A novel series of piperazinyl-pyridine ureas as antagonists of the purinergic P2Y12 receptor

Article abstract of DOI:10.1016/j.bmcl.2011.03.088

A novel series of P2Y₁₂ antagonists for development of drugs within the antiplatelet area is presented. The synthesis of the piperazinyl-pyridine urea derivatives and their structure-activity relationships (SAR) are described. Several compounds

Full text of DOI:10.1016/j.bmcl.2011.03.088

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2877–2881
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel series of piperazinyl-pyridine ureas as antagonists of the
purinergic P2Y12 receptor
Peter Bach a, , Jonas Boström a, Kay Brickmann b, J. J. J. van Giezen c, Ragnar Hovland b, Annika U. Petersson b,
⇑
Asim Ray b, Fredrik Zetterberg a
a Department of Medicinal Chemistry, AstraZeneca R&D, Pepparedsleden 1, S-43183 Mölndal, Sweden
b Department of Lead Generation, AstraZeneca R&D, Pepparedsleden 1, S-43183 Mölndal, Sweden
c Department of Bioscience, AstraZeneca R&D, Pepparedsleden 1, S-43183 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of P2Y12 antagonists for development of drugs within the antiplatelet area is presented. The
synthesis of the piperazinyl-pyridine urea derivatives and their structure–activity relationships (SAR) are
described. Several compounds showed P2Y12 antagonistic activities in the sub-micromolar range.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 31 January 2011
Revised 21 March 2011
Accepted 22 March 2011
Available online 30 March 2011
Keywords:
P2Y12
Platelet activation
Thrombosis
Acute coronary syndrome
Piperazinyl-pyridines
The 7-TM, G-protein-coupled receptor P2Y12, also known as
platelet ADP receptor, plays an important role in the amplification
phase of platelet aggregation. Upon platelet activation, ADP re-
leased from the dense-granules interacts with the P2Y12 receptor
resulting in down regulation of adenylyl-cyclase, stabilizing the
formed aggregates.1 Inhibiting the P2Y12 receptor has been proven
to reduce the occurrence of secondary myocardial infarction in pa-
tients with acute coronary syndrome.2 The first class of compounds
used in clinical trials was thienopyrimidine pro-drugs, which ac-
tive metabolite binds irreversibly to the receptor.3 Recently, data
from a large phase III trial comparing ticagrelor, the first reversible
direct acting P2Y12 antagonist,4 with clopidogrel was published.5
Ticagrelor was discovered via a medicinal chemistry program
starting from adenosine triphosphate (ATP), the natural antagonist
of the P2Y12 receptor.6 Recently reported series of P2Y12 antago-
nists include piperazinyl-glutamate-pyrimidines,7 thienopyrimi-
dines,8 anthroquinones,9 adenosine analogs,10 and dinucleoside
polyphosphates and nucleotides.11 The current letter describes
the discovery of a novel class of P2Y12 antagonists.
nyl-pyridine scaffold, as exemplified by the ‘hit’ compound 112
(Fig. 1, binding: IC50 = 0.33 M, GTP S: IC50 = 0.68
M13).
l
c
l
The ‘hit’ structure was an attractive starting point for a hit-to-
lead campaign, since SAR investigations around both the pyridine
ring (A) and the phenyl (C) could be made by parallel synthesis
on chemically tractable building blocks, while retaining the piper-
azinyl ring (B) throughout the study. SAR exploration around the
A-ring was facilitated by the commercial availability of 2- and 6-
chloropyridines. Exploration of the C-ring could be made by reac-
tion of the piperazinyl-pyridine scaffold (A–B) with isocyanates.
Compounds 5 and 6 (Scheme 1) were synthesized from com-
mercially available 6-chloropyridines 2 and 3, respectively, by a
2-step procedure from the ethyl ester with optional purification
of the crude intermediate. Hydrolysis of compound 5 gave carbox-
ylic acid 9, transesterification gave methyl ester 10, and N-methyl-
ation gave 17. Compound
6
was subjected to Pd-catalyzed
O
O
N
The novel class of antagonists was identified by a high-through-
put-screening (HTS) of the AstraZeneca compound collection
against the human P2Y12 receptor and is featured by a piperazi-
A
N
F3C
N
H
B
N
N
C
O
1
⇑
Corresponding author. Tel.: +46 31 70 64 795; fax +46 31 63 798.
E-mail address: Peter.Bach@astrazeneca.com (P. Bach).
Figure 1. Hit compound from HTS, composed of a pyridine (A), a piperazinyl (B),
and a phenyl (C).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.088

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P. Bach et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2877–2881
O
O
O
c
a, b
Cl
N
Cl
Cl
Cl
HO
O
O
N
N
N
H
N
H
N
N
N
N
9
2
5
O
O
d
e
O
Cl
N
O
O
O
Cl
N
N
O
O
H
N
N
N
10
O
N
N
N
17
O
O
O
O
N
Br
N
Br
Cl
f, g, h
HO
O
i
N
N
H
N
N
N
H
N
N
3
6
4
O
Scheme 1. Reagents and conditions: (a) piperazine, 1.2 equiv, TEA, 1.2 equiv, EtOH, microwave oven, single node heating, 120 °C, 10 min (61%); (b) PhNCO, 1.2 equiv, MeCN,
rt, 20 h (94%), (c) LiOH, 1 M (aq), 10 equiv, THF, rt, 16 h (91%); (d) H2SO4, MeOH, reflux, 24 h (8%); (e) NaH, 5.0 equiv, MeI, 3.0 equiv, DMF, 0 °C, 5 min (38%); (f) H2SO4, EtOH,
reflux, 5 h (78%); (g) piperazine, 1.2 equiv, TEA, 1.2 equiv, EtOH, microwave oven, single node heating, 120 °C, 10 min (66%); (h) PhNCO, 1.2 equiv, MeCN, rt, 16 h (95%); (i)
KCN, 5.0 equiv, Pd(OAc)2, 0.2 equiv, 1,5-bis(diphenylphosphino)pentane (DPPPE), 0.4 equiv, TMEDA, 4.0 equiv, toluene, 120 °C, 16 h (26%).
Table 1
O
Effects on potency by variation of substituents on pyridine (R1, R2, R4) and by N-
Cl
methylation (R5)
O
a
9
9
N
N
R2
R1
R4
N
H
N
N
11
R5
N
N
O
N
O
O
b
Compd
R1
R2
R4
R5
Binding IC50
M)
GTP
c
S IC50
Cl
N
R
N
(l
(lM)
R'
N
H
1
4
CF3
H
COOEt
COOEt
COOEt
COOEt
COOEt
H
CN
CN
Cl
Br
H
CN
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
0.32
0.92
0.88
1.8
0.68
2.6
1.7
2.2
3.8
>33
>33
8.6
8.5
ND
ND
ND
ND
>33
9.7
N
N
5
H
O
6
H
12: R = R' = H
13: R = H, R' = Et
14: R = R' = Et
7
H
4.1
8
9
CF3
H
>33
>33
9.8
COOH
COOMe
COO-i-Pr
C(O)NH2
C(O)NH(Et)
C(O)N(Et)2
10
11
12
13
14
15
16
17
H
H
H
H
H
H
H
H
15: R = H, R' = SO2Me
2.4
>33
>33
>33
>33
>33
9.6
O
O
N
Cl
O
Br
Cl
S
Br
N
S
c
O
C(O)NHSO2Me
SO2NEt2
Cl
Br
Cl
N
N
H
N
N
COOEt
18
16
O
ND = not determined.
Scheme 2. Reagents and conditions: (a) i-PrOH, 1.1 equiv, DCC, 1.1 equiv, DMAP,
0.1 equiv, DCM, rt, 16 h (33%); (b) 12: NH3, excess, DCC, 2.0 equiv, DMAP, 0.2 equiv,
DCM, rt, 35 min (22%); 13: NH2Et, 1.1 equiv, DCC, 1.1 equiv, DMAP, 0.1 equiv, DCM,
rt, 7 h (8%); 14: SOCl2, 17 equiv, DCM, rt, 4.5 h; concentrate, then add NHEt2, excess,
cyanation14 to give 4. Compounds 7 and 8 (Table 1) were synthe-
sized from commercial starting materials in a manner similar to
compound 5 in yields of 20% and 9% over two steps, respectively.
Carboxylic acid 9 (Scheme 2) was activated by DCC or thionyl chlo-
ride and reacted with the appropriate alcohol, amine or sulfon-
amide to give isopropyl ester 11, amides 12–14, and
acylsulfonamide 15. Sulfonamide 16 was synthesized in three
steps from commercial sulfonyl chloride 18. Piperazinyl-pyridine
scaffolds 23 and 24 to be used for parallel synthesis were made
as outlined in Scheme 3. Commercial b-ketoester 19 was converted
by a two-steps Hantzsch-type reaction15 via the 3-oxybutanone
2016 into pyridone intermediate 21. Subsequent 2-chlorination
and reaction with piperazine presented compound 23. Scale-up
DCM, rt, 1 h (62%,
2 steps); 15: H2NSO2Me, 2.0 equiv, DCC, 2.0 equiv, DMAP,
2.0 equiv, DCM, rt, 4 h (36%); (c) NHEt2, 3.3 equiv, TEA, 1.5 equiv, THF, 0 °C, 2.5 h;
concentrate, then add piperazine, 1.2 equiv, TEA, 1.5 equiv, microwave oven, single
node heating, 120 °C, 10 min; concentrate, then add PhNCO, 1.6 equiv, TEA, excess,
rt, 30 min (13%, 3 steps).
of 24 was made from 2 by reaction with piperazine. Reactions of
compounds 23 and 24 with a range of isocyanates gave ureas in
a typical yield of 60% after purification by HPLC.
Compounds were screened in vitro, for receptor affinity using a
radio-ligand binding assay and for potency in inhibiting receptor
signaling using a GTPcS assay and cell membranes expressing re-

P. Bach et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2877–2881
2879
O
O
O
N
O
b
a
O
O
O
F3C
19
F3C
O
N
H
21
O
N
F3C
20
O
O
O
O
N
N
O
O
N
d
f
c
F3C
O
N
N
H
N
F3C
N
N
NH
F3C
N
R
Cl
23
O
22
25-41 and 59-69
O
O
O
O
O
Cl
Cl
Cl
Cl
N
e
O
f
N
N
N
H
N
N
N
NH
R
24
2
O
42-58 and 70-80
Scheme 3. Synthesis and reactions of compounds 23 and 24, prepared for variation of the R group. Reagents and conditions: (a) HC(OEt)3, 1.5 equiv, Ac2O, 3.0 equiv, 120 °C,
2 h, then 140 °C, 5 h (64% of E/Z mixture); (b) H2NC(O)CH2CN, 1.0 equiv, NaOEt, 1.0 equiv, EtOH, rt, 16 h (83%); (c) (COCl)2, 5.0 equiv, DMF, 0.1 equiv, DCM, reflux, 14 h (91%);
(d) piperazine, 3.2 equiv, TEA, 2.1 equiv, EtOH, microwave oven, single node heating, 170 °C, 20 min (67%); (e) piperazine, 1.2 equiv, TEA, 1.2 equiv, EtOH, microwave oven,
single node heating, 120 °C, 10 min (61%); (f) R-NCO, 1.2 equiv, THF, rt, 14 h (typical yield: 60%).
Table 2
Effects on potency by variation of pyridine substituents and of aryl group (R6)
O
O
N
Cl
N
O
O
F3C
N
N
N
H
H
6
N
N
6
N
N
R
R
O
O
5 and 42-58
1 and 25-41
R6
Compd
Binding IC50
(lM)
GTP
c
S IC50
(l
M)
Compd
Binding IC50
(lM)
GTPcS IC50 (lM)
Phenyl
1
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
0.33
1.8
2.3
1.9
1.7
0.49
0.21
1.8
0.77
0.31
1.1
2.7
6.8
2.3
0.19
NA
0.68
1.3
1.2
1.0
1.5
0.21
0.18
0.95
0.32
0.30
1.06
6.4
5.5
1.1
0.23
NA
0.083
0.96
5
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
0.88
2.7
1.4
1.4
3.2
0.17
0.24
NA
1.7
3.7
2.6
2.0
1.6
0.28
0.42
NA
0.96
0.85
1.1
3.0
NA
2-Cl-phenyl
2-Me-phenyl
2-i-Pr-phenyl
2-OMe-phenyl
3-Cl-phenyl
3-Me-phenyl
3-CN-phenyl
3-OMe-phenyl
4-Cl-phenyl
4-Me-phenyl
4-i-Pr-phenyl
4-CN-phenyl
3,4-Di-F-phenyl
3,4-Di-Cl-phenyl
1-Naphthyl
1.7
0.60
0.60
6.6
NA
5.4
4.6
1.1
0.10
0.38
1.8
1.2
0.11
0.38
5.7
2-Naphthyl
2-Phenylethyl
0.19
3.1
NA = compound not available.
combinant human P2Y12 receptors.17 The designed compounds,
used to obtain an initial SAR around the pyridine ring (A), are listed
in Table 1. Electron withdrawing groups like cyano (4) or chloro (5)
in the pyridine 5-position (R4) increased the potency, compared to
hydrogen (7). The 3-ethoxycarbonyl substituent was important for
potency (1 vs 8). The analogous carboxylic acid 9 was found to be
inactive while the ester homologs 10 and 11 showed reduced po-
tency, compared to ethyl ester 5. Amide analogs such as carboxam-
ides (12–14), acyl sulfonamide (15), and sulfonamide (16) were all
inactive.
The importance of the urea N–H was investigated by making the
N-methyl analog (17) of compound 5. Compound 17 showed a 5-
to 11-fold lower potency compared to 5, see Table 1. Thus the urea
N–H was found to be important for potency and was retained in
the subsequent urea library. The low solubility18 (<0.1
lM) of 1
could be improved by shifting from 2-CF3/5-CN to 2-H/5-Cl pyri-
dines. For example, compounds 4, 5, and 6 (Table 1) had solubili-
ties of 90, 40, and 1.7 lM, respectively.
Given the SAR of the pyridine ring (A), two piperazinyl-pyridine
scaffolds (A–B) were selected for variations of the C-ring.

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P. Bach et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2877–2881
Compound 1 was found to be the most potent of the tested
compounds with A-ring variations (Table 1), however with low
solubility (<0.1 lM) as a drawback.
Compound 5 presented a combination of reasonable solubility
and potency. Thus, the A–B scaffolds of compounds 1 and 5 were
used as starting materials (compounds 23 and 24) and reacted
with isocyanates in a parallel format to form a library of ureas
aimed to explore the SAR of the C-ring.
effect on potency.19 The potency difference between compounds
1 and 5 was also reflected in the two subseries of compounds, that
is, 2-H/5-Cl pyridines (5, 42–58 and 70–80) are generally less po-
tent than 2-CF3/5-CN pyridines (1, 25–41 and 59–69). 1-Naphthyl
(56) and 2-naphthyl (40 and 57) compounds were more potent
than the unsubstituted phenyl compounds (1 and 5) that were
equipotent with the unsubstituted benzyls (59 and 70) and
branched benzyls (68 and 79). Elongation to 2-phenethyl (41 and
58) gave equipotency to the phenyl compounds 1 and 5 in the
functional assay, but lowered the binding affinity 7- to 10-fold.
For both scaffolds in the phenyl series, monosubstitution in the
2-position of the phenylureas with either lipophilic (Cl, Me, i-Pr),
bulky (i-Pr), or electron withdrawing (Cl) or donating (OMe)
groups gave compounds with lower or similar potency to com-
pounds with the unsubstituted phenyl (1 and 5). Monosubstitution
with lipophilic groups (Cl or Me) in the 3-position gave 3–6 times
The BigPicker program19 was used to select 80 isocyanates, that
would provide a urea-library of high diversity with regard to struc-
tural and physicochemical properties. Additional criteria were in-
house availability of the isocyanates as well as chemical feasibility.
Parallel synthesis gave a set of 141 compounds, of which 67 origi-
nated from compound 23 and 74 from compound 24. There were
63 matched-pairs, out of the 141 compounds, providing an excel-
lent opportunity for a detailed analysis. Potency in both binding
and GTP
c
S assays were determined for a total of 125 compounds.
higher GTPcS potency than their unsubstituted phenyl analogs. In
The library compounds showed first-order proportionality be-
tween the IC50 values from the two assays.20 Selected non-benzylic
ureas are included in Table 2 while all synthesized benzylic ureas
are listed in Table 3.
the 4-position of the phenylureas, introduction of lipophilic groups
(Cl or Me) had minor effects on potency, while branching (i-Pr) or
an electron withdrawing group (CN) gave reduced potency. 3,4-
Bis-substitution with electron withdrawing, but less lipophilic
groups (F) gave reduced potency, while substitution with elec-
tron-withdrawing, but more lipophilic groups (Cl), gave increased
or similar potency, which showed that lipophilicity is a required
The library compounds showed a potency range from 0.1 to
>33
lM in both the affinity assay (binding) and functional activity
(GTPcS) assay. This means that C-ring substituents have a major
Table 3
Effects on potency of benzyl ureas by variation of pyridine substituents and of benzyl group (R7, R8, R9)
O
O
N
Cl
N
O
O
R7
R7
F3C
N
N
N
H
H
N
N
N N
O
O
R9
R9
70-80
59-69
R8
R8
R7
R8
R9
Compd
Binding IC50
(
lM)
GTP
c
S IC50
(lM)
Compd
Binding IC50
(lM)
GTPcS IC50 (lM)
H
2-Cl
2-Me
3-F
3-Me
4-F
4-Me
4-OMe
3,4-Di-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
59
60
61
62
63
64
65
66
67
68
69
0.58
0.50
0.51
0.82
0.75
0.47
0.32
0.43
0.48
0.33
0.59
0.27
0.33
0.35
0.24
0.45
0.11
0.18
0.64
0.27
0.19
0.49
70
71
72
73
74
75
76
77
78
79
80
2.4
NA
1.1
2.7
1.7
1.6
0.97
4.6
0.78
0.95
0.61
1.7
NA
0.70
3.0
1.1
2.0
0.46
5.0
0.45
1.4
H
(S)-Me
Me
3-C(CH3)@CH2
0.58
NA = compound not available.
Table 4
Effects on potency of 2-CF3, 5-CN and 2-H, 5-Cl pyridines by variation of the R6 substituent
O
R4
O
R1
N
N
H
N
6
R
N
O
R1
R4
R6
Compd
Binding IC50
1.0
(l
M)
GTP
c
S IC50
(
lM)
R1
R4
Cl
R6
Compd
Binding IC50
1.9
(l
M)
GTP
cS IC50 (lM)
S
CF3
CN
81
0.76
ND
H
H
H
84
2.3
S
O
N
CF3
CF3
CN
CN
82
83
>33
>33
Cl
Cl
85
86
5.6
10
2.1
29
O
ND
ND = not determined.

P. Bach et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2877–2881
2881
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.088.
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9. Baqi, Y.; Atzler, K.; Köse, M.; Glänzel, M.; Müller, C. E. J. Med. Chem. 2009, 52,
3784.
feature in this region. This observation is underlined by the po-
tency of the 1- and 2-naphthyls (40, 56 and 57) mentioned above.
The substitutions made on the aryl group in the benzyl series (Ta-
ble 3) had little or no effect on potency. Examples of variation of
the C-ring beyond phenyls, benzyls, and naphthyls are shown in
Table 4. Phenyl-substituted cyclopropyl 81 retained the potency
levels of 2-phenylethyl 41. The potency of compounds with hetero-
aromatic C-rings spanned from no potency (e.g., isoxazole 82) to
moderate potency (e.g., thiophenes 84 and 85). Non-aromatic sub-
stituents in this position in general gave compounds with no or
low potency, exemplified by i-propyl compound 83 and tetrahy-
dropyranyl compound 86.
These data suggest that 1-naphthyl, 2-naphthyl, or lipophilic 3-
substituted phenyl groups are preferred as C-ring, and this could
be further investigated. The SAR is summarized in Figure 2 below.
In vitro clearance in rat liver microsomes was determined for a
selection of potent compounds with different types of C-rings: 1,
33, 40, and 64. All showed high clearance (i.e., low metabolic sta-
bility) with CLint values of 713, 519, 432 and 720
respectively. In vitro clearance in human liver microsomes for
compounds 40 and 64 were 280 and 565 M/min/mg, respectively.
lM/min/mg,
10. Douglass, J. G.; deCamp, J. B.; Fulcher, E. H.; Jones, W.; Mahanty, S.; Morgan, A.;
Smirnov, D.; Boyer, J. L.; Watson, P. S. Bioorg. Med. Chem. Lett. 2008, 18, 2167.
11. Douglass, J. G.; Patel, R. I.; Yerxa, B. R.; Shaver, S. R.; Watson, P. S.; Bednarski, K.;
Plourde, R.; Redick, C. C.; Brubaker, K.; Jones, A. C.; Boyer, J. L. J. Med. Chem.
2008, 51, 1007.
l
Metabolite studies showed the corresponding carboxylic acid as
the only metabolite of compound 1 in both rat liver microsomes
and human liver microsomes.21
12. Purchased from Maybridge Chemical Company, Cornwall UK.
13. Potencies were determined in a P2Y12 receptor filtration binding assay and a
In summary, we have identified a new series of piperazinyl-pyr-
idine ureas, with several compounds showing sub-micromolar
potencies towards P2Y12. Our SAR investigations showed that the
3-ethoxycarbonyl substituent on the pyridine ring, the urea N–H
of the linker, and the aromatic ring (C-ring) contribute significantly
to potency. Furthermore, we have shown that solubility could be
increased by shifting from 2-CF3/5-CN to 2-H/5-Cl pyridines. Our
efforts to further improve the properties, including solubility and
metabolic stability, of compounds containing the piperazinyl-pyr-
idine scaffold will be reported in due course.
P2Y12 receptor GTP
cS signalling assay. See Supplementary data for details on
the screening assays.
14. Sundmeier, M.; Zapf, A.; Beller, M.; Sans, J. Tetrahedron Lett. 2001, 42, 6707.
15. Mosti, L.; Menozzi, G.; Schenone, P.; Dorigo, P.; Gaion, R. M.; Belluco, P. Farmaco
1992, 47, 427.
16. (a) Jones, R. G. J. Am. Chem. Soc. 1951, 73, 3684; (b) Palanki, M. S. S.; Erdman, P.
E.; Gayo-Fung, L. M.; Shevlin, G. I.; Sullivan, R. W.; Goldman, M. E.; Ransone, L.
J.; Bennett, B. L.; Manning, A. M.; Suto, M. J. J. Med. Chem. 2000, 43, 3995.
17. Screening values are given as single values or as a mean of two measurements
with maximum four times difference between highest and lowest screening
value. In the SAR discussion, a difference in potency between compounds P3
times is considered significant. See Supplementary data for details on the
screening assays.
18. See Supplementary data for screening procedure for solubility.
19. Blomberg, N.; Cosgrove, D. A.; Kenny, P. W.; Kolmodin, K. J. Comput. Aided Mol.
Des. 2009, 23, 513.
Acknowledgments
Assistance from the plate purification group at AstraZeneca
R&D, Mölndal, is gratefully acknowledged. Pia Berntsson, Britt-
Marie Wissing and Linda Nilsson are gratefully thanked for per-
forming the screening assays. Proof-reading by Ruth Bylund and
Thomas Antonsson is highly appreciated.
20. See Supplementary data for graph.
21. Compound (initial concentration 10
protein.
lM) after 60 min at 1 mg compound/mL