126
N. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 123–126
Table 3
Fold selectivitya of selected compounds for the hEP3 receptor versus other prostanoid
receptors.
2400
1900
1400
900
Compound hIP versus
hEP3
hFP versus
hEP3
hEP4 versus hEP2 versus hEP1 versus
hEP3
hEP3
hEP3
7y
7r
838
31555
227
671
401
1053
521
445
596
3290
1682
1293
1474
5741
1401
950
1656
1094
ND
1452
6230
1074
7625
2110
3604
442
ND
13090
563
3726
ND
10769
ND
12261
ND
5189
3520
5421
950
1861
1492
5282
2034
1768
2391
1360
1363
4218
8875
21391
13421
1496
1233
9672
2900
7ab
7u
7ad
7aa
7t
400
2243
2816
4481
1058
1298
7298
2709
3350
2171
5138
1518
2151
ND
-100
-4
-3
-2
-1
0
1
2
300
Log Cmpd [8aa] µM
7h
8s
1031
2120
384
2029
1871
728
614
570
5540
1088
Figure 2. Dose–response curve showing IC50 for hEP3 binding assay in normal
buffer (–j–) and in the presence of 10% human serum (–.–).
8aa
7p
8am
7al
7f
7a
7n
7m
478
713
2867
1099
In conclusion, the 3,4-disubstituted indole template yielded a
series of potent and selective hEP3 antagonists. In our SAR studies,
we have successfully addressed both plasma protein binding and
metabolic liability of the lead candidates from this series. Com-
pounds 7aa and 8aa displayed full antagonistic behavior against
the target hEP3 receptor (IC50 = 0.3 and 1 nM for 7aa and 8aa,
respectively) in the cell-based assay. These molecules also dis-
played good potency in the platelet aggregation studies and were
selected for in vivo efficacy studies.
4176
3730
ND, not determined.
a
Ratio (IC50 receptor shown/IC50 hEP3 receptor).
Table 4
Metabolic stability versus rat and human liver microsomal preparations (in vitro).a
References and notes
Compound
7p
8p
7ad
8ad
7y
8y
7aa
8aa
Human
Rat
37
0
79
72
52
14
75
98
23
35
60
68
46
74
69.7
68.8
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Kiryiama, A.; Ushukubi, K.; Kobayashi, T.; Hirata, M.; Sugimoto, Y.; Narumiya, S.
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a
Each compound at 5 lM was incubated with liver microsomes representing
0.8 mg/mL protein concentration and the percent parent remaining at 30 min, as
determined by LC–MS/MS is reported. For comparison, compound 1 under these
assay conditions provided 68% and 2% parent remaining for rat and human liver
microsomes, respectively.
4. Chen, M. C. Y.; Amirian, D. A.; Toomey, M.; Sanders, M. J.; Soll, A. H.
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N.; Lamontagne, S.; Metters, K. M.; Sawyer, N.; Slipetz, D.; Truchon, J. F.;
Labelle, M. Bioorg. Med. Chem. Lett. 2003, 13, 3813; (b) Gallant, M.; Carriere, M.-
C.; Chateauneuf, A.; Denis, D.; Gareau, Y.; Godbout, C.; Greig, G.; Juteau, H.;
Lachance, N.; Lamontagne, S.; Metters, K. M.; Rochette, C.; Ruel, R.; Slipetz, D.;
Sawyer, N.; Tremblay, N.; Labelle, M. Bioorg. Med. Chem. Lett. 2002, 12, 2583; (c)
Juteau, H.; Gareau, Y.; Labelle, M.; Sturino, C. F.; Sawyer, N.; Tremblay, N.;
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2001, 11, 747.
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Table 5
Primary and secondary assay results for the sulfide and sulfone pair, 7aa and 8aa.
hEP3 (nM)a
mEP3 (nM)a
Rat platelet aggregation IC50 (nM)
b
Compound
7aa
8aa
1.7
4.7
15.5
2.0
358
136
a
IC50 from displacement binding assays using 3H-PGE2.
The assay was performed using PGE2 and collagen as coaggregants in the
b
presence of 20% rat serum.
Sulfide/sulfone derivatives 7aa and 8aa showed (i) high affinity
for the hEP3 and mEP3 receptor (Table 5), (ii) consistent stability in
both rat and human liver microsomes assay and (iii) good selectiv-
ity against the family of prostanoid receptors, were further evalu-
ated ex vivo using a platelet aggregation assay (rat-platelet rich
plasma, PRP).17 Both analogs afforded full antagonism of the hEP3
receptor in the cAMP functional assay. The sulfone 8aa gave 2.5-
fold better activity in the rat platelet aggregation assay compared
to the corresponding sulfide 7aa, and the sulfone 8aa showed bet-
ter activity versus mouse receptor in the binding assay as well. (Ta-
ble 5). Compound 8aa also showed low fold-shift in the presence of
human serum. The hEP3 binding curve in the normal buffer and in
the presence of 10% human serum for is shown graphically in Fig-
ure 2.
15. Beveridge, S.; Harris, R. L. N. Aust. J. Chem. 1971, 24, 1229.
16. Attempted formation of limiting amounts of the oxidizing agents generally
lead to mixtures of sulphide and sulphone which indicated that this series of
sulphoxides are more prone to subsequent chemical oxidation versus their
sulphide counter parts.
The molecule 8aa was further selected for the in vivo efficacy
studies.
17. Kawasaki, T.; Ozeki, Y.; Igawa, T.; Kambayashi, J. Stroke 2000, 31, 591.