COMPOUNDS, COMPOSITIONS AND METHODS FOR CONTROLLING

BIOFILMS

The present invention relates to substituted 2-aminoimidazoles and their imidazo[l,2-a]pyrirnidinium salts precursors being active against biofilm formation. The present invention also relates to antimicrobial compositions comprising a microbial biofilm formation inhibiting amount of such substituted 2-aminoimidazoles or imidazo[l,2-a]pyrimidinium salts in combination with excipients. The present invention also relates to methods for inhibiting or controlling microbial biofilm formation in a plant, a body part of a human or an animal, or a surface with which a human or an animal may come into contact.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions and methods for controlling biofilms and microbial, in particular bacterial, growth and for reducing bacterial colonization. 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 inhibitor 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. 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 present invention are in particular useful for preventing biofilm formation in a tissue to prevent or control a chronic bacterial infection or sepsis, and also useful for sanitation when applied to a substrate with which a human or an animal may come into contact. BACKGROUND OF THE INVENTION

Biofilms are defined in the art as structured communities or aggregates of microorganisms (e.g. bacterial cells) in which cells adhere to each other and/or to a living or inert (non-living) surface. These adherent cells are frequently embedded within 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 planctonic 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.

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 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.

Biofilms are known to provide cells with an array of advantages as compared with planktonic cells including the ability to resist challenges from predators, antibiotics, disinfectants and host immune systems. Biofilms offer a selective advantage to a microorganism to ensure its survival, or allow it a certain amount of time to exist in a dormant state until suitable growth conditions arise which for instance provide bacteria protection from antibiotics and from a host's immune system. This causes serious problems and costs in medical, hospital and industrial settings.

Biofilms are believed to be involved in 80% of human bacterial infections such as periodontitis, gingivitis, urethritis, endocarditis, benign prostatic hyperplasia, chronic prostatitis, biliary tract infections, urinary tract infections, cystitis, lung infections, sinus infections, ear infections (e.g. otitis media, otitis externa), acne and other skin infections, rosacea, dental caries, dental plaque formation, nosocomial infections, open wounds, chronic wounds and device-associated infections. There is thus a need in the art to control biofilm formation. A biofilm inhibitor may be useful to clear the way for a bactericide to penetrate the affected cells and eradicate the infection or it can provide an alternative treatment approach for certain types of infections.

Biofilms can contribute to contaminating food (food industry), decreasing flow through pipelines by colonization of the pipe interior or mild steel corrosion (oil industry), initiating biofouling on vessel hulls (shipping industry), and infesting hospitals, in particular surgery rooms and medical devices used therein. Biofilm formation in combination with mineral deposition may reduce heat transfer in water based cooling plants There is thus also a need in the art for compounds and methods for preventing biofilm formation regulated mineral deposition. Valuable plants such as those producing fruits and vegetables, forestry crops, corn, cotton, rice, soybeans and wheat are affected in a deleterious manner by biofilm formation and hence need protection from biofilm formation.

US 2003/100593 teaches, but without biological evidence, that certain substituted

2-aminoimidazoles are useful for treating various diseases including cardiovascular disorders, Kaposi sarcoma, cancer, bacterial infections, migraine, allergy and osteoporosis.

WO 2009/070304 teaches 2-aminoimidazoles useful for controlling biofilms and bacterial infections in plants. WO 2009/123753 teaches 2-aminoimidazoles useful for inhibiting bacterial biofilms.

Organic Letters (2006) 8:5781-4 and J. Org. Chem. (2008) 73:6691-7 disclose a few imidazo[l,2-a]pyrimidinium salts as precursors of substituted 2-aminoimidazoles, but do not attribute any utility to these compounds.

One object of the present invention is to address one or more of the above listed needs by providing efficient agents for reducing, controlling or inhibiting biofilm formation, such agents being either specifically acting on biofilm formation without influencing the planktonic growth of microorganisms, or being acting both on biofilm formation and planktonic growth in the same concentration range. SUMMARY OF THE INVENTION

The present invention relates to a broad group of substituted imidazo[l,2- a]pyrimidinium salts, substituted 2-aminoimidazoles and substituted 2-amino- imidazolines, most of them being unknown in the literature, which exhibit outstanding utility in controlling microbial biofilm formation, especially against a wide range of bacteria, and therefore can be formulated into anti-microbial compositions for administration to humans and animals and for application to plants and to inert surfaces susceptible to infection by microbial biofilms.

A first aspect of the present invention thus relates to a group of substituted imidazo[l,2-a]pyrimidinium salts and substituted 2-aminoimidazoles which are not known in the literature. Within this aspect the present invention specifically relates to a compound as defined in claim 1.

In view of the biological properties that have been evidenced in these compounds, a second aspect of the present invention relates to anti-microbial compositions comprising a biologically effective amount of a substituted imidazo[l,2-a]pyrimidinium salt or a substituted 2-aminoimidazole such as defined in the first aspect of the invention hereinabove, or a compound of the same group being known from the literature but without an associated utility, or a substituted 2-aminoimidazoline known from the literature but without an associated utility. Within this second aspect the present invention relates to an antimicrobial composition as defined in claim 9.

A third aspect of the present invention relates to the use of a substituted imidazo[l,2-a]pyrimidinium salt, a substituted 2-aminoimidazole or a substituted 2- aminoimidazoline as defined in the second aspect hereinabove as an inhibitor of microbial biofilm formation. Depending upon the target microbe, the use may be for inhibiting bacterial biofilm formation, or protozoal biofilm formation, or fungal biofilm formation, or algal biofilm formation. Although in most situations the compounds of this invention are only acting on microbial biofilm formation, some of them may also influence the planktonic growth of microorganisms in the same or a similar concentration range. Thus the present invention also relates to a method as defined in claim 24, i.e. making use of an anti-microbial composition as defined in the second aspect of the invention.

Each main aspect of the present invention will now be described with reference to a certain number of specific embodiments and working examples. Terms used in claims 1, 9 and 24 defining the broadest concept for each main aspect of the present invention are defined below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of certain compounds of the invention on planktonic growth at the IC50 concentration for biofilm inhibition.

DEFINITIONS

The term "optionally substituted" indicates that the specified group is either unsubstituted, or substituted by one or more suitable substituents. A "substituent" as defined herein is a monovalent atom or group of atoms replacing a hydrogen atom on a hydrocarbon chain or cycle (ring) of an organic molecule, for example halogen, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloaliphatic, heterocyclo, aryl (in particular phenyl), heteroaryl, alkoxy, amino, amido, sulfhydryl, alkylthio, alkylsulfonyl, nitro, carbonyl, carboxy, amino-acid (both natural and synthetic) and peptido, or a divalent atom replacing two hydrogen atoms on the same carbon atom of a hydrocarbon chain, for instance oxo or thioxo. The number of admissible substituents depends upon the number of hydrogen atoms that can be replaced, thus the chain length, the type of substituent and parameters such as steric hindrance which are well known to the skilled person.

The term "aliphatic" as used herein refers to "Alkyl", "Alkenyl" or "Alkynyl". The term "alkyl" or "saturated aliphatic" as used herein, and unless otherwise specified, refers to a straight or branched hydrocarbon chain containing from 1 to 20 (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) carbon atoms, hence the notation C _1-20 alkyl. Representative examples of C _1-20 alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, ieri-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n- decyl, and the like. Such alkyl groups may optionally be substituted with one or more (e.g. 2, 3 or 4) substituents as defined hereinabove.

"Alkenyl," as used herein, refers to a straight or branched hydrocarbon chain containing from 2 to 20 carbon atoms, hence the notation C2-20 alkenyl, and containing at least one carbon-carbon double bond. Representative examples of "alkenyl" include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5- hexenyl, 2-heptenyl, 2-methyl-l-heptenyl, 3-decenyl, butadienyl, hexadienyl and the like. Such alkenyl groups may optionally be substituted with one or more (e.g. 2, 3 or 4) substituents as defined hereinabove.

"Alkynyl" as used herein, refers to a straight or branched hydrocarbon chain containing from 2 to 20 carbon atoms, hence the notation C2-20 alkynyl, and containing at least one carbon-carbon triple bond. Representative examples of C2-20 alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 1-butynyl and the like. Such alkynyl groups may optionally be substituted with one or more (e.g. 2, 3 or 4) substituents as defined hereinabove.

The term "cycloaliphatic", as used herein and unless otherwise specified, refers to a saturated or ethylenically unsaturated monocyclic or polycyclic hydrocarbon group containing from 3 to 12 (e.g. 4, 5, 6, 7, 8, 9 or 10) carbon atoms, which is not aromatic, hence the notations C _3-12 cycloalkyl (saturated) and C _3-12 cycloalkenyl (unsaturated). Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tricyclodecyl, cyclododecyl, adamantyl, nornornyl, 5,6-trimethylenenorborn-2-yl and cyclooctyl. Such cycloaliphatic groups may optionally be substituted with one or more (e.g. 2, 3 or 4) substituents as defined hereinabove. Representative examples of cycloalkenyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, cyclodecenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3- cycloheptadienyl, 1 ,5-cyclooctadienyl.

The term "heterocyclic", as used herein, refers to a saturated or ethylenically unsaturated but not aromatic monocyclic or polycyclic (e.g. bicyclic) ring system comprising at least one heteroatom preferably selected from the group consisting of nitrogen, oxygen and sulfur in at least one ring. Monocyclic heterocyclic ring systems are exemplified by any 3 to 8 (e.g. 4, 5 or 6 or 7) member ring containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of O, N, and S. A 5 member ring has from 0 to 2 double bonds, and a 6 member ring has from 0-3 double bonds. Depending upon the number of double bonds, the heterocyclic ring system may be heteroaromatic (see specific definition below) or not. Representative examples of monocyclic non-aromatic heterocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, 1,2-dioxane, 1,3-dioxane, 1,4-dioxane, 1 ,2-dithiane, 1,3-dithiane, 1,4-dithiane, imidazoline, imidazolidine, isothiazoline, isothiazolidine, isoxazoline, isoxazolidine, morpholine, oxadiazoline, oxadiazolidine, oxazoline, oxazolidine, piperazine, piperidine, pyrazine, pyrazoline, pyrazolidine, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, thiadiazoline, thiadiazolidine, thiazoline, thiazolidine, thiomorpholine, thiomorpholine sulfone, thiomorpholine sulfoxide, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl or cycloalkyl or heterocyclic group as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3- benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like.

"Aromatic" as used herein refers to a ring system having one or more aromatic rings, which may be homoaromatic or heteroaromatic.

"Homoaromatic" or "aryl" as used herein refers to an aromatic ring system in which no carbon atoms have been replaced with heteroatoms. The homoaromatic group can be unsubstituted or substituted with from 1 to 5 suitable substituents as defined hereinabove, and wherein two adjacent substituents may be linked to form a cycle such as methylenedioxy. Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and mono- and polysubstituted versions thereof. "Heteroaromatic" or "heteroaryl" as used herein refers to an aromatic ring system in which one or more carbon atoms have been replaced with heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur in at least one ring. Examples of heteroaryl include, but are not limited to, pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl, indolizinyl, triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, isothiazolyl, isoxazolyl, oxazolyl, dioxazolyl, pyrazolyl, tetrazinyl, tetrazolyl, thiadiazolyl, thiazolyl, triazinyl and benzo[b]thienyl. The heteroaryl group may be optionally substituted with one or more, e.g. 1 to 4, suitable substituents as defined hereinabove.

The term " C _1-20 alkoxy " as used herein refers to substituents wherein a carbon atom of a C _1-20 alkyl group (such as defined herein), is attached to an oxygen atom through a single bond such as, but not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, ieri-butoxy, pentoxy, 3-pentoxy, or rc-hexyloxy. The term " Ci_ ₂ o alkylthio " as used herein refers to substituents wherein a carbon atom of a C _1-20 alkyl group (such as defined herein), is attached to an sulfur atom through a single bond such as, but not limited to, methylthio, ethylthio, etc.

The term " aryloxy " as used herein refers to substituents wherein a carbon atom of an aryl group (such as defined herein), is attached to an oxygen atom through a single bond such as, but not limited to, phenoxy, naphthoxy, etc.

The term " arylthio " as used herein refers to substituents wherein a carbon atom of an aryl group (such as defined herein), is attached to an sulfur atom through a single bond such as, but not limited to, phenylthio,

As used herein and unless otherwise stated, the term halogen means any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.

The term " amino acid " as used herein refers to any " natural amino acid " (Alanine (ala), Arginine (Arg), Asparagine (asn), Aspartic acid (Asp), Cysteine (cys), Glutamine (gin), Glutamic acid (glu), Glycine (gly), Histidine (his), Hydroxylysine (Hyl), Hydroxyproline (Hyp), Isoleucine (ile), Leucine (leu), Lysine (lys), Methionine (met), Phenylalanine (phe), Proline (pro), Serine (ser), Threonine (thr), Tryptophan (trp), Tyrosine (tyr), Valine (val)) in D or L conformation, as well as to " non-natural (or synthetic) amino acids " (e.g., but not limited to, phosphoserine, phosphothreonine, phosphotyrosin, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, a-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, ieri-butylglycine, and amino-acids bearing sulfonic and/or phosphonic groups. This term also comprises natural and non-natural amino acids being protected at their carboxylic terminus, e.g. as a Ci- ₂ o alkyl, phenyl or benzyl ester or as an amide, such as for example, a mono-Ci-20 alkyl or di-(Ci- ₂ o alkyl) amide, or another suitable carboxy- protecting group are known to those skilled in the art e.g. from T.W. Greene, Protecting Groups In Organic Synthesis, Wiley, New York, (1981) and references cited therein, the content of which is incorporated herein by reference).

The term " peptide " as used herein refers to a sequence of 2 to 100 amino-acids as defined hereinabove.

The term "microorganism", as used herein, refers to unicellular or cell-cluster microscopic organisms including eukaryotes such as fungi and protists, and prokaryotes, especially microorganisms that are susceptible to cause a disease in humans, but excluding a virus or a prion. These microorganisms can be organized in the form of a biofilm, thus the term "microbial biofilm".

The term "sepsis", as used herein, refers to a systemic inflammatory response syndrome associated to an infection. Septic shock is characterized namely by (a) hypotension persisting despite adequate fluid resuscitation, and (b) abnormalities related to hypoperfusion or organ dysfunction.

DETAILED DESCRIPTION OF THE INVENTION

The first aspect of the present invention will now be described with respect to a number of specific valuable embodiments.

One specific embodiment of this aspect of the present invention relates to a substituted 2-aminoimidazole compound wherein R3 is as defined in claim 2, in combination with the aforementioned definitions of Rl, R2 and R4.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 2, and wherein Rl is hydrogen, in combination with the aforementioned definitions of R2 and R4.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 2, and wherein R4 is hydrogen, in combination with the aforementioned definitions of Rl and R2.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 2, and wherein Rl and R4 are hydrogen, in combination with the aforementioned definition of R2.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound wherein R2 is as defined in claim 3, in combination with the aforementioned definitions of Rl, R3 and R4.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 3, and wherein Rl is hydrogen, in combination with the aforementioned definitions of R3 and R4.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 3, and wherein R4 is hydrogen, in combination with the aforementioned definitions of Rl and R3.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 3, and wherein Rl and R4 are hydrogen, in combination with the aforementioned definition of R3.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound wherein R4 is as defined in claim 4, in combination with the aforementioned definitions of Rl, R3 and R2. Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 4, and wherein Rl is hydrogen, in combination with the aforementioned definitions of R2 and R3.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being represented by the structural formula (I), wherein each of Rl, R3 and R4 is hydrogen, in combination with the aforementioned definition of R2.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 2, wherein Rl and R4 are hydrogen, and wherein R2 is as defined in claim 3.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 2, wherein Rl is hydrogen, and wherein R4 is as defined in claim 4.

Another specific embodiment of the present invention relates to a substituted 2- aminoimidazole compound being as defined in claim 5.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being represented by the structural formula (II) as defined in claim 1 and wherein X ^" is an inorganic or organic anion, in combination with the aforementioned definitions of Rl, R2, R3, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being as defined in claim 6, in combination with the aforementioned definitions of Rl, R2, R3, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being as defined in claim 6, and wherein each of R6, R7 and R8 is hydrogen, in combination with the aforementioned definitions of Rl, R2, R3, R4 and R5.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt wherein Rl is as defined in claim 7, in combination with the aforementioned definitions of X ^" , R2, R3, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being as defined in claim 7, and wherein each of R6, R7 and R8 is hydrogen, in combination with the aforementioned definitions of X ^" , R2, R3, R4 and R5. Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt wherein R3 is as defined in claim 8, in combination with the aforementioned definitions of X ^" , Rl, R2, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being as defined in claim 8, and wherein each of R6, R7 and R8 is hydrogen in combination with the aforementioned definitions of X ^" , Rl, R2, R4 and R5.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being represented by the structural formula (II), wherein R4 is selected from the group consisting of hydrogen, methyl, benzyl, 4-methoxybenzyl, phenyl and 4-tolyl, in combination with the aforementioned definitions of X ^" , Rl, R2, R3, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being represented by the structural formula (II), wherein R4 is selected from the group consisting of hydrogen, methyl, benzyl, 4-methoxybenzyl, phenyl and 4-tolyl, and wherein each of R6, R7 and R8 is hydrogen, in combination with the aforementioned definitions of X ^" , Rl, R2, R3 and R5.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being selected from the group consisting of l-isopropyl-2-hydroxy-2- (4-methylphenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-iu m bromide, 1-cyclo- propyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -cyclopropyl-2-hydroxy-2-(4-bromophenyl)-2,3 -dihydro- 1 H-imidazo [ 1 ,2-a] - pyrimidin-4-ium bromide, l-butyl-2-hydroxy-2-(4-bromophenyl)-2,3-dihydro-lH- imidazo[l,2-a]pyrimidin-4-ium bromide, l-butyl-2-hydroxy-2-(4-fluorophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, 1 -butyl-2-hydroxy-2-(diphenyl- methyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, l-cyclopentyl-2- hydroxy-2-(4-biphenylyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimi din-4-ium bromide, 1- hexyl-2-hydroxy-2-phenyl-3-methyl-2,3-dihydro-lH-imidazo[l,2 -a]pyrimidin-4-ium bromide, l-hexyl-2-hydroxy-2-(4-nitrophenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin- 4-ium bromide, l-cyclohexyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imi dazo- [l,2-a]pyrimidin-4-ium bromide, l-cyclohexyl-2-hydroxy-2-(4-methylthiophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, 1 -cyclohexyl-2-hydroxy-2-(4- cyanophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, l-cyclohexyl-2- hydroxy-2-(4-methoxyphenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyr imidin-4-ium bromide,

1- cyclohexyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imida zo[l,2-a]pyrimidin-4- ium bromide, l-phenyl-2-hydroxy-2-(4-bromophenyl)-3-methyl-2,3-dihydro-lH - imidazo[l,2-a]pyrimidin-4-ium bromide, l-benzyl-2-hydroxy-2-(4-iodophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, l-benzyl-2-hydroxy-2-(4- nitrophenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, l-benzyl-2- hydroxy-2-(phenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, l-(2-(3- methoxyphenyl)ethyl)-2-hydroxy-2-(3,4-dichlorophenyl)-2,3-di hydro-lH-imidazo[l,2-a]- pyrimidin-4-ium bromide, 1 -(2-(3-methoxyphenyl)ethyl)-2-hydroxy-2-(4-biphenylyl)- 2,3-dihydro-lH-imidazo[l ,2a]pyrimidin-4-ium bromide, l-(2-(3-methoxyphenyl)ethyl)-

2- hydroxy-2-(4-nitrophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrim idin-4-ium bromide, 1- (2-(3,4-dimethoxyphenyl)ethyl)-2-hydroxy-2-(4-nitrophenyl)-2 ,3-dihydro-lH-imidazo-

[1 ,2-a]pyrimidin-4-ium bromide, 1 -(4-methoxybenzyl)-2-hydroxy-2-(4-nitrophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, l-(4-methoxybenzyl)-2-hydroxy-2- (4-bromophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, 1-piperonyl- 2-hydroxy-2-(4-fluorophenyl)-2,3-dihydro-lH-imidazo[l,2-a]py rimidin-4-ium bromide, l-piperonyl-2-hydroxy-2-(3,4-methylenedioxyphenyl)-2,3-dihyd ro-lH-imidazo[l,2-a]- pyrimidin-4-ium bromide, l-cyclododecyl-2-hydroxy-2-(3-nitrophenyl)-2,3-dihydro-lH- imidazo[l,2-a]pyrimidin-4-ium bromide, l-cyclododecyl-2-hydroxy-2-(4-nitrophenyl)- 2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, and l-(tricyclo[3.3.1.13,7]dec- l-yl)-2-hydroxy-2-(4-nitrophenyl)-2,3-dihydro-lH-imidazo[l,2 -a]pyrimidin-4-ium bromide.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being selected from the group consisting of l-hexyl-2-hydroxy-2- phenyl-2,3-dihydro-lH-irnidazo[l,2-a]pyrimidin-4-ium bromide, l-heptyl-2-hydroxy-2- phenyl-2,3-dihydro- lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -octyl-2-hydroxy-2- phenyl-2, 3-dihydro- 1 H-imidazo [ 1 ,2-a]pyrimidin-4-ium bromide, 1 -nonyl-2-hydroxy-2- phenyl-2,3-dihydro- lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -decyl-2-hydroxy-2- phenyl-2,3-dihydro-lH-irnidazo[l,2-a]pyrimidin-4-ium bromide, l-undecyl-2-hydroxy-2- phenyl-2,3-dihydro-lH-irnidazo[l,2-a]pyrimidin-4-ium bromide, l-dodecyl-2-hydroxy-2- phenyl-2,3-dihydro- lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -tridecyl-2-hydroxy-2- phenyl-2,3-dihydro- lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -tetradecyl-2-hydroxy- 2-phenyl-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, l-cycloheptyl-2- hydroxy-2-phenyl-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-iu m bromide, 1-cyclooctyl -2-hydroxy-2-phenyl-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4 -ium bromide, 1-hexyl- 2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro- lH-imidazo[l ,2-a]pyrimidin-4-ium bromide,

1- heptyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, l-octyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imidazo[ l,2-a]- pyrimidin-4-ium bromide, l-nonyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH- imidazo[l,2-a]pyrimidin-4-ium bromide, l-decyl-2-hydroxy-2-(4-chlorophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, l-undecyl-2-hydroxy-2-(4-chloro- phenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, l-dodecyl-2-hydroxy-

2- (4-chlorophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-iu m bromide, 1-tridecyl- 2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro- lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, l-tetradecyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imi dazo[l,2-a]pyrimidin-4- ium bromide, l-cycloheptyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-im idazo[l,2- a]pyrimidin-4-ium bromide, l-cyclooctyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH- imidazo[l,2-a]pyrimidin-4-ium bromide, l-hexyl-2-hydroxy-2-(4-nitrophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, 1 -octyl-2-hydroxy-2-(4-nitro- phenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, 1 -decyl-2-hydroxy-2- (4-nitrophenyl)-2,3 -dihydro- 1 H-imidazo [ 1 ,2-a]pyrimidin-4-ium bromide, 1 -octyl-2- hydroxy-2-(4-fluorophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyri midin-4-ium bromide, 1- decyl-2-hydroxy-2-(4-fluorophenyl)-2,3-dihydro-lH-imidazo[l, 2-a]pyrimidin-4-ium bromide, 1 -octyl-2-hydroxy-2-(3 ,4-dichlorophenyl)-2,3-dihydro- 1 H-imidazo [1 ,2- a]pyrimidin-4-ium bromide, l-decyl-2-hydroxy-2-(3,4-dichlorophenyl)-2,3-dihydro-lH- imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -octyl-2-hydroxy-2-(4-methoxyphenyl)-2,3- dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -decyl-2-hydroxy-2-(4- methoxyhenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, l-octyl-2- hydroxy-2-(4-methylthiophenyl)-2,3-dihydro-lH-imidazo[l,2-a] pyrimidin-4-ium bromide, 1 -decyl-2-hydroxy-2-(4-methylthiophenyl)-2,3-dihydro- lH-imidazo[l ,2-a]- pyrimidin-4-ium bromide, l-octyl-2-hydroxy-2-(l-naphthalenyl)-2,3-dihydro-lH- imidazo[l,2-a]pyrimidin-4-ium bromide, l-decyl-2-hydroxy-2-(l-naphthalenyl)-2,3- dihydro- 1 H-imidazo[ 1 ,2-a]pyrimidin-4-ium bromide, 1 -octyl-2-hydroxy-2-(4- methylsulfonylphenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin -4-ium bromide, 1-decyl- 2-hydroxy-2-(4-methylsulfonylphenyl)-2,3-dihydro-lH-imidazo[ l,2-a]pyrimidin-4-ium bromide, l-octyl-2-hydroxy-2-(4'-nitro[l, -biphenylyl]-4-yl)-2,3-dihydro-lH- imidazo[ 1 ,2-a]pyrimidin-4-ium bromide, 1 -decyl-2-hydroxy-2-(4 ' -nitro [ 1 , 1 ' -biphenylyl] - 4-yl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, l-octyl-2-hydroxy-2-(3- bromophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, l-decyl-2- hydroxy-2-(3-bromophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrim idin-4-ium bromide, 1- octyl-2-hydroxy-2-([l,l':4',l"-terphenyl]-4-yl)-2,3-dihydro- lH-imidazo[l,2-a]pyrimidin- 4-ium bromide, l-octyl-2-(4-methoxyphenyl)-imidazo[l,2-a]pyrimidin-l-ium perchl orate, l-octyl-2-(4-fluorophenyl)-imidazo[l,2-a]pyrimidin-l-ium perchlorate, l-octyl-2-(3,4- dichlorophenyl)-imidazo[l,2-a]pyrimidin-l-ium perchlorate, l-octyl-2-(3-bromophenyl)- imidazo[l,2-a]pyrimidin-l-ium perchlorate, l-octyl-2-(l-naphthalenyl)-imidazo[l,2-a]- pyrimidin- 1 -ium perchlorate, 1 -octyl-2-(4-nitrophenyl)-imidazo[ 1 ,2-a]pyrimidin- 1 -ium perchlorate, 1 -octyl-2-(4' -nitro [1 , Γ -biphenylyl] -4-yl)-imidazo [ 1 ,2-a]pyrimidin-l -ium perchlorate, l-octyl-2-(4-methylsulfonylphenyl)-imidazo[l,2-a]pyrimidin-l -ium perchlorate, l-octyl-2-(4-methylthiophenyl)-imidazo[l,2-a]pyrimidin-l-ium perchlorate, l-octyl-2-([l, 1 ' :4' ,1 "-terphenyl]-4-yl)-imidazo[l ,2-a]pyrimidin-l-ium perchlorate, 1- decyl-2-(4 ' ' -nitro([ 1 , 1 ' :4' , 1 "-terphenyl] -4-yl)-imidazo [ 1 ,2-a]pyrimidin- 1 -ium perchlorate.

Another specific embodiment of the present invention relates to an imidazo[l,2- a]pyrimidinium salt being selected from the group consisting of_l-methyl-2-hydroxy-2- phenyl-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, l-methyl-2-hydroxy-2- (4-chlorophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-iu m bromide, l-methyl-2- hydroxy-2-(4-fluorophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyri midin-4-ium bromide, 1- methyl-2-hydroxy-2-(3-fluorophenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin-4-ium bromide, l-(3,4-methylenedioxyphenylmethyl)-2-hydroxy-2-(4-fluorophen yl)-2,3- dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, l-(3,4-methylenedioxyphenyl- methyl)-2-hydroxy-2-(3,4-methylendioxyphenyl)-2,3-dihydro-lH -irnidazo[l,2-a]- pyrimidin-4-ium bromide, l-ethyl-2-hydroxy-2-(4-nitrophenyl)-2,3-dihydro-lH-imidazo- [l,2-a]pyrimidin-4-ium bromide, l-(3-methoxyphenylethyl)-2-hydroxy-2-(4-fluoro- phenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, 1 -isopropyl-2-hydroxy- 2-phenyl-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, 1 -isopropyl-2- hydroxy-2-(4-nitrophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrim idin-4-ium bromide, 1- butyl-2-hydroxy-2-phenyl-2,3-dihydro-lH-imidazo[l,2-a]pyrimi din-4-ium bromide, 1- butyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imidazo[l, 2-a]pyrimidin-4-ium bromide, l-butyl-2-hydroxy-2-(4-nitrophenyl)-2,3-dihydro-lH-imidazo[l ,2-a]pyrimidin- 4-ium bromide, l-butyl-2-hydroxy-2-(4-methoxyphenyl)-2,3-dihydro-lH-imidazo [l,2- a]pyrimidin-4-ium bromide, l-isobutyl-2-hydroxy-2-phenyl-2,3-dihydro-lH-imidazo[l,2- a]pyrimidin-4-ium bromide, l-isobutyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH- imidazo[l,2-a]pyrimidin-4-ium bromide, 1-isobutyl -2-hydroxy-2-(3-bromophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, 1-isobutyl -2-hydroxy-2-(4- nitrophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, 1-isobutyl -2- hydroxy-2-(3,4-dichlorophenyl)-2,3-dihydro-lH-imidazo[l,2-a] pyrimidin-4-ium bromide, 1 -ieri-butyl-2-hydroxy-2-(4-nitrophenyl)-2,3 -dihydro- 1 H-imidazo [ 1 ,2-a] - pyrimidin-4-ium bromide, l-pentyl-2-hydroxy-2-phenyl-2,3-dihydro-lH-irnidazo[l,2- a]pyrimidin-4-ium bromide, l-pentyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH- imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -pentyl-2-hydroxy-2-(4-bromophenyl)-2,3- dihydro-lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, 1 -decyl-2-hydroxy-2-(4-methoxy- phenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide, 1 -decyl-2-hydroxy-2- (3,4-dichlorophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin- 4-ium bromide, 1- tetradecyl-2-hydroxy-2-(4-chlorophenyl)-2,3-dihydro-lH-imida zo[l,2-a]pyrimidin-4-ium bromide, 1 -cyclopropyl-2-hydroxy-2-(4-nitrophenyl)-2,3 -dihydro- 1 H-imidazo [ 1 ,2-a] - pyrimidin-4-ium bromide, 1 -cyclobutyl-2-hydroxy-2-phenyl-2,3-dihydro- 1 H- imidazo[l ,2-a]pyrimidin-4-ium bromide, 1 -cyclohexyl-2-hydroxy-2-phenyl-2,3-dihydro- lH-imidazo[l,2-a]pyrirnidin-4-ium bromide, and l-adamantyl-2-hydroxy-2-(4- nitrophenyl)-2,3-dihydro-lH-imidazo[l,2-a]pyrimidin-4-ium bromide.

All the novel compounds of the first aspect of the present invention as defined in claim 1 can be produced in good yield according to a synthetic procedure comprising an initial step of forming a substituted imidazo[l,2-a]pyrimidinium salt being represented by the structural formula (II) wherein ring A is an imidazolinyl structure represented by formula B, and wherein X ^" is the bromine anion, with the aforementioned definitions of Rl, R2, R3, R4, R5, R6, R7 and R8, including all specific meanings for each substituent. This initial step proceeds as shown in tables 1, 3 and 4 of J. Org. Chem. (2008) 73:6691- 6697 and in the left part of the synthetic scheme of the examples below, i.e. by reacting a 2-aminopyrimidine wherein the amino group is mono-substituted with Rl, and wherein the pyrimidinyl ring is optionally substituted with one, two or three substituents R6, R7 and R8, with an a-bromocarbonyl compound represented by the structural formula R ₄ CHBr-CO-R3 wherein R4 and R3 are as defined in claim 1.

When R3 is hydrogen, said a-bromocarbonyl compound is a a-bromoaldehyde such as, but not limited to, 2-bromoacetaldehyde. When R3 is not hydrogen, said a- bromocarbonyl compound is an a-bromoketone. When R3 is not hydrogen and R4 is not hydrogen, said α-bromocarbonyl compound is a 1 ,2-disubstituted bromoketone. When R3 is aryl and R4 is hydrogen, said α-bromocarbonyl compound is an optionally substituted a-bromoacetophenone. Suitable optionally substituted 2-bromoacetophenones for this reaction (thus providing the R3 substituent in the substituted imidazo[l,2-a]pyrimidinium salt ) include, but are not limited to, commercial products such as bromo-acetophenone, 2,3 ' -dibromoacetophenone, 2-bromo-4' -fluoroacetophenone, 2-bromo-4' -chloro- acetophenone, 2-bromo-4'-nitroacetophenone, 2-bromo-4'-cyanoacetophenone, 2-bromo- 4' -(trifluoromethyl)acetophenone, 2-bromo-4 ' -methoxyacetophenone, 2-bromo-3 ' - methoxyacetophenone, 2-bromo-2' -methoxyacetophenone, bromomethyl 2- naphthylketone, 2-bromo-4'-methylacetophenone, 2-bromo-4'-hydroxyacetophenone, 2- bromo-3 ' -methylacetophenone, 2-bromo-2' -methylacetophenone, 2-bromo-4 ' -ethyl - acetophenone, 2-bromo-4'-propylacetophenone, 2-bromo-4'-isopropylacetophenone, 2- bromo-4' -77-butylacetophenone, 2-bromo-4'-(methylthio)acetophenone, 2-bromo-2', 5'- dimethoxyacetophenone, 2-bromo-3',4',5'-trimethoxyacetophenone and 2-bromo-4'- phenylacetophenone. If a desirable substituted 2-bromoacetophenone is not commercially available, it can easily be prepared by bromination of the correspondingly substituted acetophenone while using techniques well known in the art.

This initial step of the synthetic procedure proceeds readily in an apolar solvent such as, but not limited to, acetonitrile, and under temperature conditions ranging from about 70°C to about 150°C during 0.5 to about 8 hours. If desirable to achieve a substituted imidazo[l,2-a]pyrirnidinium salt being represented by the structural formula (II) wherein ring A is an imidazolinyl structure represented by formula B, and wherein X ^" is an organic or inorganic anion other than the bromine anion, the bromide salt formed in the initial step may be submitted to a classical anion exchange reaction with shifting equilibrium towards the less soluble salt. In this way bromine may be replaced with the anion derived from hydrochloric acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, and glutamic acid.

If desirable to achieve a substituted irnidazo[l,2-a]pyrirnidinium salt being represented by the structural formula (II) wherein ring A is an imidazolyl structure represented by formula C, the bromide salt formed in the initial step may be submitted to dehydration by any suitable means such as, but not limited to, short heating in polyphosphoric acid and addition of an inorganic acid such as perchloric acid, as also shown in J. Org. Chem. (2008) 73:6691-6697. Such substituted imidazo[l,2- a]pyrimidinium salt being represented by the structural formula (II) wherein ring A is an imidazolyl structure represented by formula (C) are useful intermediates for making unsubstituted 2-aminoimidazole compounds as for instance disclosed in WO 2009/1223753, but they also are active for inhibiting biofilm formation as disclosed herein.

Novel substituted 2-aminoimidazole compounds represented by the structural formula (I) may then be produced in a second step by reacting the substituted imidazo[l,2-a]pyrimidinium salt being represented by the structural formula (II) wherein ring A is an imidazolinyl structure represented by formula B and wherein X ^" is the bromine anion, which is the result of the initial step, with a molar excess of hydrazine for a short period of time at a temperature range from about 70°C to about 100°C, as shown in table 4 and scheme 3 of J. Org. Chem. (2008) 73:6691-6697. No interconversion occurs between the formed 1 -unsubstituted 2-(substituted amino)imidazole compounds and their 1 -substituted 2-aminoimidazole isomers. However the signals of C-4 and C-5 of the imidazole ring of the formed 1 -unsubstituted 2-(substituted amino)imidazole compounds usually appear as broad peaks or are even not detected, presumably to a fast proton exchange between N-l and N-3.

Compounds according to the first aspect of the present invention as defined in claim 1 may form hydrates with water, or solvates with an organic solvent such as, but not limited to, a nitrile, an ester, an alcohol or an ether, using solvation methods well known in the art. When an assymetric carbon present, all possible stereoisomers or enantiomers are encompassed by the present invention.

The second aspect of the present invention as defined in claim 9 will now be described with respect to a number of specific valuable embodiments.

For instance it has been demonstrated that 2-(substituted amino)imidazole compounds according the structural formula (B) wherein Rl, R2, R3 and R4 are as defined in table 1 (wherein Ph stands for phenyl, Me for methyl, etc.) below are capable of inhibiting biofilm formation.

Table 1

H 2-(3-MeOPh)Ethy p-N0 ₂ -Ph double

It has also been demonstrated that 2-(substituted amino)imidazoline compounds according the structural formula (C) wherein Rl, R2, R3 and R4 are as defined in table 2 below are capable of inhibiting biofilm formation.

Table 2

It has also been demonstrated that 2-(substituted amino)imidazole compounds according the structural formula (B) wherein Rl, R2, R3 and R4 are as defined in table 3 (wherein Ph stands for phenyl.) below are capable of acting as anti-bacterial agents.

Table 3

Substituted 2-aminoimidazoles represented by the structural formula (I), wherein ring A is an imidazolinyl structure represented by formula (C) may be included in the anti-microbial compositions of this invention. Such compounds may be produced by performing a sequence of method steps well known in the art. One synthetic procedure is schematically shown below.

In a first step (reaction A), an optionally substituted iodobenzonitrile is condensed with an optionally substituted thiophenol (wherein the optional substituent is designated as Y in the above scheme) under basic conditions and in the presence of a suitable condensation catalyst, as disclosed in Eur. J. Org. Chem. (2008) 640-3. Although not shown in the above scheme, a similar procedure may be performed starting from an optionally substituted phenol. A representative but non-limiting detailed procedure for reaction A is as follows: to a solution of KOH (1.5 mmole) in 1 mL water, were added 1 mmole tetrabutylammonium iodide, 1 mole copper (I) iodide, 1.1 mmole iodobenzonitrile and 1 mmole of the optionally substituted thiophenol. The reaction mixture was stirred at 80°C during 10 hours. After cooling down, dichloromethane was used to extract the water phase (3 x 2 mL). The organic phase was washed 3 times with brine and dried. The solvent was evaporated under reduced pressure and the residue was purified with chromatography on silica with a mixture petroleum ether / ethyl acetate 10: 1 as eluent to provide the condensation product in good yield.

In a second step (reaction B), the cyano group of the condensation product from reaction A is converted into an aminomethyl group using reaction conditions well known in the art, e.g. from Eur. J. Org. Chem. (2008) 3314-3327. A representative but non- limiting detailed procedure for reaction B is as follows: 1 mmole of the benzonitrile derivative is added to 7 mL of dry THF. The solution is cooled down to 0°C and flushed with N ₂ then with argon. 2 mmoles LiAlH ₄ in dry THF are added slowly within 1 hour. The reaction mixture is slowly warmed up to room temperature and then stirred during 5 hours. The reaction is then quenched with water (15 mL) and the resulting biphasic solution is extracted with dichloromethane (3 x 5 mL). The organic phase is washed with NAOH 10% and brine (15 mL), dried, filtrated and evaporated under vacuum to provide the aminomethyl aromatic derivative in good yield.

In a third step (reaction C), the aminomethyl aromatic derivative is reacted with a 2-(methylthio)-4,5-dihydro-imidazole derivative which may be substituted as required at positions 1 , 4 and/or 5 of the imidazolyl ring, using reaction conditions well known in the art, e.g. from J. Pharmacy & Pharmacology (2006) 58: 1415-1420. A representative but non-limiting detailed procedure for reaction C is as follows: In 25 mL acetonitrile, 1 mmole of the aminomethyl aromatic derivative from step B and 1 mmole of 2- (methylthio)-4,5-dihydro-imidazole derivative are stirred under reflux during 36 hours. After cooling down, the mixture is evaporated under strong vacuum, triturated with diethylether and filtrated. The solvent is evaporated under vacuum and the residue is purified on chromatography. One specific embodiment of this aspect of the present invention relates to a composition wherein said compound is a known substituted 2-aminoimidazole as defined in CLAIM 10.

Another specific embodiment of the present invention relates to a composition wherein R3 is as defined in CLAIM 11, in combination with the aforementioned definitions of Rl, R2 and R4.

Another specific embodiment of the present invention relates to a composition wherein R3 is as defined in CLAIM 11, and wherein Rl is hydrogen, in combination with the aforementioned definitions of R2 and R4.

Another specific embodiment of the present invention relates to a composition wherein R3 is as defined in CLAIM 11 , and wherein R4 is hydrogen, in combination with the aforementioned definitions of Rl and R2.

Another specific embodiment of the present invention relates to a composition wherein R3 is as defined in CLAIM 11, and wherein Rl and R4 are hydrogen, in combination with the aforementioned definition of R2.

Another specific embodiment of the present invention relates to a composition wherein R2 is as defined in CLAIM 12, in combination with the aforementioned definitions of Rl, R3 and R4.

Another specific embodiment of the present invention relates to a composition wherein R2 is as defined in CLAIM 12, and wherein Rl is hydrogen, in combination with the aforementioned definitions of R3 and R4.

Another specific embodiment of the present invention relates to a composition wherein R2 is as defined in CLAIM 12, and wherein R4 is hydrogen, in combination with the aforementioned definitions of Rl and R3.

Another specific embodiment of the present invention relates to a composition wherein R2 is as defined in CLAIM 12, and wherein Rl and R4 are hydrogen, in combination with the aforementioned definition of R3.

Another specific embodiment of the present invention relates to a composition R2 is as defined in CLAIM 13. Another specific embodiment of the present invention relates to a composition wherein said compound is a known (commercial) substituted 2-aminoimidazoline as defined in CLAIM 14.

Another specific embodiment of the present invention relates to a composition wherein said compound is an imidazo[l,2-a]pyrimidinium salt being represented by the structural formula (II) and wherein X ^" is an inorganic or organic anion, in combination with the aforementioned definitions of Rl, R2, R3, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to a composition wherein said compound is as defined in CLAIM 15, in combination with the aforementioned definitions of Rl, R2, R3, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to a composition as defined in CLAIM 16.

Another specific embodiment of the present invention relates to a composition wherein said compound is a salt wherein Rl is as defined in CLAIM 17, in combination with the aforementioned definitions of X ^" , R2, R3, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to a composition wherein said compound is a salt wherein Rl is as defined in CLAIM 17, and wherein each of R6, R7 and R8 is hydrogen, in combination with the aforementioned definitions of X ^" , R2, R3, R4 and R5.

Another specific embodiment of the present invention relates to a composition wherein said compound is a salt wherein R3 is as defined in CLAIM 18, in combination with the aforementioned definitions of X ^" , Rl, R2, R4, R5, R6, R7 and R8.

Another specific embodiment of the present invention relates to a composition wherein said compound is a salt wherein R3 is as defined in CLAIM 18, and wherein each of R6, R7 and R8 is hydrogen, in combination with the aforementioned definitions of X ^" , Rl, R2, R4 and R5.

The antimicrobial compositions of the second aspect of this invention may, either for improved efficiency 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 anti-microbial peptide moiety.

The antimicrobial compositions of the second aspect of this invention 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.

When the antimicrobial compositions of this invention are formulated as liquids, at least one excipient may be a solvent for the biologically effective imidazo[l,2- a]pyrimidinium salt or substituted 2-aminoimidazole. Said solvent may be (CLAIM 20), 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.

For an effective treatment, since the imidazo[l,2-a]pyrimidinium salt or a substituted 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 as defined in CLAIM 21, or in CLAIM 22.

The anti-microbial 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).

Preferably the antimicrobial compositions of this invention are formulated for long-term 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.

The third aspect of the present invention will now be described with respect to a number of specific valuable embodiments.

The method of this aspect of the invention is effective against a wide selection of microbes. For instance one specific embodiment of this invention relates to a method as defined in CLAIM 25.

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 as defined in CLAIM 27. 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.

Another specific embodiment of the present invention relates to a method for disinfecting or sterilizing a surface ex-vivo to remove a biofilm or prevent biofilm growth.

Another specific embodiment of the present invention relates to a method as defined in CLAIM 28 (either simultaneous or sequential co-administration).

Without wishing to be bound by theory, it is believed that the active compounds represented by the structural formula (I) or the structural formula (I) are able to perform biofilm inhibition in the method of treatment of this invention through one or more of the following mechanisms of action:

Activation of the PhoPQ regulon,

Downregulation of csgD, a master regulator for biofilm formation, both in a PhoPQ dependent manner, and in a PhoPQ independent manner,

Decreasing motility of microorganisms,

Interfering with nucleotide biosynthesis, in particular purine nucleotide biosynthesis and pyrimidine nucleotide biosynthesis, thus potentially decreasing bioavailability of the messenger molecule c-di-GMP which is crucial for biofilm formation.

The biofilm inhibitor of this invention may be capable of complexing, or reversibly binding to, the organic matrix material, thereby rendering the organic matrix water insoluble. The biofilm inhibitor may exhibit greater binding affinity for functional groups in cellular proteins of microorganisms. When a microorganism contacts the anti-biofilm material of this invention, the organic material engages or disrupts at least the outer portion of the lipid bilayer of the microorganism's cell membrane sufficiently to permit insinuation of the biofilm inhibitor into the microorganism, where cell proteins or proteins in the lipid bilayer compete effectively for the biofilm inhibitor due to favourable binding constants. Stated another way, the biofilm inhibitor binds to or forms a complex with the organic material in which the association between the organic material and biofilm inhibitor is sufficiently strong that the layer or film does not elute anti-biofilm amounts of the biofilm inhibitor into a contacting solution. However, the biofilm inhibitor preferentially binds to certain proteins in the microorganism and thus is transferred from the matrix to the microorganism. The result is a contact-biofilm preventing delivery system that selectively transfers the biofilm inhibitor or into the microorganism's cell membrane upon contact, without elution or dissolution of the biofilm inhibitor into solution, thereby maintaining the long term anti-biofilm efficacy of the composition.

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.

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.

The compositions and methods of this invention are especially useful for reducing the risk of bacterial infection in a human, wherein the pathogenic bacteria are selected from the group consisting of pneumococcal species, methicillin-resistant Staphylococcus aureus, multi-drug resistant Pseudomonas species, Nesseria sp., Hemophilus sp., Proteus sp., Klebsiella sp. and Escherichia coli.

The compositions and methods of this invention are also useful for reducing 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. cholerae-suis, 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 group P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans or P. straminea. 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.

EXAMPLE 1 - production method (general procedure) for substituted imidazo|T,2- alpyrimidinium salts and substituted 2-aminoimidazoles

The compounds of the present invention - both substituted imidazo[l,2- a]pyrimidinium salts having formula (II) and substituted 2-aminoimidazoles having formula (I) - can be produced by applying the synthetic method known from J. Org. Chem. (2008) 73:6695-6694, and schematically shown below:

A representative but non-limiting methodology is as follows. In a 30 mL microwave vial were successively brought acetonitrile (15 mL), 2-alkylaminopyrimidine 1 (5 mmoles), a-bromoketone 2 (6 mmoles) and a catalytic amount of 4- dimethylaminopyridine (0.05 mmole). The reaction tube was sealed, and irradiated in a microwave reactor at a temperature of 80°C and 150 W maximum power for 30 to 45 minutes. After the reaction mixture was cooled with an air flow for 15 minutes, the resulting precipitate was washed with acetone (25 mL), ether (20 mL) and dried in vacuum to afford the imidazo[l,2-a]pyrirnidinium salt 3 as a white powder.

Then, to a suspension of the imidazo[l,2-a]pyrimidinium salt 3 (2 mmoles) in acetonitrile (5 mL), hydrazine hydrate (0.7 mL, 14 mmoles of a 64% solution) was added, and the mixture was irradiated in the sealed reaction tube for 10 minutes at a temperature of 100°C at 150 W maximum power. After cooling down, hydrazine hydrate was evaporated with toluene (3x20 mL). The resulting residue was purified by column chromatography (silica gel; MeOH-DCM 1:4 v/v with 5% of 6M NH ₃ in MeOH) to afford the substituted 2-amino-lH-imidazole 4 as an amorphous solid. EXAMPLE 2 - anti -bacterial activity of substituted 2-amino-lH-imidazoles

Evaluation of antibacterial-inhibitory activity of some substituted 2-amino-lH- imidazoles of the invention was carried out by diluting an overnight culture of Salmonella Typhimurium ATCC 14028 into a liquid medium. 200 μΐ of the diluted overnight culture was added to each well of a microtiter plate together with different concentrations of the test compound (1/2 dilution series of the compound). After an incubation of 24 hours the optical density at 600 nm of each well was determined with a microtiter plate reader. From these measurements the concentration of the test compound at which 50% inhibition was observed (GIC ₅₀ value) was calculated. The compounds of the invention had a good antibacterial inhibitory. The IC ₅₀ values of representative compounds for antibacterial activity are shown in the following Table 4.

Table 4

a GIC50: 50% inhibitory concentration for growth inhibition: determined by measuring the OD600 after 24 hours of growth in the presence of different concentrations of the compound.

EXAMPLE 3 - biofilm formation inhibiting activity of substituted 2-amino-lH- imidazoles

Evaluation of the biofilm inhibitory activity of some substituted 2-amino-lH- imidazoles of the invention was carried out by growing up biofilms of S. Typhimurium ATCC 14028 or SL1344 or Pseudomonas aeruginosa PA 14 in the presence of different concentrations (1/2 dilution series) of biofilm inhibitors, by using the Calgary biofilm device. After an incubation period of 24 hours at 25°C or 37°C or 48 hours at 16°C the biofilms were visualized by crystal violet staining. In the next step the stain was extracted from the biofilm with 30% acetic acid. The absorbance of the resolubilized stain at 570 nm is a measure of the amount of biofilm formed. From these measurements the concentration of the test compound at which 50% biofilm inhibition was observed (IC ₅₀ value) was calculated. The IC ₅₀ values of representative compounds for biofilm inhibition are shown in the following Tables 5 to 7 below and in the table 8 of the appended figure (wherein Ph stands for phenyl).

Table 5 - biofilm inhibition of 5. Tvphimurium ATCC14028 at 25°C

IC50 for biofilm inhibition

Rl R2 R3 R4 (25°C) (μΜ)

H Methyl H p-F-Ph 102

H i-Propyl H p-Me-Ph 99

H Butyl H Ph 25

H Butyl H p-F-Ph 16

H Butyl H p-Br-Ph 7

H Butyl H p-MeO-Ph 49

H cyclopentyl H p-Br-Ph 95

H cyclopentyl H Ph 53

H cyclopentyl H p-CI-Ph 4

H cyclopentyl H p-Br-Ph 12

H cyclopentyl H p-N02-Ph 12

H cyclopentyl H Bi-Ph 46

H cyclopentyl H 3,4-diCI-Ph 6

H Cyclohexyl H p-N02-Ph 44

H Benzyl H p-CI-Ph 31

H piperonyl H p-F-Ph 68

H 2-(3-MeO-Ph)Ethyl p-N0 ₂ .Ph 15 Table 6 - Pseudomonas aeruginosa PA 14 biofilm formation at 25 °C

Table 7 - biofilm inhibittion of S. Tvphimurim ATCC14028 and SL1344 by substituted 2- aminoimidazolines at 16°C

specs3 s ecs3-1

specs3-3 specs3-4

Some of the tested compounds show a concentration range in which biofilm formation is inhibited but no effect on planktonic growth is observed. These compounds are particularly interesting for prevention of biofilm formation since the development of resistance against these compounds is less likely. Table 8 indicates whether the different compounds have an effect on the planktonic growth of the bacteria at the IC50 for biofilm inhibition. To construct Table 8, the effect of the tested compounds on the growth curve of the bacteria was determined at a concentration equal to the IC50 for biofilm inhibition, by using the Bioscreen system available from Oy Growth Curves Ab Ltd).

In Table 8: a x means: no influence on planktonic growth at the IC50 for biofilm inhibition ^b nd means: not determined ^c o means: planktonic growth is retarded at the IC50 for biofilm inhibition ^d Sl means:

In examples 2 and 3, the following materials and methods were used. Stock solutions of all compounds assayed for biological activity were prepared in DMSO or EtOH and stored at -20 °C. The amount of solvent used in biofilm inhibition screens and growth inhibition screens did not exceed 2% (by volume).

Static peg assay for prevention of Salmonella Typhimurium biofilm formation

The device used for biofilm formation is a platform carrying 96 polystyrene pegs

(Nunc no. 445497) that fits as a microtiter plate lid with a peg hanging into each microtiter plate well (Nunc no. 269789). Biofilm formation assays were carried out at

16°C, 25°C and 37°C.

For biofilm assays at 16°C and 25°C, two-fold serial dilutions of the compounds in 100 μΐ liquid TSB 1/20 broth per well were prepared in the microtiter plate.

Subsequently, an overnight culture of S. Typhimurium ATCC 14028 or SL1344 (grown up in LB medium) was diluted 1: 100 into TSB 1/20 broth and 100 μΐ (ca. 10 ⁶ cells) was added to each well of the microtiter plates, resulting in a total amount of 200 μΐ medium per well. The pegged lid was placed on the microtiter plate and the plate was incubated for 48 hours at 16°C or for 24 hours at 25°C without shaking. During this incubation period biofilms were formed on the surface of the pegs. In case of the assay at 16°C, after 24 hours the lid was transferred into a new plate with medium and the specific molecules used for testing. The optical density at 600 nm (OD600) was measured for the planktonic cells in the first plate using a VERSAmax microtiter plate reader (Molecular Devices) and the growth retarding concentration IC50 for growth inhibition was determined as the concentration that decreases the OD600 of the planktonic cells 50%. For quantification of biofilm formation, the pegs were washed once in 200 μΐ phosphate buffered saline (PBS). The remaining attached bacteria were stained for 30 minutes with 200 μΐ 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 μΐ distilled water per well. After the pegs were air dried (30 min), the dye bound to the adherent cells was extracted with 30% glacial acetic acid (200 μΐ). The OD570 of each well was measured using the VERSAmax microtiter plate reader. The IC50 value for each compound was determined from concentration gradients in two or three independent experiments (with 2 or 3 repeats per experiment), by using the GraphPad software of Prism.

For biofilm assays at 37°C, two-fold serial dilutions of the compounds in 50 μΐ liquid CFA broth per well were prepared in the microtiter plate. Subsequently, an overnight culture of S. Typhimurium ATCC14028 was diluted 1: 100 into CFA broth and 100 μΐ (ca. 10° cells) was added to each well of the microtiter plates, resulting in a total amount of 150 μΐ medium per well. The pegged lid was placed on the microtiter plate and the plate was incubated for 24 hours at 37°C without shaking. During incubation the plates were placed in a humidity controlled container to prevent evaporation of the growth medium. Quantification of biofilm formation was performed similarly as described above for the assays at 16°C and 25 °C. Static peg assay for prevention of Pseudomonas aeruginosa biofilm formation The device used for Pseudomonas aeruginosa biofilm formation is the same platform carrying polystyrene pegs as described above for Salmonella Typhimurium.

Two-fold serial dilutions of the compounds in 100 μΐ liquid broth per well were prepared in the microtiter plate. Subsequently, an overnight culture of P. aeruginosa PA 14 (grown up in TSB medium) was diluted 1 :100 into liquid broth and 100 μΐ was added to each well of the microtiter plates, resulting in a total amount of 200 μΐ medium per well. The pegged lid was placed on the microtiter plate and the plate was incubated for 24 hours at 25 °C, 30°C or 37°C without shaking. During this incubation period biofilms were formed on the surface of the pegs. The liquid broth used for the tests at 25°C was TSB 1/20. The tests at 37°C were performed in CFA and/or LBNS broth. During incubation at 30°C and 37°C the plates were placed in a humidity controlled container to prevent evaporation of the growth medium. Quantification of biofilm formation was performed similarly as described previously for the S. Typhimurium biofilm assays.

Bioscreen assay for measuring growth inhibition

The Bioscreen device from Oy Growth Curves Ab Ltd was used for measuring the influence of the chemical compounds on the planktonic growth of Salmonella Typhimurium and Pseudomonas aeruginosa. The Bioscreen is a computer controlled incubator/reader/shaker that uses 10x10 well microtiter plates and measures light absorbance of each well at a specified wave length in function of time.

An overnight culture of S. Typhimurium ATCC14028 or SL1344 (grown up in LB medium) or Pseudomonas aeruginosa PA 14 (grown up in TSB medium) was diluted 1:200 in liquid broth. The broth used was TSB 1/20 for the experiments at 16°C and 25°C. For experiments at 37°C CFA medium was used for S. Typhimurium and LBNS medium for P. aeruginosa. 300 μΐ of the diluted overnight culture was added to each well of the 10x10 well microtiter plate. Subsequently serial dilutions of the chemical compounds were prepared in DMSO or EtOH. 3 μΐ of each diluted stock solution was added to the wells (containing the 300 μΐ bacterial culture) in 3-fold. As a control 3 μΐ of the appropriate solvent (DMSO or EtOH) was also added to the plate in 3- or 4-fold. The microtiter plate was incubated in the Bioscreen device at 16°C, 25°C or 37°C for at least 36 hours, with continious medium shaking. The absorbance of each well was measured at 600 nm each 15 minutes. Excel was used to generate the growth curves for treated the treated wells and the untreated control wells.

Measuring antibacterial properties

Starting from a 40 mM stock solution of each compound in DMSO, two-fold serial dilutions in 100 μΐ liquid broth per well were prepared in dublicate in sterile transparant microtiter plates. The broth used was TSB 1/20 for measurements at 25 °C and CFA (in case of Salmonella Typhimurium) and LBNS (in case of Pseudomonas aeruginosa) for measurements at 37°C. Subsequently an overnight culture of the bacteria (S. Typhimurium ATCC 14028 or P. aeruginosa PA 14) was diluted 1/200 into the corresponding liquid broth and 100 μΐ was added to each well of the microtiterplate resulting in a total amout of 200 μΐ medium per well. After an incubation of 24 hours the optical density at 600 nm of each well was determined with a microtiter plate reader (VERSAmax, Molecular Devices). From these measurements the concentration of the test compound at which 50% inhibition was observed (GIC ₅₀ value) was calculated by using the GraphPad software of Prism.

EXAMPLE 4 - anti-bacterial activity of substituted imidazori,2-alpyrimidinium bromide salts

Evaluation of antibacterial-inhibitory activity of some substituted imidazo[l,2- a]pyrimidinium bromide salts of the invention was carried out by diluting an overnight culture of Salmonella Typhimurium ATCC14028 or Pseudomonas aeruginosa PA141/200 into liquid medium. 200 μΐ of the diluted overnight culture was added to each well of a microtiterplate together with different concentrations of the test compound (1/2 dilution series of the compound). After an incubation of 24 hours the optical density at 600 nm of each well was determined with a microtiter plate reader. From these measurements the concentration of the test compound at which 50% inhibition was observed (GIC ₅₀ value) was calculated. The GIC ₅₀ values of representative compounds for antibacterial activity are shown in the following Tables 9 and 10 (wherein Ph stands for phenyl, Me for methyl):

Table 9 - Effect against Salmonella Typhimurium ATCC 14028 at 25 °C

Table 10 - Effect against Pseudomonas aeruginoa PA 14 at 25 °C o

u

(J

Cycloperityl 4-biphenylyl H 27.11 Cyclohexyl 4-CI-Ph H 61.3

Benzyl 4-l-Ph H 51.68

cyclododecyl 4-N0 ₂ .Ph H 8.891

EXAMPLE 5 - biofilm inhibitory activity of substituted imidazori,2-alpyrimidinium bromide salts

Evaluation of the biofilm inhibitory activity of some substituted imidazo[l,2- a]pyrimidinium bromide salts of the invention was carried out by growing up biofilms of S. Typhimurium ATCC14028 or SL1344 or Pseudomonas aeruginosa PA14 in the presence of different concentrations (1/2 dilution series) of biofilm inhibitors, by using the Calgary biofilm device. After an incubation period of 24 hours at 25 °C or 37 °C or 48 hours at 16°C the biofilms were visualized by crystal violet staining. In the next step the stain was extracted from the biofilm with 30% acetic acid. The absorbance of the resolubilized stain at 570 nm is a measure of the amount of biofilm formed. From these measurements the concentration of the test compound at which 50% biofilm inhibition was observed (IC ₅₀ value) was calculated. The IC ₅₀ values of representative compounds for biofilm inhibitory activity are shown in the following Tables 11 to 14 (wherein Ph stands for phenyl, Me for methyl, and Bn for benzyl). Table 11 -biofilm formation of 5. Typhimurium ATCC14028 at 25°C

Table 12 - P. aeruginosa PA 14 biofilm formation at 25 °C

Table 13 - P. aeruginosa PA 14 biofilm formation at 37 °C

Some of the compounds show a concentration range in which biofilm formation is inhibited but no effect on the planktonic growth is observed. Table 14 indicates if the different compounds have an effect on the planktonic growth of the bacteria at the IC50 for biofilm inhibition. To construct this table the effect of the compounds on the growth curve of the bacteria was determined at a concentration equal to the IC50 for biofilm inhibtion, by using the Bioscreen system from Oy Growth Curves Ab Ltd. Table 14 - effect on planktonic growth at the IC50 for biofilm inhibition (25 °C)

dioxy-Ph

Cyclododecyl 3-N0 ₂ Ph H <6.25 0 nd nd cyclododecyl 4-N0 ₂ Ph H <6.25 o 6.316 o tricyclo[3.3.1.1

3,7Jdec-l-yl 4-N0 ₂ Ph H 8.801 X nd nd ^a x means: no influence on the planktonic growth at the IC50 for biofilm inhibition ^b nd means: not determined ^c o means: planktonic growth is retarded at the IC50 for biofilm inhibition

EXAMPLE 6 - synthesis and characterization of substituted imidazori,2-alpyrirnidinium perchlorate salts

The synthesis of substituted imidazo[l,2-a]pyrimidinium perchlorate salts was performed according to the scheme and experimental procedure below.

A mixture of 84% polyphosphoric acid (3 g) and a bromide salt 3 (3 mmole) was heated in 50 ml beaker upon intensive stirring at 150°C for 15 minutes. After cooling to the room temperature the resulting viscous mass was dissolved in 30 ml of water and 1 ml (10 mmol) of 70% HCIO ₄ was added dropwise upon mild stirring. The resulting white precipitate was washed with distilled water (3x10 mL), ether (2x10 mL) until a neutral reaction of a pH paper, and then dried over P ₂ O ₅ to afford the perchlorate salt 4 as fine white crystals in good yield.

Analytical data (proton and carbon nuclear magnetic resonance, and mass spectrum) of some perchlorate salts are listed below:

2-(4-Methoxyphenyl - 1 -octylimidazoi 1 ,2-alpyrimidin- 1 -ium perchlorate: H NMR (300 MHz, DMSO- ): δ = 9.36 (m, 1H), 9.14 (m, 1H), 8.51 (s, 1H), 7.75 (m, 3H), 7.23 (d, 7 = 8.2 Hz, 2H), 3.98 (s, 3H), 3.84 (t, / = 7.5 Hz, 2H), 1.67 (m, 2H), 1.32 (m, 10H), 0.9 (t, / = 7.5 Hz, 3H). ¹³ C NMR (75.5 MHz, DMSO-d ₆ ): δ : 161.6, 157.4, 143.1, 138.4, 137.8, 131.5 (x2), 117.5, 115.3 (x2), 114.7, 110.5, 55.9, 43.2, 31.4, 29.7, 29.0, 28.4, 26.7, 23.1 , 14.3. MS (m/z) 225 [(M - C10 ₄ - Octyl)] ⁺ .

2-(4-Fluorophenyl - 1 -octylimidazoi! ,2-alpyrimidin- 1 -ium perchlorate: H NMR (300 MHz, DMSO-d ₆ ): δ = 9.39 (m, 1H), 9.16 (m, 1H), 8.57 (s, 1H), 7.81 (m, 3H), 7.54 (t, / = 8.2 Hz, 2H), 3.83 (t, / = 7.5 Hz, 2H), 1.65 (m, 2H), 1.30 (m, 10H), 0.86 (t, / = 7.5 Hz, 3H). ¹³ C NMR (75.5 MHz, DMSO-d ₆ ): δ = 165.3, 162.2, 157.1, 143.2, 137.6, 136.9, 132.4 (x2), 122.1, 117.2, 116.7 (x2), 114.8, 111.3, 43.3, 31.7, 29.7, 29.1, 29.0, 26.6, 22.6, 14.1. MS (m/z) 213 [(M - C10 ₄ - Octyl)] ⁺ .

2-(3 ,4-Dichlorophenyl - 1 -octylimidazoi 1 ,2-alpyrimidin- 1 -ium perchlorate: H NMR (300 MHz, DMSO-d ₆ ): δ = 9.41 (m, 1H), 9.18 (m, 1H), 8.60 (s, 1H), 7.98-7.65 (m, 4H), 3.81 (t, / = 7.5 Hz, 2H), 1.69 (m, 2H), 1.33 (m, 10H), 0.91 (t, / = 7.5 Hz, 3H). ¹³ C NMR (75.5 MHz, DMSO-d ₆ ): δ = 158.2, 153.2, 151.8, 148.5, 148.3, 143.2, 138.7, 135.8, 122.9, 119.2, 115.0, 112.0, 43.4, 31.5, 29.2, 29.1, 29.0, 26.6, 22.6, 14.1. MS (m/z) 263 [(M - C10 ₄ - Octyl)] ⁺ .

2-(3-Bromophenyl)- 1 -octylimidazoi 1 ,2-alpyrimidin- 1 -ium perchlorate ¾ NMR (300 MHz, DMSO-d ₆ ): δ = 9.39 (m, 1H), 9.18 (m, 1H), 8.60 (s, 1H), 7.90 (m, 2H), 7.79 (m, 1H), 7.73 (m, 2H), 3.81 (t, / = 7.5 Hz, 2H), 1.65 (m, 2H), 1.33 (m, 10H), 0.91 (t, / = 7.5 Hz, 3H). ¹³ C NMR (75.5 MHz, DMSO-d ₆ ): δ = 158.1, 143.2, 138.7, 136.8, 132.9, 131.9, 125.2, 124.8, 114.8, 111.4, 43.1, 31.5, 29.2, 29.0, 28.9, 26.4, 22.3, 14.2. MS (m/z) 273 [(M - CIO ₄ - Octyl)] ⁺ .

2-(4-Methylthiophenyl)- 1 -octylimidazoi 1 ,2-alpyrimidin- 1 -ium perchlorate. H NMR (300 MHz, DMSO-d ₆ ): δ = 9.36 (m, 1H), 9.14 (m, 1H), 8.56 (s, 1H), 7.77 (m, 1H), 7.75 (d, / = 8.3 Hz, 2H), 7.54 (d, / = 8.3 Hz, 2H), 3.99 (s, 3H), 2.58 (s, 3H). ¹³ C NMR (75.5 MHz, DMSO-d ₆ ): δ = 157.6, 143.2, 142.9, 138.5, 137.6, 130.2 (x2), 126.3 (x2), 121.4, 112.5, 42.4, 31.0, 29.2, 29.0, 28.6, 26.7, 22.1, 14.5. MS (m/z) 241 [(M - C10 ₄ - Octyl)] ⁺ .

EXAMPLE 7 - biofilm formation inhibiting activity of substituted imidazori ,2-al- pyrimidinium bromide and perchlorate salts Evaluation of the biofilm inhibitory activity of some substituted imidazo[l,2-a]- pyrimidinium bromide and perchlorate salts produced according to examples 3 and 5 was carried out by growing up biofilms of S. Typhimurium ATCC14028 or SL1344 or Pseudomonas aeruginosa PA 14 in the presence of different concentrations (1/2 dilution series) of biofilm inhibitors, by using the Calgary biofilm device. After an incubation period of 24 hours at 25 °C the biofilms were visualized by crystal violet staining. In the next step the stain was extracted from the biofilm with 30% acetic acid. The absorbance of the resolubilized stain at 57 _{bd C2C3} oun-0 nm was taken as a measure of the amount of biofilm formed. From these measurements the concentration of the test compound at which 50% biofilm inhibition was observed (IC ₅₀ value) was calculated. The IC ₅₀ values of representative imidazo[l,2-a]-pyrimidinium salts for anti-biofilm activity are shown in Table 15 wherein (1)IC50 refers to 5. Typhimurium ATCC14028, and (2)IC50 refers to P. aeruginosa PA14, ND stands for "not determined", and wherein Ph stands for phenyl, n-hep stands for n-heptyl, c-hep stands for cycloheptyl, etc.

Table 15

(1) (2)

m

cc

IC50 IC50

(μΜ) ³ (μΜ)

n-Hexyl OH Ph single Br ^" 102 3,13

n-Hep OH Ph single Br ^" 23,36 5,887

n-Octyl OH Ph single Br ^" 9,774 11,22

n-Nonyl OH Ph single Br ^" 8,255 4,852

n-Decyl OH Ph single Br ^" 15,01 3,946

n-Und OH Ph single Br ^" 24,61 28,63

n-Dod OH Ph single Br ^" 27,52 9,635

n-Trd OH Ph single Br ^" 81,64 17,04

n-Tet OH Ph single Br ^" 179,6 NA ^C

c-Hep OH Ph single Br ^" 84,15 5,811

c-Octyl OH Ph single Br ^" 58,36 4,629

n-Hexyl OH 4-CIPh single Br ^" 13,69 20,23

n-Hep OH 4-CIPh single Br ^" 6,667 - 12.46

n-Octyl OH 4-CIPh single Br ^" 6,982 11,7

n-Non OH 4-CIPh single Br ^" 18,32 60,47

n-Decyl OH 4-CIPh single Br ^" 51,33 192,8 n-Und OH 4-CIPh sing le Br ^" 61,85 205 n-Dod OH 4-CIPh sing le Br ^" 122,1 119,2 n-Trd OH 4-CIPh sing le Br ^" 316,1 59 c-Hep OH 4-CIPh sing le Br ^" 27,87 63,77 c-Octyl OH 4-CIPh sing le Br ^" 12,62 15,55 n-Hexyl OH 4-N0 ₂ Ph sing le Br ^" 54,69 10,86 n-Octyl OH 4-N0 ₂ Ph sing le Br ^" 6,62 7,65 n-Decyl OH 4-N0 ₂ Ph sing le Br ^" 9,946 ND ^d n-Octyl OH 4-FPh sing le Br ^" 7,575 7,124 n-Decyl OH 4-FPh sing le Br ^" 20,01 ND n-Octyl OH 3,4-diCIPh sing le Br ^" 7,094 - 12.29 n-Decyl OH 3,4-diCIPh sing le Br ^" 32,4 ND n-Octyl OH 4-MeOPh sing le Br ^" 10,53 13,57 n-Decyl OH 4-MeOPh sing le Br ^" 18,6 ND n-Octyl OH 4-MeSPh sing le Br ^" - 13.21 29 n-Decyl OH 4-MeSPh sing le Br ^" 45,74 ND n-Oct OH 1-naptnyl sing le Br ^" 14,72 16,66 n-Dec OH 1-napthyl sing le Br ^" 68,85 ND n-Octyl OH 4-(4'-N0 ₂ Ph)Ph sing le Br ^" 31,03 118,6 n-Decyl OH 4-(4'-N0 ₂ Ph)Ph sing le Br ^" 200 ND n-Octyl OH 4-S0 ₂ MePh sing le Br ^" 60,29 11,46 n-Decyl OH 4-S0 ₂ MePh sing le Br ^" 21,87 ND

[1,1':4',1"- n-Octyl OH terphenyl]-4-yl sing le Br ^" - 52.81 26,76 n-Octyl OH 3-BrPh sing le Br ^" 7,133 - 13.24 n-Decyl OH 3-BrPh sing le Br ^" 33,77 ND

4"-N0 ₂ [l,l':4',l"- n-Decyl OH terphenyl]-4-yl single Br ^" 525,2 ND n-Octyl NP ^b 4-OMePh double CI0 ₄ ^" 34,75 8,732 n-Octyl NP 4-FPh double CIO ₄ ^" 35,67 19,51 n-Octyl NP 3,4-diFPh double CIO ₄ ^" 3,84 12,69 n-Octyl NP 3-BrPh double CI0 ₄ ^" 7,332 6,996 n-Octyl NP 1-napthalenyl double CI0 ₄ ' 6.600 6,835 n-Octyl NP 4-(4'N0 ₂ Ph)Ph double CI0 ₄ ^" 1,492 1,04

n-Octyl NP 4-S0 ₂ MePh double CI0 ₄ ^" 81,95 49,63

n-Octyl NP 4-SMePh double CIO _a 20,65 9,295

[1,1':4',1"-

-Octyl NP terphenyl]-4-yl double ND

EXAMPLE 8 - biofilm formation inhibiting activity of substituted imidazo|T,2-al- pyrimidinium bromides

We herein assayed the influence of some 2-hydroxy-2-phenyl-2,3-dihydro- imidazopyrimidinium bromides (compounds 1-50 in table 16) for their ability to prevent the biofilm formation of S. Typhimurium ATCC 14028 and P. aeruginosa PA 14 at 25 °C. As shown in table 16 (wherein nd stands for "not determined"), these salts do reduce the biofilm formation to a certain extent.

Table 16

S.

Typhimurium P. aeruginosa

Compound Rl R IC50 ^a ^M) IC50 (μΜ)

1 Me H >800 211.8

2 i-Pr H 540.5 >400

3 n-Bu H 409.5 >400

4 i-Bu H 131.2 289.4

5 n-Pen H 278.2 565.4

6 n-Hex H 102.0 405.8

7 n-Hep H 23.4 24.8 8 n-Oct H 9.8 18.9

9 n-Non H 8.3 55.7

10 n-Dec H 15.0 47.0

11 n-Und H 24.6 >800*

12 n-Dod H 27.5 >800*

13 n-Trd H 81.6 >800*

14 n-Tet H 179.6 >800*

15 Me 4-CI 225.0 nd

16 n-Bu 4-CI 29.7 24.9

17 i-Bu 4-CI 31.4 83.4

18 n-Pen 4-CI 32.6 163.9

19 n-Hex 4-CI 13.7 79.8

20 n-Hep 4-CI 6.7 36.9

21 n-Oct 4-CI 7.0 21.7

22 n-Non 4-CI 18.3 21.0

23 n-Dec 4-CI 51.3 66.4

24 n-Und 4-CI 61.9 >800*

25 n-Dod 4-CI 122.1 >800

26 n-Trd 4-CI 316.1 >800

27 n-Tet 4-CI >800 >800

28 Me 4-N0 ₂ 698.3 nd

29 Et 4-N0 ₂ 578.8 >800

30 i-Pr 4-N0 ₂ 466.5 191.9

31 n-Bu 4-N0 ₂ 292.8 145.2

32 i-Bu 4-N0 ₂ 322.5 191.7

33 t-Bu 4-N0 ₂ 211.1 41.5

34 n-Hex 4-N0 ₂ 54.7 12.4

35 n-Oct 4-N0 ₂ 6.6 19.7

36 n-Dec 4-N0 ₂ 9.9 22.3

37 Me 4-F >800 nd

38 n-Bu 4-F 163.0 nd

39 n-Oct 4-F 7.6 107.7

40 n-Dec 4-F 20.0 nd

41 n-Bu 4-Br 90.1 nd

42 i-Bu 4-Br 5.2 nd

43 n-Pen 4-Br 1.7 nd

44 Me 3-N0 ₂ 563.8 nd

45 n-Bu 4-MeO 314.4 nd

46 n-Oct 4-MeO 10.5 90.2

47 n-Dec 4-MeO 18.6 nd

48 i-Bu 3,4-diCI 8.0 nd

49 n-Oct 3,4-diCI 7.1 11.6

50 n-Dec 3,4-diCI 32.4 nd *Although IC50 > 800 μΜ, the compound inhibits biofilm formation to a certain extent, but the dose response curve reaches a steady state level of 25 to 45 % biofilm inhibition starting from concentrations between 25 and 50 μΜ.

Since salts with a n-octyl chain substituted at the 1 -position have the highest activity against Salmonella biofilms and also have a high activity against Pseudomonas biofilms, we decided to synthesize additional l-octyl-2-hydroxy-2-phenyl-2,3-dihydro- imidazopyrimidinium salts 51-56 with more variation in the substitution pattern of the 2- phenyl ring. As depicted in Table 17 showing influence on biofilm formation of S. Typhimurium ATCC 14028 and P. aeruginosa PA14 at 25°C, these compounds generally inhibit biofilm formation of S. Typhimurium at low concentrations.

Table 17

S. Typhimurium aeruginosa

IC50 ^a IC50

Compound R (μΜ) (μΜ)

51 4-SMe ~ 13.2 36.5

52 14.7 15.1

53 4-(4'-N02Ph) 31 215.2

54 4-S02Me 60.3 99.9

4-([l,l'-biphenylyl]-4-

55 yi) ~ 52.8 336.1

56 3-Br 7.1 29

We also synthesized a series of 2-hydroxy-2-phenyl-2,3-dihydro-imidazo- pyrimidinium salts with a broad variety of cycloalkyl substituents at the 1 -position, with lengths ranging from 3 to 12 carbon atoms (compounds 57-60 in Table 18) which do have a very strong effect against Salmonella biofilms and Pseudomonas biofilms at 25 °C.

Table 18

S. Typhimurium P. aeruginosa

Compound Rl R IC50 ^a ^M) IC50 (μΜ)

57 c-Bu H 493.0 >400

58 c-Hex H 139.0 87.0

59 c-Hep H 84.2 250.0

60 c-Oct H 58.4 176.3

61 c-Pr 4-CI 82.7 179.2

62 c-Bu 4-CI 120.8 371.5

63 c-Pen 4-CI 41.8 162.8

64 c-Hex 4-CI 33.0 10.0

65 c-Hep 4-CI 27.9 76.2

66 c-Oct 4-CI 12.6 22.7

67 c-Dod 4-CI 5.6 6.8

68 c-Pr 4-N0 ₂ 125.1 nd ^b

69 c-Dod 4-N0 ₂ <6.3 6.9

70 Adamantyl 4-N0 ₂ 8.8 14.4

Finally we synthesized an array of 2-hydroxy-2-phenyl-2,3-dihydro-imidazo- pyrimidinium salts, which are substituted at the 1-position with a benzyl (compounds 71- 73), para-methoxybenzyl (compound 74), 3-methoxyphenethyl (compounds 75-78) or piperonyl (compounds 79-80) group (Table 19 showing influence on biofilm formation of S. Typhimurium ATCC 14028 at 25°C). Table 19

IC50 ^a

Compound R (μΜ) Effect on growth at: ^b

Έ Έ Έ Έ Έ

o

o o o o

71 H 211.9

72 4-N0 ₂ 174.3

73 4-1 36.66 o - -

74 4-Br 109.8

75 4-N0 ₂ 123.5

76 4-F 105.2

77 3,4-diCI 7.229 o +

78 4-([l,l'-biphenylyl]-4-yl) 12.44 o O o

79 4-F 17.19 o - -

80 3,4-MethylenedioxyPh 53.73

EXAMPLE 9 - biofilm formation inhibiting activity of substituted 2-aminoimidazoles

We synthesized an array of 4(5)-phenyl-2-aminoimidazoles 2N-substituted with either a short n- or iso-alkyl chain (C1-C5) or a n-octyl chain. As depicted in table 20, a broad diversity of (substituted) 4(5)-phenyl groups were included such as phenyl (compounds 81-84), para-chlorophenyl (compounds 85-89), para-nitrophenyl (compounds 90-95), para-fluorophenyl (compounds 96-100), para-bromophenyl (compounds 101-105), para-methoxyphenyl (compounds 106-108), 3,4-dichlorophenyl (compounds 109-111), 3,4-difluorophenyl (compound 112), para-hydroxyphenyl (compound 113), naphtyl (compound 114), para-methylsulphonylphenyl (115-116) and meta-bromophenyl (117).

Table 20 shows the influence of 2N-alkylated 2-aminoimidazoles 81-117 on the biofilm formation and the planktonic growth of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25°C.

Table 20

S. Typhimurium P. aeruginosa

Effect on growth

Compound Rl R IC50 (μΜ) ³ Effect on growth at: IC50 (μΜ) ³

i. i. ι. Έ Έ Έ Έ Έ Ξ. Έ Έ Έ Έ

5 =1 =1 =1 =5. Έ o o

81 H H 130.2 - 72.6 -

82 i-Pr H >800 261.2

83 n-Bu H 25.3 - 31.8

84 i-Bu H 4.9 - - 1.2 -

85 H CI 16.0 - 3.5 -

86 i-Bu CI 2.0 + - 0.9 -

87 n-Pe n CI >400 -6.25 *

88 n-Oct CI >400 -1.6*

89 n-Non CI >800 nd ^c

90 H N0 ₂ 17.6 o - - 34.5 +

91 Me N0 ₂ 170.4 56.1 -

92 Et N0 ₂ 171.3 103.9

93 n-Bu N0 ₂ 701.5 - >800

94 i-Bu N0 ₂ 3.8 - - 22.9 -

95 n-Oct N0 ₂ 10.9 - - - -25.0*

96 H F 84.4 - 15.0 -

97 Me F 101.7 25.2 -

98 n-Bu F 15.5 - - 10.8 -

99 n-Hex F >400 -12.5 *

100 n-Oct F >400 -3.1*

101 H Br 47.9 - 3.2 + - -

102 n-Bu Br 7.1 - - 9.8 -

103 i-Bu Br 2.9 + - 1.2 -

104 n-Pen Br 3.1 + + 10.2 -

105 n-Oct Br 1.9 >800

106 H 4-OMe 119.7 186.3

107 n-Bu 4-OMe 52.6 - - - 46.3 -

108 n-Oct 4-OMe -400* >800

109 Pr 3,4-diCI 10.9 - - 27.7

110 n-Pen 3,4-diCI 2.2 0 + 0.7 -

111 n-Oct 3,4-diCI 118.3* >800

112 Me 3,4-diF 601.9 nd

113 Me 4-OH 160.3 - nd

114 n-Oct 238.4* >800

115 H 4-S0 ₂ Me >800 >800

116 n-Oct 4-S0 ₂ Me 41.8 - - -3.1* -

117 n-Oct 3-Br 44.64 - - >800 We also synthesized an array of 2-aminoimidazoles substituted at the 2N-position with cyclo-alkyl groups with intermediate length, i.e. cyclo-pentyl (compounds 117-121) and cyclo-hexyl (compounds 122-126). Table 21 below shows the Influence of 2N-cyclo- alkyl-2-aminoimidazoles 117-126 on the biofilm formation an the planktonic growth of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25°C

Table 21

S. Typhimurium P. aeruginosa

Effect on growth Effect on

Ι050 (μΜ)" at: ^b IC50 (μΜ) ³ growth at:'

Έ Έ Έ Έ Έ Έ Έ Έ Έ

:± =_ =_ =_ =_ i i i i

o o o o o o o -n o

117 c-Pen H 52.9 - - 33.8

118 c-Pen 4-CI 4.4 13.5

119 c-Pen 4-N0 ₂ 11.8 - - 20.2

120 c-Pen 4-Br 12.1 - - 7.2

121 c-Pen 3,4-diCI 5.7 - - 7.9

122 c-Hex 4-CI >800 ~25*

123 c-Hex 4-NO2 36.7 ~25*

124 c-Hex 3-NO2 629.5 4.4

125 c-Hex 4-OMe ~ 377.6 54.8

126 c-Hex 4-SMe 191.3 15.8

Finally we synthesized an array of 4(5)-phenyl-2-aminoimidazoles, which are substituted at the 2N-position with a benzyl (compound 127), para-methoxybenzyl (compound 128), 3- methoxyphenethyl (compounds 129-131) or piperonyl group (compounds 132-133) which (Table 22) were tested against both Salmonella and Pseudomonas biofilm formation at 25°C.

Table 22

S. Txphimuriian P. aeruginosa

Compound Ι€50 (μΜ) ³ Effect on growth at: IC50 (μΜ) ³ o o

127 4-CI 30.73 26.27

128 4-N0 ₂ >800 >800

129 4-N0 ₂ 19.07 3.704

130 3,4-diCI >800 >800

131 4-([l,l'-biphenylyl]-4-yl) >800 >800

132 4-F 66.59 455.5

133 261 10.52

Effect on | growth at: ^b

o 0

■3-