Compounds for their use as drugs for the treatment and/or the prevention of infection(s) caused by biofilm-forming bacteria

Abstract

The present invention relates to compounds of the following formula (I), wherein: m represents an integer being equal to 0, 1, 2, 3, 4, 5 or 6, X represents a simple bond or a radical CHR.sub.1 wherein R.sub.1 represents:a hydrogen atom, ora linear or branched, possibly interrupted by up to 3 heteroatoms selected from O, S or N and/or possibly substituted, (C.sub.1-C.sub.12)-alkyl, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other:a hydrogen atom, ora linear or branched (C.sub.1-C.sub.12-alkyl or (C.sub.1-C.sub.12)-acyl R5 represents:a hydrogen atom, ora linear or branched, possibly substituted, (C.sub.1-C.sub.13)-alkyi possibly substituted and possibly interrupted by up to 3 heteroatoms selected from O, S or N, R.sub.6 represents:a hydrogen atom, ora linear or branched possibly substituted (C.sub.1-C.sub.12)-alkyl, possibly substituted and possibly interrupted by up to 3 heteroatoms selected from O, S or N, for their use as antibacterial drugs for the treatment and/or the prevention of infection(s) caused by biofilm-forming bacteria.

Claims

1. A method of treatment and/or of prevention of biofilm formation, comprising the administration of at least one compound of the following formula I: ##STR00108## wherein: m represents an integer being equal to 0, 1, 2, 3, 4, 5 or 6, X represents a simple bond, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, R.sub.5 represents: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.13)-alkyl, or R.sub.6 represents: a hydrogen atom, or a linear or branched optionally substituted (C.sub.1-C.sub.12)-alkyl, or a linear or branched optionally substituted (C.sub.1-C.sub.12)-acyl, or a linear or branched, optionally substituted (C.sub.1-C.sub.12)-alkyl interrupted by up to 3 heteroatoms selected from O, S or N, or a linear or branched, optionally substituted (C.sub.1-C.sub.12)-acyl interrupted by up to 3 heteroatoms selected from O, S or N, or R.sub.5 and R.sub.6 represent together with the N atom to which R.sub.6 is bound and the carbon atom to which R.sub.5 is bound an alkylene radical Z having 2 to 5 carbon atoms which is optionally substituted and/or optionally interrupted by up to 3 heteroatoms selected from O, S, N and/or terminated by an heteroatom selected from O, S, N, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixture of same.

2. The method of treatment and/or of prevention of biofilm formation according to claim 1, wherein the said compound corresponds to a compound of the following formula I: ##STR00109## wherein: m represents an integer being equal to 0, 1, 2, 3, 4, 5 or 6, X represents a simple bond, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, R.sub.5 represents: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.13)-alkyl, R.sub.6 represents: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, optionally substituted by up to 3 radicals selected from: OH, O-linear (C.sub.1-C.sub.12)-alkyl,a halogen atom chosen among Br, Cl, I, F, optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom chosen among Br, Cl, I, F, optionally salified or esterified carboxy, optionally salified or esterified carboxy (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, optionally substituted by up to 3 radicals selected from: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom chosen among Br, Cl, I, F, optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom chosen among Br, Cl, I, F, optionally salified or esterified carboxy, optionally salified or esterified carboxy (C.sub.1-C.sub.12)-alkyl, and R.sub.6 can be optionally interrupted by up to 3 heteroatoms selected from O, S or N, or R.sub.5 and R.sub.6 represent together with the N atom to which R.sub.6 is bound and the carbon atom to which R.sub.5 is bound an alkylene radical Z having 2 to 5 carbon atoms which is optionally interrupted by 1 or 2 heteroatoms selected from O, S, N and/or terminated by an heteroatom selected from O, S, N and/or optionally substituted by up to three radicals selected from: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom chosen among Br, Cl, I, F, optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom chosen among Br, Cl, I, F, optionally salified or esterified carboxy, optionally salified or esterified carboxy (C.sub.1-C.sub.12)-alkyl, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same.

3. The method of treatment and/or of prevention of biofilm formation according to claim 1, wherein the said compound corresponds to a compound of the following formula I: ##STR00110## wherein: m represents an integer being equal to 0, 1, 2, X represents a simple bond, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, R.sub.5 represents: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.13)-alkyl, R.sub.6 represents: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, optionally substituted by up to 3 radicals selected from: OH, O-linear (C.sub.1-C.sub.12)-alkyl,a halogen atom F, optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom F, optionally salified or esterified carboxy, or a linear or branched (C.sub.1-C.sub.12)-acyl, optionally substituted by up to 3 radicals selected from: OH, O-linear (C.sub.1-C.sub.12)-alkyl,a halogen atom F, optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom F, optionally salified or esterified carboxy, or R.sub.5 and R.sub.6 represent together with the N atom to which R.sub.6 is bound and the carbon atom to which R.sub.5 is bound an alkylene radical Z having 2 to 5 carbon atoms which is optionally interrupted by 1 or 2 heteroatoms selected from 0 and N and/or terminated by an heteroatom selected from 0 and N and/or optionally substituted by up to three radicals selected from: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom F, optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: OH, O-linear (C.sub.1-C.sub.12)-alkyl, a halogen atom F, optionally salified or esterified carboxy, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same.

4. The method of treatment and/or of prevention of biofilm formation according to claim 1, wherein the said compound corresponds to a compound of the following formula II: ##STR00111## wherein: m represents an integer being equal to 0, 1, 2, 3, 4, 5 or 6, X represents a simple bond, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, Z represents an alkylene radical having 2 to 5 carbon atoms which is optionally substituted and/or optionally interrupted by up to 3 heteroatoms selected from O, S, N and/or terminated by an heteroatom selected from O, S, N, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same.

5. The method of treatment and/or of prevention of biofilm formation according to claim 1, wherein the said compound corresponds to a compound of the following formula XII: ##STR00112## wherein R5 represents: a linear or branched (C.sub.1-C.sub.13)-alkyl, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same.

6. The method of treatment and/or of prevention of biofilm formation according to claim 1, wherein the said compound corresponds to a compound of the following formula II: ##STR00113## wherein: m represents 1, X represents a simple bond, R.sub.2, R.sub.3 and R.sub.4 represent a hydrogen atom, R.sub.5 and R.sub.6 represent together with the N atom to which R.sub.6 is bound and the carbon atom to which R.sub.5 is bound an alkylene radical Z having 2 to 5 carbon atoms which is optionally interrupted by 1 or 2 heteroatoms selected from O, S, N, and/or terminated by an heteroatom selected from O, S, N and/or optionally substituted by up to three radicals selected from: optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl possibly substituted by: O-linear (C.sub.1-C.sub.12)-alkyl, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same.

7. The method of treatment and/or of prevention of biofilm formation according to claim 1, wherein the said compound corresponds to a compound having the following formula: ##STR00114## ##STR00115## ##STR00116## ##STR00117##

8. Compounds of the following formula III: ##STR00118## wherein: m represents an integer being equal to 1, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, R.sub.5 represents: a linear or branched (C.sub.1-C.sub.13)-alkyl, optionally substituted by up to 3 radicals selected from: O-linear (C.sub.1-C.sub.12)-alkyl, optionally substituted by an oxo group, optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: O-linear (C.sub.1-C.sub.12)-alkyl, and R.sub.5 can be optionally interrupted by up to 3 heteroatoms selected from 0 or N, with the proviso that R.sub.5 cannot be a linear C.sub.4-alkyl if R.sub.2, R.sub.3 and R.sub.4 are hydrogen atom, R.sub.6 represents a hydrogen atom, or R.sub.5 and R.sub.6 represent together with the N atom to which R.sub.6 is bound and the carbon atom to which R.sub.5 is bound an alkylene radical Z having 2 to 5 carbon atoms which is optionally interrupted by 1 or 2 heteroatoms selected from O, S, N and/or terminated by an heteroatom selected from O, S, N and substituted by up to three radicals selected from: optionally salified or esterified carboxy, an oxo group, an aromatic or heteroaromatic aryl optionally substituted by: O-linear (C.sub.1-C.sub.12)-alkyl, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same, with the proviso that the following compounds is excluded: ##STR00119##

9. Compounds according to claim 8, the said compounds having the following formula: ##STR00120##

10. Composition comprising as active ingredient one or more of the compounds of the following formula I: ##STR00121## wherein: m represents an integer being equal to 0, 1, 2, 3, 4, 5 or 6, X represents a simple bond, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, R.sub.5 represents: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.13)-alkyl, or a linear or branched, optionally substituted (C.sub.1-C.sub.13)-alkyl interrupted by up to 3 heteroatoms selected from O, S or N, R.sub.6 represents: a hydrogen atom, or a linear or branched optionally substituted (C.sub.1-C.sub.12)-alkyl, or a linear or branched optionally substituted (C.sub.1-C.sub.12)-acyl, or a linear or branched, optionally substituted (C.sub.1-C.sub.12)-alkyl interrupted by up to 3 heteroatoms selected from O, S or N, or a linear or branched, optionally substituted (C.sub.1-C.sub.12)-acyl interrupted by up to 3 heteroatoms selected from O, S or N, or R.sub.5 and R.sub.6 represent together with the N atom to which R.sub.6 is bound and the carbon atom to which R.sub.5 is bound an alkylene radical Z having 2 to 5 carbon atoms which is optionally substituted and/or optionally interrupted by up to 3 heteroatoms selected from O, S, N and/or terminated by an heteroatom selected from O, S, N, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same, and further comprising at least one antibiotic.

11. Composition comprising as active ingredient one or more of the compounds of the following formula I: ##STR00122## wherein: m represents an integer being equal to 0, 1, 2, 3, 4, 5 or 6, X represents a simple bond, R.sub.2, R.sub.3 and R.sub.4 represent independently from each other: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.12)-alkyl, or a linear or branched (C.sub.1-C.sub.12)-acyl, R.sub.5 represents: a hydrogen atom, or a linear or branched (C.sub.1-C.sub.13)-alkyl, or R.sub.6 represents: a hydrogen atom, or a linear or branched optionally substituted (C.sub.1-C.sub.12)-alkyl, or a linear or branched optionally substituted (C.sub.1-C.sub.12)-acyl, or a linear or branched, optionally substituted (C.sub.1-C.sub.12)-alkyl interrupted by up to 3 heteroatoms selected from O, S or N, or a linear or branched, optionally substituted (C.sub.1-C.sub.12)-acyl interrupted by up to 3 heteroatoms selected from O, S or N, or R.sub.5 and R.sub.6 represent together with the N atom to which R.sub.6 is bound and the carbon atom to which R.sub.5 is bound an alkylene radical Z having 2 to 5 carbon atoms which is optionally substituted and/or optionally interrupted by up to 3 heteroatoms selected from O, S, N and/or terminated by an heteroatom selected from O, S, N, and the pharmaceutically acceptable salts, enantiomers, diastereoisomers of same, as well as mixtures of same, and further comprising at least one disinfecting agent.

12. A method of treatment and/or of prevention of infection(s) caused by biofilm-forming bacteria, comprising the administration of at least one composition according to claim 10.

13. A method of treatment and/or of prevention of biofilm formation, comprising the administration of at least one composition according to claim 10.

14. A method of treatment and/or of prevention of biofilm formation, comprising the administration of at least one composition according to claim 11.

15. A method for disinfecting comprising the use of a composition comprising as active ingredient one or more of the compounds according to claim 11.

16. The composition according to claim 10, wherein said at least one antibiotic is selected from the group consisting of aminoglycosides, quinolones, cephalosporins, ureidopenicillines, carbapenems, polymyxins and monobactams.

17. The composition according to claim 10, wherein said at least one antibiotic is selected from the group consisting of Amikacin, Ciprofloxacin, Gentamicin, Piperacilin, Tobramycin, and Ceftazidime.

18. The composition according to claim 11, wherein said at least one disinfecting agent is selected from the group consisting of antioxidants, phosphates, colouring agents, EDTA, and combinations thereof.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents the kinetic representation of biofilm formation by P. aeruginosa cultured in the trypticase soy broth (TSB). The abscissa represents the time (in hours), and the ordinate represents the value of absorbance (in au.).

(2) Congo red staining was used for measuring biofilm formation. Plot depicts mean valuesSD of absorbance [=492 nm]. The average was calculated on the basis of three replicates (n=3). A high amount of biofilm was observed after 24 hours, followed by plateau and a typical breakdown of biofilm growth after 72 hours of the culture.

(3) FIG. 2A represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis in the early stages of P. aeruginosa biofilm formation, on strain PAR 5. The abscissa represents the time (in hours), and the ordinate represents the value of absorbance (in a.u.).

(4) Biofilm formation measured by Red Congo staining at different time points. Results depict mean valuesSD calculated from three replicates (n=3). In both experiments P. aeruginosa in TSB broth alone was used as a culture control.

(5) FIG. 2B represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis in the early stages of P. aeruginosa biofilm formation, on strain PAR 20. The abscissa represents the time (in hours), and the ordinate represents the value of absorbance (in a.u.).

(6) Biofilm formation measured by Red Congo staining at different time points. Results depict mean valuesSD calculated from three replicates (n=3). In both experiments P. aeruginosa in TSB broth alone was used as a culture control.

(7) FIG. 2C represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis in the early stages of P. aeruginosa biofilm formation, on strain PAR 50. The abscissa represents the time (in hours), and the ordinate represents the value of absorbance (in a.u.).

(8) Biofilm formation measured by Red Congo staining at different time points. Results depict mean valuesSD calculated from three replicates (n=3). In both experiments P. aeruginosa in TSB broth alone was used as a culture control.

(9) FIG. 3A represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis on the mature P. aeruginosa biofilm, on strain P AR 5. The abscissa represents the time (in hours), and the ordinate represents the value of absorbance (in a.u.).

(10) Compounds were added to the bacteria culture 48 h after induction of biofilm. Biofilm development monitored by Red Congo staining at different time points. Results depict mean valuesSD calculated from three replicates (n=3). In both experiments P. aeruginosa in TSB broth alone was used as a culture control

(11) FIG. 3B represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis on the mature P. aeruginosa biofilm, on strain P AR 20. The abscissa represents the time (in hours), and the ordinate represents the value of absorbance (in a.u.).

(12) Compounds were added to the bacteria culture 48 h after induction of biofilm. Biofilm development monitored by Red Congo staining at different time points. Results depict mean valuesSD calculated from three replicates (n=3). In both experiments P. aeruginosa in TSB broth alone was used as a culture control

(13) FIG. 3C represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis on the mature P. aeruginosa biofilm, on strain P AR 50. The abscissa represents the time (in hours), and the ordinate represents the value of absorbance (in a.u.).

(14) Compounds were added to the bacteria culture 48 h after induction of biofilm. Biofilm development monitored by Red Congo staining at different time points. Results depict mean valuesSD calculated from three replicates (n=3). In both experiments P. aeruginosa in TSB broth alone was used as a culture control

(15) FIG. 4A represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis in the early stages and mature P. aeruginosa biofilm formation, strain PAR 5. The abscissa represents the time (in hours) in order to differentiate early biofilm and mature biofilm, and the ordinate represents the percentage of the biofilm reduction.

(16) The percentage of the reduction in biofilm formation by P. aeruginosa is showed after 8 hours, 24 hours, 48 hours and 72 hours of incubation.

(17) FIG. 4B represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis in the early stages and mature P. aeruginosa biofilm formation, strain PAR 20. The abscissa represents the time (in hours) in order to differentiate early biofilm and mature biofilm, and the ordinate represents the percentage of the biofilm reduction.

(18) The percentage of the reduction in biofilm formation by P. aeruginosa is showed after 8 hours, 24 hours, 48 hours and 72 hours of incubation.

(19) FIG. 4C represents the effect of the tested compounds (1, 2, 3 and 4) on exopolysaccharide biosynthesis in the early stages and mature P. aeruginosa biofilm formation, strain PAR 50. The abscissa represents the time (in hours) in order to differentiate early biofilm and mature biofilm, and the ordinate represents the percentage of the biofilm reduction.

(20) The percentage of the reduction in biofilm formation by P. aeruginosa is showed after 8 hours, 24 hours, 48 hours and 72 hours of incubation.

EXAMPLES

Example 1: Preparation of -1-C-hexyl-1,5-dideoxy-1,5-imino-D-xylitol (Compound 1) and -1-C-(2,3-dihydroxypropyl)-1,5-dideoxy-1,5-imino-L-arabinitol (Compound 2)

(21) Compounds 1 and 2 are two compounds of formulas I, with the following structure:

(22) ##STR00103##

(23) This synthesis of compounds 1 and 2 has been published under the following reference: Biela A, Ouladi F, Gallienne E, Grecki M, Frelek J, Martin O R. An improved methodology for the synthesis of 1-C-allyl imino-D-xylitol and -L-arabinitol and their rapid functionalization. Tetrahedron 69, 3348-3354 (2013). This document is incorporated herein by reference in its entirety.

Example 2: Preparation of -1-C-propyl-1,4-dideoxy-1,4-imino-L-arabinitol (Compound 4)

(24) Compound 4 is a compound of formula III, with the following structure:

(25) ##STR00104##

(26) Compound 4 is obtained by hydrogenation of the precursor of compound 3: the compound 5, as showed below:

(27) ##STR00105##

(28) Compound 4 ((1R)-1-C-propyl-1,4-dideoxy-1,4-imino-L-arabinitol) can be obtained as follows:

(29) To a solution of compound 5 (302 mg, 0.52 mmol) in iPrOH (5 mL) was added 1M HCl (2 mL) and 10% Pd on charcoal (78 mg).

(30) The mixture was placed under an atmosphere of hydrogen and vigorously stirred for 18 h; the catalyst was then removed by filtration through a membrane, washed with iPrOH, and the solution was concentrated under reduced pressure.

(31) The crude product was dissolved in water (5 mL), treated with Amberlite IRA-400 (OH.sup.) ion exchange resin, the resin was filtered and the solvent evaporated to provide homogeneous 4 (79 mg, 86%).

(32) Data for 4: R.sub.f=0.12 (pet. ether:ethyl acetate 7:3); [].sub.D 30.1 (c 0.92, MeOH). .sup.1H NMR (400 MHz, CD.sub.3OD): 3.84 (d, J=3.3 Hz, 1H, H3), 3.77 (d, J=3.6 Hz, 1H, H2), 3.67 (d, J=4.8 Hz, 2H, H5), 3.08 (dt, J=3.6 Hz, 6.8 Hz, 1H, H), 2.94-2.91 (m, 1H, H4), 1.62-1.39 (m, 4H, H6, H7), 0.97 (t, 1=7.2, 3H, H8). .sup.13C NMR (100 MHz, CD.sub.3OD): 81.53 (C3), 79.27 (C2), 68.68 (C4), 63.30 (C5), 62.65 (C1), 31.67 (C6), 21.36 (C7), 14.66 (C8). ESI-HRMS: calculated for C.sub.8H.sub.18NO.sub.3 [M+H].sup.+: 176.12812; found: 176.12819.

(33) Compound 5 was obtained as described in the following reference: A. Chronowska, E. Gallienne, C. Nicolas, A. Kato, I. Adachi, O. R. Martin, An expeditious synthesis of an analogue of ()-steviamine by way of the 1,3-dipolar cycloaddition of a nitrile oxide with a 1-C-allyl iminosugar Tetrahedron Left. 52, 2011, 6399-6402. This document is incorporated herein by reference in its entirety.

Example 3: Preparation of (1S,2S,3S,5S,9R)-1,2-dihydroxy-3-hydroxymethyl-5-p-methoxybenzylindolizidine (Compound 3)

(34) Compound 3 is a compound of formula IV, with the following structure:

(35) ##STR00106##

(36) Compound 3 can be obtained by a catalytic hydrogenolysis under aqueous acidic conditions of the compound A.

(37) ##STR00107##

(38) This synthesis has been described in the following reference: A. Chronowska, E. Gallienne, C. Nicolas, A. Kato, I. Adachi, O. R. Martin, An expeditious synthesis of an analogue of ()-steviamine by way of the 1,3-dipolar cycloaddition of a nitrile oxide with a 1-C-allyl iminosugar Tetrahedron Lett. 52, 2011, 6399-6402. This document is incorporated herein by reference in its entirety.

Example 4: Effects of the Iminosugars on Biofilm Formation and on the Number of Viable Bacteria

(39) Materials and Methods

(40) 1. Tested Compounds

(41) The above-mentioned compounds 1, 2, 3 and 4 have been used for the tests.

(42) 2. Bacterial Cultures

(43) All tests were performed on Pseudomonas aeruginosa strains coded: PAR 5, PAR 20, PAR 50, isolated from suppurated wounds of three male patients with chronic diabetic foot infections.

(44) The strains were propagated in 10 ml of Trypticase-Soy Broth (TSB, Difco) at 37 C. for 24 hours in aerobic conditions. Then the cultures were centrifuged (2000 rpm; 10 min) and washed with 10 ml of saline. Stock suspensions of the strains (110.sup.9 CFU/ml) were prepared by serial diluting of bacteria in saline using MacFarland's scale.

(45) Drug resistance of the P. aeruginosa strains were tested using the disk diffusion method. Interpretation of the drug resistance of the Pseudomonas aeruginosa strains was done in accordance with the EUCAST standards (see the reference below for more information): http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint tables/Breakpoint_table_v_3.1.pdf.

(46) To determine the drug resistance of the strains, the following antibiotics were used: amikacin, ciprofloxacin, gentamicin, piperacillin, tobramycin, ceftazidime (Oxoid, Basingstoke, UK). The results are provided in the Table 1 below. R means resistant to the antibiotic and S means sensitive to the antibiotic.

(47) TABLE-US-00001 TABLE 1 Susceptibility of the tested P. aeruginosa strains: PAR 5, PAR 20, PAR 50 to selected antibiotics Antibiotic susceptibility tests (according to EUCAST) Strain number PAR 5 PAR 20 PAR 50 Amikacin [30 g] S S R Ciprofloxacin [5 g] S S S Gentamicin [10 g] S S R Piperacillin [100 g] S S R Tobramycin [10 g] S S R Ceftazidime [30 g] R S S

(48) 3. Growth Conditions and Measurement of Biofilm Formation by P. aeruginosa

(49) P. aeruginosa biofilm was set-up in sterile plastic 96-well plates with adherent surface (Greiner Bio-One, USA).

(50) Twenty microliter quantities of the bacterial stock suspensions, prepared as described above, were added to each well followed with 180 l of sterile TSB. Final concentration of the bacteria was 110.sup.8 CFU/ml.

(51) The plates were centrifuged for 10 minutes at 2000 rpm to sediment bacteria on the bottom of each well and then incubated for 72 hours (37 C., aerobic conditions).

(52) Biofilm quantity was determined using Congo red dye according to a modified procedure described by Allison et al. (Allison G C, Sutherland I W. A staining technique for bacteria and its correlation to extracellular carbohydrate production. J Microbiol Meth 2, 93-99 (1984)).

(53) Briefly, at different time points (0, 6, 18, 24, 48 and 72 hours), the culture medium was gently removed from wells by using the pipette and immediately 200 l of 0.1% Congo red solution was added. The plates were left for 30 minutes at room temperature and then 10 minutes before the end of staining, the plate was centrifuged for 10 minutes at 2000 rpm and then washed twice with buffered saline to remove unbound dye. Absorbance was measured at =492 nm wavelength using spectrophotometer (Awarness Technology Inc., Palm City, Fla., USA). All measurements were performed in triplicates and mean valuesSD are given.

(54) Results are provided in FIG. 1. A high amount of biofilm was observed after 24 hours, followed by plateau and a typical breakdown of biofilm growth after 72 hours of the culture.

(55) 4. Biofilm Formation

(56) The experiments on biofilm were performed in two different stages of the biofilm formation: early and late (mature).

(57) Early biofilm model: formation of early biofilm under influence of the tested substances was observed by filling wells of a 96-well plate with 20 l of P. aeruginosa suspension and 180 l of TSB, as described above. Tested iminosugars (compounds 1, 2, 3 and 4) were added immediately after setting-up the bacteria at a final concentration of 0.9 mM. Then, the plate was gently mixed and incubated for 48 hours at 37 C.

(58) Biofilm formation and the number of viable bacteria were checked in parallel in the following time intervals: 0, 0.5, 8, 24, 48 hours, in accordance with the procedure described above. Biofilm thickness was estimated by staining it with Cango Red and measuring optical density while number of viable bacteria was performed using the standard viable count method. (Allison G C, Sutherland I W. A staining technique for bacteria and its correlation to extracellular carbohydrate production. J Microbiol Meth 2, 93-99 (1984)).

(59) P. aeruginosa suspension cultures in TSB broth alone were used as a control.

(60) Mature biofilm model: the tested substances were added 48 hours after adherence of bacteria to the wells in a 96-well plate. The growth conditions of P. aeruginosa were exactly the same, as described above for the early biofilm model.

(61) After the addition of iminosugars (compounds 1, 2, 3 and 4), the biofilm formation and the number of viable bacteria were checked in the following time intervals: 48, 48.5, 56, 72, 96 hours.

(62) 5. Number of Viable Bacteria

(63) Numbers of viable bacteria contained in the early and mature biofilm were estimated on separate plates with wells filled with tested P. aeruginosa strains as described above (PAR 5, PAR 20, PAR 50).

(64) At the same time intervals, biofilm content in each well was removed and mixed by multiple pippeting and transferred to sterile tubes. Then decimal dilutions of the bacterial suspension were made in TSB broth, plated on McConkey Agar (Oxoid), and incubated at 37 C. for 24 hours.

(65) Numbers of colonies grown on platter were counted and total numbers of the viable bacteria calculated as colony forming units (CFU) per ml.

Results

1. Early Biofilm

(66) Tests have been achieved on the three P. aeruginosa strains PAR 5, PAR 20, PAR 50, at an early stage of the biofilm formation (from 0 hours to 48 hours).

(67) Results are as follows:

(68) Results of PAR 5 are provided in FIG. 2A.

(69) With the culture control, the absorbance is about 1.9 a.u. at 8 hours; about 2.1 a.u. at 24 hours and about 2.25 a.u. at 48 hours.

(70) On the contrary, at 8 hours, 24 hours and 48 hours, the absorbance is always about 0.25 a.u. for compounds 1, 2, 3 and 4.

(71) Thus, there is no significative difference between the compounds 1, 2, 3 and 4 on strain PAR 5.

(72) Moreover, there is also no biofilm formation at 8 hours, 24 hours and 48 hours with compounds 1, 2, 3 and 4.

(73) This proves that compounds 1, 2, 3 and 4 inhibit the formation of an early biofilm, in comparison with culture control. Thus, compounds 1, 2, 3 and 4 can prevent the biofilm formation.

(74) Results of PAR 20 are provided in FIG. 2B.

(75) With the culture control, the absorbance is about 1.4 a.u. at 8 hours; about 1.6 a.u. at 24 hours and about 1.6 a.u. at 48 hours.

(76) On the contrary, with the compound 1, the absorbance is about 1.0 a.u. at 8 hours; about 1.0 a.u. at 24 hours and about 0.75 a.u. at 48 hours.

(77) With the compound 2, the absorbance is about 0.75 a.u. at 8 hours; about 0.9 a.u. at 24 hours and about 0.9 a.u. at 48 hours.

(78) With the compound 3, the absorbance is about 0.75 a.u. at 8 hours; about 0.7 a.u. at 24 hours and about 0.7 a.u. at 48 hours.

(79) With the compound 4, the absorbance is about 0.6 a.u. at 8 hours; about 1.0 a.u. at 24 hours and about 0.75 a.u. at 48 hours.

(80) This proves that compounds 1, 2, 3 and 4 inhibit the formation of an early biofilm, in comparison with culture control. Thus, compounds 1, 2, 3 and 4 can prevent the biofilm formation.

(81) Results of PAR 50 are provided in FIG. 2C.

(82) With the culture control, the absorbance is about 1.6 a.u. at 8 hours; about 2.6 a.u. at 24 hours and about 2.0 a.u. at 48 hours.

(83) On the contrary, with the compound 1, the absorbance is about 0.5 a.u. at 8 hours; about 0.8 a.u. at 24 hours and about 0.9 a.u. at 48 hours.

(84) With the compound 2, the absorbance is about 0.8 a.u. at 8 hours; about 0.7 a.u. at 24 hours and about 1.1 a.u. at 48 hours.

(85) With the compound 3, the absorbance is about 0.8 a.u. at 8 hours; about 0.25 a.u. at 24 hours and about 0.25 a.u. at 48 hours.

(86) With the compound 4, the absorbance is about 0.8 a.u. at 8 hours; about 1.4 a.u. at 24 hours and about 0.9 a.u. at 48 hours.

(87) This proves that compounds 1, 2, 3 and 4 inhibit the formation of an early biofilm, in comparison with culture control. Thus, compounds 1, 2, 3 and 4 can prevent the biofilm formation.

2. Mature Biofilm

(88) Tests have been achieved on the three P. aeruginosa strains PAR 5, PAR 20, PAR 50, at a mature stage of the biofilm formation (from 48 hours to 96 hours).

(89) Results are as follows:

(90) Results of PAR 5 are provided in FIG. 3A.

(91) With the culture control, the absorbance is about 0.7 a.u. at 48 hours; about 1.75 a.u. at 56 hours, about 1.25 a.u. at 72 hours and about 0.5 a.u. at 96 hours.

(92) With the compound 1, the absorbance is about 0.7 a.u. at 48 hours; about 1.1 a.u. at 56 hours, about 1.25 a.u. at 72 hours and about 0.5 a.u. at 96 hours.

(93) With the compound 2, the absorbance is about 0.75 a.u. at 48 hours; about 1.0 a.u. at 56 hours, about 1.25 a.u. at 72 hours and about 1.5 a.u. at 96 hours.

(94) With the compound 3, the absorbance is about 0.75 a.u. at 48 hours; about 1.25 a.u. at 56 hours, about 1.25 a.u. at 72 hours and about 1.25 a.u. at 96 hours.

(95) With the compound 4, the absorbance is about 0.5 a.u. at 48 hours; about 1.25 a.u. at 56 hours, about 1.25 a.u. at 72 hours and about 0.5 a.u. at 96 hours.

(96) This shows a weak influence of compounds 1, 2, 3 and 4 on mature biofilm formation.

(97) Results of PAR 20 are provided in FIG. 3B.

(98) With the culture control, the absorbance is about 0.6 a.u. at 48 hours; about 1.0 a.u. at 56 hours, about 1.1 a.u. at 72 hours and about 2.5 a.u. at 96 hours.

(99) With the compound 1, the absorbance is about 0.75 a.u. at 48 hours; about 0.6 a.u. at 56 hours, about 1.5 a.u. at 72 hours and about 1.0 a.u. at 96 hours.

(100) With the compound 2, the absorbance is about 0.6 a.u. at 48 hours; about 0.7 a.u. at 56 hours, about 1.1 a.u. at 72 hours and about 2.0 a.u. at 96 hours.

(101) With the compound 3, the absorbance is about 0.6 a.u. at 48 hours; about 1.1 a.u. at 56 hours, about 0.75 a.u. at 72 hours and about 1.5 a.u. at 96 hours.

(102) With the compound 4, the absorbance is about 0.5 a.u. at 48 hours; about 0.75 a.u. at 56 hours, about 1.25 a.u. at 72 hours and about 1.75 a.u. at 96 hours.

(103) This shows a weak influence of compounds 1, 2, 3 and 4 on mature biofilm formation.

(104) Results of PAR 50 are provided in FIG. 3C.

(105) With the culture control, the absorbance is about 1.5 a.u. at 48 hours; about 1.5 a.u. at 56 hours, about 2.4 a.u. at 72 hours and about 3.2 a.u. at 96 hours.

(106) With the compound 1, the absorbance is about 1.6 a.u. at 48 hours; about 1.5 a.u. at 56 hours, about 2.4 a.u. at 72 hours and about 1.9 a.u. at 96 hours.

(107) With the compound 2, the absorbance is about 2.0 a.u. at 48 hours; about 1.75 a.u. at 56 hours, about 1.75 a.u. at 72 hours and about 2.0 a.u. at 96 hours.

(108) With the compound 3, the absorbance is about 1.25 a.u. at 48 hours; about 1.0 a.u. at 56 hours, about 2.0 a.u. at 72 hours and about 2.5 a.u. at 96 hours.

(109) With the compound 4, the absorbance is about 1.4 a.u. at 48 hours; about 1.4 a.u. at 56 hours, about 1.5 a.u. at 72 hours and about 1.75 a.u. at 96 hours.

(110) This shows a weak influence of compounds 1, 2, 3 and 4 on mature biofilm formation.

3. Number of Viable Bacteria

(111) The effect of the tested compounds (1, 2, 3 and 4) on the number of viable P. aeruginosa populations in early (A) and mature (B) biofilm has also been studied.

(112) Early biofilm stage ends at 48 hours and mature biofilm stage starts at 48 hours. The measures for the mature biofilm began 8 hours after the beginning of this stage (at 56 hours).

(113) The results depict logs of CFU/ml for three P. aeruginosa strains: PAR 5, PAR 20 and PAR 50 measured at different time intervals (0 hours, 8 hours, 24 hours, 48 hours, 56 hours, 72 hours and 96 hours). The detection limit is 100 CFU/ml.

(114) The results are provided in Table 2 below.

(115) TABLE-US-00002 TABLE 2 Effect of the tested compounds on the number of viable P. aeruginosa populations in early (A) and mature (B) biofilm Early biofilm Mature biofilm 0 h 8 h 24 h 48 h 56 h 72 h 96 h PAR 5 PAR 5 Culture 2 10.sup.7 3 10.sup.8 2 10.sup.8 3 10.sup.8 3 10.sup.9 5 10.sup.9 3 10.sup.9 control PAR 20 PAR 20 3 10.sup.6 3 10.sup.8 1 10.sup.9 2 10.sup.9 1 10.sup.8 1 10.sup.8 1 10.sup.9 PAR 50 PAR 50 5 10.sup.7 2 10.sup.8 2 10.sup.9 3 10.sup.8 7 10.sup.9 9 10.sup.9 2 10.sup.9 PAR 5 PAR 5 Compound 1 2 10.sup.6 3 10.sup.7 9 10.sup.7 1 10.sup.8 3 10.sup.8 2 10.sup.8 3 10.sup.8 PAR 20 PAR 20 4 10.sup.6 7 10.sup.7 1 10.sup.8 4 10.sup.8 6 10.sup.7 5 10.sup.7 1 10.sup.8 PAR 50 PAR 50 8 10.sup.7 2 10.sup.8 4 10.sup.8 3 10.sup.8 2 10.sup.9 1 10.sup.9 1 10.sup.9 PAR 5 PAR 5 Compound 2 8 10.sup.6 2 10.sup.7 1 10.sup.7 1 10.sup.7 4 10.sup.8 2 10.sup.8 5 10.sup.8 PAR 20 PAR 20 2 10.sup.6 4 10.sup.7 3 10.sup.8 1 10.sup.9 4 10.sup.8 3 10.sup.7 3 10.sup.8 PAR 50 PAR 50 3 10.sup.7 2 10.sup.8 7 10.sup.8 1 10.sup.8 6 10.sup.9 1 10.sup.9 9 10.sup.9 PAR 5 PAR 5 Compound 3 6 10.sup.6 4 10.sup.7 1 10.sup.7 2 10.sup.6 3 10.sup.8 2 10.sup.8 1 10.sup.9 PAR 20 PAR 20 5 10.sup.6 3 10.sup.7 5 10.sup.8 3 10.sup.9 3 10.sup.8 7 10.sup.7 7 10.sup.7 PAR 50 PAR 50 4 10.sup.7 1 10.sup.8 6 10.sup.8 6 10.sup.8 4 10.sup.8 5 10.sup.9 8 10.sup.9 PAR 5 PAR 5 Compound 4 9 10.sup.6 3 10.sup.6 9 10.sup.7 3 10.sup.7 4 10.sup.8 3 10.sup.8 2 10.sup.9 PAR 20 PAR 20 5 10.sup.6 2 10.sup.7 4 10.sup.8 3 10.sup.9 9 10.sup.8 8 10.sup.7 4 10.sup.8 PAR 50 PAR 50 1 10.sup.7 2 10.sup.8 7 10.sup.8 1 10.sup.8 3 10.sup.9 9 10.sup.9 1 10.sup.9

(116) There is no significant effect of the tested compounds on the viable bacterial number.

4. Reduction of Biofilm Growth

(117) The effect of the tested compounds (1, 2, 3 and 4) on the reduction of biofilm growth has also been studied on the three P. aeruginosa strains PAR 5, PAR 20, PAR 50.

(118) Results of PAR 5 are provided in FIG. 4A.

(119) With the culture control, there is no modification of the biofilm growth at 8 hours, 24 hours, 48 hours and 72 hours.

(120) With the compound 1, the reduction of biofilm growth is about 90% at 8 hours; about 90% at 24 hours; about 25% at 48 hours and about 10% at 72 hours.

(121) With the compound 2, the reduction of biofilm growth is about 90% at 8 hours; about 90% at 24 hours; about 25% at 48 hours and about 10% at 72 hours.

(122) With the compound 3, the reduction of biofilm growth is about 90% at 8 hours; about 90% at 24 hours; about 25% at 48 hours and about 10% at 72 hours.

(123) With the compound 4, the reduction of biofilm growth is about 90% at 8 hours; about 90% at 24 hours; about 25% at 48 hours and about 10% at 72 hours.

(124) This proves that the compounds 1, 2, 3 and 4 can reduce the biofilm growth, notably an early biofilm.

(125) Results of PAR 20 are provided in FIG. 4B.

(126) With the culture control, there is no modification of the biofilm growth at 8 hours, 24 hours, 48 hours and 72 hours.

(127) With the compound 1, the reduction of biofilm growth is about 30% at 8 hours; about 40% at 24 hours; about 40% at 48 hours and about 0% at 72 hours.

(128) With the compound 2, the reduction of biofilm growth is about 45% at 8 hours; about 45% at 24 hours; about 0% at 48 hours and about 0% at 72 hours.

(129) With the compound 3, the reduction of biofilm growth is about 50% at 8 hours; about 60% at 24 hours; about 0% at 48 hours and about 20% at 72 hours.

(130) With the compound 4, the reduction of biofilm growth is about 60% at 8 hours; about 35% at 24 hours; about 45% at 48 hours and about 0% at 72 hours.

(131) This proves that the compounds 1, 2, 3 and 4 can reduce the biofilm growth, notably an early biofilm.

(132) Results of PAR 50 are provided in FIG. 4C.

(133) With the culture control, there is no modification of the biofilm growth at 8 hours, 24 hours, 48 hours and 72 hours.

(134) With the compound 1, the reduction of biofilm growth is about 30% at 8 hours; about 30% at 24 hours; about 0% at 48 hours and about 0% at 72 hours.

(135) With the compound 2, the reduction of biofilm growth is about 50% at 8 hours; about 70% at 24 hours; about 0% at 48 hours and about 20% at 72 hours.

(136) With the compound 3, the reduction of biofilm growth is about 45% at 8 hours; about 45% at 24 hours; about 5% at 48 hours and about 20% at 72 hours.

(137) With the compound 4, the reduction of biofilm growth is about 60% at 8 hours; about 35% at 24 hours; about 45% at 48 hours and about 30% at 72 hours.

(138) This proves that the compounds 1, 2, 3 and 4 can reduce the biofilm growth, notably an early biofilm.

5. Conclusion

(139) When iminosugars (compounds 1, 2, 3 and 4) were added at the early stage of biofilm formation, an inhibition of the biofilm development was observed. This result has been observed with all P. aeruginosa strains PAR 5, PAR 20 and PAR 50.

(140) This effect was observed already in 8 hours after starting the experiments and persisted for the next 24 and 48 hours.

(141) Thus, compounds 1, 2, 3 and 4 can prevent the biofilm formation.

(142) However, weak influence on biofilm formation was observed when the imminosugars were added to the already formed, mature biofilm of P. aeruginosa strains.

(143) Besides, no significant effect on the viable bacterial number was observed, when the biofilm is early and when the biofilm is mature.

(144) In early biofilm model, average reduction of biofilm formation compared to control culture (expressed in %), for strains PAR 5, PAR 20 and PAR 50 were: 89%, 43.5% and 60%, respectively, while in mature biofilm model reduction rate was much lower and was for PAR 57.7%, PAR 2012.8% and for PAR 5012.5%. (Percent decrease in biofilm thickness was obtained as a mean value from 3 consecutive measurements of the Congo Red stained biofilm using spectrophotometer after addition of the tested iminosugars in comparison to control regarded as 100%).

(145) Thus, compounds 1, 2, 3 and 4 can inhibit the growth of the biofilm, notably the growth of an early biofilm.