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Title:
PRIMARY HYDROXYLAMINES AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2015/114031
Kind Code:
A1
Abstract:
The present invention relates to primary hydroxylamines, to pharmaceutical compositions comprising them, to their use in the treatment and/or prevention of bacterial infections and to their use in the prevention of formation or reduction of formed biofilms of bacterial origin.

Inventors:
TORRENTS SERRA EDUARD (ES)
MIRET CASALS LAIA (ES)
ALBERICIO PALOMERA FERNANDO (ES)
Application Number:
PCT/EP2015/051755
Publication Date:
August 06, 2015
Filing Date:
January 29, 2015
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
FUNDACIÓ INST DE BIOENGINYERIA DE CATALUNYA IBEC (ES)
FUNDACIÓ INST DE RECERCA BIOMÉDICA IRB BARCELONA (ES)
International Classes:
C07C239/10; A61K31/15; A61L2/00; C07C239/12
Foreign References:
US20120234316A12012-09-20
Other References:
C.GEREZ AND M. FONTECAVE: "Reduction of the small subunit of Escherichia Coli Ribonucleotide reductase by hydrazines and hydroxylamines", BIOCHEMISTRY, vol. 31, 1992, pages 780 - 786, XP002726262
E. TORRENTS ET AL.: "Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical", PNAS, vol. 102, 2005, pages 17946 - 17951, XP002726263
DATABASE REGISTRY [online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; 20 February 2014 (2014-02-20), XP002737520, retrieved from stn Database accession no. 1550846-16-6
PETER A. S. SMITH ET AL: "Equilibrium in the Behrend rearrangement of nitrones", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 40, no. 17, 1 August 1975 (1975-08-01), pages 2504 - 2508, XP055177988, ISSN: 0022-3263, DOI: 10.1021/jo00905a018
PETER A. S. SMITH ET AL: "Oxidation of dibenzylhydroxylamines to nitrones. Effects of structure and oxidizing agent on composition of the products", THE JOURNAL OF ORGANIC CHEMISTRY, vol. 40, no. 17, 1 August 1975 (1975-08-01), pages 2508 - 2512, XP055177990, ISSN: 0022-3263, DOI: 10.1021/jo00905a019
GORDON I M ET AL: "Solvolysys of substituted benzyl azoxyarenesulfonates ; characterisation of the transition state and the selectivity of benzylic intermediates in 50% aqueous 2,2,2-trifluoroethanol", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 2, CHEMICAL SOCIETY. LETCHWORTH, GB, vol. 2, 1 January 2001 (2001-01-01), pages 2059 - 2062, XP002324847, ISSN: 1472-779X, DOI: 10.1039/B106887N
"Supplementary Material (ESI) for Perkin Transactions This journal is The Royal Society of Chemistry 2001", 18 October 2001 (2001-10-18), XP055178030, Retrieved from the Internet [retrieved on 20150320]
Attorney, Agent or Firm:
CARBONELL VALLÈS, Enric (S.L.Avda. de Burgos, 16, Edificio Euromor Madrid, ES)
Download PDF:
Claims:
CLAIMS

A compound of formu

(I)

wherein

the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R1 is selected from the group consisting of hydrogen, fluorine, methyl group, trifluoromethyl group and methoxy group

R2 is selected from the group consisting of hydrogen, fluorine, methyl group, trifluoromethyl group and methoxy group

R3 is selected from the group consisting of hydrogen, fluorine, methyl group, trifluoromethyl group and methoxy group

R4 is selected from the group consisting of hydrogen, fluorine, methyl group, methoxy group and trifluoromethyl group

with the proviso that when n is 1 and Cyc is benzene then at least one of R1, R2, R3 and R4 is different from hydrogen

or stereoisomers or pharmaceutically acceptable salts thereof,

for use in the therapeutic treatment or prevention of bacterial infections.

The compound of formula (I) for use according to claim 1 wherein R1 is selected from the group consisting of hydrogen, fluorine and methoxy group, R2 is selected from the group consisting of hydrogen, methyl group and methoxy group; R3 is selected from the group consisting of hydrogen and trifluoromethyl group and R4 is selected from the group consisting of hydrogen and trifluoromethyl group. The compound of formula (I) for use according to any one of the preceding claims, wherein R3 and R4 are both hydrogen atoms.

The compound of formula (I) for use according to any one of the preceding claims, wherein n has a value of 1.

The compound of formula (I) for use according to any one of the preceding claims, wherein R3 and R4 are both hydrogen atoms.

The compound of formula (I) for use according to claim 1 which is selected from the group consisting of:

- /V-Phenethyl-hydroxylamine

- /V-Cyclohexylmethyl-hydroxylamine

- A/-(4-Fluorobenzyl)-hydroxylamine

- A/-(2,4-Dimethoxybenzyl)-hydroxylamine

- A/-(4-Methoxy-2-methylbenzyl)-hydroxylamine

- A/-(3,5-Bis-trifluoromethylbenzyl)-hydroxylamine

- A/-[3,5-difluorobenzyl]hydroxylamine

- A/-(4-fluoro-2-(trifluoromethyl)benzyl)hydroxylamine

- A/-(4-fluoro-2-methylbenzyl)hydroxylamine

or a stereoisomer or pharmaceutically acceptable salt thereof.

The compound of formula (I) for use according to any one of the preceding claims wherein the treatment or prevention of infections is carried out in patient suffering from a chronic pulmonary infection.

The compound of formula (I) for use according to any one of the preceding claims, wherein bacteria are selected among those belonging to the genera Corynebacterium, Mycobacterium, Chlamydia, Bacillus, Streptococcus, Staphylococcus, Listeria, Clostridium, Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Yersinia, Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Mycoplasma.

The compound of formula (I) for use according to any one of the preceding claims, wherein bacteria are selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa. 10. A compound of formula (II):

)

wherein

- the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R1 is selected from the group consisting of fluorine and methoxy group R2 is selected from the group consisting of methyl group, trifluoromethyl group and methoxy group

or a stereoisomer or salt thereof,

with the proviso that when the six-membered cycle labeled "Cyc" is benzene, then n is 2. 11. The compound of formula (II) according to claim 10 wherein R2 is selected from the group consisting of methyl group and methoxy group.

12. A compound of formula (III):

wherein

the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R3 is selected from the group consisting hydrogen, fluorine and trifluoromethyl group

R4 is selected from the group consisting of fluorine, methyl and methoxy or a stereoisomer or salt thereof,

with the proviso that when the six-membered cycle labeled "Cyc" is benzene, n is 1 and R4 is fluorine, then R3 is neither hydrogen nor trifluoromethyl..

13. A compound as defined in any one of claims 10 to 12 or a stereoisomer or pharmaceutically acceptable salt thereof, for use as a medicament.

14. A composition comprising a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof, and one or more excipients. 15. A composition according to claim 14 for use in the therapeutic treatment or prevention of bacterial infections comprising a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof, and one or more pharmaceutically acceptable excipients. 16. The non-therapeutic use of a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof, to inhibit growth of bacteria outside the human body.

17. The non-therapeutic use of a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof, to inhibit the formation of bacterial biofilms or to reduce the amount of already-formed bacterial biofilms outside the human body.

18. The non-therapeutic use of a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof, to treat a medical device thereby depositing an amount of the said compound onto said device. 19. The non-therapeutic use according to any one of claims 16 to 18, wherein bacteria are selected among those belonging to the genera Corynebacterium, Mycobacterium, Chlamydia, Bacillus, Streptococcus, Staphylococcus, Listeria, Clostridium, Enterococcus, Ricketsia, Treponema, Aeromonas, Enter obacter, Erwinia, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Yersinia, Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter,

Pseudomonas, Stenotrophomonas, Burkholderia, Mycoplasma.

20. The compound of formula (I) for use according to any one of the preceding claims, wherein bacteria are selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Pseudomonas aeruginosa,

Staphylococcus epidermidis and Enterococcus faecalis.

21. A method for treating or preventing bacterial infections in patient in need thereof by administering to said patient a compound as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof.

22. A method according to claim 21 wherein the treatment or prevention of infections is carried out in patient suffering from a chronic pulmonary infection. 23. A method according to any one of claims 21 to 22 wherein bacterial infections are selected among those caused by bacteria belonging to the genera Corynebacterium, Mycobacterium, Chlamydia, Bacillus, Streptococcus, Staphylococcus, Listeria, Clostridium, Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Yersinia, Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Mycoplasma.

24. A method according to any one of claims 21 to 23 wherein bacterial infections are selected among those caused by bacteria selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa.

25. A method to inhibit growth of bacteria outside the human body comprising the application of a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof.

26. A method to inhibit the formation of bacterial biofilms or to reduce the amount of already-formed bacterial biofilms outside the human body by application of a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof.

27. A method to treat a medical device by depositing onto said device a compound of formula (I) as defined in any one of claims 1 to 6 or a stereoisomer or salt thereof.

28. A method according to any one of claims 21 to 27, wherein bacteria are selected among those belonging to the genera Corynebacterium, Mycobacterium,

Chlamydia, Bacillus, Streptococcus, Staphylococcus, Listeria, Clostridium, Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Yersinia, Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Mycoplasma.

Description:
PRIMARY HYDROXYLAMINES AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to primary hydroxylamines, to compositions comprising them, to their use in the treatment and/or prevention of bacterial infections and to their use in the prevention of formation or reduction of formed biofilms of bacterial origin.

BACKGROUND OF THE INVENTION

The advantages offered by antibiotics in the treatment of infectious diseases are compromised due to the increase in the number of antibiotic-resistant bacterial strains. This reduces the efficiency of antibiotic treatments and poses a serious health and economical problem. Currently, the need for novel antibiotics is becoming increasingly apparent (Davies D., Davies J., Microbiol. Mol. Biol. Rev. 2010, 74(3), 417; Kesselheim A. S., Outterson K., Health Aff. 2010, 29, 1689, Butler MS, Blaskovich MA, Cooper MA., J Antibiot 2013 66(10):571-91, Iwamoto M, Mu Y, Lynfield R, Bulens SN, Nadle J, Aragon D, Petit S, Ray SM, Pediatrics. 2013, 132(4) e817-24, 1: Watkins RR, Papp-Wallace KM, Drawz SM, Bonomo RA., Front Microbiol. 2013;4:392). It has been reported that every year 25000 patients die in the European Union from a bacterial infection which is multiresistent to the presently existing drugs. Thus, there is an urgent need to discover new antibacterial agents against bacteria that have become resistant to other drugs, in particular those directed towards new or under-exploited targets or working through new mechanisms.

Control of bacterial infection using chemotherapeutic principles and antibiotics is based on inhibition of cell wall synthesis, protein synthesis and other metabolic pathways. Since genetic material in the form of DNA is common to all microorganisms, inhibition of DNA synthesis is an attractive principle to suppress bacterial proliferation.

Ribonucleotide reductases are essential enzymes for microorganisms and are responsible for the supply of all four deoxyribonucleotides required for de novo DNA synthesis and repair. They are ubiquitous in almost all DNA based organisms and notably among bacteria.

Torrents E. et al (Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical, PNAS, December 2005, V. 102, Nr. 50, pp 17946-17951) have reported that the tyrosyl radical generated in Ribonucleotide reductases is a possible target for tailored antibacterial drugs and, being extremely sensitive to hydroxylamine and to /V-methylhydroxylamine.

Gerez, C. et al. (Reduction of the Small Subunit of E. coli Ribonucleotide Reductase by Hydrazines and Hydroxylamines; Biochemistry, 1992, V. 31, pp 780-786) tested the ability of 6 primary hydroxyl amines (namely, hydroxylamine, N- methylhydroxylamine, /V-isopropylhydroxylamine, /V-terc-butylhydroxylamine, N- phenylhydroxyl-amine and /V-benzylhydroxylamine) to reduce the redox centers of E. coli ribonucleotide reductase enzyme. The study reported at protein level the rate of reaction in terms of t½ (time required to reduce 50% of the redox centers). The results showed that hydroxylamine, /V-methylhydroxylamine and /V-phenylhydroxylamine react faster than /V-benzylhydroxylamine, /V-isopropylhydroxylamine and /V-terc- butylhydroxylamine.

It has also been previously suggested (Torrents E. et al; Antibacterial activity of radical scavengers against class lb ribonucleotide reductase from Bacillus anthracis, Biol. Chem., 2010, V. 391, pp 229-234) to use hydroxylamine and methyl hydroxylamine as antibacterial agents.

The present invention seeks to provide alternative hydroxylamine compounds showing a spectrum of action spanning both Gram-positive and Gram-negative bacteria with a high potency to arrest and inhibit bacterial growth and proliferation.

The inventors have now discovered a family of hydroxylamines which are capable of arresting bacterial growth at relatively low concentrations. The compounds of the invention have been shown to have a minimum inhibitory concentration (MIC) against several bacteria (both Gram-positive and Gram-negative), which is substantially lower than the MICs of /V-benzylhydroxylamine and other hydroxylamines of similar structure. The hydroxylamines of the present invention are particularly effective against Bacillus anthracis, Staphylococcus epidermis, Burkholderia cenocepacea, Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus.

Additionally the inventors have found that the hydroxylamines of the present invention are capable of inhibiting the formation of bacterial biofilms and to reduce the amount of already formed bacterial biofilms being critical in bacterial chronic infections.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a compound of formula (I):

(I)

wherein

the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

- n is i or 2

R 1 is selected from the group consisting of hydrogen, fluorine, methyl group, trifluoromethyl group and methoxy group

R 2 is selected from the group consisting of hydrogen, fluorine, methyl group, trifluoromethyl group and methoxy group

- R 3 is selected from the group consisting of hydrogen, fluorine, methyl group trifluoromethyl group and methoxy group

R 4 is selected from the group consisting of hydrogen, fluorine, methyl group, methoxy group and trifluoromethyl group

with the proviso that when n is 1 and Cyc is benzene then at least one or R 1 , R 2 , R 3 and R 4 is different from hydrogen

or stereoisomers or salts thereof,

for use in the treatment or prevention of bacterial infections.

In a second aspect, the present invention relates to a compound of formula (II):

wherein

the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R 1 is selected from the group consisting of fluorine and methoxy group

R 2 is selected from the group consisting of methyl group, trifluoromethyl group and methoxy group

or a stereoisomer or salt thereof,

with the proviso that when the six-membered cycle labeled "Cyc" is benzene, then n is 2.

.In a third aspect, the present invention relates to a compound of formula (III):

wherein

the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R 3 is selected from the group consisting hydrogen, fluorine and trifluoromethyl group

R 4 is selected from the group consisting of fluorine, methyl and methoxy or a stereoisomer or salt thereof,

with the proviso that when the six-membered cycle labeled "Cyc" is benzene, n is 1 and R 4 is fluorine, then R 3 is neither hydrogen nor trifluoromethyl.. I n a fourth aspect, the present invention relates to a compound of formulae (I I) or (I I I) as defined in the second aspect or a stereoisomer or pharmaceutically acceptable salts thereof, for use as a medicament.

I n a fifth aspect, the present invention relates to a composition comprising a compound of formula (I) as defined in the first aspect or a stereoisomer or salt thereof, and one or more excipients.

I n a sixth aspect, the present invention relates to the non-therapeutic use of a compound of formula (I) as defined in the first aspect or a stereoisomer or salt thereof, to inhibit growth of bacteria outside the human body.

I n a seventh aspect, the present invention relates to the non-therapeutic use of a compound of formula (I) as defined in the first aspect or a stereoisomer or salt thereof, to inhibit the formation of bacterial biofilms or to reduce the amount of already- formed bacterial biofilms outside the human body.

I n an eighth aspect, the present invention relates to the non-therapeutic use of a compound of formula (I) as defined in the first aspect or a stereoisomer or salt thereof, to treat a medical device thereby depositing an amount of the said compound onto said device.

DESCRIPTION OF THE INVENTION

Description of the Figures

Fig. 1 shows the capacity of various concentrations of /V-phenethyl- hydroxylamine, /V-cyclohexylmethyl-hydroxylamine, A/-(4-fluorobenzyl)-hydroxylamine and A/-(2,4-dimethoxybenzyl)-hydroxylamine to inhibit P. aeruginosa from forming biofilms.

Fig. 2 shows the capacity of various concentrations of /V-phenethyl- hydroxylamine, /V-cyclohexylmethyl-hydroxylamine, A/-(4-fluorobenzyl)-hydroxylamine and A/-(2,4-dimethoxybenzyl)-hydroxylamine to inhibit 5. aureus from forming biofilms.

Fig. 3 shows the capacity of various concentrations of /V-cyclohexylmethyl- hydroxylamine to reduce already formed P. aeruginosa biofilms. The effect of two consecutive /V-cyclohexylmethyl-hydroxylamine treatments (2 days) on formed biofilm is shown.

Fig. 4 shows the capacity of various concentrations of /V-cyclohexylmethyl- hydroxylamine, A/-(4-fluorobenzyl)-hydroxylamine and A/-(2,4-dimethoxybenzyl)- hydroxylamine to reduce already formed 5. aureus biofilms.

Fig. 5 shows the capacity of various concentrations of /V-phenethyl- hydroxylamine to reduce already formed 5. aureus biofilms. The effect of two consecutive /V-phenethyl-hydroxylamine treatments (3 days) on formed biofilm is shown.

- Fig. 6 shows the capacity of various concentrations of A/-(3,5-Bis- trifluoromethylbenzyl)-hydroxylamine to reduce already formed 5. aureus biofilms. The effect of two consecutive A/-(3,5-Bis-trifluoromethylbenzyl)-hydroxylamine treatments (3 days) on formed biofilm is shown. Definitions

As used herein, the term "salt" embraces salts with an acid or a base, which are synthesized from the pa rent compound which contains an acidic moiety by addition of a base, or which are synthesized from the parent compound which contains a basic moiety by addition of an acid. The acids or bases used to form the salts of the compounds do not always need to be pharmaceutically acceptable since the compounds are not only useful to control bacterial growth as part of a medicinal product but they may also be used for example to inhibit bacterial growth or to control bacterial biofilms in non biological surfaces. However, when the products of the invention are to be used in the treatment of animals including men, it is preferable that they are in the form of "pharmaceutically acceptable salts" that is that the acids or bases used to form the salts of the compounds are "pharmaceutically acceptable". Pharmaceutically acceptable acids include both inorganic acids, for example, hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic, hydroiodic, and nitric acid, and organic acids, for example, citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulfonic (mesylate), ethanesulfonic, benzenesulfonic (besylate), or p-toluenesulfonic (tosylate) acid. Pharmaceutically acceptable bases include alkali metal (e.g., sodium or potassium) and alkali earth metal (e.g., calcium or magnesium) hydroxides and organic bases, such as alkyl amines, arylalkyi amines, and heterocyclic amines. For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound which contains a basic or an acid moiety by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free base or free acid forms of these compounds with a stoichiometric amount of the appropriate acid or base, respectively, in water or in an organic solvent or in a mixture of the two. Preferably, the pharmaceutically acceptable salt is the mesylate.

As used herein, the term "stereoisomer" makes reference to compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which are not interchangeable, depending on the presence of chiral centers, such as enantiomers, or diastereomers, or depending on the presence of multiple bonds, such as Z and E isomers. The single isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention. If mixtures of stereoisomers are obtained, these isomers may be separated by conventional techniques such as preparative chromatography. If there are chiral centers the compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution.

Unless otherwise stated, the compounds of the invention are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon or 15 N-enriched nitrogen are within the scope of this invention.

The terms "treating" and "treatment", as used herein, mean reversing, alleviating, inhibiting the progress of infections caused by bacteria (which are known to express ribonucleotide reductase), or one or more symptoms associated with said diseases. The terms "preventing" and "prevention", as used herein, means inhibiting the onset of infections caused by bacteria (which are known to express ribonucleotide reductase), or one or more symptoms associated with said diseases.

As used herein, the term "medical device" makes reference to an instrument, apparatus, implant or related article that is used to diagnose, prevent, or treat a disease or another medical condition, and which does not achieve its purposes through chemical action within or on the body. In use a medical device will be held into contact with some part of the body.

As used herein, the term "bacterial biofilm" defines a group of bacteria in which bacterial cells stick to each other on a surface. Frequently the bacteria in said biofilms are embedded within a self-produced matrix of extracellular polymeric substance. Bacterial biofilms may form on living or non-living surfaces.

As used herein, the term "chronic pulmonary infection" is used to designate bacterial infections that happen in the lungs, chest, sinuses, nose and throat and that occur repeatedly over time. As used herein the term "chronic pulmonary infection" also encompasses lung conditions which are caused by, aggravated by or associated to bacterial infections of the lung. Examples of chronic pulmonary infections include: tuberculosis, non-tuberculous mycobacterium infection, chronic pneumonia, bronchiectasis, chronic granulomatous disease, cystic fibrosis and chronic obstructive pulmonary disease (COPD).

Compounds of formula (I) and their pharmacological activity

The compounds of formula (I) of the present invention inhibit the replication of bacteria.

Thus, in the first aspect, the present invention provides a compound of formula (I) as defined above, or a stereoisomer or a salt thereof, for use in the treatment or prevention of infections caused by bacteria, in particular those belonging to the genera Corynebacterium, Mycobacterium, Chlamydia (such as C. trachomatis), Bacillus (such as B. anthracis, B. cereus, B. thuringensis), Streptococcus (such as 5. sanguinis, 5. mutants), Staphylococcus (such as S. aureus, S. epidermidis), Listeria, Clostridium (such as C. difficile, C. botulinum, C. perfringens), Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia (such as E. coli), Klebsiella (such as K. pneumonia), Proteus, Salmonella (such as S. enterica), Serratia, Shigella, Yersinia (such as Y. pestis, Y. pseudotuberculosis, Y. enterocolitica), Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas (such as P. aeruginosa, P. putida, P. syringae), Stenotrophomonas, Burkholderia (such as B. mallei, B. cenocepacia), Mycoplasma (such as M. genitallium, M. pneumonia), more particularly bacteria selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa.

The present invention also relates to the use of a compound of formula (I) as defined above, or a stereoisomer or a salt thereof, for manufacturing a medicament for the treatment or prevention of infections caused by bacteria, in particular belonging to the genera Corynebacterium, Mycobacterium, Chlamydia (such as C. trachomatis), Bacillus (such as B. anthracis, B. cereus, B. thuringensis), Streptococcus (such as S. sanguinis, S. mutants), Staphylococcus (such as S. aureus, S. epidermidis), Listeria, Clostridium (such as C. difficile, C. botulinum, C. perfringens), Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia (such as E. coli), Klebsiella (such as K. pneumonia), Proteus, Salmonella (such as S. enterica), Serratia, Shigella, Yersinia (such as Y. pestis, Y. pseudotuberculosis, Y. enterocolitica), Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas (such as P. aeruginosa, P. putida, P. syringae), Stenotrophomonas, Burkholderia (such as B. mallei, B. cenocepacia), Mycoplasma (such as M. genitallium, M. pneumonia), more particularly bacteria those selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa.

The invention also relates to a method of treatment or prevention of infections caused by bacteria, in particular belonging to the genera Corynebacterium, Mycobacterium, Chlamydia (such as C. trachomatis), Bacillus (such as B. anthracis, B. cereus, B. thuringensis), Streptococcus (such as S. sanguinis, S. mutants), Staphylococcus (such as S. aureus, S. epidermidis), Listeria, Clostridium (such as C. difficile, C. botulinum, C. perfringens), Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia (such as E. coli), Klebsiella (such as K. pneumonia), Proteus, Salmonella (such as S. enterica), Serratia, Shigella, Yersinia (such as Y. pestis, Y. pseudotuberculosis, Y. enterocolitica), Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas (such as P. aeruginosa, P. putida, P. syringae), Stenotrophomonas, Burkholderia (such as B. mallei, B. cenocepacia), Mycoplasma (such as M. genitallium, M. pneumonia), more particularly bacteria those selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa, in a subject in need thereof of, which comprises the administration of a therapeutically effective amount of a compound of formula (I) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof.

In an embodiment according to the first aspect the present invention provides a compound of formula (I) for use as defined above, wherein R 1 is selected from the group consisting of hydrogen, fluorine and methoxy group, R 2 is selected from the group consisting of hydrogen, methyl group and methoxy group; R 3 is selected from the group consisting of hydrogen and trifluoromethyl group and R 4 is selected from the group consisting of hydrogen and trifluoromethyl group.

In an embodiment according to the first aspect, the present invention provides a compound of formula (I) for use as defined above, wherein the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane, n is 1 or 2, R 1 is selected from the group consisting of hydrogen, fluorine and methoxy group, R 2 is selected from the group consisting of hydrogen, methyl group and methoxy group and R 3 and R 4 are both hydrogen atoms.

In an embodiment according to the first aspect, the present invention provides a compound of formula (I) for use as defined above, wherein n has a value of 1.

In another embodiment according to the first aspect, the present invention provides a compound of formula (I) for use as defined above wherein the six- membered cycled labeled "Cyc" is benzene, n has a value of 1 and R 1 is not hydrogen. In a particular embodiment according to the first aspect, the present invention provides a compound of formula (I) for use as defined above, wherein R 3 and R 4 are both hydrogen atoms.

In a preferred embodiment according to the first aspect, the present invention provides a compound of formula (I) for use as defined above, which is selected from the group consisting of:

- /V-Phenethyl-hydroxylamine

- /V-Cyclohexylmethyl-hydroxylamine

- A/-(4-Fluorobenzyl)-hydroxylamine

- A/-(2,4-Dimethoxybenzyl)-hydroxylamine

- A/-(4-Methoxy-2-methylbenzyl)-hydroxylamine

- A/-(3,5-Bis-trifluoromethylbenzyl)-hydroxylamine

- A/-[3,5-difluorobenzyl]hydroxylamine

- A/-(4-fluoro-2-(trifluoromethyl)benzyl)hydroxylamine

- A/-(4-fluoro-2-methylbenzyl)hydroxylamine

or a stereoisomer or salts thereof.

In another aspect the invention relates to the non-therapeutic use of a compound of formula (I) as defined above to inhibit growth of bacteria outside the human body.

In another aspect the invention relates to the non-therapeutic use of a compound of formula (I) as defined above to inhibit the formation of bacterial biofilms or to reduce the amount of already-formed bacterial biofilms outside the human body.

In a particular embodiment of the invention the above mentioned non- therapeutic uses involve bacteria belonging to the genera Corynebacterium, Mycobacterium, Chlamydia (such as C. trachomatis), Bacillus (such as B. anthracis, B. cereus, B. thuringensis), Streptococcus (such as S. sanguinis, S. mutants), Staphylococcus (such as S. aureus, S. epidermidis), Listeria, Clostridium (such as C. difficile, C. botulinum, C. perfringens), Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia (such as E. coli), Klebsiella (such as K. pneumonia), Proteus, Salmonella (such as S. enterica), Serratia, Shigella, Yersinia (such as Y. pestis, Y. pseudotuberculosis, Y. enterocolitica), Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas (such as P. aeruginosa, P. putida, P. syringae), Stenotrophomonas, Burkholderia (such as B. mallei, B. cenocepacia), Mycoplasma (such as M. genitallium, M. pneumonia), more particularly bacteria selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Pseudomonas aeruginosa, Staphylococcus epidermidis and Enterococcus faecalis.

In another aspect the invention relates to the non-therapeutic use of a compound of formula (I) as defined above to treat a medical device thereby depositing an amount of the said compound onto said device.

In an embodiment the present invention relates to a composition as defined in the fifth aspect of the invention for use in the therapeutic treatment or prevention of bacterial infections comprising a compound of formula (I) as defined above or a stereoisomer or salt thereof, and one or more pharmaceutically acceptable excipients.

Compounds of formula (II) and (III)

The invention also provides compounds of formula (I):

(I)

wherein

the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R 1 is selected from the group consisting of hydrogen, fluorine and methoxy group

R 2 is selected from the group consisting of hydrogen, methyl group, trifluoromethyl group and methoxy group R 3 is selected from the group consisting of hydrogen, fluorine and trifluoromethyl group

R 4 is selected from the group consisting of hydrogen, fluorine, methyl, methoxy and trifluoromethyl group

with the proviso that when n is 1 and Cyc is benzene then at least one of R 1 , R 2 , R 3 and R 4 is different from hydrogen

wherein

a) when R 3 and R 4 are hydrogen atoms, the R 1 is selected from the group consisting of fluorine and methoxy group and R 2 is selected from the group consisting of methyl group, trifluoromethyl group and methoxy group with the proviso that when the six- membered cycle labeled "Cyc" is benzene, then n is 2 and

b) when R 1 and R 2 are hydrogen atoms, then R 3 is selected from the group consisting hydrogen, fluorine and trifluoromethyl group and R 4 is selected from the group consisting of fluorine, methyl and methoxy with the proviso that when the six- membered cycle labeled "Cyc" is benzene, n is 1 and R 4 is fl uorine, then R 3 is neither hydrogen nor trifluoromethyl;

or stereoisomers or salts thereofjn the second aspect, the present invention provides a compound of formula (II) wherein

the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R 1 is selected from the group consisting of fluorine and methoxy group R 2 is selected from the group consisting of methyl group, trifluoromethyl group and methoxy group

or a stereoisomer or salt thereof,

with the proviso that when the six-membered cycle labeled "Cyc" is benzene, then n is

2.

In an embodiment according to the second aspect, the present invention provides a compound of formula (II), wherein R 2 is selected from the group consisting of methyl group and methoxy group. In a third aspect, the present invention provides a compound of formula (III)

wherein

- the six-membered cycle labeled "Cyc" is selected from the group consisting of benzene and cyclohexane

n is 1 or 2

R 3 is selected from the group consisting hydrogen, fluorine and trifluoromethyl group

- R 4 is selected from the group consisting of fluorine, methyl and methoxy or a stereoisomer or salt thereof,

with the proviso that when the six-membered cycle labeled "Cyc" is benzene, n is 1 and R 4 is fluorine, then R 3 is neither hydrogen nor trifluoromethyl.

The compounds of formulae (II) and (III) fall under the definition of the compounds of formula (I). As explained above, these compounds are inhibitors of the replication bacteria.

Thus, in another aspect, the present invention provides a compound of formulae

(II) or (III) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof, for use as a medicament.

The present invention also provides the use of a compound of formulae (II) or

(III) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof, in the manufacture of a medicament.

The present invention provides a compound of formulae (II) or (III) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof, for use in the treatment or prevention of infections caused by bacteria.

The present invention also provides the use of a compound of formulae (II) or (III) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof, for manufacturing a medicament for the treatment or prevention of infections caused by bacteria belonging to the genera Corynebacterium, Mycobacterium, Chlamydia (such as C. trachomatis), Bacillus (such as B. anthracis, B. cereus, B. thuringensis), Streptococcus (such as S. sanguinis, S. mutants), Staphylococcus (such as S. aureus, S. epidermidis), Listeria, Clostridium (such as C. difficile, C. botulinum, C. perfringens), Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia (such as E. coli), Klebsiella (such as K. pneumonia), Proteus, Salmonella (such as S. enterica), Serratia, Shigella, Yersinia (such as Y. pestis, Y. pseudotuberculosis, Y. enterocolitica), Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas (such as P. aeruginosa, P. putida, P. syringae), Stenotrophomonas, Burkholderia (such as B. mallei, B. cenocepacia), Mycoplasma (such as M. genitallium, M. pneumonia), more particularly bacteria, in particular those selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa.

The invention also relates to a method of treatment or prevention of infections caused by bacteria belonging to the genera Corynebacterium, Mycobacterium, Chlamydia (such as C. trachomatis), Bacillus (such as B. anthracis, B. cereus, B. thuringensis), Streptococcus (such as S. sanguinis, S. mutants), Staphylococcus (such as S. aureus, S. epidermidis), Listeria, Clostridium (such as C. difficile, C. botulinum, C. perfringens), Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia (such as E. coli), Klebsiella (such as K. pneumonia), Proteus, Salmonella (such as S. enterica), Serratia, Shigella, Yersinia (such as Y. pestis, Y. pseudotuberculosis, Y. enterocolitica), Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas (such as P. aeruginosa, P. putida, P. syringae), Stenotrophomonas, Burkholderia (such as B. mallei, B. cenocepacia), Mycoplasma (such as M. genitallium, M. pneumonia), more particularly bacteria, in particular those selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa, in a subject in need thereof of, which comprises the administration of a therapeutically effective amount of a compound of formulae (II) or (III) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof.

Pharmaceutical compositions/formulations and administration

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

The term "pharmaceutically acceptable excipient" refers to a vehicle, diluent, or adjuvant that is administered with the active ingredient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and similar. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 21st Edition, 2005.

Compounds of the invention may be administered by the oral, sublingual, parenteral, subcutaneous, intramuscular, intravenous, transdermal, intranasal, intraocular, and/or rectal routes. The compounds may be administered alone or in combination with one or more other compounds of the invention or one or more other drugs.

Synthesis of compounds of formula (I), formula (II) and formula (III)

The compounds of formulae (II) and (III) represent a particular subgroup of the compounds of formula (I). Thus, the methods described hereinafter in relation to the preparation of compounds of formula (I) may also be used to prepare compound of formulae (II) or (III)

Scheme 1 shows the synthetic pathway for obtaining compounds of formula (I): 1. 5 M LiCI0 4 in EtOEt

Scheme 1

The starting material used for the synthesis compounds of formula (I) accor to the present invention is an aldehyde compound of formula (IV). These compounds are commercially available or may be synthetized by methods well known to the skilled in the a rt.

An equivalent of aldehyde of formula (IV) is dissolved in a 1.0 to 8.0 M solution of an alkaline perchlorate salt (p.e. LiCI0 4 ) in an organic solvent such as penta ne, hexa ne, cyclohexane, benzene, toluene, chloroform, dichloromethane, tetrahydrofuran and ethyl acetate or diethylether (LPDE solution) and from 1 to 10 equivalents of O- trimethylsilylhydroxylamine (V) are added. The reaction mixture is stirred up to 2 hours and trimethylsilyl chloride is added (1 to 10 equivalents). After stirring the mixture for a period up to 24 hours, a reducing agent selected from diborane, decaborane, borane-tetrahydrofuran (BTH F), borane-dimethyl sulfide (BMS), sodium borohydride, sodium tetrahydroborate and/or borane/triethylamine complex, (i.e. BH 3 -N Et 3; 1 to 10 equivalents) is added and the reaction mixture is reacted for a period up to 24 hours affording mono /V-alkyl-O-trimethylsilylhydroxylamine. The former steps are carried out in the form of a one pot reaction, i.e. without isolating the products resulting from each reaction step. The reaction is quenched with an aqueous saturated solution of an alkaline salt such as a bicarbonate, sulphate or phosphate (i.e. NaHC0 3 ) to obtain the mono /V-substituted hydroxylamine and then extracted with dichloromethane (DCM), another halogen-containing organic solvent or ethyl acetate. The organic extracts are combined, dried (MgS0 4 or other drying agent) and concentrated. Finally, the crude product is flash chromatographed (Si0 2 ). The following examples represent specific embodiments of the present invention. They do not intend to limit in any way the scope of the invention defined in the present description.

The following abbreviations are employed herein:

- DCM : dichloromethane

- EtOEt or Et 2 0: diethyl ether

- EtOAc: ethyl acetate

LDPE: lithium perchlorate (LiCI04) in diethyl ether

- NMR: nuclear magnetic resonance

- PBS: Phosphate buffered saline

- TSB: Tryptic soy broth

In another aspect the present invention relates to a method for treating or preventing bacterial infections in patient in need thereof by administering to said patient a compound as defined in the first aspect of the present invention or a stereoisomer or salt thereof.

In one embodiment said method for treating or preventing bacterial infections in a patient in need thereof is carried out in patient suffering from a chronic pulmonary infection.

In another embodiment the above mentioned method is used for treating or preventing bacterial infections selected among those caused by bacteria belonging to the genera Corynebacterium, Mycobacterium, Chlamydia, Bacillus, Streptococcus, Staphylococcus, Listeria, Clostridium, Enterococcus, Ricketsia, Treponema, Aeromonas, Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Yersinia, Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Mycoplasma. In a particular embodiment the bacterial infections are selected among those caused by bacteria selected from the group consisting of Bacillus anthracis, Staphylococcus aureus, Burkholderia cenocepacea, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa. In another aspect the present invention relates to a method to inhibit growth of bacteria outside the human body comprising the application of a compound of formula (I) as defined in the first aspect of the invention or a stereoisomer or salt thereof.

In another aspect the present invention relates to a method to inhibit the formation of bacterial biofilms or to reduce the amount of already-formed bacterial biofilms outside the human body by application of a compound of formula (I) as defined in the first aspect of the invention or a stereoisomer or salt thereof.

In yet another aspect the present invention relates to a method to treat a medical device by depositing onto said device a compound of formula (I) as defined in the first aspect of the invention or a stereoisomer or salt thereof.

In one embodiment the above mentioned methods to inhibit growth of bacteria outside the human body, the method to inhibit the formation of bacterial biofilms or to reduce the amount of already-formed bacterial biofilms outside the human body and the method to treat a medical device are characterized in that bacteria are selected among those belonging to the genera Corynebacterium, Mycobacterium, Chlamydia, Bacillus, Streptococcus, Staphylococcus, Listeria, Clostridium, Enterococcus, Ricketsia, Treponema, Aeromonas, Enter obacter, Erwinia, Escherichia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Yersinia, Coxiella, Legionella, Actinobacillus, Haemophilus, Pasteurella, Acinetobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Mycoplasma.

Examples

Materials and methods

General procedure for the synthesis of /V-substituted hydroxy/amines

To a mixture of aldehyde (1 g, 1 eq) in a solution of 5 M LiCI0 4 /Et 2 0 (10 mL) was added o-(trimethylsilyl)hydroxylamine (1.5 eq) at room temperature. The mixture was stirred for 10 minutes and (CH 3 ) 3 SiCI (1.5 eq) was added. After stirring the mixture for 8 hours BH 3 .NEt 3 (1.5 eq) was added and the mixture was allowed to stir at room temperature overnight. The reaction was set up for 24 hours and was quenched with aq saturated NaHC0 3 solution, and the product was extracted with DCM. The organics extracts were combined, dried with MgS0 4 and concentrated. The crude product was flash chromatographed (Si0 2 ) using a hexane-EtOAc gradient from 1:0 to 0:1.

The compounds shown below were synthetized using the method described in the previous section.

H, 13 C and 19 F NMR spectra were recorded on a Varian MERCURY 400 (400 MHz for H NMR, 101 MHz for 13 C NMR and 376 MHz for 19 F NMR) spectrometer. Chemical shifts (δ) are expressed in parts per million downfield from the deuterated solvent signal was used as reference. Coupling constants are expressed in Hertz.

Example 1: Synthesis of /V-phenethyl-hydroxylamine

The product was obtained in a 45.6 % yield. H NMR (400 MHz, CDCI 3 ) δ ppm 7.33-7.26 (m, 2H, ArH), 7.26-7.18 (m, 3H ArH), 4.22-3.92 (m, 2H, NHOH), 3.21 (t, J = 7.1 Hz, 2H, CH 2 ), 2.90 (t, J = 7.1 Hz, 2H, CH 2 ). 13 C NMR (101 MHz, CDCI 3 ) δ ppm 139.02 (s), 128.82 (2d), 128.60 (2d), 126.39 (d), 54.75 (t), 33.05 (t).

Example 2: Synthesis of /V-cyclohexylmethyl-hydroxylamine

The product was obtained in a 70.2 % yield. H NMR (400 MHz, CDCI 3 ) δ ppm 6.35 (bs, 2H, NHOH), 2.77 (d, = 6.6 Hz, 2H, CH 2 ), 1.65 (m, 6H), 1.19 (m, 3H), 0.92 (m, 2H). 13 C NMR (101 MHz, CDCI 3 ) δ ppm 60.52 (t), 35.14 (d), 31.32 (2t), 26.50 (t), 25.93 (2t).

The product was obtained in a 42.4 % yield. H NMR (400 MHz, CDCI 3 ) δ ppm 7.34-7.29 (m, 2H, ArH), 7.06-7.00 (m, 2H, ArH), 4.01 (s, 2H, CH 2 ). 13 C NMR (101 MHz, CDCI 3 ) δ ppm 162.3 (d), 132.63 (s), 130.8 (2d), 115.27 (2d), 57.23 (t). 19 F NMR (376 MHz, CDCI 3 ) δ ppm -114.8 (tt, J = 8.8, 5.4 Hz, IF, CF).

Example 4: A/-(2,4-dimethoxybenzy -hvdroxylamine

The product was obtained in a 22 % yield. H NMR (400 MHz, CDCI 3 ) δ ppm 7.16 (d, J = 7.9 Hz, IH, ArH), 6.46-6.43 (m, 2H, ArH), 4.03 (s, 2H, CH 2 ), 3.82 (s, 3H, CH 3 ), 3.80 (s, 3H, CH 3 ). 13 C NMR (101 MHz, CDCI 3 ) δ ppm 160.62 (s), 158.78 (s), 131.82 (d), 116.87 (s), 103.83 (d), 98.37 (d), 55.23 (q), 55.12 (q), 53.37 (t).

The product was obtained in a 32.8 % yield. H NMR (400 MHz, CDCI 3 ) δ ppm 7.18 (d, J = 8.2 Hz, IH, ArH), 6.73 (d, J = 2.6 Hz, IH, ArH), 6.70 (dd, J = 8.2, 2.767 Hz, IH, ArH), 4.01 (s, 2H, CH 2 ), 3.78 (s, 3H, CH 3 ), 2.35 (s, 3H, CH 3 ). 13 C NMR (101 MHz, CDCI 3 ) δ ppm 158.86 (s), 138.40 (s), 131.28 (d), 126.59 (s), 115.75 (d), 110.78 (d), 54.87 (q), 54.86 (t), 19.07 (q). Example 6: A/-(3,5-bis-trifluoromethylbenzyl)-hvdroxylamine

The product was obtained in a 51.6 % yield. IH NMR (400 MHz, CDCL3) δ ppm 7.83 (s, 2H, ArH), 7.82 (s, IH, ArH), 5.58 (bs, 2H, NHOH), 4.13 (s, 2H, CH 2 ). 13 C NMR (101 MHz, CDCI 3 ) δ ppm 140.32 (s), 131.72 (2s), 129.05 (2d), 123.29 (2q), 121.51 (d), 56.87 (t). 19 F NMR (376 MHz, CDCI 3 ) δ ppm -62.9 (s, 6F, CF 3 .

Example 7: /V-[3,5-difluorobenzyllhvdroxylamine F

The product was obtained in a 49.8 % yield. H NMR (400 MHz, D 2 0) δ ppm 7.25-7.03 (m, 3H, ArH), 4.47 (s, 2H, CH 2 ). 13 C NMR (101 MHz, D 2 0) δ ppm 162.80 (d), 162.66 (d), 132.00 (s), 113.66 (2d), 105.24 (d), 53.74 (t). 19 F NMR (376 MHz, D 2 0) δ ppm -109.33- (-109.46) (m, 2F,CF).

Example 8: /V-(4-fluoro-2-(trifluoromethvl)benzvl)hvdroxylamine)

The product was obtained in a 46.7 % yield. H NMR (400 MHz, CD 3 OD) δ ppm 7.89 (dd, J = 8.46, 5.26 Hz, 1H, ArH), 7.63 (dd, J = 8.95, 2.72 Hz, 1H, ArH), 7.55 (td, J = 8.46, 2.72 Hz, 1H, ArH), 4.93 (bs, 2H, NHOH), 4.61 (s, 2H, CH 2 ). 13 C NMR (101 MHz, CD 3 OD) δ ppm 164.32 (d), 137.16 (d), 133.24 (s), 124.72 (s), 124.54 (q), 120.91 (d), 115.53 (d), 52.12 (t). 19 F NMR (376 MHz, CD 3 OH) δ ppm -60.22 (s, 3F, CF 3 ), -110.52-(-110.57) (m, IF, CF).

Example 9: A/-(4-fluoro-2-methylbenzyl)hydroxylamine

The product was obtained in a 55.2 % yield. H NMR (400 MHz, D 2 0) δ ppm 7.43 (dd, J = 8.52, 5.93 Hz, 1H, ArH), 7.11 (dd, J = 10.04, 2.74 Hz, 1H, ArH), 7.05 (td, J = 8.52, 2.74 Hz, 1H, ArH), 4.41 (s, 2H, CH 2 ), 2.42 (s, 3H, CH 3 ). 13 C NMR (101 MHz, D 2 0) δ ppm 163.12 (d), 141.65 (s), 133.51 (d), 122.84 (s), 117.41 (d), 113.23 (d), 51.47(t), 18.52 (q). 19 F NMR (376 MHz, D 2 0) δ ppm -112.40-(-112.49) (m, IF, CF).

Comparative Example 10: /V-Benzyl-hydroxylamine hydrochloride

The compound has been obtained from Sigma Aldrich Comparative Example 11: /V-(2,6-Dimethoxvbenzvl)-hvdroxylamine H N MR (400 M Hz, DMSO-G( 6 ) :

The product was obtained in a 32.8 % yield. H N MR (400 M Hz, CDCI 3 ) δ ppm 7.18 (d, J = 8.2 Hz, 1H, ArH), 6.73 (d, J = 2.6 Hz, 1H, ArH), 6.70 (dd, J = 8.2, 2.7 Hz, 1H, ArH), 4.01 (s, 2H, CH 2 ), 3.78 (s, 3H, CH 3 ), 2.35 (s, 3H, CH 3 ). 13 C NM R (101 MHz, CDCI 3 ) δ ppm 158.86 (s), 138.40 (s), 131.28 (d), 126.59 (s), 115.75 (d), 110.78 (d), 54.87 (q), 54.86 (t), 19.07 (q).

Biological Testing

Bacterial strains and growth conditions

Wild-type Pseudomonas aeruginosa ATCC 15692 (CECT 4122), Staphylococcus aureus ATCC 12600 (CECT 86), Staphylococcus epidermidis ATCC 1798 (CECT 231), Enterococcus faecalis ATCC 19433 (CECT 481) were obtained from the Spanish Type Culture Collection (CECT) and Bacillus anthracis Sterne 7700 pX01-/pX02- were obtained from the Swedish Defense Research Agency. These strains were stored at -80°C as glycerol stocks. To obtain inocula for examination, the strains were cultured overnight on trypticase soy agar (TSA; Conda-Pronadisa, Spain) or tryptic soy broth (TSB; Sharlau, Spain) medium at 37°C.

Evaluation of the Minimum inhibitory concentration (MIC)

Minimal inhibitory concentration (M IC) assays were determined by a microtiter broth dilution method as described by Cole et al. (Cole AM, Weis P, Diamond G. J Biol Chem 1997. 272:12008-12013) and modified by Beckloff et al. (Beckloff N, Laube D, Castro T, Furga ng D, Park S, Perlin D, Clements D, Ta ng H, Scott RW, Tew GN, Dia mond G. Antimicrob Agents Chemother 2007. 51:4125-4132). I n brief, 100 μΙ of bacteria at a optical density (A 550 ) of 0.1 (lxlO 6 cfu/ml) in TSB broth (Sharlau Spain) was inoculated into the wells of 96-well assay plates (tissue culture-treated polystyrene; Nunc™ MicroWell™ Plates with Nunclon™ Delta Surface) in the presence of different concentration of compound in TSB at final concentrations (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 mM). The inoculated microplates were incubated at 37°C for 9 h at 150 rpm in an Infinite 200 Pro microplate reader (Tecan) with 15 min read at OD 550 . Because the water solubility of some compounds is low, we used 50% methanol. Experiments were carried out in triplicate. The M IC was determined as the lowest concentration that completely inhibited bacterial growth.

The M IC values were calculated for each compound. These assays were done in triplicate and the results shown in Table 1 represent the mean value of these triplicates.

Table 1 below shows M IC (minimum inhibitory concentration) value ranges of compounds tested in this assay. Results in table 1 expressed by the following data:

- "A" means 0.0625 mM≤ M IC < 0.125 mM,

- "B" means 0.125 mM≤ M IC < 0.25 mM,

- "C" means 0.25 mM≤ M IC < 0.50 mM,

- "D" means 0.50 mM≤ M IC < 1.00 mM,

- "E" means 1.00 mM≤ M IC < 2.00 mM,

- "F" means 2.00 mM≤ M IC < 4.00 mM, and

- "G" means 4.00 mM≤ M IC

- "n.d.". means not determined

Table 1

StaphyloPseudomonas Enterococcus Staphylococcus

Bacillus

Example coccus aeruginosa faecalis epidermis anthracis

aureus

1 C C D E n.d.

2 C C B D C

3 A D E D B 4 B D E D B

5 A G E C C

6 A C E D B

7 n.d. D D n.d. n.d.

8 n.d. E E n.d. n.d.

9 n.d. C D n.d. n.d.

Comp 10 G G G n.d. n.d.

Comp 11 G G G n.d. n.d.

The results summa rized in table 1 show that the hydroxylamines of the present invention (Examples 1 to 9) are more potent and/or have a broader spectra of action than similar compounds having a different substitution pattern in the benzene ring (Comparative examples 10 and 11).

Evaluation of capacity to inhibit the formation of bacterial biofilms

Overnight cultures grown in TSB were diluted to 10 6 CFU/ml. A 100-μΙ aliquot was transferred to a 96-well microtiter plate, and 100 μΙ of different compound concentration, dissolved in TSB, was added. After incubation at 37°C for 48 h without agitation, the bacterial viability was quantified. Essentially after incubation, the medium was removed and the wells were washed three-times with PBS + Polysorbate 20 0.05% and the biofilm attached to the wall of the different wells were scratched off with a sterile tip in 200 μΙ PBS + Polysorbate 20 0.05%. The suspension was serially diluted and plated onto a TSA plate to measure the viable count (cfu/ml) after incubation.

As seen in Fig. 1 and Fig. 2 /V-phenethyl-hydroxylamine, /V-cyclohexylmethyl- hydroxylamine, A/-(4-fluorobenzyl)-hydroxylamine and A/-(2,4-dimethoxybenzyl)- hydroxylamine inhibit the capacity of P. aeruginosa and 5. aureus biofilm formation.

Evaluation of capacity to reduce already formed bacterial biofilms Microtiter plates were filled with 200 μΙ of 10 6 CFU/ml in TSB and incubated at 37°C for 24 h or 3 days without shaking. After incubation, the medium was removed and the wells were rinsed twice with lx phosphate buffered saline (PBS, Gibco) . Different compound concentrations (200 μΙ) dissolved in TSB were added. After cultivation at 37°C for 24 h, the supernatant was discarded and washed three-times with PBS+Polysorbate 20 0.05% and the biofilm attach to the wall of the different wells were scratched off with a sterile tip in 200 μΙ PBS + Polysorbate 20 0.05%. The suspension was serially diluted and plated onto a TSA plate to measure the via ble count (cfu/ml) after incubation.

I n Fig. 3. the effect of various concentrations of /V-cyclohexylmethyl- hydroxylamine on P. aeruginosa formed biofilm is shown. The effect of two consecutive (2 days) /V-cyclohexylmethyl-hydroxylamine treatments on formed biofilm is shown.

I n Fig. 4. the effect of various concentrations of /V-cyclohexylmethyl- hydroxylamine, A/-(4-fluorobenzyl)-hydroxylamine and A/-(2,4-dimethoxybenzyl)- hydroxylamine on 5. aureus formed biofilm is shown.

I n Fig. 5 the effect of various concentrations of various concentrations of N- phenethyl-hydroxylamine on 5. aureus fomed biofilm is shown. The effect of three consecutive (3 days) /V-phenethyl-hydroxylamine treatments on formed biofilm is shown.

I n Fig. 6 the effect of various concentrations of various concentrations of (3,5-bis-trifluoromethylbenzyl)-hvdroxylamine on 5. aureus fomed biofilm is shown. The effect of three consecutive (3 days) /V-(3,5-bis-trifluoromethylbenzyl)- hydroxylamine treatments on formed biofilm is shown.

Evaluation of cytotoxicity

The murine macrophage J774 cell line was maintained in Dulbecco's Modified Eagle's medium (DM EM) with L-glutamine (Gibco BRL, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Lonza Ltd., Switzerland), containing 100 U/ml penicillin G (La b ERN, Barcelona, Spain) and 100 μg/ml streptomycin (Lab Reig Jofre, Barcelona, Spain) (complete medium), at 37^C in a humidified atmosphere with 5% C0 2 .

Macrophage (6xl0 4 per well) were seeded onto 96-well tissue culture plates in complete medium without antibiotics, in the presence of different concentration of compound, or left untreated. Cell viability was assessed by using a 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma). After, 24 and 48 h of exposure to the different compounds, culture supernatants were removed and 10% of MTT in complete medium was added to each well and incubated for 3 hours at 37^C. Then, water-insoluble dark blue formazan was dissolved by adding acidic isopropanol. Absorbance was measured at 550 nm using an ELISA reader (I nfinite M200 Microplate Reader, Tecan).

Table 2

Cytoxicity (IC 5 o)

Example

mM

1 1.89

2 1.33

3 1.30

4 2.27

5 2.18

6 2.07

7 n.d.

8 n.d.

9 n.d.

Comp 10 >4

Comp 11 n.d.