Discovery of α,γ-Diketo Acids as Potent Selective and Reversible Inhibitors of Hepatitis C Virus NS5b RNA-Dependent RNA Polymerase

Article abstract of DOI:10.1021/jm0342109

α,γ-Diketo acids (DKA) were discovered from screening as selective and reversible inhibitors of hepatitis C virus NS5b RNA-dependent RNA polymerase. The diketo acid moiety proved essential for activity, while substitution on the γ position was necessary for selectivity and potency. Optimization led to the identification of a DKA inhibitor of NS5b polymerase with IC₅₀ = 45 nM, one of the most potent HCV NS5b polymerase inhibitors reported.

Full text of DOI:10.1021/jm0342109

14
J . Med. Chem. 2004, 47, 14-17
Discover y of r,γ-Dik eto Acid s a s P oten t
Selective a n d Rever sible In h ibitor s of
Hep a titis C Vir u s NS5b RNA-Dep en d en t
RNA P olym er a se
F igu r e 1. DKA lead 1 (depicted in the keto form) for HCV
NS5b polymerase.
Vincenzo Summa,* Alessia Petrocchi, Paola Pace,
Victor G. Matassa,† Raffaele De Francesco,
Sergio Altamura, Licia Tomei, Uwe Koch, and
Philippe Neuner
of the protein,14 and for some of them X-ray structures
of enzyme-inhibitors complexes are also available.15
Random screening of more than 200 000 samples
identified the R,γ-diketo acid 1 (Figure 1) as a low
micromolar, specific, and reversible inhibitor of HCV
NS5b polymerase with IC50 ) 5.7 µM. At 500 µM, it did
not inhibit the Klenow DNA polymerase and poliovirus
RNA-dependent RNA polymerase. A weak inhibitory
effect was observed on HIV-RT (IC50 ) 54 µM). Recently,
we have reported that the diketo acid (DKA) mechanism
of inhibition is distinct from all the other known
inhibitors of HCV polymerase reported so far in the
literature; they are capable of interacting directly with
metal ions present in the enzyme active site of HCV
NS5b polymerase. They can be considered as product-
like inhibitors showing a mechanism of action similar
to that of foscarnet, a known pyrophosphate analogue.4
DKAs have been also reported as potent HIV inte-
grase16,17 and RNAse-H18 inhibitors with HIV antiviral
activity in cell-based assays. Similar inhibitors of in-
fluenza viruses are also known.19
Resu lts a n d Discu ssion . Initial SAR showed that
the aryl ring is absolutely required for activity. Its
replacement with aliphatic groups such as methyl, t-Bu,
and benzydryl group leads to compounds displaying IC50
> 50 µM (Table 1, 2-4). Phenyl can be replaced with
thiophene to yield 5 showing activity similar to that of
the original lead 1. However, more polar heterocycles
such as 3- or 4-pyridine as well as 2-furan were not
tolerated (Table 1, 6-8).
Departments of Medicinal Chemistry and Biochemistry,
IRBM-MRL Rome, Via Pontina, Km 30.600,
00040 Pomezia, Rome, Italy
Received October 3, 2003
Abstr act: R,γ-Diketo acids (DKA) were discovered from screen-
ing as selective and reversible inhibitors of hepatitis C virus
NS5b RNA-dependent RNA polymerase. The diketo acid
moiety proved essential for activity, while substitution on the
γ position was necessary for selectivity and potency. Optimiza-
tion led to the identification of a DKA inhibitor of NS5b
polymerase with IC50 ) 45 nM, one of the most potent HCV
NS5b polymerase inhibitors reported.
In tr od u ction . Hepatitis C virus (HCV) is a member
of the flaviviridae viruses and is now recognized as the
major cause of both parenterally transmitted and com-
munity-acquired non-A, non-B hepatitis (NANB-H). It
was identified at the molecular level at the end of
1980s.1 Although the number of new infections has been
significantly reduced by the introduction of reliable
blood testing, it has been estimated that at least 1% of
the world population is affected by the disease. More
than half of the affected individuals become chronically
infected, and liver cirrhosis develops in at least 20% of
this group. In addition, increased incidence of hepato-
cellular carcinoma in NANB-H patients suggests that
HCV also plays a role in the process of hepatocarcino-
genesis.
Ta ble 1. Replacement of Phenyl Moiety of DKA Lead
There is as yet no effective therapy for HCV-associ-
ated chronic hepatitis. Treatments with interferon R
(IFN-R) alone or in combination with ribavarin are
effective in controlling the disease only in a fraction of
the patients. A high proportion of the infected individu-
als fail to respond to IFN-R therapy, and relapse after
cessation of the treatment is not uncommon. There is
thus an obvious need to develop effective therapeutic
strategies to cure HCV-associated hepatitis.2,3
The most studied targets for anti-HCV therapy are
the NS3 protease and the NS5b polymerase.4 Several
classes of NS3 protease inhibitors have been reported
by us5-8 and others,9-12 mostly with a peptidic charac-
ter. In the case of HCV NS5b polymerase, both nucleo-
side13 and non-nucleoside inhibitors have appeared
recently in the literature. The latter class contains
different scaffolds that interact in allosteric binding sites
compd
R1
IC50 (µM)a
1
2
3
4
5
6
7
8
Ph
Me
t-Bu
5.7 ( 0.2
>50
>50
>50
(Ph)2CH
3-thiophene
3-pyridine
4-pyridine
2-furan
4.5 ( 0.2
>50
>50
>50
a
For polymerase assay, see Supporting Information. Data are
the mean of two to four independent experiments.
The DKA moiety was also explored, and it proved
essential for the inhibitory activity. Simple modification
led to the complete loss of inhibitory activity against
the HCV NS5b polymerase (Table 2, 9-11). Structure
1 is therefore the minimal scaffold capable of inhibiting
the HCV NS5b polymerase. By use of this compound
as a template, substitutions on the phenyl ring were
introduced singularly or in tandem to explore structure-
activity relationship.
* To whom correspondence should be addressed. Phone: +39(0)-
691093680. Fax: +39(0)691093654. E-mail: Vincenzo_summa@merck.
com.
† Present address: Graffinity Pharmaceutical AG Im, Neuenheiner
Feld 518, Heidelberg 69120, Germany.
10.1021/jm0342109 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2003

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 15
Ta ble 2. DKA Acid Moiety Essential for Activity
compd
X
Y
Z
IC50 (µM)a
F igu r e 2. Ortho ether substituted DKA A in a self-assembled
conformation B.
1
9
10
11
O
O
O
H2
O
O
OH
O
H
Me
H
5.7 ( 0.2
>50
>50
The meta position of the phenyl ring appears to be
more tolerant of size and nature of the substituents
compared to the ortho and para positions. Simple
O-alkyl side chains did not have the same effect
obtained in the ortho position. Aromatic moieties were
preferred, and different functional groups were toler-
ated. In the first selection of substituents, the choice
was made to obtain similar size and shape (Table 5);
benzoic amide 20, phenyl sulfonamide 21, benzylamine
22, and benzyl ether 23 were compared. The resulting
activities were all in the same range with the exception
of 23, which showed single-digit micromolar activity
(IC50 ) 8 µM). 24, which had an inverse benzyl ether
function with respect to 23, showed a reduction in
potency.
H
>50
a
For polymerase assay, see Supporting Information. Data are
the mean of two to four independent experiments.
Initial studies on the para position revealed that only
small substituents were tolerated, such as methyl 12
that retains the same potency, while the introduction
of an ethyl 13 or t-Bu 14 resulted in a considerable loss
of activity (Table 3).
Ta ble 3. Para-Substituted DKA (Figure 1)
compd
R
IC50 (µM)a
1
12
13
14
H
4-Me
4-Et
5.7 ( 0.2
5.0 ( 1.0
47.0 ( 3.0
>50
4-t-Bu
Ta ble 5. Meta-Substituted DKA (Figure 1)
a
For polymerase assay, see Supporting Information. Data are
the mean of two to four independent experiments.
compd
R
IC50 (µM)a
1
20
21
22
23
24
H
5.7 ( 0.2
11.0 ( 1.0
16.0 ( 1.5
16.7 ( 1.5
8.0 ( 0.9
30 ( 4.0
3-NHCOPh
3-NHSO2Ph
3-NH-Bn
3-O-Bn
3-CH2OPh
In contrast, substituents in the ortho position showed
a significant improvement of activity with alkyl ethers,
which were found to be the optimum functional group.
Simple aliphatic chain ethers with a length of three
carbon atoms (15) were preferred, and the potency
dropped upon increasing chain length (16, 17). Side
chains containing aromatic moieties were not tolerated
(18). Propyl side chains having a variety of terminal
functional groups were explored and are allowed with-
out a considerable improvement of activity against the
NS5b polymerase (data not shown). The only exception
was the nitrile 19, which resulted in a 20-fold improve-
ment of inhibitory activity with respect to the original
screening lead, displaying IC50 ) 350 nM (Table 4).
a
For polymerase assay, see Supporting Information. Data are
the mean of two to four independent experiments.
On the basis of these results, the benzyl ether was
chosen as a fixed fragment for further optimization. We
explored the possibility of exploiting the same interac-
tions in the meta-substituted series which render the
o-3-cyanopropoxy substituted DKA 19 such a potent
inhibitor. Molecular modeling indicated that the phenyl
ring of the benzyl moiety prefers a coplanar arrange-
ment relative to the DKA phenyl ring (Figure 3).
Ta ble 4. Ortho-Substituted DKA (Figure 1)
compd
R
IC50 (µM)a
1
15
16
17
18
19
H
5.7 ( 0.2
3.9 ( 0.5
10.0 ( 1.0
25.0 ( 2.0
44.0 ( 3.0
0.35 ( 0.04
2-O-propyl
2-O-butyl
2-O-pentyl
2-O-Bn
2-OCH2CH2CH2CN
F igu r e 3. Superimposition of DKA 19 (gray) and 25 (green).
Of four almost isoenergetic conformations of 25, only the one
most similar to 19 is shown.
a
For polymerase assay, see Supporting Information. Data are
the mean of two to four independent experiments.
Ortho ethers may adopt two different conformations.
In one (Figure 2, A), the ortho aryl oxygen is close to
the carbonyl oxygen of the DKA. In the second confor-
mation (B), the ortho aryl oxygen is oriented toward the
C3-H. This relative orientation (B) was consistent with
results from NMR experiments probably because of
electrostatically unfavorable interactions between the
ether and C4 oxygen atoms in conformation A. NOE
experiments indicated a distance larger than 4 Å
between the proton in the ortho position of the phenyl
ring and the vinylic proton C3-H of the enolic form of
the DKA.
Introduction of a cyano group in position 2 of the
benzyl moiety should place the cyano group in a position
similar to that of 19 (Figure 3). Indeed, the optimal
substituent was the 2-cyano benzyl ether 25 showing
IC50 ) 230 nM, 1.5-fold more potent than 19.
The isomeric 3-cyano benzyl ether 26 was 15-fold less
active with respect to 25. 27 with a simple, more flexible
aliphatic side chain having the same length from the
oxygen of the ether to the nitrile proved 11-fold less
active.
The nitrile group was left constant at the 2 position
of the benzyl ether, and a variety of substituents were

16 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Letters
Sch em e 1. Synthesis of 1-8 and 12-30a
added to this moiety to improve the potency (Table 6).
Introducing a single bromine in the 5-position gave a
2-fold improvement in potency 28 (IC50 ) 100 nM), while
3-chloro 29 was 1.5-fold more active (IC50 ) 110 nM).
The combination of two halogens generated 30, which
showed IC50 ) 45 nM against the NS5b polymerase. 30
is one of the most potent inhibitors of HCV NS5b
polymerase reported in the literature. 30 was highly
selective with regard to the R,â and γ human DNA
polymerases and HIV RT, showing IC50 > 5.0 µM and
IC50 > 50.0 µM, respectively.
a
Reagents and conditions: (a) Br-R or Cl-R, Cs2CO3 DMF,
80 °C, 12 h for X ) O, NH; RCOCl, THF, N(Et)3, room temp for X
) NH; RSO2Cl, Py, room temp, 8 h for X ) NH; (b) diethyl oxalate,
NaOEt, THF, room temp; (c) NaOH in MeOH.
Ta ble 6. Meta-Substituted Benzyl Ether
acetophenone generated the suitable acetophenone pre-
cursors of 20-22. Displacement with phenol of the
3-bromotoluolacetophenone gave the precursor for 24.
Bromination of suitably substituted toluene derivatives
afforded the bromo derivative used for the alkylation
of 3-hydroxyacetophenone precursors of 25-26 and
28-30. All compounds were fully characterized by
1H NMR, LC-MS, and HRMS.
compd
R2
2-CNPh
IC50 (µM)a
25
26
27
28
29
30
0.23 ( 0.02
6.70 ( 0.8
2.50 ( 0.5
0.100 ( 0.005
0.110 ( 0.010
0.045 ( 0.008
3-CNPh
CH2CH2CN
2-CN-5-BrPh
2-CN-3-ClPh
2-CN-3,5-Cl2Ph
Con clu sion . By screening, we have discovered a
novel class of HCV NS5b polymerase inhibitors selective
against other RNA and DNA polymerases. Essential
structural features of DKAs necessary for activity were
identified, and the lead 1 that showed low micromolar
activity was converted into 30 with IC50 ) 45 nM, one
of the most potent HCV NS5b polymerase inhibitors
reported in the literature.
a
For polymerase assay, see Supporting Information. Data are
the mean of two to four independent experiments.
Meta ethers do not have the same self-assembled
conformation as seen with the ortho ether, and conse-
quently, the side chain could be located in either region
of space with respect to the DKA moiety. To exclude the
possibility that the side chains of 19 and 25 explore
different regions of space generating different contacts
with NS5b polymerase, a 2,5 disubstituted compound,
31 (Figure 4), was prepared bearing the best substitu-
tion found in both positions. It was essentially inactive
(IC50 > 50 µM), suggesting that the two side chains
occupy the same region as in Figure 3.
Ack n ow led gm en t. The authors thank Nadia Gen-
nari and Sergio Serafini for the determination of the
IC50 values for NS5b, and Kara Stillmock for determin-
ing the IC50 values for human polymerases. We also
thank Prof. Luciano Cellai, Marina Taliani, and Alberto
Mochi Onori for the synthesis of some known diketo
acids and the original lead, Fabio Bonelli for the HRMS,
and Silvia Pesci and Edith Monteagudo for NMR
experiments. This work was supported in part by a
grant from the MIUR.
Su p p or tin g In for m a tion Ava ila ble: General synthetic
procedures, 1H NMR, HRMS data, and biological evaluation
for compounds listed in all tables. This material is available
free of charge via the Internet at http://pubs.acs.org.
F igu r e 4. Disubstituted DKA 31.
Syn th esis. Scheme 1 depicts a general procedure for
the synthesis of DKA. The appropriate substituted
acetophenone is reacted with diethyl oxalate in the
presence of sodium ethoxide in THF, and subsequent
hydrolysis of the ethyl ester with sodium hydroxide in
methanol gives the desired DKA. The two steps can be
carried out separately or in one pot.
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