WO2018211148A1

WO2018211148A1 - Bisquinolium derivatives for preventing or treating ebv-related cancers

Info

Publication number: WO2018211148A1
Application number: PCT/EP2018/063410
Authority: WO
Inventors: Marc Blondel; Cécile VOISSET; Maria-José LISTA; Robin Fahraeus; Marie-Paule Teulade-Fichou
Original assignee: Universite De Bretagne Occidentale; Centre Hospitalier Regional Universitaire De Brest; Institut National De La Sante Et De La Recherche Medicale (Inserm); Universite Paris Diderot Paris 7; Universite Paris Descartes; Univ Paris Xiii Paris-Nord Villetaneuse; Centre National De La Recherche Scientifique (Cnrs); Institut Curie; Universite Paris-Sud
Priority date: 2017-05-19
Filing date: 2018-05-22
Publication date: 2018-11-22

Abstract

The present invention relates to bisquinolinium derivatives of formula (I): (I) With Y₁, Y₂, Z₁, Z₂, X ^2-and L as defined in the claims, useful for treating or preventing cancers associated with the Epstein-Barr Virus (EBV-related cancers).

Description

BISQUINOLIUM DERIVATIVES FOR PREVENTING OR TREATING EBV-RELATED

CANCERS

FIELD OF INVENTION

The present invention relates to bisquinolinium derivatives useful for treating or preventing cancers associated with the Epstein-Barr Virus (EBV-related cancers) by interfering with the interaction between the host cell protein nucleolin (NCL) and the virus-encoded EBNA1 mRNA.

TECHNOLOGICAL BACKGROUND

The Epstein-Barr virus (EBV) is the first oncogenic virus discovered in human and has been linked to various cancers that include Burkitt and Hodgkin lymphomas and 10% of gastric cancers. Another example is the nasopharyngeal carcinoma which is particularly frequent among men in China and Tunisia. Altogether, these EBV-related specific cancers represent roughly 1 to 2-3% cancers worldwide. Interestingly, in these cancers, most of tumoral cells are EBV-infected whereas only a small subset of non-tumoral cells are infected. Like all the gammaherpesviruses, EBV evades the host immune system but has an Achilles heel: its genome maintenance protein (GMP) EBNA1 . Indeed, EBNA1 is essential for EBV genome replication and maintenance and as such expressed in all dividing EBV-infected cells. On the other hand, EBNA1 is highly antigenic and CD8+ T cells directed towards EBNA1 epitopes exist in all infected individuals. Hence, EBV has evolved a mechanism to limit EBNA1 production to the minimal level required for the viral genome replication and, at the same time, to minimize the production of EBNA1 -derived antigenic peptides presented to the cytotoxic T cells through the MHC class I pathway. The central glycine-alanine repeat (GAr) of EBNA1 plays a critical role in this mechanism of immune evasion as it is able to self-inhibit the translation of its own mRNA in cis. The high level of EBNA1 protein and the efficient T cell response following the infection by an EBV strain encoding a truncated version of EBNA1 in which GAr has been deleted (EBNAIAGAr) demonstrates the critical role of GAr in EBNA1 immune evasion. Of note, a polymorphism in the length of GAr exists and, importantly, the effect of GAr is length-dependent as a longer domain displays a stronger inhibitory effect on both mRNA translation and antigen presentation.

The GAr-encoding mRNA sequence is GC rich and forms predicted G-quadruplex (G4) structures that have been implicated in the regulation of EBNA1 synthesis in vitro (Murat et al Curr Opin Genet Dev 2014, 25, 22-29). G4 are particular secondary structures of nucleic acid formed by the stacking of G-quartets which correspond to a planar arrangement of 4 guanines connected by Hoogsteen hydrogen bonds. G4 structures within G-rich DNA or RNA sequences have been implicated in gene regulation where they can affect transcription, alternative splicing and translation. G4 modes of action are still relatively unknown but cellular factors that can interact with these structures are emerging.

GAr-based EBNA1 immune evasion is considered a relevant therapeutic target to treat EBV- related cancers as most tumor cells from EBV-related cancers are infected by EBV whereas, in healthy individuals, the latent infection by EBV is primarily restricted to a specific small pool of memory B cells. Hence, overcoming GAr-based self-inhibition of EBNA1 translation should unveil EBV-carrying tumor cells to cytotoxic T cells without having significant effect on the vast majority of healthy host cells.

A yeast-based (Saccharomyces cerevisiae) assay that recapitulates all the aspects of the GAr- based inhibition of translation, including the GAr-length dependency, has been developed, that allowed understanding the mechanisms of GAr-mediated mRNA translation - suppression in cis, as well as the cellular factors involved (Lista et al. Biotechnol J 2015, 10, 1670-1681 ). This assay was successfully used to identify small molecular weight compounds that can stimulate EBNA1 expression both in yeast and mammalian cells and that relieve GAr-based limitation of antigen presentation (Voisset et al. Dis Model Mech 2014, 7, 435-444).

There however remains a need for identifying new therapeutic targets, which would disrupt the GAr-based EBNA1 immune evasion of EBVn when interacted with by therapeutic agents. Such therapeutic agents, able to interact with these new targets, would thus be useful in the treatment and/or prevention of EBV-related cancers.

SUMMARY OF THE INVENTION

Nucleolin (NCL) is a multifunctional DNA/RNA-binding protein widely conserved among eukaryotes. It is involved in RNA metabolism, in particular in rRNA maturation. NCL binds to G-rich sequences in coding and non-coding regions of various mRNA, many of which encode cancer-related proteins, and enhance their translation. In addition, NCL binds to some G4 structures within DNAs and RNAs. For example, it has been recently shown that NCL binds to and stabilizes G4 structures formed within the LTR promoter of HIV, thereby silencing the provirus transcription (Tosoni et al. Nucleic Acids Res 2015, 43, 8884-8897). NCL also affects the transcription of c-MYC by binding to and stabilizing G4 present in the promoter of this oncogene and that negatively regulate its activity.

Based on the yeast assay mentioned above, the Inventors have performed a genetic screen to identify host cell genes involved in the GAr-mediated inhibition of translation. This way, the yeast NSR1 gene encoding the orthologue of human NCL was identified, and it was shown that NCL is critically involved in GAr-based limitation of translation and antigen presentation, and thus in EBNA1 immune evasion.

As a result, the NCL-EBNA1 mRNA interaction appeared as a relevant therapeutic target for the treatment and/or prevention of EBV-related cancers.

The Inventors further identified compounds able to prevent NCL from binding to G4 formed in the GAr mRNA sequence, and to stimulate GAr-limited translation and antigen presentation.

Therefore, in a first aspect, the present invention relates to a compound of formula (I), or a hydrate or a solvate thereof, for use as a drug for preventing and/or treating an Epstein-Barr- Virus (EBV)-related cancer:

wherein

Yi and Y₂ may be identical or different and are each independently CH or NR⁺;

Zi is CH or NR⁺, provided that when Yi is CH, then Zi is NR⁺, and when Yi is NR⁺, then Zi is

CH;

Z₂ is CH or NR⁺, provided that when Y₂ is CH, then Z₂ is NR⁺, and when Y₂ is NR⁺, then Z₂ is CH;

R is Ci-C₆ alkyl, optionally substituted with a OH group or a 0-(Ci-C4)alkyl group,

X^2" is one or a plurality of pharmaceutically acceptable anion(s), selected so as to obtain an overall electrically neutral salt,

L is (A), (Α'), (B), (C), (D) or (E), preferably L is (A), (Α'), (B), (D) or (E):

(Α'),

m, n, p, t, u and v may be identical or different and are each independently an integer selected from 0 to 2;

q, r and s may be identical or different and are each independently an integer selected from 0 to 3;

Ri to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a C₃-C₈ cycloalkyl group, a 0(CrC₆)alkyl group, a NR10R11 group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl or a 5- to 8-membered heterocycloalkenyl, said CrC₆ alkyl group, C₃-C₈ cycloalkyl group, 0(Ci-C₆)alkyl group, C₂-C₆ alkenyl group, C₅-C₈ cycloalkenyl group, 3- to 8-membered heterocycloalkyl or 5- to 8- memebered heterocycloalkenyl being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group;

Ri o and Rn may be identical or different and are each independently:

- a hydrogen atom,

- a d-C-6 alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)R'- group,

- a (Ci-C₆)alkyl-(OCH₂CH)i-(Ci -C6)alkyl-NHC(0)-R', with i an interger between 1 and 6 (preferably 2, 3 or 4);

or NR10R11 , taken together, form a 3- to 8-membered heterocycloalkyl or 5- to 8-membered heterocycloalkenyl;

R' is:

- a (CrC₆)alkyl group optionally substituted with an azido group, a biotinyl group or a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C6)alkyl)₂ group or a N((Ci-C₆)haloalkyl)₂ group,

- a C₂-C₆ alkynyl group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((CrC₆)alkyl)₂ group, a N((CrC₆)haloalkyl)₂ group or a C(0)-(5- to 10-membered)aryl group, or

- a (Ci-C₆)alkyl-(OCH₂CH)j optionally substituted with an azido group or a (CrC₆)alkyl group, wherein j being an interger between 1 and 6, preferably between 2 and 4 and wherein said (CrC₆)alkyl group is optionally substituted with a halogen atom, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((CrC₆)alkyl)₂ group or a N((CrC₆)haloalkyl)₂ group or a biotinyl group. For the purpose of the invention, the term "pharmaceutically acceptable" is intended to mean what is useful to the preparation of a pharmaceutical composition, and what is generally safe and non-toxic, for a pharmaceutical use.

The term "pharmaceutically acceptable salt" is intended to mean, in the framework of the present invention, a salt of a compound which is pharmaceutically acceptable, as defined above, and which possesses the pharmacological activity of the corresponding compound.

In another aspect, the present invention relates to the use of a compound of formula (I) as defined above or below, or a hydrate or a solvate thereof, for the manufacture of a medicament for preventing or treating an EBV-related cancer.

In another aspect, the present invention relates to a method for preventing or treating an EBV- related cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) as defined above or below, or a hydrate or a solvate thereof.

In another aspect, the present invention further concerns a pharmaceutical composition comprising a compound of any of formula (I) as described above, or a hydrate or a solvate thereof, as active substance, and a pharmaceutically acceptable carrier for use as a drug for preventing and/or treating an Epstein-Barr- Virus (EBV)-related cancer.

In another aspect, the present invention relates to a composition comprising:

- as active ingredient, the compound of formula (I) as defined above or below, and optionally another therapeutic agent preferably selected from antibiotics, anticancer agents, steroidal and non-steroidal anti-inflammatory drugs, and

- a pharmaceutically acceptable excipient,

for use as a drug for treating an EBV-related cancer.

In another aspect, the present invention relates to a kit comprising at least:

- a first composition comprising the compound of formula (I) as defined above or below, and a pharmaceutically acceptable excipient, and

- a second composition comprising another therapeutic agent preferably selected from antibiotics, anticancer agents, steroidal and non-steroidal anti-inflammatory drugs, for simultaneous, staggered or sequential use as a combination product for treating an EBV- related cancer. DETAILED DESCRIPTION

Compounds of formula (I)

The compounds of the invention may be prepared by methods known in the art, as described for instance in WO 2009/072027 and De Cian et al. {J. Am. C em. Soc. 2007, 129, 1856- 1857).

In particular, compounds wherein m, n, p, q, r, s, t, u, or v is not 0 may easily be obtained from the corresponding dimethyl oxo derivative, which is easily converted into the dimethyl halogenated derivative (in particular the dimethyl chlorinated derivative), oxidized into the corresponding dicarboxylic acid halogenated (advantageously chlorinated) derivative.

Said derivative may then be subjected to a peptide coupling to form the corresponding bisquinoline halogenated (advantageously chlorinated) derivative. Formation of the corresponding bisquinoilium derivative of the invention takes place under conditions known in the art.

Where appropriate, the bisquinoline halogenated (advantageously chlorinated) derivative is subjected to a cross coupling reaction, in particular a Buchwald Hartwig reaction (for instance with an amine or alcohol), followed by an alkylation reaction to form the corresponding bisquinoilium derivative. In the case of an amino or an alkoxy derivative, simply heating the bisquinoline halogenated (advantageously chlorinated) derivative in the presence of the required amine or alcohol leads to the corresponding amino or alkoxy bisquinoline, which is then subjected to n alkylation reaction to yield the corresponding bisquinoilium derivative. Such a synthetic scheme is illustrated for instance in example 1 .

In a particular embodiment, Yi is CH and Z^ is NR⁺. In another embodiment, Yi is NR⁺ and Z^ is CH.

In a particular embodiment, Y₂ is CH and Z₂ is NR⁺. In another embodiment, Y2 is NR⁺ and Z₂ is CH.

In a particular embodiment, Yi and Z₂ are CH, while Y₂ and Z^ are each independently NR⁺. In another embodiment, Y₂ and Z^ are CH, while Yi and Z₂ are each independently NR⁺.

In a preferred embodiment, Yi and Y₂ are identical, and Z^ and Z₂ are identical. Thus, in this embodiment, Yi and Y₂ are CH, while Z^ and Z₂ are each independently NR⁺. Preferably however, Z^ and Z₂ are CH, while Yi and Y₂ are each independently NR⁺. In this embodiment where Yi and Y₂ are identical, and Zi and Z₂ are identical, the compound of formula (I) may be symmetrical.

R

In a particular embodiment, R is a linear CrC₆ alkyl optionally substituted with a OH group or a linear 0-(Ci -C4)alkyl group. Preferably, R is a linear C1 -C4 alkyl optionally substituted with a OH group or a linear 0-(Ci -C-4)alkyl group. For instance, R is a linear C1 -C4 alkyl optionally substituted with a OH group or a linear 0-(CrC₂)alkyl group.

In some embodiments, R is Ci -C₆ alkyl, preferably a C1 -C4 alkyl. In these embodiments, R is preferably linear. It may be a methyl or an ethyl group.

For instance, R is CH₃, CH₂CH₃, CH₂CH₂OH or CH₂CH₂OCH₃. Preferably, R is CH₃ or CH₂CH₂OH , for instance it is CH₃.

As stated above, X^2" is one or a plurality of pharmaceutically acceptable anion(s), selected so as to obtain an overall electrically neutral (pharmaceutically acceptable) salt.

The nature of the anion may vary, provided that it is pharmaceutically acceptable, and yields a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable anions may be prepared from the corresponding base of an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, hydroxybutyric, salicylic, galactaric and galacturonic acid, the salts of the compounds of formula (I) may be prepared by conventional methods from the corresponding compound by reacting, for example, the appropriate acid, optionally in the form of a metallic or alkaline or alkaline earth base, with any of the compounds of the invention.

Typically, X^2" represents one or a plurality (generally 2) anion (s) with two negative charges. Advantageously, X^2" represents 2 anions selected from the group consisting of a halogenide, a carboxylate, a CrC₆alkylsulfonate, a Ci -C₆haloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate. For instance, X^2" represents 2 anions selected from the group consisting of CI^", Br, I^", and CHF3SO3-, such as I-, and CHF3SO3-.

L

In a particular embodiment:

m, n, p, u and v may be identical or different and are each independently 0, 1 or 2 provided that:

- at least one of m, n and p is not 0;

- at least one of u and v is not 0.

s is 1 , 2 or 3;

t is 1 or 2;

q and r may be identical or different and are each independently an integer selected from 0 to 3, provided that at least one of q and r is not 0.

Preferably, in this embdodiment, Ri to R₉ are each independently a halogen atom or a NR10R11 group;

with R10 as Rn as defined above or below.

R10 and R11 may be identical or different and are each independently:

- a hydrogen atom,

- a C1-C-6 alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group, a NHC(0)-R' group,

- a (Ci-C₆)alkyl-(OCH₂CH)i-(Ci -C6)alkyl-NHC(0)-R' group, with i an interger between 1 and 6 (preferably 2, 3 or 4);

with R' and R" as defined above or below.

For example, R10 is H and Rn is:

- a C1-C-6 alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group (such as a OCH₃ group), a NH-(CrC₆)alkyl group, a NHC(0)-R' group; or

- a (Ci-C₆)alkyl-(OCH₂CH)i-(Ci -C6)alkyl-NHC(0)-R', with i an interger between 1 and 6 (preferably 2, 3 or 4).

Advantageously, when Rn is a CrC₆ alkyl group substituted with a NHC(0)-R' group, R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a Ci -

C₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci-C₆)alkyl)₂ group or a N((CrC₆)haloalkyl)₂ group,

- a C₂-C₆ alkynyl group,

- a (Ci-C₆)alkyl-(OCH₂CH)j optionally substituted with an azido group or a (CrC₆)alkyl group, wherein j being an interger between 1 and 6, preferably between 2 and 4 and wherein said (CrC₆)alkyl group is optionally substituted with a halogen atom, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C6)alkyl)2 group or a N((Ci -C₆)haloalkyl)2 group or a biotinyl group, preferably substituted with a a halogen atom or a 0(CrC₆)alkyl group;

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C₆)alkyl)₂ group, a N((Ci -C₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group;

Advantageously, when Rn is a a (Ci -C₆)alkyl-(OCH₂CH)i-(Ci -C6)alkyl-NHC(0)-R' group, R' is a (CrC₆)alkyl group optionally substituted with an azido group, a C₂-C₆ alkynyl group, a biotinyl group or a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8- membered heterocycloalkenyl, a N((CrC₆)alkyl)₂ group or a N((CrC₆)haloalkyl)₂ group, preferably R' is a (CrC₆)alkyl group optionally substituted with a biotinyl group.

For instance, Ri o is H and Rn is:

- a (CrC₆)alkyl (preferably a CH₂CH₂CH₂CH₂- group) optionally substituted with a NHC(0)R', with R' as defined above or below and preferably representing:

- a (C₂-C₆)alkynyl group; a (CrC₆)alkyl group optionally substituted with an azido group, a biotinyl group; a 5- to 10-membered aryl group substituted with a N((Cr

C₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group; a (Ci -C₆)alkyl-(OCH₂CH₂)j substituted with an azido group; or

- a CH₂CH₂CH₂-(OCH₂CH)₃-CH₂CH₂- group substituted with a NHC(0)R', with R' as defined above or below and representing preferably a (C₂-C₆)alkynyl group; a (Ci - C₆)alkyl group optionally substituted with an azido group, a biotinyl group; a 5- to 10- membered aryl group substituted with a N((CrC₆)haloalkyl)₂ group or a C(0)-(5- to 10- )aryl group.

In a specific embodiment, L is (A), (Α'), (B), (D) or (E).

In a particular embodiment, L is (D) or (E). In another particular embodiment, L is (A), (Α'), (B) or (C) .

m, n, p, t, u and v may be identical or different and are each independently an integer selected from 0 to 2. For instance, m, n, p, t, u and v may be identical or different and are each independently are independently 0 or 1 .

q, r and s may be identical or different and are each independently an integer selected from 0 to 3. For instance, q, r and s may be identical or different and are each independently 0, 1 , or 2. Preferably, q, r and s may be identical or different and are each independently 0 or 1 . In Ri to Ri i as defined above or bleow, each CrC₆ alkyl group, C₂-C₆ alkenyl group, C₂-C₆ alkynyl group, 0(Ci -C₆)alkyl, NH-(Ci -C₆)alkyl group, N( (Ci -C₆)alkyl)₂ group or a N( (Ci - C₆)haloalkyl)₂ group is preferably linear.

Advantageously, in all embodiments, in Ri to Rn and R', each CrC₆ alkyl group, C₂-C₆ alkenyl group, C₂-C₆ alkynyl group, 0(CrC₆)alkyl, NH-(CrC₆)alkyl group, N( (CrC₆)alkyl)₂ group or a N( (CrC₆)haloalkyl)₂ group, is a (preferably linear) Ci -C₄ alkyl group, C₂-C4 alkenyl group, C₂- C₄ alkynyl group, 0(CrC₄)alkyl, NH-(Ci -C₄)alkyl group, N( (CrC₄)alkyl)₂ group or a N( (Ci - C₄)haloalkyl)₂ group.

Further advantageously, in all embodiments, in Ri to Rn and R', the 5- to 10-membered aryl group is a 6- to 10-membered aryl group (such as a phenyl, a pyridyl, or a naphthyl group), even more preferably it is a 6- to 10-membered aromatic group, preferably a phenyl group. Further advantageously, in all embodiments, in Ri to Rn and R', the C₃-C₈ cycloalkyl group is a C₃-C₆ cycloalkyl group, such as a cyclopropyl, a cyclobutyl, a cyclopentyl or a cyclohexyl group.

Further advantageously, in all embodiments, in Ri to Rn and R', the C₅-C₈ cycloalkenyl group is a C₅-C₆ cycloalkyl group, such as a a cyclopentenyl or a cyclohexenyl group.

Further advantageously, in all embodiments, in Ri to Rn and R', the 3- to 8-membered heterocycloalkyi group is a 3- to 6-membered heterocycloalkyi, such as a.

Further advantageously, in all embodiments, in Ri to Rn and R', the 5- to 8-membered heterocycloalkenyl group is a 5- to 6-membered heterocycloalkenyl, such as a dihydrofuran, a dihydrothiophene, a pyrrolin, a tetrahydropyrindine, a dihyrothiapyran.

In a particular embodiment, Ri to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a NRi ₀Rn group, or a 3- to 8- membered heterocycloalkyi said CrC₆ alkyl group, 0(CrC₆)alkyl group and 3- to 8-membered heterocycloalkyi being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group,

Ri o and Rn may be identical or different and are each independently a hydrogen atom or a Ci - C-6 alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group,

or NR10R11 , taken together, form a 3- to 8-membered heterocycloalkyi,

and R' is as defined above or below.

In some embodiments, R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a Ci - C₆ alkyl group, a 0(CrC₆)alkyl group, a 3- to 8-membered heterocycloalkyl, a N((Ci-C₆)alkyl)₂ group or a N((Ci-C₆)haloalkyl)2 group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a 3- to 8-membered heterocycloalkyl, a N((Ci-C₆)alkyl)₂ group, a N((Ci-C₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group.

In a particular embodiment, Ri to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a NRi ₀Rn group, said CrC₆ alkyl group, 0(CrC₆)alkyl group, being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' ;

Ri o and Rn may be identical or different and are each independently a hydrogen atom or a Ci - C₆ alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group;

R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a Ci - C-6 alkyl group, a 0(CrC₆)alkyl group, a N((Ci -C6)alkyl)2 group or a N((Ci-C₆)haloalkyl)2 group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a N((Ci-C₆)alkyl)₂ group, a N((Ci-C₆)haloalkyl)₂ group or a

C(0)-(5- to 10-)aryl group.

Preferably, Ri to R₉ may be identical or different and are each independently a halogen atom, a NH(CrC₆)alkyl group or a NH-(CrC₆)alkyl-NHC(0)-R' group, with R' as defined above or below.

In particular, R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a N((Ci-C₆)alkyl)₂ or N((CrC₆)haloalkyl)₂, or

- a 5- to 10-membered aryl group optionally substituted with a C(0)-(5- to 10-)aryl group;

such as the In some embodiment, L is (A) or (Α'), preferably (A). In this embodiment, Ri , R₂ and R₃ are as defined above or below, and preferably m, n and p are identical or different and are each independently 0 or 1 . More preferably:

- m and p are 0, and n is 0 or 1 , or

- m and n are 0, and p is 0 or 1 , or

- n is 0, and m and p are identical and are 0 or 1 .

In a specific embodiment, L is (A) or (Α'), preferably (A), and m and p are 0, n is 0 or 1 , and R₂ is as defined above, preferably a halogen atom such as F or Br.

In another specific embodiment, L is (A) or (Α'), preferably (A), and m and n are 0 and R₃ is as defined above, preferably a linear NH(C -(Ci-C₆)alkyl-NHC(0)-R'

group, with R' as defined above, preferably

In another specific embodiment, L is (A) or (Α'), preferably (A), and n is 0, and m and p are identical or different and are each independently preferably 0 or 1 . Ri and R₃ may be identical or different and are then each independently as defined above, preferably a halogen atom, a NH(CrC₆)alkyl group, more preferably a NH(CrC4)alkyl group.

In some embodiments, L is (B) or (C). In these embodiments, q, r and s as well as R₄, R₅ and R₆ are as defined above.

In a particular emdodiment, L is (C) and s is preferably 0 or 1 . In a particular emdodiment, L is (C) and s is 0. In another emdodiment, L is (C) and s is 1 . In this emdodiment, R₆ is preferably a halogen atom or a NH(CrC₆)alkyl group or a NH-(Ci-C₆)alkyl-NHC(0)-R' group, with R' being:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a N(Ci-C₆alkyl)₂ or N(CrC₆haloalkyl)₂, or

- a 5- to 10-membered aryl group optionally substituted with a C(0)-(5- to 10-)aryl group.

In another particular embodiment, L is (B), and q and r are preferably each independently 0 or 1 . In a particular embodiment, a and r are 0. In another emdodiment, q is 0 and r is 1 , or q is 1 and r is 0, or q and r are 1 . In these latter emdodiments, R4 and R₅ are idneitcal or different and are preferably each independently a halogen atom or a NH(CrC₆)alkyl group or a NH-(Ci- C₆)alkyl-NHC(0)-R' group, with R' being: - a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a N(Ci -C₆alkyl)₂ or N(Ci -C₆haloalkyl)₂, or

Combinations

In a particular embodiment, the compound of formula (I) is characterized in that:

Zi is CH or NR⁺, provided that when Yi is CH , then Z^ is NR⁺, and when Yi is NR⁺, then Z^ is CH ;

Z₂ is CH or NR⁺, provided that when Y₂ is CH , then Z₂ is NR⁺, and when Y₂ is NR⁺, then Z₂ is CH ;

R is Ci -C₆ alkyl, optionally substituted with a OH group or a 0-(Ci -C4)alkyl group,

L is (A), (Α'), (B), (C) , (D) or (E), preferably (A), (B), (C), (D) or (E) or (A), (Α') (B), (D) or (E), more preferably (A), (B), (D) or (E);

Ri to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a C₃-C₈ cycloalkyl group, a 0(CrC₆)alkyl group, a NRi ₀Rn group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl or a 5- to 8-membered heterocycloalkenyl, said CrC₆ alkyl group, C₃-C₈ cycloalkyl group, 0(Ci -C₆)alkyl group, C₂-C₆ alkenyl group, C₅-C₈ cycloalkenyl group, 3- to 8-membered heterocycloalkyl or 5- to 8- memebered heterocycloalkenyl being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group;

Ri o and Rn may be identical or different and are each independently:

- a hydrogen atom,

- a Ci-C₆ alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group, a

NH-(CrC₆)alkyl group or a NHC(0)R' group,

or NR10R11 , taken together, form a 3- to 8-membered heterocycloalkyi or 5- to 8-membered heterocycloalkenyl;

R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a Ci - C-6 alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a C₂- C-6 alkynyl group, a 3- to 8-membered heterocycloalkyi, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C6)alkyl)2 group or a N((Ci-C₆)haloalkyl)2 group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8- membered heterocycloalkyi, a 5- to 8-membered heterocycloalkenyl, a N((Ci-C₆)alkyl)₂group, a N((Ci-C₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group. In a particular embodiment, the compound of formula (I) is characterized in that:

R is a C1-C-6 alkyl, preferably a methyl,

X^2" is one or a plurality of pharmaceutically acceptable anion(s), selected so as to obtain an overall electrically neutral salt, preferably selected from the group consisting of a halogenide, a carboxylate, a CrC₆alkylsulfonate, a Ci-C₆haloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate

L is (A), (Α'), (B), (C), (D) or (E), preferably (A), (B), (C), (D) or (E) or (A), (Α') (B), (D) or (E), more preferably (A), (B), (D) or (E);

Ri to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a NR10R11 group, or a 3- to 8-membered heterocycloalkyi said C1-C-6 alkyl group, 0(CrC₆)alkyl group and 3- to 8-membered heterocycloalkyi being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group; Ri o and Rn may be identical or different and are each independently a hydrogen atom or a Ci - C₆ alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group,

or NR10R11 , taken together, form a 3- to 8-membered heterocycloalkyi;

R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a Ci - C-6 alkyl group, a 0(CrC₆)alkyl group, a 3- to 8-membered heterocycloalkyi, a N((Ci-C₆)alkyl)₂ group or a N((Ci-C₆)haloalkyl)₂ group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a 3- to 8-membered heterocycloalkyi, a N((Ci-C₆)alkyl)₂ group, a N((Ci-C₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group.

In yet another particular embodiment, the compound of formula (I) is characterized in that: R is C1-C-6 alkyl, optionally substituted with a OH group or a 0-(Ci-C-4)alkyl group,

X^2" is one or a plurality of pharmaceutically acceptable anion(s), selected so as to obtain an overall electrically neutral salt, preferably selected from the group consisting of a halogenide, a carboxylate, a CrC₆alkylsulfonate, a Ci-C₆haloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate L is preferably (A), (Α'), (B), (D) or (E), more preferably (A), (B), (D) or (E);

Ri and R₃ may be identical or different and are each independently a CrC₆ alkyl group, a 0(Ci - C₆)alkyl group, a NR10R11 group, or a 3- to 8-membered heterocycloalkyi said CrC₆ alkyl group, 0(CrC₆)alkyl group and 3- to 8-membered heterocycloalkyi being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group;

R₂ and R4 to R₉ may be identical or different and are each independently a halogen atom, a C1-C-6 alkyl group, a 0(CrC₆)alkyl group, a NR10R11 group, or a 3- to 8-membered heterocycloalkyi said CrC₆ alkyl group, 0(CrC₆)alkyl group and 3- to 8-membered heterocycloalkyi being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group;

R10 and Rn may be identical or different and are each independently:

- a hydrogen atom, provided that when R10 is H, Rn is not H, and when Rn is H, R10 is not H, or - a C4-C6 alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group or a NHC(0)R' group, or

- a (Ci-C₆)alkyl-(OCH₂CH)i-(Ci -C6)alkyl-NHC(0)-R', with i an integer between 1 and 6 (preferably 2, 3 or 4);

Ft' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a Ci - C₆ alkyl group, a 0(CrC₆)alkyl group, a 3- to 8-membered heterocycloalkyl, a N((Ci-C₆)alkyl)₂ group or a N((Ci-C₆)haloalkyl)₂ group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a 3- to 8-membered heterocycloalkyl, a N((Ci-C₆)alkyl)₂ group, a N((CrC₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group.

In yet another particular embodiment, the compound of formula (I) is characterized in that: R is Ci-C₆ alkyl, optionally substituted with a OH group or a 0-(Ci-C-4)alkyl group,

Ri is a hydrogen atom;

R₂ to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a NR10R11 group, or a 3- to 8-membered heterocycloalkyl said C1-C-6 alkyl group, 0(CrC₆)alkyl group and 3- to 8-membered heterocycloalkyl being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' group;

- a (Ci-C₆)alkyl-(OCH₂CH)i-(Ci-C₆)alkyl-NHC(0)-R', with i an integer between 1 and 6 (preferably 2, 3 or 4).

Advantageously, when Rn is a CrC₆ alkyl group substituted with a NHC(0)-R' group, R' is: - a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a Ci - C₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C₆)alkyl)₂ group or a N((Ci -C₆)haloalkyl)₂ group,

- a C₂-C₆ alkynyl group,

- a (Ci -C₆)alkyl— (OCH₂CH)j optionally substituted with an azido group or a (CrC₆)alkyl group, wherein j being an interger between 1 and 6, preferably between 2 and 4 and wherein said (CrC₆)alkyl group is optionally substituted with a halogen atom, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C6)alkyl)₂ group or a N((Ci -C₆)haloalkyl)₂ group or a biotinyl group, preferably substituted with a halogen atom or a 0(CrC₆)alkyl group;

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((CrC₆)alkyl)₂ group, a N((CrC₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group;

In yet another particular embodiment, the compound of formula (I) is characterized in that: R is a linear Ci -C₄ alkyl, optionally substituted with one OH , preferably ethyl or methyl or CH₂CH₂OH , more preferably methyl,

X^2" is one or a plurality of pharmaceutically acceptable anion(s), selected so as to obtain an overall electrically neutral salt, preferably selected from the group consisting of a halogenide, a carboxylate, a CrC₆alkylsulfonate, a Ci -C₆haloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate, L is (A), (Α'), (B), or (C) , preferably L is (A), (Α') or (B) or (A) or (B);

m, n, and p, may be identical or different and are each independently 0 or 1 ;

q, r and s may be identical or different and are each independently 0 or 1 ;

Ri to R₉ may be identical or different and are each independently a halogen atom, a NH(Ci -

C₆)alkyl group or a NH-(Ci -C₆)alkyl-NHC(0)-R' group;

R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl (preferably aromtic) group (such as a phenyl group), wherein said 5- to 10-membered aryl group is optionally substituted with a N(CrC₆alkyl)₂ or N(CrC₆haloalkyl)₂, or

- a 5- to 10-membered aryl group optionally substituted with a C(0)-(5- to 10-)aryl (preferably aromtic) group (such as a phenyl group).

In yet another particular embodiment, the compound of formula (I) is characterized in that: R is a linear Ci-C₄ alkyl, preferably ethyl or methyl, more preferably methyl,

X^2" is one or a plurality of pharmaceutically acceptable anion(s), selected so as to obtain an overall electrically neutral salt, preferably selected from the group consisting of a halogenide, a carboxylate, a CrC₆alkylsulfonate, a Ci-C₆haloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate, L is (Α), (Α'), (B), or (C);

m, n, and p, may be identical or different and are each independently 0 or 1 ;

q, r and s may be identical or different and are each independently 0 or 1 ;

Ri to R₉ may be identical or different and are each independently a halogen atom, a NH(Ci- C₆)alkyl group or a NH-(Ci-C₆)alkyl-NHC(0)-R' group;

R' is:

- a (CrC₆)alkyl group optionally substituted with a 5- to 10-membered aryl (preferably aromatic) group (such as a phenyl group), wherein said 5- to 10-membered aryl group is optionally substituted with a N(Ci-C₆alkyl)₂ or N(Ci-C₆haloalkyl)₂, or

- a 5- to 10-membered aryl group optionally substituted with a C(0)-(5- to 10-)aryl

(preferably aromtic) group (such as a phenyl group).

Any combination of particular, advantageous or preferred embodiment is encompassed by the present invention.

with X^2" as defined above, especially representing 2 anions selected from the group consisting of a halogenide, a carboxylate, a Ci-C₆alkylsulfonate, a Ci-C₆haloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate foror instance, 2 anions selected from the group consisting of CI^", Br, I^", and CHF3SO3-, such as I-, and CHF3SO3-.

Preferably, the compound of formula (I) is

(PhenDC6) X^2",

(BipyDC6) X^2",

with X^2" as defined above, especially representing 2 anions selected from the group consisting of a halogenide, a carboxylate, a Ci-C₆alkylsulfonate, a Ci- Cehaloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate foror instance, 2 anions selected from the group consisting of CI^", Br, I^", and CHF3SO3^", such as I^", and CHF3SO3^". Advantageously, the compound of the invention is (Br-PhenDC6) X^2", (PhenDC3-C4-C) X^2", (PhenDC3-C4-Bn) X² , or (PhenDC3-Bisalk1 ) X^2", with X^2" as defined above, especially representing 2 anions selected from I^", and CHF3SO3^".

particular embodiment, the compound of form

representing 2 anions selected from I^", and CHF3SO3^". In another particular embodiment, the

X^2" as defined above, especially representing 2 anions selected from such as I^", and CHF₃S0₃ ^" Miscellaneous

The compounds of formula (I) as described above may exist in tautomeric, diastereomeric or enantiomeric forms. The present invention contemplates all such compounds, including cis- and trans-diastereomers, E- and Z-stereomers, R- and S-enantiomers, diastereomers, d- isomers, l-isomers, the racemic mixtures thereof and other mixtures thereof. Pharmaceutically acceptable salts of such tautomeric, diastereomeric or enantiomeric forms are also included within the invention. The terms "cis" and "trans", as used herein, denote a form of geometric isomerism in which two carbon atoms connected by a double bond will each have a hydrogen atom on the same side of the double bond ("cis") or on opposite sides of the double bond ("trans"). Some of the compounds described contain alkenyl groups, and are meant to include both cis and trans or "E" and "Z" geometric forms. Furthermore, where the compounds described contain one or more stereocentersn the present invention includes R, S, and mixtures of R and S forms for each stereocenter present.

The present invention encompasses only stable compounds.

Pharmaceutical compositions

The pharmaceutically acceptable excipient is selected, according to the dosage form and mode of administration desired, from the typical excipients known to persons skilled in the art. The pharmaceutical compositions according to the invention can be administered parenterally (such as intravenously or intradermally), topically, orally or rectally.

The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intravesical or infusion techniques. Preferably, the term "parenteral" refers to infusion techniques.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.

For therapeutic purposes, formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions.

Preferably, the compositions of the invention are administered via oral route.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compound is ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents such as sodium citrate, or magnesium or calcium carbonate or bicarbonate. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

In order to selectively control the release of the active compound to a particular region of the gastrointestinal tract, the pharmaceutical compositions of the invention may be manufactured into one or several dosage forms for the controlled, sustained or timed release of one or more of the ingredients, as known in the art.

The amount of the compound of the invention that may be combined with the excipient materials to produce a single dosage of the composition will vary depending upon the subject and the particular mode of administration, as known in the art.

To improve the solubility of the compounds of the invention into aqueous solutions, and in particular into body fluids, said compounds may be formulated as cyclodextrine inclusion complexes, in particular as inclusion complexes with α-, β- or γ-cyclodextrins.

In a particular embodiment, the pharmaceutical composition of the invention further comprises another one or more therapeutic compounds. Another aspect of the present invention encompasses a combination of a compound of formula (I) as described above, with one or more therapeutic compounds.

The therapeutic compound is preferably selected from antibiotics, anticancer agents, steroidal and non-steroidal anti-inflammatory drugs, advantageously it is an anticancer agent.

The antibiotic is preferably selected from the group consisting of beta-lactams, aminoglycosides, tetracyclines, glycylcyclines, macrolides, azalides, ketolides, synergistins, lincosanides, fluoroquinolones, phenicols, rifamycins, sulfamides, trimethoprim, glycopeptides, oxazolidinones, nitromidazoles and lipopeptides.

The non-steroidal anti-inflammatory drug is preferably selected from the group consisting of salicylate and salts thereof, Celecoxib, Diclofenac and salts thereof, Diflunisal, Etodolac, Fenoprofen, Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen, Meclofenamate, Mefenamic acid, Meloxicam, Nabumetone, Naproxen, Oxaprozin, Piroxicam, Rofecoxib Salsalate, Sulindac, Tolmetin, and Valdecoxib.

The steroidal anti-inflammatory drug is preferably selected from the group consisting of Prednisone, Methylprednisolone, Prednisolone, aldosterone, Cortisol, cortisone, hydrocortisone, corticosterone, tixocortol, ciclesonide, prednicarbate Triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, halcinonide, Hydrocortisone- 17-valerate, halometasone, alclometasone, betamethasone, prednicarbate, clobetasone-17-butyrate, clobetasol-17- propionate, fluocortolone, fluocortolone, fluprednidene acetate, dexamethasone, and mixtures thereof, and the corresponding salts or hydrates thereof.

The anticancer agent is preferably cisplatine, methotrexate, cyclophosphamide, doxorubicin, fluorouracil.

In some embodiments, the combination comprises 1 , 2, 3, 4, or 5 therapeutic compounds, preferably one therapeutic compound.

In another aspect, the present invention relates to a kit comprising at least:

- a second composition comprising another therapeutic agent, preferably selected from antibiotics, anticancer agents, steroidal and non-steroidal anti-inflammatory drugs, advantageously an anticancer agent.

for simultaneous, staggered or sequential use. The kit of the invention is used as a combination product for treating an EBV-related cancer. The antibiotics, anticancer agent, steroidal and non-steroidal anti-inflammatory drug is in particular as listed above in connection with the pharmaceutical composition for use of the invention.

The other therapeutic compound is preferably selected from antibiotics, anticancer agents, steroidal and non-steroidal anti-inflammatory drugs. More preferably, it is an anticancer agent.

Therapeutic use

The compound or the composition or the kit of the invention is useful as a drug, in particular for preventing and/or treating an EBV-related cancer.

As used herein, an "EBV-related cancer" is understood as a cancer which develops after and/or triggered by infection by the Epstein-Barr virus, which distinghuishes them from cancers in general. In particular, an "EBV-related cancer" is understood as a cancer wherein more than 50%, typically more than 90%, in particular more than 95% of tumoral cells are infected by EBV, whereas most non tumoral cells are advantageously not infected by EBV. Typically, only some B-cells (memory cells) are also infected by EBV in an EBV-related cancer. Such a profile of infection by EBV explains the specificity of the compounds of the invention in e method for treating EBV-related cancers.

The prior art (E. Largy et al., Methods, vol 57, n°1 , 2012, pp129-137; Matjaz et al., Nucleic Acids Research, vol 43, n°21 , 2015, pp10376-10386; Assitan et al., Biochimie, vol 94, n°12, pp2559-2568, 2012; Verga D et al., Angewandte Chemie, Vol 53, n°4, 2014, pp994-998; Larsen et al., Chemistry, vol 18, n°35, 2012, pp10892-10902) discloses comounds with G- quadruplex ligands. However, if G-quadruplex ligands are known to be useful in the treatment of certain cancers, they are not known to be active in the treatment of EBV-related cancer. Indeed, the compounds of the invention act through a new mode of action: they prevent NCL from binding to G4 formed in the GAr mRNA sequence, thus stimulating GAr-limited translation and antigen presentation. In other words, the compounds of the invention, by rendering EBV "visible", stimulate the host immune system, which will then "attack" the EBV-infected tumoral cells, thus leading to tumoral cell death, and overall treatment of cancer.

Therefore, in a particular embodiment, the compound or the composition or the kit of the invention is used in combination, simultaneously, separately or sequentially, with ionizing or non-ionizing radiations or hyperthermia.

In particular, said EBV-related cancer is: Hodgkin's lymphoma, Burkitt's lymphoma, Nasopharyngeal carcinoma, some gastric cancers (about 10% are related to EBV infection), lymphomas in immunosuppressed patients (such as AIDS-suffering patients, post-transplant patients), T/NK cell lymphomas (such as nasal T/NK lymphoma, aggressive NK-cell leukaemia, T cell lymphoproliferative disorder of childhood).

The "effective dose" of a compound of the invention varies as a function of numerous parameters such as, for example, the route of administration and the weight, the age, the sex, the advancement of the pathology to be treated and the sensitivity of patient to be treated.

As used herein, "patient" includes any mammal, and is preferably a human being.

DEFINITIONS

The term "halogen", as used in the present invention, refers to a fluorine, bromine, chlorine or iodine atom, preferably a chlorine, bromine or fluorine atom.

The term "azido", as used in the present invention, refers to a -N₃ group.

The term "biotinyl group", as used in the present invention, refers to the following group:

The term "(d-Ce lkyl", as used in the present invention, refers to a straight or branched saturated hydrocarbon chain containing from 1 to 6 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

The term "(Ci-C6)haloalkyl", as used in the present invention, refers to a straight or branched saturated hydrocarbon chain containing from 1 to 6 carbon atoms substituted with halogen atoms, such as chlorine, bromine, iodine or fluorine atoms, preferably chlorine or fluorine atoms. Examples of (Ci-C₆)haloalkyl include, but are not limited to, CH₂CI, CH₂Br, CH₂I, CH₂F, CHF₂, CF₃, CH₂CH₂CI, CH₂CH₂BR, CH₂CH₂I, CH₂CH₂F, and the like.

The term "(Cp-CB)alkenyl", as used in the present invention, refers to a straight or branched unsaturated hydrocarbon chain containing from 2 to 6 carbon atoms and comprising at least one double bond including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.

The term "(Cp-CB)alkynyl", as used in the present invention, refers to a straight or branched unsaturated hydrocarbon chain containing from 2 to 6 carbon atoms and comprising at least one triple bond, preferably comprising only one unsaturation (i.e.a triple bond), including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like. The term "(Cs-Cstevcloalkyl", as used in the present invention, refers to a hydrocarbon monocyclic or bicyclic (fused) ring having 3 to 8 carbon atoms including, but not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like.

The term "(C₅-Cio)cvcloalkenyl", as used in the present invention, refers to a hydrocarbon monocyclic or bicyclic (fused) ring having 5 to 10 carbon atoms and comprising at least one double bond including, but not limited to, cyclopentenyl, cyclohexenyl and the like.

The term "heterocvcloalkyl", as used in the present invention, refers to a hydrocarbon monocyclic or bicyclic (fused) ring having 3 to 8 ring atoms, containing at least one heteroatom, preferably 1 or 2 heteratoms, in the ring. The heteroatom is preferably selected from O, N or S, and the S atom may be mono or dioxidized, i.e. the sulphur atom may be S, S(O) or S0₂. heterocycloalkyls include, but are not limited to, epoxide, aziridine, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl.

The term "heterocvcloalkenyl", as used in the present invention, refers to a hydrocarbon monocyclic or bicyclic (fused) ring having 5 to 8 ring atoms, containing at least one heteroatom, preferably 1 or 2 heteratoms, in the ring, and comprising at least one double bond. The heteroatom is preferably selected from O, N or S, and the S atom may be mono or dioxidized, i.e. the sulphur atom may be S, S(O) or S0₂. heterocycloalkenyls include, but are not limited to, pyrrolyl, dihydrofuranyl, dihydrothiophenyl, dihydropyranyl, tetrahydropyridinyl, dihydrooxazinyl, oxindolyl, benzothiazinyl, benzothiazinonyl, phthalimidyl, indolinyle, isoindolinyle.

As used herein, an "aryl group" may be an aromatic or heteroaromatic group.

The term "aromatic group" as used herein alone or as part of another group denotes optionally substituted homocyclic aromatic groups, preferably monocyclic or bicyclic (fused) groups, containing from 6 to 10 carbons in the ring portion, such as phenyl, naphthyl and indenyle. Phenyl and naphthyl are the most preferred aromatic groups.

The term "heteroaromatic" as used herein alone or as part of another group denotes optionally substituted 5- to 10-membered aromatic groups having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring. The heteroaromatic group preferably has 1 to 3 heteroatoms preferably selected from O, N and S in the ring, and may be bonded to the remainder of the molecule through a carbon or heteroatom. Exemplary heteroaromatics include furyl, thiophenyl, pyrrolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, triazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, indolyl, isoxindolyl, chromene-2-onyle (or coumarinyl), benzoxazolyl, benzothiazolyl, benzotriazolyl, quinolinyl, or isoquinolinyl and the like. Preferably, the heteroaromatic group is selected from a pyrrolyl, thiophenyl, isoxazolyl, triazolyl, oxazolyl, thiazolyl, benzothiazolyl, benzotriazolyl, pyrindinyl and pyrazinyl, in particular pyrrolyl, isoxazolyl,1 ,3-oxazolyl, 1 ,3-thiazolyl, 1 ,2,3-triazolyl, benzothiazolyl, benzotriazolyl, pyrindinyl and pyrazinyl.

As used herein, the term "alkylaryl" refers to a (Ci-C₆)alkyl-aryl group. Preferably, the alkylaryl group is a (Ci-C₆)alkyl-aromatic group such as a benzyl group.

Also, in the present invention, Me stands for methyl and Ph stands for phenyl. More generally, the abbreviations used to refer to chemical groups have the meaning commonly known in the art.

DESCRIPTION OF THE FIGURES

Figure 1 : Identification and confirmation of the critical role of nucleolin in GAr-based translation inhibition in yeast.

(a) Rationale of the yeast-based genetic screen. Contrary to yeast cells expressing ADE2gere (left panel) that forms white colonies on rich medium (YPD), those expressing the 43GAr-ADE2 fusion (right panel) form pink colonies due to the ability of GAr to self-limit the translation of its own mRNA in yeast as in human cells. Based on this model, we looked for yeast genes whose overexpression from high copy number plasmids leads to a redder phenotype, suggesting an exacerbation of the GAr-based translation inhibition.

(b) Effect of NSR1 overexpression on 43GAr-Ade2p protein level. The overexpression of NSR1 gene, which encodes the yeast nucleolin, led to a redder phenotype associated to a decrease in 43GAr-Ade2p level as evidenced by SDS-PAGE and western blot analysis (left panel). GAPDH was used as a loading control. The mean 43GAr-Ade2p/GAPDH ratio from 3 independent experiments is shown in the right panel and the results compared using the Student's t-test. ^***/x0.001 .

(c) Effect of NSR1 gene deletion on 43GAr-Ade2p and Ade2p protein level. SDS-PAGE and western blot analysis showing that deletion of NSR1 (nsrIA) had no effect on Ade2p level, whereas it strongly increased the level of 43GAr-Ade2p. GAPDH was used as a loading control. The mean 43GAr-Ade2p/GAPDH or Ade2p/GAPDH ratios from 3 independent experiments are shown in the right panel and the results compared using the Student's t-test. (^***/x0.001 ; ns: not significant).

(d)Human nucleolin (NCL) is able to complement NSR1 deletion. SDS-PAGE and western blot analysis of the yeast nsrIA strain expressing 43GAr-Ade2p (left) or Ade2p (right) and overexpressing (right lanes), or not (left lanes), HA-tagged human nucleolin (HA-NCL). GAPDH was used as a loading control. The 43GAr-Ade2p/GAPDH or Ade2p/GAPDH ratio are indicated below the gels. Blots represent n≥3 Figure 2: Overexpression of NCL exacerbates GAr effect on protein expression whereas its downregulation reduces its inhibitory effect on translation in mammalian cells.

(a) SDS-PAGE and western blot analysis of the level of endogenous EBNA1 in the EBV- infected B cell lineMutu-1 overexpressing (right lane), or not (left lane), HA-tagged nucleolin (HA-NCL). Actin was used as a loading control. EBNA1 /actin ratios are indicated below the gels. Blot represents n≥3.

(b) SDS-PAGE and western blot analysis of the level of endogenous EBNA1 in the Mutu- 1 cells in response to NCL knockdown. Mutu-1 cells were transfected with control siRNA or siPtNA targeting NCL, as indicated. GAPDH was used as a loading control. EBNA1 /GAPDH ratios are indicated below the gels. The efficiency of NCL downregulation was estimated by determining the NCL/GAPDH ratio in cells treated by siRNA targeting NCL versus cells treated by control siRNA. The mean EBNA1 /GAPDH ratio from 3 independent experiments is shown in the right panel and the results obtained with siRNA targeting NCL compared with control siRNA using the Student's f-test (^**p<0.01 ).

(c) SDS-PAGE and western blot analysis of the level of EBNA1 or EBNAIAGAr in response to NCL knockdown. H1299 cells were transfected with EBNA1 or EBNAIAGAr expressing vectors and with control siRNA or siRNA targeting NCL, as indicated. GAPDH was used as a loading control. EBNA1/GAPDH or EBNA1AGAr/GAPDH ratios are indicated below the gels. The efficiency of NCL downregulation was estimated by determining the level of remaining NCL in cells treated by siRNA targeting NCL versus cells treated by control siRNA. Blot represents n≥3 and a representative result is shown.

(d) Same experiment as in (c) except that H1299 cells were transfected with chicken ovalbumin (OVA) or 235-GAr-OVA. Blot represents n≥3 and a representative result is shown.

(e) Autoradiographs showing relative mRNA translation efficiencies of 235GAr-OVA versus OVA in response to NCL knockdown. H1299 cells transfected with 235GAr-OVA (upper panel) or OVA (lower panel) constructs and with control siRNA or siRNA targeting NCL, as indicated, were pulse-labelled with [³⁵S] methionine, and lysates were subjected to immunoprecipitation with antibodies raised against OVA (left lanes) or IgG as a control (right lanes), and subjected to SDS-PAGE and autoradiography. Quantification of 235GAr-OVA or OVA signals are indicated.

Figure 3: NCL downregulation activates GAr-limited antigen presentation and recognition by specific T lymphocytes.

(a) Effect of NCL knockdown on antigen presentation. H1299 were transfected with 235GAr-OVA and murine MHC class I K^b plasmids and with control siRNA or siRNA targeting NCL, as indicated, and the levels of Kb/OVA-derived antigenic peptide complexes determined using FACS analysis. These experiments were performed three times and the mean quantification of the complex level obtained in cells treated by si-NCL or by si-control are shown and compared using the Student's /-test (^***/x0.001 ).

(b) Same experiment as in (a) except that H1299 cells were transfected with OVA plasmid. (^*/x0.05).

(c) Effect of NCL knockdown on T cell proliferation. H1299 cells were transfected with mouse K^b and 235GAr-OVA plasmids and control siRNA (left) or siRNA targeting NCL (right) and then mixed with naive OVA257-264 (SIINFEKL) specific CD8⁺ T cells isolated from peripheral and mesenteric lymph-nodes of mice and stained with CellTrace™ Violet. The proliferation of these SIINFEKL-specific T cells was determined by FACS analysis. Quantification of proliferating T lymphocytes when incubated in presence of cells treated by siRNA targeting NCL (si-NCL) or with control siRNA-treated cells (si-control) are shown on the right. The results were compared using the Student's /-test (^*p<0.05).

(d) Same experiment as in (c) except that H1299 were transfected with OVA plasmid (ns: not significant).

Figure 4: NCL directly interacts with G4 formed in the GAr-encoding mRNA.

(a) Schematic representation of a G-quadruplexe (G4) structure. Left) self-assembly of 4 guanines held together by Hoogsteen hydrogen bonds (dashed lines) giving a G-quartet in presence of K⁺ and schematic representation depicted by grey rectangles. Several G-quartets stack to form G4. Right) the three main topologies adopted by G4 classified as function of strand orientation (indicated by arrows) and differing by loop arrangements. G4 RNA mostly adopt the parallel topology.

(b) RNA pull-down using G4 forming RNA oligonucleotides covalently linked to biotin and streptavidin-coupled sepharose beads. Lysate from H1299 cells was applied to the following matrices: streptavidin-coupled beads either alone (Empty), or together with GQ (containing the most probable G4 of GAr mRNA), GM (same sequence except that G critical for G4 formation were replaced by adenines or uridines) or ARPC2 (containing a G4 present in ARPC2 mRNA and that has been shown to bind NCL) RNA oligonucleotides. The sequence of these oligonucleotides is given in the Methods section. The proteins still bound to the beads after an 800 mM KCI wash were eluted and analyzed by SDS-PAGE and western blot.

(c) Same experiment as in (b) except that recombinant NCL was used instead of H 1299 cell lysate.

Figure 5: NCL-EBNA1 mRNA interaction takes place in the nucleus,

(a) Endogenous NCL interacts in cellulo with endogenous EBNA1 mRNA in the nucleus. Proximity ligation assay (PLA) experiment to determine if NCL interacts with EBNA1 mRNA in cellulo was performed in the EBV-infected Burkitt lymphoma cells Mutu-1 . The green dots (PLA signal) indicate an interaction. PLA signals was shown together with DAPI staining (top left) or alone (top right). A zoom on the nucleus of a Mutu-1 cell is shown. (b) In cellulo nuclear interaction between NCL and EBNA1 mRNA is reduced upon treatment by 0.75 μΜ PhenDC3. Same experiment than in (a) except that Mutu-1 cells were treated by DMSO (top) or PhenDC3 (bottom).

(c) The mean number of PLA dots per cell in DMSO- (vehicle) or 0.75 μΜ PhenDC3- treated Mutu-1 treated cells shown in (b) are indicated and compared using the Student's t- test (^**p<0.01 ).

(d) In cellulo nuclear interaction between NCL and EBNA1 mRNA is GAr-dependent. PLA experiments were performed in H1299 cells transfected with EBNA1 or EBNAIAGAr plasmids and treated or not with 0.75 μΜ PhenDC3. The green dots indicate an interaction.

(e) The number of PLA dots per cells obtained in (d) treated by DMSO (vehicle) or 0.75 μΜ PhenDC3 are indicated and compared using the Student's /-test (^**p<0.01 ).

Figure 6: PhenDC3 prevents GAr inhibition of protein expression and NCL binding to GAr's G4.

(a) SDS-PAGE and western blot analysis of the level of 235GAr-OVA or OVA in response to pyridostatin (PDS) treatment. H1299 cells transfected with 235GAr-OVA (left) or OVA (right) plasmids were treated with 5 μΜ PDS (right lanes) or, as a control, with DMSO (left lanes). GAPDH was used as a loading control and 235GAr-OVA/GAPDH and OVA/GAPDH ratios are indicated below the gels. The chemical structure of PDS is depicted on the right. Blot represents n≥3.

(b) SDS-PAGE and western blot analysis of the level of 235GAr-OVA or OVA in response to PhenDC3 treatment. Same experiments as in (a) except that cells were treated with 5 μΜ PhenDC3 which chemical structure is depicted on the right. Blot represents n≥3.

(c) The effect of 5 μΜ PhenDC3 treatment on 235GAr-OVA or OVA mRNA level was determined by qRT-PCR. The results were compared using the Student's Hest (ns: not significant).

(d) PhenDC3 competes for the binding of NCL on GAr and ARPC2 G4. Same experiment than in Figure 4 (c) in the presence of 10 μΜ PhenDC3 or DMSO (vehicle) as indicated.

(e) PhenDC3 increases endogenous EBNA1 expression in EBV-infected cells. The level of endogenous EBNA1 in Mutu-1 (EBV-infected B cells, left panel) and NPC-6661 (EBV- infected cells from nasopharyngeal carcinoma, right panel) cells in response to 1 μΜ PhenDC3 was determined by SDS-PAGE followed by western blot. Actin was used as a loading control and EBNA1/Actin ratio are indicated below the gels. Blots represent n≥3.

Figure 7: PhenDC3 activates GAr-limited antigen presentation.

(a) PhenDC3 increases T cell proliferation. Same experiment as in Figure 3 (c) & (d) except that 235GAr-OVA (upper panels) and OVA (lower panels) expressing H 1299 cells were treated with 5 μΜ PhenDC3 or, as control, with DMSO as indicated. Quantification of proliferating T lymphocytes following PhenDC3 treatment as compared to DMSO-treated cells is shown in the graph on the right. The results were compared using the Student's /-test (^**p<0.01 ; ns: not significant).

(b) SDS-PAGE and western blot analysis of cells used in (a).

Figure 8: Identification and confirmation of the critical role of nucleolin in GAr-based translation inhibition in yeast.

(a) Effect of NSR1 overexpression on Ade2p level. The overexpression of NSR1 gene, which encodes the yeast nucleolin, has no effect on the white color of ADE2 expressing yeast cells and on the Ade2p protein level as evidenced by SDS-PAGE and western blot analysis (left panel). GAPDH was used as a loading control. The mean Ade2p/GAPDH ratios from 3 independent experiments are shown in the right panel and the results compared using the Student's /-test (ns: not significant).

(b) Overexpression of NSR1 has no effect on 43GAr-ADE2 and ADE2 mRNA level in yeast. Relative levels of 43GAr-ADE2 or ADE2 mRNA as compared to actin mRNA in NSR1- overexpressing cells were determined by quantitative RT-PCR. The results were compared using the Student's /-test (ns: not significant).

(c) Deletion of NSR1 has no effect on 43GAr-ADE2 and ADE2 mRNA level in yeast. Relative levels of 43GAr-ADE2 or ADE2 mRNA as compared to actin mRNA in lAT or nsrIA cells were determined by quantitative RT-PCR. The results were compared using the Student's /-test (ns: not significant).

(d) Complementation of NSR1 deletion by the NSR1 gene expressed from a plasmid. SDS- PAGE and western blot analysis of the yeast nsrIA strain expressing 43GAr-Ade2p (left) or Ade2p (right) and expressing (right lanes), or not (left lanes), yeast NSR1. GAPDH was used as a loading control. The 43GAr-Ade2p/GAPDH or Ade2p/GAPDH ratios are indicated below the gels.

Figure 9: Overexpression of NCL exacerbates GAr effect on protein expression whereas its downregulation suppresses its inhibitory effect on translation.

(a) SDS-PAGE and western blot analysis of the level of endogenous EBNA1 in two EBV- infected B cell lines (B95.8 and Raji as indicated) overexpressing (right lanes), or not (left lanes), HA-tagged nucleolin (HA-NCL). Actin was used as a loading control. EBNA1 /actin ratios are indicated below the gels. Blots represent n≥3.

(b) SDS-PAGE and western blot analysis of the level of 235GAr-OVA or OVA in response to the overexpression of NCL. HCT1 16 cells were transfected with 235GAr-OVA or OVA and with NCL plasmids, as indicated. GAPDH was used as a loading control. The OVA/GAPDH or 235GAr-OVA/GAPDH protein level ratios are indicated below the gels. Blot represents n≥3. (c) SDS-PAGE and western blot analysis of the level of endogenous EBNA1 and NCL in the three EBV-infected B cell lines used in this study. GAPDH was used as a loading control. (d) Quantification of the experiments shown in Figure 2b. The mean EBNA1 /GAPDH and EBNA1AGAr/GAPDH protein level ratios from 3 independent experiments are shown. The results were compared using the Student's /-test (^*p<0.05).

(e) Quantification of the experiments shown in Figure 2c. The mean 235GAr-OVA /GAPDH and OVA/GAPDH protein level ratios from 3 independent experiments are shown.

The results were compared using the Student's /-test (^**/x0.01 ).

(f) siRNA-mediated NCL knockdown has no effect on EBNA1 and EBNAIAGAr mRNA level in H1299 cells. H 1299 cells were transfected with EBNA1 or EBNAIAGAr and with control siRNA or siRNA against NCL, as indicated. Relative levels of EBNA1 or EBNAIAGAr mRNA as compared to actin mRNA in cells treated with siRNA targeting NCL or control siRNA as indicated were determined by quantitative RT-PCR. The results were compared using the Student's /-test (ns: not significant).

(g) Same experiment as in (d) except that H1299 cells were transfected with chicken ovalbumin (OVA) or 235GAr-OVA whose relative mRNA levels compared to actin mRNA were assessed by quantitative RT-PCR. The results were compared using the Student's /-test (ns: not significant).

Figure 10: NCL downregulation activates antigen presentation and recognition by T lymphocytes.

(a) SDS-PAGE and western blot analysis of the H1299 cells used in Figure 3a & b.

(b) SDS-PAGE and western blot analysis of the H1299 cells used in Figure 3c & d.

Figure 11 : PDS does not prevent NCL binding to both GAr's and ARPC2 G4s and has a lower affinity than PhenDC3 for GAr's G4.

(a) Same experiment than in Figure 4b except that DMSO (vehicle) or PDS were added as indicated.

(b) HT-G4-FID Plots for PhenDC3 and PDS (see Methods section for description of the assay). The ability of both compounds to displace thiazole orange (TO) was assessed. Bis- DC3 was used as a negative control. The DC₅o calculated from the obtained curves are Phen DC3 : 0.26, PDS : 0.47.

BisDCO

(c) Treatment with 1 μΜ PhenDC3 has no effect on EBNA1 mRNA level in Mutu-1 cells. Relative levels of EBNA1 mRNA as compared to actin mRNA in PhenDC3- or DMSO-treated cells were determined by quantitative RT-PCR. The results were compared using the Student's /-test (ns: not significant).

(d) Toxicity of various concentration of PhenDC3 on Mutu-1 cells was assessed using MTT assay. The results obtained with the various concentrations of PhenDC3 were compared to the result obtained with cells treated by DMSO (compound vehicle) using the Student's Hest (^*/x0.05; ^***p<0.001 ; ns: not significant).

(e) PDS has no effect on endogenous EBNA1 level in Mutu-1 cells. Same experiment than in Fig. 6e, except that Mutu-1 cells were treated with 3, 6 or 9 μΜ PDS. The results were compared using the Student's Hest (ns: not significant).

EXAMPLES

Example 1 - Compounds

1. Synthesis and characterization of PhenDC3-C4-R analogs

PhenDC₃-az R = CH₂-(0-CH₂-CH₂)3-N₃

PhenDC₃-C4-C R = ^ ^{^}Y^l

Scheme S1 : a) Trimethyl orthoacetate, Meldrum's acid, 1 10 °C, r.t. 18 h; b) Diphenylether, 230 °C, 1 h, c) P(0)CI₃ 90 °C d) NCS, benzoic acid, DCE, 70 °C, 24 h; e) H₂S0₄ 95 °C, 3 h, f)

3-aminoquinoline, EDCI , HOBt, DMF, 0 °C to r. 1. 18 h, g) 1 ,4-diaminobutane, 100 °C, 30 min, MW h) for PhenDC₃-alk: 4-pentynoic acid, EDCI, Et₃N, HOBt DMF, r. t., 18 h; for PhenDC₃-az:

2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid, EDCI , Et₃N, HOBt , DMF, r. t., 18 h ; for

Phen-DC₃-C4-Bn : 4 benzoyl benzoic acid, EDCI , Et₃N, HOBt , DMF, r. t., 18 h, for Phen-

DC₃-C4-C : 4-{4-[bis(2-chloroethyl)amino]phenyl}butanoic acid, HTBU, DIPEA, DMT, r. t., 20h i) CH₃I, DMF, 40 °C, 24 h.

2,9-dimethyl-1 ,4-dihydro-1 ,10-phenantrolin-4-one (2)

Trimethylorthoacetate (30 eq) and Meldrum's acid (1 .5eq) were brought to a gentle reflux for 15 min, at 1 10 °C. The resulting yellow solution was cooled down and 2-methylquinolin-8- amine (1 .0 eq) was added. The reaction mixture was heated at reflux for 2 h and left under stirring at room temperature for 16 h. The solvent was removed under vacuum and the red oil obtained was heated at 230 °C for 1 h in diphenylether. After cooling down to 50 °C, petroleum ether was added and a dark red powder was obtained after filtration. The product was purified by column chromatography (DCM/ethanol 90/10) affording a dark red solid (41 %). ¹H NMR (300 MHz, CDCI₃): δ (ppm) 10.15 (m, 1 H), 8.29 (d, J = 9.0 Hz, 1 H), 8.12 (d, J = 8.5 Hz, 1 H), 7.58 (d, J = 9.0 Hz, 1 H), 7.47 (d, J = 8.5 Hz, 1 H), 6.36 (s, 1 H), 2.80 (s, 3H), 2.56 (s, 3H).

¹³C NMR (75 MHz, CDCI₃): δ (ppm) 178.6, 158.1 , 147.2, 137.9, 136.4, 136.3, 127.0, 124.3, 123.3, 122.1 , 121 .4, 1 12.4, 25.0, 20.4.

LR-MS (ESI-MS) m/z = 224.9 [M+H]⁺

4-chloro-2,9-dimethyl-1 ,10-phenantroline (3)

In a round-bottomed flask, phosphoryl chloride (43 eq) was stirred under argon atmosphere before addition of 2,9-dimethyl-1 ,4-dihydro-1 ,10-phenantrolin-4-one (1 .0 eq). The reaction mixture was stirred as 90 °c for 3 h 30. While still hot, the reaction mixture was carefully added to ice and water. After 15 min stirring, chloroform was added and the resulting two-layer system was brought to pH 13-14 thanks to NaOH solution addition. The aqueous layer was extracted by DCM. The combined organic layers were washed by NaOH solution, dried over MgS04 and concentrated under vacuum to afford the desired product (86 %) as light tan crystals.

¹H NMR (300 MHz, CDCI₃): δ (ppm) 8.17 (d, J = 9.0 Hz, 1 H), 8.16 (d, J= 8.5 Hz, 1 H), 7.81 (d, J= 9.0 Hz, 1 H), 7.61 (s, 1 H), 7.54 (d, J= 8.5 Hz, 1 H), 2.96 (s, 3H), 2.93 (s, 3H).

1³C NMR (75 MHz, CDCI₃): δ (ppm) 160.0, 159.2, 146.4, 144.9, 142.6, 136.4, 126.9, 126.4, 124.1 , 123.6, 121 .1 , 25.9, 25.7.

LR-MS (ESI-MS) m/z = 265.2 [M+Na]⁺

4-chloro-2,9-bis(trichloromethyl)-1 ,10-phenantroline

4-chloro-2,9-dimethyl-1 ,10-phenantroline (1 .0 eq) was dissolved in dichloroethane before addition of N-chlorosuccinimide (7.2 eq) and benzoic acid (0.02 eq). The reaction mixture was heated at reflux, at 70 °C for one day. After cooling down to room temperature, the mixture was filtered and the filtrate was concentrated under vaccum. The product was taken up in DCM and washed by saturated aqueous Na₂C0₃ solution three times. The combined organic layers were dried over MgS04 and concentrated under vacuum. The desired product (82 %) was obtained after purification by column chromatography (cyclohexane/DCM 60/40).

1H NMR (300 MHz, CDCI₃): δ (ppm) 8.50-8.35 (m, 4H), 8.07 (d, J= 9.0 Hz, 1 H).

¹³C NMR (75 MHz, DMSO-d⁶): δ (ppm) 158.5, 157.9, 144.5, 144.4, 143.1 , 138.4, 129.3, 128.6, 127.5, 123.7, 121 .2, 120.8, 98.1 , 97.3.

LR-MS (ESI-MS) m/z = 446.9 [M+H]⁺ 4-chloro-1 ,10-phenantroline-2,9-dicarboxylic acid (4)

Sulfuric acid (1 mL) and 4-chloro-2,9-bis(trichloromethyl)-1 ,10-phenantroline (1 .0 eq) are introduced in a round-bottomed flask and stirred at 95 °C for 3 h. The reaction mixture was then cooled down to room temperature, before careful addition of water. The resulting solution was refluxed for 1 h and a large amount of water was added. The precipitate was filtered, washed by water and dried by diethylether, affording the desired compound (89 %).

1H NMR (300 MHz, DMSO-d⁶): δ (ppm) 8.79 (d, J = 8.4 Hz, 1 H), 8.50 (s, 1 H), 8.44 (d, J = 8.4 Hz, 1 H), 8.41 (d, J = 9.0 Hz, 1 H), 8.36 (d, J= 9.0 Hz, 1 H).

¹³C NMR (75 MHz, DMSO-d⁶): δ (ppm) 166.1 , 165.3, 149.0, 148.4, 145.9, 144.4, 142.9, 138.4, 130.3, 130.0, 127.8, 124.0, 123.5, 123.2.

LR-MS (ESI-MS) m/z= 303.1 [M+H]⁺ PhenDC₃n-CI (5)

3-aminoquinoline (2.15 eq) and HOBt (2 eq) were successively added to a solution of 4-chloro- 1 ,10-phenantroline-2,9-dicarboxylic acid (1 .0 eq) suspended in DMF, under an argon atmosphere. The reaction mixture was then cooled to 0°C before addition of EDCI (2.2 eq). The reaction mixture was slowly allowed to reach room temperature over 1 h and was stirred overnight. The pale yellow precipitate obtained was filtrated, washed with water and diethyl ether to afford PhenDC₃n-CI (51 %).

¹H NMR (300 MHz, DMSO-d⁶ at 80°C): δ (ppm) 1 1 .66 (s, 2H), 9.64 (s, 2H), 9.03 (m, 2H), 8.89 (d, J = 8.5 Hz, 1 H), 8.69 (m, 2H), 8.43 (q, J = 9.0 Hz, 2H), 8.07 (t, J = 9.0 Hz, 4H), 7.74 (t, J = 7.2 Hz, 2H), 7.65 (t, J = 7.2 Hz, 2H).

LR-MS (ESI-MS) m/z= 555.5 [M+H]⁺

PhenDC₃n-NH₂ (6)

A sealed vial containing PhenDC₃n-CI (1 .0 eq) and butane-1 ,4-diamine (100 eq) under argon was heated up to 120°C for 1 h in microwave to give an homogeneous clear orange mixture. The expected compound was precipitated upon addition of acetonitrile. The solid was then filtered, washed with acetonitrile and diethyl ether to afford PhenDC₃n-NH₂ (85 %).

Ή NMR (300 MHz, DMSO-d⁶): δ (ppm) 1 1 .79 (bs, 1 H), 9.67 (s, 2H), 9.13 (d, J = 9.3 Hz, 2H), 8.79 (d, J = 8.3 Hz, 1 H), 8.59 (d, J = 8.3 Hz, 1 H), 8.55 (d, J = 9.3 Hz, 1 H), 8.09 (m, 5H), 7.92 (bs, 1 H), 7.69 (m, 5H), 3.47 (m, 2H), 2.69 (m, 2H), 1 .81 (m, 2H), 1 .58 (m, 2H)

LR-MS (ESI-MS) m/z= 607 [M+H]⁺

General protocol for analog Phen-DC3n-C4-R

Carboxylic acid (1 .3 eq) was dissolved in DMF with PhenDC₃n-NH₂ 6 (1 eq), HOBt (0.5 eq) and EDCI (1 .3 eq). Triethylamine (1 .3 eq) was added and the reaction mixture was stirred overnight at room temperature, with protection from light. DMF was then removed under vacuum and the crude mixture was purified by flash chromatography (DCM/ethanol/TEA 90/9/1 ) to afford the PhenDC₃n-C4-R compound. General for protocol for analog PhenDC3-C4-R

A solution of the compound PhenDC3n-C4-R (1 .0 eq) in DMF was heated at 40 °C before methyl iodide (45 eq) was added dropwise. The reaction was performed under argon atmosphere for 24h. The solvent was evaporated to dryness and the expected compound was taken from ethanol as a solid.

PhenDC₃n-alk

4-pentynoic acid (1 .3 eq) was dissolved in DMF with PhenDC₃n-NH₂ (1 eq), HOBt (0.5 eq) and EDCI (1 .3 eq). Triethylamine (1 .3 eq) was added and the reaction mixture was stirred overnight at room temperature, with protection from light. DMF was then removed under vacuum and the crude mixture was purified by flash chromatography (DCM/ethanol/TEA 90/9/1 ) to afford the PhenDC₃n-alk compound as a yellow powder (38 %).

¹H NMR (300 MHz, DMSO-d⁶): δ (ppm) 1 1 .82 (s, 1 H), 1 1 .79 (s, 1 H), 9.67 (s, 2H), 9.13 (d, J = 9.0 Hz, 1 H), 8.77 (d, J = 8.5 Hz, 1 H), 8.59 (d, J = 8.5 Hz, 1 H), 8.56 (d, J = 9.0 Hz, 1 H),8.10 (m, 5H), 7.97 (m, 1 H), 7.72 (m, 5H), 3.50 (m, 2H), 3.18 (m, 3H), 2.75 (m, 1 H), 2.29 (m, 3H), 1 .80 (m, 2H), 1 .62 (m, 2H).

LR-MS (ESI-MS) m/z= 688.16 [M+H]⁺

PhenDCs-alk

A solution of the starting compound PhenDC₃n-alk (1 .0 eq) in DMF was heated at 40 °C before methyl iodide (45 eq) was added dropwise. The reaction was performed under argon atmosphere for 24h. The solvent was evaporated to dryness and the expected compound was taken from ethanol as a yellow solid (67%).

¹H NMR (300 MHz, DMSO-d⁶): δ (ppm) 12.12 (s, 2H), 10.30 (s, 2H), 9.89 (s, 2H), 8.88 (d, J = 8.5 Hz, 1 H), 8.68 (d, J = 8.5 Hz, 1 H),8.65-8.45 (m,5H), 8.30-8.20 (m,3H),8.08 (m, 2H), 7.98 (m, 1 H), 7.69 (s, 1 H), 4.73 (s, 6H), 3.48 (under water, 2H), 3.18 (m, 2H), 2.76 (s, 1 H), 2.37 (m, 2H), 2.29 (m, 2H), 1 .80 (m, 2H), 1 .62 (m, 2H).

¹³C NMR (500MHz, DMSO-d⁶): δ (ppm) 170.2, 164.4, 163.7, 152.3, 148.5, 147.9, 145.8, 145.7, 144.2, 138.8, 135.5, 135.4, 134.7, 134.4, 133.8, 133.7, 132.8, 132.8, 130.9, 130.3, 130.2, 129.9, 129.9, 129.1 , 125.2, 122.9, 121 .5, 1 19.5, 1 19.2, 99.5, 83.8, 71 .3, 46.0, 45.8, 42.5, 38.2, 34.3, 27.0, 25.1 , 14.3

LR-MS (ESI-MS) m/z= 358.1 [(M-2l)/2+H]⁺

HR-MS (ESI+) m/z = 843.2268 calculated for C43H40N8O3I ; found, 843.2266 PhenDC₃n-az

2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (1 .2 eq) was dissolved in DMF with PhenDC₃n-NH₂ (1 eq), HOBt (0.5 eq) and EDCI (1 .5 eq). Triethylamine (1 .5 eq) was added and the reaction mixture was stirred overnight at room temperature, with protection from light. DMF was then removed under vacuum and the crude mixture was purified by flash chromatography (DCM/ethanol/TEA 90/9/1 ) to afford the PhenDC₃n-azide compound as a yellow powder (69 %).

¹H NMR (300 MHz, DMSO-d⁶): δ (ppm) 1 1 .83 (s, 1 H), 1 1 .79 (s, 1 H), 9.68 (t, J = 2.2 Hz, 2H), 9.13 (dd, J = 2.2 Hz and J = 9.0 Hz, 2H), 8.79 (d, J = 8.5 Hz, 1 H), 8.60 (d, J = 8.5 Hz, 1 H), 8.56 (d, J = 9.0 Hz, 1 H), 8.1 1 (m,5H), 7.70 (m, 6H), 7.64 (s, 1 H), 3,90 (s, 2H), 3.55(m, 10H), 3.50 (m, 2H), 3.26 (m, 3H), 1 .79 (m, 2H), 1 .67 (m, 2H).

LR-MS (ESI-MS) m/z= 823.4 [M+H]⁺

PhenDC₃-az

A solution of the starting compound PhenDC₃n-az (1 .0 eq) in DMF was heated at 40 °C before methyl iodide (100 eq) was added dropwise. The reaction was performed under argon atmosphere for 24h. The solvent was evaporated to dryness and the expected compound was taken from ethanol as a yellow solid (67%).

¹H NMR (300 MHz, DMSO-d⁶): δ (ppm) 12.1 1 (s, 2H), 10.30 (s, 2H), 9.88 (s, 2H), 8.88 (d, J = 8.3 Hz, 1 H), 8.69 (d, J = 8.3 Hz, 1 H), 8.62 (d, J = 9.2 Hz, 1 H), 8.53 (m, 4H), 8.25 (m, 2H), 8.21 (d, J = 9.0 Hz, 1 H), 8.08 (m, 2H), 7.95 (s, 1 H), 7.76 (t, J = 5.84 Hz, 1 H), 7.70 (s, 1 H), 4.73 (s, 3H), 4.72 (s, 3H), 3,89 (s, 2H), 3.58 (m, 6H), 3.55 (m, 4H), 3.37 (m, 2H), 3.25 (m, 2H) 3,1 1 (m, 1 H), 1 .80 (m, 2H), 1 .67 (m, 2H), 1 .18 (m, 1 H).

¹³C NMR (500MHz, DMSO-d⁶): δ (ppm) 169.2, 163.7, 147.9, 145.7, 145.7, 145.6, 138.8, 135.5, 135.4, 134.7, 134.5, 133.9, 133.8, 132.8, 130.9, 130.2, 129.9, 129.9, 129.1 , 122.9, 121 .6, 1 19.2, 99.6, 70.2, 70.0, 69.7, 69.6, 69.5, 69.2, 49.9, 46.0, 42.6, 37.7, 27.0, 25.1

LR-MS (ESI-MS) m/z= 425.7 [M-2l/2+H]⁺

HR-MS (ESI+) m/z = 978.2912 calculated for

; found, 978.2913

Phen-DC3n-Bn

4-benzoylbenzoic acid (1 eq) was dissolved in DMF with PhenDC₃n-NH₂ (1 eq), HOBt (0.4 eq) and EDCI (1 eq). Triethylamine (2.5 eq) was added and the reaction mixture was stirred overnight at room temperature, with protection from light. DMF was then removed under vacuum and the crude mixture was purified by flash chromatography (DCM 100 -> DCM/methanol 95/5) to afford the PhenDC₃n-Bn compound as a yellow powder (60 %).

Phen-DC3-Bn

A solution of the starting compound PhenDC₃n-Bn (1 .0 eq) in DMF was heated at 40 °C before methyl iodide (100 eq) was added dropwise. The reaction was performed under argon atmosphere for 24h. The solvent was evaporated to dryness and the expected compound was taken from ethanol as a yellow solid.

HR-MS (ESI+) m/z = 844.3486 calculated for C52H44N8O4; found, 844.3444

PhenDC3n-C4-C 4-{4-[bis(2-chloroethyl)amino]phenyl}butanoic acid (2 eq) was dissolved in DMF with PhenDC₃n-NH₂ (1 eq), DIPEA (5 eq). HBTU (2 eq) was added and the reaction mixture was stirred overnight at room temperature, with protection from light. DMF was then removed under vacuum and the crude mixture was purified by flash chromatography (DCM 100 -> DCM/methanol 96/4) to afford the PhenDC₃n-C4-C compound as a yellow powder (29 %). Ή NMR (300 MHz, DMSO-d⁶): δ= 1 1 .84 (m,2H), 9.67(bs,2H), 9.14 (m,2H), 8.78(m,1 H), 8.61 - 8.55(m,2H), 8.09(m, 4H), 7.87-7.66(m,7H), 6.98(d,9Hz,2H), 6.59(d,6Hz,2H), 3.65(s,8H), 3.51 (m, 3H), 2.09(m,3H), 1 .77-1 .62(m, 6H), 0.85(m,2H) ppm.

LR-MS (ESI-MS): m/z= 892[M+H]⁺

PhenDC3-C4-C

A solution of the starting compound PhenDC₃n-C4-C (1 .0 eq) in DMF was heated at 40 °C before methyl iodide (100 eq) was added dropwise. The reaction was performed under argon atmosphere for 16h. The solvent was evaporated to dryness and the expected compound was taken from ethanol as an orange powder (99%).

1H NMR (300 MHz, DMSO-d⁶): δ= 12.14 (bs, 2H), 10.32 (s,2H), 9.88 (s,2H), 8.90 (m,1 H), 8.70- 8.51 (m,5H), 8.24-8.05 (m,10H), 7.00 (d,9Hz,2H), 6.60 (m,2H), 4.72 (s,6H), 3.66 (s,8H), 3.24- 3.16 (m,5H), 2.09 (m,2H), 1 .78-1 .62 (m,7H) ppm.

LR-MS (ESI-MS): m/z= 462

2. Synthesis and characterizati -biotine

Scheme S2 : g) 4,7,10-trioxa-1 ,13-tridecanediamine, 100 °C, 30 min, MW h) 2,5- dioxopyrrolidin-1 -yl 5-{2-oxo-hexahydro-1 H-thieno[3,4-d]imidazolidin-4-yl}, DMF, r. t., 16 h i) CH₃I, DMF, 40 °C, 24 h.

Compound 7

A sealed vial containing PhenDC₃n-CI (1 .0 eq) and 4,7,10-trioxa-1 ,13-tridecanediamine (100 eq) under argon was heated up to 100°C for 30 min in microwave. The expected compound was precipitated upon addition of acetonitrile. The solid was then filtered, washed with acetonitrile and diethyl ether to afford compound 7.

PhenDCsn-Biotine

The compound 7 (1 eq) and 2,5-dioxopyrrolidin-1 -yl 5-{2-oxo-hexahydro-1 H-thieno[3,4- d]imidazolidin-4-yl}(1 ,6 eq) in DMF was stirred at room temperature for 16h. DMF was then removed under vacuum and the crude mixture was purified by flash chromatography (DCM/ethanol 80/20) to afford the PhenDC₃n-Biotine compound as a bright yellow powder (36 %).

LR-MS (ESI-MS): m/z= 965.1 [M + H]⁺, Tr=7.01 min

PhenDCs-Biotine

A solution of the starting compound PhenDC₃n-Biotine (1 .0 eq) in DMF was heated at 40 °C before methyl iodide (45 eq) was added dropwise. The reaction was performed under argon atmosphere for 48h. The solvent was evaporated to dryness and the expected compound was taken from ethanol as an orange brown powder (100%).

LR-MS (ESI-MS): m/z= 497.2 [^] , Tr=4.13 min

3. Synthesis and characterization of PhenDC3-Bisalk1

11 PhenDC₃-Bisalk1

Scheme S3: a) P(0)CI3 90 °C, 3h30 b) NCS, benzoic acid, DCE, 70 °C, 16 h; c) H2S04 95 °C, 3 h, d) 3-aminoquinoline, EDCI , HOBt, DMF, 0 °C to r. t. 18 h, e) butylamine, 100 °C, 10 days f) CH₃I, DMF, 50 °C, 16 h.

Compound 11 was already described in this paper Larsen et al., Chem. Eur. J. 2012, 18, 10892 - 10902.

Phen-DC3n-Bisalk1

A sealed vial containing compound 11 (1 eq) and butylamine (135 eq) under argon was heated at 100°C for 10 days. H₂0 was added and the precipitate was filtered to give a yellow powder (69%).

LR-MS (ESI-MS): m/z= 663.1 [M + H]⁺

PhenDC3-Bisalk1

A solution of the starting compound PhenDC₃n-Bisalk1 (1 .0 eq) in DMF was heated at 40 °C before methyl iodide (140 eq) was added dropwise. The reaction was performed under argon atmosphere for 16h. The precipitate was filtered off and dried with Et₂0 affording product as a yellow-orange powder.

LR-MS (ESI-MS): m/z= 819.2 [M - /]+ 4. Synthesis and characterization of Br-PhenDC3

5-bromo-2,9-dimethyl-1 ,10-phenanthroline (2)

In a 100 ml flask previously dried under vacuum, 2, 9-dimethyl-1 ,10-phenanthroline (1 , 1 ,790 g, 8,60 mmol) was introduced in an heavy wall tube with a Teflon screw. The reaction vessel was cooled down with ice and oleum (20%, 6 ml) and Bromine (0,244 ml, 4,73 mmol) were added. The mixture was stirred at 135°C for 20h. The reaction mixture was cooled down to room temperature, poured over ice and neutralized with NH₄OH. The mixture was extracted with EtOAc, dried over MgS0₄, and evaporated to dryness to afford 5-bromo-2,9-dimethyl- 1 ,10-phenanthroline (2,103 g, 7,32 mmol, 85 % yield) as final product.

M. p. = 172°C;

1H NMR (300 MHz, CDCI₃): δ= 8.53 (d, J = 8.5 Hz, 1 H), 8.04 (d, J = 8.2 Hz, 1 H), 8.03 (s, 1 H),

7.58 (d, J = 8.5 Hz, 1 H), 7.50 (d, J = 8.2 Hz, 1 ), 2.97 (s, 3H), 2.93 (s, 3H) ppm;

¹³C NMR (75 MHz, CDCI₃): δ= 160.18, 159.88, 145.74, 144.70, 136.05, 135.32, 128.58,

127.03, 126.00, 124.25, 124.10, 1 19.65, 25.91 , 25.62 ppm.;

LC-MS (ESI-MS): m/z= 287.1 [M+], t = 4.45 min;

HRMS (ESI-MS) Ci₄Hn BrN₂: 287.0186 (calculated: 287.0184 Ci₄Hi₂BrN₂ ⁺).

5-bromo-1 ,10-phenanthroline-2,9-dicarboxylic acid (4)

In a 100 ml round-bottomed flask, 5-bromo-2,9-dimethyl-1 ,10-phenanthroline (2, 2.00 g, 6.96 mmol), N-Chlorosuccinimide (7.44 g, 55.7 mmol), and a portion of benzoic acid (29.7 mg, 0.244 mmol) were dissolved in CCI₄ (Ratio: 4.00, Volume: 40 ml) and CHCI₃ (Ratio: 1 .00, Volume: 10 ml). The mixture was refluxed for 16h, cooled to RT and then filtered. Filtrate was washed with three portions of saturated aq Na2C0₃, dried over MgS0₄, and the solvent removed under reduced pressure. The product was purified by column chromatography in cyclohexane/DCM, gradient from 90:10 to 60:40 affording 5-bromo-2,9-bis(trichloromethyl)-1 ,10-phenanthroline (3, 2.030 g, 4.1 1 mmol, 59 % yield). Due to the low stability of compound 3, after isolation ¹ H NMR and LRMS were recorded and the product submitted to the next reaction.

1H NMR (300 MHz, CDCI₃): δ= 8.84 (d, J = 8.8 Hz, 1 H), 8.41 - 8.27 (m, 4 H) ppm;

LC-MS (ESI-MS): m/z= 490.8 [M+] C14H5BrCI6N2, t = 8.52 min.

In a 250 mL round-bottomed flask, 5-bromo-2,9-bis(trichloromethyl)-1 ,10-phenanthroline (3, 2.030 g, 4.1 1 mmol) was added portion wise to sulfuric acid (10 ml, 189 mmol) 98% and the mixture was heated at 80-90°C for 6h. The reaction mixture was cooled to room temperature before careful and slow addition of drops of water, until a fine white solid precipitate. The heterogeneous mixture was refluxed for 2h, cooled to room temperature and then the precipitation of the product was induced by slow addition of water. The precipitate was collected, washed with H₂0 and Et₂0 affording 5-bromo-1 ,10-phenanthroline-2,9-dicarboxylic acid (4, 1 .298 g, 3.74 mmol, 91 % yield) as white powder.

Deo 239 °C;

¹H NMR (300 MHz, DMSO-d₆): δ= 8.84 (d, J = 9.0 Hz, 1 H), 8.71 (s, 1 H), 8.68 (d, J = 9.0 Hz, 1 H), 8.50 (d, J = 9.0 Hz, 1 H), 8.40 (d, J = 9.0 Hz, 1 H) ppm;

¹³C NMR (75 MHz, CDCI₃): δ= 166.12, 165.85, 149.06, 148.84, 145.31 , 144.24, 137.43, 131 .71 , 130.55, 129.15, 124.47, 124.08, 39.85, 39.68, 39.51 , 39.34, 39.18 ppm;

LC-MS (ESI-MS): m/z= 345.0 [M-], t = 5.35 min;

HRMS (ESI-MS) C14H7BrN204: 346.9673 (calculated: 346.9667 C14H8BrN204+).

5-bromo-N2,N9-di(quinolin-3-yl)-1 ,10-phenanthroline-2,9-dicarboxamide (5)

In a 100 ml flask, 5-bromo-1 ,10-phenanthroline-2,9-dicarboxylic acid (4, 100 mg, 0,288 mmol) and 3-aminoquinoline (87 mg, 0,605 mmol) were dissolved in DMF (5 ml). HOBt (7,79 mg, 0,058 mmol) and EDC (1 16 mg, 0,605 mmol) were added. No precipitation occurred and the reaction mixture was stirred at room temperature overnight. The product was precipitated by addition of H20. The precipitate was filtered, washed with CH₂CI₂, with H₂0, with 1 % NaHC0₃ solution and with Et20 affording 5-bromo-N2,N9-di(quinolin-3-yl)-1 ,10-phenan throline-2,9- dicarboxamide (5, 153 mg, 0,255 mmol, 89 % yield) as a green powder.

Stable till 300 °C;

¹H NMR (300 MHz, DMSO-d₆): δ= 1 1.87 (bs, 2 H), 9.67 (s, 2 H), 9.15 (s, 2 H), 9.04 (d, J = 9.0 Hz, 1 H), 8.89-8.84 (m, 2 H), 8.80 (d, J = 9.0 Hz, 1 H), 8.70 (d, J = 9.0 Hz, 1 H), 8.1 1 (m, 4 H), 7.76-7.67 (m, 4 H) ppm;

LC-MS (ESI-MS): m/z= 599.8 [M+], 299.9 [M2+], t = 7.66 min;

HRMS (ESI-MS) C32H19BrN602: 599.0835 (calculated: 599.0831 C32H20BrN6O2+).

3,3'-((5-bromo-1 ,10-phenanthroline-2,9-dicarbonyl)bis(azanediyl))bis(1-methylquin 1-ium) trifluoromethanesulfonate (BrPhenDC3)

In a 100 ml flask, 5-bromo-N2,N9-di(quinolin-3-yl)-1 ,10-phenanthroline-2,9-dicarboxamide (5, 60 mg, 0,100 mmol) was suspended in CICH₂CH₂CI (20 ml) under Argon. After refluxing for 2h, methyl trifluoromethanesulfonate (0,4 ml, 3,53 mmol) was added dropwise over 2 min and mixture was refluxed for 16h. The formed precipitate was filtered, washed with Et₂0 affording 6,6'-((5-bromo-1 ,10-phenanthroline-2,9-dicarbonyl)bis(azanediyl))bis(1 -methylquinolin-1 -ium) trifluoromethanesulfonate (BrPhenDC3, 79 mg, 0,085 mmol, 85 % yield) as a dark yellow powder.

Dec. > 237 °C;

1H NMR (300 MHz, DMSO-d₆): δ= 10.30 (s, 2 H), 9.90 (s, 2 H), 9.12 (d, J = 9.0 Hz, 1 H), 8.97- 8.93 (m, 2 H), 8.88 (d, J = 9.0 Hz, 1 H), 8.80 (d, J = 9.0 Hz, 1 H), 8.58-8.53 (m, 4 H) 8.30-8.25 (m, 2H), 8.12-8.07 (m, 2 H), 7.45 (s, 6 H) ppm;

¹³C NMR (75 MHz, DMSO-d₆): δ= 165.27, 165.01 , 150.86, 150.62, 147.58, 147.50, 146.34, 145.24, 140.59, 140.54, 137.50, 137.46, 136.88, 136.70, 135.90, 135.86, 134.55, 134.48, 134.16, 133.39, 132.20, 132.02, 131 .89, 131 .03, 125.15, 124.71 , 124.14, 123.85, 121 .28, 121 .1 1 , 47.96, 42.00, 41 .91 , 41 .83, 41 .74, 41 .66, 41 .57, 41 .50, 41 .41 , 41 .24, 41 .07, 40.91 ppm;

LC-MS (ESI-MS): m/z= 314 [M2+-2Triflate], t = 4.25 min;

HRMS (ESI-MS) C36H25BrF6N608S2: 777.0712 (calculated: 777.0743 C35H25BrF3N605S+). Example 2 - Biological Results

1. Materials and Methods

Yeast strains and culture media

All the yeast strains used in this study are derived from the W303 WT strain (Bondel et al. Genetics 2000, 155, 1033-1044): MATa, Ieu2-3, 112 trp1- 1 can1- 100 ura3-1 ade2-1 his3- 11, 15. The ade2A strain genotype is: MATa, Ieu2-3, 112 trpl- 1 can1-100 ura3- 1 ade2- 1::his5 S. pombe. Yeast cells were grown and used as previously described (Blondel et al, EMBO J 2005, 24, 1440-1452). The media used for yeast growth were: YPD [1 % (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose], i/₃ YPD [0.33% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose]. Yeast minimal media w/o uracil [6.7% (w/v) yeast nitrogen base, 0.77% (w/v) amino acids without uracil, 2% (w/v) glucose]. Yeast minimal media w/o uracil and tryptophan [6.7% (w/v) yeast nitrogen base, 0.72% (w/v) amino acids without uracil, 2% (w/v) glucose]. Solid media contained 2% (w/v) agar.

Yeast-based genetic screen for isolation of enhancers of GAr-based translation inhibition

A yeast genomic DNA library (a kind gift by F. Lacroute) constructed by inserting ~4 kb genomic DNA fragments (obtained by Sau3A partial digestion) at the unique BamHI site in the replicative 2 μ multicopy pFL44L vector containing l//¾43-marker, was transformed into 43GAr- Ade2p pink yeast strain using standard lithium acetate procedures (Fukuda et al. J Bacteriol 1983, 153, 163-168). This multicopy plasmid is present at ~ 50-100 copies per yeast cell. Transformants were selected on uracil-free minimal solid medium and a positive selection was carried out based on the redder color phenotype. Out of -20,000 transformants growing on uracil-free medium, 39 gave a redder phenotype. Plasmids originated form the pFL44-based library were extracted from these 39 redder transformants, purified and amplified in E. coli and then re-transformed into 43GAr-Ade2p yeast strain for confirmation of the redder phenotype. The extremities of the confirmed clones were sequenced using the following primers: F- 5' GTGCTGCAAGGCGATTAAGT 3' and R- 5TGTGGAATTGTGAGCGGATA 3'. Two confirmed clones contained overlapping genomic fragments containing the yeast NSR1 gene.

Plasmid constructions

All vectors were constructed using standard cloning procedures. T4 DNA ligase and restriction enzymes were purchased from New England Biolabs. Plasmid maintenance was carried out in TOP10 E.coli strain. The p416 (GPD) containing NSR1 gene was constructed as follows: NSR1 coding sequence was amplified from genomic DNA of the S. cerevisiae W303 WT strain using the following primers:

NSR1 -F 5' CGCGGATCCATGGCTAAGACTACTAAAGTAAAAGGTAAC 3' and

NSR1 - R 5' CCGCTCGAGCGGTTAATCAAATGTTTTCTTTGAACCAG 3'.

The corresponding PCR fragment was cloned into BamHI and Xhol cloning sites of p416 (GPD) centromeric vector. In order to introduce a HA tag in frame with human NCL, its coding sequence was PCR-amplified from cDNA extracted from HEK293T cells using HA-NCL F- 5' CGCGGATCCATGTACCCATACGATGTTCCAGATTACGCTGTGAAGCTCGCGAAGGCAG

3' and NCL R- 5' CCGCTCGAGCGGCTATTCAAACTTCGTCTTCTTTCC 3' primers and cloned into pCDNA3 vector (Invitrogen) using BamHI and Xhol cloning sites. HA-NCL was then sub-cloned into the S.cerevisiae vector p414 (GPD). All generated constructs were amplified in the TOP10 E.coli strain, and sequenced by the Sanger method.

Generation of nsrIA yeast strains

NSR1 gene deletion was carried out by replacement with kanMX6 cassette amplified from PFA6a-kanMX6 vector (Longtine et al. Yeast 1998, 14, 953-961 ), using the following primers:

F-5'

ACCAATTTCGGATCACTCAACCCAGGCAGGATAAAATAAGCGGATCCCCGGGTTAATTA

A 3'

And R-5'

AAGAGAAAAAATTGAAATTGAAATTCATTTCATTTTCTCAGAATCCGAGCTCGTTTAAAC

3'.

Then the PCR fragment was transformed into W303 ade2A, 43GAr-ADE2 and W303 ade2A, ADE2 yeast strains using standard lithium acetate procedures (Fukuda et al. J Bacteriol 1983, 153, 163-168). The transformed cells were spread on YPD + 100 μς/ηηί kanamycin plates which were then incubated 5 days at 29°C, after which the plates were replicated on fresh YPD + 100 ^g/mL kanamycin plates, and the deletion of NSR1 gene in kanamycin-resistant colonies was checked by PCR, 367 using the following primers: nsrIA 368 Fbis-5' GTACTTAAGTGTAGCTGTTGC 3' and nsrIA Rbis-5' TAGAGATGGTGAATGAAAGG 3'.

Yeast protein extracts

5 mL of 0,8-1 ,0 ODeoonm exponentially growing cells were harvested and cell pellets were resuspended into 300 μί of lysis buffer (25 mM Tris-HCI pH 6.8; 10% glycerol; 5% β- mercaptoethanol; 5% SDS; 8 M Urea; 0.02 % Bromophenol Blue).

Mammalian cells protein extracts Whole cells were harvested 48 hours post-transfection and lysed in 20 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1 % Igepal containing protease inhibitors (Roche, Germany). Samples were centrifuged at 16,000 g during 20 min at 4°C and protein concentrations were measured using a Bradford assay.

Western blotting

Equal protein quantities and volumes of all samples were loaded onto 10% NuPAGE® Bis-Tris gels (Invitrogen), and transferred onto 0.45 μηη nitrocellulose membranes (GE Healthcare). Membranes were blocked during 1 hour at room temperature in PBS 1 X containing 0.1 % Igepal and 3% BSA. Membranes were analyzed using the following antibodies: anti-HA serum (1 :2500); anti-Nsrl p mouse monoclonal antibody (Abeam, 1 :5000), anti-NCL rabbit polyclonal antibody (Abeam, 1 :5000), anti-GAPDH (Sigma, 1 :5000), anti-EBNA1 mouse monoclonal antibody (OT1 X, 1 :2000), anti-OVA rabbit polyclonal antibody (Sigma, 1 :2500), anti-actin (Sigma, 1/5000). The membranes were then washed with fresh PBS 1 X + 0.1 % Igepal and incubated for 45 min with swine anti rabbit or goat anti-mouse secondary antibodies (Dako) conjugated to horseradish peroxidase at a 1 :3000 dilution, and analyzed by enhanced chemiluminescence (ECL, GE Healthcare) using a Vilbert-Lourmat Photodocumentation 393 Chemistart 5000 imager. All the experiments were repeated at least three times. Relative protein levels for each sample were normalized to GAPDH or Actin protein levels as indicated, using Fusion-Capt Advance software.

Cell culture, Transfectlon.

H1299 cells are derived from metastatic lymph node from lung carcinoma. Raji cells are type III latency Burkitt's lymphoma. HCT1 16 cells are derived from colorectal carcinoma. B95.8 cells are derived from cotton-top Tamarin Monkey peripheral blood lymphocyte. Mutu-1 cells are derived from an EBV-positive Burkitt's lymphoma biopsy specimen from a Kenyan patient. NPC- 6661 cell line was established from a xenografted NPC in the early 90's 35 and was kindly provided by Prof. Kwok-Wai Lo from the Chinese University of Hong Kong. H1299, Raji, B95.8 and Mutu-1 cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. HCT1 16 cells were cultured in McCoy's 5A Glutamax media supplemented with 10% FBS, and NPC-6661 cells in RPMI-1640 media supplemented with 25 mM HEPES (Gibco) and 2 mM L-glutamine and 10% FBS. All cells were cultured at 37°C with 5% C02. Transient transfections were performed using Genejuice reagent (Merck Bioscience) according to the manufacturer's protocol or electroporation using Gene PulserXL system (Biorad).

RNA extraction and quantitative real-time PCR Total yeast, H1299 and Mutu-1 cellular RNA was extracted using RNAeasy and RNAase-free, DNase kits (Qiagen). cDNA synthesis was carried out using 1 μg of DNA-free RNA using M- MLV reverse transcriptase (Invitrogen) and Oligo-dT primer. Triplicated cDNA samples were analysized by quantitative PCR using PERFECTA SYBR fastmix (Quanta Bioscience). The relative abundance of target mRNA was normalized using Actin as an endogenous control.

Quantification of gene expression was determined using the -2 ^Ct method. The primers used for PCR were \D£2-forward: 5 419 '-ATTGTGCAAATGCCTAGAGGTG-3', \D£2-reverse: 5'- AATCATAA - GCGCCAAGCAGTC-3'; Actin-forward: 5 -ATGGTNGGNATGGGNCARAAR-3', Actin-reverse: 5 -CTCCATRTCRTCCCAGTTGGT-3'; EBNA /-forward: 5'- GGCAGTGGACCTCAAAGAAGAG-3'; EBNA 1- reverse: 5'-CAATGCAACTTGGACGTTTTT- 3'; OVA-forward: 5'-G AGG AGGCTTGG AACCTAT-3' ; OVA reverse: 5'- CAGTTTGAGAATCCACGGAG-3'. All the experiments were performed in triplicates and were repeated at least three times.

NCL siRNA downregulation

1 , 75 x 105 H 1299 cells were transfected with 0.75 g of EBNA1 , EBNAIAGAr, 235GAr-OVA or OVA vectors using standard procedures and incubated at 37°C for 8 hours. Cells were then transfected either with 40 nM of control siRNA or FlexiTube GeneSolution for NCL (Qiagen). siRNA transfection were performed using HiPerFect transfection reagent (Qiagen) following the manufacturer's protocol. 40 hours after siRNA transfection, cells were collected for western blot or flow cytometry analyses.

Mutu-1 cells were electroporated using SG Cell Line 4D-Nucleofector® X Kit from Lonza (V4XC-3012) following manufacturer's instructions and 300 nM of control siRNA or FlexiTube GeneSolution for NCL (Qiagen). 40 hours after siRNA transfection, cells were collected for western blot analyses.

Flo w cytometry analysis

48 hours after the transfection, cells were collected using trypsin and washed twice with 1 X PBS. Cells were suspended in 50 μί of 1X PBS and incubated with 0.4 μg of anti-Mouse OVA257-264 (SIINFEKL) peptide antibody bound to H-2Kb PE or Anti-Mouse MHC Class I (H-2Kb) antibody bound to PE (Ebioscience) for 30 min at room temperature. Cells were then washed with 1 X PBS and analyzed by FACS on a CANTO II flow cytometer (BD Biosciences, USA).

RNA pull-down experiments

For the preparation of whole cell extracts, confluent H1299 cells were harvested after trypsin treatment and washed twice with 1 X PBS (Gibco). Cells were suspended in 500 μί of lysis buffer (20 mM Tris-HCI pH 7.5; 200 mM NaCI and 0.1 % Igepal) containing 1 X protease inhibitor cocktail (Roche). Cell lysis was performed by 5 series of vortex followed 445 by 10 min incubation on ice, and 3 series of 3 sec sonication at 20% amplitude. After lysis cells were centrifuged at 4°C for 15 min at 16,000g, and the supernatant was quantified by Bradford. The whole cell extracts or recombinant GST-NCL (Abnova) were used for pull-down assays with the following G-quadruplex forming oligonucleotides: GQ- 5'-GGGGCAGGAGCAGGAGGA- 3'Biotin TEG, ARPC2- 5' AGCCGGGGGCUGGGCGGGGACCGGGCUUGU-3'Biotin TEG. The negative control for EBNA1 G quadruplex was the GM- 5' GAGGCAGUAGCAGUAGAA- 3'Biotin TEG oligonucleotide which, according to the GQRS-H predictor software, is unable to form G4 structures. To avoid unspecific binding, high-affinity streptavidin sepharose beads (GE Healthcare) were incubated in 1 mL blocking buffer containing 10 mM Tris-HCI pH 7.5; 100 mM KCI; 0.1 mM EDTA; 1 mM DTT; 0.01 % Triton X-100; 0.1 % BSA; 0.02% S. cerevisiae tRNAs (Sigma), for 1 hour at 4°C on a rotating wheel. 10 pg of each folded biotinylated RNA oligos were incubated with 50 μΙ_ of solution containing the streptaviding sepharose beads for 90 min at 4°C on a rotating wheel. 500 μg of cell extract or 200 ng of recombinant GST-NCL were incubated with the RNA oligonucleotides bound to the streptavidin beads during 90 min at room temperature. Beads were washed with increasing KCI concentration (200-800 mM). Protein still bound to beads after the washes were eluted using 2X SDS loading buffer and analyzed by western blotting against NCL, as previously described. In the input lane of the western blots was loaded a quantity of extract which corresponds to half of the quantity that was incubated with the beads for each condition.

Proximity Ligation Assay (PLA)

Cells were fixed with 4% paraformaldehyde in PBS 1 X for 20 min and permeabilized with 0.4% Triton X-100, 0.05% CHAPS for 5 minutes at room temperature. 50 ng of EBNA1 -digoxigenin mRNA probe (5' CTTTCCAAACCACCCTCCTTTTTTGCGCCTGCCTCCATCAAAAA 3') or control sense probe were denaturated 5 minutes at 80 °C and the hybridization reaction was carried out overnight at 37 °C in 40 μΙ_ hybridization buffer (10% formamide; 2X SSC, 0.2 mg/mL E. coli tRNAs, 0.2 mg/mL sheared salmon sperm DNA and 2 mg/mL BSA. A blocking solution of 3% BSA 0.1 % saponine in 1 X PBS was added for 30 min followed by 2 hours incubation at room temperature with the primary antibodies (anti-digoxigenin 1/200 -Sigma- and anti-NCL 1/1000 -Abeam-) diluted in PBS 1 X, 0,3% BSA, 0.1 % saponine. The proximity ligation assay (PLA) was carried out using the Duolink PLA in situ kit, PLA probe Anti-Rabbit Plus, the Duolink in situ PLA probe Anti-Mouse MINUS and the in situ detection reagent FarRed (all from Sigma) following the manufacturer's protocol. PLA results were visualized using a Zeiss LSM780 confocal microscope. All the PLA experiments were performed at least three times independently and, each time, PLA dots were counted in 50 to 100 cells. For each PLA experiment the following controls were performed: w/o mRNA probe, w/o antibodies and with the control sense probe.

³⁵S methionine pulse-labeling

8 X 105 cells were transiently transfected with 4 μg of 235GAr-OVA or OVA vectors and, 8 hours later, NCL silencing was performed using 40 nM of NCL siRNA or control siRNA (as previously described). 40 hours after the transfection cells were incubated 30 min in a methionine-free medium. After incubation, 25 μΜ of MG132 proteasome inhibitor was added to the medium and cells were incubated for 45 min. Cells were then cultured in a medium containing 0.15 mCi/mL ³⁵S-methionine (Perkin Elmer, Boston, USA) for i hour and harvested. Cell pellets were suspended in 20 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1 % Igepal and treated as described above. Lysates were pre-cleared with 1 μg normal rabbit serum (Dako) bound to protein G-Sepharose magnetic beads (GE Healthcare) for 30 min at 4°C and further immunoprecipitated with 1 μg of anti-OVA polyclonal antibody (Sigma) or IgG-rabbit (Dako), pre-bound to protein G-Sepharose magnetic beads overnight at 4°C. Beads were then washed and proteins eluted using 2X SDS loading buffer. Immunoprecipitates were analyzed by SDS- PAGE using 10% precast NUPAGE gels (Invitrogen). The amount of radiolabeled proteins was visualized using a Storm Phosphorimager (GE Healthcare).

T cell proliferation assay

Naive OVA257-264 specific CD8⁺ T cells were isolated by negative selection from peripheral and mesenteric lymph-nodes of 12-week-old female OT1 mice using the CD8+ T cell isolation kit (Miltenyi Biotec, Germany). Afterwards, CD8⁺ T cells were stained with CellTraceTM Violet (Thermo Fisher Scientific, USA) according to the manufacturer's protocol and mixed with H1299 cells cotransfected with mouse k^b expression vector and OVA or GAr-OVA constructs. For all the assays, 10⁵ H1299 cells were harvested 48h after transfection and co-incubated with 4 x 10⁵ stained T cells at 37°C in humidified air/C0₂ atmosphere in 1 497 ml of RPMI medium containing 10% FBS, 4 mM L-glutamine, 100 U/ml penicillin, 100 μg ml streptomycin, 5 mM HEPES and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich). After 3 days, cells were harvested, stained with hamster anti-mouse CD3-APC (Miltenyi Biotec) and fixable viability dye eFluor® 780 (eBioscience, USA) and analyzed by FACS on a CANTO II flow cytometer (BD Biosciences, USA). Cells were gated for live CD3⁺ cells (10.000 events collected) and data were analyzed using BD FACSDiva software version 8.0.1 . The percentage of proliferating T cells was considered for statistical analysis.

MTT assay

A total of 30,000 Mutu-1 cells were plated in 0.1 ml in 96-well flat bottom plates and exposed to PhenDC3 at the indicated concentrations or DMSO (vehicle). After 24 hours, 10 μΙ of 5 mg/ml MTT solution (CT01 -5, Merck Millipore) in PBS pH 7.4 were added to each well and incubated for 4 h. 0.1 mL of isopropanol-HCI 0.1 N-Triton X-100 10% were added to each well to dissolve the formazan crystals. The absorbance was then measured at 540 nm.

Statistical analyses

Data shown are mean ± s.d. of minimum three independent experiments. Two-tailed unpaired Student's t-test was performed by comparing data to the corresponding reference point or as indicated and p values are shown. ^*p<0.05; ^**p<0.01 ; ^***p<0.001 ; ns: not significant.

Fluorescence Intercalator Displacement (FID) assay for G4 ligands

G4-FID assay is performed in a 96-well Non-Binding Surface Black with black bottom polystyrene microplates (Corning). Every ligand is tested on a line of the microplate, in duplicate (in other plate). The microplate is filled with (a) K+100 solution (qs for 200 μΙ_) (b) 10 μΙ_ of a solution of pre-folded oligonucleotides (5 μΜ) and TO (10 μΜ - 2 molar equiv.) and (c) an extemporaneously prepared 5 μΜ ligand solution in K+100 buffer (0 to 100 μΙ_ along the line of the microplate, i.e., from column A to column H: 0, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 1.0, 1.25, 1.5, 2.0 and 2.5 μΜ). After 5 min of orbital shaking at 500 rpm, fluorescence is measured using the following experimental parameters; positioning delay: 0.5 s, 20 flashes per well, emission/excitation filters for TO: 485/520, gain adjusted at 80% of the fluorescence from the most fluorescent well (i.e., a well from column A). The percentage of TO displacement is calculated from the fluorescence intensity (F), using: % TO displacement = 1 -F/F₀ (F₀ being the fluorescence from the fluorescent probe bound to DNA without added ligand. The percentage of displacement is then plotted as a function of the concentration of added ligand. The DNA affinity is evaluated by the concentration of ligand required to decrease the fluorescence of the probe by 50%, noted DC50, and determined after non-linear fitting of the displacement curve.

2. Results

Yeast nucleolin Nsrl p is required for GAr-based inhibition of protein expression in yeast and human nucleolin NCL can functionally replace Nsrl p

The yeast assay used in this genetic screen is based on a fusion between the yeast Ade2p reporter protein and a GAr domain of 43 amino acid (43GAr). Because GAr is able to self- inhibit the translation of its own mRNA in yeast, this leads to a reduction in Ade2p level. This can easily be monitored in yeast as cells which express Ade2p at a functional level form white colonies, whereas cells that do not express Ade2p readily form red colonies and any intermediate level of Ade2p leads to the formation of pink colonies whose intensity of coloration is inversely proportional to the level of Ade2p expressed. Hence, a yeast strain expressing the 43GAr-ADE2 construct from the constitutive ADH promoter forms pink colonies, whereas a control strain expressing ADE2 from the same promoter forms white colonies (Fig. 1 a). The 43GAr- ADE2 strain was used to identify yeast genes whose overexpression leads to a redder phenotype meaning that they potentially exacerbate GAr-based inhibition of translation. For this purpose the 43GAr-ADE2 yeast strain was transformed by a yeast genomic DNA library consisting of small genomic fragments (~4kb) cloned into a yeast 2 μ multicopy plasmid which is present at -50 to 100 copies per yeast cell, hence potentially allowing to assess the effect of overexpressing the vast majority of yeast gene on GAr-based inhibition of translation. This way, two independent clones were isolated, bearing overlapping genomic fragments that, among a few other genes, contained the yeast NSR1 gene. We then subcloned NSR1 gene alone under the control of the strong constitutive GPD promoter in a low copy number vector (CEN) which confirmed its ability, when overexpressed, to both confer a redder phenotype and exacerbate the ability of GAr to decrease 43GAr-Ade2p protein expression whereas having no significant effect on Ade2p protein in the control strain (Fig. 1 b and 8a) nor on 43GAr-ADE2 or ADE2 mRNA levels (Fig. 8b).

Then, the effect of NSR1 downregulation on GAr-based inhibition of protein expression was determined. As NSR1 is not essential in yeast, this gene was deleted in both 43GAr-ADE2 and ADE2 strains and it was observed that its absence completely abolished the GAr inhibitory effect on 43GAr- Ade2p expression (first two lanes of the western blot in Fig. 1 c). This effect was GAr-dependent as NSR1 deletion had no effect on Ade2p protein level in the control strain (last two lanes of the western blot in Fig. 1 c). As controls, it was checked that NSR1 deletion had no significant effect on 43GAr-ADE2 and ADE2 mRNA levels (Fig. 8c).

Next, the potential ability of NCL, the gene encoding the human nucleolin, to functionally complement the deletion of NSR1 in yeast was assessed, and it was found that the expression of NCL led to a decrease in 134 43GAr-Ade2p level whereas having no effect on Ade2p level (Fig. 1 d). Of note, the human NCL was as efficient as the yeast Nsrl p (Fig. 8d).

Taken together, these results show that Nsrl p, the yeast orthologue of nucleolin, is critically involved in the GAr-based inhibition of protein expression in yeast. As NCL, the human nucleolin, is able to fully complement the deletion of the yeast NSR1 gene, this suggests that NCL represents a host cell factor important for the GAr-mediated self-limitation of EBNA1 expression in EBV-infected human cells.

NCL also controls GAr-dependent EBNA1 expression in EBV-infected cells

Newt, the role of human nucleolin (NCL) on GAr-based self-inhibition of translation in human cells was assessed. For this purpose, HA-tagged NCL (HA-NCL) was first overexpressed in three EBV infected B cell lines: Mutu-1 , B95.8 and Raji. As shown in Figure 2a for Mutu-1 and in Figure 9a for B95.8 and Raji, overexpression of HA-NCL led to a significant decrease in EBNA1 endogenous level in all these three cell lines, as compared to actin. Similar to yeast, this effect is GAr-dependent as overexpression of HA-NCL also decreased the level of transfected 235GAr-OVA (ovalbumin), the fusion protein that is used to assess the effect of GAr on MHC class l-restricted antigen presentation (see below), whereas having no significant effect on OVA alone, the control protein (Fig. 9b). The relative levels of endogenous ABNA1 and NCL were also determined in B95.8, Mutu-1 and Raji celles (Figure 9c).

The effect of downregulating endogenous NCL on endogenous EBNA1 level in EBV-infected Burkitt lymphoma Mutu-1 cells was then determined using siRNA. As observed by Chen et al. (Proc Natl Acad Sci U S 2014, 1 1 1 , 243-248), it was not possible to knockdown more than -40-50% the expression of NCL, probably because it is an essential gene in mammalian cells. However, this partial downregulation led to a significant increase (-50%) in EBNA1 level (Fig. 2b) demonstrating that endogenous NCL expression restricts endogenous EBNA1 expression in EBV-infected cells.

To determine if the impact of NCL level on EBNA1 expression is GAr-dependent, the effect of downregulating endogenous NCL on GAr-dependent suppression of protein expression in H1299 cells was then determined. Again it was not possible to knockdown more than -40-50% the expression of NCL. However, this partial downregulation led to a significant increase in both EBNA1 (Fig. 2c) and 235GAr-OVA (Fig. 2d) protein levels to about the same extent (-80% and -50% respectively) than the increase of endogenous EBNA1 in Mutu-1 cells, whereas having no effect on EBNAIAGAr or on OVA alone. Quantifications of three independent experiments are shown in Figure 9d and e. Next a metabolic 35S methionine pulse labeling experiment was performed, and an increase in newly synthesized 235GAr-OVA following NCL down-regulation was observed (Fig. 2e, upper panel). In contrast, no increase in newly synthesized OVA was observed (Fig. 2e, lower panel), nor any 160 significant effect on the levels of GAr-carrying mRNAs (Fig. 9f and 9g respectively). Altogether, these results demonstrate that NCL downregulation does interfere with GAr-based suppression of translation.

These findings confirmed that, as in yeast, NCL represents a host cell factor critically involved in the GAr-dependent suppression of EBNA1 synthesis, a mechanism at the basis of EBV immune evasion in latently infected cells.

NCL downregulation overrides GAr-restricted antigen presentation

We next tested if downregulating NCL has an effect on GAr-restricted MHC class I antigen presentation. Indeed, as NCL downregulation led to a GAr-dependent increase in protein expression, it was also expected to stimulate antigen presentation. For this purpose, we determined the effect of siRNA-mediated NCL knockdown on the GAr-restricted presentation of the ovalbumin-derived SIINFEKL antigenic peptide (OVA257-264) in complex with the murine kb MHC class I receptor using a specific monoclonal antibody. FACS analysis revealed that NCL knockdown significantly increased (+ 42.1 % ± 1 .37, p=0.0005) the formation of this complex in 235GAr-OVA-expressing cells (Fig. 3a). In contrast, NCL knockdown only had a modest effect on OVA-expressing cells (+ 19.7% ± 6.1 1 , p=0.043; Fig. 3b). In all cases, the efficiency of siRNA mediated NCL downregulation and its effect on 235GAr-OVA or OVA expression were determined (Fig. 10a).

It was then tested if the observed increase in antigen presentation following NCL downregulation does have an effect on T cell activation. For this purpose, the proliferation of naive CD8+ T cells (OT1 cells) recognizing specifically the OVA257-264 SIINFEKL epitope on the murine kb MHC class I molecule was determined. The OT1 cells were isolated from peripheral and mesenteric lymph-nodes of 12-week-old mice and stained with the CellTraceTM Violet fluorescent dye. Then, OT1 cells were mixed with H1299 cells expressing 235GAr-OVA and the k^b molecule. As a control, H1299 cells expressing OVA and the kb molecule were used. As expected and due to the GAr inhibitory effect on both translation 186 and antigen presentation, 235GAr-OVA-expressing H1299 cells (Fig. 3c, left panel) led to a much weaker activation of OT1 cells as compared to OVA-expressing H1299 cells (Fig. 3d, left panel), as determined by evaluating the number of dividing OT1 cells by FACS analysis. However, siRNA-mediated NCL knockdown in 235GAr-OVA expressing H1299 cells significantly increased proliferation of OT1 cells (Fig. 3c, right panels) whereas it had no effect in OVA- expressing H1299 cells (Fig. 3d, right panels). The efficiency of siRNA-mediated NCL downregulation and its effect on 235GAr-OVA or OVA expression are shown in Fig. 10b.

NCL directly interacts in the nucleus with G4 present in GAr-encoding mRNA sequence NCL has been reported to bind to some G-quadruplexes (G4) formed in both DNA (Gonzalez et al. J Biol Chem 2009, 284, 23622-23635) and RNA sequences (von Hacht et al. Nucleic Acids Res 2014, 42, 6630-6644). G4 are composed and stabilized by the stacking of guanine tetrads which are assembled in a planar arrangement by Hoogsteen hydrogen bonding (Fig. 4a) and have been involved in the regulation of gene expression, DNA replication and telomere maintenance. The G-rich sequence of GAr-encoding mRNA contains a cluster of 13 predicted G4. Hence, the ability of NCL to bind to these structures was determined. For this purpose, a pulldown assay recently developed to identify RNA G4 binding proteins (von Hacht et al.) was adapted to an 18 nt long oligonucleotide containing the most probable G4 that can form in the GAr-encoding mRNA sequence. Briefly, this oligonucleotide (GQ) was linked to biotin and pulldown experiments using streptavidin-conjugated sepharose beads were performed. As a negative control, an oligonucleotide (GM) with a similar sequence except that the four guanines forming the G4 were replaced by adenines or uridines was used in order to completely abolish the G4 structure, as predicted using the GQRS-H predictor software (Frees et al. Hum Genomics 2014, 8, 8). As a positive control, ARPC2 30 nt-long oligonucleotide was used which corresponds to a G4 found in the ARPC2 mRNA and that has been shown to bind NCL (von Hacht et al.). As shown in Figure 4b, NCL was precipitated from H1299 cell extracts when using GQ or ARPC2 oligonucleotides, but no 21 1 t when using GM or empty beads showing that NCL binds to G4 formed in the GAr mRNA sequence.

Next the same pulldown experiment was performed using recombinant NCL instead of cell lysate. Similar results were obtained (Fig. 4c) showing that NCL directly binds GAr most probable G4. Finally, to check if, and where, the NCL-GAr G4 interaction occurs in cellulo, a proximity ligation assay (PLA) was performed to assess if endogenous NCL associates with endogenous EBNA1 mRNA in EBV-infected Mutu-1 cells. We observed nuclear PLA dots (Fig. 5a) indicating that endogenous NCL does interact with endogenous EBNA1 mRNA and that this interaction mostly takes place in the nucleus. In contrast, no PLA dots were observed in the various controls (without the probe specific for EBNA1 mRNA or without antibodies, not shown). To assess if this interaction is GAr-dependent, we repeated this PLA in H1299 cells expressing EBNA1 or EBNAIAGAr-. Again, nuclear PLA dots in cells expressing the full-length EBNA1 mRNA were observed (Fig. 5d and e). In contrast, almost no dots were detected in cells expressing EBNAIAGAr (Fig. 5e) as well as in the various controls (not shown) indicating that the ability of NCL to interact in the nucleus with EBNA1 mRNA is Gar-dependent.

Taken together, these results indicate that NCL directly interacts with the Gar's G4 of the EBNA1 mRNA in the nuclear compartment.

PhenDC3 and analogs prevent NCL-EBNA1 mRNA interaction and GAr-based inhibition of protein expression

Next, the effect of various G4 ligands were tested on GAr-based inhibition of protein expression. Among the reported G4 ligands, pyridostatin (PDS, molecular structure depicted in Fig. 6a) and PhenDC3 (molecular structure depicted in Fig. 6b) are the best benchmark probes compatible with cellular assays. Indeed, PDS and PhenDC3 at micromolar concentrations have been shown to efficiently target various G4 in cell-based experiments (Muller et al. Expert Rev Clin Pharmacol 2014, 7, 663-679). As, at the same range of concentrations (1-5 μΜ), PDS has been shown to exacerbate GAr-based inhibition of protein expression in an in vitro coupled transcription-translation system 29, the effect of PDS was first tested on the level of 235GAr-OVA in H1299 cells. However, at the same concentration (5 μΜ), no clear effect on 235GAr-OVA or OVA expression was observed (Fig. 6a) suggesting that PDS may not be able to interfere with GAr-mediated inhibition of EBNA1 expression in a cellular context. PhenDC3 was then tested at the same concentration (5 μΜ) and was found to lead to a significant increase in the steady-state level of 235GAr-OVA in H1299 cells (Fig. 6b, left panel).

This effect is GAr-dependent since PhenDC3 had no significant effect on OVA expression (Fig. 6b, right panel) and is not due to an effect on the level of the corresponding RNA (Fig. 6c). Hence, one possibility is that PhenDC3 prevents the binding of NCL on EBNA1 mRNA G4. To test this hypothesis, the same G4 oligonucleotide pulldown assay as in Figure 4c was performed in the presence or absence of 10 μΜ PhenDC3 and it was observed that PhenDC3 does prevent the binding of NCL on GAr G4 (Fig. 6d), readily explaining its effect on 235GAr- OVA expression. Of note, PhenDC3 prevents the binding of NCL on ARPC2 G4 (Fig. 6d).

In contrast, PDS had no effect on the binding of NCL on both types of G4 (Supplementary Fig. 4a). We also checked that PDS and PhenDC3 are both able to bind to the most probable G4 that can form in the GAr-encoding mRNA sequence by determining the ability of these two compounds to displace the thiazole orange (TO) fluorescent probe from the GAr's G4 oligonucleotide used in the pulldown experiments. As shown in Fig. 1 1 b, both PhenDC3 and PDS bind the GAr's G4 but the affinity of PhenDC3 (DC50 =0.26 μΜ) is higher than that of PDS (DC50 =0.47 μΜ). Taken together these results suggest that PhenDC3, but not PDS, is also able to prevent the binding of NCL on these G4 structures by a competitive mechanism. This difference, which may be due, at least in part, to the lower affinity of PDS for GAr's G4, is consistent with the fact that PhenDC3 does interfere with the GAr self-inhibitory effect on protein expression whereas PDS is inactive.

To confirm that PhenDC3 prevents the binding of NCL on EBNA1 mRNA in more physiological settings, the effect of PhenDC3 in the PLA experiment on EBV-infected Mutu-1 cells was tested. As shown in Fig. 5b and c, the number of nuclear PLA dots per cell was significantly reduced (-3 fold) when Mutu-1 cells were treated with 0.75 μΜ PhenDC3 confirming the ability of PhenDC3 to interfere with this interaction in a cellular context. The same result was obtained when using H1299 cells expressing transfected EBNA1 (Fig. 5d and e).

Next, PhenDC3 effect on endogenous EBNA1 expression in EBV infected cells was tested and it also increased the endogenous EBNA1 level in Mutu-1 (EBV-infected B-cells; Fig. 6e left panel) and NPC-6661 (EBV infected cells derived from a nasopharyngeal carcinoma (Hui et al. Cancer Genet Cytogenet 1998, 101, 83-88); Fig. 6e right panel) cells. Importantly, PhenDC3 had no effect on EBNA1 mRNA level in Mutu-1 cells (Fig. 1 1 c).

It was also checked that PhenDC3 is not significantly toxic on Mutu-1 cells when used at a concentration range (0.5-1 μΜ) in which it increases the expression of EBNA1 (Fig. 1 1 d). Finally, we found that PDS had no effect on endogenous EBNA1 level in Mutu-1 cells (Fig. 1 1 e) confirming that, contrary to PhenDC3, PDS is not able to interfere with the GAr-based self-inhibition of protein expression.

To conclude, the PhenDC3 G4 ligand prevents the binding of NCL on GAr's G4 and, at the same time, leads to an increase in EBNA1 and 235GAr-OVA expression, thereby supporting the crucial role of NCL in GAr-based self-inhibition of translation by binding to G4 formed in the EBNA1 mRNA.

The same results were observed with the diiodide or dimesylate salt of the following compounds: PhenDC6, BipyDC3, BipyDC6, PDC3, PDC6, F-PhenDC3, Br-PhenDC3, Br- PhenDC6, PhenDC3-C4-C, PhenDC3-C4-Bn and PhenDC3-Bislalk1 .

PhenDC3 activates GAr-limited antigen presentation

Finally, to assess the ability of PhenDC3 to interfere with GAr-based immune evasion, the same OT1 T cell proliferation assay as in Figure 3c & d was performed in the presence or absence of 5 μΜ PhenDC3. As shown in Figure 7a, PhenDC3 significantly increased (two-fold change) the proliferation of OT1 T cells added to 235GAr-OVA-expressing H1299 cells whereas it had no effect on OT1 cells added to OVA-expressing cells. A western blot analysis confirmed that PhenDC3 at 5 μΜ increases the level of 235GAr-OVA whereas it has no effect on OVA (Fig. 7b).

3. Discussion

In this study nucleolin was identified as a host cell factor critically involved in GAr based EBNA1 immune evasion via its ability to bind G-quadruplexes formed in the GAr-encoding sequence of the EBNA1 mRNA. First, thanks to a genetic screen performed in a yeast model that recapitulates all the aspects of GAr-based self-inhibition of translation, the yeast nucleolin Nsrl p was isolated as a critical host cell factor involved in GAr-based inhibition of protein expression in yeast. Indeed, the overexpression of Nsr1 p exacerbates the GAr effect whereas the deletion of NSR1 gene fully abrogates GAr ability to self-inhibit translation. Second, as the human NCL gene, which encodes human nucleolin, is able to fully complement the effect of NSR1 deletion on GAr in yeast, the role of NCL in GAr-based inhibition of protein expression in human cells was tested and its overexpression and downregulation had the same effect on GAr than in yeast cells. Then in line with its ability to interfere with GAr-mediated translation inhibition, it was checked that NCL downregulation also increases antigen presentation and T cell proliferation. It was also shown that NCL directly interacts with G4 present in GAr encoding sequence of the EBNA1 mRNA in vitro and observed this interaction in cellulo in the nucleus of EBV infected cells. This suggests that the binding of NCL on EBNA1 mRNA G-quadruplexes is per se important for GAr-based translation inhibition and thereby for EBNA1 immune evasion. In line with this model, it was shown that the G4 ligand PhenDC3 both prevents the binding of NCL on G4 of the EBNA1 mRNA, and increases EBNA1 expression and GAr- dependent antigen presentation.

This indicates that the interaction between NCL and G4 of the EBNA1 mRNA is a relevant and druggable therapeutic target to treat EBV-related cancers. Interestingly the G4 ligand pyridostatin (PDS) had no effect on EBNA1 expression in cellulo indicating that only some G4 ligands are able to interfere with NCL-EBNA1 mRNA interaction, which can be attributed to off target binding and/or differences in pharmacological properties (cell penetration, intracellular distribution etc.). Of note, in vitro, PDS affinity for PDS affinity for GAr's G4 appears significantly weaker than that of PhenDC3 which may contribute, at least in part, to the differential activity of these two G4 ligands on GAr.This also points out two different possible mechanisms of action for G4 ligands whose binding on G4 may either stabilize them or prevent the binding of cellular partners, by direct competition or by modifying G4 structure. In line with these hypotheses, several closely related PhenDC3 analogs were tested, and ot was and observed that they were active on EBNA1 expression. Of note, PDS has been found to suppress EBNA1 expression in an in vitro coupled transcription translation assay (Murat et al. Nat Chem Biol 2014, 10, 358-364) but it is not known, in this experiment, if this effect is related to a change in EBNA1 mRNA level or to G4 stabilization that may exacerbate the GAr- dependent translation inhibition.

This brings out an intriguing point regarding the role of NCL in GAr-based self-limitation of EBNA1 expression and antigenic presentation. Indeed, NCL has been also positively involved in EBV episome maintenance and transcription. Hence, NCL appears to positively control both EBV episome maintenance and transcription on the one hand and the self-limitation of the EBV GMP expression on the other. As for EBV, one can consider it makes sense to have the same host cell protein regulating these two key aspects of EBV's latency. Indeed, if NCL level is low, then the maintenance and transcription of EBV episome should be compromised but, as a result of NCL role in GAr-based limitation of EBNA1 expression, EBNA1 mRNA should be more efficiently translated, which may compensate for its reduced level and favor the maintenance of EBV genome. On the contrary, if NCL level is high, then EBV episome will be efficiently maintained and transcribed, hence leading to a high level of EBNA1 mRNA, but then the increased NCL could further downregulate its translation, thereby limiting the level of EBNA1 and therefore its detection by the immune system. Importantly, the role of NCL in EBNA1 immune evasion involves its ability to interact with G4 structures present in EBNA1 mRNA, whereas its role in episome maintenance and transcription involves its ability to interact with EBNAI 's N-terminal 100 amino acids (hence upstream of the GAr domain of EBNA1 protein). Therefore, targeting the NCL-EBNA1 mRNA interaction should specifically affect EBNA1 immune evasion.

What could be NCL mechanism of action in GAr-based self-inhibition of translation and antigen presentation? EBNA1 G4 may constitute a recognition signal for NCL that is, itself, directly or indirectly, responsible for translation inhibition by interfering with either translation initiation and/or elongation machinery. Alternatively, NCL could stabilize G4 that, themselves, may inhibit the ribosome progression. Of note, as NCL-EBNA1 mRNA interaction occurs in the nucleus, either of these two possible mechanisms would explain why EBNA1 mRNA is translated mainly in mitosis, at a time when the nuclear envelope has been disaggregated. In either case, it is unlikely that the virus has developed a novel mechanism to exploit NCL for controlling gene expression. Rather, it is likely that this reflects a more general evolutionary conserved cellular pathway. The fact that NCL effect on GAr-based limitation of protein expression is also operant in yeast strengthens this hypothesis as yeast has no common evolutionary history with EBV.

Hence, the therapeutic effect of PhenDC3 and more generally the compounds of formula (I) as defined herein for treating and/or preventing EBV-related cancers has been successfully demonstrated. This is clearly novel regarding the already known use of some G4 ligands as general anticancer agents through their ability to bind to G4 that form in telomeric DNA, hence destabilizing telomeres ultimately thereby leading to apoptosis of cancer cells. In the present invention, we describe an effect of some particular G4-ligands, including PhenDC3 that, through binding to G4 that form in the GAr-encoding sequence of EBNA1 mRNA, prevent the interaction of the host cell protein nucleolin (NCL) with these G4, thereby interfering with the GAr-dependent immune evasion of EBV. Hence, the effect of some G4-ligands presented here is at the level of RNA (and not DNA) and is due to the ability of these G4 ligands to interfere with an original mechanism of EBV immune evasion, and has therapeutic applications for the treatment of a specific sub-class of cancers, the cancers linked to EBV that roughly represent 1 to 2-3% of cancers worldwide, by unveiling tumour cells from these particular cancers to the immune system of the host.

Claims

1. A compound of formula (I), or a hydrate or a solvate thereof, for use as a drug for preventing and/or treating an Epstein-Barr- Virus (EBV)-related cancer:

wherein

CH;

L is (A), (A'), (

q, r and s may be identical or different and are each independently an integer selected from 0 to 3; Ri to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a C₃-C₈ cycloalkyl group, a 0(CrC₆)alkyl group, a NR10R11 group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl or a 5- to 8-membered heterocycloalkenyl, said CrC₆ alkyl group, a C₃-C₈ cycloalkyl group, 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl or a 5- to 8-memebered heterocycloalkenyl being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' ;

Ri o and Rn may be identical or different and are each independently:

- a hydrogen atom,

NH-(CrC₆)alkyl group or a NHC(0)R'- group,

R' is:

- a C₂-C₆ alkynyl group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((CrC₆)alkyl)₂ group, a N((CrC₆)haloalkyl)₂ group or a C(0)-(5- to 10-)aryl group, or

- a (Ci-C₆)alkyl-(OCH₂CH)j optionally substituted with an azido group or a (Ci-C₆)alkyl group, wherein j being an interger between 1 and 6, preferably between 2 and 4 and wherein said (CrC₆)alkyl group is optionally substituted with a halogen atom, a 0(CrC₆)alkyl group, a C₂- C-6 alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8- membered heterocycloalkenyl, a N((CrC₆)alkyl)₂ group or a N((CrC₆)haloalkyl)₂ group or a biotinyl group.

2. A compound of formula (I), or a hydrate or a solvate thereof,

wherein

Zi is CH or NR⁺, provided that when Yi is CH , then Zi is NR⁺, and when Yi is NR⁺, then Zi is CH ;

- at least one of m, n and p is not 0;

- at least one of u and v is not 0.

s is 1 , 2 or 3;

t is 1 or 2;

q and r may be identical or different and are each independently an integer selected from 0 to 3, provided that at least one of q and r is not 0;

Ri to R₉ may be identical or different and are each independently a halogen atom, a CrC₆ alkyl group, a C₃-C₈ cycloalkyl group, a 0(CrC₆)alkyl group, a NRi ₀Rn group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl or a 5- to 8-membered heterocycloalkenyl, said CrC₆ alkyl group, a C₃-C₈ cycloalkyl group, 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl or a 5- to 8-memebered heterocycloalkenyl being optionally substituted with one to three halogen atoms, a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)-R' ;

Ri o and Rn may be identical or different and are each independently:

- a hydrogen atom,

- a Ci -C₆ alkyl group optionally substituted with a OH group, a 0-(CrC₆)alkyl group, a NH-(CrC₆)alkyl group or a NHC(0)R'- group,

- a (Ci -C₆)alkyl-(OCH₂CH)i-(Ci -C6)alkyl-NHC(0)-R', with i an interger between 1 and 6

(preferably 2, 3 or 4);

R' is:

- a (CrC₆)alkyl group optionally substituted with an azido group, a biotinyl group or a

5- to 10-membered aryl group, wherein said 5- to 10-membered aryl group is optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C6)alkyl)2 group or a N((Ci -C₆)haloalkyl)2 group,

- a C2-C-6 alkynyl group, or

- a 5- to 10-membered aryl group optionally substituted with a halogen atom, a CrC₆ alkyl group, a 0(CrC₆)alkyl group, a C₂-C₆ alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((CrC₆)alkyl)2 group, a N((CrC₆)haloalkyl)2 group or a C(0)-(5- to 10-)aryl group, or

- a (Ci -C6)alkyl-(OCH₂CH)j optionally substituted with an azido group or a (CrC₆)alkyl group, wherein j being an integer between 1 and 6, preferably between 2 and 4 and wherein said (CrC₆)alkyl group is optionally substituted with a halogen atom, a 0(CrC₆)alkyl group, a C2-C-6 alkenyl group, a C₅-C₈ cycloalkenyl group, a 3- to 8-membered heterocycloalkyl, a 5- to 8-membered heterocycloalkenyl, a N((Ci -C6)alkyl)2 group or a N((Ci -C₆)haloalkyl)2 group or a biotinyl group.

3. The compound for use of claim 1 or the compound of claim 2, wherein Yi and Y2 are identical, and Zi and Z₂ are identical. 4. The compound for use of claim 1 or 3 or the compound of claim 2 or 3, wherein R is a linear C1-C4 alkyl, preferably an ethyl or a methyl group.

5. The compound for use of any of claims 1 , 3 and 4 or the compound of any of claims 2 to 4, wherein X^2- represents two anions selected from the group consisting of a halogenide, a carboxylate, a CrC₆alkylsulfonate, a Ci-C₆haloalkylsulfonate and an alkylarylsulfonate, preferably a halogenide, a methanesulfonate, a trifluoromethanesulfonate or a tosylate.

6. The compound for use of any of claims 1 and 3 to 5 or the compound of any of claims 2 to 5, wherein L is (A) or (Α'), and

- m and p are 0, and n is 0 or 1 , or

- m and n are 0, and p is 0 or 1 , or

- n is 0, and m and p are identical and are 0 or 1 .

7. The compound or the compound for use of claim 6, wherein m and p are 0, n is 0 or 1 , and R₂ is a halogen atom such as F or Br. 8. The compound or the compound for use of claim 6, wherein m and n are 0 and R3 is a linear NH(CrC₆)alkyl group or a NH-(Ci-C₆)alkyl-NHC(0)-R' group, with R' as defined above

in claim 1 , preferably

9. The compound or the compound for use of claim 6, wherein n is 0 and Ri and R3 may be identical or different and are each independently a halogen atom, a NH(CrC₆)alkyl group, preferably a NH(CrC4)alkyl group.

10. The compound for use of any of claims 1 and 3 to 5 or the compound of any of claims 2 to 5, wherein L is (B) or (C), preferably L is (B).

11. The compound or the compound for use of claim 10, wherein q, r and s are 0.

12. The compound for use of claim 1 , wherein it is:

14. A composition comprising : - as active ingredient, the compound of formula (I) as defined in any of claims 1 to 13, and optionally another therapeutic agent selected from antibiotics, anticancer agents, steroidal and non-steroidal anti-inflammatory drugs, and

- a pharmaceutically acceptable excipient,

for use for treating an Epstein-Barr-Virus (EBV)-related cancer.

15. The compound for use of any of claims 1 and 3 to 12, or the composition for use of claim 14, wherein the EBV-related cancer is a Hodgkin's lymphoma, a Burkitt's lymphoma, a nasopharyngeal carcinoma, a gastric cancer, lymphomas in immunosuppressed patients, T/NK cell lymphomas.