Novel nucleic acid binding compounds and uses

Abstract

The present invention relates to compounds of formula (I) as defined in claim 1: TIFF2025540566000073.tif33167 and salts thereof, and their uses, particularly in PCR applications.

Description

[Technical Field]

[0001] The present invention relates to novel compounds that can bind to nucleic acids, in particular single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). This binding can correspond to the binding of the compound to the nucleic acid structure, for example by weak bonds, in particular by hydrogen or ionic bonds, or any other weak bonds. The compounds according to the present invention exhibit a change in fluorescence resulting from the binding of the compound to nucleic acids, typically ssDNA or dsDNA. [Background technology]

[0002] Compounds that can bind or intercalate into DNA strand conformation, resulting in changes in fluorescence, are useful tools for studying nucleic acids. These compounds, known as nonspecific DNA-binding dyes, can be used to detect and quantify DNA and RNA in a variety of environments, including solutions, cell extracts, electrophoresis gels, microarray chips, living or fixed cells, dead cells, and environmental samples. These compounds have also been used in real-time polymerase chain reaction (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. Because melt curve analysis is specific and allows for gene identification, it is useful for identifying gene mutations.

[0003] 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 proposed to be suitable for DNA detection. However, the present inventors have reproduced certain examples from these patents, and the results obtained indicate that these examples have potentially improved fluorescence or stability. It is interesting that this class of compounds has stability that allows for their storage under conditions comparable to those commonly used in PCR, i.e., aqueous, slightly alkaline conditions. Thiols are commonly used in PCR media for reverse transcriptase PCR (RT-PCR) and are also present in buffers used for storing PCR enzymes. Therefore, thiols may be present during PCR, and some of the compounds described in these patents are not very stable in the presence of thiols. Furthermore, unlike other compounds described in US 7,387,887, Example A10 in US 7,387,887 cannot be produced by the method described; production begins exclusively with urea or thiourea.

[0004] WO2008 / 052742, published by Roche Diagnostics GmbH, proposes a new class of fluorescent dyes capable of emitting fluorescence when appropriately excited while specifically bound to double-stranded nucleic acids. This new class of fluorescent dyes contains a pyrimidinyl ring, the 5- and 6-positions of which form additional aromatic structures, particularly quinazolinyl structures. This fluorescent dye moiety is presented at the end of page 6 of this patent application WO2008 / 052742 as essential for obtaining interesting excitation and emission spectra. The patent application states that the proposed compounds have improved thermal and chemical stability, but that the only concern in stability studies is photochemical stability. In addition, this fused aromatic structure significantly affects the stability and fluorescence of the molecule. The inventors of the present patent application have shown that thiazole orange and SybrGreen, which contain quinolinyl and thus have a structure similar to that described in WO2008 / 052742, are not satisfactorily stable both under PCR conditions and during storage in aqueous, slightly alkaline media. In the present invention, the inventors propose a new structure in which the pyrimidine does not form part of an additional aromatic structure.

[0005] Patent US9,682,970 in the name of BIOTIUM describes many molecules containing fluorescent nucleic acid dyes and methods for their use, including nucleic acid detection, nucleic acid amplification reactions, and high-resolution melt curve analysis. A wide variety of formulas have been proposed for these molecules, and these formulas encompass a wide variety of structures. Substituents in the proposed structures can contain positively charged moieties, which have the effect of enhancing the nucleic acid binding affinity of the molecule through electrostatic interactions between the negatively charged phosphate backbone of nucleic acids and the positive charge of the moiety. The positively charged moiety can be covalently attached to the molecule by various types of arms originating from an N, O, S, or C atom (see the definitions of L1 and L2 in column 15 of US9,682,970). There are no examples of molecules of this type containing pyrimidinium groups. The exceptions include molecules that possess highly basic guanidino groups. One example is the following molecule:

[0006] [ka]

[0007] There is no description of a method for preparing this compound, and as a result, it is not publicly available. Furthermore, in this patent application, column 53 states that "The higher degree of protonation of guanidino and amidino than amine side chains also makes guanidino and amidino more effective than amine side chains in enhancing the nucleic acid binding of the compounds of the present invention through electrostatic interactions. Furthermore, amidino and guanidino in particular contain a large number of nitrogen atoms and acidic protons, which make them good hydrogen bond acceptors and donors. Thus, in addition to electrostatic interactions, hydrogen bonds between guanidino or amidino and the nucleic acid backbone may also play a role in enhancing the nucleic acid binding affinity of the compound. In the context of nucleic acid dyes, these collective properties of guanidino and amidino make them excellent alternatives to amine side chains."

[0008] Several compounds belonging to the class of non-specific DNA-binding dyes and devices for DNA detection and / or analysis are commercially available: Thiazole Orange, SYBR® Green I, LCGreen® Plus (Clinical Chemistry Vol. 52: No. 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). However, improvements in various aspects of nucleic acid-binding dyes are still needed, including, but not limited to, detection limit, dynamic range of detection, and compatibility with different detection formats and devices. The present invention aims to address some of these needs. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,456,281 [Patent Document 2] U.S. Patent No. 7,387,887 [Patent Document 3] U.S. Patent No. 7,582,429 [Patent Document 4] International Publication No. 2008 / 052742 [Patent Document 5] U.S. Patent No. 9,682,970 [Non-patent literature]

[0010] [Non-Patent Document 1] Clinical Chemistry Vol. 52: No. 3, 2006, Mark G. Herrmann et al., “Amplicon DNA melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dye” Summary of the Invention [Problem to be solved by the invention]

[0011] In this context, the object of the present invention is to provide novel nucleic acid-binding compounds, more specifically DNA-binding compounds (especially dsDNA-binding compounds), which exhibit enhanced, detectable fluorescence when bound to nucleic acids (typically DNA, especially dsDNA). The object of the present invention is to propose such novel compounds with overall improved properties. In particular, the object of the present invention is to provide compounds with both stable and fluorescent properties suitable for nucleic acid detection, more specifically DNA detection. In particular, with regard to stability, the compounds according to the present invention have excellent stability at pH 9 in tests carried out at 40°C (this test is an accelerated test related to the evaluation of stability at ambient temperature). Furthermore, other results indicate their stability in the presence of thiols in aqueous media. Due to their particularly suitable stability and fluorescence stability, the compounds according to the present invention provide high performance in nucleic acid detection, nucleic acid amplification reactions, and high-resolution melt curve analysis. Furthermore, they can be formulated and stored in aqueous media, which facilitates their rapid implementation in targeted nucleic acid detection methods, typically using PCR technology. [Means for solving the problem]

[0012] (Summary of the Invention) The present invention relates to a compound of formula (I):

[0013] [ka] [In the formula, - n is equal to 0, 1, 2 or 3, Ri, Rj and Rk are the same or different and are hydrogen and C 1~6 independently selected from the group consisting of alkyl, X is oxygen, sulfur, selenium, tellurium or C(CH3)2, Re is an alkyl or piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group attached to the remainder of the molecule by one of its carbon atoms or Re is -(CH2) k1 -Y1, where k1 is 1, 2, 3, 4, 5 or 6, Y1 is hydroxy, C 1~6 Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N + R1'R1"-(CH2) p1 -] m1 -G1' group (m1 is 1, 2 or 3, G1' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p1 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R1' and R1" are the same or different, C 1~6 alkyl, and preferentially methyl or ethyl, - Z is a halogen atom and 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)O alkyl, -P(O2)O -, -P(O2)Oalkyl, -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR and -CONHR groups, -CONHR being preferred, and which are fused monocyclic or polycyclic aromatic or nitrogen-containing heteroaromatic rings, optionally substituted by one or several identical or different substituent(s) A, where R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group linked to the rest of the molecule by one of its carbon atoms, or -(CH2) k2 -Y2, where k2 is 1, 2, 3, 4, 5 or 6, Y2 is hydroxy, C 1~6 Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N + R2'R2"-(CH2) p2 -] m2 -G2' group (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, and preferentially methyl or ethyl, - R1 is alkyl or -(CH2) k3 -Y3, where k3 is 1, 2, 3, 4, 5 or 6, Y3 is hydroxy, C 1~6 Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N + R3'R3"-(CH2) p3 -] m3-G3' group (m3 is 1, 2 or 3, G3' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p3 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R3' and R3" are the same or different, C 1~6 alkyl, and preferentially methyl or ethyl, R2, R3 and R4 are the same or different and are selected from hydrogen, alkyl groups, aryl groups, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the remainder of the molecule through one of its carbon atoms, and -(CH2) k4 -Y groups, wherein: k4 is 1, 2, 3, 4, 5, 6, Y4 is aryl, hydroxy, C 1~6 Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N + R4'R4"-(CH2) p4 -] m4 -G4' group (m4 is 1, 2 or 3, G4' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p4 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R4' and R4" are the same or different, C 1~6 alkyl, and preferentially methyl or ethyl, provided that at least one of the substituents A, R2, R3, and R4 comprises a secondary amine, a tertiary amine, or a quaternary ammonium. and at least one anion, particularly a halide anion, typically Cl - , Br - and I -trifluoroacetate, acetate, formate; sulfonate, e.g., methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, e.g., methylsulfate; phosphate, pyrophosphate, and triphosphate.

[0014] According to particular embodiments of the compounds of formula (I) and salts thereof, Z is selected from the group consisting of halogen atoms, alkyl, alkenyl, alkynyl, aryl, -CF, -NO, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -S(O)O. - , -S(O2)O alkyl, -P(O2)O - , P(O2)Oalkyl, -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR and -CONHR groups, -CONHR being preferred, and which are fused monocyclic or polycyclic aromatic or nitrogen-containing heteroaromatic rings, optionally substituted by one or several identical or different substituent(s) A, where R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group linked to the rest of the molecule by one of its carbon atoms, or -(CH2) k2 -Y2, where k2 is 1, 2, 3, 4, 5 or 6, Y2 is hydroxy, C 1~6 Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N + R2'R2"-(CH2) p2 -] m2-G2' group (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, and preferentially methyl or ethyl.

[0015] The compounds of the present invention are nucleic acid-binding compounds. They have a specific core structure with one or several substituents containing functional groups selected from secondary amines, tertiary amines, and quaternary ammonium. Secondary amines, tertiary amines, and quaternary ammonium contain or can contain positively charged moieties, which serve to provide binding affinity sites for nucleic acids. Indeed, electrostatic interactions can occur between the negatively charged phosphate backbone of nucleic acids and the positive charge of functional groups selected from secondary amines, tertiary amines, and quaternary ammonium. The positively charged functional groups can be protonated amines (i.e., secondary or tertiary amines) or quaternary ammonium, typically trialkylammonium. Secondary and tertiary amines are bases whose basicity increases from secondary to tertiary. Thus, the fraction of these protonated forms varies and increases from primary to tertiary and is a function of the pH of the medium in which they are present. In the targeted applications, the pH is typically in the range of 7.5 to 9.5, typically 8 to 9, and in both cases, a protonated fraction is present. Quaternary ammonium, typically trialkylammonium, is a completely and permanently positively charged moiety, independent of the pH of the medium.These functional groups, selected from secondary amine, tertiary amine and quaternary ammonium, are introduced into the compound according to the present invention by specific bonding and at specific positions.All of these choices result in compounds that not only exhibit high fluorescence and stability when used in amplification techniques, particularly PCR techniques, but also show great stability when stored in aqueous, slightly alkaline medium, and even in the presence of thiol entities.

[0016] According to a particular embodiment, the compound according to the invention has formula (II):

[0017] [ka] [In the formula, X, R, R, R, R, R, R, R, R, and n are as previously defined for formula (I), - Ra, Rb, Rc and Rd are the same or different and are hydrogen and halogen atoms and 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)O alkyl, -P(O2)O - , P(O2)Oalkyl, -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR and -CONHR groups, -CONHR being preferred and R being a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms, or -(CH2) k2 -Y2, where k2 is 1, 2, 3, 4, 5 or 6, Y2 is hydroxy, C 1~6 Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N + R2'R2"-(CH2) p2 -] m2 -G2' group (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6alkyl, and preferentially methyl or ethyl, provided that at least one of the substituents Ra, Rb, Rc, Rd, R2, R3 and R4 comprises a secondary amine, a tertiary amine or a quaternary ammonium, and in particular two of the substituents Ra, Rb, Rc, Rd, R2, R3 and R4 comprise a secondary amine, a tertiary amine or a quaternary ammonium. and at least one anion, particularly a halide anion, typically Cl - , Br - and I - trifluoroacetate, acetate, formate; sulfonate, for example, methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, for example, methylsulfate; phosphate, pyrophosphate, and triphosphate.

[0018] According to a particular embodiment of the compounds of formula (II) and their salts, Ra, Rb, Rc and Rd are identical or different and are selected from the group consisting of hydrogen and halogen atoms, as well as alkyl, alkenyl, alkynyl, aryl, -CF3, -NO2, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -S(O2)O. - , -S(O2)O alkyl, -P(O2)O - , P(O2)Oalkyl, -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR and -CONHR groups, -CONHR being preferred and R being a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms, or -(CH2) k2 -Y2, where k2 is 1, 2, 3, 4, 5 or 6, Y2 is hydroxy, C 1~6Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N + R2'R2"-(CH2) p2 -] m2 -G2' group (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, and preferentially methyl or ethyl.

[0019] Typically, in the compounds of formula (II) and their salts, at least two (particularly two) of the substituents Ra, Rb, Rc, Rd, R2, R3 and R4 comprise a secondary amine, a tertiary amine or a quaternary ammonium, and in particular at least two (typically two) of the substituents Ra, Rb, Rc, Rd, R2, R3 and R4 comprise a quaternary ammonium, such as a trialkylammonium, typically trimethylammonium. Advantageously, Rc and R2 are two substituents comprising a quaternary ammonium, such as a trialkylammonium, typically trimethylammonium.

[0020] According to a particular embodiment, the compounds according to the invention (corresponding to formula (I) or (II)) comprise one of the following characteristics or any combination of the following characteristics, and advantageously, when these characteristics are not mutually exclusive, they have all the following characteristics: at least one Y2 or Y4 group, in particular only one Y2 or Y4 group, or two Y2 and Y4 groups, comprises or is trialkylammonium, typically trimethylammonium, k2 is 3, 4, 5 or 6, typically 3; k3 is 2, 3, 4, 5 or 6, typically k3 is 2 or 3; k4 is 4, 5 or 6, typically dialkylamino, trialkylammonium or -[N + R4'R4"-(CH2) p4 -] m4 -G4' is 4; Ri, Rj and Rk are hydrogen; - R4 = H, R1 and R3 are the same or different, C 1~6 alkyl, in particular methyl or ethyl, and according to a particular embodiment, R4 = H, R1 is methyl or ethyl and R3 is methyl; X is oxygen or sulfur, in particular sulfur.

[0021] By selecting the value of n, it is possible to adjust the fluorescence characteristics obtained after the compound of the present invention binds to a nucleic acid amplicon. For example, compared to a compound with n=0, a compound with n=1, 2, or 3 can detect the amplification and melting of the amplicon at higher excitation and emission wavelengths. This is useful for reducing the fluorescence background of materials used in nucleic acid detection / analysis, such as plastics, and can thereby increase the sensitivity of fluorescence detection resulting from the binding of the compound of the present invention to a nucleic acid, or more precisely, to a nucleic acid amplicon.

[0022] According to a particular embodiment, in particular in compounds according to the invention where n=0, the compounds of the invention have the formula (III):

[0023] [ka] (wherein R1, R2, R3, R4, Ra, Rb, Rc, Rd and Re are as defined for formula (I) or (II), and at least one anion, particularly a halide anion, typically Cl - , Br - and I -trifluoroacetate, acetate, formate; sulfonate, for example, methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, for example, methylsulfate; phosphate, pyrophosphate, and triphosphate.

[0024] Typically, in a first alternative of the compounds according to the invention (comprising formulae (I), (II) and (III)) and salts thereof, at least one (typically one) of the R2 and R3 groups is -(CH2) k4 -Y4, where k4 is 1, 2, 3, 4, 5 or 6, Y4 is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N + R4'R4"-(CH2) p4 -] m4 -G4' (m4 is 1, 2 or 3, G4' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p4 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R4' and R4" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl, and Y4 is preferentially trialkylammonium, typically trimethylammonium, or -[N + Me2-(CH2) p4 -] m4 -N + Me3 (p4 and m4 are as described above), Other groups that are R2 or R3 are C 1~6 It is alkyl.

[0025] In particular, R2 is as described above -(CH2) k4 -Y4 and R3 is C 1~6 It is an alkyl, typically a methyl group.

[0026] According to a particular embodiment of this first alternative, the compounds of the invention have formula (IIa):

[0027] [ka] In particular, the formula (IIIa):

[0028] [ka] [In the formula, - Rc is a hydrogen or halogen atom, or an 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)O alkyl, -P(O2)O - , P(O2)O alkyl, a group selected from among -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR and -CONHR groups, the -CONHR group being preferred, R being a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms or a -(CH2) k2 -Y2, where: k2 is 1, 2, 3, 4, 5 or 6, typically k2 is 3, 4, 5 or 6, Y2 is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C1~6 alkyl, preferentially methyl or ethyl, and Y2 is preferentially trialkylammonium, typically trimethylammonium or -[N + Me2-(CH2) p2 -] m2 -N + Me3 (wherein p2 and m2 are 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. and at least one anion, particularly a halide anion, typically Cl - , Br - and I - , trifluoroacetate ion, acetate ion, formate ion; sulfonate ions, for example, methylsulfonate ion, trifluoromethylsulfonate ion, and tosylate ion; sulfate ions, for example, methylsulfate ion; phosphate ion, pyrophosphate ion, and triphosphate ion.

[0029] According to a particular embodiment of this first alternative, in particular in formula (IIa) or (IIIa), Rc is a hydrogen or halogen atom, or is alkyl, alkenyl, alkynyl, aryl, -CF3, -NO2, -CN, -C(O)alkyl, -Salkyl, -Oalkyl, -NHalkyl, -NHC(O)H, -S(O2)O - , -S(O2)O alkyl, -P(O2)O - , P(O2)O alkyl, a group selected from the group consisting of -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR and -CONHR, the -CONHR group being preferred, where R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms or -(CH2) k2 -Y2, where: k2 is 1, 2, 3, 4, 5 or 6, typically k2 is 3, 4, 5 or 6, Y2 is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl, and Y2 is preferentially trialkylammonium, typically trimethylammonium or -[N + Me2-(CH2) p2 -] m2 -N + Me3 (p2 and m2 are as described above).

[0030] According to a particular embodiment of this first alternative, in particular in formula (IIa) or (IIIa), Rc is a hydrogen atom, a halogen atom, in particular Br, -NHC(O)alkyl, in particular -NHCOMe, -NHCOR or -CONHR, and R is -(CH2) k2 -Y2, where k2 is 3, 4, 5 or 6, in particular k2 is 3, Y2 is trialkylammonium or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, G2' is trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl, and Y2 is preferentially trialkylammonium, typically trimethylammonium or -[N + Me2-(CH2) p2 -]m2 -N + Me3 (p2 and m2 are as described above, in particular m2=1 and p2=2 or 3).

[0031] According to a particular embodiment of this first alternative, in particular in formula (IIa) or (IIIa), R4 = H, R1 and R3 are identical or different, and C 1~6 alkyl, especially methyl or ethyl, and R2 is -(CH2) k4 -Y4 (k4 is 4, 5 or 6, Y4 is trialkylammonium, especially trimethylammonium), Rc is hydrogen, or -NHCOR or -CONHR [R is -(CH2) k2 -Y2, k2 is 3, 4, 5 or 6, Y2 is trialkylammonium, especially trimethylammonium or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, G'2 is trialkylammonium, in particular trimethylammonium, p2 is 2 or 3, R2' and R2" are the same or different, C 1~6 is alkyl, typically methyl.

[0032] According to a second alternative, in the compounds according to the invention and their salts, Z of formula (II), (IIa), (III) and (IIIa), or a corresponding group, is substituted with at least one substituent selected from among -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR or -CONHR, -CONHR being preferred, in which R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group linked to the rest of the molecule by one of its carbon atoms or -(CH2) k2-Y2, where 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' (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl, and Y2 is preferentially trialkylammonium, typically trimethylammonium or -[N + Me2-(CH2) p2 -] m2 -N+Me3 (where p2 and m2 are as described above). In other words: In the compounds of formula (II) and (III) and their salts, at least one substituent Ra, Rb, Rc or Rd is selected from among the groups -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR or -CONHR, -CONHR being preferred, in which R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group linked to the rest of the molecule by one of its carbon atoms or -(CH2) k2 -Y2, where 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' (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl, and Y2 is preferentially trialkylammonium, typically trimethylammonium or -[N + Me2-(CH2) p2 -] m2 -N + Me3 (wherein p2 and m2 are as described above), in the compounds of formula (IIa) and (IIIa) and their salts, Rc is selected from among -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR or -CONHR, -CONHR being preferred and R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group linked to the rest of the molecule by one of its carbon atoms or -(CH2) k2 -Y2, where 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' (m2 is 1, 2 or 3, G2' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl, and Y2 is preferentially trialkylammonium, typically trimethylammonium or -[N + Me2-(CH2) p2 -]m2 -N + Me3 (p2 and m2 are as described above).

[0033] According to a particular embodiment of this second alternative, the compound of the invention has formula (IIa):

[0034] [ka] In particular, formula (IIIa):

[0035] [ka] wherein Rc is -CH=NOR, -C(CH3)=NOR, -CH=N-NH-C(O)-R, -C(CH3)=N-NH-C(O)-R, -CH=NOC(O)-R, -C(CH3)=NOC(O)-R, -NHCOR or -CONHR, -CONHR being preferred, and R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms or -(CH2) k2 -Y2, where 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' (m2 is 1, 2 or 3, G'2 is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl, and Y2 is preferentially trialkylammonium, typically trimethylammonium, X, Re, R1, R2, R3, R4, Ri, Rj, Rk and n are as defined for formula (I) and previously for the second alternative. and at least one anion, particularly a halide anion, typically Cl - , Br - and I - trifluoroacetate, acetate, formate; sulfonate, for example, methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, for example, methylsulfate; phosphate, pyrophosphate, and triphosphate.

[0036] According to a particular embodiment of this second alternative, R4 = H, R1 and R3 are identical or different, and C 1~6 alkyl, especially methyl or ethyl; R2 is hydrogen, C 1~6 alkyl, in particular methyl or ethyl, or -(CH2) k4 -Y4 (k4 is 4, 5 or 6, Y4 is trialkylammonium, especially trimethylammonium), Rc is -NHCOR or -CONHR, and R is -(CH2) k2 -Y2, where k2 is 3, 4, 5 or 6, and Y2 is trialkylammonium, especially trimethylammonium, or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, G'2 is trialkylammonium, especially trimethylammonium, p2 is 2 or 3, R2' and R2" are the same or different, C 1~6 alkyl, typically methyl.

[0037] According to another particular embodiment of the compounds according to the invention (including the first and second alternatives and all disclosed embodiments), R4 = H, R1 and R3 are the same or different, and C 1~6 alkyl, especially methyl or ethyl, and R2 is -(CH2) k4-Y4, where 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' (m4 is 1, 2 or 3, G4' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p4 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R4' and R4" are the same or different, C 1~6 alkyl, preferentially methyl or ethyl; and Y4 is preferentially trialkylammonium, typically trimethylammonium.

[0038] According to another particular embodiment of the compounds according to the invention (including the first and second alternatives and all disclosed embodiments), R4 = H, R1 is methyl or ethyl, and R3 is methyl.

[0039] Depending on the considered substituents, secondary amine, tertiary amine or quaternary ammonium (provided as defined) may correspond to the following groups in the compounds of formulae (I), (II), (IIa), (III) and (IIIa): piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, morpholinyl, alkylamino, dialkylamino and trialkylammonium groups, which include -[N + R2'R2"-(CH2) p2 -] m2 -G2' and -[N + R4'R4"-(CH2) p4 -] m4 Groups present within the group -G4' are also included.

[0040] According to a particular embodiment of all compounds described in the present invention, Re is typically C 1~6It is alkyl, in particular methyl. In particular, when the compounds described in the present invention contain one or several quaternary ammonium groups, this quaternary ammonium group is trimethylammonium.

[0041] According to certain embodiments of the compounds according to the invention (including the first and second alternatives and all disclosed embodiments), R2 is -(CH2) k4 -Y4 (k4 is 4, 5 or 6, and Y4 is trialkylammonium, especially trimethylammonium).

[0042] According to certain embodiments of the compounds according to the present invention (including the first and second alternatives and all disclosed embodiments), Rc is -NHCOR or -CONHR [R is -(CH2) k2 -Y2, k2 is 3, 4, 5 or 6, Y2 is trialkylammonium, particularly trimethylammonium, or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, G'2 is trialkylammonium, especially trimethylammonium, p2 is 2 or 3, R2' and R2" are the same or different, C 1~6 is alkyl, typically methyl.

[0043] According to a particular embodiment of all the compounds described in the present invention, they contain at least one quaternary ammonium, preferentially trialkylammonium, typically trimethylammonium.

[0044] According to a particular embodiment of all the compounds described in the present invention, they are in the form of trifluoroacetate salts.

[0045] Particular compounds of the present invention are compounds of the formula:

[0046] [ka] wherein n is as defined for formula (I), n is typically equal to 0, 1 or 2, and Ri, Rj and Rk are as defined for formula (I), typically Ri=Rj=Rk=H, in particular formula (IIIa)

[0047] [ka] [In the formula, Re is methyl; - R4=H, R1 and R3 are alkyl groups, in particular methyl or ethyl, typically R1 and R3 are methyl; R2 and Rc are defined as follows: i) Rc is H, a halogen atom, typically Br, or -NHCOalkyl, typically -NHCOMe, and R2 is -(CH2) k4 -Y4 (k4 is 4, 5 or 6 and Y4 is trialkylammonium, especially trimethylammonium), typically R2 is -(CH2)4-N + Me3, or ii) R2 is alkyl, typically methyl, and Rc is -NHCOR or -CONHR [R is -(CH2) k2 -Y2, k2 is 3, 4, 5 or 6, Y2 is trialkylammonium, especially trimethylammonium or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, G'2 is trialkylammonium, especially trimethylammonium, p2 is 2 or 3, R2' and R2" are the same or different, C 1~6 R is alkyl, typically methyl. ) and more specifically, R is alkyl, typically methyl, and R is -CONHR [R is -(CH) k2 -Y2, k2 is 3, 4, 5 or 6, Y2 is trialkylammonium, especially trimethylammonium or -[N + R2'R2"-(CH2) p2-] m2 -G2' (m2 is 1, G'2 is trialkylammonium, especially trimethylammonium, p2 is 2 or 3, R2' and R2" are the same or different, C 1~6 Rc is -CONH-(CH2)3-N, 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 + is Me3, or iii) R2 is -(CH2) k4 -Y4 (k4 is 4, 5 or 6, and Y4 is trialkylammonium, especially trimethylammonium), Rc is -NHCOR or -CONHR [R is -(CH2) k2 -Y2, k2 is 3, 4, 5 or 6, Y2 is trialkylammonium, especially trimethylammonium or -[N + R2'R2"-(CH2) p2 -] m2 -G2' (m2 is 1, G'2 is trialkylammonium, especially trimethylammonium, p2 is 2 or 3, R2' and R2" are the same or different, C 1~6 ) and typically R2 is -(CH2)4-N + Me3, Rc is -CONH-(CH2)3-N + Me3, -CONH-(CH2)3-N + Me2-(CH2)3-N + Me3 or -NHCO-(CH2)3-N + Me3.] and at least one anion, in particular a halide anion, typically Cl - , Br - and I -trifluoroacetate, acetate, formate; sulfonate, for example, methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, for example, methylsulfate; and salts thereof with anions selected from phosphate, pyrophosphate, and triphosphate, typically trifluoroacetates.

[0048] By way of example, compounds according to the invention include:

[0049] [ka] TIFF2025540566000013.tif206163TIFF2025540566000014.tif196163TIFF2025540566000015.tif109164, and at least one anion, in particular a halide anion, typically Cl - , Br - and I - trifluoroacetate, acetate, formate; sulfonate, for example, methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, for example, methylsulfate; phosphate, pyrophosphate, and triphosphate, and salts thereof with anions selected from these, particularly trifluoroacetate salts thereof.

[0050] The present invention also relates to the use of these compounds in nucleic acid detection and analysis, as well as to the 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 previously given in the disclosure of the compound or a salt of this compound. In particular, compounds of formula (I), (II), (IIa), (III), (IIIa), or any other formula falling within the scope of formula (I) given in the specification, are used in the uses, methods, mixtures, and kits of the present invention in the form of a salt as described in the specification.

[0051] The use of the compounds according to the invention for detecting a target nucleic acid, which may be a single-stranded or double-stranded nucleic acid, is another object of the present invention.

[0052] The present invention also relates to a method for detecting a target nucleic acid, which is a single-stranded or double-stranded nucleic acid, comprising the step of mixing a compound according to the present invention with a sample comprising the target nucleic acid or an amplicon of the target nucleic acid.

[0053] In the uses and methods according to the invention, the following steps may be carried out: - amplifying the target nucleic acid to produce an amplicon; - adding a compound according to the invention to a sample containing the target nucleic acid and / or amplicon before, during or after the amplification step, - monitoring the fluorescence from the compound according to the invention during or after the amplification step.

[0054] According to a particular embodiment, in the uses and methods according to the invention, the following steps are carried out: - amplifying the target nucleic acid in the presence of a compound according to the invention, in particular by PCR, to produce an amplicon; and - monitoring the fluorescence of the compound according to the invention during amplification, resulting from the binding of the compound to the amplicon.

[0055] According to a particular embodiment, the amplification step is followed by a step of melting the generated amplicons while monitoring the fluorescence from the compound according to the invention to obtain a melting curve.

[0056] When the target nucleic acid is a double-stranded nucleic acid, particularly dsDNA, it is possible to obtain a melting curve.In particular, the melting curve can be obtained using any double-stranded nucleic acid that can melt and hybridize with a complementary nucleic acid by Watson-Crick base pairing, such as DNA, DNA-RNA hybrids, and also includes nucleotide analogs (e.g., BrdU) and / or non-phosphodiester internucleoside bonds (e.g., peptide nucleic acid (PNA) or thiodiester bonds).

[0057] According to a particular embodiment, the uses and methods according to the invention may comprise a step of quantifying the target nucleic acid 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 according to the invention with a sample comprising an amplicon of the target nucleic acid, the amount of target nucleic acid initially present in the sample obtained in the quantifying step is the amount of target nucleic acid present in the sample used in the amplification step to obtain the amplicon.

[0058] The present invention also provides - mixing the compound according to the invention with a sample comprising a target nucleic acid, a polymerase and a pair of primers suitable for amplifying a portion of the target nucleic acid and generating at least an amplicon, to obtain a PCR mixture; - amplifying the target nucleic acid from the PCR mixture to produce at least an amplicon; and - monitoring the fluorescence from the compound according to the invention during or after the amplification step. The present invention also relates to a method for PCR analysis of a target nucleic acid, comprising:

[0059] According to some embodiments, such methods of PCR analysis further comprise detecting the presence of amplicons from the monitored fluorescence.

[0060] In these PCR analysis methods, the monitoring step may occur after amplification and comprises generating a melting curve, which, according to certain embodiments, is used to genotype the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism and / or epigenetic mutation.

[0061] According to a particular embodiment, the PCR analysis method according to the invention may comprise a step of quantifying the target nucleic acid initially present in the sample.

[0062] Illustratively, the method for PCR analysis of a target nucleic acid according to the present invention comprises: - mixing a compound according to the invention with a sample comprising a target nucleic acid and at least one pair of primers suitable for amplifying a portion of the target nucleic acid and generating an amplicon, to obtain a PCR mixture; - amplifying the target nucleic acid from the PCR mixture to produce at least an amplicon; - monitoring the fluorescence of the compound according to the invention resulting from binding of the compound to the amplicon during the amplification step, - at the end of the amplification step, melting the amplicons produced to obtain a melting curve, and - using the shape of the melting curve to identify the genotype or polymorphism of the target nucleic acid. may include:

[0063] According to certain embodiments, in any of the methods of the present invention, the amplification step can include multiple temperature cycles including at least a denaturation temperature and an extension temperature, each cycle having a cycle time of less than 90 seconds, the polymerase being provided at a concentration of at least 0.005 μM or 0.02 U / μL, and the primers being provided at a concentration of at least 0.1 μM each. According to certain embodiments, the amplification step can include multiple temperature cycles including at least a denaturation temperature and an extension temperature, each cycle having a cycle time of less than 20 seconds, the polymerase being provided at a concentration of at least 0.5 μM or 1.9 U / μL, and the primers being provided at a concentration of at least 2 μM each.

[0064] Another object of the present invention is a PCR reaction mixture comprising: - target nucleic acid, - a pair of primers suitable for amplifying a portion of the target nucleic acid to produce an amplicon; - polymerases, in particular thermostable polymerases, - a compound according to the invention.

[0065] The PCR reaction mixture according to the invention may be in a buffer solution of pH 7.5-9.5, preferentially 8-9.

[0066] Another object of the present invention is to provide a kit for detecting a target nucleic acid, comprising: - a pair of primers suitable for amplifying a portion of the target nucleic acid to produce an amplicon; polymerases, in particular thermostable polymerases, and - Compounds according to the invention The kit includes:

[0067] The kit according to the invention typically comprises a buffer solution of pH 7.5-9.5, preferentially pH 8-9.

[0068] In particular, in such kits, the compounds according to the invention are provided in buffer solutions. DETAILED DESCRIPTION OF THE INVENTION

[0069] definition In the compounds according to the invention, the meanings of the substituents are as usual unless otherwise specified.

[0070] The term "alkyl," as used herein, refers to a monovalent saturated hydrocarbon moiety containing 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms. Alkyl groups may be straight-chained or branched, and illustrative examples include methyl (Me), ethyl, propyl, butyl, dodecyl, 4-ethylpentyl, and the like. 1~6 Alkyl refers to alkyl containing 1, 2, 3, 4, 5 or 6 carbon atoms, typically methyl.

[0071] The term "alkenyl," as used herein, refers to a monovalent hydrocarbon moiety containing 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms, and containing at least one carbon-carbon double bond, each of which can have an E or Z configuration. The term "alkynyl," as used herein, refers to a monovalent hydrocarbon moiety containing 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms, and containing at least one carbon-carbon triple bond. Alkenyl and alkynyl groups can be linear or branched. The double and triple bonds of alkenyl and alkynyl groups, respectively, can be in any position. 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.

[0072] The term "aryl" or aromatic ring (which may be monocyclic or polycyclic and is fused in the definition of Z), as used herein, refers to a cyclic aromatic hydrocarbonated moiety, illustratively including, but not limited to, phenyl (Ph) and naphthyl. Phenyl is an exemplary aryl group, and in Z, it is referred to as a fused benzo. As used in the definition of Z, nitrogen-containing aromatic rings refer to pyrrolo, pyrazolo, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, and the like. In particular, according to the definition of Z, typically, when fused with other rings presented in formula (I), Z can form an optionally substituted benzoxazolium ring or benzothiazolium ring, or an optionally substituted naphthoxazolium ring or naphthothiazolium ring.

[0073] Amino means -NH2. Alkylamino means -NHR' (where R' is alkyl, especially C 1~6 Alkyl is typically methyl or ethyl. Thus, alkylamino corresponds to a secondary amine.

[0074] Dialkylamino is —NR′R″ (where R′ and R″ are independently alkyl, especially C 1~6 The term "dialkylamino" refers to a group consisting of alkyl, typically methyl or ethyl. Often, R'=R". Thus, dialkylamino corresponds to a tertiary amine. The most common dialkylamino groups exemplified herein are -NMe2 and -NEt2.

[0075] Trialkylammonium is -N + R'R"R"' (R', R" and R'" are independently alkyl, particularly C 1~6 alkyl, typically methyl or ethyl. Often, R'=R"=R"'. The most common dialkylamino groups exemplified herein are -N + Me3 and -N+ It is Et3.

[0076] Unless otherwise specified, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups (referred to as nitrogen groups) can be covalently bonded to the rest of the molecule through one of their carbon atoms or through their nitrogen atom. When these nitrogen groups are covalently bonded to the core of the molecule through one of their carbon atoms, the piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups include the corresponding unsubstituted and corresponding substituted groups, and when substituted, the nitrogen atom of the piperidinyl, piperazinyl, pyrrolidinyl, or morpholinyl group may be bonded at the N-position to one or two C 1~6 The pyridinyl group is substituted with an alkyl group (typically methyl or ethyl), and the nitrogen atom of the pyridinyl group is substituted with one C 1~6 It is substituted with an alkyl group (typically methyl or ethyl) so that it is in the ammonium form.

[0077] When these nitrogen groups are covalently bonded to the core of the molecule by these nitrogen atoms, the piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups include the corresponding unsubstituted and corresponding substituted groups, and when they contain substituents, the nitrogen atom of the piperidinyl, piperazinyl, pyrrolidinyl or morpholinyl group may have one C 1~6 It is substituted with an alkyl group (typically methyl or ethyl) so that it can be in the ammonium form.

[0078] The compounds of the present invention are cyanine derivatives having a pyrimidinium core structure, where X is, in particular, oxygen or sulfur, and the moiety Z represents an optionally substituted fused benzo to form an optionally substituted benzoxazolium or benzothiazolium ring, or an optionally substituted fused naphth to form an optionally substituted naphthoxazolium or naphthothiazolium ring.

[0079] It is recognized that the compounds of Formula (I), (II), (IIa), (III), or (IIIa) and salts thereof described herein may contain one or several chiral centers. In such cases, all stereoisomers are considered 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 amounts, pure diastereoisomers, and mixtures of diastereoisomers containing any relative amounts of one or more stereoisomeric configurations.

[0080] It is also recognized that the compounds of Formula (I), (II), (IIa), (III), or (IIIa) herein and their salts may contain geometric centers. In these cases, all geometric isomers are considered 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 as various mixtures of geometric configurations. It is also understood that, depending on 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 can interconvert between cis and trans, or between E and Z configurations, depending on conditions such as solvent composition, solvent polarity, ionic strength, etc. Thus, the two forms cis / trans or E / Z are often in equilibrium. In the formulae (III), (IIIa), (I.1) to (I.9) of the present invention, even if the double bond is presented in a particular geometrical form, all possible isomeric forms are in equilibrium.

[0081] The compounds according to the invention are in the form of salts: the compounds of formula (I), (II), (IIa), (III) or (IIIa) may be N +The compounds of the present invention are positively charged by (Re). When the compounds of the present invention contain quaternary ammonium groups, they can contain additional charges. Salts of compounds of formula (I), (II), (IIa), (III) or (IIIa) contain several anions (typically identical) corresponding to the number of positive charges on the compound of formula (I), (II), (IIa), (III) or (IIIa). When the compounds of formula (I), (II), (IIa), (III) and (IIIa) are positively charged, several resonance structures of these compounds can exist. Typically, the positive charge is formally located on the nitrogen atom N as shown in formula (I), (II), (IIa), (III) and (IIIa). + The charge may be located on (Re) or alternatively, the charge may be located on the pyrimidinyl group.

[0082] "Nucleic acid," as used herein, refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA, RNA, or a DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, capable of hybridizing to a complementary nucleic acid via Watson-Crick base pairing. Nucleic acids of the present invention can also contain nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside 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.

[0083] Preparation of compounds according to the invention When n=0 and X=S, compounds according to the invention can be obtained by the route presented in Scheme 1, where Z, Ri, Re, R1, R2, R3 and R4 are as defined for (I).

[0084] [ka]

[0085] The inventors have 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 at the position corresponding to R1.

[0086] In Scheme 1, an appropriately substituted amidine (IX) is reacted with an appropriately substituted diacetone (VIII) under strongly alkaline conditions (e.g., in the presence of potassium carbonate), typically in a water / ethanol mixture, at room temperature (typically 22°C) for 1-6 weeks to give pyrimidine molecule (VII), which can be purified by column chromatography or liquid / liquid extraction. Pyrimidine molecule (VII) is alkylated at the N1 position by reacting with an excess of alkylating agent (VI) (P-R1, where P is Cl, Br, or I or a tosyl group, and R1 is as defined for (I)), typically in acetonitrile at 50-90°C in a sealed tube for 1-6 days to give pyrimidinium compound (V). After evaporation of the remaining alkylating agent (VI), compound (V) is reacted with compound (IV) (typically a benzothiazolium derivative), typically in a mixture of acetonitrile, ethanol, and triethylamine, at room temperature (typically 22°C) for several minutes to produce the expected unsymmetrical cyanine (I), which is then purified by reverse-phase chromatography. Examples of alkylating agents (VI) P-R1 are MeI, EtI, and Br(CH2)3N(Me3). When R2 = H, alkylating agent (VI) can also be R1-tosyl.

[0087] Compounds (IX), (VIII) and (VI) are commercially available or are prepared from commercially available compounds by common practice.

[0088] Compound (IV) may also be commercially available. If compound (IV) is not commercially available, especially if it is substituted at the Z ring, these compounds can be prepared as described in Scheme 2 below (Routes 1-3 relate to the synthesis of certain compounds of formula (IV)), or by methods analogous thereto. Scheme 2 relates to compounds (IV) where Z is a fused benzo and Re is Me, and describes different ways of introducing the substituent Rc into 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.

[0089] [ka]

[0090] According to Route 1, commercially available ester 4 is hydrolyzed under alkaline conditions to give carboxylic acid 5, which is activated in the form of hydroxysuccinimidyl ester 6. This hydroxysuccinimidyl ester 6 can be conveniently substituted with an amine, for example amine 7. Total alkylation of the nitrogen atoms in the alkyl chain and in the benzothiazole ring is advantageously carried out simultaneously with an alkylating agent at elevated temperature to give the expected product VI.1. Route 3 is similar to Route 1, but with the addition of two ammonium groups (one -N in the illustrative example). + (Me)3 and -N(Me) + They differ in that they incorporate more complex Rc groups, including those with 2-

[0091] According to Route 2, commercially available aminocarboxylic acid 11 was activated in the form of hydroxysuccinimidyl ester 12 and conjugated to aminobenzothiazole 13, as described in Routes 1 or 3, followed by peralkylation of the amino atoms in the alkyl chain and in the benzothiazole ring to give compound VI.2.

[0092] Other compounds of formula (IV) can be easily prepared using other routes, which are adapted or similar to Routes 1 to 3. Depending on the desired compound, a person skilled in the art can select and perform more appropriate steps.

[0093] 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 reaction presented in Scheme 3, where Z, Re, R1, R2, R3 and R4 are as defined for (I).

[0094] [ka]

[0095] As shown in Scheme 3, a suitably substituted methylbenzothiazole (X) can be N-alkylated to give compound (IX) in the presence of an alkylating agent Re-P (where P is a leaving group, e.g., Cl, Br, or I, or a tosyl group, and Re is as defined for (I)) at elevated temperatures (e.g., 130-160 °C) without solvent. The leaving group most suitable for the Re group is selected. When Re = Me, MeOT can be used. The resulting activated compound (IX) can then be reacted with a masked aldehyde as bisphenylimine (XII) in the presence of acetic anhydride and acetic acid, or by fusion alone, to give the corresponding acetylated hemicyanine (XIII), which can be purified by reverse-phase chromatography using acetonitrile / water / TFA as the eluent. The hemicyanine (XIII) can then be reacted with an activated pyrimidinium (V.1) under slightly alkaline conditions to give the expected compound (I).

[0096] When X=O or when X=C(CH), the same routes as those described in Schemes 1 and 3 can be followed, except that the initial benzothiazole compound (X) is replaced with benzoxazole or dimethylindole, respectively, as the starting compound.

[0097] Compounds of formula (IX) can be prepared in a manner similar to that described for compounds of formula (IV). Compounds (X) and (XII) are commercially available or are prepared from commercially available compounds according to common practice.

[0098] Use of the compounds according to the invention The compounds of the present invention can be used for a variety of different applications. In particular, they can be used for detecting target nucleic acids, which can be single-stranded or double-stranded. The compounds of the present invention have the ability to bind to nucleic acids, particularly double-stranded nucleic acids, typically single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and typically exhibit this binding ability when these compounds are in an aqueous medium with a pH of 5 to 11, i.e., in an aqueous medium commonly used as a PCR medium, with a pH of 7.5 to 9.5, typically pH 8 to 9. The compounds of the present invention also have the ability to bind to RNA. This binding to nucleic acids can correspond to weak bonds, particularly hydrogen or ionic bonds, or any other bond with a weak bond. The compounds of the present invention exhibit a change in fluorescence generated from their binding to nucleic acids, particularly double-stranded nucleic acids, typically ssDNA or preferentially dsDNA. The compounds of the present invention can interact with nucleic acids (i.e., RNA or DNA strands), typically double-stranded DNA, particularly the minor groove of the DNA double helix, and more generally bind to DNA strands through several types of weak bonds, resulting in a change in fluorescence that can be monitored. As a result, these compounds are useful tools for the study of nucleic acids. After they bind to nucleic acids, the compounds according to the invention behave as fluorescent dyes.

[0099] Thus, the present invention encompasses uses and methods for detecting a target nucleic acid, which is a single-stranded or double-stranded nucleic acid, comprising mixing a compound according to the present invention with a sample containing the target nucleic acid.

[0100] The present invention also relates to a method for detecting a target nucleic acid, which is a single-stranded or double-stranded nucleic acid, comprising the step of mixing a compound according to the present invention with a sample comprising the target nucleic acid or an amplicon of the target nucleic acid.

[0101] In certain embodiments, the compounds according to the present invention are used to detect double-stranded nucleic acids in real time during nucleic acid amplification reactions and / or after amplification via melting curve analysis or end-point analysis.In this context, the compounds according to the present invention are part of the reaction mixture for amplification reactions, preferably PCR reactions, and may already be present at the start of the amplification reaction.As shown by the inventors, the compounds according to the present invention do not significantly interfere with the efficiency of such amplification (preferably PCR) reactions.In particular, the compounds according to the present invention do not significantly inhibit amplification (preferably PCR) even when present at a concentration that provides a high fluorescent signal relative to a certain amount of nucleic acid, typically relative to the amount of dsDNA generated by PCR in the absence of the compounds according to the present invention.

[0102] In the use or method according to the invention, the following steps may be carried out: - amplifying the target nucleic acid to produce an amplicon; - adding a compound according to the invention to a sample containing the target nucleic acid and / or amplicon before, during or after the amplification step, - monitoring the fluorescence from the compound according to the invention during or after the amplification step.

[0103] According to a particular embodiment, in the uses and methods according to the invention, the following steps are carried out: - amplifying the target nucleic acid in the presence of a compound according to the invention, in particular by PCR, to produce an amplicon; and - monitoring the fluorescence of the compound according to the invention during amplification, resulting from the binding of the compound to the amplicon.

[0104] According to a particular embodiment, the amplification step is followed by a step of melting the generated amplicons while monitoring the fluorescence from the compound according to the invention to obtain a melting curve.

[0105] It will be appreciated that in the methods and uses according to the present invention, the compounds of the present invention and the amplicons are placed in a sample suitable for obtaining their binding and fluorescence. Typically, the sample is an aqueous medium with a pH of 7.5 to 9.5, more particularly 8 to 9. Thus, the methods and uses according to the present invention include a step of contacting the generated amplicons with the compounds of the present invention under conditions that allow binding via these bonds, particularly weak bond(s). If the compounds are present during amplification, typical amplification conditions, particularly PCR amplification conditions, allow this binding. Generally, when the target nucleic acid is a double-stranded nucleic acid, typically dsDNA, binding of the compounds of the present invention to the amplicons occurs at a temperature below the melting temperature of the amplicons. The temperature during binding is, for example, in the range of 20 to 60°C. Samples containing the generated amplicons and the compounds of the present invention often contain one or several salts commonly used in PCR media, such as NaCl and MgCl2.

[0106] According to a particular embodiment, in the use or method according to the invention, the following steps may be carried out: - amplifying the target nucleic acid in the presence of a compound according to the invention, in particular by PCR, typically by real-time PCR, to generate an amplicon, - optionally during the amplification step, monitoring the fluorescence of the compound according to the invention resulting from binding of the compound to the amplicon, and - optionally after the amplification step, monitoring the fluorescence resulting from binding of the compound according to the invention to the amplicon via end-point analysis or while melting the amplicon to obtain a melting curve.

[0107] It is also possible to carry out the amplification step first and add the compound according to the invention later, especially if the aim is to obtain a melting curve or if an end-point analysis of fluorescence is the goal.

[0108] Whether the compound according to the invention is present during amplification or added later, the melting step allows for the analysis of the target nucleic acid, e.g., the identification of a particular genotype or polymorphism. According to a particular embodiment, the melting curve is used to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism and / or epigenetic mutation, in order to identify the genotype of the target nucleic acid.

[0109] A melting curve (also called a melt curve) is generated by slowly denaturing (melting) the generated amplicon. The generation of melting curves and their use for analyzing nucleic acids are known in the art. More precisely, when melting curve analysis is used for detecting or quantifying a target double-stranded nucleic acid (preferably dsDNA), a mixture containing the generated amplicon and a compound according to the present invention is subjected to a thermal gradient. Preferably, the gradient is a continuous gradient, but a step gradient is also possible. Most preferably, the gradient is a linear gradient. In one particular embodiment, the sample is subjected to an increasing temperature, thereby generating a dissociation curve. According to another embodiment, the double-stranded nucleic acid (preferably dsDNA) is first thermally denatured to become single-stranded, and the temperature dependence of fluorescence is monitored during subsequent renaturation.

[0110] In the methods and uses according to the invention, the amplification of the target nucleic acid, in particular the target DNA, can be carried out by different techniques, in particular by enzymatic amplification reactions. By "enzymatic amplification reaction" it is understood that a process is understood in which multiple copies of a target nucleotide fragment are generated by the action of at least one enzyme. Such amplification reactions (isothermal or not) are well known to those skilled in the art, and the following techniques may be mentioned in particular: all types of PCR (polymerase chain reaction) (e.g. standard PCR, real-time PCR, quantitative PCR, digital PCR, multiplex PCR, asymmetric PCR, nested PCR, semi-nested PCR, LATE-PCR, touchdown PCR, hot start PCR, COLD-PCR, assembly PCR), LCR (ligase chain reaction), RCR (repair PCR), and the like. These include the following: 3SR (Self-sustained Sequence Replication) from 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) from U.S. Pat. No. 5,399,491, and LAMP (Loop-Mediated Isothermal Amplification) from U.S. Pat. No. 6,410,278, RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification). When the enzymatic amplification reaction is PCR, more specifically RT-PCR (RT stands for "reverse transcription") is mentioned, where the amplification step is preceded by a step of reverse transcription of messenger RNA (mRNA) into complementary DNA (cDNA). When PCR is quantitative, it is performed using qPCR or RT-qPCR.

[0111] These techniques use a pair of primers (consisting of two primers), typically one forward primer and one reverse primer. In the present specification, the use of a pair of primers in the present methods, uses, kits, and mixtures is described, meaning that one or several pairs of primers can be used. A primer is a nucleotide fragment that may consist of 5 to 100 nucleotides, preferably 15 to 30 nucleotides, and possesses specificity for hybridization with a target nucleic acid sequence under conditions determined for the initiation of enzymatic polymerization, for example, in an enzymatic amplification reaction of the target nucleic acid sequence. For example, when one reverse primer and several forward primers, or alternatively, one forward primer and several reverse primers, are used for amplification, they form several pairs of primers. When it is desired to simultaneously detect variants or different target nucleic acids from the same sample and the same amplification, several pairs of primers are used, thereby generating several amplicons. This is multiplex amplification, which is commonly used in conjunction with PCR methods. For this purpose, the methods, uses, kits, and mixtures according to the present invention contain more than one primer pair: one pair for each target nucleic acid.

[0112] PCR assay methods using the compounds according to the invention are of particular interest.

[0113] The present invention also relates to a method for PCR analysis of a target nucleic acid comprising the steps of: - mixing the compound according to the invention with a sample comprising a target nucleic acid, a polymerase and a pair of primers suitable for amplifying a portion of the target nucleic acid and generating at least an amplicon, to obtain a PCR mixture; - amplifying the target nucleic acid from the PCR mixture to produce at least an amplicon; and - monitoring the fluorescence from the compound according to the invention during or after the amplification step.

[0114] According to some embodiments, such PCR analysis methods further comprise detecting the presence of amplicons from the monitored fluorescence.

[0115] In these PCR analysis methods, the monitoring step may occur after amplification and may involve generating a melting curve or an end-point analysis of fluorescence. According to certain embodiments, the melting curve, and in particular its shape, is used to genotype the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially a single nucleotide polymorphism (SNP) and / or epigenetic mutation. When an end-point analysis of fluorescence is performed, the total amount of fluorescence is analyzed at the end of the amplification stage, i.e., once all amplification cycles are completed; the amount of fluorescence is monitored and analyzed after each amplification cycle, rather than during amplification, as is commonly done in real-time PCR.

[0116] It is also possible for the method of the invention to comprise the quantification of the target nucleic acid initially present in the sample, the amount of target nucleic acid initially present in the sample corresponding to the amount of nucleic acid present in the initial sample used, meaning the sample used before any step of amplification.

[0117] Quantification of the initial amount of nucleic acid in the sample can be done by any method traditionally known by a person skilled in the art and can be applied during or after any amplification method, preferably PCR, qPCR or LAMP.

[0118] One method for quantifying a target nucleic acid is by determining the Cp (also called Ct for crossing point - cycle threshold) and comparing the Cp to a standard or control.

[0119] Absolute quantification involving qPCR amplification frequently uses the standard curve approach. In this approach, a standard curve is generated by plotting Cp values ​​obtained from amplification, preferentially real-time PCR, against a single reference template (also called a standard or control) of known quantity, and the resulting regression line can be used to extrapolate the amount of target nucleic acid in the sample of interest. Serial dilutions (typically 10-fold dilutions) of the reference template are placed alongside the sample containing the specific target nucleic acid to be quantified. Several separate reactions are run, usually one for each level of the reference target and one for each sample of interest. Because assay-specific differences in PCR efficiency often affect quantification, separate standard curves using separate reference templates are run to quantify different gene targets.

[0120] As described in WO2017 / 165269, it is also possible to use the single-point Cp of a single quantification standard using an imported calibration curve or quantification curve. When a single-point Cp is used, these methods can include only one quantification standard nucleic acid provided at a known concentration, obtain the Cp for this standard, and then import the regression line corresponding to this standard (standard curve) and place it on the single-point Cp for calibration, and then use the calibrated standard curve to perform quantification of the target nucleic acid.

[0121] Thus, a method for detecting a target nucleic acid or a method for PCR analysis of a target nucleic acid may correspond to a method for performing quantitative amplification, preferably PCR, on a sample. In that case, the method may comprise amplifying the sample in an amplification mixture, the amplification mixture comprising a pair of target primers designed to amplify a target that may be present in the sample, the amplification mixture further comprising a plurality of quantitative standard nucleic acids each provided at different known concentrations and at least one pair of quantitative standard primers, the quantitative standard primers designed to amplify the quantitative standard nucleic acid, generating a standard curve from the quantitative standard amplicons, and using the standard curve to quantify the target nucleic acid.

[0122] In the present invention, when quantitative amplification is performed, an external or internal quantitative standard nucleic acid can be used to quantify the target nucleic acid. When the standard nucleic acid is external, it means that it is separated and not present in the same reaction mixture (also called the sample) containing the target nucleic acid to be quantified. When the standard nucleic acid is internal, it is present in the same reaction mixture (also called the sample) as the target nucleic acid to be quantified. The internal standard nucleic acid(s) are generally amplified and quantified simultaneously with the target nucleic acid, but this can also be done in advance, and the resulting standard curve can be saved and imported at the time of target nucleic acid quantification.

[0123] Quantitation standards can be synthetic or natural substances. Calibration or quantification can be performed on known naturally occurring microorganisms with known concentrations, or on other naturally occurring nucleic acid templates, such as viruses, yeasts or bacteriophages, and / or synthetic particles that can mimic membrane and / or capsid and / or envelope structures, or even housekeeping genes.

[0124] As an alternative to determining Cp by absolute or normalized amplification data, melting curves can also be used to quantify target nucleic acids.

[0125] In particular, when the target nucleic acid is a double-stranded nucleic acid, preferably dsDNA, quantification may mean the generation of a melting curve, more precisely the generation of several melting curves.

[0126] Quantification methods using melting curves are known to those skilled in the art. For example, the Livak method can be used. It is also possible to use the maximum value of the negative first derivative of fluorescence intensity with respect to temperature (maximum value of -(d fluorescence intensity / d temperature)), which gives the melting temperature and then the amount of target nucleic acid. This method can further include determining a value for the melting curve and identifying the amplification cycle at which the value for the melting curve exceeds a predetermined value, thereby determining Cp. The value can be determined by the peak height or peak area of ​​the negative derivative of the melting curve. A series of negative derivative melting curves can be used, with the flattest curve representing the earliest cycle and the area under the curve increasing over several cycles. It is expected that such derivative melting curves obtained at multiple cycles during amplification can be used to determine Cp. For each cycle, the transition height of each melting curve, or the area under the negative first derivative of the melting curve, can be determined. The cycle at which this value exceeds a predetermined threshold can then be assigned Cp.

[0127] Other methods for determining Cp can be applied. For example, a melting detector can be used (see US6,387,621; US6,730,501; and US7,373,253, which are incorporated herein by reference). The detector examines the curve shape and background noise to determine whether generated amplicons, preferably PCR-derived amplicons, are present in the sample. The melting detector can be used to increase the sensitivity of the system (Poritz et al., PLos One 6(10):e26047). Optionally, additional filters can be applied to melting curve analysis, such as windowing the melting transitions to analyze only those melting curves whose melting transitions, represented as melting peaks, are within a set temperature range, thereby increasing the specificity of the system. Such methods are expected to provide more accurate Cp (see WO2014 / 039963).

[0128] The method of continuous temperature and fluorescence monitoring is illustratively used for relative quantification using a compound according to the present invention as a dsDNA-binding dye in a single reaction with a control or reference nucleic acid. A multiplex amplification (preferably PCR) reaction is provided containing a known initial concentration of the control or reference nucleic acid and an unknown concentration of the target nucleic acid. Primers for amplifying the control or reference nucleic acid are present at the same initial concentration as the primers for amplifying the target nucleic acid. Additionally, the control or reference nucleic acid is preferably selected so that its melting temperature is sufficiently distant from the melting temperature of the target nucleic acid so that the melting of each of these nucleic acids can be distinguished from the other melting temperatures. It is understood that multiple target nucleic acids of unknown concentrations can be multiplexed in a reaction, with the desirably resulting melting curve for each nucleic acid being distinguishable from the melting curves of the other target nucleic acids and the control or reference nucleic acid.

[0129] In PCR, the fluorescent signal obtained from the binding of the compounds of the present invention to dsDNA allows the generation of amplification curves for a reference or control nucleic acid and a target nucleic acid to be quantified. However, sometimes the signals from the control and target combine to generate a single amplification curve, preventing the identification of information about the amplification of each individual nucleic acid. To avoid this, a series of melting curves can be generated during PCR cycling, using continuous data collection. Provided that the melting temperatures of the control or reference nucleic acid and the target nucleic acid are sufficiently different, the melting profiles of the two reactions can be distinguished. To generate a corrected amplification curve for a control or reference nucleic acid, the integral of the negative first derivative of the melting curve within a predefined melting window can be calculated for each cycle and plotted as a function of cycle number, with Cp being determined as the cycle at which each value exceeds a predetermined value. Similarly, a corrected amplification curve for a target nucleic acid can be generated by integrating the negative first derivative of the melting curve over a predefined melting window for the target, as described in WO 2014 / 039963, which is incorporated by reference.

[0130] Other methods for converting melt curves to values ​​are known in the art, such as using the peak height of the negative first derivative, and it is understood that the default value is selected according to the method being used.

[0131] For example, a PCR analysis method according to the present invention may include the steps of: mixing a compound according to the present invention with a sample containing an unknown initial amount of target nucleic acid and primers designed to amplify the target nucleic acid to form a mixture; amplifying the target nucleic acid in the presence of a compound according to the present invention to produce an amplicon; monitoring the fluorescence resulting from binding of a compound according to the present invention to the amplicon over a range of temperatures during multiple amplification cycles to generate multiple melting curves; and quantifying the initial amount of target nucleic acid using the melting curves.

[0132] Illustratively, a method for PCR analysis of a target nucleic acid according to the present invention may include the following steps: - mixing a compound according to the invention with a sample comprising a target nucleic acid and at least one pair of primers suitable for amplifying a portion of the target nucleic acid and generating an amplicon, to obtain a PCR mixture; - amplifying the target nucleic acid from the PCR mixture to produce at least an amplicon; - monitoring the fluorescence of the compound according to the invention resulting from binding of the compound to the amplicon during the amplification step, - at the end of the amplification step, melting the amplicons produced to obtain a melting curve, and - Using the shape of the melting curve to identify the genotype or polymorphism of the target nucleic acid.

[0133] According to certain embodiments, in any method according to the present invention, the amplification step can include multiple temperature cycles including at least a denaturation temperature and an extension temperature, each cycle having a cycle time of less than 90 seconds per cycle, the polymerase being provided at a concentration of at least 0.005 μM, and the primers being provided at a concentration of at least 0.1 μM, respectively. According to certain embodiments, the amplification step can include multiple temperature cycles including at least a denaturation temperature and an extension temperature, each cycle having a cycle time of less than 20 seconds per cycle, the polymerase being provided at a concentration of at least 0.5 μM, and the primers being provided at a concentration of at least 2 μM, respectively. These concentrations relate to an amplification mixture, particularly a PCR mixture.

[0134] PCR techniques are often classified by the time required for PCR and the amount of primers used. Further details are given in US Pat. Nos. 7,387,887 and 9,932,634. Conventional or standard PCR is fairly slow, occurring in approximately 90 seconds or less per cycle; rapid PCR occurs in less than 60 seconds per cycle, e.g., between 20 and 60 seconds per cycle; and fast, ultrafast, 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 less than 2 seconds for extreme PCR. Faster PCR speeds require increased concentrations of primers and polymerase, which allows PCR efficiency and yield to be maintained. The primer concentration is in the range of at least 0.1 μM for conventional or standard PCR and at least 2 μM for extreme PCR, i.e. at least 0.1 μM, at least 0.2 μM, at least 0.4 μM, at least 0.6 μM, at least 0.8 μM, at least 1 μM, at least 1.2 μM, at least 1.4 μM, at least 1.6 μM, at least 1.8 μM or at least 2 μM. The polymerase concentration is in the range of at least 0.005 μM for conventional or standard PCR and at least 0.5 μM for extreme PCR, i.e. at least 0.005 μM, at least 0.01 μM, at least 0.02 μM, at least 0.04 μM, at least 0.06 μM, at least 0.08 μM, at least 0.1 μM, at least 0.2 μM, at least 0.3 μM, at least 0.4 μM or at least 0.5 μM. Any of these types of PCR can be used according to the present invention. Herein, 1 μM of polymerase corresponds to 3.8 U / μL. All these concentrations are relative to the PCR mixture.

[0135] Further details on methods for nucleic acid analysis 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.

[0136] The present invention also provides a PCR reaction mixture, also called a PCR mix, comprising: - target nucleic acid, - a pair of primers suitable for amplifying a portion of the target nucleic acid to produce an amplicon; - polymerases, in particular thermostable polymerases, - Compounds according to the invention The present invention relates to a PCR reaction mixture comprising:

[0137] The primer pairs are 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 primer pair can be used, particularly for multiplex PCR, in which case more than one target sequence will be amplified.

[0138] The target nucleic acid is typically total genomic DNA or alternatively total cellular RNA or total cellular mRNA. In the case of RNA, the thermostable DNA polymerase may be a DNA polymerase or a mixture of polymerases that contain reverse transcriptase activity.

[0139] Typically, such PCR reaction mixtures also contain a mix of deoxynucleoside triphosphates, usually dA, dG, dC and dT, or dA, dG, dC and dU.

[0140] Such PCR reaction mixtures traditionally contain a buffer. In particular, PCR reaction mixtures are buffered at pH 7.5 to 9.5, preferentially pH 8 to 9. Such PCR reaction mixtures may also contain a thiol, typically selected from dithiothreitol, β-mercaptoethanol, and thioglycerol, which is typically reserved for RT-PCR analysis.

[0141] The conventional components of the mixture are used in concentrations readily determined by one skilled in the art according to common practice.

[0142] In the methods, uses and mixtures of the present invention, the concentration of the compound according to the invention is typically 1 to 20 μmol / L (μM), preferably 2 to 10 μmol / L, which corresponds to the concentration of the compound according to the invention in the sample used for monitoring the fluorescence of the compound according to the invention resulting from binding of the compound to the amplicon.

[0143] In certain embodiments, the compounds according to the present invention are used for the detection of double-stranded nucleic acids during melting curve analysis, as disclosed for other compounds known in the art. More precisely, as described in WO2008 / 052742, double-stranded DNA fragments are subjected to a thermal gradient in the presence of the compounds according to the present invention. Preferably, the gradient is a continuous gradient, but a step gradient is also possible. Most preferably, the gradient is a linear gradient. In one specific embodiment, the sample is subjected to a temperature increase resulting in the generation of a dissociation curve. In another embodiment, the target double-stranded nucleic acid is first thermally denatured into single strands, and the temperature dependence of fluorescence is monitored during subsequent renaturation. The first derivative of the melting curve can be generated, and the characteristic temperature of nucleic acid dissociation can be obtained. These techniques are well known in the art, and further details can be found, for example, in the article by Carl T. Wittwer, Chemistry, 1997, Vol. 245, pp. 154-160.

[0144] The concentration of the compound according to the present invention added before, during, or after the amplification step and then used in the resulting mixture / sample used to monitor fluorescence is typically 1-20 μmol / L, preferably 2-10 μmol / L. This concentration corresponds to the concentration of the compound according to the present invention in the sample used to monitor the fluorescence of the compound according to the present invention resulting from binding of the compound to the amplicon. Preferably, the double-stranded DNA to be analyzed is derived from a PCR amplification reaction. Additionally, amplification can be monitored in real time using the compound according to the present invention, which in some embodiments can be followed by subsequent melting curve analysis or end-point analysis of fluorescence using the compound.

[0145] The present invention also provides a kit for detecting a target nucleic acid, comprising: - at least one pair of primers suitable for amplifying a portion of the target nucleic acid to produce an amplicon; - polymerases, in particular thermostable polymerases, - Compounds according to the invention The present invention relates to a kit comprising:

[0146] When the kit is used to obtain a melting curve, the kit allows for the analysis and identification of the target nucleic acid.

[0147] Such kits also traditionally contain a buffer, in particular a buffer resulting in a pH of 7.5 to 9.5, preferentially 8 to 9. Buffers suitable for PCR (e.g., Tris) are commercially available and can be used. Such buffers, typically used for RT-PCR analysis, may also contain a thiol, such as a thiol selected from dithiothreitol, β-mercaptoethanol, and thioglycerol. The compound according to the invention can also be in the form of a buffer, e.g., in the form of such a buffer in the kit.

[0148] The polymerase can also be stored in the form of a buffer resulting in a pH between 7.5 and 9.5, preferentially between 8 and 9. Optionally, this buffer may also contain a thiol, typically chosen from among dithiothreitol, β-mercaptoethanol and thioglycerol. [Brief explanation of the drawings]

[0149] [Figure 1] Stability and fluorescence data are presented for Examples 1-4 and 6, 7 and 10 (compounds I.1-I.4 and I.6, I.7 and I.10) and other comparison compounds. [Figure 2] Although not part of the present invention, stability and fluorescence data for compounds that are differently substituted at positions corresponding to R1 are presented. [Figure 3]The stability and fluorescence obtained using Examples 1 to 3 according to the present invention and Comparative Example 8 are compared. [Figure 4] Stability in the presence of thiols is presented for Examples 1-4 and for prior art compound N7. [Figure 5A] The resulting fluorescence (RFU) is presented as a function of temperature (°C) (left panel) and the change in fluorescence (-d(RFU) / dT(T)) as a function of temperature (°C) (right panel) obtained in the following evaluations for Examples 1 to 5 and 10 of the present invention (compounds I.1 to I.5 and I.10) in the presence or absence of a model DNA duplex, (-DUPLEX) or (+DUPLEX), respectively. [Figure 5B] The resulting fluorescence (RFU) is presented as a function of temperature (°C) (left panel) and the change in fluorescence (-d(RFU) / dT(T)) as a function of temperature (°C) (right panel) obtained in the following evaluations for Examples 1 to 5 and 10 of the present invention (compounds I.1 to I.5 and I.10) in the presence or absence of a model DNA duplex, (-DUPLEX) or (+DUPLEX), respectively. [Figure 5C] The resulting fluorescence (RFU) is presented as a function of temperature (°C) (left panel) and the change in fluorescence (-d(RFU) / dT(T)) as a function of temperature (°C) (right panel) obtained in the following evaluations for Examples 1 to 5 and 10 of the present invention (compounds I.1 to I.5 and I.10) in the presence or absence of a model DNA duplex, (-DUPLEX) or (+DUPLEX), respectively. [Figure 5D] The resulting fluorescence (RFU) is presented as a function of temperature (°C) (left panel) and the change in fluorescence (-d(RFU) / dT(T)) as a function of temperature (°C) (right panel) obtained in the following evaluations for Examples 1 to 5 and 10 of the present invention (compounds I.1 to I.5 and I.10) in the presence or absence of a model DNA duplex, (-DUPLEX) or (+DUPLEX), respectively. [Figure 5E] The resulting fluorescence (RFU) is presented as a function of temperature (°C) (left panel) and the change in fluorescence (-d(RFU) / dT(T)) as a function of temperature (°C) (right panel) obtained in the following evaluations for Examples 1 to 5 and 10 of the present invention (compounds I.1 to I.5 and I.10) in the presence or absence of a model DNA duplex, (-DUPLEX) or (+DUPLEX), respectively. [Figure 5F] The resulting fluorescence (RFU) is presented as a function of temperature (°C) (left panel) and the change in fluorescence (-d(RFU) / dT(T)) as a function of temperature (°C) (right panel) obtained in the following evaluations for Examples 1 to 5 and 10 of the present invention (compounds I.1 to I.5 and I.10) in the presence or absence of a model DNA duplex, (-DUPLEX) or (+DUPLEX), respectively. [Figure 6] For Examples 1 and 3 of the present invention (compounds I.1 and I.3), respectively, the following are presented: PCR amplification curves of the biological targets (Panel A), melting curves of the generated amplicons (Panel B) and the first derivatives of the melting curves to accurately determine the melting temperatures of the amplicons (Panel C). [Figure 7] For Examples 1 and 3 of the present invention (compounds I.1 and I.3), respectively, the following are presented: PCR amplification curves of the biological targets (Panel A), melting curves of the generated amplicons (Panel B) and the first derivatives of the melting curves to accurately determine the melting temperatures of the amplicons (Panel C). [Figure 8A] Functional assays under high-throughput real-time PCR conditions of the compounds described in Examples 1 to 4 of the present invention (compounds I.1 to I.4) are presented in comparison with prior art compound N7. [Figure 8B] Panel A provides further details on the maximum fluorescence value (Max Fluo) obtained at the end of PCR, Panel B on the Cp (cycle number) and Panel C on the Tm (melting temperature) measurements of the amplicons, showing the values ​​obtained with compounds I.1 to I.4 compared to prior art compound N7. [Figure 9] The excitation and emission maxima of compounds I.1-I.7 and I.10 are presented. [Example]

[0150] Abbreviation: Me = methyl; Et = ethyl TFA - = Trifluoroacetate TFA = trifluoroacetic acid TEA = Triethanolamine ACN = acetonitrile DCM = dichloromethane DSC = N,N'-disuccinimidyl carbonate NHS = N-hydroxysuccinimide DIPEA = N,N-diisopropylethylamine Ts = Tosil Ac = acetyl Room temperature=20℃

[0151] I. Preparation of Compound (I) In the examples hereafter, all compounds correspond to formulas (I), (II) and (III). These are trifluoroacetic acid salts (TFA - ) salt.

[0152] For the analysis, the following equipment was used: UPLC-Mass Spectrometry Analysis: 0–95% acetonitrile in 10 mM TFA pH 2 for 5 min at 0.5 ml / min using a UPLC BEH C18 1.7 μm 2.1 × 50 mm column on a UPLC-MS system Alliance (Waters). 1 H NMR and 13 CNMR: Bruker System 400MHz.

[0153] 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 -

[0154] Formula (I) and specific (II) where 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, k4=4 and Y4=-N + (Me)3.

[0155] [ka]

[0156] a) Preparation of 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium, 1

[0157] [ka]

[0158] 2-(Methylthio)benzo[d]thiazole (10 g; 55.16 mmol) was placed in a 250 mL round-bottom flask and methyl p-toluenesulfonate (75 mL; 496.47 mmol) was added. The mixture was stirred and heated at 145 °C (fusion reaction) for 2 hours. The mixture turned orange. The mixture was then allowed to cool to room temperature. 180 mL of diethyl ether was added to precipitate the resulting compound, and the resulting white precipitate was filtered and washed twice with diethyl ether. Compound 1 was obtained as a solid by evaporating the remaining 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)

[0159] b) Preparation of 4-(4,6-dimethylpyrimidin-2-yl)-N,N-dimethylbutan-1-amine, 2

[0160] [ka]

[0161] Acetylacetone (1.16 g; 6.4 mmol) was placed in a 50 mL round-bottom flask and water (9.5 mL) was added. Then, 0.996 g (4.61 mmol) of dimethylaminoacetamidine (Chemspace Riga, Latvia) and K2CO3 (2.95 g; 221.37 mmol) were added. The mixture was stirred at room temperature for 4 weeks. HCl 6 M was added until pH 2, and the resulting aqueous phase was washed with DCM for 2 hours. The aqueous phase was then neutralized to pH 12 with NaOH 1 M, and the resulting compound 2 was extracted three times with dichloromethane. The DCM was evaporated on a rotary evaporator. Yield=43%. Mass spectrometry (ESI-Q(+)): M / Z=208.1 1 H 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)

[0162] c) Preparation of 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium, diiodine, 3

[0163] [ka]

[0164] In a glass tube with a screw cap, 526 mg (2.54 mmol) of compound 2 was placed and dissolved in 8 mL of acetonitrile, followed by the addition of 1.145 g (8.07 mmol) of methyl iodide. The mixture was heated to 85 °C and stirred at 600 rpm in a thermomixer for 17 hours. ACN and excess CHCl were evaporated using a rotary evaporator to give the expected crude compound 3. Yield = 94.5%. Mass spectrometry (ESI-Q(+)): M / Z=118.5

[0165] d) Compound I.1, 2TFA - Preparation of

[0166] [ka]

[0167] 129.9 mg (0.336 mmol) of 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium (Compound 1) was placed in a 100 mL round-bottom flask, and 3 mL of a 50 mg / mL solution of 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium, diiodine (Compound 3, 150 mg; 0.305 mmol) in acetonitrile was added. 9 mL of acetonitrile and 0.8 mL of ethyl alcohol were added sequentially. Finally, 42.6 μL (30.9 mg; 0.305 mmol) of triethylamine was added dropwise. The mixture was then stirred at room temperature for 25 min. The solvent was evaporated and the resulting solid was purified by flash chromatography on a 20 g C18 column (eluent A: 10 mM TFA in water, eluent B: 90% ACN and 10% 10 mM TFA in water, 5-65% gradient of eluent B in 20 min) to give compounds I.1, I.2, I.3, I.4, I.5, I.6, I.7, I.8, I.9, I.10, I.11, I.12, I.13, I.14, I.15, I.16, I.17, I.18, I.19, I.20, I.21, I.22, I.23, I.24, I.25, I.26, I.27, I.28, I.29, I.30, I.31, I.32, I.33, I.34, I.35, I.36, I - Yield = 53%. Mass spectrometry (ESI-Q(+)): M / Z=192.1

[0168] 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 certain (II) (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 and R=-(CH2) k2 -Y2, k2=3 and Y2=-N + (Me)3.

[0169] [ka]

[0170] a) Preparation of 2-(methylthio)benzo[d]thiazole-6-carboxylic acid, 5

[0171] [ka]

[0172] 200 mg (0.789 mmol) of ethyl 2-(methylthio)benzo[d]thiazole-6-carboxylate (compound 4, Chemspace Riga, Latvia) was placed in a 50 mL round-bottom flask, and 20 mL of dichloromethane and 7.2 mL of NaOH (167 mM in methanol) were added. The mixture was stirred at room temperature for 20 h. A suspension formed, and the solvent was evaporated using a rotary evaporator. The crude product obtained was washed with diethyl ether and filtered. The powder obtained was dissolved in 20 mL of water (3 M HCl), adjusted to pH 1-2, and then extracted four times with 20 mL of dichloromethane. The organic phase was dried over MgSO4 and evaporated on a rotary evaporator to give compound 5. Yield = 55% - 100 mg. 1 H NMR (200 MHz; MeOD; δ = 3.4 ppm) δ (ppm): 8.65 (s, 1H); 8.20 (dd, 1H); 7.94 (d, 1H) and 2.94 (s, 3H). 13 C 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).

[0173] b) Preparation of 2,5-dioxopyrrolidin-1-yl 2-(methylthio)benzo[d]thiazole-6-carboxylate, 6

[0174] [ka]

[0175] 160 mg (0.71 mmol) of 2-(methylthio)benzo[d]thiazole-6-carboxylic acid (compound 5) was dissolved in 10 mL of 1,4-dioxane, and 161 mg (0.78 mmol) of dicyclohexylcarbodiimide was added. The mixture was stirred at room temperature for 30 minutes, then completed with 90 mg (0.78 mmol) of N-hydroxysuccinimide, and stirring was maintained for 20 hours. The suspension was filtered, and the filtrate was evaporated on a rotary evaporator. The evaporation residue was dissolved in 20 mL of acetylacetate and washed three times with 10 mL of saturated bicarbonate solution, followed by three times with 10 mL of brine. The organic phase was dried over MgSO4 and then evaporated on a rotary evaporator, resulting in compound 6. Yield=95%—220 mg. 1 H 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).

[0176] c) Preparation of N-(3-(dimethylamino)propyl)-2-(methylthio)benzo[d]thiazole-6-carboxamide, 8

[0177] [ka]

[0178] In a 100 mL round-bottom flask, 390 mg (1.21 mmol) of 2,5-dioxopyrrolidin-1-yl 2-(methylthio)benzo[d]thiazole-6-carboxylate (compound 6) was dissolved in 20 mL of 1,4-dioxane, and 305 μL (247 mg; 2.42 mmol) of N,N-dimethylpropane-1,3-diamine (compound 7) was added. The mixture was stirred at room temperature for 20 hours. The crude product was filtered, and the filtrate was evaporated using a rotary evaporator. The residue was purified by normal-phase flash chromatography using a mixture of CH₂Cl₂ / MeOH / TEA: 94 / 3 / 3, v / v / v, to give compound 8. Yield=80%—300 mg. 1 H 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 (quintet, 2H). 13 C 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).

[0179] d) Preparation of 3-methyl-2-(methylthio)-6-((3-(trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, ditosylate, IV.1

[0180] [ka]

[0181] N-(3-(dimethylamino)propyl)-2-(methylthio)benzo[d]thiazole-6-carboxamide (compound 8, 114 mg; 0.368 mmol) was placed in a 10 mL round-bottom flask and methyl p-toluenesulfonate (151 mg; 0.81 mmol) was added. The mixture was stirred and heated at 145 °C (fusion reaction) for 4 hours. The mixture was then allowed to cool to room temperature. The crude product was washed several times with diethyl ether, and the remainder of the solvent was evaporated using a rotary evaporator to obtain compound IV.1 as a powder in the form of a ditosylate salt. Yield = 225 mg. 1 H 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).

[0182] e) Preparation of 2,4,6-trimethylpyrimidine, 9

[0183] [ka]

[0184] Acetylacetone (11 g; 60.68 mmol) was placed in a round-bottom flask and water (95 mL) was added. Methylacetamidine (10 g; 43.86 mmol) and K2CO3 (28 g; 202.60 mmol) were added. The mixture was stirred at room temperature for 3 weeks. Colorless crystals were observed on the surface and a white precipitate was observed at the bottom of the round-bottom flask. The solution was orange. The crystals on top of the mixture were filtered and washed with cold water. These crystals are expected to be compound 9, but some compound 9 remained dissolved in water. A portion of K2CO3 was added to the mixture to saturate the water, and liquid extraction was performed. The organic phase was collected and the DCM was evaporated at 500 mbar using a rotary evaporator. Some DCM remained. This mixture contained compound 9 along with the imine. Purification was performed on a normal-phase chromatography column. The resulting mixture was placed on top of the column, and the eluent (DCM / MeOH 98:2) was added. After evaporation of all the DCM, pure compound 9 was obtained. Yield=14%. Mass spectrometry (ESI-Q(+)): M / Z=123.1

[0185] f) Preparation of 1-ethyl-2,4,6-trimethylpyrimidin-1-ium, iodide 10

[0186] [ka]

[0187] 150 mg (1.23 mmol) of 2,4,6-trimethylpyrimidine 9 was placed in a round-bottom flask and dissolved in acetonitrile to obtain a 0.15 M solution. 574 mg (3.69 mmol) of iodoethane was then added. The mixture was stirred at reflux for 20 h. The solvent was evaporated, and the resulting pyrimidinium 10, in the form of its iodide salt, was purified by flash chromatography on a C18 column. Yield = 50%. Mass spectrometry (ESI-Q(+)): M / Z=151.1

[0188] g) Compound I.2, 2TFA - Preparation of

[0189] [ka]

[0190] 94.3 mg of previously prepared 3-methyl-2-(methylthio)-6-((3-(trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, ditosylate IV.1 was placed in a 25 mL round-bottom flask, and 6 mL of acetonitrile and 0.5 mL of ethyl alcohol were added. 27 mg (0.102 mmol) of previously prepared 1-ethyl-2,4,6-trimethylpyrimidin-1-ium, iodide 10 was dissolved in 3.155 mL of acetonitrile. 108 mg (0.511 mmol) of trimethylamine was added, and the mixture was stirred at room temperature for 25 minutes. The solvent was evaporated, and compound I.2, 2TFA, was purified by flash chromatography using a C18 column, 12 g. - was purified (Eluent A: 10 mM TFA in water, Eluent B: 90% ACN and 10% 10 mM TFA in water. 5-65% gradient of Eluent B in 20 min). Yield=47%. Mass spectrometry (ESI-Q(+)): M / Z=220.7

[0191] 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 especially (II) (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, k4=4 and Y4=-N + (Me)3; Rc = -NHCOR and R = -(CH2) k2 -Y2, k2=3 and Y2=-N + (Me)3.

[0192] [ka]

[0193] a) Preparation of 4-((2,5-dioxopyrrolidin-1-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-aminium chloride, 12

[0194] [ka]

[0195] (3-Carboxypropyl)trimethylammonium chloride (compound 11, as the chloride salt, 1.2 g; 6.61 mmol) was placed in a 250 mL round-bottom flask, and 100 mL of anhydrous acetonitrile and 8 equivalents of anhydrous pyridine were added under a nitrogen stream. The mixture was stirred for 10 minutes, and then DSC (5.086 g; 19.85 mmol) was added. After 12 hours at room temperature, the solvent was evaporated and the crude product was precipitated in diethyl ether. The remaining diethyl ether was evaporated using a rotary evaporator to dry the resulting solid, yielding compound 12. Yield=100%. Mass spectrometry (ESI-Q(+)): M / Z=243.1

[0196] b) Preparation of N,N,N-trimethyl-4-((2-(methylthio)benzo[d]thiazol-6-yl)amino)-4-oxobutan-1-aminium chloride, 14

[0197] [ka]

[0198] 4-((2,5-Dioxopyrrolidin-1-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-aminium chloride (Compound 12, 675 mg; 2.42 mmol) was placed in a 250 mL round-bottom flask and 38 mL of anhydrous acetonitrile was added. The mixture was vigorously stirred under a stream of nitrogen. 184.0 mg of 2-(methylthio)benzo[d]thiazol-6-amine (Chemspace ref CSC000183586; Compound 13; 0.94 mmol) was added to the round-bottom flask, followed immediately by the addition of 1.1 equivalents of anhydrous DIPEA until the pH reached 5.8. The mixture was stirred at 80 °C for 24 h. The solvent was evaporated, and liquid-liquid extraction with water and DCM was performed. The aqueous phase was basified with DIPEA to reach a pH of 8–9, followed by three extractions with DCM. The aqueous phase was collected and the water was evaporated using a rotary evaporator to give compound 14. Yield=100%. Mass spectrometry (ESI-Q(+)): M / Z=324.1

[0199] c) Preparation of 3-methyl-2-(methylthio)-6-(4-(trimethylammonio)butanamido)benzo[d]thiazol-3-ium tosylate chloride, IV.2

[0200] [ka]

[0201] 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 added to a 10 mL round-bottom flask 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 allowed to cool to room temperature. 6 mL of acetone was added, and the resulting mixture was vigorously mixed and transferred to a 50 mL tube containing diethyl ether. The flask was rinsed with 6 mL of acetone, then diluted with 30 mL of ether, vigorously shaken, and centrifuged at 1500 rpm. The supernatant was discarded. The precipitate was washed 10 times with 40 mL of a 1 / 9 (v / v) acetone / ethyl ether mixture. The solid obtained was dried by evaporating the remaining solvent using a rotary evaporator, yielding compound IV.2. Yield=61%. Mass spectrometry (ESI-Q(+)): M / Z=169.6

[0202] d) Compound I.3, 3TFA - Preparation of

[0203] [ka]

[0204] 296 mg of 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium diiodine (compound 3 previously prepared in Example 1) was placed in a 50 mL round-bottom flask, and 10 mL of acetonitrile and 0.5 mL of ethyl alcohol were added. 150 mg of compound IV.2 previously prepared was dissolved in 3 mL of acetonitrile. One 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 solvent was evaporated, and compound I.3, 3TFA, was obtained. - was purified by flash chromatography on a 12 g C18 column (eluent A: 10 mM TFA in water, eluent B: 90% ACN and 10% 10 mM TFA in water. 5-65% gradient of eluent B in 20 min). Yield=17%. Mass spectrometry (ESI-Q(+)): M / Z=175.78

[0205] 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 especially (II) (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, k4=4 and Y4=-N + (Me)3.

[0206] [ka]

[0207] a) Preparation of 6-bromo-3-methyl-2-(methylthio)benzo[d]thiazol-3-ium, tosylate, 15

[0208] [ka]

[0209] 6-Bromo-2-(methylthio)benzo[d]thiazole (500 mg; 1.92 mmol) was placed in a 10 mL round-bottom flask and methyl p-toluenesulfonate (3.040 g; 16.3 mmol) was added. The mixture was stirred and heated at 145 °C (fusion reaction) for 1 hour and 30 minutes. The mixture was then allowed to cool to room temperature. 18 mL of diethyl ether was added, and the mixture was vigorously mixed. This was transferred to a 50 mL tube, the flask was rinsed with 6 mL of diethyl ether, vigorously shaken, and centrifuged at 1500 rpm. This process was repeated three times, after which the supernatant was discarded. The precipitate was then washed three times with 18 mL of acetone. The remaining solvent was evaporated using a rotary evaporator, and the resulting solid was dried to give compound 15 as the tosylate salt. Yield: 35%. Mass spectrometry (ESI-Q(+)): M / Z = 273.9 and 275.9 NMR 1 H (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).

[0210] b) Compound I.4, 2TFA - Preparation of

[0211] [ka]

[0212] 200.0 mg (0.450 mmol) of 6-bromo-3-methyl-2-(methylthio)benzo[d]thiazol-3-ium tosylate, 15, was placed in a 100 mL round-bottom flask, and 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium, diiodine (previously prepared compound 3; 170 mg; 0.346 mmol), 5 mL of acetonitrile, and 1 mL of ethyl alcohol were added sequentially. Finally, 210 μL (152 mg; 1.505 mmol) of trimethylamine was added dropwise. The mixture was stirred at room temperature for 25 min. After evaporation of the solvent and purification by flash chromatography using a C18 column (TFA / ACN / water eluent, as in previous Example 3), compound I.4, 2TFA, 2H NMR (3.0 MHz, 10.0 Hz), δ 1.0 Hz, ... - The yield was 90%. Mass spectrometry (ESI-Q(+)): M / Z = 231.1 and 232.1

[0213] 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 especially (II) (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, k4=4 and Y4=-N + (Me)3.

[0214] [ka]

[0215] a) Preparation of 2,3-dimethylbenzo[d]thiazol-3-ium tosylate 16

[0216] [ka]

[0217] 2-Methylbenzothiazole (1 g; 6.63 mmol) was placed in a 100 mL round-bottom flask and methyl p-toluenesulfonate (9.9 g; 52.8 mmol) was added. The mixture was stirred and heated at 145 °C for 2 h. The mixture was then allowed to cool to room temperature. 50 mL of ethyl ether was added to precipitate the crude product. The supernatant was discarded, and the resulting solid was washed three times with 50 mL of diethyl ether. The resulting precipitate was then washed three times with 350 mL of acetone. The resulting solid was dried by evaporating the remaining solvent using a rotary evaporator to give a gray powder corresponding to compound 16 as the tosylate salt (2.15 g, yield = 96%). 1 H 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, H ortho-TsO - ), 7.26 (d, 2H, H meta 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.

[0218] b) Preparation of 3-methyl-2-(2-(phenylamino)vinyl)benzo[d]thiazol-3-ium, tosylate, 17

[0219] [ka]

[0220] 2,3-Dimethylbenzo[d]thiazol-3-ium, tosylate salt, 16 (167.9 mg; 0.50 mmol) was placed in a 50 mL round-bottom flask and N,N'-diphenylformamidine (196.2 mg; 1.00 mmol) was added. The mixture was stirred and heated at 150 °C (fusion reaction) for 2 h 30 min. The mixture was then allowed to cool to room temperature. After liquid extraction in water / dichloromethane and evaporation of the organic phase, final purification was performed by reverse-phase flash chromatography (eluent A: 10 mM TFA in water; eluent B: 90% ACN and 10% 10 mM TFA in water, gradient 20-100% of eluent B in 30 min) to afford compound 17 as the tosylate salt after purification. Yield = 67%. Mass spectrometry (ESI-Q(+)): M / Z=267.2

[0221] c) 3-methyl-2-(2-(N-phenylacetamido)vinyl)benzo[d]thiazol-3-ium, 18

[0222] [ka]

[0223] The previously prepared 3-methyl-2-(2-(phenylamino)vinyl)benzo[d]thiazol-3-ium tosylate salt, 17 (127.1 mg; 0.334 mmol), was placed in a 10 mL round-bottom flask and acetic anhydride (800 mg; 7.84 mmol) was added. The mixture was stirred and heated at 100 °C for 2 h 30 min. The solvent was evaporated and co-evaporated with ACN using a rotary evaporator for 2 h to give compound 18. Finally, there was no odor of acetic acid. Yield = 100%. Mass spectrometry (ESI-Q(+)): M / Z=309.1

[0224] 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 -

[0225] [ka]

[0226] 70.5 mg (0.167 mmol) of 3-methyl-2-(2-(N-phenylacetamido)vinyl)benzo[d]thiazol-3-ium 18 was placed in a 10 mL round-bottom flask, and 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium diiodide (compound 3, previously prepared; 164.1 mg; 0.334 mmol), 3 mL of acetonitrile, and 0.5 mL of ethyl alcohol were added sequentially. Finally, 16.9 mg (0.167 mmol) of triethylamine was added dropwise. The mixture was stirred at room temperature for 25 min. After evaporation of the solvent and purification by PREP chromatography using a C18 column (eluent A: 10 mM TFA in water, eluent B: 90% ACN and 10% 10 mM TFA in water, 10-50% gradient of eluent B in 15 min), compound I.5, 2TFA was obtained. - The yield was 46%. Mass spectrometry (ESI-Q(+)): M / Z=205.1

[0227] 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, 4TFA- Formula (I) and especially (II), (Z=substituted fused benzo; X=S; n=0; Ri=H; Re=Me; Ra=Rb=Rd=H; Rc=-C(O)NHR, where R is -(CH2) k2 -Y2, k2=3 and Y2 is -[N + R2'R2"-(CH2) p2 -] m2 -G2', p2=3, m2=1, R2'=R2"=Me and G2'=-N + (Me)3; R1 = R3 = Me; R4 = H; R2 = -(CH2) k4-Y4, k4=4 and Y4=-N + (Me)3.

[0228] [ka]

[0229] a) Preparation of N-(3-((3-(dimethylamino)propyl)amino)propyl)-2-(methylthio)benzo[d]thiazole-6-carboxamide, 21

[0230] [ka]

[0231] This compound was prepared following the same procedure as for compound 8 in 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

[0232] b) Preparation of 6-((3-(dimethyl(3-(trimethylammonio)propyl)ammonio)propyl)carbamoyl)-3-methyl-2-(methylthio)benzo[d]thiazol-3-ium, tritosylate (IV.3)

[0233] [ka]

[0234] This compound was prepared from compound (21) following the same procedure as for compound IV.1 in Example 2. Mass spectrometry (ESI-Q(+)): M / Z+2TFA-=651.2.

[0235] c) Preparation of I.6

[0236] [ka]

[0237] 75 mg (0.099 mmol) 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 mmol) of 1,4,6-trimethyl-2-(4-trimethylammonio)butyl)pyrimidin-1-ium, diiodine (compound 3 prepared previously in Example 1), 6 mL of acetonitrile, and 0.5 mL of ethyl alcohol were added sequentially to a 25 mL round-bottom flask. Finally, 13.8 μL (10.0 mg; 0.099 mmol) of trimethylamine was added dropwise. The mixture was stirred at room temperature for 25 min. After evaporation of the solvent and purification by flash chromatography on a C18 column (TFA / ACN / water eluent, as in previous Example 3), compound I.6, 4TFA - The yield was 3%. Mass spectrometry (ESI-Q(+)): M / Z+3TFA-=952.4

[0238] 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 especially (II) (Z=substituted fused benzo; X=S; n=0; Ri=H; Re=Me; Ra=Rb=Rd=H; Rc=-C(O)NHR, where R is -(CH2) k2 -Y2, k2=3 and Y2 is -N + (Me)3; R1 = R3 = Me; R4 = H; R2 = -(CH2) k4 -Y4, k4=4 and Y4=-N + (Me)3.

[0239] [ka]

[0240] 140.5 mg (0.185 mmol) of 3-methyl-2-(methylthio)-6-((3-(trimethylammonio)propyl)carbamoyl)benzo[d]thiazol-3-ium, ditosylate (Example 2 of compound IV.1) and 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium, diiodine (compound 3 previously prepared in Example 1; 100 mg; 0.204 mmol), 6 mL of acetonitrile, and 0.5 mL of ethyl alcohol were added successively to a 25 mL round-bottom flask, followed by the dropwise addition of 77.4 μL (56.2 mg; 0.555 mmol) of triethylamine. The mixture was stirred at room temperature for 25 minutes. The solvent was evaporated. The crude product was purified by precipitation in 6 mL of acetonitrile, and the precipitate was washed four times with 1.5 ml of acetonitrile to give compound I.7, 3TFA. - The product was purified by flash chromatography on a C18 column (TFA / ACN / water eluent, as in previous Example 3). Yield=21%. Mass spectrometry (ESI-Q(+)): M / Z=175.8

[0241] H. Example 8 Formula (I) and especially (II) (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, k4=4 and Y4=-N + (Me)3.

[0242] [ka]

[0243] I. Example 9 Formula (I) and especially (II) (Z=substituted fused benzo; X=S; n=0; Ri=H; Re=Me; Ra=Rb=Rd=H; Rc=-C(O)NHR, where R is -(CH2) k2-Y2, k2=3 and Y2 is -[N + R2'R2"-(CH2) p2 -] m2 -G2', p2=3, m2=1, R2'=R2"=Me and G2'=-N + (Me)3; R1=Et; R2=R3=Me; R4=H.

[0244] [ka]

[0245] 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, 2TFA - , Compound I.10, 2TFA - Formula (I) and especially (II) (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, k4=4 and Y4=-N + (Me)3.

[0246] [ka]

[0247] a) Preparation of N-(2-(methylthio)benzo[d]thiazol-6-yl)acetamide (22)

[0248] [ka]

[0249] Compound (13) 2-(methylthio)benzo[d]thiazol-6-amine-methane (1.842 g, 9.4 mmol) was added to a 50 mL round-bottom flask along with 30 mL of DCM and acetic anhydride (1.151 g, 11.3 mmol). The mixture was then stirred at room temperature for 24 hours. The mixture was evaporated 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).

[0250] b) 6-acetamido-3-methyl-2(methylthio)benzo[d]thiazol-3-ium, TsO - Preparation of (23)

[0251] [ka]

[0252] Compound (22) (2.065 g, 8.66 mmol) was added to a round-bottom flask along with p-toluenesulfonic acid monohydrate (1.780 g, 9.53 mmol). The mixture was then stirred at 130° C. for 3 hours. The heat was turned off, the mixture was allowed to cool to room temperature, and the crude product was then triturated with 20 mL of acetone four more times to obtain a red powder. The remaining solvent was evaporated 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.

[0253] c) Preparation of compound (I.10), 2TFA-

[0254] [ka]

[0255] 134.2 mg (0.316 mmol) of 6-acetamido-3-methyl-2(methylthio)benzo[d]thiazol-3-ium, TsO - (Compound 23) was poured into a 100 mL round-bottom flask along with 818 μL of a 75 mM solution of 1,4,6-trimethyl-2-(4-(trimethylammonio)butyl)pyrimidin-1-ium, diiodine in acetonitrile (Compound 3, 150 mg; 0.316 mmol), and 30 mL of acetonitrile and 10 mL of ethyl alcohol were added sequentially. The suspension mixture was then stirred and heated at 80° C. until dissolution was complete. The solution was cooled at room temperature, and then 88.1 μL (64 mg; 0.634 mmol) of triethylamine was added dropwise. The mixture was then stirred at room temperature for 25 minutes. The solvent was evaporated and the resulting solid was purified by preparative chromatography using a PrepPure C18 column from Buchi (ref 11068740) (eluent A: 10 mM TFA in water, eluent B: 90% ACN and 10% 10 mM TFA in water, 5-80% gradient of eluent B in 40 min) to give compound (I.10), 2TFA - Yield = 35%. Mass spectrometry (ESI-Q(+)): M / Z=220.63

[0256] II. Evaluation of Compounds According to the Invention A. Stability in PCR-mimicking conditions and fluorescence experiments

[0257] 1) Protocol a) Accelerated stability test under alkaline conditions (Figures 1, 2 and 3) Compounds were prepared at 10 μM or 1× in Tris-HCl 20 mM pH 9 and stored at 40°C. 20 μL aliquots were periodically injected into a UPLC (Alliance chain from Waters (USA) equipped with mass and UV detectors, performed on a UPLC BEH C18 1.7 μm 2.1 × 50 mm column (Waters) using Method A: 0–95% acetonitrile, 10 mM TFA pH 2 for 5 min at 0.5 mL / min, with detection at maximum plot or specific wavelength) to reach the compound's half-life (T1 / 2 in days) under these conditions.

[0258] b) Analytical fluorescence evaluation of compounds (Figures 1, 2 and 3 and 9) A 20 μL solution was prepared using 10 μM compounds in a typical PCR mix formulation (20 mM Tris pH 8.4, 10 mM NaCl, 4× 0.3 mM dNTPs, 4 mM MgCl2, 1 U / μL TAQ polymerase, and 550 ng / μL BSA). An 80-base double-stranded amplicon, roughly equivalent to the amount of amplicon obtained in a standard PCR reaction, was added to the vial to a final concentration of 0.13 μg / μL (5 μM). The solution was then injected into a microplate reader, and the maximum fluorescence emission was recorded using a fluorescence spectrometer reader (TECAN Austria) at an excitation wavelength (λ absorption maximum -30 nm) with a gain of 60, allowing comparison of different compounds with each other.

[0259] 2) Comparison with other compounds FIG. 1 shows stability and fluorescence data for Examples 1-4 and 6, 7 and 10 (compounds I.1-I.4, I.6, I.7 and I.10) as trifluoroacetate salts and for the following comparative examples: Comparative Example 1 (Compound 1):

[0260] [ka] Corresponding to the acetate form of D6 in US 7,387,887 Comparative Example 2 (Compound 2):

[0261] [ka] Corresponding to N7 in acetate form in US 7,387,887 Comparative Example 3 (Compound 3): Thiazole Orange - Comparative Example 4 (Compound 4): SybrGreen Comparative Example 5 (Compound 5):

[0262] [ka] Comparative Example 9: Compound present in the commercial kit Resolight® from Roche diagnostics. Its mass spectrometry (ESI-Q(+)) gives M / Z=240,13 (Z=2), which corresponds to the mass of Example 6 (R27) or Example 7 (R28) of WO2008 / 052742.

[0263] The data presented in Figure 1, representing the ambient temperature or simulated behavior of the tested compounds, demonstrate that when compounds according to the present invention interact with nucleic acid sequences, both are highly stable and highly fluorescent under PCR conditions. Other tested compounds, including prior art compounds, either exhibit low stability (half-life ≤ 20 days), low fluorescence (fluorescence < 35,000 RFU), or both stability and fluorescence. These results also reveal that the compounds in the Resolight® kit obtained from Roche diagnostics are less stable and produce lower fluorescence than all of the compounds according to the present invention. Using the same conditions, compound I.5 (n = 1) produces a fluorescence of 9,476 RFU. While this fluorescence is lower than that obtained with the compound according to the present invention (n = 0), the measured fluorescence amount allows for the use of the compounds in nucleic acid analysis. A strict comparison of the fluorescence obtained between compounds (n = 0, 1, 2, or 3) is not appropriate. The interest of these various compounds lies in the possibility of obtaining absorption and emission over a wide range of wavelengths. Figure 9 shows the modulation of the maximum absorption and emission wavelengths, particularly with the choice of n.

[0264] 3) Study on the influence of alkyl chain at 1-position Further tests were carried out to study the effect of the presence of an alkyl chain at the 1-position as an alternative to the aryl group of Example A10 of US 7,387,887. Figure 2 compares the stability and fluorescence obtained with the following two compounds:

[0265] [ka]

[0266] Methyl at the 1-position of pyrimidine (the N atom corresponding to the substituent R1 in formula (I)) appears to induce better stability and better fluorescence than phenyl.

[0267] 4) The effect of the bonding method of the ammonium group on the compound The compound of Example R6 of US Pat. No. 7,387,887 (two ammonium groups -N + Other studies were carried out to study the effect of the presence of ammonium bonded directly to the Z moiety of (Me)3) via its nitrogen atom.

[0268] FIG. 3 compares the stability and fluorescence obtained with compounds 1 to 3 according to the invention (compounds I.1 to I.3 as trifluoroacetates) and the following compounds:

[0269] [ka]

[0270] The resulting fluorescence is significantly reduced compared to Comparative Example 8, making this compound less suitable for PCR applications. Stability may be increased or decreased for compounds according to the invention, but in any case this does not alter the suitability of the compound for use in PCR.

[0271] B. Stability in the presence of thiols Compounds were prepared at 10 μM solutions in Tris 20 mM pH 9 and 100 μM in thioglycerol and stored at 20° C. 20 μL aliquots were periodically injected onto the UPLC as partially described in A1)a).

[0272] The results obtained for Examples 1-4 (corresponding to compounds I.1-I.4 as trifluoroacetates) and for the prior art compound N7 are presented in Figure 4. These results show that the compounds of the present invention, in contrast to N7, are stable in the presence of thiols, since these compounds retain a sulfur group at position 2 of the pyrimidinium ring. Although not shown, compound I.10 is also stable in the presence of thiols.

[0273] C. Analytical Melting Measurements A 10 μM compound solution was prepared in a total volume of 20 μL in a typical PCR mix formulation (20 mM Tris pH 8.4, 10 mM NaCl, 1× proprietary stabilizing buffer, 4× 0.3 mM dNTPs, 4 mM MgCl2, 1 U / μL TAQ polymerase, and 550 ng / μL BSA) either alone (-DUPLEX) or in the presence of two complementary DNA strands ((+DUPLEX) 80 bases long, 5 μM each). Typical melting curves were obtained by placing the solution in a 200 μL PCR polypropylene cuvette and recording the emitted fluorescence (CFX Maestro from Biorad Laboratories) as a function of temperature from 20°C to 90°C (0.5°C / min), using a pre-denaturation step at 95°C for 1 min. The appropriate fluorescence channel was used as a function of the compound's fluorescence signature (FAM was used for all compounds except for I.5, where the Hex channel was used). The melting temperatures of the duplexes were accurately determined by calculating the first derivative of the melting curves and plotting it as a function of temperature. Figures 5A-5F show that all of the compounds of the present invention can be used for detecting the melting of duplexes (Tm) with very good sensitivity (Tm peak height), as summarized in Table 1 herein below. The data in Table 1 also show that in the FAM channel, the prior art compounds D6 and N7 are less fluorescent than the compounds of the present invention.

[0274] [Table 1]

[0275] D. Functional Assay (PCR) Compound I.1 of Example 1 (Figure 6) and I.3 of Example 3 (Figure 7) were used at 10 μM and 5 μM, respectively, in standard PCR amplification reactions, here 10 μM of the biological model: S. saccharomyces (S. cerevisiae). e 5;10 e 4;10 e 3 and 10 eTwo PCR reactions (two replicates) were performed using a LightCycler® 480 Instrument II (Roche) equipped with a (440 / 488) filter. The presence of the compounds according to the present invention allowed for real-time amplification monitoring (Panel A), and after PCR completion, melting experiments were performed by plotting fluorescence = f(temperature) to determine the melting curve of the amplicon (Panel B). From this melting curve, the melting temperature was further extracted by plotting the first derivative of emitted fluorescence = f(temperature), as shown in Panel C. This demonstrates that the novel compounds according to the present invention are highly efficient and useful for monitoring real-time PCR and determining the melting temperature of specific amplicons with great sensitivity.

[0276] E. Functional Assay (Fast PCR Conditions) Compound I.1 of Example 1, I.2 of Example 2, I.3 of Example 3 and I.4 of Example 4 each had a 2.5×10 8A biological model with Cp / PCR was used for fast PCR amplification reactions (five replicates). A standard 30-cycle fast cycling PCR protocol, such as that described in the Qiagen Fast Cycling PCR Handbook (October 2012), was used. For comparison, prior art compound N7 was used as a control. Figure 8A shows real-time PCR curves obtained with four compounds of the present invention, demonstrating their ability to detect amplicons under ultrafast cycling conditions. Panels A, B, and C of Figure 8B show details of Max Fluo, Cp, and Tm measurements, respectively, demonstrating the high reproducibility of these experiments and the ability of these compounds to sensitively detect the presence of specific amplicons. The results also demonstrate that the dyes of the present invention (I.1–I.4) provide approximately twice as much fluorescence (Max Fluo) as compound N7. Therefore, these dyes are much more efficient and valuable in detecting a given target with high sensitivity. In addition, the Cp and Tm were similar to those of N7 (around + / - 1 Cp and + / - 2°C of the Cp obtained by N7, respectively), demonstrating that the dyes of the present invention do not inhibit PCR.

Claims

1. Formula (I): 【Chemistry 1】 [In the formula, n is equal to 0, 1, 2 or 3, Ri, Rj and Rk are the same or different and are hydrogen and C １～６ independently selected from the group consisting of alkyl, - X is oxygen, sulfur, selenium, tellurium or C(CH ３ ) ２ and Re is an alkyl, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atoms and -(CH ２ ) ｋ１ -Y １ is a group selected from the group k1 is 1, 2, 3, 4, 5 or 6, ・Y １ is hydroxy, C １～６ Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N ＋ R １ 'R １ "-(CH ２ ) ｐ１ -] ｍ１ -G １ ' group (m1 is 1, 2 or 3, G １ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p1 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R １ ' and R １ " are the same or different, C １～６ alkyl, and preferentially methyl or ethyl; Z is a halogen atom, an alkyl, an alkenyl, an alkynyl, an aryl, or —CF ３ , -NO ２ , —CN, —C(O)alkyl, —Salkyl, —Oalkyl, —NHalkyl, —NHC(O)H, —NHC(O)phenyl, —NHC(O)alkyl, —S(O ２ ) O － , -S(O ２ )O alkyl, -P(O ２ ) O － , P(O ２ ) O alkyl, —CH═N—O—R, —C(CH ３ )=N-O-R, -CH=N-NH-C(O)-R, -C(CH ３ )=N-NH-C(O)-R, -CH=N-O-C(O)-R, -C(CH ３ )=N—O—C(O)—R, —NHCOR and —CONHR groups, where —CONHR is preferred, is a fused monocyclic or polycyclic aromatic or nitrogen-containing heteroaromatic ring, optionally substituted by one or several identical or different substituent(s) A, and R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group linked to the rest of the molecule by one of its carbon atoms, or is a —(CH ２ ) ｋ２ -Y ２ where: k2 is 1, 2, 3, 4, 5 or 6, ・Y ２ is hydroxy, C １～６ Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N ＋ R ２ 'R ２ "-(CH ２ ) ｐ２ -] ｍ２ -G ２ ' group (m2 is 1, 2 or 3, G ２ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ２ ' and R ２ " are the same or different, C １～６ alkyl, and preferentially methyl or ethyl; -R １ is alkyl or -(CH ２ ) ｋ３ -Y ３ is a group, where k3 is 1, 2, 3, 4, 5 or 6, ・Y ３ is hydroxy, C １～６ Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N ＋ R ３ 'R ３ "-(CH ２ ) ｐ３ -] ｍ３ -G ３ ' group (m3 is 1, 2 or 3, G ３ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p3 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ３ ' and R ３ " are the same or different, C １～６ alkyl, and preferentially methyl or ethyl; -R ２ , R ３ and R ４ are the same or different and are selected from hydrogen, alkyl groups, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups bonded to the remainder of the molecule by one of its carbon atoms, and -(CH ２ ) ｋ４ -Y ４ are independently selected from the group ・k4 is 1, 2, 3, 4, 5, 6, ・Y ４ is aryl, hydroxy, C １～６ Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N ＋ R ４ 'R ４ "-(CH ２ ) ｐ４ -] ｍ４ -G ４ ' group (m4 is 1, 2 or 3, G ４ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p4 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ４ ' and R ４ " are the same or different, C １～６ alkyl, and preferentially methyl or ethyl; However, the substituents A and R ２ , R ３ and R ４ and at least one of the groups comprises a secondary amine, a tertiary amine, or a quaternary ammonium. and at least one anion, particularly a halide anion, typically Cl － ,Br － and I － trifluoroacetate, acetate, formate; sulfonate, e.g., methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, e.g., methylsulfate; phosphate, pyrophosphate, and triphosphate.

2. Formula (II): 【Chemistry 2】 [In the formula, - X, Re, R １ , R ２ , R ３ , R ４ , Ri, Rj, Rk and n are as defined for claim 1; Ra, Rb, Rc and Rd are the same or different and are selected from hydrogen and halogen atoms as well as alkyl, alkenyl, alkynyl, aryl, -CF ３ , -NO ２ , —CN, —C(O)alkyl, —Salkyl, —Oalkyl, —NHalkyl, —NHC(O)H, —NHC(O)phenyl, —NHC(O)alkyl, —S(O ２ ) O － , -S(O ２ )O alkyl, -P(O ２ ) O － , P(O ２ ) O alkyl, —CH═N—O—R, —C(CH ３ )=N-O-R, -CH=N-NH-C(O)-R, -C(CH ３ )=N-NH-C(O)-R, -CH=N-O-C(O)-R, -C(CH ３ )=N—O—C(O)—R, —NHCOR and —CONHR groups, —CONHR being preferred, where R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms, or —(CH ２ ) ｋ２ -Y ２ where: k2 is 1, 2, 3, 4, 5 or 6, ・Y ２ is hydroxy, C １～６ Alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, and morpholinyl groups, and -[N ＋ R ２ 'R ２ "-(CH ２ ) ｐ２ -] ｍ２ -G ２ ' group (m2 is 1, 2 or 3, G ２ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ２ ' and R ２ " are the same or different, C １～６ alkyl, and preferentially methyl or ethyl; However, the substituents Ra, Rb, Rc, Rd, and R ２ , R ３ and R ４ At least one of the substituents Ra, Rb, Rc, Rd, R ２ , R ３ and R ４ contains a secondary amine, a tertiary amine or a quaternary ammonium. and at least one anion, particularly a halide anion, typically Cl － ,Br － and I － 3. The compound of claim 1, including salts thereof with anions selected from: trifluoroacetate, acetate, formate; sulfonate, e.g., methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, e.g., methylsulfate; phosphate, pyrophosphate, and triphosphate.

3. At least one Y comprises or is a trialkylammonium, typically trimethylammonium. ２ group or Y ４ groups, especially two Y ２ Group and Y ４ 3. The compound of claim 1 or 2, comprising a group.

4. The compound according to any one of claims 1 to 3, wherein k2 is 3, 4, 5 or 6.

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

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

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

9. n=0, in particular of formula (III): 【Transformation 3】 (In the formula, R １ , R ２ , R ３ , R ４ , Ra, Rb, Rc, Rd and Re are as defined in any one of claims 1 to 6. and at least one anion, particularly a halide anion, typically Cl － ,Br － and I － 9. The compound according to any one of claims 1 to 8, including salts thereof with anions selected from: trifluoroacetate, acetate, formate; sulfonate, for example, methylsulfonate, trifluoromethylsulfonate and tosylate; sulfate, for example, methylsulfate; phosphate, pyrophosphate and triphosphate.

10. R ２ Groups and R ３ At least one of the groups is —(CH ２ ) ｋ４ -Y ４ where: k4 is 1, 2, 3, 4, 5 or 6, ・Y ４ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N ＋ R ４ 'R ４ "-(CH ２ ) ｐ４ -] ｍ４ -G ４ ' (m4 is 1, 2 or 3, G ４ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p4 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ４ ' and R ４ " are the same or different, C １～６ alkyl, preferentially methyl or ethyl; ４ is preferentially a trialkylammonium, typically trimethylammonium, Other R ２ group or R ３ The base is C １～６ The compound of any one of claims 1 to 9, which is alkyl.

11. Formula (IIa): 【Chemistry 4】 and especially (IIIa): 【Transformation 5】 [In the formula, Rc is a hydrogen or halogen atom, or an alkyl, alkenyl, alkynyl, aryl, —CF ３ , -NO ２ , —CN, —C(O)alkyl, —Salkyl, —Oalkyl, —NHalkyl, —NHC(O)H, —NHC(O)phenyl, —NHC(O)alkyl, —S(O ２ ) O － , -S(O ２ )O alkyl, -P(O ２ ) O － , P(O ２ ) O alkyl, -CH=N-O-R, -C(CH ３ )=N-O-R, -CH=N-NH-C(O)-R, -C(CH ３ )=N-NH-C(O)-R, -CH=N-O-C(O)-R, -C(CH ３ )═N—O—C(O)—R, —NHCOR and —CONHR groups, the —CONHR group being preferred, R being a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms or a —(CH ２ ) ｋ２ -Y ２ is selected from, where: k2 is 1, 2 or preferentially 3, 4, 5 or 6, - Y ２ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N ＋ R ２ 'R ２ "-(CH ２ ) ｐ２ -] ｍ２ -G ２ ' (m2 is 1, 2 or 3, G ２ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ２ ' and R ２ " are the same or different, C １～６ alkyl, preferentially methyl or ethyl; ２ is preferentially a trialkylammonium, typically trimethylammonium or -[N ＋ Me ２ - (CH ２ ) ｐ２ -] ｍ２ -N ＋ Me ３ (wherein p2 and m2 are as described above), - X, Re, R １ , R ２ , R ３ , R ４ , Ri, Rj, Rk and n are as defined for any one of claims 1 to 10. at least one anion, particularly a halide anion, typically Cl － ,Br － and I － 11. The compound of claim 10, including salts thereof with anions selected from: trifluoroacetate, acetate, formate; sulfonate, e.g., methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, e.g., methylsulfate; phosphate, pyrophosphate, and triphosphate.

12. Z or the corresponding group in formula (II) and (III) is -CH=N-O-R, -C(CH ３ )=N-O-R, -CH=N-NH-C(O)-R, -C(CH ３ )=N-NH-C(O)-R, -CH=N-O-C(O)-R, -C(CH ３ )═N—O—C(O)—R, —NHCOR or —CONHR, with —CONHR being preferred, where R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms or —(CH ２ ) ｋ２ -Y ２ where k2 is 1, 2, or preferentially 3, 4, 5 or 6; ２ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N ＋ R ２ 'R ２ "-(CH ２ ) ｐ２ -] ｍ２ -G ２ ' (m2 is 1, 2 or 3, G ２ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ２ ' and R ２ " are the same or different, C １～６ alkyl, preferentially methyl or ethyl; ２ is preferentially a trialkylammonium, typically trimethylammonium or -[N ＋ Me ２ - (CH ２ ) ｐ２ -] ｍ２ -N ＋ Me ３ (p2 and m2 are as defined above).

13. Formula (IIa): 【Transformation 6】 and, in particular, (IIIa): 【Transformation 7】 [Wherein, Rc is —CH═N—O—R, —C(CH3)═N—O—R, —CH═N—NH—C(O)—R, —C(CH ３ )=N-NH-C(O)-R, -CH=N-O-C(O)-R, -C(CH ３ )=N—O—C(O)—R, —NHCOR or —CONHR, with —CONHR being preferred, where R is a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl group bonded to the remainder of the molecule by one of its carbon atoms or —(CH ２ ) ｋ２ -Y ２ where k2 is 1, 2, or preferentially 3, 4, 5 or 6; ２ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N ＋ R ２ 'R ２ "-(CH ２ ) ｐ２ -] ｍ２ -G ２ ' (m2 is 1, 2 or 3, and G' ２ is H or is amino, alkylamino, dialkylamino or trialkylammonium, p2 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ２ ' and R ２ " are the same or different, C １～６ alkyl, preferentially methyl or ethyl; ２ is preferentially a trialkylammonium, typically trimethylammonium or -[N ＋ Me ２ - (CH ２ ) ｐ２ -] ｍ２ -N ＋ Me ３ (wherein p2 and m2 are as described above), X, Re, R １ , R ２ , R ３ , R ４ , Ri, Rj, Rk and n are as defined for any one of claims 1 to 10. at least one anion, particularly a halide anion, typically Cl － ,Br － and I － 13. The compound of claim 12, including salts thereof with anions selected from: trifluoroacetate, acetate, formate; sulfonate, e.g., methylsulfonate, trifluoromethylsulfonate, and tosylate; sulfate, e.g., methylsulfate; phosphate, pyrophosphate, and triphosphate.

14. R ４ = H, and R １ and R ３ are the same or different, and C １～６ alkyl, in particular methyl or ethyl; R ２ Ga-(CH ２ ) ｋ４ -Y ４ where k4 is 1, 2, 3, 4, 5, or 6; and Y ４ is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N ＋ R ４ 'R ４ "-(CH ２ ) ｐ４ -] ｍ４ -G ４ ' (m4 is 1, 2 or 3, G ４ ' is H or is amino, alkylamino, dialkylamino or trialkylammonium, p4 is 1, 2, 3, 4, 5 or 6, preferentially 2 or 3, R ４ ' and R ４ " are the same or different, C １～６ alkyl, preferentially methyl or ethyl; ４ A compound according to any one of claims 10 to 13, wherein is preferentially trialkylammonium, typically trimethylammonium.

15. Re is C １～６ A compound according to any one of claims 1 to 14, which is alkyl, in particular methyl.

16. A compound according to any one of claims 1 to 15, comprising at least one quaternary ammonium, preferentially trialkylammonium, typically trimethylammonium.

17. The compound according to any one of claims 1 to 16, in the form of a trifluoroacetate ion salt. 【Request Item 18】 【Chemistry 8】 【change】 【change】 【change】 and at least one anion, in particular a halide anion, typically Cl － ,Br － and I － 2. The compound according to claim 1, comprising salts thereof with anions selected from among: trifluoroacetate, acetate, formate; sulfonate, e.g., methylsulfonate, trifluoromethylsulfonate and tosylate; sulfate, e.g., methylsulfate; phosphate, pyrophosphate and triphosphate, in particular the trifluoroacetate salts thereof.

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

20. 20. A method for detecting a target nucleic acid, which is a single-stranded or double-stranded nucleic acid, comprising the 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. Steps below: - amplifying the target nucleic acid to produce an amplicon; - adding a compound according to any one of claims 1 to 18 to a sample containing target nucleic acids and / or amplicons before, during or after an amplification step, - monitoring the fluorescence from the compound according to the invention during or after the amplification step. The use according to claim 19 or the method according to claim 20, wherein

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

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

24. - 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 for amplifying a portion of the target nucleic acid and generating at least an amplicon, to obtain a PCR mixture, - amplifying the target nucleic acid from the PCR mixture to produce at least an amplicon; and - monitoring the fluorescence from the compound according to any one of claims 1 to 18 during or after the amplification step. A method for PCR analysis of a target nucleic acid, comprising:

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

26. 25. The method of claim 24, wherein the monitoring step occurs after amplification and comprises generating a melting curve.

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

28. The method of any one of claims 20 to 27, comprising the step of quantifying the target nucleic acid initially present in the sample.

29. - mixing a compound according to any one of claims 1 to 18 with a sample comprising a target nucleic acid and at least one pair of primers suitable for amplifying a portion of the target nucleic acid and generating an amplicon, to obtain a PCR mixture, - amplifying the target nucleic acid from the PCR mixture to produce at least an amplicon; - monitoring the fluorescence of the compound according to any one of claims 1 to 18 resulting from binding of the compound to the amplicon during the amplification step, - at the end of the amplification step, melting the amplicons produced to obtain a melting curve, and - using the shape of the melting curve to identify the genotype or polymorphism of the target nucleic acid.

24. The method for PCR analysis of a target nucleic acid according to claim 23, comprising:

30. 30. The method of any one of claims 20 to 29, wherein the amplification step comprises a plurality of temperature cycles comprising at least a denaturation temperature and an extension temperature, each cycle having a cycle time of less than 90 seconds per cycle, the polymerase being provided at a concentration of at least 0.005 μM or 0.02 U / μL, and the primers being provided at a concentration of at least 0.1 μM each.

31. 30. The method of any one of claims 20 to 29, wherein the amplification step comprises a plurality of temperature cycles comprising at least a denaturation temperature and an extension temperature, each cycle having a cycle time of less than 20 seconds per cycle, the polymerase being provided at a concentration of at least 0.5 μM or 1.9 U / μL, and the primers being provided at a concentration of at least 2 μM each.

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

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

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

35. 35. The kit according to claim 34, which also comprises a buffer solution of pH 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 a buffer solution.

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