WO2016169573A1

WO2016169573A1 - Sesquiterpene lactones as potent and broad spectrum antiviral compounds against all genotypes of hepatitis c virus (hcv)

Info

Publication number: WO2016169573A1
Application number: PCT/EG2016/000014
Authority: WO
Inventors: Mahmoud Fahmi ELSEBAI
Original assignee: Elsebai Mahmoud Fahmi
Priority date: 2015-04-20
Filing date: 2016-04-18
Publication date: 2016-10-27

Abstract

The present invention relates to potent HCV entry inhibitors (cynaropicrin and grosheimol), in addition to mono-caffoeylquinic acid derivatives which have also anti-HCV activity but less than cynaropicrin and grosheimol.

Description

SESQUITERPENE LACTONES AS POTENT AND BROAD SPECTRUM ANTIVIRAL

COMPOUNDS AGAINST ALL GENOTYPES OF HEPATITIS C VIRUS (HCV)

1- Technical Field

Hepatitis C virus (HCV) infection is a significant public health problem since it is the leading cause of chronic liver diseases such as liver cirrhosis and hepatocellular carcinoma. HCV chronic patients often demand liver transplantation and HCV infection rates increase worldwide. The two terpenoid compounds grosheimol and cynaropicrin provide outstanding opportunity to eradicate this virus since they showed potent and broad spectrum activity against all 7 major genotypes of HCV. This invention relates to products and methods for treating HCV infection. The products are the derivatives of sesquiterpene lactones such as cynaropicrin and grosheimol, and the derivatives of mono-caffeoylquinic acids such as chlorogenic acid. The method includes administering to a subject in need thereof an effective amount of one or more of these sesquiterpene lactones and /or mono- caffeoylquinic acid derivatives with or without other anti-HCV or liver supportive agents such as curcumin and epigallocatechin-3-gallate (EGCG).

2 -Background Art

Hepatitis C virus infection is a significant public health problem and infection rates increase worldwide (Beaulieu et al., 2012). In the United States, HCV is the most common chronic blood-borne infection and it appears to be the major causative factor responsible for the recent doubling of HCC which was estimated to result in ~10,000 deaths in the United States only in the year 2011 (Gonzalez et al., 2009; Ibrahim et al., 2013). As many as 4 million individuals in the United States and as many as 200 million people worldwide are infected with HCV (WHO, June 2011 ). About 3 - 4 million people are infected per year, and more than 350,000 people die yearly from hepatitis C- related diseases. The overall medical and social costs of chronic HCV infections are estimated to exceed $85 billion, and recently, it has been shown that US mortality rates from HCV now exceed those from HIV (Ibrahim et al., 2013).

There is no preventive vaccine available for HCV due to its highly mutable nature. The NS3-4A protease inhibitors telaprevir (Incivek™) and boceprevir (Victrelis™) were approved in 2011 by FDA for treatment of patients infected with genotype 1 (Moss et al., 2012). In 2013, FDA approved the use of sofosbuvir (Sovaldi™) against infection by chronic hepatitis C genotypes 1 , 2, 3 and 4, in combination with pegylated interferon and ribavirin, or with ribavirin alone (FDA December 6, 2013); simeprevir (Olysio™) was approved for use in combination with peg-interferon-a and for the treatment of chronic hepatitis C infection, genotype 1 (FDA November 22, 2013). Daclatasvir, an NS5A replication complex inhibitor, is a potent and promising direct antiviral agent (DAA) for HCV, being most effective in genotype 1 b infection (Berger et al., 2014; Miura et al., 2014). Although these drugs represent the first Direct Acting Antivirals (DAAs) approved for HCV therapy, it was found that monotherapy utilizing any of these drugs alone resulted in the rapid development of resistant strains. As a result, these drugs are only FDA approved when given in combination therapy with ribavirin and/or pegylated interferon (PEG-IFN). Moreover, the severe side effects and extremely high cost associated with PEG-IFN, in addition to their narrow spectral activity towards different genotypes of HCV, remain problematic (Farnik and Zeuzem, 2012; Ward et al., 2014). In addition, these drugs are contraindicated in patients having low platelet count, coronary artery disease, autoimmunity, advanced liver disease, seizure disorders, and in pregnancy, or who are intolerant to IFN-based therapies (Carrefio, 2014). There are seven major genotypes and more than 50 subtypes of HCV; genotype 1 is the most virulent since it is associated with more severe liver diseases and a higher risk of HCC than the other genotypes. It is spread worldwide especially in United States and Northern Europe; genotype 2 is also worldwide spread especially in United States and Japan; genotype 3 in India; genotype 4 in the Middle East and Africa; genotype 5 in South Africa; genotype 6 in Hong Kong and Southeast Asia; genotype 7 in Vietnam, Thailand, Indonesia and Burma (Nakajima et al., 2013; Salam et al., 2013).

The present invention features the unexpected characters that grosheimol and cynaropicrin are effective in neutralizing all genotypes of HCV by inhibiting cell entry, also mono-caffoeyquinic acid derivatives showed, but less, activity against HCV. Their structural elucidation was done through extensive spectroscopic data such as NMR and accurate mass measurements. Prior art:

There is no preventive vaccine available for HCV due to its highly mutable nature. The recently approved HCV NS3-4A protease inhibitors boceprivir and telaprevir although showing significantly improved efficacy, treatment with protease inhibitors showed rapid emergence of drug-resistant virus, in addition to their side effects, combined therapy with IFN and ribavirin and highly expensive, in addition to their narrow spectral activity towards different genotypes of HCV (Farnik and Zeuzem, 2012). The standard therapy pegylated interferon plus ribavirin is only effective in 50-60% of patients and is associated with serious side-effects, and half of those which respond relapse after cessation of interferon treatment. Therefore, therapeutic alternatives are of major importance. Some other sesquiterpene lactones as active agents against HCV were mentioned in the US2004/20040229936A1 patent application.

The advantages of our invention and its commerciality:

The compounds in our invention are more potent than the compounds mentioned in the US2004/20040229936A1 patent application.

The compounds in our invention are active against all major genotypes of HCV, i.e., active against the known seven genotypes 1-7 as illustrated in table .

All these compounds were present also in the Egyptian wild artichoke (WEA) (Cynara cardunculus L. var. sylvestris (Lam.) Fiori) which is a medicinal food plant worldwide and has long human consumption without serious side effects; the literature reported the safety of its ingredients.

These compounds are present in the plants of family Asteraceae, e.g. artichoke plant which is available in all markets almost in all countries, which give the first impression about the safety of these active compounds.

Many of these compounds are already presented in the pharmaceutical markets for other purposes, example chlorogenic acid which is available in many dosage forms as an antioxidant.

These compounds are water soluble and hence druggable injections are easy to be formulated which will decrease the amount of dosage form and shorten the duration of therapy and thus reducing putative side effects.

The active compounds are simple ones and are not complicated and hence they can be synthesized and marketed with affordable prices.

3- DETAILED DESCRIPTION

This invention relates to use of one or more sesquiterpene lactones and/or mono-caffoeylquinic acids or any of its derivatives for treating HCV infection. The sesquiterpene lactones and/or mono-caffoeylquinic acid derivatives can be found naturally or semi-synthesized from naturally-occurring sesquiterpene lactones and/or mono-caffoeylquinic acid derivatives, respectively, or totally synthesized in a chemical laboratory.

The sesquiterpene lactones or mono-caffoeylquinic acid derivatives mentioned herein may contain an exo- methylene double bond and one or more asymmetric centers. Thus, they can occur as racemic mixtures and racemates, individual diastereomers, single enantiomers, diastereomeric mixtures, and trans- or cis-isomeric forms. All such isomeric forms are contemplated.

The sesquiterpene lactones or the mono-caffoeylquinic acid derivatives can be modified by synthetic methods, examples: 1 )-the addition of amino acid or nitrogen containing compound (e.g., dimethylamine) or a sulfur- containing compound (such as mercaptoethanol, cystine, or a cystine-containing peptide) to the exo-methylene group of the γ-lactone ring, and/or 2)-sugar glycosylation for any free OH group. 3)- the methylene group on y- lactone ring of, for example grosheimol and/or cynaropicrin or any of the γ-lactone ring-containing compounds can be reduced to form a methyl group using a suitable reducing agent.

Within the scope of this invention also is a pharmaceutical composition contains an effective amount of at least one sesquiterpene lactone and/or mono-caffoeylquinic acid derivatives described above and a pharmaceutical acceptable carrier. This invention also covers a method of administering an effective amount of one or more sesquiterpene lactones and/or mono-caffoeylquinic acid derivatives mentioned above to treat HCV infection. Effective doses will vary, as recognized by those skilled in the art, depending on excipient usage, the route of administration, and the possibility of co- administration with other therapeutic treatment.

A sterile injectable formulations can be a suspension or solution in a non-toxic acceptable parenteral diluent or solvent, such as a solution in 1 ,3-butanediol. Among the acceptable vehicles that can be employed are water, mannitol, Ringer's solution, glucose solution, and isotonic sodium chloride solution. Fixed oils also are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or di-glycerides) such as castor oil or olive oil, especially in their polyoxyethylated versions. Fatty acid, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils; these oil solutions or suspensions can also contain a long chain alcohol dispersant or diluent, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Spans or Tweens or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.

To practice the method of the present invention, a composition having one or more sesquiterpene lactones and/or mono-caffoeylquinic acid derivatives can be administered orally, parenterally, nasally, topically, rectally, or buccally. The term "parenteral" as used herein refers to intravenous, intramuscular, subcutaneous, intracutaneous, intrasternal, or intralesional injection, as well as any suitable infusion tool.

A formulation for oral administration can be any orally acceptable dosage form including tablets, capsules, emulsions, and aqueous solutions, dispersions, and suspensions. For oral administration in a capsule form, useful diluents include lactose and/or any type of starch (corn, rice, potato etc). In the case of tablets, commonly used carriers include lactose and/or any type of starch (com, rice, potato etc). Lubricating agents, such as magnesium stearate, are also typically added. For the oral suspensions or emulsions, the active ingredient can be suspended or dissolved in an oily phase combined with suspending or emulsifying agents, which can be combined with, if desired, certain flavoring, sweetening, or coloring agents.

A formulation having one or more active sesquiterpene lactones or mono-caffoeylquinic acid derivatives can also be administered in the form of suppositories for rectal administration. A nasal aerosol or inhalation formulation can be prepared according to protocols well known in the art of pharmaceutical preparations. For example, this formulation can be prepared as a solution in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

A pharmaceutically acceptable carrier can be used with one or more active sesquiterpene lactones or mono- caffoeylquinic acid derivatives. The carrier in the pharmaceutical formulation must be compatible with the active constituents of the formulation (and preferably, capable of stabilizing the active constituents) and not deleterious to the subject to be treated. One or more solubilizing agents can be utilized as pharmaceutical excipients for delivery of an active sesquiterpene lactone and/or mono-caffoeylquinic acid derivatives. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.

Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

Materials and Methods for the spectro-analyses

Synthesis of compounds: for the synthesis of sesquiterpene lactone derivatives, as reported in (Nosse et al., 2003), for the synthesis of mono-caffoeylquinic acid derivatives, as in reference (STOCKIGT and ZENK, 1974).

RESULTS of the in vitro studies of the active compounds against

HCV

Identification of compounds with antiviral activity against HCV

We have synthesized some sesquiterpene lactones for measuring their anti-HCV activity. These compounds were detected also in the Egyptian Wild Artichoke (WEA) (Cynara cardunculus L. var. sylvestris (Lam.) Fiori), therefore we have tested its total extract for its anti-HCV activity. Anti-HCV activity of these compounds was evaluated by inoculation of the highly permissive human hepatoma cell line Huh7/Scr cells (kindly provided by F. Chisari, The Scripps Research Institute, La Jolla, CA) with firefly luciferase reporter viruses based on the intra-genotypic genotype 2a chimera Jc1 (Luc-Jc1 (Koutsoudakis et al., 2006), Fig.4A) in the presence of the compounds. Briefly, the day of infection, Huh7/Scr cells were treated with 10 or 20 μΜ of each compound or dasatinib (at the same concentrations) (Lupberger et al., 2011 ), a known HCV entry inhibitor, for 1h. Then, compounds-containing media were removed and cells were infected with fresh media that contained Luc-Jc1 and compounds. At 4h post infection media were replaced again with fresh compounds-containing media and 72h after infection cells were assayed for firefly luciferase activity. Cell viability was monitored in parallel with an ATP assay. Among all compounds tested, 7 molecules inhibited HCV cell entry and/or RNA replication (Fig. 1 B). Compounds cynaropicrin and grosheimol exhibited the greater potency by inhibiting HCV more than 90% and 80%, respectively. For this, these compounds were characterized further.

Chemical characterization of compounds grosheimol and cynaropicrin

Grosheimol is constructed from perhydro-azulen system with attached lactone ring and two exo-methylenes resulting in a sesquiterepene lactone derivative. Grosheimol was obtained as light yellow powder with a molecular formula C₁₅H₂o0₄ on the basis of accurate mass measurements (HRESI⁺MS: found= 265.1440 [M+H], calcld= 265.1472 [M+H], HRESI^'MS: found= 263.1285 [M-H], calcld= 263.1283 [M-H]; [a]_D +29.3 (c = 0.15, methanol)). Grosheimol was characterized based on its complete spectroscopic data (1 D- and 2D- N R) and accurate mass measurements.

Cynaropicrin was characterized based on its complete spectroscopic data and accurate mass measurements (HRESI⁺MS, m/z, found = 369.1318 [M + Na]⁺, calcld =263.1314 [M + Naf; [a]_D +148.42 (c = 0.38, methanol)) and comparing them to the reported data (Grabarczyk and Makowska, 1973; Scotti et al., 2007; Xiaoli et al., 2005).

Cynaropicrin and grosheimol potently inhibit HCV

To assess the potency of the cynaropicrin and grosheimol against HCV, the Luc-Jc1 virus was used to estimate the half maximal effective concentration [EC₅₀] , the half maximal cytotoxic concentration [CC₅₀] and the selectivity index [(CC₅₀ / EC₅₀), SI] by performing a dose-response infection assay. Dasatinib served again as positive control. The infection protocol and the compounds incubation period were similar to that described above. As shown in Fig. 2A-D, estimated EC₅₀ for cynaropicrin was 1.27 μΜ [CC₅₀: 16.94 μΜ, and the selectivity index (Sl): 13.33], as compared with an EC₅₀ of 1.03 μΜ for grosheimol [CC₅₀: 35.59 μΜ, SI: 35.59] and an EC₆₀ of 1.47 μΜ for dasatinib [CC₅₀: 20.31 μΜ, SI: 13.81] in this assay.

Cynaropicrin inhibits HCVcc and HCVpp cell entry while Grosheimol inhibits selectively HCVcc entry.

We then sought to determine in which step of the viral life cycle cynaropicrin and grosheimol exert their action; whether it was entry, replication/translation or assembly/release. Different HCV-based systems exist that allow us to dissect the different steps of the viral life cycle (Tellinghuisen et al., 2007). To assess if cynaropicrin and grosheimol inhibit the entry step, we used the HCV pseudoparticle system (HCVpp). HCVpp is a well-established system for the study of HCV entry and neutralization (Bartosch et al., 2003; Hsu et al., 2003). They consist of lenti- or retroviral core surrounded by an envelope displaying HCV E1 E2 envelope glycoproteins. For consistency with the previous experiments, we used HCVpp which carry glycoproteins (genotype 2a, isolate J6CH) identical to those of the Luc-Jc1 virus. The day of infections, Huh7/Scr cells were treated with total WEA extract (500 pg/ml), or synthesized compounds cynaropicrin, grosheimol or the control Dasatinib for 1 h (all at 5 μΜ). Then, cells were infected with HCVpp-compounds mix at the same concentration. Six hours post infection HCVpp-containing media was replaced with fresh media-compounds mix and 72 h after infection cells were assayed for Renilla luciferase activity. HCVpp were inhibited in the presence of total WEA extract or cynaropicrin, while grosheimol did not exhibit any inhibitory effect against HCVpp . These results indicate that the compound cynaropicrin acts at the entry level. The positive control Dasatinib also reduced infectivity levels of the HCVpp as was expected. Consistent with the results in Fig.4, grosheimol did not show cytotoxity in this concentration while cynaropicrin reduced cell viability by around 40%.

HCV entry is a multistep process that involves viral proteins and several cellular receptors. Cynaropicrin-mediated inhibition of the HCVpp could be a result of either a block in virus-cell binding, virus uptake or viral delivery into the cytoplasm. Furthermore, cynaropicrin could be acting directly on the viral particle or could be mediating an effect on the host cell, or both. In order to address these questions we performed time-of-addition experiments in which either the cells or the virus were pre-incubated for 1 h with compounds (cynaropicrin or grosheimol) prior to virus inoculation, or a virus-compounds mix was directly added to the cells or compounds was added after 4h of virus inoculation (Fig. 3B, left). Surprisingly, both compounds interfered with HCV infection when viruses were incubated with the compounds and then compounds were present during virus inoculation (Fig. 3B, right). Pre-exposition of the cells to the compounds or addition 4 h after infection did not result in HCV inhibition. For cynaropicrin, this data corroborate the previous results and suggest a mechanism of inhibition in the early steps of entry. For grosheimol, this data indicate also an entry-inhibition mechanism, specific only for HCVcc and not for HCVpp. Cynaropicrin and grosheimol neither inhibit HCV RNA replication and/or translation nor HCV particle production

To investigate the impact of cynaropicrin and grosheimol in HCV RNA translation and/or replication, we transfected Huh7/Scr cells with a subgenomic JFH1 luciferase replicon (SGR-JFH1 , Fig. 4A, top) (Kato et al., 2003). Subgenomic replicons are autonomous-replicating molecules that lack structural proteins. A reporter gene {firefly luciferase) is expressed via the HCV IRES followed by the encephalomyocarditis IRES which drives the expression of non-structrural proteins (NS3-NS5B). As a result, the viral RNA is translated and replicates but does not encapsidate or produce new virions. Briefly, Huh7/Scr cells were electroporated with the SGR-JFH1 RNA. Cynaropicrin, grosheimol or total WEA extract were added 4h later and luciferase activity was assayed after 48 hours. Changes in luciferase levels correlate with levels of HCV replication and/or translation. A 2'-modified nucleoside analog (2'-C-methyladenosine, 2'-C- et) (Carroll et al., 2003) was used as a positive control. As shown in Fig. 4A (bottom), none of the tested compounds inhibited HCV RNA replication and/or translation, while the control compound exerted a strong inhibitory effect in this assay, suggesting that these compounds do not act at these viral life-cycle steps.

Cynaropicrin and Grosheimol do not inhibit HCV particle production

To investigate a potential role of the synthesized sesquiterpene lactones in HCV particle production, we adapted and slightly modified a HCV particle production assay described in Menzel ef. al. (Menzel et al., 2012). Briefly, as shown in Fig. 4B top, Huh/Scr cells were electroporated with Luc-Jc1 virus and cultured for 40h. At this time point cells were extensively washed with PBS and fed with fresh medium containing cynaropicrin or grosheimol for 2h. The MAPK ERK kinase inhibitor U0126, which has been shown to inhibit HCV particle production, served as positive control. After this two-hour compound treatment, cells were washed again extensively and fed with fresh- medium for 6h to allow particle production post compound treatment. Finally, medium from cells was collected, clarified from cells with centrifugation and tested for particle production in naive Huh7/Scr cells, while the effect of the compounds on HCV replication/translation and their cytotoxicity was measured in the electroporated cells by luciferase assays. As shown, in Fig. 4B, bottom, neither cynaropicrin nor grosheimol inhibited HCV particle production in this assay while U0126 did inhibit without any effect on replication/translation. These data narrow down the effect of both compounds at the entry level.

Cynaropicrin and grosheimol are active against all major HCV genotypes

HCV isolates have been classified into seven major genotypes (1-7), differing in their nucleotide sequence by around 30%, and a number of subtypes (a, b, and so on) with ~20% sequence divergence (Nakano et al., 2012). HCV treatment efficacy is influenced by viral genotype and treatment decisions are made taking the HCV genotype into consideration (Lange and Zeuzem, 2013). All previous experiments in this study were performed using viruses derived from the genotype 2a. In order to determine if the synthesized compounds are also active against the other HCV genotypes, we used chimeric JFH-1 based reporter virus constructs, carrying Renilla luciferase inserted at the NS5A gene and structural proteins from all major HCV genotypes: 1a (isolate TN), 1b (isolate J4), 2b (isolate J8), 3a (isolate S52), 4a (isolate ED43), 5a (isolate SA13), 6a (isolate HK6a) and 7a (isolate QC69) (Fig. 5A) (Gottwein et al., 2011). Similar dose-response inhibition experiments as described above were performed in order to evaluate the potency of cynaropicrin and grosheimol against multiple HCV genotypes. Importantly, infectivity was reduced in this treatment condition for all different genotypes, indicating that these synthesized compounds inhibit HCV infection independently of viral genotype or subtype. Estimated EC₅os, CC₅₀s and Sis for all major HCV genotypes are presented in Table 1.

Table 1: Antiviral Activity of grosheimol and cynaropicrin across all major HCV genotypes

7a (QC69) 1.44 15.12 10.51 5.68 86.45 15.23

Note: Selectivity Index (SI) is the ratio that measures the effectiveness of an antiviral compound. When it is over 10, one may consider it for optimization studies, and this means that both compounds cynaropicrin and grosheimol are significant drug candidates as anti-HCV.

4- Brief description of the drawing

Figure 1: Screening of compounds for antiviral against HCV. (A) Schematic drawing of the Luc-Jc1 reporter virus genome used for the screening. UT : untranslated region, Flue: Firefly luciferase, E CV: encephalomyocarditis virus, IRES: internal ribosomal entry site. (B) Huh7/Scr cells were seeded on 96 well plates, 1.2 x 10⁴ cells/well, 16h prior to infections. The day of infections, cells were incubated with compounds for 1h in the indicated concentrations. Then, compounds-containing media were removed and cells were inoculated with Luc- Jc1 virus-compounds preparations at the same concentrations. Finally, virus-compounds preparations were replaced with fresh medium-compounds preparations and HCV infection efficiency was determined 72h post inoculation using Firefly luciferase assays. Cell viability was measured in parallel using an ATP assay. All data were plotted as percentage relative to D SO for both infectivity and cell viability. Data is expressed as mean values of four measurements of two biological replicates (±SEM).

Figure 2: Grosheimol and cynaropicrin inhibit HCV genotype 2a. Huh7/Scr cells were seeded on 96 well plates, 1.2 x 10⁴ cells/well, 16h prior to infections. The day of infections cells were treated with increasing concentrations of (A) cynaropicrin, (B) grosheimol or (C) total artichoke-extract for 1 h. Then, compounds- containing media were removed and cells were infected with Luc-Jc1 virus-compounds mix at a multiplicity of infection (MOI) of 0.01 TCID₅₀/cell. The tyrosine kinase inhibitor Dasatinib, a known HCV entry inhibitor, served as positive control (D). Virus-compounds mix were replaced 4 h post infection with fresh media-compounds mix and 72 h after infection cells were assayed for Firefly luciferase activity and the mean relative light units (RLU) were plotted as percentage relative to DMSO for both infectivity and cell viability. Half maximal Effective Concentration 50 (EC50) and half maximal Cytotoxic, Concentration 50 (CC50) were estimated by non-linear regression of log inhibitor vs. normalized response and used to calculate the Selectivity Index (SI) value. Data is expressed as mean values of four measurements of two biological replicates (±SEM).

Figure 3: Cynaropicrin inhibits HCVcc and HCVpp cell entry while grosheimol inhibits selectively HCVcc entry. (A) Huh7/Scr cells were seeded on 96 well plates, 1.2 x 10⁴ cells/well, 16h prior to infections. The day of infections, cells were treated with 5 μ of cynaropicrin, grosheimol or dasatinib for 1 h. Then, compounds- containing media were removed and cells were infected with HCV pseudoparticles (HCVpp)-compounds mix at the same concentration. HCVpp were carrying identical glycoproteins (genotype 2a, isolate J6CF) to the Luc-Jc1 viruses. HCVpp-compounds mix were replaced 6 h post infection with fresh media-compounds mix and 72 h after infection cells were assayed for Renilla luciferase activity and the mean relative light units (RLU) were plotted as percentage relative to DMSO for both infectivity and cell viability. (B) Huh7/Scr cells were seeded on 96 well plates, 1.2 x 10 cells/well, 16 h prior to infections. The day of infections, cells were inoculated with Luc-Jc1 reporter viruses prepared in the absence of drugs. Cynaropicrin (10 μΜ) or grosheimol (20 μΜ) were added to the cells only before inoculation (black), were added to viruses and pre-incubated with them at 37 °C prior to inoculation (stripes), or selectively directly after inoculation (white) as schematically depicted at the left. Infectivity was determined 72h later by Firefly luciferase assays and the mean relative light units (RLU) were plotted as percentage relative to DMSO for both infectivity and cell viability. Data is expressed as mean values of four measurements of two biological replicates (±SEM).

Figure 4: Neither grosheimol nor cynaropicrin inhibit HCV translation/replication or particle production and egress. (A) Huh7/Scr cells were transfected by electroporation with the subgenomic Firefly luciferase replicon depicted at the top. Electroporated cells were seeded on 96 well plates at a concentration of 1.2 x 10⁴ cells/well. Compounds were added 4 h post transfection at a final concentration of 5 μΜ and the levels of HCV RNA translation/replication were quantified by firefly luciferase assays 72 h post transfection. A 2'-modified nucleoside analog (2'-C-methyladenosine, 2'-C-Met) (Carroll et al., 2003) was used as a positive control. Data is expressed as mean values of four measurements of two biological replicates (±SEM). (B) Schematic representation of the experimental procedure is depicted at the top. Huh-7/Scr cells were electroporated with Luc-Jc1 RNA and seeded into replicate tissue culture plates. The grosheimol and cynaropicrin compounds and the known HCV particle production inhibitor U0126, were added into the medium at 40 h post electroporation at the given concentrations. Two hours later, cells were washed 3 times with PBS to remove the compounds and fed for 6 h with fresh medium. Finally, at 48 h post electroporation supernatant was harvested and cells were lysed. HCV RNA replication in cells was measured by using a firefly luciferase reporter assay (top panel). The release of infectious particles was determined by inoculation of na^'ive cells with the collected culture fluids and determination of firefly luciferase activity in naive cells 72 h after inoculation (bottom panel). Data is expressed as mean values of four measurements of two biological replicates (±SEM).

The compounds mentioned herein are predicted to have activity against HIV (Human Immune-deficiency virus), HBV (hepatitis B virus), Zika virus, Ebola virus, and Coronaviruses.

Claims

5- Claims

1. A product and/or method for treating hepatitis C virus infection, comprising administering to a subject in need rmulae:

wherein R-ι , R₂, R₃, R4, R₅, Re, R7, Re, R9 and R₁₀ independently can be H, OH, CI, Br, F, I, SR, OR, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aralkyl, aryl, heteroaryl, an amino acid moiety, a peptide moiety, NRR\ C(0)R, COOR, 0(C)OR, methylene group, a carbonyl group, or an epoxy group. The letter R or R^' independently refers to H, alkyl (such as methyl, ethyl, propyl, butyl, pentenyl, isoprenyl, galloyl, or quinic acid moiety), OH, alkanoyl group (ester group such as acetyl or fatty acid ester moiety from C4-C28), cycloalkyl, heterocycloalkyl, aralkyl, aryl, heteroaryl.

2- The products and/or methods also include any of the stereoisomers of any of the compounds in the formulae of claim 1.

3- The method of claim 1 , wherein the sesquiterpene lactones or mono-caffoeylquinic acid derivatives mentioned herein in claims 1 and 2 may contain an exo-methylene double bond and one or more asymmetric centers. Thus, they can occur as racemic mixtures and racemates, individual diastereomers, single enantiomers, diastereomeric mixtures, and trans- or cis-isomeric forms. All such isomeric forms are claimed.

4- The products and/or methods wherein one or more of the compounds in the formulae in claims 1 ,2 and 3 is concurrently administered in combination with one or more of the following therapeutic agents: curcumin, epigallocatechin-3-gallate (EGCG), EGCG-O-tetrastearate, EGCG-O-tetraeicosapentaenoate, EGCG-O- tetradocosahexaenoate, and EGCG-O-octabutyrate, ladanein, BJ486K, sulochrin, monochloro-sulochrin, deoxyfunicone, dihydro-geodin, 3-O-methyl-funicone, lectins such as scytovirin and griffithsin, lactoferrins of human, camel, bovine and sheep, tellimagrandin I , eugeniin, casuarictin, pedunculagin, lamiridosins A and B, nobiletin, rhodisin, epicatechin-3-O-gallate (ECG), 3,3'-digalloylprocyanidin B2, epicatechin, proanthocyanidins with mean degree of polymerization from 3 to 20 such as 3,3'-digalloylproprodelphinidin B2 or rhodisin and 3,3'- digalloylprocyanidin B2, coumarin derivatives 5,5'-bi(6,7-dihydroxycoumarin), daphneticin, daphnetin, and 7- hydroxy-8-methoxycoumarin, polyphenol compounds such as 1 ,2,6-tri-0-galloyl- -D-glucose, 1 ,2,3,6-tetra-O- galloyl-p-D-glucose, and 1 ,2,3,4,6-penta-0-galloyl-p-D-glucose, rutin, bergenin, 1 1 -0-(4-0-methylgalloyl)- bergenin, psammaplin A, embelin, 5-O-methylembelin, rapanone, quercetin, 7,3'-dimethoxyquercetin (D Q), 5,7,3',4'-tetramethoxyquercetin (T Q), 5-OH-3,7-dimethoxyflavone, manoalide, 19-hydroxyspruceanol-19-0-p-D- glucopyranoside, lucidone, mellein, Sch 351633, Sch 68631 , moracin P, moracin O, ursolic acid and oleanolic acid, acetyl ursolic acid, malonyl ursolic acid hemiester, rhododendroglycosides l-lll, wedelolactone, luteolin and apigenin, excoecariphenol D, corilagin, geraniin, chebulagic acid, SCH 644343, SCH 644342, cyclosporine A, sanglifehrins A-D, xanthohumol, artemisinin, parthenolide, costunolide, dehydrocostus lactone, helenalin, and alantolactone, hispitolides A-E, ambrosanolide-B, genkwanine M, naringenin, a-glucosidase such as 1- deoxynojirimycin (DNJ) castanospermine (CAST), celgosivir, N-nonyl deoxynojirimycin, glycyrrhizin, schisandra, silymarin, ascorbic acid, lipoic acid, L-glutathione and a-tocopherol, selenium and N-acetyl-L-cysteine, cynarin or any of its structural- and stereo-isomers, caffeic acid, chlorogenic acid or any of its structural- and stereo-isomers, luteolin, ursodeoxycholic acid (UDCA, ursodiol), tauroursodeoxycholic, glycyrrhizin, glycyrrhizic acid, glycyrrhetic acid, isoliquiritigenin, glycycoumarin, silymarin, silybin or silibinin, silybin A and silybin, isosilybin, silychristin, isosilychristin, taxifolin, 2,3-dehydrosilybin, and silydianin, disuccinate derivative of silybin, emodin and ω- hydroxyemodin, Spirulina platensis, honokiol, flueggrene A and flueggrene B, oxymatrine and matrine, myriberine A, gomisin A, biyouxanthones A and B, shikonin, schizandronic acid, helioxathin, Interferon-a, Interferon Omega, PEG-INTRON, Roferon A, Pegasys, Wellferon, Omniferon, Albuferon-a, Rebif, Rebetron, Symmetrel, Heptazyme, VX-497, Viramidine, and Levovirin), Actimmune (IFN-©), IP-501 , T67, IDN-6556, CellCept, Civacir, boceprivir, sovaprevir, Metisazone, Aciclovir, Idoxuridine, Vidarabine, Ribavirin, Ganciclovir, Famciclovir, Valaciclovir, Cidofovir, Penciclovir, Valganciclovir, Brivudine, Ribavirin, Rimantadine, Tromantadine, Foscarnet, Fosfonet, Saquinavir, Ritonavir, Nelfinavir, Amprenavir, Fosamprenavir, Atazanavir, Tipranavir, Darunavir, Telaprevir, Boceprevir, Faldaprevir, Simeprevir, Asunaprevir, Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir, Tenofovir disoproxil, Adefovir dipivoxil, Alisporivir, Emtricitabine, Telbivudine, Clevudine, Nevirapine, Delavirdine, Efavirenz, Etravirine, Entecavir, Rilpivirine, Zanamivir, Oseltamivir, Moroxydine, Lysozyme, Inosine pranobex, Pleconaril, Enfuvirtide, Raltegravir, araviroc, aribavir, Elvitegravir, Dolutegravir, Umifenovir, Daclatasvir, Sofosbuvir, Zadazin and / or Ceplene.

5- The products and/or methods of claim 1 , wherein one or more of the compounds in the formulae in claims 1 , 2 and 3 include the compounds themselves, as well as their prodrugs and their salts, if applicable. A salt, for example, can be formed between an anion and a positively charged group such as amino, on a sesquiterpene lactone or mono-caffoeylquinic acid derivative. Suitable anions include iodide, bromide, chloride, sulfate, nitrate, phosphate, bisulfate, sulfamate, methanesulfonate, trifluoroacetate, maleate, succinate, citrate, tartrate, fumarate, salicylate, acetate, naphthalenesulfonate, and lactate. Likewise, a salt can also be formed between a cation and a negatively charged group such as carboxylate on a sesquiterpene lactone and/or mono-caffoeylquinic acid derivative or any of their derivatives mentioned in claim 1. Suitable cations include potassium, sodium, calcium, magnesium, and an ammonium cation such as tetramethylammonium ion. Any of the derivatives mentioned in claim 1 also include those salts containing quaternary nitrogen atoms. Examples of prodrugs include esters, methylated derivatives and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active sesquiterpene lactones and/or mono-caffoeylquinic acid derivatives.

6. The method of claims 1-5, wherein the sesquiterpene lactone is

- The method of claims 1-5 wherein the sesquiterpene lactone is

- The method of claims 1-5 wherein the sesquiterpene lactone is

ims 1-5 wherein the sesquiterpene lactone is

- 16-

5. The method of claims 1-5 wherein the compound is

16. The method of claims 1-5, wherein the mono-caffeoylquinic acid derivative is

17. The compounds mentioned in claims 1-16 are also claimed for activity against HIV (Human Immune-deficiency virus), Zika virus, HBV (hepatitis B virus), Ebola virus, and Coronaviruses.