New azole antagonists with high affinity for the P2Y1 receptor

Article abstract of DOI:10.1016/j.bmcl.2013.04.041

Five-membered-ring heterocyclic urea mimics have been found to be potent and selective antagonists of the P2Y₁ receptor. SAR of the various heterocyclic replacements is presented, as well as side-chain SAR of the more potent thiadiazole ring sy

Full text of DOI:10.1016/j.bmcl.2013.04.041

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3519–3522
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New azole antagonists with high affinity for the P2Y1 receptor
,
⇑
Réjean Ruel , Alexandre L’Heureux à, Carl Thibeault §, Jean-Paul Daris, Alain Martel , Laura A. Price,
Qimin Wu, Ji Hua, Ruth R. Wexler, Robert Rehfuss, Patrick Y. S. Lam –
Research and Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2013
Revised 9 April 2013
Accepted 16 April 2013
Available online 25 April 2013
Five-membered-ring heterocyclic urea mimics have been found to be potent and selective antagonists of
the P2Y1 receptor. SAR of the various heterocyclic replacements is presented, as well as side-chain SAR of
the more potent thiadiazole ring system which leads to thiadiazole 4c as a new antiplatelet agent.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Purinergic receptor
P2Y1 antagonists
Platelet aggregation
Five-membered-ring heterocyclic urea
mimics
Thiadiazoles
Adenosine diphosphate receptors have become compelling tar-
gets for antiplatelet therapy.1 These P2 purinergic receptors on the
platelet membrane are classified as either the ligand-gated cation
channels (P2X) or the G-protein coupled receptors (P2Y).2 Two dis-
tinct GPCRs from the latter group, namely the P2Y1 and the P2Y12
receptors, are important regulators of thrombosis and hemostasis.
Mice lacking either receptor have for example been shown to exhi-
bit significant reduction in ADP-induced platelet aggregation.3
Inhibition of the P2Y12 receptor has resulted in several marketed
drugs.4 The thienopyridine analog clopidogrel (PlavixÒ), for exam-
ple, is in world-wide clinical use to treat acute coronary syndrome,
and several new P2Y12 antagonists, such as prasugrel and ticagrelor
were recently approved.4 More recently, synthetic agonists5 and
antagonists6 for the P2Y1 receptor have been identified. Among
them, MRS-2500 completely blocked the ADP-induced platelet
aggregation and reduced arterial thrombosis with a concomitant
moderate bleeding time. Furthermore, the combination of MRS-
2500 with clopidogrel led to increased antithrombotic activity
compared to each alone.6a The inhibition of P2Y1 may therefore
represent a relevant alternative over P2Y12 blockade7 and new
small molecule P2Y1 antagonists need to be developed to evaluate
their relative benefits.
We have previously described potent and selective urea7d
antagonists of the P2Y1 receptor and we now wish to report herein
our efforts aimed at the identification of five-membered ring het-
erocyclic urea isosteres8 with high affinity for the P2Y1 receptor.
Figure 1 shows the structure of biarylurea 1, a lead compound
which showed a high affinity for the P2Y1 receptor (Ki = 6 nM).7d
The compound suffered however from high lipophilicity (HPLC
logP = 5.7), poor aqueous solubility (<1 lg/mL at pH 6.5) and high
human protein binding (99.3%). While the affinity for the receptor
(Ki) was measured in vitro in washed platelets, the functional assay
was run in platelet-rich plasma and, presumably because of the
high protein binding, the compound activity was significantly de-
creased in the ADP-induced aggregation assay (IC50 = 2.1
[ADP] = 2.5 M).
lM at
l
We have explored a number of heterocyclic urea mimics exem-
plified by general structure 3 (Fig. 1). Since the various heterocyclic
rings explored in 3 bear no substitution on the phenyl attached to
the central ring, the analogs thereafter should be compared with
the unsubstituted urea 2a (Ki = 41 nM). Common to all heterocy-
cles reported in Table 1 is the presence of the hydrogen bond donor
of the 3-amino substituent which was shown to be essential for
activity in the biarylurea series.7d
Four- to six-membered ring heterocyclic urea mimics were ex-
plored, and the five-membered ring containing analogs were found
to be most active.9 Table 1 shows the structures of the five-mem-
bered ring urea isosteres studied along with their Ki values. Exam-
ples of novel non-urea five-membered ring heterocycles that retain
high affinity on the P2Y1 receptor include thiazoles,10 oxazoles,10
thiadiazoles, oxadiazoles, isoxazoles and N-methyl pyrazoles.
⇑
Corresponding author. Tel.: +1 514 343 6111x53053; fax: +1 514 343 7780.
E-mail address: rejean.ruel@umontreal.ca (R. Ruel).
Present address: IRIC, University of Montreal, Montreal, QC, Canada.
Present address: OmegaChem, St-Romuald, QC, Canada.
Present address: Boehringer-Ingelheim, Laval, QC, Canada.
Present address: Lam Drug Discovery Consulting LLC, PA, USA.
à
§
–
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.041

3520
R. Ruel et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3519–3522
H
N
H
N
3b was about fourfold less potent on the P2Y1 receptor with a Ki
H
N
value = 59 nM (entry 3).10 It is noteworthy that while two regioiso-
meric thiadiazoles 3c and 3d showed high affinity on the P2Y1
receptor, the regioisomer 3e which bears both nitrogen atoms in
the cyclic guanine arrangement was significantly less potent.
The same trend was also observed with the oxadiazoles 3f and
3g. The oxazoline 3h however showed significantly reduced affin-
ity on the P2Y1 receptor (entry 9). Interestingly, the regioisomeric
isoxazoles 3i–3j were found to have similar affinity for the P2Y1
receptor but only one of the two regioisomeric N-methyl pyrazoles
3k showed high affinity on P2Y1. The N-methyl pyrazole 3k, bear-
ing the methyl group on the nitrogen distal to the NH linker,
showed a similar level of affinity to isoxazoles 3i and 3j. We
hypothesize that steric hindrance caused by the methyl group in
the regioisomer 3l may prevent the essential interaction between
the pyridine 3-amino substituent and the receptor. The presence
of the methyl group in regioisomer 3k is clearly beneficial; in com-
parison, the unsubstituted pyrazole 3m was about 20-fold less ac-
tive (870 nM).
Het.
O
N
Bu-t
O
R
N
Bu-t
O
1; R = OCF3
3
P2Y1Ki = 6 nM
PA IC50 = 2.1 µM (2.5 µM ADP)
2a; R = H
P2Y1Ki = 41 nM
PA % Ctrl = 95%
2b; R = CF3
P2Y1Ki = 15 nM
PA IC50 = 12.6 µM (2.5 µM ADP)
Figure 1. Urea lead compounds and proposed heterocyclic urea mimics.
Table 1
Five-membered ring heterocyclic urea mimics. SAR on azole analogs 3 and urea 2a
H
N
H
N
R
O
O
N
N
O
t-Bu
t-Bu
The more potent thiadiazoles were explored further and Table 2
summarizes the results obtained from the variation of side-chain
‘R’ in 4. Unlike thiazoles, only one substituent can be attached to
the thiadiazole ring but the diaza analogs were attractive targets
nevertheless based on their overall increased polarity. In fact,
based on HPLC logP measurements, thiadiazole 3c (HPLC
logP = 5.01) was one log unit lower than thiazole 3a (HPLC
logP = 5.98). The unsubstituted 1,3,4-thiadiazole 4a showed signif-
icantly decreased affinity against the P2Y1 receptor presumably
due to a lack of hydrophobic interactions (Table 2, entry 1). In fact,
the more lipophilic compounds generally displayed better binding
affinity but the para-substitution of the phenyl ring had minimal
3
2a
a
Entry
1
Compound
R
P2Y1 (Ki, nM)
2a
—
41
H
N
2
3
3a Ref.9
3b Ref.9
15
S
Ph
Ph
Ph
N
N
N
N
H
N
O
59
11
H
N
S
N
4
5
3c
Table 2
Thiadiazole side-chain SAR
H
N
S
N
3d
25
H
N
S
N
Ph
R
H
N
N
N
t-Bu
O
N
S
6
7
3e
3f
1500
70
Ph
Ph
Ph
N
N
N
H
N
O
N
4
H
N
a
Entry
Compound
R
P2Y1 (Ki, nM)
N
O
8
3g
3h
3i
410
2500
19
1
2
4a
4b
H
2730
10
H
N
OCF3
O
9
Ph
Ph
CF3
NMe2
3
4
4c
7
N
N
H
N
4d
27
10
O
N
N
5
6
7
4e
4f
55
250
670
H
N
NMe2
NMe2
11
12
13
3j
23
45
Ph
Ph
O
N
H
N
3k
3l
4g
NMe2
H
N
8
9
4h
4i
18
21
>36000
Ph
Ph
N
N
N
H
N
N
14
3m
870
N
H
10
11
4j
340
275
N
N
a
4k
Ki values are reported as the mean of at least three individual determinations.
Variability around the mean was <50%.
12
13
4l
4m
–COOH
–COOEt
2900
90
a
Among the most active analogs were the thiazole 3a10 and the
thiadiazole 3c (entries 2 and 4). By comparison, the oxazole analog
Ki values are reported as the mean of at least three individual determinations
(refer to assay conditions). Variability around the mean was <50%.

R. Ruel et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3519–3522
3521
S
C
N
S
C
Ph
H
N
Ph
H
N
H
N
NH
N
HN
7
N
a
S
a
O
O
N
b
N
O
SMe
O
N
N
H
S
N
O
b
N
O
N
5
O
N
O
N
O
5
3c
16
3i
Ph
NH2
O
O
H
N
N
S
Cl
+
N
NH
c
C
N
O
NH
Ph
N
N
O
N
N
HN
S
18
c
d
O
S
N
NH
O
N
O
17
N
3j
3d
Scheme 3. Synthesis of isoxazoles. Reagent and conditions: (a) (i) PhCOCH3 (15),
NaH, DMF, (ii) MeI, THF, 45%; (b) NH2OH, EtOH, 85 °C, 6 h, sealed tube, 16%; (c) 14 +
n-BuLi, THF, 0 °C, 20 min, then 18 was added, 45%.
9
5
Scheme 1. Synthesis of 1,3,4- and 1,2,4-thiadiazoles. Reagent and conditions: (a)
PhCONHNH2 (6), CH2Cl2, 23 °C, 3 days; (b) H2SO4, 23 °C, 1 h, 87%, two steps; (c)
PhC(@NH)NH2 (8), DMF, 23 °C, 30 min; and then (d) DEAD (one pot) 23 °C, 30 min,
46% two steps
S
C
Ph
H
N
N
a
b
O
S
H
N
Ph
H
N
N
H
N
N
5
O
N
N
O
N
H
NH2
O
a
O
b
O
Ph
N
O
N
N
O
19
10
12
Ph
+
3f
H
N
Ph
H
N
c
N
N
N
N
H
N
H
N
N
Ph
N
O
N
O
O
H
N
N
O
Ph
S
d
N
O
N
O
3k
3l
Scheme 4. Synthesis of N-methylpyrazoles. Reagent and conditions: (a) PhCOCH3
(15), LiHMDS, THF, 51%; (b) CH3NHNH2 (20), EtOH, AcOH, 85 °C, 67%.
3g
13
Scheme 2. Synthesis of oxadiazoles. Reagent and conditions: (a) CDI,
PhC(O)NHNH2 (11), THF, 20%; (b) PPh3, Cl3CCCl3, Et3N, AcCN, 76%; (c) PhC(O)NCS
(14), THF, 60%; (d) (i) NaH, MeI, THF, (ii) NH2OH, EtOH, 10%.
The compounds disclosed in Tables 1 and 2 were tested against
P2Y12, the other receptor implicated in platelet aggregation, and
they were all found inactive (>40 lM). Finally, Schild plot analysis
effect on Ki as shown by analogs 4b–4d, which are essentially equi-
potent to phenyl analog 3c (Table 1, entry 4). Basic substituents on
the phenyl ring were poorly tolerated resulting in a >10-fold de-
crease in binding affinity; see compounds 4f and 4g compared to
4d and 4e. The 2-pyridine 4i showed similar affinity to the phenyl-
thiadiazole 3c, while the 3- and 4-pyridines 4j and 4k, showed de-
creased affinity for the receptor. Side-chain SAR was fairly
promiscuous and tolerated large groups such as the naphthyl 4h
(Ki = 18 nM) and a large number of other phenyl substituted ana-
logs not shown here. Finally, the carboxylic acid 4l also showed re-
duced binding affinity by comparison to the ethyl ester 4m as was
the case in the thiazole series.9
with 2-MeS ADP agonist resulted in right shifts of the inhibitory
concentration curves indicating that the reported compounds be-
haved as competitive antagonists of the P2Y1 receptor.
The synthesis of these five-membered ring heterocyclic isosteres
has been carried out according to literature precedent.11 For exam-
ple, the thiadiazoles 3c and 3d were prepared from a common iso-
thiocyanate intermediate 5 as described in Scheme 1. Treatment
of 5 with phenylhydrazide (6) provided adduct 7 which gave rise
to the 1,3,4-thiadiazole 3c in 87% overall yield upon treatment with
sulfuric acid. Conversely, the regioisomeric 1,2,4-thiadiazole 3d was
obtained from the treatment of benzamidine (8) with isothiocya-
nate 5 which provided 9 to which diethylazodicarboxylate was di-
rectly added to afford thiadiazole 3d in 46% overall yield.
Scheme 2 describes the synthesis of oxadiazoles 3f and 3g. 3-
Amino pyridine 10 was first treated with CDI and phenylhydrazide
(11) to afford 12 in low yield. Treatment of 12 with triphenylphos-
phine and hexachloroethane12 gave 1,3,4-oxadiazole 3f in 76%
yield. The same intermediate amine 10 was also converted to inter-
mediate 13 upon treatment with benzoyl isothiocyanate (14)
which was converted in low yield to regioisomeric oxadiazole 3g.
In the ADP-induced platelet aggregation assay run in platelet-
rich plasma, the p-trifluoromethyl phenyl analog 4c was the most
potent, with an IC50 of 7.5
the corresponding urea analog 2b had an IC50 of 12.6
[ADP] = 2.5 M) whereas, under the same conditions, the unsubsti-
tuted phenyl analog 3c had an IC50 of 13 M. In the thiadiazole ser-
ies, a much weaker binder, 4-pyridinyl 4k (Ki = 275 nM) translated
to an IC50 of 29 M in the functional assay.
l
M (at [ADP] = 2.5
l
M). For comparison,
lM (at
l
l
l

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R. Ruel et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3519–3522
2. (a) Kahner, B. N. Front. Biosci. 2008, 13, 433; (b) Kahner, B. N.; Shankar, H.;
The synthesis of regioisomeric isoxazoles 3i and 3j is outlined in
Murugappan, S.; Prasad, G. L.; Kunapuli, S. P. J. Thromb. Haemost. 2006, 4, 2317;
(c) Illes, P.; Ribeiro, J. A. Eur. J. Pharmacol. 2004, 483, 5.
Scheme 3. The reaction of acetophenone anion on isothiocyanate 5
afforded, after treatment with methyl iodide, the intermediate Mi-
chael acceptor 16 which was reacted with hydroxylamine to pro-
vide isoxazole 3i. The synthesis of the isomeric isoxazole 3j first
3. (a) Cattaneo, M. In Platelets; Michelson, A. D., Ed.; Academic Press: San Diego,
2006; (b) Fabre, J. E.; Nguyen, M.; Latour, A.; Keifer, J. A.; Audoly, L. P.; Coffman,
T. M.; Koller, B. H. Nat. Med. 1999, 5, 1199; (c) Leon, C.; Hechler, B.; Freund, M.;
Eckly, A.; Vial, C.; Ohlmann, P.; Dierich, A.; LeMeur, M.; Cazenave, J.-P.; Gachet,
C. J. Clin. Invest. 1999, 104, 1731; (d) Foster, C. J.; Prosser, D. M.; Agans, J. M.;
Zhai, Y.; Smith, M. D.; Lachowicz, J. E.; Zhang, F. L.; Gustafson, E.; Monsma, F. J.;
Wiekowski, M. T.; Abbondanzo, S. J.; Cook, D. N.; Bayne, M. L.; Lira, S. A.;
Chintala, M. S. J. Clin. Invest. 2001, 107, 1591.
required the preparation of
a-chloroisoxazole 18 which was
formed via a [3 + 2] cycloaddition of benzonitrile oxide and ethyl-
ene dichloride, followed by elimination with triethylamine. Substi-
tution of the chloride with the lithium salt of amine 17 then
provided isoxazole 3j in 45% yield.
The N-methyl pyrazoles 3k and 3l were obtained as a mixture
according to the sequence described in Scheme 4. The same isocy-
anate intermediate 5 was treated with the lithium anion of aceto-
phenone to give 19 in 51% isolated yield. Upon treatment with
methylhydrazine (20), a (1:1) regioisomeric mixture was obtained
and separated using preparative reverse-phase HPLC. The structure
of each regioisomer was assigned based on NOE experiments.
Using hydrazine instead of methylhydrazine afforded the pyrazole
analog 3m (Table 1, entry 14).
In summary, a number of five-membered ring heteroaryls were
examined as replacements for the urea linkage in 1. Several hetero-
aromatic systems, for example, the thiazoles, oxazoles, thiadiazoles
and isoxazoles (3a–3d, 3f, 3i–3k), inhibited P2Y1 with comparable
potency to the urea. Further exploration of the thiadiazole side
chain led to the identification of compound 4c with a PA IC50 of
4. For a recent review see: Giorgi, M. A.; Arazi, H. C.; Gonzalez, C. D.; Di Girolamo,
G. Expert Opin. Pharmacother. 2011, 12, 1285. and references cited therein.
5. (a) Chhatriwala, M.; Ravi, R. G.; Patel, R. I.; Boyer, J. L.; Jacobson, K. A.; Harden,
T. K. J. Pharmacol. Exp. Ther. 2004, 311, 1038. and references cited therein; (b)
Norambuena, A.; Poblete, M. I.; Donoso, M. V.; Espinoza, C. S.; Gonzalez, A.;
Huidobro-Toro, J. P. Mol. Pharmacol. 2008, 74, 1666.
6. (a) Hechler, B.; Nonne, C.; Roh, E. J.; Cattaneo, M.; Cazenave, J.-P.; Lanza, F.;
Jacobson, K. A.; Gachet, C. J. Pharmacol. Exp. Ther. 2006, 316, 556. and references
cited therein; (b) Pfefferkorn, J. A.; Choi, C.; Winters, T.; Kennedy, R.; Chi, L.;
Perrin, L. A.; Lu, G.; Ping, Y.-W.; McClanahan, T.; Schroeder, R.; Leininger, M. T.;
Geyer, A.; Schefzick, S.; Atherton, J. Bioorg. Med. Chem. Lett. 2008, 18, 3338; (c)
Morales-Ramos, A. I.; Mecom, J. S.; Kiesow, T. J.; Graybill, T. L.; Brown, G. D.;
Aiyar, N. V.; Davenport, E. A.; Kallal, L. A.; Knapp-Reed, B. A.; Li, P.; Londregan,
A. T.; Morrow, D. M.; Senadhi, S.; Thalji, R. K.; Zhao, S.; Burns-Kurtis, C. L.;
Marino, J. P. Bioorg. Med. Chem. Lett. 2008, 18, 6222; (d) Houston, D.; Costanzi,
S.; Jacobson, K. A.; Harden, T. K. Comb. Chem. High Through Screen. 2008, 11,
410; (e) Thalji, R. K.; Aiyar, N.; Davenport, E. A.; Erhardt, J. A.; Kallal, L. A.;
Morrow, D. M.; Senadhi, S.; BurnsKurtis, C. L.; Marino, J. P., Jr. Bioorg. Med. Chem.
Lett. 2010, 20, 4104.
7. (a) Bird, J. E.; Wang, X.; Smith, P. L.; Barbera, F.; Huang, C.; Schumacher, W. A. J.
Thromb. Thrombolysis 2012, 34, 199; (b) Wong, P. C.; Crain, E. J.; Jiang, X.;
Bostwick, J. S.; Thibault, C.; Ruel, R.; Rehfuss, R.; Schumacher, W. A.; Ogletree,
M. L. Circulation 2006, 114. II_248, abstract 1310; (c) Schumacher, W. A.;
Bostwick, J. S.; Ogletree, M. L.; Stewart, A.; Steinbacher, T. E.; Hua, J.; Price, L. A.;
Wong, P. C.; Rhefuss, R. J. Pharmacol. Exp. Ther. 2007, 322, 1; (d) Chao, H.; Turdi,
H.; Herpin, T. F.; Roberge, J. Y.; Liu, Y.; Schnur, D.; Poss, M. A.; Rehfuss, R.; Hua,
J.; Wu, Q.; Price, L. A.; Abell, L. M.; Schumacher, W. A.; Bostwick, J. S.;
Steinbacher, ; Stewart, A. B.; T, E.; Ogletree, M. R.; Huang, C. S.; Chang, M.;
Cacace, A. M.; Arcuri, M. J.; Celani, D.; Wexler, R. R.; Lawrence, R. M. J. Med.
Chem. 2013, 56, 1704.
7.5
lM, comparable to its urea counterpart. Compared to its bind-
ing affinity (Ki = 7 nM), the weaker activity of 4c in the ADP-in-
duced aggregation assay (IC50 = 7.5
l
M
at [ADP] = 2.5 lM)
suggests that the thiadiazole suffers from the same liabilities13 re-
ported for compound 1. The SAR reported herein nevertheless al-
lowed us to identify the most promising azole rings for further
optimization. Our work to enhance functional potency in ADP-in-
duced platelet aggregation will be described in due course.
8. The compounds reported herein were disclosed in: Sutton, J.; Pi, Z.; Ruel, R.;
L’Heureux, A.; Thibeault, C.; Lam, P. Y. S. U.S. 2006/0173002A1.
9.
A
limited number of six-membered ring analogs were moderately active
against P2Y1. These included phenyl and pyridine analogs. Unpublished results.
Acknowledgments
10. Pi, Z.; Sutton, J.; Lloyd, J.; Hua, J.; Price, L.; Wu, Q.; Chang, M.; Zheng, J.; Rehfuss,
R.; Huang, C. S.; Wexler, R. R.; Lam, P. Y. S. Bioorg. Med. Chem. Lett. submitted for
publication.
11. Dhar, T. G. M.; Liu, C.; Pitts, W. J.; Guo, J.; Watterson, S. H.; Gu, H.; Fleener, C. A.;
Rouleau, K.; Sherbina, N. Z.; Barrish, J. C.; Hollenbaugh, D.; Iwanowicz, E. J.
Bioorg. Med. Chem. Lett. 2002, 12, 3125.
12. James, C. A.; Poirier, B.; Grisé, C.; Martel, A.; Ruediger, E. H. Tetrahedron Lett.
2006, 47, 511.
13. For 4c: HPLC logP = 6.00; solubility <1 lg/mL at pH 6.5).
We wish to thank Dr. Christopher Barr for NOE experiments on
N-methyl pyrazoles and Dr. Gerry Everlof is also acknowledged for
HPLC logP measurements. Dr. Jennifer X. Qiao is also gratefully
acknowledged for her help during the preparation of the
manuscript.
References and notes
1. (a) Cattaneo, M. Drug News Perspect. 2006, 19, 253; (b) Murugappan, S.;
Kunapuli, S. P. Front. Biosci. 2006, 11, 1977; (c) Herbert, J. M. Expert Opin.
Investig. Drugs 2004, 13, 457.