FR3101875A1 - FLUORESCENT ANALOGUE OF VANCOMYCIN - Google Patents

Abstract

The present invention relates to a novel fluorescent analogue of vancomycin and its use for the detection of Gram + bacteria, in particular in a rapid diagnostic method for identifying the presence of Gram + bacteria in vivo, for example infectious keratitis, or even in vitro on fluids or tissues taken from a subject, human or animal.

Description

FLUORESCENT ANALOG OF VANCOMYCIN

FIELD OF THE INVENTION

The present invention relates to a novel fluorescent analogue of vancomycin and its use for the detection of Gram+ bacteria, in particular in a rapid diagnostic method for identifying the presence of Gram+ bacteria in vivo , for example infectious keratitis, or else in vitro on fluids or tissues taken from a subject, human or animal.

BACKGROUND OF THE INVENTION
Vancomycin is a well-known antibiotic whose spectrum is limited to Gram-positive bacteria (staphylococci, streptococci, for example). It inhibits the synthesis of peptidoglycans constituting their wall. Colored or fluorescent derivatives of vancomycin are also known, used in particular as indicator probes for the presence of colonies of Gram+ bacteria (Kocaoglu & Carlson, 2016, Gao & al., 2006). Mention will also be made of the product BODIPY (registered trademark) FL Vancomycin marketed by the company ThermoFisher Scientific as a laboratory reagent.

These state-of-the-art analogues present stability and purification problems and none of these analogues is suitable or indicated for use in a simplified in vitro or in vivo diagnostic method, and in particular in vivo , and in particular to rapidly detect infectious keratitis.

BRIEF DESCRIPTION OF THE INVENTION
The invention relates to a new fluorescent analogue of vancomycin of formula BL-CO-V in which
B is a fluorophore derived from BODIPY,
L is a link arm, and
-CO-V is vancomycin, the bond of L-CO-V being a bond on the carboxylic acid residue of vancomycin, in particular an amide bond.
The invention also relates to a composition, in particular a sterile composition, comprising said fluorescent analogue of vancomycin and a vehicle suitable for its use, in particular for its topical application.

The invention also relates to a method for detecting the presence of Gram+ bacteria in a biological sample, said method comprising adding an appropriate quantity of said fluorescent analog of vancomycin to the biological sample and then observing the persistence of an emission fluorescence, marker of the presence of Gram+ bacteria in the biological sample. The persistence of this fluorescence emission comes from the fact that the fluorescent analogue of vancomycin has bound to the cell wall of the Gram+ bacteria present.

represents a “green” derivative of vancomycin according to the invention.

represents the diagram for the preparation of the derivative of FIG. 1 according to the invention.

represents a “red” derivative of vancomycin according to the invention.

represents the diagram for the preparation of the derivative of FIG. 3 according to the invention.

represents the Bodipy Compound of the COMBO AP343A (state of the art).

DETAILED DESCRIPTION OF THE INVENTION

The novel fluorescent analog of vancomycin B-L-CO-V according to the invention comprises a fluorophore derived from BODIPY (B) linked to the carboxylic acid group of vancomycin (V) by a linker arm (L) also called "Linker".

The fluorophores derived from BODIPY (dipyrromethene-boron) are known to those skilled in the art. Mention will be made in particular of the fluorophores described in application WO 2006/0875459 and in particular hydrophilic derivatives as described in applications WO 2010/076516 and WO 2014/013205. These are in particular compounds B of formula I

(I)

in which

S ¹ represents a group of formula -CΞC-L'-A, where L' is a linking group which is a single bond (in which case S ¹ is a group of formula -CΞC-A); or a divalent hydrocarbon group chosen from the group consisting of linear or branched alkylenes, optionally comprising one or more oxygen, nitrogen or sulfur within their chain (such as poly(ethylene glycol), poly(ethoxy) or poly(propoxy ), For example) ; linear or branched alkenylenes; linear or branched alkynylenes, and arylenes; or a divalent hydrocarbon-based chain consisting of a sequence of at least two divalent hydrocarbon-based groups of the aforementioned type; and A is a polar functional group selected from sulfonate, sulfate, phosphate, ammonium, carboxylate, hydroxyl, phosphonate, alkylammonium sulfate and polyoxyethylene groups;

S ² represents a group -CΞC-L'-A identical to or different from S ¹ , where L' and A have the above meanings; F; H; or a hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by one or more oxygen, nitrogen or sulfur atoms, optionally cyclized in whole or in part, optionally aromatic, and optionally functionalized, and

each of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ or R ⁷ denotes, independently of the others, H or a hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by one or more atoms oxygen, optionally cyclized in whole or in part, optionally aromatic and optionally functionalized, it being understood that all or part of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ , can be linked together to form a bridged form (i.e. at least two of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ can together form a ring with the carbons to which they are connected) .

According to a particular embodiment of the invention, S ¹ and S ² are identical or different, chosen from F, or the alkyne derivatives of formulas

n being 1, 2, or 3, in particular 1. Advantageously, S ¹ and S ² are identical.

The bond of the BODIPY derivative with the linker arm L takes place via one of the substituents R ¹ to R ⁷ , in particular via one of R ¹ , R ³ or R ⁶ , more particularly R ¹ . In particular, a substituent as defined above carrying a carbonyl group will be chosen for this bond, the bond with the linker taking place via an ester, amide or thioester bond.

According to another particular embodiment of the invention, the connection of the linker with the substituent of BODIPY is done by an alkynyl bond (-CΞC-).

In particular, the BL- (fluorophore-linker) bond is made via one of R ¹ , R ³ or R ⁶ , more particularly R ¹ of formula -Ar-CO-L- or -Ar-L-, in which Ar is a divalent aromatic hydrocarbon radical, with 1 to 4 fused or unfused rings, having in particular from 4 to 16 carbon atoms, preferably from 4 to 10 carbon atoms, and which may contain in their ring nitrogen, sulfur or oxygen (heterocycles). The aryl groups are optionally substituted by one or more alkyl or alkoxy groups, in particular by methyl or methoxy. Among the aryl radicals, mention may in particular be made of the phenyl, naphthyl, pyrenyl, perylenyl, anthracenyl, thienyl, pyrolyl, pyridinyl or furanyl radical, in particular phenyl or thienyl.

R ² and R ⁵ are generally identical, in particular chosen from H, methyl or ethyl groups.

R ³ , R ⁴ , R ⁶ and R ⁷ , which are identical or different, are generally chosen from H, alkyl, in particular methyl, ethyl or propyl groups, and aryls of formula

in which, R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and R ¹³ represent, independently of one another, the H, alkyl or alkoxy groups comprising from 1 to 10 carbon atoms, in particular methyl, ethyl, propyl .

According to a particular mode of the invention, R ³ and R ⁶ are identical and/or R ⁴ and R ⁷ are identical.

Among the preferred BODIPY derivatives, the derivatives described in applications WO 2006/087459, WO 2010076516 and WO 2014/013205 and their examples will be used.

Linker L is a bifunctional derivative that allows binding to both the BODIPY derivative B and the carboxylic acid group of vancomycin V. It can be represented by the formula -Z ¹ -R ⁸ -Z ² -.

R ⁸ is a divalent hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by one or more oxygen atoms, optionally cyclized in whole or in part. R ⁸ is more particularly chosen from linear or branched divalent alkylene chains, optionally comprising one or more oxygen, nitrogen or sulfur atoms within their chain (such as poly(ethylene glycol), poly(ethoxy) or poly( propoxy), for example); linear or branched alkenylenes; linear or branched alkynylenes, and arylenes; or a divalent hydrocarbon-based chain consisting of a sequence of at least two divalent hydrocarbon-based groups of the aforementioned type. Mention will more particularly be made of C ₃ to C ₆ alkylene groups, in particular ethylene, 1,3-propylene, 1,4-butylene and 1,5-pentylene groups, optionally substituted, or alternatively -alk ₁ -( O-alk ₂ ) _n -O-alk ₃ - n ranging from 1 to 10, in particular from 1 to 5, more particularly 1, 2 or 3, each alk1 and alk3 group being independently of one another chosen from a methylene or ethylene group and alk2 being an alkylene chosen from ethylene and propylene (1,2- or 1,3-).

Z ¹ is the group allowing the bond with the BODIPY derivative described above, either directly or via a carbonyl group carried by one of the substituents as described above. Z ¹ is particularly chosen from the groups -CΞC-, -O-, -NH- and -S-, more particularly -CΞC- and -NH-.

Z ² is the group allowing the bond with the carboxylic acid of vancomycin. Mention will be made in particular of the groups -O-, -NH- and -S-, more particularly -NH to form an amide bond.

Preferably Z ¹ and Z ² are -NH- groups. The connecting arm L is in particular chosen from the following divalent groups

-HN-(CH ₂ -CH ₂ ) _n -CH ₂ -NH-

-HN-CH ₂ -(O-CH ₂ -CH ₂ ) _n -O-CH ₂ -NH-, and

-CΞC-( _CH2 - _CH2 ) _n - _CH2 -NH-

n being an integer from 1 to 6, preferably 1, 2 or 3, more preferably 1.

The invention also relates to a BLH intermediate for the preparation of an analogue of vancomycin, B and L being defined above. In particular, the BLH intermediate according to the invention is represented by formula II, in which one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ is a substituent on which the connecting arm L is attached, and more particularly of the formula -Ar-CO-Z ¹ -R ⁸ -Z ² -H or -Ar-Z ¹ -R ⁸ -Z ² -H in which Ar, Z ¹ , R ⁸ and Z ² are defined above. Preferably, the BL bond takes place via the R ¹ substituent.

According to a particular mode, the intermediate is represented by the formula II

(II)

wherein L, S ¹ , S ² , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are described above.

According to a preferred embodiment, R ¹ is a substituent of formula –Ar-CO-Z ¹ -R ⁸ -Z ² -H.

A representative example of preferred intermediates is represented by formula III

(III)

where Z is -Z ¹ -R ⁸ -Z ² - and Z ¹ , Z ² , S ¹ , S ² , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are defined plus high.

The fluorescent analogue of vancomycin according to the invention is prepared by the usual methods of coupling the various parts which constitute it, in particular by coupling the intermediate B-L-H above with vancomycin so as to graft the intermediate onto the carboxylic acid group of vancomycin. For these coupling methods, Silverman & al. (2016).

The invention also relates to a composition, in particular a sterile composition, comprising said fluorescent analogue of vancomycin and a vehicle suitable for its use, in particular for its topical application.

The composition, its constituents, in particular the vehicle, and its physicochemical properties, in particular viscosity and transparency, will depend on the use of the composition.

The composition is advantageously a composition transparent to the radiation of the fluorophore. They will advantageously be aqueous or hydroalcoholic solutions. These solutions can be packaged in a multidose form, for repeated use, or single dose for a single use.

For topical application to the skin or mucous membranes of a subject in order to rapidly identify the presence or absence of Gram+ bacteria, the composition will be a sterile composition which has the characteristics of a pharmaceutical composition, in particular as regards sterility and safety with respect to the subjects.

The composition for topical use according to the invention may comprise the usual additives of this type of composition, such as thickening agents or even pH regulators, buffers or even acids or bases, salts, or antioxidants or other agents for stability or more conservatives. Said additives will be chosen so as not to interfere in a substantial manner, alone or in combination, with the radiation of the fluorophore but also with the ability of vancomycin to bind to Gram+ bacteria.

According to a particular embodiment of the invention, the composition for topical use and in particular for application in the eye, does not include preservatives.

Viscosity of solution will depend on site of application. Thus, for application in the eye, the composition will advantageously be in the form of eye drops. Its viscosity can range from 1.5-2mPa.s (low viscosity) to 10 ⁵ -10 ⁶ mPa.s (high viscosity) through a semi-viscosity (viscosity 10 to 100 times higher than low viscosity eye drops )

For application to the skin, the composition is advantageously a solution with a viscosity of 1,000 to 100,000 mPa.s.

The composition for topical use according to the invention advantageously comprises from 0.01 to 3% by weight of fluorescent analogue of vancomycin of formula B-L-CO-V, preferably from 0.5 to 1% by weight, and from 'water.

The invention also relates to a method for detecting the presence of Gram+ bacteria in a biological sample, said method comprising adding an appropriate quantity of said fluorescent analog of vancomycin to the biological sample and then observing the presence of an emission fluorescence, marker of the presence of Gram+ bacteria in the biological sample. The persistence of this fluorescence emission comes from the fact that the fluorescent analogue of vancomycin has bound to the cell wall of the Gram+ bacteria present.

This detection can be made in vitro , on biological samples previously taken from humans or animals, or even in vivo on a human or animal subject, on sites, skin or mucous membranes where the presence of Gram+ bacteria responsible for pathologies.

Mention will be made in particular of Gram+ bacteria sensitive to vancomycin and capable of being pathogenic for humans and animals, such as, for example, the Staphylococcus genus, in particular S. aureus or coagulase-negative staphylococci, the Streptococcus genus, in particular S. pneumoniae , S. viridans , S. pyogenes or S. agalactiae , but also Corynebacterium spp , Propionibacterium acnes or Actinomyces spp .

Thus, the invention relates to a method for detecting the presence or absence of Gram+ bacteria liable to be responsible for infectious keratitis in a human or animal subject, which consists in applying to the eye of the subject an appropriate quantity of fluorescent analogue of vancomycin then to observe the persistence of a fluorescence emission, a marker of the presence of Gram+ bacteria.

The observation of the persistence of the fluorescence emission is done by usual methods well known to those skilled in the art.

Depending on the application of the compound, the fluorescent analogue of vancomycin can be detected using standard laboratory fluorescence microscopes in the case of in vitro use, or using a suitable medical device that can be used in the clinic, equipped with an excitation light source like the ophthalmology slit lamp used in current practice for the observation of fluorescein or the in vivo confocal fluorescence microscope used in dermatology but also suitable for ophthalmology.

The invention also relates to the use of a fluorescent analog according to the invention to control the absence of Gram + bacteria, and in particular of pathogenic Gram + bacteria on food. It relates to a method for detecting the presence or absence of Gram+ bacteria liable to contaminate food, which consists in applying to said food, testing an appropriate quantity of fluorescent analogue of vancomycin and then observing the persistence of a fluorescence emission, marker of the presence of Gram+ bacteria.

The images obtained may be post-processed by software or an algorithm (management of contrasts, spatial filtering) in order to eliminate background noise or any non-specific coloring generated by the analog, in particular by using spatial filtering passes Butterworth type top.

The auto-fluorescence of biological tissues or cells (composed mainly of water and hemoglobin) can generate a strong background noise of auto-fluorescence, thus masking a real fluorescence signal. To rule out such auto-fluorescence, the usual methods of extinguishing this auto-fluorescence can be used. For example, we can cite the addition of Trypan blue, a dye known to extinguish the auto-fluorescence of biological tissues or cells.

EXAMPLES

Example 1: Preparation of a "green" analogue of vancomycin - VancomBod FL

Fluorophore: 4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1, 2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzoic acid

The steps in the preparation of this green analog are shown in Figure 2.

Step 1: Preparation of 2,5-dioxopyrrolidin-1-yl 4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-1,3,7,9-tetramethyl -5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzoate

A mixture of 4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-1,3,7,9-tetramethyl-5H-4l4,5l4- dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzoic (405 mg, 0.71 mmol, 1 eq), N-hydroxysuccinimide (173 mg, 1.4 mmol, 2 eq), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (224 mg, 1.4 mmol, 2 eq) and 4-(Dimethylamino)pyridine (176 mg, 1.4 mmol, 2 eq) in Dry DCM (100 ml) at room temperature for 16 h. Water is added and the mixture is extracted with DCM. The organic phase is washed twice with water, dried over MgSO ₄ , filtered and concentrated. The residue is purified by column chromatography (SiO ₂ ; DCM/AcOEt: 8/2) to obtain the desired product in the form of an orange powder (455 mg; 100%).

¹ H NMR (400 MHz, Chloroform-d) δ ppm: 8.28 (d, J =8.3 Hz, 2 H), 7.54 (d, J =8.1 Hz, 2 H), 6.04 (s, 2 H), 4.22 ( s, 4H), 3.63 - 3.73 (m, 4H), 3.52 - 3.60 (m, 4H), 3.38 (s, 6H), 2.96 (s, 4H), 2.74 (s, 6H), 1.37 (s, 6H).

Step 2: Preparation of N-(2-aminoethyl)-4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-1,3,7,9-tetramethyl- 5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzamide

Ethylene diamine (0.09 mL, 1.3 mmol, 6 eq) is added. to a solution of 2,5-dioxopyrrolidin-1-yl 4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-1,3,7,9-tetramethyl- 5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzoate (140 mg, 0.22 mmol, 1 eq) obtained at step 1 in dry DMF. The mixture is mixed at room temperature for 4 hours. Water is added and the organic phase is extracted with ethyl acetate, washed five times with water, dried over MgSO ₄ and filtered. The solvent is then removed under reduced pressure to obtain an orange powder (127 mg; 97%) used without further purification in the next step.

¹ H NMR (300 MHz, Chloroform-d) δ ppm: 7.96 (d, J =7.9 Hz, 2 H), 7.41 (d, J =7.9 Hz, 2 H), 7.01 (s, 1 H), 6.01 ( s, 2H), 4.20 (s, 4H), 3.62 - 3.71 (m, 4H), 3.51 - 3.60 (m, 4H), 3.36 (s, 6H), 3.01 (s, 2H), 2.73 (s, 6H), 1.92 (s, 4H), 1.34 (s, 6H).

Step 3: Coupling with vancomycin hydrochloride

N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (20 mg, 0.05 mmol, 1.5 eq) and DIEA (30 μl, 0.17 mmol, 5 eq) to a mixture of Vancomycin hydrochloride (50 mg, 0.03 mmol, 1 eq) and N-(2-aminoethyl)-4-(5,5-bis(3-(2-methoxyethoxy)prop- 1-yn-1-yl)-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin- 10-yl)benzamide (43 mg, 0.07 mmol, 2 eq) obtained in step 2 in a dry DMF/DMSO mixture (2 mL, 1/1, v/v). The mixture is left under stirring at room temperature for 16 h. After evaporation of the DMF, ACN is added. The precipitate is centrifuged, washed three times with ACN then 3 times with DCM and once with diethyl ketone. The crude product is dissolved in dry DMF (3 mL) and Ps-trisamine (70 mg) is added. The suspension obtained is stirred for 64 h. The suspension is then filtered through a sintered glass filter and the retentate is washed with dry DCM. The solvent is removed by evaporation under reduced pressure to obtain an orange powder (38 mg, 55%).

ESI-HRMS, m/z calculated for C ₁₀₀ H ₁₁₇ BCl ₂ N ₁₃ O ₂₉ : 1015.8848[M+H] ⁺ ; found: 1015.8835 [M+H] ⁺ .

Example 2: Preparation of a "red" analogue of vancomycin - VancomBod red

Fluorophore: 4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-2,8-dimethyl-3,7-di(thiophen-2-yl)-5H acid -4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzoic acid

The steps in the preparation of this green analog are shown in Figure 4.

Step 1: Preparation of N-(2-aminoethyl)-4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-2,8-dimethyl-3,7- di(thiophen-2-yl)-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzamide

A mixture of 4-(5,5-bis(3-(2-methoxyethoxy)prop-1-yn-1-yl)-2,8-dimethyl-3,7-di(thiophen-2- yl)-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzoic (116 mg, 0.16 mmol, 1 eq), N-Hydroxysuccinimide (38 mg, 0.33 mmol, 2 eq), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (52 mg, 0.33 mmol, 2 eq) and 4-Dimethylaminopyridine (41 mg, 0.33 mmol , 2 eq) in dry DCM (5 mL) for 16 h at room temperature. Hydrochloric acid (1M) is then added and the organic phase is extracted 3 times with DCM. The combined organic phases are washed with water (3 times), dried over MgSO ₄ , filtered and the solvent is then removed under reduced pressure. The residue obtained is purified by column chromatography (SiO ₂ ; DCM/EtOH: 85/15 to 80/20) to obtain a blue wax (54 mg; 45%).

¹ H NMR (500 MHz, Chloroform- d ) δ ppm: 7.97 (d, J =7.9 Hz, 2 H), 7.84 (dd, J =3.4, 0.9 Hz, 2 H), 7.64 (d, J =8.2 Hz , 2 H), 7.45 (dd, J =4.9, 0.9 Hz, 2 H), 7.12 (dd, J =4.9, 3.7 Hz, 2 H), 7.04 (t, J =5.2 Hz, 1 H), 6.57 ( s, 2H), 3.93 (s, 4H), 3.59 (q, J =5.5Hz, 4H), 3.43 - 3.48 (m, 4H), 3.37 - 3.42 (m, 4H), 3.34 (s , 6 H), 3.02 (t, J =5.8 Hz, 3 H), 2.08 (s, 6 H).

Step 2: Coupling with vancomycin hydrochloride

N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (27 mg, 0.07 mmol, 1.5 eq) and DIEA (30 μl, 0.17 mmol, 3 eq) to a mixture of Vancomycin hydrochloride (70 mg, 0.05 mmol, 1 eq) and N-(2-aminoethyl)-4-(5,5-bis(3-(2-methoxyethoxy)prop- 1-yn-1-yl)-2,8-dimethyl-3,7-di(thiophen-2-yl)-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborinin-10-yl)benzamide (53 mg, 0.07 mmol, 1.5 eq) obtained in step 1 in a dry DMF/DMSO mixture (2 mL, 1/1, v/v). The mixture is left under stirring at room temperature for 16 h. The crude product is precipitated by the addition of DCM. The precipitate is centrifuged, washed 3 times with DCM. The residue is dissolved in dry DMF (3 mL) and Ps-trisamine (210 mg) is added. The suspension obtained is stirred for 120 h. The suspension is then filtered through a sintered glass filter and the retentate is washed with dry DCM and the filtrate concentrated to 1-2 mL. DCM is added to the concentrated filtrate to precipitate the product. The solid is washed with DCM (3 times) and once with pentane to obtain a dark blue powder (40 mg; 39%).

Example 3: Photo-bleaching measurements

The tests are carried out with the analog of example 1 (VancomBod FL) on the one hand and the product BODIPY™ FL Vancomycin marketed by the company ThermoFisher Scientific (VancoBODIPY Fisher) on the other hand.

The measurements were carried out with a confocal microscope (Olympus, FV1200) equipped with a 473nm excitation laser and a 490-525nm bandpass emission filter. The laser power was set at 20% throughout the experiment. Gain and Offset remained unchanged.

Measurements at 1 μg / mL : A series of 50 images per field was taken. Laser intensity for imaging the bacterium marked with VancomBod FL: 600/1000 and that marked with VancoBODIPY Fisher: 500/1000.

The fluorescence decay obeys a first-order exponential decay law. Under identical illumination conditions, the VancoBODIPY Fisher has a faster whitening dynamic. It loses 95% of its fluorescence in 29.4 s against 38.7 s for the VancomBod FL.

Measurements at 10 μg / mL : A series of 100 images per field was taken. Laser intensity for imaging the bacterium marked with VancomBod FL: 600/1000 and that marked with VancoBODIPY Fisher: 500/1000.

Under identical lighting conditions, the VancoBODIPY Fisher has a whitening dynamic 2 times faster. It loses 95% of its fluorescence in 52.5 s against 115.8 s for the VancomBod FL.

Example 4: Specificity of the labeling – in vitro test on bacterial culture

Wild Type S. aureus (Gram +) ATCC 29213 bacteria (reference strain) and Wild Type E. Coli (Gram -) DH5α bacteria (reference strain) are cultured on agar for 24 hours at 37°C. An isolated colony of each bacterium is incubated in nutrient culture medium without antibiotics for 18 h at 37° C. with shaking. Then, a bacterial solution is prepared at approximately 10 ⁸ CFU/mL (optical density at 0.5 at 600 nm). The bacteria are then labeled for 20 min at room temperature either with the vancomycin analogue at a final concentration of 1 and 10 μg/mL, or with the product BODIPY™ FL Vancomycin marketed by the company ThermoFisher Scientific at a final concentration of 1 and 10µg/mL. After centrifugation for 10 min at 8000 g , the supernatant is eliminated and the pellet is taken up in 1 mL of culture medium. The bacteria are observed in confocal microscopy (Olympus FV1200)

Like the product marketed by ThermoFisher Scientific, VancomBod FL is specific for Gram + bacteria with labeling at the level of the bacterial wall (corona labeling) whatever the concentration. No labeling of E. Coli bacteria is observed. The position of B on the carboxylic acid group of Vancomycin does not alter its specificity.

Example 5: Ex-vivo tests on corneas

Trial 1

Wild Type S. aureus (Gram +) ATCC 29213 bacteria (reference strain) are cultured according to the protocol set out in example 4. A cornea rebue from the bank preserved in organoculture is selected. Using a 30G ½'' needle, scratches are made in order to generate lesions of the epithelium. The cornea is then placed epithelium side down on a concave plastic holder and the bacterial solution is placed between the cornea and the holder. The cornea and the support are then placed in a humid chamber at 37°C for 2 hours. Then, the cornea is cut in half. One half is labeled with VancomBod FL at 1 μg/mL + Hoechst 33342 (5 μg/mL) + Ethidium (4 μM) for 20 min at 37°C and the other half with the product BODIPY™ FL Vancomycin marketed by the company ThermoFisher Scientific at 1µg/mL + Hoechst 33342 (5µg/mL) + Ethidium (4µM) for 20min at 37°C. After rinsing, the pieces are observed by epifluorescence microscopy (Olympus IX81) and by confocal microscopy (Olympus FV1200).

The VancomBod FL makes it possible to image bacteria intensely at high magnifications (x400 or x600) despite the non-specific staining localized in the cytoplasm of dead cells (labeled with Ethidium) present especially in lesions. This undesirable coloration is also observable with the commercial compound. It can in most cases be eliminated by post-treatment. If it is too intense to be eliminated by post-treatment, the bacteria labeled with VancomBod FL remain easily visible. An attenuation of the non-specific staining by Trypan Blue is possible and has been tested (see test 2).

Test 2: Adding Trypan Blue

Trypan blue is a dye known to quench the auto-fluorescence of biological tissues or cells (composed mainly of water and hemoglobin) which can generate a significant background noise of auto-fluorescence, thus masking a real signal of fluorescence (PMID ref: 21777584).

The preparation protocol for S. aureus bacteria and the corneal infection protocol remain the same as for test 1. Before labeling the epithelium with the compound VancoBod FL at 1µg/mL + Hoechst 33342 (5µg/mL) + Ethidium (4µM), the infected cornea is cut in half. One half is marked beforehand with 0.4% Trypan blue for 2 min. After rinsing, the pieces are observed by epifluorescence microscopy (Olympus IX81) and by confocal microscopy (Olympus FV1200).

The images without Trypan blue show, as in test 1, a non-specific coloration of the cells which is sometimes not eliminable by post-processing. The addition of Trypan blue before staining with VancomBod FL quenches the fluorescence in these cells and makes it easier to visualize the labeled bacteria.

Example 6 Labeling of an Ulcer on Uninfected Human Cornea with the Analog Described in Example 2 and a Bodipy Compound (AP343A)

The Bodipy Compound of the COMBO AP343A is shown in Figure 5.

A cornea rejected from the bank preserved in organoculture is selected. Using a 30G ½'' needle, a lesion of about 1mm is made. The cornea is then placed on an artificial Moria chamber (Moria Surgical) then imaged without prior staining using a 633nm excitation source and an ultra-sensitive EMCCD (Electron Multiplying Charge Coupled Device) camera (Ixon Ultra 888 1024x1024px, ANDOR). Exposure time is set to 300ms/frame. Then the cornea is marked for 20 min away from light with the red VancomBod of example 2 at 1 mg/mL. Since no infection was present, no labeling was detected with the EMCCD (even when making a succession of 10 compiled images). The same cornea is then labeled for 20 min away from light with a “red” Bodipy (AP343A, abs: 591 nm and em: 646 nm in H ₂ O) at 500 μg/mL then imaged again.

The images obtained in the absence of labeling, or with a labeling by the “red” analogue of example 2 are therefore compared to the images obtained with a labeling by a “red” BODIPY AP343A.

The results show an absence of marking of the walls of the ulcer with the analog according to the invention with images comparable to those obtained in the absence of marking, without or with post-image processing.

On the other hand, the images obtained with the BODIPY AP343A show a marking of the walls of the ulcer.

This example clearly confirms the specificity of the product, which does not report false positives in the absence of Gram+ bacteria.

REFERENCES

Kocaoglu & Carlson, 2016;

Gao & al., 2006

WO 2006/087459, WO 2010076516 and WO 2014/013205.

Claims (14)

Fluorescent analogue of vancomycin of formula BL-CO-V in which
B is a fluorophore derived from BODIPY,
L is a link arm, and
-CO-V is vancomycin, the bond of L-CO-V being a bond on the carboxylic acid residue of vancomycin. Analog according to Claim 1, characterized in that the fluorophore B is a compound of formula I
(I)
in which
S ¹ represents a group of formula -CΞC-L'-A, where L' is a linking group which is a single bond (in which case S ¹ is a group of formula -CΞC-A); or a divalent hydrocarbon group selected from the group consisting of linear or branched alkylenes, optionally comprising one or more oxygen, nitrogen or sulfur atoms within their chain (such as poly(ethylene glycol), poly(ethoxy) or poly(propoxy), for example); linear or branched alkenylenes; linear or branched alkynylenes, and arylenes; or a divalent hydrocarbon-based chain consisting of a sequence of at least two divalent hydrocarbon-based groups of the aforementioned type; and A is a polar functional group selected from sulfonate, sulfate, phosphate, ammonium, carboxylate, hydroxyl, phosphonate, alkylammonium sulfate and polyoxyethylene groups;
S ² represents a group -CΞC-L'-A identical to or different from S ¹ , where L' and A have the above meanings; F; H; or a hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by one or more oxygen, nitrogen or sulfur atoms, optionally cyclized in whole or in part, optionally aromatic, and optionally functionalized, and
each of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ or R ⁷ denotes, independently of the others, H or a hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by one or more atoms oxygen, optionally cyclized in whole or in part, optionally aromatic and optionally functionalized, it being understood that all or part of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ , can be linked together to form a bridged form (i.e. at least two of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ can together form a ring with the carbons to which they are connected) . Analog according to one of Claims 1 or 2, characterized in that L is a bifunctional derivative of formula
^Z1 - ^R8 - ^Z2- .
In which
R ⁸ is a divalent hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by one or more oxygen atoms, optionally cyclized in whole or in part,
Z ¹ is the group allowing the bond with the fluorophore B, particularly chosen from the groups -CΞC-, -O-, -NH- and -S-, more particularly -CΞC- and -NH-, and
Z ² is the group allowing the bond with the carboxylic acid of vancomycin, in particular chosen from the groups -O-, -NH- and -S-, more particularly -NH-. Analog according to one of Claims 1 to 3, characterized in that L is chosen from the following divalent groups
-HN-(CH ₂ -CH ₂ ) _n -CH ₂ -NH-
-HN-CH ₂ -(O-CH ₂ -CH ₂ ) _n -O-CH ₂ -NH-, and
-CΞC-( _CH2 - _CH2 ) _n - _CH2 -NH-
n being an integer from 1 to 6, preferably 1, 2 or 3, more preferably 1. Analog according to one of Claims 2 to 4, characterized in that the BL- bond is made via R ¹ of formula -Ar-CO-L- or -Ar-L-, in which Ar is a divalent aromatic hydrocarbon radical, from 1 to 4 rings fused or not, having in particular from 4 to 16 carbon atoms, preferably from 4 to 10 carbon atoms, and possibly containing in their cycle nitrogen, sulfur or oxygen atoms (heterocycles). Derivative of BODIPY of formula B-L-H, B and L being defined according to one of Claims 1 to 5. BODIPY derivative according to claim 6 of formula II
(II)
in which L, S ¹ , S ² , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are defined according to one of claims 2 to 4. Liquid composition characterized in that it comprises a fluorescent analogue of vancomycin according to one of Claims 1 to 5 and a suitable vehicle for its use. Composition according to Claim 8, characterized in that it is in a form suitable for its application to the skin and the mucous membranes. Composition according to Claim 9, characterized in that it is eye drops for application to the eye. Use of a fluorescent analogue of vancomycin according to one of Claims 1 to 5 for the detection of Gram+ bacteria in a biological sample. Use according to claim 11, characterized in that the Gram+ bacteria are pathogenic bacteria of the Staphylococcus genus. Method for detecting the presence of Gram+ bacteria in a biological sample, characterized in that it comprises the addition of an appropriate quantity of a fluorescent analogue of vancomycin according to one of Claims 1 to 5 to the biological sample then to observe the persistence of a fluorescence emission, a marker of the presence of Gram+ bacteria. Method according to claim 13, characterized in that the Gram+ bacteria are pathogenic bacteria of the Staphylococcus genus.