WO2024110392A1

WO2024110392A1 - New nucleic acid binding compounds and uses

Info

Publication number: WO2024110392A1
Application number: PCT/EP2023/082402
Authority: WO
Inventors: Alain Laurent; Arnaud Burr
Original assignee: bioMérieux
Priority date: 2022-11-22
Filing date: 2023-11-20
Publication date: 2024-05-30

Abstract

The invention concerns compounds of formula (I) and their salts, as defined into claim 1 and their uses, in particular in PCR applications.

Description

Title: New nucleic acid binding compounds and uses

FIELD

The invention is related to new compounds that can bind to nucleic acids, and in particular to DNA single strand (ssDNA) or DNA double strand (dsDNA). This binding corresponds to the binding of the compound to the acid nucleic structure, illustratively by weak bonds, in particular hydrogen or ionic bonds or may correspond to any other binding by weak bonds. The compounds according to the invention exhibit a change in fluorescence resulting from the binding of said compounds to the nucleic acids, typically ssDNA or dsDNA.

BACKGROUND OF THE INVENTION

Compounds which are able to bind or insert in DNA strands geometry and to lead to a change in fluorescence are useful tools for studying nucleic acids. These compounds, known as non-specific DNA binding dyes, can be used to detect and quantify DNA and RNA in a variety of environments, including solutions, cell extracts, electrophoretic gels, micro-array chips, live or fixed cells, dead cells, and environmental samples. These compounds have also been used in real-time polymerase chain reaction (real-time PCR or rt-PCR), a common gene detection technique widely used in both research and diagnostics. These compounds have also been used in melt curve analysis, a post-PCR DNA analysis technique. The melt curve analysis is specific and allows the identification of the gene, and so is useful for identifying gene mutation detection.

The patents US 7,456,281, US 7,387,887 and US 7,582,429 in the name of Idaho Technology and the University of Utah Research Foundation describe a broad class of compounds which are presented as suitable for detection of DNA. Nevertheless, the inventors of the present invention have reproduced certain examples of these patents and the obtained results show that these examples have fluorescence or stability that could be improved. It is interesting to have stability for this kind of compounds that allows their storage in aqueous and slightly alkaline conditions, corresponding to common conditions used in PCR. Thiols are commonly used in PCR media of reverse transcriptase PCR (RT-PCR) and are also present in buffered solutions used for the storage of PCR enzymes. Therefore, thiols can be present during PCR and some compounds described in these patents are poorly stable in the presence of thiols. Additionally, unlike other compounds described in US 7,387,887, the production of example A10 of US 7,387,887 cannot be carried out by the described process, which exclusively starts from ureas or thioureas. WO 2008/052742 in the name of Roche Diagnostics Gmbh proposes a new class of fluorescent dyes which are capable of emitting fluorescence when they are excited appropriately in the status of being specifically bound to a double stranded nucleic acid. This new class of fluorescent dyes comprises a pyrimidinyl ring in which the 5- and 6- positions of the pyrimidinyl ring are an integral part of a further aromatic structure, forming in particular a quinazolinyl structure. This part of the fluorescent dyes is presented as mandatory in this patent application WO 2008/052742, at the end of page 6, in order to obtain interesting excitation and emission spectra. In this patent application, it is stated that the proposed compounds have improved thermal and chemical stability, but the only stability study concerns the photochemical stability. In addition, this fused aromatic structure greatly influences the stability and the fluorescence of the molecules. The inventors of the current patent application showed that thiazole orange and SybrGreen, which include a quinolinyl, and so have a structure close to the ones described in WO 2008/052742, are not satisfactorily stable, both in PCR conditions and during storage in aqueous and slightly alkaline medium. In the present invention, the inventors propose new structures which do not include a pyrimidine forming an integral part of a further aromatic structure. The patent US 9,682,970 in the name of BIOTIUM describes numerous molecules including fluorescent nucleic acid dyes, and methods for use that includes nucleic acid detection, nucleic acid amplification reactions, and high-resolution melt curve analysis. A very broad formula is proposed for these molecules, which includes a large variety of structures. The substituents of the proposed structures may comprise a positively charged moiety, which has the effect of enhancing the nucleic acid binding affinity of the molecule via electrostatic interaction between the negatively charged phosphate backbone of nucleic acid and the positive charge of the moiety. This positively charged moiety may be covalently attached to the molecule by various kinds of arms starting from an N, O, S, or C atom (see the definition of L₁ and L₂ in column 15 of US 9,682,970). There is no example of this type of molecules including a pyrimidinium group, except molecules carrying a guanidino group, which is highly basic. An example is the following one:

There is no description of a method of preparation of this compound which, as a result, is not available to the public. Additionally, in this patent application, it is stated in column 53 that “The higher degree of protonation of guanidino and amidino than an amine side chain also makes guanidino and amidino more effective than the latter in enhancing the nucleic acid binding of compounds in the present invention via electrostatic interaction. Furthermore, amidino and guanidino, in particular, comprise multiple nitrogen atoms and acidic protons, which make amidino and guanidino good hydrogen bond acceptors and donors. Thus, in addition to electrostatic interaction, hydrogen bonding between the guanidino or amidino and the nucleic acid backbone may also play a role in enhancing the nucleic acid binding affinity of the compounds. These collective properties of guanidino and amidino make them superior alternative to an amine side chain in the context of nucleic acid dyes”. Several compounds belonging to the class of non-specific DNA binding dyes and instruments for DNA detection and/or analysis are commercially available: thiazole orange, SYBR® Green I, LCGreen® Plus (Clinical Chemistry 52:3, 2006, Mark G. Herrmann et al. “Amplicon DNA melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dye”) and LightCycler® 480 High Resolution Melting Dye (04909640001, Roche). Nevertheless, there remains a need for improvement in various aspects of nucleic acid binding dyes including but not limited to detection limit, dynamic range of detection and compatibility with different detection formats and instruments. It is the intention of the present invention to address some of these needs. In this context, an object of the invention is to provide new nucleic acid binding compounds, and more specifically DNA binding compounds (and in particular dsDNA binding compounds), which exhibit, in their form bound to nucleic acid (typically DNA, and in particular dsDNA), enhanced and detectable fluorescence. The purpose of the invention is to propose such new compounds with properties globally improved. In particular, the purpose of the invention is to provide compounds with both properties of stability and fluorescence, which are suitable for application in nucleic acid detection, and more particularly DNA detection. In particular, with respect to stability, the compounds according to the invention have a great stability at pH 9, in a test performed at 40°C (which is an accelerated test relevant for evaluation of the stability at ambient temperature). Additionally, other results show their stability in presence of thiols in an aqueous medium. With their particularly suitable stability and fluorescence stability, the compounds according to the invention lead to high performances in nucleic acid detection, nucleic acid amplification reactions, and high-resolution melt curve analysis. Additionally, they can be formulated and stored in aqueous media and this facilitates their quick implementation in the targeted nucleic acid detection methods, typically with the use of PCR techniques. SUMMARY OF THE INVENTION The invention concerns compounds having the formula (I):

wherein: - n is equal to 0, 1, 2 or 3; - Ri, Rj and Rk are identical or different and are independently selected from the group consisting of hydrogen and C_1-6alkyl; - X is oxygen, sulfur, selenium, tellurium or C(CH₃)₂, - Re is an alkyl, or a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or Re is – (CH₂)_k1-Y₁, in which: o k1 is 1, 2, 3, 4, 5 or 6, and o Y₁ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₁’R₁’’-(CH₂)_p1-]_m1-G₁’, with m1 being 1, 2 or 3, G₁’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p1 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₁’ and R₁’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; - Z is a fused mono or polycyclic aromatic or nitrogen-containing heteroaromatic ring, optionally substituted by one or several substituent(s) A identical or different selected from among halogen atoms and the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, - NHC(O)phenyl, -NHC(O)alkyl, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, -P(O₂)Oalkyl, – CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N- O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, –CONHR being preferred, R being either a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: ok2 is 1, 2, 3, 4, 5 or 6, and oY₂ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; - R₁ is an alkyl or –(CH₂)_k3-Y₃, in which: ok3 is 1, 2, 3, 4, 5 or 6, and oY₃ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₃’R₃’’-(CH₂)_p3-]_m3-G₃’, with m3 being 1, 2 or 3, G₃’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p3 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₃’ and R₃’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; - R₂, R₃ and R₄ are identical or different and are independently selected from among hydrogen, alkyl groups, aryl groups, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, and the groups –(CH₂)_k4-Y₄, in which: ok4 is 1, 2, 3, 4, 5, 6, and oY₄ is a group selected from among aryl, hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’, with m4 being 1, 2 or 3, G₄’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₄’ and R₄’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; with the proviso that at least one of the substituents A, R₂, R₃ and R₄ includes a secondary amine, a tertiary amine or a quaternary ammonium, including their salts with at least one anion, in particular, chosen from among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate. According to specific embodiments of the compounds of formula (I) and their salts, Z is a fused mono or polycyclic aromatic or nitrogen-containing heteroaromatic ring, optionally substituted by one or several substituent(s) A identical or different selected from among halogen atoms and the groups alkyl, alkenyl, alkynyl, aryl, -CF - 3, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -S(O₂)O , -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl, –CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)- R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR and – CONHR, –CONHR being preferred, R being either a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: ok2 is 1, 2, 3, 4, 5 or 6, and oY₂ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl. The compounds of the invention are nucleic acid binding compounds. The compounds according to the invention have a specific core structure which carries one or several substituents that include a function selected from among secondary amines, tertiary amines and quaternary ammoniums. Secondary amines, tertiary amines and quaternary ammoniums comprise or may comprise a positively charged moiety, which has the effect to provide binding affinity site for the nucleic acid. Indeed, electrostatic interaction may occur between the negatively charged phosphate backbone of nucleic acid and the positive charge of the functions selected from among secondary amines, tertiary amines and quaternary ammoniums. The positively charged functions may be a protonated amine (i.e. secondary or tertiary amine), or a quaternary ammonium, typically a trialkylammonium. Secondary and tertiary amines are bases whose basicity increases from secondary to tertiary. So, the fraction of their protonated form differs and increases from primary to tertiary and is a function of the pH of the medium where they are located. In targeted applications, illustratively, the pH will be in the range from 7.5-9.5, typically from 8- 9, and there will be a protonated fraction, in any case. A quaternary ammonium, typically a trialkylammonium, is a fully and permanently positively charged moiety independent of the pH of the medium. These functions selected from among secondary amines, tertiary amines and quaternary ammoniums are introduced by a specific link and on specific positions of the compounds according to the invention. All of these choices lead to compounds which exhibit both high fluorescence and stability when used in amplification techniques, in particular PCR techniques, but also great stability when stored in aqueous and slightly alkaline media, and even in the presence of a thiol entity. According to specific embodiments, the compounds according to the invention have the formula (II):

wherein: - X, Re, R₁, R₂, R₃, R₄, Ri, Rj, Rk and n are as defined for formula (I), previously; - Ra, Rb, Rc and Rd, identical or different, are selected from among hydrogen and halogen atoms and the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -NHC(O)phenyl, - NHC(O)alkyl, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl, –CH=N-O-R, - C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, - C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, –CONHR being preferred, R being either a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: o k2 is 1, 2, 3, 4, 5 or 6 and o Y₂ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; with the proviso that at least one of the substituents Ra, Rb, Rc, Rd, R₂, R₃ and R₄ includes a secondary amine, a tertiary amine or a quaternary ammonium; and in particular two substituents Ra, Rb, Rc, Rd, R₂, R₃ and R₄ include a secondary amine, a tertiary amine or a quaternary ammonium ; including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate. According to specific embodiments of the compounds of formula (II) and their salts, Ra, Rb, Rc and Rd, identical or different, are selected from among hydrogen and halogen atoms and the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl, –CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)- R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, –CONHR being preferred, R being either a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or – (CH₂)_k2-Y₂, in which: o k2 is 1, 2, 3, 4, 5 or 6 and o Y₂ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl. Typically, in compounds of formula (II) and their salts, at least two (and in particular two) of the substituents Ra, Rb, Rc, Rd, R₂, R₃ and R₄ include a secondary amine, a tertiary amine or a quaternary ammonium; and in particular at least two (typically two) substituents Ra, Rb, Rc, Rd, R₂, R₃ and R₄ include a quaternary ammonium, such as a trialkylammonium, typically a trimethylammonium. Advantageously, Rc and R₂ are the two substituents that include a quaternary ammonium, such as a trialkylammonium, typically a trimethylammonium. According to specific embodiments, the compounds according to the invention (corresponding to formula (I) or (II)) include one of the following features or any combination of the following features, and advantageously, when they do not exclude each other, all the following features: - at least one group Y₂, or Y₄, and in particular only one group Y₂ or Y₄ or the two groups Y₂ and Y₄, include(s) or is(are) a trialkylammonium, typically a trimethylammonium; - k2 is 3, 4, 5 or 6, and typically 3; - k3 is 2, 3, 4, 5 or 6; typically k3 is 2 or 3; - k4 is 4, 5 or 6, and typically 4, when Y₄ is a dialkylamino, a trialkylammonium, or -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’ as defined for formula (I); - Ri, Rj and Rk are hydrogen; - R₄ = H, R₁ and R₃ are identical or different and are C_1-6alkyl, and in particular methyl or ethyl; according to specific embodiments, R₄ = H, R₁ is methyl or ethyl and R₃ is methyl; - X is oxygen or sulfur, and in particular sulfur. By selecting the value of n, it is possible to adjust the properties of fluorescence obtained after the binding of a compound of the invention, with an amplicon of a nucleic acid. For instance, by comparison with compounds wherein n=0, compounds wherein n=1, 2 or 3 allow the detection of the amplification and the melting of the amplicon at higher excitation and emission wavelengths. This may be useful to reduce the fluorescent background of materials used in the detection/analysis of nucleic acids, such as plastic for example and, as a result, to increase the sensitivity of fluorescence detection, resulting from the binding of the compounds of the invention to nucleic acids, or more precisely to amplicons of nucleic acids. According to specific embodiments, in the compounds according to the invention n=0, and, in particular, the compounds of the invention have the formula (III):

(III), wherein R₁, R₂, R₃, R₄, Ra, Rb, Rc, Rd and Re are as defined for formula (I) or (II); including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate. Typically, in a first alternative of compounds according to the invention (including formula (I), (II) and (III)), and their salts, at least one (and typically one) of the groups R₂ and R₃ is –(CH₂)_k4-Y₄, in which: o k4 is 1, 2, 3, 4, 5 or 6 and o Y₄ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’, with m4 being 1, 2 or 3, G₄’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₄’ and R₄’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₄ being preferentially a trialkylammonium, typically a trimethylammonium, or -[N⁺Me₂-(CH₂)_p4-]_m4-N⁺Me₃ with p4 and m4 as described above; and the other group which is R₂ or R₃ is a C_1-6alkyl. In particular, R₂ is –(CH₂)_k4-Y₄ as described above and R₃ is a C_1-6alkyl, typically a methyl group. According to specific embodiments of this first alternative, the compounds of the invention have the formula (IIa):

and, in particular, the formula (IIIa), (IIIa), wherein: - Rc is a hydrogen or halogen atom or a group selected from among the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -NHC(O)phenyl, -NHC(O)alkyl, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl,–CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)- R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, the –CONHR group being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: o k2 is 1, 2, 3, 4, 5 or 6; and typically k2 is 3, 4, 5 or 6; o Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium, or -[N⁺Me₂-(CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above; - X, Re, R₁, R₂, R₃, R₄, Ri, Rj, Rk and n are as defined for formula (I), and previously for the first alternative; including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate. According to specific embodiments of this first alternative, and in particular in formula (IIa) or (IIIa), Rc is a hydrogen or halogen atom or a group selected from among the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, - Oalkyl, -NHalkyl, -NHC(O)H, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl,–CH=N-O- R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, - C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, the –CONHR group being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: o k2 is 1, 2, 3, 4, 5 or 6; and typically k2 is 3, 4, 5 or 6; o Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium, or -[N⁺Me₂-(CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above. According to specific embodiments of this first alternative, and in particular in formula (IIa) or (IIIa), Rc is a hydrogen atom, an halogen atom, in particular Br, - NHC(O)alkyl, in particular -NHCOMe, –NHCOR or –CONHR, with R being –(CH₂)_k2-Y₂, in which: o k2 is 3, 4, 5 or 6; in particular k2 is 3; o Y₂ is a trialkylammonium or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, G₂’ being a trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium, or -[N⁺Me₂-(CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above, and in particular m2 = 1 and p2 = 2 or 3. According to specific embodiments of this first alternative, and in particular in formula (IIa) or (IIIa), R₄ = H, R₁ and R₃ are identical or different and are C_1-6alkyl, and in particular methyl or ethyl, and R₂ is –(CH₂)_k4-Y₄, in which k4 is 4, 5 or 6 and Y₄ is a trialkylammonium, in particular trimethylammonium and Rc is hydrogen or – NHCOR or –CONHR, with R being –(CH₂)_k2-Y₂, in which k2 is 3, 4, 5 or 6 and Y₂ is a trialkylammonium, in particular a trimethylammonium, or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, G’₂ being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3, and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, typically a methyl. According to a second alternative, in the compounds according to the invention, and their salts, Z, or the corresponding group in formula (II), (IIa), (III) and (IIIa), is substituted by at least one substituent selected from among –CH=N-O-R, - C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, - C(CH₃)=N-O-C(O)-R, –NHCOR or –CONHR, –CONHR being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which k2 is 1, 2, 3, 4, 5 or 6 (and typically k2 is 3, 4, 5 or 6) and Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium or -[N⁺Me₂-(CH₂)_p2-]_m2-N+Me₃ with p2 and m2 as described above. In other words: - in compounds of formula (II) and (III), and their salts, at least one substituents Ra, Rb, Rc or Rd is selected from among the groups –CH=N-O-R, -C(CH₃)=N-O- R, -CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O- C(O)-R, –NHCOR or –CONHR, –CONHR being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which k2 is 1, 2, 3, 4, 5 or 6 (and typically k2 is 3, 4, 5 or 6) and Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium or -[N⁺Me₂-(CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above; - and in compounds of formula (IIa) and (IIIa) and their salts, Rc is selected from among –CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR or –CONHR, –CONHR being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which k2 is 1, 2, 3, 4, 5 or 6 (and typically k2 is 3, 4, 5 or 6) and Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2- G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium or -[N⁺Me₂- (CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above. According to specific embodiments of this second alternative, the compounds of the invention have the formula (IIa):

and in particular the formula (IIIa), (IIIa), wherein Rc is –CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR or –CONHR, –CONHR being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which k2 is 1, 2, 3, 4, 5 or 6 (typically k2 is 3, 4, 5 or 6) and Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G’₂ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium ; and X, Re, R₁, R₂, R₃, R₄, Ri, Rj, Rk and n are as defined for formula (I), and previously for the second alternative, including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate. According to specific embodiments of this second alternative, R₄ = H, R₁ and R₃ are identical or different and are C_1-6alkyl, and in particular methyl or ethyl, and R₂ is hydrogen, C_1-6alkyl, and in particular methyl or ethyl, or –(CH₂)_k4-Y₄, in which k4 is 4, 5 or 6 and Y₄ is a trialkylammonium, in particular trimethylammonium and Rc is –NHCOR or –CONHR, with R being –(CH₂)_k2-Y₂, in which k2 is 3, 4, 5 or 6 and Y₂ is a trialkylammonium, in particular a trimethylammonium, or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, G’₂ being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3, and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, typically a methyl. According to other specific embodiments of the compounds according to the invention (including the first and the second alternatives and all the disclosed embodiments), R₄ = H, R₁ and R₃ are identical or different and are C_1-6alkyl, and in particular methyl or ethyl, and R₂ is –(CH₂)_k4-Y₄, in which k4 is 1, 2, 3, 4, 5 or 6 (typically k4 is 4, 5 or 6) and Y₄ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’, with m4 being 1, 2 or 3, G₄’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₄’ and R₄’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₄ being preferentially a trialkylammonium, typically a trimethylammonium. According to other specific embodiments of the compounds according to the invention (including the first and the second alternatives and all the disclosed embodiments), R₄ = H, R₁ is methyl or ethyl and R₃ is methyl. Depending on the substituent considered, the secondary amine, tertiary amine or quaternary ammonium (defined in the proviso) may correspond to the following groups in the compounds of formula (I), (II), (IIa), (III) and (IIIa): piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, morpholinyl, alkylamino, dialkylamino and trialkylammonium groups, including the ones present in the groups -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’ and -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’, as defined in the present specification. According to specific embodiments of all the compounds described in the invention, Re is typically C_1-6alkyl, in particular methyl. In particular, when the compounds described in the invention include one or several quaternary ammonium, this quaternary ammonium is trimethylammonium. According to specific embodiments of the compounds according to the invention (including the first and the second alternatives and all the disclosed embodiments), R₂ is –(CH₂)_k4-Y₄, in which k4 is 4, 5 or 6 and Y₄ is a trialkylammonium, in particular trimethylammonium. According to specific embodiments of the compounds according to the invention (including the first and the second alternatives and all the disclosed embodiments), Rc is –NHCOR or –CONHR, with R being –(CH₂)_k2-Y₂, in which k2 is 3, 4, 5 or 6 and Y₂ is a trialkylammonium, in particular a trimethylammonium, or -[N⁺R₂’R₂’’-(CH₂)_p2- ]_m2-G₂’, with m2 being 1, G’₂ being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3, and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, typically a methyl. According to specific embodiments of all the compounds described in the invention, they include at least one quaternary ammonium, preferentially a trialkylammonium, typically a trimethylammonium. According to specific embodiments of all the compounds described in the invention, they are in the form of a trifluoroacetate salt. Specific compounds of the invention are compounds of the following formula:

(IIa), with n as defined for formula (I) and n being typically equal to 0, 1 or 2, Ri, Rj and Rk as defined for formula (I) and typically Ri=Rj=Rk=H,

and in particular the formula (IIIa), (IIIa), wherein: - Re is methyl, - R₄=H, - R₁ and R₃ are an alkyl group, in particular, methyl or ethyl, and typically R₁ and R₃ are methyl, - and R₂ and Rc are defined as follows: i) Rc is H, an halogen atom, typically Br, or –NHCOalkyl, typically –NHCOMe, and R₂ is –(CH₂)_k4-Y₄, in which k4 is 4, 5 or 6 and Y₄ is a trialkylammonium, in particular trimethylammonium, typically R₂ is – (CH₂)₄-N⁺Me₃; or, ii) R₂ is an alkyl, typically methyl, and Rc is –NHCOR or –CONHR, with R being –(CH₂)_k2-Y₂, in which k2 is 3, 4, 5 or 6 and Y₂ is a trialkylammonium, in particular a trimethylammonium, or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, G’₂ being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3, and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, typically a methyl; more specifically, R₂ is an alkyl, typically methyl, and Rc is –CONHR with R being –(CH₂)_k2-Y₂, in which k2 is 3, 4, 5 or 6 and Y₂ is a trialkylammonium, in particular a trimethylammonium, or -[N⁺R₂’R₂’’-(CH₂)_p2- ]_m2-G₂’, with m2 being 1, G’₂ being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3, and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, typically a methyl ; typically R₂ is methyl and Rc is –CONH- (CH₂)₃-N⁺Me₃, –CONH-(CH₂)₃-N⁺Me₂-(CH₂)₃-N⁺Me₃ or –NHCO-(CH₂)₃-N⁺Me₃; or iii) R₂ is –(CH₂)_k4-Y₄, in which k4 is 4, 5 or 6 and Y₄ is a trialkylammonium, in particular trimethylammonium and Rc is –NHCOR or –CONHR with R being – (CH₂)_k2-Y₂, in which k2 is 3, 4, 5 or 6 and Y₂ is a trialkylammonium, in particular a trimethylammonium, or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, G’₂ being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3, and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, typically a methyl ; typically R₂ is -(CH₂)₄-N⁺Me₃ and Rc is –CONH-(CH₂)₃-N⁺Me₃, –CONH- (CH₂)₃-N⁺Me₂-(CH₂)₃-N⁺Me₃ or –NHCO-(CH₂)₃-N⁺Me₃, and their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate; and typically their trifluoroacetate salts. As way of examples, the compounds according to the invention are selected from among:

including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate, in particular their trifluoroacetate salt. The invention also concerns the uses of these compounds in acid nucleic detection and analysis and corresponding methods, mixtures and kits, as defined in the section “Uses of the compounds according to the invention”. In these cases, “compound” means a compound of any formula given previously in the disclosure of the compounds or a salt of this compound. In particular, compounds of formula (I), (II), (IIa), (III), (IIIa) or of any other formula given in the specification falling in the scope of formula (I) is used in the form of a salt as described in the specification, in the uses, methods, mixtures and kits of the invention. The use of a compound according to this invention, for the detection of a target nucleic acid which is a single stranded or double stranded nucleic acid, is another object of the invention. The invention is also relating to a method for detecting a target nucleic acid which is a single stranded or double stranded nucleic acid comprising a step of mixing a compound in accordance with the invention, with a sample comprising the target nucleic acid or an amplicon of the target nucleic acid. In the uses and the methods according to the invention, the following steps may be carried out: - amplifying the target nucleic acid to generate the amplicon, - adding a compound according to the invention to the sample comprising the target nucleic acid and/or the amplicon, before, during or after the amplifying step, - monitoring fluorescence from the compound according to the invention during or subsequent to the amplifying step. According to particular embodiments, in the use and the method according to the invention, the following steps are carried out: - amplifying the target nucleic acid, in the presence of the compound according to the invention, in particular by PCR, to generate the amplicon, and - during the amplification, monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon. According to specific embodiments, subsequent to the amplification step, a step of melting the generated amplicon is carried out, while monitoring the fluorescence from the compound according to the invention, to obtain a melting curve. It is possible to obtain a melting curve when the target nucleic acid is a double stranded nucleic acid, in particular a dsDNA. In particular, a melting curve can be obtained with any double stranded nucleic acid that can melt and that is capable of hybridization to a complementary nucleic acid by Watson-Crick base pairing, such as DNA, DNA-RNA hybrid, which could also include nucleotide analogs (e.g. BrdU) and/or non-phosphodiester internucleosidic linkages ( e.g., peptide nucleic acid (PNA) or thiodiester linkages). According to specific embodiments, the use and the method according to the invention may include a step of quantifying the target nucleic acid, which is initially present in the sample. When the method for detecting a target nucleic acid according to the invention comprises a step of mixing a compound in accordance with the invention, with a sample comprising an amplicon of the target nucleic acid, the quantity of target nucleic acid initially present in the sample obtained by the step of quantifying is the quantity of the target nucleic acid which is present in the sample used for obtaining the amplicon, by an amplifying step. The invention also concerns a method of PCR analysis of a target nucleic acid comprising the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid, a polymerase, and a pair of primers suitable to amplify a portion of the target nucleic acid and generate at least an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture and generating at least an amplicon, and - monitoring the fluorescence from the compound according to the invention during or subsequent to the amplifying step. According to some embodiments, such a method of PCR analysis further comprises detecting the presence of the amplicon from the monitored fluorescence. In these methods of PCR analysis, the monitoring step may occur subsequent to amplification and includes generating a melting curve. According to specific embodiments, the said melting curve is used to identify the genotype of the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism, and/or epigenetic variation. According to specific embodiments, a method of PCR analysis according to the invention may include a step of quantifying the target nucleic acid, which is initially present in the sample. Illustratively, a method of PCR analysis of a target nucleic acid according to the invention may comprise the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid and at least a pair of primers suitable to amplify a portion of the target nucleic acid and generate an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture, and generating at least an amplicon, - during the amplifying step, monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon, - at the end of the amplifying step, melting the generated amplicon, to obtain a melting curve, and - identifying the genotype or polymorphism of the target nucleic acid using a shape of the melting curve. According to particular embodiments, whatever the methods according to the invention, the amplifying step may include a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 90 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.005 µM or 0.02 U/µL and primers are each provided at a concentration of at least 0.1 µM. According to specific embodiment, the amplifying step includes a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 20 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.5 µM or 1.9 U/µL and primers are each provided at a concentration of at least 2 µM. Another object of the invention is a PCR reaction mixture comprising: - a target nucleic acid, - a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, - a polymerase, in particular a thermostable polymerase, - a compound in accordance with the invention. The PCR reaction mixture according to the invention, may be in a buffer of pH from 7.5 to 9.5, preferentially from 8 to 9. Another object of the invention is a kit for detecting a target nucleic acid, comprising: - a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, - a polymerase, in particular a thermostable polymerase, and - a compound in accordance with the invention. The kit according to the invention, typically, includes a buffer of pH from 7.5 to 9.5, preferentially from 8 to 9. In particular, in such a kit, the compound in accordance with the invention is provided in the buffer. DEFINITIONS In the compounds according to the invention, the meaning of the substituents is usual, if it is not specified otherwise. The term “alkyl” as used in the invention refers to a monovalent saturated hydrocarbon moiety comprising from 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms. An alkyl group may be linear or branched and, illustratively includes methyl (Me), ethyl, propyl, butyl, dodecyl, 4-ethylpentyl, and the like. C_1-6alkyl refers to alkyl comprising 1, 2, 3, 4, 5 or 6 carbon atoms and typically to methyl. The term "alkenyl", as used herein, refers to monovalent hydrocarbon moieties comprising from 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms, which contain at least one carbon-carbon double bond, wherein each double bond can have E- or Z-configuration. The term "alkynyl", as used herein, refers to monovalent hydrocarbon moieties comprising from 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms, which contain at least one carbon-carbon triple bond. The alkenyl and alkynyl groups can be linear or branched. Double bonds and triple bonds in alkenyl groups and alkynyl groups respectively can be present in any positions. Examples of alkenyl and alkynyl are ethenyl, prop-1-enyl, prop-2-enyl, but-2-enyl, 2- methylprop-2-enyl, 3-methylbut-2-enyl, hex-3-enyl, hex-4-enyl, prop-2-ynyl, but-2- ynyl, but-3-ynyl, hex-4-ynyl or hex-5-ynyl. The term “aryl” or aromatic cyclic ring (that can be mono or polycyclic and which are fused in the definition of Z) as used in the invention refers to a cyclic aromatic hydrocarbonated moiety, illustratively including but not limited to phenyl (Ph) and naphthyl. Phenyl is the illustrative aryl group and in Z is named as fused-benzo. Nitrogen containing aromatic ring used in the definition of Z refers to pyrrolo, pyrazolo, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazalinyl, and the like. In particular, according to the definition of Z, typically when fused with the other cycle ring presented in formula (I), Z can form an optionally substituted benzoxazolium or benzothiazolium ring, or an optionally substituted naphthoxazolium or naphthothiazolium ring. Amino means –NH₂. Alkylamino means –NHR’ with R’ being an alkyl, in particular a C_1-6alkyl, typically methyl or ethyl. So, alkylamino corresponds to a secondary amine. Dialkylamino means –NR’R” with R’ and R” being, independently, an alkyl, in particular a C_1-6alkyl, typically methyl or ethyl. Most of the time, R’ = R”. So, dialkylamino corresponds to a tertiary amine. The most common dialkylamino groups illustrated herein are –NMe₂ and -NEt₂. Trialkylammonium means –N⁺R’R”R”’ with R’, R” and R’” being, independently, an alkyl, in particular a C_1-6alkyl, typically methyl or ethyl. Most of the time, R’ = R” = R’’’. The most common dialkylamino groups illustrated herein are –N⁺Me₃ and -N⁺Et₃. If it is not specified otherwise, the piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups (named nitrogen groups) can be covalently bonded to the rest of the molecules by one of their carbon atoms or by their nitrogen atom. When the covalent bond of these nitrogen groups to the core of the molecule is made by one of their carbon atoms, the piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups include the unsubstituted corresponding groups and the substituted corresponding groups where the nitrogen atom of the piperidinyl, piperazinyl, pyrrolidinyl or morpholinyl group is substituted in the N-position by one or two C_1-6alkyl group(s) (typically a methyl or an ethyl), and the nitrogen atom of the pyridinyl group is substituted in the N-position by one C_1-6alkyl group (typically a methyl or an ethyl) and is then in an ammonium form. When the covalent bond of these nitrogen groups to the core of the molecule is made by their nitrogen atom, the piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups include the unsubstituted corresponding groups and the substituted corresponding groups where the nitrogen atom of the piperidinyl, piperazinyl, pyrrolidinyl or morpholinyl group is substituted in the N-position by one C_1-6 alkyl group (typically a methyl or an ethyl) and so could be in an ammonium form. The compounds of the invention are cyanine derivatives, having a pyrimidinium core structure, wherein X is, in particular, oxygen or sulfur and the moiety Z represents an optionally-substituted fused benzo, forming an optionally substituted benzoxazolium or benzothiazolium ring, or an optionally-substituted fused naphtho, forming an optionally substituted naphthoxazolium or naphthothiazolium ring. It is appreciated that the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts described herein may contain one or several chiral centers. In those cases, all stereoisomers are understood to be included in the description of these compounds, unless otherwise indicated. Such stereoisomers include pure enantiomers, racemic mixtures, mixtures of enantiomers in any relative amount, pure diastereoisomers and mixtures of diastereoisomers containing any relative amount of one or more stereoisomeric configurations. It is also appreciated that the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts herein may contain geometric centers. In those cases, all geometric isomers are understood to be included in the description of the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts, unless otherwise indicated. Such geometric isomers include cis, trans, E and Z isomers, either in pure form or in various mixtures of geometric configurations. It is also understood that depending upon the nature of the double bond contained in the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts, such double bond isomers may interconvert between cis and trans, or between E and Z configurations depending upon the conditions, such as solvent composition, solvent polarity, ionic strength, and the like. So, the two forms cis/trans or E/Z are most of the time, in equilibrium. In the presented formula (III), (IIIa), (I.1) to (I.9), even if the double bond is presented in a specific geometric form, all possible isomeric forms are in equilibrium. The compounds according to the invention are in the form of a salt: the compound of formula (I), (II), (IIa), (III) or (IIIa) are positively charged due to the N⁺(Re). They may include an additional charge when they include a quaternary ammonium group. The salts of the compounds of formula (I), (II), (IIa), (III) or (IIIa) include a number of anions (typically which are identical) corresponding to the number of positive charges on the compound of formula (I), (II), (IIa), (III) or (IIIa). As the compounds of formula (I), (II), (IIa), (III) and (IIIa) are positively charged, several resonance structures of these compounds may exist. Typically, a positive charge may be formally localized on the nitrogen atom N⁺(Re) as depicted in Formula (I), (II), IIa), (III) and (IIIa), or alternatively, the charge may be localized on the pyrimidinyl group. "Nucleic acid" as used herein refers to a naturally occuring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleosidic linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof. PREPARATION OF THE COMPOUNDS ACCORDING TO THE INVENTION When n=0 and X=S, the compounds according to the invention can be obtained by the route presented on Scheme 1, in which Z, Ri, Re, R₁, R₂, R₃ and R₄ are as defined for (I).

The inventors had developed a new route for the preparation of these compounds, which differs from the route described in US 7,387,887. In particular, when R₂ is not H, this new route is not suitable for introducing an aryl group in the position corresponding to R₁. In Scheme 1, a conveniently substituted amidine (IX) is reacted with a conveniently substituted dicetone (VIII) in strongly alkaline conditions (for instance, in the presence of potassium carbonate), typically in a mixture of water/ethanol for 1 to 6 weeks at room temperature (typically 22°C) to obtain the pyrimidine molecule (VII) which can be purified by column chromatography or by liquid/liquid extraction. The pyrimidine molecule (VII) is alkylated at the N1 position by reaction with an excess of an alkylating agent (VI) (P-R₁, with P being Cl, Br or I or a tosyl group and R₁ as defined for (I)), typically in acetonitrile at 50-90°C in a closed tube for 1-6 days to give the pyrimidinium compound (V). After evaporation of the remaining alkylating agent (VI), the compound (V) is reacted with a compound (IV) (typically a benzothiazolium derivative), typically in a mixture of acetonitrile and ethanol and triethylamine at room temperature (typically 22°C), for a few minutes to yield the expected asymmetric cyanine (I), which may be purified by reverse phase chromatography. Examples of the alkylating agent (VI) P-R₁ are MeI, EtI, Br(CH2)3N(Me3). If R₂ = H, the alkylating agent (VI) can also be R₁-Tosyl. The compounds (IX), (VIII) and (VI) are commercially available or prepared according to common practice from commercially available compounds. The compounds (IV) can also be commercially available. When the compounds (IV) are not commercially available, in particular, when they are substituted on the Z cycle, they can be prepared as described hereafter on Scheme 2 (routes 1 to 3 concerning the synthesis of certain compounds of formula (IV)), or according to methods similar to the described ones. Scheme 2 concerns compounds (IV) where Z is a fused-benzo and Re is Me and describes different ways to introduce a substituent Rc, on this fused-benzo. In route 1, Rc is –CONH-(CH₂)₃-N⁺(Me)₃. In route 2, Rc is – NHCO-(CH₂)₃-N⁺(Me)₃. In route 3, Rc is –CONH-(CH₂)₃-N⁺(Me)₂-(CH₂)₃-N⁺(Me)₃.

According to route 1, commercial ester 4 is hydrolyzed in alkaline conditions to yield the carboxylic acid 5 which is activated under the form of a hydroxysuccinimidyl ester 6. This hydroxysuccinimidyl ester 6 can be conveniently substituted by an amine, such as amine 7. Peralkylation of the nitrogen atoms in the alkyl chain and in the benzothiazole ring are, advantageously, done at the same time by an alkylating agent at elevated temperature to yield expected products VI.1. Route 3 is similar to route 1, but with the introduction of a more complex group Rc including two ammonium group (with one -N⁺(Me)₃ and –N(Me)+₂- in the illustrative example). According to route 2, the commercial amino carboxylic acid 11 is activated under the form of a hydroxysuccinimidyl ester 12 and conjugated to the amino benzothiazole 13, before peralkylation of the amino atoms in the alkyl chain and in the benzothiazole ring as described in route 1 or 3 to obtain the compound VI.2. Other compounds of formula (IV) can easily be prepared with other routes, adapted or close to routes 1 to 3. Depending on the wished compound, the skilled person will choose the more appropriate step to be conducted. When n=1, 2 or 3, X=S and Ri =Rj=Rk= H, the compounds according to the invention can be obtained by the synthesis reactions presented in Scheme 3 in which Z, Re, R₁, R₂, R₃ and R₄ are as defined for (I).

( ) As shown on Scheme 3, conveniently substituted methyl benzothiazoles (X) are N- alkylated to obtain the compound (IX), in particular at high temperature (for instance from 130 to 160°C) without solvent in the presence of an alkylating agent Re-P with P being a leaving group, for instance a Cl, Br or I or a tosyl group and Re as defined for (I). The leaving group the most suitable for the Re group will be chosen. When Re=Me, MeOTs can be used. The obtained activated compounds (IX) can then react with a masked aldehyde as bis phenyl imines (XII), to yield in the presence of acetic anhydride and acetic acid, or only by fusing, the corresponding acetylated hemicyanines (XIII) which can be purified by reverse phase chromatography using a acetonitrile/water/TFA eluents. The hemicyanine (XIII) can then be reacted in slightly alkaline conditions with activated pyrimidinium (V.1), to obtain the expected compounds (I). When X=O, or when X= C(CH₃)₂, the same route as described in Schemes 1 and 3 can be followed, except that the starting benzothiazole compound (X) is replaced by respectively a benzoxazole or a dimethyl indole as starting compound. The compounds of formula (IX) can be prepared in a similar way as described for compounds of formula (IV). The compounds (X) and (XII) are commercially available or prepared according to common practice from commercially available compounds. USES OF THE COMPOUNDS ACCORDING TO THE INVENTION The compounds of the present invention may be used for a variety of different applications. In particular, they may be used for the detection of a target nucleic acid which is a single stranded or double stranded nucleic acid. The compounds according to the invention have the ability to bind to nucleic acids, and in particular to double strand nucleic acids, typically to single strand DNA (ssDNA) or double strand DNA (dsDNA), typically when they are in aqueous media of pH 5-11, and as a result in aqueous medium of pH 7.5-9.5, typically of pH 8-9, generally used in PCR media. The compounds according to the invention have also the ability to bind to RNA. This binding to nucleic acid corresponds to weak bonds, in particular hydrogen or ionic bonds or may correspond to any other binding by weak bond. The compounds according to the invention exhibit a change in fluorescence resulting from their binding to a nucleic acid, and in particular to double strand nucleic acid, typically ssDNA or preferentially dsDNA. The compounds according to the invention are able to interact with nucleic acid (i.e. RNA or DNA strands), typically with double stranded DNA, in particular the minor groove of a DNA double helix, and more generally to bind to the DNA strands by several kinds of weak binding and to lead to a change in fluorescence, which can be monitored. As a result, they are useful tools for studying nucleic acids. After their binding with nucleic acids, the compounds according to the invention behave as fluorescent dyes. So, the invention encompasses uses and methods for detecting a target nucleic acid which is a single stranded or double stranded nucleic acid comprising a step of mixing a compound according to the invention, with a sample comprising the target nucleic acid. The invention is also related to a method for detecting a target nucleic acid which is a single stranded or double stranded nucleic acid comprising a step of mixing a compound in accordance with the invention, with a sample comprising the target nucleic acid or an amplicon of the target nucleic acid. In particular embodiments, the compounds according to the present invention are used for the detection of double stranded nucleic acids during a nucleic acid amplification reaction in real time and/or subsequent to amplification via a melting curve analysis or end-point analysis. In this context, the compound of the invention will be a part of an amplification, preferably a PCR, reaction mixture and it can already be present at the beginning of the amplification reaction. As it has been shown by the inventors, the compounds according to the invention do not significantly interfere with the efficiency of such a amplification (preferably PCR) reaction. In particular, the compounds according to the invention do not significantly inhibit amplification (preferably PCR) when present at concentrations that provide high fluorescence signal for an amount of nucleic acid, and typically dsDNA generated by PCR, in the absence of the compound according to the invention. In the use or the method according to the invention, the following steps may be carried out: - amplifying the target nucleic acid to generate an amplicon, - adding a compound according to the invention to the sample comprising the target nucleic acid and/or the amplicon, before, during or after the amplifying step, - monitoring fluorescence from the compound according to the invention during or subsequent to the amplifying step. According to particular embodiments, in the use and the method according to the invention, the following steps are carried out: -amplifying the target nucleic acid, in the presence of the compound according to the invention, in particular by PCR, to generate an amplicon, and - during the amplification, monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon. According to specific embodiments, subsequent to the amplification step, a step of melting the generated amplicon is carried out, while monitoring the fluorescence from the compound according to the invention, to obtain a melting curve. Of course, in the methods and uses according to the invention, the compound of the invention and the amplicon are placed in a sample, which is suitable for their binding and the fluorescence obtaining. Typically, this sample is an aqueous medium of pH 7.5-9.5, more specifically of pH 8-9. So, the methods and uses according to the invention include a step of contacting the generated amplicon and the compound according to the invention, in conditions which enable their binding, in particular by weak bond(s). When the compound is present during the amplification, the usual conditions of amplification, in particular of PCR, enable this binding. In general, the binding of the compound of the invention with an amplicon occurs at a temperature lower than the melting temperature of the amplicon, when the target nucleic acid is a double stranded nucleic acid, typically dsDNA. The temperature during the binding, is, for instance, in the range from 20 to 60°C. The sample containing the generated amplicon and the compound according to the invention, often, includes one or several salts commonly used in PCR medium like NaCl and MgCl₂. According to specific embodiments, in the use or the method according to the invention, the following steps may be carried out: - amplifying of the target nucleic acid, in the presence of the compound according to the invention, in particular by PCR, and typically by real time PCR, to produce an amplicon, - optionally during the amplifying step, monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon, and - optionally subsequent to the amplifying step, monitoring the fluorescence resulting from the binding of the compound according to the invention to the amplicon, via an end-point analysis or while melting the amplicon to obtain a melting curve. It could also be possible to carry out the amplifying step first and to add the compound according to the invention later, in particular if the purpose is to obtain a melting curve or if an end-point analysis of the fluorescence is aimed. Whether the compound according to the invention is present during amplification or is added subsequently, the melting step allows the analysis of the targeted nucleic acid, for instance the identification of a specific genotype or polymorphism. According to specific embodiments, the said melting curve is used to identify the genotype of the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism, and/or epigenetic variation. A melting curve (also called melt curve) is generated by slowly denaturing (melting) the generated amplicon. The generation of melting curves and the use for analysis of nucleic acid are known in the art. More precisely, when a melting curve analysis is used for the detection or the quantification of a target double stranded nucleic acid (preferably dsDNA), the mixture containing the generated amplicon and the compound according to the invention is subjected to a thermal gradient. Preferably, the gradient is a continuous gradient, but step gradients are also possible. Most preferably, the gradient is a linear gradient. In one particular embodiment, the sample is subjected to a temperature increase which results in the generation of a dissociation curve. According to other embodiments, the double stranded nucleic acid (preferably dsDNA) is first thermally denatured into single strands and temperature dependence of fluorescence is monitored during subsequent renaturation. In the method and uses according to the invention, the amplification of the target nucleic acid, and in particular of the target DNA, can be carried out by different techniques, and in particular by enzymatic amplification reaction. By «enzymatic amplification reaction», it should be understood a process generating multiple copies of a target nucleotide fragment, by the action of at least one enzyme. Such amplification reactions (isothermal or not) are well known to one skilled in the art and the following techniques may be mentioned in particular: PCR (Polymerase Chain Reaction) of all kind (for instance, standard PCR, RealTime-PCR, quantitative PCR, digital PCR, multiplex PCR, asymetric PCR, nested PCR, semi-nested PCR, LATE-PCR, Touchdown PCR, Hot-Start PCR, COLD-PCR, assembly PCR), LCR (Ligase Chain Reaction), RCR (Repair Chain Reaction), 3SR (Self Sustained Sequence Replication) with the patent application WO-A-90/06995, NASBA (Nucleic Acid Sequence-Based Amplification), SDA (Strand Displacement Amplification), MDA (Multiple Displacement Amplification), RPA (Recombinase Polymerase Amplification), HDA (Helicase Dependent Amplification, RCA (Rolling Circle Amplification), TMA (Transcription Mediated Amplification) with U.S. Pat. No. 5,399,491, and LAMP (Loop mediated isothermal amplification) with the U.S. Pat. No. 6,410,278, RT-LAMP (Reverse Transcription-Loop-mediated isothermal Amplification). When the enzymatic amplification reaction is a PCR, we will talk more particularly of RT-PCR (RT standing for «reverse transcription»), when the amplification step is preceded by a messenger RNA reverse-transcription step (mRNA) into complementary DNA (cDNA), and from qPCR or RT-qPCR when PCR is quantitative. These techniques use a pair of primers (consisting of two primers), typically one forward primer and one reverse primer. Herein, when in the methods, uses, kits and mixtures, it is mentioned that a pair of primer is used, that means that one or several pairs of primers can be used. A primer is a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably of 15 to 30 nucleotides, and possesses a specificity of hybridization with a target nucleic acid sequence, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleic acid sequence. For instance, when one reverse primer and several forward primers or alternatively one forward primer and several reverse primers are used in an amplification, they form several pairs of primers. When it is desired to detect variants or different target nucleic acids simultaneously from the same sample and the same amplification, several pairs of primers will be used and so several amplicons are produced. This is the multiplex amplification and generally this is used with the PCR method. For this purpose, the methods, uses, kits and mixtures according to the invention will include more than one pair of primers: one for each target nucleic acid. Methods of PCR analysis using a compound according to the invention are particularly interesting. The invention also concerns a method of PCR analysis of a target nucleic acid comprising the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid, a polymerase, and a pair of primers suitable to amplify a portion of the target nucleic acid and generate at least an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture and generating at least an amplicon, and - monitoring the fluorescence from the compound according to the invention during or subsequent to the amplifying step. According to some embodiments, such a method of PCR analysis further comprises detecting the presence of the amplicon from the monitored fluorescence. In these methods of PCR analysis, the monitoring step may occur subsequent to amplification and may include generating a melting curve or end-point analysis of the fluorescence. According to specific embodiments, the said melting curve, and in particular its shape, is used to identify the genotype of the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism (SNP), and/or epigenetic variation. When end-point analysis of the fluorescence are done, the total amount of fluorescence is analyzed at the end of the amplification phase, when all the amplification cycles are completed and not during the amplification as it is generally done in Real-Time PCR where the amount of fluorescence is monitored and analyzed after each cycle of amplification. It is also possible that a method of the invention includes the quantification of the target nucleic acid, which is initially present in the sample. The quantity of target nucleic acid, which is initially present in the sample, corresponds to the amount of nucleic acid which is present in the initial sample used, that means the sample used before any step of amplification. Quantification of the initial amount of nucleic acid in the sample could be carried out by any method classically known by those in the art and it could be applied during or after any amplification method, preferably PCR, qPCR or LAMP. One method for quantifying a target nucleic acid is by determining Cp (Crossing point – also named Ct for Cycle Threshold) and comparing the Cp to a standard or to a control. Absolute quantification, including amplification by qPCR, frequently uses a standard curve approach. In this approach, a standard curve generated from plotting the Cp values obtained from amplification, preferentially real-time PCR, against known quantities of a single reference template (also called standard or control) provides a regression line that can be used to extrapolate the quantities of the target nucleic acid in a sample of interest. Serial dilutions (generally 10-fold dilutions) of the reference template are set up alongside samples containing the specific target nucleic acid that needs to be quantified. Various separate reactions are run, usually one for each level of the reference target and one each for the samples of interest. Also, since assay-specific differences in PCR efficiencies often affect quantification, separate standard curves, with separate reference templates, may be set up to quantify different gene targets. It is also possible to use a single point of Cp of a single quantification standard as it is described in WO 2017/165269 using an imported calibration or quantification curve. When a single point of Cp is used, these methods could comprise only one quantification standard nucleic acid provided at a known concentration, the Cp for this standard is obtained and then a regression line corresponding to this standard (standard curve) is imported and placed on the single point Cp for the calibration and then the quantification of the target nucleic acid can be operated using the calibrated standard curve. So, the method for detecting a target nucleic acid or the method of PCR analysis of a target nucleic acid may corresponds to a methods of performing quantitative amplification, preferably PCR, on a sample. In that cases, the methods may comprise amplifying the sample in an amplification mixture, the amplification mixture comprising a pair of target primers configured to amplify a target that may be present in the sample, the amplification mixture further comprising a plurality of quantification standard nucleic acids each provided at a different known concentration and at least one pair of quantification standard primers, the quantification standard primers configured to amplify quantification standard nucleic acids, generating a standard curve from the quantification standard amplicons, and quantifying the target nucleic acid using the standard curve. In the present invention, when quantitative amplification is performed, either external or internal quantification standard nucleic acids maybe be used for the quantification of the target nucleic acid. This means that if the standard nucleic acid is external, it is separated and not in the same reaction mixture (also called sample) as the one containing the target nucleic acid to quantify. If the standard nucleic acid is internal, it is in the same reaction mixture (also called sample) as the target nucleic acid to be quantified. The internal standard nucleic acid(s) is(are) generally amplified at the same time as the target nucleic acid to quantify but this can also be done previously and the standard curve obtained can be stored and imported at the moment of the quantification of the target nucleic acid. Quantification standard can be synthetic or natural. The calibration or quantification can be performed against a known natural microorganism with known concentrations or against other naturally occurring nucleic acid templates. It could be for examples a yeast or bacteriophages for viruses and/or synthetic particles able to mimic membrane and/or capsid and/or envelope structures but also housekeeping genes. As an alternative to determining Cp by absolute or normalized amplification data, the quantification of the target nucleic acid could also be done using the melting curves. In particular, when the target nucleic acid is a double stranded nucleic acid, preferably dsDNA, the quantification may imply the generation of a melting curve, and more precisely of several melting curves. Methods of quantification using a melting curve are known from those skilled in the art. Livak method for example is usable. It is also possible to use the maximum of the negative first derivative of the intensity of the fluorescence and of the temperature (max of – (dIntensity of fluorescence/dTemperature) which gives the melting temperature and then the quantity of the target nucleic acid. This method may further include determining a value for the melting curve, and determining a Cp by identifying the amplification cycle in which the value for the melting curve exceeds a predetermined value. The value may be determined by peak height or peak area of a negative derivative of the melting curve. A set of negative derivative melting curves can be used, wherein the flattest curves represent the earliest cycles and the area under the curve increases through a number of cycles. It is expected that such derivative melting curves acquired at a plurality of cycles during amplification can be used to determine Cp. The height of the transition for each melting curve or the area under the negative first derivative of the melt curve can be determined for each cycle. The Cp may then be assigned to the cycle at which this value exceeds a pre-determined threshold. Other methods for determining Cp may be applied. For example, a melt detector may be used (see U.S. 6,387,621; US 6,730,501; and US 7,373,253, herein incorporated by reference). The detector would interrogate curve shape and background noise to determine if the produced amplicon, preferably the amplicon obtained by PCR, is present in the sample. The use of a melt detector could be used to increase the sensitivity of the system (See Poritz, et al., PLos One 6(10):e2604 7). Optionally, additional filters could be applied to the melting curve analysis to window the melt transition to increase the specificity of the system, by analyzing only those melting curves having a melting transition, displayed as a melt peak, within a set temperature range. It is expected that such methods would result in a more accurate Cp (see WO 2014/039963). Methods of continuous monitoring of temperature and fluorescence are used for relative quantification, illustratively using a compound according to the invention, as dsDNA binding dye, in a single reaction with a control or standard nucleic acid. A multiplexed amplification (preferably PCR) reaction is provided, containing a control or standard nucleic acid at a known initial concentration and a target nucleic acid at an unknown concentration. Primers for amplification of the control or standard nucleic acid are present at the same initial concentration as primers for amplification of the target nucleic acid. In addition, it is desirable if the control or standard nucleic acid is selected such that its melting temperature is sufficiently well separated from the melting temperature of the target nucleic acid, so that melting of each of these nucleic acids is discernable from melting of the other. It is understood that multiple target nucleic acids of unknown concentration may be multiplexed in the reaction, noting that it is desirable that the melting curve for each nucleic acid is distinguishable from the others and from the control or standard nucleic acid. In PCR, the signals of fluorescence obtained from the binding of the compound of the invention to the dsDNA allow to produce amplification curves for the standard or control nucleic acid and the target nucleic acid to quantify. However, sometimes signals from the control and the target combine to generate a single amplification curve and information about the amplification of the individual nucleic acids is not discernable. To avoid this, with continuous data acquisition, a series of melting curves can be generated during PCR cycling. Provided that the melting temperatures of the control or standard nucleic acid and the target nucleic acid are sufficiently separated, the melting profile of each of the two reactions can be distinguished. To generate a corrected amplification curve for the control or standard nucleic acid, at each cycle the integral of the negative first derivative of the melt curve over a pre- defined melt window can be computed and plotted as a function of the cycle number, with the Cp determined as the cycle at which each value exceeds a predetermined value. Similarly, a corrected amplification curve for the target nucleic acid may be generated by integrating the negative first derivative of the melting curve over the pre-defined melt window for the target as it is described in WO 2014/039963 which is incorporated by reference. Other methods for converting the melt curve to a value are known in the art, such as using peak height of the negative first derivative. It is understood that the predetermined value should be selected according to method used. For instance, a method of PCR analysis, according to the invention, may comprise the steps of mixing the compound according to the invention, with a sample comprising an unknown initial quantity of a target nucleic acid and primers configured for amplifying the target nucleic acid, to form a mixture, amplifying the target nucleic acid in the presence of the compound according to the invention to generate an amplicon, monitoring fluorescence of monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon throughout a temperature range during a plurality of amplification cycles to generate a plurality of melting curves, and using the melting curves to quantify the initial quantity of the target nucleic acid. Illustratively, a method of PCR analysis of a target nucleic acid according to the invention may comprise the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid and at least a pair of primers suitable to amplify a portion of the target nucleic acid and generate an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture, and generating at least an amplicon, - during the amplifying step, monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon, - at the end of the amplifying step, melting the generated amplicon, to obtain a melting curve, and - identifying the genotype or polymorphism of the target nucleic acid using a shape of the melting curve. According to particular embodiments, whatever the methods according to the invention, the amplifying step may include a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 90 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.005 µM and primers are each provided at a concentration of at least 0.1 µM. According to specific embodiment, the amplifying step includes a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 20 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.5 µM and primers are each provided at a concentration of at least 2 µM. These concentrations are related to the amplification mixture, in particular to the PCR mixture. The PCR techniques are often classified according to the time required for the PCR and according to the quantity of primers which is used. More details are given in US 7387887 and US 9932634. Classical or standard PCR are quite slow and occur in approximately 90 seconds or less per cycles, rapid PCR occur in less than 60 seconds per cycle, for example between 20 and 60 seconds per cycle, fast, ultra fast and extreme PCR occur in less than 20 seconds, preferentially less than 12 seconds for fast PCR, less than 6 seconds for ultrafast PCR and in less than 2 seconds for extreme PCR. As the PCR speeds become faster, the concentrations of primers and polymerase are increased. This allows maintaining PCR efficiency and yield. The concentrations of primers range from at least 0.1µM for the classical or standard PCR to at least 2µM for extreme PCR, that is at least 0.1µM, at least 0.2µM, at least 0.4µM, at least 0.6µM, at least 0.8µM, at least 1µM, at least 1.2µM, at least 1.4µM, at least 1.6µM, at least 1.8 µM or at least 2µM. The concentrations of polymerase range from at least 0.005µM for classical or standard PCR to at least 0.5µM for extreme PCR, that is at least 0.005 µM, at least 0.01µM, at least 0.02µM, at least 0.04µM, at least 0.06µM, at least 0.08 µM, at least 0.1µM, at least 0.2µM, at least 0.3µM, at least 0.4µM or at least 0.5 µM. Any kinds of these PCR can be used, according to the invention. Herein, 1 µM of polymerase corresponds to 3.8 U/µL. All these concentrations are related to the PCR mixture. More details on the method for the analysis of nucleic acids which can be used with the compounds according to the invention can be found in US 9,682,970, WO 2008/052742, WO 2006/121423, US 7,456,281, US 7,387,887 and US 7,582,429. The invention also concerns a PCR reaction mixture, also named PCR mixture, comprising: - a target nucleic acid, - a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, - a polymerase, in particular a thermostable polymerase, - a compound according to the invention. Said pair of primers is designed to amplify a specific sequence of interest in the target nucleic acid according to standard methods known in the art of molecular biology. More than one pair of primers can be used, in particular for multiplex PCR, where more than one target sequence must be amplified. The target nucleic acid is typically total genomic DNA or alternatively total cellular RNA or total cellular mRNA. In case of RNA, the thermostable DNA polymerase may be a DNA polymerase or a mixture of polymerases comprising reverse transcriptase activity. Typically, such a PCR reaction mixture also includes a mix of deoxynucleoside triphosphates which is usually dA, dG, dC and dT, or dA, dG, dC and dU. Such a PCR reaction mixture classically includes a buffer. In particular, the PCR reaction mixture is buffered at pH from 7.5 to 9.5, preferentially from 8 to 9. Such a PCR reaction mixture may also include a thiol, typically selected from among the dithiothreitol, the beta mercaptoethanol and the thioglycerol, typically when it is dedicated to a RT-PCR analysis. The classical components of the mixture will be used in a concentration, easily determined by the person skilled in the art, according to common practice. In the method, use and mixture of the invention, the concentration of the compound according to the present invention is, typically, from 1 to 20 µmol/L (µM), and preferably from 2 to 10 µmol/L. This concentration corresponds to the concentration of the compound according to the invention in the sample which is used for monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon. In a specific embodiment, a compound according to the present invention is used for detection of double stranded nucleic acids during a melting curve analysis as disclosed for other compounds known in the art. More precisely, as described in WO 2008/052742, a double stranded DNA fragment is subjected to a thermal gradient in the presence of a compound according to the present invention. Preferably, the gradient is a continuous gradient, but step gradients are also possible. Most preferably, the gradient is a linear gradient. In one particular embodiment, the sample is subjected to a temperature increase which results in the generation of a dissociation curve. In another embodiment, the target double stranded nucleic acid is first thermally denatured into single strands and temperature dependence of fluorescence is monitored during subsequent renaturation. A first derivative of the melting curve may be generated and a characteristic temperature of the nucleic acid dissociation is obtained. These techniques are well known in the art and more details can be found, for instance in the Article of Carl T. Wittwer Chemistry 1997, 245, 154- 160. The concentration of the compound according to the present invention added before, during or after the amplification step and then used in the obtained mixture/sample used for monitoring the fluorescence is, typically, from 1 to 20 µmol/L, and preferably from 2 to 10 µmol/L. This concentration corresponds to the concentration of the compound according to the invention in the sample which is used for monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon. Preferably the double stranded DNA which is analyzed is derived from a PCR amplification reaction. In addition, amplification can be monitored in real time using a compound according to the invention and, in some embodiments, can be followed by subsequent melting curve analysis or end-point analysis of the fluorescence using said compound. The invention also concerns a kit for detecting a target nucleic acid, comprising: − at least a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, − a polymerase, in particular a thermostable polymerase, − a compound according to the invention. If the kit is used to obtain a melting curve, the kit allows the analysis and the identification of the target nucleic acid. Such a kit classically includes, also, a buffer, in particular, a buffer leading to a pH from 7.5 to 9.5, preferentially from 8 to 9. Suitable buffers for PCR (Tris for instance) are commercially available and can be used. Such a buffer may also include a thiol, for instance selected from among the dithiothreitol, the beta mercaptoethanol and the thioglycerol, typically when it is dedicated to a RT-PCR analysis. The compound according to the invention can also be in a buffer, for instance in such a buffer in the kit. The polymerase may, also, be stored in a buffer leading to a pH from 7.5 to 9.5, preferentially from 8 to 9. In some cases, this buffer may also include a thiol, typically selected from among the dithiothreitol, the beta mercaptoethanol and the thioglycerol. BRIEF DESCRIPTION OF THE FIGURES Figure 1 presents the stability and fluorescence data for the examples 1 to 4 and 6, 7 and 10 (compounds I.1 to I.4 and I.6, I.7 and I.10) and for other comparative compounds. Figure 2 presents the stability and fluorescence data of compounds not belonging to the invention, but differently substituted in the position corresponding to R₁. Figure 3 compares the stability and the fluorescence obtained with the examples 1 to 3 according to the invention and a comparative example 8. Figure 4 presents the stability for the examples 1 to 4 and for a compound N7 of the prior art, in the presence of a thiol. Figures 5A to 5F present the obtained fluorescence (RFU), in function of the temperature (°C) (in the left part), and the difference of fluorescence (- d(RFU)/dT(T)), in function of the temperature (°C) (in the right part), obtained in the evaluation hereafter, respectively for the examples 1 to 5 and 10 of the invention (compounds I.1 to I.5 and I.10) in or without the presence of a model DNA duplex, respectively (- DUPLEX) or (+ DUPLEX). Figures 6 and 7 present respectively for example 1 and 3 of the invention (compounds I.1 and I.3): the PCR amplification curve of a biological target (panel A), the melting curve of the generated amplicon (panel B) and the first derivative of the melting curve to determine precisely the melting temperature of the amplicon (panel C). Figure 8A presents the functional assays in fast real time PCR conditions of compounds described in the examples 1 to 4 of the invention (compounds I.1 to I.4) in comparison with compound N7 of the prior art. Figure 8B describes with more details the maximal fluorescence (Max Fluo) obtained at the end of the PCR on panel A, the Cp (number of cycles) on panel B and the Tm (melting temperature) measurements of the amplicon on panel C given by compounds I.1 to I.4 in comparison with compound N7 of the prior art. Figure 9 presents the excitation and emission maxima of the compounds I.1 to I.7 and I.10. EXAMPLES Abbreviations: Me = methyl ; Et = ethyl TFA- = trifluoroacetate TFA = trifluoroacetic acid TEA = triethanolamine ACN = acetonitrile DCM = dichlomethane DSC = N,N’-disuccinimidyl carbonate NHS =N-hydroxysuccinimide DIPEA = N,N-Diisopropylethylamine Ts = tosyl Ac = acetyl room temperature = 20°C I. Preparation of Compounds (I) In the examples hereafter, all compounds correspond to formula (I), (II) and (III). They were prepared as trifluoroacetate (TFA-) salts. For the analysis, the following instruments were used: UPLC-Mass spectrometry analysis: 0 to 95% acetonitrile in 10 mM TFA pH 2 for 5 min at 0,5 ml/min on a UPLC BEH C18 1.7 µm 2.1x50 mm column on the UPCL-MS system Alliance (Waters). ¹H NMR and ¹³C NMR: Bruker System 400 MHz. A. Example 1 2-((1,6-dimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-4(1H)- ylidene)methyl)-3-methylbenzo[d]thiazol-3-ium ditrifluoroacetate, compound I.1, 2TFA- Formula (I) and particular (II), with Z = fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rc=Rd=H ; R₁=R₃=Me ; R₄ = H ; R₂ = -(CH₂)_k4-Y₄, with k4=4 and Y₄ = - N⁺(Me)₃.

a) Preparation of 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium, 1

2-(Methylthio)benzo[d]thiazole (10g ; 55.16 mmoles) was put in a round bottomed flask of 250mL and methyl p-toluenesulfonate (75 mL ; 496.47 mmoles) was added. The mixture was stirred and heated at 145°C (fusion reaction) during 2 hours. The mixture turned orange. The mixture was then left to cool down to room temperature. 180mL of diethyl ether were added to precipitate the obtained compound and the obtained white precipitate was filtered and washed with diethyl ether two times. The compound 1 was obtained, as a solid, by evaporating the rest of diethyl ether using a rotary evaporator. Yield = 90 %. Mass spectrometry (ESI-Q (+)): M/Z = 196.02 ¹H NMR (400 MHz, D₂O) δ (ppm) : 8.5(d,1H) ; 8.1(t,1H) ; 7.87(t,1H) ; 7.25(d,1H) ; 4.3(s,3H) ; 2.44(s,3H) b) Preparation of 4-(4,6-dimethylpyrimidin-2-yl)-N,N-dimethylbutan-1- amine, 2

Acetylacetone (1.16 g ; 6.4 mmoles) was put in a round bottomed flask of 50 mL and water (9.5 mL) was added. 0.996 g (4.61 mmoles) of dimethyl amino acetamidine (Chemspace Riga, Latvia) and K₂CO₃ (2.95 g ; 221.37 mmoles) were then added. The mixture was stirred at room temperature, during four weeks. HCl 6M was added until pH 2 and the obtained aqueous phase was washed 2 times with DCM. Then, the aqueous phase was neutralized with NaOH 1M until pH 12 and the obtained compound 2 was extracted 3 times with dichloromethane. The DCM was evaporated with a rotary evaporator. Yield = 43 %. Mass spectrometry (ESI-Q (+)): M/Z= 208.1 ¹H NMR (200 MHz ; DMSO ; δ = 2.5 ppm) δ (ppm) : 7.04 (s ; 1H) ; 2.75 (t ; 2H) ; 2.36 (s ; 6H) ; 2.18 (t ; 2H) ; 2.09 (s ; 6H) ; 1.7 (q ; 2H)and 1.42 (q ; 2H) c) Preparation of 1,4,6-trimethyl-2-(4- (trimethylammonio)butyl)pyrimidin-1-ium, diiodine 3

In a glass tube with screw cap, 526 mg (2.54 mmol) of compound 2 were poured and dissolved in 8 mL of acetonitrile and 1.145 g (8.07 mmoles) of methyl iodide was added. The mixture was heated at 85 °C and stirred at 600 rpm during 17 hours in a thermomixer. The ACN and excess of CH₃I were evaporated with a rotary evaporator to obtain the expected crude compound 3. Yield = 94.5 %. Mass spectrometry (ESI-Q (+)) : M/Z= 118.5 d) Preparation of compound I.1, 2TFA-

129.9 mg (0.336 mmole) of 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium (compound 1) were poured in a round bottomed flask of 100 mL, 3 mL of a solution of 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium, diiodine (compound 3, 150 mg ; 0.305 mmole) at 50 mg/mL in acetonitrile were added, and 9 mL of acetonitrile and 0.8 mL of ethyl alcohol were successively added. Finally, 42.6 µl (30.9 mg ; 0.305 mmole) of triethylamine were dropped. The mixture was then stirred at room temperature for 25 minutes. The solvents were evaporated and the obtained solid was purified by flash chromatography with C18 column of 20 g (eluent A 10 mM TFA in water, eluent B 10 mM TFA in 90% ACN and 10 % water, gradient 5 to 65 % of eluent B in 20 min), leading to compound I.1, 2TFA-. Yield = 53 %. Mass spectrometry (ESI-Q (+)) : M/Z= 192.1 B. Example 2 2-((1-ethyl-2,6-dimethylpyrimidin-4(1H)-ylidene)methyl)-3-methyl-6-((3- (trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium ditrifluoroacetate, compound I.2, 2TFA- Formula (I) and particular (II), with Z = substituted fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rd=H ; R₁=Et ; R₂=R₃=Me ; R₄ = H ; Rc = -CONHR with R = - (CH₂)_k2-Y₂, with k2=3 and Y₂ = -N⁺(Me)₃.

a) Preparation of 2-(methylthio)benzo[d]thiazole-6-carboxylic acid, 5

200 mg (0.789 mmol) of ethyl 2-(methylthio)benzo[d]thiazole-6-carboxylate (compound 4, Chemspace Riga, Latvia) were put in a round bottomed flask of 50 mL, and 20 mL dichloromethane and 7.2 mL of NaOH (167 mM in methanol) were added. The mixture was stirred during 20 hours at room temperature. A suspension appeared, and the solvents were evaporated with a rotary evaporator. The obtained crude product was washed in diethylether and filtered. The obtained powder was dissolved in 20 mL of water HCl 3M until pH 1-2 and then extracted 4 times with 20 mL of dichloromethane. The organic phases were dried with MgSO₄ and evaporated with rotary evaporator, leading to compound 5. Yield = 55 % - 100 mg. ¹H NMR (200 MHz ; MeOD ; δ = 3.4 ppm) δ (ppm) : 8.65 (s, 1H) ; 8.20 (dd, 1H) ; 7.94 (d, 1H) and2.94 (s, 3H). ¹³C NMR (50 MHz ; MeOD ; δ = 48.0 ppm) δ (ppm) : 173.44 (C) ; 167.73 (C) ; 156.01 (CH) ; 134.83 (C) ; 127.47 (CH) ; 126.44 (C) ; 123.10 (CH) ; 120.19 (CH) and 14.63 (CH₃). b) Preparation of 2,5-dioxopyrrolidin-1-yl 2- (methylthio)benzo[d]thiazole-6-carboxylate, 6

160 mg (0.71 mmole) of 2-(methylthio)benzo[d]thiazole-6-carboxylic acid (compound 5) were dissolved in 10 mL of 1,4-dioxane and 161 mg (0.78 mmole) of dicyclohexylcarbodiimide were added. The mixture was stirred during 30 minutes at room temperature, then it was completed with 90 mg (0.78 mmole) of N- hydroxysuccinimide and the stirring was maintained during 20 hours. The suspension was filtered and the filtrate was evaporated with a rotary evaporator. The residue of evaporation was dissolved in 20 mL of acetyl acetate and washed 3 times with 10 mL of saturated bicarbonate solution and then 3 times with 10 mL of brine. The organic phase was dried with MgSO₄ and then evaporated with a rotary evaporator, leading to compound 6. Yield = 95 % - 220 mg. ¹H NMR (200 MHz ; CDCl₃ ; δ = 7.25 ppm) δ (ppm) : 8.56 (d, 1H) ; 8.16 (dd, 1H) ; 7.91 (d, 1H) ; 2.91 (s, 4H) and 2.82 (s, 3H). c) Preparation of N-(3-(dimethylamino)propyl)-2- (methylthio)benzo[d]thiazole-6-carboxamide, 8

390 mg (1.21 mmole) of 2,5-dioxopyrrolidin-1-yl 2-(methylthio)benzo[d]thiazole-6- carboxylate (compound 6) were dissolved in 20 mL of 1,4-dioxane in a round bottomed flask of 100 mL and 305 µL (247 mg ; 2.42 mmoles) of N,N- dimethylpropane-1,3-diamine (compound 7) were added. The mixture was stirred during 20 hours at room temperature. The crude product was filtered and the filtrate was evaporated with a rotary evaporator. The residue was purified by normal flash chromatography with mix CH₂CL₂/MeOH/TEA : 94/3/3, v/v/v, leading to compound 8. Yield = 80 % - 300 mg. ¹H NMR (200 MHz ; CDCl₃ ; δ = 7.25 ppm) δ (ppm) : 8.57 (m, 1H) ; 8.19 (d, 1H) ; 7.75-7.60 (M, 2H) ; 3.46 (q, 2H) ; 2.69 (s, 3H) ; 2.44 (t, 2H) ; 2.23 (s, 6H) and 1.70 (quint, 2H). ¹³C NMR (50 MHz ; CDCl₃ ; δ = 77.0 ppm) δ (ppm) : 170.94 (CO) ; 166.30 (C) ; 154.92 (C) ; 135.27 (C) ; 130.42 (C) ; 124.34 (CH); 120.70 (CH) ; 120.53 (CH) ; 58.94 (CH₂) ; 45.19 (CH₃) ; 40.41 (CH₂) ; 25.06 (CH₂) and 15.84 (CH₃). d) Preparation of 3-methyl-2-(methylthio)-6-((3- (trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, ditosylate, IV.1

N-(3-(Dimethylamino)propyl)-2-(methylthio)benzo[d]thiazole-6-carboxamide (compound 8, 114 mg ; 0.368 mmole) was put in a round bottomed flask of 10 mL and methyl p-toluenesulfonate (151 mg; 0.81 mmole) was added. The mixture was stirred and heated at 145°C (fusion reaction) for 4 hours. The mixture was then left to cool down to room temperature. The crude product was washed several times with diethyl ether and the compound IV.1 under the form of a ditosylate salt was obtained as a powder by evaporating the rest of solvent using a rotary evaporator. Yield = 225 mg. ¹H NMR (200 MHz ; D₂0 ; δ = 4.8 ppm) δ (ppm) : 8.24 (d, 1H) ; 7.92 (dd, 1H) ; 7.74 (d, 1H) ; 7.56 (d, 1H) ; 7.32 (AB, 16H) ; 6.86 (d, 1H) ; 3.88 (s, 3H) ; 3.38 (M, 8H) ; 3.04 (s, 16H) ; 2.93 (s, 3H) ; 2.21 (s, 12H) and 2.05 (m, 4H). e) Preparation of 2,4,6-trimethylpyrimidine, 9

Acetylacetone (11 g ; 60.68mmol) was put in a round bottomed flask and water (95 mL) was added. The methyl acetamidine (10 g ; 43.86 mmol) and K₂CO₃ (28 g ; 202.60 mmol) were added. The mixture was stirred at room temperature, during 3 weeks. It was possible to observe uncolored crystals on the surface and a white precipitate at the bottom of the round bottomed flask. The solution was orange. The crystals at the top of the mixture were filtered and washed with cold water. These crystals were the expected compound 9, but some quantity of compound 9 remained dissolved in the water. In the mixture, some K₂CO₃ were added to saturate the water and a liquid extraction was carried out. The organic phase was recovered and the DCM at 500 mbar was evaporated using a rotary evaporator. Some DCM remained. This mixture contained compound 9, with an imine. Purification was carried out with a chromatographic column with normal phase. The obtained mixture was put at the top of the column and the eluent (DCM/MeOH 98:2) was added. The pure compound 9 was obtained, after total evaporation of DCM. Yield = 14 %. Mass spectrometry (ESI-Q (+)) : M/Z= 123.1 f) Preparation of 1-ethyl-2,4,6-trimethylpyrimidin-1-ium, iodine 10

150 mg (1.23 mmole) of 2,4,6-trimethylpyrimidine 9 were put in a round bottomed flask, and were dissolved in acetonitrile to obtain a solution of 0.15M; 574 mg (3.69 mmoles) of iodoethane were then added. The mixture was stirred at reflux during 20 hours. The solvents were evaporated and purification of the obtained pyrimidinium 10 in the form of an iodine salt was carried out, by flash chromatography with C18 column. Yield = 50 %. Mass spectrometry (ESI-Q (+)) : M/Z= 151.1 g) Preparation of compound I.2, 2TFA-

94.3 mg of previously prepared 3-methyl-2-(methylthio)-6-((3- (trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, ditosylate IV.1 were put in a round bottomed flask of 25 mL and 6 mL of acetonitrile and 0.5 mL of ethyl alcohol were added. 27 mg (0.102 mmole) of previously prepared 1-ethyl-2,4,6- trimethylpyrimidin-1-ium, iodine 10 were dissolved in 3.155 mL of acetonitrile. 108 mg (0.511 mmole) of trimethylamine were added and the mixture was stirred at room temperature for 25 minutes. The solvents were evaporated and compound I.2, 2TFA- was purified by flash chromatography with C18 column of 12 g (Eluent A 10 mM TFA in water, Eluent B 10 mM TFA in 90% ACN and 10 % water. Gradient 5 to 65 % of eluent B in 20 minutes). Yield = 47 %. Mass spectrometry (ESI-Q (+)) : M/Z= 220.7 C. Example 3 . Preparation of 2-((1,6-dimethyl-2-(4- (trimethylammonio)butyl)pyrimidin-4(1H)-ylidene)methyl)-3- methyl-6-((3 (trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, I.3, 3TFA Formula (I) and in particular (II), with Z = substituted fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rd=H ; R₁=R₃=Me ; R₄ = H ; R₂ = -(CH₂)_k4-Y₄, with k4=4 and Y₄ = -N⁺(Me)₃ ; Rc = -NHCOR with R = -(CH₂)_k2-Y₂, with k2=3 and Y₂ = - N⁺(Me)₃.

a) Preparation of 4-((2,5-dioxopyrrolidin-1-yl)oxy)-N,N,N-trimethyl-4- oxobutan-1-aminium chloride, 12

(3-Carboxypropyl)trimethylammonium chloride (compound 11, as chloride salt, 1.2 g ; 6.61 mmoles) was poured in a round bottomed flask of 250 mL and 100 mL of anhydrous acetonitrile and 8 eq of anhydrous pyridine were added under nitrogen flow. The mixture was stirred during 10 minutes before adding DSC (5.086 g ; 19.85 mmoles). After 12 hours at room temperature, the solvents were evaporated and the crude product was precipitated in diethyl ether. The obtained solid was dried by evaporation of the rest of diethyl ether using a rotary evaporator and compound 12 was obtained. Yield = 100 %. Mass spectrometry (ESI-Q (+)) : M/Z= 243.1 b) Preparation of N,N,N-trimethyl-4-((2-(methylthio)benzo[d]thiazol-6- yl)amino)-4-oxobutan-1-aminium chloride, 14

4-((2,5-dioxopyrrolidin-1-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-aminium chloride, (compound 12, 675 mg ; 2.42 mmoles) was put in a round bottomed flask of 250 mL, and 38 mL of anhydrous acetonitrile were added. The mixture was stirred vigorously under nitrogen flow. 184.0 mg of 2-(methylthio) benzo[d]thiazol-6-amine (Chemspace ref CSC000183586 ; compound 13 ; 0.94 mmole) were added into the round bottomED flask and immediately after 1.1 equivalent of anhydrous DIPEA was added, until pH = 5.8. The mixture was stirred at 80 °C during 24 hours. The solvents were evaporated and a liquid-liquid extraction with water and DCM was carried out. The aqueous phase was basified with DIPEA to reach pH 8-9 and then an extraction was carried out three times in DCM. The aqueous phase was recovered and the evaporation of water with a rotary evaporator led to compound 14. Yield = 100 %. Mass spectrometry (ESI-Q (+)) : M/Z= 324.1 c) Preparation of 3-methyl-2-(methylthio)-6-(4- (trimethylammonio)butanamido)benzo[d]thiazol-3-ium tosylate chloride, IV.2

N,N,N-trimethyl-4-((2-(methylthio)benzo[d]thiazol-6-yl)amino)-4-oxobutan-1- aminium chloride (compound 14 ; 100 mg ; 0.294 mmol) was put in a round bottomed flask of 10mL and methyl p-toluenesulfonate (982 mg; 5.26 mmol) was added. The mixture was stirred and heated at 145°C (fusion reaction) for 2 hours. The mixture was then left to cool down to room temperature. 6 mL of acetone were added and the obtained mixture was mixed vigorously and transferred to a 50 mL tube diethyl ether. The flask was rinsed with 6 mL of acetone then diluted with 30 mL of ether, shaken vigorously and the supernatant was discarded after centrifugation at 1500 rpm. The precipitate was washed 10 times in 40 mL of acetone / ethyl ether mixture 1/9, v/v. The obtained solid was dried by evaporating the rest of solvent with a rotary evaporator and compound IV.2 was obtained. Yield = 61 %. Mass spectrometry (ESI-Q (+)) : M/Z= 169.6 d) Preparation of Compound I.3, 3TFA-

296 mg of 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium diiodine (compound 3 prepared previously in example 1) were poured in a round bottomed flask of 50 mL and 10 mL of acetonitrile and 0.5 mL of ethyl alcohol were added. 150 mg of compound IV.2 previously prepared were dissolved in 3 mL of acetonitrile. 1 equivalent of triethylamine (43 µL) was added and the mixture was stirred at room temperature for 25 minutes. The mixture turned from yellow to orange. The solvents were evaporated and the Compound I.3, 3TFA- was purified by flash chromatography with C18 column of 12 g (eluent A 10 mM TFA in water, eluent B 10 mM TFA in 90% ACN and 10 % water. Gradient 5 to 65 % of eluent B in 20 minutes). Yield = 17 %. Mass spectrometry (ESI-Q (+)) : M/Z= 175.78 D. Example 4 6-Bromo-2-((1,6-dimethyl-2-(4-(trimethylammonio)butyl)pyrimidin- 4(1H)-ylidene)methyl)-3-methylbenzo[d]thiazol-3-ium, ditrifluoroacetate, I.4, 2TFA- Formula (I) and in particular (II), with Z = substituted fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rd=H ; Rc = Br ; R₁=R₃=Me ; R₄ = H ; R₂ = -(CH₂)_k4-Y₄, with k4=4 and Y₄ = -N⁺(Me)₃.

a) Preparation of 6-bromo-3-methyl-2-(methylthio)benzo[d]thiazol-3- ium, tosylate 15

6-Bromo-2-(methylthio)benzo[d]thiazole (500 mg ; 1.92 mmoles) was put in a round bottomed flask of 10 mL and methyl p-toluenesulfonate (3.040 g ; 16.3 mmoles) was added. The mixture was stirred and heated at 145°C (fusion reaction) for 1 hour and 30 minutes. The mixture was then left to cool down to room temperature. 18 mL of diethyl ether were added and the mixture was mixed vigorously. It was transferred to 50 mL tube and the flask was rinsed with 6 mL of diethyl ether, shaken vigorously and the supernatant was discarded after centrifugation at 1500 rpm, repeated 3 times. Then, the precipitate was washed 3 times with 18 mL of acetone. The obtained solid was dried by evaporating the rest of solvent using a rotary evaporator, to obtain compound 15 as a tosylate salt . Yield = 35 %. Mass spectrometry (ESI-Q (+)) : M/Z= 273.9 and 275.9 NMR ¹H (200 MHz ; DMSO ; δ = 2.5 ppm) δ (ppm): 8.65 (sd ; 1H) ; 8.15 (d ; 1H) ; 8.03 (dd ; 1H) ; 4.08 (s ; 3H) and 2.29 (s ; 3H). b) Preparation of compound I.4, 2TFA-

200.0 mg (0.450 mmole) of 6-bromo-3-methyl-2-(methylthio)benzo[d]thiazol-3-ium, tosylate 15 were put in a round bottomed flask of 100 mL, 1,4,6-trimethyl-2-(4- (trimethylammonio)butyl)pyrimidin-1-ium, diiodine (compound 3 previoulsy prepared ; 170 mg ; 0.346 mmole), 5 mL of acetonitrile and 1 mL of ethyl alcohol were successively added. Finally, 210 µL (152 mg ; 1.505 mmole) of trimethylamine were dropped. The mixture was stirred at room temperature for 25 minutes. The solvents were evaporated and the compound I.4, 2TFA- was obtained, after purification by flash chromatography with C18 column (TFA/ACN/water eluents, as in previous example 3). Yield = 90 %. Mass spectrometry (ESI-Q (+)) : M/Z= 231.1 and 232.1 E. Example 5 2-3-(1,6-dimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-4(1H)- ylidene)prop-1-en-1-yl)-3-methylbenzo[d]thiazol-3-ium, ditrifluoroacetate, I.5, 2TFA- Formula (I) and in particular (II), with Z = fused-benzo ; X=S ; n=1 ; Ri=Rj=Rk=H ; Re = Me ; Ra=Rb=Rc=Rd=H ; R₁=R₃=Me ; R₄ = H ; R₂ = -(CH₂)_k4-Y₄, with k4=4 and Y₄ = -N⁺(Me)₃.

a) Preparation of 2,3-dimethylbenzo[d]thiazol-3-ium, tosylate 16

2-Methylbenzothiazole (1 g ; 6.63 mmoles) was put in a round bottomed flask of 100 mL and methyl p-toluenesulfonate (9.9 g ; 52.8 mmoles) was added. The mixture was stirred and heated at 145°C for 2 hours. The mixture was then left to cool down to room temperature. 50 mL of ethyl ether were added, the crude product was precipitated, supernatant was discarded and the obtained solid was washed 3 more times in 50 mL of diethyl ether. Then, the obtained precipitate was washed 3 times with 50 mL of acetone. The obtained solid was dried by evaporating the rest of solvent with a rotary evaporator to obtain a grey powder (2.15 g, Yield = 96 %), corresponding to compound 16 as a tosylate salt. ¹H NMR (D₂O, 400 MHz) : δ (ppm) 8.05 (d, 1H, H1), 7.88 (d, 1H, H2), 7.76 (td, 1H, H3), 7.64 (td, 1H, H4), 7.58 (d, 2H, HorthoTsO-), 7.26 (d, 2H, Hmeta TsO-), 4.02 (s, 3H, H6), 3.00 (s, 3H, H5), 2.30 (s, 3H, CH₃-TsO-). Mass spectrometry (ESI-Q (+)) : M/Z= 164.1. b) Preparation of 3-methyl-2-(2-(phenylamino)vinyl)benzo[d]thiazol-3- ium, tosylate 17

2,3-Dimethylbenzo[d]thiazol-3-ium, tosylate 16 (167.9 mg ; 0.50 mmole) was put in a round bottomed flask of 50 mL and N,N’-diphenylformamidine (196.2 mg; 1.00 mmole) was added. The mixture was stirred and heated at 150°C (fusion reaction) for 2 hours and 30 minutes. The mixture was then left to cool down to room temperature. Compound 17 as a tosylate salt was obtained after purification, by extraction liquid in water / dichloromethane and evaporation of the organic phase before final purification by reverse flash chromatography (eluent A 10 mM TFA in water, eluent B 10 mM TFA in 90% ACN and 10 % water. Gradient 20 to 100 % of eluent B in 30 minutes). Yield = 67 %. Mass spectrometry (ESI-Q (+)) : M/Z= 267.2 c) 3-methyl-2-(2-(N-phenylacetamido)vinyl)benzo[d]thiazol-3-ium, 18

3-Methyl-2-(2-(phenylamino)vinyl)benzo[d]thiazol-3-ium, tosylate 17 (127.1mg ; 0.334 mmole) prepared previously was put in a round bottomed flask of 10 mL and acetic anhydride (800 mg ; 7.84 mmoles) was added. The mixture was stirred and heated at 100 °C during 2 hours and 30 minutes. The solvent was evaporated and co-evaporation 2 times with ACN using a rotary evaporator was carried out, to obtain compound 18. At the end, there is no odor of acetic acid. Yield = 100 %. Mass spectrometry (ESI-Q (+)) : M/Z= 309.1 d) 2-3-(1,6-dimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-4(1H)- ylidene)prop-1-en-1-yl)-3-methylbenzo[d]thiazol-3-ium, I.5, 2TFA-

70.5 mg (0.167 mmole) of 3-methyl-2-(2-(N-phenylacetamido)vinyl)benzo[d]thiazol- 3-ium 18 were put in a round bottomed flask of 10 mL, 1,4,6-trimethyl-2-(4- (trimethylammonio)butyl)pyrimidin-1-ium diiodine (compound 3 previoulsy prepared ; 164.1 mg ; 0.334 mmole), 3 mL of acetonitrile and 0.5 mL of ethyl alcohol were successively added. Finally, 16.9 mg (0.167 mmole) of triethylamine were dropped. The mixture was stirred at room temperature for 25 minutes. The solvents were evaporated and compound I.5, 2TFA- was obtained after purification by PREP chromatography with C18 column (eluent A 10 mM TFA in water, EluEnt B 10 mM TFA in 90% ACN and 10 % water. Gradient 10 to 50 % of eluent B in 15 minutes). Yield = 46 %. Mass spectrometry (ESI-Q (+)) : M/Z= 205.1 F. Example 6: (Z)-6-((3-(dimethyl(3- (trimethylammonio)propyl)ammonio)propyl)carbamoyl)-2-((1,6- dimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-4(1H)- ylidene)methyl)-3-methylbenzo[d]thiazol-3-ium, 4 TFA- Formula (I) and in particular (II), with Z = substituted fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rd=H ; Rc = -C(O)NHR, in which R is –(CH₂)_k2-Y₂ with k2 = 3 and Y₂ being -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’ with p₂=3, m₂=1, R₂’=R₂”=Me and G₂’=- N⁺(Me)₃; R₁=R₃=Me ; R₄ = H ; R₂ = -(CH₂)_k4-Y₄, with k4=4 and Y₄ = -N⁺(Me)₃.

a) Preparation of N-(3-((3-(dimethylamino)propyl)amino)propyl)-2- (methylthio)benzo[d]thiazole-6-carboxamide, 21

This compound was prepared, according to the same way as for compound 8 in the Example 2 except that the amine used was N1-(3-aminopropyl)-N3,N3- dimethylpropane-1,3-diamine, 20. Mass spectrometry (ESI-Q (+)) : M/Z= 366,15 b) Preparation of 6-((3-(dimethyl(3- (trimethylammonio)propyl)ammonio)propyl)carbamoyl)-3-methyl- 2-(methylthio)benzo[d]thiazol-3-ium, tritosylate (IV.3)

This compound was prepared from compound (21), according to the same way as for the compound IV.1 of the Example 2. Mass spectrometry (ESI-Q (+)) : M/Z + 2 TFA-= 651.2. c) Preparation of I.6

75 mg (0.099 mmole) of 6-((3-(dimethyl(3- (trimethylammonio)propyl)ammonio)propyl)carbamoyl)-3-methyl-2- (methylthio)benzo[d]thiazol-3-ium, tritosylate (compound VI.3), 55.8 mg (0.114 mmole) of 1,4,6-trimethyl-2-(4-trimethylammonio)butyl)pyrimidin-1-ium, diiodine (compound 3 previously prepared in example 1), 6 mL of acetonitrile and 0.5 mL of ethyl alcohol were successively added in a round bottomed flask of 25 mL. Finally, 13.8 µL (10.0 mg ; 0.099 mmole) of trimethylamine were dropped. The mixture was stirred at room temperature for 25 minutes. The solvents were evaporated and the compound I.6, 4TFA- was obtained, after purification by flash chromatography with C18 column (TFA/ACN/water eluents, as in previous example 3). Yield = 3 %. Mass spectrometry (ESI-Q (+)) : M/Z + 3 TFA-= 952.4 G. Example 7 (Compound I.7) (Z)-2-((1,6-dimethyl-2-(4- (trimethylammonio)butyl)pyrimidin-4(1H)-ylidene)methyl)-3-methyl- 6-((3-(trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, 3TFA- Formula (I) and in particular (II), with Z = substituted fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rd=H ; Rc = -C(O)NHR, in which R is –(CH₂)_k2-Y₂ with k2 = 3 and Y₂ being -N⁺(Me)₃; R₁=R₃=Me ; R₄ = H ; R₂ = -(CH₂)_k4-Y₄, with k4=4 and Y₄ = -N⁺(Me)₃.

140.5 mg (0.185 mmole) of 3-methyl-2-(methylthio)-6-((3- (trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, ditosylate (compound IV.1 of example 2), and 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1- ium, diiodine (compound 3 previously prepared in example 1 ; 100 mg ; 0.204 mmole), 6 mL of acetonitrile and 0.5 mL of ethyl alcohol were successively added in a round bottomed flask of 25 mL followed by the drop of 77.4 µL (56.2 mg ; 0.555 mmole) of triethylamine. The mixture was stirred at room temperature for 25 minutes. The solvents were evaporated. The crude product was purified by precipitation in 6 mL of acetonitrile, the precipitate was washed 4 times with 1.5 ml of acetonitrile to obtain the compound I.7, 3TFA-. This product was purified by flash chromatography with a C18 column (TFA/ACN/water eluents, as in previous example 3). Yield = 21 %. Mass spectrometry (ESI-Q (+)) : M/Z = 175.8 H. Example 8 Formula (I) and in particular (II), with Z = fused-benzo ; X=S ; n=2 ; Ri=Rj=Rk=H ; Re = Me ; Ra=Rb=Rc=Rd=H ; R₁=R₃=Me ; R₄ = H ; R₂ = -(CH₂)_k4-Y₄, with k4=4 and Y₄ = -N⁺(Me)₃.

I. Example 9 Formula (I) and in particular (II), with Z = substituted fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rd=H ; Rc = -C(O)NHR, in which R is –(CH₂)_k2-Y₂ with k2 = 3 and Y₂ being -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’ with p₂=3, m₂=1, R₂’=R₂”=Me and G₂’=- N⁺(Me)₃ ; R₁=Et ; R₂=R₃=Me ; R₄ = H.

J. Example 10: (Z)-6-acetamido-2-((1,6-dimethyl-2-(4- (trimethylammonio)butyl)pyrimidin-4(1H)-ylidene)methyl)-3- methylbenzo[d]thiazol-3-ium, 2 TFA-, compound I.10, 2TFA- Formula (I) and in particular (II), with Z = substituted fused-benzo ; X=S ; n=0 ; Ri=H ; Re = Me ; Ra=Rb=Rd=H ; Rc = -NHC(O)Me ; R₁=R₃=Me ; R₄ = H ; R₂ = - (CH₂)_k4-Y₄, with k4=4 and Y₄ = -N⁺(Me)₃.

a) Preparation of N-(2-(methylthio)benzo[d]thiazol-6-yl)acetamide (22)

The compound (13) 2-(methylthio)benzo[d]thiazol-6-amine-methane (1.842 g, 9.4 mmoles) was poured in a round bottomed flask of 50 mL with 30 mL of DCM and acetic anhydride (1.151 g, 11.3 mmol). The mixture was then stirred at room temperature for 24 h. The mixture was evaporated by using a rotary evaporator. Yield = 92 %. Mass spectrometry (ESI-Q (+)): M/Z= 239.1 1H NMR (200 MHz, CDCl3) δ (ppm): 8.33 (d,1H) ; 7.75 (d,1H) ; 7.17 (d,1H) ; 2.76 (s,3H) ; 2.20 (s,3H). b) Preparation of 6-acetamido-3-methyl-2(methylthio)benzo[d]thiazol- 3-ium, TsO- (23)

Compound (22) (2.065 g, 8.66 mmol) was poured in a round bottomed flask with p- toluenesulfonic acid monohydrate (1.780 g, 9.53 mmol). The mixture was then stirred at 130 °C for 3 h. The heating was turned off and the mixture was cooled down to room temperature then the crude product was triturated with 20 mL of acetone and was tritured 4 more times until obtaining a red powder. The residue of solvent was evaporated by using a rotary evaporator. Yield = 74 %. Mass spectrometry (ESI-Q (+)): M/Z= 253.1 1H NMR (200 MHz, D2O) δ (ppm): 8.1 (d,1H) ; 7.57 (d,1H) ; 7.44 (d, 1H) ; 7. 38 (d, 2H) ; 7.04 (d, 2H) ; 3.79 (s, 3H) ; 2.86 (s, 3H) ; 2.12 (s, 3H) ; 2.06 (s, 3H). RMN 13C (50MHz ; D2O) δ (ppm): 180.07 ; 172.69 ; 142.02 ; 139.37 ; 138.56 ; 136.63 ; 129.12 ; 128.68 ; 125.06 ; 121.80 ; 115.15 ; 113.50 ; 35.80 ; 23.10 ; 20.34 ; 17.49. c) Preparation of compound (I.10), 2TFA-

134.2 mg (0.316 mmole) of 6-acetamido-3-methyl-2(methylthio)benzo[d]thiazol-3- ium, TsO- (compound 23) was poured in a round bottomed flask of 100 mL with 818 µL of a solution of 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium, diiodine at 75 mM in acetonitrile (compound 3, 150 mg ; 0.316 mmole) and 30 mL of acetonitrile and 10 mL of ethyl alcohol were successively added. The suspension mixture was then stirred and heated at 80 °C until complete dissolution. The solution was cooled at room temperature before 88.1 µl (64 mg ; 0.634 mmole) of triethylamine was dropped. The mixture was then stirred at room temperature for 25 minutes. The solvents were evaporated and the obtained solid was purified by preparative chromatography with a PrepPure C18 column from Buchi (ref 11068740), (eluent A 10 mM TFA in water, eluent B 10 mM TFA in 90% ACN and 10 % water, gradient 5 to 80 % of eluent B in 40 min), leading to compound (I.10), 2TFA-. Yield = 35 %. Mass spectrometry (ESI-Q (+)) : M/Z= 220.63 II. Evaluation of the compounds according to the invention A. Stability in PCR mimic conditions and Fluorescence studies 1) Protocols a) Accelerated stability testing in alkaline conditions (Figures 1, 2 and 3) The compounds were prepared at 10 µM or 1X in a solution of Tris HCl 20 mM pH 9 and stored at 40°C. 20 µL aliquots were injected regularly on UPLC (Alliance chain from Waters (USA) equipped with mass and UV detector, using Method A: 0 to 95% acetonitrile in 10 mM TFA pH 2 for 5 min at 0.5 mL/min on a UPLC BEH C18 1.7 µm 2.1x50 mm column (Waters) - Max plot detection or at specific wavelengths) to reach the half-life of the compound in these conditions (T1/2 in days). b) Analytical fluorescence evaluation of the compounds (Figures 1, 2 and 3 and 9) A solution of 20 µL was made with 10 µM compound, in a typical PCR mix formulation (Tris pH 8.4 20 mM, NaCl 10 mM , dNTP(4x0.3 mM), MgCl2 4 mM, TAQ polymerase 1 U/µL and BSA 550 ng/µL). A double stranded duplex amplicon of 80 mers was added at a final concentration of 0.13 µg/µL (5 µM) in the vial that corresponded roughly to the generated amplicon quantity of a standard PCR reaction. Then the solution was poured in a micro plate and the maximum fluorescence emission was recorded with a spectrofluorometer reader (TECAN Austria) upon excitation at: (λ absorption max -30 nm) with a gain of 60 in order to be able to compare the different compounds with each other. 2) Comparison with other compounds The Figure 1 shows the stability and fluorescence data for the examples 1 to 4 and 6, 7 and 10 (compounds I.1 to I.4, I.6, I.7 and I.10) as trifluoroacetate salts and for the following comparative examples:

- comparative example 1 (Comp.1) :

, corresponding to D6 which is in acetate form in US 7,387,887, - comparative example 2 (Comp.2) :

corresponding to N7 which is in acetate form in US 7,387,887, - comparative example 3 (Comp.3) : thiazole orange - comparative example 4 (Comp.4) : SybrGreen - comparative example 5 (Comp.5) :

- comparative example 9 : compound which is present in the commercial kit Resolight® from Roche diagnostic. Its mass spectrometry analysis (ESI-Q(+)) gives a M/Z= 240,13 (with Z = 2) , which corresponds to the mass of example 6 (R27) or example 7 (R28) of WO 2008/052742. The data presented on Figure 1, obtained or simulating the behavior at ambient temperature of the tested compound, show that the compounds according to the invention are both highly stable in PCR conditions and highly fluorescent, when they interact with nucleic acid sequences. The other tested compounds, including compounds of the prior art, are either less stable (half life ≤ 20 days), or less fluorescent (Fluorescence < 35000 RFU) or both less stable and less fluorescent. It is also apparent from these results, that the compound from the kit Resolight® from Roche diagnostic is less stable and lead to less fluorescence, than all the compounds according to the invention. With the same conditions, the compound I.5 (n=1) leads to a fluorescence of 9476 RFU. This fluorescence is lower than the fluorescence obtained with the compounds of the invention where n=0, but the quantity of measured fluorescence enables the use of the compound in the analysis of nucleic acids. It is not suitable to strictly compare the fluorescence obtained between compounds where n=0, 1, 2 or 3. The interest of these various compounds relies on the possibility of obtaining absorption and emission on a broad range of wavelengths. Figure 9 shows the modulation of the maximum absorption and maximum emission wavelengths, in particular by the choice of n. 3) Study of the influence of the alkyl chain in position 1 Other tests were carried out to study the influence of the presence of an alkyl chain in position 1, instead of an aryl group as in example A10 of US 7,387,887. The Figure 2 compares the stability and the fluorescence obtained with the two following compounds:

TFA- comparative 6 (Comp. 6) and

TFA- comparative 7 (Comp. 7) It appears that methyl in position 1 (N atom corresponding to substituent R₁ in formula (I)) of the pyrimidine induces a better stability and a better fluorescence than phenyl. 4) Influence on the way of bonding ammonium groups on the compound Other tests were carried out for studying the influence of the presence of an ammonium directly attached by its nitrogen atom to the Z part of the compound, as in example R6 of US 7,387,887, which carries two ammonium groups –N⁺(Me)₃. The Figure 3 compares the stability and the fluorescence obtained with the compounds 1 to 3 according to the invention (compounds I.1 to I.3 as trifluoroacetate salts) and with the following compound:

3TFA- comparative 8 (Comp. 8) It appears that the obtained fluorescence is greatly reduced for comparative example 8 rendering this compound not suitable for PCR application, with respect to the stability, for the compounds according to the invention, it is imposable or reduced, but remains suitable for the use of the compound in a PCR in any case. B. Stability in the presence of a thiol Compounds were prepared at 10 µM in a solution of tris 20 mM pH 9 and thioglycerol at 100 µM and stored at 20°C. 20 µL aliquots were injected regularly on UPLC as described in Part A 1) a). The obtained results for the examples 1 to 4 (corresponding to compounds I.1 to I.4, as trifluoroacetate salt) and compound N7 of the prior art are presented on Figure 4. They show that the compounds of the present invention are stable in the presence of a thiol as they do not bear a sulfur group at the 2 position of the pyrimidinium ring contrary to N7. Even if not represented, compound I.10 is also stable in the presence of thiols. C. Analytical melting measurements 10 µM compounds solutions were prepared in a typical PCR mix formulation (Tris pH 8.4 20 mM, NaCl 10 mM, 1X proprietary stabilization buffer, dNTP(4x0.3 mM), MgCl2 4 mM, TAQ polymerase 1 U/µL and BSA 550 ng/µL) alone (- DUPLEX) or in the presence of two complementary DNA strands ((+ DUPLEX) 80 mers, 5 µM each)) for a total volume of 20 µL. The solutions were placed in PCR polypropylene cuvettes of 200 µL and the emitted fluorescence was recorded as a function of temperature from 20°C to 90°C (0.5°C/min) using a preliminary denaturing step during 1 minute at 95°C (CFX Maestro from Biorad Laboratories) to obtain a typical melting curve. The appropriate fluorescence channel was used as a function of the fluorescence characteristics of the compound (FAM for all compounds except for I.5 where Hex channel was used). The first derivative of this melting curve was calculated and plotted as a function of the temperature to precisely determine the melting temperature of the duplex. The Figures 5A to 5F show that all the compounds of the present invention can be used for the detection of the melting of a duplex (Tm) with a very good sensitivity (Tm peak height) as summarized in Table 1 hereinafter. The data of Table 1 also show that compounds D6 and N7 from the prior art are less fluorescent than the compounds of the invention on the FAM channel.

Table 1

D. Functionnal assays (PCR) The compounds, I.1 of example 1 (figure 6) and I.3 of example3 (figure 7) were used at respectively 10 µM and 5 µM in a standard PCR reaction amplification using the biological model: S.cerevisiae at 10^e5 ; 10^e4 ; 10^e3 and 10^e2 cp/PCR reaction (duplicats) on the LightCycler® 480 Instrument II (Roche) equipped with a (440/488) filter. The presence of the compounds according to the invention allowed to monitor real time amplification (panel A) and to do a melting experiment after PCR completion by plotting the fluorescence = f(Temperature) in order to determine the melting curve of the amplicon (panel B), from which it is further extracted the melting temperature by plotting the first derivative of the emitted fluorescence = f(Temperature) as seen on panel C. This showed that the new compounds of the present invention are very efficient and useful to monitor real time PCR and to determine the melting temperature of a dedicated amplicon with a great sensitivity. E. Functionnal assays (fast PCR conditions) The compounds, I.1 of example 1, I.2 of example 2 , I.3 of example 3 and I.4 of example 4 were used at respectively 10 µM in a fast PCR reaction amplification using a biological model at 2.5. 10⁸ Cp/PCR (5 replicates) with 30 cycles of standard fast cycling PCR protocol, such as the one described in the Qiagen Fast Cycling PCR Handbook (October 2012). By comparison, compound N7 of the prior art was used as a control. The Figure 8A shows the real time PCR curves obtained with the 4 compounds of the invention and demonstrates the ability of these compounds to be used to detect an amplicon in very fast cycling conditions. The panel A, B and C of Figures 8B show respectively the details of the Max Fluo, Cp and Tm measurements showing the high reproducibility of these experiments and the ability of these compounds to detect with a great sensitivity the presence of a dedicated amplicon. is the obtained results also show that the dyes of the invention (I.1 to I.4) provide roughly twice more fluorescence (Max Fluo) than compound N7. Therefore, these dyes are much more efficient and valuable in detecting a given target with a high sensitivity. In addition, both Cp and Tm are in the same order as with N7 (respectively +/- 1Cp around the Cp given by N7 and +/- 2°C around Cp given by N7), demonstrating that the dyes of the invention do not inhibit PCR.

Claims

CLAIMS 1. Compounds having the formula (I) :

wherein : -n is equal to 0, 1, 2 or 3; -Ri, Rj and Rk are identical or different and are independently selected from the group consisting of hydrogen and C_1-6alkyl; -X is oxygen, sulfur, selenium, tellurium or C(CH₃)₂, -Re is a group selected from among alkyl, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, and the groups –(CH₂)_k1-Y₁, in which: ok1 is 1, 2, 3, 4, 5 or 6 and oY₁ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₁’R₁’’-(CH₂)_p1-]_m1-G₁’, with m1 being 1, 2 or 3, G₁’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p1 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₁’ and R₁’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; - Z is a fused mono or polycyclic aromatic or nitrogen-containing heteroaromatic ring, optionally substituted by one or several substituent(s) A identical or different selected from among halogen atoms and the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, - NHC(O)phenyl, -NHC(O)alkyl, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl, –CH=N- O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, –CONHR being preferred, R being either a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: ok2 is 1, 2, 3, 4, 5 or 6 and oY₂ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; -R₁ is an alkyl or a group –(CH₂)_k3-Y₃, in which: ok3 is 1, 2, 3, 4, 5 or 6 and oY₃ a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₃’R₃’’-(CH₂)_p3-]_m3-G₃’, with m3 being 1, 2 or 3, G₃’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p3 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₃’ and R₃’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; -R₂, R₃ and R₄ are identical or different and are independently selected from among hydrogen, and the groups alkyl piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, and –(CH₂)_k4-Y₄, in which: ok4 is 1, 2, 3, 4, 5, 6 and oY₄ is a group selected from among aryl, hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’, with m4 being 1, 2 or 3, G₄’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₄’ and R₄’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; with the proviso that at least one of the substituents A, R₂, R₃ and R₄ includes a secondary amine, a tertiary amine or a quaternary ammonium, including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate. 2. The compounds according to claim 1, having the formula (II) :

wherein : -X, Re, R₁, R₂, R₃, R₄, Ri, Rj, Rk and n are as defined for claim 1; -Ra, Rb, Rc and Rd, identical or different, are selected from among hydrogen and halogen atoms and the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -NHC(O)phenyl, - NHC(O)alkyl, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl, –CH=N-O-R, -C(CH₃)=N- O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, –CONHR being preferred, R being either a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: ok2 is 1, 2, 3, 4, 5 or 6 and oY₂ is a group selected from among hydroxy, C_1-6alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1,

2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl; with the proviso that at least one of the substituents Ra, Rb, Rc, Rd, R₂, R₃ and R₄ includes a secondary amine, a tertiary amine or a quaternary ammonium; and in particular two substituents Ra, Rb, Rc, Rd, R₂, R₃ and R₄ include a secondary amine, a tertiary amine or a quaternary ammonium ; including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate. 3. The compounds according to claim 1 or 2, wherein it comprises at least one group Y₂ or Y₄, and in particular two groups Y₂ and Y₄, which include(s) or is(are) a trialkylammonium, typically a trimethylammonium. 4. The compounds according to any one of claims 1 to 3, wherein k2 is 3, 4, 5 or 6. 5. The compounds according to any one of claims 1 to 4, wherein k3 is 2,

3,

4,

5 or 6; preferentially k3 is 2 or 3.

6. The compounds according to any one of claims 1 to 5, wherein k4 is 4, 5 or 6.

7. The compounds according to any one of claims 1 to 6, wherein Ri, Rj and Rk are hydrogen.

8. The compounds according to any one of claims 1 to 7, wherein X is oxygen or sulfur.

9. The compounds according to any one of claims 1 to 8, wherein n=0,

and, in particular, having the formula (III): (III), wherein R₁, R₂, R₃, R₄, Ra, Rb, Rc, Rd and Re are as defined in anyone of claims 1 to 6; including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate.

10. The compounds according to any one of claims 1 to 9, wherein at least one of the groups R₂ and R₃ is–(CH₂)_k4-Y₄, in which: ok4 is 1, 2, 3, 4, 5 or 6 and oY₄ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’, with m4 being 1, 2 or 3, G₄’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₄’ and R₄’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₄ being preferentially a trialkylammonium, typically a trimethylammonium; and the other group R₂ or R₃ is a C_1-6alkyl.

11. The compounds according to claim 10 of formula (IIa):

( ), - Rc is a hydrogen or halogen atom or a group selected from among the groups alkyl, alkenyl, alkynyl, aryl, -CF₃, -NO₂, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -NHC(O)phenyl, -NHC(O)alkyl, -S(O₂)O-, -S(O₂)Oalkyl, -P(O₂)O-, P(O₂)Oalkyl,–CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)- R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR and –CONHR, the group – CONHR being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which: - k2 is 1, 2, or preferentially 3, 4, 5 or 6 and - Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium, or -[N⁺Me₂-(CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above; - X, Re, R₁, R₂, R₃, R₄, Ri, Rj, Rk and n are as defined for any one of claims 1 to 10; including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate.

12. The compounds according to any one of claims 1 to 9, wherein Z or the corresponding group in formula (II) and (III), is substituted by at least one –CH=N-O-R, -C(CH₃)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR or –CONHR, –CONHR being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which k2 is 1, 2, or preferentially 3, 4, 5 or 6 and Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G₂’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium, or -[N⁺Me₂-(CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above.

13. The compounds according to claim 12 of formula (IIa):

wherein Rc is –CH=N-O-R, -C(CH3)=N-O-R, –CH=N-NH-C(O)-R, -C(CH₃)=N-NH-C(O)-R, –CH=N-O-C(O)-R, -C(CH₃)=N-O-C(O)-R, –NHCOR or –CONHR, –CONHR being preferred, with R being selected from piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the rest of the molecule by one of its carbon atom, or –(CH₂)_k2-Y₂, in which k2 is 1, 2, or preferentially 3, 4, 5 or 6 and Y₂ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₂’R₂’’-(CH₂)_p2-]_m2-G₂’, with m2 being 1, 2 or 3, G’₂ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₂’ and R₂’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₂ being preferentially a trialkylammonium, typically a trimethylammonium, or -[N⁺Me₂-(CH₂)_p2-]_m2-N⁺Me₃ with p2 and m2 as described above ; and X, Re, R₁, R₂, R₃, R₄, Ri, Rj, Rk and n are as defined for any one of claims 1 to 10, including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate.

14. The compounds according to any one of claims 10 to 13, wherein R₄ = H, R₁ and R₃ are identical or different and are C_1-6alkyl, and in particular methyl or ethyl, and R₂ is –(CH₂)_k4-Y₄, in which k4 is 1, 2, 3, 4, 5 or 6 and Y₄ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N⁺R₄’R₄’’-(CH₂)_p4-]_m4-G₄’, with m4 being 1, 2 or 3, G₄’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R₄’ and R₄’’, identical or different, being a C_1-6alkyl, preferentially a methyl or an ethyl, Y₄ being preferentially a trialkylammonium, typically a trimethylammonium.

15. The compounds according to any one of claims 1 to 14, wherein Re is C_1-6alkyl, in particular methyl.

16. The compounds according to any one of claims 1 to 15, wherein it includes at least one quaternary ammonium, preferentially a trialkylammonium, typically a trimethylammonium.

17. The compounds according to any one of claims 1 to 16, wherein it is in the form of a trifluoroacetate salt.

18. The compounds according to claim 1 selected from among:

including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate, in particular their trifluoroacetate salt.

19. Use of a compound according to any one of claims 1 to 18, for the detection of a target nucleic acid which is a single stranded or double stranded nucleic acid.

20. Method for detecting a target nucleic acid which is a single stranded or double stranded nucleic acid comprising a step of mixing a compound according to any one of claims 1 to 18, with a sample comprising the target nucleic acid or an amplicon of the target nucleic acid.

21. The use according to claim 19 or the method according to claim 20, wherein the following steps are carried out: - amplifying the target nucleic acid to generate the amplicon, - adding a compound according to any one of claims 1 to 18 to the sample comprising the target nucleic acid and/or the amplicon, before, during or after the amplifying step, - monitoring fluorescence from the compound according to the invention during or subsequent to the amplifying step.

22. The use or the method according to claim 21, wherein the following steps are carried out: -amplifying the target nucleic acid, in the presence of the compound according to any one of claims 1 to 18, in particular by PCR, to generate the amplicon, and - during the amplification, monitoring the fluorescence of the compound according to any one of claims 1 to 18, resulting from the binding of the compound to the amplicon.

23. The use according to claim 19 or the method according to claim 20 or 20, wherein subsequent to the amplification step, a step of melting the generated amplicon is carried out, while monitoring the fluorescence from the compound according to any one of claims 1 to 18, to obtain a melting curve.

24. A method of PCR analysis of a target nucleic acid comprising the steps of: - mixing a compound according to any one of claims 1 to 18 with a sample comprising a target nucleic acid, a polymerase, and a pair of primers suitable to amplify a portion of the target nucleic acid and generate at least an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture and generating at least an amplicon, and - monitoring the fluorescence from the compound according to any one of claims 1 to 18 during or subsequent to the amplifying step.

25. The method of claim 24, further comprising detecting the presence of the amplicon from the monitored fluorescence.

26. The method of claim 24, wherein the monitoring step occurs subsequent to amplification and includes generating a melting curve.

27. The method of claim 25 wherein the melting curve is used to identify the genotype of the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism, and/or epigenetic variation.

28. The method of any one of claims 20 to 27 wherein it includes a step of quantifying the target nucleic acid, which is initially present in the sample.

29. The method of PCR analysis of a target nucleic acid according to claim 23, comprising the steps of: - mixing a compound according to any one of claims 1 to 18 with a sample comprising a target nucleic acid and at least a pair of primers suitable to amplify a portion of the target nucleic acid and generate an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture, and generating at least an amplicon, - during the amplifying step, monitoring the fluorescence of the compound according to any one of claims 1 to 18 resulting from the binding of the compound to the amplicon, - at the end of the amplifying step, melting the generated amplicon, to obtain a melting curve, and - identifying the genotype or polymorphism of the target nucleic acid using a shape of the melting curve.

30. The method according to anyone of claims 20 to 29, wherein the amplifying step includes a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 90 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.005 µM or 0.02 U/µL and primers are each provided at a concentration of at least 0.1 µM.

31. The method according to anyone of claims 20 to 29, wherein the amplifying step includes a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 20 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.5 µM or 1.9 U/µL and primers are each provided at a concentration of at least 2 µM.

32. A PCR reaction mixture comprising: -a target nucleic acid, -at least a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, -a polymerase, in particular a thermostable polymerase, -a compound according to any one of claims 1 to 18.

33. The PCR reaction mixture according to claim 32, wherein it is in a buffer of pH from 7.5 to 9.5, preferentially 8 to 9.

34. A kit for detecting a target nucleic acid, comprising: -at least a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, -a polymerase, in particular a thermostable polymerase, and -a compound according to any one of claims 1 to 18.

35. The kit according to claim 34, also including a buffer of pH from 7.5 to 9.5, preferentially 8 to 9.

36. The kit according to claim 34 or 35, wherein the compound according to any one of claims 1 to 18 is provided in the buffer.