Fluorescent beta-lactamase substrates and related detection methods

Abstract

The present invention relates to probes for the detection of β-lactamase-type enzyme activity. In particular, the present invention relates to novel fluorogenic substrates for detecting the presence of catalytically active β-lactamases and detection methods using such substrates.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to probes for the detection of β-lactamase-type enzyme activity. In particular, the present invention relates to novel fluorescent substrates for detecting the presence of catalytically active β-lactamases and detection methods using such substrates. [Background technology] Beta-lactam antibiotics are a well-known class of antibiotics that contain a beta-lactam ring in their structure. However, bacteria tend to develop resistance to these antibiotics by producing beta-lactamases, enzymes that promote the hydrolysis of the antibiotic's beta-lactam ring, rendering the drug ineffective. Bacterial resistance has become a global problem. Among antibiotic-resistant bacteria, those that develop resistance to carbapenems are currently considered untreatable. In fact, in hospitals, patients carrying these bacteria have a 50% chance of dying from this infection.

[0002] Therefore, in such situations, it is useful to detect β-lactamase activity, particularly carbapenemase activity, to identify bacteria that are resistant to β-lactam antibiotics.

[0003] Detecting this activity by capturing the fluorescence emitted by the probe is a much more sensitive method than collecting white light residue during simple absorption by the probe; i.e., the detection threshold is much lower. Detecting fluorescence emission is very easy to implement, and as a result, fluorescent molecules are very attractive tools for life science. For example, a class of fluorophores that provide intramolecular proton transfer in the excited state, called ESIPT ("excited-state intramolecular proton transfer"), is described, inter alia, in a) Ormson, S. M., et al. Progress in Reaction Kinetics (1994) 19, 45-91; b) Legourrierec, D., et al. Progress in Reaction Kinetics (1994), 19, 211-275; and c) Zhao, I., Ji, S., Chen, Y., Guo, H., & Yang, P. (2012). Excited-State Intramolecular Proton Transfer (ESIPT): From Photophysical Principles to the Development of New Chromophores and Applications in Fluorescent Molecular Probes and Luminescent Materials. Physical Chemistry Chemical Physics, 14 (25), 8803. The first interpretation of the increased fluorescence found in certain phenolic compounds as an ESIPT phenomenon can be attributed to Weller (for methyl salicylate: Weller, A. (1961). Fast Reactions of Excited Molecules. Progress in Reaction Kinetics and Mechanism 1, 187) and Heller and Williams (for hydroxyphenylbenzoxazole: Heller A., ​​et Williams, DL, 1. Phys. Chem. (1970) 74, 4473-4480).

[0004] The ESIPT fluorophore class is particularly attractive to life science researchers due to its superior properties compared to traditional fluorophores. These exceptional properties of ESIPT fluorophores include: (a) The large Stokes shift often exceeds 130 nm and can reach values ​​of 250 nm, which allows for an instrument selection that maximizes the sensitivity of detection; this is by escaping / ignoring autofluorescence due to cellular and tissue components; (b) the ability to engineer fluorophores that emit brilliant fluorescence in the solid state (a rare property among all known fluorophores). This last property allows for the generation of a high-intensity signal at the activation site with minimal dilution caused by diffusion; (c) the ability to design ESIPT fluorophores that emit in the red or near-infrared (600–850 nm) wavelengths, where tissue transparency is greatest; probes using such fluorophores are also particularly suitable for imaging in living animals; and finally, (d) The ability to design non-fluorescent substrates by replacing the hydrogen atom of the hydroxyl of the ESIPT fluorophore with a substituent that has specific reactivity with respect to a chemical or biochemical analyte, and whose cleavage causes the appearance of fluorescence.

[0005] The sensitivity level of methods for detecting enzymatic activity using substrates that result in the production of fluorescence is closely related to (i) the rate of photobleaching, (ii) the extent of accumulation of the fluorescent signal at the site of its production (hence the rate of diffusion from that site and the question of whether the fluorophore precipitates), (iii) the actual extinguishing / lighting mode in which the substrate functions (the absence of background fluorescence from untransformed substrate), and (iv) the degree of excitation and emission spectral stacking (their separation at baseline is the most favorable configuration; see point a) above). Point (iv) is of particular importance because complete separation at baseline offers the opportunity for a very wide choice of filters for the light source (to excite the fluorophore at all possible wavelengths), but even more importantly, for the detector (to collect photons from all wavelengths emitted by the fluorophore). Point (iv) also minimizes interference of the detection method with tissue autofluorescence (characterized by a weak Stokes shift of the native fluorophore), a frequent problem encountered with established fluorophores, most of which themselves exhibit a weak Stokes shift.

[0006] Among the important classes of ESIPT fluorophores, dichloro-HPQ (6-chloro-2-(5-chloro-2-hydroxyphenyl)-4(3H)-quinazolinone; CAS number: 28683-92-3) is of particular interest considering that it is completely insoluble in aqueous / physiological media while being highly fluorescent in the solid state, and furthermore only in the solid state and not in solution.

[0007] Several probes have been developed to detect β-lactamases. However, these probes are either very expensive or do not guarantee a high level of fluorescent signal accumulation at the site of production. In fact, genotypic detection (PCR of β-lactamase messenger RNA) seems to be the current diagnostic industry's preferred method for detecting bacterial resistance, but it should be criticized due to its high cost and long analysis time.

[0008] In such situations, it would be useful to provide improved probes capable of detecting β-lactamase activity, and in particular carbapenemase activity.

[0009] One of the aims of the present invention is to propose novel β-lactamase substrates that are stable in aqueous media, remain non-fluorescent or only mildly fluorescent at wavelengths significantly different from the wavelength at which the released fluorophore is itself fluorescent, but react rapidly with β-lactamase to fragment into tracer molecules, including small fluorescent molecules.

[0010] [overview] The present invention primarily relates to compounds of formula (I).

[0011] [ka]

[0012] where: -W is -O- or -NR 13 - and R 13 is C1-C4 alkyl or a hydrogen atom; -R1 is such that HWR1 obtained after cleavage of the -C(O)-WR1 bond present in formula (I) belongs to the class of fluorophores, preferably the class of fluorophores that bring about intramolecular proton transfer in the excited state; R2, R3 and R4 are defined as: o R2 is C1-C4 alkyl, R3 is C1-C4 alkyl or a hydrogen atom, and R4 is C1-C4 alkyl; Or, R3 is C1-C4 alkyl or a hydrogen atom, and R2 and R4 are bonded to each other and form -(CH2) in the direction from R2 to R4. p -Y q -(CH2) r -Forming chains, ● Y is O, NR 14 , N(R 14 )2 + or S, preferably NR 14 or N(R 14 )2 + and ● p=0, 1, 2, 3, 4 or 5, ● q=0 or 1, ● r=0, 1, 2, 3, 4 or 5, ● p+q+r=3, 4, 5, or 6, ● Each R 14 are independently a hydrogen atom, a C1-C6 alkyl, an amino protecting group, or -(L) q represents -GP, where q is equal to 0 or 1, L is a linking arm, and GP is a hydrophilic group; or R2 is C1-C4 alkyl, and R3 and R4 are joined together to form an aliphatic carbocyclic ring together with the carbon atoms to which they are attached; R5 and R6 are the same or different and each independently represent a hydrogen atom, a C1-C4 alkyl, or a C5-C 10 represents aryl; -R7 is a hydrogen atom or a group selected from C1-C4 alkyl and C1-C4 alkoxy; -R8 represents a hydrogen atom; -V represents an oxygen atom or a sulfur atom; -n is 0 or 1; -Z is -S-, -SO-, or -CR9R 10 - and R9 and R 10 are the same or different and each independently represent a hydrogen atom or a C1-C4 alkyl; -Q is H, a cation, or R16, where R16 is C1-C6 alkyl, optionally substituted with aryl or O—(CO)—R, and R is independently selected from H, C1-C6 alkyl, and C3-C6 cycloalkyl; -R 11 is an organyl group, an organylamino group, an organyloxy group, or an organylthio group, -X is a bond, or

[0013] [ka]

[0014] represents a group selected from

[0015] Applicants have developed a family of β-lactamase substrates with the following properties: - Rapid response to the activity of extended-spectrum β-lactamase (ESBL) members or carbapenemase activity by generating strong fluorescence; - release of a fluorophore, preferably an ESIPT fluorophore, containing the "ELF97 alcohol" upon enzymatic conversion, which has been recognized for its unique property of signal retention at the enzyme active site; and - In the absence of target enzyme activity, they are largely or completely non-fluorescent, thus maximizing detection sensitivity through favorable signal-to-background ratios.

[0016] Thus, the compound (I) according to the invention reveals the presence of β-lactamase activity by the generation of fluorescence.

[0017] More specifically, the probe is invisible before encountering the target β-lactamase enzyme and may therefore be called a "stealth probe." However, when it is chemically modified by the enzyme (hydrolytic opening of the β-lactam ring), it fragments through a cascade reaction, releasing a detectable fluorophore. The probe contains three molecular components: i) a self-insoluble spacer; ii) a cephalosporin or carbapenem group at one end that serves as a substrate for the target enzyme; and iii) a WR1 group at the other end that belongs to the fluorophore class when released as HWR1 upon fragmentation.

[0018] The spacer choice in this invention offers two substantial advantages over the corresponding molecular probes: (a) its chemical bond with WR1 is stable against spontaneous hydrolysis and therefore stable against fluorophore release and false-positive signal generation, and (b) its chemical bond with the cephalosporin or carbapenem unit is of carbamate nature, which not only ensures hydrolytic stability but also the small size of the bond is crucial, as it ensures sufficient molecular recognition by the enzyme and therefore an efficient turnover rate.

[0019] Two ways of pre-organizing the spacer for cyclization consist of introducing two alkyl substituents on the α-carbon of the -NC(V)-O- group (or forming a carbocyclic ring) or including a bond between the nitrogen group -NC(V)-O- and its α-carbon in the heterocyclyl ring, facilitating the fragmentation process.

[0020] The present invention therefore relates to compounds of formula (I) for the detection of β-lactamase or carbapenemase activity in in vitro diagnostic tests, including live cell assays (bacteria), with or without the implementation of the variants described in this patent application. The compounds of formula (I) according to the invention can also be used for the in vivo detection of β-lactamases in animals. The present invention therefore also relates to compounds of formula (I) according to the invention for the in vivo detection of β-lactamases in humans.

[0021] More particularly, the present invention relates to a method for detecting the presence of β-lactamase activity in vitro or ex vivo, comprising the steps of: - placing the sample to be analyzed in contact with compound (I); applying appropriate conditions to cleave the covalent bond between -C(=V) and NR7, followed by cleavage of -C(O)-WR1, allowing the formation of a fluorescent precipitate by binding and resulting in the release of HWR1; - quantitative or qualitative analysis of fluorescent precipitates, -Correlation of quantitative or qualitative analysis of fluorescent precipitate with the presence or absence of β-lactamase in the sample.

[0022] The precipitates obtainable using compounds of formula (I) according to the present invention by cleaving the covalent bond between C(=V) and NR7, followed by cleavage of -C(O)-WR1, resulting in the release of HWR1 after cyclization of the spacer, are strongly fluorescent, whereas the corresponding compounds of formula (I) are only mildly or not at all fluorescent. The compounds according to the present invention are β-lactamase substrates that operate in an off / on mode, thus enabling probing of this enzyme activity without the need to wash away excess probe before readout ("no-wash assay"). Thus, in the absence of the enzyme (off mode), the compounds according to the present invention are not or only mildly fluorescent, whereas in the presence of the β-lactamase enzyme, the compounds are fragmented, releasing a detectable fluorophore (on mode).

[0023] In particular, the detection method according to the invention can be carried out under physiological conditions, in particular in an aqueous medium buffered to pH 7.4.

[0024] The present invention also relates to compounds of formula (II) which are intermediates in the synthesis of compounds of formula (I):

[0025] [ka]

[0026] where: -R2, R3, R4, R5, R6, R7, R8, R 11 , Z, n, Q, X, and V are as defined for compound (I); -R 12 represents a hydrogen atom or an amine functional protecting group.

[0027] The present invention also relates to a method for preparing compound (I), which comprises the steps of: - the implementation of compound (II) as defined herein - Implementation of the compound of formula (III)

[0028] [ka]

[0029] wherein R1 is as defined for compound (I) and M represents a leaving group, preferably selected from a halide atom, in particular Cl, an imidazolyl group, a triazolyl group, and para-nitrophenoxyl, preferably M represents para-nitrophenoxyl.

[0030] - Obtaining compound (I) by addition reaction of said compound (II) to compound (III).

[0031] The various compounds according to the present invention can be found in all possible optical isomeric forms, and in some cases in the form of mixtures in all proportions, at least unless otherwise specified. According to certain embodiments, compounds according to the present invention containing asymmetric carbon atoms are found in racemic form, with R and S configurations in approximately equal proportions. According to other embodiments, the compounds of formula (I) of the present invention are either diastereomers or enantiomers, and can be found in isomer-enriched form with a diastereomeric or enantiomeric excess of more than 80%, or more than 95%, or in pure isomeric form, i.e., with a diastereomeric or enantiomeric excess of more than 99%.

[0032] The compounds can be isolated in diastereomeric or enantiomerically enriched form by classical separation techniques, for example fractional crystallization of racemic salts with optically active acids or bases, the principles of which are well known or, most frequently, classical chromatographic techniques on chiral or non-chiral phases.

[0033] Where applicable, the compounds according to the invention can be found in the form of a salt, in particular a hydrochloride, acetate, hydrotrifluoroacetate, sodium salt or ammonium salt.

[0034] The present invention will now be described in more detail. First, certain terms that will be used will be defined.

[0035] [Detailed explanation] [Definition] By "aliphatic heterocycle" is understood in the context of the present invention a substituted or unsubstituted saturated ring containing 3 to 20 members, preferably 5 to 10 members, more preferably 5, 6, 7 or 8 members, and containing at least one heteroatom such as O, N or S.

[0036] "Aliphatic carbocyclic ring" is understood in the context of the present invention to mean a substituted or unsubstituted saturated ring containing 3 to 30 members, preferably 5 to 10 members, more preferably 5, 6, 7 or 8 members, composed solely of carbon atoms.

[0037] "Alkyl" in the context of the present invention is understood to be a saturated hydrocarbon chain which may be linear or branched. Preferably, the term alkyl, unless otherwise specified, refers to an alkyl group containing at least 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, in particular an alkyl(C1-C4) group. Methyl, ethyl, n-propyl, isopropyl, and tert-butyl are examples of (C1-C4) alkyl groups (alkyl having 1 to 4 carbon atoms).

[0038] By "alkylene" is understood a divalent alkyl group.

[0039] "Heteroalkyl" in the context of this invention is understood to be a straight or branched hydrocarbon chain consisting of 1 to 6 carbon atoms and 1 to 3 heteroatoms selected from the group consisting of O, N, Si and S. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S may be located at any interior position of the heteroalkyl group or at the position of the remainder of the molecule that is attached to the alkyl group.

[0040] "Aryl" is understood to be a monocyclic, bicyclic, or polycyclic ring, and unless otherwise specified, is understood to be an unsaturated hydrocarbon having at least 5 to 24, 5 to 20, or 5 to 15 members, preferably containing single and double bond modifications, and containing at least one aromatic ring. Examples of optional aryl groups include phenyl, naphthyl, anthracenyl, phenanthrenyl, and cinnamyl groups. The term aryl also includes monocyclic, bicyclic, or polycyclic, unsaturated hydrocarbon rings in which one of the constituent carbons is found in the form of -C(O)carboxy, such as 1H-phenalen-1-one (CAS No. 548-39-0).

[0041] By "arylene" is understood a divalent aryl group.

[0042] Unless otherwise specified, the term "heteroaryl" is understood to mean a monocyclic, bicyclic, or polycyclic carbocyclic ring containing at least 5 to 24 members, preferably 6 to 20 members, and more preferably 6 to 15 members, and containing at least one aromatic group and at least one heteroatom selected from oxygen, nitrogen, or sulfur atoms incorporated into the carbocyclic ring. Examples of heteroaryl groups include 2-, 3-, or 4-pyridinyl, 2- or 3-fluoyl, 2- or 3-thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, benzimidazolyl, benzothiazolyl, oxazolyl, benzoxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl, tetrazolyl, thiadazolyl, oxadiazolyl, triazolyl, pyridazinyl, indolyl, oxanyl, 4(1H)-quinolinonyl, dibenzothiophenyl, dibenzofuranyl, and 9H-carbazolyl. The term heteroaryl also comprises said groups in which one of the constituent carbon atoms is found in the form of carboxy-C(O), such as 4(3H)-pyrimidinonyl, 4(3H)-quinazolinonyl or 4(1H)-quinolinone.

[0043] Without further specification, when group is considered to be substituted, this means that it is substituted by one or several substituents selected from among chlorine, bromine, iodine or fluorine atom, cyano, alkyl, trifluoroalkyl, trifluoromethyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, alkylamino, diacrylamino, hydroxy, alkoxy, aryloxy, alkoxycarbonyl, aryloxycarbonyl group, and means that said group itself can be substituted.The terms used to define these substituents are generally recognized by those skilled in the art.

[0044] By "alkoxy" and "aryloxy" are understood -O-alkyl and -O-aryl groups respectively, with alkyl and aryl being defined in the context of the present invention.

[0045] By "haloalkyl" is understood a saturated, straight or branched hydrocarbon chain in which at least one hydrogen atom has been replaced by a halogen atom.

[0046] By "β-lactamase" is understood a β-lactamase enzyme that has the ability to catalyze the hydrolysis of β-lactams to open the β-lactam ring.

[0047] By "β-lactam" is understood a four-membered cyclic amide.

[0048] Classically, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond and, unless otherwise specified, having from 2 to 20 carbon atoms, preferably from 2 to 6 carbon atoms.

[0049] In the context of the present invention, the term "alkenylene" refers to a divalent alkenyl group.

[0050] The term "alkynyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond and, unless otherwise specified, having from 2 to 12 carbon atoms, preferably from 2 to 6 carbon atoms.

[0051] By "alkynylene" is understood a divalent alkynyl group.

[0052] A "linking arm" is understood to be a divalent group that covalently links two parts of a compound.

[0053] An "organyl group" is understood to be any organic substituent, regardless of functional type, having one free valence at a carbon atom that is used to attach said organyl group to a compound.

[0054] An "organylthio group" is understood to be any organic substituent, regardless of functional type, having one free valence at the sulfur atom that is used to attach said organyl group to a compound. An "organyloxy group" is understood to be any organic substituent, regardless of functional type, having one free valence on the oxygen atom that is used to attach said organyl group to a compound.

[0055] An "organylamino group" is understood to be any organic substituent, regardless of functional type, having one free valence on the nitrogen atom that is used to attach said organyl group to a compound.

[0056] "Water-solubilizing group" or "hydro solubilizing group" is understood to be a hydrophilic group that makes it possible to improve the solubility of a probe in aqueous media, and in particular relates to probes that differ from it only by substituting a hydrogen atom for the hydrophilic group. In particular, said hydrophilic group is capable of modifying the electrostatic properties of the probe.

[0057] As used herein, the term "protecting group" refers to a chemical substituent that can be selectively removed by readily available reagents that do not attack the regenerated or other functional groups in a molecule. Suitable protecting groups are known in the art and continue to be developed. Suitable protecting groups can be found, for example, in Wutz et al. ("Greene's Protective Groups in Organic Synthesis, Fourth Edition," Wiley-Interscience, 2007). Protecting groups for protecting amino groups such as those described by Wutz et al. (pages 696-927) are used in certain embodiments. Representative examples of amino protecting groups include, but are not limited to, t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), carboxybenzyl (Cbz), benzyl (Bn), allyl, and trifluoroacetyl.

[0058] "Fluorescence" is the property of a molecule to emit light of a longer wavelength when excited by light of a given wavelength. Fluorescence is a phenomenon resulting from the interaction of a fluorophore with an incident photon. This process is also called excitation. The absorption of a photon results in the transition of an electron in the fluorophore from its base state to a higher energy level. The electron then returns to its original state by emitting a photon. This process is called fluorescence. The fluorophore then emits light of a wavelength longer than the wavelength of the absorbed photon. This is simply due to the fact that the energy of the emitted photon is less than the energy of the absorbed photon due to energy dissipation during the lifetime of the excited state. This is the definition given in patent application WO 2004 / 058787.

[0059] Compound (I) according to the present invention is called a "β-lactamase substrate" because it is converted into another substance during a chemical reaction, particularly hydrolysis, catalyzed by β-lactamase. During this reaction in aqueous medium, compound (I) (also called the "probe") is cleaved under the action of the target β-lactamase, which results in fragmentation of the probe, including the formation of a compound that is highly insoluble and precipitates in situ; upon adoption in the solid state, it begins to emit strong fluorescence when excited by light of the appropriate wavelength.

[0060] A "spacer" in the context of the present invention is a fragment of compound (I) which has at one end ii) a β-lactam group which serves as a substrate for the target enzyme, and at the other end iii) a WR1 group which, when released as HWR1 by said fragmentation, belongs to the class of fluorophores, more particularly to the class of solid fluorophores.

[0061] [Compound of formula (I)] The present invention relates to compounds of formula (I):

[0062] [ka]

[0063] where: -W is -O- or -NR 13 - and R 13 is C1-C4 alkyl or a hydrogen atom; -R1 is such that HWR1 obtained after cleavage of the -C(O)-WR1 bond present in formula (I) belongs to the class of fluorophores, preferably the class of fluorophores that bring about intramolecular proton transfer in the excited state; R2, R3 and R4 are defined as: o R2 is C1-C4 alkyl, R3 is C1-C4 alkyl or a hydrogen atom, and R4 is C1-C4 alkyl; Or, R3 is C1-C4 alkyl or a hydrogen atom, and R2 and R4 are bonded to each other and form -(CH2) in the direction from R2 to R4. p -Y q -(CH2) r -Forming chains, ● Y is O, NR 14 , N(R 14 )2 + or S, preferably NR 14 or N(R 14 )2 + and ● p=0, 1, 2, 3, 4 or 5, ● q=0 or 1, ● r=0, 1, 2, 3, 4 or 5, ● p+q+r=3, 4, 5, or 6, ● Each R 14 are independently a hydrogen atom, a C1-C6 alkyl, an amino protecting group, or -(L) q represents -GP, where q is equal to 0 or 1, L is a linking arm, and GP is a hydrophilic group; or R2 is C1-C4 alkyl, and R3 and R4 are joined together to form an aliphatic carbocyclic ring together with the carbon atoms to which they are attached; R5 and R6 are the same or different and each independently represent a hydrogen atom, a C1-C4 alkyl, or a C5-C 10 represents aryl; -R7 is a hydrogen atom or a group selected from C1-C4 alkyl and C1-C4 alkoxy; -R8 represents a hydrogen atom; -V represents an oxygen atom or a sulfur atom; -n is 0 or 1; -Z is -S-, -SO-, or -CR9R 10 - and R9 and R 10 are the same or different and each independently represent a hydrogen atom or a C1-C4 alkyl; -Q is H, a cation, or R16, where R16 is C1-C6 alkyl, optionally substituted with aryl or O—(CO)—R, and R is independently selected from H, C1-C6 alkyl, and C3-C6 cycloalkyl; -R 11 is an organyl group, an organylamino group, an organyloxy group, or an organylthio group, -X is a bond, or

[0064] [ka]

[0065] represents a group selected from

[0066] In certain embodiments, compound (I) is of formula (Ia):

[0067] [ka]

[0068] where R1, R2, R3, R4, R5, R6, R7, R8, R 11 , Q, W, X and V are as defined for compound (I).

[0069] In another embodiment, compound (I) is of formula (Ib):

[0070] [ka]

[0071] where R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , Q, W, X and V are as defined for compound (I).

[0072] The compounds of formula (Ib) are particularly useful for detecting carbapenemase activity.

[0073] The R1 group is selected so that the resulting fluorescent precipitate corresponding to HWR1, released after cleavage of the -C(O)-WR1 bond, belongs to the class of fluorophores, preferably those that result in intramolecular proton transfer in the excited state (ESIPT).

[0074] ESIPT fluorophores exhibit Stokes shifts exceeding 100 nm, often approaching 200 nm. All ESIPT fluorophores lose this fluorescence emission, corresponding to a Stokes shift of more than 100 nm, when alkylated, acylated, or otherwise functionalized so that the phenolic WH group causes intramolecular excited-state proton transfer. This functionalization prevents hydrogen atom transfer during excitation by irradiation, thus preventing the emission of fluorescence characteristic of proton transfer processes.

[0075] Incorporation of HWR1 into the carbamate or urea group of compounds of formula (I) prevents proton transfer. Intramolecular proton transfer can therefore occur using the group obtained after cleavage of the -C(O)-WR1 bond.

[0076] Most frequently, the R1 group corresponds to a phenyl group, unsubstituted or substituted and / or possibly containing heteroatoms such as nitrogen, fused with one or more unsaturated carbocyclic rings. When W is O, the -OR1 phenoxy derivative, when not bound to a substrate, corresponds to its protonated form, the HO-R1 phenolic derivative, belonging to the ESIPT class of fluorophores.

[0077] Reference may be made in particular to specifications WO2013 / 045854, WO2014 / 020285, and WO2015 / 197981, which provide examples of such ESIPT fluorophores that may be used in the present invention.

[0078] Preferably, R1 comprises an aromatic group containing one or more aromatic rings, substituted or unsubstituted, which rings may contain one or more heteroatoms selected from nitrogen, oxygen or sulfur atoms, and / or one or more carbon atoms in the form of a C=O carbonyl.

[0079] WR1 may be an aromatic -OR1 according to formula (A1).

[0080] [ka]

[0081] where: -X2 is an oxygen atom, and X1 is -NH2, -OH, -SH, C1-C 20 Alkyl, C5-C 24 Aryl, C2-C6 alkenyl, -O-(C1-C 20 alkyl), -O-phenyl, -NH-(C1-C 20 alkyl), -NH-phenyl, -S-(C1-C 20 alkyl), or -S-(C5-C 24 aryl groups), wherein said alkyl, aryl, alkenyl, and phenyl groups are optionally substituted; or X2 represents a nitrogen atom and is bonded to X1, which represents CH, O, S, N, or NH, and is an optionally substituted C5-C 24 forming a heteroaryl; -

[0082] [ka]

[0083] is C5-C 24 Aryl or C5-C 24 Heteroaryl groups of the formula:

[0084] [ka]

[0085] Selected from the group is optionally substituted; X3 represents S, O or NRd, and Rd represents a hydrogen atom or a C1-C4 alkyl group.

[0086] Advantageously, -OR1 is an aryloxy species, preferably corresponding to one of the following preferred structures (A2), (A3) or (A4): -

[0087] [ka]

[0088] where: ○ T is -NH-C(O)-, -S-, -O-, -NH-, -N(C1-C 20 alkyl)- or -N(C5-C 24 aryl)-; Re is a hydrogen atom or an electron-withdrawing group such as -CN or -COORh, and Rh represents a C1-C4 alkyl group, or Re is -CONRiRj, where Ri and Rj are the same or different, and Ri and Rj represent a hydrogen atom or a C1-C4 alkyl group, or Re is -CF3, C2-C6 alkenyl, or heteroaryl, wherein said heteroaryl and alkenyl are optionally substituted; Rf is a hydrogen atom, a chlorine, bromine, iodine or fluorine atom, -OH, -NH2, -NRkRI, -NHRk or -ORk, where Rk and RI are the same or different, and Rk and RI each independently represent a C1-C4 alkyl group; or Re and Rf are each joined to each other to form a hydrocarbon chain containing 4 or 5 members, which may be saturated or unsaturated, substituted or unsubstituted, and separated by one or more heteroatoms selected from among N, S and O; o Rg is a hydrogen atom, Br, Cl, I or F atom; -

[0089] [ka]

[0090] where: ○ T' is -NH2, -OH, C5-C 24 an aryl group, a C1-C4 alkyl group, -SH, -NHR'g, -OR'g, -NR'gRh', -SR'g, or an optionally substituted C2-C6 alkenyl group, or a heteroaryl, where R'g and Rh' are the same or different and are a C1-C4 alkyl group or a C5-C 24 represents an aryl group; R'e is a hydrogen atom or an electron-withdrawing group such as -CN or -COOR'i, where R'i represents a C1-C4 alkyl group, or R'e is -CONR'jR'k, where R'j and R'k are identical or different and represent a hydrogen atom or a C1-C4 alkyl group, or R'e is -CF3, or a 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-benzimidazolyl, 4-pyrimidinon-2-yl or quinazolinon-2-yl group; R'f is a hydrogen atom, a chlorine, bromine, iodine, or fluorine atom, -OH, -NH2, -NR'IR'm, or OR'I, where R'I and R'm are the same or different and represent a C1-C4 alkyl group; or R'e and R'f are each joined to each other to form a hydrocarbon chain containing 4 or 5 members, which may be saturated or unsaturated, substituted or unsubstituted, and which may be separated by one or more heteroatoms selected from among N, S and O; -

[0091] [ka]

[0092] where: -X'2 is an oxygen atom, and X'1 is -NH2, -OH, -SH, C1-C 20 Alkyl, C5-C 24 Aryl, C2-C6 alkenyl, -O-(C1-C 20 alkyl), -O-phenyl, -NH-(C1-C 20 alkyl), -NH-phenyl, -S-(C1-C 20 alkyl), or -S-(C5-C 24 aryl groups), wherein the alkyl, aryl, alkenyl, and phenyl groups are optionally substituted; or X2 represents a nitrogen atom and is bonded to X1, which represents CH, O, S, N, or NH, and is an optionally substituted C5-C 24 forming a heteroaryl; -

[0093] [ka]

[0094] is an optionally substituted C5-C 10 Aryl or C5-C 10 represents heteroaryl.

[0095] In formulas (A1) to (A4), when it is stated that a substituent is optionally substituted, the substituent may be optionally substituted with one or more substituents. The one or more substituents are preferably halide, —CN, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C5-C 10 Cycloalkyl, C5-C 10 Aryl, C5-C 10 Heteroaryl, C5-C 24and selected from heterocycloalkyl, -NH, -NH(C-C alkyl), -N(C-C alkyl), hydroxy, oxo, aryloxy, alkoxycarbonyl, and aryloxycarbonyl groups, wherein said cycloalkyl, aryl, heteroaryl, and heterocycloalkyl may be substituted by halide, C-C alkyl, C-C haloalkyl, C-C alkoxy, oxo, -NH, -NH(C-C alkyl), or -N(C-C alkyl).

[0096] According to certain embodiments of the present invention, WR1 is an aromatic group having —OR1 responding to one of the following formulas (A5) or (A6):

[0097] [ka]

[0098] The very large Stokes shift of such fluorophores (approximately 170 nm for A6), or any analog of HPQ, contributes to the excellent sensitivity of the probe and makes the emitted fluorophore easily distinguishable from native fluorescence that may originate from the biological sample being analyzed.

[0099] According to one embodiment, R1 is selected from the group consisting of fluoresceins (including rhodamines and rhodols), coumarins (including 7-amino- and 7-hydroxy-coumarins), cyanines, phenoxazines, and acridinones.

[0100] According to one embodiment of the present invention, R2 is (C1-C4) alkyl, R3 is (C1-C4) alkyl or a hydrogen atom, and R4 is (C1-C4) alkyl. According to another embodiment, R2, R3, and R4, whether identical or different, represent a (C1-C4) alkyl group, for example, methyl or ethyl. According to a specific embodiment, R2 = R3 = R4 = -CH3.

[0101] According to another embodiment, R2 is C1-C4 alkyl, and R3 and R4 are bonded together to form an aliphatic carbocyclic ring with the atoms to which they are bonded, e.g., R3 and R4 are bonded together to form a -(CH2)m- chain, where m=3, 4, 5, or 6.

[0102] Advantageously, R3 is a hydrogen atom or a C1-C4 alkyl, preferably a hydrogen atom, and R2 and R4 are linked together to form a -CH2CH2-Y-CH2- chain in the direction from R2 to R4, where Y is -CH2-, -NR 14 - or N(R 14 )2 + - represents R 14 is a hydrogen atom or -(L) q represents -GP, where q is equal to 0 or 1, L is a linking arm, and GP is a hydro solubilizing group.

[0103] In a specific embodiment, R3 is C1-C4 alkyl or a hydrogen atom, R2 and R4 are bonded to each other, and the direction from R2 to R4 is -(CH2) p -Y q -(CH2) r -Forming chains, ● Y is O, NR 14 , N(R 14 )2 + or S, preferably NR 14 or N(R 14 )2 + and ● p=0, 1, 2, 3, 4 or 5, ● q=0 or 1, ● r=0, 1, 2, 3, 4 or 5, ● p+q+r=3, 4, 5, or 6, ● Each R 14 are independently a hydrogen atom, a C1-C6 alkyl, an amino protecting group, or -(L) q -GP, where q is equal to 0 or 1, L is a linking arm, and GP is a hydrophilic group.

[0104] For example, when R2 and R4 are bonded to each other and the bond is -(CH2) p -Y q -(CH2) r -Forming chains, ● Y is NR 14 or N(R 14 )2 + , ● p=2, 3, 4 or 5, ● q=1, ● r=0, 1, 2, 3, 4 or 5, ● p+q+r=3, 4, 5, or 6, and ● Each R 14 are independently a hydrogen atom, a C1-C6 alkyl, an amino protecting group, or -(L) q -GP, where q is equal to 0 or 1, L is a linking arm, and GP is a hydrophilic group, or R2 and R4 are bonded to each other, and -(CH2) p -Y q -(CH2) r -Forming chains, ● p=2, 3, 4 or 5, ● q=0, ● r=0, 1, 2, 3, 4 or 5, ● p+q+r=3, 4, 5, or 6.

[0105] In specific embodiments, Y is N(R 14 )2 + where the positive charge is on the N atom, making it ammonium. In this case, a counterion is present. The counterion can be selected from the group consisting of halides, trifluoroacetates, and acetates.

[0106] In a specific embodiment, q=1 and L is a linking arm, in particular a -(L1)m1-(L2)m2-(L'1)m'1- arm (towards piperazine -> GP group): -L1 and L'1 are the same or different and are selected from among -O-, -NH-, -N(C1-C6)alkyl)-, -N(phenyl)-, -N(aryl)-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)-O-, -NHC(O)-O-, -OC(O)-NH-, -NHC(O)-NH-, -S-, -SOC-, -N=N-, -NHC(O)- and -CONH-; -L2 is selected from the following divalent groups: (C1-C 20 ) alkylene, (C1-C 20 ) alkenylene, (C1-C 20 ) alkynylene, (C6-C 24 ) arylene, (C7-C 44 ) alkylarylene, (C7-C 44 ) alkenylarylene, (C7-C 44 ) alkynylarylene, (C7-C 44 ) alkylcycloalkylene, (C7-C 44 ) alkenylcycloalkylene, (C7-C 44 ) alkynylcycloalkylene, (C7-C 44 ) alkylheterocycloalkylene, (C7-C 44 ) alkenylheterocycloalkylene, (C7-C 44 ) alkynylheterocycloalkylene; said groups are separated or terminated by triazole groups and can be substituted or unsubstituted, in particular (C-C 10 ) alkoxy, (C1-C 10 ) alkyl, (C6-C 10 ) aryl, amide, imide, phosphide, nitride, (C1-C 10 ) alkenyl, (C1-C 10 ) alkynyl, and -OH, which may be substituted or unsubstituted by one or more substituents selected from among: m, m'1 and m2 are the same or different and are equal to 0 or 1;

[0107] The L arm, if present, is selected to extend the GP group from the piperazine or for synthetic reasons. According to a preferred embodiment, L represents -(L1)m1-(L2)m2-(L'1)m'1, where L1=-C(O)-, m1=m2=1, m'1=1 or 0, and L2 and L'1 are as defined above. In particular, L is -C(O)-(CH2) p represents -L3-, where p is equal to 1, 2, 3 or 4, and L3 is a triazole group, in particular a 1H-1,2,3 triazole group.

[0108] GP is a hydrophilic group. Examples of hydrophilic groups include groups that can form charged species in aqueous solution. Examples of water-soluble GP groups include: - an F1 functional group selected from among an amine (primary, secondary, tertiary, or quaternary), an amidine, a guanidine, or a tetrazole; F2 cationic or anionic functional groups, in particular groups of the ammonium, carboxylate, sulfonate or phosphate type; - groups containing one or more of these F1 and / or F2 functional groups; -Polyethylene glycol; -Sugars or polysaccharides such as glucose, galactose and mannose; peptide groups such as polylysine, polyarginine, TAT peptide, and -amino acid Some examples include:

[0109] Examples of amine functional groups include -NH2, -NH(C1-C4) alkyl, and dialkylamine, where the alkyl groups are the same or different and contain from 1 to 4 carbon atoms.

[0110] These two methods of pre-organizing the spacer for cyclization consist of either introducing two alkyl substituents on the α-carbon of the -NC(V)-O- group (or forming a carbocyclic ring) or including a bond between the nitrogen -NC(V)-O- group and its α-carbon in the heterocyclyl ring, facilitating the immolation process.

[0111] According to one embodiment, R5 and R6 are identical and represent a hydrogen atom. According to one embodiment, R7 represents a hydrogen atom or a (C1-C4)alyl group such as methyl, and preferably a hydrogen atom.

[0112] According to one embodiment, R5, R6 and R7 each represent a hydrogen atom.

[0113] X is a bond, or

[0114] [ka]

[0115] represents a group selected from

[0116] For the present invention, the double bond can have any configuration (Z or E). According to one embodiment, X is a bond, or

[0117] [ka]

[0118] Preferably, X is a bond.

[0119] R 11 is C1-C6 alkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, heterocyclyl having 5 to 10 ring atoms, C5-C 10 Aryl, C7-C16 and -NR"R""; wherein said alkyl, cycloalkyl, heteroalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl are independently selected from oxo, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, heterocyclyl having 5 to 10 ring atoms, C5-C6 10 aryl, heteroaryl having 5 to 10 ring atoms, optionally substituted with one or more substituents selected from -OH, -NR"R"", -NO2, -CN, -O-(CO)-R, and -(CO)-R; each R is independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, and -NR"R"", and each R" and R"' is independently selected from H and C1-C6 alkyl.

[0120] Preferably, R 11 is selected from C1-C6 alkyl, NR''R'''', and C1-C6 heteroalkyl, wherein the alkyl and heteroalkyl are selected from oxo, heteroaryl having 5 to 10 ring atoms, C5-C 10 and optionally substituted with one or more substituents independently selected from aryl, C-C heteroalkyl, —O—(CO)—R, and —OH; R is selected from H and C-C alkyl, and R″ and R′″ are independently selected from H and C-C alkyl, more preferably R 11 teeth

[0121] [ka]

[0122] is selected from.

[0123] Cation Q is Na + , K. + , Li + , NH4 + You can choose from.

[0124] According to a particular embodiment, Z is S and n is 1. According to another particular embodiment, Z is -CR9R 10 -, n is 0; R and R 10 are the same or different and independently represent a hydrogen atom or a C1-C4 alkyl.

[0125] According to a particular embodiment, compound (I) is of formula (Ic):

[0126] [ka]

[0127] Here, R1, R 11 , Z, Q, n, X, and Y, and n are as defined for compounds of formula (I).

[0128] According to a particular embodiment, compound (I) is of formula (Id):

[0129] [ka]

[0130] Here, R1, R 11 , Q, X, and Y are as defined for compounds of formula (I).

[0131] The compound of formula (Id) can be of formula (Id'), (Id'') or (Id'''):

[0132] [ka]

[0133] As defined for compounds of formula (I).

[0134] The compound of formula (Id) may be selected from the following compounds:

[0135] [ka] TIFF0007791815000024.tif46169

[0136] According to a particular embodiment, compound (I) is of formula (Ie):

[0137] [ka]

[0138] where R1, R9, R 10 , R 11 , Q, X and Y are as defined for compounds of formula (I).

[0139] The compound of formula (Ie) can be of formula (Ie'), (Ie'') or (Ie'''):

[0140] [ka]

[0141] wherein Y is as defined for compounds of formula (I).

[0142] The compound of formula (Ie) can be selected from the following compounds:

[0143] [ka] TIFF0007791815000028.tif42169

[0144] According to one embodiment, in the compound of formula (I): -W is -O-; -R1 is such as HOR1 obtained after cleavage of the -C(O)-OR1 bond present in formula (I), HOR1 belonging to the class of fluorophores, preferably the class of fluorophores that bring about intramolecular proton transfer in the excited state, such that -OR1 corresponds to a group of formula (A1):

[0145] [ka]

[0146] where: -X2 is an oxygen atom, and X1 is -NH2, -OH, -SH, C1-C 20 Alkyl, C5-C 24 Aryl, C2-C6 alkenyl, -O-(C1-C 20 alkyl), -O-phenyl, -NH-(C1-C 20 alkyl), -NH-phenyl, -S-(C1-C 20 alkyl), or -S-(C5-C 24 aryl groups), wherein said alkyl, aryl, alkenyl, and phenyl groups are optionally substituted; or X2 represents a nitrogen atom and is bonded to X1, which represents CH, O, S, N, or NH, and is an optionally substituted C5-C 24 forming a heteroaryl; -

[0147] [ka]

[0148] is C5-C 24 Aryl or C5-C 24 Heteroaryl groups of the formula:

[0149] [ka]

[0150] Selected from the group is optionally substituted; X3 represents S, O, or NRd, where Rd represents a hydrogen atom or a C1-C4 alkyl group; R2, R3 and R4 are defined as follows: R3 is C1-C4 alkyl or a hydrogen atom, R2 and R4 are bonded to each other, and the direction from R2 to R4 is -(CH2) p -Y q -(CH2) r -Forming chains, ● Y is O, NR 14 , N(R 14 )2 + or S, preferably NR 14 or N(R 14 )2 + and ● p=0, 1, 2, 3, 4 or 5, ● q=0 or 1, ● r=0, 1, 2, 3, 4 or 5, ● p+q+r=3, 4, 5, or 6, ● Each R 14 are independently a hydrogen atom, a C1-C6 alkyl, an amino protecting group, or -(L) q represents -GP, where q is equal to 0 or 1, L is a linking arm, and GP is a hydrophilic group; R5 and R6 are the same or different and each independently represent a hydrogen atom, a C1-C4 alkyl, or a C5-C 10 represents aryl; -R7 is a hydrogen atom or a group selected from C1-C4 alkyl and C1-C4 alkoxy; -R8 represents a hydrogen atom; -n is 0 or 1; -Z is -S- or -CR9R 10 - and R9 and R 10 are the same or different and each independently represent a hydrogen atom or a C1-C4 alkyl; -Q is H or a cation, R 11is C1-C6 alkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, heterocyclyl having 5 to 10 ring atoms, C5-C 10 Aryl, C7-C 16 aralkyl; The alkyl, cycloalkyl, heteroalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl, and aralkyl are independently selected from oxo, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, heterocyclyl having 5 to 10 ring atoms, C5-C 10 optionally substituted with one or more substituents selected from aryl, heteroaryl having 5 to 10 ring atoms, -OH, -NR''R'''', -NO2, -CN, and -(CO)-R; each R is independently selected from H, C-C alkyl, C-C alkoxy, and -NR"R""; each R" and R"' is independently selected from H and C1-C6 alkyl; -X is a bond, or

[0151] [ka]

[0152] represents a group selected from

[0153] [Compound of formula (II)] The present invention also relates to compounds of formula (II):

[0154] [ka]

[0155] where: -R2, R3, R4, R5, R6, R7, R8, R 11, Z, n, Q, X and V are as defined for compound (I); -R 12 represents a hydrogen atom or an amine functional protecting group.

[0156] The compound of formula (II) is a synthetic intermediate of the compound of formula (I) by the amine function protecting group. The amine function protecting group is understood to be, for example, the protecting group described in Organic Synthesis, Greene TW et Wuts PGN, ed. John Wiley and Sons, 2006, and the protecting group described in Kocienski PJ, 1994, Georg Thieme Verlag.

[0157] According to one embodiment, R 12 is an amine functional protecting group. For example, R 12 represents an amine function protecting group selected from among an aryl group or a carbamate group, such as a tert-butoxycarbonyl (Boc) group, a fluorophenylmethoxycarbonyl (Fmoc) group, an aryloxycarbonyl (Alloc) group, or a 2,2,2-trichloroethoxycarbonyl (Troc) group.

[0158] According to a particular embodiment, R 12 represents a hydrogen atom.

[0159] In certain embodiments, the compound of formula (II) is of formula (IIa):

[0160] [ka]

[0161] where R3, R4, R5, R6, R7, R8, R 11 , Q, X, and Y are as defined for compounds of formula (I), and R 12 is as defined for compounds of formula (II).

[0162] In another particular embodiment, the compound of formula (II) is of formula (IIb):

[0163] [ka]

[0164] where R3, R4, R5, R6, R7, R8, R9, R 10, R 11 , Q, X and Y are as defined for compounds of formula (I), and R 12 is as defined for compounds of formula (II).

[0165] Advantageously, the compound of formula (II) is of formula (IIc):

[0166] [ka]

[0167] where R 11 , Z, Q, n, X and Y are as defined for compounds of formula (I), and R 12 is as defined for compounds of formula (II).

[0168] In certain embodiments, the compound of formula (II) is of formula (IId):

[0169] [ka]

[0170] where R 11 , Q, X and Y are as defined for compounds of formula (I), and R 12 is as defined for compounds of formula (II).

[0171] In another particular embodiment, the compound of formula (II) is of formula (IIe):

[0172] [ka]

[0173] where R 11 , Q, X and Y are as defined for compounds of formula (I), and R 12 is as defined for compounds of formula (II).

[0174] [Method for preparing compounds of formula (I)] The present invention also relates to a method for preparing a compound of formula (I) comprising the steps of: - carrying out compound (II); - carrying out the compound of formula (III):

[0175] [ka]

[0176] wherein R1 is as defined for compound (I), M represents a leaving group, preferably selected from a halogen atom, in particular Cl, an imidazolyl group, a triazolyl group, and para-nitrophenoxy, more preferably M represents para-nitrophenoxyl; - Obtaining compound (I) by addition reaction of said compound (II) to compound (III).

[0177] - Obtaining compound (I) by addition reaction of compound (II) to compound (III). This method is particularly suitable when X is a bond.

[0178] According to one embodiment, the addition reaction of compound (II) to compound (III) is carried out by reacting R 12 is carried out using compound (II) in which is a hydrogen atom.

[0179] According to another embodiment, R 12is not a hydrogen atom, and the step of deprotecting the amine functionality of compound (II) can be carried out by adding compound (II) to compound (III) to form R 12 This is carried out before the reaction to obtain compound (II), such as =H.

[0180] When V=O, compound (II) can be advantageously obtained according to the following steps: - the preparation of a compound (V) of the formula:

[0181] [ka]

[0182] - carrying out the compound of formula (VI)

[0183] [ka]

[0184] - Compound (VI) is added to Compound (V) to obtain Compound (II), wherein R, R 11 , Q, X and Z are as defined in the context of the present invention, and K represents a leaving group, in particular a halogen, in particular chlorine, or an imidazolyl group or a para-nitrophenyl group.

[0185] According to a particular embodiment, the compound of formula (V) is a compound of formula (Va):

[0186] [ka]

[0187] Here, R8, R 11 , Q, Z, X and K are as defined in the context of the invention.

[0188] According to another particular embodiment, the compound of formula (V) is of formula (Vb):

[0189] [ka]

[0190] Here, R8, R9, R 10 , R 11 , Q, X and K are as defined in the context of the invention.

[0191] The present invention also relates to a compound in which X is

[0192] [ka]

[0193] In this case, the compound can be prepared using the Sonogashira coupling reaction, which is a reaction well known to those skilled in the art. The method can include the following steps: - carrying out a compound of formula (VII):

[0194] [ka]

[0195] - carrying out the compound of formula (VIII)

[0196] [ka]

[0197] and reacting said compound (VII) with compound (VIII) to obtain compound (I), where R1, R2, R3, R4, R5, R6, R7, R8, V, R 11 , Q, W, Z, and n are as defined in the context of the present invention, and LG represents a leaving group, preferably selected from halogen and trifluoromethanesulfonate (triflate).

[0198] This process can be carried out using standard conditions such as a palladium catalyst and a copper cocatalyst.

[0199] The present invention also relates to a compound in which X is

[0200] [ka]

[0201] In this case, the compound can be prepared using the Suzuki coupling reaction, which is a reaction well known to those skilled in the art. The method can include the following steps: - carrying out a compound of formula (VII):

[0202] [ka]

[0203] - carrying out the compound of formula (IX)

[0204] [ka]

[0205] and reacting said compound (VII) with compound (IX) to obtain compound (I), where R1, R2, R3, R4, R5, R6, R7, R8, V, R 11 , Q, W, Z, and n are as defined in the context of the present invention, and LG represents a leaving group, preferably selected from halogen and trifluoromethanesulfonate (triflate), 15 represents OH, or each R 15 are bonded to each other and, together with the B atoms to which they are attached, form a heterocycle having 5 to 10 ring atoms. Examples of R15 groups bonded to each other include pinacol, catechol, and methyliminodiacetate.

[0206] This process can be carried out under standard conditions such as palladium catalysis and base.

[0207] The present invention also relates to a compound in which X is

[0208] [ka]

[0209] In this case, the compound can be prepared using the Suzuki coupling reaction, which is a reaction well known to those skilled in the art. The method can include the following steps: - carrying out a compound of formula (VII)

[0210] [ka]

[0211] - carrying out the compound of formula (X)

[0212] [ka]

[0213] and reacting said compound (VII) with compound (X) to obtain compound (I), where R1, R2, R3, R4, R5, R6, R7, R8, V, R 11 , Q, W, Z, and n are as defined in the context of the present invention, LG represents a leaving group, preferably selected from halogen and trifluoromethanesulfonate (triflate), R 15 represents OH, or each R 15 are bonded to each other and, together with the B atoms to which they are attached, form a heterocycle having 5 to 10 ring atoms. Examples of R15 groups bonded to each other include pinacol, catechol, and methyliminodiacetate.

[0214] This process can be carried out under standard conditions, such as palladium catalysis and base.

[0215] Compounds of formula (X) can be obtained from compounds of formula (VIII) by hdroboration of the triple bond. The double bond in compounds of formula (X) can be E or Z.

[0216] [Detection of the presence of β-lactamase] The present invention also relates to a method for detecting β-lactamase in vitro or ex vivo, which comprises the steps of: - placing the sample to be analyzed in contact with compound (I); applying appropriate conditions to cleave the covalent bond between -C(=V) and NR7, followed by cleavage of -C(O)-WR1, allowing the formation of a fluorescent precipitate by binding and resulting in the release of HWR1; - quantitative or qualitative analysis of fluorescent precipitates, - Correlating quantitative or qualitative analysis of the fluorescent precipitate with the presence or absence of β-lactamase in the sample.

[0217] The present invention also relates to a method for detecting antibiotic-resistant bacteria in vitro or ex vivo, which comprises the steps of: - placing the sample to be analyzed in contact with compound (I); applying appropriate conditions to cleave the covalent bond between -C(=V) and NR7, followed by cleavage of -C(O)-WR1, allowing the formation of a fluorescent precipitate by binding and resulting in the release of HWR1; - quantitative or qualitative analysis of fluorescent precipitates, - Correlating quantitative or qualitative analysis of fluorescent precipitates with the presence or absence of antibiotic-resistant bacteria in the sample.

[0218] The present invention also relates to a kit for detecting β-lactamase, which comprises compound (I).

[0219] The present invention also relates to a device for detecting β-lactamase, comprising compound (I). Preferably, said device is an in-vitro diagnostic medical device (IVD).

[0220] According to one embodiment, the β-lactamase is a carbapenemase.

[0221] The present invention also relates to a method for detecting carbapenemase in vitro or ex vivo, which comprises the steps of: - placing the sample to be analyzed in contact with compound (I); applying appropriate conditions to cleave the covalent bond between -C(=V) and NR7, followed by cleavage of -C(O)-WR1, allowing the formation of a fluorescent precipitate by binding and resulting in the release of HWR1; - quantitative or qualitative analysis of fluorescent precipitates, -Correlate quantitative or qualitative analysis of fluorescent precipitates with the presence or absence of carbapenemase in the sample.

[0222] The compounds of formula (I) according to the present invention may also be used to detect β-lactamases in vivo, in animals or humans.

[0223] Administration of the compounds of formula (I) can be accomplished, for example, by intravenous or intraperitoneal injection, or transdermally by using a spray containing the molecule in solution.

[0224] Analysis of the fluorescence of the compounds of formula (I) can be carried out in an imaging chamber using tomography techniques of the fluorescence or epifluorescence type.

[0225] The present invention also relates to a method for detecting the presence of β-lactamases in vitro or ex vivo by means of compounds (I) according to the invention.

[0226] The sample may be any suitable biological sample derived from a human, animal, plant, or microorganism. In the case of a sample derived from a human or animal, it may in particular be a sample of a biological fluid, in particular whole blood, serum, plasma, urine, a tissue sample, or a sample of isolated cells, in particular a sample of cell culture medium. In the case of a sample derived from a plant, it may be a plant extract, an extract of a fungus or algae, an extract of living cells, in particular an extract of cell culture medium. The sample may also directly contain the plant. In the case of a sample derived from a microorganism, the microorganism may be a bacterium, a virus, a fungus, or a yeast, or may be a microflora. The sample may directly contain the microorganism, or an extract of the latter, or even the medium in which the microorganism was cultured. In all cases, the sample may be used as is or subjected to a concentrated or cultured species preparation known to those skilled in the art before being placed in the presence of the probe.

[0227] Analysis of compounds or fluorescent precipitates - exposing the fluorescent precipitate to a light source capable of producing light at an absorption wavelength of the fluorescent precipitate; and - detecting the fluorescence of the resulting precipitate.

[0228] The analysis may also include a step of sorting the analyzed sample based on the signal provided by the fluorescent precipitate following the step of detecting the fluorescence. The sorted sample may be a spatially separated microbial colony, such as a microbial culture dish. The sorted sample may also be a small object, liquid, solid, gelatinous, or heterogeneous composition, containing either microbial biomolecules or colonies. When detection is performed on several samples in parallel, sorting can be performed by diverting the sample stream, which is set to move in a device capable of sorting according to an optical signal representative of the emitted fluorescence, such as a flow cytometer or a digital millifluidic or microfluidic device.

[0229] The present invention makes the activity of β-lactamases accessible through fluorescence imaging using a fluorophore, preferably an ESIPT fluorophore. Advantageously, no background noise due to spontaneous degradation (i.e., in the absence of the target β-lactamase in physiological media) was observed. The probe itself is slightly or not at all fluorescent, specifically at the emission wavelength of the fluorophore fiber where the detection / imaging instrument is set. Thus, the probe functions in an on / off mode and can be used to develop assays with maximum sensitivity.

[0230] The probes according to the invention are interesting for several sensitive applications in the life sciences, in particular: (1) high-yield targeting of β-lactamase activity expressed by bacterial colonies on agar plates (colony analysis); (2) in vitro detection of β-lactamases in biological fluids (hematology and others); (3) visualization of β-lactamase activity at the level of simple cells in flow cytometry; (4) detection of intracellular β-lactamases in cultured cells (confocal fluorescence microscopy); (5) histochemical detection of β-lactamases (at the tissue level); and finally (6) in vivo imaging of whole animals.

[0231] Thus, the compounds of formula (I) as β-lactamase substrates according to the present invention have numerous potential applications. Examples of these applications include the design of bacterial colony assays. These are currently performed on agar plates (Petri dishes), where up to 3,000 colonies can be identified without the need to actively separate them into separate compartments, such as the wells contained in a multi-well dish. It is therefore possible to (1) design tests for clinical samples that allow the identification of pathogenic strains of interest among bacterial strains, and (2) complete large-scale parallel testing of banks of self-produced proteins expressed by classical bacterial hosts (often commercial). This collection of proteins can be understood to include specific proteins of interest, such as β-lactamases with selectivity for specific β-lactam groups or β-lactamases that hydrolyze β-lactams. In particular, in the field of directed evolution of β-lactamases or enzymes, it is easy to identify 10 6 There is a strong need for an effective and sensitive assay for sifting through a large number of protein variants exceeding 10 ...

[0232] The probes according to the invention can also be used for macroscopic fluorescence imaging, i.e., whole organisms, where the probe penetrates the cell wall to reach the activity of interest.

[0233] The examples relating to the accompanying drawings may illustrate the invention in a non-limiting manner.

[0234] [Example] Example 1: Synthesis of Compound 15

[0235] [ka]

[0236] (ELF-97) A solution of anthranilamide (2.000 g, 11.7 mmol, 1.0 equiv.) in dry EtOH (20 mL) is treated with 5-chlorosalicylaldehyde (1.831 g, 11.7 mmol, 1.0 equiv.) and the mixture is refluxed for 30 minutes. Next, para-toluenesulfonic acid (PTSA) (40 mg, 0.234 mmol, 0.02 equiv.) is added and refluxing is continued for an additional hour. The reaction mixture is cooled to room temperature and treated in portions with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (2.678 g, 11.8 mmol, 1.01 equiv.). Stirring is continued overnight. The resulting crude suspension is filtered, and the filter cake is washed twice with EtOH and twice with diethyl ether. ELF-97 (3.41 g, 11.11 mmol, 95%) is obtained as a light beige powder and is used in the subsequent step without further purification.

[0237] 1 H-NMR (300 MHz, CDCl3): δ (ppm) = 13.38 (s, 1H), 12.64 (s, 1H), 8.29 (s, 1H), 8.10 (s,1H), 7.88 (q, J =7.8 Hz, 2H), 7.49 (d, J =7.6 Hz,1H), 7.05 (d, J = 8.8 Hz, 1H) Spectral data are based on literature values ​​(M. Prost, L. Canaple, J. Samarut, J. Hasserodt. Tagging Live Cells that Express Specific Peptidase Activity with Solid-State Fluorescence. ChemBioChem 2014, 15, 1413-1417).

[0238] (Compound 4) A solution of 2-aminomethylpiperidine (1) (3.0 g, 26.3 mmol, 1.0 equiv.) in toluene (50 mL) was treated portionwise with phthalic anhydride (3.89 g, 26.3 mmol, 1.0 equiv.), followed by the dropwise addition of triethylamine (550 μL, 3.95 mmol, 0.15 equiv.). The mixture was refluxed for 2 h using a Dean-Stark apparatus. The mixture was filtered, and the filtrate was reduced to dryness under reduced pressure. The product 2 (5.42 g, 22.2 mmol, 85%) was obtained as a pale yellow solid and used in the next step without further purification.

[0239] An ice-cold solution of compound 2 (5.42 g, 22.2 mmol, 1.0 equiv.) in ethanol (45 mL) is treated with potassium carbonate (3.99 g, 28.9 mmol, 1.3 equiv.), tetra-n-butylammonium iodide (820 mg, 2.2 mmol, 0.10 equiv.), and aryl bromide (2.50 mmL, 28.9 mmol, 1.3 equiv.). The cooling bath is removed, and the mixture is stirred for 36 h. Upon verifying completion of the reaction, the mixture is filtered over a pad of Celite, and the filtrate is evaporated to dryness under reduced pressure. The oily residue is dissolved in EtOAc and washed with a saturated aqueous solution of NH4Cl. The two layers are separated, and the organic phase is washed twice with saturated aqueous NH4Cl. The combined aqueous phases are extracted three times with EtOAc. The combined organic phases are dried over Na2SO4, filtered, and evaporated to dryness. The crude oil is purified by silica gel column chromatography (PE / EtOAc 80 / 20 to 60 / 40 v / v) to give 3 (3.582 g, 12.6 mmol, 57%) as a pale yellow oil.

[0240] 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 7.89-7.82 (m, 2H), 7.75-7.68 (m, 2H), 3.68 (d, J = 4 Hz, 2H), 3.13-3.05 (m, 1H), 2.98-2.88 (m, 1H), 2.64-2.54 (m, 1H), 1.87-1.78 (m, 1H), 1.76-1.68 (m, 1H), 1.63-1.54 (m, 1H), 1.45-1.34 (m, 2H), 1.30-1.15 (m, 1H).

[0241] 13 C-NMR (75 MHz, CDCl3): δ (ppm) = 168.4, 133.7, 131.9, 123.0, 55.5, 46.4, 43.4, 30.6, 26.1, 24.1. HRMS: ESI: [M+H] + m / z found 245.1290, calc. 245.1290. An ice-cold solution of 3 (3.582 g, 12.6 mmol, 1.0 equiv.) in iPrOH / HO 6 / 1 v / v (175 ml) is treated portionwise with sodium borohydride (665 mg, 17.58 mmol, 5.0 equiv.). The cooling is removed, and the mixture is stirred at room temperature overnight (o / n). The pH is then adjusted to 1 with concentrated HCl. The resulting mixture is filtered, and the filtrate is heated to 80 °C for 2 h. The isopropyl alcohol is removed under reduced pressure, and the resulting aqueous solution is washed five times with diethyl ether, basified with 2 M aqueous NaOH, and extracted with diethyl ether. The combined organic extracts are dried over NaSO, filtered, and evaporated to dryness to give 4 as a pale yellow oil (1.939 g, 12.6 mmol, quantitative yield).

[0242] 1H -NMR (300 MHz, CDCl3): δ (ppm) = 5.99-5.86 (m, 1H), 5.24-5.13 (m, 2H),3.41(ddt, J = 14 Hz, J = 6Hz, J = 1.5 Hz, 1H), 3.04-2.90 (m,3H), 2.74 (dd, J = 13 Hz, J = 3 Hz, 1H), 2.21 (tt, J = 9.6 Hz, J = 3.3H, 2 Hz), 1.80-1.71 (m, 1H), 1.68-1.43 (m, 2H), 1.39-1.25 (m, 3H). 13 C -NMR (75 MHz, CDCl3): δ (ppm) = 157.34, 134.50, 117.71, 58.62, 56.29, 51.90, 42.31, 28.85, 24.92, 23.58.

[0243] [ka]

[0244] HRMS: ESI: [M+H] + m / z found 155.1543, calc. 155.1548. (Compound 6) A solution of 7-aminocephalosporanic acid (5) (2.000 g, 7.345 mmol, 1.0 equiv.) in HO / MeOH (20 mL, 1 / 1 v / v) at −20°C is treated with 10 M NaOH (2 mL), and the resulting mixture is stirred at −20°C for 30 min. The pH is adjusted to 3 with concentrated HCl. The temperature is then brought to 0°C, and the pale yellow precipitate thus formed is filtered off, washed with MeOH, acetone, and ether, and then dried. The desired alcohol 6 is obtained as an off-white powder (1.451 mg, 6.302 mmol, 86%).

[0245] 1H -NMR (300 MHz, DMSO): δ (ppm) = 4.83 (AB system, Δμ = 59 Hz, J = 5 Hz, 2H), 4.21 (AB system, Δμ =17 Hz, J = 13 Hz, 2H), 3.52 (AB system, Δμ = 26 Hz, J =18 Hz, 2H). Spectral data are in accordance with literature values ​​(S. Desgranges, CC Ruddle, LP Burke, TM McFadden, JE O'Brien, D. Fitzgerald-Hughes, H. Humphreys, TP Smyth, M. Devocelle. β-Lactam-host defense peptide conjugates as antibiotic prodrug candidates targeting resistant bacteria. RSC Advances 2012, 2, 2480).

[0246] A solution of benzophenone hydrazone (8) (4.906 g, 25.00 mmol, 1.0 equiv.) in PE (30 mL) is treated with mercury(II) oxide (25.469 g, 25.25 mmol, 1.01 equiv.), and the resulting mixture is stirred at room temperature for 6 h under the exclusion of daylight. The resulting purple mixture is filtered to remove mercury-containing residues, and the resulting solution is evaporated under reduced pressure. The purple liquid containing the target reagent diazodiphenylmethane 9 (4.570 g, 23.53 mmol, 94%) is dissolved in EtOAc (15 mL) and used immediately in the next step without further purification.

[0247] A solution (80 mL) of compound 6 (5.000 g, 21.72 mmol, 1.0 equiv.) in dimethylacetamide (DMAC) was treated with bis(trimethylsilyl)acetamide (BSA) (13.3 mL, 54.29 mmol, 2.5 equiv.), and the resulting mixture was stirred at room temperature for 30 min. The clear solution was cooled to -30 °C, and 2-thiopheneacetyl chloride (3.48 mL, 28.23 mmol, 1.3 equiv.) was added dropwise. The resulting mixture was stirred at -20 °C for 2 h, poured onto ice water, and extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, and their volume was adjusted to 80 mL under reduced pressure. The solution was cooled to 0 °C and treated with a solution of the above diazodiphenylmethane 9 (4.429 g, 22.80 mmol, 1.05 equiv.) in EtOAc until a purple color was obtained. The volume of the resulting solution was reduced in vacuo and then added dropwise to a pentane solution (300 mL), which resulted in the precipitation of a pale yellow solid, which was filtered off to give the doubly protected product 10 (3.957 g, 7.601 mmol, 35% over two steps).

[0248] 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 9.23-9.12 (m, 1H), 7.54-7.46 (m, 11H), 7.01-6.88 (m, 3H), 5.89-5.67 (m, 1H), 5.20-5.02 (m, 2H), 4.21 (d, J = 4 Hz, 1H), 3.78 (s, 2H), 3.62 (s, 1H), 2.95 (s, 1H), 2.79 (s, 1H). Spectral data are in accordance with literature values ​​(S. Desgranges, CC Ruddle, LP Burke, TM McFadden, JE O'Brien, D. Fitzgerald-Hughes, H. Humphreys, TP Smyth, M. Devocelle. β-Lactam-host defense peptide conjugates as antibiotic prodrug candidates targeting resistant bacteria. RSC Advances 2012, 2, 2480).

[0249] [ka]

[0250] (Compound 15) An ice-cold solution of alcohol 10 (1.000 g, 1.921 mmol, 1.0 equiv) in DCM (50 mL) was treated with 4-nitrophenyl chloroformate (775 mg, 3.842 mmol, 2.0 equiv), pyridine (155 μL, 1.921 mmol, 1.0 equiv), and 4-dimethylaminopyridine (DMAP) (24 mg, 0.192 mmol, 0.1 equiv). After stirring at room temperature for 2 h, the mixture was washed with water, dried over NaSO, and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography (PE / EtOAc 70 / 30) to give the desired carbonate 11 as a pale yellow solid (685 mg, 0.999 mmol, 52%).

[0251] 1H -NMR (300 MHz, CDCl3): δ (ppm) = 8.26 (d, J = 9 Hz, 2H), 7.43 (d, J = 7 Hz, 2H), 7.40-7.23 (m, 11H), 7.03-6.98 (m, 1H), 6.96 (d, J = 11 Hz, 2H), 6.65 (d, J = 9 Hz, 1H), 5.89 (dd, J = 9 Hz, J = 5 Hz, 1H), 5.26 (d, J = 13 Hz, 1H), 5.04-4.95 (m, 2H), 3.84 (s, 2H), 3.52 (AB system, Δμ = 55 Hz, J = 19 Hz, 2H).

[0252] Spectral data are in accordance with literature values ​​(S. Desgranges, CC Ruddle, LP Burke, TM McFadden, JE O'Brien, D. Fitzgerald-Hughes, H. Humphreys, TP Smyth, M. Devocelle. β-Lactam-host defense peptide conjugates as antibiotic prodrug candidates targeting resistant bacteria. RSC Advances 2012, 2, 2480).

[0253] A solution of 11 (100 mg, 0.146 mmol, 1.0 equiv.) in DCM (2 mL) was treated with primary amine 4 (25 mg, 0.160 mmol, 1.2 equiv.). The stirred mixture was cooled in an ice bath, and then DIPEA (127 μL, 0.729 mmol, 5.0 equiv.) was added. After 5 min, the ice bath was removed, and the solution was stirred at 30 °C overnight. The mixture was then washed with saturated aqueous Na2CO3 (twice) and NaHCO3 (twice), dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting crude oil was purified by silica gel column chromatography (DCM / MeOH, 99 / 1 v / v) to give the desired carbamate 12 as a yellow oil (39 mg, 0.056 mmol, 38%).

[0254] 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 7.57-7.48 (m, 1H), 7.42-7.22 (m, 11H), 7.09-6.84 (m, 3H), 5.94-5.75 (m, 1H), 5.25-4.94 (m, 3H), 3.94-3.63 (m, 2H), 3.49-3.12 (m, 4H), 3.04-2.74 (m, 2H), 2.53-1.98 (m, 4H), 1.81-1.37 (m, 6H). ESI: [M+H] + m / z found 701.2, calc. 701.2. To a solution of 12 (39 mg, 0.056 mmol, 1.0 equiv.) in dry DCM (1.5 mL) was added 1,3-dimethylbarbituric acid (DMBA) (43 mg, 0.278 mmol, 5.0 equiv.), and the resulting mixture was degassed using an argon flux before being treated with tetrakis(triphenylphosphine)palladium(0)Pd(PPh3)4 (1 mg, 0.0006 mmol, 0.01 equiv.). After completion of the reaction (typically about 4 h), the reaction mixture was evaporated to dryness and purified by silica gel chromatography (DCM / MeOH, 99 / 1 v / v) to give the desired secondary amine 13 as a yellow oil (15 mg, 0.023 mmol, 41%).

[0255] 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 7.46-7.17 (m, 10H), 7.04-6.84 (m, 2H), 5.53-5.32 (m, 1H), 5.08-4.92 (m, 1H), 4.30-4.03 (m, 1H), 4.00-3.88 (m, 2H), 3.79-3.71 (m, 2H), 3.55-3.40 (m, 2H), 3.40-2.85 (m, 4H), 2.85-2.59 (m, 1H), 1.88-1.45 (m, 6H).

[0256] ESI: [M+H] +m / z found 661.2, calc. 661.2. To an ice-cooled suspension of ELF-97 (7 mg, 0.023 mmol, 1.0 equiv.) in dry DCM (1 mL) was added N,N-diisopropylethylamine (DIPEA) (20 μL, 0.113 mmol, 5.0 equiv.) dropwise under argon, followed by a solution of triphosgene (20 mg, 0.068 mmol, 3.0 equiv.) in dry DCM (1 mL). The mixture was stirred at 0 °C for 1 h and then at room temperature overnight. The next morning, the mixture was reduced to dryness under reduced pressure while trapping volatiles in a liquid-nitrogen trap. The latter contents were subsequently destroyed by the addition of ethanolic sodium hydroxide. The resulting chloroformate of ELF-97 (solid residue) was used in the next step without further purification.

[0257] To an ice-cold suspension of the above-prepared chloroformate of ELF-97 (1.0 equiv.) in dry DCM (1 mL) under argon, a clear solution of secondary amine 13 (15 mg, 0.023 mmol, 1.0 equiv.) is added dropwise. Stirring is continued at 0 °C for an additional 30 min, then at room temperature overnight. The reaction mixture is washed three times with saturated NaHCO , and the organic phase is dried over Na SO , filtered, and evaporated under reduced pressure. The crude product is purified by silica gel column chromatography (PE / EtOAc, 8 / 2 v / v) to give the desired protected probe 14 as an off-white solid (12 mg, 0.012 mmol, 53%).

[0258] ESI: [M+H] + m / z found 992.0, calc. 992.3.

[0259] An ice-cold solution of 14 (12 mg, 0.012 mmol, 1.0 equiv.) in dry DCM (1 mL) is treated dropwise with TFA (500 μL, excess) and anisole (7.2 μg, 0.66 mmol, 5.5 equiv.). The stirred mixture is warmed to room temperature and monitored by mass spectrometry to determine the completion point (1-2 h). All nonvolatiles are removed under reduced pressure. The crude residue is subjected to preparative purification by HPLC (ACN / HO 0 / 100-50 / 50 v / v) to obtain the desired compound 15 as a white powder (1.5 mg, 0.0018 mmol, 15%) after lyophilization.

[0260] ESI: [M+H] + m / z found 826.3, calc. 826.1. Example 2: Detection of fluorescence of compound 15 The fluorescence of compound 15 was evaluated in the presence or absence of β-lactamase. The test was performed in a microwell plate (75 μM, 37 °C, 10 U mL -1 ) and the fluorescence was measured over time using a plate fluorometer. The results are shown in Figure 1.

[0261] The results demonstrate that the compounds according to the present invention can detect β-lactamase activity by emitting fluorescence (fluorogenic probe). In the presence of β-lactamase, compound 15 is hydrolyzed, leading to the fragmentation of the compound and the release of a small, highly fluorescent molecule (ELF 97). However, in the absence of enzyme activity, no change in fluorescence was observed over a 2-hour period, thus demonstrating the stability of probe 15 in the (physiological) incubation medium.

[0262] Example 3: Synthesis of Compound 25

[0263] [ka]

[0264] To an ice-cold solution of aldehyde 16 (1 g, 4.3 mmol) in methanol (20 mL) was added sodium borohydride (1 equiv., 4.3 mmol, 164 mg). The solution was stirred at 0 °C for 20 min, after which acetone (2 mL) was added. The volatiles were removed under reduced pressure, and the slide residue was dissolved in ethyl acetate / water (50 mL / 20 mL). The mixture was transferred to a separatory funnel, the aqueous phase was removed, and the organic phase was washed with brine, dried over sodium sulfate, and filtered to give the crude alcohol 17 in substantially pure form as a pale yellow solid.

[0265] The crude alcohol 17 was dissolved in anhydrous DCM (20 mL) and p-nitrophenyl chloroformate (1.05 equiv., 912 mg) was added. The flask was placed in an ice bath and pyridine (2 equiv., 8.6 mmol, 0.7 mL) was added dropwise. The reaction was then stirred at room temperature for 16 h, then diluted with diethyl ether and filtered over Celite. Celite (20 g) was added to the resulting solution and the solvent was removed under reduced pressure. The Celite-adsorbed crude mixture was subjected to silica gel flash chromatography (petroleum ether / ethyl acetate 8:2) to afford the activated carbonate 18 (1.06 g, 2.67 mmol, 62% over two steps) as a pale yellow solid.

[0266] 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 8.35 - 8.16 (m, 2H), 7.85 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.41 - 7.33 (m, 2H), 5.31 (s, 2H), 1.35 (s, 12H).

[0267] [ka]

[0268] To a solution of N-methyl-N'-allyl-aminomethylpiperidine 19 (1.0 equiv., 0.89 mmol, 240 mg) in anhydrous dichloromethane (5 mL) was added carbonate 19 (1.05 equiv., 0.93 mmol, 373 mg) and potassium carbonate (5 equiv., 4.45 mmol, 615 mg). MS (M+H + Upon completion of the reaction, as determined by HPLC (HPLC yield: 20 = 530.4), the reaction mixture was diluted with a 1:1 mixture of petroleum ether and diethyl ether (15 mL), filtered through Celite, and the Celite was rinsed with 100 mL of a 1:1 mixture of PE / EtO. The filtrate was concentrated under reduced pressure to give essentially pure carbamate 20 as a pale yellow solid, which was used directly in the next step. Alternatively, it could be purified by silica gel flash chromatography using EtO as the eluent for characterization.

[0269] 20: 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 7.79 (d, J = 7.7 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 5.94 - 5.58 (m, 1H), 5.25 - 4.96 (m, 4H), 3.81 - 3.02 (m, 8H), 2.97 (s, 3H), 2.70 (brm, 2H), 2.39 (brm, 1H), 1.44 (s, 9H), 1.33 (s, 12H).

[0270] Crude 20 was placed in a round-bottom flask and 1,3-dimethylbarbituric acid (3 equiv., 2.67 mmol, 416 mg) was added, followed by DCM (8 mL). The solution was purged with argon for 10 min, Pd(PPh3)4 (1 mol%, 10 mg) was added, and the mixture was stirred at room temperature under argon for 20–60 min. After completion of the reaction as determined by MS, the solvent was evaporated and the crude mixture containing 21 was used with further purification in the next step.

[0271] ELF-97 (1.3 equiv., 1.157 mmol, 355 mg) was placed in a round-bottom flask under argon, followed by triphosgene (1.3 equiv., 1.157 mmol, 343 mg) and DCM (10 mL). The solution was cooled to 0 °C, and pyridine (6 equiv., 0.43 mL) was added dropwise. The ice bath was removed, and the solution was stirred at room temperature for 20 min. The volatiles were removed under reduced pressure, DCM (5 mL) was added, and the volatiles were removed again under reduced pressure. The resulting solid ELF chloroformate was suspended in DCM (5 mL), the flask was cooled in an ice bath, and crude product 21 (1 equiv., 0.89 mmol) was added in DCM (10 mL) followed by DIPEA (3 equiv., 0.47 mL). The reaction was stirred at room temperature for 3 h until no 21 was detected by MS. The reaction was then diluted with EtO, cooled to 0 °C, and allowed to settle. Aqueous NaHCO (10 mL) was added. The mixture was transferred to a separatory funnel, and the organic phase was washed successively with water and brine, dried over NaSO, filtered, and concentrated under reduced pressure. The solid residue was partially dissolved in EtO and filtered through a silica pad pretreated with 2.5 wt% triethylamine to remove excess unreacted ELF-97, and the silica was rinsed with EtO (200 mL). The solution was then concentrated under reduced pressure and subjected to silica gel flash chromatography (eluent DCM / EtO 1:0 to 3:7) to afford pure boronate 22 as a glassy pale yellow solid (536.6 mg, 0.65 mmol, 73%) (M+H + 22=822.4).

[0272] twenty two: 1H -NMR (300 MHz, CDCl3): δ (ppm) = 8.27 - 8.20 (m, 1H), 8.01 - 7.88 (m, 1H), 7.76 (appt, J = 7.5 Hz, 1H), 7.70 (d, J = 1.9 Hz, 2H), 7.39 - 7.26 (m, 4H), 7.18 - 7.01 (m, 1H), 5.23 - 4.76 (m, 2H), 4.70 - 4.37 (m, 1H), 4.20 - 3.81 (m, 3H), 3.70 (m, 1H), 3.38 - 3.11 (m, 2H), 3.13 - 2.68 (m, 6H), 1.46 (m, 9H), 1.39 - 1.26 (m, 12H).

[0273] [ka]

[0274] Using published conditions for coupling aryl-pinacol-boronic esters with intermediate 23 (Chem. Eur. J. 2020, 26, 3647-3652), compound 22 can be coupled with enol triflate 23 to give compound 24, which can be deprotected using the conditions described in the above reference to give compound 25.

[0275] Example 4: Synthesis of Compound 32

[0276] [ka]

[0277] Trisopropylacetylene 26 (1 equiv., 20 mmol, 3.64 g) was placed in a dry round-bottom flask under argon and dissolved in anhydrous THF (40 ml). The flask was placed at -78 °C, and n-BuLi (1.5 equiv., 30 mmol) was added dropwise over 10 min. The flask was then placed at 0 °C for 30 min. Paraformaldehyde (3 g) was then added in one proton ion, and the reaction was stirred at room temperature for 14 h. The mixture was cooled to 0 °C, and saturated aqueous NH4Cl was added. The mixture was extracted with Et2O, washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The crude oil was subjected to silica gel flash chromatography (EP / Et2O 1:0 to 1:1) to give pure alcohol 27 (3.3 g, 15.5 mmol, 77%) as a colorless oil.

[0278] 27: 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 4.30 (d, J = 5.6 Hz, 2H), 1.07 (s, 21H). Alcohol 27 (3.3 g, 15.5 mmol, 1 equiv.) was dissolved in anhydrous DCM (30 mL) and p-nitrophenyl chloroformate (1.05 equiv., 3.29 g) was added. The flask was placed in an ice bath, and pyridine (2.5 equiv., 38.75 mmol, 3.2 mL) was added dropwise. After stirring at room temperature for 16 h, the mixture was diluted with diethyl ether, filtered through Celite, and rinsed with EtO. The solvent was removed under reduced pressure, and the crude mixture was subjected to silica gel flash chromatography (eluent EP / CHCl 1:0 to 0:1) to afford activated carbon-boronate 28 (5.56 g, 14.7 mmol, 95%) as a colorless oil.

[0279] 28: 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 8.42 - 8.18 (m, 2H), 7.45 - 7.34 (m, 2H), 4.85 (d, J = 5.4 Hz, 2H), 1.07 (s, 21H).

[0280] [ka]

[0281] To a solution of N-methyl-N'-allyl-aminomethylpiperidine 19 (1.0 equiv., 0.868 mmol, 233 mg) in anhydrous dichloromethane (5 mL) was added carbonate 28 (1.1 equiv., 0.96 mmol, 362 mg) and potassium carbonate (5 equiv., 4.4 mmol, 621 mg). MS (M+H + Upon completion of the reaction, as judged by HCl (29 = 508.7), the reaction mixture was diluted with a 1:1 mixture of petroleum ether and diethyl ether (15 mL), filtered over Celite, and the Celite was rinsed with 100 mL of a 1:1 mixture of PE / EtO. The filtrate was concentrated under reduced pressure to give essentially pure carbamate 29 as a pale yellow oil, which was used directly in the next step.

[0282] The crude carbamate 29 was placed in a round-bottom flask and 1,3-dimethylbarbituric acid (3 equiv., 2.6 mmol, 406 mg) was added, followed by DCM (8 mL). The solution was purged with argon for 10 min, Pd(PPh3)4 (1 mol%, 10 mg) was added, and the mixture was stirred at room temperature under argon for 20–60 min. In parallel, ELF-97 (1.3 equiv., 1.13 mmol, 347 mg) was reacted with triphosgene (1.3 equiv., 1.13 mmol, 335 mg) and pyridine (6 equiv., 5 mmol, 0.41 mL), followed by sequential evaporation / dissolution in DCM and subsequent placement in DCM (10 mL) in an ice-cooled plating bath, to prepare ELF chloroformate, as in Example 3. After completion of the deallylation reaction, as determined by MS, the solution containing deallylated 29 was cannulated onto a cold solution of ELF chloroformate in DCM, and the flask was rinsed twice with DCM (2 + 2 mL). The reaction was stirred at room temperature for 14 h, placed in an ice bath, diluted with EtO (50 mL), and saturated aqueous NaHCO (20 mL) was added. 30 was extracted with EtO, and the organic phase was washed with water, brine, dried over NaSO, filtered, and concentrated under reduced pressure. Purification of the crude mixture, as in Example 3, by initial filtration over an EtN-impregnated silica pad followed by flash chromatography on silica gel (DCM / EtO 0:1 to 1:0) afforded pure 30 as a glassy, ​​colorless solid (456 mg, 0.57 mmol, 67%, 3 steps). (M+H + 30=799.3).

[0283] 30: 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 8.32 - 8.17 (m, 1H), 8.02 (d, J = 2.7 Hz, 1H), 7.72 (pseudoq, J = 2.5, 2.1 Hz, 2H), 7.56 - 7.45 (m, 1H), 7.25 - 7.08 (m, 1H), 4.79 - 4.29 (m, 3H), 4.26 - 3.58 (m, 5H), 3.36 - 2.70 (m, 7H), 1.48 (s, 9H), 1.05 (s, 21H). Pure 30 (0.47 mmol, 376 mg) was placed in a flask, dissolved in technical-grade THF (15 mL), and placed in an ice bath. Next, a solution of TBAF (1 M in THF, 1.02 equiv., 479 μL) was added dropwise, and the reaction was stirred at room temperature for 16 h. The flask was placed in an ice bath, and saturated aqueous NaHCO3 (10 mL) was added dropwise. 31 was extracted with Et2O, washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification of the crude mixture by flash chromatography on silica gel (DCM / Et2O 0:1 to 1:0) afforded pure 31 as a colorless glassy solid (294 mg, 0.46 mmol, 95%). (M+H + 31=644.3).

[0284] 31: 1 H -NMR (300 MHz, CDCl3): δ (ppm) = 1 H NMR (300 MHz, Chloroform-d) δ 8.28 - 8.16 (m, 1H), 8.02 - 7.89 (m, 1H), 7.80 - 7.63 (m, 2H), 7.54 -7.43 (m, 1H), 7.25 - 7.12 (m, 1H), 4.87 - 4.35 (m, 3H), 4.33 - 3.54 (m, 4H), 3.31 - 3.10 (m, 1H), 3.10 - 2.68 (m, 6H), 2.46 - 2.22 (m, 1H), 1.47 (s, 9H).

[0285] [ka]

[0286] Compound 31 can undergo a Pd / Cu co-catalyzed Sonogashira coupling to give a coupled intermediate that can be deprotected to give compound 32, as in Example 3. [Brief explanation of the drawings]

[0287]

Claims

1. Compounds of formula (I): 【Chemistry 1】 Where: -W is -O- or -NR １３ - and R １３ is C １ -C ４ is an alkyl or hydrogen atom; -R １ is selected from the group consisting of fluoresceins, coumarins, cyanines, phenoxazines, and acridinones; or WR １ is an aromatic group -OR １ and: 【Chemistry 2】 Where: -X ２ is an oxygen atom, and X １ Ha-NH ２ , -OH, -SH, C １ -C ２０ Alkyl, C6-C ２４ Aryl, C ２ -C ６ alkenyl, —O—(C １ -C ２０ alkyl), —O-phenyl, —NH—(C １ -C ２０ alkyl), —NH-phenyl, —S—(C １ -C ２０ alkyl), or —S—(C6-C ２４ aryl groups), wherein said alkyl, aryl, alkenyl, and phenyl groups are optionally substituted; Or X ２ represents a nitrogen atom, and X represents CH, O, S, N, or NH. １ and optionally substituted C ５ -C ２４ forming a heteroaryl; － 【Transformation 3】 is an optionally substituted phenyl group, an optionally substituted naphthyl group, or 【Chemistry 4】 Selected from the group is optionally substituted; X ３ represents S, O or NRd, where Rd is a hydrogen atom or C １ -C ４ represents an alkyl group; -R ２ , R ３ and R ４ is defined as either: ○ R ２ is C １ -C ４ alkyl, and R ３ is C １ -C ４ alkyl or hydrogen atom, R ４ is C １ -C ４ Is alkyl; ○ or R ３ is C １ -C ４ alkyl or hydrogen atom, R ２ and R ４ and are bonded to each other, and R ２ From R ４ In the direction of -(CH ２ ) ｐ -Y ｑ - (CH ２ ) ｒ - forming chains, ● Y is O, NR １４ , N(R １４ ) ２ ＋ or S, ● p = 0, 1, 2, 3, 4 or 5; ● q = 0 or 1, ● r = 0, 1, 2, 3, 4 or 5; ● p + q + r = 3, 4, 5, or 6; ● Each R １４ represents a hydrogen atom; ○ or R ２ is C １ -C ４ alkyl, and R ３ and R ４ and are bonded to each other to form an aliphatic carbocyclic ring together with the carbon atoms to which they are attached; -R ５ and R ６ are the same or different, and each independently represents a hydrogen atom, C １ -C ４ Alkyl, or C6-C １０ represents aryl; -R ７ is a hydrogen atom or C １ -C ４ Alkyl and C １ -C ４ is a group selected from alkoxy; -R ８ represents a hydrogen atom; -V represents an oxygen atom or a sulfur atom; - n is 0 or 1; -Z is -S-, -SO-, or -CR ９ R １０ - and R ９ and R １０ are the same or different and each independently represent a hydrogen atom or C １ -C ４ represents alkyl; -Q is H, a cation or R16, where R16 is C １ -C ６ alkyl, optionally substituted with aryl or O—(CO)—R, where R is independently H, C １ -C ６ Alkyl and C ３ -C ６ cycloalkyl; -R １１ teeth, 【Transformation 5】 is selected from -X is a bond, or 【Transformation 6】 represents a group selected from

2. The compound (I) according to claim 1, which is of formula (Ia): 【Transformation 7】 Here, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R １１ , Q, W, X and V are as defined in claim 1.

3. The compound (I) according to claim 1, which is of formula (Ib): 【Transformation 8】 Here, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ , R １１ , Q, W, X and V are as defined in claim 1.

4. -OR １ is an aryloxy species and corresponds to one of the following structures (A2), (A3) or (A4): － 【Chemistry 9】 where: ○ T is -NH-C(O)-, -S-, -O-, -NH-, -N(C １ -C ２０ alkyl)- or -N(C6-C ２４ aryl)-; Re is a hydrogen atom or a group selected from the group consisting of -CN or -COORh, and Rh is C １ -C ４ Re represents an alkyl group, or Re is -CONRiRj, where Ri and Rj are the same or different, and Ri and Rj are hydrogen atoms or C １ -C ４ Re represents an alkyl group, or Re is —CF ３ , C ２ -C ６ alkenyl, or heteroaryl, wherein said heteroaryl and alkenyl are optionally substituted; Rf is a hydrogen atom, a chlorine, bromine, iodine or fluorine atom, -OH, -NH ２ , -NRkRI, -NHRk or -ORk, where Rk and RI are the same or different, and Rk and RI are each independently C １ -C ４ represents an alkyl group; or Re and Rf are each linked to one another to form a hydrocarbon chain containing 4 or 5 members, which may be saturated or unsaturated, substituted or unsubstituted, and which may be separated by one or more heteroatoms selected from among N, S and O; － 【Chemistry 10】 where: ○ T' is -NH ２ , —OH, C6-C ２４ Aryl group, C １ -C ４ an alkyl group, —SH, —NHR′g, —OR′g, —NR′gRh′, —SR′g, or an optionally substituted C ２ -C ６ alkenyl group, or heteroaryl, R'g and Rh' are the same or different, C １ -C ４ Alkyl group or C6-C ２４ represents an aryl group; R'e is a hydrogen atom or a group selected from the group consisting of -CN or -COOR'i, and R'i is C １ -C ４ R'e represents an alkyl group, or R'e is -CONR'jR'k, where R'j and R'k are the same or different and are a hydrogen atom or a C １ -C ４ represents an alkyl group, or R'e is -CF ３ or a 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-benzimidazolyl, 4-pyrimidinon-2-yl or quinazolinon-2-yl group; ○ R'f is a hydrogen atom, chlorine, bromine, iodine, fluorine atom, -OH, -NH ２ , -NR'IR'm, or OR'I, where R'I and R'm are the same or different, and C １ -C ４ represents an alkyl group; or R'e and R'f are each linked to each other to form a hydrocarbon chain containing 4 or 5 members, which may be saturated or unsaturated, substituted or unsubstituted, and which may be separated by one or more heteroatoms selected from among N, S and O; － 【Chemistry 11】 where: -X' ２ is an oxygen atom, and X' １ Ha-NH ２ , -OH, -SH, C １ -C ２０ Alkyl, C6-C ２４ Aryl, C ２ -C ６ alkenyl, —O—(C １ -C ２０ alkyl), —O-phenyl, —NH—(C １ -C ２０ alkyl), —NH-phenyl, —S—(C １ -C ２０ alkyl), or —S—(C6-C ２４ aryl groups), wherein the alkyl, aryl, alkenyl, and phenyl groups are optionally substituted; Or, X' ２ represents a nitrogen atom, and X' represents CH, O, S, N or NH. １ and optionally substituted C ５ -C ２４ forming a heteroaryl; － 【Chemistry 12】 is an optionally substituted C6-C １０ Aryl or C ５ -C １０ represents heteroaryl.

5. The compound (I) according to any one of claims 1 to 4, which is represented by formula (Ic): 【Chemistry 13】 Here, R １ , R １１ , Z, Q, X and n are as defined in any one of claims 1 to 4, and Y is -CH ２ -, -NR １４ -, or -N(R １４ ) ２ ＋ -, and each R １４ represents a hydrogen atom.

6. The compound (I) according to any one of claims 1 to 5, which is represented by formula (Id): 【Chemistry 14】 Here, R １ , R １１ , X and Q are as defined in any one of claims 1 to 5, and Y is as defined in claim 5.

7. The compound (I) according to any one of claims 1 to 6, which is represented by formula (Ie): 【Chemistry 15】 Here, R １ , R ９ , R １０ , R １１ , X and Q are as defined in any one of claims 1 to 5, and Y is as defined in claim 5.

8. A method for detecting β-lactamase in vitro or ex vivo, comprising the steps of: - placing the sample to be analyzed in contact with a compound (I) according to any one of claims 1 to 3, -C(=V) and NR ７ and subsequently cleaving the covalent bond between —C(O)—WR １ is cleaved and bound, and HWR １ applying appropriate conditions to allow the formation of a fluorescent precipitate by resulting in the release of - quantitative or qualitative analysis of fluorescent precipitates, - Quantitative or qualitative analysis of the fluorescent precipitate is correlated with the presence or absence of β-lactamase in the sample.

9. A method for detecting antibiotic-resistant bacteria in vitro or ex vivo, comprising the steps of: - placing the sample to be analyzed in contact with a compound (I) according to any one of claims 1 to 3, -C(=V) and NR ７ and subsequently cleaving the covalent bond between —C(O)—WR １ is cleaved and bound, and HWR １ applying appropriate conditions to allow the formation of a fluorescent precipitate by resulting in the release of - quantitative or qualitative analysis of fluorescent precipitates, - Quantitative or qualitative analysis of the fluorescent precipitate is correlated with the presence or absence of antibiotic-resistant bacteria in the sample.

10. A kit for detecting β-lactamase, comprising a compound according to any one of claims 1 to 7.

11. A device for detecting β-lactamase, comprising a compound according to claims 1 to 7.

12. The compound (I) according to any one of claims 1 to 7 for the in vivo detection of β-lactamase in humans.

13. Compound of formula (II): 【Chemistry 16】 Where: -R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R １１ , Z, n, Q, X and V are as defined in any one of claims 1 to 7; -R １２ represents a hydrogen atom or a group selected from the group consisting of a tert-butoxycarbonyl group, a fluorophenylmethoxycarbonyl group, an aryloxycarbonyl group, and a 2,2,2-trichloroethoxycarbonyl group.

14. A process for preparing compounds of formula (I) according to claims 1 to 7, comprising the steps of: -R １２ represents a hydrogen atom, reacting a compound (II) according to claim 13 with a compound of formula (III) 【Chemistry 17】 Here, R １ is as defined in any one of claims 1 to 7, wherein M represents a group selected from a halogen atom, an imidazolyl group, a triazolyl group, and para-nitrophenoxy: - Obtaining compound (I) by addition reaction of said compound (II) to compound (III).

15. X is [Chemistry 18] A process for preparing compounds of formula (I) according to claims 1 to 7, comprising the steps of: a compound of the formula (VII): 【Chemistry 19】 with a compound of formula (VIII) 【Chemistry 20】 and reacting said compound (VII) with compound (VIII) to obtain compound (I), Here, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , V, R １１ , Q, W, Z and n are as defined in any one of claims 1 to 7, and LG represents a group selected from halogen and trifluoromethanesulfonate.

16. X is 【Chemistry 21】 A process for preparing compounds of formula (I) according to claims 1 to 7, comprising the steps of: a compound of the formula (VII): 【Chemistry 22】 with a compound of formula (IX) 【Chemistry 23】 and reacting said compound (VII) with compound (IX) to obtain compound (I), Here, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , V, R １１ , Q, W, Z, and n are as defined in any one of claims 1 to 7, LG represents a group selected from halogen and trifluoromethanesulfonate (triflate), R １５ represents OH, or each R １５ are bonded to each other and together with the B atom to which they are attached form a heterocycle having 5 to 10 ring atoms.

17. X is 【Chemistry 24】 A process for preparing compounds of formula (I) according to claims 1 to 7, comprising the steps of: a compound of the formula (VII): 【Chemistry 25】 with a compound of formula (X) 【Chemistry 26】 - reacting said compound (VII) with compound (X) to obtain compound (I); Here, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , V, R １１ , Q, W, Z and n is as defined in any one of claims 1 to 10, LG represents a group selected from halogen and trifluoromethanesulfonate (triflate), R １５ represents OH, or each R １５ are bonded to each other and together with the B atom to which they are attached form a heterocycle having 5 to 10 ring atoms.

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