2-(2-Thienyl)-5,6-dihydroxy-4-carboxypyrimidines as inhibitors of the hepatitis C virus NS5B polymerase: Discovery, SAR, modeling, and mutagenesis

Article abstract of DOI:10.1021/jm051064t

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral RNA. We recently disclosed dihydroxypyrimidine carboxylates 2 as novel, reversible inhibitors of the HCV NS5B polymerase. This series was further developed into 5,6-dihydroxy-2-(2- thienyl)pyrimidine-4-carboxylic acids such as 34 (EC₅₀ 9.3 μM), which now show activity in the cell-based HCV replication assay. The structure-activity relationship of these inhibitors is discussed in the context of their physicochemical properties and of the polymerase crystal structure. We also report the results of mutagenesis experiments which support the proposed binding model, which involves pyrophosphate-like chelation of the active site Mg ions.

Full text of DOI:10.1021/jm051064t

J. Med. Chem. 2006, 49, 1693-1705
1693
2-(2-Thienyl)-5,6-dihydroxy-4-carboxypyrimidines as Inhibitors of the Hepatitis C Virus NS5B
Polymerase: Discovery, SAR, Modeling, and Mutagenesis
Uwe Koch,*,† Barbara Attenni,† Savina Malancona,† Stefania Colarusso,† Immacolata Conte,† Marcello Di Filippo,†
Steven Harper,† Barbara Pacini,† Claudia Giomini,† Steven Thomas,‡ Ilario Incitti,† Licia Tomei,† Raffaele De Francesco,†
Sergio Altamura,† Victor G. Matassa,§ and Frank Narjes*,†
Istituto Di Ricerche Di Biologia Molecolare, P. Angeletti S.p.A. (Merck Research Laboratories, Rome), Via Pontina Km 30,600,
00040 Pomezia, Italy, and Merck Sharp & Dohme Research Laboratories, The Neuroscience Research Centre,
Terling’s Park, Harlow, Eastwick Road, Essex, CM20 2QR, United Kingdom
ReceiVed October 21, 2005
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies
are in urgent demand. The polymerase of HCV is responsible for the replication of viral RNA. We recently
disclosed dihydroxypyrimidine carboxylates 2 as novel, reversible inhibitors of the HCV NS5B polymerase.
This series was further developed into 5,6-dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic acids such as 34
(EC50 9.3 µM), which now show activity in the cell-based HCV replication assay. The structure-activity
relationship of these inhibitors is discussed in the context of their physicochemical properties and of the
polymerase crystal structure. We also report the results of mutagenesis experiments which support the proposed
binding model, which involves pyrophosphate-like chelation of the active site Mg ions.
Introduction
core of the HCV replication machinery. This protein synthesizes
both a complementary (-)-strand of HCV RNA and, using this
as a template, the (+)-stranded HCV RNA genome itself.18,19
NS5B has been characterized by biochemical methods and
by X-ray crystallography. It adopts a tertiary structure which
resembles a right hand, a motif common to most nucleotide
polymerizing enzymes. The catalytic activity of the enzyme is
mediated by two magnesium ions in the active site, which serve
to activate the 3′-OH of the elongating RNA and position the
incoming nucleotide-triphosphate for nucleophilic attack.20-24
Until very recently, it was not possible to propagate the virus
in cell culture,25,26 and as a consequence a subgenomic replicon
system had been adapted for efficient RNA replication in the
human hepatoma Huh-7 cell-line, allowing the study of the
activity of key enzymes such as the NS3 protease-helicase and
the NS5B polymerase.27,28 This assay has been validated by the
observation that an inhibitor of the NS3 protease, which was
also a potent inhibitor of the replicon system, elicited a strong
reduction in viral load in a phase 1b clinical trial.29-31
NS5B is normally not expressed in uninfected cells and
presents an attractive target for the development of safe antiviral
drugs.32-34 Several laboratories, including ours, have disclosed
allosteric inhibitors, which inhibited HCV replication in the
replicon system. Several of these allosteric inhibitors have been
reported to block the initiation step of viral replication either
by preventing binding of RNA to the polymerase or by blocking
a necessary conformational change.35-41
Apart from allosteric inhibitors, two classes of active-site
inhibitors have been described. The first are modified chain-
terminating nucleoside (substrate) analogues, which were shown
to block RNA replication in the replicon system.42-44 The
second class are pyrophosphate (product) analogues, namely
diketo acids 1 (Figure 1). We have recently shown that 1 inhibits
selectively the HCV RdRP elongation step.45 Kinetic experi-
ments indicated that 1 and Foscarnet, a known pyrophosphate
mimic and an approved antiviral drug46 interact at a common
binding site.47 Due to the reactivity of the diketo moiety, 1 was
further elaborated into 5,6-dihydroxypyrimidine-4-carboxylic
acids such as 2.48,49 Intensive SAR studies directed at improving
Hepatitis C virus (HCV) infection constitutes a global health
problem, which affects more than 170 million individuals.1,2 If
untreated, more than 60% of these individuals will develop
chronic liver disease, which in 15-20% of the cases can lead
to chronic hepatitis, liver cirrhosis and even hepatocellular
carcinoma.3 In contrast to hepatitis B virus infections, no vaccine
or general therapy is available. Current therapy is based upon
a combination of pegylated R-interferon (IFN) and ribavirin.4-6
Although more efficacious than previous treatments, which were
based on IFN monotherapy,4 50% of patients infected with the
genotype 1 form of the virus fail to show a sustained virological
response.7,8 Another drawback of this treatment is its poor
tolerability, which is associated with severe flu-like symptoms,
depression and anemia, causing about 20% of patients to
discontinue treatment.9 This has stimulated intense research
programs in the pharmaceutical industry to find a broadly
effective antiviral therapy. By analogy to HIV research, most
efforts to develop antiviral agents for HCV have focused on
inhibition of the essential viral enzymes.10-16
HCV is a small, enveloped virus, belonging to the Flaviviri-
dae, a family of positive strand RNA viruses. Its genome is
approximately 9600 nucleotides in length and encodes a single
polyprotein of about 3000 amino acids.17 This polyprotein
undergoes maturational processing in the cytoplasm or in the
endoplasmic reticulum of infected cells to generate structural
and nonstructural (NS) viral proteins. Among the NS proteins
are two viral enzymes, which have been shown to be crucial
for viral replication and consequently were selected as potential
targets for antiviral therapy. The NS3 protease-helicase processes
the viral polyprotein downstream of it and liberates the NS5B
RNA dependent RNA polymerase (NS5B RdRp), the catalytic
* Corresponding authors. U.K., Tel.: +39 0691093644, e-mail:
uwe_koch@merck.com; F.N., Tel. +39 0691093625, Fax: +39 0691093654,
e-mail: frank_narjes@merck.com.
† Merck Research Laboratories, Rome.
‡ Merck Sharp & Dohme Research Laboratories, United Kingdom.
§ Current address: Almirall SA, Calle Treball 2-4, Sant Just Desvern,
Barcelona 08960, Spain.
10.1021/jm051064t CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/25/2006

1694 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Koch et al.
inhibitors are expected to develop resistance mutations less fast
than allosteric inhibitors. Finally, we also expect that combina-
tion therapy will be required for an effective treatment, and an
active site inhibitor together with an allosteric inhibitor would
present a good combination, as is the case in the treatment of
HIV infection.
In this paper we describe our efforts to further improve the
potency of the dihydroxypyrimidine series, with the aim to arrive
at compounds which would show activity in the subgenomic
replicon assay. We also provide further evidence which supports
the binding model of dihydroxypyrimidines as pyrophosphate
isosteres.
Biology. The compounds described were routinely assessed
for activity against the purified ∆C55 NS5B enzyme56 in the
presence of a homopolymeric template RNA. Selected com-
pounds were also tested on the full-length and ∆C21 enzyme
forms confirming potency (data not shown, except for 34).
Expression in E. coli BL21(DE3) and purification of the HCV
1b-BK strain NS5B∆C55 (residues 1-536), NS5B∆C21 (resi-
dues 1-570), mutants ∆C55-R158M and ∆C55-R158K were
carried out as already described.56,57 The full-length HCV NS5B
protein was purified from Sf9 cells infected with a recombinant
baculovirus as described.56
Figure 1.
Inhibition of replication of subgenomic HCV RNA was
measured in Huh-7 cells using a modification of the procedure
of Bartenschlager27 in the presence of 10% fetal calf serum.36
Figure 2.
the potency of 2, led to substitution in the ortho or meta position
of the phenyl group on the 2-position of the pyrimidine with
amides, ureas and sulfonamides, preferentially terminating in
an aryl residue.50,51 Compound 5 (IC50 0.48 µM), with a 60-
fold improvement in potency over 2, is an example of this effort.
Exchanging the phenyl group for a 2-thienyl residue also gave
an order of magnitude increase in potency (3, IC50 2.6 µM),
while the pyridine 4 (IC50 > 50 µM) was inactive.52 None of
the compounds 1-5 showed any activity in the replicon system.
Diketo acids have been proposed to inhibit HIV-integrase by
a mechanism involving chelation of active site magnesium
ions.53 By analogy it was hypothesized that the dianion formed
by deprotonation of the acid and the 5-hydroxy group in 2 could
be chelating one or both of the magnesium ions in the active
site (A in Figure 2) and thus act as a pyrophosphate mimic,
thereby blocking the elongation step of RNA synthesis. This
was proven by biochemical studies which established that
arylpyrimidines are competitive with diketo acids and other
classical pyrophosphate analogues.47 A more indirect proof came
from SAR studies, where it was shown that the free carboxylic
acid and the 5-hydroxy group on the pyrimidine are essential
for activity.49,52
Results and Discussion
SAR in the 2-Position of the Pyrimidine. The lack of
activity of dihydroxypyrimidines in the replicon assay was
thought to be due to two factors, lack of intrinsic potency, and
the physiochemical properties of the compounds. At physi-
ological pH the dihydroxypyrimidines can exist as a mono- or
dianion, depending on the pKa of the hydroxy group, which
reduces their ability to cross cell membranes. Since previous
studies had shown that the 5,6-dihydroxy-4-carboxylic acid
moiety is crucial for activity, our initial efforts concentrated on
improving the potency of these inhibitors. The exchange for a
phenyl group to a thiophene had produced a 10-fold gain in
potency, and consequently we expanded the SAR in the
2-position. As the results in Table 1 demonstrate, an aryl
substituent directly attached to the pyrimidine is required for
activity, since substituents such as cyclohexyl (7, IC50 > 50
µM), n-butyl (8, IC50 > 50 µM) or benzyl (9, IC50 > 50 µM)
lost significant in activity. Preferred are five-membered aromatic
heterocycles, with furan 10 being equipotent to thiophene 3,
and the electron-poor thiazole 11 gaining about 4-fold (IC50 0.76
µM). Relatively basic heterocycles such as imidazole 13, as well
as its precursor, N-benzyl imidazole 12 are at least 10-fold less
active, as was already seen with pyridine 4. Conversion of the
pyridine into the less polar pyrazine, which has a much reduced
dipole moment compared to pyridine, results in compound 14
(IC50 5.3 µM), which is nearly as potent as 3. The ben-
zothiophene derivative 15 is 4-fold less potent than 3, indicating
steric constraints in the binding pocket.
Dihydroxypyrimidine A can exist in the tautomeric pyrimi-
done form B (Figure 2), which in principle is also capable of
magnesium ion chelation. Presently, it is not known if A or B
or a mixture of them is responsible for the observed inhibition
of NS5B RdRP or if a bis- or monoanion is the active species.
That pyrimidone B is a possible inhibitor was shown with
N-methyl pyrimidone 6 (IC50 4.7 µM), an equipotent analogue
of dihydroxypyrimidine 3.52
We have continued to develop our dihydroxypyrimidine series
of NS5B inhibitors for several reasons. The genetic variability
of the virus is high. It can be classified in six major genotypes,
each of which is subdivided into several subtypes.54 Allosteric
inhibitors have been shown to be highly specific for one
genotype.55 In contrast, due to the structural constraints neces-
sary to maintain the catalytic function, the active site is more
conserved and active site inhibitors are potent against a majority
of viral genotypes.55 For the same reason mechanism-based
This set of results seemed to indicate that an electron-
withdrawing effect, going from thiophene to the more electron-
withdrawing thiazole is beneficial for potency. To test this
hypothesis, we prepared the sulfide 16, which shows 55%
inhibition at 50 µM. Oxidation to the more electron-withdrawing
sulfone gives compound 17, which now has an IC50 of 2.4 µM.
The Hammett constant, which measures the effect of substituents
in the para-position of benzoic acids on the pKa, changes from
0 for the sulfide to 0.72 for the sulfone. Similarly, the thienyl-

Inhibitors of the Hepatitis C Virus NS5B Polymerase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1695
Table 1. Inhibition of NS5B RdRP by 2-Substituted Dihydroxypyrimidines 3-17
a IC50 values are the average of at least three independent determinations.
Table 2. Inhibition of NS5B RdRP by 2-(2-Thienyl)dihydroxypyrimidines
a IC50 values are the average of at least two independent determinations. The standard error is within (20% of the average.
substituted pyrimidine 3 has a larger Hammett constant (0.05)
than the cyclohexyl-substituted 7 (-0.22).58 These results
suggest that the importance of the 2-substituent being electron-
withdrawing is due to their effect on the pKa of the pyrimidine.
In addition, the polarity of the substituent, steric limitations and
its conformational preference with respect to the pyrimidine
seem to be important. However, none of these compounds
showed any inhibition of the replicon assay at a fixed concen-
tration of 100 µM.
studies of the side-chain SAR with thiophene 3, since it allows
investigation of SAR in three different positions.
It became immediately apparent that substitution in the 5′-
and the 4′-position of the thiophene ring of 3 were detrimental,
since residues such as methyl or nitro in these positions led to
a significant loss in potency (18, IC50 48.6 µM; 20, IC50 16
µM; 21, IC50 9.4 µM, Table 2). Previously a thienyl acetamide
residue in the meta position of the phenyl ring in 2 had given
a 10-fold improvement in activity,50 but in the 4′-position of
the thiophene more than 4-fold loss (23, IC50 13.8 µM) was
observed. This left only the 3′-position, where a methyl group
leads to a 2-fold loss (19, IC50 5.6 µM), but the electron-
withdrawing nitro group gave a 4-fold gain in potency (22, IC50
We then proceeded to investigate the possibility of incorpo-
rating the potency enhancing side-chains found in the phenyl
series. Although thiazole 11 was the most potent compound in
this preliminary round of SAR, we decided to conduct the

1696 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Koch et al.
Table 3. Inhibition of NS5B RdRP by 2-(2-Thienyl)dihydroxypyrimidines
R
compd
IC50 (µM)a
EC50 (µM)a,b
compd
IC50 (µM)a
EC50 (µM)a,b
compd
IC50 (µM)a
EC50 (µM)a,b
Ph
(CH2)2Ph
CH2(2-Cl-Ph)
CH2(2-Cl-6-Me-Ph)
CH2(2,6-Cl2-Ph)
CH2-3-indolyl
CH2-3-benzothienyl
CH2-1-naphthyl
CH2-(2-Ph-3-indolyl)
29
31
34
37
-
-
40
-
0.58
0.62
0.15
0.09
>50
-
30
33
36
-
0.80
0.58
0.21
>50
>50
32
35
-
38
-
-
42
-
0.46
0.14
>50
>50
9.3 ( 2.8
13.6 ( 3.2
31.5 ( 6.4
53.3 ( 6.3
0.19
0.14
18.6 ( 2.3
13.6 ( 2.3
-
39
41
-
0.20
0.23
>50
>50
0.22
30.0 ( 4.1
-
43
0.28
>50
a IC50 and EC50 values are the average of at least three independent determinations. Standard deviations for the IC50 are within 30% of the average.
b Compounds were initially tested in the replicon assay at a fixed concentration of 100 µM. EC50 values were determined only for compounds showing
>50% inhibition.
0.68 µM) with respect to 3, making this compound equipotent
to thiazole 11. Converting the nitro group to the electron-
donating amine 24 (IC50 1.9 µM) reduces activity to the levels
of 3, again underscoring the importance of the electronic
properties of the 2-substituent on the activity of the pyrimidines.
Acylation of amine 24 with a selection of side-chains
previously described in the phenyl series50,51 gave another order
of magnitude in potency for the benzyl urea 25 (IC50 0.23 µM)
and the corresponding carbamate 26 (IC50 0.29 µM), confirming
the SAR already observed in the phenyl series. The SAR was
different for the sulfonamide and the amide linking groups.
Naphthylsulfonamide 27 (IC50 1.42 µM) gave only a 1.5-fold
gain in activity instead of the 10-fold observed in the phenyl
series (IC50 2.6 µM),51 while for amide 28 (IC50 0.38 µM) the
opposite was observed. The amide linkage had no impact on
activity in the phenyl series, where the corresponding compound
was equipotent with 2 (IC50 33 µM).50 None of these compounds
inhibited the replicon assay to any significant extent at a
concentration of 100 µM.
Further SAR then concentrated on ureas, carbamates and
amides. Reducing or elongating the chain gave a 2-fold loss in
potency across all linking groups (Table 3, see compounds 29-
33). Chlorination of the aromatic residue led to a small
improvement in intrinsic potency and, more important, to
activity in the replicon assay. Cell-based activity was best in
the urea series, where compound 34 had an EC50 of 9.3 µM
and showed no cytotoxicity up to 100 µM. Carbamate 35 and
amide 36 were 3- and 5-fold less active, despite similar IC50
values.
In an attempt to further improve potency we used a rapid
analogue approach to prepare a diverse set of ureas, carbamates
and amides. Residues R (Table 3) were initially chosen to
maximize diversity among them and between the three linking
groups. Despite a large number of compounds made, no major
improvement in potency could be achieved, and the SAR
remained rather flat. Aromatic residues were always preferred
over aliphatic ones (results not shown), and a simple methylene
group was the preferred spacer between the aromatic group and
the linker. Moving the chlorine on the aromatic ring of 34 from
the ortho- to the meta- or the para-position gave a 2-3-fold
loss in potency (results not shown). Further substitution on the
aromatic group with methyl groups or halogens led to a slight
increase in intrinsic potency, but not to an improvement in cell-
based activity (see urea 37, IC50 0.09 µM, EC50 13.6 µM). The
same was observed for substitution in the benzylic position,
where a variety of groups were accepted, but activity in the
replicon system remained poor (results not shown). In summary,
a large number of aromatic groups and substitution patterns are
accepted and, irrespective of the linking group, gave potent
enzyme inhibitors. Cell-based activity in the low micromolar
range was best in the urea series, followed by carbamates,
whereas amides were significantly less active.
From this investigation the urea 34 emerged as one of the
more active compounds. It loses 60-fold in potency in the cell-
based assay with respect to the enzyme assay. This is not due
to the enzyme form used in our in vitro assay, the ∆C55
polymerase, since 34 inhibits the ∆C21 (IC50 0.045 µM) and
the full-length version of the NS5B polymerase (IC50 0.13 µM)
with similar IC50s. It was also active on the ∆C21 NS5B
polymerase of genotype 2a (IC50 0.044 µM) and 3a (IC50 0.048
µM), thus showing that this class of inhibitors has the potential
to be active against a broad range of viral isolates.
The side-chain on the thiophene does not only increase the
potency of the inhibitors, but renders them also more lipophilic,
as evidenced by the distribution coefficient (log D) at pH 7.4
of unsubstituted 3 (-1.95) versus urea 34 (+0.37). This certainly
aids cell-penetration. More polar compounds, such as thiazole
11 (log D -2.12) or carbamate 32 (-0.43), are only 3-5-fold
less active in the enzyme assay, but show less than 20%
inhibition of the replicon at 100 µM. A further increase in
lipophilicity can be achieved by substitution on the aryl ring
(37, log D +0.6), or by its conversion into a naphththalene or
other bicyclic heteroaramatic groups (39, calculated log D 1.93;
42 calculated log D 1.09). This led to good or even improved
intrinsic potency, but not improved replicon potency with respect
to 34. We believe that the loss of potency in the replicon assay
is due to a large extent to the polar anionic character of the
compound and to some smaller extent to protein binding. This
is reflected by running the replicon assay in the absence of
serum, where an EC50 of 3.1 µM resulted, still 20-fold above
its IC50. In line with this, 34 showed poor permeability in Caco-2
cell line, both in the apical-basal and the basal-apical direction,
and is highly bound to plasma proteins (0.2% free in human
plasma).
In an attempt to reduce the polar character of our compounds,
we investigated substitutions on the N-1 nitrogen of the

Inhibitors of the Hepatitis C Virus NS5B Polymerase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1697
Table 4. Inhibition of NS5B RdRP by N-Alkylpyrimidinones
Table 5. IC50 and pKa of the 5-hydroxy Group of Selected Pyrimidines
a
compd
IC50 (µM) Mg2+ (Mn2+
)
pKa
2
3
6
30 (2.3)
2.6 (0.24)
4.2 (0.46)
>50 (>25)
2.9 (0.24)
0.76 (0.21)
0.68 (0.065)
0.15 (0.036)
1.2 (0.08)
8.6 ( 0.2
7.7 ( 0.2
9.1 ( 0.5
8.7
7.4 ( 0.1
6.9 ( 0.3
6.9 ( 0.2
6.6 ( 0.1
9.2 ( 0.4
7
10
11
22
34
47
no.
R1
R2
IC50 (µM)a
22
44
45
46
47
H
Me
allyl
Bn
NO2
NO2
NO2
NO2
0.68
8.7
69.3
79.3
1.2
a Average of three independent determinations. Data aquired using a
Sirius Profiler SGA (see Experimental Section).
Me
NH(CO)NH-(2-ClPh)
a Values are the average of at least two independent determinations.
to the related diketo acids, which have been shown to inhibit
NS5B by the same mechanism involving Mg-chelation,59 we
assume the change of the pKa to influence the Mg-chelating
capability. To test this assumption we determined the apparent
pKa value of the 5-hydroxy group of some selected compounds.
Importantly the pKa of the phenolic hydroxyl group of all
compounds with low micromolar activity is close to seven with
inhibition being measured at pH 7.4. Thus the pH is close to
the inflection point of the pH-dependent dissociation curve and
consequently small variations of the pKa can significantly change
the ratio of the mono- and bis-anionic form under the assay
conditions. Depending on which protonation state corresponds
to the bio-active form this influences the measured inhibition.
From the results presented in Table 5 it becomes clear that
generally a more electron-withdrawing substituent in position
2 of the pyrimidine lowers the pKa of the 5-hydroxy group.
The most active compounds, thiazole 11 and nitrothiophene 22
also have the lowest pKa value. This means that decreasing pKa
increases the amount of the bis-anionic form under our assay
condition (pH 7.4). To test whether an increase in the amount
of the doubly deprotonated species leads to an increase in
potency the IC50 of the same compound was measured at
different pH values. We observed for example a 2-fold gain in
potency for thiazole 11 when the pH is raised to 8.0 (IC50 0.34
µM instead of 0.76 µM), and a drop of 2-3-fold at pH 7.0
(IC50 1.9 µM). Taken together these results provide evidence
for the hypothesis that the doubly deprotonated pyrimidine is
the bioactive species. Possibly this is due to its better capability
to chelate Mg ions.
Introduction of the urea substituent 34 leads to the most potent
compound in this series. The urea also causes a significant
increase in acidity compared to the compound with an unsub-
stituted thiophene 3. This strong effect is somewhat unexepected
since the urea is not directly attached to the pyrimidine and its
electron-withdrawing effect has to be transmitted through the
thiophene. A possible explanation comes from the modeling
which suggests for 34 a planar conformation (Figures 3a and
3b). This type of arrangement with a contact between a hydrogen
bond acceptor and donor within a six-membered ring is similar
to that observed in the enolic form of 1,3-diketones and could
help to increase acidity by stabilizing the bis-anion. In fact the
urea 34 has the lowest pKa in this series. 34 is also the most
potent compound in this series. Affinity seems to be further
increased by hydrophobic interaction of the terminal aryl group
in a hydrophobic pocket.
Figure 3. (a) Superposition of models of 34 (green) and the
N-methylpyrimidone 47. (b) Possible hydrogen bonding network in the
syn orientation of urea and acid.
pyrimidine, also based on the precedence that the N-methyl
pyrimidone 6 is as active as the dihydroxypyrimidine 3. As
Table 4 shows, alkylation of nitrothienyl pyrimidine 22 leads
to a loss of potency, which increases with the size of the
substituent. The O-alkylated compounds did not show activity.
The N-methyl group is tolerated, and so we converted compound
44 to the urea analogue 47. Compared to the dihydroxypyri-
midine 34, the pyrimidone 47 is about 1 order of magnitude
less potent in the enzyme assay and does not inhibit the replicon
system at 100 µM. Modeling suggests that the loss of potency
is due to the position of the side-chain, which as a consequence
of the twist induced by the methyl group at N-1, now occupies
a different region of space. The minimum energy conformer,
obtained in water using an MMFF force field, shows a twist
with an angle between thiophene and pyrimidone of 70° ((20°)
(Figure 3a).
Modeling for the dihydroxypyrimidine 34 on the other hand,
showed a planar compound, despite the presence of a substituent
in the 3-position of the thiophene. The syn and anti orientation
of the urea and the carboxylate, obtained by a 180° rotation
around the pyrimidine thiophene axis, are almost isoenergetic.
For both orientations the planar arrangement is only possible
with the inner urea NH forming a close intramolecular contact
to either to the N-3 or N-1 nitrogen atoms of the pyrimidine.
Amides and carbamates can have similar interactions. In the
syn conformation it is possible for the urea to form a second
close contact between the outer urea NH and the carboxylic
acid (Figure 3b), an arrangement which we term intramolecular
hydrophilic collapse, which would enforce the planar arrange-
ment. We speculate that this effect could explain the enhanced
activity of ureas in the replicon assay with respect to the
equipotent carbamates and amides (see compound 34 versus
35 and 36, Table 3).
Introduction of an N-methyl group to generate the 2-thieno-
substituted N-methyl pyrimidone 6 increases the pKa by more
than one unit without having a significant effect on potency.
According to the pKa of 6 only a small quantity of this
compound should be present in its bis-anionic form at pH 7.4.
Thus, at least for the thieno-substituted compound, the monoan-
ionic form of the N-methyl pyrimidone has the same potency
The SAR suggests that the substituent in the C-2 position
has an influence on the pKa of the 5-hydroxy group. In analogy

1698 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Koch et al.
Table 6. Activity of 3, 11, and 35 in HCV NS5B Containing
as the corresponding, doubly deprotonated pyrimidine. We
speculate that in case of the pyrimidines it is the higher affinity
of the doubly charged form for the Mg ion which is responsible
for the pKa dependence of the SAR. After N-methylation only
a single tautomer is possible which by itself in its monoanionic
form may have a high affinity for the Mg ion. This implies the
importance of the charge to be due to its effect on chelation
not on a direct interaction with the protein. That chelation is of
central importance for the affinity of these compounds is in
agreement with the strong effect on affinity observed when Mg
is exchanged for Mn in the NS5B assay (see Table 5).49
Site-Directed Mutations
IC50 (µM), Km for UTP
compd
wt
R158Ma
R158Ka
3
11
35
2.6
9.3
2.77
0.34
15.0
92.0
16.0
0.90
20.0
0.76
0.14
6.0
UTP
a Measured in duplicate.
The N-methyl group of the pyrimidone 47 sterically prevents
a planar arrangement of the 2-thienyl group and the pyrimidine
(Figure 3a) and as a consequence the 2-substituent occupies an
area different from the same substituent of 34 after superposition
of the pyrimidine moiety. Interestingly, we do not observe any
decrease in the pKa compared to the unsubstituted pyrimidone
6, confirming the hypothesis that the formation of the close
intramolecular contact between the urea NH and the pyrimidine
helps to increase acidity.
Molecular Modeling and Mutagenesis. To gain further
insight into the mechanism of inhibition we have generated a
binding model for the pyrimidines on the basis of the existing
crystal structures of the NS5B protein. The active site of NS5b
binds two Mg ions which have been shown crystallographically
to mediate the binding of the triphosphate of a cocrystallized
nucleotide.23 During elongation, we assume, in analogy to other
polymerases,60-62 one Mg ion to chelate the primer ribose
hydroxy group and the R-phosphate of the incoming nucleoside.
The second Mg ion is bound between the â- and γ-phosphate
of the incoming nucleotidetriphosphate (NTP), which after
addition of this nucleotide to the primer is cleaved off as the
pyrophosphate.
SAR and biochemical data suggest that pyrimidines have a
similar binding mode as the pyrophosphate. Accordingly we
propose the pyrimidine to interact with the Mg ion chelating
the â- and γ-phosphate groups of the triphosphate. We have
used crystallographic information about the standard Mg-
oxygen interaction geometry to generate a binding model of
the pyrimidine bound to the Mg ion in the crystal structure of
NS5B. The position and orientation of the pyrimidine was
optimized to reduce steric interactions with the protein while
maintaining the chelation geometry.
The model suggests the 2-thiophene of the pyrimidine to be
stacked against Arg158 which in the crystal structure with GTP
forms hydrogen bonds to the R-phosphate.23 By analogy to the
T7 DNA polymerase and HIV reverse transcriptase,60,62 which
both contain a conserved basic residue in contact with the
R-phosphate, we expect Arg158 to play a critical role in
activating the R-phosphate for nucleophilic attack by the 2′-
OH of the primer and at the same time interacting lipophilically
with the base of the incoming nucleosidetriphosphate. To test
the importance of this residue for inhibition by the pyrimidines
we suggested two mutations of Arg158.
Since the interactions between Arg158 and the thiophene
seem to be mainly of lipophilic nature a mutation into Met, a
more lipophilic amino acid with similar size but without a
charge, should maintain the affinity of the pyrimidine. Mutation
into the more polar Lys however should decrease the capability
to interact lipophilically with the thiophene while maintaining
a positive charge. We observe a decrease of inhibition for
pyrimidines 3, 11, and 35 for both mutations (Table 6).
However, in the case of thiophene 3 the decrease in potency is
significantly larger for the Arg158Lys mutation (35-fold) than
Figure 4. Model of active 2′-substituted 2-thienyl-pyrimidines in the
active site together with a primer and template. The pyrimidines
correspond to those in Table 3. The two Mg ions are indicated as violet
spheres, carbon atoms are shown in green, oxygen in red, nitrogen in
blue and sulfur in yellow.
for the Arg158Met (3-fold) suggesting that the lipophilic
component of the interaction between Arg158 and the 2-thiophene
is particularly important. The same kind of behavior is observed
for thiazole 11, and also for compound 35, with a potency
enhancing side-chain. Here the loss in potency on the Arg158Lys
mutant is less severe (6-fold), but still larger than on the
Arg158Met mutant (2-fold). The Km for the substrate UTP has
been measured and does not show significant differences
between wt and mutant enzymes (Table 6). These results
confirm a mode of binding of pyrimidines which involves
chelation of Mg ions and lipophilic interactions of the substituent
in position 2 of the pyrimidine with Arg158.
In agreement with SAR this model shows that the space
available to substituents in positions 4 and 5 of the thiophene
is limited. The substituents in position 3 are bound in a cavity
characterized by the presence of a number of basic and lipophilic
residues (Figure 4). Interaction with these residues is responsible
for the increase in potency of some compounds containing
lipophilic substituents in this position. The binding site is further
characterized by the presence of a number of basic amino acids
explaining the lack of activity of the 2-imidazolo substituted
pyrimidines 12 and 13. The conformational change introduced
by the N-methyl group of pyrimidone 47 is not compatible with
the binding mode described for the dihydroxypyrimidine. In the
twisted conformation the o-chlorobenzyl moiety cannot be
accommodated in the binding pocket requiring either a confor-
mational change or a less optimal Mg-chelation geometry, both
of which accompanied by a loss of binding energy and thus
affinity.
Conclusion
We have described the further development of the dihydroxy-
pyrimidine class of NS5B inhibitors, which were inactive in
the cell-based HCV replicon assay. We have identified different

Inhibitors of the Hepatitis C Virus NS5B Polymerase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1699
Scheme 2a
Scheme 1. Synthesis of Pyrimidine Carboxylates 3, 7-15,
18-22a
a Reagents and conditions: (a) H2NOH‚HCl, Na2CO3, EtOH, H2O, rt;
(b) DMAD, CHCl3, rt to reflux; (c) xylenes, 140-150 °C; (d) NaOH, 80
°C; (e) for 52g: PvCl, DMAP, pyridine, rt; HCO2NH4, Pd/C, MeOH, rt;
then step d.
properties which contribute to binding. Modulation of the pKa
by substituents in position 2 of the pyrimidine influence affinity
by their effect on Mg chelation. We have described the
physicochemical and conformational properties of the substit-
uents attached to the pyrimidine which favor affinity. Molecular
modeling studies were used to suggest a mode of binding of
pyrimidines, on the basis of which site directed mutations have
been designed which confirm the proposed model. This model
involves pyrophosphate like chelation of the active site Mg ions
and identifies a lipophilic pocket which can be used as the basis
for the development of more potent inhibitors. SAR data confirm
the binding mode of the dihydroxypyrimidine. Urea 34 emerged
from these studies as one of the most potent compounds, which
showed the same potency on a variety of polymerases from
different viral isolates and also inhibited the cell-based replicon
assay. This makes the dihydroxypyrimidine class of inhibitors
a promising starting point for future development of more active
compounds.
Chemistry. For the synthesis of 2-substituted dihydroxypy-
rimidine carboxylates 3-15 and 18-22 the method of Culbert-
son was used.63 Briefly, amidoximes 49a-n, obtained from
nitriles 48a-n, either commercially available or prepared as
described in the Experimental Section, were reacted with
dimethylacetylene dicarboxylate (DMAD) to yield adducts
50a-n as a mixture of geometrical isomers (Scheme 1). Usually
these adducts were not isolated, but cyclized directly in refluxing
xylene to yield the desired pyrimidine methyl esters 52. In most
cases the esters precipitated from the reaction mixture and were
isolated by filtration generally in good purity and yields ranging
from 3 to 60%. Polymerization of more sensitive substrates such
as 48d (9%) and 48e (3%) under the reaction conditions was
the major side-reaction. As a side-product imidazole 53 was
observed in several cases in the LC-MS trace of the crude
mixtures and isolated and characterized in the case of 53n. It is
assumed that the reaction from 50 to 52 follows a Claisen-type
rearrangement via intermediate 51 shown in Scheme 1,63 which
after tautomerization of the amine could cyclize onto the ketone
to give the imidazole or onto one ester group to yield 52.
Hydrolysis of the ester gave the final products in about 70-
90% purity. Imidazole 52g was first acylated with pivaloyl
chloride, which made the compound soluble enough for the
subsequent hydrogenation to give 13.
a Reagents and conditions: (a) Pd/C, H2, MeOH, rt; (b) PvCl, 4-DMAP,
py, rt; (c) NaOH, 80 °C; Method A: RCNO, py, rt;, temp.; then (c); Method
B: RCO2H, CDI, DMF, add 54, rt; then (c); Method C: PhOC(O)Cl, DCM,
rt, add RNH2, rt; then (c); Method D: RCO2H, BOP-Cl, DCM, NEt3, 0 °C,
1 h; add 56, NEt3; then (c); Method E: ROH, DSC, CH3CN, Et3N, rt,
overnight; add 24, CH3CN, H2O, rt.
hydrogenated to amine 54. Reaction with isocyanates then led
to the desired ureas. Addition of 54 to a solution of carboxylic
acids, previously activated with CDI and triethylamine, led to
the amides. Reactions with amine 54 were sometimes capricious,
due to acylation of the 5-hydroxy group and due to the low
solubility of some of the products, which made a workup from
organic solvents difficult. Protection of the 5-hydroxy group as
its pivaloate ester solved both of these problems and amine 56
was used for the rapid analogue synthesis. Ureas were prepared
either by reaction with isocyanates or via the intermediate
phenylcarbamate.64 Amides were accessed by the BOP-Cl
mediated coupling of acids.65,66 After workup and deprotection
of the ester groups products usually precipitated upon acidifica-
tion of the hydrolysis mixture and were isolated by filtration.
They were then purified to >95% purity by reversed phase
HPLC.
Carbamates were also accessible from 56, but in some cases
hydrolysis of the carbamate occurred under the harsh conditions
employed for methyl ester hydrolysis. To avoid this amino acid
24 was reacted directly with the appropriate alcohol in the
presence of N,N′-disuccinidyl carbonate.67 For the synthesis of
sulfonamide 27 a cyclic methylene acetal was used to block
the reactive diol functionality.51
Alkylation of 55 with alkylhalides produced a mixture of the
products of N- and O-alkylation, which were easily separated
by column chromatography. The synthesis then proceeded as
described above for the dihydroxypyrimidines (Scheme 3).
Sulfide 16 and sulfone 17 were prepared from dichloropyrimi-
dine 59 as shown in Scheme 4.
Experimental Section
Synthesis and Characterization of Inhibitors. General Pro-
cedures. Solvents and reagents were used as received from
commercial sources.
Melting points were determined in open capillaries using a
Buechi 535 apparatus and are reported uncorrected. Nuclear
magnetic resonance spectra (1H NMR recorded at 400 or 300 MHz,
Scheme 2 illustrates the chemistry used for the investigation
of the side-chain SAR in the thiophene series. Ester 52n was

1700 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Scheme 3. Synthesis of N-Alkylpyrimidones 44-47a
Koch et al.
General Procedure for the Synthesis of 2-Substituted 5,6-
Dihydroxypyrimidine-4-carboxylic Acids (3, 11, 15, 19, 22). The
procedure from Culbertson was followed.63 Usually amidoximes
49 and Michael adducts 50 were used without further purification.
The preparation of 5,6-dihydroxy-2-(3-nitro-2-thienyl)pyrimidine-
4-carboxylic acid (22) is a representative example.
3-Nitro-2-thiophenamidoximate (49n). Nitrile 48n (86.93
mmol, 13.4 g) was suspended in water (360 mL) and ethanol (48
mL). Sodium carbonate (15.7 g, 147.8 mmol) and hydroxylamine
(18.7 g, 287 mmol) were added, and the mixture was left at room
temperature for 24 h. The orange solid was isolated by filtration,
washed with a small portion of diethyl ether and dried. The
amidoxime (14.0 g, 86%) was obtained as an orange solid and
existed as a mixture E/Z of about 4:1* as determined by 13C-
a Reagents and conditions: (a) Cs2CO3, THF, 50 °C; add electrophile;
57a Me2SO4; 57b allyl bromide; 57c benzyl bromide; (b) Pd/C, H2, MeOH,
rt; DCM, py, 2-chlorobenzyl isocyanate; (c) NaOH, 80 °C.
1
spectroscopy. H NMR (400 MHz, DMSO-d6) δ, 9.95 (bs, 1 H),
7.68 (d, J ) 5.3 Hz, 1H), 7.60 (d, J ) 5.3 Hz, 1H) 6.08 (bs, 2H);
13C NMR (100 MHz, DMSO-d6) δ 161.5, 145.5, 144.6*, 139.5*,
136.6, 127.5*, 127.0, 124.2, 123.5*; m/z 188 (M + H); recrystal-
lization from dichloromethane/n-pentane, mp 201-202 °C; Anal.
(C5H5N3O3S) C, H, N, S.
Scheme 4. Synthesis of Inhibitors 16 and 17
Dimethyl 2-({[1-Amino(3-nitro-2-thienyl)methylene]amino}-
oxy)but-2-enedioate (50n). Amidoxime 49n (11.87 g, 63.90 mmol)
was suspended in dichloromethane (250 mL). Triethylamine (0.5
mL) and dimethyl acetylenedicarboxylate (9.53 g, 67.1 mmol) were
added. The mixture was refluxed for 3 h and became homogeneous
during this time. Evaporation of the dichloromethane gave 20.88 g
of a red oil, which was dissolved in ethyl acetate (400 mL). After
washing with water and brine, the organic phase was dried over
sodium sulfate and evaporated. The reddish oil (20.5 g, 97%) was
1
used without further purification. H NMR (CDCl3, two diaster-
ereomers 2.5:1*) δ 7.61*, 7.58 (d, J ) 5.6 Hz, 1H), 7.38*, 7.33
(d, J ) 5.6 Hz, 1H) 6.10, 5.84* (bs, 2H), 5.94, 5.88* (s, 1H), 3.90*,
3.83 (s, 3H), 3.71, 3.68* (s, 3H); 13C NMR (100 MHz, CDCl3) δ
165.4*, 163.9, 161.6, 158.3*, 153.1, 149.1, 148.8*, 144.7*, 131.1,
130.9*, 126.2*, 125.9, 124.3*, 124.2, 104.3, 95.4*, 52.0*, 51.8,
50.7, 50.5*; m/z 330 (M + H).
a Reagents and conditions: (a) NaOMe, MeOH, 90%; (b) NaSMe, NMP,
50 °C, 70%; (c) BBr3, DCM, then RP-HPLC, 50% (d) H2O2, AcOH, then
RP-HPLC, 24%.
13C NMR recorded at 100 or 75 MHz) were obtained on Bruker
AMX spectrometers and are referenced in ppm relative to TMS or
the solvent signal. Low-resolution mass spectra (m/z) were recorded
on a Perkin-Elmer API 100 (electrospray ionization) mass spec-
trometer. High-resolution mass measurements were performed on
a Finnigan TSQ Quantum Ultra AM in selected ion monitoring
(SIM) mode. Elemental analyses were performed by the analytical
chemistry group at IRBM.
Methyl 5,6-dihydroxy-2-(3-nitro-2-thienyl)pyrimidine-4-car-
boxylate (52n). To a flask containing adduct 50n (30.7 g, 93.28
mmol) was added xylene (212 mL), and the reaction was left at
140 °C until the disappearance of the starting material. The reaction
mixture was stored in a refrigerator at 4 °C overnight and the
precipitate isolated by filtration. The brown solid was washed with
ethyl acetate and petrol ether and dried under vacuum to give the
Thin-layer chromatography was performed on aluminum plates
precoated with Merck silica gel F254, which were visualized by the
quenching of the UV fluorescence (λmax 254 nm) or by staining
with 5% phosphomolybdic acid in 95% ethanol. Flash chromatog-
raphy was accomplished on Merck silica gel (200-400 mesh).
Reversed phase analytical HPLC was performed on a Waters
Alliance separation module 2695, using H2O/0.1% TFA (A) and
CH3CN/0.1% TFA (B) as eluents. Peaks were detected with a
Waters 996 PDA detector using the Maxplot option (210-400 nM)
contained in the PDA program. Inhibitor purity was determined
using the following methods: Method A: Waters Symmetry C18
column (150 × 3.9 mm, 5 µm), flow rate 1 mL/min, gradient: 90%
A (or 100% for polar compounds), 0.5 min isocratic, linear to 0%
A in 10 min, then isocratic; Method B: Phase Separations
Spherisorb S5 ODS2 column (25 × 4.6 mm, 5 µm), flow 1.5 mL/
min, gradient: 90%A, 3 min isocratic, linear to 50%A in 15 min;
Method C: Waters X-terra C18 column (50 × 2.5 mm, 3.5 µm),
flow rate 0.5 mL/min, gradient: 100% A, 2.0 min isocratic, linear
to 0% A in 7 min. Preparative HPLC was conducted on a Waters
Symmetry C18 column (150 × 19 mm, 7 µm) or on Waters Prep-
NovaPak column (HR C18, 40 × 100 mm, 6 micron) using H2O/
0.1% TFA (A) and CH3CN/0.1% TFA (B) as eluents; detection at
220 nm with a Waters 486 absorbance detector. All reactions were
run under inert atmosphere (nitrogen or argon) in oven or flame
dried glassware.
1
product as a beige powder (13.3 g, 48%). H NMR (300 MHz,
DMSO-d6) δ 13.2 (bs, 1H), 7.88 (d, J ) 5.5 Hz, 1H), 7.72 (d, J )
5.5 Hz, 1H), 3.82 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 165.6,
158.9, 147.2, 145.2, 139.3, 135.4, 129.6, 128.5, 124.2, 52.7; m/z
296 (M - H); Anal. (C10H7N3O6S) C, H, N.
Evaporation of the mother liquor under reduced pressure and
purification of the residue by flash chromatography on silica gel
(PE/EtOAc 1:1) gave a brown solid which was recrystallized from
methanol and water to give dimethyl 2-(3-nitro-2-thienyl)-1H-
imidazole-4,5-dicarboxylate (53n) as a yellow powder in 4% yield.
1H NMR (400 MHz, DMSO-d6) δ 14.0 (bs, 1H), 7.90 (d, J ) 5.5
Hz, 1H), 7.75 (d, J ) 5.5 Hz, 1H) 3.85 (s, 6H); 13C NMR (75
MHz, DMSO-d6) δ 160.2, 144.0, 138.2, 130.7, 130.3, 128.9, 124.2,
51.9; m/z 312 (M + H); HRMS calc. for C11H10N3O6S (M + H)
312.0285, fd. 312.0269; HPLC: A: 4.25 min, 97.6%; B: 6.81 min,
98.1%.
5,6-Dihydroxy-2-(3-nitro-2-thienyl)pyrimidine-4-carboxylic Acid
(22). To a suspension of 52n (72 mg, 0.24 mmol) in methanol (1
mL) and water (1 mL) was added 0.5 N NaOH (2 equiv), and the
mixture was heated to 80 °C. After 15 min hydrolysis was complete,
as indicated by HPLC. The solution was cooled to room temper-
ature, and then 1 N HCl was added dropwise until a pH of 2 was
reached. The resulting solid was isolated by filtration and washed
with water, MeOH and EtOAc and then dried under vacuum to
1
The following compounds were prepared according to described
procedures: 1,3-thiazole-2-carbonitrile68 (48f), 1-benzyl-1H-imi-
dazole-2-carbonitrile69 (48g).
give 22 as a yellow powder (29 mg, 43%). H NMR (400 MHz,
DMSO-d6) δ 13.3 (bs, 1H), 11.8 (bs, 1H), 7.89 (d, J ) 5.4 Hz,
1H), 7.71 (d, J ) 5.4 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ

Inhibitors of the Hepatitis C Virus NS5B Polymerase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1701
169.5, 159.1, 149.6, 145.6, 139.7, 135.9, 129.9, 129.2, 124.5; m/z
282 (M - H); Anal. (C9H5N3O6S) C, H, N.
the residue was treated with HCl (1 N, 8 mL) and stirred for 10
min. The solution was diluted with AcCN and purifed by Prep
HPLC using a Waters Prep-NovaPak column. Lyophilization gave
16 (33 mg, 50%) as a colorless powder. 1H NMR (300 MHz,
DMSO) δ 2.47 (s, 3H); 13C NMR (75 MHz, DMSO) δ 169.37,
158.91, 148.94, 145.98, 128.68, 13.22; m/z 201 (M - H); HRMS
calc. for C6H7N2O4S (M + H) 203.0121, fd. 203.0125; HPLC: A
(100%): 2.13 min, 98.1%; B: 3.38, 97.8%.
5,6-Dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic Acid (3).
Adduct formation and cyclization gave 52a as a brown powder
(30%); hydrolysis and purification by RP-HPLC gave 3 as a beige
1
powder (65%). H NMR (400 MHz, DMSO-d6) δ 12.4 (bs, 1H),
7.84 (d, J ) 3.5 Hz, 1H), 7.61 (d, J ) 4.9 Hz, 1H) 7.09 (dd, J )
3.5, 4.9, 1H); 13C NMR (75 MHz, DMSO-d6) δ 168.8, 158.5, 146.4,
142.0, 136.7, 130.9, 128.7, 128.2, 127.9; m/z 239 (M + H); Anal.
(C9H6N2O4S‚0.5 H2O‚0.5 TFA) C, H, N.
5,6-Dihydroxy-2-(methylsulfonyl)pyrimidine-4-carboxylic Acid
(17). A solution of 16 (33 mg, 50%) (0.022 g, 0.11 mmol) in glacial
acetic acid (1 mL) was treated with a 35% aqueous solution of
hydrogen peroxide (0.5 mL). The resulting solution was stirred at
room temperature for 16h then diluted with water/AcCN (1:1) and
lyophilized. The crude material was purifed by Prep HPLC using
a Waters Prep-NovaPak column. Lyophilization gave 17 (0.006 g,
5,6-Dihydroxy-2-(1,3-thiazol-2-yl)pyrimidine-4-carboxylic Acid
(11). Adduct formation and cyclization gave 52f as a brown powder
(35%); hydrolysis and purification by RP-HPLC yielded 11 as a
1
beige solid (71%). H NMR (400 MHz, DMSO-d6) δ 12.51 (bs,
1H), 7.96 (d, J ) 2.95, 1H), 7.91 (d, J ) 2.95, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 168.3, 161.5, 159.2, 154.3, 143.5, 137.1,
127.0, 123.8; m/z 238 (M - H); m/z 240 (M + H); HRMS calc.
for C8H6N3O4S (M + H) 240.0074, fd. 240.0072; HPLC: A
(100%): 3.22 min, 95.0%; B: 4.39 min; 96.2%.
1
24%) as acolorless powder. H NMR (300 MHz, DMSO) δ 3.27
(s, 3H); 13C NMR (75 MHz, DMSO) δ 39.46 (SO2Me); m/z 233
(M - H); HRMS calc. for C6H5N2O6S (M - H) 233.0098, fd.
233.0069; HPLC (from 100% A): A: 1.1 min, 99%; B: 2.3 min,
98.7%.
2-(1-Benzothien-2-yl)-5,6-dihydroxypyrimidine-4-carboxy-
lic Acid (15). Adduct formation and cyclization gave 52i as a pale
yellow solid (92%); hydrolysis and precipitation gave 15 as a beige
solid after extensive washing with water, MeOH and EtOAc (46%).
1H NMR (400 MHz, DMSO-d6) δ 13.2 (bs, 1H), 8.27 (s, 1H), 7.97
(d, J ) 2.1 Hz, 1H), 7.86 (d, J ) 2.1 Hz, 1H) 7.37-7.46 (m, 2H);
m/z 289 (M + H); Anal. (C13H8N2O4S‚0.5 H2O) C, H, N.
5,6-Dihydroxy-2-(3-methyl-2-thienyl)pyrimidine-4-carboxy-
lic Acid (19). Adduct formation and cyclization gave 24% of 52k;
hydrolysis and purification by RP-HPLC gave 19 (24%). 1H NMR
(400 MHz, DMSO-d6) δ 12.2 (bs, 1H), 7.53 (d, J ) 4.0 Hz, 1H),
7.93 (d, J ) 4.0 Hz, 1H) 2.36 (s, 3H); 13C NMR (75 MHz, DMSO-
d6) δ 169.0, 161.3, 157.4, 136.9, 131.0, 128.4, 126.3, 14.9; m/z
251 (M - H); Anal. (C10H8N2O4S‚0.5 H2O) C, H, N.
Methyl 2-(3-Amino-2-thienyl)-5,6-dihydroxypyrimidine-4-
carboxylate (54). To a solution of 52n (1.5 g, 5.05 mmol) in ethyl
acetate/methanol (2:1, 200 mL) was added Pd/C (10% Pd, 1.5 g)
and the reaction stirred under an atmosphere of hydrogen for 5 h
at ambient temperature. The catalyst was removed by filtration and
washed with hot ethyl acetate and methanol. After evaporation 54
(1.08 g, 80%) was obtained as a yellow solid, which was used
without further purification. 1H NMR (400 MHz, DMSO-d6) δ 8.80
(bs, 3H), 7.48 (d, J ) 5.2 Hz, 1H), 6.67 (d, J ) 5.2 Hz, 1H) 3.85
(s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 166.04, 160.32, 145.82,
144.34, 142.52, 130.11, 128.94, 122.82, 110.07, 52.85; m/z 268
(M + H).
Synthesis of 5,6-Dihydroxy-2-(methylthio)pyrimidine-4-car-
boxylic Acid (16). 2,6-Dichloro-5-methoxypyrimidine-4-car-
boxylate (59). N,N-dimethylaniline (0.856 mL, 6.76 mmol) was
added to a stirred solution of methyl 2,6-dihydroxy-5-methoxypyr-
imidine-4-carboxylate70 (1.0 g, 5 mmol) in POCl3 (34.5 mL). After
refluxing overnight POCl3 was evaporated and the residue was
poured on ice water and extracted into diethyl ether. The combined
ethereal layers were washed with brine, dried over sodium sulfate
and evaporated in vacuo. Purification by flash chromatography
(SiO2; petroleum ether/EtOAc 4:1) gave methyl 2,6-dichloro-5-
methoxypyrimidine-4-carboxylate (59) (1.07 g, 90%) as a beige
(3-Amino-2-thienyl)-5,6-dihydroxypyrimidine-4-carboxylic Acid
(24). A solution of methyl 2-(3-amino-2-thienyl)-5-[(2,2-dimeth-
ylpropanoyl)oxy]-6-hydroxypyrimidine-4-carboxylate (56) (0.080
g, 0.22 mmol) in 0.5 N NaOH (1.2 mL) was heated at 90 °C for
30 min. The cooled reaction mixture was acidified with 1 N HCl
to pH 1 allowing the formation of a precipitate. The solid was
washed with water, acetone and diethyl ether and then dried under
vacuum. 24 (30 mg, 30%) was obtained as the hydrochloride salt.
1H NMR (400 MHz, D2O-NaOH, pH ) 7) δ 7.65 (d, J ) 4.9 Hz,
1H), 6.62 (d, J ) 4.9 Hz, 1H); 13C NMR (100 MHz, 330K, DMSO-
d6) δ 169.5, 159.5, 150.5, 146.7, 144.0, 128.9, 128.4, 121.8, 102.9;
m/z 252 (M - H).
1
solid. H NMR (300 MHz, DSMO) δ 3.95 (s, 3H), 3.93 (s, 3H);
13C NMR (75 MHz, DMSO) δ 161.97, 157.79, 151.25, 151.14,
148.47, 62.40, 53.45; m/z 238.0 (M + H).
Methyl 5-[(2,2-Dimethylpropanoyl)oxy]-6-hydroxy-2-(3-nitro-
2-thienyl)pyrimidine-4-carboxylate (55). 52n (3.0 g, 10 mmol)
was dissolved in anhyd pyridine (30 mL) at room temperature. A
catalytic amount of 4-DMAP (0.12 g, 1 mmol) was added, followed
by the dropwise addition of neat pivaloyl chloride (1.29 mL, 10.5
mmol). After 3 h at room temperature, the pyridine was removed
in vacuo, the residue dissolved in ethyl acetate and washed with 1
N HCl (2×) and brine. Drying over Na2SO4 and removal of the
solvents gave a brown solid, which was crystallized from EtOAc
Methyl 5,6-Dimethoxy-2-(methylthio)pyrimidine-4-carbox-
ylate (60). Powdered sodium methoxide (0.14 g, 2.5 mmol) was
added to a stirred solution of 59 (0.5 g, 2.1 mmol) in anhydrous
methanol (5 mL) at room temperature and stirred for 30 min. The
reaction mixture was then diluted with ethyl acetate and washed
with water and with brine. The organic layer was dried over sodium
sulfate and evaporated to afford pure methyl 2-chloro-5,6-
dimethoxypyrimidine-4-carboxylate (0.44 g, 90%) as a white solid.
1H NMR (400 MHz, DMSO) δ 4.04 (s, 3H), 3.89 (s, 3H), 3.83 (s,
3H); 13C NMR (75 MHz, DMSO) δ 165.13, 162.81, 150.74, 148.45,
139.71, 61.26, 55.55, 52.94; m/z 232 (M + H). A solution of this
compound (0.100 g, 0.43 mmol) in anhydrous NMP (1 mL) was
treated with sodium methanethiolate (0.033 g, 0.47 mmol) in one
portion and stirred at 50 °C for 16 h. The reaction mixture was
diluted with ethyl acetate, washed with water and brine, dried over
sodium sulfate and evaporated in vacuo, leaving methyl 5,6-
dimethoxy-2-(methylthio)pyrimidine-4-carboxylate (60) (0.073 g,
1
to give 55 (2.5 g, 66%) as a beige solid. H NMR (400 MHz,
DMSO-d6) δ 13.9 (bs, 1H), 7.96 (d, J ) 5.5 Hz, 1H), 7.73 (d, J )
5.5 Hz, 1H), 3.83 (s, 3H), 1.30 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ 173.9, 162.2, 157.2, 147.6, 145.2, 142.2, 137.7, 133.5,
130.0, 123.7, 52.6, 38.2, 26.3; m/z (ES+) 382 (M + H); Anal.
(C15H15N3O7S) calc. C 47.24, H 3.96, N 11.02; fd. C 46.61, H 3.71,
N 10.75.
Methyl 5-[(2,2-Dimethylpropanoyl)oxy]-6-hydroxy-2-(3-amino-
2-thienyl)pyrimidine-4-carboxylate (56). 55 (1.47 g, 3.8 mmol)
was dissolved in MeOH/AcOEt (1:1 v/v, 45 mL) and hydrogenated
at atmospheric pressure in the presence of 10% Pd/C (0.7 g). After
2 h, the catalyst was removed by filtration and extensively washed
with hot MeOH and EtOAc. Evaporation of the filtrate gave the
1
70%) as a white solid. H NMR (400 MHz, DMSO) δ 4.01 (s,
3H), 3.88 (s, 3H), 3.76 (s, 3H), 2.51 (s, 3H); m/z 244.8 (M + H).
5,6-Dihydroxy-2-(methylthio)pyrimidine-4-carboxylic Acid
(16). A solution of 60 (0.080 g, 0.33 mmol) in anhydrous DCM (1
mL) was treated with BBr3 (1 M solution in DCM, 1.98 mL, 0.198
mmol). The resulting solution was allowed to warm to room
temperature and stirred for 4 h. The volatiles were evaporated, and
1
amine (1.19 g, 89%) as an off-white solid. H NMR (300 MHz,
DMSO) δ 9.12 (bs, 2H), 7.55 (d, J ) 5.2 Hz, 1H), 6.65 (d, J ) 5.2
Hz, 1H), 3.85 (s, 3H), 1.29 (s, 9H); 13C NMR (100 MHz, DMSO-

1702 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Koch et al.
d6) δ 175.0, 163.3, 159.9, 154.3, 151.6, 143.2, 141.5, 131.0, 121.5,
103.3, 52.9, 30.7, 26.8; m/z 352 (M + H).
Method C: Synthesis of Ureas from Amine 56. The prepara-
tion of 2-[3-({[(2-chloro-6-methylbenzyl)amino]carbonyl}amino)-
2-thienyl]-5,6-dihydroxypyrimidine-4-carboxylic acid (37) is a
representative example. Amine 56 (1.6 g, 4.55 mmol) was dissolved
in dry THF under nitrogen. After cooling at 0 °C, pyridine (0.5
mL, 5.69 mmol) and phenyl chloroformate (0.6 mL, 4.78 mmol)
were added. The ice bath was removed and the reaction was stirred
2 h at RT. The mixture was diluted with EtOAc and washed with
HCl (1 N), water, saturated NaHCO3 and brine. Evaporation of
the solvent gave methyl 5-[(2,2-dimethylpropanoyl)oxy]-6-hydroxy-
2-{3-[(phenoxycarbonyl)amino]-2-thienyl}-pyrimidine-4-carbox-
Method A. Synthesis of Ureas from Amine 54. The preparation
of 2-[3-({[(2-chlorobenzyl)amino]carbonyl}amino)-2-thienyl]-
5,6-dihydroxypyrimidine-4-carboxylic acid (34) is a representa-
tive example. Amine 54 (300 mg, 1.12 mmol) was dissolved in
pyridine (10 mL). 2-Chloro-benzylisocyanate was added dropwise
at room temperature and the reaction monitored by analytical HPLC.
When all the amine was consumed, the solvent was evaporated
and the residue was taken into a large volume of ethyl actetate,
washed with hydrochloric acid (1 N), brine, and dried over sodium
sulfate. The methyl ester obtained after evaporation was hydrolyzed
using sodium hydroxide (4.5 mL, 1 N) and methanol (3 mL) at 80
°C for 30 min. The solution was then cooled in an ice-bath and
acidified with hydrochloric acid (1 N) until a pH of 2 was obtained.
The precipitated solid was isolated by filtration, washed with water
and diethyl ether and dried. It was boiled once in ethyl acetate,
filtered and washed again with ethyl acetate and diethyl ether, and
dried under vacuum to give 270 mg of crude product. This was
purified by preparative reversed phase HPLC using a Waters Prep-
NovaPak column to give 131 mg of 34 (28%) as a colorless powder.
1H NMR (DMSO-d6) δ 12.7 (bs, 1H), 11.05 (bs, 1H), 7.83 (d, J )
5.4 Hz, 1H), 7.61 (d, J ) 5.4 Hz, 1H), 7.42-7.50 (m, 2H), 7.27-
7.38 (m, 2H), 7.00 (bs, 1H), 4.42 (bd, J ) 5.9 Hz, 2H); 13C NMR
(75 MHz, DMSO/HCl, 330K) δ 167.8, 159.3, 153.9, 142.1, 142.0,
140.6, 136.6, 131.6, 128.6, 128.6, 128.1, 127.2, 126.8, 122.1, 112.3,
40.6; m/z 419, 421 (M - H); HRMS calc. for C17H14ClN4O5S
421.0373, fd. 421.0228; HPLC: A: 6.77 min, 100%, B: 10.34,
99%; Anal. (C17H13N4O5S‚H2O) C, H, N, S.
1
ylate in 97% yield. H NMR (DMSO-d6) δ 11.68 (bs, 1H), 7.85
(d, J ) 5.4 Hz, 1H), 7.80 (d, J ) 5.4 Hz, 1H), 7.45 (m, 2H), 7.29
(m, 3H), 3.83 (s, 3H), 1.31 (s, 9H); m/z 470 (M - H). This
compound (100 mg, 0.21 mmol) was suspended in 0.5 mL of
DMSO. 2-Chloro-6-methylbenzylamine (34 mg, 0.22 mmol) was
added and the reaction mixture heated at 60 °C for 3 h. After cooling
to room temperature, the reaction was diluted with EtOAc and
washed with HCl (1 N), water, NaOH (1 M), brine, dried over
sodium sulfate and evaporated. The crude compound was dissolved
in methanol (2 mL) and treated with NaOH (1M, 0.5 mL). The
reaction was stirred 1 h at 80 °C. The solution was then cooled in
an ice-bath and acidified with hydrochloric acid (1 N) until a pH
of 2 was obtained. The precipitated solid was isolated by filtration,
washed with water and diethyl ether and dried affording 37 (19
mg, 15%). 1H NMR (DMSO-d6) δ 12.72 (bs, 1H), 11.01 (bs, 1H),
7.86 (d, J ) 5.7 Hz, 1H), 7.61 (bs, 1H), 7.2-7.3 (m, 3H), 6.40 (s,
1H), 4.48 (d, J ) 4.43 Hz, 2H), 2.45 (s, 3H); m/z 435 (M + H);
HRMS calc. for C18H16ClN4O5S (M + H) 435.0530, fd. 435.0506;
HPLC: A: 7.09 min, 96.1%; B: 11.4 min, 95.2%.
2-(3-{[(Benzylamino)carbonyl]amino}-2-thienyl)-5,6-dihy-
1
Method D: Synthesis of Amides from Amine 56. A solution
of the carboxylic acid (0.2 mmol) and triethylamine (0.034 mL,
0.22 mmol) in DCM (0.7 mL) was cooled to 0 °C and treated with
BOP-Cl (56 mg, 0.22 mmol) in one portion. After 30 min amine
56 (70 mg, 0.20 mmol) and triethylamine (0.034 mL, 0.22 mmol)
were added sequentially and the reaction mixture was allowed to
warm to RT and stirred overnight. The reaction mixture was then
diluted with EtOAc, washed with 1 N HCl, saturated NaHCO3 and
brine, and dried over Na2SO4. The crude residue obtained after
evaporation of the volatiles was dissolved in THF and water and
treated with triturated with 1 N NaOH (0.5 mL). The reaction
mixture was heated at 80 °C for 1 h and then cooled to RT and
acidified with 1 N HCl. The precipitate formed was filtered off,
washed with H2O and with diethyl ether and further purified by
RP-HPLC.
droxypyrimidine-4-carboxylic Acid (25). H NMR (DMSO-d6)
δ, 12.8 (bs, 1H), 11.05 (bs, 1 H), 7.85 (d, J ) 5.4 Hz, 1H), 7.61
(d, J ) 5.4 Hz, 1H), 7.30-7.41 (m, 4H), 7.18-7.27 (m, 1H), 6.93
(bs, 1H), 4.36 (bd, J ) 5.7 Hz, 2H); m/z 387 (M + H), 385 (M -
H); HRMS calc. for C17H15N4O5S (M + H) 387.0763, fd. 387.0728;
HPLC: A: 6.1 min, 96.2%; B: 9.1 min, 98.7%.
Method B: Synthesis of Amides from Amine 54. A solution
of the carboxylic acid (0.27 mmol) and 1,1-carbonyldiimidazole
(43 mg, 0.27 mmol) in anhydrous DMF (0.5 mL) was stirred at
room temperature under nitrogen overnight. Amine 54 (60 mg, 0.22
mmol) was added as a solid and stirring continued for 24 h. HCl
(1 N, 2 mL) was then added to the reaction mixture and the
precipitate formed was isolated by centrifugation, washed with H2O
(2 × 2 mL) and dried. The solid was dissolved in THF/H2O (1:1
v/v, 2 mL) and treated with 1 N NaOH (0.66 mmol) for 1 h at 80
°C. The reaction mixture was acidified with 1 N HCl and the
precipitate isolated by filtration and purified by RP-HPLC.
5,6-Dihydroxy-2-{3-[(phenylacetyl)amino]-2-thienyl}pyrimidine-
4-carboxylic Acid (28). 1H NMR (400 MHz, DMSO) δ 12.90 (bs,
1H), 12.52 (bs, 1H), 7.96 (d, J ) 7.0 Hz, 1H), 7.63 (d, J ) 5.1 Hz,
1H), 7.38-7.31 (m, 2H), 7.30 (t, J ) 7.5 Hz, 2H), 7.20 (m, 1H),
3.71 (s, 2H); m/z 370 (M - H); HRMS calc. for C17H14N3O5S (M
+ H) 372.0654, fd. 372.0660; HPLC: A: 6.47 min, 96.0%; B:
9.39 min, 96.2%.
5,6-Dihydroxy-2-(3-{[(2-phenyl-1H-indol-3-yl)acetyl]amino}-
2-thienyl)pyrimidine-4-carboxylic Acid (43). 48 mg (50%) of a
1
yellow powder obtained; 96% pure by HPLC. H NMR (DMSO-
d6) δ, 13.1 (bs, 1H), 12.65 (s, 1H), 11.3 (bs, 1H), 8.01 (d, J ) 5.4
Hz, 1H), 7.82 (d, J ) 7.5, 2H); 7.71 (d, J ) 5.4 Hz, 1H), 7.59 (d,
J ) 7.9 Hz, 1H), 7.49 (t, J ) 7.6 Hz, 2H), 7.33-7.42 (m, 2H),
7.10 (t, J ) 7.7 Hz, 1H), 6.95 (t, J ) 7.4 Hz, 1H), 4.04 (s, 2H);
m/z 485 (M - H); HRMS calc. for C25H19N4O5S (M + H)
487.1183, fd. 487.1189; HPLC: A: 7.46 min, 95.2%; B: 11.5 min,
95.6%.
Method E: Synthesis of Carbamates from 24. The alcohol
(0.38 mmol) was dissolved in CH3CN (1.0 mL) and treated with
DSC (97.4 mg, 0.38 mmol) and Et3N (0.13 mL, 0.95 mmol) at
room temperature,67 and the resulting solution was stirred overnight.
Amine 24 (55 mg, 0.19 mmol) dissolved in 1:1 mixture of CH3CN
and H2O (1 mL) and Et3N (0.080 mL, 0.57 mmol) was added, and
the resulting mixture was stirred at room temperature overnight.
The volatiles were evaporated, and the residue was treated with
HCl (1 N, 2 mL × 3) and washed respectively with H2O and diethyl
ether and further purified by RP-HPLC.
2-(3-{[(2-Chlorophenyl)acetyl]amino}-2-thienyl)-5,6-dihy-
droxypyrimidine-4-carboxylic Acid (36). 33 mg (89%) of a yellow
1
powder obtained; 96% pure by HPLC. H NMR (DMSO-d6) δ,
13.0 (bs, 1H), 12.5 (s, 1 H), 11.05 (bs, 1H), 7.96 (d, J ) 5.4 Hz,
1H), 7.71 (d, J ) 5.4 Hz, 1H), 7.42-7.47 (m, 2H), 7.30-7.35 (m,
2H), 3.98 (s, 2H); m/z 404, 406 (M - H); HRMS calc. for C17H13-
ClN3O5S (M + H) 406.0264, fd. 406.0277; HPLC: A: 6.74 min,
97.0%; B: 9.95 min, 96.6%.
5,6-Dihydroxy-2-{3-[(1H-indol-3-ylacetyl)amino]-2-thienyl}-
pyrimidine-4-carboxylic Acid (39). 1Η ΝΜR (300 MHz, DMSO)
δ 12.42 (bs, 1H), 10.92 (bs, 1H), 8.01 (d, J ) 5.2 Hz, 1H), 7.68
(d, J ) 5.2 Hz, 1H), 7.63 (d, J ) 7.9 Hz, 1H), 7.33 (m, 2H), 7.06
(t, J ) 7.6 Hz, 1H), 6.96 (t, J ) 7.6 Hz, 1H) 3.86 (s, 2H); 13C
NMR (75 MHz, DMSO/HCl) δ 169.09, 168.76, 160.06, 145.78,
145.27, 140.66, 136.46, 128.80, 128.32, 127.39, 124.41, 122.52,
121.38, 119.07, 118.84, 113.79, 111.755, 108.37, 34.60; m/z 409
(M - H); Anal. (C19H14N4O5S‚H2O) C, H, N, S.
2-(3-{[(Benzyloxy)carbonyl]amino}-2-thienyl)-5,6-dihydroxy-
pyrimidine-4-carboxylic Acid (26). 1H NMR (400 MHz, DMSO-
d6) δ 11.29 (s, 1H), 7.78 (d, J ) 4.7 Hz, 1H), 7.70 (d, J ) 4.7 Hz,
1H) 7.48-7.27 (m, 5H), 5.20 (s, 2H); 13C NMR (300 MHz, DMSO-
d6) δ 168.8, 159.7, 153.0, 146.0, 144.8, 139.7, 136.7, 128.6, 128.3,
128.0, 127.6, 122.0, 113.9, 66.3; m/z 386 (M - H); m/z 389 (M +

Inhibitors of the Hepatitis C Virus NS5B Polymerase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1703
H); HRMS calc. for C17H14N3O6S (M + H) 388.0603, fd. 388.0581;
HPLC A: 7.18 min, 98.0%; B: 10.93 min, 99.1%.
isocyanate (40 mg, 1.1 equiv) were added at 0 °C. The resulting
solution was stirred overnight at room temperature, and then another
1.1 equiv of isocyanate was added. The reaction was brought to
reflux for 5 h and cooled to room temperature. After dilution with
ethyl acetate, the organic phase was washed with hydrochloric acid
(1 N), water, sat. aq sodium hydrogen carbonate, and brine. The
crude product, obtained after evaporation of the solvents, was
directly hydrolyzed using sodium hydroxide (0.5 M, 3 equiv) in
methanol (2 mL) and THF (1.5 mL) at 80 °C for 1 h. The solution
was cooled to room temperature and acidified to pH 2 with
hydrochloric acid (1 N). The crude product was purified by RP-
HPLC. After lyophilization 47 (36 mg, 38%) was obtained as a
colorless powder. 1H NMR (400 MHz; DMSO-d6) δ 8.88 (bs, 1H),
7.66 (d, J ) 5.4 Hz, 1H), 7.53 (d, J ) 5.4 Hz, 1H), 7.42 (d, J )
6.7 Hz, 1H), 7.22-7.35 (m, 3H), 6.73 (t, J ) 5.8 Hz, 1H), 4.31 (d,
J ) 5.8 Hz, 2H), 3.32 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
169.1, 159.4, 154.8150.1, 147.2, 143.3, 138.7, 137.4, 132.4, 129.6,
129.3, 129.1, 127.7, 127.0, 122.7, 113.6, 41.2, 33.7; m/z 433, 435
(M - H); HRMS calc. for C18H16ClN4O5S (M + H) 435.0524, fd.
435.0521; A: HPLC: 6.61 min, 98.8%; B: 12.7 min, 97.5%.
Determination of the pKa. Data were acquired using the Sirius
Profiler SGA (Sirius Analytical, Forest Row, East Sussex, UK) and
the built-in software. A solution of the compound in DMSO 10
mmol, 20 µL) was diluted to 2 mL using deionized water and
infused at 0.25 mL/min into a 1 mL/min pH gradient. Changes in
the UV spectrum were recorded with the diode array detector during
the 2 min run with the pH changing from 2 to 12 and used to
calculate the apparent pKa of the compound.
Molecular Modeling. Conformational analysis was performed
with MacroModel71 generating 1000 initial conformers for each
compound using the Monte Carlo algorithm. The conformers were
subsequently energy-minimized with the MMFF method.72,73
Information about the geometric details of magnesium chelation
were obtained from small molecule crystal structures as deposited
in the CSD.74 This information was used to place the pyrimidine
oxygen atoms into positions corresponding to a typical chelation
geometry. Finally the compound was optimized in the active site
of the protein. The binding of the primer-template duplex and the
nucleoside triphosphate was inferred from the HIV RT ternary
complex as described by Bressanelli.62,75
2-[3-({[(2-Chlorobenzyl)oxy]carbonyl}amino)-2-thienyl]-5,6-
dihydroxypyrimidine-4-carboxylic Acid (35). 1Η ΝΜR (300
MHz, 300K, DMSO-d6) δ 11.46 (bs, 1H), 7.79 (d, J ) 5.52 Hz,
1H), 7.69 (d, J ) 5.31 Hz, 2H), 7.61-7.58 (m, 1H), 7.52-7.48
(m, 1H), 7.44-7.37 (m, 2H), 5.28 (s, 2H); m/z 420 (M - H). HRMS
calc. for C17H13ClN3O6S (M + H) 422.0214, fd. 422.0228; HPLC:
A: 7.80 min, 96.6%; B: 12.5 min, 95.2%.
2-[3-({[(2,6-Dichlorobenzyl)oxy]carbonyl}amino)-2-thienyl]-
5,6-dihydroxy-pyrimidine-4-carboxylic Acid (38). 1H NMR (400
MHz, DMSO-d6) δ 11.13 (s, 1H), 7.85-7.69 (m, 2 H), 7.57 (d, J
) 7.6 Hz, 2 H) 7.52-7.45 (m, 1H), 5.44 (s, 2H); 13C NMR (300
MHz, DMSO-d6) δ 168.6, 159.6, 152.4, 139.0, 136.0, 131.4, 130.9,
128.7, 128.1, 121.7, 61.6; m/z 455 (M - H); m/z 457 (M + H);
HRMS calc. for C17H12Cl2N3O6S (M + H) 455.9824, fd. 455.9799;
HPLC: A: 8.51 min, 97.2%; B: 12.6 min, 96.3%.
5,6-Dihydroxy-2-(3-{[(1-naphthylmethoxy)carbonyl]amino}-
2-thienyl)pyrimidine-4-carboxylic Acid (42). 1H NMR (400 MHz,
DMSO-d6) δ 11.35 (s, 1H), 8.17-8.07 (m, 1 H), 8.01-7.89 (m,
2H) 7.83-7.74 (m, 1H), 7.73-7.47 (m, 5H), 5.69 (s, 2H); m/z 436
(M - H); HRMS calc. for C21H16N3O6S 438.0760 (M + H), fd.
438.0766; HPLC: A: 7.98 min, 95.0%; B: 13.1 min, 94.2%.
Methyl 5-[(2,2-Dimethylpropanoyl)oxy]-1-methyl-2-(3-ni-
trothien-2-yl)-6-oxo-1,6-dihydro-pyrimidine-4-carboxylate (57a).
Pyrimidine 55 (0.8 g, 2.10 mmol) was dissolved in THF (10 mL).
Cesium carbonate (1.03 g, 1.5 equiv) was added, and the mixture
was heated to 50 °C. After 5 min neat dimethyl sulfate (0.53 g, 2
equiv) was added dropwise and the reaction was then stirred for 1
h at this temperature. After cooling to room temperature, the reaction
mixture was diluted with ethyl acetate and the organic phase washed
with NH4Cl (2 M) and brine. Flash chromatography (PE/EA 3:1)
gave the O-methylated compound 58a (0.33 g, 39.8%) as a colorless
1
solid. H NMR (400 MHz, CDCl3) δ 7.44 (d, J ) 5.4 Hz, 1H),
7.41 (d, J ) 5.4 Hz, 1H), 4.01 (s, 3H), 3.96 (s, 3H), 1.40 (s, 9H);
13C NMR (100 MHz, CDCl3) δ 175.0, 163.5, 162.6, 154.1, 147.4,
146.5, 136.5, 132.6, 127.8, 124.2, 55.5, 53.1, 39.2, 26.9; m/z 396
(M + H); recrystallization from dichloromethane/n-pentane, mp
112-114 °C; Anal. (C16H17N3O7S) C, H, N.
The eluent was then changed to PE/EA 1:1 to yield 57a (0.45 g,
1
54.7%) as a colorless solid. H NMR (400 MHz, CDCl3) δ 7.71
Acknowledgment. We thank Silvia Pesci and Renzo Bazzo
for NMR experiments. We are grateful to Francesca Naimo and
Anna Alfieri for analytical chemistry, to Fabio Bonelli and
Vincenzo Pucci for HRMS determination, and to Nadia Gennari,
Monica Bisbocci, Sergio Serafini and Mauro Cerretani for the
inhibition data on the NS5B enzyme and the replicon. We are
indebted to Marco Poma for the resynthesis of compound 3.
We thank Michael Rowley and Joerg Habermann for proofread-
ing of the manuscript and for helpful discussions. This work
was supported in part by a grant from the MIUR.
(d, J ) 5.5 Hz, 1H), 7.57 (d, J ) 5.5 Hz, 1H), 3.92 (s, 3H), 3.40
(s, 3H), 1.43 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 175.0, 162.5,
157.8, 147.8, 146.0, 140.3, 139.4, 134.3, 127.9, 123.7, 53.1, 39.3,
33.5, 27.0; m/z 396 (M + H); recrystallization from dichlo-
romethane/n-pentane, mp 159-162 °C; Anal. (C16H17N3O7S) C,
H, N.
5-Hydroxy-1-methyl-2-(3-nitrothien-2-yl)-6-oxo-1,6-dihydro-
pyrimidine-4-carboxylic Acid (44). Pyrimidone 57a (90 mg) was
hydrolyzed using sodium hydroxide as described for compound 22.
Purification by RP-HPLC gave 44 (30 mg, 44%) as a colorless
1
powder. H NMR (400 MHz; DMSO-d6) δ 7.97 (d, J ) 5.5 Hz,
1H), 7.78 (d, J ) 5.5 Hz, 1H), 3.25 (s, 3H); 13C NMR (100 MHz;
DMSO-d6) δ 168.1, 158.2, 147.7, 145.0, 139.6, 135.5, 129.5, 126.7,
122.8, 32.8; m/z 296 (M - H); HRMS calc. for C10H8N3O6S (M +
H) 298.0128, fd. 298.0123; HPLC: 4.32 min, 100%; C: 4.62,
99.2%.
Supporting Information Available: Synthetic procedures for
the preparation of nitriles 48i, 48n, and inhibitors 21, 23, 27, 45
and 46. NMR and MS data for compounds 7-10, 12-14, 18, 20,
29-33, 40 and 41. Protocols for biological assays. This material
is available free of charge via the Internet at http://pubs.acs.org.
2-[3-({[(2-Chlorobenzyl)amino]carbonyl}amino)-2-thienyl]-
5-hydroxy-1-methyl-6-oxo-1,6-dihydropyrimidine-4-carboxyl-
ic Acid (47). Pyrimidone 57a was dissolved in methanol (0.02 M).
Pd/C (10% Pd, 20% in weight) was added, and the reaction stirred
for 4 h under an atmosphere of hydrogen. Removal of the catalyst
by filtration, exhaustive washing of the catalyst and evaporation
of the solvents gave methyl 5-[(2,2-dimethylpropanoyl)oxy]-1-
methyl-2-(3-aminothien-2-yl)-6-oxo-1,6-dihydropyrimidine-4-car-
boxylate (93%) as a yellow powder, which was used without further
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1
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The amine (80 mg, 0.22 mmol) was dissolved in dichloromethane
(2 mL) and pyridine (26 mg, 1.5 equiv) and neat 2-chlorobenzyl

1704 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
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