HCV NS5b RNA-dependent RNA polymerase inhibitors: From α,γ- diketoacids to 4,5-dihydroxypyrimidine- or 3-methyl-5- hydroxypyrimidinonecarboxylic acids. Design and synthesis

Article abstract of DOI:10.1021/jm0494669

A new class of the HCV NS5b RNA-dependent RNA polymerase inhibitors, the dihyroxypyrimidinecarboxylic acid derivative, was designed from a diketoacid and meconic acid derivative discovered by screening. Mechanism of action and essential moieties required for activity were identified. The corresponding N-methylpyrimidinone was also prepared; both classes are novel, reversible, and selective inhibitors of the HCV NS5b polymerase with improved druglike characteristics.

Full text of DOI:10.1021/jm0494669

5336
J . Med. Chem. 2004, 47, 5336-5339
HCV NS5b RNA-Dep en d en t RNA
P olym er a se In h ibitor s: F r om
r,γ-Dik etoa cid s to
4,5-Dih yd r oxyp yr im id in e- or 3-Meth yl-5-
h yd r oxyp yr im id in on eca r boxylic Acid s.
Design a n d Syn th esis
F igu r e 1. Leads for HCV NS5b polymerase.
Vincenzo Summa,* Alessia Petrocchi,
Victor G. Matassa, Marina Taliani, Ralph Laufer,
Raffaele De Francesco, Sergio Altamura, and
Paola Pace
Recently, we reported the discovery by random screen-
ing of R,γ-diketoacid 1 (DKA) as potent, reversible, and
selective inhibitors of HCV NS5b RNA-dependent RNA
polymerase with IC50 ) 5.7 µM.18 The R,γ-diketoacid
functionality proved to be essential for enzyme inhibi-
tion in this class of inhibitors. It can be considered as
an active site anchor because it interacts with the Mg2+
ions, i.e., the catalytic center of the polymerase. The
aromatic portion of the molecules proved to be essential
for specificity and potency (Figures 1, 1).
In a second random screening the monoethylester of
meconic acid 2 (Figure 1, 2) was discovered as a new
class of selective and reversible inhibitor of the HCV
NS5b polymerase, having an IC50 ) 2.25 µM.19
Departments of Medicinal Chemistry and Biochemistry
IRBM-MRL Rome, Via Pontina, Km 30.600,
00040 Pomezia (Rome), Italy
Received J uly 5, 2004
Abstr a ct: A new class of the HCV NS5b RNA-dependent RNA
polymerase inhibitors, the dihyroxypyrimidinecarboxylic acid
derivative, was designed from a diketoacid and meconic acid
derivative discovered by screening. Mechanism of action and
essential moieties required for activity were identified. The
corresponding N-methylpyrimidinone was also prepared; both
classes are novel, reversible, and selective inhibitors of the
HCV NS5b polymerase with improved druglike characteristics.
The two screening leads share a very similar chelating
portion formed by a carbonyl, one acid hydroxyl func-
tion, and a carboxylic acid. A displacement competition
experiment between the DKA 1 and the meconic acid
derivative 2 indicated that the two compounds inhibit
the enzyme in a mutually exclusive fashion, indicating
a common mechanism of action. Kinetic competition
experiments confirmed this result. However, there was
concern for the development of these compounds as
chemotherapeutic agents due to their intrinsic chemical
and biological instability.20 Therefore, we sought viable
replacements of these core structures with more drug-
like characteristics. Several possible classes of inhibitors
were designed by keeping the distances fixed between
the chelating functions and the other moieties that
generate the specificity. Particularly useful in assigning
the correct distances between the different atoms that
should interact with the metals in the active site was
the X-ray structure of the T7 DNA polymerase cocrys-
tallized with a nucleoside triphosphate chelating the two
metals.21 The distance between the two oxygens of the
triphosphate engaged in a Mg2+ chelation is 3.1 Å. In
the CSD is also available the crystal structure of a
diketo compound chelating Mg2+ with the distance
between the two oxygens being 2.8 Å. On the basis of
these distances, several possible chelation patterns were
considered, and some of them were prepared and tested
without success. However, the dihydroxypyrimidinecar-
boxylic acid 3 designed as hybrid of DKA 1 and meconic
acid derivatives 2 (Figure 2) was particularly attractive.
Superimposition of DKA 1 and dihydroxypyrimidine-
carboxylic acid 3 shows a good overlap of the chelating
moieties and the aromatic ring of the two inhibitors
(Figure 3, A). Even better overlap of the chelating
moiety was obtained with the meconic acid derivative
2 and 3 (Figure 3, B).
Hepatitis C virus (HCV) was identified in 1989 as the
pathogen responsible for non-A and non-B hepatitis
(NANB-H).1 It has been estimated that 1-3% of the
world population is infected. Seventy to eighty percent
of infections become chronic and may progress to
cirrhosis. In addition, increased incidence of hepatocel-
lular carcinoma in NANB-H patients suggests that HCV
plays a role in the progression of hepatocarcinogenesis.
The recommended therapies are based on interferon-R
(IFN-R) alone or in combination with the broad spec-
trum antiviral ribavirin. Only in a fraction of the patient
population are these therapies effective.2,3 They also
cause severe side effects that significantly reduce com-
pliance to the therapy. There is thus an obvious need
to develop an efficacious and well tolerated anti HCV
agent.4,5
HCV is a member of the flaviviridae viruses and is a
small enveloped positive-stranded RNA virus that en-
codes a polyprotein of 3000 amino acids divided into four
structural and six nonstructural proteins.6 The HCV
NS5b RNA-dependent RNA polymerase is a pivotal
enzyme in the replication of the virus. Therefore, NS5b
has become a prime target for both the screening and
the design of small molecules inhibitors of viral replica-
tion. Recently, different classes of NS5b inhibitors have
appeared in the literature: they can be divided by their
mechanism of action into three major classes:7 nucleo-
side,8,9 non-nucleoside inhibitors acting at allosteric
binding sites,10-17 and last non-nucleoside inhibitors
capable of interacting with the pyrophosphate binding
site discovered in this laboratory.
* To whom correspondence should be addressed. Address: Depart-
ment Medicinal Chemistry IRBM MRL Rome, Via Pontina Km 30.600
Pomezia (RM) Italy. Phone +39(0)691093680. Fax +39(0)691093654.
E-mail: Vincenzo_summa@merck.com.
Present address: Graffinity Pharmaceutical AG Im, Neuenheiner
Feld 518, Heidelberg, Germany 69120.
An aryl moiety was preferred to the carboxylic ester
as a fragment to insert in the hybrid to improve the
10.1021/jm0494669 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/16/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5337
F igu r e 4. Two of the possible tautomers of the dihyroxypy-
rimidine 3.
F igu r e 2. Design of dihydroypyrimidinecarboxylic acid de-
rivative 3 from 1 and 2.
F igu r e 5. N-Methylpyrimidinone 11.
on the phenyl ring of 3. The benzyl ether moiety was
introduced in all three aromatic positions. While the o-
and p-benzyl ether-substituted analogues 3 led to inac-
tive compounds (data not shown), the meta-substituted
benzyl ether derivative 6 gave a compound 2-fold more
potent showing an IC50 ) 15 µM (Mg2+). 6 was also
kinetically competitive with DKA 1. Deprotection of the
benzyl ether moiety in 6 by hydrogenation led to 7
having the desired phenolic function in the meta posi-
tion. 7 was even more potent than the previous com-
pound, showing an IC50 ) 5.8 µM (Mg2+) and submi-
F igu r e 3. (A) Superimposition of dihydroxypyrimidine (green)
and DKA 1 (blue). (B) Superimposition of dihydroxypyrimidine
and meconic acid derivative 2 (blue).
Ta ble 1.
cromolar activity in the presence of Mn2+
.
The moieties essential for chelation were investigated
to understand the minimal requirement for the activity
of this new class of HCV NS5b polymerase inhibitors.
O-Methylation of the oxygen in the 4 and 5 positions of
the pyrimidine core (Table 1, compounds 8 and 9) led
compd
R
R1
R2 R3 IC50 Mg/µMa IC50 Mn/µMa
3
4
5
6
7
8
9
10
H
CO2H
CO2Me
CO2H
H
H
H
H
H
H
H
H
30
>100
29
2.3
30
1.4
1.0
0.6
4-Cl
4-Cl
3-OBn CO2H
3-OH
3-OH
4-Cl
15
5.8
to inactive compounds, even in the presence of Mn2+
,
CO2H
CO2H
CO2H
H
H
H
H
Me
H
Me
H
H
>50
>50
>50
>50
>50
>50
indicating that the chelating properties were completely
abolished. To complete the definition of the pharma-
cophore of the chelating moiety, 8 was decarboxylated
to give 10, which was also inactive even in the presence
of Mn2+. Thus, the carboxylic acid was the third es-
sential moiety of the active site anchor (Table 1,
compound 10). All compounds were also tested in the
HCV replicon assay, and they were neither active nor
toxic up to 50 µM. The log D of compounds 5 and 6 are
-1.05 and -0.22, respectively: this property could
influence the cell permeability of these molecules in the
cell-based assay. In addition, the lack of activity in this
system is most likely due to the intrinsically modest
enzyme inhibition.
The dihydroxypyrimidine carboxylic acid has several
possible tautomeric forms in solution. The 1H NMR
spectra revealed that in DMSO the compounds are
present mainly in one form (dihydroxy pyrimidine), but
this does not exclude the possibility that upon binding
with Mg2+ or Mn2+ the equilibrium shifts toward a
pyrimidinone form (Figure 4, 3B) and that this is the
active tautomer.
3-OH
a
Polymerase assay: see Supporting Information.
druglike character of the new molecules. Searching
commercially available samples, we found the methyl
dihydroxypyrimidinecarboxylic ester derivative 4 (Table
1) that corresponded very well to our designed molecule.
This compound under our routine screening conditions
resulted inactive at 100 µM in the presence of Mg2+ but
showed an IC50 ) 30 µM in the presence of Mn2+ (Table
1). These results indicated that 4 was capable of
inhibiting the HCV NS5b polymerase. The methyl ester
of 4 was hydrolyzed to corresponding acid 5 that was
active both in the presence of Mg2+ and Mn2+showing
IC50’s of 29 µM and 1.4 µM, respectively (Table 1). The
biochemical behavior of this new class of inhibitors was
identical to the other leads 1 and 2, showing an
increasing inhibitory potential in the presence of Mn2+
with respect to Mg2+. Kinetic competition experiments
between the DKA 1 and the new lead 5 confirmed a
mutually exclusive inhibition.
The unsubstituted compound 3, prepared as a refer-
ence compound, was equipotent to the lead 5 under both
assay conditions (Mg2+ or Mn2+), indicating that an
electron-withdrawing group in the para position of the
aryl moiety is a neutral substitution.
On the basis of the experience with the DKAs, in
which a benzyl ether or a simple phenolic function
enhanced the inhibitory activity with respect to the
original DKA 1, these two moieties were incorporated
To establish if the hydroxypyrimidinone 3B is able
to chelate the Mg2+ in the active site, the N-3 meth-
ylpyrimidinone of the most active compound 7 was
prepared giving 11 that showed IC50 ) 6.0 µM (Mg2+
and IC50 ) 1.0 µM (Mn2+) (Figure 5).
)
The shift in potency in the presence of the two metals
indicated that the N-methylpyrimidinone has the same
behavior of DKA 1, meconic acid derivative 2, and
dihydroxypyrimidine 3, suggesting that all four classes
share the same mechanism of action. What remains

5338 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Letters
Sch em e 1. Synthesis of Compounds 3, 6, 7, 10a
Ta ble 2.
compd HIV RT IC50/µMa HIV Int IC50/µMb Klenow IC50/µMa
3
5
6
7
>100
23
>100
>100
>50
>50
>50
>50
>100
>100
>100
>100
a
b
Polymerase assay: see Supporting Information. See ref 22.
unproven is if the dihydroxypyrimidine is present in the
pyrimidinone form 3B on binding with the cations Mg2+
or Mn2+ in the enzyme active site.
Active compounds were also tested against HIV-RT,
HIV-integrase, and Klenow DNA-dependent DNA poly-
merase to measure their selectivity (Table 2). The
original lead 5 was unselective, having similar activity
on HIV-RT and HCV NS5b Pol. This undesirable
property disappeared with all the other compounds.
While aryl-DKAs have been reported as potent HIV
integrase inhibitors,22 our compounds showed no detect-
able inhibition against this target at tested concentra-
tion. These very preliminary results show that the
substitution of aryl group in this new class of inhibitors
can modulate the selectivity and potency. These results
are in agreement with the function of the aryl moiety
in DKA series, where it performs the same role.
It was then established whether the new class of
inhibitors was more chemically or biologically stable
than DKA’s and meconic acid derivatives. Both 3 and 5
were completely stable in 1 N HCl solution and in
aqueous media at pH ) 7.4 after 24 h at room temper-
ature; in contrast, 1 and 2 were almost completely
decarboxylated in acid media. The stability of 5 and 7
were also measured in the presence of glutathione. On
incubation for 2 h with and without GSH, in the same
buffer and temperature conditions, the compounds
proved to be completely stable. In contrast the DKA 1
was more than 50% consumed in the same experiment.
Finally 3[H]-3 was used to measure the irreversible
covalent binding of this inhibitor to rat liver microsomes
in the presence of NADPH. In both cases, after 4 h of
incubation, the residual radioactivity was less than 2
pmol/mg protein, indicating that compound 3, the
prototype of the series, does not bind irreversibly to rat
liver microsome protein. In contrast the DKA showed a
high level of irreversible binding (>400 pmol/mg pro-
tein).
Scheme 1 depicts a general procedure for the synthe-
sis of dihydroxypyrimidines.23 The appropriately sub-
stituted benzamide oxime was reacted with dimethyl
acetylendicarboxylate to afford the Michael adduct as
a mixture of cis-trans isomers which, on heating in
refluxing xylene, probably underwent a Claisen rear-
rangement and cyclization to afford the methyl-5,6-
dihydroxy-2-arylpyrimidine-4-carboxylate. This was hy-
drolyzed to the carboxylic acid 3 (R ) H) or 6 (R )
3-OBn) by heating with an excess of NaOH. Hydrogena-
tion of the benzyl ether of 6 gave 7, which when heated
with 6 N HCl afforded the 2-aryl-4,5-pyrimidinediol 10.
The synthesis of the methylated derivatives 8 and 11
started with the 3-p-methoxybenzyl-protected dihy-
droxypyrimidine 14 (Scheme 2), which was prepared
according to the above procedure.
a
Reagents and conditions: (a) DMAD (1.2 equiv), CHCl3 or
MeOH, 50 °C, 2 h; (b) xylene, 140 °C, 12-18 h; (c) 2N NaOH (3
equiv), MeOH, 50 °C; (d) Pd/C, MeOH, H2, rt; (e) 6 N HCl, reflux,
30 min.
Sch em e 2. Synthesis of Compounds 8, 9, and 11a
a
Reagents and conditions: (a) (PhCO)2O (1.2 equiv), Pyr (3
equiv), CH2Cl2; (b) CH3I (3 equiv), CsCO3 (2 equiv), THF, 40 °C,
5 h; (c) 2M TMSCHN2 soln in hexane (1.1 equiv × 5), MeOH, 4
°C, 12 h; (d) CH2Cl2: TFA (8:2 v/v), 30 min; (e) 2 N NaOH (3 equiv),
MeOH, 50 °C.
16 (60-70% yield, 40:60 ratio), which were purified by
flash chromatography. The p-methoxybenzyl group was
cleaved by trifluorocetic acid in dichloromethane, and
subsequent basic hydrolysis afforded 8 and 11. The
reaction of 4 with TMS-diazomethane in methanol
followed by basic hydrolysis afforded the 5-O-methyl
derivative 9.
1
All compounds were fully characterized by H NMR
and LC-MS and HRMS. For unambiguous structural
elucidation of compound 8, 9, and 11, HMBC NMR was
performed.
Two novel classes of HCV NS5b polymerase inhibitors
were discovered: 4,5-dihydroxypyrimidine-6-carboxylic
acid derivative 3 was designed from the other two leads,
DKA 1 and meconic acid derivative 2, originally discov-
ered by screening. The corresponding N3-methylpyri-
midinone was synthesized in order to investigate the
importance of keto-enol tautomerism of the 5-hydroxy
group. The compounds were also tested against other
RNA and DNA polymerases and HIV integrase proving
that it is possible to achieve selectivity. Essential
structural features of these new classes of HCV poly-
merase inhibitors necessary for activity were identified;
the mechanism of action is similar to the other two leads
from screening. The novel series offers considerable
improvements over the original leads in druglike char-
acteristics, being stable in acid and in the presence of
GSH, and not covalently binding to rat liver microsomes.
After benzoylation of the 5-hydroxy group, reaction
with methyl iodide and cesium carbonate in THF
afforded a mixture of the methylated derivatives 15 and

Letters
Ack n ow led gm en t. The authors thank Nadia Gen-
nari and Sergio Serafini for determination of the IC50
values for all assays, Kara Stillmock for testing in HIV-
Int assay, Uwe Koch for the minimization and super-
imposition of the lead, Silvia Pesci for NMR experi-
ments, and Fabio Talamo for the HRMS. This work was
supported in part by a grant from the MIUR.
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5339
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