NMM, HOBt, DCM/DMF.

1390
J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1388–1394
H
N
N
O
O
N
11
NH
N
O
N
C5H11
O
O
OtBu
i, iii
iv, ii
R
N
N Cbz
HN
10
Cl
N
O
O
i, ii
O
O
N
1c
N
12
NH
NH
N
N
O
O
N
N
C5H11O
C5H11O
O
O
OtBu
OH
Scheme 2. Synthesis of substituted piperazine-4-pyridine analogs 12. Reagents and conditions: (i) excess mono-substituted piperazine, TEA, DMSO, 100 °C; (ii) 10% TFA/
DCM; (iii) H2, Pd/C, methanol; (iv) 2 equiv electrophile, TEA, DCM.
Table 1
Binding and PRP activity data for a representative set of 4-substituted piperidine-4-pyridine analogs 3–65
R
N
3 R1=Pr
4 R1=Bu
5 R1=Pent
O
6 R1=Hex
O
N
NH
N
O
N
R1O
O
OH
b
Compd
R1
R
Kia (nM)
IC50
3.9
(lM)
Relative potency to AZD6140c
I
Pent
Pent
15
15
3.3
OMe
NH2
5a
0.78
0.62
4b
5b
Bu
Pent
4.0
1.5
0.91
0.71
1.0
0.99
NHMe
NHEt
NMe2
NEt2
4c
5c
Bu
Pent
3.8
1.5
1.3
0.95
1.4
1.2
4d
5d
Bu
Pent
3.2
1.9
0.89
1.0
1.3
1.3
4e
5e
Bu
Pent
7.5
4.0
2.4
2.2
2.1
2.7
4f
5f
Bu
Pent
1.5
1.1
0.80
0.74
1.0
0.93
N
OMe
NH2
5g
5h
Pent
Pent
11
2.7
3.5
2.8
0.86
0.66
NMe2
5i
Pent
12
0.39
0.54
5j
Pent
8.0
0.75
0.59
N

J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1388–1394
1391
Table 1 (continued)
b
Compd
R1
R
Kia (nM)
IC50
6.8
(lM)
Relative potency to AZD6140c
O
5k
4l
Pent
25
3.9
1.5
N
H
O
Bu
0.79
1.1
N
O
5m
5n
Pent
Pent
6.5
7.9
2.4
1.6
0.90
0.86
NH2
O
O
O
O
NHMe
NHEt
NHiPr
NMe2
4o
5o
Bu
Pent
0.68
0.58
1.2
0.65
0.90
1.0
4p
5p
Bu
Pent
4.3
6.0
0.62
0.75
0.82
0.64
4q
5q
Bu
Pent
2.6
0.98
0.63
0.60
0.53
0.72
3r
4r
5r
6r
Pr
Bu
Pent
Hex
12
1.1
4.0
4.8
2.0
0.43
0.52
0.82
2.3
0.71
0.65
1.7
O
O
NEt2
N
5s
Pent
1.2
0.41
0.64
4t
5t
Bu
Pent
2.1
2.8
0.36
0.40
0.54
0.65
O
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
IC50 value of compound/IC50 value of AZD-6140 control from same plate = normalized IC50 ratio.
l
M ADP.
c
Replacing the piperidine with a piperazine ring at the 4-position
majority of these compounds were slightly less potent in the PRP as-
of the pyridine was explored and a representative set of analogs is
shown in Table 2. Only the pentyl carbamates of the piperazine tail
(R1) are listed, as there was no significant difference in PRP activity
between the butyl carbamates and the corresponding pentyl carba-
mates. The unsubstituted piperazine analog 12a had good binding
and PRP potency while substitution of the nitrogen with alkyl
chains of length beyond a methyl tended to decrease the potency
as demonstrated with the propyl analog 12c. Substitution with a
hydroxyethyl 12d had slightly improved potency relative to the
unsubstituted analog 12a, but capping the hydroxy with an alkyl
group 12e,f tended to slightly decrease the PRP potency. Extending
a tertiary amine two to three carbons away from the 4-position of
the piperidine 12h,i increased the PRP potency. The small aceta-
mides, both primary 12k and the dimethyl tertiary 12l, were some
of the most potent 4-substituted piperazines. The amides 12o–q
and the ureas 12r–u were slightly less active than the unsubstitut-
ed analog 12a. Overall, these compounds had good potency
compared to candidate I and relatively flat SAR across a variety
of functionalities. Substituted homopiperazines at the 4-position
of the pyridine were also investigated and similar SAR was ob-
served with no advantage in terms of potency (data not shown).
The 3-substituted piperidine regioisomers were explored and a
representative set of analogs is shown in Table 3. In most cases, the
diastereomeric mixtures were tested and if the mixture warranted,
then the corresponding enantiomers were prepared. Overall, the
say than their corresponding 4-substituted piperidine regioisomers.
Substitution with a hydroxy had similar activity to candidate I with
no significant difference in activity between the two enantiomers
7a and 7b. The methoxy analog 7c was less active than the hydroxy.
One of the most dramatic differences in activity was observed with
7d, theregioisomerof candidate I, whichwasconsiderably less active
in the PRP assay. A trend similar to that seen with the 4-substituted
analogs was observed with the 3-substituted analogs, where replac-
ing the hydroxy or methoxy with an amine tended to increase both
the binding and PRP activity, as illustrated with compounds 7e,f.
Extending the amino group by an additional methylene provided
compounds that were slightly more potent in the PRP assay (7g).
The primary 7j and tertiary 7k amides were considerably less active
in both the binding and PRP assays as compared to the corresponding
4-substituted piperidines 5m and 5r, respectively (Table 1).
Replacing the piperazine ring of candidate I with other ring
sizes was investigated. A representative set of pyrrolidine analogs
is shown in Table 4. The majority of these compounds were slightly
less potent in the PRP assay than their corresponding 4-substituted
piperidine analogs. Several pure diastereomers of the amide ana-
logs were prepared and none showed any significant difference
in activity. The glycine amide ethers 8b and 8c showed exceptional
PRP potency but unfortunately had high in vivo rat clearance val-
ues (Table 6). Reducing the ring size by an additional methylene
provided the 3-substituted azetidine analogs (Table 5). These ana-

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J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1388–1394
Table 2
Table 3
Binding and PRP activity data for a representative set of 4-substituted piperazine-4-
Binding and PRP activity data for a representative set of 3-substituted piperazine-4-
pyridine analogs 125
pyridine analogs 75
R
N
R
N
N
O
O
N
O
O
N
12
7
NH
N
NH
N
O
N
O
N
C5H11
O
C5H11O
O
OH
O
OH
b
Compd
R
Kia (nM)
IC50
(l
M)
Relative potency
to AZD6140c
b
Compd
R
Kia
(nM)
IC50
M)
Relative potency to
AZD6140c
(
l
7ad
7be
7c
OH
18
16
31
2.4
4.4
5.2
3.7
4.9
6.3
12a
12b
12c
12d
12e
12f
12g
12h
12i
H
Me
Pr
6.5
15
29
1.9
2.3
5.0
1.8
1.1
1.5
1.1
0.51
0.52
0.68
2.3
2.5
6.3
1.1
2.1
3.1
1.6
0.70
0.72
0.81
OMe
NH2
7d
7e
22
11
—
OMe
(CH2)2OH
13
5.5
1.8
2.2
(CH2)2OMe
(CH2)2OPr
(CH2)2NH2
(CH2)2NMe2
(CH2)3NMe2
8.3
6.3
4.5
8.5
8.4
8.9
7f
11
3.3
3.3
1.5
N
N
7g
7.3
1.7
2.9
12j
(CH2)2SO2Me
O
O
12k
12l
1.7
4.9
5.9
0.51
0.69
2.9
1.8
1.1
3.2
NH2
O
7h
7i
9.9
10
3.4
3.8
N
H
O
NMe2
O
2.6
N
12m
NEt2
O
O
O
7j
17
26
7.8
7.7
7.3
4.2
NH2
NEt2
12n
5.2
1.6
2.2
N
7k
O
O
12o
12p
22
16
2.3
5.0
2.7
6.6
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
IC50 value of compound/IC50 value of AZD-6140 control from same
l
M ADP.
c
O
plate = normalized IC50 ratio.
d
12q
12r
2.8
6.3
0.71
1.9
2.5
1.8
(S,S) diastereomer.
(S,R) diastereomer.
NMe2
e
O
NH2
O
logs showed similar SAR as the 4-substituted piperidines, but were
slightly less active in the PRP assay.
12s
12t
5.9
12
3.0
3.2
3.6
4.6
The potency optimization described above resulted in many
analogs with improved potency over candidate I. The next goal
was to identify a compound with similar or improved pharmacoki-
netic characteristics. 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 micro-
somal assays. Many compounds were evaluated for their in vivo
pharmacokinetic characteristics in the rat, and a representative
set of pharmacokinetic profiles is shown in Table 6.6
Several compounds from the 4-substituted piperazine-4-pyri-
dine series (12) were evaluated, which included acylated (12o)
and aliphatic (12b) substitution at the 4-nitrogen of the piperazine
ring. All of the compounds evaluated in this series had unaccept-
able clearance values. The most potent analogs from the 3-substi-
NMe2
O
NEt2
O
12u
10
6.1
14
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
l
M ADP.
IC50 value of compound/IC50 value of AZD-6140 control from same
plate = normalized IC50 ratio.
c

J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1388–1394
1393
Table 4
Binding and PRP activity data for a representative set of 3-substituted pyrrolidine-4-pyridine analogs 85
R
N
O
O
N
8
NH
N
O
N
C5H11O
O
OH
R4
H
Kia (nM)
IC50
7.1
(lM)
Relative potency to AZD6140c
b
Compd
R
8ad
OH
13
3.9
O
O
8be
8cf
H
5.0
3.5
0.66
0.33
0.75
O
NMe2
NEt2
H
H
0.38
O
8dd
8ed
21
31
29
11
10
4.1
OH
OMe
H
H
7.3
8ge
NMe2
26
24
3.0
3.6
8ff
3.8
8he
8if
H
1.7
2.6
4.8
6.6
4.6
5.0
O
NEt2
8je
H
1.7
1.4
2.6
2.2
2.8
1.7
O
8kf
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 AZD-6140 control from same plate = normalized IC50 ratio.
Mixture of diastereomers.
(S,S) diastereomer.
(S,R) diastereomer.
c
d
e
f
tuted pyrrolidine-4-pyridine series (8), which included the glycine
amide ethers (8b,c), also had high clearance values. It was previ-
ously found that compounds with 4-piperidine substituted with
a side-chain containing a basic amine usually had high clearance
values, and this same trend was observed for the 3-substituted
piperidines (7g). However, replacing the basic amine group with
a methoxy resulted in compounds with acceptable clearance val-
ues, although a slight loss in potency was observed. These studies
led to the previously reported discovery of candidate I.3 Another
strategy to reduce the basicity was converting the amine into an
amide, in particular the amides with the carbonyl on the methy-
lene linker which provided compounds with exceptional potency.
This also had a profound effect on the clearance values. The pri-
mary amide 5m still had a high clearance value, but the secondary
(5o) and tertiary (4r,5r,5s) amides had acceptable to good clear-
ance values. This led to the discovery of compounds 4t and 5t, both
pyrrolidine amides, with single digit clearance values and good
bioavailability. The dog pharmacokinetic profiles are shown in Ta-
ble 7 and are consistent with the rat in regards to the low clearance
with slightly lower bioavailability.6
were evaluated in the rat safety studies (single dose and 7-day re-
peat dose) without significant toxicological findings. Both com-
pounds had good solubility.7 In vitro receptor binding, signaling,
and functional studies have shown that these pyridine analogs
are high affinity, selective, and competitive antagonists at P2Y12
receptors. Schild analysis indicates that 4t and 5t are competitive
inhibitors of ADP in the platelet aggregation assay. All of the com-
pounds were inactive at 10 lM against the other purinergic recep-
tors tested, including the closest homologue, P2Y13 (48% homology
to P2Y12), and a second platelet purinergic GPCR, P2Y1 (19% homol-
ogy to P2Y12). The potency of compounds 4t and 5t was deter-
mined by repeated measurements of their IC50 in the PRP assay.8
Further evaluation of the potency of compounds 4t and 5t in three
human donors using 20
the 4-well Chronolog PRP aggregometry assay found the average
IC50 to be 0.255 0.156 M and 0.325 0.267 M, respectively.
lM ADP-stimulated PRP aggregation with
l
l
The Kb for compounds 4t and 5t in the human PRP platelet aggre-
gation assays is 49 nM and 68 nM, respectively. Both compounds
4t and 5t showed only a slightly reduced binding affinity when
0.4% human serum albumin was added to the binding assay, result-
ing in Ki’s of 6.3 nM and 6.7 nM, respectively. This is consistent
with their measured plasma protein binding data.9
Further evaluation of compounds 4t and 5t showed they had
acceptable margins versus hERG activity (IC50’s > 90 lM) and a sat-
isfactory CYP inhibition profile. No safety issues were observed as
both were inactive in the Ames and Micronucleus assays and both
In conclusion, we have described our efforts at refining the SAR
for more potent analogs with improved pharmacokinetic properties.

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J. J. Parlow et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1388–1394
Table 5
Table 7
Binding and PRP activity data for a representative set of 3-substituted azetidine-4-
Dog pharmacokinetic profiles of selected P2Y12 antagonistsa
pyridine analogs 95
Compd
CL (mL/min/kg)
Vdss (L/kg)
T1/2
,
(h)
Foral (%)
eff
R
Ib
1
2
3
2
0.14
0.12
0.22
0.18
1.5
0.7
0.8
1.0
93
51
31
36
5rb
4tc
5tc
N
a
Male beagle dogs (n = 2–4 dogs).
Dose: iv infusion at 0.2 mg/kg; po at 0.2 mg/kg. Vehicle: 50% PEG400/40% PBS/
b
O
O
N
10% ethanol.
9
NH
c
Dose: iv infusion at 0.5 mg/kg; po at 1.0 mg/kg. Vehicle: 50% PEG400/40% PBS/
N
10% ethanol.
O
N
C5H11
O
Exploring cyclic amines of varying ring sizes at the 4-position of the
pyridine ring determined that piperidines were the optimal ring size
and that substitution at the 4-position was favored. It was found that
modulation of the potency and pharmacokinetic properties could be
achieved through various substitutions at the 4-position of the
piperidine ring. 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, acceptable
clearance values could be achieved. Non-basic groups, such as 4-pip-
eridinecarboxamides, provided low clearance compounds. In partic-
ular, the 4-pyrrolidineamide piperidine compounds 4t and 5t
exhibited exceptional potency with single-digit nanomolar P2Y12
O
OH
b
Compd
R
Kia
(nM)
IC50
M)
Relative potency to
(
l
AZD6140c
9a
9b
9c
OH
OMe
NMe2
2.5
2.0
5.5
8.6
>10
2.7
9.2
—
—
9d
9e
3.3
7.1
1.4
1.3
—
N
O
1.9
NHMe
O
9f
0.64
1.7
1.9
2.0
1.8
—
binding Ki’s and sub-micromolar PRP IC50’s (vs 20 lM ADP) with
NH2
low clearance in both rat and dog and good bioavailability in both
species. The potency, selectivity, safety, and pharmacokinetic pro-
files for these two compounds support further evaluation.
O
O
9g
9h
—
NHMe
NEt2
2.0
1.5
References and notes
1. Gachet, C. Thromb. Haemost. 2001, 86, 222.
a
Membranes from CHO cells expressing recombinant human P2Y12 receptors
2. (a) Meadows, T. A.; Bhatt, D. L. Circ. Res. 2007, 100, 1261; (b) Savi, P.; Pereillo, J.
M.; Uzabiaga, M. F.; Combalbert, J.; Picard, C.; Maffrand, J. P.; Pascal, M.; Herbert,
J. M. Thromb. Haemost. 2000, 84, 891.
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.
3. (a) Parlow, J. J.; Burney, M. W.; 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; (b) Parlow, J. J.; Burney, M. W.; 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,
6148; (c) Parlow, J. J.; Burney, M. W.; 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. J. Med. Chem., in press.
c
IC50 value of compound/IC50 value of AZD-6140 control from same
plate = normalized IC50 ratio.
Table 6
Rat pharmacokinetic profiles of selected P2Y12 antagonistsa,b
4. 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.
5. These compounds showed significantly reduced binding affinity when 0.4%
human serum albumin was added to the binding assay. In most cases, the
difference in Ki value from the binding assay to the IC50 value of the functional
assay was attributed to protein binding.
Compd
CL (mL/min/kg)
Vdss (L/kg)
T1/2
,
(h)
Foral (%)
eff
I
5a
5i
5m
5o
4r
5r
5s
4t
3
0.3
2.6
5.1
2.2
0.3
0.9
2.5
0.2
0.7
0.5
10
0.7
0.4
0.4
0.6
0.3
0.4
0.6
0.3
1.5
1.1
0.8
0.4
0.4
0.2
0.3
89
0
1
2
—
24
42
6
89
100
—
70
145
44
11
24
13
9
6. The Pfizer Institutional Animal Care and Use Committee reviewed and approved
the animal use in these studies. The animal care and use program is fully
accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care, International.
5
5
5t
7g
8b
8c
12b
12o
141
70
47
41
87
2.4
1.8
0.8
2.1
—
—
—
—
7. Compound 4t solubility: 921
Compound 5t solubility: 299
l
l
M at pH 2.0; 195
M at pH 2.0; 48
l
M at pH 6.5; 969
l
l
M at pH 9.0.
M at pH 9.0.
lM at pH 6.5; 1291
8. The IC50’s for compounds 4t and 5t were determined from geometric mean of
seven IC50’s in the PRP assay.
9. Definitive human PPB data: 4t Fu 3.4% at 0.2
10 M; 5t Fu 5.3% at 0.2 M, 5.7% at 2 M, and 6.1% at 10
concentration-dependent PPB.
l
M, 4.4% at 2
lM, and 3.3% at
a
l
l
l
lM. Neither showed
Male Sprague–Dawley rats (n = 2–4 rats).
Dose: iv infusion at 2 mg/kg; po at 5 mg/kg. Vehicle: 50% PEG400/40% PBS/10%
b
ethanol.