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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 Abstract—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 DOI: 10.1134/S1068162012020124
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 [1]. To date, about 170 million people are Mg2+ binding site (Fig. 1) and a hydrophobic group infected with HCV [2], 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 [3]. 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 [4]. 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 [6],while dihydroxypyrimidines act as competitive inhibi Viral RNA dependent RNA polymerase (NS5B, tors with regard to triphosphates [7].
EC 2.7.7.48) 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 [5]. 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 [8]. 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 Fig. 1. 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 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 aminogroup 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 methylenediphospho nic acid aminoalkyl derivatives were synthesized by Scheme 2.
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 Fig. 2. 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 ≥ 0.9.
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 n = 1 NH2C1 PCPOHn = 2 NH2C2 PCPOHn = 3 NH2C3 PCPOH Fig. 3. 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, [9]). 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 Fig. 4. 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 J 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 7.2 Hz, 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 nate (iPrPCPOH). 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 ). The
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), assay. 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 121 Hz).
isolate). The enzyme was isolated and purified as wedescribed previously [11].
Syntheses of bisphosphonates (MePCPOH,
EtPCPOH, PrPCPOH, iPrPCPOH, PivPCPOH,
PhC2 PCPOH) (scheme 2, [10]). Corresponding car
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.
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