20-F-20-C-Methyl Nucleosides and Nucleotides for the Treatment of Hepatitis C Virus: from Discovery to the Clinic - Accounts in Drug Discovery Case Studies in Medicinal Chemistry - page 243

O>>CH 2

Cytosine >> Guanosine

Base

W

HO

No compatible
substitution
α -OH only

X

CH 3 >CH 2 F

Z

Y

F>OH>H

Figure 11.2 SAR overview of anti-HCV activity for the 2 0 -F-2 0 -C-methyl class of

nucleosides is shown, thus demonstrating the unique specificity of this
class of nucleosides.

atom. Overall, this study demonstrated that the 3 0 -a-hydroxyl group was cri-
tical for activity, no substitution at 4 0 was tolerated, the carbocyclic analog
was inactive, and the only natural base or base analog that afforded workable
activity was the cytosine base. The closest 2 0 -F-2 0 -C-methyl nucleoside
analog that showed any activity was the guanosine analog, for which the
activity was 10-fold less than PSI-6130. 14-16 The second line of investigation

maintained the cytosine base and the 3 0 - and 4 0 -substitutions as in PSI-6130,
but varied the 2 0 -a- and 2 0 -b-substitution. Again, PSI-6130 was the most
potent analog by far. Although the b-CH 2 F derivative showed some activity,
no other substitution at the 2 0 -a-orb-positions provided active HCV inhibi-
tors. 14-16 Based on these SAR studies, the 2 0 -F-2 0 -C-methyl class of nucleosides
afforded limited opportunity to identify more effective nucleosides beyond
PSI-6130.

B

11.2.2 Mechanism of Action of PSI-6130
With PSI-6130 identified as the lead development candidate, a complete
understanding of the metabolism of this nucleoside was required to determine
the mechanism of action. For inhibition of HCV RNA replication to occur,
metabolism of the nucleoside to the corresponding active 5 0 -triphosphate form
was required. After incubating either replicon cells or primary human hepa-
tocytes with radiolabeled PSI-6130, the expected 5 0 -mono-, di-, and tripho-
sphate derivatives of PSI-6130 were identified. 17,18 As shown in Figure 11.3, the
deaminated metabolite of PSI-6130, b-D-2 0 -F-2 0 -methyluridine, PSI-6206 (8),
and its corresponding 5 0 -mono-, di-, and triphosphate derivatives, were iden-
tified. 17,18 Phosphorylation of PSI-6130 to its corresponding 5 0 -monopho-
sphate (PSI-6130-MP) was shown to be catalyzed by human deoxycytidine
kinase (k cat /K m ¼ 1.9 10 4 mM 1 s 1 ). 18 PSI-6130-MP was subsequently
phosphorylated to the corresponding 5 0 -diphosphate (PSI-6130-DP) and 5 0 -
triphosphate (PSI-6130-TP) by UMP-CMP kinase (k cat /K m ¼ 0.012 mM 1 s 1 )
and nucleoside diphosphate kinase (k cat /K m ¼ 0.015 mM 1 s 1 ), respectively. 18
To demonstrate that PSI-6130-TP was an inhibitor of the HCV RNA-depen-
dent RNA polymerase (RdRp), it was tested using membrane-associated sub-
cellular fractions containing HCV replication complexes (replicase assay) or