Benzofuran-substituted urea derivatives as novel P2Y1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.05.072

Benzofuran-substituted urea analogs have been identified as novel P2Y₁ receptor antagonists. Structure-activity relationship studies around the urea and the benzofuran moieties resulted in compounds having improved potency. Several analogs were

Full text of DOI:10.1016/j.bmcl.2010.05.072

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4104–4107
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzofuran-substituted urea derivatives as novel P2Y1 receptor antagonists
b
c
d
c
Reema K. Thalji a, , Nambi Aiyar , Elizabeth A. Davenport , Joseph A. Erhardt , Lorena A. Kallal ,
*
Dwight M. Morrow e, Shobha Senadhi b, Cynthia L. Burns-Kurtis b, Joseph P. Marino Jr. f
a Department of Chemistry, Infectious Diseases Centre for Excellence in Drug Discovery, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
b Department of Biology, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
c Department of Biological Reagents and Assay Development, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
d Cancer Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
e Stem Cell Discovery Performance Unit, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
f Department of Chemistry, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzofuran-substituted urea analogs have been identified as novel P2Y1 receptor antagonists. Structure–
activity relationship studies around the urea and the benzofuran moieties resulted in compounds having
improved potency. Several analogs were shown to inhibit ADP-mediated platelet activation.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 12 April 2010
Revised 14 May 2010
Accepted 18 May 2010
Available online 9 June 2010
Keywords:
P2Y1
Purinergic
Thrombosis
Antiplatelet
Urea
Purinergic receptor antagonists have recently demonstrated ther-
apeutic potential for the treatment of a variety of diseases, including
thrombosis, diabetes, cystic fibrosis, and cancer.1 The P2Y1 and P2Y12
receptors play key roles in platelet aggregation and thrombus forma-
tion.2 The nucleotide adenosine diphosphate (ADP) acts as the
endogenous activator for both of these receptors on the platelet sur-
face. The binding of ADP to the P2Y1 receptor results in an agonist
response followed by a transitory increase in intracellular Ca+, and fi-
nally platelet shape change and reversible aggregation. Activation of
the P2Y12 receptor reduces cyclic adenosine monophosphate (cAMP)
levels causing an amplification of the platelet response and stabiliza-
tion of the resulting aggregates.
Inhibition of the P2Y12 receptor is a well-established strategy
for anti-thrombotic therapy; PlavixÒ (clopidogrel), an irreversible
P2Y12 receptor antagonist, is the number one selling drug on the
market for antiplatelet therapy.3 However, P2Y1 is a relatively
new target being explored for anti-thrombotic therapies. Several
studies have shown that exclusive inhibition of the P2Y1 receptor
can effectively prevent platelet aggregation and thrombus forma-
tion both in vitro4 and in vivo.5 Therefore, P2Y1 receptor antago-
nists offer great potential as novel anti-thrombotic agents.
A number of potent nucleotide-based inhibitors of P2Y1 such as
16 (Fig. 1) have been reported, and a few non-nucleotide inhibitors
have been disclosed.7 In an effort to identify new non-nucleotide-
based P2Y1 antagonists, we conducted a high throughput screen of
the GSK compound collection using a FLIPR-based cellular assay.8
This afforded tetrahydro-quinolinamine 2, (the lead optimization
of which we have discussed in a previous publication7a) and pyri-
dyl-urea analog 3 (Fig. 1) as micromolar hits. Interestingly, com-
pound 3 is structurally related to previously reported aryl-urea
antagonists of the P2Y1 receptor such as 4.7b Based on the promising
FLIPR activity observed for 3, we initiated lead optimization efforts
to identify novel urea-based inhibitors with improved potency. To-
wards this end, we designed benzofuran analogs 5 (Fig. 1) in order
to probe the effect of constraining the aryl-ether group of 3. Herein,
we present a structure–activity relationship (SAR) study of these no-
vel P2Y1 receptor antagonists.
The benzofuran analogs 5a–m were prepared according to the
sequence outlined in Scheme 1.9 Selective ortho-iodination of
2-nitrophenol (6) was achieved using thallium acetate.10 Sona-
gashira coupling of 7 with the appropriate alkyne11 proceeded
smoothly to form intermediate 8. In situ cyclization afforded the
desired nitro-benzofuran intermediate 9. Reduction of the nitro-
group using SnCl2–H2O, followed by addition of the appropriate
isocyanate afforded benzofuran analogs 5a–m.
All compounds were initially evaluated in the P2Y1 FLIPR assay.8
Hits were then followed up in a competitive binding assay employ-
ing radiolabeled ADP ([33P]-2-SMe-ADP) to confirm P2Y1 specific
activity (see Tables 1 and 2).8 SAR studies around R1 when
* Corresponding author.
E-mail address: reema.k.thalji@gsk.com (R.K. Thalji).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.072

R. K. Thalji et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4104–4107
4105
Ph
HN
O
P
Me
Et
HO
HO
O
N
H
N
N
N
O
Me
O
P
N
N
O
HO
OH
O2N
I
2: HTS lead
1
FLIPR IC50 = 1.6 µM
H
N
H
N
H
N
H
N
H
N
H
N
2
R
O
O
O
N
O
N
Cl
O
OCF3
O
Ph
N
1
Me
R
4
5
3: HTS lead
FLIPR IC50 = 0.79 µM
Figure 1. P2Y1 receptor antagonists.
NO2
NO2
1
OH
R
NO2
H
a
OH
I
b
OH
1
R
7
6
8
NO2
NH2
H
N
H
N
2
R
d
c
O
O
O
O
1
1
R
R
1
R
9
10
5a-m
Scheme 1. Reagents and conditions: (a) Tl(OAc)2, I2, CH2Cl2, 25 °C; (b) (PPh3)2PdCl2, CuI, piperidine, DMF, 60 °C; (c) SnCl2–2H2O, 25 °C; (d) R2NCO, CH2Cl2, 25 °C.
R2 = 4-OCF3-Ph indicate that ortho-substitution of the phenyl ring
is essential for activity (Table 1). The 3-CF3 analog 5a was inactive
while the 2-CF3 analog 5b displayed weak activity. Larger groups at
the ortho-position seem to improve activity, and this trend is most
evident in the binding assay. The 2-i-Pr (5c) and 2-t-Bu (5d) deriv-
atives showed roughly a fivefold improvement in binding activity
over the 2-CF3 analog 5b. The 2-Cl derivative 5e is inactive, most
likely due to the relatively small size of the chlorine atom. A similar
enhancement of P2Y1 activity with ortho-substitution was also re-
ported for a structurally similar aryl-ether series (4).7b In the ben-
zofuran series, electronics also seem to play an important role, as
the bulky but more electron-donating 2-i-PrO analog 5f is inactive.
Due to the potency and ease of synthesis of the 2-i-Pr analog 5c,
the 2-i-Pr group at the R1 position was maintained in conducting
SAR studies around R2 (Table 2). Several new analogs (5g–k) were
found to be more potent than 5c in the cellular assay. Polar substit-
uents such as NMe2 (5l) result in significant reduction of activity,
while non-polar groups such as n-pentyl (5h) and t-Bu (5i) im-
prove both the cellular and binding activity relative to 5c. The
observation that polarity can disrupt activity is also seen in com-
paring the more polar BuO analog 5j to the analogous n-pentyl
derivative 5h.
The effect of several benzofuran analogs on ADP-mediated
platelet alpha-granule release was measured by detecting surface
expression of P-selectin (CD62P) using flow cytometry.8 The results
are reported in Table 3. Analogs 5c, 5g, and 5i all show significant
inhibition of P-selectin expression demonstrating that this class of
P2Y1 inhibitors is functionally active.
In conclusion, we have identified benzofuran-ureas (5) as a no-
vel class of P2Y1 receptor antagonists. SAR studies showed that a

4106
R. K. Thalji et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4104–4107
3. For PlavixÒ, see: (a) Plosker, G. L.; Lyseng-Williamson, K. A. Drugs 2007, 67, 613;
Table 1
For other P2Y12 inhibitors, see: (b) Duggan, S. T.; Keating, G. M. Drugs 2009, 69,
1707; (c) Husted, S. Eur. Heart J. Suppl. 2007, 9, D20.
4. (a) Boyer, J. L.; Adams, M.; Ravi, R. G.; Jacobson, K. A.; Harden, T. K. Br. J.
Pharmacol. 2002, 135, 2004; (b) Waldo, G. L.; Corbitt, J.; Boyer, J. L.; Ravi, G.;
Kim, H. S.; Ji, X. D. Mol. Pharmacol. 2002, 62, 1249.
Cellular and binding assay results for compounds 5a–f
H
N
H
N
O
OCF3
O
5. León, C.; Freund, M.; Ravanat, C.; Baurand, A.; Cazenave, J. P.; Gachet, C.
Circulation 2001, 103, 718.
1
6. (a) Kim, H. S.; Ohno, M.; Xu, B.; Kim, H. O.; Choi, Y.; Ji, X. D.; Maddileti, S.;
Marquez, V. E.; Harden, T. K.; Jacobsen, K. A. J. Med. Chem. 2003, 46, 4974; (b)
Jacobson, K. A.; De Castro, S. World Patent WO2009152431A1, 2009.; (c)
Houston, D.; Costanzi, S.; Jacobson, K. A.; Harden, T. K. Comb. Chem. High
Throughput Screening 2008, 11, 410; (d) Raboisson, P.; Baurand, A.; Cazenave, J.-
P.; Gachet, C.; Retat, M.; Spiess, B.; Bourguignon, J.-J. J. Med. Chem. 2002, 45,
962; (e) Nandanan, E.; Camaioni, E.; Jang, S.-Y.; Kim, Y.-C.; Cristalli, G.;
Herdewijn, P.; Secrist, J. A.; Tiwari, K. N.; Mohanram, A.; Harden, T. K.; Boyer, J.
L.; Jacobsen, K. A. J. Med. Chem. 1999, 42, 1625; (f) Camaioni, E.; Boyer, J. L.;
Mohanram, A.; Harden, T. K.; Jacobsen, K. A. J. Med. Chem. 1998, 41, 183.
7. For tetrahydro-quinolinamine inhibitors, see: (a) Morales-Ramos, A. I.; Mecom,
J. S.; 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., Jr. Bioorg. Med. Chem. Lett. 2008, 18, 6222; For aryl-urea inhibitors, see: (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) Sutton, J.
C.; Pi, Z.; Ruel, R.; L’Heureux, A.; Thibeault, C.; Lam, P. Y. S. World Patent
WO2006078621A2, 2006.; (d) Chao, H. J.; Tuerdi, H.; Herpin, T.; Roberge, J. Y.;
Liu, Y.; Lawrence, R. M.; Rehfuss, R. P.; Clark, C. G.; Qiao, J. X.; Gungor, T.; Lam, P.
Y. S.; Wang, T. C.; Ruel, R.; L’Heureux, A. L.; Thibeault, C.; Bouthillier, G.; Schnur,
D. M. World Patent WO2005113511A1, 2005.; (e) Tuerdi, H.; Chao, H. J.; Qiao, J.
X.; Wang, T. C.; Gungor, T. U.S. Patent 2005261244A1, 2005.; (f) Herpin, T. F.;
Morton, G. C.; Rehfuss, R. P.; Lawrence, R. M.; Poss, M. A.; Roberge, J. Y.; Gungor,
T. World Patent WO2005070920A1, 2005.
R
a
Compound
R1
FLIPR IC50
(l
M)
Kia
(lM)
5a
5b
5c
5d
5e
5f
3-CF3
2-CF3
2-i-Pr
2-t-Bu
2-Cl
>25
6.3
4.0
4.0
>25
>25
nd
2.5
0.6
0.4
nd
nd
2-i-PrO
a
Values are means of at least three determinations with a standard devia-
tion 6 0.3 log units (nd, not determined).
Table 2
Cellular and binding assay results for compounds 5g–m
H
N
H
N
2
R
O
O
8. Assay protocols: P2Y1 FLIPR assay: HEK-293 MSRII cells endogenously
expressing P2Y1 were maintained in DMEM/F12 medium supplemented with
10% fetal bovine serum, 1% L-glutamine, 15 mM Hepes, and 1% penicillin–
streptomycin at 37 °C in a humidified 5% CO2 incubator. Cells were seeded at a
density of 20,000 cells/well (384-well format) and cultured for 48 h prior to
experiment. On the day of the experiment, growth media was removed and the
cells were loaded with calcium 3 dye (Molecular Devices) in HBSS, pH 7.4,
containing 2.5 mM probenecid for 1 h at 37 °C. The dye loaded cells were then
incubated with compound for 10 min prior to challenge with an EC80
concentration of ADP (determined daily; typically 2–6 nM). Intracellular
a
Compound
R2
FLIPR IC50
(l
M)
Kia
(lM) or %I at 10 lM
5g
5h
5i
5j
5k
5l
3-CF3-4-Me-Ph
4-n-Pentyl-Ph
4-t-Bu-Ph
4-BuO-Ph
4-Cl-Ph
1.6
0.63
1.3
2.0
2.0
69%
0.14
72%
0.64
0.76
nd
calcium fluxes were measured on
a fluorescence imaging plate reader
4-Me2N-Ph
3-CF3-Ph
15.8
5.0
(FLIPR). Compound IC50 values were subsequently determined by non-linear
regression analysis using activity base.
5m
60%
a
P2Y1 binding assay: Membranes were prepared from BacMam-transduced U2OS
Values are means of at least three determinations with a standard devia-
cells expressing human P2Y1. [33P]-2-MeS-ADP was utilized as the radioligand.
tion 6 0.3 log units (nd, not determined).
Binding reactions were performed in 96-well plates in a volume of 130
lL
containing 150 pM hP2Y1 expressing U2OS cell
[
33P]-2-MeS-ADP, 0.5
lg
membranes pre-bound to 0.5 mg of WGA-SPA (wheat germ agglutinin-
coupled scintillation proximity assay) beads in 15 mM Hepes, 145 mM NaCl,
0.1 mM MgCl2, 5 mM EDTA, 5 mM KCl binding buffer, and various
concentrations of test compound or dimethylsulfoxide vehicle control.
Reactions were allowed to proceed to completion at room temperature for
1 h. Following centrifugation (2000g), supernatants were counted on a Perkin-
Elmer Topcounter.
P-selectin assay: Platelets were obtained by standard venepuncture technique
from healthy human volunteers who had not taken aspirin or non-steroidal
anti-inflammatory drugs for 10 days. Blood was collected into EDTA (10 mM)
anticoagulant and centrifuged at 300g for 4 min to obtain platelet-rich plasma
(PRP). Prior to assay, PRP was diluted 1:50 in Hepes Modified Tyrodes Buffer
(HMTB: 12 mM Na bicarbonate, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCL,
and 10 mM HEPES, pH 7.4), in order to minimize compound binding to plasma
protein binding. Platelets were incubated with compounds or
dimethylsulfoxide vehicle and immediately activated with ADP (300 nM) for
5 min. Following a 15 min incubation with phycoerythrin (PE) conjugated anti-
CD62P antibody, platelets were fixed for 30 min in 2% paraformaldehyde,
diluted in PBS, and analyzed with a BD FACScalibur according to standard
procedures.
Table 3
P-selectin assay results
Compound
%I at 10 lM
5c
5g
5i
49%
79%
a
100% (300 nM IC50
)
a
Value is a mean for n = 2 independent donors with a standard deviation 6 0.25
log units.
large, relatively non-polar ortho-phenyl substituent on the benzo-
furan ring is required for optimal activity. We also observed that
alkyl-substituted aryl groups are optimal substituents on the urea.
Finally, we have demonstrated that the benzofuran-substituted
urea analogs are functional P2Y1 inhibitors that affect ADP-medi-
ated platelet activation.
9. Representative experimental procedure: A two-neck round bottom flask was
charged with a solution of 2-iodo-6-nitrophenol (Ref. 10, 100 mg, 0.377 mmol)
in DMF (0.4 mL). Under a stream of nitrogen, piperidine (37 lL, 0.377 mmol),
(Ph3P)2PdCl2 (5.3 mg, 0.0075 mmol) and CuI (2.9 mg, 0.015 equiv) were then
added. The mixture was heated to 60 °C and a solution of the 1-ethynyl-2-(1-
methylethyl)benzene (Ref. 11, 65.3 mg, 0.453 mmol) in DMF (0.9 mL) was
added dropwise over 1 h. The resulting solution was stirred for another 2 h,
cooled to room temperature and then quenched with water. The product was
extracted three times with Et2O. The organic layers were combined and washed
with brine, dried over Na2SO4, filtered, and concentrated. The residue was
purified by reverse-phase HPLC (Sunfire, 40–100% MeCN/H2O, 0.1% TFA,
10 min) to afford 2-[2-(1-methylethyl)phenyl]-7-nitro-1-benzofuran (9) as a
yellow solid (70 mg, 66% yield). 1H NMR (CDCl3) d 1.37 (d, J = 6.9 Hz, 6H), 3.49
(sept, J = 6.9 Hz, 1H), 6.97 (s, 1H), 7.33 (t, J = 7.3 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H),
7.46–7.53 (m, 2H), 7.70 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 8.16 (d,
References and notes
1. (a) Kellerman, D.; Evans, R.; Mathews, D.; Shaffer, C. Adv. Drug Delivery Rev.
2002, 54, 1463; (b) Amisten, S.; Melander, O.; Wihlborg, A. K.; Berglund, G.;
Erlinge, D. Eur. Heart J. 2007, 28, 13; (c) Herbert, J. M.; Savi, P. Semin. Vasc. Med.
2003, 3, 113.
2. For reviews, see: (a) Hechler, B.; Cattaneo, M.; Gachet, C. Semin. Thromb.
Hemost. 2005, 312, 150; (b) Gachet, C. Annu. Rev. Pharmacol. Toxicol. 2006, 46,
277; (c) Oury, C.; Toth-Zsamboki, E.; Vermylen, J.; Hoylaerts, M. F. Curr. Pharm.
Des. 2006, 12, 859. and reference therein.

R. K. Thalji et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4104–4107
a solution of 9 (61 mg, 0.217 mmol, 1 equiv) in EtOAc
methylethyl)phenyl]-1-benzofuran-7-yl}-N0-{4-
4107
J = 8.1 Hz, 1H). To
(1.1 mL) was added SnCl2–2H2O (245 mg, 1.08 mmol). The mixture was stirred
overnight and then quenched with ice and 1 N NaOH (aq). The aqueous layer
was extracted two times with EtOAc and the organic layers were combined,
dried over Na2SO4, filtered and concentrated to afford 2-[2-(1-
methylethyl)phenyl]-1-benzofuran-7-amine (10). A solution of this residue
[(trifluoromethyl)oxy]phenyl}urea (5c) as the TFA salt (60 mg, 61% yield over
two steps). 1H NMR (CDCl3) d 1.27 (d, J = 6.9 Hz, 6H), 3.38 (sept, J = 6.9 Hz, 1H),
6.85 (s, 1H), 7.19 (d, J = 8.8 Hz, 2H), 7.29 (m, 1H), 7.39–7.49 (m, 6H), 7.57 (d,
J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H).
10. Cambie, R. C.; Larsen, D. S.; Rutledge, P. S.; Woodgate, P. D. Aust. J. Chem. 1997,
50, 767.
and 1-isocyanato-4-[(trifluoromethyl)oxy]benzene (36 lL, 0.239 mmol) in
CH2Cl2 (0.43 mL) was stirred at room temperature overnight. The solvent
was removed in vacuo and the residue was purified by reverse-phase HPLC
(Sunfire, 40–100% MeCN/H2O, 0.1% TFA, 10 min) to afford N-{2-[2-(1-
11. Alkynes that were not commercially available were prepared according to the
following paper: Tobisu, M.; Nakai, H.; Chatani, N. J. Org. Chem. 2009, 74, 5471.