FLUOROGENIC BETA-LACTAMASE SUBSTRATE AND ASSOCIATED DETECTION METHOD

Abstract

Probes for the detection of β-lactamase-type enzymatic activity. In particular, novel fluorogenic substrates for detecting the presence of a catalytically active β-lactamase and a detection method using such substrates.

Claims

1. A compound of formula (I): ##STR00062## in which: W is —O— or —NR.sub.13—, with R.sub.13 being a C.sub.1-C.sub.4 alkyl or a hydrogen atom; R.sub.1 is such that HWR.sub.1, obtained after cleavage of the —C(O)—WR.sub.1 bond present in formula (I), belongs to the class of fluorophores; R.sub.2, R.sub.3 and R.sub.4 are defined as follows: either R.sub.2 is a C.sub.1-C.sub.4 alkyl, R.sub.3 is a C.sub.1-C.sub.4 alkyl or a hydrogen atom, and R.sub.4 is a C.sub.1-C.sub.4 alkyl; or R.sub.3 is a C.sub.1-C.sub.4 alkyl or a hydrogen atom and R.sub.2 and R.sub.4 are bonded to each other and form a —(CH.sub.2).sub.p—Y.sub.q—(CH.sub.2).sub.r— chain in direction of R.sub.2 toward R.sub.4, wherein Y is O, NR.sub.14, N(R.sub.14).sub.2.sup.+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,and each R.sub.14 represents independently a hydrogen atom, a C.sub.1-C.sub.6 alkyl, an amino protecting group, or -(L).sub.q-GP, with q which is equal to 0 or 1, L which is a linking arm and GP which is a hydro solubilizing group; or R.sub.2 is a C.sub.1-C.sub.4 alkyl and R.sub.3 and R.sub.4 are bonded together and form, with the carbon atom to which they are bonded, an aliphatic carbocycle; R.sub.5 and R.sub.6 are identical or different and represent, independently of each other, a hydrogen atom, a C.sub.1-C.sub.4 alkyl, or a C.sub.5-C.sub.10 aryl; R.sub.7 is a hydrogen atom, or a group selected from C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.4 alkoxy; R.sub.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.sub.9R.sub.10—; with R.sub.9 and R.sub.10 being identical or different and representing, independently of each other, a hydrogen atom or a C.sub.1-C.sub.4 alkyl; Q is H, a cation or R16, where R16 is selected from C.sub.1-C.sub.6 alkyl optionally substituted with an aryl or a —O(CO)—R, with R being independently selected from H, C.sub.1-C.sub.6 alkyl and C.sub.3-C.sub.6 cycloalkyl; and R.sub.11 is an organyl group, an organylamino group, an organyloxy group, or an organylthio group, X represents a bond, or a group selected from ##STR00063##

2. Compound (I) according to claim 1 of formula (Ia): ##STR00064## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.11, Q, W, X and V are as defined in claim 1.

3. Compound (I) according to claim 1 of formula (Ib): ##STR00065## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, Q, W, X and V are as defined in claim 1.

4. Compound (I) according to claim 1, wherein R.sub.1 comprises an aromatic group comprising one or more aromatic rings, optionally 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 C═O carbonyl.

5. Compound (I) according to claim 1, wherein WR.sub.i is an aromatic group —OR.sub.1 according to formula (A1): ##STR00066## in which: either X.sub.2 is an oxygen atom and X.sub.1 is a —NH.sub.2, —OH, —SH, C.sub.1-C.sub.20 alkyl, C.sub.5-C.sub.24 aryl, C.sub.2-C.sub.6 alkenyl, —O-(C.sub.1-C.sub.20 alkyl), —O-phenyl, —NH-(C.sub.1-C.sub.20 alkyl), —NH-phenyl, —S-(C.sub.1-C.sub.20 alkyl) or —S-(C.sub.5-C.sub.24 aryl group), said alkyl, aryl, alkenyl and phenyl groups being optionally substituted; or X.sub.2 represents a nitrogen atom and is bound to X.sub.1 which then represents CH, O, S, N or, NH to form a C.sub.5-C.sub.24 heteroaryl, optionally substituted; ##STR00067## represents a C.sub.5-C.sub.24 aryl or a C.sub.5-C.sub.24 heteroaryl, optionally substituted, ##STR00068## or —OR.sub.1 is of the aryloxy type and corresponds to one of the following structures (A2), (A3) or (A4): ##STR00069## in which: T is —NH—C(O)—, —S—, —O—, —NH—, —N(C.sub.1-C.sub.20 alkyl)- or —N(C.sub.5-C.sub.24 aryl)-; Re is a hydrogen atom or an electron-withdrawing group such as —CN or —COORh, with Rh which represents a C.sub.1-C.sub.4 alkyl group, or Re is —CONRiRj, with Ri and Rj, identical or different, which represent a hydrogen atom, or a C.sub.1-C.sub.4 alkyl group, or Re is —CF.sub.3, a C.sub.2-C.sub.6 alkenyl, or a heteroaryl, said heteroaryl and alkenyl being optionally substituted; Rf is a hydrogen atom, a chlorine, bromine, iodine or fluorine atom, —OH, —NH.sub.2, —NRkRI, —NHRk or —ORk, with Rk and RI, identical or different, which each, independently, represent a C.sub.1-C.sub.4 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; and Rg is a hydrogen, Br, Cl, I or F atom; ##STR00070## in which: T′ is —NH.sub.2, —OH, a C.sub.5-C.sub.24 aryl group, a C.sub.1-C.sub.4 alkyl group, —SH, —NHR′g, —OR′g, —NR′gRh', —SR′g, or an optionally substituted C.sub.2-C.sub.6 alkenyl group, or a heteroaryl, R′g and Rh′, identical or different, representing a C.sub.1-C.sub.4 alkyl or C.sub.5-C.sub.24 aryl group; R′e is a hydrogen atom or an electron-withdrawing group such as —CN or —COOR′i, with R′i which represents a C.sub.1-C.sub.4 alkyl group, or R′e is —CONR′jR′k, with R′j and R′k, identical or different, which represent a hydrogen atom, or a C.sub.1-C.sub.4 alkyl group, or R′e is —CF.sub.3, or a 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-benzo imidazolyl, 4-pyrimidinone-2-yl or quinazolinone-2-yl group; R′f is a hydrogen, chlorine, bromine, iodine or fluoride atom, —OH, —NH.sub.2, -NR′IR′m or —OR′I, with R′I and R′m, identical or different, which represent a C.sub.1-C.sub.4 alkyl group; or R′e and R′f 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 ##STR00071## in which either X′.sub.2 is an oxygen atom and X′.sub.1 is a —NH.sub.2, —OH, —SH, C.sub.1-C.sub.20 alkyl, C.sub.5-C.sub.24 aryl, C.sub.2-C.sub.6 alkenyl, —O-(C.sub.1-C.sub.20 alkyl), —O-phenyl, —NH-(C.sub.1-C.sub.20 alkyl), —NH-phenyl, —S-(C.sub.1-C.sub.20 alkyl) or —S-(C.sub.5-C.sub.24 aryl group), said alkyl, aryl, alkenyl and phenyl groups being optionally substituted; or X′.sub.2 represents a nitrogen atom and is bound to X.sub.i which then represents CH, O, S, N or, NH to form a C.sub.5-C.sub.24 heteroaryl, optionally substituted; ##STR00072## represents a C.sub.5-C.sub.10 aryl or a C.sub.5-C.sub.10 heteroaryl, optionally substituted.

6. Compound (I) according to claim 1, wherein 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.

7. Compound (I) according to claim 1, wherein R.sub.11 is selected from C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, heterocyclyl having 5 to 10 ring atoms, C.sub.5-C.sub.10 aryl, heteroaryl having 5 to 10 ring atoms, C.sub.7-C.sub.16 aralkyl, and NR″R″′; said alkyl, cycloalkyl, hetroalkyl, haloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl and aralkyl being optionally substituted with one or more substituents independently selected from oxo, halogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.1-C.sub.6 haloalkyl, heterocyclyl having 5 to 10 ring atoms, C.sub.5-C.sub.10 aryl, heteroaryl having 5 to 10 ring atoms, —OH, —NR″R″′, —NO.sub.2, —CN, —O—(CO)—R and —(CO)—R; with each R being independently selected from H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy and —NR″R′′; and with each R″ and R″′ being independently selected from H and C.sub.1-C.sub.6 alkyl.

8. Compound (I) according to claim 1 of formula (Ic): ##STR00073## where R.sub.1, R.sub.11, Z, Q, X and n are as defined in claim 1, and Y represents —CH.sub.2—, —NR.sub.14—, or —N(R.sub.14).sub.2.sup.+—, with each R.sub.14 represents independently a hydrogen atom, a C.sub.1-C.sub.6 alkyl, an amino protecting group or -(L).sub.q-GP, with q which is equal to 0 or 1, L which is a linking arm and GP which is a hydro solubilizing group.

9. Compound (I) according to claim 1 of formula (Id): ##STR00074## where R.sub.1, R.sub.11, X and Q are as defined in claim 1, and Y represents —CH.sub.2—, —NR.sub.14—, or —N(R.sub.14).sub.2.sup.+—, with each R.sub.14 represents independently a hydrogen atom, a C.sub.1-C.sub.6 alkyl, an amino protecting group or -(L).sub.q-GP, with q which is equal to 0 or 1, L which is a linking arm and GP which is a hydro solubilizing group.

10. Compound (I) according to claim 1 of formula (Ie): ##STR00075## where R.sub.1, R.sub.9, R.sub.10, R.sub.11, X and Q are as defined in claim 1, and Y represents —CH.sub.2—, —NR.sub.14—, or —N(R.sub.14).sub.2.sup.+—, with each R.sub.14 represents independently a hydrogen atom, a C.sub.1-C.sub.6 alkyl, an amino protecting group or -(L).sub.q-GP, with q which is equal to 0 or 1, L which is a linking arm and GP which is a hydro solubilizing group.

11. Method for the in vitro or ex vivo detection of a β-lactamase, comprising the steps of: putting a sample to be analyzed 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 C(═V) and NR.sub.7, followed by a cleavage of the —C(O)—WR.sub.1, bond, leading to the release of HWR.sub.1, quantitative or qualitative analysis of the fluorescent precipitate, and correlate the quantitative or qualitative analysis of the fluorescent precipitate to the presence or absence of β-lactamase in the sample.

12. Method for the in vitro or ex vivo detection of antibiotic-resistant bacteria comprising the steps of: putting a sample to be analyzed 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 C(═V) and NR.sub.7, followed by a cleavage of the —C(O)—WR.sub.1, bond, leading to the release of HWR.sub.1, quantitative or qualitative analysis of the fluorescent precipitate, and correlate the quantitative or qualitative analysis of the fluorescent precipitate to the presence or absence of antibiotic-resistant bacteria in the sample.

13. A kit for detecting a β-lactamase, said kit comprising a compound according to claim 1.

14. A device for detecting a β-lactamase, said device comprising a compound according to claim 1.

15. A method for the in vivo detection, in animals or in human beings, of a β-lactamase, using a compound (I) according to claim 1.

16. A compound of formula (II): ##STR00076## in which: R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.11, Z, n, Q, X and V are as defined in claim 1, R.sub.12 represents a hydrogen atom, or an amine function protecting group.

17. Process for the preparation of a compound of formula (I) according to claim 1 comprising the following steps: implementation of a compound (II), ##STR00077## in which: R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.11, Z, n, Q, X and V are as defined in claim 1, where R.sub.12 represents a hydrogen atom; implementation of a compound (III) of formula ##STR00078## with R.sub.1 as defined in claim 1 and M representing a leaving group, obtaining compound (I) by addition reaction of said compound (II) to compound (III).

18. Process for the preparation of a compound of formula (I) according to claim 1, where X is ##STR00079## comprising the following steps: implementation of a compound (VII) of the following formula: ##STR00080## implementation of a compound (VIII) of formula ##STR00081## and obtaining compound (I) by reaction of said compound (VII) with compound (VIII), where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, V, R.sub.11, Q, W, Z and n are as defined in claim 1, and LG represents a leaving group.

19. Process for the preparation of a compound of formula (I) according to claim 1, where X is ##STR00082## comprising the following steps: implementation of a compound (VII) of the following formula: ##STR00083## implementation of a compound (X) of formula ##STR00084## and obtaining compound (I) by reaction of said compound (VII) with compound (IX), where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, V, R.sub.11, Q, W, Z and n are as defined in claim 1, LG represents a leaving group, and R.sub.15 represents OH, or both R.sub.15 are bonded to each other and forms, together with the B atom to which they are bonded, a heterocycle having from 5 to 10 ring atoms.

20. Process for the preparation of a compound of formula (I) according to claim 1, where X is ##STR00085## comprising the following steps: implementation of a compound (VII) of the following formula ##STR00086## implementation of a compound (X) of formula ##STR00087## and obtaining compound (I) by reaction of said compound (VII) with compound (X), where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, V, R.sub.11, Q, W, Z and n are as defined in claim 1, LG represents a leaving group, and R.sub.15 represents OH, or both R.sub.15 are bonded to each other and forms, together with the B atom to which they are bonded, a heterocycle having from 5 to 10 ring atoms.

Description

EXAMPLES

Example 1: Synthesis of Compound 15

[0322] ##STR00053##

ELF-97

[0323] A solution of anthranilamide (2.000 g, 11.7 mmol, 1.0 eq.) in dry EtOH (20 mL) is treated with 5-chlorosalicylaldehyde (1.831 g, 11.7 mmol, 1.0 eq.), and the mixture is refluxed for 30 minutes. Then, para-toluenesulfonic acid (PTSA) (40 mg, 0.234 mmol, 0.02 eq.) is added, and refluxing is continued for another hour. The reaction mixture is brought down to room temperature and treated portionwise with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (2.678 g, 11.8 mmol, 1.01 eq.). Stirring is continued overnight. The resulting crude suspension is filtered and the filter cake washed 2 times with EtOH and 2 times with diethyl ether. ELF-97 (3.41 g, 11.11 mmol, 95%) is obtained as a light beige powder and used without further purification in a later step.

[0324] .sup.1H-NMR (300 MHz, CDCl.sub.3): 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)

[0325] Spectral data are in accordance with 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).

Compound 4

[0326] A solution of 2-aminomethylpiperidine (1) (3.0 g, 26.3 mmol, 1.0 eq.) in toluene (50 mL) is treated portionwise with phthalic anhydride (3.89 g, 26.3 mmol, 1.0 eq.), followed by dropwise addition of triethylamine (550 μL, 3.95 mmol, 0.15 eq.). The mixture is refluxed for 2 h using a Dean-Stark apparatus. The mixture is filtered and the filtrate reduced to dryness under reduced pressure. The product 2 (5.42 g, 22.2 mmol, 85%) is obtained as a light yellow solid and used in the next step without further purification.

[0327] An ice-cold ethanol solution (45 mL) of compound 2 (5.42 g, 22.2 mmol, 1.0 eq.) is treated with potassium carbonate (3.99 g, 28.9 mmol, 1.3 eq.), tetra-n-butylammonium iodide (820 mg, 2.2 mmol, 0.10 eq.), and allyl bromide (2.50 mmL, 28.9 mmol, 1.3 eq.). The cooling bath is removed and the mixture stirred for 36 h. Upon verification that the reaction is complete, the mixture is filtered over a pad of Celite and the filtrate evaporated to dryness under reduced pressure. The oily residue is taken up in EtOAc and washed with a saturated aqueous solution of NH.sub.4Cl; the two layers are separated, and the organic phase is washed twice with saturated aqueous NH.sub.4Cl. The combined aqueous phases are extracted 3 times with EtOAc. The combined organic phases are dried over Na.sub.2SO.sub.4, filtered and evaporated to dryness. The crude oil is purified via column chromatography on silica gel (PE/EtOAc 80/20 to 60/40 v/v) to yield 3 (3.582 g, 12.6 mmol, 57%) as a light yellow oil.

[0328] .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

[0329] .sup.13C-NMR (75 MHz, CDCl.sub.3): δ(ppm)=168.4, 133.7, 131.9, 123.0, 55.5, 46.4, 43.4, 30.6, 26.1, 24.1.

[0330] HRMS: ESI: [M+H].sup.+ m/z found 245.1290, calc. 245.1290.

[0331] An ice-cold solution of 3 (3.582 g, 12.6 mmol, 1.0 eq.) in iPrOH/H.sub.2O 6/1 v/v (175 mL) is treated portionwise with sodium borohydride (665 mg, 17.58 mmol, 5.0 eq.). Cooling is removed, and the Mixture stirred overnight (o/n) at room temperature. The pH is then adjusted to 1 using concentrated HCl. The resulting mixture is filtered and the filtrate heated to 80° C. for 2 h. The isopropyl alcohol is removed under reduced pressure and the resulting aqueous solution washed 5 times with diethyl ether, basified With 2 M NaOH aqueous solution, and extracted With diethyl ether. The combined organic extracts are dried over Na.sub.2SO.sub.4, filtered and evaporated to dryness to obtain 4 as a light yellow oil (1.939 g, 12.6 mmol, quantitative yield).

[0332] .sup.1H -NMR (300 MHz, CDCl.sub.3): 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, 11H), 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).

[0333] .sup.13C -NMR (75 MHz, CDCl.sub.3): δ(ppm)=157.34, 134.50, 117.71, 58.62, 56.29, 51.90, 42.31, 28.85, 24.92, 23.58.

##STR00054##

[0334] HRMS: ESI: [M+H].sup.+ m/z found 155.1543, calc. 155.1548.

Compound 6

[0335] A solution of 7-aminocephalosporanic acid (5) (2.000 g, 7.345 mmol, 1.0 eq.) in H.sub.2O/MeOH (20 mL, 1/1 v/v) at −20° C. is treated with 10 M NaOH (2 mL) and the resulting mixture stirred at −20° C. for 30 min. The pH is adjusted to 3 with concentrated HCl. The temperature is now brought to 0° C. and the light 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%).

[0336] .sup.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).

[0337] Spectral data are in accordance with literature values (S. Desgranges, C. C. Ruddle, L. P. Burke, T. M. McFadden, J. E. O'Brien, D. Fitzgerald-Hughes, H. Humphreys, T. P. Smyth, M. Devocelle. β-Lactam-host defense peptide conjugates as antibiotic prodrug candidates targeting resistant bacteria. RSC Advances 2012, 2, 2480).

[0338] A solution of benzophenone hydrazone (8) (4.906 g, 25.00 mmol, 1.0 eq.) in PE (30 mL) is treated with mercury oxide (II) (25.469 g, 25.25 mmol, 1.01 eq.) and the resulting mixture stirred for 6 h at room temperature and under daylight exclusion. The resulting purple mixture is filtered to remove mercury-containing residues and the resulting solution evaporated under reduced pressure. The purple liquid containing the target reagent diazodiphenylmethane 9 (4.570 g, 23.53 mmol, 94%) is taken up in EtOAc (15 mL) and quickly used in the next step without further purification.

[0339] A solution of compound 6 (5.000 g, 21.72 mmol, 1.0 eq.) in dimethylacetamide (DMAC) (80 mL) is treated with bis(trimethylsilyl)acetamide (BSA) (13.3 mL, 54.29 mmol, 2.5 eq.) and the resulting mixture stirred for 30 min at room temperature. The clear solution is cooled to −30° C. and 2-thiopheneacetyl chloride (3.48 mL, 28.23 mmol, 1.3 eq.) is added dropwise. The resulting mixture is stirred for 2 h at −20° C., poured onto ice water and extracted with EtOAc. The combined organic phases are washed with brine, dried over Na.sub.2SO.sub.4 and their volume adjusted to 80 mL under reduced pressure. The solution is cooled to 0 ° C. and treated with the above diazodiphenylmethane 9 solution in EtOAc (4.429 g, 22.80 mmol, 1.05 eq.) until the purple color persists. The volume of the resulting solution is reduced under vacuum before being added dropwise to a solution of pentane (300 mL), thus causing the precipitation of a light yellow solid. The latter is filtered off to give the doubly protected product 10 (3.957 g, 7.601 mmol, 35% over two steps).

[0340] .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

[0341] Spectral data are in accordance with literature values (S. Desgranges, C. C. Ruddle, L. P. Burke, T. M. McFadden, J. E. O′Brien, D. Fitzgerald-Hughes, H. umphreys, T. P. Smyth, M. Devocelle. +-Lactam-host defence peptide conjugates as antibiotic Prodrug candidates targeting resistant bacteria. RSC Advances 2012, 2, 2480).

##STR00055##

Compound 15

[0342] An ice-cold solution of alcohol 10 (1.000 g, 1.921 mmol, 1.0 eq.) in DCM (50 mL) is treated with 4-nitrophenyl chloroformate (775 mg, 3.842 mmol, 2.0 eq.), pyridine (155 μL, 1.921 mmol, 1.0 eq.) and 4-dimethylaminopyridine (DMAP) (24 mg, 0.192 mmol, 0.1 eq.). The resulting mixture is stirred for 2 h at room temperature, then washed with water, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The crude residue is purified by flash column chromatography on silica gel (PE/EtOAc 70/30) to furnish the desired carbonate 11 as a light yellow solid (685 mg, 0.999 mmol, 52%).

[0343] .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

[0344] Spectral data are in accordance with literature values (S. Desgranges, C. C. Ruddle, L. P. Burke, T. M. McFadden, J. E. O'Brien, D. Fitzgerald-Hughes, H. Humphreys, T. P. Smyth, M. Devocelle. β-Lactam-host defence peptide conjugates as antibiotic prodrug candidates targeting resistant bacteria. RSC Advances 2012, 2, 2480).

[0345] A solution of 11 (100 mg, 0.146 mmol, 1.0 eq.) in DCM (2 mL) is treated with primary amine 4 (25 mg, 0.160 mmol, 1.2 eq.)), and the stirred mixture cooled in an ice-bath before addition of DIPEA (127 μL, 0.729 mmol, 5.0 eq.). After 5 min, the ice bath is removed and the solution stirred at 30° C. overnight. The mixture is then washed using saturated Na.sub.2CO.sub.3 (2 times) and NaHCO.sub.3 (2 times) aqueous solutions, dried over Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure. The resulting crude oil is purified via column chromatography on silica gel (DCM/MeOH, 99/1 v/v) to furnish the desired carbamate 12 as a yellow oil (39 mg, 0.056 mmol, 38%).

[0346] .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

[0347] ESI : [M+H].sup.+ m/z found 701.2, calc. 701.2.

[0348] To a solution of 12 (39 mg, 0.056 mmol, 1.0 eq.) in dry DCM (1.5 mL) is added 1,3-dimethylbarbituric acid (DMBA) (43 mg, 0.278 mmol, 5.0 eq.), the resulting mixture is degassed using an argon flux, before being treated with tetrakis(triphenylphosphine)palladium(0) Pd(PPh.sub.3).sub.4 (1 mg, 0.0006 mmol, 0.01 eq.). After completion of the reaction (typically around 4 h), the reaction mixture is evaporated to dryness and purified via chromatography on silica gel (DCM/MeOH, 99/1 v/v) to furnish the desired secondary amine 13 as a yellow oil (15 mg, 0.023 mmol, 41%).

[0349] .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

[0350] ESI: [M+H].sup.+ m/z found 661.2, calc. 661.2.

[0351] To an ice-cold suspension of ELF-97 (7 mg, 0.023 mmol, 1.0 eq.) in dry DCM (1 mL) under an argon atmosphere is added dropwise N,N-diisopropylethylamine (DIPEA) (20 μL, 0.113 mmol, 5.0 eq.), followed by a solution of triphosgene (20 mg, 0.068 mmol, 3.0 eq.) in dry DCM (1 mL). The mixture is stirred for 1 h at 0° C. and overnight at r.t. Next morning, it is reduced to dryness under reduced pressure while the volatiles are trapped in a liquid-nitrogen trap. The latter's contents are subsequently destroyed by addition of ethanolic sodium hydroxyde). The resulting chloroformate of ELF-97 (solid residue) is used without further purification in the next step.

[0352] To an ice-cold suspension of the above-prepared chloroformate of ELF-97 (1.0 eq.) in dry DCM (1 mL) and under argon is added dropwise a clear solution of the secondary amine 13 (15 mg, 0.023 mmol, 1.0 eq.). Stirring is continued for another 30 min at 0° C. and then at r.t. overnight. The reaction mixture is washed three times with saturated NaHCO.sub.3 and the organic phase dried over Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure. The crude product is purified via column chromatography on silica gel (PE/EtOAc, 8/2 v/v) to furnish the desired protected probe 14 as an off-white solid (12 mg, 0.012 mmol, 53%).

[0353] ESI: [M+H].sup.+m/z found 992.0, calc. 992.3.

[0354] An ice-cold solution of 14 (12 mg, 0.012 mmol, 1.0 eq.) in dry DCM (1 mL) is treated dropwise with TFA (500 μL, excess) and anisole (7.2 μg, 0.66 mmol, 5.5 eq). The stirred mixture is allowed to warm to r.t., and monitored by mass spectrometry to determine the point of completion (1-2 hours). All volatiles are the removed under reduced pressure. The crude residue is subjected to purification by prep. HPLC (ACN/H.sub.2O 0/100 to 50/50 v/v) to furnish the target compound 15 as a white powder after freeze-drying (1.5 mg, 0.0018 mmol, 15%).

[0355] ESI: [M+H].sup.+ m/z found 826.3, calc. 826.1.

Example 2: Detection of Fluorescence of Compound 15

[0356] The fluorescence of compound 15 was evaluated with and without a β-lactamase. The test was performed by incubation/chemical reaction in a microwell plate (75 μ, 37° C., 10 U.mL.sup.−1). The fluorescence was measured over time by a plate fluorimeter. The obtained results are shown in FIG. 1.

[0357] The results show that the compounds according to the invention can detect β-lactamase activity by generating fluorescence (fluorogenic probes). In the presence of β-lactamase, compound 15 is hydrolyzed which leads to the fragmentation of the compound, and the release of a small fluorescent molecule (ELF 97) which generates intense fluorescence. In the absence of the enzyme activity however, no change in fluorescence is observed over 2 hours, thus proving the stability of probe 15 in the incubation medium (physiological).

Example 3: Synthesis of Compound 25

[0358] ##STR00056##

[0359] To an ice-cold solution of aldehyde 16 (1 g, 4.3 mmol) in methanol (20 mL) was added sodium borohydride (1 eq, 4.3 mmol, 164 mg). The solution was stirred at 0° C. for 20 minutes before 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 into a separatory funnel, the aquous phase removed and the organic phase was washed with brine, dried over sodium sulfate and filtered, yielding the crude alcohol 17 in essentially pure form as a yellow pale solid.

[0360] The crude alcohol 17 was dissolved in anydrous 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 16h, and then diluted with diethyl ether and filtered on celite. To the resulting solution was added Celite (20 g) and the solvents were removed under reduced pressure. The celite adsorbed crude mixture was subjected to flash chromatography on silica gel (Petroleum ether/ethyl acetate 8:2) to give the activated carbonate 18 (1.06 g, 2.67 mmol, 62% over 2 steps) as a yellow pale solid.

[0361] .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

##STR00057##

[0362] To a solution of N-methyl-N′-allyl-aminomethylpiperidine 19 (1.0 equiv, 0.89 mmol, 240mg) in anhydrous dichloromethane (5mL) was added carbonate 19 (1.05 equiv., 0.93 mmol, 373 mg) and potassium carbonate (5 equiv., 4.45 mmol, 615 mg). Upon completion of the reaction as judged by MS (M+H.sup.+20=530.4), the reaction mixture was diluted with a 1:1 mixture of petroleum ether and diethyl ether (15 mL), and filtered through celite, the celite rinsed with 100 mL of PE/Et.sub.2O 1:1 mixture. The filtrate was concentrated under reduced pressure to yield essentially pure carbamate 20 as a yellow pale solid, that was used directly in the next step. It could alternatively be purified by flash chromatography on silica gel using Et.sub.2O as eluent for characterization.

[0363] 20: .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

[0364] Crude 20 was placed in a round bottom flask to which 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 minutes, Pd(PPh.sub.3).sub.4 (1 mol %, 10 mg) was added, and the mixture stirred at room temperature under argon for 20-60 min. After completion of the reaction as judged by MS, the solvent was evaporated and the crude mixture containing 21 was used without further purification in the next step.

[0365] ELF-97 (1.3 equiv, 1.157 mmol, 355 mg) was placed in a round bottom flask under argon, followed by triphosgen (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 stirred at rt for 20 min. Volatiles were removed under reduced pressure, DCM (5 mL) was added, and volatiles were removed again under reduced pressure. The solid ELF chlorformate obtained was suspended in DCM (5 mL), the flask cooled in an ice bath, and the crude product 21 (1 equiv., 0.89 mmol) was added in solution in DCM (10 mL), followed by DIPEA (3 equiv., 0.47mL). The reaction was stirred at room temperature for 3 hours until no more 21 was detected in MS. The reaction was then diluted in Et.sub.2O and cooled to 0° C. before sat. Aqueous NaHCO.sub.3 (10 mL) was added. The mixture was transferred in a separatory funnel, the organic phase washed successively with water and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under educed pressure. The solid residue was partially dissolved in Et.sub.2O and filtered through a pad of silica pretreated with 2.5 w/w % of triethlamine to remove the excess of unreacted ELF-97, rinsing the silica with Et.sub.2O (200 mL). The solution was then concentrated under reduced pressure and subjected to flash chromatography on silica gel (Eluent DCM/Et.sub.2O 1:0 to 3:7) to give pure boronate 22 as a glassy yellow pale solid (536.6 mg, 0.65 mmol, 73%) (M+H.sup.+22=822.4).

[0366] 22: .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

##STR00058##

[0367] Compound 22 can be coupled with enol triflate 23 using conditions published (Chem. Eur. J. 2020, 26, 3647-3652) for the coupling of Aryl-pinacol-boronate esters with intermediate 23, giving Compound 24, that can be deprotected in conditions described in the above reference to give compound 25.

Example 4: Synthesis of Compound 32

[0368] ##STR00059##

[0369] 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 minutes, before the flask was placed at 0° C. for 30 minutes. Paraformaldehyde (e quiv., 3 g) was then added in one portion and the reaction stirred at room temperature for 14 h. The mixture was cooled to 0° C. and aqueous sat. NH.sub.4Cl was added. The mixture was extracted with Et.sub.2O, washed with water, brine, dried over Na.sub.2SO.sub.4 and concentrated under educed pressure. The crude oil was subjected to flash chromatography on silica gel (EP/Et.sub.2O 1:0 to 1:1) to give pure alcohol 27 (3.3 g, 15.5 mmol, 77%) as a colorless oil.

[0370] 27: .sup.1H -NMR (300 MHz, CDCl.sub.3): δ (ppm)=4.30 (d, J=5.6 Hz, 2H), 1.07 (s, 21H).

[0371] Alcohol 27 (3.3 g, 15.5 mmol, 1 equiv.) was dissolved in anydrous DCM (30mL) 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. The reaction was then stirred at room temperature for 16 h, and then diluted with diethyl ether and filtered on celite, rinsing with Et.sub.2O. The solvents were removed under reduced pressure and the crude mixture was subjected to flash chromatography on silica gel (eluent EP/CHCl.sub.3 1:0 to 0:1) to give activated carbonate 28 (5.56 g, 14.7 mmol, 95%) as a colorless oil.

[0372] 28: .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

##STR00060##

[0373] 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). Upon completion of the reaction as judged by MS (M+H.sup.+29=508.7), the reaction mixture was diluted with a 1:1 mixture of petroleum ether and diethyl ether (15 mL), and filtered on celite, the celite rinsed with 100 mL of PE/Et2O 1:1 mixture. The filtrate was concentrated under reduced pressure to yield essentially pure carbamate 29 as a yellow pale oil, that was used directly in the next step.

[0374] The crude carbamate 29 was placed in a round bottom flask to which 1,3-dimethylbarbituric acid (3 equiv., 2.6 mmol, 406 mg) was added, followed by DCM (8mL). The solution was purged with argon for 10 minutes, Pd(PPh.sub.3).sub.4 (1 mol %, 10 mg) was added, and the mixture stirred at room temperature under argon for 20-60 min. In parallel, ELF chlorofrmate was preapared as for example 3, by reaction of ELF-97 (1.3 equiv., 1.13 mmol, 347 mg) with triphosgen (1.3 equiv., 1.13 mmol, 335 mg) and pyridine (6 equiv., 5 mmol, 0.41 mL), successive evaporation/dissolution in DCM, before being placed in DCM (10 mL) in an ice-cold bath. After completion of the deallylation reaction, as judged by MS, the solution containing deallylated 29 was cannulated on the cold solution of ELF chloroformate in DCM and the flask rinsed twice with DCM (2+2 mL). The reaction was stirred at room temperature for 14 h and placed in an ice bath, diluted with Et.sub.2O (50 mL) and sat aqueous NaHCO.sub.3 (20 mL) was added. 30 was extracted with Et.sub.2O, the organic phase washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduce pressure. Purification of the crude mixture as for example 3, by a first filtration on a pad of Et.sub.3N-impregnated silica followed by flash chromatography over silicagel (DCM/Et.sub.2O 0:1 to 1:0), afforded pure 30 as a glassy colorless solid (456 mg, 0.57 mmol, 67%, 3 steps). (M+H.sup.+30=799.3).

[0375] 30: .sup.1H -NMR (300 MHz, CDCl.sub.3): 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).

[0376] 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. A solution of TBAF (1 M in THF, 1. 02 equiv., 479 μL) was then added dropwise and the reaction stirred 16h at room temperature. The flask was placed in an ice bath and aqueous sat. NaHCO.sub.3 (10 mL) was added dropwise. 31 was extracted with Et.sub.2O, washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduce pressure. Purification of the crude mixture flash chromatography over silicagel (DCM/Et.sub.2O 0:1 to 1:0), afforded pure 31 as a glassy colorless solid (294 mg, 0.46 mmol, 95%). (M+H.sup.+31=644.3).

[0377] 31: .sup.1H -NMR (300 MHz, CDCl.sub.3): δ (ppm)=.sup.1H 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).

##STR00061##

[0378] Compound 31 can undergo Sonogashira coupling using Pd/Cu co-catalysis to give an coupled intermediate that can be deprotected as for example 3 to give compound 32.