Tetrahydro-4-quinolinamines identified as novel P2Y1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.09.102

High-throughput screening of the GSK compound collection against the P2Y₁ receptor identified a novel series of tetrahydro-4-quinolinamine antagonists. Optimal substitution around the piperidine group was pivotal for ensuring activity. An exemplar analog from this series was shown to inhibit platelet aggregation.

Full text of DOI:10.1016/j.bmcl.2008.09.102

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6222–6226
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetrahydro-4-quinolinamines identified as novel P2Y1 receptor antagonists
a
c
c
a
Ángel I. Morales-Ramos a, , John S. Mecom , Terry J. Kiesow , Todd L. Graybill , Gregory D. Brown ,
Nambi V. Aiyar b, Elizabeth A. Davenport d, Lorena A. Kallal d, Beth A. Knapp-Reed a, Peng Li a,
Allyn T. Londregan a, Dwight M. Morrow d, Shobha Senadhi b, Reema K. Thalji a, Steve Zhao a,
*
Cynthia L. Burns-Kurtis b, Joseph P. Marino Jr. a,
*
a Department of Chemistry, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, 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 Chemistry, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
d Department of Biochemistry, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening of the GSK compound collection against the P2Y1 receptor identified a novel
series of tetrahydro-4-quinolinamine antagonists. Optimal substitution around the piperidine group was
pivotal for ensuring activity. An exemplar analog from this series was shown to inhibit platelet
aggregation.
Received 18 August 2008
Revised 27 September 2008
Accepted 29 September 2008
Available online 2 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
P2Y1 receptor
Antagonist
Platelet aggregation
Thrombosis
Tetrahydro-4-quinolinamine
Adenosine diphosphate (ADP) is a key activator of platelets and
plays a central role in hemostasis and thrombosis. ADP activates
platelets through interactions with two G-protein coupled P2
receptors, P2Y1 and P2Y12, producing two separate intracellular
signals which synergize together to produce complete platelet acti-
vation.1 Activation of the Gq-coupled P2Y1 receptor leads to rapid
Ca2+ entry and mobilization of intracellular Ca2+ stores resulting in
platelet shape change and weak, transient aggregation. Activation
of the Gi-coupled P2Y12 receptor leads to the inhibition of cAMP
production, resulting in the amplification of the platelet response
and complete, irreversible aggregation.
Taken together, these data suggest that a P2Y1 antagonist could
have significant utility in the treatment of a variety of thrombotic
disorders. To date, there are few publications which describe selec-
tive, non-nucleotide, small molecule antagonists of P2Y1,5 there-
fore, an effort to discover such molecules was initiated.
High-throughput screening of the GSK compound collection in a
FLIPR-based, HEK-293 cellular assay8 identified tetrahydro-4-quin-
olinamine 1 as an antagonist of P2Y1 with low micromolar activity
(Fig. 1). Compound 1 also demonstrated low micromolar affinity in
The P2Y1 receptor plays an integral role in thrombosis as
evidenced by studies utilizing transgenic mice ðP2Yꢀ1 =ꢀÞ, as well
as rodents treated with nucleotide P2Y1 antagonists (such as
MRS2179 and MRS2500).2 In both types of studies, ADP-induced
platelet shape change, Ca2+ mobilization, and aggregation was
abolished and the platelet response to other agonists was impaired.3
Further, these animals were protected in models of systemic vascu-
lar thromboembolism as well as models of localized arterial and
venous thrombosis.3,4
NH
CH3
CH3
N
O
O2N
1
FLIPR IC50 = 1.6 μM
Binding Ki = 0.5 μM
* Corresponding authors.
E-mail address: joseph.p.marino@gsk.com (J.P. Marino).
Figure 1. P2Y1 antagonist, 1, high-throughput screening hit.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.102

Á. I. Morales-Ramos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6222–6226
6223
a radioligand ([33P]-2-SMe-ADP) binding assay8 using U2OS cell
membranes expressing recombinant human P2Y1 receptor.
Given the moderate potency of this new tetrahydroquinoline
hit, early lead optimization chemistry efforts focused on SAR stud-
ies and confirmation of functional activity in a human platelet
aggregation assay.
stereochemistry for analogs 9 and 10. The synthesis of analog 13
was initiated via the acylation of dihydroquinolinone 11. -Alkyl-
a
ation and stereoselective reduction of ketone 11 produced quino-
line 12. Conversion of the cyclic benzylic alcohol to an azide
using diphenylphosphoryl azide (DPPA), followed by reduction to
the amine, and cross-coupling with phenyl bromide, produced tet-
rahydroquinoline 13. Unsubstituted tetrahydroquinoline 15 was
obtained in two steps from dihydroquinolinone 11. N-Acylation
followed by TiCl4 mediated reductive amination with aniline pro-
vided target 15.
Given the potential for nitroarenes to generate reactive metab-
olites, lead optimization efforts focused on replacing this function-
ality in lead compound 1. The SAR around the 4-nitro-phenyl
substituent is summarized in Table 1. The 4-methoxy-phenyl, 4-
chloro-phenyl, 4-trifluoromethoxy-phenyl, and 4-bromo-phenyl
analogs (5b, 5c, 5d, and 5e) were found to be suitable replacements
for nitrophenyl, with all demonstrating slightly improved potency
and binding affinity over compound 1. Removal of the para-substi-
tuent resulted in a complete loss in activity as evidenced by ana-
logs 5a and 5f.
Phenyl ureas 6a–i demonstrated comparable activity to the
benzamides (Table 2). In contrast to the benzamide series, substi-
tution at the meta-position consistently offered the greatest po-
tency. 3-Bromo-phenyl (6f), 3-chloro-phenyl (6b), and 3-
methoxy-phenyl (6d) all demonstrated comparable potency to
lead compound 1. Interestingly, the unsubstituted phenyl analog,
6g, offered similar potency to the meta-substituted analogs. The
2-chloro-phenyl analog, 6a, was found to be inactive and the activ-
ity of the 4-methoxy-phenyl analog (6e) was attenuated relative to
the 3-methoxy-analog (6d). 3,5-Disubstitution of the phenyl group
resulted in good potency, as evidenced by 6h and 6i.
SAR exploration around the piperidine group focused on the
various alkyl substituents as well as their relative stereochemistry
(Table 3). Removal of the methyl group at C3 led to a slight de-
crease in potency for the cis isomer 9. There exists a strong prefer-
ence for the C2–C4 relative stereochemistry to be cis (9) based on
the loss of activity observed for the trans isomer (10). The ethyl
group at C2 also proved to be important for activity as evidenced
by the 10-fold decrease in potency observed for analog 13. Not
The synthesis of the p-nitro-aniline screening hit
1 was
achieved following the sequence outlined in Scheme 1. A tandem
condensation/cyclization of aniline and propionaldehyde resulted
in the formation of N-phenyl-1,2,3,4-tetrahydro-4-quinolinamine
4 as a single diastereomer.6 Quinolamine 4 was treated with excess
p-nitrobenzoyl chloride and polymer-bounded N-methylmorpho-
line to afford the tetrahydroquinoline 1.
The efficient preparation of intermediate 4 facilitated rapid ana-
loging and SAR studies around the benzamide moeity. Tetrahydro-
quinoline 4 was treated with commercially available benzoyl
chlorides in the presence of polymer-bounded N-methylmorpho-
line to provide amides 5a–f (Scheme 1). In the case of the 4-meth-
oxy-benzamide analog 5b, the secondary aniline was methylated
by treatment with sodium hydride, followed by methyl iodide to
provide 5g. In addition, replacement of the amide group with urea
was also investigated. Intermediate 4 was treated with commer-
cially available aryl isocyanates in the presence of triethylamine
to produce phenylureas 6a–i.
Attempts were made to reduce the structural complexity
around the piperidine ring by eliminating chiral centers. Scheme
2 illustrates the synthetic routes to the des-3-methyl analogs 9/
10, the des-2-ethyl-analog 13, and tetrahydroquinoline 15 in which
the 2 and 3 positions are unsubstituted. Ethyl lithium addition to
quinoline 7, followed by acylation of the resulting amine produced
the corresponding 1,2-dihydroquinoline. Hydrobromination of the
olefin, followed by dehalogenation of the bromohydrin, led to the
formation of alcohol 8 as a single diastereomer. Treatment of the
alcohol with iodotrimethylsilane provided an iodide intermediate
which was rapidly treated with aniline under basic conditions to
afford diastereomers 9 and 10. The diastereomeric mixture was
easily separated by reverse phase HPLC to cleanly provide the cis
(9) and trans (10) C2–C4 isomers. Two dimensional NMR (nOe)
studies highlighting the methine hydrogens confirmed the relative
NH
NH
4
3
CH3
CH3
CH3
i
ii
O
2
+
CH3
CH3
H
N
N
NH2
H
4
3
2
O
R
1; R=
5 a-f
p
-NO2
iii
iv
NH
N
CH3
CH3
N
N
CH3
R
N
O
CH3
H
6 a-i
O
MeO
5 g
Scheme 1. Reagents and conditions: (i) EtOH, rt; (ii) a—R-Ph-COCl, PS-NMM, CH2Cl2, rt; b—PS-trisamine; (iii) R-Ph-NCO, NEt3, CH2Cl2, rt; (iv) NaH, MeI, DMSO, 150 °C lwave,
5 min.

6224
Á. I. Morales-Ramos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6222–6226
OH
N
NH
4
2
i,ii,iii,iv
v
Et
N
N
Et
O
O
7
MeO
MeO
8
9; cis C2-C4
10; trans C2-C4
OH
N
O
NH
CH3
CH3
4
ii, vi,vii
viii, ix, x
2
N
H
N
O
O
11
12
13
MeO
MeO
O
NH
N
xi
ii
N
O
O
MeO
14
15
MeO
Scheme 2. Reagents and conditions: (i) EtLi, THF, ꢀ78 °C, 15 min; (ii) 4-MeO-Ph-COCl, NMM, CH2Cl2, rt; (iii) NBS, DMSO/H2O, rt; (iv) AIBN (5 mol%), Bu3SnH, toluene, 80 °C;
(v) a—TMSI, CH2Cl2, 0 °C; b—aniline, Ba2CO3, THF, rt; (vi) LHMDS, THF, ꢀ78 °C, then MeI; (vii) -selectride, THF, ꢀ78 °C; (viii) DPPA, DBU (5 mol%), CH2Cl2, 0 °C to rt; (ix) PPh3,
THF/H2O, 70 °C; (x) Ph-Br, Pd2(dba)3 (3 mol%), NaOt-Bu, X-PHOS (6 mol%), t-BuOH, 120 °C wave, 10 min; (xi) a—aniline, TiCl4, CH2Cl2; b—NaBH4, MeOH.
L
l
Table 1
Table 3
SAR of endocyclic amides 1 and 5a–f
Relative stereochemistry and substitution of the piperidine ring
a
Compound
R
FLIPR IC50
(
lM)
P2Y1 Kia
(lM)
1
4-NO2
3-OCF3
4-OMe
4-Cl
4-OCF3
4-Br
1.6
>25
0.8
1.3
1.0
0.8
25
0.5
ND
0.4
0.4
0.1
0.1
ND
R1
N
5a
5b
5c
5d
5e
5f
R2
4
3
2
R3
R 4
N
H
1
O
ND, not determined.
a
Values are means of at least three determinations with a standard devia-
tion 6 0.3 log units.
MeO
a
Compound
R1
R2
R3
R4
FLIPR IC50
(lM)
P2Y1 Kia
(lM)
5b
5g
9
10
13
15
H
CH3
H
H
H
CH3
CH3
H
H
CH3
H
Et
Et
Et
H
H
H
H
H
H
Et
H
H
0.8
>25
3.2
>25
10
0.4
ND
0.8
ND
ND
ND
Table 2
SAR of endocyclic phenyl ureas 6a–i
H
>25
a
Compound
R
FLIPR IC50
(lM)
P2Y1 Kia
(lM)
ND, not determined.
a
Values are means of at least three determinations with a standard devia-
tion 6 0.3 log units.
6a
6b
6c
6d
6e
6f
6g
6h
6i
2-Cl
3-Cl
4-Cl
3-OMe
4-OMe
3-Br
>25
1.0
2.0
1.6
6.0
1.0
1.6
0.8
0.6
ND
0.3
0.3
0.5
ND
0.1
0.4
0.2
0.07
too surprisingly removal of both the 2-ethyl and 3-methyl groups
led to a loss of activity as evidenced by analog 15. Methylation of
the aniline nitrogen of 5b also led to a complete loss in activity
(analog 5g), suggesting that the aniline hydrogen may play a role
in hydrogen bonding or in achieving
conformation.
H
3,5-OMe
3,5-Cl
a favorable binding
ND, not determined.
a
The importance of absolute stereochemistry on P2Y1 activity is
Values are means of at least three determinations with a standard devia-
highlighted in Table 4. Separation of enantiomers for racemate 6i
tion 6 0.3 log units.

Á. I. Morales-Ramos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6222–6226
6225
Table 4
2. (a) Camaioni, E.; Boyer, J. L.; Mohanram, A.; Harden, T. K.; Jacobson, K. A. J. Med.
Chem. 1998, 41, 183; (b) Kim, H. S.; Ohno, M.; Xu, B.; Kim, H. O.; Choi, Y.; Ji, X. D.;
Maddileti, S.; Marquez, V. E.; Harden, T. K.; Jacobson, K. A. J. Med. Chem. 2003, 46,
4974.
3. (a) Fabre, J.-E.; Nguyen, M.; Latour, A.; Keifer, J. A.; Audoly, L. P.; Coffman, T. M.;
Koller, B. H. Nat. Med. 1999, 510, 1199; (b) Léon, C.; Hechler, B.; Freund, M.;
Eckly, A.; Vial, C.; Ohlmann, P.; Dierich, A.; LeMeur, M.; Cazenave, J.-P.; Gachet,
C. J. Clin. Invest. 1999, 10412, 1731; (c) Baurand, A.; Raboisson, P.; Freund, M.;
Leon, C.; Cazenave, J.; Bourguignon, J.; Gachet, C. Eur. J. Pharmacol. 2001, 412,
213; d Cattaneo, M.; Lecchi, A.; Ohno, M.; Joshi, B. V.; Besada, P.; Tchilibon, S.;
Lombardi, R.; Bischofberger, N.; Harden, T. K.; Jacobson, K. A. Biochem.
Pharmacol. 2004, 68, 1995.
4. (a) León, C.; Freund, M.; Ravanat, C.; Baurand, A.; Cazenave, J. P.; Gachet, C.
Circulation 2001, 103, 718; (b) Lenain, N.; Freund, M.; León, C.; Cazenave, J. P.;
Gachet, C. J. Thromb. Haemost. 2003, 1, 11440; (c) 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.
Data for individual enantiomers of analog 6i
Compound Absolute
FLIPR
P2Y1 Kia Inhibition of platelet
a
b
stereochemistry IC50
(nM)
aggregation IC50 (nM)
(% purity)
(nM)
16
17
2R,3S,4S (99.5)
2S,3R,4R (97.8)
600
>25,000
70
ND
504 103
ND
ND, not determined.
a
Values are means of at least three determinations with a standard devia-
tion 6 0.3 log units.
b
Values are means SEM for n = 3 independent donors.
5. (a) 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.; (b) Tuerdi, H.; Chao, H. J.; Qiao, J. X.; Wang, T. C.;
Gungor, T. U. S. Patent 2005261244A1, 2005.; c Herpin, T. F.; Morton, G. C.;
Rehfuss, R. P.; Lawrence, R. M.; Poss, M. A.; Roberge, J. Y.; Gungor, T. World
Patent WO2005070920A1, 2005.; d Sutton, J. C.; Pi, Z.; Ruel, R.; L’Heureux, A.;
Thibeault, C.; Lam, P. Y. S. World Patent WO2006078621A2, 2006.; (e)
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.
6. Funabashi, M.; Masaharu, I.; Juji, Y. Bull. Chem. Soc. Jpn. 1969, 42, 2885.
7. For details of the platelet aggregation assay, see: Wilson, R. J.; Giblin, G. M. P.;
Roomans, S.; Rhodes, S. A.; Cartwright, K. A.; Shield, V. J.; Brown, J.; Wise, A.;
Chowdhury, J.; Pritchard, S.; Coote, J.; Noel, L. S.; Kenakin, T.; Burns-Kurtis, C. L.;
Morrison, V.; Gray, D. W.; Giles, H. Br. J. Pharmacol. 2006, 1483, 326.
8. Experimental procedure for the preparation of compound 16.Preparation of (rac)-
2-ethyl-3-methyl-N-phenyl-1,2,3,4-tetrahydro-4-quinolinamine (4): In a 250 mL
round bottom flask, aniline (18.6 g, 200 mmol) was dissolved in absolute ethanol
(50 mL). The solution was cooled to 0 °C. To this solution was added
propionaldehyde (11.6 g, 200 mmol) dropwise. Once the addition was
completed, the reaction was allowed to warm to room temperature and stirred
overnight (ꢁ14 h). The resulting yellow precipitate was filtered, washed with cold
ethanol, and then allowed to dry at RT (under vacuum) for 24 h. The title
compound was obtained as a white solid (9.3 g, 35 mmol, 35% yield). 1H NMR
(400 MHz, CHCl3-d) dppm 7.16–7.23 (3H, m) 7.02 (1H, t, J = 7.6 Hz) 6.62–6.68 (3H,
m) 6.61 (1H, d, J = 1.0 Hz) 6.51 (1H, dd, J = 8.0, 1.1 Hz) 4.31 (1H, t, J = 9.0 Hz) 3.78–
3.86 (2H, m) 3.13 (1H, td, J = 7.8, 3.4 Hz) 1.84–1.91 (1H, m) 1.68–1.75 (1H, m)
1.57–1.63 (1H, m) 1.08 (3H, d, J = 6.8 Hz) 0.98 (3H, t, J = 7.4 Hz).
Figure 2. Inhibition of ADP-induced platelet aggregation by example 16.
was carried out by chiral HPLC. Assignment of absolute configura-
tion was determined by vibrational circular dichroism (VCD). All
P2Y1 activity is derived from a single enantiomer, as the 2R, 3S, 4
S (16) isomer was active in all assays tested, and the 2S, 3R, 4 R
(17) isomer was inactive in the primary FLIPR screen.
Preparation of (2R,3S,4S)-2-ethyl-3-methyl-N-[3,5-(chloro)phenyl]-4-(phenyla-
mino)-3,4-dihydro-1(2H)-quinolinecarboxamide (16): In a 100 mL round bottom
flask (rac)-2-ethyl-3-methyl-N-phenyl-1,2,3,4-tetrahydro-4-quinolinamine
4
(1 g, 3.76 mmol) was dissolved in dichloromethane (13 mL). To this solution
was added 3,5-dichlorophenylisocyanate (0.85 g, 4.51 mmol) followed by
triethylamine (785 lL, 5.64 mmol) at room temperature. The reaction mixture
was stirred overnight (ꢁ14 h). The resulting mixture was diluted with
dichloromethane (50 mL) and quenched with water (25 mL). The phases were
separated, and the aqueous phase was back-extracted with dichloromethane
twice (15 mL each). The combined organic phases were dried over MgSO4 and
purified by flash chromatography (ISCO, 40 g SiO2 cartridge, 0–15% ethyl acetate/
hexanes as the eluents). Thefractions corresponding to (rac)-2-ethyl-3-methyl-N-
[3,5-(chloro)phenyl]-4-(phenylamino)-3,4-dihydro-1(2H)-quinolinecarboxamide
(6i) were combined and concentrated under reduced pressure to provide the title
compound as the racemate (1.2 g, 2.65 mmol, 70% yield).
The functional activity of compound 16 was subsequently eval-
uated in a platelet aggregation assay. Human washed platelets
were incubated with compound for 5 min prior to the addition of
10 lM ADP and aggregation was monitored by standard light
transmittance aggregometry.7 Compound 16 inhibited ADP-in-
duced aggregation with an IC50 of 504 103 nM (Fig. 2).
In summary, a new series of tetrahydroquinoline P2Y1 antago-
nists have been identified. para-Substituted benzamides and
meta-substituted phenylureas provided enhanced activity. Within
the piperidine ring of the tetrahydroquinoline, the cis stereochem-
istry for the C2–C4 substituents, and the trans stereochemistry for
the C2–C3 and C3–C4 substituents also enhanced potency. In addi-
tion, P2Y1 activity appears to reside in a single enantiomer
(2R,3S,4S). One of the most potent analogs identified, 16, demon-
strated functional activity in a human platelet aggregation assay.
Thus far poor aqueous solubility has hindered pharmacokinetic
studies with urea 16 and similar analogs. Therefore future lead
optimization efforts to improve in vivo exposure will be necessary
prior to the evaluation of tetrahydro-4-quinolamines in thrombo-
sis models.
The racemate (0.67 g) was separated by chiral HPLC (SFC with 25% MeOH as the
modifier solvent; 10
280 nm).
lm Chiralpak OD, 10 mm ꢂ 250 mm, 10 ml/min, UV at
Rt = 7.1 min for 2R,3S,4S-enantiomer (99.4% purity), and Rt = 9.0 min for 2S,3R,4R-
enantiomer (97.8% purity).
The absolute configuration for the (2R,3S,4S)-enantiomer was assigned by
vibrational circular dichorism (VCD) studies (Bomem Chiral IR VCD spectrometer
at 4 cmꢀ1; frequency range = 2000–800 cmꢀ1; dual PEM calibrated at 1400 cmꢀ1
;
PEM1 = 0.250k; PEM2 = 0.275k; Scan Method: single block scan 180 min; CCl4 as
solvent; Concentration 10 mg/125 L).
l
(2R,3S,4S)-2-Ethyl-3-methyl-N-[3,5-(chloro)phenyl]-4-(phenylamino)-3,4-dihydro-
1(2H)-quinolinecarboxamide (16): LC/MS: (M+H) = 454.0; 1H NMR (400 MHz,
CHCl3-d) d ppm 7.39–7.44 (3H, m) 7.36 (1H, d, J = 7.6 Hz) 7.24–7.31 (3H, m) 7.17–
7.23 (2H, m) 7.02 (1H, m) 6.97 (1H, br s) 6.74 (1H, t, J = 7.3 Hz) 6.62 (2H, d, J = 7.8 Hz)
4.41 (1H, dt, J = 7.8, 5.6 Hz) 3.99 (1H, d, J = 9.6 Hz) 1.65–1.72 (1H, m) 1.59 (1H, ddd,
J = 13.6, 7.6, 5.6 Hz) 1.35–1.39 (1H, m) 1.30 (3H, d, J = 6.7 Hz) 0.92 (3H, t, J = 7.4 Hz).
Screening Assay Protocols.
P2Y1 FLIPR: HEK-293 MSRII cells endogenously expressing P2Y1 were maintained in
DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% L-glutamine,
References and notes
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
wasremovedandthecellswereloadedwithCalcium3dyefromMolecularDevicesin
HBSS,pH7.4containing2.5 mMprobenecidfor1 hat37 °C.Thedyeloadedcellswere
then incubated with compound for 10 min prior to challenge with an EC80
1. 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.

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Á. I. Morales-Ramos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6222–6226
33P]-2-SMe-ADP, 0.5
g hP2Y1 expressing U2OS cell membranes pre-bound to
concentration of ADP (determined daily; typically 2–6 nM). Intracellular calcium
[
l
fluxes were measured on a Fluorescence Imaging Plate Reader (FLIPR). Compound
IC50 values were subsequently determined by non-linear regression analysis using
Activity Base.
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 mMKClbinding
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.
P2Y1 Binding: Membranes were prepared from BacMam transduced U2OS cells
expressing human P2Y1. [33P]-2-MeS-ADP was utilized as the radioligand. Binding
reactions were performed in 96-well plates in a volume of 130 lL containing 150 pM