FLUOROGENIC GLYCOSIDASE SUBSTRATE AND ASSOCIATED DETECTION METHOD

Abstract

The invention relates to novel glycosidase substrates of formula (I), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R9, V, X, Y and Z are as defined in claim 1, and a method for detecting the presence of a catalytically active glycosidase by means of one of said substrates.

##STR00001##

Claims

1. Compounds of formula (I): ##STR00037## in which: R1 is such that HOR1, obtained after cleavage of the C(O)OR1 bond present in formula (I), belongs to the class of fluorophores leading to an intramolecular proton transfer in an excited state, called ESIPT, R2, R3 and R4 are defined as follows: either R2 is an (C1-C4) alkyl, R3 is an (C1-C4) alkyl or a hydrogen atom, and R4 is an (C1-C4) alkyl, or R3 is an (C1-C4) alkyl or a hydrogen atom and R2 and R4 are bonded together and form, with the carbon and nitrogen atoms to which they are bonded, an aliphatic heterocycle which can be substituted by a water-solubilizing group, or R2 is an (C1-C4) alkyl and R3 and R4 are bonded together and form, with the carbon atom to which they are bonded, an aliphatic carbocycle, R5 and R6 are identical or different and represent, independently of each other, a hydrogen atom, an (C1-C4) alkyl, or an (C5-C10)aryl, R7 is a hydrogen atom, or a group chosen from among the (C1-C4) alkyls and (C1-C4) alkoxys, R8 represents a hydrogen atom or an (C1-C10) alkyl group, substituted or non-substituted, or a-D1-D2-D3 group with: D1 representing a triazolyl or CH2-triazolyl group, D2 representing an (C1-C10) alkylene, (C1-C10) alkenylene or (C1-C10) alkynylene group, said groups possibly being interrupted by one or more hetero-atoms chosen from among O or N, a divalent glycosyl group, an O(CHRCHR)n- or N(CHRCHRO)n-group, n being an integer varying from 1 to 20, R and R, identical or different, representing H or CH3 upon condition that R and R are not simultaneously CH3, an amino acid or a peptide, or a combination of these groups, D3 representing a maleimidocaproyl motif, amino acid, peptide, folic acid, antibody or antibody fragment bonded to D2, by a carboxylic acid function comprised in it, forming an ester or amide bond, R9 and R9, identical or different, represent a hydrogen atom, or an electron-withdrawing group, such as a halogen atom, or a group chosen from among NO2, CN, or a group chosen from among NHC(O)CH2-Ab, with Ab representing an antibody, V represents an oxygen atom or a sulfur atom, X, Y and Z are such that: either X represents CR10, Y represents CR10 and Z represents OR0, or X represents CR10, Y represents COR0 and Z represents R10, or X represents CR10, Y represents a nitrogen atom and Z represents OR0, or X represents a nitrogen atom, Y represents COR0 and Z represents R10 with: R0 representing a glycosyl group bound by its anomeric carbon atom to the rest of the molecule of formula (I), and R10 and R10, identical or different, representing a hydrogen atom or an electron-donating group such as an (C1-C20) alkyl, an (C5-C24) aryl, or an (C1-C20) alkoxy, in the form of a mixture of optical isomers according to all proportions, or in an optical isomer enriched form.

2. Compounds (I) according to claim 1, wherein R3 is a hydrogen atom or an (C1-C4) alkyl, and R2 and R4 are bonded to each other and form a (CH2)m- chain with m=3, 4 or 5.

3. Compounds (I) according to claim 1, wherein R3 is a hydrogen atom or an (C1-C4) alkyl, and R2 and R4 are bonded to each other and form a CH2CH2-NR11-CH2- chain in the direction of R2 toward R4, R11 representing a hydrogen atom or -(L)n-GP with n which is equal to 0 or 1, L a linking arm and GP a hydro solubilizing group.

4. Compounds (I) according to claim 1, wherein R2, R3 and R4, identical or different, represent an (C1-C4) alkyl group, for example, methyl or ethyl.

5. Compounds (I) according to claim 1, wherein R1 is an aromatic group comprising one or more aromatic rings, substituted or not substituted, which rings can comprise one or more hetero-atoms chosen from among the nitrogen, oxygen or sulfur atoms and/or one or more carbon atoms in the form of a CO carbonyl.

6. Compounds (I) according to claim 1, wherein R1 is an aromatic group with OR1 according to formula (A1): ##STR00038## in which: either X2 is an oxygen atom and X1 is a NH2, OH, SH, (C1-C20) alkyl, (C5-C24) aryl, O(C1-C20) alkyl, O-phenyl, NH(C1-C20) alkyl or NH-phenyl, S(C1-C20) alkyl or S(C5-C24) aryl group, said alkyl and phenyl groups being possibly substituted or non-substituted, Or X2 represents a nitrogen atom and is bound to X1 which then represents CH, O, S, N or, NH to form a (C5-C24) hetero-aryl, substituted or not substituted, ##STR00039## represents an (C5-C24) aryl or a (C5-C24) hetero-aryl, substituted or non-substituted, for example, chosen from among the phenyl, naphtyl groups, and: ##STR00040## said groups being able to be substituted or non-substituted, with X3 which represents S, O or NRd and Rd which represents a hydrogen atom or an (C1-C4) alkyl group, and OR1 is of the phenoxy type and corresponds to one of the following structures (A2) or (A3): ##STR00041## in which: T is NHC(O), S, O, NH, N((C1-C20) alkyl)- or N(C5-C24)aryl)-, Re is a hydrogen atom or an electron-withdrawing carbon substitute such as CN or COORh, with Rh which represents an (C1-C4) alkyl group, or Re is CONRiRj, with Ri and Rj, identical or different, which represent a hydrogen atom, or an (C1-C4) alkyl group, or Re is CF3, or a 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-benzo imidazolyl, 4-pyrimidinone-2-yl or quinazolinone-2-yl group, Rf is a hydrogen atom, a chlorine, bromine, iodine or fluorine atom, OH, NH2, NRkRI, NHRk or ORk, with Rk and RI, identical or different, which each, independently, represent an (C1-C4) alkyl group, Or Re and Rf are bonded to each other to form a hydrocarbon chain comprising 4 or 5 members, saturated or unsaturated, substituted or non-substituted, possibly interrupted by one or more hetero-atoms chosen from among N, S and O, Rg is a hydrogen, Br, Cl, I or F atom, ##STR00042## in which: T is NH2, OH, an (C5-C24) aryl group, an (C1-C4) alkyl group, SH, NHRg, ORg, NRgRh or SRg, Rg and Rh, identical or different, representing an (C1-C4) alkyl or aryl group, Re is a hydrogen atom or an electron-withdrawing carbon substitute such as CN or COORi, with Ri which represents an (C1-C4) alkyl group, or Re is CONRjRk, with Rj and Rk, identical or different, which represent a hydrogen atom, or an (C1-C4) alkyl group, or Re is CF3, or a 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-benzo imidazolyl, 4-pyrimidinone-2-yl or quinazolinone-2-yl group, Rf is a hydrogen, chlorine, bromine, iodine or fluoride atom, OH, NH2, NRlRm or ORl, with Rl and Rm, identical or different, which represent an (C1-C4) alkyl group, or Re and Rf are bonded to each other to form a hydrocarbon chain comprising 4 or 5 members, saturated or unsaturated, substituted or non-substituted, possibly interrupted by one or more hetero-atoms chosen from among N, S and O.

7. Compounds (I) according to claim 1, wherein R1 is an aromatic group with OR1 according to one of the following formulas (A4) or (A5): ##STR00043##

8. Compounds (I) according to claim 1, wherein R0 is cleavable from the rest of compound (I) by the catalytic action of a glycosidase.

9. Compounds (I) according to claim 1, wherein R0 is a group that is cleavable under the action of a glycosidase, chosen from among N-acetyl--galactosaminidase; N-acetyl--glucosaminidase; -amylase; -arabinofuranosidase, -arabinosidase; -cellobiosidase; -chitobiosidase; -galactosidase; -galactosidase; -glucosidase; -glucosidase; -glucuronidase; -maltosidase; -mannosidase; -mannosidase; -xylosidase; -D-fucosidase; -L-fucosidase, -L-fucosidase; L-iduronidase or cellulase; and R0 is a mono-glycosylated group bound by its anomeric carbon atom chosen from among galactosyl, glucosyl, mannosyl, gulosyl, allosyl, altrosyl, idosyl, talosyl, fucosyl, fructosyl, arabinosyl, lyxosyl, ribosyl, xylosyl, glucuronyl and N-acetyl-hexosaminyl or a polyglycosylated group constituted of several of these monoglycosylated groups, identical or different.

10. Compounds (I) according to claim 1, wherein R5 and R6 are identical and represent a hydrogen atom.

11. Compounds according to claim 1, wherein R7 represents a hydrogen atom or an (C1-C4) alkyl.

12. Compounds (I) according to claim 1, wherein R8 represents a hydrogen atom.

13. Compounds (I) according to claim 1, wherein V represents an oxygen atom.

14. Compounds (I) according to claim 1, wherein X, Y and Z are such that: either X represents CR10, Y represents CR10 and Z represents OR0, or X represents CR10, Y represents COR0 and Z represents R10, with R10, R10 and R0 as defined in claim 1.

15. Compounds (I) according to claim 1, wherein at least one of groups R9 or R9 represents a halogen atom, a NO2 group, or a CN group.

16. Compounds (I) according to claim 1, wherein R10 and, if applicable, R10 are identical and represent a hydrogen atom.

17. Compounds (I) according to claim 1 of formula (Ia): ##STR00044## where R1, R2, R3, R4, R5, R6, R7, R8, R0 and V are as defined in claim 1 in the form of a mixture of optical isomers according to all proportions, or in an enriched form in an optical isomer.

18. Compounds (I) according to claim 1 of formula (Ib): ##STR00045## where R1, R2, R3, R4, R9, R9, X, Y and Z are as defined in claim 1, in the form of a mixture of optical isomers according to all proportions, or in an enriched form in an optical isomer.

19. Compounds (I) according to claim 1 of formula (Ic): ##STR00046## where R0 and R1 are as defined in claim 1, in the form of a mixture of optical isomers according to all proportions, or in an enriched form in an optical isomer.

20. Compounds (I) according to any claim 1 for the in vivo detection, in human beings, of a glycosidase.

21. Method for the in vitro or ex vivo detection of a glycosidase, comprising the steps of: putting a sample thought to contain said glycosidase into contact with a compound (I) according to claim 1, applying suitable conditions in order to make possible the formation of a fluorescent precipitate by cleavage of the covalent bond between O and R0, followed by a cleavage of the C(O)OR1, bond, leading to the release of HOR1, and quantitative or qualitative analysis of the fluorescent precipitate.

22. Method according to claim 21, wherein the analysis of the fluorescent precipitant comprises: a step of exposing the fluorescent precipitate to a light source capable of producing light at an absorption wavelength of the fluorescent precipitate, and a step of detecting the fluorescence of the resulting precipitate.

23. Compounds of formula (II): ##STR00047## in which: R2, R3, R4, R5, R6, R7, R8, R9, R9 and V are as defined in claim 1, R12 represents a hydrogen atom, or an amine functions protecting group, X, Y and Z are such that: either X represents CR10, Y represents CR10 and Z represents OR0, or X represents CR10, Y represents COR0 and Z represents R10, or X represents CR10, Y represents a nitrogen atom and Z represents OR0, or X represents a nitrogen atom, Y represents COR0 and Z represents R10, with R0 representing a R0 group of which all of the alcohol functions are protected by a protecting group, and R0, R10 et R10 are as defined in claim 1, in the form of an optical isomer according to all proportions, or in an optical isomer enriched form.

24. Compound (II) according to claim 23, wherein R12 represents a hydrogen atom.

25. Compound (II) according to claim 23, wherein R12 represents an amine protecting group.

26. Process for the preparation of a compound of formula (I) according to claim 1 comprising the following steps: availability of a compound (II) according to any one of claims 23 to 25, availability of a compound (III) of formula ##STR00048## with R1 as defined in any one of claims 5, 7 and M representing a leaving group, obtaining compound (IV) by addition reaction of said compound (II) to compound (III), said compound (IV) having the formula: ##STR00049## in which R1, R2, R3, R4, R5, R6, R7, R8, R9, R9, V, X, Y and Z are as defined in any one of claims 1 to 16 and 23, and deprotecting the alcohol functions present in the R0 group of said compound (IV) in order to obtain said compound (I).

Description

[0206] Examples, in relation to the annexed figures, make it possible to illustrate the invention, but not in a limitative way.

[0207] FIG. 1 is a diagram showing the degradation mechanism, using formula (I) compounds, calling on the pair of spacers according to the invention.

[0208] FIG. 2 is an evolution curve of the solid-state fluorescence for probes 13 and 27 of examples 1 and 3 and for probe 28 of the prior art at 37 C. (concentration: 10 M).

[0209] FIG. 3 represents evolutions curves for solid state fluorescence for probe 21 of example 2 at 37 C. (concentrations: 0 M, 5 M, 10 M, 25 M et 50 M).

[0210] FIG. 4 represents the light signal controlling for the cellulase activity produced by a micro-organism.

[0211] FIG. 5 is a graph representing the detection kinetics of cellulase activity in a micro-organism culturing medium.

EXAMPLES

[0212] General Information

[0213] Column chromatography was conducted on 60-mesh silica gel (40-63 pm). The RMN spectra for 1H and 13C were recorded at 300 MHz and at 75 or 125 MHz, respectively, in deuterated chloroform, deuterated DMSO or deuterated methanol. Chemical displacements (5) are indicated in ppm and noted in reference to tetramethylsilane or according to residual solvent signals; the abbreviations s=singlet, d=doublet, t=triplet, m=multiplet, b=large are used. RMN (J) coupling constants are indicated in Hertz. Fluorescent analyses were conducted in 96-well black polypropelyne plates (Corning Costar, Corning, Inc.), and registered on a microplate fluorimeter (EnSpire plate reader by Perkin Elmer). Except when specified chemical products were purchased with analytic reactive quality and used without other purification.

[0214] Commercial dry DCM was dried and purified by passing it through an activated aluminum column under argon (GT S100 Solvent Station System). TEA was distilled using calcium hydride and stored in KOH pellets. The other reagents noted as dry were dried on molecular sieves. If not otherwise noted, all reactions were conducted under atmospheric air with solvents and commercial reagents, without additional drying or purification. Millipore water obtained using an Elga Purelab purification system was used in all experiments.

[0215] The following abbreviations are used:

DIPEA=diisopropylehtylamine
TEA=triethylamine
py=pyridine
DCM=dichloromethane
Yld.=yield
DMSO=dimethyl sulfoxide
TFA=trifluoroacetic acid
rt=room temperature.

Example 1

[0216] Compound 13 is prepared as described in Diagram 1 below.

Diagram 1: Chemical Synthesis of Compound 13

[0217] ##STR00033##

Preparation of Compound 2

[0218] To a solution of 2-aminoethylpiperidine 1 (3.0 g, 26.3 mmol, 1.0 eq.) in 100 mL of toluene was added, little by little, phthalic anhydride (3.89 g, 26.3 mmol, 1.0 eq.) and, drop by drop, triethylamine (550 L, 3.95 mmol, 0.15 eq.). The mixture was then heated under reflux for 2 h using a Dean-Stark device. Then, the mixture was filtered and the solvent was evaporated under reduced pressure. Compound 2 (6.605 g, 24.7 mmol, yld: 94%) was obtained in the form of a light yellow solid and used without purification.

[0219] 1H-NMR (300 MHz, CDCl3): (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).

[0220] 13C-NMR (75 MHz, CDCl3): (ppm)=168.4, 133.7, 131.9, 123.0, 55.5, 46.4, 43.4, 30.6, 26.1, 24.1.

[0221] HRMS: ESI: [M+H]+m/z found 245.1290, calc. 245.1290

Preparation of Compound 3

[0222] To a solution of compound 2 (6.605 g, 24.7 mmol, 1.0 eq.) in 80 mL of ethanol, cooled in an ice bath, potassium carbonate (4.36 g, 31.6 mmol, 1.3 eq.), tetra-n-butylammonium iodide (912 mg, 2.47 mmol, 0.10 eq.) and allyl bromide (2.73 mL, 31.6 mmol, 1.3 eq.) were added. The ice bath was removed and the mixture was stirred for 36 h. At the end of the reaction, the mixture was filtered with Celite and evaporated under reduced pressure. The residual oil was dissolved in EtOAc and an aqueous solution saturated in NH4CI was added. The two phases were separated and the organic phase was washed two times with an aqueous solution saturated in NH4CI. The combined aqueous phases were extracted 3 times with EtOAc. The combined organic phases were dried with Na2SO4, filtered and concentrated under reduced pressure. The raw product was purified by column chromatography on silica gel (pure DCM, then DCM: MeOH: Et3N/99:0.5:0.5/v:v:v) in order to obtain compound 3 in the form of a yellow oil that crystallizes (2.24 g, 7.89 mmol, yield: 35%).

[0223] 1H-NMR (300 MHz, CDCl3): (ppm)=7.84-7.78 (m, 2H), 7.72-7.65 (m, 2H), 5.95-5.81 (m, 1H), 5.22-5.08 (m, 2H), 3.93 (dd, 1H, J=13 Hz, J=5 Hz), 3.73 (dd, 1H, J=13 Hz, J=8 Hz), 3.42 (dd, 1H, J=14 Hz, J=6 Hz), 3.21 (dd, 1H, J=14 Hz, J=6 Hz), 2.88-2.76 (m, 2H), 2.37-2.28 (m, 2H), 1.74-1.47 (m, 4H), 1.40-1.25 (m, 2H).

[0224] 13C-NMR (75 MHz, CDCl3): (ppm)=168.3, 135.5, 133.8, 132.0, 123.0, 117.2, 57.6, 57.3, 50.3, 38.4, 28.1, 24.7, 22.0.

[0225] HRMS: ESI: [M+H]+m/z found 285.1595, calc. 285.1603

Preparation of Compound 4

[0226] To a solution of 3 (1.312 g, 4.6 mmol, 1.0 eq.) in iPrOH: H2O/6:1/v:v (50 mL) cooled in ice was added, little by little, sodium borohydride (874 mg, 23 mmol, 5.0 eq.). After having been stirred at room temperature for one night, the pH was acidified to pH=1, using an aqueous solution of HCl at 37%. The mixture was filtered and then heated to 80 C. for 2 hours. iPrOH was evaporated under reduced pressure and the resulting aqueous solution was washed 5 times with diethyl ether and then lyophilized Compound 4 was obtained in the form of a white powder (1.045 g, 4.6 mmol, quantitative yld).

[0227] RMN of the basic product (1-allyl-2-(aminomethyl) piperidine): H-NMR (300 MHz, CDCl3): (ppm)=5.99-5.86 (m, 1H), 5.24-5.13 (m, 2H), 3.41 (ddt, 1H, J=14 Hz, J=5.7 Hz, J=1.5 Hz), 3.04-2.90 (m, 3H), 2.74 (dd, 1H, J=13 Hz, J=3.3 Hz), 2.21 (tt, 2H, J=9.6 Hz,=3.3 Hz), 1.80-1.71 (m, 1H), 1.68-1.43 (m, 2H), 1.39-1.25 (m, 3H).

[0228] 13C-NMR (75 MHz, CDCl3): (ppm)=134.93, 116.87, 61.47, 56.23, 51.93, 42.97, 28.28, 24.95, 23.66.

[0229] HRMS: ESI: [M+H]+m/z found 155.1543, calc. 155.1548

Preparation of Compound 6

[0230] Acetobromogalactose 5 (700 mg, 1.70 mmol, 1.0 eq.), of 4-hydroxy-3-nitrobenzaldehyde (313 mg, 1.87 mmol, 1.1 eq.) and Ag2O (1.300 g, 5.61 mmol, 3.3 eq.) were dissolved in acetonitrile and stirred for one night at room temperature. The reactional mixture was then filtered with Celite and concentrated under reduced pressure. The resulting oil was purified by column chromatography on silica gel (petroleum ether: ethyl acetate/4:6/v:v) in order to obtain compound 6 in the form of a light yellow solid (669 mg, 1.34 mmol, yld: 79%).

[0231] 1H-MR (300 MHz, DMSO-d6): (ppm)=9.98 (s, 1H), 8.45 (d, J=2 Hz, 1H), 8.26 (dd, J=9 Hz, J=2 Hz, 1H), 7.60 (d, J=9 Hz, 1H), 5.80 (d, J=8 Hz, 1H), 5.40 (bs, 1H), 5.30-5.27 (m, 2H), 4.55 (td, J=6 Hz, J=1 Hz, 1H), 4.15 (d, J=6 Hz, 2H), 2.16 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.96 (s, 3H).

[0232] 13C-NMR (75 MHz, CDCl3): (ppm)=188.55, 170.24, 170.07, 169.16, 153.45, 141.25, 133.96, 131.48, 126.84, 118.80, 100.09, 71.82, 70.35, 67.59, 66.58, 61.36, 20.66, 20.61, 20.59, 20.55.

[0233] HRMS: ESI: [M+Na]+m/z found 520.1052, calc. 520.1067

Preparation of Compound 7

[0234] To a solution of compound 6 (636 mg, 1.28 mmol, 1.0 eq.) in CHCl3: iPrOH 5:1 v:v (12 mL) in an ice bath, was added sodium borohydride (53 mg, 1.41 mmol, 1.1 eq.).

[0235] The reaction was stirred for 1 hour and halted by the addition of an aqueous solution saturated in NH4CI.

[0236] After 5 minutes of stirring, the phases were separated and the aqueous phase was extracted 2 times with dichloromethane. The combined organic phases were dried with Na2SO4, filtered and evaporated under reduced pressure to give compound 7 in the form of a white powder.

[0237] (605 mg, 1.22 mmol, yld: 95%) which was used in the next step without purification.

[0238] 1H-NMR (300 MHz, DMSO-d6): (ppm)=7.80 (d=2 Hz, 1H), 7.63 (dd, J=9 Hz, J=2 Hz, 1H), 7.37 (d, J=9 Hz, 1H), 5.56 (d, J=7 Hz, 1H), 5.43 (t, J=6 Hz, 1H), 5.37 (d, J=3 Hz, 1H), 5.31-5.19 (m, 2H), 4.53-4.45 (m, 3H), 4.19-4.08 (m, 2H), 2.16 (s, 3H), 2.04 (s, 6H), 1.95 (s, 3H).

[0239] 13C-NMR (125 MHz, DMSO-d6): (ppm)=170.54, 170.46, 170.13, 169.47, 147.70, 140.84, 138.90, 132.41, 122.80, 118.30, 99.34, 71.35, 70.54, 68.34, 67.72, 61.94, 61.87, 21.10, 20.98, 20.92.

[0240] HRMS: ESI: [M+Na]+m/z found 522.1209, calc. 522.1224

Preparation of Compound 8

[0241] To a solution of compound 7 (120 mg, 0.240 mmol, 1.0 eq.) in dry DCM (5 mL), cooled in ice was added, successively, 4-nitrophenyl chloroformate (107 mg, 0.53 mmol, 2.2 eq.) and pyridine (48 L, 0.60 mmol, 2.5 eq.) drop by drop. The reaction mixture was stirred at 0 C. for 30 min and at room temperature for 2 h. At the end of the reaction, the reaction was stopped using an HCl 1 M aqueous solution and the phases were separated. The organic phase was washed with a solution of HCl 1 M, and the combined aqueous phases were extracted with DCM. The organic phases were dried with Na2SO4, filtered and evaporated under reduced pressure. The raw product was purified by column chromatography on silica gel (petroleum ether gradient: ethyl acetate/85:15 to 50:50/v:v) in order to obtain compound 7 in the form of a white solid (111 mg, 0.18 mmol, yld: 75%).

[0242] 1H-NMR (300 MHz, CDCl3): (ppm)=8.32 (d, J=9 Hz, 2H), 7.94 (d, J=2 Hz, 1H), 7.64 (dd, J=9 Hz, J=2 Hz, 1H), 7.44-7.39 (m, 3H), 5.59 (dd, J=10 Hz, J=8 Hz, 1H), 5.52 (d, J=3 Hz, 1H), 5.33 (s, 2H), 5.17-5.14 (m, H), 4.30 (dd, J=11 Hz, J=7 Hz, 1H), 4.23-4.14 (m, 2H), 2.23 (s, 3H), 2.17 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H).

[0243] 13C-NMR (125 MHz, CDCl3): =171.0, 137.8, 135.1, 129.4, 128.9, 128.9, 128.8, 128.4, 127.3, 127.2, 80.1, 62.8, 54.2, 53.0, 49.7, 43.9, 41.0, 40.1, 28.5 ppm.

[0244] MS: ESI: [M+Na]+m/z found 687.1263, calc. 687.1286

Preparation of Compound 9

[0245] To a suspension of compound 4 (44 mg, 0.20 mmol, 1.3 eq.) in DCM (3 mL), was added compound 8 (100 mg, 0.150 mmol, 1.0 eq.). The reactional mixture was cooled in an ice bath and DIPEA (81 qL, 0.47 mmol, 3.1 eq.) was added. After 5 minutes, the ice bath was removed and the reactional mixture was heated to 30 C. for one night. The mixture was then washed with aqueous solutions saturated with Na2CO3 et NaHCO.sub.3, dried with Na2SO4, filtered and evaporated under reduced pressure. The raw product was purified by column chromatography on silica gel in order to obtain compound 9 in the form of a white solid (56 mg, 0.083 mmol, yld: 55%).

[0246] NMR: 1H-NMR (300 MHz, CDCl3): (ppm) J=7.82 (d, J=2 Hz, 1H), 7.53 (dd, J=9 Hz, J=2 Hz, 1H), 7.35 (d, J=9 Hz, 1H), 5.93-5.75 (m, 1H), 5.56 (dd,=10 Hz, J=8 Hz, 1H), 5.48 (d,=J/3 Hz, 1H), 5.32 (bs, 1H), 5.22-5.06 (m, 6H), 4.29-4.14 (m, 2H), 4.11-4.06 (m, 1H), 3.43-3.22 (m, 3H), 2.99-2.8 (m, 2H), 2.43-2.36 (m, 1H), 2.21 (s, 3H), 2.14 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H), 1.76-1.25 (m, 6H).

[0247] 13C-NMR (125 MHz, CDCl3): (ppm)=170.31, 170.19, 170.13, 169.40, 156.26, 148.92, 141.30, 134.68, 133.19, 133.14, 124.59, 119.83, 117.75, 100.82, 71.47, 70.57, 67.85, 66.75, 64.74, 61.37, 58.30, 56.36, 51.99, 42.51, 28.97, 25.00, 23.73, 20.70, 20.67, 20.59.

[0248] HRMS: ESI: [M+H]+m/z found 680.2683, calc. 680.2667

Preparation of Compound 10

[0249] A solution of compound 9 (20 mg, 0.029 mmol, 1.0 eq.) and of 1,3-dimethylbarbituric acid (37 mg, 0.24 mmol, 8.0 eq.) in dry DCM (3 mL) was degassed with an argon flow. Then, palladium (0) tetrakis (triphenylphosphine) (0.6 mg, 0.0005 mmol, 2 mol %) was added. At the end of the reaction, the reactional mixture was dry evaporated and purified by column chromatography on silica gel in order to obtain compound 10 in the form of a white solid (13 mg, 0.020 mmol, yld: 70%).

[0250] 1H-NMR (300 MHz, CDCl3): (ppm)=7.82 (d, J=2 Hz, 1H), 7.52 (dd, J=9 Hz, J=2 Hz, 1H), 7.35 (d, J=9 Hz, 1H), 5.56 (dd, J=10 Hz, J=8 Hz, 1H), 5.48 (d, J=3 Hz, 1H), 5.32-5.26 (m, 1H), 5.14-5.06 (m, 4H), 4.30-4.15 (m, 2H), 4.11-4.06 (m, 1H), 3.29-3.20 (m, 1H), 3.11-3.00 (m, 2H), 2.72-2.58 (m, 2H), 2.20 (s, 3H), 2.14 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H), 1.87-1.77 (m, 1H), 1.68-1.59 (m, 2H), 1.42-1.34 (m, 2H), 1.31-1.26 (m, 1H).

[0251] 13C-NMR (125 MHz, CDCl3): (ppm)=170.32, 170.19, 170.14, 169.40, 156.15, 148.92, 141.30, 133.14, 133.10, 124.59, 119.84, 100.82, 71.48, 70.57, 67.85, 66.73, 64.73, 61.36, 56.03, 46.88, 46.68, 30.26, 26.47, 24.26, 20.70, 20.67, 20.59.

[0252] HRMS: ESI: [M+H]+m/z found 640.2337, calc. 640.2354

Preparation of Compound 12

[0253] To suspension of compound 10 (13 mg, 0.020 mmol, 1.0 eq.) in dry DCM (2 mL) under an argon atmosphere and cooled in ice, was added, drop by drop, a solution of compound 11 (8 mg, 0.021 mmol, 1.05 eq.) and of DIPEA (10 qL, 0.060 mmol, 3.0 eq.). After the addition, the reactional mixture was mixed at 0 C. for 30 min, then at room temperature for one night. The reactional mixture was then washed with an aqueous solution saturated with NaHCO.sub.3, and the organic phase was dried with Na2SO4, filtered and evaporated under reduced pressure. The raw product was purified by column chromatography on silica gel in order to obtain compound 12 in the form of a white powder (10 mg, 0.010 mmol, yld: 52%).

[0254] 1H-NMR (300 MHz, CDCl3): (ppm)=10.41-10.29 (m, 1H), 8.24 (bs, 1H), 8.16-8.05 (m, 1H), 7.85-7.63 (m, 2.5H), 7.57-7.46 (m, 2H), 7.45-7.37 (m, 0.5H), 7.25-7.08 (m, 2H), 6.23-6.10 (m, 0.5H), 5.83-5.75 (m, 0.5H), 5.57 (dd, J=10 Hz, J=8 Hz, 1H), 5.49 (d, J=3 Hz, 1H), 5.17-4.96 (m, 2H), 4.94-4.69 (m, 1H), 4.58 (bs, 1H), 4.33-3.99 (m, 4H), 3.79-3.63 (m, 1H), 3.39-3.01 (m, 2H), 2.21 (s, 3H), 2.16 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 1.82-1.24 (m, 6H).

[0255] 13 C-NMR (125 MHz, CDCl3): (ppm)=170.36, 170.20, 170.14, 169.39, 161.09, 156.42, 152.66, 149.05, 147.37, 140.98, 139.32, 135.29, 133.30, 132.64, 132.37, 130.70, 129.72, 127.87, 125.88, 125.39, 125.04, 124.39, 124.03, 122.30, 119.77, 114.09, 100.76, 71.43, 70.62, 67.84, 66.73, 64.44, 61.31, 40.84, 29.72, 29.40, 25.38, 22.72, 20.69, 20.60, 18.89.

[0256] HRMS: ESI: [M+H]+m/z found 972.2077, calc. 972.2109

Preparation of Compound 13

[0257] To a solution of compound 12 (10 mg, 0.010 mmol, 1.0 eq.) in dry methanol, in an ice bath, was added sodium methoxyde (1.1 mg, 0.020 mmol, 2.0 eq.). The reactional mixture was stirred for 1 h at 0 C. Then the reaction was stopped with Dowex@50X8-100 resin, then filtered and concentrated under reduced pressure. Product 13 was obtained in the form of a white resin (8 mg, 0.010 mmol, quantitative ryld). A high purity was obtained using HPLC in reverse phase (isocratic, water: acetonitrile, 1:1 v:v with 0.1% TFA) to give compound 13 in the form of a white powder.

[0258] 1H-NMR (300 MHz, CD3OD): (ppm)=8.07 (m, 1H), 7.73-7.69 (m, 2H), 7.67-7.60 (m, 2H), 7.50-7.31 (m, 2H), 7.31-7.23 (m, 1H), 7.13-7.05 (m, 1H), 5.02-4.93 (m, 1H), 4.93-4.82 (m, 2H), 4.51-4.40 (m, 1H), 4.13-4.03 (m, 1H), 3.83-3.78 (m, 1H), 3.77-3.71 (m, 1H), 3.68-3.58 (m, 3H), 3.53-3.44 (m, 2H), 3.12-3.01 (m, 1H), 2.94-2.83 (m, 1H), 1.59-1.25 (m, 6H).

[0259] 13C-NMR (125 MHz, CD3OD): (ppm)=163.06, 158.56, 154.58, 152.42, 151.11, 149.16, 148.62, 141.78, 136.22, 134.26, 134.07, 132.97, 132.68, 132.09, 131.01, 130.43, 129.97, 126.45, 126.12, 125.42, 125.22, 123.48, 118.91, 103.10, 96.37, 77.36, 74.86, 71.96, 70.10, 65.76, 62.32, 53.19, 52.68, 41.16, 27.00, 26.27, 19.86.

[0260] HRMS: ESI: m/z [M+H]+ found: 804.1671 calc. 804.1687

Example 2

[0261] Compound 21 is prepared as described in Diagram 2 below.

Diagram 2: Chemical Synthesis of Compound 21

[0262] ##STR00034##

Preparation of Compound 15

[0263] A method analogous to that used for the preparation of compound 6 was used using acetobromo-D-cellobiose 14 (2 g, 2.86 mmol, 1.0 eq.), of 4-hydroxy-3-nitrobenzaldehyde (478 mg, 2.86 mmol, 1.0 eq.) and Ag2O (729 mg, 3.15 mmol, 1.1 eq.)) in order to obtain compound 15 in the form of a light yellow solid (1.864 g, 2.37 mmol, yld: 83%).

[0264] 1H-NMR (300 MHz, CDCl3): (ppm)=9.98 (s, 1H), 8.30 (d, J=2 Hz, 1H), 8.07 (dd, J=9 Hz, J=2 Hz, 1H), 7.43 (d, J=9 Hz, 1H), 5.35-5.05 (m, 5H), 4.95 (t, J=8 Hz, 1H), 4.64-4.57 (m, 2H), 4.38 (dd, J=13 Hz, J=4 Hz, 1H), 4.16-3.98 (m, 3H), 3.94-3.87 (m, 1H), 3.74-3.67 (m, 1H), 2.12 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H).

[0265] 13C-NMR (125 MHz, CDCl3): (ppm)=189.23, 170.88, 170.54, 170.46, 170.11, 169.71, 169.68, 169.47, 153.68, 141.40, 134.58, 131.65, 127.10, 118.64, 101.22, 98.98, 76.24, 73.60, 73.19, 72.39, 71.93, 70.97, 68.11, 61.91, 21.02, 20.88, 20.86.

[0266] HRMS: ESI: [M+Na]+m/z found 808.1874, calc. 808.1912

Preparation of Compound 16

[0267] A method analogous to that used for the preparation of compound 7 was used using compound 15 (650 g, 0.83 mmol, 1.0 eq.), and NaBH4 (34 mg, 0.91 mmol, 1.1 eq.) in order to obtain compound 16 in the form of a white powder pour (580 mg, 0.74 mmol, yld: 89%), which was used in the next reaction without purification.

[0268] 1H-NMR (300 MHz, CDCl3): (ppm)=7.82 (d, J=2 Hz, 1H), 7.54 (dd, J=9 Hz, J=2 Hz, 1H), 7.32 (d, J=9 Hz, 1H), 5.31-5.06 (m, 5H), 4.97 (t, J=8 Hz, 1H), 4.74 (d, J=6 Hz, 1H), 4.64-4.57 (m, 2H), 4.40 (dd, J=13 Hz,=4 Hz, 1H), 4.15-4.07 (m, 2H), 4.01-3.95 (m, 1H), 3.84-3.78 (m, 1H), 3.74-3.68 (m, 1H), 2.14 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.87 (t, J=6 Hz, 1H).

[0269] 13C-NMR (125 MHz, CDCl3): (ppm)=170.48, 170.20, 170.12, 169.79, 169.54, 169.32, 169.11, 148.15, 141.10, 137.49, 131.78, 123.00, 119.23, 100.71, 99.61, 76.01, 73.04, 72.86, 72.23, 71.95, 71.60, 70.85, 67.83, 63.04, 61.63, 61.57, 20.60, 20.54, 20.41.

[0270] HRMS: ESI: [M+Na]+m/z found 810.2022, calc. 810.2069

Preparation of Compound 17

[0271] A method analogous to that used for the preparation of compound 8 was used using compound 16 (400 mg, 0.51 mmol, 1.0 eq.), 4-nitrophenyl chlorformate (225 mg, 1.07 mmol, 2.2 eq.) and pyridine (102 L, 1.27 mmol, 2.5 eq.) in order to obtain compound 17 in the form of a white powder (380 mg, 0.40 mmol, yld: 79%).

[0272] 1H-NMR (300 MHz, CDCl3): (ppm)=8.32 (d, J=9 Hz, 2H), 7.92 (d, J=2 Hz, 1H), 7.63 (dd, J=9 Hz, J=2 Hz, 1H), 7.41 (d, J=9 Hz, 2H), 7.35 (d, J=9 Hz, 2H), 5.32-5.07 (m, 5H), 4.97 (t, J=8 Hz, 1H), 4.67-4.58 (m, 2H), 4.64-4.57 (m, 2H), 4.40 (dd, J=13 Hz, J=4 Hz, 1H),4.15-4.06 (m, 2H), 4.03-3.97 (m, 1H), 3.87-3.81 (m, 1H), 3.74-3.68 (m, 1H), 2.14 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H).

[0273] 13C-NMR (75 MHz, CDCl3): (ppm)=170.51, 170.07, 170.05, 169.68, 169.33, 169.28, 169.03, 155.27, 152.18, 149.47, 145.42, 140.92, 133.94, 130.03, 125.35, 125.29, 121.71, 119.05, 100.75, 99.22, 75.98, 73.09, 72.80, 72.09, 71.91, 71.54, 70.70, 68.81, 67.74, 61.56, 61.53, 0.60, 20.55, 20.42.

[0274] HRMS: ESI: [M+Na]+m/z found 975.2103, calc. 975.2131

Preparation of Compound 18

[0275] A method analogous to that used for the preparation of compound 9 was used, using compound 17 (100 mg, 0.11 mmol, 1.0 eq.), 4 (30 mg, 0.20 mmol, 1.3 eq.) and DIPEA (40 L, 0.23 mmol, 2.1 eq.) in order to obtain compound 18 in the form of a white solid (68 mg, 0.070 mmol, yld: 67%).

[0276] 1H-NMR (300 MHz, CDCl3): (ppm)=7.82 (d, J=2 Hz, 1H), 7.53 (dd, J=9 Hz, J=2 Hz, 1H), 7.28 (d, J=9 Hz, 1H), 5.94-5.80 (m, 1H), 5.44-5.06 (m, 9H), 4.96 (t, J=8 Hz, 1H), 4.64-4.56 (m, 2H), 4.40 (dd, J=13 Hz, J=4 Hz, 1H), 4.15-4.06 (m, 2H), 4.01-3.95 (m, 1H), 3.84-3.78 (m, 1H), 3.73-3.67 (m, 1H), 3.44-3.24 (m, 3H), 3.05-2.91 (m, 2H), 2.42 (bs, 1H), 2.22 (t,=11 Hz, 1H), 2.13 (s, 3H), 2.12 (s, 3H), 2.08 (s, 6H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.77-1.26 (m, 6H).

[0277] 13C-NMR (75 MHz, CDCl3): (ppm)=170.49, 170.18, 170.12, 169.72, 169.47, 169.29, 169.02, 156.20, 148.73, 141.14, 134.66, 133.19, 132.93, 124.56, 119.18, 117.69, 100.80, 99.37, 75.94, 73.01, 72.86, 72.18, 72.06, 71.59, 70.84, 67.74, 64.64, 61.51, 61.48, 58.28, 56.31, 51.93, 42.47, 28.93, 24.94, 23.67, 20.69, 20.64, 20.54, 20.52. HRMS: ESI: [NI+H]+m/z found 968.3545, calc. 968.3512

Preparation of Compound 19

[0278] A method analogous to that used for the preparation of compound 10 was used, using compound 18 (68 mg, 0.070 mmol, 1.0 eq.), 1, 3- dimethylbarbituric acid (86 mg, 0.56 mmol, 8.0 eq.) and palladium(0) tetrakis (triphenylphosphine) (1.6 mg, 0.0014 mmol, 2 mol %) in order to obtain compound 19 in the form of a white solid (49 mg, 0.053 mmol, yld: 77%).

[0279] 1H-NMR (300 MHz, CDCl3): (ppm)=7.79 (d, J=2 Hz, 1H), 7.51 (dd, J=9 Hz, J=2 Hz, 1H), 7.27 (d, J=9 Hz, 1H), 5.49-5.42 (m, 1H), 5.29-5.04 (m, 7H), 4.95 (t, J=8 Hz, 1H), 4.63-4.55 (m, 2H), 4.39 (dd, J=13 Hz, J=4 Hz, 1H), 4.14-4.04 (m, 2H), 4.00-3.93 (m, 1H), 3.84-3.78 (m, 1H), 3.73-3.67 (m, 1H), 3.30-3.21 (m, 1H), 3.12-3.02 (m, 2H), 2.70-2.62 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.08 (s, 6H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.85-1.12 (m, 6H).

[0280] 13C-NMR (75 MHz, CDCl3): (ppm)=170.50, 170.20, 170.12, 169.72, 169.48, 169.30, 169.02, 156.10, 148.74, 141.15, 133.18, 132.84, 124.56, 119.20, 100.82, 99.36, 75.93, 73.02, 72.87, 72.19, 72.08, 71.60, 70.85, 67.75, 64.68, 61.53, 61.47, 56.00, 46.83, 46.63, 30.22, 26.42, 24.22, 20.70, 20.66, 20.55, 20.53.

[0281] HRMS: ESI: [M+H]+m/z found 928.3230, calc. 928.3199

Preparation of Compound 20

[0282] A method analogous to that used for the preparation of compound 12 was used using compound 19 (49 mg, 0.053 mmol, 1.0 eq.), 11 (21 mg, 0.054 mmol, 1.05 eq.) and DIPEA (28 L, 0.16 mmol, 3.0 eq.) in order to obtain compound 20 in the form of a white powder. (29 mg, 0.023 mmol, yld: 43%).

[0283] 1H-NMR (300 MHz, CDCl3): (ppm)=10.52 (bs, 1H), 8.21 (bs, 1H), 8.10-7.97 (m, 1H), 7.76-7.64 (m, 2.5H), 7.56 (m, 0.5H), 7.50-7.43 (m, 2H), 7.18-7.09 (m, 2H), 6.17-6.07 (m, 0.5H), 5.76 (bs, 0.5H), 5.30-5.04 (m, 5H), 4.95 (t, =8 Hz, 1H), 4.91-4.72 (m, 2H), 4.63-4.48 (m, 3H), 4.40 (dd, J=13 Hz, J=4 Hz, 1H), 4.19-4.04 (m, 3H), 4.00-3.93 (m, 1H), 3.83-3.57 (m, 3H), 3.37-3.18 (m, 1.5H), 3.11-2.98 (m, 0.5H), 2.12 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.06 (s, 6H), 2.03 (s, 3H), 2.01 (s, 3H), 1.81-1.44 (m, 6H).

[0284] 13C-NMR (75 MHz, CDCl3): (ppm)=170.52, 170.21, 170.18, 169.74, 169.50, 169.31, 169.04, 161.13, 156.33, 154.23, 152.63, 149.11, 147.57, 147.34, 140.89, 135.28, 133.22, 132.80, 132.24, 130.59, 129.54, 127.82, 126.91, 125.85, 125.24, 124.84, 124.17, 122.22, 119.11, 100.85, 99.30, 75.97, 72.98, 72.90, 72.24, 72.20, 72.08, 71.61, 70.85, 67.76, 64.47, 61.53, 61.48, 51.46, 40.79, 26.03, 25.27, 20.70, 20.67, 20.54, 18.84.

[0285] HRMS: ESI: [M+H]+m/z found 1260.2914, calc. 1260.2954

Preparation of Compound 21

[0286] A method analogous to that used for the preparation of compound 13 was used, using compound 20 (23 g, 0.018 mmol, 1.0 eq.), and sodium methoxyde (1.0 mg, 0.036 mmol, 2.0 eq.) in order to obtain compound 21 in the form of a white powder (17 mg, 0.017 mmol, yld: 97%).

[0287] 1H-NMR (300 MHz, CDCl3): (ppm)=8.19 (bs, 1H), 7.85-7.81 (m, 2H), 7.79-7.72 (m, 2H), 7.62-7.43 (m, 2H), 7.40-7.33 (m, 1H), 7.27-7.17 (m, 2H), 5.14-5.04 (m, 1.5H), 4.99-4.94 (m, 1H), 4.62-4.52 (m, 0.5H), 4.47 (d, =8 Hz, 2H), 4.26-4.16 (m, 1H), 3.97-3.82 (m, 3H), 3.74-3.56 (m, 5H), 3.44-3.35 (m, 3.5H), 3.30-2.95 (m, 3H), 3.11-2.98 (m, 0.5H), 1.72-1.15 (m, 6H).

[0288] 13C-NMR (75 MHz, CDCl3): (ppm)=161.65, 157.10, 153.33, 153.05, 150.98, 149.35, 148.93, 147.75, 147.23, 140.47, 134.82, 132.84, 132.67, 131.57, 130.77, 129.62, 129.05, 128.55, 125.05, 123.86, 122.08, 117.46, 103.16, 100.77, 78.53, 76.75, 76.50, 75.55, 74.94, 73.51, 73.00, 72.99, 69.98, 64.34, 61.05, 60.19, 51.29, 39.52, 25.73, 24.79, 18.46.

[0289] HRMS: ESI: [M+H]+m/z found: 966.2201, calc. 966.2215

Example 3

[0290] Compound 21 is prepared as described in Diagram 3 below.

Diagram 3: Chemical Synthesis of Compound 27

[0291] ##STR00035##

Preparation of Compound 22

[0292] To a solution of 3 (335 mg, 1.48 mmol, 1.0 eq.) in 5 mL in dichloromethane were added potassium carbonate (636 mg, 4.6 mmol, 3.1 eq.) and, drop by drop, methyl chlorformate (115 L, 1.48 mmol, 1.0 eq.). After stirring for 10 minutes at room temperature, the solvent was evaporated under reduced pressure. The raw product was purified by column chromatography on silica gel in order to obtain compound 22 in the form of a light, yellow oil (282 mg, 1.33 mmol, yld: 90%).

[0293] 1H-NMR (300 MHz, CDCl3): (ppm)=5.96-5.82 (m, 1H), 5.24-5.16 (m, 3H), 3.69 (s, 3H), 3.41-3.30 (m, 3H), 3.01-2.91 (m, 2H), 2.40 (m, 1H), 2.23-2.17 (m, 1H), 1.75-1.70 (m, 1H), 1.60-1.56 (m, 2H), 1.54-1.40 (m, 2H), 1.37-1.24 (m, 1H).

[0294] 13C-NMR (75 MHz, CDCls): (ppm)=157.34, 134.50, 117.71, 58.62, 56.29, 51.90, 42.31, 28.85, 24.92, 23.58.

[0295] HRMS: ESI: [NI+H]+m/z found 213.1601, calc. 213.1603

Preparation of Compound 23

[0296] To a solution of lithium tetrahydro aluminate (2.64 mmol, 2.0 eq.) in 5 mL of tetrahydrofuran, 22 (280 mg, 1.32 mmol, 1.0 eq.) was added, drop by drop, and the medium was stirred at 40 C. for one night. The solvent was evaporated and product 23 was used without purification.

[0297] H-NMR (300 MHz, CDCl3): (ppm)=5.97-5.82 (m, 1H), 5.24-5.08 (m, 2H), 3.43-3.33 (m, 1H), 2.98-2.84 (m, 2H), 2.71-2.53 (m, 3H), 2.46-2.30 (m, 3H), 2.23-2.12 (m, 2H), 1.83-1.23 (m, 8H).

[0298] 13C-NMR (75 MHz, CDCl3): (ppm)=127.43, 124.84, 61.65, 58.05, 55.71, 52.99, 52.19, 50.49, 48.26, 45.28, 34.14, 27.96, 26.19, 21.68, 21.43, 20.96, 20.02.

Preparation of Compound 24

[0299] A method analogous to that used for the preparation of compound 9 was used using compound 8 (150 mg, 0.23 mmol, 1.0 eq.), 23 (100 mg, 0.42 mmol, 1.8 eq.) and DIPEA (300 qL, 1.72 mmol, 7.6 eq.) in order to obtain compound 24 in the form of a white solid (84 mg, 0.12 mmol, yld: 53%).

[0300] 1H-NMR (500 MHz, CDCl3): (ppm)=7.75 (s, 1H), 7.48 (dd, J=9 Hz, J=2 Hz, 1H), 7.30 (d, =9 Hz, 1H), 5.90-5.72 (m, 1H), 5.50 (dd, J=10 Hz, J=8 Hz, 1H), 5.42 (d, J=3 Hz, 1H), 5.14 (d, J=6 Hz, 1H), 5.12-4.99 (m, 6H), 4.25-4.11 (m, 2H), 4.11-4.01 (m, 1H), 3.58-3.47 (m, 1H), 3.35-3.19 (m, 2H), 3.09-2.96 (m, 1H), 2.88 (d, J=3 Hz, 3H), 2.81-2.71 (m, 1H), 2.68-2.58 (m, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.02 (s, 3H), 1.97 (s, 3H), 1.70-1.36 (m, 6H).

[0301] 13C-NMR (125 MHz, CDCl3): (ppm)=170.29, 170.18, 170.11, 169.38, 156.04, 148.92, 141.16, 135.08, 133.27, 133.23, 124.60, 119.66, 117.51, 100.69, 71.41, 70.53, 67.81, 66.74, 65.25, 61.36, 57.46, 57.10, 51.26, 49.88, 35.68, 30.92, 29.67, 28.20, 24.90, 22.60, 20.64.

[0302] LRMS: ESI: [M+H]+m/z found 694.2, calc. 694.2823.

Preparation of Compound 25

[0303] A method analogous to that used for the preparation of compound 10 was used using compound 24 (84 mg, 0.12 mmol, 1.0 eq.), 1,3-Dimethylbarbituric acid (95 mg, 0.61 mmol, 5.0 eq.) and palladium(0) tetrakis (triphenylphosphine (1 mg, 0.0012 mmol, 1 mol %) in order to obtain compound 25 in the form of a white solid (49 mg, 0.07 mmol, yld: 62%).

[0304] 1H-NMR (500 MHz, CDCl3): (ppm)=7.78 (d, J=9 Hz, 1H), 7.49 (d, J=8 Hz, 1H), 7.30 (d, J=9 Hz, 1H), 5.51 (dd, J=10 Hz, J=8 Hz, 1H), 5.43 (d, J=3 Hz, 1H), 5.32-5.26 (m, 1H), 5.13-4.98 (m, 4H), 4.24-4.19 (m, 1H), 4.16-4.10 (m, 1H), 4.08-4.03 (m, 1H), 3.26-3.19 (m, 1H), 3.18-3.12 (m, 1H), 3.06-3.01 (m, 1H), 2.93 (d, J=7 Hz, 3H), 2.82-2.68 (m, 1H), 2.61-2.51 (m, 1H), 2.15 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H), 1.79-1.75 (m, 1H), 1.61-1.50 (m, 2H), 1.42-1.20 (m, 3H).

[0305] 13C-NMR (125 MHz, CDCl3): (ppm)=170.34, 170.22, 170.16, 169.42, 156.23, 148.93, 141.26, 133.30, 133.20, 124.59, 119.74, 100.77, 71.47, 70.57, 67.86, 66.77, 65.28, 61.39, 55.55, 55.12, 46.80, 36.00, 30.56, 26.30, 24.35, 20.72, 20.70, 20.62.

[0306] HRMS: ESI: [M+H]+m/z found 654.2484, calc. 654.2504

Preparation of Compound 26

[0307] A method analogous to that used for the preparation of compound 12 was used using compound 25 (25 mg, 0.04 mmol, 1.0 eq.), 11 (21 mg, 0.04 mmol, 1.0 eq.) and DIPEA (33 L, 0.19 mmol, 5.0 eq.) in order to obtain compound 26 in the form of a white powder (10 mg, 0.01 mmol, yld: 27%).

[0308] 1H-NMR (300 MHz, CDCl3): (ppm)=10.65-10.32 (m, 1H), 8.27-8.14 (m, 1H), 8.06-7.90 (m, 1H), 7.86-7.67 (m, 3H), 7.57-7.46 (m, 1H), 7.46-7.34 (m, 1H), 7.34-7.21 (m, 1H), 7.11-7.03 (m, 1H), 5.56-5.49 (m, 1H), 5.46 (br s, 1H), 5.13-4.98 (m, 2H), 4.98-4.67 (m, 1H), 4.55 (br s, 1H), 4.31-3.97 (m, 4H), 3.93-3.78 (m, 1H), 3.28-3.03 (m, 2H), 3.01-2.88 (m, 3H), 2.19 (s, 3H), 2.12 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.78-1.23 (m, 6H).

[0309] 13C-NMR (75 MHz, CDCls): (ppm)=170.41, 170.30, 170.25, 169.52, 160.53, 156.49, 153.10, 149.15, 147.52, 141.32, 139.29, 135.29, 133.31, 132.92, 132.28, 130.79, 129.79, 127.87, 126.09, 125.49, 125.13, 124.45, 124.38, 122.55, 119.83, 114.28, 100.84, 71.54, 70.67, 67.94, 66.82, 65.99, 61.42, 40.78, 29.83, 29.46, 26.68, 25.40, 20.78, 20.70, 20.54, 19.04.

[0310] HRMS: ESI: [M+H]+m/z found 986.2219, calc. 986.2260

Preparation of Compound 27

[0311] A method analogous to that used for the preparation of compound 13 was used, using compound 26 (10 g, 0.01 mmol, 1.0 eq.), and sodium methoxyde (2.0 mg, 0.04 mmol, 3.5 eq.) in order to obtain compound 27 in the form of a white powder (3.57 mg, 0.004 mmol, yld: 43%).

[0312] Resolution of the RMN spectra is too low to be useful.

[0313] HRMS: ESI: [M+H]+m/z found: 818.1838, calc. 818.1838

Example 4

[0314] Probes 13, 21 and 27 according to the invention were evaluated by incubation with the target enzyme, -galactosidase (EC 3.2.1.23; b-gal; commercial) in an in vitro medium in multi-well micro-plates designed for fluorescence readers. The probes were evaluated using the following criteria: [0315] detection of the elevated fluorescence intensity generated by the presence of enzyme activity (on), [0316] detection of the complete absence (off) of fluorescence in samples that do not contain the target enzyme (no intrinsic fluorescence), [0317] detection of the absence of any hydrolytic degradation of the probe over time, demonstrating the robustness of the probe at pH 7 in an aqueous medium (no false positive signal), [0318] detection of the rapidity of response to the presence of enzyme activity making it possible to reach a maximum signal quickly, [0319] detection of improved kinetics of the two-spacer probe, [0320] detection of high photo-stability of the solid fluorophore generated under extended irradiation by the fluorescence reader.

[0321] These results were compared with those obtained with a probe from the prior art (compound I. 1 of application WO 2014/020285) comprising a cyclizing type spacer (28):

##STR00036##

Protocol for the Detection of Fluorescence:

[0322] 10 mM probe parent solutions in MeOH were diluted with PBS (Dulbecco's Phosphate Buffer Saline, Invitrogen Corp.) in order to obtain solutions with concentration ranges from 50 M to 1 mM. Ten L of each of these solutions was added to 80 L of PBS in a 96-well black plate, and heated to 37 C. before the addition of the purified enzyme. Final probe concentrations were in the range of 5 L to 100 L. The plate was then incubated at 37 C. (or 25 C.) and fluorescence was measured over time by a fluorescence reader (EnSpire, Perkin Elmer; acquisition wavelengths: .sub.ex=355 nm, .sub.em=530 nm). The resulting curves are the mean of the duplicates.

Results:

[0323] The results obtained are presented in FIGS. 2 and 3.

[0324] Compared to probe 28, probe 13 according to the invention comprising a pair of eliminating/cyclizing spacers makes it possible to reap the benefits of greater response speed while conserving the high stability of the probe in the absence of the target enzyme (false positive signal). Thus, under the same temperature, pH and concentration conditions, probe 13, based on a pair of spacers, has an enzymatic response that is 5 times more rapid than that of probe 28 which comprises only a single spacer. In addition, in the absence of enzyme, probe 13 is stable for more than 15 h and does not generate any measurable fluorescence.

[0325] Probes 21 and 27 according to the invention make it possible to benefit from quicker response time while conserving the probe's high stability in the absence of the target enzyme (lack of false positive signal).

[0326] Probe 21 was tested at different concentrations: 5 M, 10 M, 25 M and 50 M. The fluorescence measured is proportional to the concentration of the probe.

[0327] Fluorescence can be detected at a 5 M content of the probe.

Example 5

[0328] A supernatant of a yeast strain culture, not secreting (A) or secreting (B) a B-glucosidase, was added to probe 21 according to the invention responding to this enzymatic activity. To do this, yeast cells bearing a plasmid that provides hygromycin resistance and bearing, or not, an expression cassette for a secreted beta-glucosidase were cultured for 86 h at 30 C. in 5 mL of a YPD rich medium (10 g of Bacto Peptone Difco, 10 g of Bacto Yeast Extract Difco, 20 g of Glucose, 20 g of Bacto Agar, qsp 1 L distilled water) containing 200 g/mL of hygromycin. The culture was then centrifuged at 4000 tr/min on an Allegra 25R centrifuge (Beckman/Coulter), in a swash plate TS-5.1-500) at 20 C. and 20 L of supernatant are taken and added to 180 L of a PBS1X solution containing the probe substrate at 50 M. The mixture was then homogenized and incubated 30 minutes at 37 C. The, 10 L of this mixture were deposited between a microscope slide and cover glass and observed by fluorescence microscope with a 340 nm excitation filter and a 525 nm emission filter.

[0329] The photographs, represented in FIG. 4, were obtained at 100 magnification at immersion on a Zeiss, AX10 microscope.

[0330] Fluorescent precipitants (white dots) appear distinctly, which makes it possible to forecast use with high throughput imaging (automated segmentation and quantification).

Example 6

[0331] The detection kinetics of cellulase activity in a micro-organism culturing medium was evaluated by optical reading on a MITHRAS LB 940 device of the enzymatic activity of a supernatant of a yeast cells culture that secrete (Supernatant) or which do not secrete (Control) p-glucosydase.

[0332] Yeast culturing is conducted according to the protocol described in example 5 until the obtaining of 20 L of supernatant. This supernatant is then added to 180 L of a PBS1X solution containing probe 21 according to the invention at 50 M in opaque background microplate wells. Signal reading is executed over time on a MITHRAS LB 940 device, after excitation of the probe to 340 nm and collection of the emission at 535 nm.

[0333] The results, shown in FIG. 5, show that the invention makes it possible to detect glycosidase activity secreted into the supernatant after 45 minutes of incubation, that the signal is maximum after 3 h45 m of incubation, and that the signal to noise ratio is about 55 nm.