R. M. Scarborough et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1805–1808
1807
parent unacylatedtricyclic ring analogue 19 (Table 2). It
is not readily apparent from this limited set of analo-
gues what factors may be influencing the reactivity of
the 2-aminobenzothiazole with chlorosulfonylacetyl
chloride or what promotes further acylation of the tri-
cyclic ring system 4.
non-nucleoside based antagonists of the P2Y12 receptor
andmake them useful reagents to stu yd this newly
characterizedandimportant platelet ADP receptor.
Acknowledgements
Analogues of 2 exhibit a very stringent structure–activity
relationship (Table 1). Only analogues with small sub-
stitutents on the benzo ring of the tricyclic nucleus that
have the ability to participate in hydrogen bonding
retain goodreceptor affinity (analogues 2, 7, and 9).
Other electron withdrawing or bulky substituents
attachedto the benzo ring greatly re du ce the P2Y
The authors thank Bonnie Loo, Laurent Gousset and
Fahimeh Raoufi for technical assistance with the bio-
assays.
References and Notes
1
2
receptor binding affinity of analogues. One of the two
unacylatedbenzothiazolo[2,3- c]thiadiazines prepared
retainedlittle functional activity (analogue 19, Table 2),
and displayed extremely weak inhibition in the binding
assay. Thus, from this limitedset of analogues, activity
in this series of P2Y12 antagonists optimally requires a
relatively small substituent R in the benzo ring that is
capable of forming a potential H-bondwith the recep-
tor. Additionally, a 2-oxo-ethanesulfonamide group
attachedto the tricyclic nucleus is requiredto retain
high affinity for the P2Y12 platelet ADP receptor (2 and
1
2
4
3
4
. Awtry, E. H.; Loscalzo, J. Circulation 2000, 101, 1206.
. Scarborough, R. M.; Gretler, D. D. J. Med. Chem. 2000,
3, 3453.
. Quinn, M. J.; Fitzgerald, D. J. Circulation 1999, 100, 1667.
. (a) Daniel, J. L.; Dangelmaier, C.; Jin, J.; Ashby, B.; Smith,
J. B.; Kunapuli, S. P. J. Biol. Chem. 1998, 273, 2024. (b) Jant-
zen, H. M.; Gousset, L.; Bhaskar, V.; Vincent, D.; Tai, A.;
Reynolds, E. E.; Conley, P. B. Thromb. Haemost. 1999, 82, 111.
5. Hollopeter, G.; Jantzen, H. M.; Vincent, D.; Li, G.; Eng-
land, L.; Ramakrishnan, V.; Yang, R. B.; Nurden, P.; Nurden,
A.; Julius, D.; Conley, P. B. Nature 2001, 409, 202.
6. Savi, P.; Pereillo, J. M.; Uzabiaga, M. F.; Combalbert, J.;
Picard, C.; Maffrand, J. P.; Pascal, M.; Herbert, J. M.
Thromb. Haemost. 2000, 84, 891.
7
–17 versus 18 and 19).
To assess the selectivity of these series for the P2Y12
receptor, the most potent compound, CT50547, was
7
. (a) Humphries, R. G.; Tomlinson, W.; Clegg, J. A.; Ingall,
A. H.; Kindon, N. D.; Leff, P. Br. J. Pharmacol. 1995, 115,
110. (b) Humphries, R. G.; Robertson, M. J.; Leff, P. Trends
evaluatedfor inhibition of the clonedP2Y
andthe other recently de scribedplatelet ADP receptor,
receptor
2
1
1
Pharmacol. Sci. 1995, 16, 179. (c) Ingall, A. H.; Dixon, J.;
Bailey, A.; Coombs, M. E.; Cox, D.; McInally, J. I.; Hunt,
S. F.; Kindon, N. D.; Teobald, B. J.; Willis, P. A.; Humphries,
R. G.; Leff, P.; Clegg, J. A.; Smith, J. A.; Tomlinson, W. J.
Med. Chem. 1999, 42, 213.
9
P2Y . CT50547 (also referredto as C1330-7 ) blocked
1
the human P2Y12 receptor expressedin Xenopus oocytes
5
with an IC50 value of 40 nM. Washedhuman platelets
or Jurkat cells transfectedwith the clonedhuman P2Y 1
receptor were loaded with Fura-2 and peak elevations of
intracellular calcium ions in response to P2Y receptor
8
. Savi, P.; Laplace, M. C.; Maffrand, J. P.; Herbert, J. M. J.
Pharmacol. Exp. Ther. 1994, 269, 772.
. Characterization of 2 (also known as C1330-7, see ref 5)
HRMS for C H N O S : M+H expected597.0242; M+H
1
9
stimulation were determined by fluorescence spectro-
4
b
22 20
4
1
8 4
scopy. The IC50 value for CT50547 in each of these
assays was >100 mM, indicating a 1000-fold selectivity
6
obtained: 597.0248. H NMR (DMSO-d ) d 7.95–7.97 (d,
J=8.8 Hz, 1H, aromatic H), 7.33 (d, J=2.4 Hz, 1H, aromatic
H), 7.27 (d, J=2.4 Hz, 1H, aromatic H), 6.93–6.95 (d, J=9.0,
for P2Y12 over P2Y (data not shown).
1
1
H, aromatic H), 6.81–6.83 (dd, J=2.4, 9.0 Hz, 1H, aromatic
H), 6.49–6.51 (dd, J=2.6, 9.2 Hz, 1H, aromatic H), 4.65 (s,
2H, CH ),
), 3.93–4.01 (qÂ2, J=7.0, 7.0 Hz, 4H, ethyl CH
). C NMR
) d 189.84, 167.82, 159.13, 158.95, 156.04, 155.46,
While CT50547 is a potent andnovel antagonist of the
platelet P2Y12 receptor, the stringent requirements for
activity in this series limits the useful SAR andthe
potential to further manipulate these compounds into
drug candidates. However, CT50547 and analogues
are the first reversible, purinergic receptor-specific,
2
2
1
3
1.27–1.31 (tÂ2, J=7.0, 7.0 Hz, 6H, ethyl CH
3
(
DMSO-d
6
1
1
1
1
31.09, 130.43, 126.84, 122.83, 119.52, 114.99, 113.53, 113.38,
07.89, 107.74, 101.41, 64.16, 63.93, 63.11, 15.12, 15.06. IR
À1
588/1553 cm
0. Alternate possible structure:
.
Table 2. Benzothiazolo[2,3-c]thiadiazine analogues 18–19
Analogues
R
[3H]2-MeS-ADP
binding inhibition
Aggregation
inhibition
IC50 (mM)
a
a
IC50 (mM)
1
1
8
9
7-CF
5-CH
3
O
>120
77 (Æ4.7)
>50
>120
1
1. The final compounds were analytically pure as determined
1
3
O
1
by HPLC, H NMR andMS. Representative synthesis: 2, N -
(6-Ethoxy-1,3-benzothiazol-2-yl-2-(7-ethoxy-4-hydroxy-2,2-dioxo-
2H-2] benzo[4,5][1,3]thiazolo[2,3-c][1,2,4] thiadiazin-3-yl)-2-oxo-
aValues are means of 2–3 experiments performedin du plicate, stan-
dard deviation is given in parentheses.
6