Non-hydrolysable analogues of inorganic pyrophosphate as inhibitors of hepatitis C virus RNA-dependent RNA-polymerase

Article abstract of DOI:10.1134/S1068162012020124

Inorganic pyrophosphate (PP_i) is the product of the polymerization reaction catalyzed by DNA-and RNA-polymerases. A number of novel non-hydrolsable PP_i analogues was synthesized; some of them inhibited the polymerization reaction catalyzed by hepatitis C virus RNA-dependent RNA-polymerase (NS5B). A new pharmacophore based on a non-hydrolysable methylenediphosphonate backbone has been developed. The structure-activity relationship analysis of 12 bisphosphonates is presented and the structural features crucial for NS5B polymerase activity inhibition are stated. Pleiades Publishing, Ltd., 2012.

Full text of DOI:10.1134/S1068162012020124

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 2, pp. 224–229. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © D.V. Yanvarev, A.N. Korovina, N.N. Usanov, S.N. Kochetkov, 2012, published in Bioorganicheskaya Khimiya, 2012, Vol. 38, No. 2, pp. 257–262.
NonꢀHydrolysable Analogues of Inorganic Pyrophosphate
as Inhibitors of Hepatitis C Virus RNAꢀDependent RNAꢀPolymerase
D. V. Yanvarev1, A. N. Korovina, N. N. Usanov, and S. N. Kochetkov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
Received May 25, 2011; in final form, June 22, 2011
Abstract—Inorganic pyrophosphate (PPi) is the product of the polymerization reaction catalyzed by DNAꢀ
and RNAꢀpolymerases. A number of novel nonꢀhydrolsable PPi analogues was synthesized; some of them
inhibited the polymerization reaction catalyzed by hepatitis C virus RNAꢀdependent RNAꢀpolymerase
(NS5B). A new pharmacophore based on a nonꢀhydrolysable methylenediphosphonate backbone has been
developed. The structureꢀactivity relationship analysis of 12 bisphosphonates is presented and the structural
features crucial for NS5B polymerase activity inhibition are stated.
Keywords: hepatitis C virus, pyrophosphate, analogues, RNAꢀdependent RNAꢀpolymerase (NS5B), methyleneꢀ
diphosphonates
DOI: 10.1134/S1068162012020124
1
INTRODUCTION
enzyme. Nonꢀnucleoside inhibitors can be subdivided
into two classes by the principle of action. They are
compounds, which bind polymerase outside the cataꢀ
lytic site, representing “allosteric” noncompetitive
inhibitors, and Mg2+ꢀchelating agents, which contain a
Mg2+ꢀbinding site (Fig. 1) and a hydrophobic group
some distance from it.
Hepatitis C virus (HCV) causes a wideꢀspread viral
disease. Its chronic form leads to a number of such
strong liver affections as cirrhosis or hepatocellular
carcinoma [1]. To date, about 170 million people are
infected with HCV [2], thereby attaching high social
significance to the search for antiviral drugs. The curꢀ
rent hepatitis C therapy is based on interferon alpha
and a nonspecific antiviral nucleoside analog ribavirin
combination [3]. However, the clinical use of this drug
combination is usually complicated by toxic side
effects and is not efficient in the case of a number of
HCV genotypes [4]. These factors are responsible for
the critical need for new drug families with less promꢀ
inent side effects and better acceptability.
In spite of the seeming structural consistency of
Mg2+ꢀchelating NS5B inhibitors, the mechanism of
their actions differs. Thus,
α,γꢀdiketo acids and
4,5ꢀdihydroxypyrimidine carboxylic acids imitate
inorganic pyrophosphate in the catalytic site of the
enzyme (Fig. 1). However, α,γꢀdiketo acids are nonꢀ
competitive inhibitors of the HCV polymerase with
regard to RNAꢀsubstrate and also to nucleotides [6],
while dihydroxypyrimidines act as competitive inhibiꢀ
tors with regard to triphosphates [7].
NS5B chelating inhibitors, which bind Mg2+ in the
corresponding region of the catalytic site, are mimetics
of the inorganic pyrophosphate, which is one of the
reaction products. At that they have lots of structural
differences. At the same time, the data on NS5B inhibiꢀ
tion by PPi structural analogues are almost absent,
though such compounds efficiently inhibited a number
of viral and cellular polymerases [8]. We report the synꢀ
thesis of a number of PPi analogues, and their action as
NS5B polymerase inhibitors.
Viral RNAꢀdependent RNAꢀpolymerase (NS5B,
EC 2.7.7.48) is a promising target for an antiꢀHCV
drug design as nonꢀinfected liver cells do not bear this
enzyme, suggesting low cytotoxicity of specific inhibꢀ
itors of NS5B polymerase activity [5]. To date, many
such inhibitors are known, however none of them is in
clinical use.
It is possible to subdivide known NS5B inhibitors
into nucleoside and nonꢀnucleoside inhibitors
according to their structure. Nucleoside inhibitors
undergo phosphorylation to corresponding triphosꢀ
phates in the cell, which function as terminating subꢀ
strates. Their incorporation into growing RNA chains
leads to the premature chain termination. Triphosꢀ
phates of nucleoside inhibitors compete with native
nucleotides for the nucleotide binding site of the
RESULTS AND DISCUSSION
At the first stage of the investigation, NS5B inhibiꢀ
tion by the simplest PPi mimetics such as phosphoꢀ
noacetic and phosphonoformic acids, a number of
derivatives which are used in the treatment of various
1
Corresponding author: phone: +7 (499) 135ꢀ60ꢀ65; fax:
+7 (499) 135ꢀ14ꢀ05; eꢀmail: Yanvariov@mail.ru.
224

NONꢀHYDROLYSABLE ANALOGUES OF INORGANIC PYROPHOSPHATE
HO OH
225
O
O
OH
HO
O
OH
P
P
R
OH
N
HO
OH
O
O
N
O
O
R
4,5ꢀDihydroxypyrimidine derivatives
Inorganic pyrophosphate
α,γꢀDiketo acid derivatives
Fig. 1. Structural formulas of known RdRp inhibitors.
bone pathologies (MePCPOH, NH2C2ꢀPCPOH, and synthesis scheme 1, modified by using the correspondꢀ
ing amides instead of carboxylic acids.
NH2C3ꢀPCPOH), and also some new compounds
(PivPCPOH, PhC2ꢀPCPNH2), were studied (table).
The synthesis of such compounds is thoroughly
described in the literature [9, 10], and all of the comꢀ
pounds used in the work were obtained according to
one of two schemes.
HO
P(O)(OH)2
P(O)(OH)2
1)
RCOOH + H3PO3 + PCl3
2)
R
1) PhSO3H, 65–70
°
C, 14 h 2) 1 M HClaq, boiling, 4 h
Scheme 1.
The synthesis scheme 2 gave higher product yields
and did not require high temperatures and the use of
ionic liquids as a solvent. However, this method is not
applicable for synthesis of compounds with the amino
group in the side chain, as it is necessary to use acyl
chlorides on the first stage of the synthesis [10]. That is
why all the compounds except methylenediphosphoꢀ
nic acid aminoalkyl derivatives were synthesized by
scheme 2. Bisphosphonates containing the amino
group at the methylenediphosphate scaffold
RCOCl
+
P(OEt)3
O O
0°C, 4 h
OEt
OEt
RC P
NH(iPr)
2
HOP(OEt)
HO
HO
P(O)(OH)2
P(O)(OH)2
P(O)(OEt)2
P(O)(OEt)2
1. BrSi(Me)
2. MetOH
3
2
0°C, 6 h
R
R
Scheme 2.
To estimate the inhibitor activity of pyrophosphate
(
MePCPNH2 and PhC2ꢀPCPNH2) were obtained by analogues and preliminary structureꢀactivity relationꢀ
Compounds under investigation
Structure
R1
R2
Abbreviation
PFA
HOOCꢀP(O)(OH)2
–
–
–
–
HOOC(CH2)ꢀP(O)(OH)2
PAA
H
H
PCP
R1
P(O)(OH)2
OH
Me
Et
MePCPOH
EtPCPOH
PrPCPOH
P(O)(OH)2
R2
''
''
n
Pr
''
i
Pr
iPrPCPOH
''
(CH3)3C
PivPCPOH
''
''
CH2NH2
(CH2)2NH2
(CH2)3NH2
(CH2)4NH2
CH3
NH2C1ꢀPCPOH
NH2C2ꢀPCPOH
NH2C3ꢀPCPOH
NH2C4ꢀPCPOH
MePCPNH2
''
''
NH2
OH
NH2
(CH2)2Ph
(CH2)2Ph
PhC2ꢀPCPOH
PhC2ꢀPCPNH2
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226
YANVAREV et al.
100 100 96 98
100
97
99
96
100
50
0
88
84
17
100
Fig. 2. Remaining NS5B polymerase activity under the action of inhibitors at a concentration of 500
µ
M. The rate of radiolabelled
UMP incorporation in the absence of inhibitors was taken as 100% activity. The data represented as the average value the value
of confidence interval for 0.9
p
≥
.
ship, the remaining RNAꢀdependent RNAꢀpolymerase aminoꢀ and chelatorꢀgroup led to insignificant
activity values under action of inhibitors at the concenꢀ changes in inhibition. The most active was bisphosꢀ
tration of 500
µM were measured. Recombinant phonate with NH2ꢀgroup on the linker equivalent to
enzyme and poly(rA)ꢀoligo(U) as a primerꢀtemplate two methylene groups. It should be noted, that all the
complex were used in this experiment. The absence of compounds containing the NH2ꢀgroup on the methylꢀ
the polymerase reaction suppression at such a high ene linker vail, in their inhibition activity to the bisꢀ
inhibitor concentration served as a criterion to admit phosphonate with NH2ꢀgroup, directly bind to the
the compound inactive. The concentration of inhibitors PCPꢀscaffold of the molecule. Another important
was 8ꢀfold lower than Mg2+ concentration, excluding observation was that the inhibitor activity increases in
inhibition by means of Mg2+ concentration decrease the case of compounds with the aromatic substituent
due to its chelation by bisphosphonates.
(PhC2ꢀPCPOH).
These facts served as a basis for the synthesis of a
compound comprising the NH2ꢀgroup and an aroꢀ
matic substituent at the PCPꢀscaffold. Figure 4 shows
that this modification increased the inhibitor activity
compared with initial compounds.
At the first stage, the simplest inorganic pyrophosꢀ
phate derivatives were synthesized and studied. The
remaining polymerase activity data at the inhibitor
concentration equal to 500 µM are shown in Fig. 2. It
can be seen from the plot that only bisphosphonates
with amino group (MePCPNH2) or aromatic substitꢀ
uent (PhC2ꢀPCPOH) have shown somewhat signifiꢀ
cant activity.
The data obtained have given evidence that the
inhibitory activity of the methylenediphosphonic
acid derivatives strongly depend upon the compound
structure.
At the next stage, we decided to synthesize a numꢀ
ber of bisphosphonates with the NH2ꢀgroup on polyꢀ
The mechanism of their action is not limited to
methylene linkers of different length at the chelator Mg2+ꢀchelation. Their activity depends significantly
(methylenediphosphonic) backbone. The data on this on the chelating group structure and also on the subꢀ
compound family are shown in Fig. 3. As can be seen stituent in the side chain; this allows us to state the
from the figure, the variation of distance between specificity of this inhibitor family. The simplicity of the
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NONꢀHYDROLYSABLE ANALOGUES OF INORGANIC PYROPHOSPHATE
227
100
90
89
82
(CH2)n
[P]
OH
[P]
65
[P] = H2PO3
H2N
50
n
n
n
n
= 1 NH2C1ꢀPCPOH
= 2 NH2C2ꢀPCPOH
= 3 NH2C3ꢀPCPOH
= 4 NH2C4ꢀPCPOH
17
0
MePCPNH2
NH2C2ꢀPCPOH NH2C4ꢀPCPOH
NH2C1ꢀPCPOH NH2C3ꢀPCPOH
Fig. 3. Inhibitor activity of bisphosphonates depending on the distance between NH ꢀgroup and chelating PCP scaffold. Inhibꢀ
2
itors concentrations were 500 µM.
introduction of substituents into methylenediphosꢀ at 65–70°С, after which heating was stopped. After
phonic scaffold makes promising pharmacophores of
this class of inhibitors. Potential antihepatitis agents
can be created on this basis.
the reaction mixture reached room temperature, 1M
HCl in the amount of 50 mL (cooled to 0–5 ) was
added and the homogeneous solution was boiled for
4 h with a reflux condenser. Then heating was stopped,
and after the reaction mixture reached room temperaꢀ
ture it was poured into 50 mL of cold water, the pH was
adjusted to 4–3 by 50% aqueous NaOH solution. Then
°С
EXPERIMENTAL
All reagents used were purchased from Acros
Organics (Belgium) and were used without further
the reaction mix was left overnight at +5
formed were washed with 96% ethanol (
°
2
С
. The crystals
α
ꢀ32P] uridine triphosphate was a kind
× 40 mL) and
purification. [
gift of Dr. Yu. S. Skoblov. To analyse the incorporation
of
ꢀ32P]UTP, anion exchange filters were used
recrystallized from hot water (60 mL). The formed
crystals were filtered and dried for 4 h in vacuum
(1 mm Hg, 60°С).
[α
(Whatman DE81 diameter 23 mm). All NMR spectra
were recorded on an AMX IIIꢀ400 (Bruker) spectroꢀ
(2ꢀAminoꢀ1ꢀhydroxyethylidene) bisphosphonate
NH2C1ꢀPCPOH). Yield 44%.
1
meter at 400 MHz for H, at 162 MHz for 31P (with
(
phosphorusꢀproton interaction decoupling, 85%
Н3РО4 as an external standard) and at 100.6 MHz for
13C (with carbonꢀproton interaction decoupling). In
all NMR experiments D2O was used as a solvent.
Radioactivity was measured on a LSꢀcounter SLꢀ4000
Intertechnique (France) by the Cherenkov method.
The concentrations of inhibitors were measured by
100
96
88
1H NMR with 5
Bisphosphonates (NH2C1ꢀPCPOH, NH2C2ꢀ
PCPOH, NH2C3ꢀPCPOH, NH2C4ꢀPCPOH,
MePCPOH, EtPCPOH, PrPCPOH, PrPCPOH)
µL of (CH3)3OD as a reference.
50
i
(scheme 1, [9]). Into a twoꢀnecked 150 mL roundꢀ
bottomed flask, equipped with a thermometer, magꢀ
netic stir bar and dropping funnel, 0.1 mol carboxylic
acid, 30 g benzenesulfonic acid and 0.1 mol (8.4 g)
H3PO3 were added. The flask was filled with dry Ar,
17
2
and the reaction mix was heated to 65–70°С for
0
PhC2ꢀPCPNH2
20 min before melting. After heating was stopped,
0.2 mol PCl3 (17.5 mL) was added, from the dropping
funnel with an addition rate that allows keeping the
MePCPNH2
PhC2ꢀPCPOH
MePCPOH
reaction mixture temperature not higher than 70
(25–30 min). The reaction mixture was stirred for 20 h
°С
Fig. 4. Inhibitor activity of bisphosphonates depending on
2+
the structure of Mg ꢀchelating PCP group.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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228
YANVAREV et al.
1H NMR:
31P NMR :
(3ꢀAminoꢀ1ꢀhydroxypropylidene) bisphosphonate
NH2C2ꢀPCPOH). Yield 78%.
1H NMR :
2.88 (t, 7.6 Hz, 2H), 1.92–2.04
(m, 2H). 31P NMR : 18.5 (s).
δ
1.71 (t,
14.8 (s) (pH 2)
J
13.5 Hz)
phase (gradient of methanol 0–5%). The solvents were
removed in vacuum and 5 mM trimethylbromosilane
(5 mmol) was added to the residue at . In an hour,
methanol (100 mL) was added, and reaction mixture
was left at overnight. Crystals formed were filtered
and washed up with cold methanol ( 30 mL) folꢀ
, 1 mm Hg).
δ
0°С
(
0°С
δ
J
2
×
lowed by drying in vacuum (60°С
(4ꢀAminoꢀ1ꢀhydroxybutylidene) bisphosphonate
NH2C3ꢀPCPOH). Yield 71%.
(1ꢀHydroxyethylidene)
bisphosphonate
(
(MePCPOH). Yield 89%, 1H NMR:
J
δ
1.0 (t,
1H NMR:
δ 2.78 (t, J 7.3 Hz, 2H), 1.79–1.91
16.3 Hz), 31P NMR:
72.3 (t, 152 Hz).
δ
19.9 (s). 13С NMR
:
δ
21.5 (s),
(m, 2H), 1.40 (m, 2H). 31P NMR : 18.9 (s).
J
(5ꢀAminoꢀ1ꢀhydroxypentylidene) bisphosphonate
(1ꢀHydroxybutylidene)
bisphosphonate
1.0 (t, 7.2 Hz,
19.9 (s).
(
NH2C4ꢀPCPOH). Yield 62%.
1H NMR:
2.62 (t, 7.2 Hz, 2H), 1.77–1.90
(m, 2H), 1.52–1.60 (m, 2H), 1.42 (m, 2H).
31PꢀNMR:
19.1 (s).
(PrPCPOH). Yield 78%, 1H NMR:
δ
J
3H), 1.7 (m, 2H), 1.0 (m, 2H), 31P NMR:
δ
δ
J
(2ꢀMethylꢀ1ꢀhydroxypropylidene) bisphosphoꢀ
δ
1
nate (
i
PrPCPOH). Yield 92%, H NMR:
δ 1.22
Syntheses of bisphosphonates bearing NH2ꢀgroup
in their scaffold (MePCPNH2, PhC2ꢀPCPNH2). The
ratio of reagents, temperature and reaction conditions
were identical to those described above, but the correꢀ
sponding amides were used instead of carboxylic acids.
The reaction time was 5 h and the purification was the
same as described above.
(s, 6H), 2.5 (m, 1H), 31P NMR:
δ
19.8 (s).
(2,2,2ꢀTrimethylꢀ1ꢀhydroxypropylidene) bisphosꢀ
phonate (PivPCPOH). Yield 84%, 1H NMR:
δ
1.20 (s), 31P NMR:
δ
19.8 (s).
(3ꢀPhenylꢀ1ꢀhydroxypropylidene) bisphosphonate
(MePCPOH). Yield 79%, 1H NMR:
.1ꢀ7.2
δ
7
(3ꢀPhenylꢀ1ꢀaminopropylidene) bipshosphonate (m, 5H), 2.78 (m, 2H), 2.1ꢀ2.2 (m, 2H), 31P NMR:
PhC2ꢀPCPNH2). Yield 77%.
19.7 (s), 13С NMR:
143 (s), 129 (s), 129.5 (s),
127 (s), 73.8 (t, 147 Hz).
(
δ
δ
1H NMR :
7.3 (m, 5H), 2.93–2.88 (m, 2H),
δ
J
2.34–2.23 (m, 2H). 31P NMR :
21 (s) (pH 10); 13 (s)
(pH 3). 13C NMR :
144 (s), 132 (s), 131 (s), 129 (s),
60 (t, 123 Hz), 37 (s), 33 (s).
(1ꢀAminoethylidene) bisphosphonate (MePCPNH2).
Yield 80%.
1H NMR :
13С NMR
58.0 (t,
Syntheses of bisphosphonates (MePCPOH,
EtPCPOH, PrPCPOH, PrPCPOH, PivPCPOH,
PhC2ꢀPCPOH) (scheme 2, [10]). Corresponding carꢀ
bon acid chloroanhydrides (except acetyl chloride and
pivaloyl chloride, which were from commercial
sources) were prepared by boiling carbon acids for two
hours with three equivalents of SOCl2. The excess of
SOCl2 was removed in vacuum and chloroanhydrides
were distilled in vacuum (20 mm Hg). Yields varied in
the range of 89–94%.
δ
RNAꢀdependent RNA polymerase (NS5B) activity
assay. The expression plasmid (pETꢀ21dꢀ2cꢀMꢀ5B55)
encoded NS5B protein (65 kDa) without 55 Cꢀtermiꢀ
nal amino acid residues. Two additional amino acid
residues (Met, Asn) were located on the protein
Nꢀterminus as against the native protein (RB01 HCV
isolate). The enzyme was isolated and purified as we
described previously [11].
δ
J
δ
1.7 (t,
J
13.2 Hz), 31P NMR :
J 121 Hz).
δ 13.8 (s),
:
δ
HCV RNAꢀdependent RNA polymerase inhibiꢀ
tion was tested by radiolabelled UMP incorporation
method in poly(rA)ꢀoligo(U) primerꢀtemplate sysꢀ
i
tem. The standard reaction mixture contained 0.3
of NS5B 55, 100 g/mL poly(rA), 25 g/mL oligo(U),
10 M UTP, and 1 Ci L buffer
ꢀ32P]UTP in 20
(20 mM TrisꢀHCl, pH 7.5, 20 mM KCl, 4 mM MgCl2
µg
Δ
µ
µ
µ
µ
[α
µ
,
and 1 mM dithiothreitol). The mixtures were incuꢀ
bated for 30 min at 30°C and applied onto DEꢀ81 filꢀ
ters. The filters were washed four times with 0.5 M
potassium phosphate buffer (pH 7.0), once with ethꢀ
anol and dried on air. The radioactivity was measured
by the Cherenkov method. HCV RdRp was inhibited
by the addition of a solution of the compound under
investigation to the reaction mixture up to the final
Chloroanhydrides (1 mmol) were dissolved in
10 mL of dry benzene and slowly added from a dropꢀ
ping funnel to an intensively stirred solution of triethyl
phosphite (1 mmol) in 10 mL of dry benzene at
The rate of addition of chloroanhydrides should not
allow the reaction mixture to heat above +5 . After
addition of chloroanhydrides, the reaction mixture was
stirred for 2 hours at , then diethyl phosphite
0°С.
°С
concentration of 500
µM.
0°С
(1 mmol) and diisopropyl amine (0.1 mmol) were added,
and the reaction mixture was stirred for 4–5 hours at
ACKNOWLEDGMENTS
+5°С. Then the solvents were removed in vacuum and
This work was supported by the Russian Foundaꢀ
the residue was purified by column chromatography tion for Basic Research, Project nos. 09ꢀ04ꢀ01221ꢀa,
on silica gel with chloroformꢀmethanol as the mobile 12ꢀ04ꢀ00958ꢀa, and 11ꢀ04ꢀ12035ꢀofiꢀmꢀ2011 and by
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NONꢀHYDROLYSABLE ANALOGUES OF INORGANIC PYROPHOSPHATE
229
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