EP1883635A2

EP1883635A2 - Light-emitting biomarker

Info

Publication number: EP1883635A2
Application number: EP06794461A
Authority: EP
Inventors: Pierre-Yves Renard; Anthony Romieu; Marc Massonneau
Original assignee: Universite de Rouen; Quidd
Current assignee: Universite de Rouen; Quidd
Priority date: 2005-05-27
Filing date: 2006-05-24
Publication date: 2008-02-06
Also published as: FR2886292A1; US7968735B2; WO2006129036A3; WO2006129036A2; CN101233126A; US20090041669A1; JP2008545682A; FR2886292B1; IL187675A0; CA2609782A1; WO2006129036B1

Abstract

The invention concerns novel 1,2-dioxetane derivatives of general formula (I) as defined in the description, capable of emitting a detectable luminescent signal, their use in a method for detecting and/or quantizing a physical, chemical or biological, in particular enzymatic, phenomenon, as well as a kit for implementing said method.

Description

Biomarker with luminescent emission

The present invention relates to novel compounds capable of emitting a detectable luminescent signal, to their use in a method for detecting and / or quantifying a physical, chemical or biological phenomenon, in particular enzymatic, as well as to a kit enabling the implementation of the method.

Many detection methods used in biochemistry, or cell biology, rely on the generation of a light signal, for example fluorescence.

Thus, many chromogenic, fluorescent or luminescent tests are described in vitro to detect the activities of various enzymes. Most of these tests rely on the use of donor / acceptor or donor / quencher pairs of resonance fluorescence energy transfer (FRET) or the use of pro-fluorophore entities.

Unfortunately, this method of detection satisfactory in terms of in vitro analysis is generally not exploitable in vivo analysis. Thus, in vivo application, it is necessary to provide a near-infrared excitation. The choice of donor / acceptor or donor / quencher pairs for which the excitations do not overlap then becomes extremely limited (FUNOVICS et al., Anal Bioanal Chem., 2003, 377: 956-963).

In order to overcome this need to excite the fluorescence donor in the near infrared, it has been proposed to use bioluminescent or chemiluminescent compounds, that is to say whose luminescence is generated at the same time. from a chemical or biological reaction (LAXMAN et al., Proc Natl Acad Sci.

USA, 2002, 99: 16551-16555).

Chemiluminescent compounds, their preparations and their uses have been known for a long time. These molecules called "high energies" contain enough energy to generate carbonyl groups in an excited electronic state, responsible for the phenomenon of chemiluminescence observed.

As representative of chemiluminescent compounds, mention may be made of compounds derived from dioxetane and more particularly derivatives of 1,2-dioxetane. These compounds are thermally labile and can decompose over a wide temperature range by emitting a light signal. In general, these compounds have the following general formula:

wherein each R substituent corresponds to a hydrocarbon radical optionally comprising one or more heteroatoms, and two adjacent R's or an R adjacent to Ar-XW and one of the other two R or Ar may be joined, X may be O, S or N and W can represent a detachable group, if necessary loaded.

Numerous 1,2-dioxetane derivatives have been synthesized, among which compounds comprising a 3-bonded spiroadamantyl radical, already used as chemiluminescent substrates. Such derivatives are in particular described in the documents WO 96/24849, WO 90/07511 and US 5,330,900. These 1,2-dioxetane derivatives may comprise a recognition site for an enzyme and may be activated by an enzyme recognizing this site to generate a luminescent signal.

More recently, other dioxetane derivatives have also been developed, described in JP 2002-02038, EP 1 342 724, and US 2004/0077018, in which various radicals are used to stabilize the dioxetane ring.

The principle of the chemiluminescence reaction of these compounds rests on an electron exchange (CIEEL) between the phenolate function and the stretched dioxetane ring located in para of the phenol aromatic ring (MATSUMOTO, J. Photochem. Photobiol C: Photochem Rev., 2004, 5: 27-53). The generation of a compound comprising an unstable phenolate leads to the rupture of the stretched 1,2-dioxetane unit, stabilized by the radicals R, following an electron exchange with the phenolate by the CIEEL phenomenon, for result in the appearance of an activated ester returning to the ground state by emitting a luminescent signal.

Phenol, in itself, as well as its esters with carboxylic acids are the most commonly used precursors for generating an oxygen anion

(oxyanion) phenol in an aqueous alkaline solution. However, other derivatives of this type of 1,2-dioxetane have also been synthesized in which this same oxygen atom has been replaced by a thiol group, an amino group or a carbon

(MATSUMOTO, J. Photochem Photobiol, C: Photochem Rev., 2004, 5: 27-53, SABELLE et al, J Am Chem Soc, 2002, 124 (17), 4874-4880, MATSUMOTO et al.

Tetrahedron Lett, 2004, 45: 3779-82; WO 96/24849; WO 00/44719). However, these The latter derivatives have a much lower quantum yield and are not practical.

The object of the present invention is to propose new compounds derived from 1,2-dioxetane capable of generating, directly or by fluorescence energy transfer at intramolecular resonance (FRET), a luminescent signal that can be detectable through of a layer of biological tissue layers.

Another object of the present invention is to provide compounds derived from 1,2-dioxetane which can be activated by any chemical, physical or biological phenomenon, and in particular enzymatic activity. The invention thus has the advantage of translating information resulting from this activation, for example a hydrolysis-type information in the context of an enzymatic activity, into the appearance of a phenolate or a thiophenate, and to confer on the derived from spectroscopic properties distinct from those originally assigned to them.

Another object of the present invention is to provide compounds derived from 1,2-dioxetane which can be administered in vivo in order to be used in medical imaging methods.

The inventors thus observed, against all odds, that it was possible to associate with a 1,2-dioxetane backbone, as defined previously, a reagent arm activated by a physical, chemical or biological phenomenon, and especially by a enzymatic activity, without prejudicing the emission of a light signal.

The reactive arm considered according to the invention ensures, thanks to an intramolecular rearrangement, the transfer of the electrons necessary for the activation of the light signal.

Thus, according to one of its first objects, the present invention relates to a compound of general formula (I):

S-B-A in which

S represents a labile structure activatable by a physical, chemical or biological phenomenon, - B is a reactive arm whose chemical structure is such that the activation of the labile structure S induces its intramolecular rearrangement in a form conducive to the release of a molecule A ^" , A is a chromophore of the general formula (IIa)

in which

O symbolizes the covalent bond to B, a) R ₁ , R ₂ and R ₃ represent, independently of each other, a radical as defined below, such as, for example, a hydrogen atom or an alkyl radical; an alkoxy radical, an aryl radical, a hydroxyl radical, an oxo unit, an amino group, a silyl group, a halogen atom, b) R ₂ and R ₃ may form an oxo unit, c) R ₁ and R ₂ can be bonded to form a condensed ring with the dioxetane ring, as defined below, d) R ₂ and R ₃ or R ₁ and Ar can be linked to form a spiro ring with the carbon of the dioxetane entity that carry them, as defined below,

Ar represents an arylene radical as defined in particular below,

X is O, N or S, and derivatives thereof, such as salts, esters or derivatives obtained by functionalizing a compound of general formula (I) with at least one targeting moiety. According to yet another subject, the present invention relates to a compound of general formula (Va):

in which, S is a labile structure and X, Ar, R ₁ , R ₂ , R ₃ , R ₅ and R ₆ are as defined below.

According to another of its objects, the present invention relates to a compound derived from the functionalization of a compound according to the invention by at least one structural unit favoring, for example, targeting to a particular cell or tissue.

According to yet another of its objects, the present invention relates to a use for the purpose of detecting and / or quantifying a physical, chemical or biological phenomenon of at least one compound according to the present invention in which S is a structure labile activatable by said phenomenon. According to yet another subject, the present invention relates to the use of a compound according to the invention, and in particular of general formula (Va), for the preparation of a pharmaceutical composition intended for the implementation of an in vivo diagnostic method.

According to yet another of its objects, the present invention relates to a kit for the detection and / or quantification of a biological entity, in particular an enzyme, comprising a compound of general formula (I), and in particular of general formula

(Va), wherein S is a labile structure activatable by said biological entity to be detected and / or quantified.

According to yet another of its objects, the present invention relates to a method for detecting and / or quantifying a biological entity, in particular an enzyme, comprising at least the steps of: contacting at least one effective amount of at least one compound of general formula (I), and in particular of general formula (Va), in which S is a labile structure activatable by said biological entity, in particular an enzyme, to detect and / or quantify in a medium presumed to contain said biological entity, and

measurement of the luminescent signal generated.

According to yet another of its objects, the present invention relates to an in vivo diagnostic method, in particular for medical imaging purposes, comprising the detection and / or the quantification of a biological entity, in particular an enzyme, by means of of a compound of general formula (I), and in particular of general formula (Va) in which S is a labile structure activatable by said biological entity to be detected and / or quantified. DEFINITIONS

For the purposes of the present invention, the term "alkyl radical" is intended to mean a linear, branched or cyclic, condensed or non-condensed, saturated or unsaturated hydrocarbon radical containing from 1 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms and more particularly 4 carbon atoms, may be substituted by radicals as defined below.

By way of example, include in this definition radicals such as methyl, ethyl, isopropyl, n-butyl, t-butyl, t-butylmethyl, n-propyl, pentyl, n-hexyl, 2-ethylbutyl, heptyl, octyl , nonyl, decyl, undecyl, dodecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl or icosanyl.

For the purposes of the present invention, the term "cyclic alkyl radical" means an alkylene ring of 4 to 10 carbon atoms, such as cyclopropyl, cyclopentyl, cyclohexyl, cyclohexylmethyl or cycloheptyl.

The term "polycyclic alkyl radical" means a polycyclic alkylene of 4 to 20 carbon atoms, in particular of 6 to 12 carbon atoms, optionally substituted with one to 10 radicals independently chosen from a C ₁ -C ₁₀ alkyl radical, in particular C ₂ -C ₆ alkoxy radical C ₁ -C _10, especially C ₂ -C ₆ alkylamino C ₁ -C _10, especially C ₂ -C ₆ alkylthio C ₁ -C ₁₀ , especially C ₂ -C ₆ , a halogen atom and a C ₁ -C ₁₀ haloalkyl radical, especially C ₂ -C ₆ . As an example of a polycyclic radical that is suitable for the implementation of the invention, mention may be made of the adamantyl radical or the bicyclo [2.2.1] heptyl radical.

For the purposes of the present invention, the term "alkoxy radical" means a radical - OR in which the alkyl radical is a linear, branched or cyclic hydrocarbon radical, condensed or not, saturated or unsaturated, as defined above.

By way of example, mention may be made of methoxy, ethoxy, propoxy, butoxy, n-butoxy, isobutoxy, sec-butoxy, n-pentoxy, isopentoxy, sec-pentoxy, t-pentoxy, hexyloxy, methoxyethoxy and methoxypropoxy. ethoxyethoxy, ethoxypropoxy and the like.

For the purposes of the present invention, the term "acyl radical" means a linear, branched or cyclic hydrocarbon radical, condensed or not, saturated or unsaturated, comprising a C = O function and having 1 to 20 carbon atoms, in particular from 2 to 12 carbon atoms and preferably 3 to 6 carbon atoms and more particularly 4 carbon atoms, for example, a formyl radical, an acetyl radical, a succinyl radical, a benzoyl radical, a 1-naphthoyl or 2-naphthoyl radical; .

The hydrocarbon chain of the aforementioned radicals can be interrupted by one or more heteroatoms chosen from O, N and S, to form, for example, a heteroalkyl radical such as an alkyl ether radical, an alkyl ester radical or a heterocycle.

For the purposes of the present invention, the term "heterocyclic radical" is understood to mean, for example, and without limitation, a furanyl radical, a thiophenyl radical, a pyrolyl radical, an oxazolyl radical, an isoxazolyl radical, a thiazolyl radical or a radical. isothiazolyl, an imidazolyl radical, a pyrazolyl radical, a furazanyl radical, a pyranyl radical, a pyridinyl radical, a pyridadinyl radical, a pyrimidinyl radical or a pyradinyl radical.

For the purposes of the present invention, the term "aryl radical" means an aromatic ring comprising from 1 to 5 aromatic rings, optionally fused, of 6 to 30 carbon atoms, in particular of 6 to 10 carbon atoms, optionally comprising one or more heteroatoms chosen from O, N and S. As examples of aryl radicals suitable for the implementation of the invention, it is possible to mention the phenyl radical, the naphthyl radical, the anthryl radical, and all the aromatic rings comprising a plurality of heteroatoms selected from O, N and S, such as for example pyridine, thiophene, pyrrole, furan, quinoline. For the purposes of the present invention, the term "arylene radical" means an aryl radical, as defined above, which is divalent.

By way of examples of substituents which may be used for substituting the abovementioned radicals, mention may be made of a hydroxyl radical, an oxo unit, a thio group, an amino group, a halogen atom, a alkyl radical, a carboxy radical, an acyl radical, an amido radical, an alkoxy radical, an alkyl- or dialkylamino radical, an alkylthio radical or a haloalkyl radical such as, for example, perfluoroalkyl, a alkyl-, dialkyl- or trialkyl-silyl radical, an alkyl-, dialkyl- or trialkyl-siloxy radical, an aryl radical, as defined above.

For the purposes of the present invention, the term "halogen atom", an F, Cl, Br or I atom. The halogen atoms advantageously used in the present invention are fluorine and chlorine.

For the purposes of the present invention, the term "haloalkyl radical" means an alkyl radical as defined previously substituted with one or more halogen atoms as defined above, and in particular, by way of non-limiting example, perfluoromethyl.

DERIVATIVES OF 1.2-DIOXETANE

The compounds according to the present invention are of general formula (I)

S-B-A and include derivatives thereof, such as salts, esters or derivatives functionalized with structural elements, especially for targeting purposes.

Reaction Arm B

In the general formula (I) of the compounds in accordance with the invention, B is different from a simple chemical bond and represents a reactive hydrocarbon arm whose chemical structure is such that the activation of the labile structure S by a chemical phenomenon, physical or biological causes the intramolecular rearrangement of B in a form conducive to the release of a molecule A ^" .

Advantageously B is represented by an alkyl radical C ₁ -C ₈ , in particular C ₂ -C ₆ , and in particular C ₃ -C ₄ , optionally interrupted by a C ₆ -C ₁₄ arylene radical, especially C ₆ -C ₁₀ and / or by one or more heteroatoms chosen from O, N, and S, and optionally substituted by one or more substituents as defined above and in particular selected from a hydroxyl radical, an oxo unit, an amino group, a halogen, an alkyl radical, an alkoxy radical, a carboxy radical, an alkylamino radical, a dialkylamino radical, an aryl radical or an aryloxy radical. Advantageously, the alkyl radical comprises two carbon atoms.

The arylene radical may also be substituted with one or more substituents as defined above. According to one embodiment variant, the arylene radical is chosen from a phenylene or a naphthylene. On the phenylene radical, the positions of the two bonds of the radical may be in ortho, meta or para. Advantageously they are in ortho. According to one embodiment, the reactive arm B can be chosen from: a benzylidene radical,

a radical DN (CH ₃ ) - (CH ₂ ) ₂ -N (CH ₃ ) -C (O) -O, in which D and O respectively symbolize the covalent bond with the labile structure S and the chromophore A, or a derivative represented by the general formula (III):

in which

D and O symbolize, respectively, the covalent bond with the labile structure S and the chromophore A, j is an integer ranging from 0 to 2, and advantageously is equal to 1, R ₄ is a C ₁ -C ₂₀ hydrocarbon-based chain, in particular C ₂ -C ₁₂ and in particular C ₃ -C ₆ , optionally interrupted by one or more heteroatoms and / or unit (s) -CO and / or -N (alkyl) -, placed in ortho, meta or para of the radical D- (CH ₂ ) J-, and in particular chosen from radicals - (CH ₂ ) _k -O, - (CH ₂ ) _k -C (O) -O, -CH ₂ -OC (O) -N (CH ₃ ) - (CH ₂ ) ₂ -N (CH ₃ ) -C (O) -O, where k is 1 or 2, and advantageously 1,

R ₅ and R ₆ are, independently of one another, a hydrogen atom or a linear, branched or cyclic, saturated or unsaturated C ₁ -C ₂₀ , especially C ₂ -C ₁₂ , hydrocarbon-based chain, and in particular C ₃ -C ₆ chosen from an alkyl radical, an alkoxy radical, an alkyl- or dialkylamino radical, an alkylthio radical, or an aromatic C ₆ -C ₃₀ , especially C ₆ -C ₁₀ ring chosen from an aryl radical; , an aryloxy radical, an arylamino radical, an arylthio radical, especially as defined above. According to one particular embodiment, the radicals R ₅ and R ₆ are, independently of one another, chosen from: a hydrogen atom, a C ₁ -C ₆ alkyl radical, an alkenyl radical,

C ₂ -C ₆ , a C ₃ -C ₆ alkinyl radical, branched or unbranched, a C ₃ -C ₆ cycloalkyl radical, which may or may not be substituted with one or more substituents as defined above and chosen in particular from an atom of halogen, an alkyl radical, an alkoxy radical, an aryl radical such as, for example, a benzyloxy radical or a benzyl radical, a cyclic, aliphatic, aromatic or heteroaromatic hydrocarbon-based chain, comprising one or more heteroatoms chosen from O, N and S, C ₃ -C ₈ , in particular C ₄ -C ₇ , optionally substituted with one or more substituents as defined above and in particular selected from a halogen atom, an alkyl radical, an alkoxy radical and an aryl radical such as, for example, a benzyloxy radical, a benzyl radical, a phenyl radical or a naphthyl radical.

According to a particular embodiment, when R ₄ is chosen from - (CH ₂ ) k-C (O) -O and -CH ₂ -OC (O) -N (CH ₃ ) - (CH ₂ ) ₂ -N ( CH ₃ ) -C (O) -O, then R ₄ is in para with the radical D- (CH ₂ ) _r , j = k = 1 and R ₅ = R ₆ = H.

According to another particular embodiment, R ₄ can be represented by - (CH ₂ ) k -O, positioned in ortho with the radical D- (CH ₂ ) _j -, and j = k = 1 and R ₅ = R ₆ = -OCH ₃ .

Advantageously, the reagent arm B may be as defined in US Pat. No. 6,271,345, the content of which is incorporated herein by reference.

According to a particular embodiment, the intramolecular rearrangement of B leads to the formation of a lactam or a lactone.

Chromophore A In the general formula (I) compounds derived from 1,2-dioxetane according to the invention, the chromophore A is represented by the general formula (IIa):

0-0

O - X-Ar R ₃

in which

O symbolizes the covalent bond with B, a) R ₁ , R ₂ and R ₃ represent, independently of one another, a hydrogen atom, a hydroxyl radical, an amino radical, a thio radical, a halogen atom or a C ₁ hydrocarbon-based chain; -C ₂₀ , especially C ₂ -C ₁₂ , and in particular C ₃ -C ₆ selected from an alkoxy radical, an alkyl radical, a heteroalkyl radical, a cycloalkyl radical, a heterocycloalkyl radical, an ether radical, an alkenyl radical; an alkynyl radical, an alkylamino radical, a dialkylamino radical, an alkyl-, dialkyl- or trialkyl-silyl radical, an alkyl-, dialkyl- or trialkyl-siloxy radical, an alkylthio radical, a haloalkyl radical or a C aromatic ring; ₆ -C ₃₀ , in particular C ₆ -C ₁₀ , selected from an aryl radical, and a heteroaryl radical, optionally substituted, b) R ₂ and R ₃ may together represent an oxo unit, c) R ₁ and R ₂ can be linked so as to form a C ₄ -C ₈ ring condensed with the dioxetane ring, if necessary interrupted by one or more heteroatoms chosen from O, N and S, and which may be substituted by one or more radicals as defined previously in a) for R ₁ , R ₂ and R ₃ , d) R ₂ and R ₃ or R ₁ and Ar may be linked so as to form a C ₄ -C ₂₀ spiro ring, in particular C ₆ -C ₁₀ , with the carbon of the dioxetane entity which carries them, this cycle possibly being monocyclic or polycyclic, saturated or unsaturated, aromatic, condensed or not, and if appropriate incorporate one or more heteroatoms selected from O, N and S and optionally substituted with one or more radicals as defined above in a) for R ₁ , R ₂ and R ₃ ,

Ar represents a C ₆ -C ₃₀ arylene radical, in particular C ₆ -C ₁₀ arylene, and being optionally substituted with one or more substituents chosen in particular from a hydroxyl radical, a halogen atom, an oxo unit, an amino radical, an alkylamino radical, a dialkylamino radical, a thio radical, an alkyl-, dialkyl- or trialkylsilyl radical, an alkyl-, dialkyl- or trialkyl-siloxy radical, an alkyl radical, an alkoxy radical, an alkylthio radical or a radical; carboxy, a formyl radical, an alkyl ester radical, an alkyl ketone radical, a haloalkyl radical, an aryl radical, an arylester radical, an arylketone radical, an arylamino radical, a diarylamino radical, and

X can represent O, NH or S. Advantageously, X can represent O. The compound A ^" generated following the activation of the S-labile structure and the intramolecular rearrangement of the reactive arm B is unstable and reacts by emitting a light signal through a chemiluminescence reaction.

According to a particular embodiment, R ₁ may be a fluorescent radical or an alkyl radical, as defined above, substituted by a fluorescent radical.

According to another embodiment, Ar may be substituted by a fluorescent radical or a radical capable of imparting a fluorescent character to the entire structure or a radical capable of modifying its fluorescent properties.

According to another particular embodiment, Ar and R ₁ and / or R ₂ and R ₃ may be combined to form a cyclic or polycyclic or spiro-fused alkyl radical which may: either comprise at least one double bond or at least one triple bond in the ring or in a side chain, and optionally one or more heteroatoms selected from O, N and S, be connected to, or condensed with, a substituted aromatic ring or not. When Ar and R ₁ are not linked together, Ar may be an aryl radical, such as a phenyl, naphthyl or anthryl radical, where appropriate substituted, in particular with radicals as defined previously and R ₁ may be such that previously defined. Such compounds are described in more detail in US 2004/0077018, the contents of which are incorporated herein by reference.

According to another embodiment, R ₂ and R ₃ are linked together to form an adamantyl radical spiro carbon dioxetane ring which carries them, optionally substituted, in particular by the radicals R ₈ and R ₉ defined below . According to another embodiment, R ₁ and R ₂ may be linked together to form a ring fused with the dioxetane ring, and optionally comprising one or more heteroatoms selected from O, N, and S. Advantageously, R ₁ and R ₂ form an oxacyclopentane condensed with dioxetane. Such compounds are in particular described in application EP 1 342 724, the content of which is also incorporated herein by reference. In another embodiment, R ₁ and R ₂ may be linked together to form a polycyclic ring fused with dioxetane. Such compounds are described in JP 2004-262817, the contents of which are incorporated herein by reference. According to a particular variant embodiment, the chromophore A is represented by the general formula (IIb):

in which :

O symbolizes the covalent bond with B, and

R ₁ is as defined above. Advantageously, R ₁ may be chosen from an alkyl radical, a fluorescent group and an alkyl radical substituted by a fluorescent group. The alkyl radical is as defined above. Advantageously, R ₁ may be a linear, branched or cyclic, saturated or unsaturated C ₁ -C ₆ alkyl or alkoxy radical, optionally comprising one or more heteroatoms chosen from O, N and S, and optionally substituted. R ₁ may be substituted with radicals as defined above. Advantageously, R ₁ is a C ₁ -C ₃ alkyl or alkoxy radical, preferably a methyl or methoxy radical.

R ₈ and R ₉ are, independently of one another, a hydrogen atom or an electroactive radical,

X represents O, N or S. Advantageously X represents O, and Ar represents an arylene radical, as defined previously, or a radical of general formula (IVa):

in which Δ symbolizes the covalent bond with X and symbolizes the covalent bond with the dioxetane ring. Δ and * can be arranged in ortho, meta or para. Advantageously Δ and * may be arranged in meta,

R ₁₀ represents a hydrogen atom, an electroactive radical or a fluorescent radical. Advantageously, the electroactive radical may be able to modify the fluorescent properties of the radical Ar in the image, for example, of an alkyl-, dialkyl- or trialkyl-silyl radical, a radical NO ₂ , an anthryl radical or a naphthyl radical. (see for example MATSUMOTO et al., Tetrahedron Lett, 2002, 43: 8955-58).

R ₁₁ is chosen from a hydrogen atom or an aryl radical, for example C ₆ -C ₁₀ , condensed with the phenylene radical, in particular a phenyl radical to form a naphthylene radical.

For the purposes of the present invention, the term "electroactive group" is intended to mean an electroacceptor or electron donor group According to one particular variant, the chromophore A is such that at least one of the groups R _8, R ₉ or R ₁₀ is an electroactive radical promoting the solubilization of the compound of general formula (I) according to the invention in an aqueous solution.

By way of example of groups promoting the solubilization of the compound of general formula (I) in an aqueous solution, mention may be made of ammonium, phosphonium, sulphonium, carboxylic acid, sulphonic acid, trifluoromethylsulfonyl, methylsulfonyl, cyano and hydroxy.

According to another embodiment, R ₈ and R ₉ are, independently of one another, an electroactive radical chosen from a hydroxyl radical, a halogen atom, a cyano radical, an amide radical or an alkoxy radical, a halogenated alkyl radical, a phenyl radical, an alkoxyphenyl radical.

According to another alternative embodiment, R ₁₀ is a hydrogen atom or an electroactive radical advantageously chosen from a halogen atom, especially a chlorine, a fluorine, a cyano radical, a nitro radical or a mono or disubstituted amide radical. an alkyl radical, an alkoxy radical, a trialkylammonium radical, an alkylamido radical, an alkylcarbamoyl radical, an ester radical, an alkylsulphonamido radical, a trifluroromethyl radical, an alkyl-, dialkyl- or trialkylsilyl radical, an alkyl-, dialkyl- or trialkylsiloxy, an alkylamidosulfonyl radical, an alkylsulfonyl radical, a radical a alkylthioether, a haloalkyl radical, a C ₆ -C ₁₀ radical chosen from an aryloxy radical, an arylamido radical, an arylcarbamoyl radical, an arylsulphonamido radical, an aryl radical, a heteroaryl radical, a triaryl- or alkylaryl-silyl radical, a triarylsiloxy radical, an arylamidosulfonyl radical, an arylsulfonyl radical and an arylthioether radical.

According to a particular embodiment, R ₈ , R ₉ and R ₁₀ are advantageously a hydrogen atom.

According to another particular embodiment, R ₁ or R ₁₀ may be a fluorescent radical in order to modify, by intramolecular FRET, the emission wavelength of the luminescent compound according to the invention.

For example, these fluorescent radicals may be chosen, in a non-limiting manner, from phenyl and its derivatives, naphthalene and its derivatives such as 5-dimethylaminonaphthalene-1-sulfonic acid and hydroxynaphthalenes, acrrhrenes and its derivatives such as 9,10-diphenylanthracene, 9-methylanthracene, pyrene and its derivatives such as N- (1-pyrene) iodoacetamide, hydroxypyrenes, biphenyl and its derivatives, acridine and its derivatives such as hydroxyacridines and 9 methylacridine, coumarin and its derivatives such as 7-dialkylamino-4-methylcoumarin, 4-bromomethyl-7-methoxycoumarin, xanthene and its derivatives, phthalocyanine and its derivatives, stilbene and its derivatives such as 6, 6'-dibromostilbene and hydroxystilbenes, furan and its derivatives, oxazole and its derivatives, oxadiazole and its derivatives, nitrobenzoxadiazole and its derivatives such as hydroxynitrobenzoxadiazoles, benzothiazole and its derivatives, fluorescein and its derivatives such as 5-iodoacetamidofluorescein, fluorescein-5-maleimide, rhodamine and its derivatives such as tetramethylrhodamine, tetraethylrhodamine, rhodols, BODIPY and its derivatives, eosin and its derivatives, erythrosine and its derivatives such as hydroxyerythrosines, 5-iodocetamidoerythrosine, resorufin and its derivatives such as hydroxyrescufines, quinoline and its derivatives such as 6-hydroxyquinoline and 6-aminoquinoline, carbazole and its derivatives such as N-methylcarbazole, fluorescent cyanines and derivatives such as hydroxycyanines, carbocyanine and its derivatives such as phenylcarbocyanine, pyridinium salts and derivatives such as 4 (4-dialkyldiamidostyryl) -N-methylpyridinium iodate, a fluorescent complex of lanthanides and its derivatives, fluorescent proteins, such as Green Fluorescent Protein (GFP) and its mutants or quantum dots.

Advantageously, R ₁ and R ₁₀ can advantageously be chosen from molecules whose fluorescence emission wavelength is in the range from 400 to 900 nanometers, in particular ranging from 500 to 900, and more particularly ranging from

600 to 900 nanometers, such as rhodamine and its derivatives, BODIPY and its derivatives, cyanines and derivatives or quantum dots.

According to another particular embodiment, the compounds in accordance with the present invention may be of general formula (Va):

wherein S is a labile structure, and X, Ar, R ₁ , R ₂ , R ₃ , R ₅ and R ₆ are as defined above.

Advantageously, R ₂ and R ₃ are bonded to form an adamantyl radical in spiro with the carbon of the dioxetane ring which carries them, optionally substituted by radicals as defined above, in particular by radicals such as R ₈ and R ₉ . Advantageously, R ₁ , R ₅ and R ₆ are methoxy radicals.

According to another particular embodiment, the compounds in accordance with the present invention may be of general formula (Vb): in which,

S is a labile structure, and Ar, R ₁ , R ₂ , R ₃ , R ₅ , R ₆ , R ₁₀ and R ₁₁ are as defined above.

Advantageously, R ₂ and R ₃ are fused to form an adamantyl radical in spiro with the carbon of the dioxetane ring which carries them, optionally substituted with radicals as defined above, in particular with radicals such as R ₈ and R ₉ .

Advantageously, R ₁ , R ₅ and R ₆ are methoxy radicals.

R ₁₀ and R ₁₁ are advantageously a hydrogen atom, or R ₁₀ and a hydrogen atom and R ₁₁ forms with the phenylene radical a naphthylene radical.

According to another particular embodiment, the compounds in accordance with the present invention may be of general formula (Vc):

wherein S is the labile structure and R ₁ , R ₅ , R ₆ , R ₈ , R ₉ , R ₁₀ and R ₁₁ are as previously defined.

According to another particular embodiment, the compounds in accordance with the present invention may be represented by the general formula (Vc) in which R ₈ = R ₉ = R ₁₀ = R ₁₁ = H and R ₁ = R ₅ = R ₆ = -OCH ₃ .

S labile structure

In the general formula (I) of the compounds according to the invention, S represents a labile structure activatable by a phenomenon or a chemical, physical, or biological entity.

By "physical phenomenon" is meant for the purposes of the present invention, for example, a temperature variation, a pressure variation.

By "chemical phenomenon" is meant for the purpose of the present invention a phenomenon involving a chemical entity modifying the chemical properties of the environment, such as, for example, a pH variation by an increase or decrease in protons, a variation salinity by an increase or decrease in the salt content (s), a variation of the redox potential, an appearance or disappearance of chemical species, for example ions such as F ^" , S ^" , and ions of alkaline earth metals such as Ca ²⁺ . When S represents a labile structure activatable by a chemical phenomenon, S may advantageously be, for example an ammonium, an amide, an amine, an ester, a silylane, a thiol, a carbamate, or a carbonate.

For the purposes of the present invention, the term "biological phenomenon" is intended to denote a phenomenon involving the activity of a biological entity, for example an enzymatic activity, such as, for example, a hydrolysis, a transfer of an amino group, an isomerization.

Thus, during the activation of the compounds according to the invention by a biological phenomenon, the compounds are subjected to the action of a biological entity. By way of illustration of a biological entity capable of activating the compounds according to the invention, mention may be made, in a nonlimiting manner, of enzymes, ribozymes and abzymes. By way of example of enzymes capable of activating the compounds in accordance with the invention, mention may be made of transferases, such as aminotransferases; hydrolases such as esterases (alkaline phosphatase for example), glucosidases, proteases (trypsin, metalloproteinases or caspases, for example); Isomerases When S is a structure activatable by a biological entity, S may advantageously be of general formula (VI):

P-L-D in which:

D symbolizes the covalent link with B, - P is a recognizable substrate by said entity,

L represents an oxygen, a sulfur, a nitrogen unit, a carbonyl or a -CO-Q- function, with Q being O, S or NH.

According to one embodiment, S may represent a labile structure by the action of an enzyme, and especially by the action of a hydrolase.

Advantageously, when S represents a labile structure by action of a protease type hydrolase, L represents -CO-Q, with Q being NH, and P represents a substrate recognized by a protease, such as for example a peptide or pseudopeptide residue. By way of example of proteases that may be suitable for the implementation of the present invention, mention may be made, without limitation, of caspases such as caspase-3, acylaminoacyl peptidase, Faminopeptidase M, penicillinase G acylase, thermolysin, cathepsins B, G, L, metalloproteinases, elastase, subtilisin, plasminogen activator, urokinase. According to one embodiment, when P is a peptide residue, it comprises at least two amino acid residues, in particular at least four, in particular at month six and more particularly at least eight amino acid residues.

These amino acids can be of L or D configuration, natural or synthetic. These peptides can be obtained by chemical synthesis or molecular biology according to the usual methods of those skilled in the art. As an example of a peptide residue suitable for the implementation of the present invention, mention may be made of the tetrapeptide sequence DEVD (amino acid written in a one-letter code) which is selectively hydrolyzed after the last aspartate residue, by the caspase-3.

According to another variant embodiment, S may represent a labile structure by the action of an esterase hydrolase, such as a carboxyesterase, a phosphatase or a lipase.

In this case, L may represent -CO-Q with Q being O, and P representing a carbon residue (of the alkyl chain type such that the PL group constitutes a carboxylic acid residue, in particular a fatty acid residue). recognized by an esterase or glucosidase enzyme or a phosphate group recognized by a phosphatase enzyme.

When P is a phosphate group, it may be of formula (VII)

O

II - PO M + 1 - ^{M +} in which M ⁺ represents a cation such as an alkali metal, such as Na ⁺ or K ⁺ , an ammonium, or an N (R) ₄ ⁺ quaternary ammonium cation in which each R can be an alkyl C ₁ -C ₂ , aralkyl, such as benzyl, or form part of a heterocycle, such as pyridinium. Such structures are advantageously hydrolysed by an alkaline phosphatase.

According to another variant embodiment, S may represent a labile structure by the action of a glucosidase type enzyme. In this case, L may advantageously be an oxygen atom and P represents a substrate recognized by a glucosidase, such as a sugar.

By way of example of a sugar which may be suitable for the implementation of the invention, mention may be made of α-D- or β-D-galactoside, α-D- or β-D-glucoside , α-D- or β-D-mannoside and α-D or β-D-fructofuranoside. According to another embodiment S may represent a labile structure by action of an aminotransferase.

In this case, L may represent a carbonyl and P may represent a substrate recognized by an enzyme such as, for example, and without limitation, an alanine aminotransferase or an aspartate aminotransferase.

TARGETING

According to one embodiment, a compound according to the invention may further comprise, at the level of the labile structure S, a targeting structure D. Thus, a compound may have the following general formula (VII):

D-S-B-A in which:

D is a targeting structure, and S, B and A are as defined above. The targeting structure D, or ligand, is an element capable of recognizing or binding preferentially cells expressing a particular cellular element, such as, for example, and in a nonlimiting manner, a receptor, an enzyme, a structural protein, a glycoprotein, a sugar, a lipid or a phospholipid. Advantageously, the cellular element is a membrane element, preferably having a part of its structure arranged in the extracellular medium.

According to one embodiment, this cellular element may be specific for a physiological or pathological state of the cell or tissue surrounding the cell or organ comprising the cell. Thus, and without limitation, the cellular element may be specific for the cell's growth state, the position in the cell cycle, an inflammatory response of the cell or tissue, an apoptosis, tissue degeneration.

The expression "preferentially recognize or bind" aims to indicate that the targeting structure D has a particular affinity for the cells or tissues under consideration, even if a non-specific or less important binding with other cells or other tissues does not occur. can be totally excluded in vivo or ex vivo. The preferential bond, however, allows targeting of the compounds according to the invention to the sites of interest, reducing the dissemination of the compound according to the invention to tissues and / or cells of lesser interest.

Thus, the targeting structure D can be chosen, for example and without limitation, from a lectin, an antibody or a fragment thereof recognizing an element present on the surface of cells such as for example a protein, a sugar a lipid; a ligand of a cellular receptor such as, for example, a peptide such as neuropeptide Y, a catecholamine such as adrenaline, an antagonist such as, for example, a β-adrenergic receptor antagonist such as propranolol, a growth factor, an acid amine or a derivative thereof, such as, for example, glutamate, a cytokine such as, for example, interleukin, interferon or TNF, a hormone, a vitamin, an apolipoprotein, cholesterol; a ligand capable of interacting with a membrane lipid or phospholipid such as, for example, an annexin; a ligand capable of interacting with a sugar present on the surface of the cells.

The term "cellular receptor" is intended to denote any cellular element capable of transducing information from the outside to the inside of the cell.

It is within the skill of those skilled in the art to determine the nature of the structure D appropriate to the desired targeting, where appropriate by using test methods customarily practiced in the field.

According to one embodiment, the targeting structure D may be a ligand, for example a polypeptide, capable of binding to the surface of cells typically or specifically present in a particular tissue or pathology.

Targeting structure D can also bind to the surface of tumor cells or present in tumor tissue, or on the surface of cells present in inflammatory tissue. The targeting structure D may, for example, bind to the surface of cells involved in an apoptosis process, which expose negatively charged lipids, such as phosphatidylserine, on their surface.

According to an alternative embodiment, the targeting structure D can be a protein or a fragment of a protein such as a peptide or a polypeptide or a pseudopeptide.

Among the proteins capable of binding to the membranes exhibiting negatively charged lipids, mention may be made, in a nonlimiting manner, of the family of Annexins, families of proteins comprising a Cl or C2 domain, such as blood coagulation factors V and VIII, protein families comprising a PH domain or a FYVE domain, or proteins comprising an identical domain or homologous to the domain Β2-glycoproteins-I (βGP-I). These proteins or domains derived from their sequences can be used as a targeting structure in compounds according to the invention.

According to another embodiment, to a targeting structure D may be fixed one or more compounds of general formula (I), in order to increase, in particular, the intensity of the emitted signal. Thus, at least one, especially at least two, in particular at least 3, in particular at least 4, and more particularly at least 5 compounds of formula S-B-A may be fixed per targeting element D.

DETECTION METHOD As mentioned above, the subject of the present invention is also the use, for the purposes of detection and / or quantification of a chemical, physical or biological phenomenon, as specified above, of at least one compliant compound. to the invention, wherein S is a labile structure activatable by said phenomenon.

Advantageously, the compound according to the invention used in this use may be of general formula (Va) or (Vb) as defined above.

In particular, the present invention aims to also provide a method for detecting and / or quantifying a biological entity.

The process according to the invention comprises at least the steps of contacting at least one effective amount of at least one compound according to the present invention, and in particular of general formula (Va) or (Vb) in which S a labile structure activatable by said biological entity to be detected and / or quantified with a presumed medium comprises said entity and the measurement of the generated luminescent signal. The method according to the invention can be conducted in vitro, ex vivo or in vivo.

When placing in the presence of a medium, in which a chemical, physical or biological phenomenon is likely to occur, with an effective amount of at least one compound according to the invention, the entity S of which is an activatable labile structure by said phenomenon, under conditions conducive to the realization of said phenomenon, the activation of the structure S leads to the emission of a luminescent signal.

By detecting the presence or absence of the luminescent signal, it is possible to highlight the presence or absence of said phenomenon and by measuring the intensity of the signal, the magnitude of the phenomenon can also be determined.

According to a particular embodiment, the biological phenomenon to be detected / quantified may be an activity of a biological entity, in particular an enzyme.

In this particular implementation, the labile structure S is a substrate recognized by said enzyme. The action of the enzyme on the substrate S, especially on the L group of the general formula (II), under conditions conducive to the reactivity of the enzyme leads to the emission of a luminescent signal.

Thus, in the context for example of a hydrolase, the hydrolysis of an enzymatically hydrolysable group, in particular represented by L in the general formula (II), under conditions conducive to the reactivity of the enzyme, leads to the emission of a luminescent signal.

By detecting the presence or absence of a light signal, it is possible to demonstrate the presence or absence of the enzyme and by measuring the signal intensity, the concentration and / or the kinetic characteristics can also be determined. .

Advantageously, the medium which may be suitable for carrying out the process according to the present invention may be a biological, synthetic or natural medium such as a sample of biological fluid.

It may be advantageous to use, in vitro or, if appropriate, ex vivo, together with a compound according to the invention, at least one light amplifier capable of increasing the luminescent signal resulting from the degradation of said compound according to the invention. 'invention.

Such compounds are already known. As an example of these enhancers, mention may be made of fluorescein, bovine albumin, human albumin, and quaternary onium polymer salts such as polyvinylbenzyltrimethylammonium chloride (TMQ), polyvinylbenzyltributylammonium (TBQ) chloride (Sapphire-II ™), polyvinylbenzyldimethylammonium chloride (BDMQ) (Sapphire-I ™), polyvinylbenzyltributylphosphonium chloride, polyvinylbenzyltributylsulfonium chloride, poly (benzyldimethylvinylbenzyl) ammonium chloride, a sodium salt, fluorescein sodium (Emerald ™), poly (benzyltributyl) ammonium, and fluorescein sodium salt (Emerald II ™).

When the process according to the invention is carried out in vitro, the detection of the fluorescence signal can be done by any apparatus usually used by those skilled in the art in this field. For example, a sample placed in contact with a compound according to the invention may, optionally after an incubation period, be subjected to an analytical method comprising or not a step of separating the elements making up the sample, for example by chromatography (For example a high performance liquid chromatography, HPLC), and a spectral analysis step (for example by UV spectroscopy) of the different fractions from the separation step. The detection and measurement of the specific spectroscopic signal of a compound according to the invention (for example the height and the surface of a chromatography peak) may be correlated with the presence, and / or the intensity / quantity of the phenomenon. to detect, such as for example the presence, quantity, and / or kinetic activity of an enzyme.

The method and the use according to the present invention can also be advantageously implemented in vivo.

The compounds according to the present invention may be previously administered to a living being, such as for example an animal or a human being, and then the detection of the fluorescence signal can be carried out in vivo according to the means usually used in the field.

Advantageously, the implementation in vivo of a use according to the invention makes it possible to establish images of the tissues or organs likely in which said phenomenon to be detected / quantified is likely to occur. According to a particular embodiment it is possible to establish images of the tissues or organs capable of expressing a biological entity, and in particular an enzyme, to be detected and / or quantified. Thus, according to a particular embodiment, the method according to the invention can be implemented in medical imaging methods.

According to another embodiment, the method according to the invention is an in vivo diagnostic method comprising the detection and / or the quantification of a biological entity, in particular an enzyme, by means of a compound of general formula (Va) or

(Vb) wherein S is a labile structure activatable by said biological entity to be detected and / or quantified.

The method according to the invention can be applied, for example and without limitation, to medical, experimental, clinical or pre-clinical diagnostic targets in humans or in animals such as laboratory animals or animals used. in agriculture, such as rat, mouse, guinea pig, non-human primate, guinea pig, or pig.

According to a particular embodiment, the biological entity capable of being detected and / or quantified by a method according to the invention may be an enzyme, chosen in particular, in a non-limiting manner, from an aminotransferase or a hydrolase, such as esterase, protease or glucosidase.

According to another embodiment, when the enzyme to be detected and / or to be quantified is a protease, it may advantageously be chosen, in a non-limiting manner, from caspases, penicillinases, carboxypeptidases, trypsin, acylaminoacylpeptidase, aminopeptidases, cathepsins, metalloproteinases, elastase, subtilisin, thermolysin, plasminogen activator, urokinase, isomerases.

In the context of the in vivo implementation, the compounds in accordance with the invention may be shaped so as to be suitable for oral, or parenteral, in particular intravenous, intraarterial, intracardiac, intracerebroventricular, and intraperitoneal administration. , intratumoral, or for pulmonary, nasal, ophthalmic and possibly rectal, vaginal or topical administration.

The compounds in accordance with the invention can thus be used in a formulation adapted to the detection method to be carried out and to the chosen administration voice. For example, the compounds according to the invention may be prepared in the form of a tablet, a capsule or an aqueous solution, concentrated or otherwise. This aqueous solution can be advantageously sterile.

According to another embodiment, the compounds in accordance with the invention may be in solid form, for example in powder form, and be prepared in aqueous solution, advantageously sterile, just before their administration.

Thus, according to one of its aspects, the present invention also relates to the use of a compound according to the invention, in particular of general formula (Va) or (Vb) for the manufacture of a pharmaceutical composition intended for use in in vivo diagnostic method, including a medical imaging method.

Thus according to another of its aspects, the present invention also relates to a pharmaceutical composition comprising at least an effective amount of at least one compound according to the present invention, in particular of general formula (Va) or (Vb). Advantageously, the implementation of a compound according to the invention for the preparation of a pharmaceutical composition may be in the form of a derivative, such as a pharmaceutically acceptable salt or ester. Thus, the present invention also relates to the salts and esters of the compounds according to the invention. By way of example of an ester of a compound according to the invention, mention may be made of a succinate, a hemisuccinate, a malate, a tartrate, a glycolate of a compound according to the invention. As an example of a salt of a compound according to the invention, mention may be made of a sulfate, a phosphate, a sodium salt or a calcium salt of a compound according to the invention.

The adjustment of the appropriate effective amounts of the compound (s) according to the invention, to be implemented in a method or a use according to the invention, depends on the phenomenon, or the entity, to be detected / quantified (chemical, physical , biological) and the environment in which the method according to the invention is conducted (in vitro, ex vivo or in vivo).

In vivo, the effective amounts can also be adjusted according to the size, the weight of the individual to whom the process according to the invention is applied, or in which the use according to the invention is implemented, as well as depending on the organ or tissue targeted.

Adjustments can be made by any method usually practiced by those skilled in the art. For the purposes of the present invention, the term "effective amount" means the quantity necessary and sufficient to obtain the desired effect, namely the detection and / or quantification of a chemical, physical or biological phenomenon, or a biological entity. According to another particular embodiment, the present invention relates to a kit for detecting and / or quantifying by generating a luminescent signal of a chemical, physical or biological phenomenon, said kit comprising at least one compound according to the present invention wherein S is a labile structure activatable by said physical, chemical or biological phenomenon to detect and / or quantify.

According to one particular embodiment, the present invention relates to a kit for the detection and / or quantification of a biological entity, in particular an enzyme, comprising a compound according to the present invention, and in particular of general formula (Va). or (Vb), wherein S is a labile structure activatable by said biological entity to be detected and / or quantified.

According to one particular embodiment, the kit according to the present invention may also comprise at least one light amplifier as defined above.

The present invention will be better understood from the following examples.

These examples are presented as illustrations of the invention and should not be construed as limiting the scope of the present invention.

EXPERIMENTAL SECTION

High Performance Liquid Chromatography (HPLC) Separation Several chromatographic systems have been used for analytical experiments and purification steps:

System A: reverse phase HPLC (C 18, HYPERSIL GOLD, 5 μ, 4.6 × 150) in triethylammonium acetate and acetonitrile buffer (100 mm TEAA, pH 7.0) [75% TEAA (2) min), then a linear gradient of 25 to 100% (30 min) of acetonitrile] at a flow rate of 1.0 ml / min. Dual UV detection was performed at 254 and 285 nm. - System B: reverse phase HPLC (Cl 8, Nucelosil, 5 μ, 10 × 250) in a demineralized water and acetonitrile mixture [75% water (5 min), then 25 to 55% linear gradient (15 min and 55-90% (70 min) acetonitrile] at a flow rate of 5.0 ml / min. UV detection was performed at 260 nm. - System C: reverse phase HPLC (C 18, HYPERSIL GOLD, 5 μ, 4.6 x

150) in triethylammonium acetate and acetonitrile buffer (100 mm TEAA, pH 7.0) [90% TEAA (2 min), then 10 to 90% linear gradient (40 min) of acetonitrile] at a rate 1.0 ml / min. Dual UV detection was performed at 254 and 285 nm.

Example 1: Reaction Arm

Synthesis of 4,5-dimethoxy-2 - [(phenylacetamino) methyl] phenylacetic acid (7).

r \

Potassium hydroxide (2 g, 36 mmol), finely pulverized was added to a solution of phenylacetonitrile 13 (2 mL, 17 mmol) in tert-butyl alcohol (20 mL) with stirring. The reaction mixture is refluxed for 45 minutes, then cooled to room temperature and poured into an aqueous solution of sodium chloride.

(50 ml). The product is extracted with chloroform (CHCl ₃ ). The organic phase is dried over MgSO ₄ and concentrated under reduced pressure to give phenylacetamide 12 (2.1 g, 94%) as a white powder. Mp 152 ° C. ¹ H NMR (CDCl ₃ , 300 MHz) δ

7.40-7.10 (m, 5H), 5.88 and 5.39 (br s, NH ₂ ), 3.50 (s, 2H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ

173.8, 134.9, 129.5, 129.1, 127.5, 43.4.

A mixture of phenylacetamide 12 (2 g, 15 mmol) in a solution of potassium carbonate (4%, 2 ml, 0.6 mmol) and an aqueous solution of formaldehyde (37%, 2 ml, 20 mmol) It was refluxed for 15 minutes (until complete dissolution). The mixture was cooled and extracted with CH ₂ Cl ₂ (2 x 30 mL). The combined organic phases were dried over MgSO ₄ and concentrated under reduced pressure to give a crude product (1.35 g), recrystallization from toluene (20 ml) gave N-hydroxymethylphenylacetamide 11 (1.1 g). , 44%) in the form of a powder white. Mp 78 ° C. ¹ H NMR (CDCl ₃ , 300 MHz) δ 7.50-7.20 (m, 5H), 6.48 (br s, NH), 4.69 (t, J = 7.0 Hz, 2H), 3.67 (t, J = 7 Hz, OH ), 3.60 (s, 2H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ 172.9, 134.4, 129.5, 129.0, 127.5, 64.2, 43.6.

A solution of homovanillic acid (2 g, 11 mmol), sulfuric acid (500 ml, 9.4 mmol) in methanol (100 ml) was refluxed for 3 hours. After cooling, a solution of sodium bicarbonate was added to the reaction mixture. The product was extracted with CH2Cl2 (2 x 50 mL). The combined organic layers were dried over MgSO ₄ and concentrated under reduced pressure to give methyl 4-hydroxy-3-methoxy-phenylacetate (2 g, 94%) as a colorless oil. ¹ H NMR (CDCl ₃ , 300 MHz) δ 7.00-6.70 (m, 3H), 5.82 (br s, OH), 3.87 (s, 3H), 3.70 (s, 3H), 3.56 (s, 2H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ 172.6, 146.6, 144.8, 125.7, 122.1, 114.5, 111.8, 55.9, 52.1, 40.8.

Cesium carbonate (6.6 g, 20.2 mmol) and dimethyl sulfate (2 mL, 21 mmol) were added to a solution of methyl 4-hydroxy-3-methoxyphenylacetate (2 g, 10.2 g). mmol) in anhydrous DMF (80 ml). The reaction mixture was heated at 90 ° C for three hours. After cooling, water (20 ml) was added to the mixture and the product was extracted with diethyl ether (2 x 50 ml).

The combined organic phases were washed with aqueous NaCl (20 mL), dried over MgSO ₄ and concentrated under reduced pressure. Chromatography on silica gel using cyclohexane / ethyl acetate (1: 1) as eluent gave methyl 2- (3,4-dimethoxyphenyl) acetate (1.7 g, 80%) as the eluent. form of a colorless oil. ¹ H NMR (CDCl ₃ , 300 MHz) δ 6.80-6.50 (m, 3H), 3.71 (s, 3H), 3.69 (s, 3H), 3.40 (s, 2H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ 172.2, 148.8, 148.0, 126.4, 121.3, 112.3, 111.1, 55.8, 55.7, 51.9, 40.6. A solution of pre-cooled acetic acid (20 ml) and sulfuric acid (730 μl; 14 mmol) was added to a mixture of N-hydroxymethylphenylacetamide 11 and methyl 2- (3,4-dimethoxyphenyl) acetate 8 ( 1.5 g, 7.2 mmol). The suspension was stirred for 2 hours at 0 ° C and then at room temperature for 12 hours. The reaction mixture was neutralized with 5M sodium hydroxide solution at 0 ° C. The product was extracted with ethyl acetate (2 x 30 ml). The combined organic layers were dried over MgSO ₄ and concentrated to give methyl 4,5-dimethoxy-2 - [(phenylacetamino) methyl] phenylacetate 6 (2 g, 78%) as a colorless oil that has crystallized. Mp 118 ° C. ¹ H NMR (CDCl ₃ , 300 MHz) δ 7.40-7.15 (m, 5H), 6.68 (s, 1H), 6.59 (s, 1H), 6.24 (br s, NH), 4.28 (d, J = 5.6 Hz). , 2H), 3.76 (s, 3H), 3.71 (s, 3H), 3.57 (s, 3H), 3.51 (s, 2H), 3.47 (s, 2H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ 172.8, 170.8, 148.4 (2C), 135.0, 129.7 (2C), 129.2, 127.5 (2C), 124.9, 113.8, 113.2, 56.3 (2C), 52.7, 44.2, 41.7 , 38.3. IR (CHCl ₃ ) 3017, 1732, 1660, 1520, 1466, 1275. Anal. Calculated for C ₂₀ H ₂₃ NO ₅ : C, 67.21; H, 6.49; N, 3.92. Measured C, 66.88; H, 6.56; N, 3.97.

An aqueous solution of lithium hydroxide (0.5 M, 56 ml, 28 mmol) was added to a solution of methyl 4,5-dimethoxy-2 - [(phenylacetamino) methyl] phenylacetate 6 (2 g; 6 mmol) in methanol (50 ml). The reaction mixture was stirred at room temperature for 12 hours, then neutralized with an aqueous solution of HCl (2N) and a precipitation of the product was observed. Filtration gave 4,5-dimethoxy-2 - [(phenylacetamino) methyl] phenylacetic acid 7 (1.5 g, 78%) as a white powder. Mp 186 ° C. ¹ H NMR ((CD ₃ ) ₂ SO, 300 MHz) δ 8.40 (br s, 1H), 7.30-7.20 (m, 6H), 6.80 (s, 1H), 6.73 (s, 1H), 4.18 (d, J = 5.6 Hz, 2H), 3.70 (s, 3H), 3.59 (s, 3H), 3.55 (s, 2H), 3.44 (s, 2H). ¹³ C NMR ((CD ₃ ) ₂ SO, 75 MHz) δ 172.8, 170.0, 147.4, 147.3, 136.6, 129.9, 129.0 (2C), 128.3 (2C), 126.4, 125.2, 114.6, 111.9, 55.6, 55.4, 42.5 , 39.6, 37.5. Anal. Calculated for C ₁₉ H ₂₁ NO ₅ : C, 66.46; H, 6.16; N, 4.08. Measured C, 66.52; H, 5.92; N, 4.06.

Example 2: Precursor of dioxetane

Synthesis of 2- [l- (3-hydroxy) phenyl) -l-methoxymethylene] tricyclo [3.3.1.1 ^s' ^7] decane (3a)

Tert-Butyldimethylsilyl chloride (2.7 g, 18 mmol) was added to a solution of methyl 3-hydroxybenzoate 4a (2.3 g, 15 mmol), and imidazole (2.6 g; 5 mmol) in anhydrous DMF (5 ml). The reaction mixture was stirred at room temperature for 14 hours under an argon atmosphere. The mixture was diluted with water (20 ml) and the product was extracted with p-pentane (2 x 20 ml). The phases The combined organic compounds were dried over MgSO ₄ and concentrated under reduced pressure. Chromatography on silica gel using cyclohexane / ethyl acetate as eluent (v / v: 95/5) gave the methyl ester of 3- (tert-butyldimethylsilyloxy) benzoic acid 4b (3.7%). 93%). ¹ H NMR (CDCl _3, 300 MHz) δ 7.42 (d, J = 8 Hz, IH), 7.28 (t, J = 2 Hz, IH), 7.07 (t, J = 8 Hz, IH), 6.81 (dd , J = 8 Hz, 2 Hz, 1H), 3.68 (s, 3H), 0.78 (s, 9H), 0.1 (s, 6H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ 166.9, 155.8, 131.6,

129.4, 124.9, 122.7, 121.1, 52.2, 25.7, 18.2, -4.4.

LiAlH ₄ (800 mg) was slowly added to a suspension of TiCl ₃ (THF) ₃ (16.5 g, 44.6 mmol) in dry THF (15 mL) at 0 ° C. After 10 minutes at 0 ° C, triethylamine (3.5 mL) was added and the mixture was refluxed for one hour. A solution of 2-adamantanone (822 mg, 5.47 mmol) and methyl benzoate ester 4b previously prepared (1.2 g, 444.50 mmol) in anhydrous THF (10 mL) was added dropwise. drop by drop to the mixture. The reaction mixture was refluxed for 3 hours. Water was cautiously added to the mixture at 0 ° C, which was then diluted with diethyl ether. The organic phase was washed with saturated NaCl solution, then dried over MgSO ₄ and concentrated under reduced pressure. Chromatography on silica gel using Cyclohexane / EtOAc as eluent (v / v: 99/1) afforded 2- [1- (3-tert-butyldimethylsilyloxy) phenyl) -1-methoxymethylene] tricyclo [ 3.3.1.1] decane 3b (796 mg, 66%) as a colorless oil. ¹ H NMR (CDCl ₃ , 300 MHz) δ 7.09 (t, J = Hz, 1H), 6.81 (d, J = Hz, 1H), 6.70 (m, 2H), 3.20 (s, 3H), 3.14 (br. s, 1H), 2.54 (br s, 1H), 1.87-1.67 (m, 12H), 0.91 (s, 9H), 0.14 (s, 6H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ 155.5, 143.6, 136.9, 131.2, 129.0, 122.6, 121.2,

119.5, 57.6, 39.3, 39.2, 38.9, 37.4, 32.9, 30.5, 25.8, 18.3, -4.70.

A solution of W-Bu ₄ NF in THF (2.1 mL, 2.1 mmol, 1.0M) was added to a solution of 2- [1- (3-tert-butyldimethylsilyloxy) phenyl) -1-methoxymethylene ] tricyclo [3.3.1.1 ³ ' ⁷ ] decane 3b (740 mg, 1.91 mmol) in dry THF

(12 ml). The reaction mixture was stirred for 15 minutes at room temperature. The mixture was diluted in diethyl ether (30 ml) and washed with saturated NaCl solution.

The organic phase was dried over MgSO ₄ and concentrated under reduced pressure to give a crude product of 2- [1- (3-hydroxy) phenyl) -1-methoxymethylene] tricyclo [3.3.1.1 ³ ' ⁷ ] decane 3a under form of a yellow oil. ¹ H NMR (CDCl ₃ , 300 MHz) δ 7.19-6.67 (m, 4H), 3.67 (s, 3H), 3.30 (br s, 1H), 3.1 (br s, 1H), 2.50 (br s, 2H), 1.87- 1.64 (m, 10H).

Example 3 Dioxetane

Synthesis of the chiomoluminescent derivative according to the invention (1)

2- [1- (3-hydroxy) phenyl) -1-methoxymethylene] tricyclo [3.3.1.1 ³ ' ⁷ ] decane 3a (520 mg), triethylamine (800 μl) and benzotriazol-1-yloxytris hexafluorophosphate. (Dimethylamino) phosphonium (BOP) (950 mg) was added to a solution of 4,5-dimethoxy-2 - [(phenylacetamino) methyl] phenylacetic acid (656 mg, 1.91 mmol) in anhydrous DMF ( 10 ml). The reaction mixture was stirred at room temperature for 6 hours. The mixture was concentrated in vacuo. Chromatography on silica gel using cyclohexane / EtOAc (v / v: 40/60) as eluent gave the enol ester 3- (adamantan-2-ylidene-methoxy-methyl) -phenyl ether of the [4,5-dimethoxy-2- (phenylacetylamino-methyl) -phenyl] -acetic acid 2 (606 mg, 53%) as a white powder. Mp 95 ° C. ¹ H NMR (CDCl ₃ , 300 MHz) δ 7.21 (t, J = 7.9 Hz, 1H), 7.15-7.00 (m, 6H), 6.91 (t, J = 2.3Hz, 1H), 6.84 (ddd, J = 7.9 Hz, 2.3 Hz, 0.8 Hz, 1H), 6.80 (s, 1H), 6.77 (s, 1H), 4.44 (d, J = 5.3 Hz, 2H), 3.88 (s, 3H), 3.85 (s, 2H). ), 3.81 (s, 3H), 3.53 (s, 2H), 3.30 (s, 3H), 3.25 (br s, 1H), 2.66 (br s, 1H), 1.90-1.55 (m, 12H). ¹³ C NMR (CDCl ₃ , 75 MHz) δ 150.6, 148.6, 148.5, 142.6, 137.3, 134.9, 132.9, 129.4, 129.1, 128.9, 127.3, 127.1, 124.1, 122.1, 120.4, 113.5, 112.9, 58.0, 56.1, 56.0 , 43.9, 41.4, 39.3, 39.1, 38.3, 37.2, 32.2, 30.3, 28.3. MS (MALDI-TOF, positive mode) m / z 634.27 [M + K] ⁺ , 618.30 [M + Na] ⁺ . Anal. Calculated for C ₃₇ H _4! No. ₆ : C, 74.60; H, 6.94; N, 2.35. Measured: C, 74.25; H, 7.09; N, 2.41. Ozone was bubbled through a cooled solution (-78 ° C.) of freshly distilled triphenyl phosphite (37 μl, 0.14 mmol) in 9 ml of anhydrous CH 2 Cl 2 until a slight blue color appeared. The reaction medium was then purged with argon until the blue color disappeared. The previously synthesized enol ether 2 (21 mg, 0.035 mmol) dissolved in 1 ml of anhydrous CH 2 Cl 2 was added at -78 ° C. The resulting reaction mixture was stirred at -30 ° C for 1.5 hours and allowed to warm to room temperature for 2 hours. After a control HPLC (System A), the reaction mixture was evaporated to dryness. The oily residue obtained was purified by semi-preparative HPLC (system B). The fractions containing the product (recovery time TR 48-50 min) were partially evaporated and then lyophilized to give dioxetane 1 as a white powder (about 1 mg, 5% yield). HPLC (System C) TR 23.8 min. UV (recorded during HPLC analysis) Xm _8x 235 and 281 nm. MS (ESI, positive mode m / z 650.0 (M + Na) ⁺ (calculated molecular weight: 627.74 for C ₃₇ H ₄₁ NO ₈ ).

Claims

1. Compound of general formula (I):

S-B-A wherein - S represents a labile structure activatable by a chemical or biological entity,

B is a reactive arm, whose chemical structure is such that the activation of the labile structure S induces its intramolecular rearrangement in a form suitable for the release of a molecule A ^" , - A is a chromophore of general formula (IIa) :

0-0

RR ₂

0 - X-Ar R ₃

in which - 0 symbolizes the covalent bond with B, a) R ₁ , R ₂ and R ₃ represent, independently of one another, a hydrogen atom, a hydroxyl radical, an amino radical, a thio radical, a halogen atom, a C ₁ -C ₂₀ , especially C ₂ -C ₁₂ , and in particular C ₃ -C ₆ hydrocarbon-based chain chosen from an alkyl radical, an alkoxy radical, a heteroalkyl radical or a cycloalkyl radical; a heterocycloalkyl radical, an ether radical, an alkenyl radical, an alkynyl radical, an alkylamino radical, a dialkylamino radical, an alkyl-, dialkyl- or trialkyl-silyl radical, an alkyl-, dialkyl- or trialkyl-siloxy radical, a radical alkylthio, a haloalkyl radical, a C ₆ -C ₃₀ , especially C ₆ -C ₁₀ , aromatic ring chosen from an aryl radical, a heteroaryl radical, an aralkyl radical, where appropriate substituted, b) R ₂ and R ₃ may be together for an oxo motif, c) R ₁ and R ₂ may be linked to form a C ₄ -C ₈ ring fused with the dioxetane ring, if necessary interrupted by one or more heteroatoms chosen from O, N and S, and which may be substituted by one or more radicals as defined above in a) for R ₁ , R ₂ and R ₃ , d) R ₂ and R ₃ or R ₁ and Ar can be linked so as to form a C ₄ -C ₂₀ spiro ring, in particular C ₆ -C ₁₀ , with the carbon of the dioxetane entity which carries them; ring that may be monocyclic or polycyclic, saturated or unsaturated, aromatic, condensed or not, and where appropriate incorporate one or more heteroatoms selected from O, N and S and may be optionally substituted with one or more substituents as defined above in a) for R ₁ , R ₂ and R ₃ ,

Ar represents a C ₆ -C ₃₀ arylene radical, in particular C ₆ -C ₁₀ arylene, and being optionally substituted with one or more substituents chosen in particular from a hydroxyl radical, a halogen atom, an oxo unit, an amino radical, an alkylamino radical, a dialkylamino radical, a thio radical, an alkyl-, dialkyl- or trialkylsilyl radical, an alkyl-, dialkyl- or trialkylsiloxy radical, an alkyl radical, an alkoxy radical, an alkylthio radical, a carboxy radical or a radical; formyl, an alkyl ester radical, an alkyl ketone radical, a haloalkyl radical, an aryl radical, an arylester radical, an arylketone radical, an arylamino radical, a diarylamino radical, and - X represents O, NH or S, and their derivatives.

2. Compound according to claim 1, characterized in that R ₁ is a fluorescent radical.

3. Compound according to claim 1 or 2, in which B is represented by a C ₁ -C ₈ , especially C ₂ -C ₆ , and especially C ₃ -C ₄ , alkyl radical, optionally interrupted by an arylene radical. C ₆ -C ₁₄ , in particular C ₆ -C ₁₀ and / or by one or more heteroatoms chosen from O, N, and S, and optionally substituted with one or more substituents chosen from a hydroxyl radical, an oxo unit, a radical amino, a halogen atom, an alkyl radical, an alkoxy radical, a carboxy radical, an alkylamino radical, a dialkylamino radical, an aryl radical and an aryloxy radical.

A compound according to any one of the preceding claims wherein B is represented by the general formula (III):

in which :

D and 0 symbolize, respectively, the covalent bond with the labile structure S and the chromophore A, j is an integer from 0 to 2, and advantageously is equal to 1,

R ₄ is a C ₁ -C ₂₀ hydrocarbon-based chain, in particular C ₂ -C ₁₂ and in particular C ₃ -C ₆ , where appropriate interrupted by one or more heteroatoms and / or CO-and / or -CO motif (s); N (alkyl) -, placed in ortho, meta or para of the radical D- (CH ₂ ) J-, and in particular chosen from radicals - (CH ₂ ) _k -O, - (CH ₂ ) _k -C (O) -O, -CH ₂ -OC (O) -N (CH ₃ ) - (CH ₂ ) ₂ -N (CH ₃ ) -C (O) -O, where k is 1 or 2, and advantageously equal to 1

R ₅ and R _e are, independently of one another, a hydrogen atom or a linear, branched or cyclic, saturated or unsaturated C ₁ -C ₂₀ hydrocarbon-based chain, in particular C ₂ -C _12, and in particular C ₃ -C ₆ chosen from an alkyl radical, an alkoxy radical, an alkyl- or dialkylamino radical, an alkylthio radical, or an aromatic C ₆ -C ₃₀ , especially C ₆ -C ₁₀ ring chosen from an aryl radical; an aryloxy radical, an arylamino radical and an arylthio radical.

5. A compound according to any one of the preceding claims wherein Ar represents an arylene radical of general formula (IVa):

in which : Δ symbolizes the covalent bond with X and * symbolizes the covalent bond with the dioxetane ring,

R ₁₀ represents a hydrogen atom, an electroactive radical, or a fluorescent radical, and R ₁₁ is chosen from a hydrogen atom or a C ₆ aryl radical;

C ₁₀ , condensed with the phenylene radical.

6. Compound according to any one of the preceding claims wherein R ₁ is a linear or branched or cyclic, saturated or unsaturated C ₁ -C ₆ alkyl or alkoxy radical, optionally comprising one or more atoms chosen from O, N and S and optionally substituted.

7. A compound according to any one of the preceding claims wherein when R ₁ and / or R ₁₀ are a fluorescent radical, they are chosen from phenyl and its derivatives, naphthalene and its derivatives, anthracene and its derivatives, the pyrene and its derivatives, biphenyl and its derivatives, acridine and its derivatives, coumarin and its derivatives, xanthene and its derivatives, phthalocyanine and its derivatives, stilbene and its derivatives, furan and its derivatives, oxazole and its derivatives, oxadiazole and its derivatives, nitrobenzoxadiazole and its derivatives, benzothiazole and its derivatives, fluorescein and its derivatives, rhodamine and its derivatives, BODIPY and its derivatives, eosin and its derivatives, Erythrosine and its derivatives, resorufin and its derivatives, quinoline and its derivatives, carbazole and its derivatives, fluorescent cyanines and derivatives, carbocyanine and its derivatives, pyridinium salts and derivatives, a Fluorescent mplex of lanthanides and its derivatives, fluorescent proteins and quantum dots.

8. A compound according to any one of claims 4 to 7 wherein R ₄ is represented by the radical - (CH ₂ ) Ic-O positioned ortho with the radical D- (CH ₂ ) J-, and j = k = l and R ₅ = R ₆ = -O-CH ₃ .

9. A compound according to any one of claims 5 to 8 wherein R ₁₀ is an electroactive radical chosen from a halogen atom, a cyano radical, a nitro radical, a mono or disubstituted amide radical, an alkyl radical or a radical. alkoxy, a trialkylammonium radical, an alkylamido radical, an alkylcarbamoyl radical, an ester radical, an alkylsulphonamido radical, a trifluroromethyl radical, an alkyl-, dialkyl- or trialkyl-silyl radical, an alkyl-, dialkyl- or trialkyl-siloxy radical, an alkylamidosulfonyl radical, an alkylsulfonyl radical, a radical an alkylthioether, a haloalkyl radical, a C ₆ -C ₁₀ radical chosen from an aryloxy radical, an arylamido radical, an arylcarbamoyl radical, an arylsulphonamido radical, an aryl radical, a heteroaryl radical, a triaryl or alkylarylsilyl radical, a triarylsiloxy radical, an arylamidosulfonyl radical or an arylsulphonyl radical; and an arylthioether radical.

A compound according to any one of the preceding claims wherein X is O.

11. Compound according to any one of the preceding claims, characterized in that it corresponds to the general formula (Va):

in which :

S, X, Ar, R ₁ , R ₂ , R ₃ , R ₅ , and R ₆ are as defined in any one of the preceding claims.

12. Compound according to any one of the preceding claims of general formula (Vb):

wherein S, R ₁ , R ₂ , R ₃ , R ₅ , R ₆ , Rio and Rn are as defined in any one of the preceding claims.

13. Compound according to the preceding claim wherein Ri ₀ = Rn = H, R ₁ = R ₅ = R ₆ = -OCH ₃ , and R ₂ and R ₃ are bonded to form an adamantyl radical in spiro with the carbon of the dioxetane ring who wear them, if necessary substituted.

A compound according to any one of the preceding claims, wherein when S is a structure activatable by a biological entity, S is of general formula (VI):

P-L-D in which:

D symbolizes the covalent link with B, P represents a substrate recognizable by said entity,

L represents oxygen, sulfur, nitrogen, carbonyl or -CO-Q-, with Q being O, S or NH.

15. Compound according to the preceding claim wherein S represents a labile structure by the action of an enzyme.

16. A compound according to the preceding claim wherein when S represents a labile structure by action of a protease, L represents -CO-Q, with Q being NH, and P represents a substrate recognized by said protease.

17. Compound according to any one of the preceding claims, characterized in that it is a derivative of general formula (VII):

D-S-B-A in which

D is a targeting structure, and S, B and A are as defined in the preceding claims.

18. A compound according to the preceding claim wherein the targeting structure D can be selected from a lectin, an antibody or a fragment thereof; a ligand of a cellular receptor; a ligand capable of interacting with a membrane lipid or phospholipid; a ligand capable of interacting with a sugar present on the surface of the cells.

19. A pharmaceutical composition comprising at least one effective amount of at least one compound as defined in any one of claims 1 to 18.

20. Use for the purpose of detecting and / or quantifying a physical, chemical or biological phenomenon of at least one compound according to any one of claims 1 to 18 wherein S is a labile structure activatable by said phenomenon.

21. A method for detecting and / or quantifying a biological entity comprising at least the steps of: bringing into contact at least one effective amount of at least one compound as defined according to any one of claims 1 to 18 wherein S is a labile structure activatable by said biological entity to detect and / or quantify with a medium presumed to include said entity, and measure the generated luminescent signal.

22. Method according to the preceding claim, characterized in that said compound is as defined in claims 11 to 18.

23. The method of claim 21 or 22 carried out in vitro comprising the step of adding a light amplifier selected from fluorescein, bovine albumin, human albumin, quaternary onium polymer salts such as chloride of polyvinylbenzyltrimethylammonium (TMQ), polyvinylbenzyltributylammonium chloride (TBQ), polyvinylbenzyldimethylammonium chloride (BDMQ), polyvinylbenzyltributylphosphonium chloride, polyvinylbenzyltributylsulfonium chloride, poly (benzyldimethylvinylbenzyl) ammonium chloride, a sodium salt of fluorescein, poly (benzyltributyl) ammonium and the fluorescein sodium salt.

24. A method according to any one of claims 21 to 23 wherein said biological entity to be detected and / or quantified is an enzyme and in that S of the compound of general formula (I) is a substrate activatable by said enzyme.

25. Kit for detection and / or quantification by detection of a biological entity comprising at least one compound as defined in any one of claims 1 to 18 wherein S is a labile structure activatable by said biological entity to be detected. and / or quantify.

26. Use of a compound as defined in any one of claims 1 to 18 for the manufacture of a pharmaceutical composition for the implementation of an in vivo diagnostic method.