5-ARYL-2-aminoimidazoles as Anti-bacterials

Abstract

The invention relates to substituted 5-aryl-2-aminoimidazoles represented by the structural formula (I), wherein,R1 is H, R2 is a C2-20 alkyl substituted with a polar group selected from the group consisting of an amine, an alcohol, an ester, an amide, reverse amide, carboxylic acid, and a thiol, R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, nitro, C1-12 alkoxy, C1-12 alkyl, hydroxyl and methyl sulfonyl.

Description

[0001] 5-ARYL-2-AM I NOIMIDAZOLES AS ANTI-BACTERIALS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to 5-aryl-2-aminoimidazoles compounds, compositions and methods for controlling biofilms and microbial, in particular bacterial, growth and for reducing bacterial colonization.

[0004] BACKGROUND OF THE INVENTION

[0005] Biofilms are complex, condition-dependent, surface-associated communities of microorganisms (e.g. bacterial cells) embedded in a self-produced matrix of extracellular polymeric substance. Biofilms represent a prevalent mode of microbial life in natural, industrial and hospital settings. The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism. Biofilm cells exhibit profound changes in gene expression and cell physiology compared with planktonic cells, and multiple genetic pathways mediate the regulation of biofilm formation. Microorganisms in biofilms form microbial colonies or condominiums that make it easy to carry out chemical reactions that are impossible for individual microbial cells. Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae.

[0006] Within biofilms bacteria are up to 1000 times more tolerant to antibiotics, disinfectants and other stress factors, which strongly impedes antimicrobial treatment. Hence persistent biofilm infections and contaminations often occur and cause a tremendous amount of problems in various sectors, including medicine, food industry, household and agriculture.

[0007] The use of effective antimicrobial compositions to avoid biofilm formation is recommended for any surface in contact with water, such as swimming pool liners, water cooling surfaces, hoses, water dispensers, water storage and distribution systems for drinking water, horticulture or aquaculture, and for surfaces of medical devices such as catheters, medical implants, wound dressings and the like, especially when intended for patients with metabolic disorders.

[0008] In the medical sector, biofilms are often associated with implantable devices. Staphylococci are the principal microorganisms that colonize these devices. They comprise up to two-third of all pathogens in orthopaedic implant infections, where they can cause septic arthritis and osteomyelitis, resulting in the inflammatory destruction of bones and joints. These infections can occur as a result of direct contamination during the operation, or as a result of microbiological spread from chronic infections elsewhere in the body. Unfortunately, the lack of a suitable treatment often leaves extraction of the contaminated device as the only viable option for eliminating the biofilm. Afterwards a prolonged antimicrobial treatment is required to make sure that the infection is eradicated.

[0009] Given the extent of problems caused by such biofilms, there has been a strong effort to develop novel anti-biofilm strategies [Bjarnsholt et al. (2010) Expert Rev Mol Med 12, ell; Landini et al. (2010) Appl Microbiol Biotechnol 86, 813-823; Lynch & Abbanat et al. (2010) Expert Opin Ther Pat 20, 1373-1387]. One of the most promising approaches are compounds able to prevent or eradicate biofilms, without affecting the planktonic growth of the microorganisms [Ren et al. (2005) Appl Environ Microbiol 71, 4022-4034; Rogers et ai. (2009) J Am Chem Soc 131, 9868-9869]. These specific anti-biofilm compounds are believed to be less prone to resistance development. Indeed, in vitro evolution experiments with Salmonella showed that resistance to the specific anti-biofilm compounds does not develop after 100 days of exposure, while under the same conditions resistance to classical antimicrobials developed within a few days [Dieltjens et al. (2020) Nat Common 11, 107]. The explanation can be found in the public good character of the targeted slimy biofilm matrix. Microbes that acquire resistance to the inhibitor and produce the slime again will share this slime- and therefore the benefit of their resistance- with the susceptible microbes around. They do however not share the energy cost of slime production and are therefore outcompeted by the susceptible microbes which experience a relative benefit [Dieltjens et al. (2020) Nat Common 11, 107],

[0010] Previously, we have reported the development of several series of specific antibiofilm compounds, based on the 2-aminoimidazole (2AI) scaffold. These series include the mono-substituted 5-Aryl-2Als (5-Ar-2Als) [Steenackers et al. (2011 J Med Chem 54, 472-484], Nl-substituted 5-Ar-2Als [Steenackers et at. (2011) J Med Chem 54, 472-484], 2N-substituted 5-Ar-2Als [Steenackers et at. (2011) Bioorg Med Chem 19, 3462-3473], 4,5-di-substituted 2Als [Steenackers et al. (2011) J Med Chem 54, 472-484], 1,4,5- trisubstituted 2Als [Ermolat'Ev et at. (2010) Angew Chemie - Int Ed 49, 9465-9468], 2AI-triazole-conjugates [Ermolat'Ev et al. (2010) Angew Chemie - Int Ed 49, 9465-9468], N1,2N- disubstituted 5-Ar-2Als [Peeters et al. (2016) Antimicrob Agents Chemother 60, 6483-6497] and 5-Ar-2Als dimers [Trang et al. (2018) Bioorganic Med Chem 26, 1470-1480]. These compounds were shown to display a preventive activity against either biofilms of Salmonella Typhimurium, one of the most important causes of foodborne infections worldwide and a notorious biofilm former both inside and outside the host, and of P. aeroginosa, a Gram-negative opportunistic pathogen that can infect immunocompromised people such as cystic fibrosis patients and cause life-threatening chronic lung infections [Folkesson et al. (2012) Nat Rev Microbiol 10, 841-851] or against biofilms of Staphylococcus aureus, one of the most common pathogens found in medical device associated infections, depending on the structure of the 5-Ar-2Als [Peeters et al. (2016) Antimicrob Agents Chemother 60, 6483-606497]. During the last decade, several synthetic methodologies leading to diversely substituted 2-Als have been published [Steenackers et at. (2011) J Med Chem 54, 472-484; Ermolat'Ev et at. (2010) Angew Chemie - Int Ed 49, 9465-9468; Steenackers et al. (2014) Org Biomol Chem 12, 3671-3678; Peeters et al. (2016) Antimicrob Agents Chemother 60, 6483-6497;Trang et al. (2018) Bioorganic Med Chem 26, 1470-1480; Guo et al. (2015) Org Lett 17, 1157-1159; Ermolat'ev et a / . (2006) Org Lett 8, 5781-5784; Steenackers et al. (2011) Bioorg Med Chem 19, 3462-3473; Lissens et ai. (2022) FEMS Microbiol Rev 46, fuac019].

[0011] Previously (W02017070755), it was shown that 5-Ar-2Als substituted at both the Nl- and 2N position combine the activity against Gram-positive bacteria, of the Nl-substituted compounds, with the low toxicity of the 2N-substituted compounds. However, biofilm infections / contaminations are caused by both Grampositive and Gram-negative bacteria so broad spectrum anti-biofilm compounds are of interest.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to the treatment and prevention of infectious diseases caused by microbial biofilm formation, in particular to antimicrobial prophylactic and therapeutic compositions containing an effective amount of a biofilm formation inhibiting compound to reduce or eliminate colonization with potentially pathogenic microorganisms, more particularly bacteria (including bacterial strains resistant to many or most common antimicrobial agents), thereby reducing the risk of subsequent disease occurrence.

[0014] Furthermore, the present invention relates to compounds, and to compositions and methods involving these compounds, for inhibiting, reducing or preventing the formation of a biofilm on a surface of a medical device such as a catheter, or on a tissue such as teeth, urethra or lungs of a human (e.g. a cystic fibrosis patient). These compounds, compositions and methods of the present invention are in particular useful for preventing biofilm formation in a tissue to prevent or control a chronic bacterial infection or sepsis. These compounds, compositions and methods are equally useful, to control bacterial plant pathogens in irrigation water systems and also useful for sanitation when applied to a substrate with which a human, plant or an animal may come into contact. An example hereof are irrigation systems used in horticulture.

[0015] Herein, it is shown that 5-Ar-2Als where part of the 2N alkyl chain is substituted with a polar group show higher anti-biofilm activity and a more broad activity spectrum, e.g. activity against both Gram-positive and Gram-negative bacteria, compared to the corresponding compound without polar group. So, the present invention provides said polar-2N-disubstituted 5-Ar-2Als, their use in the preparation of antimicrobial solutions, pharmaceutical or veterinary preparations as well as methods for the elimination of biofilms involving the use of said compounds.

[0016] Furthermore, as part of the present invention, it was shown that 5-Ar-2Als coupled to iron oxide nanoparticles enhance the activity and biofilm-specificity of the compounds.

[0017] The present discloses a selected group of substituted 5-aryl-2-aminoimidazoles which exhibit a broad microbial biofilm activity, against both Gram-negative and Gram-positive bacteria, and therefore can be formulated into antimicrobial compositions for administration to humans and animals and for application to inert surfaces susceptible to infection by microbial biofilms. The present invention also provides a surface, such as the surface of a medical implant, coated with the compounds of the present invention. The present invention also provides a method for suppressing, reducing, inhibiting, controlling, treating or preventing the development of a microbial biofilm on a biotic or abiotic surface or in a subject, which comprises the step of exposure or administration of such a composition on said surface or to the subject.

[0018] The invention is further summarised in the following statements

[0019] 1. A substituted 5-aryl-2-aminoimidazole represented by the structural formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or polymorphic form thereof, wherein,

[0020] R1 is H,

[0021] R2 is a C2-20 alkyl substituted with a polar group selected from the group consisting of an amine, an alcohol, an ester, an amide, reverse amide, carboxylic acid, and a thiol,

[0022] R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, nitro, C1-12 alkoxy, C1-12 alkyl, hydroxyl and methyl sulfonyl.

[0023] Herein alkyl chains may be linear or branched.

[0024] 2. The compound according to statement 1, wherein

[0025] R1 is H,

[0026] R2 is a C2-20 alkyl substituted with a polar group selected from the group consisting of an amine, an alcohol, an ester, an amide, a reverse amide, carboxylic acid, and a thiol,

[0027] R3 is a halogen or hydrogen,

[0028] R4 is a halogen or hydrogen.

[0029] 3. The compound according to statement 1 or 2, wherein R3 is hydrogen.

[0030] 4. he compound according to any one of statements 1 to 3, wherein the polar group is an amine. The compound according to any one of statements 1 to 4, wherein the polar group is terminally located.

[0031] Alternatively the polar group can be a secondary amine in the alkyl chain.

[0032] This is for example by R.2 represented by -(CH2)a-NH-(CH2)b-CH3, wherein a is 1 to 19 and b is 0 to 18, wherein a + b is maximally 20. The compound according to any one of statements 1 to 5, wherein R.2 is (C H2)2-20-N H 2. The compound according to statement any one of statements 1 to 6, wherein R4 is Br or F. The compound according to any one of statements 1 to 7, wherein R2 is (C H2)2-16-N H 2 . The compound according to any one of statements 1 to 8, wherein R2 is (C H2)2-12-N H 2. The compound according to any one of statements 1 to 8, wherein R2 is (C H2)2-4- N H 2. The compound according to any one of statements 1 to 10, wherein, R1 is H,

[0033] R2 is a C4-12 alkyl substituted with an amine group, typically (CH2)4-i2- NH2,

[0034] R3 is hydrogen,

[0035] R4 is hydrogen or a halogen. The compound according to statement 1, selected from the group consisting of:

[0036] Nl-(5-phenyl-lH-imidazol-2-yl)butane-l,4-diamine, N-butylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine, Nl-(5-phenyl-lH-imidazol-2-yl)hexane-l,6-diamine , N-hexylamine-5-(4-bromophenyl)-lH-imidazol-2-amine, Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)hexane-l,6-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)octane-l,8-diamine , N-octylamine-5-(4-bromophenyl)-lH-imidazol-2-amine, Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)octane-l,8-diamine, N-octylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine, Nl-(5-phenyl-lH-imidazol-2-yl)dodecane-l,12-diamine, Nl-(5-phenyl-lH-imidazol-2-yl) penta ne-l,5-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)heptane-l,7-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)decane-l,10-diamine and Nl-(5-phenyl-lH-imidazol-2-yl)undecane-l,ll-diamine. 13. A composition comprising a compound according to any one of statements 1 to 12, for use in the treatment of a gram-negative or gram-positive bacterial infection.

[0037] 14. The composition for use according to statement 13, wherein the bacterial infection is an infection of P aeruginosa or S. aureus.

[0038] 15. The composition for use according to statement 13 or 14, wherein the bacterial infection is a biofilm.

[0039] 16. A substituted 5-aryl-2-aminoimidazole according to any one of statements 1 to 12 for use as a medicament.

[0040] 17. An in vitro use of a composition comp rising a compound according to any one of statements 1 to 12, for preventing or treating bacterial growth.

[0041] 18. The in vitro use according to statement 18, for preventing or treating a bacterial biofilm.

[0042] 19. The in vitro use according to statement 18, for preventing or treating bacterial growth in water used for irrigation in horticulture.

[0043] 20. A surface or particle coated with a substituted 5-aryl-2-aminoimidazoles according to any one of statements 1 to 12.

[0044] 21. The coated surface or particle according to statement 20, wherein the coating occurs via the NH2 group of R.2.

[0045] 22. The coated particle according to statement 21 or 22, which is a magnetic particle.

[0046] BRIEF DESCRIPTION OF DRAWINGS

[0047] Fig. 1. Structure of 5-Ar-2AI based compounds

[0048] Compound 1 : N-butyl-5-phenyl-lH-imidazol-2-amine

[0049] Compound 2: N-butyl-5-(4-fluorophenyl)-lH-imidazol-2-amine

[0050] Compound 3: N-hexyl-5-phenyl-lH-imidazol-2-amine

[0051] Compound 4: 5-(4-bromophenyl)-N-hexyl-lH-imidazol-2-amine

[0052] Compound 5: 5-(4-chlorophenyl)-N-hexyl-lH-imidazol-2-amine

[0053] Compound 6: N-octyl-5-phenyl-lH-imidazol-2-amine

[0054] Compound 7: 5-(4-bromophenyl)-N-octyl-lH-imidazol-2-amine

[0055] Compound 8: 5-(4-chlorophenyl)-N-octyl-lH-imidazol-2-amine

[0056] Compound 9: 5-(4-fluorophenyl)-N-octyl-lH-imidazol-2-amine

[0057] Compound 10: N-dodecyl-5-phenyl-lH-imidazol-2-amine

[0058] Fig. 2. Structure of novel 5-Ar-2AI compounds

[0059] Compound 11 : Nl-(5-phenyl-lH-imidazol-2-yl)butane-l,4-diamine dihydrochloride Compound 12: Nl-(5-(4-fluorophenyl)-lH-imidazol-2-yl)butane-l,4-diamine dihydrochloride

[0060] Compound 13: Nl-(5-phenyl-lH-imidazol-2-yl)hexane-l,6-diamine dihydrochloride

[0061] Compound 14: Nl-(5-(4-bromophenyl)-lH-imidazol-2-yl)hexane-l,6-diamine dihydrochloride

[0062] Compound 15: Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)hexane-l,6-diamine dihydrochloride

[0063] Compound 16: Nl-(5-phenyl-lH-imidazol-2-yl)octane-l,8-diamine dihydrochloride

[0064] Compound 17: Nl-(5-(4-bromophenyl)-lH-imidazol-2-yl)octane-l,8-diamine dihydrochloride

[0065] Compound 18: Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)octane-l,8-diamine dihydrochloride

[0066] Compound 19: Nl-(5-(4-fluorophenyl)-lH-imidazol-2-yl)octane-l,8-diamine dihydrochloride

[0067] Compound 20: Nl-(5-phenyl-lH-imidazol-2-yl)dodecane-l,12-diamine dihydrochloride

[0068] Compound 21 : Nl-(5-phenyl-lH-imidazol-2-yl)pentane-l,5-diamine dihydrochloride

[0069] Compound 22: Nl-(5-phenyl-lH-imidazol-2-yl)heptane-l,7-diamine dihydrochloride

[0070] Compound 23: Nl-(5-phenyl-lH-imidazol-2-yl)decane-l,10-diamine dihydrochloride

[0071] Compound 24: Nl-(5-phenyl-lH-imidazol-2-yl)undecane-l,ll-diamine dihydrochloride

[0072] Fig. 3. Synthesis of novel 2-AI compounds

[0073] A) Schematic representation of the synthesis of 2-aminoimidazoles. I Chloropyrimidine is coupled with an amine bearing the desired polar group R1 II The newly obtained aminopyrimidine is reacted with a bromoacetophenone. Ill The resulting imidazopyridinium salt is treated with hydrazine to provide the desired 2-aminoimidazole. B) Schematic representation of the synthesis of the aminated analogues.

[0074] Fig. 4. Activity of novel 2-AI compounds compared to corresponding previously reported compounds

[0075] Antibiofilm effect of 2N-substituted compounds with polar group (grey bars) compared to corresponding 2N-substituted compounds without polar group (black bars). Bars represent the BICso values with the standard deviation of the three repeats. BICso is defined as the concentration needed to inhibit the biofilm formation by 50%. a) compound 1 vs. compound 11; b) compound 2 vs. compound 12; c) compound 3 vs. compound 13; d) compound 4 vs. compound 14; e) compound 5 vs. compound 15; f) compound 6 vs. compound 16; g) compound 7 vs. compound 17; h) compound 8 vs. compound 18; i) compound 9 vs. compound 19; j) compound 10 vs. compound 20.

[0076] Fig. 5 Antibiofilm effect of 2N-substituted compounds with polar group. Bars represent the BICso values with the standard deviation of the three repeats. BICso is defined as the concentration needed to inhibit the biofilm formation by 50%. a) compound 21; b) compound 22, c) compound 23 and d) compound 24.

[0077] Fig. 6 Synthesis of NP-coupled 2-AIs.

[0078] Schematic representation of the functionalization of the iron oxide nanoparticles. A) The inhibitor is coupled with the modified PEG chain by CDI coupling. B) The double bond of the PEG chain is coupled with a thiol at the end of a silane by thiolene click chemistry. C) The silanes are connected to the nanoparticles surface by hydrolysis and polycondensation of the siloxanes.

[0079] Fig. 7 Activity of NP-coupled 2-AIs

[0080] A) Effect of novel compound 17 on the total biofilm mass on a panel of monospecies bacterial biofilms, determined by crystal violet staining. The bars represent the 95% confidence intervals of three independent experiments. The BIC50 and IC50 represent the concentration of each compound needed to inhibit respectively biofilm formation and planktonic growth by 50%. B) Effect of novel compound 17 covalently bound to iron oxide nanoparticles (synthesized from a starting ratio of 10% allylPEG-5-Ar-2AIs and 90% hydroxypolyethoxy allyl ether ) on the total biofilm mass on a panel of monospecies bacterial biofilms, determined by crystal violet staining. The bars represent the 95% confidence intervals of three independent experiments. C) Effect of novel compound 17 covalently bound to iron oxide nanoparticles (synthesized from a starting ratio of 20% allylPEG-5-Ar- 2AIs and 80% hydroxypolyethoxy allyl ether ) on the total biofilm mass on a panel of monospecies bacterial biofilms, determined by crystal violet staining. The bars represent the 95% confidence intervals of three independent experiments.

[0081] Fig. 8 Effect of magnetic forces on the preventive effect of 5-Ar-2AIs functionalized to iron oxide nanoparticles against S. Typhimurium biofilms.

[0082] The percentage of CFU / cm2of S. Typhimurium biofilm cells treated with functionalized iron oxide nanoparticles containing 10% PEG-5-Ar-2AIs in presence of a magnet underneath the biofilm is shown as compared to the controls where no magnet was applied. Data represent the mean ± SEM of three repeats (***, p < 0.001).

[0083] Fig. 9 Effect of heating on the preventive effect of 5-Ar-2AIs functionalized to iron oxide nanoparticles against S. Typhimurium biofilms

[0084] The percentages of CFU of S. Typhimurium without heating (black bars), with heating (white bars) and a combination of heating and magnetic forces (grey bars) after treatment with functionalized iron oxide nanoparticles containing either no inhibitor (100% PEG-OH) or 10% PEG-INH1 is shown as compared to the control cells where no compound was added. Data represent the mean ± SEM of 3 biological repeats (****, p < 0.0001).

[0085] Fig. 10 FIC indices for combinations of conventional antibiotics of different antibiotic classes (Erythromycin (ERY), Chloramphenicol (CHL), Cefuroxime (CXM), Amoxicillin (AX), Moxifloxacin (MOX), Gentamicin (GEN) and Minocycline (MIN)) with a 2N-polar substituted 5-Ar-2AI (Nl-(5-phenyl-lH-imidazol-2- yl)heptane-l,7-diamine (2-AI)) and more particularly for combinations of 2-AI and ERY in a ratio of 73:1, 2-AI and CHL in a ratio of 30: 1, 2-AI and CXM in a ratio of 50: 1, 2-AI and AX in a ratio of 9: 1, 2-AI and MOX in a ratio of 2509:1, 2-AI and GEN in a ratio of 7: 1 and 2-AI and MIN in a ratio of 29: 1.

[0086] FIG. 11 Reduction of the percentage of plants with symptoms of hairy root disease caused by the inhibition of the plant pathogen Agrobacterium rhizogenes by 2N- polar substituted 5-Ar-2AIs.

[0087] A) The percentage of plants with symptoms of hairy root disease during the 22 weeks of the growing trial. B) The percentage of plants with symptoms of hairy root disease on the final day of the growing trial. Negative control: no induced infection of Agrobacterium rhizogenes, no treatment. Positive control: induced infection of Agrobacterium rhizogenes, no treatment; 100 pM 2-AI: induced infection of Agrobacterium rhizogenes, treatment with 100 pM of the 5-Ar-2AI compound (Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)octane-l,8-diamine) at the start and addition of 50pM every three weeks; 5-Ar-2AI + 50ppm H2O2: induced Agrobacterium infection, treatment with 100 pM of the 5-Ar-2AI compound at the start and addition of 50pM every three weeks and continuous addition of H2O2 to a constant concentration of 50ppm.7

[0088] FIG. 12 Embodiments of 5-aryl-2-aminoimidazoles with alternative polar groups on the R2 substituent. DETAILED DESCRIPTION OF THE INVENTION

[0089] As compared to prior art substituted 2-aminoimidazoles (W02011080132 and W02017070755), the present invention provides a selection of novel compounds wherein said compounds show a surprisingly better activity and activity spectrum, e.g. activity against both Gram-positive and Gram-negative bacteria and synergistic effect when combined with antibiotics.

[0090] The invention relates to substituted 5-aryl-2-aminoimidazole compounds represented by the structural formula (I) wherein R1 is hydrogen; R2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide, , reverse amide, carboxylic acid, or a thiol polar functional groups; and R3 and R4 are each independently selected from the group comprising hydrogen, halogen, nitro, C1-12 alkoxy, C1-12 alkyl, hydroxyl and methyl sulfonyl. Herein alkyl chain can be linear or branched.

[0091] These substituted 5-aryl-2-aminoimidazole compounds are represented by the structural formula (I) wherein R1 is hydrogen; R2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide, reverse amide, carboxylic acid, or a thiol polar functional groups and R3 and R4 are each independently selected from the group consisting of hydrogen halogen, nitro, methoxy, methyl, hydroxyl and methyl sulfonyl. More specifically, R.3 is hydrogen, or R3 is hydrogen, and R4 is hydrogen or a halogen.

[0092] Typically, the compounds of the present invention also include pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or polymorphic forms thereof. In a preferred embodiment of the present invention, said compound is selected from the group comprising Nl-(5-phenyl-lH-imidazol-2-yl)butane-l,4-diamine, N-butylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine, Nl-(5-phenyl-lH- imidazol-2-yl)hexane-l,6-diamine, N-hexylamine-5-(4-bromophenyl)-lH- imidazol-2-amine, N l-(5-(4-chlorophenyl)- 1H -imidazol-2-y I) hexane- 1,6-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)octane-l,8-diamine, N-octylamine-5-(4- bromophenyl)-lH-imidazol-2-amine, Nl-(5-(4-chlorophenyl)-lH-imidazol-2- yl)octane-l,8-diamine, N-octylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine ,Nl-(5-phenyl-lH-imidazol-2-yl)dodecane-l,12-diamine, Nl-(5-phenyl-lH- imidazol-2-yl)pentane-l,5-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)heptane-l,7- diamine, Nl-(5-phenyl-lH-imidazol-2-yl)decane-l,10-diamine, Nl-(5-phenyl-lH- imidazol-2-yl)undecane-l,ll-diamine.

[0093] Alternatively, the invention provides compounds represented by the structural formula (II) and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or polymorphic forms thereof wherein R1 is hydrogen; R2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide, reverse amide, carboxylic acid, or a thiol polar functional groups; and R3, R4, R5, R6 and R7 are each independently selected from the group comprising hydrogen, halogen, nitro, C1-12 alkoxy, C1-12 alkyl, hydroxyl and methyl sulfonyl. Herein alkyl chain can be linear or branched.

[0094] More specifically, 5-aryl-2-aminoimidazole compounds are represented by the structural formula (II) wherein R1 is hydrogen; R2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide, reverse amide, carboxylic acid, or a thiol polar functional groups and R3, R4, R5, R6 and R7 are each independently selected from the group consisting of hydrogen halogen, nitro, methoxy, methyl, hydroxyl and methyl sulfonyl. More specifically, R3, R4, R5, R6 and R7 are hydrogen or a halogen. Typically, the compounds of the present invention as described herein are used in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject. Preferably, said microbial associated condition or infection is a bacterial, fungal or yeast biofilm associated condition or infection. Said microbial biofilm may also comprise a combination of bacterial and / or fungal and / or yeast infection.

[0095] Furthermore, the compounds of the present inventions as described herein are used in the preventive or curative treatment of microbial biofilm associated contamination of industrial installations, water distribution systems for cooling systems, horticulture and aquaculture.

[0096] The invention further provides iron oxide nanoparticles coupled with any of the compounds according to the first object of the present invention.

[0097] Als disclosed herein is that said compound can be linked to the iron oxide nanoparticle via an amide bond between a free amine at R2 and a carboxyl group of a linker molecule covalently bound to the iron oxide nanoparticle.

[0098] Preferably, said compounds are coupled to said iron oxide nanoparticles at a concentration of 1 to 40 mol %, more preferably at a concentration of 5to 30% mol %, even more preferably at a concentration of 1 to 30 mol %.

[0099] The invention relates to composition for use in treatment or prevention of a pathological condition associated with a microbial infection or for decreasing or eradicating bacterial growth in an animal or human, wherein said composition comprises one or more excipients and microbial biofilm inhibiting amount of a compound selected from the group consisting of substituted 5-aryl-2- aminoimidaoles represented by the structural formula (I) wherein R1 is hydrogen; R.2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide, reverse amide, carboxylic acid, or a thiol polar functional groups; and R3 and R4 are each independently selected from the group comprising hydrogen, halogen, nitro, C1-12 alkoxy, C1-12 alkyl, hydroxyl and methyl sulfonyl. More specific R3 is hydrogen, or R3 is hydrogen, and R4 is hydrogen or a halogen.

[0100] As disclosed herein is that said composition comprises one or more excipients and a biofilm inhibiting amount of a compound selected from the group consisting of substituted 5-aryl-aminoimidazoles represented by the structural formula (I) wherein R1 is hydrogen; ; R2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide, reverse amide, carboxylic acid, or a thiol polar functional groups and R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, nitro, methoxy, methyl, hydroxyl and methyl sulfonyl. More specifically, R.3 is hydrogen, or R.3 is hydrogen, and R4 is hydrogen or a halogen.

[0101] Typically, the compositions of the present invention also include pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or polymorphic forms thereof. In a preferred embodiment, said composition comprises a compound selected from the group comprising Nl-(5-phenyl-lH-imidazol-2-yl)butane-l,4-diamine, N- butylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine, Nl-(5-phenyl-lH-imidazol- 2-yl)hexane-l,6-diamine, N-hexylamine-5-(4-bromophenyl)-lH-imidazol-2- amine, N l-(5-(4-chlorophenyl)- 1 H -imidazol-2-y I) hexane- 1,6-diamine, Nl-(5- phenyl-lH-imidazol-2-yl)octane-l,8-diamine, N-octylamine-5-(4-bromophenyl)- lH-imidazol-2-amine, Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)octane-l,8- diamine, N-octylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine ,Nl-(5-phenyl- lH-imidazol-2-yl)dodecane-l,12-diamine, Nl-(5-phenyl-lH-imidazol-2- yl) penta ne-l,5-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)heptane-l,7-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)decane-l,10-diamine, Nl-(5-phenyl-lH-imidazol- 2-yl)undecane-l,ll-diamine.

[0102] Typically, said compositions of the present invention as described herein are used in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject. Preferably, said microbial associated condition or infection is a bacterial, fungal or yeast biofilm associated condition or infection. Said microbial biofilm may also comprise a combination of bacterial and / or fungal and / or yeast infection.

[0103] Typically, the said compositions of the present invention as described herein may, either for improved efficacy or for controlling several types of microbes in the same or a vicinal locus of a human, an animal or a plant, further comprise an effective amount of another anti-microbial (e.g. antibacterial, antiprotozoal or antifungal) entity or agent. Such combination of active agents may be in the form of a kit wherein each agent is kept separate until effective use. The other anti-microbial entity may be a biocide, an antibiotic agent or another specific therapeutic entity. Suitable antibiotic agents include, without limitation, penicillin, quinoline, vancomycin, sulfonamides, ampicillin, ciprofloxacin, and sulfisoxazole. The specific therapeutic entity can include a targeting moiety coupled to an antimicrobial peptide moiety.

[0104] Typically, said compositions of the present invention as described herein may, depending upon the desired mode of administration or application, be formulated in very different forms such as, but not limited to, liquids, gels, foams, semi-solids and solids. Practically these compositions can be in the form of an oral tablet, a capsule, a nasal aerosol, a liquid, such as throat wash, mouth wash or gargle, a tooth-paste or a topical ointment. They can be in the form of tampons, rinses, creams or aerosols, soaps, hair shampoos, antiperspirants, facial tissues, skin cleansers, component of a wound dressing or any device suitable for sanitation or hygienic treatment.

[0105] When the antimicrobial compositions of this invention are formulated as liquids, at least one excipient may be a solvent for the biologically effective substituted 5- aryl-2-aminoimidazole. Said solvent may be dimethylformamide, tetra hydrofuran, acetonitrile, dichloromethane, N-methylpyrrolidone, acetone, chloroform, dimethylsulfoxide and mixtures thereof, but is not limited thereto. The respective proportions of the active compound and the solvent in the liquid formulation are mainly determined by the solubility limit of the active compound in the relevant solvent, which can readily be determined by the skilled person. A liquid antimicrobial composition of this invention may also be in the form of a kit where the active compound and the solvent are kept separately until effective use.

[0106] For an effective treatment, since the substituted 5-aryl-2-aminoimidazole may become toxic above a certain concentration, it is necessary for safety reasons to provide administration or application in the form of a composition comprising one or more excipients. Particularly preferred are compositions comprising an excipient which is agriculturally acceptable for application to a plant, or an excipient which is pharmaceutically or veterinary acceptable for administration to, or contact with, a human or an animal.

[0107] The compositions of the present invention can include one or more non-active excipients or ingredients, e.g., ingredients that do not interfere with the biofilm inhibiting function of the active compound. The non-active ingredient can be a powder, an encapsulated solid, or an aqueous carrier. In one embodiment, the compositions of the present invention in oral form may include, without limitation, thickening materials, humectants, water, buffering agents, surfactants, titanium dioxide, flavouring systems, sweetening agents, colouring agents, and mixtures thereof. Pharmaceutically acceptable excipients, ingredients and carriers are well known, and one skilled in the pharmaceutical art can easily select them for any particular route of administration (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).

[0108] Generally, the antimicrobial compositions of the invention are formulated for longterm storage such as concentrated solutions or lyophilized powder preparations. They may also be included in vesicles, e.g. liposomes, or be formulated as controlled release systems, using controlled release technologies known in the art. When the antimicrobial compositions of this invention are formulated as gels or foams, they may be enclosed within a dispensing device for gel or foam.

[0109] When intended for a human or an animal such as, but not limited to, a mammal, a domestic animal or cattle, the method of treatment of this invention may be by administering the anti-microbial composition intravesicularly, topically, orally, rectally, ocularly, otically, nasally, parenterally, vaginally, intravenously, topically to an infected body part of said human or animal. Said body part may be an epithelial surface or a mucosal surface. Said mucosal surface may be a buccal cavity, vagina, gastrointestinal tract or oesophageal tract. When intended for disinfecting a surface which may come into contact with a human or an animal such as, but not limited to, a medical device or an implantable device (e.g. a prosthetic device, a heart valve, a pacemaker, a dental device, a stent or a catheter or a prosthetic bladder material), the method of treatment of this invention may be by dipping said medical device into the anti-microbial composition. The surface to be disinfected may be e.g. a biological surface or an inert solid industrial or domestic surface such as a heat exchanger, an air-filtering device, a component of an aquaculture system, kitchenware or a pipeline, or a surface in a hospital such as in a surgery unit where sanitization is essential. The material from which said surface is made is not a critical parameter of the method of the invention, as soon as it is susceptible to biofilm formation. The surface can include a plastic such as a silicone or another type of polymeric material.

[0110] The present invention presents methods for treating a microbial infection or for inhibiting microbial biofilm formation in a plant, or on a surface with which a human or an animal may come into contact, by applying to said plant or surface, an antimicrobial compound or composition according to any one of previous objects of the present invention.

[0111] The present invention relates to the use of a composition for disinfecting or sterilizing a surface ex vivo to decrease or eradicate a biofilm or prevent biofilm growth, wherein said composition comprises a biofilm inhibiting amount of a compound selected from the group consisting of substituted 5-aryl-2- ami. noimidazoles represented by the structural formula (I) wherein R1 is hydrogen; R.2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide , reverse amide, carboxylic acid, or a thiol polar functional groups; and R.3 and R.4 are each independently selected from the group comprising hydrogen, halogen, nitro, C1-12 alkoxy, C1-12 alkyl, hydroxyl and methyl sulfonyl. More specific R3 is hydrogen, or R3 is hydrogen, and R4 is hydrogen or a halogen.

[0112] Also disclosed herein is that said composition can comprise a biofilm inhibiting amount of compound selected from the group consisting of substituted 5-aryl-2- aminoimidazole compound is represented by the structural formula (I) wherein R1 is hydrogen; ; R2 is selected from the group comprising C2-12 alkyl substituted with a polar group, comprising amine, alcohol, ester, amide, reverse amide, carboxylic acid, or a thiol polar functional groups and R.3 and R4 are each independently selected from the group consisting of hydrogen halogen, nitro, methoxy, methyl, hydroxyl and methyl sulfonyl. More specific R3 is hydrogen, or R3 is hydrogen, and R4 is hydrogen or a halogen.

[0113] Typically, the compositions of the present invention also include pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or polymorphic forms thereof. In a preferred embodiment, said composition comprises a compound selected from the group comprising Nl-(5-phenyl-lH-imidazol-2-yl)butane-l,4-diamine, N- butylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine, Nl-(5-phenyl-lH-imidazol- 2-yl)hexane-l,6-diamine, N-hexylamine-5-(4-bromophenyl)-lH-imidazol-2- amine, N l-(5-(4-chlorophenyl)- 1 H -imidazol-2-y I) hexane- 1,6-diamine, Nl-(5- phenyl-lH-imidazol-2-yl)octane-l,8-diamine, N-octylamine-5-(4-bromophenyl)- lH-imidazol-2-amine, Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)octane-l,8- diamine, N-octylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine ,Nl-(5-phenyl- lH-imidazol-2-yl)dodecane-l,12-diamine, Nl-(5-phenyl-lH-imidazol-2- yl) penta ne-l,5-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)heptane-l,7-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)decane-l,10-diamine, Nl-(5-phenyl-lH-imidazol- 2-yl)undecane-l,ll-diamine.

[0114] In another preferred embodiment, said composition comprises one or more excipients. In another preferred embodiment, said excipient is a solvent for said compound. In an even more preferred embodiment, said solvent is selected from the group comprising dimethylformamide, tetrahydrofuran, acetonitrile, dichloromethane, N-methylpyrrolidone, acetone, chloroform, and dimethylsulfoxide.

[0115] Typically, the compositions of the present invention as described herein may be co-administered or co-applied with one or more other antibacterial agents.

[0116] The compositions and methods of this invention are especially useful for treating or preventing a pathologic condition associated with a microbial infection or for decreasing bacterial growth in an animal or a human in need of such treatment. The compositions and methods of this invention are especially useful for treating a human with a wound selected from the group consisting of an ulcer, a laceration, a deep penetrating wound and a surgical wound.

[0117] The compositions and methods of this invention are also useful for reducing the risk of bacterial infection or sepsis in a person colonized with pathogenic bacteria. This is especially relevant to immuno-compromised patients affected with leukaemia, lymphoma, carcinoma, sarcoma, allogenic transplant, congenital or acquired immunodeficiency, cystic fibrosis, and AIDS.

[0118] The compositions and methods of this invention are especially useful for reducing or eradicating the risk of bacterial infection in a human, wherein the pathogenic bacteria are gram positive bacteria selected from the group consisting of pneumococcal species, methicillin-resistant Staphylococcus aureus, S. epidermidis, S. hominis, S. haemolyticus, S. capitis, S. warneri, multi-drug resistant, Streptococcus spp., Enterococcus spp., Propionibacterium acnes.

[0119] The compositions and methods of this invention are also useful for reducing or eradicating the risk of bacterial infection in a person, wherein the pathogenic bacteria are gram negative bacteria selected from the group consisting of Salmonella, e.g. S. Typhimurium, S. enteritidis, S. arizonae, S. bongori, S. choleraesuis, S. choleraesuis, S. enterica, S. paratyphi, S. pullorum, S. subterranea, and S. typhi or Pseudomonas, e.g; a bacterium of the Pseudomonas aeruginosa group such as P. aeruginosa, P. aicaiigenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans or P. straminea, or Neisseria sp.. Hemophilus sp., Proteus sp., Klebsiella sp., Escherichia coli or other bacteria such as Serratia liquefaciens, Burkholdeia cepacia, Porphyromonoas gingivaiis or yeasts such as C. albicans.

[0120] The compositions and methods of this inventions are also useful for reducing or eradicating a biofilm consisting of a combination of any one of the bacterial and / or yeast species as described above.

[0121] Yet another embodiment of present invention is a process for imparting microbial control properties to a fluid composition, said process comprising adding an antimicrobial composition as defined hereinabove to said fluid composition. EXAMPLES

[0122] Materials and Methods

[0123] General procedure for the synthesis of substituted 2-aminoimidazoles.

[0124] The synthesis of aminated compounds of the present invention was adapted from [Steenackers et al. (2011) Bioorg Med Chem 19, 3462-3473] and performed according to the scheme in Figure 3B and the experimental procedure below.

[0125] A representative but not-limiting methodology is as follow. Chloro-pyrimidine (1 equiv) and alkyl diamine 1 (3 equiv) were dissolved in ethanol (2 ml / mmol) and triethylamine (1,5 equiv) was added. The mixture was stirred at 85° C for 16 hours. Afterwards the volatiles were removed in vacuo. The crude was dissolved in dichloromethane and washed with water and brine. The organic phase was dried over sodium sulphate. Boc-anhydride (2 equiv) was added portion wise and let react for 1 hour at 45° C. After this time the volatiles were evaporated, and the resulting residue was purified by column chromatography (silica gel; hepatane- ethylacetate 7:3 v / v) to afford the Boc-co-N-2-alkylaminopyrimidine 2 as a yellow liquid.

[0126] After the Boc-co-N-2-alkylaminopyrimidine (1 equiv) is dissolved in acetonitrile (4 ml / mmol) and a-bromoketone 3 is added. The mixture was let react for 3 hours at 85°C and hydrazine hydrate (7 equiv) is added. The reaction mixture was stirred for 1 hours at 85°C and then let cool down to room temperature. The crude is diluted with dichloromethane and washed with water three time and finally with brine. The organic phase is dried over sodium sulphate and the solvent evaporated under reduced pressure. The mixture was purified by column chromatography (silica gel; ethylacetate-methanol 85: 15 v / v) to afford the Boc-co-N-2-alkyl- aminoimidazole 4 as a dark yellow-orange liquid. Finally, the product was treated with HCI in dioxane 4N (10 equiv) under N2 atmosphere at 0° C. The mixture was let react at room temperature for 4 hours. Afterwards the volatiles were evaporated, and the crude was washed three times with acetone and dried under reduced pressure to afford Boc-co-N-2-alkyl-aminoimidazole HCI salt 5 as pale yellow-white powder.

[0127] N l-(5-phenyl-lH-imidazol-2-yl)butane-l,4-diamine dihydrochloride

[0128] XH NMR (400 MHz, DMSO-de) 6 13.03 (s, 1H), 12.72 (s, 1H), 8.27 - 8.13 (m, 3H), 7.97 (t, J = 6.1 Hz, 1H), 7.77 (d, J = 7.0 Hz, 1H), 7.55 - 7.45 (m, 2H), 7.43 (s, 1H), 7.35 - 7.30 (m, 1H), 7.16 - 7.08 (m, 1H), 3.45 (q, J = 6.5 Hz, 2H), 2.87 - 2.78 (m, 2H), 1.73 - 1.60 (m, 4H).13C NMR (101 MHz, DMSO-cfe) 6 148.18, 129.30, 128.37, 128.28, 127.19, 124.95, 110.05, 66.82, 42.63, 26.39, 24.55. N l-(5-(4-fluorophenyl)-lH-imidazol-2-yl)butane-l,4-diamine dihydrochloride

[0129] XH NMR (400 MHz, DMSO-c / e) 6 12.93 (s, 1H), 12.58 (s, OH), 8.18 - 7.99 (m, 3H), 7.81 (dd, J = 8.8, 5.4 Hz, 1H), 7.45 (s, 1H), 7.34 - 7.26 (m, 1H), 3.41 (d, J = 6.3 Hz, 5H), 2.82 (d, J = 6.3 Hz, 2H), 1.73 - 1.56 (m, 4H).13C NMR (101 MHz, DMSO- d&) 6 148.11, 127.27, 127.19, 126.43, 124.91, 124.88, 116.43, 116.21, 110.00,

[0130] 42.60, 26.28, 24.57.

[0131] N l-(5-phenyl-lH-imidazol-2-yl)pentane-l,5-diamine dihydrochloride

[0132] XH NMR (400 MHz, DMSO-c / e) 6 12.96 (s, 1H), 12.66 (s, 1H), 8.16 (d, J = 19.5 Hz, 3H), 7.96 (t, J = 6.1 Hz, 1H), 7.79 - 7.73 (m, 1H), 7.46 (d, J = 10.9 Hz, 1H), 7.43 (d, J = 1.4 Hz, 1H), 7.41 (s, 1H), 7.35 - 7.30 (m, 1H), 7.16 - 7.08 (m, 1H), 3.48 - 3.34 (m, 2H), 2.82 - 2.73 (m, 2H), 1.70 - 1.53 (m, 4H), 1.49 - 1.33 (m, 2H).13C NMR (101 MHz, DMSO-c / e) 6 148.25, 129.30, 128.36, 128.29, 127.15, 124.93, 110.04, 66.82, 43.00, 40.61, 40.40, 40.19, 39.98, 39.78, 39.57, 39.36, 31.17, 28.67, 26.87, 23.22.

[0133] N l-(5-phenyl-lH-imidazol-2-yl)hexane-l,6-diamine dihydrochloride

[0134] XH NMR (400 MHz, DMSO-c / e) 6 12.96 (s, 1H), 12.66 (s, 1H), 8.20 - 8.07 (m, 3H), 7.94 (t, J = 6.0 Hz, 1H), 7.78 - 7.73 (m, 1H), 7.49 - 7.42 (m, 2H), 7.41 (s, 1H), 7.35 - 7.29 (m, 1H), 7.13 (ddd, J = 10.0, 6.7, 2.4 Hz, 1H), 3.41 (q, J = 6.8 Hz, 2H), 2.76 (td, J = 8.4, 7.8, 4.1 Hz, 2H), 1.64 - 1.52 (m, 4H), 1.36 (dd, J = 6.9, 3.6 Hz, 4H).13C NMR (101 MHz, DMSO-cfe) 6 148.29, 129.30, 128.36, 128.28,

[0135] 127.60, 127.11, 124.91, 110.03, 66.82, 43.08, 29.12, 27.22, 25.92, 25.88.

[0136] N l-(5-(4-bromophenyl)-lH-imidazol-2-yl)hexane-l,6-diamine dihydro- chloride

[0137] XH NMR (400 MHz, Methanol-c / 4) 6 7.63 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.28 (s, 1H), 3.41 (t, J = 7.1 Hz, 2H), 2.97 (t, J = 7.7 Hz, 2H), 1.73 (d, J = 7.2 Hz, 4H), 1.51 (t, J = 3.7 Hz, 4H).13C NMR (101 MHz, Methanol-c / 4) 6 148.02, 131.92, 126.94, 126.73, 126.22, 121.86, 109.43, 42.81, 39.26, 28.58, 27.08, 25.83, 25.73.

[0138] N l-(5-(4-chlorophenyl)-lH-imidazol-2-yl)hexane-l,6-diamine dihydro- chloride

[0139] XH NMR (400 MHz, Methanol-c / 4) 6 7.64 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.27 (s, 1H), 3.41 (d, J = 7.1 Hz, 2H), 2.97 (t, J = 7.7 Hz, 2H), 1.78 - 1.69 (m, 4H), 1.56 - 1.47 (m, 4H).13C NMR (101 MHz, Methanol-c / 4) 6 147.97, 133.83, 128.89, 126.82, 126.32, 126.02, 109.35, 42.85, 39.28, 28.56, 27.03, 25.79, 25.70.

[0140] N l-(5-phenyl-lH-imidazol-2-yl)heptane-l,7-diamine dihydrochloride

[0141] XH NMR (400 MHz, DMSO-c / e) 6 12.96 (s, 1H), 12.65 (s, 1H), 8.12 (s, 3H), 7.92 (t, J = 6.0 Hz, 1H), 7.79 - 7.72 (m, 2H), 7.48 (s, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.35 - 7.28 (m, 1H), 3.40 (q, J = 6.6 Hz, 2H), 2.74 (d, J = 7.1 Hz, 2H), 1.57 (d, J = 6.5 Hz, 4H), 1.36 - 1.26 (m, 6H).13C NMR (101 MHz, DMSO-c / e) 6 148.29, 129.31, 128.36, 128.26, 127.10, 126.29, 124.89, 110.02, 66.82, 43.18, 29.23,

[0142] 28.60, 28.52, 27.30, 26.25.

[0143] Nl-(5-phenyl-lH-imidazol-2-yl)octane-l,8-diamine dihydrochloride

[0144] XH NMR (400 MHz, DMSO-c / e) 6 12.65 (s, 1H), 8.14 (s, 3H), 7.92 (t, J = 6.0 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.48 (s, 1H), 7.42 (t, J = 7.7 Hz, 2H), 7.32 (t, J 7.1 Hz, 1H), 7.13 (dd, J = 9.5, 5.2 Hz, 1H), 3.39 (q, J = 6.4 Hz, 2H), 2.73 (d, J

[0145] 6.9 Hz, 2H), 1.57 (dt, J = 13.5, 6.7 Hz, 4H), 1.30 (d, J = 9.5 Hz, 8H).13C NMR (101 MHz, DMSO-c / e) 6 148.28, 129.31, 128.96, 128.36, 128.26, 127.09, 126.29,

[0146] 124.89, 110.02, 66.82, 43.22, 29.31, 28.87, 28.84, 27.31, 26.30, 26.24.

[0147] N l-(5-(4-bromophenyl)-lH-imidazol-2-yl)octane-l,8-diamine dihydrochloride

[0148] XH NMR (400 MHz, Methanol-d4) 6 7.63 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.28 (s, 1H), 3.38 (t, J = 7.2 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H), 1.74 - 1.66 (m, 4H), 1.47 - 1.42 (m, 8H).13C NMR (101 MHz, Methanol-d4) 6 131.93, 126.21,

[0149] 121.89, 109.44, 42.94, 39.35, 28.81, 28.78, 27.20, 26.29, 26.04.

[0150] N l-(5-(4-chlorophenyl)-lH-imidazol-2-yl)octane-l,8-diamine dihydrochloride

[0151] XH NMR (400 MHz, DMSO-c / e) 6 13.04 (s, 1H), 12.67 (s, 1H), 8.12 (s, 2H), 7.96 (t, J = 6.0 Hz, 1H), 7.84 - 7.76 (m, 2H), 7.54 (s, 1H), 7.52 - 7.45 (m, 2H), 3.39 (q, J = 6.4 Hz, 2H), 2.77 - 2.67 (m, 2H), 1.55 (d, J = 7.0 Hz, 4H), 1.35 - 1.24 (m, 8H). 13C NMR (101 MHz, DMSO-c / e) 6 148.37, 132.67, 129.30, 127.28, 126.65, 126.08, 110.80, 66.82, 43.25, 29.31, 28.87, 27.32, 26.30, 26.24.

[0152] N l-(5-(4-fluorophenyl)-lH-imidazol-2-yl)octane-l,8-diamine dihydro- chloride

[0153] XH NMR (400 MHz, Methanol-d4) 6 7.63 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.28 (s, 1H), 3.38 (t, J = 7.2 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H), 1.74 - 1.66 (m, 4H), 1.47 - 1.42 (m, 8H).13C NMR (101 MHz, Methanol-c / 4) 6 131.93, 126.21, 121.89, 109.44, 42.94, 39.35, 28.81, 28.78, 27.20, 26.29, 26.04.

[0154] N l-(5-phenyl-lH-imidazol-2-yl)decane-l,10-diamine dihydrochloride

[0155] XH NMR (400 MHz, DMSO-c / e) 6 7.97 (s, 3H), 7.73 (d, J = 7 A Hz, 1H), 7.56 - 7.50 (m, 1H), 7.49 - 7.41 (m, 2H), 7.36 (s, 1H), 7.17 (q, J = 7.9, 6.1 Hz, 1H), 3.38 - 3.31 (m, 2H), 2.74 (q, J = 7.6, 6.9 Hz, 2H), 1.65 - 1.49 (m, 4H), 1.28 (s, 12H).13C NMR (101 MHz, DMSO-c / e) 6 126.31, 110.12, 66.82, 29.01, 27.41, 26.50, 26.32.

[0156] N l-(5-phenyl-lH-imidazol-2-yl)undecane-l,ll-diamine dihydrochloride

[0157] XH NMR (400 MHz, DMSO-c / e) 6 12.96 (s, 1H), 12.64 (s, 1H), 8.13 (s, 3H), 7.91 (t, J = 5.9 Hz, 1H), 7.79 - 7.72 (m, 1H), 7.48 (s, 1H), 7.46 - 7.36 (m, 2H), 7.35 - 7.28 (m, 1H), 7.17 - 7.07 (m, 1H), 3.44 - 3.32 (m, 2H), 2.72 (d, J = 6.6 Hz, 2H), 1.55 (t, J = 7.5 Hz, 4H), 1.26 (d, J = 9.8 Hz, 14H).13C NMR (101 MHz, DMSO- d&) 6 148.29, 130.33, 129.30, 128.35, 128.27, 127.09, 124.88, 110.01, 66.82, 43.23, 29.39, 29.34, 29.31, 29.28, 29.14, 29.01, 27.35, 26.45, 26.34, 26.32.

[0158] N l-(5-phenyl-lH-imidazol-2-yl)dodecane-l,12-diamine dihydrochlorideXH NMR (400 MHz, DMSO-c / e) 6 8.21 (s, 2H), 7.89 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 5.8 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.18 (s, 1H), 3.97 (t, J = 7.2 Hz, 2H), 2.07 - 1.93 (m, 2H), 1.73 (d, J = 2.4 Hz, 2H), 1.65 - 1.53 (m, 4H), 1.35 (t, J = 7.1 Hz, 2H), 1.18 (s, 2H), 1.03 (d, J = 15.9 Hz, 6H), 0.81 (t, J = 7.2 Hz, 4H).13C NMR (101 MHz, DMSO-c / e) 6 147.19, 135.07, 129.63, 129.36, 127.98, 113.21, 55.18, 42.74, 32.54, 31.50, 28.76, 28.48, 27.94, 25.67, 23.87, 22.47, 14.38.

[0159] Coupling of biofilm inhibitors with PEG chain

[0160] To increase the water solubility and chain length, the biofilm inhibitors were first coupled to a PEG chain. The COOH functionality of the PEG chain was coupled with the amine functionality of the inhibitors using 1,1-carbonyldiimidazole. This molecule is frequently used in organic synthesis for the introduction of carbonyl groups and the coupling of amino acids resulting in polypeptides. In general, a modified version of hydroxy polyethoxy allyl ether with a COOH end group 8 (0.0512g, 0.10 mmol) was added to a mixture of 1,1-carbonyldiimidazole (0.0178 g, 0.11 mmol) and 1 ml dichloromethane. The resulting solution was placed on a shaker for 5 min. Afterwards, 0.10 mmol of compound 17 was added to the solution and shaken overnight at rt. Finally, the solution was dried in vacuo at 80°C and PEG chain coupled compound 17 was obtained as a yellow oil. The coupling was confirmed by Fourier transform infrared (FT-IR) measurements.

[0161] Functionalization of iron oxide nanoparticles with biofilm inhibitors and stabilizing ligands

[0162] To functionalize the nanoparticle surface with the PEG functionalized inhibitor, thiol-ene click chemistry was used in which a thiol silane is connected to the allyl end group of our PEG chain by hydrothiolation. In this way, silanol molecules could be formed by hydrolysis of alkoxysilanes and covalently bonded to the nanoparticle surface with a very high density. However, to ensure that our nanoparticle is water dispersible, other stabilizing ligands were added to the surface. A ratio of 20% allylPEG-5-Ar-2AIs and 80% hydroxypolyethoxy allyl ether (=allylPEG-OH) seems to be the maximum inhibitor loading to ensure water dispersibility and efficiency of the nanoparticle. 10% allylPEG-5-Ar-2AIs is taken as an example in the synthesis protocol.

[0163] In general, allylPEG-5-Ar-2AIs (0.05 mmol, 1 eq) was added to a mixture of allylPEG-OH (0.224 g, 0.45 mmol, 4.5 eq), 2,2-dimethoxy-2-phenylacetophenone (6.4 mg, 0.025 mmol) and 3-mercaptopropyl trimethoxysilane (0.093 ml, 0.5 mmol) in 0.5 ml chloroform, leading to a mixture of 20% PEG-5-Ar-2AIs and 80% PEG-OH siloxanes. This mixture was placed in a UV chamber, equipped with 3 LEDs (365 nm, output power 200 mW), for 1 h on top of a stirring plate.

[0164] The developed silanes were then mixed with 50 mg iron oxide nanoparticles, 0.025 ml water, 1.25 ml triethylamine and 25 ml toluene. This solution was placed in the ultrasonic bath for 5 h at a temperature of 50°C.

[0165] Afterwards, the solution was placed on a magnet to precipitate the functionalized particles and the precipitate was washed three times with heptane and acetone. Finally, the functionalized nanoparticles were dispersed in water with a concentration of 5 mg / ml. The functionalization was confirmed by FT-IR. The loading of the biofilm inhibitors on the nanoparticle surface was confirmed by CHN element analysis. Chemistry: reagents and analysis. All solvents and reagents were purchased from commercial sources and were used without prior purification. TLC analysis was performed on aluminium backed plates. The products were purified by silica gel (200-300 mesh) column chromatography. All NMR spectra were recorded on a Bruker Avance III HD 400 spectrometer at 400 MHz (XH) and at 101 MHz (13C). TheXH and13C chemical shifts are reported in parts per million relative to tetramethylsilane using the residual solvent signal as the internal reference. The following abbreviation were used to designate chemical shift multiplicities: s=singlet, d=doublet, dd=doublet of doublets, t=triplet, dt=doublet of triplets, q=quartet, p=pentet and m = multiplet. The13C NMR spectra are proton decoupled. 2-aminoimidazoles 1-10 were synthesized according to Steenackers et al. (2011) Bioorg Med Chem 19, 3462-3473.

[0166] Strains and growth media.

[0167] The strains Agrobacterium rhizogenes ST15.13 / 097, Salmonella enterica serovar Typhimurium ATCC1402837, P. aeruginosa PA14, Escherichia coli TGI, S. aureus SH1000 and a clinical Staphylococcus epidermidis strain, isolated from a patient with osteomyelitis were used in this study. Overnight cultures of A. rhizogenes ST15.13 / 097, S. Typhimurium ATCC1402837, P. aeruginosa PA14, Escherichia coli TGI, S. aureus SH1000 and S. epidermidis were grown with aeration in lysogeny broth (LB) at 37 °C. Phosphate-buffered saline (PBS) was prepared by combining 8.8 g liter 1 NaCI, 1.24 g liter 1 K2HP04, and 0.39 g liter KH2P0 4 (pH 7.4).

[0168] Anti-biofilm assay: inhibition of bacterial biofilms.

[0169] A static peg assay [Steenackers et al. (2011) Bioorg Med Chem 19, 3462-3473a; Steenackers et al. (2014) Molecules 19, 16707-16723], was used for bacterial biofilm formation. The Calgary Biofilm Device consists of a platform carrying 96 polystyrene pegs (Nunc no. 445497) that fits as a microtiter plate lid with one peg hanging into each microtiter plate well (Nunc no. 269789). Twofold serial dilutions of the compounds or nanoparticles (dissolved in 100% DMSO) in 100 pl liquid broth (TSB diluted 1 / 20 or TSB) per well were prepared in the microtiter plate in duplicate or triplicate, with a maximum concentration of 1600 pM for inhibitors or 1600 pg / ml for nanoparticles and a minimum concentration of 0.8 pM for inhibitors or 0.8 pg / ml for nanoparticles. Subsequently, an overnight culture of P. aeruginosa PA14, Escherichia coli TGI, S. aureus SH1000 or S. epidermidis was diluted 1 : 100 into TSB 1 / 20 (for P. aeruginosa PA14 and Escherichia coli TGI) or TSB (for S. aureus SH1000 and S. epidermidis / Next 100 pl was added to each well of the microtiter plate, resulting in a total volume of 200 pl medium per well (final concentration of compounds ranges from 800 mM or pg / ml (2% DMSO) to 0.4 mM or pg / ml (0.001% DMSO)). In the next step, the pegged lid was placed on the microtiter plate and the plate was incubated for 24h at 25°C (for P. aeruginosa PA14 and Escherichia coll TGI) or 48 h at 37 C (for S. aureus SH1000 and S. epidermidis)' without shaking. At 37 °C the plates were placed in a sealed container with wet towels on the bottom to prevent evaporation of the growth medium. During this incubation period, biofilms were formed on the surface of the pegs. After incubation, the optical density at 600 nm (ODeoo) was measured for the planktonic cells in the microtiter plate using a Synergy MX multimode reader (Biotek, Winooski, VT). This gives a first indication of the effect of the compounds on the planktonic growth. For quantification of biofilm formation, the pegs were washed once in 200 pl PBS. The remaining attached bacteria were stained for 30 min with 200 pl 0.1% (w / v) crystal violet in an isopropanol / methanol / PBS solution (v / v 1 :1 : 18). Excess stain was rinsed off by placing the pegs in a 96-well plate filled with 200 pl distilled water per well. After air drying the pegs (30 min), the dye bound to the adherent biofilm was extracted with 30% glacial acetic acid (200 pl per well of a 96-well plate). The optical densities at 570 nm (OD570) of each well was measured using a Synergy M X multimode reader (Biotek, Winooski, VT). The BICso and IC50 values for each compound or nanoparticle were determined from the concentration gradient by using nonlinear curve fitting (GraphPad Prism 10; Graphpad Software, Inc., La Jolla, CA). BICso is defined as the concentration of compound or nanoparticle needed to inhibit biofilm formation by 50%. In the same assay the effect on planktonic growth was evaluated. ICso is defined as the concentration of compound or nanoparticle needed to inhibit planktonic growth by 50%. Data represent the means of at least 3 repeats with three replicates for each repeat.

[0170] Magnetic forces during S. Typhimurium biofilm growth

[0171] In order to determine the effect of magnetic forces on the inhibitory effect of the nanoparticles on the number of living cells in the Salmonella biofilm, the colony forming units (CFU) per cm2biofilm surface were counted, both in absence and presence of a magnet underneath the surface. A concentration range of nanoparticles in 100 pl TSB diluted 1 / 20 per well was prepared in a microtiter plate in triplicate. Subsequently, an overnight culture of S. Typhimurium ATCC14028 was diluted 1: 100 into TSB 1 / 20. Next 100 pl was added to each well of the microtiter plate, resulting in a total volume of 200 pl medium per well. In the next step, a lid was placed on the microtiter plate and it was incubated for 24 h at 25°C without shaking. During this incubation period, a magnet of 1.3 Tesla was placed underneath the microtiter plate wherein biofilms were grown. After incubation the planktonic phase was removed and tenfold dilution series were made in PBS. Next, 200 pl fresh PBS was added to the wells containing the biofilm and cells were scraped from the bottom with a pipet tip. Afterwards, the cells were passed 5 times through a 25 gauge syringe to break up bacterial clumps and tenfold dilution series were made in PBS. Both the planktonic and the biofilm dilution series were plated out on LB agar plates, and after overnight incubation at 37°C, respectively CFU / ml and CFU / cm2, were determined.

[0172] Magnetic nanoparticle heating

[0173] To determine the effect of magnetic heating of iron oxide nanoparticles on the inhibition of S. Typhimurium ATCC14028 biofilms, an in-house developed magnetic setup was used. First, an overnight culture of S. Typhimurium ATCC14028 was diluted 1 : 100 in 0.92 ml TSB diluted 1 / 20 that was poured into the cap of a closed 15 mL Greiner Bio-One plastic falcon tube by turning the tube upside down. 0.08 mL of nanoparticle stock solution was added to obtain a final concentration of 400 pg / ml nanoparticles. The tube was incubated at 25°C for 48h without shaking and biofilm was formed on the cap of the tube. After incubation, the tube was placed in the center of the copper coil (diameter 2 cm) of the magnetic set-up onto which an AC magnetic field was applied (200 Gauss, 240 kHz) for 30 min. Next, the top of the plastic tube was removed from the cap and the cap was washed with 1 ml PBS to remove the cells that were not in the biofilm. Then, the biofilm was scraped off from the cap in 1 ml PBS and passed 5 times through a 25 gauge syringe to break up bacterial clumps. Tenfold dilution series were made in PBS and both the planktonic and the biofilm dilution series were plated out on LB plates. Finally, after overnight incubation at 37°C, CFU / cm2were determined.

[0174] Checkerboard assay

[0175] In order to determine possible synergistic interactions between conventional antibiotics on one hand and the novel 2N-polar substituted 5-Ar-2AIs on the other hand, a checkerboard assay was used. To a combination of antibiotics (Erythromycin, Chloramphenicol, Cefuroxime, Amoxicillin, Moxifloxacin, Gentamicin and Minocycline) and a 2N-polar substituted 5-Ar-2AI (Nl-(5-phenyl- lH-imidazol-2-yl)heptane-l,7-diamine) two-fold diluted across rows and columns of a microplate respectively, a bacterial suspension of approximately 5*105CFU / mL was added. After 24 h of incubation at 37° C., growth of the bacteria was quantified by measuring the ODsgsnm. Synergism was determined by FICI (fractional inhibitory concentration index) calculations. The FICI was calculated by the formula FICI = ICc-A / ICm-A + ICc-B / ICm-B. ICC-A and ICC-B are the IC values of the azole or quaternary ammonium compound in combination and ICm-A and ICm-B are the IC values of the antibiotic or 2N-polar substituted 5-Ar-2AIs A and B alone. IC stands for growth inhibition concentration which is the minimal concentration of a compound that causes complete growth inhibition. The interaction was defined as synergistic for a value of FICI<1.

[0176] Horticulture pilot plant

[0177] One 2N-polar substituted 5-Ar-2AI (Nl-(5-(4-chlorophenyl)-lH-imidazol-2- yl)octane-l,8-diamine) was tested in a horticulture pilot plant to evaluate the efficacy in Agrobacterium rhizogenes removal, a plant pathogen known to cause hairy root disease in tomatoes. The pilot plant consisted of four separate water distribution systems, each with their own water reservoir, pumping installation and tubing. Each water distribution system provided water to 18 tomato plants (Marinice-Maxifort) on rockwool substrates via a sprinkling system. After watering the plants, the water was recollected and re-used. All plants were housed in the same room under comparable environmental conditions. The four water distribution systems were used as following : (i) negative control: no induced Agrobacterium infection, no treatment; (ii) positive control: induced Agrobacterium infection, no treatment; (iii) 5-Ar-2AI treatment: induced Agrobacterium infection, treatment with 100 pM of the 5-Ar-2AI compound at the start and addition of 50pM every three weeks; (iv) 5-Ar-2AI + H2O2 treatment: induced Agrobacterium infection, treatment with 100 pM of the 5-Ar-2AI compound at the start and addition of 50pM every three weeks and continuous addition of H2O2 to a constant concentration of 50ppm. To induce an infection of Agrobacterium, 103CFU / mL of the bacterium was added to the system. The system ran for 22 weeks. Every two weeks, plants were visually evaluated for the presence of hairy roots and scored a value between one (no hairy roots) and five (100% hairy roots). A score above three is considered as an infected plant. Results are expressed as percentage of plants with hairy roots symptoms. Example 1. Novel compounds have higher activity and broader activity spectrum compared to corresponding non-substituted 2-AI compounds.

[0178] We tested ten novel anti-biofilm compounds that have a polar group substituted on the carbon chain on the 2N position of the 5-Ar-2AIs (Figure 2) and compared their activity to the corresponding 2N-substituted 5-Ar-2AIs without a polar constituent (Figure 1), by using a crystal violet assay. BICso is defined as the concentration of compound needed to inhibit biofilm formation by 50%.

[0179] The preventive anti-biofilm activity of the novel 2N-polar substituted 5-Ar-2AIs was evaluated against a panel of Gram-positive and Gram-negative bacterial biofilms. Interestingly, as indicated in Figure 4, all novel compounds showed lower BICso, values, and thus better anti-biofilm activity, compared to the corresponding 2N-substituted 5-Ar-2AIs (without a polar constituent). At low carbon chain lengths on the 2N position, compounds without a polar constituent show no or very poor activity against Staphylococci and longer chain lengths are needed to inhibit these bacterial biofilms. On the contrary, a reverse relationship between the carbon chain length at the 2N-position at the anti-biofilm activity against P. aeruginosa is observed for the compounds without polar constituents, e.g. compounds with longer chain lengths show lower activity against P. aeruginosa. For the novel compounds with a polar constituent on the 2N carbon chain - in this case an amine functional group - all compounds are active against all bacterial biofilms tested (E. coli, P. aeruginosa, S. aureus and S. epidermidis). Although, a positive increase in activity is observed in function of 2N-carbon chain length.

[0180] We tested four additional compounds that have a polar group substituted on the carbon chain on the 2N position of the 5-Ar-2AIs. These compounds also showed broad spectrum anti-biofilm activity against all bacteria tested (E. coli, P. aeruginosa, S. aureus and S. epidermidis) as shown in Figure 5.

[0181] Example 2. NP-coupled 2-AIs show higher anti-biofilm activity and higher biofilm specificity compared to the dissolved 2-AI inhibitor.

[0182] First, the different inhibitors covalently attached to the iron oxide nanoparticles are evaluated against biofilm mass production and planktonic growth of a broad panel of bacteria, by means of crystal violet staining in the Calgary Biofilm Device. Both nanoparticles with 10% and 20% PEG functionalized inhibitor are evaluated. As shown in Figure 7, nanoparticles with 10% compound 17 are very active against E. coli biofilms (BIC50 is 1.9 pg / ml). They also have a moderate antibiofilm activity against S. Typhimurium (BICso is 38.0 pg / ml) and to a lesser extent against P. aeruginosa and S. aureus (BICso is 102.9 pg / ml and 109.0 pg / ml respectively). If the amount of compound 17 on the surface of the nanoparticles is increased to 20%, the nanoparticles are very active against S. Typhimurium, E. coli and P. aeruginosa (BICso between 1.2 and 10.2 pg / ml), and to a lesser extent to the Gram-positives S. aureus and S. epidermidis (BICso is 41.7 and 70.5 pg / ml respectively). Overall, these results indicate that increasing the ratio of PEGylated inhibitor on the surface from 10 to 20% in general strongly enhances the activity. In general the activity spectra of the inhibitors coupled to the nanoparticles turn out to be more narrow than the inhibitors in solution, which are highly active against both Gram-negative and Gram-positive bacteria. A remarkable finding is that the BICso (pg / ml) of the nanoparticles with 20% PEGylated inhibitor against several species is similar or lower than the BICso (pg / ml) of the dissolved inhibitor. Given the fact the majority of the mass of the nanoparticles is constituted by the iron oxide core and the PEG chains, this indicates that the same anti-biofilm activity is achieved with a much lower overall concentration of biofilm inhibitor. Consequently, the functionalized nanoparticles actually show a relatively better activity since less inhibitor is present per volume unit. This could be explained by the high surface to volume ratio of nanoparticles and mass loading of antibiofilm compounds which significantly increases the local inhibitor concentration. Alternatively, a synergistic effect between the iron oxide nanoparticles and the 2- aminoimidazole biofilm inhibitors is possible. Another striking finding is that the biofilm-specificity of the inhibitors seems to be enhanced when covalently bound to the nanoparticle surfaces. The ICso values are generally much higher compared to those of the inhibitors in solution. Overall, these results confirm that covalent attachment to the nanoparticles enhances the anti-biofilm activity and biofilmspecificity of the 5-Ar-2-AIs compared to the 5-Ar-2-AIs in solution.

[0183] Example 3. Effect of magnetic enrichment of functionalized nanoparticles at the surface on S. Typhimurium biofilm inhibition

[0184] To assess the effect of magnetic forces on S. Typhimurium biofilms, a magnet of 1.3 Tesla is placed underneath the microtiter plate wherein biofilms are grown on the bottom of the wells, as described in Material & Methods (section 'Magnetic forces during S. Typhimurium biofilm growth'). Concentrations with little or no inhibitory effect against biofilm cells in the absence of the magnet, are tested in order to determine the effect of concentrating the nanoparticles (functionalized with 10% PEG-5-Ar-2-AIs) at the surface in a preventive manner, in comparison to a biofilm where no magnetic forces are applied. Figure 8 shows that the inhibitory effect of the functionalized nanoparticles on the biofilm can be enhanced when a magnet is placed underneath the growing biofilm. Especially at a concentration of 25 pg / ml a significant (P < 0.001) reduction of biofilm cells is observed when the magnetic field is applied, probably by enrichment of nanoparticles at the biofilm prone surface. In conclusion, proof of concept is delivered for the possibility to magnetically concentrate iron oxide nanoparticles functionalized with biofilm inhibitors at the surface to achieve increased biofilm inhibition.

[0185] Example 4. Effect of magnetic particle heating of nanoparticles in combination with magnetic enrichment on S. Typhimurium biofilm inhibition

[0186] Magnetic nanoparticles can locally produce high temperatures in the presence of an applied AC magnetic field. Here we assess the effect of this magnetic nanoparticle heating as well as the combination with coupled biofilm inhibitors on S. Typhimurium biofilms. As described in Materials & Methods, biofilms are first grown in a tube for 48 hours after which the tube is placed in the center of a copper coil onto which an AC magnetic field is applied for 30 min. The percentages of biofilm cells treated from the start with magnetic nanoparticles functionalized with 0% (100% PEG-OH) and 10% PEG-5-Ar-2-AIs are determined as compared to an untreated control (Figure 9). A direct comparison is made with biofilms to which the AC magnetic field is not applied. Figure 9 indicates that the magnetic particle heating only offers a small additional inhibitory effect both in case of the PEG-OH functionalized iron oxide nanoparticles and the PEG-INH1 functionalized nanoparticles.

[0187] The effect of particle heating can potentially be increased when the nanoparticles are enriched at the biofilm prone surface by means of magnetic forces underneath the biofilm, as described in the previous section. To validate this, a magnet of 1.3 Tesla is placed underneath the tubes in which the biofilms are grown prior to the magnetic particle heating treatment. A further decrease in CFU / mL is observed using this combination in comparison to the biofilms where only magnetic heating was used, both for the nanoparticles with 0% PEG-5-Ar-2-AIs (100% PEG-OH) and 10% PEG-5-Ar-2-AIs. This leads to an increase of more than 10% biofilm inhibition for the nanoparticles functionalized with 0% 5-Ar-2-AIs (100% PEG-OH) and 10% PEG-5-Ar-2-AIs, in comparison to the biofilm where no magnetic heating nor magnetic forces are used (Figure 9). This effect can potentially be further improved by optimizing the concentration of magnetic nanoparticles, magnetic field intensity and heating time. In summary, magnetic particle heating in combination with magnetically concentrating of iron oxide nanoparticles at the surface can be a promising tool to use in combination with inhibitors of biofilm formation.

[0188] Example 5: 2N polar substituted 5-Ar-2-AIs show synergistic activity with antibiotics

[0189] The bars in Figure 10 represent the FIC indices for the different combinations. Synergy (FIC <1) was observed for the combinations of conventional antibiotics of different antibiotic classes (Erythromycin (ERY), Chloramphenicol (CHL), Cefuroxime (CXM), Amoxicillin (AX), Moxifloxacin (MOX), Gentamicin (GEN) and Minocycline (MIN)) with a 2N-polar substituted 5-Ar-2AI (Nl-(5-phenyl-lH- imidazol-2-yl)heptane-l,7-diamine) and more particularly for combinations of 2- AI and ERY in a ratio of 73:1, 2-AI and CHL in a ratio of 30: 1, 2-AI and CXM in a ratio of 50: 1, 2-AI and AX in a ratio of 9:1, 2-AI and MOX in a ratio of 2509: 1, 2- AI and GEN in a ratio of 7: 1 and 2-AI and MIN in a ratio of 63: 1.

[0190] Example 6: 2N polar substituted 5-Ar-2-AIs can inhibit plant pathogens in irrigation water systems of horticulture plants.

[0191] We tested a 2N polar substituted 5-Ar-2-AI compound (Nl-(5-(4-chlorophenyl)- lH-imidazol-2-yl)octane-l,8-diamine) against the plant pathogen Agrobacterium rhizogenes , which causes hairy roots disease in tomato plant, in a pilot irrigation water system for horticulture tomato plants. The addition of 100 pM 2N polar substituted 5-Ar-2-AI compound at the start of the trial and subsequent addition of 50 pM (every three weeks) completely reduces the percentage of plant pathogens with symptoms of hairy roots disease (Figure 11).

Claims

CLAIMS1. A substituted 5-aryl-2-aminoimidazole represented by the structural formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or polymorphic form thereof,wherein,R1 is H,R2 is a C2-20 alkyl substituted with a polar group selected from the group consisting of an amine, an alcohol, an ester, an amide, reverse amide, carboxylic acid, and a thiol, R3 is a halogen or hydrogen, R4 is a halogen or hydrogen.

2. The compound according to claim 1, wherein the polar group is an amine.

3. The compound according to claim 1 or 2, wherein R2 is (CH2)2-2o-NH2.

4. The compound according to claim any one of claims 1 to 3, wherein R4 isBr or F.

5. The compound according to any one of claims 1 to 4, wherein R.2 is (C H2)2-i6-N H 2, or wherein R.2 is (CH 2)2-i2- N H 2.

6. The compound according to any one of claims 1 to 5, wherein,R1 is H,R2 is (CH2)4-i2-NH2,R.3 is hydrogen,R4 is hydrogen or a halogen.

7. The compound according to any one of claims 1 to 6, selected from the group consisting of:Nl-(5-phenyl-lH-imidazol-2-yl)butane-l,4-diamine, N-butylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine, Nl-(5-phenyl-lH-imidazol-2-yl)hexane-l,6-diamine , N-hexylamine-5-(4-bromophenyl)-lH-imidazol-2-amine, Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)hexane-l,6-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)octane-l,8-diamine , N-octylamine-5-(4-bromophenyl)-lH-imidazol-2-amine, Nl-(5-(4-chlorophenyl)-lH-imidazol-2-yl)octane-l,8-diamine, N-octylamine-5-(4-fluorophenyl)-lH-imidazol-2-amine, Nl-(5-phenyl-lH-imidazol-2-yl)dodecane-l,12-diamine, Nl-(5-phenyl-lH-imidazol-2-yl) penta ne-l,5-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)heptane-l,7-diamine, Nl-(5-phenyl-lH-imidazol-2-yl)decane-l,10-diamine and Nl-(5-phenyl-lH-imidazol-2-yl)undecane-l,ll-diamine.

8. A composition comprising a compound according to any one of claims 1 to 7, for use in the treatment of a gram-negative or gram-positive bacterial infection.

9. The composition for use according to claim 8, wherein the bacterial infection is an infection of P aeruginosa or S. aureus.

10. The composition for use according to claim 8 or 9, wherein the bacterial infection is a biofilm.

11. A substituted 5-aryl-2-aminoimidazole according to any one of claims 1 to7 for use as a medicament.

12. An in vitro use of a composition comp rising a compound according to any one of claims 1 to 7, for preventing or treating planktonic bacterial growth, or for preventing or treating a bacterial biofilm.

13. A surface or particle coated with a substituted 5-aryl-2-aminoimidazoles according to any one of claims 1 to 7.

14. The coated surface or particle according to claim 13, wherein the coating occurs via the NH2 group of R.2.

15. The coated particle according to claim 13 or 14, which is a magnetic particle.

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