M. O. Clarke et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3568–3572
3571
21, for example, was found to have F = 0.4% (10.7 mg/Kg po dose)
and CL = 1.72 L/h/Kg (0.4 mg/Kg iv dose) in rats.
acid 27 was found to be almost 7-fold less potent than acyclic
o-MeO benzyl phosphinic acid 28, while there was no significant
difference between the corresponding macrocyclic benzyl phos-
phinic acids (compounds 31 and 33).
The next series of phosphinic acids to be explored was the phe-
nyl and benzyl substituted phosphinic acids (Table 3). It was again
found that di-substitution of the carbon attached to phosphorous is
very poorly tolerated, as phenyl phosphinic acid 25 had a double
digit IC50 and a micromolar EC50. The simplest benzyl phosphinic
acid, compound 26, was equipotent to compound 21 on the
enzymatic level, but lost about 2.5-fold activity in the cell based
assay. More importantly, however, pharmacokinetic evaluation
showed that oral absorption had been achieved (F (dog) = 4.6%),
although clearance remained intermediate, to high (CL (rat) =
2.2 L/h/Kg; CL (dog) = 0.64 L/h/Kg). Further exploration of the
substitution of the aryl ring in the benzyl phosphinic acids found
that, while the enzymatic activity could be kept constant, efficacy
in the cell based replicon could be both eroded (o-Cl benzyl phos-
phinic acid 27) and improved (o-MeO benzyl phosphinic acid 28).
Expanding upon the advances gained with benzyl phosphinic
acids, macrocyclization to form a 15-membered ring was em-
ployed to further increase the potency of these compounds.16 This
macrocyclization tactic resulted in a dramatic increase in the po-
tency of the phosphinic acids, particularly in the cell based assay
(Table 4). Notably, large differences in EC50’s between acyclic
ortho-substituted benzyl phosphinic acids were lost upon macro-
cyclization. By way of an example, acyclic o-Cl benzyl phosphinic
It has previously been reported that saturation of the macrocy-
cles in carboxylic acid based NS3 protease inhibitors can have a
dramatic effect on their pharmacokinetic profile.17 Saturation of
the macrocyclic benzyl phosphinic acids resulted in a maintenance,
or slight improvement, in EC50 values, while also having a pro-
nounced effect on their pharmacokinetic profile (Table 5). The
clearance of 31, after an iv administration to dogs, was found to
be 0.56 L/h/Kg, while the saturated analog 32 was found to be
0.19 L/h/Kg. This same trend was also seen in the o-MeO macrocy-
clic analogs: CL (dog) = 1.04 L/h/Kg for 33 and CL (dog) = 0.32 L/h/Kg
for 34. Compound 30 was found to have a similar clearance (CL
(dog) = 0.36 L/h/Kg) to that of the other saturated macrocycles,
while maintaining the bioavailability (F (dog) = 6.4%) seen in com-
pound 26.
In summary, a new, potent class of phosphinic acid derived
HCV NS3 protease inhibitors has been discovered. Advancement
from alkyl phosphinic acids to benzyl phosphinic acids resulted
in improved pharmacokinetic profiles, notably the achievement
of oral bioavailability with compound 26. Macrocyclization of
the benzyl phosphinic acids resulted in single digit nanomolar
inhibitors. Saturation of the macrocycle of benzyl phosphinic
acids resulted in improved clearances, and in the case of com-
pound 30, a single digit nanomolar inhibitor with measurable oral
bioavailability. The optimization of these promising macrocyclic
benzyl phosphinic acids is continuing, in an effort to discover a
therapeutic agent for HCV.
Table 4
IC50/EC50 measurements of macrocyclic, benzyl phosphinic acids
Acknowledgments
The authors thank Huiling Yang, Margaret Robinson and Scott
Hluhanich for determining biological activities.
References and notes
a
b
Compd
R
Macrocycle HCV NS3 IC50
M)
HCV replicon EC50
M)
(l
(l
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29
30
31
32
33
Bn
Bn
Olefin
Saturated
Olefin
Saturated
Olefin
0.003
0.006
0.001
0.003
0.003
0.010
0.005
0.005
0.005
0.009
o-Cl-Bn
o-Cl-Bn
o-MeO-
Bn
o-MeO-
Bn
34
Saturated
0.003
0.007
a
IC50 determined by enzymatic assay using a HCV genotype 1b NS3/4A protein.
EC50 determined by cell based assay using Huh-7 cells harboring subgenomic
b
HCV genotype 1b replicon.
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Table 5
PK (dog) parameters of select compounds
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Compd
Macrocycle
F (po)
CL (1 mg/Kg iv)
2
No
No
Saturated
Olefin
Saturated
Olefin
0.5%a
4.6%a
6.4%b
NA
NA
NA
0.67 L/h/Kg
0.64 L/h/Kgc
0.36 L/h/Kg
0.56 L/h/Kg
0.19 L/h/Kg
1.04 L/h/Kg
0.32 L/h/Kg
26
30
31
32
33
34
Saturated
NA
12. Pyun, H.-J.; Chaudhary, K.; Somoza, J. R.; Sheng, X. C.; Choung, U. Tetrahedron
Lett. 2009, 50, 3833.
13. Casarez, A.; Chaudhary, K.; Cho, A.; Clarke, M.; Doerffler, E.; Fardis, M.; Kim, C.
U.; Pyun, H.-J.; Sheng, X. C.; Wang, J. WO Patent 2008/005565 A2.
a
b
c
The dose used was 3 mg/Kg po.
The dose used was 4 mg/Kg po.
The dose used was 0.5 mg/Kg iv.