Adenosine analogues as inhibitors of P2Y12 receptor mediated platelet aggregation

Article abstract of DOI:10.1016/j.bmcl.2008.01.038

Modified adenosine derivatives may lead to the development of P2Y₁₂ antagonists that are potent, selective, and bind reversibly to the receptor. Analogues of 2′,3′-trans-styryl acetal-N6-ureido-adenosine monophosphate were prepared by modificat

Full text of DOI:10.1016/j.bmcl.2008.01.038

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2167–2171
Adenosine analogues as inhibitors of P2Y12 receptor
mediated platelet aggregation
James G. Douglass, J. Bryan deCamp, Emilee H. Fulcher, William Jones, Sanjoy Mahanty,
*
´
Anna Morgan, Dima Smirnov, Jose L. Boyer and Paul S. Watson
Inspire Pharmaceuticals, Inc., Department of Medicinal Chemistry, 4222 Emperor Boulevard, Suite 200, Durham, NC 27703, USA
Received 15 November 2007; revised 8 January 2008; accepted 10 January 2008
Available online 13 January 2008
Abstract—Modified adenosine derivatives may lead to the development of P2Y12 antagonists that are potent, selective, and bind
reversibly to the receptor. Analogues of 20,30-trans-styryl acetal-N6-ureido-adenosine monophosphate were prepared by modifica-
tion of the 50-position. The resulting analogues were tested for P2Y12 antagonism in a platelet aggregation assay.
Ó 2008 Elsevier Ltd. All rights reserved.
PlavixÒ (clopidogrel bisulfate tablets), an inhibitor of
ADP-induced platelet aggregation,1 acts by irreversible
inhibition of activation of the P2Y12 receptor.2 It has
been shown clinically that the incidence of myocardial
infarction and stroke are reduced by irreversible inhibi-
tion of ADP-induced platelet aggregation.3
for the discovery of new chemotypes that antagonize
the P2Y12 receptor. In fact, this approach has led to
the discovery of a novel reversible short-acting antag-
onist, cangrelor (1),6 and AZD6140 (2),7 a reversible
orally available antagonist discovered by AstraZeneca
scientists (Fig. 1).
However, several limitations regarding the use of irre-
versible P2Y12 receptor antagonists have been identi-
fied which include (1) a low level of inhibition in
approximately one third of the patient population,
(2) variability of the response from patient to patient
with the available dosing regimens,4 (3) the current
drugs require hepatic activation, and (4) irreversible
inhibition complicates the drug’s usefulness when pro-
cedures, such as coronary artery bypass graft surgery
(CABG), become necessary in a patient’s treatment
protocol.5 In the past decade, there have been several
efforts to design and develop improved irreversible
P2Y12 receptor antagonists and to explore the useful-
ness of reversible intravenous and orally administered
P2Y12 receptor antagonists. Most of these efforts focus
on the fact that adenosine triphosphate (ATP) acts as
an antagonist of the P2Y12 receptor. This invites the
possibility of exploring ATP mimetics as a foundation
Although the ATP mimetic approach has been suc-
cessful, several other reversible P2Y12 antagonists have
been described that do not mimic the ATP structural
components necessary for inhibition (Fig. 1).8,9 Work
in our own laboratories led to the discovery of
INS50589 (3), an adenosine monophosphate derivative
with 20,30-cyclic acetal and N6-urea modifications
(Fig. 2).10
Compound 3 inhibits platelet aggregation with an IC50
of 16 nM. This compound was evaluated in early clinical
trials as an intravenously delivered drug for cardiovas-
cular use.
This letter describes our initial efforts to discover non-
phosphate50-substituentsthatwouldmaintain thepotency
of our phosphate analogues and might also be useful for
developing an orally active reversible antagonist.
Based on previous results in the phosphate series, we
decided to focus on derivatives that contain the trans-
acetal derived from the reaction of the 20- and 30-hydro-
xyl groups of adenosine with trans-cinnamaldehyde and
an N6-ethyl urea. Our previous work demonstrated
Keywords: Adenosine derivatives; P2Y12 antagonists; Platelet aggre-
gation assay.
*
Corresponding author. Tel.: +1 919 287 1268; fax: +1 919 941
9797; e-mail: pwatson@inspirepharm.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.038

2168
J. G. Douglass et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2167–2171
SMe
NH2
HN
N
N
N
N
N
N
OH OH OH
Cl
Cl
NaO
ONa ONa
HO
O
O
O
CF3
NaO
O
O
N
P
P
P
S
N
O
P
O
P
P
O
O
O
O
O
O
HO
OH
HO
OH
1, cangrelor
ATP
CO2H
F
F
O
N
Me
Me
HN
O
N
H
N
N
N
N
Me
HO
Me
N
N
S
O
EtO
N
O
BX 048
O
2, AZD6140
HO
OEt
OH
O
OH
OH
O
O
S
O
O
EtO
S
N
N
N
H
S
S
N
CT 50547
O
Figure 1. Potent P2Y12 antagonists in preclinical and clinical development.
NHEt
these functional groups were important for both P2Y12
potency and selectivity. Conveniently, the acetal also
provided the synthetic advantage of protecting the sec-
ondary alcohols of adenosine from interfering with
N6- and 50-alcohol modifications. The synthesis of start-
ing material 4 has been described elsewhere.11
HN
N
O
N
N
N
ONa
NaO
O
P
O
O
O
O
50-Amides and amines. Alcohol 4 was reacted with para-
toluenesulfonyl chloride in pyridine at 0 °C to afford the
tosylate 5 in high yield (Scheme 1, 95%). Tosylate 5 was
displaced with sodium azide to give azide 6, which was
then reduced using Staudinger’s conditions to provide
the 50-amine 7 in 80% yield. Reductive aminations of 7
using an aldehyde and sodium triacetoxyborohydride
in dichloroethane (DCE) afforded secondary and ter-
tiary amines. In addition, amine 7 was used to construct
several amides by treating the amine with commercially
available acids in the presence of EDC, HOBt, and
DMF (Table 1).
3, INS50589
NH2
N
N
N
Ph
OH OH OH
NHEt
O
N
HO
O
O
O
P
P
P
O
HN
O
O
O
N
N
N
HO
OH
N
ATP
R
O
O
O
Ph
50-Sulfides and sulfones. Tosylate 5 was also treated
with several thiols under basic conditions to afford
Figure 2. Simple ATP mimetics as P2Y12 antagonists.
Me
HN
Me
O
Me
O
HN
HN
HN
HN
HN
O
N
N
N
N
N
N
N
N
N
N
N
N
HO
R
H2N
a
b
O
O
O
O
O
O
O
O
O
Ph
Ph
Ph
4
7
R = OTs, 5
R = N3, 6
Scheme 1. Reagents and conditions: (a) TsCl, pyridine, 0 °C then NaN3, DMF, 100 °C, 85% for two steps; (b) PPh3, THF, H2O, 80%.

J. G. Douglass et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2167–2171
2169
Table 1. Effect of selected amines and amides on the relative potency
to inhibit platelet aggregation in a washed platelet assay
sulfide derivatives 8 (Scheme 2). The sulfides were
then oxidized to the corresponding sulfones 9 using
potassium hydrogen persulfate (oxoneÒ) at 0 °C in
methanol and sodium bicarbonate (pH 5) (see
Table 2).
NHEt
HN
N
O
N
N
N
50-Ethers and acids. Alcohol 4 was also oxidized directly
(Scheme 3) to either the aldehyde (10) or the corre-
sponding acid (11). Aldehyde 10 was then homologated
using either a stabilized or unstabilized Wittig reagent.
Subsequent modifications provided chain extended car-
boxylic acids. For example, aldehyde 10 was reacted
with the anion of (EtO)2POCH2CO2Me in THF, re-
duced and hydrolyzed to provide acid 12. In addition,
alcohol 4 was also alkylated with reactive electrophiles
to provide a variety of alkyl and aryl ether derivatives
(see Table 3). Experimental conditions for all derivatives
reported in this letter are provided in the Supplemental
materials section.
R3HN
O
O
O
Ph
Compound R3
IC50 lM (n = 3) % Inhibition
at 30 lM
7
H
4.73 1.94
—
EtO2C
13
4.33 1.40
—
As part of the design of the acute use P2Y12 receptor
antagonist INS50589 (3), the phosphate group contrib-
utes to the high binding potency yet is readily cleaved
in situ by endogenous ectonucleotidases to a 100-fold
less potent metabolite (4, IC50 = 1.27. 0.41 lM).
Our goal in this study was to determine what type
of functional groups could regain this loss in potency
and serve as potential leads for chronic orally active
P2Y12 antagonists. Initially we examined the potency
of the 50-alcohol (4), amine (7), and thiomethyl ether
(27). None of these derivatives provided any gain in
potency relative to 4. Thiopropyl (28) and thioheptyl
(30) derivatives were also found to be inactive. Substi-
tution of the propyl chain with hydroxyl groups (33
and 35) proved to be better than the alkyl derivatives,
and (for 35) comparable in potency to alcohol 4. Re-
moval of the terminal methyl group of 33, hydroxyl
ethyl derivative 31, led to a loss of potency. The cor-
responding sulfones of 28, 31, 33, and 35 (29, 32, 34,
and 36, respectively) offered no improvement. The
highly charged terminal sulfate (37) was prepared
and found to be modestly potent. On the other hand,
acetic acid derivative 38 and its respective sulfone
derivative 39 were both found to be comparable in po-
tency to 4.
EtO2C
HO2C
14a
15
—
46 4.2
—
2.70 0.95
HO2C
16a
17
0.58 0.11
—
OH
F
—
70
—
7
18
2.55 0.68
F
19a
—
NR
N
S
20
21
—
—
73 12
70 12
Me
EtO2C
O
22
23
0.513 0.16
2.95 0.32
—
—
HO
HO
Based on these findings we focused our efforts on
replacing the phosphate charge with a carboxylic acid
moiety. Preparation of the one, two, and three car-
bon analogues, 11, 40, and 12, respectively, resulted
in improved potency when compared to 4 (1.27 lM
vs 0.201, 0.663, and 0.495 lM, respectively). The
nitrogen and oxygen versions of sulfide 38 (amine
22, 0.513 lM and ether 41, 0.243 lM) proved to be
fairly potent, with the ether comparable in potency
to the one carbon acid derivative 11 (0.243 vs
0.201 lM, respectively). The slightly more rigid a,b-
cyclopropane amino acids (15 and 16), 5 atoms in
length, were found to be close in potency to 4. Inter-
estingly, one of their corresponding esters (13) did
not lose much in binding potency. In the amine series
O
24
25
4.86 0.91
2.23 0.44
—
—
O
S
O
O
Me
O
Me
26
—
53
7
Me
N
H
O
a Trisubstituted amine derivative.

2170
J. G. Douglass et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2167–2171
Me
O
Me
O
Me
O
HN
HN
HN
HN
HN
HN
N
N
N
N
N
N
N
N
N
N
N
N
R3S
R3O2S
TsO
O
a
b
O
O
O
O
O
O
O
O
5
8
9
Scheme 2. Reagents and conditions: (a) R3SH, t-BuOK, DMSO; (b) 8, oxone, NaHCO3, MeOH, 0 °C.
Table 2. Effect of selected 50-sulfides and sulfones on the relative
potency to inhibit platelet aggregation in a washed platelet assay
Me
O
Me
O
HN
HN
HN
HN
NHEt
HN
N
O
N
N
N
N
N
N
HO
O
N
N
N
N
N
R3
O
R
a or b
c
O
O
4
O
O
O
O
O
O
Ph
Ph
Ph
R = CHO, 10
R = CO2H, 11
12
Compound
R3
IC50 lM
(n = 3)
% Inhibition
at 30 lM
Scheme 3. Reagents and conditions: (a) Dess–Martin periodinane,
CH2Cl2 for 10; (b) NaClO2, NaHPO4, DMSO, H2O, 0 °C for 11; (c)
(EtO)2POCH2CO2Me, NaH, THF, then 10; CuSO4, NaBH4, MeOH;
LiOH, THF, H2O, 22% for three steps.
27
28
29
30
31
32
–SCH3
—
—
—
—
—
—
63
52
65
38
63
57
6
5
7
9
2
3
–SCH2CH2CH3
–SO2CH2CH2CH3
–SCH2(CH2)5CH3
–SCH2CH2OH
–SO2CH2CH2OH
OH
modestly potent. Recognizing that the 50-heteroatom
of choice was oxygen, aryl groups were tolerated
and acids provided some increase in potency (ꢀ6-
fold) respective to alcohol 4, we decided to examine
50-benzyl ethers that differed in the location of a car-
boxylic acid on the ring. It was eventually found that
in the series, ortho (42), meta (43), and para (44) the
potency to inhibit platelet aggregation was measured
to be 0.040, 0.616, and 1.31 lM, respectively. Benzoic
acid 42 was only 2-fold less potent than our starting
phosphate clinical candidate.
33
34
35
36
—
76
—
—
14
4
S
Me
OH
Me
O
S
O
2.49 0.8
OH
OH
9.9
2
S
OH
OH
O
S
—
—
5
6
O
37
38
39
–SCH2(CH2)2SO3H
–SCH2CO2H
–SO2CH2CO2H
90
—
—
3.8 0.8
2.48 0.6
Using the acetal/urea ribose backbone established from
INS50589, we have discovered non-phosphate 50-modi-
fications that have achieved similar low nanomolar
activity. We were able to identify a 2-carboxybenzyl
group as a bioisostere for a phosphate group. Addi-
tional studies are planned to evaluate the pharmacoki-
netic and pharmacodynamic effects of this new
functional group in a series of antagonists as well as
the effects of modifications of the acetal and urea sub-
stituents on P2Y12 potency.
we prepared and examined the potency of a ran-
domly selected list of benzyl/heteroaryl amines (17–
20) and heteroaryl amides (24–26) without much suc-
cess in improving potency but not completely elimi-
nating it either. Amide 23 was found to be

J. G. Douglass et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2167–2171
Table 3. Effect of 50-acids and ethers on the relative potency to inhibit
platelet aggregation in a washed platelet assay
2171
References and notes
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NHEt
HN
N
O
N
N
N
4
R
5. Yende, S.; Wunderink, R. G. Crit. Care Med. 2001, 29,
2271.
O
O
O
6. van Giezen, J. J.; Humphries, R. G. Semin. Thromb.
Hemost. 2005, 31, 195.
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Ph
Compound
R4
IC50 lM (n = 3)
3
4
–CH2OPO(ONa)2
–CH2OH
–CO2H
0.016 0.013
1.27 0.41
11
40
12
41
0.201 0.071
0.663 0.020
0.495 0.041
0.243 0.06
–CH2CO2H
–CH2CH2CO2H
–CH2OCH2CO2H
42
0.040 0.017
-CH2OCH2
CO2H
9. Scarborough, R. M.; Laibelman, A. M.; Clizbe, L. A.;
Fretto, L. J.; Conley, P. B.; Reynolds, E.; Sedlock, D.
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43
44
0.616 0.214
1.31 0.38
-CH2OCH2
-CH2OCH2
CO2H
CO2H
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.01.038.