ISSN 1068 1620, 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.
Non Hydrolysable Analogues of Inorganic Pyrophosphate
as Inhibitors of Hepatitis C Virus RNA Dependent RNA Polymerase
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
—Inorganic pyrophosphate (PPi
) is the product of the polymerization reaction catalyzed by DNA
and RNA polymerases. A number of novel non hydrolsable PPi
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 beendeveloped. The structure activity relationship analysis of 12 bisphosphonates is presented and the structuralfeatures crucial for NS5B polymerase activity inhibition are stated.
Keywords: hepatitis C virus, pyrophosphate, analogues, RNA dependent RNA polymerase (NS5B), methylenediphosphonates
enzyme. Non nucleoside inhibitors can be subdividedinto two classes by the principle of action. They are
Hepatitis C virus (HCV) causes a wide spread viral
compounds, which bind polymerase outside the cata
disease. Its chronic form leads to a number of such
lytic site, representing “allosteric” noncompetitive
strong liver affections as cirrhosis or hepatocellular
inhibitors, and Mg2+ chelating agents, which contain a
carcinoma . To date, about 170 million people are
Mg2+ binding site (Fig. 1) and a hydrophobic group
infected with HCV , thereby attaching high social
significance to the search for antiviral drugs. The current hepatitis C therapy is based on interferon alpha
In spite of the seeming structural consistency of
and a nonspecific antiviral nucleoside analog ribavirin
Mg2+ chelating NS5B inhibitors, the mechanism of
combination . However, the clinical use of this drug
their actions differs. Thus, α,γ diketo acids and
combination is usually complicated by toxic side
4,5 dihydroxypyrimidine carboxylic acids imitate
effects and is not efficient in the case of a number of
inorganic pyrophosphate in the catalytic site of the
HCV genotypes . These factors are responsible for
enzyme (Fig. 1). However, α,γ diketo acids are non
the critical need for new drug families with less prom
competitive inhibitors of the HCV polymerase with
inent side effects and better acceptability.
regard to RNA substrate and also to nucleotides ,while dihydroxypyrimidines act as competitive inhibi
Viral RNA dependent RNA polymerase (NS5B,
tors with regard to triphosphates .
EC 188.8.131.52) is a promising target for an anti HCVdrug design as non infected liver cells do not bear this
NS5B chelating inhibitors, which bind Mg2+ in the
enzyme, suggesting low cytotoxicity of specific inhib
corresponding region of the catalytic site, are mimetics
itors of NS5B polymerase activity . To date, many
of the inorganic pyrophosphate, which is one of the
such inhibitors are known, however none of them is in
reaction products. At that they have lots of structural
differences. At the same time, the data on NS5B inhibition by PP
structural analogues are almost absent,
It is possible to subdivide known NS5B inhibitors
though such compounds efficiently inhibited a number
into nucleoside and non nucleoside inhibitors
of viral and cellular polymerases . We report the syn
according to their structure. Nucleoside inhibitors
thesis of a number of PP
analogues, and their action as
undergo phosphorylation to corresponding triphos
phates in the cell, which function as terminating substrates. Their incorporation into growing RNA chainsleads to the premature chain termination. Triphos
phates of nucleoside inhibitors compete with nativenucleotides for the nucleotide binding site of the
At the first stage of the investigation, NS5B inhibi
tion by the simplest PP
mimetics such as phospho
1 Corresponding author: phone: +7 (499) 135 60 65; fax:
noacetic and phosphonoformic acids, a number of
+7 (499) 135 14 05; e mail: [email protected]
derivatives which are used in the treatment of various
NON HYDROLYSABLE ANALOGUES OF INORGANIC PYROPHOSPHATE
Structural formulas of known RdRp inhibitors.
bone pathologies (MePCPOH, NH C2 PCPOH, and
synthesis scheme 1, modified by using the correspond
NH C3 PCPOH), and also some new compounds
ing amides instead of carboxylic acids.
(PivPCPOH, PhC2 PCPNH ), were studied (table).
The synthesis of such compounds is thoroughly
described in the literature [9, 10], and all of the com
pounds used in the work were obtained according to
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 aminogroup in the side chain, as it is necessary to use acyl
chlorides on the first stage of the synthesis . That is
why all the compounds except methylenediphospho
nic acid aminoalkyl derivatives were synthesized by
scheme 2. Bisphosphonates containing the aminogroup at the methylenediphosphate scaffold
To estimate the inhibitor activity of pyrophosphate
(MePCPNH and PhC2 PCPNH ) were obtained by
analogues and preliminary structure activity relation
Remaining NS5B polymerase activity under the action of inhibitors at a concentration of 500 µM. The rate of radiolabelled
UMP incorporation in the absence of inhibitors was taken as 100% activity. The data represented as the average value ± the valueof confidence interval for p
ship, the remaining RNA dependent RNA polymerase
amino and chelator group led to insignificant
activity values under action of inhibitors at the concen
changes in inhibition. The most active was bisphos
tration of 500 µM were measured. Recombinant
phonate with NH group on the linker equivalent to
enzyme and poly(rA) oligo(U) as a primer template
two methylene groups. It should be noted, that all the
complex were used in this experiment. The absence of
compounds containing the NH group on the methyl
the polymerase reaction suppression at such a high
ene linker vail, in their inhibition activity to the bis
inhibitor concentration served as a criterion to admit
phosphonate with NH group, directly bind to the
the compound inactive. The concentration of inhibitors
PCP scaffold of the molecule. Another important
was 8 fold 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.
At the first stage, the simplest inorganic pyrophos
These facts served as a basis for the synthesis of a
phate derivatives were synthesized and studied. The
compound comprising the NH group and an aro
remaining polymerase activity data at the inhibitor
matic substituent at the PCP scaffold. Figure 4 shows
concentration equal to 500 µM are shown in Fig. 2. It
that this modification increased the inhibitor activity
can be seen from the plot that only bisphosphonates
with amino group (MePCPNH ) or aromatic substit
The data obtained have given evidence that the
uent (PhC2 PCPOH) have shown somewhat signifi
inhibitory activity of the methylenediphosphonic
acid derivatives strongly depend upon the compound
At the next stage, we decided to synthesize a num
ber of bisphosphonates with the NH group on poly
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 sub
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
NON HYDROLYSABLE ANALOGUES OF INORGANIC PYROPHOSPHATE
= 1 NH2C1 PCPOHn
= 2 NH2C2 PCPOHn
= 3 NH2C3 PCPOH
Inhibitor activity of bisphosphonates depending on the distance between NH2 group and chelating PCP scaffold. Inhib
introduction of substituents into methylenediphos
at 65–70°С, after which heating was stopped. After
phonic scaffold makes promising pharmacophores of
the reaction mixture reached room temperature, 1M
this class of inhibitors. Potential antihepatitis agents
HCl in the amount of 50 mL (cooled to 0–5°С) was
added and the homogeneous solution was boiled for4 h with a reflux condenser. Then heating was stopped,and after the reaction mixture reached room tempera
ture it was poured into 50 mL of cold water, the pH was
All reagents used were purchased from Acros
adjusted to 4–3 by 50% aqueous NaOH solution. Then
Organics (Belgium) and were used without further
the reaction mix was left overnight at +5°С. The crystals
purification. [α 32P] uridine triphosphate was a kind
formed were washed with 96% ethanol (2 × 40 mL) and
gift of Dr. Yu. S. Skoblov. To analyse the incorporation
recrystallized from hot water (60 mL). The formed
of [α 32P]UTP, anion exchange filters were used
crystals were filtered and dried for 4 h in vacuum
(Whatman DE81 diameter 23 mm). All NMR spectra
were recorded on an AMX III 400 (Bruker) spectro
(2 Amino 1 hydroxyethylidene) bisphosphonate
meter at 400 MHz for 1H, at 162 MHz for 31P (with
phosphorus proton interaction decoupling, 85%Н РО as an external standard) and at 100.6 MHz for
13C (with carbon proton interaction decoupling). In
all NMR experiments D O was used as a solvent.
Radioactivity was measured on a LS counter SL 4000
Intertechnique (France) by the Cherenkov method.
The concentrations of inhibitors were measured by1H NMR with 5 µL of (CH ) OD as a reference.
Bisphosphonates (NH C1 PCPOH, NH C2
PCPOH, NH C3 PCPOH, NH C4 PCPOH,
MePCPOH, EtPCPOH, PrPCPOH, iPrPCPOH)
(scheme 1, ).
Into a two necked 150 mL round
bottomed flask, equipped with a thermometer, mag
netic stir bar and dropping funnel, 0.1 mol carboxylic
acid, 30 g benzenesulfonic acid and 0.1 mol (8.4 g)H PO were added. The flask was filled with dry Ar,
and the reaction mix was heated to 65–70°С for
20 min before melting. After heating was stopped,
0.2 mol PCl (17.5 mL) was added, from the dropping
funnel with an addition rate that allows keeping thereaction mixture temperature not higher than 70°С
Inhibitor activity of bisphosphonates depending on
(25–30 min). The reaction mixture was stirred for 20 h
the structure of Mg2+ chelating PCP group.
phase (gradient of methanol 0–5%). The solvents were
removed in vacuum and 5 mM trimethylbromosilane(5 mmol) was added to the residue at 0°С. In an hour,
(3 Amino 1 hydroxypropylidene) bisphosphonate
methanol (100 mL) was added, and reaction mixture
was left at 0°С overnight. Crystals formed were filtered
1H NMR : δ 2.88 (t, J
7.6 Hz, 2H), 1.92–2.04
and washed up with cold methanol (2 × 30 mL) fol
lowed by drying in vacuum (60°С, 1 mm Hg).
(4 Amino 1 hydroxybutylidene) bisphosphonate
1H NMR: δ 2.78 (t, J
7.3 Hz, 2H), 1.79–1.91
16.3 Hz), 31P NMR: δ 19.9 (s). 13С NMR: δ 21.5 (s),
(m, 2H), 1.40 (m, 2H). 31P NMR : 18.9 (s).
(5 Amino 1 hydroxypentylidene) bisphosphonate
(PrPCPOH). Yield 78%, 1H NMR: δ 1.0 (t, J
1H NMR: δ 2.62 (t, J
7.2 Hz, 2H), 1.77–1.90
3H), 1.7 (m, 2H), 1.0 (m, 2H), 31P NMR: δ 19.9 (s).
(m, 2H), 1.52–1.60 (m, 2H), 1.42 (m, 2H).
31P NMR: δ 19.1 (s).
(2 Methyl 1 hydroxypropylidene) bisphospho
PrPCPOH). Yield 92%, 1H NMR: δ 1.22
Syntheses of bisphosphonates bearing NH group
(s, 6H), 2.5 (m, 1H), 31P NMR: δ 19.8 (s).
in their scaffold (MePCPNH , PhC2 PCPNH ).
ratio of reagents, temperature and reaction conditions
(2,2,2 Trimethyl 1 hydroxypropylidene) bisphos
were identical to those described above, but the corre
sponding amides were used instead of carboxylic acids.
The reaction time was 5 h and the purification was the
(3 Phenyl 1 hydroxypropylidene) bisphosphonate
(3 Phenyl 1 aminopropylidene) bipshosphonate
(m, 5H), 2.78 (m, 2H), 2.1 2.2 (m, 2H), 31P NMR:
δ 19.7 (s), 13С NMR: δ 143 (s), 129 (s), 129.5 (s),
1H NMR : δ 7.3 (m, 5H), 2.93–2.88 (m, 2H),
2.34–2.23 (m, 2H). 31P NMR : δ 21 (s) (pH 10); 13 (s)
RNA dependent RNA polymerase (NS5B) activity
(pH 3). 13C NMR : δ 144 (s), 132 (s), 131 (s), 129 (s),
The expression plasmid (pET 21d 2c M 5B55)
60 (t, J
123 Hz), 37 (s), 33 (s).
encoded NS5B protein (65 kDa) without 55 C termi
(1 Aminoethylidene) bisphosphonate (MePCPNH ).
nal amino acid residues. Two additional amino acid
residues (Met, Asn) were located on the protein
1H NMR : δ 1.7 (t, J
13.2 Hz), 31P NMR : δ 13.8 (s),
N terminus as against the native protein (RB01 HCV
13С NMR : δ 58.0 (t, J
isolate). The enzyme was isolated and purified as wedescribed previously .
Syntheses of bisphosphonates (MePCPOH,
EtPCPOH, PrPCPOH, iPrPCPOH, PivPCPOH,
PhC2 PCPOH) (scheme 2, ).
tion was tested by radiolabelled UMP incorporation
bon acid chloroanhydrides (except acetyl chloride and
method in poly(rA) oligo(U) primer template sys
pivaloyl chloride, which were from commercial
tem. The standard reaction mixture contained 0.3 µg
sources) were prepared by boiling carbon acids for two
of NS5BΔ55, 100 µg/mL poly(rA), 25 µg/mL oligo(U),
hours with three equivalents of SOCl . The excess of
10 µM UTP, and 1 µCi [α 32P]UTP in 20 µL buffer
SOCl was removed in vacuum and chloroanhydrides
(20 mM Tris HCl, pH 7.5, 20 mM KCl, 4 mM MgCl ,
were distilled in vacuum (20 mm Hg). Yields varied in
and 1 mM dithiothreitol). The mixtures were incu
bated for 30 min at 30°C and applied onto DE 81 fil
Chloroanhydrides (1 mmol) were dissolved in
ters. The filters were washed four times with 0.5 M
10 mL of dry benzene and slowly added from a drop
potassium phosphate buffer (pH 7.0), once with eth
ping funnel to an intensively stirred solution of triethyl
anol and dried on air. The radioactivity was measured
phosphite (1 mmol) in 10 mL of dry benzene at 0°С.
by the Cherenkov method. HCV RdRp was inhibited
The rate of addition of chloroanhydrides should not
by the addition of a solution of the compound under
allow the reaction mixture to heat above +5°С. After
investigation to the reaction mixture up to the final
addition of chloroanhydrides, the reaction mixture was
stirred for 2 hours at 0°С, then diethyl phosphite(1 mmol) and diisopropyl amine (0.1 mmol) were added,
and the reaction mixture was stirred for 4–5 hours at+5°С. Then the solvents were removed in vacuum and
This work was supported by the Russian Founda
the residue was purified by column chromatography
tion for Basic Research, Project nos. 09 04 01221 a,
on silica gel with chloroform methanol as the mobile
12 04 00958 a, and 11 04 12035 ofi m 2011 and by
NON HYDROLYSABLE ANALOGUES OF INORGANIC PYROPHOSPHATE
the Program of Presidium of the Russian Academy of
6. Kozlov, M.V., Polyakov, K.M., Filipova, S.E., Evsti
Sciences “Molecular and Cellular Biology.”
feev, V.V., Lyudva, G.S., and Kochetkov, S.N., Biochemistry (Moscow)
, 2009, vol. 74, pp. 1028–1035.
7. Powdrill, M.H., Deval, J., Narjes, F., De Francesco, R.,
and Gotte, M., Antimicrob. Agents Chemother.
, 2010,vol. 54, no. 3, pp. 977–983.
1. Levrero, M., Oncogene
, 2006, vol. 25, pp. 3834–3847.
8. Crumpacker, C.S., Am. J. Med.
, 1992, vol. 92, no. 2A,
2. Global Burden of Hepatitis C Working Group, J. Clin.
9. Kieczykowski, G.R., Jobson, R.B., Melillo, D.G.,
, 2004, vol. 44, pp. 20–29.
Reinhold, D.F., Grenda, V.J., and Shinkai, I., J. Org.
3. Heathcote, J. and Main, J., J. Viral Hepat.
, 1995, vol. 60, pp. 8310–8312.
10. Nicholson, D.A. and Vaughn, H., Org. Chem.
4. NIH Consensus and State of the Science Statements,
11. Ivanov, A.V., Korovina, A.N., Tunitskaya, V.L.,
Kostyuk, D.A., Rechinsky, V.O., Kukhanova, M.K.,
5. De Francesco, R., Tomei, L., Altamura, S., Summa, V.,
and Kochetkov, S.N., Protein Expr. Purif.
, 2006, vol. 48,
and Migliaccio, G., Antiviral Res.
, 2003, vol. 58, pp. 1–16.
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