NZ751185B2 - New use of triazolo(4,5-d)pyrimidine derivatives for prevention and treatment of bacterial infection - Google Patents

Abstract

Triazolo(4,5-d)pyrimidine derivatives for use in treatment or prevention of bacterial infection in a host mammal in need of such treatment or use as inhibitor of biofilm formation on a surface of biomaterial or medical device, particularly of cardiovascular device such as prosthetic heart valve or pacemakers.

Description

New use of Triazolo(4,5-d)pyrimidine derivatives for prevention and treatment of bacterial infection The present invention relates to a new use of Triazolo(4,5-d)pyrimidine derivatives for tion and treatment of ial infection.

Bacteria are often incriminated in healthcare-associated infections (including medical device—related infections), causing increased patient morbidity and mortality, and posing huge ial burden on healthcare services. The situation has become critical since more and more bacteria are becoming resistant to antibiotics belonging to various classes such as Penicillins, Methicillins, Carbapenems, Cephalosporins, Quinolones, Amino-glycosides, and Glycopeptides, and an increasing number of infections are ng difficult to cure.

The increasing resistance to antibiotics is a growing public health concern because of the limited treatment options available for these serious infections. In Europe, antimicrobial resistance causes approximately 25,000 deaths every year. The al burden associated with antimicrobial resistance is estimated to cost approximately €15 billion per year.

At present, 0 deaths are estimated to be attributed to antimicrobial resistance globally as reported in Review on AMR, Antimicrobial resistance: Tackling a crisis for the health and wealth of nations, 2014 The use of antibiotics is not safe especially in long-term therapy or high dose therapy.

Such environmental pressure may promote ion of resistant bacteria, population, altering tion structure and increasing the risk of horizontal gene er leading to the mobility of resistant genes into the microbiome.

Antibiotic treatment s both the « good » and the « bad » bacteria.

The human gastro-intestinal tract (GI) iota is made of about trillions of microorganisms most of them ia. Microbiota and host’s defense relationship is essential for metabolic and physiological functions contributing to health. By disrupting this benefit interaction, dietary components, physical and psychological stress, drugs but also antibiotics increase incidence of several diseases like obesity, inflammation and cardiovascular es (CVD). CVD remain the first cause of death in industrial society with growing incidence in other countries.

For instance recent studies showed a direct link between long term antibiotics treatment, disruption of GI microbiota and risks of atherosclerosis in mice.

The source of bacterial ion is diverse and there is a large number of bacterial infections.

Infections caused by Gram-positive bacteria represent a major public health burden, not just in terms of morbidity and mortality, but also in terms of increased iture on t management and implementation of infection l measures.

Staphylococcus aureus and enterococci are established pathogens in the hospital environment, and their frequent multidrug resistance complicates therapy.

Staphylococcus aureus is an important pathogen sible for a broad range of clinical manifestations ranging from relatively benign skin infections to life-threatening ions such as endocarditis and osteomyelitis. It is also a commensal bacterium (colonizing approximately 30 percent of the human population).

Two major shifts in S. aureus epidemiology have occurred since the 1990s: an epidemic of community-associated skin and soft tissue infections ly driven by specific methicillin-resistant S. aureus [MRSA] strains), and an increase in the number of care-associated infections (especially infective endocarditis and prosthetic device infections). ase-negative lococci (CONS) are the most frequent constituent of the normal flora of the skin. These organisms are common contaminants in clinical specimens as well as increasingly recognized as agents of clinically icant infection, including bacteremia and endocarditis. Patients at particular risk for CoNS infection include those with prosthetic devices, pacemakers, intravascular catheters, and immunocompromised hosts.

Coagulase-negative lococci account for approximately one-third of bloodstream isolates in intensive care units, making these organisms the most common cause of nosocomial tream infection.

Enterococcal species can cause a variety of infections, including urinary tract infections, emia, endocarditis, and meningitis. Enterococci are relatively resistant to the killing effects of cell wall—active agents (penicillin, ampicillin, and vancomycin) and are impermeable to aminoglycosides.

Vancomycin-resistant cocci (VRE) are an increasingly common and difficult-to- treat cause of hospital-acquired infection.

Multiple epidemics of VRE infection have been described in diverse hospital settings (e.g., medical and surgical intensive care units, and medical and pediatric wards) and, like methicillin-resistant Staphylococcus aureus, VRE is endemic in many large hospitals.

The beta-hemolytic ococcus aga/actiae (Group B Streptococcus, GBS) is another Gram-positive bacteria. The bacteria can cause sepsis and/or meningitis in the newborn s. It is also an important cause of ity and mortality in the elderly and in immuno-compromised adults. Complications of infection include sepsis, pneumonia, osteomyelitis, endocarditis, and urinary tract infections.

The factors that make these bacteria especially adept at surviving on various erials include adherence and production of biofilm (see below).

The four above mentioned bacteria have the ability to form biofilms on any surface biotic and abiotic. The initial step of biofilm formation is the attachment/adherence to e, which is stronger in shear stress conditions. The protein mainly responsible for this adhesion is the ccharide intercellular n (PIA), which allows bacteria to bind to each other, as well as to surfaces, creating the biofilm. The second stage of biofilm formation is the development of a community structure and ecosystem, which gives rise to the mature biofilm. The final stage is the detachment from the surface with consequent spreading into other locations. In all the phases of biofilm formation the quorum sensing (QS) , mediating cell-to-cell communication, is involved.

Bacteria in the biofilm produce ellular polymeric substances (EPS) consisting mainly of polysaccharides, nucleic acids (extracellular DNA) and proteins, that t them from external s, including immune system components and antimicrobials.

Moreover, bacteria in the biofilm have a decreased metabolism, making them less susceptible to antibiotics; this is due to the fact that most antimicrobials e a certain degree of cellular activity in order to be effective. Another factor reinforcing such resistance is the impaired diffusion of the antibiotics throughout the biofilm e of the presence ofthe EPS matrix barrier.

It was also reported that in the biofilm there is higher rate of plasmid exchange increasing the chances of developing naturally ing and antimicrobial-induced resistance.

Strategies that have been developed to eliminate biofilms target 3 ent steps in the biofilm formation: inhibition of the initial stage, i.e. the adhesion of bacteria to surfaces; disrupting the biofilm ecture during the maturation process or step 2; inhibiting the Q5 system or step 3.

Because of the high resistance of these biofilms to antibiotics there is an increasing need of control and prevention of microbial growth and biofilm formation at step 2.

The treatment in case of infected medical device is either a conservative treatment or the removal of the device together with a long treatment with antibiotics, but these approaches have high failure rates and elevated economical burden.

This is the reason why clinicians try to adopt a preventive approach by istering antibiotics before implantation. Another solution could be the modification of the medical devices, e.g. surfaces coated with silver, which have antimicrobial ty or with hydrogels as well as polyurethanes, which reduce bacterial adhesion, to mention few examples.

According to Eggiman in American Society for Microbiology Press, Washington, DC 2000. p.247, pacemakers and implantable cardioverter-defibrillators [ICDs]) can become infected, with a rate of infections ranging from 0.8 to 5.7 percent.

The infection can involve subcutaneous pocket containing the device or the subcutaneous segment of the leads. Deeper infection can also occur that involves the transvenous portion of the lead, y with associated bacteremia and/or scular infection.

The device and/or pocket itself can be the source of infection, usually due to contamination at the time of implantation, or can be ary to emia from a different source.

Perioperative contamination of the pacemaker pocket with skin flora appears to be the most common source of subcutaneous ion.

Cardiac device-related ive endocarditis (CDRIE) is another life-threatening condition, with increasing incidence due to growing number of tations (81000 pacemaker implantation per year in Europe). The incidence of CDRIE reaches 0.14 percent, and is even higher after ICD implantation.

Staphylococcus aureus and coagulase-negative staphylococci (often Staphylococcus epidermidis) cause 65 to 75 percent of generator pocket infections and up to 89 percent of device-related rditis. Episodes arising within two weeks of implantation are more likely to be due to S. aureus.

Successful treatment of an infected medical device or biomaterial, regardless of the involved component, generally es removal of the entire system and administration of antibiotics targeting the causative bacteria. Importantly, medical therapy alone is associated with high mortality and risk of recurrence.

Prosthetic valve rditis (PVE) is a serious infection with potentially fatal consequences.

Bacteria can reach the valve prosthesis by direct contamination peratively or via hematogenous spread during the initial days and weeks after surgery. The bacteria have direct access to the prosthesis-annulus interface and to perivalvular tissue along suture pathways because the valve sewing ring, cardiac annulus, and anchoring sutures are not endothelialized early after valve implantation. These structures are coated with host proteins, such as fibronectin and fibrinogen, to which some organisms can adhere and te infection.

The risk of developing prosthetic valve endocarditis (PVE) is greatest during the initial three months after y, remains high through the sixth month, and then falls gradually with an annual rate of approximately 0.4 percent from 12 months postoperatively onward. The percentage of patients developing PVE during the initial year after valve replacement ranges from 1 to 3 percent in studies with active follow- up; by five years, the cumulative percentage ranges from 3 to 6 t.

The most frequently encountered pathogens in early PVE (within two months of implantation) are S. aureus and coagulase-negative staphylococci.

The most frequently encountered pathogens in late PVE (two months after valve tation) are streptococci and S. aureus, followed by coagulase-negative staphylococci and ente rococci.

The coagulase-negative staphylococci causing PVE during the initial year after surgery are almost exclusively Staphylococcus epidermidis. Between 84 and 87 percent of these organisms are methicillin resistant and thus ant to all of the beta-lactam antibiotics.

According to the 2008 French survey, PVE accounts for about 20 percent of all infective endocarditis. PVE is related to health care in about 30 t of cases. S. aureus is the first causative pathogen, being responsible for more than 20 percent of PVE.

Importantly, when comparing data from 1999, PVE-related mortality remains high, reaching about 40 percent after surgery, and 25 percent in-hospital mortality.

Periprosthetic joint infection (PJI) occurs in 1 to 2 percent of joint replacement surgeries and is a leading cause of arthroplasty failure.

Biofilms play an important role in the pathogenesis of PJIs. Bacteria within biofilm become ant to therapy; as a result, cterial therapy is often unsuccessful unless the biofilm is physically disrupted or removed by al debridement. etic joint infections are rized ing to the timing of symptom onset after implantation: early onset (<3 months after surgery), delayed onset (from 3 to 12 months after surgery), and late onset (>12 months after surgery). These infections have the following characteristics. Early-onset infections are usually acquired during implantation and are often due to virulent organisms, such as Staphylococcus , or mixed infections. Delayed-onset infections are also usually acquired during implantation. Consistent with the nt presentation, delayed infections are usually caused by less virulent bacteria, such as coagulase-negative staphylococci or enterococci. Late-onset infections resulting from hematogenous seeding are typically acute and often due to S. , or beta hemolytic streptococci.

The ment of PJIs lly consists of both surgery and antibacterial therapy.

There is therefore an urgent need in the art for a new antibacterial therapy, or at least provide the public with a useful choice.

We have surprisingly found that Triazolo(4,5-d)pyrimidine derivatives possess antibacterial activity and can be used in the treatment or prevention of bacterial infection in a host mammal.

We have also found that such Triazolo(4,5-d)pyrimidine derivatives can also be used in a method for controlling ial growth in biofilm formation at early stage such as step 1 or 2 or for killing bacteria at all steps of biofilm formation including the latest step 3 wherein the biofilm has reached its maturation stage of matrix formation and start detachment from the surface with a consequent spreading of bacteria into other locations.

In a first aspect, the invention es therefore Triazolo(4,5-d)pyrimidine derivatives for use in the treatment or prevention of bacterial infection in a host mammal in need of such treatment.

In a first particular , the present invention provides use of a triazolo(4,5- midine derivative of formula (I) [FOLLOWED BY PAGE 8A] wherein R1 is C 3-5 alkyl ally substituted by one or more halogen atoms; R2 is a phenyl group, optionally substituted by one or more halogen atoms; R3 and R4 are both hydroxyl; R is XOH, wherein X is CH2, or a bond; or a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt provided that when X is CH2 or a bond, R1 is not propyl; when X is CH2 and R1 is CH 2CH 2CF 3, butyl or pentyl, the phenyl group at R2 must be substituted by fluorine; in the manufacture of a medicament for treatment or prevention of bacterial infection.

In a second particular aspect, the t invention provides use of a triazolo(4,5- midine derivative of formula (I) [FOLLOWED BY PAGE 8B] n R1 is C3-5 alkyl optionally substituted by one or more halogen atoms; R2 is a phenyl group, optionally tuted by one or more halogen atoms; R3 and R4 are both hydroxyl; R is XOH, where X is CH2, OCH2CH 2, or a bond; or a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt provided that when X is CH2 or a bond, R1 is not propyl; when X is CH2 and R1 is CH 2CH 2CF 3, butyl or pentyl, the phenyl group at R2 must be substituted by fluorine; when X is OCH2CH 2 and R1 is propyl, the phenyl group at R2 must be substituted by fluorine; as inhibitor of biofilm formation on a surface.

In a yet further particular aspect, the present invention provides a method for ent of a bacterial infection in a non-human host mammal in need of such treatment which ses administering to the host an effective amount of the triazolo(4,5-d)pyrimidine derivative of formula(I) as defined in the first particular aspect as above.

In a yet further particular aspect, the present ion provides a method for prevention of a bacterial infection in a non-human host mammal in need of such prevention which comprises administering to the host an effective amount of triazolo(4,5-d)pyrimidine derivative of formula(I) as defined in the first ular aspect as above.

In a yet further particular aspect, the present invention provides a method of bacteria killing or prevention of bacterial growth in biofilm formation comprising using , by applying on a surface, an effective amount of triazolo(4,5-d)pyrimidine tive of a(I ) as defined in the second particular aspect as above.

[FOLLOWED BY PAGE 8C] By bacterial infection one means particularly Gram-positive bacterial infection such as for example pneumonia, septicemia, endocarditis, yelitis, meningitis, urinary tract, skin, and soft tissue infections. The source of bacterial infection is diverse, and can be caused for example by the use of implantable biomaterials.

By biomaterials, one means all implantable foreign material for al use in host s such as for prosthetic joints, pacemakers, implantable cardioverter- [FOLLOWED BY PAGE 9] defibrillators intravascular , catheters, coronary stent, prosthetic heart valves, intraocular lens, dental implants and the like.

By Triazolo(4,5-d)pyrimidine derivatives one means compounds of the following formula (I) R2 1&1 R N/ g ' "a N\ R3 $R3 wherein R1 is C3-5 alkyl optionally substituted by one or more halogen atoms; R2 is a phenyl group, optionally tuted by one or more halogen atoms; R3 and R4 are both hydroxyl; R is OH or XOH, wherein X is CH2, OCHZCHZ, or a bond; or a ceutical acceptable salt or solvate thereof, or a solvate thereof or a solvate of such a salt provided that when X is CH2 or a bond, R1 is not ; when X is CH2 and R1 CH2CH2CF3, butyl or pentyl, the phenyl group at R2 must be tuted by fluorine; when X is OCHZCHZ and R1 is propyl, the phenyl group at R2 must be substituted by fluorine.

Alkyl groups whether alone or as part of another group are straight chained and fully saturated.

R1 is a C3_5 alkyl optionally tuted by one or more fluorine atoms. ably R1 is 3,3,3, -trifluoropropyl, butyl or propyl.

R2 is phenyl or phenyl tuted by one or more n atoms. Preferably R2 is phenyl substituted by fluorine atoms. Most preferably R2 is 4-fluorophenyl or 3,4- difluorophenyl.

R is OH or XOH, where X is CH2, OCHZCHZ, or a bond ; preferably R is OH or OCHZCHZOH. When X is a bond, R is OH.

Most preferred Triazolo(4,5-d)pyrimidine derivatives are the ones including R2 as 4- fluorophenyl or 3,4-difluorophenyl and or R as OCH2 CHZOH.

Triazolo(4,5-d)pyrimidine derivatives are well known compounds . They may be obtained according to the method described in US 6,525,060 which is incorporated by reference.

Triazolo(4,5-d)pyrimidine derivatives are used as medicament against platelet adhesion and aggregation that are primary steps in arterial thrombosis.

They work by antagonizing the platelet P2Y12 receptor for ADP in a reversible manner, providing antiplatelet s after oral administration. P2Y12 is one of the two ADP receptors expressed by platelets, acting by amplifying et responses to other agonists, which stabilizes platelet aggregates and promotes thrombosis. As a consequence, P2Y12 inhibitors, alone or in combination with aspirin, significantly improve outcomes of patients with coronary artery disease and peripheral ar disease.

We have now surprisingly found that such Triazolo(4,5-d)pyrimidine derivatives have also an antibacterial effect.

Prefered Triazolo(4,5-d)pyrimidine derivatives are derivatives with R equals OH or OCHZCHZOH and /or R2 equals 4-fluorophenyl or 3,4 difluorophenyl.

Most red Triazolo(4,5-d)pyrimidine derivatives are (1R-(10L, 20L, 3B(1R*, 2*),5B))- 3-(7-((2-(3,4-difluorophenyl)cyclopropyl)amino)((3,3,3-trifluoropropyl)thio)3H-1,2,3- triazolo(4,5d)pyrimidinyl)5(hydroxy)cyclopentane-1,2-diol; (IS-(10L, 20L, 3B(1R*, 2*),5B))(7-((2-(3,4-dif|uorophenyl)cyclopropyl)amino) (propylthio)(3H-1,2,3-triazolo(4,5d)pyrimidinyl)5(2-hydroxyethoxy)cyclopentane- 1,2-diol; (15,25,3R,SS)[7-[(1R,2$)(3,4-difluorophenyl)cyclopropylamino]—5-(propylthio)-3H- [1,2,3]—triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)—1,2-cyclopentanediol); (15,25,3R,SS)[7-[(1R,2$)—2-(4-fluorophenyl)cyclopropylamino]—5-(propylthio)—3H- [1,2,3]—triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)—1,2-cyclopentanediol); (15,2R,3S,4R)—4-[7—[[(1R,2$)—2—(3,4—Dif|uorophenyl)cyclopropyl]amino]—5— (propylthio)—3H—1,2,3—triazo|o[4,5—d] pyrimidin—3—yl]—1,2,3—cyc|opentanetriol; and ceutical acceptable salt or e thereof, or a e thereof or a solvate of such a salt.

The most preferred Triazolo(4,5-d)pyrimidine derivative is (15,25,3R,SS)[7-[(1R,2$)— 2-(3,4-difluorophenyl)cyclopropylamino]—5-(propylthio)—3H-[1,2,3]-triazolo[4,5- d]pyrimidinyl]—5-(2-hydroxyethoxy)—1,2-cyclopentanediol) as defined in formula (II) and also called Triafluocyl hereafter. \l‘" K» {I a" »_ ?x" .n w r El 0 y""‘»_ N" "rm"! RSI/V Ho‘ cm, and a pharmaceutical acceptable salt or solvate thereof, or a solvate thereof or a solvate of such a salt.

Another most preferred Triazolo(4,5-d)pyrimidine derivative is ,3S,4R)-4—[7- [[(1R,2$)-2—(3,4—Difluoropheny|)cyclopropyl]amino]—5—(propylthio)—3H—1,2,3— triazo|o[4,5—d]pyrimidin—3—yl]—1,2,3—cyclopentanetrio| as defined in formula (III) and also called Fluometacyl hereafter (III) and a pharmaceutical acceptable salt or solvate f, or a solvate thereof or a solvate of such a salt.

According to the invention the Triazolo(4,5-d)pyrimidine derivative has to be administered to the patient over several days (especially in case of prevention).The Triazolo(4,5-d)pyrimidine derivative may be administered on their own or as a pharmaceutical composition, with non-toxic doses being inferior to 1.8 g per day.

A r preferred object of the invention is a pharmaceutical composition of Triazolo(4,5-d)pyrimidine derivative for use in the prevention or ent of bacterial infection, The pharmaceutical composition may be a dry powder or a liquid composition having physiological compatibility. The compositions include, in addition to triazolo(4,5- d)pyrimidine derivative, auxiliary substances, preservatives, solvents and/or viscosity modulating agents. By solvent, one means for example water, saline or any other logical solution, ethanol, ol, oil such as ble oil or a mixture thereof.

By viscosity modulating agent on means for e carboxymethylcellulose.

The Triazolo(4,5-d)pyrimidine tive of the present invention exhibits its effects through oral, intravenous, intravascular, intramuscular, parenteral, or topical administration, and can be additionally used into a composition for parenteral administration, particularly an injection composition or in a composition for topical administration. It can also be loaded in nanoparticles for nanomedicine ations. It can be used in an aerosol composition. Such aerosol ition is for example a solution, a suspension, a micronised powder mixture and the like. The composition is administered by using a nebulizer, a metered dose inhaler or a dry powder inhaler or any device designed for such an administration. es of galenic compositions include tablets, capsules, powders, pills, syrups, chewing, granules, and the like. These may be produced through well known que and with use of typical additives such as excipients, lubricants, and binders.

Suitable auxiliary substances and pharmaceutical compositions are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the composition to render the ition isotonic. Examples of pharmaceutically acceptable substances include , Ringer's solution and dextrose solution. pH ofthe solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.

A still further preferred object of the invention is a method of treatment or prevention of bacterial infection in a host mammal in need of such treatment which comprises administering to the host an effective amount of triazolo(4,5-d)pyrimidine derivatives as defined in formula (I), preferably (1R-(10L, 20L, 3B(1R*, 2*),5B))—3-(7-((2-(3,4- difluorophenyl)cyclopropyl)amino)((3,3,3-trifluoropropyl)thio)3H-1,2,3- lo(4,5d)pyrimidinyl)5(hydroxy)cyclopentane-1,2-diol; (IS-(10L, 20L, 3B(1R*, 2*),5[3))(7-((2-(3,4-difluorophenyl)cyclopropyl)amino) (propylthio)(3H-1,2,3-triazolo(4,5d)pyrimidinyl)5(2-hydroxyethoxy)cyclopentane- 1,2-diol; (15,25,3R,SS)[7-[(1R,2$)(3,4-difluorophenyl)cyclopropylamino]—5-(propylthio)-3H- [1,2,3]—triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)—1,2-cyclopentanediol); most preferably (15,25,3R,SS)[7-[(1R,2$)—2-(4-fluorophenyl)cyclopropylamino]—5- (propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)-1,2- cyclopentanediol) as defined in formula II; or most preferably ,35,4R)[7-[[(1R,2$)—2-(3,4- Difluorophenyl)cyclopropyl]amino]—5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin yl]-1,2,3-cyclopentanetriol, as defined in formula III; or a pharmaceutical acceptable salt or e thereof, or a solvate thereof or a solvate of such a salt.

In another aspect, the invention provides the use of Triazolo(4,5-d)pyrimidine derivatives, preferably (1R-(10L, 20L, 3B(1R*, 2*),5B))—3-(7-((2-(3,4- rophenyl)cyclopropyl)amino)((3,3,3-trifluoropropyl)thio)3H-1,2,3- triazolo(4,5d)pyrimidinyl)5(hydroxy)cyclopentane-1,2-diol; (IS-(10L, 20L, 3B(1R*, 2*),5[3))(7-((2-(3,4-difluorophenyl)cyclopropyl)amino) (propylthio)(3H-1,2,3-triazolo(4,5d)pyrimidinyl)5(2-hydroxyethoxy)cyclopentane- 1,2-diol; (15,25,3R,SS)[7-[(1R,2$)(3,4-difluorophenyl)cyclopropylamino]—5-(propylthio)-3H- [1,2,3]—triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)—1,2-cyclopentanediol); (15,25,3R,SS)[7-[(1R,2$)—2-(4-fluorophenyl)cyclopropylamino]—5-(propylthio)—3H- [1,2,3]—triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)—1,2-cyclopentanediol); and pharmaceutical acceptable salt or solvate thereof, or a solvate thereof or a solvate of such a salt; and most preferably (15,25,3R,SS)[7-[(1R,2$)—2-(3,4- difluorophenyl)cyclopropylamino]—5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin yl](2-hydroxyethoxy)-1,2-cyclopentanediol) or a pharmaceutical acceptable salt or solvate thereof, or a solvate thereof or a solvate of such a salt; as inhibitor of m on a surface, ularly a surface of a biomaterial or of a medical device.

The most preferred tor of biofilm on a e is (lS,2R,35,4R)[7-[[(1R,2$) (3,4-Difluorophenyl)cyclopropyl]amino]—5-(propylthio)-3H-1,2,3-triazolo[4,5- d]pyrimidinyl]—1,2,3-cyclopentanetriol, as defined in formula III (III) and pharmaceutical able salt or solvate thereof, or a e thereof or a solvate of such a salt; By surface one means any type of e such as rubber or plastic e as for example surface made of polyethylene, polypropylene, polyurethane, polyvinyl chloride, polyvinylpyrrolidone, polytetrafluoroethylene, silicone or the like, or mers but also and preferably metallic surface such as stainless steel, silver, gold, titanium, ic alloys pyrolitic carbon, and the like. It can also be used on orbable or biomaterial surface such as biological prosthesis or devices which are made of biological material such as for example porcine or bovine pericardium By tion of biofilm on a surface one means inhibition of the biofilm formation at all stages of its formation starting from a prevention or an inhibition of adherence of bacteria on the surface at step 1 but also and mainly an inhibition in bacteria grow, multiplication, and formation of microcolonies on the surface at step 2. By inhibition of biofilm one also means inhibition of the matrix at the maturation step 3 and inhibition of bacteria dispersion from the matrix in a colonisation step. By inhibition of biofilm, one also means killing bacteria at all steps ofthe biofilm formation.

By medical device one means biomaterial as defined above but also medical device requesting no bacterial contamination such as wound dressing, soft tissue fillers, root canal fillers, contact lens, blood bag and the like.

A last further aspect according to the invention, is a method for killing or controlling bacterial growth in biofilm formation on a surface sing applying lo(4,5- d)pyrimidine derivative on a surface either at a prevention step, reducing bacteria adherence and survival on the substrate or at a stage where the biofilm is already present, or even at a maturation step with a matrix formation wherein a more complex architecture of biofilm is ished ting bacteria as a r to conventional antibacterial agent.

The method of bacteria killing or tion of bacterial growth on a surface is generally d to biomaterials or medical devices.

The biomaterial or medical device are preferably implantable foreign material for clinical use in host mammals such as prosthetic devices, pacemakers, implantable cardioverter-defibrillators, intravascular catheters, coronary stent, heart valves, intraocular lens and the like but could be extended to other medical devices requesting no bacterial ination such as for example wound dressings, soft tissue fillers containing local anaesthetics, root canal fillers with ancillary medicinal substances and the like.

The method of bacteria killing or prevention of bacterial growth could also be applied to surface of experimental device in need of such antibacterial treatment.

Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ising’ and the like are to be construed in an inclusive sense as d to an exclusive or exhaustive sense; that is to say in the sense of "including but not limited to." [FOLLOWED BY PAGE 17A] In the description in this specification reference may be made to subject matter which is not within the scope of the ed claims. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the ion as defined in the appended claims.

In this specification where reference has been made to patent ications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, nce to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. ption of the Figures Figure 1 illustrates a bacteriostatic and bactericidal effect of Triafluocyl on Staphylococcus aureus. Growth curves (A) and viable counts (B) in the presence of different concentrations of Triafluocyl or DMSO as vehicle are shown.

[FOLLOWED BY PAGE 18] Figure 2 illustrates an inhibition of Staphylococcus aureus biofilm formation by Triafluocyl at stage 2.

Figure 3 illustrates a bacteriostatic and bactericidal effect of Triafluocyl on Enterococcus faecalis. Growth curves (A) and viable count (B) in the presence of different concentrations of Triafluocyl or DMSO as vehicle are shown.

Figure 4 illustrates an inhibition of Enterococcus faecalis biofilm formation by Triafluocyl at stage 2.

Figure 5 illustrates a bacteriostatic and bactericidal effect of Triafluocyl on Staphylococcus epidermidis. Growth curve (upper panel) and viable count (lower panel) in the presence of ent concentrations of Triafluocyl or DMSO as vehicle.

Figure 6 rates an inhibition of Staphylococcus epidermidis biofilm formation at stage 2 by Triafluocyl.

Figure 7 illustrates a destruction of mature biofilm (stage 3: 24-hour biofilm) by Triafluocyl. Viable count of S. epidermidis biofilm after a 24h ent with Triafluocyl (upper panel). Percentage of live cells in the biofilm (lower panel).

Figure 8 rates bactericidal activity against MRSA, GISA and VRE strains of Triafluocyl as compared to Vancomycin and Mynocycline: Figure 8A illustrates a killing curve for methilcillin-resistant S. aureus (MRSA).

Figure SB illustrates a killing curve for Glycopeptide intermediate-resistant S. aureus .

Figure 8C illustrates a killing curve for ycin resistant E. faecalis (VRE).

Figure 9 illustrates bactericidal activity of Fluometacyl t S. aureus MRSA.

Figure 10A and 10B illustrate the antibacterial effect of different concentrations of Fluometacyl on S. aureus and S. epidermidis biofilm formation respectively.

Examples: The invention is illustrated ter by the ing non limiting examples.

We have conducted in vitro experiments, using S. aureus, S. epidermidis, and E. faecalis as clinically relevant ositive bacterial strains.

The tests were performed in accordance with the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

Example 1: use of (15,25,3R,SS)[7-[(1R,2$)(3,4-difluorophenyl)cyclopropylamino]— -(propylthio)-3H-[1,2,3]—triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)-1,2- cyclopentanediol) or Triafluocyl (Cayman, item N015425).

S. aureus (American Type Culture Collection, ATCC 25904) was grown ght in Tryptic Soy Broth (TSB) medium, diluted 1:100 in fresh TSB, and incubated aerobically at 37°C until bacteria growth reached a logarithmic phase (OD600 = 0.25-0.3).

Increasing concentrations of Triafluocyl (Cayman Chemical, Item No. 15425) or vehicle (DMSO) was then added in 5 ml of bacteria suspensions. Bacterial growth was measured after different time intervals (20-100 min) by spectrophotometry (ODGOO) and by counting the colony-forming units after g appropriate culture dilutions on TS agar plates.

Bacteriostatic and bactericidal effects were ed with Triafluocyl. In Figure 1 kinetics of S. aureus growth in the presence of an increasing concentrations (lug/ml to pg/ml) of Triafluocyl were ed by turbidity measurement (upper , and viable count (lower graph). Data ent medians 1r ** range (n = 3). * p<0.05,' p<0.01, *** p<0,001, Triafluocyl vs vehicle.

As shown in Figure 1, while a concentration of 10 ug/ml Triafluocyl was able to inhibit bacterial growth, 20 ug/ml Triafluocyl displayed potent bactericidal effect.

Example 2: use of (1S,2S,3R,5S)[7-[(1R,2S)(3,4-difluorophenyl) cyclopropylamino]—5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidinyl]—5-(2- hydroxyethoxy)-1,2-cyclopentanediol) or uocyl as tor of biofilm formation.

S. aureus (ATCC 25904) was grown overnight in TSB medium, before being diluted 100 fold in fresh TSB, and ted aerobically at 37°C until bacteria culture reached an OD600 of 0.6 (corresponding to approximately 1-3x108 ). Bacteria cultures were then diluted to 1x104 CFU/ml in fresh TSB. 800 pl aliquots of diluted bacteria suspensions were distributed in each well of a l plate. Bacteria were allowed to adhere for 4 hours under static conditions at 37°C. After removing media, wells were rinsed 2 times with PBS to eliminate planktonic bacteria and re-filled with TSB supplemented with 0.5 % e Triafluocyl or DMSO as vehicle was then added at desired concentration and plates were ted at 37°C for 20 hours. After incubation, wells were washed and stained with 0.5 % (w/v) crystal violet for 30 minutes, washed again and the dye was solubilized by adding 20 % acetic acid (v/v in water) before reading absorbance at 595 S. aureus biofilms were formed on polystyrene surface in the presence of increasing concentrations of Triafluocyl or DMSO as vehicle. In figure 2, Biofilm mass is presented ** P < 0.01; *** as percentage of values obtained in the presence of DMSO (*P < 0.05; P < 0.001, Triafluocyl versus DMSO, n= 4).

Triafluocyl significantly reduces S. aureus biofilm ion at all concentrations tested. In the presence of 10 ug/ml Triafluocyl, no biofilm could form on polystyrene surface.

Example 3: use of (15,25,3R,SS)[7-[(1R,2$)(3,4-difluorophenyl)cyclopropylamino]— -(propylthio)-3H-[1,2,3]—triazolo[4,5-d]pyrimidinyl]—5-(2-hydroxyethoxy)-1,2- cyclopentanediol) or Triafluocyl (Cayman Chemical, Item No. 15425) .

E. faeca/is (ATCC 29212) was grown overnight in Brain heart infusion (BHI) medium, diluted 1:100 in fresh BHI, and incubated aerobically at 37°C until bacteria growth reached a logarithmic phase (OD600 = 0.25-0.3).

Increasing trations of Triafluocyl (Cayman Chemical, Item No. 15425) or DMSO as vehicle was then added in 5 ml of bacteria suspensions. Bacterial growth was measured after different time intervals 0 min) by spectrophotometry (ODGOO) and by ng the -forming units after plating riate culture dilutions on BHI agar plates.

Bacteriostatic and bactericidal s were measured with Triafluocyl. In Figure 3 kinetics of E.faeca/is growth in the presence of an increasing concentrations (Sug/ml to 40 ug/ml) of Triafluocyl were measured by turbidity measurement (upper graph), and viable count (lower graph). Data represent medians 1r range (n = 3).

As shown in Figure 3, while a concentration of 10 ug/ml Triafluocyl was able to inhibit bacterial growth, 20 ug/ml Triafluocyl and more importantly 40 ug /m| displayed potent bactericidal effects.

Example 4: use of Triafluocyl as inhibitor of biofilm formation.

E. is (ATCC 29212) was grown overnight in BHI medium, before being diluted 100 fold in fresh TSB, and incubated aerobically at 37°C until bacteria culture d an OD600 of 0.6 (corresponding to approximately 8 CFU/ml). Bacteria cultures were then diluted to 1x104 CFU/ml in fresh TSB. 800 pl aliquots of diluted bacteria suspensions were distributed in each well of a 24-well plate. Bacteria were allowed to adhere for 4 hours under static conditions at 37°C. After removing media, wells were rinsed 2 times with PBS to eliminate planktonic bacteria and re-filled with TSB mented with 0.5 % glucose Triafluocyl or DMSO as vehicle was then added at desired concentration and plates were incubated at 37°C for 20 hours. After incubation, wells were washed and stained with 0.5 % (w/v) crystal violet for 30 minutes, washed again and the dye was solubilized by adding 20 % acetic acid (v/v in water) before reading absorbance at 595 E. faeca/is biofilms were formed on polystyrene e in the presence of increasing concentrations of Triafluocyl or DMSO as vehicle. In figure 2, Biofilm mass is presented ** P < 0.01; *** as percentage of values obtained in the presence of DMSO (*P < 0.05; P < 0.001, Triafluocyl versus DMSO, n= 4).

Triafluocyl icantly reduces E. faeca/is biofilm formation at a starting concentration of 10 ug/ml. In the presence of 40 ug/ml Triafluocyl, no biofilm could form on polystyrene surface. e 5: ill study of Triafluocyl against S. epidermidis To evaluate Triafluocyl antibacterial effect we have tested S. epidermidis liquid growth in the presence of different Triafluocyl concentrations in logarithmic phase. In this phase usually ia are highly tible to agents with bactericidal activity because they are rapidly dividing.

A 1:100 inoculum in 50ml TSB of an O/N culture of S. epidermidis was cultured for 3hr up to its thmic phase (OD600 = 0,26 and z 3x108 CFU/ml).

Bacteria were split in several tubes containing different concentrations of DMSO as vehicle alone or in combination with Triafluocyl in TSB and grown for 100min at 37°C with 220rpm shaking, the OD600 was measured every 20min.

Compared to the growth with DMSO (0,25%) we observed a dose-dependent inhibition of S. epidermidis growth n 10ug/ml and 20ug/ml Triafluocyl (Figure 5). At 50ug/ml we observed a slight bacteriostatic activity, confirmed by the number of viable cells at 80min, 3x108 CFU/ml, equal to the number of bacteria in the untreated control at the beginning of the assay (Figure 5).

Moreover, we have tested the effect of Triafluocyl on a low-density inoculum, 0.08x106 CFU/ml, from a culture of S. epidermidis in logarithmic phase. We have ed the growth for 4hr with or without Triafluocyl and measured the ODGOO: already Bug/ml of Triafluocyl decreased the OD by 50% compared to the growth in absence of Triafluocyl at the same time point; 10ug/ml and 20ug/ml ted growth (OD value equal to OD at the beginning of the growth) (data not shown).

This means that the lower the inoculum density the lower the tration of Triafluocyl to slow down growth or kill bacteria.

Example 6: Triafluocyl prevents S. epidermidis biofilm formation To study the effect of Triafluocyl on biofilm formation, S. epidermidis in early logarithmic phase (5x108 CFU/ml) was plated in a 24-well plate and let to adhere at the bottom of the well for 4hr at 37°C in static conditions. After 4hr incubation, planktonic bacteria were removed and nt bacteria were washed twice in TSB. Fresh TSB medium mented or not with 0.25% glucose was added to the well with 5 different concentrations of Triafluocyl and incubated for 24 hours. Wells were washed 3 times with NaCl 0.9% and incubated for 1hr at RT with Crystal Violet 1% solution in dHZO to stain the m.

Wells were washed 3 times with dH20 to eliminate unbound crystal violet, then 400u| Acetic Acid 10% was added and ted at RT for 10min. Absorbance was measured in triplicate at 570nm, reflecting total s ofthe biofilm (live and dead bacteria).

Triafluocyl affected biofilm formation e 6): already at Bug/ml, in the absence of glucose, it inhibited biofilm formation by 50%, while in ce of glucose we reach 50% biofilm reduction only at 20ug/ml Triafluocyl.

The concentration of Triafluocyl that inhibits at least 90% biofilm formation is called minimum biofilm tory concentration (MBIC). Triafluocyl MBIC for S. epidermidis is 50ug/ml both in the ce and in the absence of glucose.

Example 7: Triafluocyl destroys S. epidermidis mature biofilm In another experiment we let adhere 0.5x108 CFU/ml S. epidermidis cells for 4hr and let the m form for additional 24hr in ce of 0.25% e, at this point we treated the biofilm with several concentrations of Triafluocyl for 24hr in TSB with 0.25% glucose and determined the viable count (Figure 7) as well as the percentage of live cells using the ht bacterial viability kit (Molecular Probes).

For biofilm analysis, we first washed the m to eliminate all planktonic bacteria and then the biofilm was detached mechanically using a scraper. To assure that the aggregates from the biofilm were completely dissociated, the suspension of cells was passed through a needle (0.5x16mm) and a dilution was plated on TSA plates.

Only the highest concentration of Triafluocyl, 50ug/ml, was ive in ng the number of viable cells in the biofilm with a reduction of almost 3 log (from 1.1x108 CFU/ml in the control to 1.5x105 CFU/ml).

In the same experiment we also determined the percentage of live and dead bacteria.

To do so we followed the procedure of the kit LIVE/DEAD from lar Probes.

Briefly, the biofilm was resuspended in a solution of 0.9% NaCl and cells were stained with a mixture of SYTO9 (green fluorescence) and propidium iodide (PI) (red fluorescence) for 15 min in the dark. Stained cells were transferred in a 96-well plate and fluorescence was measured using the Enspire Spectrophotometer with excitation ngth of 470nm and emission spectra in the range of 490 - 700nm. SYTO9 dye (green scence 500-520nm) penetrates all the cells (dead and live) and binds to DNA, while PI (red fluorescence in the range 610-630nm) enters only in dead cells with a damaged cell membrane. When PI and SYTO9 are in the same cell the green fluorescence intensity decreases. Therefore, in a population of cells with high percentage of dead cells there is a ion in the emission spectra of the green fluorescence, because there is more PI staining. The ratio of fluorescence intensity (green/live) is plotted against a known percentage of live cells to obtain a standard curve and the percentage of live cells in our samples is obtained by extrapolation (Figure 7). Triafluocyl, at concentrations of 20ug/ml and 50ug/ml reduced the percentage of live bacteria to 80% and 30%, respectively.

Example 8: Triafluocyl antibacterial s on S. epidermidis: determination of l Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) The Minimal tory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC) of Triafluocyl were determined in lococcus epidermidis (ATCC 35984, also known as RP62A) according to EUCAST (European Committee on crobial Susceptibility Testing) recommendations.

Briefly, a single colony grown on a Tryptic Soy Agar (TSA) plate was resuspended and cultured in Tryptic Soy Broth (TSB) overnight (O/N) in aerobic ions (37°C with 220rpm shaking), next day a 1:50 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic ions for 3hr and an inoculum of 1:100 dilution, corresponding to 3x105 CFU/ml, was incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO (vehicle). After O/N growth the OD of each culture was measured at 600nm in a spectrophotometer (ODGOO). The MIC represents the concentration at which there is no visible growth of bacteria, i.e. AOD at 600nm equal to zero (blank is the medium alone).

We have also determined the MBC, i.e. the concentration at which the liquid e, when spread on TSA plates, will not produce any colony.

The MIC for Triafluocyl against $.epidermidis is equal to 12 i 3 ug/ml and the MBC is 17 i 3 ug/ml (two biological ates, detection limit 103).

The Minimum Duration for killing 99,9% $.epidermidis (MDK99,9) by Triafluocyl, a tolerance metric according to the EUCAST, was 2 hours.

Example 9: Triafluocyl antibacterial effects on S. aureus: determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) Further ments were conducted using different strains of S. aureus, as clinically relevant Gram-positive bacterial strains: ATCC 25904, ATCC 6538, methilcillin-resistant S. aureus (MRSA) ATCC BAA-1556, Glycopeptide ediate-resistant (GISA) S. aureus Mu-50 (ATCC 700695) in order to determine the Minimal Inhibitory Concentration (MIC) which is the minimal tration required to prevent bacterial growth; the Minimal Bactericidal Concentration (MBC) which determines the lowest tration at which an antimicrobial agent kill a particular rganism and a Minimum Duration for g 99,9% bacteria (MDK99,9) which is a tolerance metric according to the EUCAST.

MIC determination: A single colony selected from the different strains of S. aureus is resuspended and cultured in the appropriate medium overnight (O/N) in aerobic conditions (37°C with 220rpm shaking), next day a 1:100 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic conditions for 3hr (OD=0,08—0,1) and an um of 1:300 dilution, corresponding to 3x105 CFU/ml, was ted in presence or absence of different concentrations of Triafluocyl in 1% DMSO. After O/N growth the OD of each culture was measured at 600nm in a spectrophotometer ). The MIC represents the concentration at which there is no visible growth of bacteria, i.e.

AOD at 600nm equal to zero (blank is the medium alone). MIC for Triafluocyl against S. aureus ATCC 25904, ATCC 6538, methilcillin-resistant S. aureus (MRSA) ATCC BAA- 1556, Glycopeptide intermediate-resistant (GISA), and S. aureus Mu-50 (ATCC 700695) were 20, 20, 15, and 20 ug/ml, respectively.

MBC and MDK99,9 determination: A single colony selected from the different strains of S. aureus is resuspended and cultured in the appropriate medium (TSB, or BHI) overnight (O/N) in aerobic conditions (37°C with 220rpm shaking), next day a 1:100 inoculum in the appropriate medium was incubated in aerobic conditions for 2h. The culture is then challenged with triafluocyl at MIC concentration or higher concentrations. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates. The concentration that kill at least 99,9% of the d um in 24h is defined as the MBC. And the real time needed is defined as the MDK99,9. MBC for Triafluocyl against S. aureus ATCC 25904, ATCC 6538, methilcillin-resistant S. aureus (MRSA) ATCC BAA-1556, Glycopeptide intermediate-resistant (GISA), and S. aureus Mu-50 (ATCC 700695) were 20 ug/ml for each of them. 9 for Triafluocyl against S. aureus ATCC 25904, ATCC 6538, cillin-resistant S. aureus (MRSA) ATCC BAA- 1556, Glycopeptide intermediate-resistant (GISA), and S. aureus Mu-50 (ATCC 700695) were 10, 6, 2, and 14 hours, respectively.

Example 10: Triafluocyl antibacterial s on E. faeca/is: determination of Minimal tory Concentration (MIC) and Minimal Bactericidal tration (MBC) Further experiments were ted using different strains of E. faeca/is, as clinically relevant Gram-positive bacterial strains: E. faeca/is ycin-resistant (VRE) ATCC BAA-2365, and E. faeca/is ATCC 29212 in order to determine the Minimal Inhibitory Concentration (MIC) which is the minimal concentration required to prevent bacterial growth; the Minimal icidal Concentration (MBC) which determines the lowest concentration at which an antimicrobial agent kill a particular microorganism and a Minimum Duration for killing 99,9% ia (MDK99,9) which is a tolerance metric according to the EUCAST.

MIC determination: A single colony ed from the different strains of E. faecalis is resuspended and cultured in the riate medium overnight (O/N) in aerobic conditions (37°C with 220rpm shaking), next day a 1:100 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic conditions for 3hr (OD=0,08—0,1) and an inoculum of 1:300 dilution, corresponding to 3x105 CFU/ml, was incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO. After O/N growth the OD of each culture was measured at 600nm in a spectrophotometer (ODGOO). The MIC represents the concentration at which there is no visible growth of bacteria, i.e.

AOD at 600nm equal to zero (blank is the medium alone). MIC for Triafluocyl against E. faeca/is vancomycin-resistant (VRE) ATCC BAA-2365, and E. faeca/is ATCC 29212 were and 40 ug/ml, respectively.

MBC and MDK99,9 ination: A single colony selected from the different strains of E. is is resuspended and cultured in the appropriate medium (TSB, or BHI) overnight (O/N) in aerobic ions (37°C with 220rpm shaking), next day a 1:100 inoculum in the appropriate medium was incubated in aerobic conditions for 2h. The culture is then challenged with triafluocyl at MIC concentration or higher concentrations. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates. The concentration that kill at least 99,9% of the started inoculum in 24h is defined as the MBC. And the real time needed is defined as the 9. MBC for Triafluocyl against E. faeca/is vancomycin-resistant (VRE) ATCC BAA-2365 was 20 ug/ml. 9 for Triafluocyl t E. faeca/is vancomycin-resistant (VRE) ATCC BAA-2365 was 24 hours.

Example 11: Triafluocyl antibacterial effects on ococcus agalactiae: determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) Further experiments were conducted using 5. agalactiae (ATCC 12386), as clinically relevant ositive bacterial strains in order to determine the Minimal Inhibitory Concentration (MIC) which is the minimal concentration required to prevent bacterial ; the Minimal icidal Concentration (MBC) which determines the lowest concentration at which an antimicrobial agent kill a particular microorganism and a Minimum Duration for killing 99,9% bacteria (MDK99,9) which is a tolerance metric according to the .

MIC determination: A single colony selected from the different strains of 5. agalactiae (ATCC 12386) is resuspended and cultured in the appropriate medium overnight (O/N) in aerobic conditions (37°C with 220rpm shaking), next day a 1:100 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic conditions for 3hr (OD=0,08-0,1) and an um of 1:300 dilution, corresponding to 3x105 CFU/ml, was incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO. After O/N growth the OD of each culture was measured at 600nm in a spectrophotometer (ODGOO). The MIC represents the concentration at which there is no visible growth of bacteria, i.e. AOD at 600nm equal to zero (blank is the medium alone). M|C for Triafluocyl against 5. agalactiae (ATCC 12386) was 40 ug/ml.

MBC and MDK99,9 determination: A single colony ed from 5. agalactiae (ATCC 12386) is resuspended and cultured in the appropriate medium (TSB, or BHI) overnight (O/N) in aerobic conditions (37°C with 220rpm shaking), next day a 1:100 inoculum in the appropriate medium was incubated in aerobic ions for 2h. The culture is then challenged with triafluocyl at M|C tration or higher concentrations.

Bacterial growth was measured after different time intervals by counting the colony- g units after plating riate culture ons on BHI agar plates. The concentration that kill at least 99,9% of the started inoculum in 24h is defined as the MBC. And the real time needed is defined as the MDK99,9. MBC for Triafluocyl against 5. agalactiae (ATCC 12386) was 40 ug/ml. MDK99,9 for Triafluocyl against 5. aga/actiae (ATCC 12386) was 1 hour.

The results of all experiments are illustrated in Table 1 and in figures 8 A,B,C, wherein the effect of Triafluocyl on resistant strains such as MRSA: methilcillin-resistant S. aureus; GISA: Glycopeptideintermediate-resistant S. aureus; VRE: vancomycin- resistant E. is is shown.

Strains Resistance MIC MBC MDK 99,9 ug/ml ug/ml Time (h) $.aureus 20 20 10 (ATCC25904) $.aureus 20 20 6 (ATCC6538) $.aureus MRSA 15 20 2 $.aureus-Mu50 GISA 20 20 14 S. midis 15 20 2 E.faecalis 40 nd nd E.faecalis VRE 20 20 24 . tiae 40 40 1 Table 1 MIC: minimal inhibitory concentration; MBC: minimal bactericidal concentration (cut-off = 99,9% reduction in CFU); MDK99,9: time(h) needed to kill 99,9% of the started inoculum; nd: not ined.

Figure 8A also illustrates a comparison between the cterial effects of Triafluocyl, Vancomycin and Minocycline on MRSA.

S. aureus MRSA (ATCC BAA-1556) was grown overnight in brain heart infusion (BHI) , diluted 1:100 in fresh BHI, and incubated aerobically at 37°C until bacteria growth reached a logarithmic phase (OD600 = 0.25-0.3).

Triafluocyl (Cayman Chemical, Item No. 15425) (20ug/ml), Vancomycin (Sigma, 4ug/ml or 10ug/ml), Minocycline (Sigma, 8pg/ml) or a solvent (DMSO) were added to 5 ml of S. aureus MRSA suspensions.

Bacterial growth for S. aureus MRSA was measured after different time intervals by counting the colony-forming units after plating appropriate e dilutions on BHI agar plates.

One y observes that Triafluocyl causes a se of S. aureus MRSA viable count as early as after the first two hours, at which time doses of Vancomycin and Minocycline equal to 10- and 8-fold MIC, respectively, were ineffective. Over the 24h- ment, the bactericidal effect of Vancomycin and Minocyclin remained less efficient than the one of Triafluocyl.

Figure SB) illustrates a comparison between the antibacterial effect of Triafluocyl and Minocycline on S. aureus GISA.

S. aureus Mu50 GISA was grown overnight in brain heart infusion (BHI) medium, diluted 1:100 in fresh BHI, and incubated aerobically at 37°C until bacteria growth reached a logarithmic phase (OD600 = 0.25-0.3).

Triafluocyl (Cayman Chemical, Item No. 15425) (20ug/ml), Minocycline (Sigma, 8pg/ml) or e (DMSO) were then added in 5 ml of bacteria suspensions. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates.

Here again Triafluocyl (20ug/ml) had a quicker and more efficient cterial effect than a high dose of Minocycline (10ug/ml).

Figure 8C rates a comparison between Triafluocyl and Minocycline on E. faecalis E. faeca/is VRE (ATCC BAA-2365) was grown overnight in brain heart infusion (BHI) medium, diluted 1:100 in fresh BHI, and incubated aerobically at 37°C until bacteria growth reached a logarithmic phase (OD600 = 0.2025).

Triafluocyl (Cayman Chemical, Item No. 15425) (20ug/ml), Minocycline (Sigma, 10ug/ml) or vehicle (DMSO) was then added in 5 ml of ia suspensions. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating riate culture dilutions on BHI agar plates.

Here Triafluocyl (20pg/ml) showed bactericidal effect while a high dose of Minocycline (10pg/ml) was only bacteriostatic.

Example 9: tacyl antibacterial effects on gram-positive bacteria strains: S. aureus, S. epidermidis, E. faecalis.

Susceptibility testing: MIC and MBC determination: The Minimal tory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC) of tacyl were ined on several gram-positive strains (Table 2) ing EUCAST (European Committee on Antimicrobial susceptibility Testing) recommendations.

For M|C determination a single colony was resuspended and cultured in the appropriate bacteria-specific medium (TSB: Tryptic Soy Broth for S. aureus atcc 25904 and S. epidermidis and BHI: brain-heart infusion medium for all the other strains) overnight (O/N) in aerobic conditions (37°C with 220rpm shaking), next day a 1:100 inoculum was incubated in Mueller-Hinton broth (MHB) in aerobic ions for 3hr (OD600=0,08-0,1). A further um, 1:300 dilution ofthe MHB culture, corresponding to 3x105 CFU/ml, was grown in presence or absence of different concentrations of Fluometacyl, 1% DMSO in MHB for 20hr.

For MBC determination a 1:100 inoculum of an O/N culture (prepared like before) was incubated in aerobic conditions for 2h in bacteria-specific . The culture was then challenged with tacyl at the MIC concentration or higher. Bacterial growth was measured after different time als by counting the colony-forming units (CFU) after plating appropriate culture dilutions on bacteria-specific medium agar plates. The concentration that kills at least 99,9% of the started inoculum in 24h is defined as the Strains Resistance MIC MBC uM uM $.aureus 30-38 38 5904) $.aureus M RSA 20-30 38 $.aureus-MU50 GISA 30-38 38 S. epidermidis 30 38 E.faeca/is VRE 38 38 Table 2: MIC and MBC determination for different strains. MRSA: methilcillin-resistant S. aureus; GISA: Glycopeptide intermediate-resistant S. aureus; VRE: vancomycin- ant Enteroccocus. MIC: minimal inhibitory concentration; MBC: minimal bactericidal tration (cut-off = 99,9% reduction in CFU). ill study of Fluometacyl against methilcillin-resistant S. aureus S. aureus MRSA (ATCC BAA-1556) was grown overnight in BHI medium, then a 1:100 inoculum was d in fresh BHI and incubated aerobically at 37°C until bacteria growth reached a logarithmic phase (OD600 = 0.25-0.3). The culture was split into two and challenged with 38uM Fluometacyl (= 18.2ug/ml) or DMSO (Ctrl). Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates. (N=2) Example 10: Fluometacyl antibacterial effects on biofilm formation Staphyloccocus aureus (ATCC 25904) or Staphyloccocus epidermidis (ATCC 35984) were grown overnight in TSB medium, before being diluted 100 fold in fresh TSB, and incubated aerobically at 37°C until bacteria culture reached an OD600 of 0.6 (corresponding to approximately 8 CFU/ml). Bacteria cultures were then diluted to 1x104 CFU/ml in fresh TSB. Aliquots of 800pl diluted bacteria suspensions were distributed in each well of a 24-well plate. Bacteria were allowed to adhere for 4 hours under static ions at 37°C. After removing the media, wells were rinsed 2 times with PBS to eliminate onic bacteria and re-filled with TSB supplemented with 0.5 % glucose containing Fluometacyl at desired concentration or DMSO alone (Ctrl). The 24-well plates were incubated at 37°C for 20 hours. Wells were then washed and d with 0.5 % (w/v) l violet for 30 minutes and rinsed with PBS 4 times. The dye was solubilized by adding 20 % acetic acid (v/v in water) before reading absorbance at 595 nm.Figure 10A and Figure 10B show Fluometacyl effect on S. aureus and S. epidermidis biofilm formation respectively at all concentrations tested. In presence of 38 pM Fluometacyl, both S. aureus and S. epidermidis could not form any biofilm.

Claims (27)

CLAIMS :

1. Use of a triazolo(4,5-d)pyrimidine derivative of formula (I) 5 wherein R1 is C 3-5 alkyl optionally substituted by one or more halogen atoms; R2 is a phenyl group, ally substituted by one or more halogen atoms; R3 and R4 are both hydroxyl; R is XOH, wherein X is CH2, or a bond; or a ceutical acceptable salt or solvate thereof, or a solvate of such a salt provided that when X is CH2 or a bond, R1 is not ; when X is CH2 and R1 is 10 CH 2CH 2CF 3, butyl or pentyl, the phenyl group at R2 must be substituted by fluorine; in the manufacture of a medicament for treatment or prevention of bacterial infection.

2. The use according to claim 1 wherein R2 is phenyl substituted by ne atoms. 15

3. The use according to claim 1 or claim 2 wherein R is OH.

4. The use according to any one of claims 1 to 3 wherein the triazolo(4,5-d) pyrimidine derivative is selected from: (1R-(1a, 2a, 3ß(1R*, 2*),5ß))(7-((2-(3,4-difluorophenyl)cyclopropyl)amino)((3,3,3- trifluoropropyl)thio)3H-1,2,3-triazolo(4,5d)pyrimidinyl)5(hydroxy)cyclopentane-1,2- diol; (1S,2R,3S,4R)[7-[[(1R,2S)(3,4-Difluorophenyl)cyclopropyl]amino](propylthio)- 5 ,3-triazolo[4,5-d]pyrimidinyl]-1,2,3-cyclopentanetriol; and a pharmaceutical acceptable salt or solvate thereof, or a e thereof or a solvate of such a salt.

5. The use according to any one of claims 1 to 4 wherein the triazolo(4,5-d) pyrimidine 10 derivative is (1S,2R,3S,4R)[7-[[(1R,2S)(3,4-Difluorophenyl)cyclopropyl]amino] lthio)-3H-1,2,3-triazolo[4,5-d]pyrimidinyl]-1,2,3-cyclopentanetriol also called Fluometacyl.

6. The use according to any one of claims 1 to 5 wherein the medicament is to be 15 administered topically.

7. Use of a triazolo(4,5-d)pyrimidine derivative of formula (I) wherein R1 is C3-5 alkyl optionally substituted by one or more halogen atoms; R2 is a 20 phenyl group, optionally substituted by one or more halogen atoms; R3 and R4 are both hydroxyl; R is XOH, where X is CH2, OCH2CH 2, or a bond; or a ceutical acceptable salt or solvate thereof, or a e of such a salt provided that when X is CH2 or a bond, R1 is not propyl; when X is CH2 and R1 is CH 2CH 2CF 3, butyl or pentyl, the phenyl group at R2 must be substituted by fluorine; when X is OCH2CH 2 and R1 is propyl, the phenyl group at R2 must be substituted by 5 fluorine; as inhibitor of biofilm formation on a surface.

8. The use according to claim 7 wherein R2 is phenyl substituted by ne atoms. 10

9. The use according to claim 7 or claim 8 wherein R is OH or OCH2 CH 2OH.

10. The use according to any one of claims 7 to 9 wherein R is OH.

11. The use according to any one of claims 7 to 10 wherein the triazolo(4,5-d) 15 pyrimidine derivative is selected from: (1R-(1a, 2a, 3ß(1R*, 2*),5ß))(7-((2-(3,4-difluorophenyl)cyclopropyl)amino)((3,3,3- trifluoropropyl)thio)3H-1,2,3-triazolo(4,5d)pyrimidinyl)5(hydroxy)cyclopentane-1,2- diol; (1S-(1a, 2a, 3ß(1R*, 2*),5ß))(7-((2-(3,4-difluorophenyl)cyclopropyl)amino) 20 (propylthio)(3H-1,2,3-triazolo(4,5d)pyrimidinyl)5(2-hydroxyethoxy)cyclopentane- 1,2-diol; (1S,2S,3R,5S)[7-[(1R,2S)(3,4-difluorophenyl)cyclopropylamino](propylthio)-3H- [1,2,3]-triazolo[4,5-d]pyrimidinyl](2-hydroxyethoxy)-1,2-cyclopentanediol); (1S,2S,3R,5S)[7-[(1R,2S)(4-fluorophenyl)cyclopropylamino](propylthio)-3H- 25 [1,2,3]-triazolo[4,5-d]pyrimidinyl](2-hydroxyethoxy)-1,2-cyclopentanediol); (1S,2R,3S,4R)[7-[[(1R,2S)(3,4-Difluorophenyl)cyclopropyl]amino](propylthio)- 3H-1,2,3-triazolo[4,5-d]pyrimidinyl]-1,2,3-cyclopentanetriol; and a pharmaceutical acceptable salt or e thereof, or a solvate thereof or a solvate of such a salt.

12. The use ing to any one of claims 7 to 9 and 11 wherein the triazolo(4,5-d) pyrimidine derivative is ,3R,5S)[7-[(1R,2S)(3,4- difluorophenyl)cyclopropylamino](propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin yl](2-hydroxyethoxy)-1,2-cyclopentanediol) also called Triafluocyl.

13. The use according to any one of claims 7 to11 wherein the triazolo(4,5-d) dine derivative is (1S,2R,3S,4R)[7-[[(1R,2S)(3,4- Difluorophenyl)cyclopropyl]amino](propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin yl]-1,2,3-cyclopentanetriol also called Fluometacyl.

14. A method for treatment of a bacterial infection in a non-human host mammal in need of such treatment which comprises stering to the host an effective amount of the triazolo(4,5-d)pyrimidine derivative of formula(I) as defined in any one of claims 1 to 6.

15.The method according to claim 14 wherein the triazolo(4,5-d)pyrimidine derivative is (1S,2R,3S,4R)[7-[[(1R,2S)(3,4-Difluorophenyl)cyclopropyl]amino](propylthio)- 3H-1,2,3-triazolo[4,5-d]pyrimidinyl]-1,2,3-cyclopentanetriol or Fluometacyl. 25

16. The method according to claim 14 or claim 15 wherein the effective amount to be administered to the host is inferior to 1.8 g per day.

17. The method according to any one of claims 14 to 16 n the triazolo(4,5- d)pyrimidine derivative is administered topically.

18. A method of tion of a bacterial infection in a non-human host mammal in 5 need of such prevention which comprises administering to the host an effective amount of Triazolo(4,5-d)pyrimidine derivative of formula(I) as defined in any one of claims 1 to 6.

19. The method according to claim 18 wherein the triazolo(4,5-d)pyrimidine derivative 10 is (1S,2R,3S,4R)[7-[[(1R,2S)(3,4-Difluorophenyl)cyclopropyl]amino](propylthio)- 3H-1,2,3-triazolo[4,5-d]pyrimidinyl]-1,2,3-cyclopentanetriol or Fluometacyl.

20. The method according to claim 18 or claim 19 wherein an effective amount to be administered to the host is inferior to 1.8 g per day.

21. A method of bacteria killing or prevention of ial growth in biofilm formation comprising using , by applying on a surface, an effective amount of triazolo(4,5- midine derivative of formula(I ) as defined in any one of claims 7 to 13. 20

22. The method according to claim 21 wherein the effective amount is between 20 and 100 µg/ml.

23. The method of bacteria killing in biofilm formation on a e according to claim 21 or claim 22 wherein the biofilm formation is at a maturation step 3 comprising 25 more than 90x106 CFU/cm 2.

24. The method according to any one of claims 21 to 23 wherein the e belongs to a biomaterial.

25. The method according to claim 24 wherein the biomaterial is a cardiovascular device, preferably a heart valve or a pacemaker.

26. The method according to claim 21 wherein the surface s to an intravascular 5 catheter.

27. The use according to claim 1 or 7, substantially as herein described with reference to any one of the Examples and/or