Thienopyrimidine-based P2Y12 platelet aggregation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.08.059

Herein we describe the design and synthesis of a novel series of potent thienopyrimidine P2Y₁₂ inhibitors and the negative impact protein binding has on the inhibition of platelet aggregation.

Full text of DOI:10.1016/j.bmcl.2009.08.059

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5919–5923
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thienopyrimidine-based P2Y12 platelet aggregation inhibitors
à
*
Steven W. Kortum , Rhonda M. Lachance, Barbara A. Schweitzer , Gopichand Yalamanchili ,
Hayat Rahman à, Michael D. Ennis, Rita M. Huff, Ruth E. TenBrink
Pfizer Global Research and Development, Pfizer Inc., St. Louis Laboratories, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the design and synthesis of a novel series of potent thienopyrimidine P2Y12 inhibitors
and the negative impact protein binding has on the inhibition of platelet aggregation.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 29 July 2009
Revised 14 August 2009
Accepted 14 August 2009
Available online 20 August 2009
Keywords:
P2Y12
Thienopyrimidine
Platelet aggregation inhibition
The success of PlavixÒ (clopidogrel) in inhibiting platelet aggre-
gation and the subsequent discovery of the P2Y12 receptor as the
mechanism of action validates the inhibition of the P2Y12 receptor
as a viable strategy for platelet aggregation inhibition.1,2 Whereas
clopidogrel is an irreversible inhibitor of the P2Y12 receptor, we
were interested in developing a reversible inhibitor.3 AZD-6140,
an orally active P2Y12 reversible inhibitor in clinical evaluation
for acute coronary syndrome (ACS), shown in Figure 1 has a total
of six chiral centers (four contiguous).4 One of our goals was to de-
velop a less complex adenosine diphosphate (ADP)-stimulated
P2Y12 antagonist while retaining the hydrophilic and hydrophobic
regions as in AZD6140.5
ride at 150 °C and then quenched in ice water to give the desired
product in >95% yield. At atmospheric pressure, thienopyrimidi-
none 4 and POCl3 with catalytic N,N-dimethylformamide (DMF)
gave yields ranging from 0% to 30%. Use of a sealed tube (150 °C)
gave improved yields (80–95%), but for larger scale reactions, phe-
nylphosphonic dichloride in place of phosphorus oxychloride al-
lowed the elimination of sealed pressure vessels while
maintaining high yields of 4,6-dichlorothienopyrimidine 5. Thio-
phene 3 is reacted with formamide at 130 °C for 12 h to give thie-
nopyrimidinone 6 in 75% yield. Conversion of thienopyrimidinone
6 to thienopyrimidine 7 was accomplished using thionyl chloride
and DMF at 80 °C in 86% yield.
We, along with others, investigated the thienopyrimidine core
as a potential candidate for platelet inhibition.6 Our efforts were
concentrated on the central theme of a hydrophobic northern re-
gion with a hydrophilic southern region as exemplified by com-
pound 21k.
Scheme 2 outlines the synthesis of the C-6 hydrogen analogs.
Displacement of the C-4 chloro group of 7 with boc-piperazine 8
was accomplished at room temperature in the presence of diiso-
propylethylamine (DIEA) to give thienopyrimidine 9 in 70–90%
yield. For exploration of the substituents at C-6, the BOC group of
The synthesis of the chloro intermediates 5 and 7 are outlined
in Scheme 1. Butyraldehyde 1 and methyl cyanoacetate 2 were
combined in the presence of elemental sulfur and triethylamine
in the classic Gewald synthesis to give aminothiophene 3 in 70%
yield.7,8
F
F
Northern Hydrophobic Region
Aminothiophene 3 reacted with potassium cyanate in acetic
acid at room temperature for 18 h to give thienopyrimidinedione
4 in 65% yield. Thienopyrimidinedione 4 was converted to the
4,6-dichlorothienopyrimidine 5 using phenylphosphonic dichlo-
HN
AZD6140
O
N
N
N
N
N
N
N
N
OH
OH
S
HO
O
O
O
S
HO
N
H
N
H
N
Southern Hydrophilic Region
* Corresponding author. Tel.: +1 636 247 3592.
E-mail address: steve.kortum@pfizer.com (S.W. Kortum).
Present address: Monsanto Corp., St. Louis, MO, USA.
21k
Figure 1. Comparison of AZD6140 to the thienopyrimidine compound 21k high-
lighting the hydrophilic and hydrophobic regions of each molecule.
à
Retired.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.059

5920
S. W. Kortum et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5919–5923
O
O
Cl
O
O
b
c
1
N
a
NH
+
S
NH2
S
N
Cl
N
H
4
O
S
5
3
O
2
N
O
d
Cl
O
e
N
NH
S
S
N
N
6
7
Scheme 1. Synthesis of the thienopyrimidine cores 5 and 7. Reagents and conditions: (a) sulfur, triethylamine, DMF, rt, 18 h (70%); (b) acetic acid, H2O, KOCN, rt, 18 h (64%);
(c) phenylphosphonic dichloride, 150 °C, 3 h (95%); (d) formamide, ammonium formate, 135 °C, 12 h (75%); (e) thionyl chloride, DMF, 80 °C, 3 h (86%).
Cl
O
O
N
O
O
N
N
N
N
H
N
N
N
S
N
Cl
O
O
Cl
N
a
N
5
N
+
a
b
S
N
N
N
H
S
N
+
S
N
Cl
S
c
N
Cl
8
7
9
O
O
12
13
N
b
N
H
8
R
N
H
N
O
N
N
O
c
N
N
N
N
N
S
S
N
N
d
N
N
10
N
11
S
N
Cl
R
S
N
N
R2
15a-l
Scheme 2. Synthesis of C-6 hydrogen-substituted thienopyrimidine analogs 11a–h.
Reagents and conditions: (a) THF, DIEA, rt, 6 h (70–90%); (b) hydrochloric acid,
methanol, rt, 3 h (quant); (c) DMF, DIEA, rt, 18 h (30–90%).
14
Scheme 3. Synthesis of the C-6 amino-substituted thienopyrimidine analogs 15a–l.
Reagents and conditions: (a) THF, DIEA, rt, 6 h (70–90%); (b) hydrochloric acid,
methanol, rt, 3 h (quant); (c) DMF, DIEA, rt, 1 h (94%); (d) DIEA, NMP, 130 °C, 18 h
(40–90%).
thienopyrimidine 9 was removed using HCl in methanol to give
thienopyrimidine 10 in quantitative yield. Thienopyrimidine 10
was either acylated with the appropriate acid chloride using DIEA
as base at room temperature to give thienopyrimidines 11a–h in
30–60% yield or coupled with the appropriate acid using O-(7-aza-
benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophos-
phate (HATU) in comparable yields.
Scheme 3 outlines the synthesis of the C-6 nitrogen analogs.
BOC deprotection of 12 and acylation of piperazine 13 were accom-
plished using the same procedures in similar yields to those of the
C-6 hydrogen analogs. The C-6 chloro group of 14 was displaced
using an appropriate amine with DIEA in 1-methyl-2-pyrrolidi-
none (NMP) at 105 °C to give the corresponding thienopyrimidines
15a–l in 40–90% yield.
Scheme 4 outlines the synthesis of urea analogs 21a–k. The C-6
chloro group of 12 is displaced with sodium azide in NMP at 130 °C
to give azide 16 in 77% yield. Azide 16 was reduced using trimeth-
ylphosphine in tetrahydrofuran (THF) to give amine 17 in 85%
yield. Amine 17 was heated in pyridine with ethyl-3-isocyanato-
propionate at 80 °C to give urea 18 (81%) followed by BOC depro-
tection to give 19 in quantitative yield. Acylation of 19 to give
the intermediate esters 20a–k in 30–60% yield and subsequent
hydrolysis was accomplished in 50–90% yield using lithium
hydroxide to give thienopyrimidines 21a–k.
branes.9 The P2Y12 binding assay with added protein, human ser-
um albumin (HSA) and alpha-1 acid glycoprotein (AGP), was
used to give a readout on the protein binding of our inhibitors be-
fore going into the human platelet rich plasma (hPRP) aggregation
functional assay.10,11 A large portion of the discrepancies between
the P2Y12 binding and functional assays, nM versus lM, are likely
due to the increased amount of protein in the hPRP aggregation
assay as compared with the P2Y12 binding assay.
In keeping with the hydrophobic nature of the northern substi-
tuent we first looked at several hydrophobic substituents on the
piperazine ring while keeping C-6 as H (Table 1). Biphenyl 11c
was the most active compound in the P2Y12 binding assay, fol-
lowed by naphthyl carbamate 11g, indicating that larger, hydro-
phobic groups were preferred. All of the compounds lacking a
C-6 substituent displayed poor activity in the hPRP aggregation
assay.
Table 2 lists analogs containing C-6 nitrogen based substituents
with the 4-biphenylacetyl group as the northern piperazine substi-
tuent. Substituents with carbonyl groups directly attached to (21k)
or one atom removed from (15h and 15k) the C-6 nitrogen were
the most active in both the P2Y12 binding and hPRP aggregation as-
says. Moving the carbonyl even further away from the C-6 nitro-
gen, as in 15c and 15g, resulted in a 10–15-fold decrease in
The P2Y12 binding assay used for this study uses recombinant
human P2Y12 transfected Chinese Hamster Ovary (CHO) cell mem-

S. W. Kortum et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5919–5923
5921
O
O
N
O
O
N
O
O
N
O
O
N
N
N
N
N
N
N
N
N
O
c
b
a
O
O
S
N
N
N
H
S
S
+
S
N
NH2
N
Cl
H
N
N
N
N
18
17
16
12
d
H
N
R
N
R
N
N
O
N
O
e
N
OH
O
f
N
O
O
N
O
O
N
O
S
N
N
N
H
S
N
N
N
S
N
N
N
H
H
H
H
H
19
20a-k
21a-k
Scheme 4. Synthesis of the C-6 urea-substituted analogs 21a–k. Reagents and conditions: (a) NMP, H2O, sodium azide, 130 °C, 18 h (77%); (b) THF, trimethylphosphine, rt,
20 h (85%); (c) pyridine, ethyl-3-isocyanatopropionate, 80 °C, 18 h (81%); (d) HCl, ethanol, rt, 1 h (quant); (e) DMF, DIEA, rt, 18 h (30–90%); (f) THF, H2O, lithium hydroxide, rt,
6 h (50–90%).
Table 2
Table 1
Table of hPRP IC50 and P2Y12 Ki values for the northern biphenyl analogs
Table of hPRP IC50, %inhibition, and P2Y12 Ki values for the C-6 hydrogen analogs
R
N
O
N
N
N
N
N
S
N
Compd
R
P2Y12 Ki (nM)
hPRP agg. IC50
M)or %inh.a,b
S
N
R
(
l
Compd
R
P2Y12
Ki (nM)
P2Y12 + protein
hPRP agg.
O
O
F
a
Kib (nM)
IC50
(lM)
11a
2990
28
0
F
F
N
H
OH
15a
19
35
75 (4X)
11
OH
11b
11c
4400
29
N
O
15b
15c
15d
15e
241 (7X)
122 (15X)
325 (1.3X)
1810 (2X)
37
OH
7
H
N
OH
8
57
O
11d
11e
2100
NT
8
O
O
F
NH2
F
18
240
839
40
N
N
F
O
11f
11g
11h
NT
4
O
O
F
F
>100
N
F
130
660
1.25
28
O
O
OH
O
15f
37
31
163 (4.4X)
203 (6.5X)
21
73
N
N
O
15g
N
a
%Inhibition values at 50
Human platelet rich plasma (hPRP), see Ref. 11.
l
M concentration (NT = not tested).
(continued on next page)
b

5922
S. W. Kortum et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5919–5923
Table 3
Table 2 (continued)
Table of hPRP IC50 and P2Y12 Ki values for the southern urea analogs
Compd
R
P2Y12
Ki (nM)
P2Y12 + protein
hPRP agg.
IC50 (lM)
R
N
a
Kib (nM)
O
N
O
N
15h
5
27
78
4
31 (6X)
4
O
O
O
N
S
O
N
N
N
Compd
R
P2Y12 Ki
P2Y12 + protein
hPRP agg.
OH
a
(nM)
Kib (nM)
IC50
(lM)
15i
15j
15k
15l
21k
297 (11X)
441 (5.6X)
21 (5X)
30
56
3
N
O
O
21a
223
36
231 (1X)
16
H
N
OH
21b
21c
21d
21e
21f
21g
21h
21i
46 (1.3X)
593 (1.1X)
161 (4.3X)
447 (2.3X)
172 (1X)
4
25
42
39
18
34
16
38
86
5
O
N
NH2
O
O
NH2
O
539
37
NH
410
397 (1X)
10 (3X)
45
5
N
N
O
O
O
3
N
H
N
H
OH
O
O
O
O
F
191
177
68
a
Human platelet rich plasma (hPRP), see Ref. 11.
Value in parenthesis is the fold shift between binding with and binding without
b
added protein.
F
F
F
potency in the hPRP aggregation assay and a 2–10-fold decrease in
P2Y12 affinity. Alcohols 15a, 15b, 15f, 15i, and 15j were 4–12-fold
less potent in the P2Y12 binding assay and 2–11-fold less potent in
the hPRP aggregation assay than 21k. For amines 15d and 15l, the
loss in affinity was on the order of 40–80-fold in the P2Y12 binding
assay and ꢀ8-fold less potent in the hPRP aggregation assay as
compared with 21k. Hydrophobic analogs such as trifluoroacetyl
15e were more than 150-fold less potent in the P2Y12 binding as-
say and more than 20-fold less potent in the hPRP aggregation as-
say than 21k. This loss of affinity for C-6 hydrophobic substituents
demonstrates the preference for hydrophilic substituents in the
southern region. In general, analogs which were potent in the
P2Y12 binding assay and displayed a smaller, less than 10-fold, shift
from the P2Y12 binding assay with added protein were among the
more potent analogs in the hPRP aggregation assay. Potent analogs
such as 15c (8 nM) with a 15-fold shift in potency with added pro-
tein (122 nM) were not as active in the hPRP aggregation assay as
compounds such as 15h which only had a sixfold shift upon addi-
tion of protein. Thus the right shift in affinity observed on going
from no protein added to added protein in the binding assay was
adequate to predict loss of potency in the hPRP aggregation assay
due to protein binding.
140 (2X)
O
O
F
295
460
213
3
367 (1.2X)
380 (0.8X)
783 (3.7X)
10 (3.3X)
O
O
O
21j
O
21k
Table 3 lists some of the analogs made where the C-6 urea was
held constant while varying the northern piperazine substituent.
Since most of the analogs listed in Table 1 were inactive in the
hPRP aggregation assay, the C-6 urea was subsequently used to
help evaluate the SAR around the northern piperazine substituent.
The comparator for this series of compounds is the 4-biphenylace-
tyl analog 21k (Table 2). Compounds such as 21c, 21e, and 21j
which have a phenyl ring appended to an alkyl chain via an ether
linkage resulted in a 60–180-fold decrease in P2Y12 affinity with
only a three to sixfold decrease in hPRP aggregation affinity. Small,
a
Human platelet rich plasma (hPRP), see Ref. 11.
Value in parenthesis is the fold shift between binding with and binding without
b
added protein.
straight-chained alkyl substituents such as 21f, 21h, and 21i also
resulted in a 60–175-fold decrease in P2Y12 binding affinity
and a three to eightfold decrease in hPRP aggregation affinity.

S. W. Kortum et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5919–5923
5923
4. (a) Angiolillo, D. J.; Bhatt, D. L.; Gurbel, P. A.; Jennings, L. K. Am. J. Cardiol. 2009,
103, 40A; (b) Cannon, C. P.; Husted, S.; Harrington, R. A.; Scirica, B. M.;
Emanuelsson, H.; Peters, G.; Storey, R. F. J. Am. Coll. Cardiol. 2007, 50, 2196.
5. 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.
6. Levy, D.E.; Smyth, M.S.; Scarborough, R.M. WO2003/022214.
7. (a) Gewald, K. Z. Chem. 1962, 305; (b) Chem. Abstr. 1963, 58, 6770.; (c) Gewald,
K. Chem. Ber. 1965, 98, 3571.
8. Aminothiazole synthesis of compound 3: to a mixture of sulfur (6.4 g, 200 mmol)
in DMF (25 mL) was added methyl cyanoacetate (19.8 g, 200 mmol). The
mixture was placed under nitrogen and triethylamine (15 mL, 108 mmol) was
added to form a burnt orange solution. Butyraldehyde (18 mL, 200 mmol) was
added dropwise at a sufficient rate as to keep the temperature of the mixture
between 45 and 50 °C. Once the addition was complete the mixture was
allowed to stir at room temperature for 18 h. The mixture was partitioned
between brine and ethyl acetate. The layers were separated and the organic
phase washed three times with brine, dried over anhydrous magnesium sulfate
and concentrated under reduced pressure. The residue was purified via flash
chromatography on silica gel (100 g) using 15% ethyl acetate in hexanes.
Fractions containing the desired product were concentrated under reduced
pressure, slurried in hexanes and collected via vacuum filtration to give 25.74 g
(70%) of the title compound: 1H NMR (300 MHz, chloroform-d) d ppm 1.11 -
1.31 (m, 3H) 2.61 (qd, J = 7.52, 1.21 Hz, 2H) 3.76–3.83 (m, 3H) 5.79 (br s., 2H)
6.62 (t, J = 1.31 Hz, 1H).
Compounds which exhibited the greatest affinity for the P2Y12
receptor were compounds which either contained a phenyl group
directly attached to the carbonyl such as 21k and 21d or analogs
which are small and branched immediately following the carbonyl
group as in the 2-methylpropanoyl analog 21b. Compared with the
biphenyl analog 21k, analogs such as 21a, and 21g, where the phe-
nyl ring is one methylene group removed from the carbonyl were
22–77-fold less potent in the P2Y12 binding assay respectively and
28–46-fold less potent in the hPRP aggregation assay. Only 21b
was equivalent in potency to 21k in the hPRP aggregation assay.
Here too, compounds with higher protein binding as indicated by
a large shift between the P2Y12 binding assay with and without
added protein such as 21d, 21g, and 21j showed weaker hPRP
aggregation inhibition.
Receptor binding studies were conducted on other closely re-
lated purinergic receptors such as P2Y1 and P2Y13, which share
19% and 48% homology with P2Y12, respectively. A subset of thie-
nopyrimidine analogs were tested and showed a greater than
100-fold selectivity for the P2Y12 receptor over both the P2Y1 and
P2Y13 receptors. The majority of compounds made were not tested
for selectivity against these receptors.
9. P2Y12 assays: dry compounds are diluted as 10 mM dimethylsulfoxide (DMSO)
stocks and are tested in a seven-point, threefold dilution series run in triplicate
beginning at 10 lM. A 1 mM DMSO intermediate stock is made in a dilution
We have demonstrated that the thienopyrimidine core can be
used in preparing potent P2Y12 inhibitors. Northern hydrophobic
groups combined with southern hydrophilic groups give low nano-
molar P2Y12 inhibitors with low micromolar inhibition of hPRP.
Compounds such as 15h (5 nM), 15k (4 nM), and 21k (3 nM) con-
taining the biphenyl northern substituent and highly polar amide
and acid substituents in the southern region exhibit the greatest
potency in the P2Y12 binding assay. These compounds also exhibit
the greatest potency in the hPRP aggregation assay at 4, 3, and
plate and from this the seven dilutions are made. The highest concentration is
diluted into water containing 0.02% Bovine serum albumin (BSA), and the
remaining six concentrations are diluted into assay buffer containing 0.02%
BSA. To a polypropylene assay plate the following are added: (a) 30
buffer containing one protease inhibitor cocktail tablet per 50 mL; (b) 30
1 nM 33P 2-MeSADP made in assay buffer containing 0.02% BSA and 12.5 mg/
mL ascorbic acid; (c) 30 L of cold 1.5 M 2-MeSADP for the positive control
wells, or assay buffer containing 0.02% BSA and 12.5 mg/mL ascorbic acid for
the negative control wells; (d) (Method a) 60 L of 10 g/well membranes,
(Method b) 60 L of 1 g/well membranes, (Method c) 60 L of 0.3 g/well
l
L of assay
lL of
l
l
l
l
l
l
l
l
membranes.Incubate the plates at room temperature for 1 h. Stop the reaction
using a cell harvester to aspirate/transfer the supernatant onto GF/B UniFilter
plates, and wash three times with wash buffer, aspirating between each wash.
The filter plates are dried for approximately 20 min in an oven at 50 °C. Back
seals are adhered to the filter plates and 25 lL of Microscint 20 scintillation
fluid is added. The filter plates are sealed, shaken for 30 min, and counted on a
Top Count.
5 lM, respectively. The highly polar acid and amide groups of
15k, 21k, and 15h help to offset protein binding attributes of the
highly hydrophobic biphenyl group. A smaller northern substitu-
ent such as the isopropyl amide of 21b with reduced protein bind-
ing improves the hPRP to P2Y12 ratio but was 10-fold less potent
than biphenyl 21k in the P2Y12 binding assay. As indicated by some
of the differences between assay results with and without added
protein, the presence of protein binding can negatively impact
the hPRP aggregation activity of the thienopyrimidines.
10. P2Y12 assays with added protein: the same as the P2Y12 assay without protein
with the following exceptions:(Method a) 60
with 5X human serum albumin (HSA) (1.75%) and 5X AGP (0.075%), (Method b)
60 of g/well membranes made with 5X HSA (1.75%) and 5X AGP
(0.075%), (Method c) 60 L of 0.3 g/well membranes made with 5X HSA
(1.75%) and 5X AGP (0.075%).
lL of 1 lg/well membranes made
lL
1 l
l
l
11. Human platelet-rich plasma (hPRP) preparation:Fifty millilitres polypropylene
centrifuge tubes were filled with whole blood and centrifuged at 910g ꢁ 10 s
and then 200g ꢁ 15 min at room temperature to obtain platelet-rich plasma
(PRP). PRP was removed using a wide bore transfer pipet avoiding disturbance
of the buffy coat (polymorphonuclear leukocytes (PMNLs)) and red blood cells,
and placed in a clean polypropylene tube. Remaining plasma, buffy coat and
red cells were centrifuged at 2380g ꢁ 15 min, again at room temperature, to
obtain platelet-poor plasma (PPP). PPP was removed, in the same manner as for
the PRP, and placed in a clean polypropylene tube. PRP platelet counts were
determined using a Z1 Coulter Particle Counter and individual PRP was diluted
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.08.059.
References and notes
to 300,000 platelets/lL with autologous PPP.hPRP aggregation assay: the 96-
1. (a) BalditSollier, C.; Berge, N.; Boval, B.; Hovsepian, L.; Drouet, L. Thromb.
Haemost. 2009, 101, 116; (b) Joseph, J. E.; Machin, S. J. Blood Rev. 1997, 11, 178;
(c) Bauer, S. M. Exp. Opin. Emergency Drugs 2003, 8, 93.
2. (a) Herbert, J.-M.; Savi, P. Semin. Vasc. Med. 2003, 3, 113; (b) Nicholas, R. A. Mol.
Pharmacol. 2001, 60, 416; (c) Andre, P.; Delaney, S. M.; LaRocca, T.; Vincent, D.;
DeGuzman, F.; Jurek, M.; Koller, B.; Phillips, D. R.; Conley, P. B. J. Clin. Invest.
2003, 112, 398; (d) Hollopeter, G.; Jantzen, H.-M.; Vincent, D.; Li, G.; England,
L.; Ramakrishnan, V.; Yang, R.-B.; Nurden, P.; Nurden, A.; Julius, D.; Conley, P. B.
Nature 2001, 409, 202.
well hPRP aggregation assay monitors aggregation based on the increase of
light transmittance through a stirring suspension of platelets after stimulation
by adenosine diphosphate (ADP) as they aggregate into large particles. Assays
were performed using SpectroMax 190 Microplate Spectrophotometer with
SoftMax Pro software from Molecular Devices. Platelets were tested for their
sensitivity to compounds in 180
18 L of 10X compound and 14
Reactions were initiated by the addition of 20
l
l
L reaction volumes containing 144
L of 10X ADP in a 96-well polystyrene plate.
M ADP. Compounds were
M. Each concentration of inhibitor was
lL PRP,
l
l
tested at a final concentration of 30
tested in duplicate.
l
3. Savi, P.; Pereillo, J. M.; Uzabiaga, M. F.; Combalbert, J.; Picard, C.; Maffrand, J. P.;
Pascal, M.; Herbert, J. M. Thrombo. Haemost. 2000, 84(1), 891.