J. Lloyd et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1436–1439
1439
hERG,10 sodium11 and L-type calcium12 ion channels. This com-
pound was crystallized and the single crystal X-ray analysis
showed the R stereochemistry in the dihydropyrimidine ring and
S stereochemistry in the pyrrolidine ring (Fig. 2).
Table 4
Inhibition of KV1.5 of 2-(4-fluorophenyl)pyrrolidine amides
a
Compds
KV1.5 inhibition IC50
(
lM)
7a
7b
7c
7d
2.1
1.8
0.15
0.05
The pharmacokinetics of 7d was investigated in rats and dogs
(Table 5).13 Compound 7d has intermediate systemic clearance in
rats. Steady-state volume of distribution was greater than total body
water, indicating significant extravascular distribution. Terminal
half-life was 0.57 h in rats. Oral bioavailability (F) was 51%. Half-life
was longer in dogs (1.5 h) with slightly lower bioavailability.
The pharmacodynamic activity of 7d was tested in a rabbit
model which measured the effective refractory period (ERP) in
both atrium and ventricle.14 Like humans, rabbits express the IKur
current in atrium but not ventricle. The compound was dosed at
0.3, 1.0, 3.0, and 10 mg/kg and prolonged atrial ERP by >20% at a
dose of 3 mg/kg. Reflecting the selectivity for KV1.5 over ventricu-
lar ion channels, 7d showed no effect on ventricular ERP up to the
highest dose of 10 mg/kg with plasma concentration of nearly
a
Values are means of 2–4 experiments.
Cl
F
Cl
N
7d
KV1.5 IC50 0.05 uM
hERG IC50 10.5 uM
O
I
Na IC50 >10 uM (38% inh)
Ca IC50 >10 uM (7% inh)
N
N
I
N
H
Figure 2. Stereochemistry and activity of 7d.
7
lM (Fig. 3). Plasma free fraction in rabbits for 7d was 3.0%.
In conclusion, pyrrolidine amides of pyrazolodihydropyrimi-
Table 5
dines were discovered as potent and selective KV1.5 blockers. A
substituent at the 2-position significantly enhanced activity and
the S-configuration is favored over the R-configuration. Aryl and
some uncharged heteroaryl substituents were well tolerated. Com-
pound 7d was chosen for more complete in vitro and in vivo eval-
uation and found to be potent in a pharmacodynamic model
measuring effective refractory period. This compound was chosen
for further pre-clinical toxicology studies and development as a
clinical candidate.
Pharmacokinetic parameters of compound 7d in rats and dogs
Rata
Boga
Dose (
lmol/kg)
10 (inf)b
10 (inf)b
20 (po)c
20 (po)c
F (%)
t1/2 (h)
Clearance (mL/min/kg)
Vdss (L/kg)
51
0.57 0.17
35
1.6 0.6
6
37
7
1.5 0.6
42 4.3
2.1 0.08
9
a
b
c
Values are means from three animals.
inf = intra-arterial infusion for 10 min.
po = oral gavage.
References and notes
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3. Nattel, S.; Khairy, P.; Roy, D.; Thibault, B.; Guerra, P.; Talajic, M.; Dubuc, M.
Drugs 2002, 62, 2377.
40
(n=5)
Atrial ERP
4. Roden, D. M. Br. J. Pharmacol. 2008, 154, 1502.
Ventricular ERP
6.95 2.8
30
20
10
0
5. Amos, G. J.; Wettwer, E.; Li, Q.; Himmel, H. M.; Ravens, U. Circulation 1994, 90, I-
581a; Li, G. R.; Feng, J.; Yue, L.; Carrier, M.; Nattel, S. Circ. Res. 1996, 78, 689.
6. Vacarro, W.; Huynh, T.; Lloyd, J.; Atwal, K. S.; Finlay, H. J.; Levesque, P. C.;
Conder, M. L.; Jenkins-West, T.; Shi, H.; Sun, L. Bioorg. Med. Chem. Lett. 2008, I18,
6381.
1.37 0.52
0.37 0.14
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75, 215.
0.13 0.03
8. Garvey, D. S.; Wasicak, J. T.; Elliott, R. L.; Lebold, S.; Hettinger, A.-M.; Carrera, G.
M.; Lin, N.-H.; He, Y.; Holladay, M. W. J. Med. Chem. 1994, 37, 4455.
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10. Zhou, Z.; Vorperian, V.; Gong, Q.; Zhang, S.; January, C. T. J. Cardiovas.
Electrophys. 1999, 10, 836.
-10
0.3
1
3
10
11. Abriel, H.; Wehrens, X. H. T.; Benhorin, J.; Kerem, B.; Kass, R. S. Circulation 2000,
102, 921.
BMS-394136 Dose (mg/kg I.V.)
12. Fearon, I. M.; Ball, S. G.; Peers, C. Br. J. Pharmacol. 2000, 129, 547.
13. Compound 7d was administered to rats and dogs, as a solution in polyethylene
glycol 200: ethanol/water (1:1:1). Plasma was prepared from each blood
sample by centrifugation and analyzed by LC/MS. Plasma concentrations
versus time data were analyzed by non-compartmental methods. The total
plasma clearance, terminal half-life (t1/2), and the steady state volume of
Figure 3. Pharmacodymanic effect of 7d on prolongation of atrial and ventricular
effective refractory period in rabbits (n = 5). Plasma concentrations (lM) are noted
at each point on the AERP versus dose plot.
distribution (Vdss
) were calculated after intra-arterial administration. The
acetate and hexane) to separate the diastereomers and chiral HPLC
(Chiracel AD, 13% isopropanol, heptane with 0.1% TFA) to separate
the enantiomers (Table 4). The most active isomer (7d) showed
excellent activity in blocking KV1.5 and very good selectivity over
absolute oral bioavailability (F, expressed as %) was estimated by taking the
ratio of dose-normalized AUC value after an oral dose to that after an intra-
arterial dose.
14. Sun, H.; Lloyd, J.; Shi, H.; Li, D.; Levesque, P. Heart Rhythm. 2008, 5, S213.