N-alkyl-2-substituted ATP analogs for use as antibacterial agents

Abstract

The invention relates to compounds of formula (I) wherein R1, R2, R3 and X have the same meaning as the meanings defined in the claims and the description, tautomers, enantiomers or diastereomers thereof. The invention also relates to the use of such compounds as inhibitors of bacterial adhesion on surfaces and biofilm formation, ex vivo methods for preventing bacterial growth in biofilm formation and the use of such compounds in the diagnosis or prognosis of bacterial infections. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the newly discovered antibacterial properties of N-alkyl-2-substituted ATP analogs for clinical use in another indication. The newly discovered antibacterial properties of the analogs can be applied to prevent or treat bacterial infections in subjects; in addition, the N-alkyl-2-substituted ATP analogs can be used ex vivo or in vitro and used to inhibit bacterial adhesion and biofilm formation on surfaces. Background Art

[0002] Cangrelor is a synthetic analog of adenosine triphosphate (ATP), which belongs to the N-alkyl-2-substituted ATP analogs and is a P2Y 12 A potent antagonist of the P2Y receptor 12 The receptor is a G protein-coupled purinergic receptor, which is an important component of platelet activation. Cangrelor is represented by the following formula II:

[0003]

[0004] or represented by its sodium salt represented by formula III:

[0005]

[0006] Cangrelor is the only intravenous platelet P2Y 12 It provides a rapid, potent and reliable antiplatelet effect. Such pharmacological properties allow overcoming oral P2Y 12 Limitations of oral P2Y inhibitors 12 Inhibitors are characterized by an inevitably delayed onset of action, which is enhanced in the high-risk short-term setting where gastrointestinal absorption is further impaired (De Luca et al., J Am Heart Assoc. 2021;10:e022125).

[0007] Antibiotics are used to prevent or treat bacterial infections. However, due to the ability of bacteria to rapidly develop resistance mechanisms and the misuse of antibiotics, we are currently facing a global spread of resistant bacteria throughout the world. Currently available antibiotics are becoming increasingly ineffective and have limited treatment options for common infections such as urinary tract infections, sepsis, sexually transmitted infections, and certain forms of diarrhea. This phenomenon also threatens the safety of surgery, chemotherapy, or other treatments.

[0008] The World Health Organization predicts that by 2050, antibiotic resistance could cause 10 million deaths each year. A study in The Lancet (2022) showed that in 2019, resistant bacteria directly caused 1.27 million deaths (Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022 Feb 12; 399(10325): 629-655. doi: 10.1016 / S0140-6736(21)02724-0).

[0009] Infections caused by Gram-positive bacteria represent a major public health burden, not only in terms of morbidity and mortality, but also in terms of increased expenses related to patient management and implementation of infection control measures. More specifically, Staphylococcus aureus and enterococci are established pathogens in hospital settings, and their frequent multidrug resistance complicates treatment.

[0010] Staphylococcus aureus is an important, highly virulent pathogen causing a wide range of clinical manifestations from relatively benign skin infections to life-threatening conditions such as endocarditis and osteomyelitis. It is also a commensal bacterium (colonizing approximately 30% of the human population).

[0011] Since the 1990s, there have been two major shifts in the epidemiology of S. aureus: an increase in the prevalence of community-associated skin and soft tissue infections (driven primarily by specific methicillin-resistant S. aureus [MRSA] strains), and an increase in the number of health care-associated infections (particularly infective endocarditis and prosthetic device infections). Patients with MRSA infections are 64% more likely to die than those with drug-susceptible infections.

[0012] Therefore, there is an urgent need in the art for new antibacterial therapeutic alternatives.

[0013] The pathogenicity of S. aureus depends on the expression of a wide range of virulence factors. Among these factors, toxins are key virulence determinants with cytotoxic properties that allow the bacteria to evade the host immune system. The carotenoid pigment staphyloxanthin helps S. aureus resist reactive oxygen species-dependent killing by host neutrophils. Bacterial virulence is also determined by the ability of bacteria to adhere to host tissues or surfaces by different mechanisms, which is an initiating step in bacterial colonization, biofilm formation, and infection.

[0014] Antibacterial therapeutic alternatives to typical antibiotics involve agents that can inhibit bacterial virulence factors (so-called anti-virulence agents). Clinical trials are currently testing such anti-virulence agents for the prevention or treatment of multi-resistant bacterial infections (Imperi F, Chen W, Smani Y. Front Microbiol. 2021; 12). These anti-virulence agents represent valuable alternatives to antibiotics (Ogawara H. J Antibiot (Tokyo). 2021; 74: 24-41). In fact, compared to antibiotics, these drugs do not exert high selective pressure on bacteria, thereby limiting the development of resistance and the spread of virulence genes.

[0015] In 2017, C. Oury et al. described in EP3509598B1 the novel use of triazolo(4,5-D)pyrimidine derivatives for preventing and treating bacterial infections, but triazolo(4,5-D)pyrimidine derivatives (such as Ticagrelor) are generally insoluble in aqueous environments or media. Summary of the invention

[0016] The present invention is based on the unexpected discovery that at least one of the above objectives can be achieved by N-alkyl-2-substituted ATP analogs that are clinically useful for another indication.

[0017] The present invention provides N-alkyl-2-substituted ATP analogs with antibacterial properties. The analogs can inhibit the hemolytic activity of clinically relevant strains of Staphylococcus aureus (e.g., clinical isolates or methicillin-resistant strains from patients with infective endocarditis). In addition, the inventors have observed that the analogs of the present invention can inhibit the production of the main bacterial pigment (staphyloxanthin). Since staphyloxanthin has antioxidant activity, it helps bacteria escape the killing of reactive oxygen species produced by phagocytic innate immune cells, so these data further show that the analogs of the present invention can reduce the virulence of Staphylococcus aureus. In addition, the inventors have observed that the analogs of the present invention can reduce bacterial adhesion. Therefore, the compounds of the present invention are suitable for use as antibacterial agents, more particularly for the treatment and / or prevention of diseases caused by bacterial infections. Antibacterial agents also refer to anti-toxic agents or anti-infective agents herein.

[0018] Compared to triazolo(4,5-D)pyrimidine derivatives (e.g., ticagrelor), the compounds of the present invention are highly soluble in aqueous environments or media. This property will enable administration via the intravenous route, resulting in a very rapid onset of action, which will be particularly important in short-term settings with a high risk of impaired gastrointestinal absorption. The water-soluble properties will also be advantageous for the use of the compounds of the present invention in water-based compositions or coatings, such as hydrogels or nanogels or nanodispersions, etc.

[0019] A first aspect of the present invention provides a compound of formula I, a tautomer, enantiomer or diastereomer thereof, or a salt or solvate thereof or a solvate of such a salt for use in preventing or treating a bacterial infection in a subject,

[0020]

[0021] in,

[0022] R 1 and R 2 are each independently hydrogen or halogen;

[0023] R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen;

[0024] R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl,

[0025] X is an acidic moiety.

[0026] The present invention also relates to a pharmaceutical composition comprising a compound of formula (I) for use in preventing or treating a bacterial infection in a subject.

[0027] Another related aspect of the invention provides the use of a compound of formula I as described herein as an antibacterial agent, more specifically, as an inhibitor of bacterial adhesion and biofilm formation on a surface. In some embodiments, the use is a non-therapeutic use.

[0028] Another related aspect of the invention provides an ex vivo method for preventing bacterial growth in biofilm formation comprising applying or grafting onto a surface an effective amount of a compound of formula I as described herein.

[0029] A further related aspect of the invention provides a compound of formula I as described herein for use in the diagnosis or prognosis of a bacterial infection.Such diagnosis or prognosis may be performed, for example, using a detectable marker or using any suitable method. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, its application, or uses.

[0031] Figure 1 Shown is a graph plotting the percentage of hemolytic activity of Staphylococcus aureus (IE strain) isolated from patients with infective endocarditis against control or 0.5 μg / ml cangrelor tetrasodium. The hemolytic activity obtained against the control was arbitrarily set to 100%.

[0032] Figure 2 Shown is a graph plotting the percentage of hemolytic activity of the methicillin-resistant JE2 (USA300) Staphylococcus aureus strain against a control or 0.5 μg / ml of cangrelor tetrasodium. The hemolytic activity obtained against the control was arbitrarily set to 100%.

[0033] Figure 3 Shown is a graph plotting the percentage of pigment production (staphyloxanthin) of IE strain of S. aureus against control or 0.5 μg / ml cangrelor tetrasodium. The pigment production obtained against the control was arbitrarily set to 100%.

[0034] Figure 4 Shown is a graph plotting the percentage of staphyloxanthin production by JE2 S. aureus in the presence of control or 1.0 μg / ml cangrelor tetrasodium. A paired t-test was performed with p-values ​​*<0.05.

[0035] Figure 5 Shown is a graph plotting the percentage of JE2 S. aureus adhesion to Von Willebrand factor (VWF) under flow in the presence of control or 1.0 μg / ml cangrelor tetrasodium.

[0036] Figure 6 Shown is a graph plotting the percentage of JE2 S. aureus adhesion to fibrinogen (Fg) in the presence of control or 1.0 μg / ml cangrelor tetrasodium.

[0037] Figure 7 Shown are the area under the curve (AUC) obtained from bacterial growth curves of JE2 S. aureus treated with control or 1 μg / ml, 20 μg / ml and 40 μg / ml cangrelor.

[0038] Figure 8 Shown are the maximum metabolic activities of JE2 S. aureus treated with control or 1 μg / ml, 20 μg / ml and 40 μg / ml cangrelor. A one-way ANOVA with Tukey's multiple comparisons was performed. DETAILED DESCRIPTION

[0039] Before describing the present invention, it should be understood that the present invention is not limited to the particular processes, methods and compounds described, as such processes, methods and compounds may of course vary. It should also be understood that the terminology used herein is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0040] When describing the compounds and methods of the present invention, the terms used are to be interpreted in accordance with the following definitions unless the context indicates otherwise.

[0041] As used in the specification and the appended claims, unless the context clearly indicates otherwise, nouns modified by unquantified words refer to both one and more than one. For example, "a compound" means one compound or more than one compound.

[0042] As used herein, the terms "comprising" and "consisting of" are synonymous with "including" or "containing" and are inclusive or open-ended and do not exclude additional non-recited members, elements, or method steps. The terms "comprising" and "consisting of" also include the term "consisting of".

[0043] The term "about" as used herein in relation to measurable values ​​such as parameters, amounts, temporal durations, etc., is intended to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less, of or relative to the specified value, within which such variations are suitable for implementation in the disclosed invention. It should be understood that the value to which the modifier "about" refers is itself also specifically and preferably disclosed.

[0044] As used herein, the term "and / or" when used with a list of two or more items means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a list is described as containing groups A, B, and / or C, the list can contain: A alone; B alone; C alone; A and B combined; A and C combined; B and C combined; or A, B, and C combined.

[0045] Numerical ranges expressed by endpoints include all integer numbers and fractions where appropriate that fall within the range (e.g., 1 to 5 may include 1, 2, 3, 4 when referring to, for example, a number of elements, and may also include 1.5, 2, 2.75, and 3.80 when referring to, for example, measured values). The expression of endpoints also includes the endpoint values ​​themselves (e.g., from 1.0 to 5.0 includes both 1.0 and 5.0). Any numerical range recited herein is intended to include all subranges contained therein.

[0046] References to "one embodiment" or "an embodiment" throughout this specification are intended to mean that the described particular features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" that appear in multiple locations throughout this specification are not necessarily all but may refer to the same embodiment. In addition, in one or more embodiments, as will become apparent to those skilled in the art from this disclosure, the particular features, structures, or characteristics may be combined in any suitable manner. In addition, although some embodiments described herein include some features but do not include other features included in other embodiments, as will be appreciated by those skilled in the art, the combination of features of different embodiments is intended to be within the scope of the present invention and to form different embodiments. For example, in the following claims, any claimed embodiment may be used in any combination.

[0047] Unless otherwise defined, all terms (including technical terms and scientific terms) used to disclose the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs. Through further guidance, the definitions of the terms used in the specification are included to better understand the teachings of the present invention.

[0048] When describing the present invention, the terms used should be interpreted in accordance with the following definitions, unless the context indicates otherwise.

[0049] The above terms and other terms used in the specification are well understood by those skilled in the art.

[0050] Whenever the term "substituted" is used herein, it is intended to indicate the replacement of one or more hydrogen atoms on the indicated atom in the expression using "substituted" with a selection from the indicated group, provided that the normal valence of the indicated atom is not exceeded and that the substitution results in a chemically stable compound, i.e., a compound sufficiently robust to survive isolation from a reaction mixture.

[0051] When a group may be substituted, such group may be substituted with one or more, and preferably one, two or three substituents. Preferred substituents may be selected from, but are not limited to, for example, the group comprising halogen, hydroxy, alkyl, alkoxy, trifluoromethyl, trifluoromethoxy, cycloalkyl, aryl, arylalkyl, heterocyclyl, heteroaryl, cyano, amino, nitro, carboxyl and mono- or di-alkylamino.

[0052] The term "halo" or "halogen" as a group or part of a group is generic for fluorine, chlorine, bromine, iodine.

[0053] The term "alkyl" as a group or part of a group refers to a group of the formula -C n H 2n+1 wherein n is a number greater than or equal to 1. The alkyl group may be straight or branched and may be substituted as indicated herein. In general, the alkyl groups of the present invention contain 1 to 6 carbon atoms, preferably 1 to 5 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 1 to 2 carbon atoms. When a subscript is used after a carbon atom herein, the subscript refers to the number of carbon atoms that the named group may contain. For example, "C 1-6 "Alkyl" includes all straight-chain or branched alkyl groups having 1 to 6 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, butyl and isomers thereof (e.g., n-butyl, isobutyl and tert-butyl); pentyl and isomers thereof, hexyl and isomers thereof. For example, "C 1-5 "Alkyl" includes all straight or branched chain alkyl groups having 1 to 5 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, butyl and isomers thereof (e.g., n-butyl, isobutyl and tert-butyl); pentyl and isomers thereof. For example, "C 1-4 "Alkyl" includes all straight or branched chain alkyl groups having 1 to 4 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, butyl and isomers thereof (e.g., n-butyl, isobutyl and tert-butyl). For example, "C 1-3 The term "alkyl" includes all straight-chain or branched alkyl groups having 1 to 3 carbon atoms and thus includes methyl, ethyl, n-propyl, isopropyl.

[0054] The term "alkoxy" or "alkyloxy" as a group or part of a group refers to a radical having the formula -OR b A group in which R bis an alkyl group as defined herein above. Some non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and hexyloxy.

[0055] The term "C 1-6 "Alkylthio" as a group or part of a group refers to a group having the formula -SR b A group in which R b is an alkyl group as defined herein above. Suitable C 1-6 Some non-limiting examples of alkylthio include methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio, and hexylthio.

[0056] The term "monoalkylamino or dialkylamino" as a group or part of a group refers to a group of the formula -N(R o )(R p ) group, wherein R o and R p Each is independently selected from hydrogen or alkyl, wherein R o or R p At least one of is an alkyl group. Thus, alkylamino includes monoalkylamino (e.g., mono-C 1-6 Alkylamino, such as methylamino and ethylamino) and dialkylamino (such as di-C 1-6 Some non-limiting examples of suitable mono- or di-alkylamino groups include n-propylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, n-hexylamino, di-n-propylamino, diisopropylamino, ethylmethylamino, methyl-n-propylamino, methyl-isopropylamino, n-butylmethylamino, isobutylmethylamino, tert-butylmethylamino, ethyl-n-propylamino, ethyl-isopropylamino, n-butylethylamino, isobutylethylamino, tert-butylethylamino, di-n-butylamino, diisobutylamino, methylpentylamino, methylhexylamino, ethylpentylamino, ethylhexylamino, propylpentylamino, propylhexylamino, and the like.

[0057] Structural isomers are a type of isomers in which molecules with the same molecular formula have different bonding patterns and atomic organizations. Tautomerism ("tautomerism") can occur when structural isomers are interconvertible via a low energy barrier. This can take the form of proton tautomerism in compounds of the invention containing, for example, imino, keto or oxime groups, or so-called valence tautomerism in compounds containing aromatic moieties. Compounds of formula I can exhibit tautomerism, such as imine-enamine tautomerism at the 6th position of adenine. The compounds also contain one or more asymmetric carbon atoms and therefore exhibit optical and / or diastereoisomerism.

[0058] The present invention includes all possible stereoisomer compounds of formula I and any subgroup thereof. When the desired compound is as a single enantiomer, such can be synthesized by stereospecificity, by the decomposition of the final product or any convenient intermediate, or obtained by chiral chromatography, because each is known in the art. The decomposition of the final product, intermediate or starting material can be achieved by any suitable method known in the art. For stereochemistry, see, for example, ELEiel, SH Wilen, and LN Mander's Stereochemistry of Organic Compounds (Wiley-Interscience, 1994), which is incorporated by reference.

[0059] Some preferred statements (features) and embodiments of the compounds and methods of the present invention are now described. Unless clearly indicated to the contrary, each statement and embodiment of the present invention so defined may be combined with any other statement and / or embodiment. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0060] A first aspect of the present invention provides a compound of formula I, a tautomer, enantiomer or diastereomer thereof, or a salt or solvate thereof or a solvate of such a salt for use in preventing or treating a bacterial infection in a subject,

[0061]

[0062] in,

[0063] R 1 and R 2 are each independently hydrogen or halogen;

[0064] R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen;

[0065] R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl,

[0066] X is an acidic moiety.

[0067] The present invention also relates to a pharmaceutical composition comprising a compound of formula I for use in preventing or treating a bacterial infection in a subject.

[0068] In addition to the compound of formula I, the pharmaceutical composition may also contain auxiliary substances, preservatives, solvents and / or viscosity modifiers. Solvents are meant, for example, water, saline solutions or any other physiological solutions, ethanol, glycerol, oils (e.g., vegetable oils), or mixtures thereof. Viscosity modifiers are meant, for example, carboxymethylcellulose.

[0069] Another related aspect of the invention provides the use of a compound of formula I as described herein as an inhibitor of bacterial adhesion and biofilm formation on a surface. In some embodiments, the use is a non-therapeutic use.

[0070] Another aspect of the present invention provides an ex vivo method for preventing bacterial growth in biofilm formation, comprising applying or grafting onto a surface an effective amount of a compound of formula I as described herein.

[0071] A further related aspect of the invention provides a compound of formula I as described herein for use in the diagnosis or prognosis of a bacterial infection.

[0072] For example, when used for diagnosis or prognosis, the compound of Formula I may contain a detectable marker. In some embodiments, the compound of Formula I contains an isotope. In some embodiments, the isotope is 18 F.

[0073] As used herein, the term "detectable marker" refers to any type of label that can be detected and thus allows the presence of a compound of Formula I to be determined. In some specific embodiments, the marker is an isotope that allows the use of a compound of Formula I as a radiotracer. In some embodiments, the isotope is 18 F.

[0074] Acidic moieties that may represent X include Bronsted-Lowry acids, i.e., moieties that act as proton donors. The acidic moiety may be a monoacid or a polyacid. In some embodiments, X is selected from -P(O)(OH)2, -SO3H, or -CO2H. In some embodiments, X is -P(O)(OH)2.

[0075] In some embodiments, R 4 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is substituted with one, two or three substituents. 4 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is substituted with one, two or three substituents independently selected from halogen. 4 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is substituted with two or three substituents selected from halogen, wherein the two or three substituents are the same.

[0076] In some embodiments, R 3 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is substituted with one, two or three substituents. 3 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is independently selected from C 1-6 In some embodiments, R 3 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is selected from C 1-6 In some embodiments, R 3 It is C 1-2 alkyl, and wherein the C 1-2 The alkyl group is substituted with one substituent selected from methylthio.

[0077] In some embodiments, R 1 and R 2 In some embodiments, R l and R 2 Same and represents Cl.

[0078] The compounds of the present invention may be in the form of salts as generally described below. Some preferred but non-limiting examples of suitable organic and / or inorganic acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citric acid, as well as other pharmaceutically acceptable acids known per se (see Stahl and Wermuth's Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002), which is incorporated herein by reference).

[0079] When the compounds of the present invention contain acidic groups as well as basic groups, the compounds of the present invention may also form internal salts, and such compounds are within the scope of the present invention. When the compounds of the present invention contain hydrogen-donating heteroatoms (e.g., NH), the present invention also encompasses salts and / or isomers formed by transferring the hydrogen atom to basic groups or atoms within the molecule.

[0080] The salt of the compound of formula I can be formed by free acid or its salt, or free base or its salt or derivative and one or more equivalents of suitable base or acid reaction. The reaction can be carried out in a salt-insoluble solvent or medium, or in a salt-soluble solvent (e.g., ethanol, tetrahydrofuran or diethyl ether), the solvent or medium can be removed in a vacuum, or removed by freeze drying or other methods known in the art. The reaction can also be a double decomposition process, or can be carried out on an ion exchange resin.

[0081] Salts of the compounds of formula I include acid addition salts and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Some examples include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, sucrose, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, and xinafoate. Suitable base salts are formed from bases that form non-toxic salts. Some examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases, such as hemisulphate and hemicalcium salts, can also be formed. For a review of suitable salts, see Stahl and Wermuth's Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002), which is incorporated herein by reference.

[0082] Salts of compounds of formula I include alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; salts of group III elements, such as aluminum salts; and ammonium salts. Salts with suitable organic bases, such as salts with hydroxylamine; lower alkylamines, such as methylamine or ethylamine; salts with substituted lower alkylamines, such as alkylamines substituted with hydroxyl; or salts with monocyclic nitrogen heterocyclic compounds, such as piperidine or morpholine; and salts with amino acids, such as salts with arginine, lysine, etc., or N-alkyl derivatives thereof; or salts with amino sugars such as N-methyl-D-glucosamine or glucosamine. In some embodiments, the salt is a pharmaceutically acceptable salt. Pharmaceutically acceptable salts constitute non-toxic physiologically acceptable salts of compounds of formula I described above.

[0083] In some embodiments, the compound is a salt; preferably the sodium salt of the compound of formula I.

[0084] In some embodiments, the compound of Formula I is:

[0085]

[0086] Also known as cangrelor tetrasodium or cangrelor tetrasodium.

[0087] One aspect of the present invention provides a compound according to formula I as described herein for use in preventing or treating a bacterial infection in a subject. In some embodiments, the bacterial infection is an infection caused by a gram-positive or gram-negative bacteria.

[0088] Therefore, the present invention also provides a method for treating a bacterial infection in a subject in need thereof, comprising administering to the subject an effective amount of a compound according to formula I as described herein.

[0089] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human host. In some specific embodiments, the infection is an infection of a body surface. Body surfaces include, but are not limited to, epidermis and mucosa. In some embodiments, the surface is a body surface. In some embodiments, the body surface is selected from an intact epidermal surface, a damaged epidermal surface, or a mucosal surface.

[0090] The damaged epidermal surface can be blistered, burned by fire, inflamed, pustular, sunburned, bitten, stung or otherwise injured skin. Some suitable examples of mucosal surfaces include the mucosa of the mouth (including the tongue), nose, eyes, throat, esophagus, stomach, vagina and rectum.

[0091] In some embodiments, the bacterial infection is an infection caused by gram-positive bacteria. Some examples of gram-positive bacteria are: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus anthracis, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Listeria monocytogenes, Nocardia sp., Streptococcus pneumoniae, Streptococcus agalactiae. In some embodiments, the bacterial infection is an infection caused by staphylococci; preferably an infection caused by Staphylococcus aureus; preferably an infection caused by methicillin-resistant Staphylococcus aureus.

[0092] In some embodiments, the bacterial infection is an infection caused by a gram-negative bacteria, such as Acinetobacter spp., such as Acinetobacter baumannii, Bordetella pertussis, Campylobacter spp.; Enterobacteriaceae, such as Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Salmonella spp., Serratia marcescens, Shigella spp., Yersinia spp.; Haemophilus influenzae, Helocobacter pylorilegionella pneumophila), Neisseria spp., Pseudomonas aeruginosa, Vibrio cholera, etc.

[0093] In some embodiments where the bacterial infection is caused by Gram-negative bacteria, the compound of Formula I as described herein is used with a membrane permeabilizing agent. In some embodiments, the membrane permeabilizing agent is selected from polymyxin B and polymyxin E.

[0094] Another related aspect of the invention provides the use of a compound of formula I as described herein as an inhibitor of bacterial adhesion and biofilm formation on a surface. In some embodiments, the use is a non-therapeutic use.

[0095] Surface means any type of surface, such as a rubber or plastic surface, such as, for example, a surface made of polyethylene, polypropylene, polyurethane, polyvinyl chloride, polyvinyl pyrrolidone, polytetrafluoroethylene, silicone, etc. or copolymers, but also and preferably a metal surface, such as stainless steel, silver, gold, titanium, metal alloys, pyrolytic carbon, etc. In some embodiments, the surface is the surface of a biomaterial of a medical device. The present invention can also be used for bioabsorbable or biomaterial surfaces, such as bioprostheses or devices made of biomaterials such as porcine or bovine pericardium.

[0096] S. aureus and coagulase-negative staphylococci (usually S. epidermidis) cause 65% to 75% of generator pocket infections and up to 89% of device-related endocarditis. Episodes occurring within two weeks of implantation are more likely to be due to S. aureus.

[0097] Prosthetic valve endocarditis (PVE) is a serious infection with potentially fatal consequences. Bacteria can reach the valve prosthesis through direct contamination during surgery or hematogenous spread in the first days and weeks after surgery. Because the valve sewing ring, cardiac annulus, and anchoring sutures are not endothelialized early after valve implantation, bacteria gain direct access to the prosthesis-ring interface and follow the suture path into the paravalvular tissue. These structures are coated with host proteins, such as fibronectin and fibrinogen, to which some organisms can adhere and initiate infection.

[0098] The risk of developing prosthetic valve endocarditis (PVE) is highest during the first three months after surgery, remains high through the sixth month, and then gradually decreases starting 12 months after surgery, with an annual incidence of approximately 0.4%. In studies with active follow-up, the percentage of patients who develop PVE during the first year after valve replacement is 1% to 3%; by five years, the cumulative percentage is 3% to 6%.

[0099] The most frequently encountered pathogens in early PVE (within two months of implantation) are Staphylococcus aureus and coagulase-negative staphylococci. The most frequently encountered pathogens in late PVE (two months after valve implantation) are Streptococci and Staphylococcus aureus, followed by coagulase-negative staphylococci and enterococci. The coagulase-negative staphylococci causing PVE during the first year after surgery are almost exclusively Staphylococcus epidermidis. 84% to 87% of these organisms are methicillin-resistant and therefore resistant to all beta-lactam antibiotics.

[0100] PVE is known to account for approximately 20% of all infective endocarditis. PVE is healthcare-associated in approximately 30% of cases. Staphylococcus aureus is the leading causative pathogen, accounting for more than 20% of PVE.

[0101] Periprosthetic joint infection (PJI) occurs in 1% to 2% of joint replacement surgeries and is the leading cause of arthroplasty failure. Biofilms play an important role in the pathogenesis of PJI. Bacteria within biofilms become resistant to treatment; therefore, antibacterial therapy is often unsuccessful unless the biofilm is physically disrupted or removed by surgical debridement. The management of PJI usually consists of both surgery and antibacterial therapy.

[0102] Therefore, the use of the compounds of formula I according to the present invention is advantageous in the treatment, prevention and management of the above mentioned conditions by applying the compounds of formula I to the surface of a medical device.

[0103] Medical devices include, but are not limited to, any device, tool, instrument, implant, etc., related to medicine or human or veterinary practice or intended for use in curing or treating a disease or condition. Medical devices may contain all natural and synthetic materials, as well as both fibrous and non-fibrous materials. For example, the material may be composed of: metal, plastic, paper, glass, ceramic, textile, rubber, polymer, composite material, or any other material or combination of materials. Some exemplary medical devices include, but are not limited to, catheters of any kind; cannulas; needles; stents of any size, shape, or placement; coils of any size, shape, or placement; contact lenses; IUDs; peristaltic pump chambers; endotracheal tubes; gastrointestinal feeding tubes; arteriovenous shunts; condoms; oxygenators and renal membranes; gloves; pacemaker guides; wound dressings; metal pins, plates, and screws; metal artificial hips; artificial knees, etc.

[0104] In some embodiments, the surface is the surface of a cardiovascular device or the surface of a catheter. In some embodiments, the surface is the surface of a prosthetic heart valve, a pacemaker, a cardioverter-defibrillator, a cardiac ablation catheter, a cardiovascular angioplasty device, a ventricular assist device, or a catheter.

[0105] Some examples of cardiovascular devices suitable for the present invention include: prosthetic heart valves, pacemakers, cardioverter-defibrillators, cardiac ablation catheters, cardiovascular angioplasty devices, ventricular assist devices (mechanical pumps), and the like.

[0106] The term "biomaterial" as used herein refers to a substance that is engineered to interact with a biological system for medical purposes. Biomaterials can be used in the following applications: joint replacements, bone plates, intraocular lenses, bone cement, artificial ligaments and tendons, dental implants, vascular prostheses, heart valves, skin repair devices (e.g., wound dressings), cochlear replacements, contact lenses, breast implants, drug delivery mechanisms, sustainable materials, vascular grafts, stents, nerve conduits, surgical sutures, clips and staples for wound closure, pins, screws for fracture stabilization, and surgical meshes, etc.

[0107] In some specific embodiments, the use according to this aspect of the invention encompasses applying a compound of formula I as described herein to said surface. In some specific embodiments, the application is ensured by spraying a composition comprising a compound of formula I on said surface.

[0108] The compound of formula I according to the present invention can be applied to the surface of the biomaterial or medical device in the form of a coating.

[0109] As used herein, a "coating" is a covering applied to a surface. Coating of a surface can be achieved by any method known in the art, such as, but not limited to, spraying or by immersing the device (or its surface) in a composition comprising a compound of Formula I.

[0110] Therefore, the present invention also provides a composition comprising:

[0111] - a compound of formula I as described herein;

[0112] -Solubilizing carrier.

[0113] Suitable solubilizing carriers for preparing compositions according to the present invention include water, water-based solutions, alcohols, dimethyl sulfoxide, and mixtures thereof. In some embodiments, the solubilizing carrier is a water-based solution. Suitable water-based solutions for preparing compositions according to the present invention include culture media (e.g., tryptic soy broth), saline solutions (particularly physiological saline solutions), physiological solutions (e.g., Frog Ringer solutions, Krebs' solutions, Tyrode solutions, Ringer-Lock solutions, DeJalen solutions, or artificial cerebrospinal fluid) and buffer solutions. In some preferred embodiments, the composition is in the form of nanodispersions or nanogels. Nanodispersions refer to compositions having at least one phase within the nanometer size range. Preferably, the compounds of formula I as described herein are in nanometer-sized phases. Nanogels refer to compositions based on polymers, in which polymers are cross-linked to form a network. Preferably, the compounds of formula I described herein are encapsulated in such a network. Advantageously, nanodispersions or nanogel compositions enhance the stability of compounds of formula I.

[0114] In some embodiments, the composition does not comprise cangrelor dihydrate:

[0115]

[0116] In some specific embodiments, the composition is a sprayable composition. Therefore, the present invention also provides a container equipped with a spraying device, such as but not limited to a spray can comprising a composition according to the present invention. In some specific embodiments, the composition (and / or container) also comprises a gas propellant.

[0117] The compounds of formula I according to the invention can also be encapsulated in polymer networks or gel-like structures, as described, for example, by C. Oury in WO2018 / 122318.

[0118] Yet another related aspect of the invention provides a medical device, preferably a cardiovascular device, most preferably a catheter, a prosthetic heart valve or a pacemaker, coated with a compound of formula I or a composition comprising a compound of formula I as described herein.

[0119] Another related aspect of the invention provides an ex vivo method for preventing bacterial growth in biofilm formation comprising applying or grafting onto a surface an effective amount of a compound of formula I as described herein.

[0120] Bacteria in biofilms produce extracellular polymeric substances (EPS), mainly composed of polysaccharides, nucleic acids (extracellular DNA) and proteins, which protect them from external threats, including immune system components and antimicrobial agents. In addition, bacteria in biofilms have reduced metabolism, making them less sensitive to antibiotics; this is due to the fact that most antimicrobials require a certain degree of cell activity in order to be effective. Another factor that enhances such resistance is that the diffusion of antimicrobial drugs throughout the biofilm is impaired due to the presence of the EPS matrix barrier.

[0121] It has been established that biofilm formation on any surface occurs in different states or steps. The initial step of biofilm formation is attachment / adhesion to the surface, which is stronger under shear stress conditions. The protein primarily responsible for this adhesion is polysaccharide intercellular adhesin (PIA), which allows bacteria to bind to each other and to the surface, producing a biofilm. The second stage of biofilm formation is the development of community structure and ecosystem, which produces a mature biofilm. The final stage is detachment from the surface, followed by spread to other locations. In all stages of biofilm formation, quorum sensing (QS) systems are involved, which mediate cell-to-cell communication.

[0122] As used herein, "preventing bacterial growth in biofilm formation" refers to inhibiting the formation of biofilms at all stages of biofilm formation, starting with step 1, prevention or inhibition of bacterial adhesion on surfaces, but also and primarily inhibiting bacterial growth, reproduction and formation of microcolonies on surfaces at step 2. Inhibition of biofilm formation during the maturation step 3 and inhibition of bacterial dispersion from the substrate in the colonization step are also contemplated in this definition. Bacteria may also be killed at all steps of biofilm formation.

[0123] Therefore, the use of compounds of formula I according to the invention is advantageous in inhibiting bacterial biofilm formation on surfaces.

[0124] In some embodiments, the method for controlling bacterial growth in biofilm formation on a surface comprises applying or grafting an effective amount of a compound of Formula I as described herein on the surface in a preventive step, or at a stage where a biofilm already exists, or even at a mature step of matrix formation (where a more complex biofilm structure is established, protecting bacteria as a barrier to conventional antibacterial agents), reducing the adhesion and survival of bacteria on the matrix or in the biofilm.

[0125] In some embodiments, methods for preventing bacterial growth may also be applied to surfaces of experimental devices in need of such antibacterial or anti-infective treatments.

[0126] Another related aspect of the present invention provides a compound of formula I as described herein for use in diagnosing or prognosing bacterial infections. Advantageously, the compound of formula I can bind to bacteria while simultaneously binding to platelets. Thus, it has a dual nature, enabling diagnosis or prognosis of bacterial infections. For example, the compound of formula I can bind to lipids present in bacterial membranes via its alkyl chain. Advantageously, by this dual binding possibility, the compound of formula I can accumulate at the site of infection and thereby act as a tracer.

[0127] The compounds of formula I according to the present invention can be prepared by methods known to those skilled in the art. In particular, the synthesis method described in WO9418216 can be used.

[0128] Example

[0129] The following examples are provided for the purpose of illustrating the present invention and should in no way be construed as limiting the scope of the present invention.

[0130] Bacterial strains and growth conditions

[0131] Staphylococcus aureus strains included previously characterized infective endocarditis (IE) clinical isolates (Liesenborghs L, Meyers S, Lox M, Criel M, Claes J, Peetermans M, Trenson S, VandeVelde G, Vanden Berghe P, Baatsen P, et al. Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heartvalves. Eur Heart J 2019; 40: 3248–3259) or JE2 (USA300MRSA). The bacteria were cultured in tryptic soy broth (TSB, Sigma) at 37°C with stirring (200 rpm). Prior to all experiments, a single colony from a TSA plate was used to inoculate 4 mL of TSB in a 15 mL polystyrene tube, and the bacteria were grown overnight. The liquid culture was then diluted 100-fold in 20 mL of fresh TSB and aliquoted in new tubes. For tetrasodium cangrelor treatment, different concentrations of the drug were added to the corresponding tubes. Tetrasodium cangrelor was dissolved in a culture medium (i.e., tryptic soy broth) containing nutrients and proteins for culturing bacteria, more specifically, the culture medium contained: casein, soy peptone, sodium chloride, dipotassium hydrogen phosphate, dextrose, water. Supernatants and bacterial precipitates were collected at designated times (exponential growth phase or stationary growth phase). The supernatant was filtered through a 0.22 μM cellulose acetate filter (VWR) and kept at -20°C until further use.

[0132] Bacterial markers

[0133] Staphylococcus aureus JE2 (USA300 MRSA) in exponential growth phase, untreated or treated with 1 μg / ml of cangrelor, was stained with 30 μg / ml of 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (Sigma-Aldrich) in phosphate-buffered saline (PBS) on a shaker at room temperature for 30 minutes. After incubation, the bacteria were centrifuged at 14,000×g for 5 minutes and the supernatant was removed. Next, a washing step was performed by resuspending the bacteria in PBS and removing the PBS after centrifugation (14,000×g for 5 minutes). The bacterial pellet was then resuspended in 100 μl of 6% bovine serum albumin (BSA). The stained bacteria were kept on ice until further use in the Von Willebrand factor (VWF) adhesion assay and fibrinogen (Fg) adhesion assay.

[0134] Preparation of red blood cell suspension and hemolysis assay

[0135] Freshly drawn citrate anticoagulated blood from healthy volunteers was centrifuged at 1,000 × g for 15 minutes. After discarding the upper layer of plasma, the red blood cells were washed once in phosphate buffered saline (PBS) and resuspended in 2% PBS. The supernatant from the overnight bacterial culture was mixed with a freshly prepared suspension of human red blood cells in a final ratio of 1:5 and incubated on a benchtop shaker (800 rpm) under gentle stirring for 30 minutes. The sample was then centrifuged at 4,000 × g for 5 minutes. The hemolytic activity in the supernatant was determined by measuring the absorbance at 570 nm in a spectrophotometer.

[0136] Analysis of staphyloxanthin production

[0137] Bacterial pellets from overnight cultures were resuspended in 400 μL methanol and incubated at 37 °C on a benchtop shaker with gentle agitation for 30 min. Samples were centrifuged at 14,000 × g for 5 min to remove cell debris. The pigment intensity in the supernatant was analyzed by measuring absorbance at 470 nm.

[0138] Example 1: Cangrelor Tetrasodium Inhibits Hemolytic Activity of Staphylococcus aureus Isolated from Patients with Infective Endocarditis

[0139] Supernatants were prepared from S. aureus IE clinical isolates grown to exponential phase in the absence (control) or presence (0.5 μg / mL) of cangrelor tetrasodium and added to erythrocyte suspensions for 30 minutes prior to assessment of hemolysis. Cangrelor tetrasodium was purchased from Bio-Techne Ltd., reference AR-C 69931 tetrasodium salt. A concentration of 0.5 μg / mL tetrasodium cangrelor corresponds to the steady-state concentration of the drug achieved in patients receiving conventional antiplatelet dosing (i.e., an intravenous bolus (30 μg / kg) followed by a 4-μg / kg / minute infusion) (Akers, WS, Oh, JJ, Oestreich, JH, Ferraris, S., Wethington, M. and Steinhubl, SR Pharmacokinetics and Pharmacodynamics of a Bolus and Infusion of cangrelor: A Direct, Parenteral P2Y12 Receptor Antagonist. The Journal of Clinical Pharmacology 2010; 50: 27-35). Figure 1 It is shown that bacteria grown in the presence of cangrelor tetrasodium resulted in a 90% loss of supernatant hemolytic activity compared to the vehicle control (set as 100%). These data demonstrate that cangrelor is able to reduce the virulence of clinical S. aureus strains.

[0140] Example 2: Cangrelor tetrasodium inhibits the hemolytic activity of methicillin-resistant Staphylococcus aureus

[0141] Supernatants were prepared from the methicillin-resistant JE2 (USA300) S. aureus strain grown to stationary phase in the absence (control) or presence (1 μg / mL) of cangrelor and added to erythrocyte suspensions for 30 minutes prior to assessment of hemolysis. Figure 2 It is shown that bacteria cultured in the presence of cangrelor resulted in a complete loss of supernatant hemolytic activity compared to vehicle control (set as 100%). These data indicate that cangrelor is able to reduce the virulence of methicillin-resistant Staphylococcus aureus strains.

[0142] Example 3: Cangrelor Tetrasodium Inhibits Staphyloxanthin Production in Staphylococcus aureus

[0143] The content of the major bacteriochrome, staphyloxanthin, was measured using protein extracts from S. aureus IE clinical isolates grown to exponential phase in the absence (control) or presence (0.5 μg / mL) of cangrelor. Figure 3 and Figure 4 It is shown that bacteria grown in the presence of Cangrelor resulted in a 90% loss of bacterial staphyloxanthin production compared to vehicle controls (set to 100%). Since staphyloxanthin has antioxidant activity, which helps bacteria escape killing by reactive oxygen species produced by phagocytic innate immune cells, these data further suggest that Cangrelor is able to reduce the virulence of S. aureus.

[0144] Example 4: Von Willebrand Factor Adhesion Assay

[0145] Glass coverslips (24×60 mm) were coated with 50 μg / ml VWF (Haemate P, CSL Behring, Mechelen, Belgium) for 4 h at room temperature. The coated coverslips were fixed in a flow chamber system (Provenzale, I., Brouns, SLN, van der Meijden, PEJ, Swieringa, F. & Heemskerk, JWM Hole blood based multiparameter assessment of thrombus formation in standard microfluidic devices to proxy in vivo haemostasis and thrombosis. Micromachines vol. 10 787 (2019). The labeled bacteria in the exponential phase were diluted to approximately 10 in TSB medium. 7 Up to 10 8 CFU / ml. Using the flow chamber system, 1 ml of the bacterial mixture was perfused onto the VWF-coated surface using a 1 ml syringe. The coverslip was fixed in the flow chamber and pumped with a high-precision Harvard pump (PHD 11plus, Harvard Apparatus, Holliston, MA, USA) at 1000 s. -1 The labeled bacteria were perfused at a shear rate of 1.54 Å for 10 min.

[0146] After perfusion, the coverslips were removed from the system and washed with PBS, followed by a 10-min fixation step with 4% paraformaldehyde (PFA, miliporefore, Merck). TMDiamond Antifade Mountant (Invitrogen) was used to fix the slides. Confocal microscopy was performed at 40× magnification and fluorescence intensity was measured using QuPath software (Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Sci. Rep. 7, 1-7 (2017)). Quantification of bacterial adhesion to VWF was determined by determining the percentage of positive pixels corresponding to the fluorescent signal and bacterial adhesion. Figure 5 It is shown that growing JE2 bacteria in the presence of 1 μg / ml Cangrelor resulted in reduced adhesion of bacteria to von Willebrand factor under flow compared to vehicle controls.

[0147] Example 5: Fibrinogen adhesion assay

[0148] A 96-well plate (cellstar, greiner) was coated with human plasma fibrinogen (Fg, Merck) at room temperature for 24 hours and then washed with PBS. Next, a blocking step of 1 hour was performed with 2% BSA, followed by a washing step with PBS. The labeled bacteria in the exponential phase were then diluted in TSB medium to obtain 109 CFU / ml of labeled bacteria. After this, the bacterial solution was incubated at 37°C for 1 hour and washed with PBS. The adherent bacteria were fixed with 4% PFA for 10 minutes and washed again. The bacterial adhesion was then quantified using a spectrophotometer (multi-mode microplate reader FilterMax F5, Molecular Devices) Ex.: 485nm and em.: 535nm, and the surface of the wells was scanned. The initial bacterial amount was plated on a TSA plate to determine the exact CFU / ml. Finally, the fluorescence signal was scaled according to the obtained CFU / ml. Figure 6 It is shown that growing JE2 bacteria in the presence of 1 μg / ml cangrelor resulted in reduced bacterial adhesion to fibrinogen compared to vehicle controls.

[0149] Example 6: Bacterial growth curve and metabolic activity

[0150] Bacterial growth curves and metabolic activity profiles of JE2 bacteria in the exponential phase were obtained after treatment with different concentrations of cangrelor (1 μg / ml, 20 μg / ml and 40 μg / ml). Bacterial growth curves were obtained by kinetic readings using a spectrophotometer, with readings at an optical density of 595 nm every 10 minutes for 7 hours under continuous shaking. The calscreener TMMetabolic activity profiles were obtained using Symcel (Symcel), which measured the metabolic response over time (15 hours) in the presence of varying concentrations of cangrelor. Figure 7 and Figure 8 It is shown that JE2 bacteria grown in the presence of different concentrations of cangrelor (including 1 μg / ml, 20 μg / ml and 40 μg / ml) did not affect bacterial growth or bacterial metabolic activity compared to vehicle controls. Although cangrelor did not inhibit bacterial growth of JE2, it could inhibit the production of virulence factors of this strain after treatment with a clinically relevant dose of 1 μg / ml.

[0151] Example 7: As a method comprising a detectable marker 18 N-alkyl-2-substituted ATP analogs of F 18 Preparation of F-cangrelor and bacterial uptake

[0152] A strategy previously described by Josse et al. in Bioorganic and Medicinal Chemistry 9 (2001) 665 and by Cheguillaume et al. in Bioorganic and Medicinal Chemistry 13 (2005) 1357 was used to synthesize the sulfhydryl group for [ 18 F]-labeled sulfurized precursor. Then prepare 18 F-Cangrelo.

[0153] 18 F-Cangrelor selective uptake in bacteria. Staphylococcus epidermidis was then analyzed; the bacteria were 18 F-cangrelo was incubated together and the relative radioactivity associated with the bacterial cells was measured.

[0154] S. epidermidis was grown overnight in tryptic soy broth (TSB) at 37°C with shaking at 250 rpm. The overnight culture was diluted to an OD of 600 0.1, and incubate until mid-exponential phase. 8 CFU were resuspended in 1 ml of RPMI 1640 cell culture medium provided by Sigma-Aldrich (R7638). Bacteria and bacteria-free controls were incubated with 2 MBq 18 F-cangrelo was incubated at 37°C for 1 hour. Bacteria were harvested by centrifugation (600×g, 5 minutes) and washed three times by successive centrifugation. After washing, cells were transferred to scintillation vials. The supernatant was also collected in a scintillation vial. The cells were counted by gamma counter (2470 Wizard 2TM (Perkin Elmer)) to count bacteria and supernatant.

[0155] The bacterial sediment was observed 18 F-cangrelor activity, thus indicating bacterial uptake and the potential utility of the tracer for diagnosis of bacterial infections.

Claims

1. A compound of formula I, a tautomer, enantiomer or diastereomer thereof, or a salt or solvate thereof or a solvate of such a salt for use in preventing or treating a bacterial infection in a subject, in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety.

2. The compound for use according to claim 1, wherein X is selected from -P(O)(OH)2, -SO3H or -CO2H.

3. The compound for use according to any one of claims 1 or 2, wherein R 4 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is substituted with one, two or three substituents.

4. The compound for use according to any one of claims 1 to 3, wherein R 3 It is C 1-6 alkyl, and wherein the C 1-6 The alkyl group is substituted with one, two or three substituents.

5. The compound for use according to any one of claims 1 to 4, wherein R 1 and R 2 same.

6. The compound for use according to any one of claims 1 to 5, wherein the compound is a salt; preferably a sodium salt of the compound of formula I.

7. The compound for use according to any one of claims 1 to 6, which is tetrasodium cangrelor of formula III 8. The compound for use according to any one of claims 1 to 7, wherein the bacterial infection is an infection caused by Gram-positive bacteria; preferably an infection caused by Staphylococcus; preferably an infection caused by Staphylococcus aureus; preferably an infection caused by methicillin-resistant Staphylococcus aureus.

9. A compound for use according to any one of claims 1 to 8, wherein the bacterial infection is an infection of a body surface.

10. The ex vivo or in vitro use of a compound of formula I, its tautomers, enantiomers or diastereomers or its salts or solvates or solvates of such salts as antibacterial agents, in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , Thio-C 1-6 Alkyl, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety.

11. Use of a compound of formula I, its tautomers, enantiomers or diastereomers or its salts or solvates or solvates of such salts as inhibitors of bacterial adhesion and biofilm formation on surfaces, in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety.

12. The use according to claim 11, wherein the surface is a surface of a biomaterial or a medical device; preferably a surface of a cardiovascular device or a surface of a catheter; most preferably a surface of a catheter, a prosthetic heart valve or a pacemaker.

13. An in vitro method for preventing bacterial growth in biofilm formation, comprising applying or grafting onto a surface an effective amount of a compound of formula I, a tautomer, enantiomer or diastereomer thereof, or a salt or solvate thereof, or a solvate of such a salt in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety.

14. A compound of formula I, a tautomer, enantiomer or diastereomer thereof, or a salt or solvate thereof, or a solvate of such a salt, for use in the diagnosis or prognosis of a bacterial infection, in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety.

15. A medical device, preferably a cardiovascular device, most preferably a catheter, a prosthetic heart valve or a pacemaker, coated with a composition comprising: a compound of formula I, a tautomer, enantiomer or diastereomer thereof, or a salt or solvate thereof, or a solvate of such a salt in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety.

16. A pharmaceutical composition comprising a compound of formula (I), a tautomer, enantiomer or diastereomer thereof, or a salt or solvate thereof, or a solvate of such a salt, for use in preventing or treating a bacterial infection in a subject, in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety.

17. A composition comprising: - a compound of formula I, its tautomers, enantiomers or diastereomers; - solubilizing carriers; The compound of formula I has the following structure: in, R 1 and R 2 are each independently hydrogen or halogen; R 3 and R 4 Each independently is phenyl or C 1-6 Alkyl; wherein each phenyl or C 1-6 The alkyl group may be substituted or unsubstituted with one or more substituents, each of which is independently selected from OR 5 , C 1-6 Alkylthio, NR 6 R 7 , phenyl, COOR 8 or halogen; R 5 , R 6 , R 7 and R 8 are independently hydrogen or C 1-6 alkyl, X is an acidic moiety; or a salt or solvate thereof, or a solvate of such a salt.

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