Parallel synthesis of pteridine derivatives as potent inhibitors for hepatitis C virus NS5B RNA-dependent RNA polymerase

Article abstract of DOI:10.1016/j.bmcl.2004.11.028

From compound library screening using an HCV NS5B RNA-dependent RNA polymerase enzymatic assay, we identified a pteridine hit compound with an IC₅₀ of 15 μM. Our SAR studies were focused on the different groups at the 6- and 7-positions, substitutions at the 4-position, and replacement of N₁ or N₃ with carbon in the pteridine ring. We found that NH or OH at 4-position is critical for the inhibitory activity. Furthermore, a hydrophobic substituent at the 4-position may help compounds permeate through the cell membrane.

Full text of DOI:10.1016/j.bmcl.2004.11.028

Bioorganic & Medicinal Chemistry Letters 15 (2005) 675–678
Parallel synthesis of pteridine derivatives as potent inhibitors
for hepatitis C virus NS5B RNA-dependent RNA polymerase
Yili Ding,* Jean-Luc Girardet, Kenneth L. Smith, Gary Larson, Brett Prigaro,
Vicky C. H. Lai, Weidong Zhong and Jim Z. Wu
Valeant Pharmaceuticals International, 3300 Hyland Avenue Costa Mesa, CA 92626, USA
Received 15 October 2004; revised 8 November 2004; accepted 12 November 2004
Available online 2 December 2004
Abstract—From compound library screening using an HCV NS5B RNA-dependent RNA polymerase enzymatic assay, we identi-
fied a pteridine hit compound with an IC50 of 15 lM. Our SAR studies were focused on the different groups at the 6- and 7-posi-
tions, substitutions at the 4-position, and replacement of N1 or N3 with carbon in the pteridine ring. We found that NH or OH at
4-position is critical for the inhibitory activity. Furthermore, a hydrophobic substituent at the 4-position may help compounds
permeate through the cell membrane.
Ó 2004 Elsevier Ltd. All rights reserved.
It is estimated that 170 million people worldwide are in-
fected with hepatitis C virus (HCV) including 4 million
in the United States.1 Within 20 years of infection, a sig-
nificant proportion of them will develop cirrhosis and
end-stage liver diseases such as hepatocellular carci-
noma.2 Current therapies based on combinations of
pegylated interferons and ribavirin have limited efficacy
in a significant proportion of patients and are associated
with some severe side effects.3 Thus there is an urgent
need to develop more efficacious and safer therapeutics
for the treatment of chronic HCV infection and associ-
ated liver diseases.4
screening and design of small molecule inhibitors for
viral replication interference.5,6
From a high throughput screening of compound li-
braries by using an HCV NS5B RdRp enzymatic assay,7
we identified an interesting hit 1a with an IC50 of 15 lM.
This compound possesses a pteridine pharmacophore.
Although it is not very potent, it bears certain resem-
blance to a benzimidazole 5-carboxylate NS5B RdRp
inhibitor 1b discovered by Boehringer Ingelheim.8 Com-
pared to the hit 1b, compound 1a is less negatively
charged and may have advantage to possess better cellu-
lar permeability. Since the hit 1b has been successfully
optimized to achieve nanomolar potency,8 we attempted
to optimize compound 1a through systematic SAR stud-
ies. In this communication, we report parallel synthesis
of pteridine derivatives of 1a and evaluation of their
inhibitory activities against HCV NS5B RdRp.
The HCV NS5B RNA-dependent RNA polymerase
(RdRp) is a central enzyme in the replication of the viral
genome and has since become a target of choice for
O
OH
N
N
5
N
OH
4
6
7
Most of the compounds described in this study were pre-
pared according to Scheme 1. Through the condensation
of 4,5-diaminopyrimidone 2 with commercially avail-
able diketones (3a–l) in refluxing acetic acid in the pres-
ence of sodium acetate, compounds 4a–l were obtained
as solids in 50–60% yields. After chlorination with
POCl3, compounds 4a–l were converted into their corre-
sponding chlorinated derivatives 5a–l with 50–70%
yields, which were then used as key intermediates for
the library synthesis. Through a parallel synthetic strat-
egy, compounds 5a–l were reacted, respectively, with
O
3
2
N
N
N
8
1
1a
1b
IC50 = 1.6 µM
IC50 = 15 µM
Keywords: HCV; Inhibitor; NS5B; Pteridine.
*
Corresponding author. Tel.: +1 714 545 0100; fax: +1 714 641
7222; e-mail: yding@icnpharm.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.028

676
Y. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 675–678
1ag and 1ah) was not tolerated for activity. However,
OH
OH
R1
R2
R1
R2
N
N
O
O
the mid-size alkyl amino derivative 1ab was 2-fold more
potent than compound 1a. Further reduction in the size
of the amino group (1aa) results in a slight decrease of
activity. From this initial SAR, it seems that a free
NH is required for maintaining activity, as substitution
by a tertiary amine, as in the case of compound 1ae,
results in a total loss of activity.
H2N
H2N
N
a
N
+
N
N
2
3a-3l
4a-4l
b
2
NR'R"
N
Cl
N
c
After the series of modifications on the 4-position, we
then evaluated the modifications at the 6,7-diaryl moi-
ety. The effects of the modifications are summarized in
Table 2. Substitutions at the para- or meta-position on
either ring with an electron donating group (4b and
4d) resulted in a considerable loss of activity. Introduc-
tion of a fluorine atom at the para-position of the aryl
group (4e) increased the potency about 30-fold with re-
spect to compound 1a with an IC50 of 0.5 lM. Surpris-
ingly, introduction of a bromine atom at the para-
position of the aryl part (4c) dropped the potency by
2-fold, suggesting that NS5B RdRp can not tolerate a
big substituent at the position. Introduction of a chlo-
rine atom at the ortho-position (4h) also resulted in loss
of the activity, indicating that a small group at the para-
position is optimal for activity. When polar heterocycle
groups such as 2-pyridine or 2-furan replaced the phenyl
groups (4i and 4j), the compounds lost inhibitory activ-
ity, suggesting that neutral substituents are preferred at
6,7-positions. Loss of activity for compounds 4k and 4l
indicated that the existence of both aryl groups is re-
quired, and the enzyme NS5B RdRp does not prefer
the planar conformation of 4l.
R1
R2
R1
R2
N
N
N
N
N
N
6ap-6lp
5a-5l
Scheme 1. Reagents and conditions: (a) 3a–l, compound 2, AcOH,
AcONa, 150 °C, 2 h; (b) 4a–l, POCl3, 75 °C, 1 h; (c) 5a–l, amines,
EtOH, 70 °C, 2 h.
different amines to provide compounds 6ap–lp. Com-
pounds 1aa–ah were obtained from the same synthetic
strategy. Their structures were confirmed by ESMS
and 1H NMR spectra. All the compounds for biological
assays are more than 90% pure by (HPLC).
The compounds described in this study were evaluated
for inhibitory activity against HCV NS5B RdRp using
a published procedure.7 Compounds were first dissolved
into DMSO, and then transferred into the assay buffer.
Each compound was tested in duplicate. IC50 (inhibitor
concentration for 50% inhibition) was determined for
each compound with a standard deviation typically
<10%.
We also examined the activity for compounds 4m, 4n
and 4o, in which the N3 and/or N1 in the pteridine ring
was replaced by carbon atom or one aryl group was re-
placed by CONH2 group to assess the importance of the
N1 and N3 moieties and the aryl part. Interestingly,
these compounds possess no activity against NS5B
RdRp, highlighting the importance of the presence of
the N1 and N3 in pteridine pharmacophore, and con-
firmed the importance of the diaryl groups for the
potency.
As shown in Table 1, substitution at the 4-position of
pteridine of 1a with a bulky amino group (1ac, 1ad,
Table 1. SAR at the 4-position of pteridine of compound 1a
R
N
N
N
N
F
OH
N
N
N
N
Compounds
R
IC50 (lM)
1aa
16
N
NH
F
1ab
6
4m
4n
NH
NH
NH
NH2
N
1ac
1ad
>100
87
N
O
H2NOC
N
N
1ae
1af
>100
29
N
4o
It is of interest to examine whether the features of p-
fluoro aryl scaffold (4e) and 4-alkylamino substitution
could be combined to further improve the potency.
The inhibitory activities of the 4-position amino deriva-
tives for compound 4e are listed in Table 3. Again, bulky
amino groups (6ec, 6ed, 6eg, 6eh, 6ej, 6eo and 6ep)
reduced the activity. Also, consistent with the SAR
HN
HN
1ag
1ah
>100
>100
CH3
F
HN

Y. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 675–678
677
Table 2. SAR at the 6-, 7-disubstitution of pteridine of compound 1a
Table 3. SAR at the 4-position for compound 4e with the IC50 of
0.5 lM
OH
R1
R2
N
N
F
N
R
N
N
N
Compounds
R1
R2
H3C
IC50 (lM)
N
N
F
4a
>100
H3C
Compounds
R
IC50 (lM)
2.9
H3CO
H3CO
4b
>100
6ea
6eb
NH
NH
1.5
4c
4d
4e
4f
30
>100
0.5
Br
Br
6ec
6ed
6ee
>100
73
NH
NH
N
H3CO
F
H3CO
F
O
68
23
O2N
6ef
3.2
9.6
HN
HN
4g
30
O2N
6eg
CH3
Cl
Cl
HN
4h
>100
6eh
6ei
26
21
F
N
N
N
4i
>100
58
O
O
4j
NH
6ej
10
4k
>100
6ek
6el
2.1
1.3
HN
HN
4l
>100
CO2Me
6em
51
N
based on compound 1a, lack of an NH group (6ee, 6ei
and 6em) was detrimental to the activity, emphasizing
the importance of a free NH group there. In this series,
compounds with a mid sized amino group (6eb) and
nonplanar cyclohexyl amino group (6el) gave the best
IC50 (1.5 and 1.3 lM). However, they are still less potent
than 4e.
O
6en
6eo
2.9
13
HN
HN
MeO
HN
6ep
80
Similar alkyl amine modifications for other disubsti-
tuted pteridine analogues were also made, and the
resulting compounds did not show much improvement
for the inhibitory activity.
In conclusion, from the compound screening using an
HCV NS5B RdRp enzymatic assay, we identified a hit
compound 1a with an IC50 of 15 lM. We explored the
structure–activity relationship for this class of inhibi-
tors. Introducing a fluorine atom at the para-position
of the aryl part increases the potency dramatically, but
addition of a bulky group at the para-, ortho- or meta-
position on the aryl group are not tolerated. Small alkyl
amines at the 4-position of the pteridine can maintain or
increase the potency. NH or OH at the 4-position and
presence of N1 and N3 in the pteridine ring is critical
for activity. Introduction of a hydrophobic amino group
at the 4-position may help compounds gain permeability
We further evaluated the compounds with IC50 under
5 lM for cellular activity using a cell-based HCV
subgenomic replicon assay. Compound 4e, which was
the most active in the NS5B RdRp assay
(IC50 = 0.5 lM), showed an EC50 of 90 lM in the repli-
con assay. However, its alkyl aminated analogue 6el
showed a much better activity in the replicon assay with
an EC50 of 18 lM and no toxicity up to 250 lM. This
result indicates that the partially negatively charged 4-
hydroxyl in 4e may be problematic for cellular perme-
ability. However, introduction of a hydrophobic amino
group at the 4-position may overcome this problem.

678
Y. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 675–678
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Current studies are focusing on elucidation of the mech-
anism of action of these pteridine inhibitors against
HCV NS5B RdRp and further improvement of their
enzymatic and cellular potency through structure-based
drug design efforts.
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