WO2013156957A1

WO2013156957A1 - Compounds for the prevention or treatment of infections with viruses of the flaviviridae family

Info

Publication number: WO2013156957A1
Application number: PCT/IB2013/053075
Authority: WO
Inventors: Jean Dubuisson; Czeslaw Wychowski; Yann GUERARDEL; Thibaut VAUSSELIN; Christophe BIOT
Original assignee: Centre National De La Recherche Scientifique; Institut Pasteur De Lille; Universite Lille 2 Droit Et Santé; Universite Des Sciences Et Technologies De Lille
Priority date: 2012-04-19
Filing date: 2013-04-18
Publication date: 2013-10-24
Also published as: FR2989588A1

Abstract

The present invention relates to a compound of the following formula (I): (I), or to a pharmaceutically acceptable salt thereof, for the use thereof in the prevention or treatment of an infection with a virus of the Flaviviridae family, in particular with the Hepatitis C virus (HCV), in a patient.

Description

COMPOUNDS FOR THE PREVENTION OR TREATMENT OF INFECTIONS WITH FLAVIVIRIDAE FAMILY VIRUSES

Field of the invention

The present invention provides compounds useful for preventing or treating infections with viruses of the family Flaviviridae, including hepatitis C virus infections.

Technical background

Viruses of the family Flaviviridae have many pathogenic species.

Among them, the hepatitis C virus (HCV) is a major cause of chronic liver disease. In fact, it is estimated that about 170 million people are infected worldwide, which represents about 3% of the world's population. However, there is no vaccine and current treatments based pegylated interferon-α (PEGASYS®, PEG-Intron®, or Viraferon-PEG®) and ribavirin are nonspecific. In addition, they are poorly tolerated by patients and are effective in only about 50% of cases. Thus, in the absence of effective curative or prophylactic treatments, infected patients have a high risk of developing cirrhosis or hepatocellular carcinoma, which necessitates an increasing use of liver transplantation.

However, new molecules specifically targeting HCV replication are beginning to enter the market, which should lead to improved treatment efficacy in the coming years. In May 2011, the US Federal Drug Agency (FDA) approved two inhibitors of HCV protease, boceprevir (Victrelis®, Merck) and telaprevir (Incivek®, Vertex) for treatment. These molecules, however, appear to be specific for genotype 1 HCV. In addition, these treatments would cause significant side effects, such as rash, anemia and pruritus (Alkhouri & Zein (2012) Cleve Clin J Med 79: 213-222).

In addition, other antiviral molecules blocking the entry of HCV into target cells by interacting with envelope proteins are being developed. The molecule ITX 5061 (iTherX Pharmaceuticals) has just entered a phase I clinical trial (Zhu et al., (2012) J. Infect Dis 205: 656). Another molecule blocking the entry of HCV has recently been described by Bristol-Myers Squibb (Baldick et al., (2010) PLoS Pathog 6: el001086). However, this molecule seems specific to genotype 1. A molecule extracted from green tea, (-) - epigallocatechin-3-gallate has also been described as having an inhibitory activity on the entry of HCV (Calland et al (2012) Hepatology 55: 720). However, these molecules have not yet demonstrated their clinical effectiveness.

In addition, HCV mutates rapidly and develops resistance to treatment. It therefore remains essential to continue identifying new anti-HCV molecules

Ferroquine is a ferrocene derivative of chloroquine that exhibits antimalarial activity in vitro and in vivo. Ferroquine is currently in clinical trials for the treatment of malaria and is well tolerated up to 1600 mg single dose or 800 mg repeated dose (Mombo-Ngoma et al (201 1) Malaria Journal 10 : 53). Several derivatives, in particular demethylated or hydroxylated, of ferroquine also exhibit anti-malarial activity (49: 2845).

ferroquine

Summary of the invention

The present invention stems from the unexpected demonstration by the inventors that ferroquine exhibited an inhibition effect of several viruses of the family of

Flaviviridae and in particular hepatitis C virus. In addition, the inventors have shown that this effect is notably underpinned by an inhibition of the entry of HCV into its target cells, by an inhibition of the replication of HCV, as well as by only by inhibition of cell-to-cell HCV transmission. Moreover, the inventors have shown that the combination of ferroquine with interferon-α or boceprevir has an additive anti-HCV effect. In addition, the inventors have shown that the antiviral effect of ferroquine is not limited to a particular genotype of HCV.

Thus, the present invention relates to a compound of formula (I) below:

(I) in which :

R ₁ and R ₂ , which are identical or different, are selected from the group consisting of a hydrogen atom, an alkyl group, especially from 1 to 10 carbon atoms, preferably methyl or ethyl, and an alkoxy group, in particular from 1 to 10 carbon atoms, from an acyl group, in particular from 1 to 10 carbon atoms, from a hydroxyalkyl group, in particular from 1 to 10 carbon atoms, from an acyloxyalkyl group, in particular from 2 to 10 atoms of carbon, an acetylalkyl group, in particular of 2 to 10 carbon atoms, and a group:

R ₃ represents CH or a heteroatom, in particular selected from the group consisting of N and P, and

R 4 represents a hydrogen atom, an alkyl group, in particular from 1 to 10 carbon atoms, preferably methyl or ethyl, an alkoxy group, especially from 1 to 10 carbon atoms, an acyl group, in particular from 1 to 10 carbon atoms; carbon atoms, a hydroxyalkyl group, in particular of 1 to 10 carbon atoms, an acyloxyalkyl group, especially of 2 to 10 carbon atoms, or an acetylalkyl group, in particular of 2 to 10 carbon atoms,

or a pharmaceutically acceptable salt thereof,

for use in the prevention or treatment of an infection of a Flaviviridae virus, including hepatitis C virus (HCV), in an individual.

The present invention also relates to the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the prevention or treatment infection with a virus of the family Flaviviridae, including hepatitis C virus (HCV), in an individual.

The present invention also relates to a method for treating an infection of a Flaviviridae virus, in particular a hepatitis C virus (HCV), in an individual, in which the individual is administered prophylactically or prophylactically. therapeutically effective of at least one compound of formula (I) as defined above, or at least one pharmaceutically acceptable salt thereof.

In a preferred embodiment of the compound for its use as defined above, the use as defined above, or the method as defined above, the compound, or the pharmaceutically acceptable salt thereof. it is associated with at least one additional compound intended for the prevention or treatment of an infection of a virus of the Flaviviridae family, in particular of the hepatitis C virus (HCV).

The present invention also relates to a pharmaceutical composition, comprising, as active substances, at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, and at least one additional compound intended the prevention or the treatment of an infection of a Flaviviridae virus, in particular with the hepatitis C virus (HCV), possibly in combination with a pharmaceutically acceptable vehicle, in particular for its use in the prevention or the treatment infection with a virus of the family Flaviviridae, including the virus of hepatitis C (HCV).

The present invention also relates to products containing:

at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, and

at least one additional compound for the prevention or treatment of an infection of a virus of the family Flaviviridae, in particular the virus of hepatitis C (HCV), as a combination product for simultaneous, separate or spread use in time for the prevention or treatment of an infection of a Flaviviridae family virus, including hepatitis C virus (HCV). Detailed description of the invention

Prevention or treatment of infections with a virus of the family Flaviviridae

The family Flaviviridae is well known to those skilled in the art. Preferably, the virus of the Flaviviridae family according to the invention is a Flavivirus, such as the yellow fever virus, the West Nile virus, or the dengue virus, a Hepacivirus, such as the virus of the hepatitis C virus (HCV), or a Pestivirus, such as the bovine viral diarrhea virus or the classical swine fever virus. Most preferably, the virus of the Flaviviridae family according to the invention is the hepatitis C virus.

As it is understood here, the prevention or treatment of hepatitis C virus infections is synonymous with the prevention or treatment of hepatitis C. "hepatitis C" or "HCV" refers to a viral species responsible for hepatitis C, formerly known as non-A, non-B hepatitis. The HCV according to the invention may especially be of one of the six genotypes. major, 1 to 6, currently referenced. These genotypes are themselves divided into different subtypes including in particular the subtypes 1a, 1b, 2a, 2b, 2c, or 3a.

The prevention or treatment of hepatitis C virus infections is intended to prevent the development of HCV viral load or symptoms of hepatitis C in an uninfected or asymptomatic individual, or to reduce or eliminate the risk of hepatitis C virus infection. HCV viral load or symptoms of hepatitis C in the individual infected or with symptoms of hepatitis C. HCV viral load refers in particular to the viral load in the blood or liver. The symptoms of hepatitis C correspond in particular to fibrosis and / or necrosis of the liver, especially in an individual with a detectable viral load in HCV. In the context of prevention, the individual according to the invention may have no HCV viral load, and neither necrosis nor fibrosis of the liver; he may be at risk for hepatitis C infection or may have been infected with HCV. As part of the treatment, the individual may suffer from acute or chronic hepatitis C. In particular, the individual may exhibit an activity, that is to say a necrosis, greater than A0 according to the Metavir classification well known to those skilled in the art (see, for example, Bedossa (1993) Ann, Pathol. 260) and / or fibrosis greater than F0 according to the Metavir classification. Preferably, the individual to be treated according to the invention has an activity greater than or equal to Al and a fibrosis greater than or equal to F2, according to the Metavir classification.

The individual according to the invention is preferably a mammal, more preferably a porcine, a bovine or a human, and particularly preferably a human. Furthermore, in a preferred embodiment of the invention, the individual according to the invention is both carrier of malaria and infection with a virus of the family Flaviviridae, including HCV. In addition, in another preferred embodiment of the invention the individual must undergo or have undergone a liver transplant.

Preferably, the compound of formula (I) according to the invention exerts its therapeutic anti-virus effect of the family Flaviviridae by inhibiting the entry of the virus into its target cells and / or by inhibiting the replication of the genome of the virus and / or inhibiting cell-to-cell virus transmission. Compound of formula (I)

As those skilled in the art are well aware, the cyclopentadienyl ring of the compound of formula (I) is free from rotation; also, as it is understood here, the offset representation of the cycles adopted for the formulas (I) to (VII) according to the invention should not be interpreted as limiting the invention to this conformation but on the contrary as also covering the forms overshadowed.

Preferably, the grouping:

represents a group selected from the group consisting of groups:

piperazino piperidino morpholino

Also preferably, one of R 1 and R ₂ is -H, -CH ₃ or -CH ₂ -CH ₃ , and the other is -CH ₃ , -CH ₂ -CH ₃ or -CH ₂ -CH ₂ -OH

Preferably, the compound of formula (I) according to the invention is the ferroquine of formula (II) below:

(Π) Also preferably, the compound of formula (I) according to the invention is a hydroxyferroquin selected from the group consisting of the following compounds of formulas (III), (IV) and (V):

(V)

Also preferably, the compound of formula (I) according to the invention is an active metabolite of ferroquine or of hydroxyferroquine, in particular selected from the group consisting of the following compounds of formulas (VI) and (VII):

(VII)

Thus, according to the invention, the compound of formula (I) is preferably selected from the group consisting of compounds of formulas (II), (III), (IV), (V), (VI) and (VII).

As used herein, a pharmaceutically acceptable salt means any salt of a compound of formula (I) which can be used for the manufacture of a medicament, that is to say having an acceptable risk / benefit ratio for the concerned individual.

Additional compound

The additional compound according to the invention is of any type which makes it possible to prevent or treat an infection with a virus of the family Flaviviridae, in particular with HCV. It may especially be interferon-α, ribavirin, an inhibitor of HCV replication, an HCV protease inhibitor, or an inhibitor of HCV cell entry. Preferably, the additional compound for the prevention or treatment of an infection of a virus of the family Flaviviridae, including the hepatitis C virus (HCV), is specific to HCV and / or is selected in the group consisting of interferon-a, including pegylated, ribavirin, boceprevir, and telaprevir. In a particularly preferred manner, the additional compound intended for the prevention or treatment of an infection of a Flaviviridae virus, in particular to the hepatitis C virus (HCV), is boceprevir or telaprevir.

Administration

The compound of formula (I) according to the invention, or its pharmaceutically acceptable salt or ester, may in particular be administered by the oral or intravenous route.

Preferably, when the compound of formula (I) according to the invention is ferroquine, it is administered at a dose of 10 mg / day to 2000 mg / day, in particular from 200 mg / day to 1600 mg / day.

The compound of formula (I) according to the invention may be combined with a pharmaceutically acceptable vehicle, that is to say any vehicle, diluent, excipient or adjuvant, likely to be administered to an individual accompanying an active pharmaceutical ingredient.

Description of figures

Figures 1 and 2

Huh-7 cells pretreated for 1 hour with the compounds were infected with JFH1-Luc (FIG. 1) or JFH1 (FIG. 2) in the presence or absence of compounds. 48 hours after infection, infected cells were lysed to quantify luciferase activity (Figure 1) or immunofluorescent treated to count cells (Figure 2). The results are expressed as a percentage of infection compared to a control infection in the absence of a compound (y-axis) as a function of the concentration of compound (abscissa axis, in μΜ). Error bars represent the standard deviation of at least three experiments. The cytotoxic concentrations of chloroquine (CQ), hydroxychloroquine (HCQ), ferroquine (FQ) and hydroxyferroquine of formula (IV) (HFQ) required to reduce cell viability by 50% (CC ₅₀ ) are respectively 19.58 μΜ,> 20 μΜ, 5.34 μΜ and 10.36 μΜ.

Figures 3, 4, 5 and 6

Figure 3 shows the times at which the FQ was added or removed.

Huh-7 cells were infected with JFH1-Luc and treated for different time periods by FQ (FIGS. 4 and 5). 48 hours after infection, cells were lysed to quantify luciferase activity. The results are expressed as a percentage of infection compared to a control infection in the absence of a compound (y-axis) at different exposure times I, II, Illa and Illb (x-axis). The dark columns represent the results for the control infection and the clear columns show the results obtained in the presence of FQ at 1 μΜ. Error bars represent the standard deviation of at least three experiments.

Figure 6 shows the antiviral effect of FQ on HCV pseudoparticles (VHCpp) containing envelope glycoproteins of isolate JFH1. Huh-7 cells were infected with HCVPs (dot) or pseudoparticles containing the envelope glycoprotein of the endogenous retro-virus RDI 14 (squares). The cells were treated for 1 hour before adding FQ and for the duration of the infection. 48 hours after infection, cells were lysed to quantify luciferase activity. The results are expressed as a percentage of infection compared to a control infection in the absence of compound (solvent only) (y-axis) as a function of the compound concentration (abscissa axis, in μΜ). Error bars represent the standard deviation of at least three experiments. Figures 7 and 8

Figure 7: RNA JFH1-AE1E2-Luc was introduced by electroporation into Huh-7 cells and 4 hours after infection, the cells were treated or not with FQ. The cells were lysed 48 hours later. The results are expressed as a percentage of replication relative to a control replication in the absence of a compound (ordinate axis) as a function of the concentration of compound (abscissa axis).

Figure 8: Huh-7 cells were inoculated with HCVcc for 2h and then cultured in the presence (clear columns) or absence (dark control columns) of FQ for 70h. The supernatants were recovered, the cells were lysed and the amount (y axis, fmol / L) intracellular (lysates) and extracellular (supernatant) of the HCV core protein was determined. The data presented are representative of three independent experiments. Error bars represent the standard deviation of at least three experiments. Turkey's multiple comparison test was used for statistical analysis, ns: non-significant differences. Figure 9

Figure 9 represents the amount of HCV RNA (ordinate axis, in Log10 of the amount of HCV RNA), relative to the total amount of RNA (g)) of uninfected or inoculated Huh-7 cells. by HCVcc without addition or in the presence of solvent, FQ at 1 or 2 μΜ or heparin. Mean values +/- standard deviation (error bars) of three different experiments are presented. Turkey's multiple comparison test was used for statistical analysis. No significant difference was found between treated and untreated cells.

Figure 10

FIG. 10 represents the number of infected cells per focal point of infection (ordinate axis) of Huh-7 cells inoculated with the JFH1 virus for 2 h and then covered by the anti-E2 3/11 monoclonal antibody (nAb) in the presence or absence of absence of FQ. 48 hours after infection, the cells were fixed and then treated with immunofluorescence to detect HCV-positive cells. The number of cells per household has been determined for at least 46 homes in each condition. The bars represent the average values. Two-factor ANOVA and Bonferroni tests were used to compare conditions in the presence and absence of FQ (p <0.0001). *** indicates a significant difference. Figures 11 and 12

Figures 11 and 12 show the isobolographic curves obtained respectively for the mixtures FQ / IFN-α (IFN) and FQ / boceprevir (BCP). The ordinate axis represents the percentage of the ICso obtained in the presence of IFN-α or boceprevir alone and the abscissa axis the percentage of the ICso obtained in the presence of the FQ alone. The dotted diagonal represents the additivity line and the IC ₅ o values mixed in different proportions of FQ / IFN-α (IFN) and FQ / boceprevir (BCP).

EXAMPLES

Materials and methods Chemical products. Ferroquine (FQ) and hydroxyfroquine of formula IV (HFQ in the following) were synthesized as previously described (Biot et al (1997) J Med Chem 40: 3715-3718, Biot et al (2006) J Med Chem 49 : 2845-2849). Chloroquine (CQ) (reference: C6628), hydroxychloroquine (HCQ) (reference: H0915) and interferon-a (IFN?) (Reference: 14784) were obtained from Sigma-Aldrich. Compounds FQ, HFQ, CQ and HCQ were prepared as 1M stock solutions in dimethylsulfoxide (DMSO).

Cell cultures. Huh-7 (14), HEK-293T (ATCC CRL-1268) and HeLa (ATCC CCL-2) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). inactivated by heat. Madin-Darby kidney bovine cells (MDBK, ATCC CCL-22) were cultured in DMEM supplemented with glutamax-I and 10% horse serum. The toxicity of the compounds was evaluated on Huh-7 cells using a commercial MTS test (Celltiter 96, reference: G5421) according to the manufacturer's instructions (Promega).

Antibody. HCV anti-glycoprotein monoclonal antibodies (A4) (Dubuisson et al (1994) J. Virol 68: 6147-6160), HCV anti-glycoprotein E2 (3/11) (Flint et al (1999) J Virol 73: 6235-6244), anti-yellow fever virus coat protein (2D12) (ATCC CRL-1689) and anti-NS3 protein of bovine viral diarrhea virus (Osc-23) (Boulanger et al. (1991) J. Gen. Virol 72: 1195-1198) were used. The secondary antibodies conjugated to fluorophores Cy3 (reference 1 15-165-062) and Alexa488 (reference: Al 101) were respectively obtained from Jackson ImmunoResearch and Invitrogen.

Virus. A modified version of the plasmid encoding the genome of strain JFH1 (genotype 2a, GenBank reference AB237837) (Wakita (2005) Nat Med 11: 791-796) was used to produce cell culture HCV (HCVcc). Briefly, HCVcc was produced in Huh-7 cells after electroporation with in vitro transcribed RNA from plasmid JFH1 containing (JFH1-Luc) or not the Renilla luciferase reporter gene and modified to expressing the A4 epitope (Goueslain et al (2010) J Virol 84: 773-787) and mutations allowing to increase the viral titre (Delgrange et al (2007) J Gen Virol 88: 2495-2503). Stocks of plasmid JFH1 were produced by amplification in Huh-7 cells. In the JFH1-Luc construct, the Renilla luciferase gene is fused with the viral open reading phase in a monocistronic configuration. It was verified that the luciferase data of this virus were within the linear limits of the assay. The plasmid JFH1-AE1E2-Luc, containing a deletion in phase in the region, and the mutant incapable of replicating JFH1-GND-Luc, have been previously described (Goueslain et al (2010) J Virol 84: 773-787, Rocha-Perugini and others (2008) PLoS ONE 3: el866). The inhibitory effect of the compounds was determined by measuring luciferase activity in cell lysates or by quantifying indirect fluorescence infectivity with the anti-E1 monoclonal antibody. For quantitative binding experiments, virus in purified form was obtained by precipitation of the supernatant of HCV-infected Huh-7 cells with 8% polyethylene glycol 6000. The precipitated virus was then loaded onto a 10-40% continuous gradient of iodixanol. Fractions of 1 ml were collected and the most infectious fractions were pooled. The stock title was 5.10 ⁶ fofu units / ml. VHC pseudotyped retroviral particles (VHCpp) expressing the firefly luciferase reporter gene were produced in HEK-293T cells as previously described (Op De Beeck et al (2004) J Virol 78: 2994-3002). In some experiments, NADL strains of bovine viral diarrhea virus and 17D of yellow fever virus were also used to test the effect of the compounds on other viruses.

Determination of inhibition of cell-to-cell transmission. Huh-7 cells plated on glass coverslips in 24-well plates were infected with HCVcc for 2h (multiplicity of infection (MOI) 0.01). The inoculum was removed and replaced in culture medium containing the anti-E2 neutralizing monoclonal antibody (50 μg ml). Two days after infection, the foci were detected by indirect immunofluorescence. Indirect immunofluorescence. Infected cells cultured on glass slides were processed to detect viral proteins by indirect immunofluorescence as previously described (Rouille et al (2006) J Virol 80: 2832-2841). The nuclei were stained with 4 ', 6'-diamidino-2-phenylindole (DAPI) at 1 μg / ml. The coverslips were observed using a Zeiss Axiophot microscope, and the fluorescent signals were recorded using a Coolsnap ES camera {Photometrix, Kew, Australia). For the quantification of antigen positive cells, images of randomly selected lamellae areas were recorded. Quantification of the HCV core protein. Huh-7 cells were inoculated for 2h with HCVcc in 6-well plates. The HCV core antigen expressed inside the cells or secreted in the supernatant was quantified using the chemiluminescent microparticles (Architect HCVAg Test, Abbott, France) as described (Alsaleh et al (2010) J. Virol 84 12515-12258).

Quantitative binding test. The amount of virus bound to Huh-7 cells was determined by real-time quantitative RT-PCR as previously described (Castelain et al (2004) J Clin Virol 31: 227-234). Detection of HCV Receptor Expression by Flow Cytometry and Western Labeling. Huh-7 cells were treated with FQ for 48 hours. After incubation with FQ, the cells were stained with antibodies to perform flow cytometry and Western labeling as previously described (Ciesek et al (2010) Gastroenterology 138: 1875-1884).

Isobologram. To determine the possible interactions of FQ with other anti-HCV molecules, the antiviral activities of combinations between FQ and IFN-α or boceprevir were determined. The IC ₅₀ values were determined for each compound alone as well as for the compounds in fixed concentration ratios. IC ₅₀ values were then used to calculate the 50% fractional inhibitory concentrations that were used to construct the isobolograms as reported in Ohrt et al. (2002) Antimicrob Agents Chemother 46: 2518-2524, which makes it possible to determine whether the combinations have a synergistic, antagonistic or additive effect. Graphs and statistics. Prism v5.0c software (GraphPad Software Inc., La Jolla, CA) was used to prepare the graphs, calculate the IC ₀ values, and to determine the statistical significance of the differences between the data groups. Example 1 Ferroquine inhibits HCV infection

The chemical structures of ferroquine (FQ), hydroxy ferroquine of formula (IV) (HFQ), chloroquine (CQ) and hydroxychloroquine (HCQ) are shown in the diagram below:

HFQ HCQ

In order to test the effect of ferroquine (FQ) on the HCV reproductive cycle, the compound was added to Huh-7 target cells before and during infection with a virus containing the luciferase reporter gene. As can be seen in Figure 1, FQ causes a dose-dependent inhibition of luciferase expression.

Importantly, similar dose-dependent inhibition of HCV expression was also observed with a virus lacking a reporter gene (Figure 2). indicating that the FQ specifically affects the reproductive cycle of HCV with an IC ₅ o estimated 0,8μΜ (± 0.26) and an IC ₉₀ of 1, 92 μΜ.

Similar results were obtained on HepG2 cells expressing CD81.

The inventors have also analyzed the potential activity of a hydroxyferroquine (HFQ). As can be seen in Figures 1 and 2, HFQ has an anti-viral effect on HCV with an intensity similar to that of FQ. In contrast, other aminoquinoline compounds such as CQ and HCQ are less effective against HCV (Figures 1 and 2).

To determine if HCV is the only member of the family Flaviviridae to be affected by FQ, the inventors tested this compound on two other members of this family of viruses, the virus of bovine viral diarrhea and the fever virus yellow. FQ also has antiviral action, but at higher concentrations than for the HCV, with IC ₅₀ values of 6.74 μΜ and 3.63 μΜ for bovine viral diarrhea virus and yellow fever virus.

All these results indicate that FQ and HFQ have an anti-viral effect against viruses of the Flaviviridae family and particularly against the hepatitis C virus.

Example 2: Ferroquine inhibits HCV entry

To determine if it has an effect on HCV entry, FQ was added or removed at different times, during, and after inoculation of Huh-7 cells by JFH1-Luc (Figure 3). As seen in Figures 4 and 5, the greatest decrease in HCVcc infection is seen when FQ is present during viral infection, and only a weak anti-viral effect was detected when FQ was been added after the infection. These results suggest that FQ inhibits an early stage of the HCV reproductive cycle, which is in all likelihood the entry stage.

In order to specify the effect of FQ on the entry of HCV, the inventors used a system implementing HCV pseudoparticles (VHCpp). These are retroviral nucleocapsids that carry HCV glycoproteins in their envelopes. In such a context, only the early stages of the virus's reproductive cycle, namely the interaction of the virus with its cellular receptors, its binding and fusion, are specific for HCV, whereas the subsequent stages are dependent on the elements of the virus. Viral retro nucleocapsid. Using this approach, FQ inhibits the entry of VHCpp pseudoparticles in a dose-dependent manner (Figure 6). In addition, this effect is specific for HCV envelope glycoproteins, since FQ has no influence on the entry of pseudoparticles containing the envelope glycoprotein of the endogenous retrovirus FDI 14. This confirms that the FQ is well an inhibitor of HCV entry.

Since FQ specifically affects the entry of HCV, this led the inventors to test its antiviral effect on different HCV genotypes / subtypes in the context of the HCV pseudoparticle system (HCVP). As shown in Table 1, FQ inhibits HCV infection of the different genotypes / subtypes tested, indicating that FQ inhibits HCV entry independently of the genotype. Genotype IC ₅₀ (μΜ)

the 0.85 ± 0.11

lb 0.49 ± 0.09

2a 0.26 ± 0.06

2b 0.28 i- 0.00

3 not determined

4 0.66 ± 0.09

0.69 ± 0.42

6

0.74 0.53

RDI 14

no inhibition

Table 1: FQ inhibits HCV entry genotype-independent

HCVPs expressing E1E2 glycoproteins of different genotypes were tested for their sensitivity to FQ and the corresponding IC _50s are indicated. The sequences of the following isolates were used to generate the VHCpp: H77 (Genbank AAB67037), UKN1B-5.23 (gtlb; AY734976), JFH1 (gt2a; AB047639), UKN2B-1.1 (gt2b; AY734982), UKN3A-1.28 ( gt3a; AY734984), U N4-11.1 (gt4; AY734986), and UKN6-5,340 (gt6; AY736194).

Example 3 Effect of ferroquine on the replication and assembly of HCV

Although the above data indicate that FQ strongly inhibits the entry of HCV, it can not be ruled out that it also has additional effects on other stages of the HCV reproductive cycle.

Thus, to analyze the effect of FQ on HCV genome replication, Huh-7 cells were electroporated in the presence of JFH1-AE1E2-RNA.

Inactive Luc for assembly that has been transcribed in vitro to bypass the entry step and avoid interference with late stages of the HCV reproductive cycle.

As seen in Figure 7, FQ also has an effect on HCV replication, but at higher concentrations (compare Figure 1 and Figure 7).

This observation makes it possible to interpret the weak antiviral effect detected when the FQ is added after infection in Example 2.

Furthermore, in order to determine if FQ could affect the assembly and secretion of HCV virion, the intracellular and extracellular amount of

HCV was determined for infected cells treated, after infection, with 1 μl of FQ for 70h. The amount of core protein in the culture supernatant reflects the amount of viral particles secreted.

As seen in Figure 8, the amounts of core protein released into the supernatant of infected cells are similar in the presence or absence of treatment with the

FQ.

These data indicate that FQ does not inhibit assembly and exit of virions from

HCV. Example 4 Ferroquine Inhibits a Post-Binding Step During Viral Entry

Since FQ exhibits antiviral activity on an early stage of the HCV reproductive cycle, the inventors have explored in more detail its mode of action on HCV entry.

To determine whether FQ directly affects the binding of virus particles to the cell surface or a subsequent stage of virus entry, the inventors analyzed the binding of the virus in the presence of FQ. Cells were inoculated with purified HCVc at 4 ° C in the presence of FQ or heparin, and the amount of bound virus was determined by quantifying genomic RNA of HCV. As expected, heparin strongly reduced HCV binding to the cell surface (Figure 9). In contrast, in the presence of FQ, no effect on virus binding was observed (Figure 9), indicating that FQ does not inhibit HCV entry by preventing virus binding to the surface of the virus. cells.

In addition, the effect of FQ on the expression of essential HCV input factors, CD81, SRB1, CLDN-1 and OCLN was examined. Huh-7 cells were treated with FQ at 1 μ§ ηύ for 48 hours, then the expression of CD81, SRB1, CLDN-1 and OCLN was determined by Western labeling or flow cytometry. The expression levels of the four factors are unchanged, indicating that the FQ does not act by regulating them negatively. Example 5 Ferroquine inhibits viral cell-to-cell transmission

Following the infection of Huh-7 cells with HCV, the viruses produced are transmitted to adjacent cells, producing outbreaks of infection. This mode of infection is insensitive to neutralization by anti-E2 antibodies (Timpe et al (2008) Hepatology 47: 17-24). In order to determine if FQ can block cell-to-cell transmission, Huh-7 cells infected at low multiplicity of infection were incubated with neutralizing monoclonal antibody 3/11 (Brimacombe et al (2011) J Virol 85: 596-605), in the presence or absence of 1 μΜ of FQ. Three days after infection, the foci were visualized by immunofluorescence and their size was measured by counting the number of cells per household. As seen in Figure 10, treatment with FQ leads to a sharp decrease in average household size. Indeed, in the control condition the foci contain on average 40 cells, whereas for the cells treated with FQ the number is reduced to 16 cells. This clearly indicates that FQ blocks cell-to-cell transmission of HCV. Example 6: Antiviral effect of FQ in combination with IFN-α or boceprevir

The inventors tested whether the FQ could be combined with other anti-HCV compounds currently used in the treatment of hepatitis C. Thus, the inventors used the boceprevir, a compound that directly targets the HCV NS3 / 4A protease. , and IFN-α, which has been used for decades in anti-HCV therapy. These combinations were analyzed using isobolograms to determine their pharmacochemical interactions (Tallarida (2006) J Pharmacol Exp Ther 319: 1-7). Each compound was combined with FQ in different fractions of their IC ₅₀ . For each fraction IC ₅ o of the FQ, the percentage of second IC ₅₀ of the compound required to obtain 100% of the total IC ₅ o was estimated and reported on a graph. In isobologram analyzes, a straight diagonal indicates that the two compounds are in an additive relationship, while concave and convex diagonals respectively indicate synergistic and antagonistic effects. Combinations of FQ with IFN-α or boceprevir clearly have additive effects (Figures 11 and 12), indicating that FQ would be effective in combination treatments.

Discussion

HCV entry is an attractive target for antiviral therapy, offering the opportunity to prevent multiple virus-receptor interactions and to prevent the fusion of plasma membrane virus. The inventors have shown that FQ exhibits an antiviral activity independent of the genotype against HCV by inhibiting a step of the entry of the virus into the cell after its binding with the plasma membrane and by blocking cell-to-cell transmission between neighboring cells. .

Although ferroquine (FQ) is an analogue of chloroquine (CQ), its mechanism of action is potentially different (Dubar et al (2012) Chem Commun (Camb) 48: 910-912). FQ indeed has lower basic properties than those of CQ (Biot et al (2005) Mol Pharm 2: 185-193). This difference may explain why FQ does not exhibit antiviral activity against viruses such as VSV, influenza virus, or Sindbis virus (Biot et al (2006) J Med Chem 49: 2845-2849) , while QC blocks the entry into the cell of these viruses as well as that of other pH-dependent viruses (Picard-Maureau et al., (2003) J. Virol 77: 4722-4730; Cassell et al. (1984) J Virol 52: 857-864, Yoshimura et al (1982) J Virol 43: 284-293).

FQ inhibits a step of HCV entry into the posterior cell at the binding step. Moreover, the effect of FQ on HCV pseudoparticles suggests that FQ affects the input functions of the HCV envelope proteins and not the virion-associated lipoprotein portion. It has recently been shown that HCV envelope proteins form large molecular complexes stabilized by intramolecular disulfide bridges (Vieyres et al (2010) J Virol 84: 10159-10168). This observation suggests that the input process requires rearrangement of these disulfide bridges so that the fusion process can take place. It is therefore possible that the oxidative properties of FQ under acidic conditions could inhibit the fusion process by affecting the reorganization of disulfide bridges within endosomes. Alternatively, it can not be ruled out that FQ modifies certain specific lipids required for the fusion of the HCV envelope with an early endosomal membrane.

In clinical studies in healthy human subjects, it could be shown that FQ was safe and well tolerated with oral doses of at least 1600 mg and that the mean estimate for apparent terminal half-life in blood was 16 days. In addition, a blood concentration of 487 ng / mL (ie 1.1 μΜ) was observed for the highest dose of FQ tested, which is slightly higher than the ICF ₅₀ calculated for HCV in cell culture, which is therefore indicative of in vivo anti-HCV activity, alone or in combination with other anti-HCV compounds.

Ferroquine may provide a novel approach to prevent HCV infections, particularly in the context of liver transplantation of patients chronically infected with HCV. Indeed, one of the main problems in liver transplants is graft reinfection, which is always observed concomitantly with an acceleration of progression of liver injury (Brown (2005) Nature 436: 973-978). Thus, the ability of FQ to inhibit HCV cell-to-cell transmission is a major advantage for an HCV entry inhibitor. In addition, FQ has antiviral activity against all HCV genotypes that have been tested, increasing its interest as a broad-spectrum anti-HCV agent. The combination of entry, replication and protease inhibitors in a multi-compound treatment context may be a way to reduce the risk of emergence of resistant viruses. The FQ could thus be an interesting option to test for the manufacture of low cost anti-HCV combination. Finally, these results highlight the importance of using FQ in individuals with both malaria and hepatitis C.

Claims

1. Compound of formula (I) below:

(I)

in which :

R 1 and R ₂ , identical or different, are selected from the group consisting of a hydrogen atom, an alkyl group of 1 to 10 carbon atoms, an alkoxy group of 1 to 10 carbon atoms, a acyl group of 1 to 10 carbon atoms, a hydroxyalkyl group of 1 to 10 carbon atoms, an acyloxyalkyl group of 2 to 10 carbon atoms, an acetylalkyl group of 2 to 10 carbon atoms, and a grouping:

- R ₃ represents CH or a heteroatom selected from the group consisting of N and P, and - R4 represents a hydrogen atom, an alkyl group of 1 to 10 carbon atoms, an alkoxy group of 1 to 10 carbon atoms, an acyl group of 1 to 10 carbon atoms, a hydroxyalkyl group of 1 to 10 carbon atoms, an acyloxyalkyl group of 2 to 10 carbon atoms or an acetylalkyl group of 2 to 10 carbon atoms,

or a pharmaceutically acceptable salt thereof,

for its use in the prevention or treatment of an infection of a Flaviviridae family virus in an individual.

2. A compound or a pharmaceutically acceptable salt thereof for use according to claim 1 of the following formula (II):

(Π)

A compound or a pharmaceutically acceptable salt thereof for use according to claim 1, selected from the group consisting of compounds of the following formulas (III), (IV) and (V):

A compound or a pharmaceutically acceptable salt thereof for use according to claim 1, selected from the group consisting of compounds of the following formulas (VI) and (VII):

(VII)

A compound or a pharmaceutically acceptable salt thereof for use as claimed in any one of claims 1 to 4 in the prevention or treatment of a hepatitis C virus (HCV) infection in an individual.

6. Compound or pharmaceutically acceptable salt thereof for use according to one of claims 1 to 5, associated with at least one additional compound for the prevention or treatment of infection with a virus of the family Flaviviridae .

A compound or a pharmaceutically acceptable salt thereof according to claim 6, wherein the additional compound for the prevention or treatment of infection with a virus of the family Flaviviridae is selected from the group consisting of interferon -a, ribavirin, boceprevir, and telaprevir.

8. Pharmaceutical composition, comprising as active substances, at least one compound of formula (I) as defined in one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and at least one additional compound for the prevention or treatment of infection with a virus of the family Flaviviridae, optionally in combination with a pharmaceutically acceptable carrier, for its use in the prevention or treatment in the prevention or treatment of an infection with a virus of the family Flaviviridae in an individual.

A pharmaceutical composition for use according to claim 8, wherein the compound of formula (I) is selected from the group consisting of compounds of formulas (II), (III), (IV), (V), (VI) and (VII).

The pharmaceutical composition for use according to claim 8 or 9, wherein the additional compound for the prevention or treatment of infection with a Flaviviridae virus is selected from the group consisting of interferon and , ribavirin, boceprevir, and telaprevir.

11. Products containing:

at least one compound of formula (I) as defined in one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and

at least one additional compound intended for the prevention or treatment of an infection with a virus of the family Flaviviridae,

as a combination product for simultaneous, separate or spread use over time for the prevention or treatment of infection with a Flaviviridae virus.

Products according to claim 11, wherein the compound of formula (I) is selected from the group consisting of compounds of formulas (II), (III), (IV), (V), (VI) and (VII) .

Products according to claim 11 or 12, wherein the additional compound for the prevention or treatment of infection with a virus of the family Flaviviridae is selected from the group consisting of interferon-α, ribavirin, boceprevir, and telaprevir.

14. Pharmaceutical composition, comprising as active substances, at least one compound of formula (I) as defined in one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and at least one additional compound for the prevention or treatment of infection with a Flaviviridae virus specific for HCV infections and / or selected from the group consisting of interferon-a, ribavirin, boceprevir, and telaprevir, optionally in combination with a pharmaceutically acceptable carrier.

A pharmaceutical composition for use according to claim 14, wherein the compound of formula (I) is selected from the group consisting of compounds of formulas (II), (III), (IV), (V), (VI) and (VII).