WO2020079051A1

WO2020079051A1 - Compounds for treatment of microbial infection

Info

Publication number: WO2020079051A1
Application number: PCT/EP2019/078044
Authority: WO
Inventors: Martin Vestergaard; Nils Aage BRÜNNER; Hanne INGMER
Original assignee: Scandion Oncology A/S
Priority date: 2018-10-16
Filing date: 2019-10-16
Publication date: 2020-04-23

Abstract

The present invention relates to treatment, amelioration and/or prevention of a microbial infection in a subject. Microbial infections of particular interest are bacterial infections caused by Gram-positive bacteria, especially anti-biotic resistant Gram- positive bacteria.

Description

Compounds for Treatment of Microbial Infection

Technical field

The present invention relates to treatment, amelioration and/or prevention of a microbial infection in a subject.

Background

Bacterial pathogens that cause disease in humans remain a serious threat to public health and antibiotics are still our primary weapons for treatment of infectious diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available antibiotics. The impact of antibiotic resistance on society is at present already tangible in terms of increased morbidity, mortality and healthcare costs. The human and economic burden of antibiotic resistance is for example in Europe estimated to be 25,000 excess deaths and the cost of extended hospital care and productivity loss exceeding 1.5 billion Euros each year. A recent report commissioned by the British Government estimated that the global burden of antibiotic resistance could become 10 million additional deaths a year by 2050. This disturbing circumstance necessitates continuous development of new antibacterial therapeutics to combat the increasing problems of antibiotic resistance. Antibiotic resistance is the ability of bacteria by any mechanism to resist the effect of an antibiotic. Either a bacterium can be intrinsically resistant to a certain antibiotic or can become resistant through spontaneous chromosomal mutations or by acquisition of resistance mechanisms by horizontal gene transfer.

As an example, Staphylococcus aureus is a serious human pathogen known to easily become resistant to antibiotics. S. aureus is an opportunistic pathogen that many of us carry naturally, but some strains are highly virulent causing for example blood stream and heart valve infections in healthy young people, often with a lethal outcome.

Particularly challenging are the methicillin resistant strains (the MRSAs) and less common the strains with resistance to vancomycin, which is considered to be the antibiotic of last resort when treating staphylococcal infections. Antibiotic resistance is common and treating Staphylococcal infections is becoming increasingly challenging. The vast majority of the antibiotics that have been clinically approved in recent years and antibiotics that are in clinical development are derivatives of existing classes of antibiotics.

Hence, there is a need in the art for developing alternative treatments of microbial infections, and in particular those infections caused by bacteria resistant to commonly employed antibiotics, such as b-lactams (e.g. methicillin) and vancomycin. Summary

The present inventors have surprisingly found that SCO-101 and structurally related compounds exhibit significant inhibitory activities against pathogenic microorganisms, for example Gram-positive bacteria such as S. aureus. In particular, SCO-101 and structurally related compounds have proven effective in killing or inhibiting growth of S. aureus being resistant to methicillin or vancomycin.

The present disclosure thus provides SCO-101 and related compounds useful for the treatment of a microbial infection.

In one aspect, a method is provided for treating, ameliorating or preventing a microbial infection, said method comprising administration to a subject in need thereof a compound of formula (Z),

or a pharmaceutically acceptable salt thereof,

wherein

R¹, R² and R³ independently of each other represent hydrogen, halo, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, aryl or heteroaryl; wherein R^a and R^b independently of each other are hydrogen or alkyl; and wherein Y is selected from the group consisting of:

,

In one aspect, a method is provided for treating, ameliorating or preventing a microbial infection, said method comprising administration to a subject in need thereof a compound of formula (I),

or a pharmaceutically acceptable salt thereof,

wherein

R¹, R² and R³ independently of each other represent hydrogen, halo, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, aryl or heteroaryl;

wherein R^a and R^b independently of each other are hydrogen or alkyl.

In a further aspect, a method is provided for treating, ameliorating or preventing a microbial infection, said method comprising administration to a subject in need thereof a compound of formula (I),

or a pharmaceutically acceptable salt thereof, wherein

R¹, R² and R³ independently of each other represent hydrogen, halo, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, phenyl or heteroaryl;

which phenyl is optionally substituted with halo, trifluoromethyl, nitro, -CO-NHR^c, -CO- 0-R^c or -CO-NR’R”;

wherein R^c is hydrogen, alkyl, or phenyl;

R’ and R” independently of each other are hydrogen or alkyl; or

R’ and R” together with the nitrogen to which they are attached form a 5- to 7- membered heterocyclic ring, which ring may optionally comprise as a ring member, one oxygen atom, and/or one additional nitrogen atom, and/or one carbon-carbon double bond, and/or one carbon-nitrogen double bond;

and which heterocyclic ring may optionally be substituted with alkyl;

R^a and R^b independently of each other are hydrogen or alkyl.

In another aspect a method for disinfecting a surface of an object or a subject is provided comprising

a) providing a compound of formula (Z) or a pharmaceutically acceptable salt thereof as defined herein, and

b) exposing the surface of the object or subject to said compound.

In one aspect, a composition is provided comprising an effective amount of a compound of formula (Z) as defined herein for use in treating, ameliorating or preventing a microbial infection in a subject.

In a further aspect, a use of a compound of formula (Z) as defined herein for the manufacture of a medicament for treating, ameliorating and/or preventing a microbial infection in a subject.

Description of Drawings

Fig. 1 : (a) Time-kill curves of S. aureus incubated with SCO-101 and marketed antibiotics at a concentration corresponding to 4x MIC. SCO-101 is bacteriostatic against stationary phase cells; (b) Time-kill curves of S. aureus incubated with SCO- 101 at increasing concentrations corresponding to 4x, 8x and 16x MIC. SCO-101 is bacteriostatic at 4x and 8x MIC, whereas it is bactericidal at 16x MIC against stationary phase cells.

Detailed description

Terms and definitions

To facilitate the understanding of the following description, a number of definitions are presented in the following paragraphs.

The term "treatment", as used anywhere herein comprises any type of therapy, which aims at terminating, preventing, ameliorating and/or reducing the susceptibility to a clinical condition as described herein. In one embodiment, the term treatment relates to prophylactic treatment (i.e. a therapy to reduce the susceptibility of a microbial infection as defined herein).

Thus, "treatment," "treating," and the like, as used herein, refer to obtaining a desired effect, such as a biological, pharmacologic and/or physiologic effect, covering any treatment of a microbial infection in a mammal, including a human. The term is also used in the context of disinfecting objects from which the microbial infection may disseminate. The effect may be prophylactic in terms of completely or partially preventing a microbial infection or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for the microbial infection and/or adverse effect attributable to the infection. That is, "treatment" includes (1 ) preventing the microbial infection from occurring or recurring in a subject, (2) inhibiting the microbial infection, such as arresting its development, (3) stopping, terminating or alleviating the microbial infection or at least symptoms associated therewith, so that the subject no longer suffers from the disorder or clinical condition or its symptoms, such as causing regression of the microbial infection or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating, or ameliorating the microbial infection, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, and/or immune deficiency.

The terms "prevent", "preventing," and "prevention", as used herein, refer to a decrease in the occurrence of symptoms or characteristics of a microbial infection. The term is also used in the context of preventing dissemination of the microbial infection, for example from objects, from which pathogenic microorganisms may disseminate. The prevention may be complete. The prevention may also be partial, such that for example the occurrence of symptoms or characteristics of a microbial infection in a subject is less than that which would have occurred without the present invention. Prevention also refers to reduced susceptibility to a microbial infection.

The terms "ameliorate", "ameliorating" and’’amelioration", are also used separately herein to refer to a reduction of the severity of the occurrence of biological effects or symptoms or characteristics of microbial infection.

The term“antibiotic resistance” as used herein refers to a bacterium, which has acquired resistance to an antibiotic that was once able to treat an infection by that bacterium. Antibiotic resistance may be dose dependent, meaning that higher doses of the antibiotic than usual are required to sufficiently treat the infection, or complete in that a certain antibiotic is without effect irrespective of dose. The non-resistant bacteria may be referred to as antibiotic sensitive bacteria.

Microbial infections

A“microbial infection”, also referred to herein as“infection”, may be defined as the invasion and multiplication of microorganisms such as bacteria, viruses, fungi and parasites that are not normally present within the body. A microbial infection according to the present disclosure may be a bacterial infection or a fungal infection.

An infection may cause no symptoms and be subclinical, or it may cause symptoms and be clinically apparent. An infection may remain localized, or it may spread through the blood or lymphatic vessels to become systemic (body wide). Microorganisms that live naturally in the body without causing clinical symptoms are not considered infections. For example, bacteria that normally live within the mouth and intestine without causing clinical symptoms are not infections.

A bacterium is a prokaryote in which the nucleus is not covered by a nuclear envelope. It has a macromolecular membrane called a capsule outside the plasma membrane and cell wall. The capsule has the function to protect the bacterial body from the immune system of the host. The structure of the cell wall varies widely depending on the species of bacteria with the flagellum being absent in some species.

A fungus is a eukaryote with subcellular organelles enclosed by membranes within the cytoplasm. Aside from the yeast-type, the structures of fungi include the mycelial form, which takes a thin thread-like shape. Some fungi have dimorphic properties, which enable them to alternate between both forms.

A“bacterial infection” according to the present disclosure may be caused by Gram- positive bacteria, Gram-negative bacteria, Mycobacteria, or a combination thereof.

Gram-positive bacteria

Gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their cell wall. Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through a microscope. This is because the thick peptidoglycan layer in the bacterial cell wall retains the stain after it is washed away from the rest of the sample in the decolorization stage of the test. Thus, although there are some exceptions, this staining result gives insight into the cell wall structure and is used to taxonomically group bacteria into the broad groups of Gram-positive and Gram-negative. The staining result coupled with microscopic information about shape (cocci or rods) and information on growth ability (aerobic, anaerobic or facultative) and media used for growth can lead to an identification of the organism.

In general, the following characteristics are present in Gram-positive bacteria: • Cytoplasmic lipid membrane.

• Thick peptidoglycan layer.

• Teichoic acids and lipoids are present, forming lipoteichoic acids, which serve as chelating agents, and also for certain types of adherence.

• Peptidoglycan chains are cross-linked to form rigid cell walls by a bacterial enzyme DD-transpeptidase.

• A much smaller volume of periplasm than that in Gram-negative bacteria.

Only some species have a capsule, usually consisting of polysaccharides. Also, only some species are flagellates, and when they do have flagella, have only two basal body rings to support them, whereas Gram-negative have four. Both Gram-positive and Gram-negative bacteria commonly have a surface layer called an S-layer. In Gram- positive bacteria, the S-layer is attached to the peptidoglycan layer. Specific to Gram- positive bacteria is the presence of teichoic acids in the cell wall. Some of these are lipoteichoic acids, which have a lipid component in the cell membrane that can assist in anchoring the peptidoglycan.

Gram-positive cocci encompass the important human pathogens

of Staphylococcus with a tendency to grow in "grape-like" clusters

and Streptococcus which tends to grow in chains. Gram-positive bacilli (rods) encompass Bacillus, Corynebacterium, Listeria and Clostridia species. The

staphylococci are further subdivided into coagulase positive (S. aureus) and coagulase negative (S. epidermidis and S. saprophyticus) species.

At least six Gram-positive genera are typically pathogenic in humans. Two of these, Streptococcus and Staphylococcus, are cocci (sphere-shaped). The remaining organisms are bacilli (rod-shaped) and can be subdivided based on their ability to form spores. The non-spore formers are Corynebacterium and Listeria (a

coccobacillus), whereas Bacillus and Clostridium produce spores. The spore-forming bacteria can again be divided based on their respiration: Bacillus is a facultative anaerobe, while Clostridium is an obligate anaerobe. Also, Rathybacter, Leifsonia, and Clavibacter are three Gram-positive genera that cause plant disease. Gram- positive bacteria are capable of causing serious and sometimes fatal infections in newborn infants. In some embodiments, the Gram-positive bacteria include, but are not limited to methicillin-susceptible and methicillin-resistant Staphylococci (including

Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, and coagulase-negative Staphylococci), glycopeptide intermediary- susceptible S. aureus (GISA), penicillin-susceptible and penicillin-resistant Streptococci (including Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. avium, S. bovis, S. lactis,

S. sangius and Streptococci Group C, Streptococci Group G and viridans

Streptococci), Enterococci (including vancomycin- susceptible and vancomycin- resistant strains such as Enterococcus faecalis and E. faecium), Clostridium difficile, C. clostridiiforme, C. innocuum, C. perfringens, C. ramosum, Listeria monocytogenes, Corynebacterium jeikeium, Bifidobacterium spp., Eubacterium aerofaciens, E. lentum, Lactobacillus acidophilus, L. casei, L. plantarum, Lactococcus spp., Leuconostoc spp., Pediococcus, Peptostreptococcus anaerobius, P. asaccarolyticus, P. magnus, P.

micros, P. prevotii, P. productus, Propionibacterium acnes, and Actinomyces spp.

In the context of the present disclosure, the bacteria Propionibacterium acnes and Cutibacterium acnes are equivalent.

In one embodiment, the method according to the present disclosure may be used in the treatment of a bacterial infection caused by Gram-positive bacteria selected from the group consisting of: Staphylococcus, Streptococcus, Enterococcus, Bacillus,

Clostridium, Corynebacterium, Listeria, Propionibacterium acnes and Actinomyces.

In one embodiment, the Gram-positive bacteria Staphylococcus according to the present disclosure may be selected from the group consisting of: S. aureus, S.

epidermidis, S. haemolyticus, S. lugdunensis, S. saprophyticus, S. hominis and S. capitis.

In one embodiment, the Staphylococcus according to the present disclosure may be selected from the group consisting of: S. aureus and S. epidermidis.

In one embodiment, the Gram-positive bacteria Streptococcus according to the present disclosure may be selected from the group consisting of: S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. suis, S. mitis, S. mutans, S. uberis and S. pneumoniae. In one embodiment, the Streptococcus is selected from the group consisting of S. uberis and S. agalactiae. In one embodiment, the Streptococcus is selected from the group consisting of S. pyogenes and S. pneumoniae.

In one embodiment, the Gram-positive bacteria Enterococcus according to the present disclosure may be selected from the group consisting of: E. faecalis and E. faecium.

Mycobacteria

Mycobacterium is a genus of Actinobacteria, given its own family, the

Mycobacteriaceae. Over 190 species are recognized in this genus. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (, Mycobacterium tuberculosis) and leprosy ( Mycobacterium leprae) in humans. The Greek prefix myco- means "fungus," alluding to the way mycobacteria have been observed to grow in a mold-like fashion on the surface of cultures. It is acid fast and cannot be stained by the Gram-stain procedure.

In one embodiment, the bacterial infection according to the present disclosure is caused by Mycobacteria selected from the group consisting of M. tuberculosis, M. leprae, M. ulcerans, M. avium and M. smegmatis.

In one embodiment, the bacterial infection according to the present disclosure is caused by Mycobacteria selected from the group consisting of M. tuberculosis, M. leprae, M. ulcerans, M. abscessus, M. avium and M. smegmatis.

Gram-negative bacteria

Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram-staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane.

Gram-negative bacteria are found everywhere, in virtually all environments on Earth that support life. The Gram-negative bacteria include the model organism Escherichia coli (E. coli), as well as many pathogenic bacteria, such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, and Yersinia pestis.

In general, Gram-negative bacteria display these characteristics:

• An inner cell membrane is present (cytoplasmic).

• A thin peptidoglycan layer is present (This is much thicker in Gram-positive bacteria).

• Has outer membrane containing lipopolysaccharides (LPS, which consists of lipid A, core polysaccharide, and O antigen) in its outer leaflet and

phospholipids in the inner leaflet.

• Porins exist in the outer membrane, which act like pores for particular

molecules.

• Between the outer membrane and the cytoplasmic membrane there is a space filled with a concentrated gel-like substance called periplasm.

• The S-layer is directly attached to the outer membrane rather than to the

peptidoglycan.

• If present, flagella have four supporting rings instead of two.

• Teichoic acids or lipoteichoic acids are absent.

• Lipoproteins are attached to the polysaccharide backbone.

• Some contain Braun's lipoprotein, which serves as a link between the outer membrane and the peptidoglycan chain by a covalent bond.

In one embodiment, the method according to the present disclosure may be used in the treatment of a bacterial infection caused by Gram-negative bacteria selected from the group consisting of: Enterobacteriaceae (E. coli, Klebsiella, Salmonella, Shigella), Pseudomonas, Vibrio, Campylobacter, Legionella, Neisseria, Haemophilus,

Acinetobacter, Helicobacter, Bartonella, Bordetella, Borrelia, Brucella, Francisella, Leptospira, Treponema, Yersinia, and Chlamydia.

Antibiotic-resistant bacteria

The methods and uses provided herein can be applied to treatment, prevention and/or amelioration of an infection with any bacteria having acquired antibiotic resistance. The anti-biotic resistant bacteria are in one embodiment to be selected from the group consisting of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin intermediate-resistant Staphylococcus aureus (VISA), vancomycin-resistant S. aureus (VRSA), extended spectrum beta-lactamase producing bacteria (ESBL), vancomycin- resistant Enterococcus (VRE) and multidrug-resistant A. baumannii (MRAB). In one embodiment, the microorganism is Klebsiella pneumoniae. In a preferred embodiment, the bacterium is MRSA.

In one embodiment, the bacterial infection is caused by an antibiotic-resistant bacterium, such as a multi-resistant bacterium.

In one embodiment, the antibiotic-resistant bacterium is selected from the group consisting of: Staphylococcus aureus, Clostridium difficile, Enterococcus,

Mycobacterium tuberculosis, Mycoplasma genitalium, Streptococcus, Campylobacter, Neisseria gonorrhoeae and Gamma proteobacteria.

In a particular embodiment, the antibiotic-resistant bacterium is methicillin resistant Staphylococcus aureus (MRSA). In a further embodiment, the antibiotic-resistant bacterium is vancomycin intermediate-resistant Staphylococcus aureus (VISA).

Methicillin-resistant Staphylococcus aureus (MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium responsible for several types of infections in humans, which are d ifficu It-to-treat. MRSA comprise any strain of Staphylococcus aureus that has developed multiple drug resistance to beta-lactam antibiotics. The strains may have gained drug resistance through horizontal gene transfer and natural selection b-lactam antibiotics are a broad spectrum group which includes some penams - penicillin derivatives such as methicillin and oxacillin, and cephems such as the cephalosporins. MRSA have evolved from horizontal gene transfer of the mecA gene to at least five distinct S. aureus lineages. Beta-lactam antibiotics permanently inactivate penicillin-binding proteins (PBPs), which are essential for bacterial life, by permanently binding to their active sites. Some forms of MRSA, however, express a PBP that will not allow the antibiotic into their active site. Acquisition of staphylococcal cassette chromosome (SCCmec) in methicillin-sensitive Staphylococcus aureus (MSSA) gives rise to a number of genetically different MRSA lineages. These genetic variations within different MRSA strains likely explain the variability in virulence and associated MRSA infections. The first MRSA strain, ST250 MRSA-1 originated from SCCmec and ST250-MSSA integration. Historically, major MRSA clones: ST2470-MRSA-I, ST239-MRSA-III, ST5-MRSA-II, and ST5-MRSA-IV were responsible for causing hospital-acquired MRSA (HA-MRSA) infections. ST239- MRSA-III, known as the Brazilian clone, was highly transmissible compared to others and distributed in Argentina, Czech Republic, and Portugal. In the UK, the most common strains of MRSA are EMRSA15 and EMRSA16. EMRSA16 has been found to be identical to the ST36:USA200 strain, which circulates in the United States, and to carry the SCCmec type II, enterotoxin A and toxic shock syndrome toxin 1 genes. Under the new international typing system, this strain is now called MRSA252. EMRSA 15 is also found to be one of the common MRSA strains in Asia. Other common strains include ST5:USA100 and EMRSA 1. These strains are genetic characteristics of HA- MRSA.

Community-acquired MRSA (CA-MRSA) strains emerged in late 1990 to 2000, infecting healthy people who had not been in contact with health care facilities. It has been proposed that CA-MRSA did not evolve from the HA-MRSA. This is supported by molecular typing of CA-MRSA strains and genome comparison between CA-MRSA and HA-MRSA, which indicate that novel MRSA strains integrated SCCmec into MSSA separately on its own. By mid-2000, CA-MRSA was introduced into the health care systems and distinguishing CA-MRSA from HA-MRSA became a difficult process. Community-acquired MRSA (CA-MRSA) is more easily treated and more virulent than hospital-acquired MRSA (HA-MRSA). The genetic mechanism for the enhanced virulence in CA-MRSA remains an active area of research. Especially the Panton- Valentine leukocidin (PVL) genes are of interest because they are a unique feature of CA-MRSA.

In the United States, most cases of CA-MRSA are caused by a CC8 strain designated ST8:USA300, which carries SCCmec type IV, Panton-Valentine leukocidin, phenol- soluble modulin (PSM)-alpha and enterotoxins Q and K, and ST1 :USA400. The ST8:USA300 strain results in skin infections, necrotizing fasciitis and toxic shock syndrome, whereas the ST1 :USA400 strain results in necrotizing pneumonia and pulmonary sepsis. Other community-acquired strains of MRSA are ST8:USA500 and ST59:USA1000. In many nations of the world, MRSA strains with different predominant genetic background types have come to predominate among CA-MRSA strains;

USA300 easily tops the list in the U.S. and is becoming more common in Canada after its first appearance there in 2004. For example, in Australia ST93 strains are common, while in continental Europe ST80 strains, which carry SCCmec type IV, predominate. In Taiwan, ST59 strains, some of which are resistant to many non-beta-lactam antibiotics, have arisen as common causes of skin and soft tissue infections in the community. In a remote region of Alaska, unlike most of the continental U.S., USA300 was found rarely in a study of MRSA strains from outbreaks in 1996 and 2000 as well as in surveillance from 2004-06.

An MRSA strain, CC398, is found in intensively reared production animals (primarily pigs, but also cattle and poultry), where it can be transmitted to humans as LA-MRSA (livestock-associated MRSA).

For CA-MRSA strains, it has been found in a study that 15 (63%) are associated with skin infections, 5 (21%) bloodstream, 3 (13%) respiratory, and 1 (4%) a catheter-site infection. One third of the patients were <18 years of age (3 were <2 years of age), and 50% were female. Nineteen (79%) patients were initially seen at hospitals, 3 (13%) at outpatient centers, and 2 (8%) at a prison. Three (13%) were considered to have healthcare-onset infections.

The term“MRSA” as used herein includes any Methicillin-resistant strain of

Staphylococcus aureus (S. aureus). The term also includes any strain regardless of how it is acquired and thus includes HA-MRSA (healthcare-associated MRSA), CA- MRSA (community-associated MRSA) and LA-MRSA (livestock-associated). Several newly discovered strains of MRSA show antibiotic resistance even to vancomycin and teicoplanin. These new evolutions of the MRSA bacterium are referred to as vancomycin intermediate-resistant Staphylococcus aureus (VISA). Thus, in one embodiment of the present disclosure, MRSA also includes VISA strains. Specifically, the methods and uses, whether therapeutic or non-therapeutic, is applied in specific embodiments for treatment, prevention and/or amelioration of a microbial infection with at least one MRSA strain selected from the group consisting of CC1 ,

CC5, CC8 (USA300), CC22, CC30, CC45, CC80 and CC398. In another embodiment, the strain is selected from the group consisting of ST250 MRSA-1 , EMRSA15 and EMRSA16/ USA200. In another embodiment, the strain is selected from the group consisting of ST250 MRSA-1 , ST2470-MRSA-I, ST239-MRSA-III, ST5-MRSA-II, ST5- MRSA-IV, ST239-MRSA-III, EMRSA15, EMRSA16, ST5:USA100, EMRSA 1 ,

ST8:USA300 (CC8), ST1 :USA400, ST8:USA500, ST59:USA1000 and CC398.

MRSA is generally obtained by touching the skin of another person who is colonized with MRSA, or by touching a contaminated surface (such as a countertop, door handle, or phone). You can develop an infection from MRSA if your skin is colonized and the bacteria enter an opening (e.g. a cut, scrape, or wound) in the skin. Infection is most commonly seen under the nostrils. The rest of the respiratory tract, open wounds, intravenous catheters, and the urinary tract are also potential sites for infection. Healthy individuals may carry MRSA asymptomatically for periods ranging from a few weeks to many years. People with compromised immune systems are at a significantly greater risk of symptomatic secondary infection. MRSA can usually be detected by swabbing the nostrils and isolating the bacteria found inside the nostrils. Combined with extra sanitary measures for those in contact with infected people, swab screening people admitted to hospitals has been found to be effective in minimizing the spread of MRSA in hospitals in the United States, Denmark, Finland, and the Netherlands. MRSA may progress substantially within 24-48 hours of initial topical symptoms. After 72 hours, MRSA can take hold in human tissues and eventually become resistant to treatment. The initial presentation of MRSA is small red bumps that resemble pimples, spider bites, or boils; they may be accompanied by fever and, occasionally, rashes. Within a few days, the bumps become larger and more painful; they eventually open into deep, pus-filled boils. About 75 percent of community-associated (CA-) MRSA infections are localized to skin and soft tissue. Infections can usually be treated effectively with conventional antibiotics. Some CA-MRSA strains display enhanced virulence, spreading more rapidly and causing illness much more severe than traditional HA- MRSA infections, and they can affect vital organs and lead to widespread infection (sepsis), toxic shock syndrome, and necrotizing pneumonia. This is thought to be due to toxins carried by CA-MRSA strains, such as PVL and PSM, though PVL was recently found not to be a factor in a study by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. It is not known why some healthy people develop CA-MRSA skin infections that are treatable while others infected with the same strain develop severe infections or die.

People are occasionally colonized with CA-MRSA and are completely asymptomatic. The most common manifestations of CA-MRSA are simple skin infections, such as impetigo, boils, abscesses, folliculitis, and cellulitis. Rarer, but more serious, manifestations can occur, such as necrotizing fasciitis and pyomyositis (most commonly found in the tropics), necrotizing pneumonia, and infective endocarditis (which affects the valves of the heart), and bone (osteomyelitis) and joint infections. CA-MRSA often results in abscess formation that requires incision and drainage.

Before the spread of MRSA into the community, abscesses were not considered contagious, because infection was assumed to require violation of skin integrity and the introduction of Staphylococci from normal skin colonization. However, newly emerging CA-MRSA is transmissible from HA-MRSA.

Diagnostic microbiology laboratories and reference laboratories use molecular techniques for identifying and characterizing MRSA have recently been developed. Normally, the bacterium must be cultured from blood, urine, sputum, or other body-fluid samples, and in sufficient quantities to perform confirmatory tests early-on. Still, because no quick and easy method exists to diagnose MRSA, initial treatment of the infection is often based upon empirical treatment/'strong suspicion' and techniques by the treating physician; these include quantitative PCR procedures, which are employed in clinical laboratories for quickly detecting and identifying MRSA strains.

Another common laboratory test is a rapid latex agglutination test that detects the PBP2a protein. PBP2a is a variant penicillin-binding protein that imparts the ability of S. aureus to be resistant to oxacillin.

MRSA infections can also be defined as illness compatible with staphylococcal disease in a patient from whom a strain of S. aureus resistant to oxacillin by disk diffusion was isolated from a clinically relevant site. Because it is suspected that community strains has entered the healthcare setting, epidemiologic risk factor data are not always useful in distinguishing community versus healthcare strains. Therefore, microbiologic definitions can be used. An MRSA isolate can be considered to be an HA-MRSA strain if it is resistant to at least 2 of the following antibiotics: trimethoprim/ sulfamethoxazole (TMP/SMX), ciprofloxacin, gentamicin, rifampin, and tetracycline. A MRSA isolate is considered to be a CA-MRSA strain if 1 ) antimicrobial susceptibility results are available for at least 2 of the following agents: TMP/SMX, ciprofloxacin, gentamicin, rifampin, tetracycline, and 2) the isolate was resistant to no more than 1 of the agents and was confirmed to be susceptible to at least 2 of these agents.

An infection can be considered to be healthcare onset if the MRSA culture is obtained >48 hours after a patient was admitted to the hospital and the patient had no evidence of the infection at the time of admission. A MRSA culture obtained within 48 hours of hospital admission or evidence of infection on admission can be considered an indication of a community-onset infection.

Skin disease was defined as a primary skin infection such as abscess, cellulitis, folliculitis, or a skin infection spreading to contiguous tissues. Surgical site infections (SSIs) were not considered to be skin disease.

Thus, the compositions, uses and methods, whether therapeutic or non-therapeutic, are in one embodiment applied to an object or subject for which S. aureus have been identified using one or more of the techniques described above, such as in particular qPCR.

Fungal infections

Pathogenic fungi are fungi that cause disease in humans or other organisms.

Approximately 300 fungi are known to be pathogenic to humans. The study of fungi pathogenic to humans is called "medical mycology". Although fungi are eukaryotic, many pathogenic fungi are microorganisms.

Because fungal spores are present in the air or in the soil, one can inhale the spores or they can land on a person. Thus fungal infections often start off in the lungs or on the skin. People with weak immune systems are very prone to fungal infections. Examples of fungal infections include:

• tinea pedis (athlete's foot)

• tinea corporis (ringworms)

· yeast infection

• onychomycosis (fungal infection of the toenails)

• tinea versicolor (fungal infection of the skin)

• tinea cruris (jock itch) In one embodiment, the method according to the present disclosure relates to the treatment, prevention and/or amelioration of a microbial infection which is a fungal infection caused by a fungus. In some embodiment, the fungus is selected from the group consisting of: Aspergillus, Candida, Cryptococcus and Pneumocystis. In one embodiment, the fungus is Candida, such as Candida albicans.

In some embodiments, the fungal infection presents itself with a fungal disease. Said fungal disease may be selected from the group consisting of: aspergillosis, blastomycosis, candidiasis, Candida auris, coccidioidomycosis (valley fever), cryptococcus neoformans infection, cryptococcus gattii infection, fungal eye infection, fungal nail infection, histoplasmosis, mucormycosis, mycetoma, pneumocystis pneumonia, ringworm, sporotrichosis and talaromycosis.

Compounds for treatment of microbial infections

It is within the scope of the present disclosure to provide a compound of formula (Z),

or a pharmaceutically acceptable salt thereof,

wherein R¹, R² and R³ independently of each other represent hydrogen, halo, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, aryl or heteroaryl;

wherein R^a and R^b independently of each other are hydrogen or alkyl; and wherein Y is selected from the group consisting of:

,

It is within the scope of the present invention to provide a compound of formula (I)

or a pharmaceutically acceptable salt thereof,

wherein

wherein R^a and R^b independently of each other are hydrogen or alkyl.

It is further within the scope of the present invention to provide a compound of formula

(I)

or a pharmaceutically acceptable salt thereof,

wherein R¹, R² and R³ independently of each other represent hydrogen, halo, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, phenyl or heteroaryl;

wherein R^c is hydrogen, alkyl, or phenyl;

R’ and R” independently of each other are hydrogen or alkyl; or

and which heterocyclic ring may optionally be substituted with alkyl;

R^a and R^b independently of each other are hydrogen or alkyl; for use in the therapeutic treatment, prevention and/or amelioration of a microbial infection with a microorganism, such as a Gram-positive bacterium, in a subject.

In one embodiment, the compounds of formula (Z) results in decreased mutation frequency of the bacteria and/or delayed development of resistance to antibiotics.

In one embodiment, the compound of formula (Z) as defined herein may be of formula (II):

or a pharmaceutically acceptable salt thereof, wherein R¹, R² and R³ are as defined for formula (I).

In some embodiments, R¹ of formula (I) represents halo. In one embodiment, R² and R³ independently of each other represent halo or trifluoromethyl.

In one embodiment, the compound of formula (I) is selected from:

/V-4-Nitrophenyl-/V^'-[4-bromo-2-(1 -/-/-tetrazol-5-yl)phenyl] urea; /V-3,5-Di(trifluoromethyl)phenyl-/V^'-[4-bromo-2-(1-/-/-tetrazol-5-yl)phenyl] urea;

L/-3-T rifluoromethylphenyl-/V-[4-(3-nitrophenyl)-2-(1 -/-/-tetrazol-5-yl)phenyl] urea;

/V-3-Trifluoromethylphenyl-/V-[4-(4-anilinocarbonylphenyl)-2-(1-/-/-tetrazol-5-yl)phenyl] urea;

/V-3-Trifluoromethylphenyl-/V-[4-(4-trifluoromethylphenyl)-2-(1-/-/-tetrazol-5-yl)phenyl] urea;

N-( 3-T rifluoromethyl-phenyl)-/V^'-[2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

N-( 3-T rifluoromethyl-phenyl)-/V^'-[4-bromo-2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

N-( 3-T rilfuoromethyl-phenyl)-/V^'-[4-phenyl-2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

/V-(3-Chloro-phenyl)-/V^'-[2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

N-( 3-T rifluoromethyl-phenyl)-/V^'-[4-amino-2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

N-( 3-T rifluoromethyl-phenyl)-/V^'-[4-acetylamino-2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

N-( 3-T rifuoromethyl-phenyl)-/V^'-[4-carbamoyl-2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

/V-(3-Trifluoromethyl-phenyl)-/V^'-[4-(/V^",/V^"-dimethylcarbamoyl)-2-(1-/-/-tetrazol-5-yl)- phenyl] urea;

3^'-(1-/-/-tetrazol-5-yl)-4^'-[3-(3-trifluoromethyl-phenyl)-ureido]-biphenyl-4-carboxylic acid; N-(Biphenyl-4-yl)-/V^'-[2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

/V-(Biphenyl-3-yl)-/V^'-[2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

/V-(3-Acetyl-phenyl)-/V^'-[2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

/V-(Biphenyl-3-yl)-/V^'-[2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

/V-[3-(Pyridin-3-yl)-phenyl]-/V^'-[2-(1-/-/-tetrazol-5-yl)-phenyl] urea;

/V-(3-Bromo-phenyl)-/V^'-[4^'-(4-methyl-piperazine-1-carbonyl)-3-(1-/-/-tetrazol-5-yl)- biphenyl-4-yl] urea;

/V-(3,5-Dichlorophenyl)-/V^'-[4-bromo-2-(1-/-/-tetrazol-5-yl)-phenyl] urea;

/V-(3,4-Dichlorophenyl)-/V^'-[4-bromo-2-(1-/-/-tetrazol-5-yl)-phenyl] urea;

N-( 2-T rifluoromethyl-phenyl)-/V^'-[4-bromo-2-(1 -/-/-tetrazol-5-yl)-phenyl] urea;

/V-(2-Fluorophenyl)-/V^'-[4-bromo-2-(1 -/-/-tetrazol-5-yl)-phenyl] urea; and

/V-(2-Ethylphenyl)-/V^'-[4-bromo-2-(1-/-/-tetrazol-5-yl)-phenyl] urea;

or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of formula (I) is selected from:

/V-4-Nitrophenyl-/V^'-[4-bromo-2-(1 -/-/-tetrazol-5-yl)phenyl] urea;

/V-3,5-Di(trifluoromethyl)phenyl-/V^'-[4-bromo-2-(1-/-/-tetrazol-5-yl)phenyl] urea;

N- 3-T rifluoromethylphenyl-/V-[4-(3-nitrophenyl)-2-(1 -/-/-tetrazol-5-yl)phenyl] urea; /V-3-Trifluoromethylphenyl-/V-[4-(4-anilinocarbonylphenyl)-2-(1-/-/-tetrazol-5-yl)phenyl] urea; and

or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the compound of formula (I) is of formula (III):

or a pharmaceutically acceptable salt thereof.

The compound of formula (III) is SCO-101 as described previously e.g. in WO

2017/198700.

In one embodiment, the compound is of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein R³ and R² are defined as for formula (I); and wherein X is halo, trifluoromethyl, nitro, -CO-NHR^c, -CO-0-R^c or -CO- NR’R”, -S0₂-NR’R”, -0-CF₃, -P0₄H₂;

wherein R^c is hydrogen, alkyl, or phenyl;

R’ and R” independently of each other are hydrogen or alkyl; or

R’ and R” together with the nitrogen to which they are attached form a 5- to 7- membered heterocyclic ring, which ring may optionally comprise as a ring member, one oxygen atom, and/or one additional nitrogen atom, and/or one carbon-carbon double bond, and/or one carbon-nitrogen double bond; and which heterocyclic ring may optionally be substituted with alkyl.

In one embodiment, the compound of formula (IV) is of formula (V):

or a pharmaceutically acceptable salt thereof, wherein R², R³, and X are as defined for formula (IV).

In one embodiment, the compound of formula (I) is of formula (Va) or (Vb):

or a pharmaceutically acceptable salt thereof.

In one embodiment, R² and R³ according to the present disclosure are independently selected from the group consisting of: halo and trifluoromethyl.

In one embodiment, the compound is selected from the group consisting of:

pharmaceutically acceptable salt thereof.

In one embodiment, the compound is selected from the group consisting of:

acceptable salt thereof.

In one embodiment, the compound is selected from the group consisting of:

pharmaceutically acceptable salt thereof. In one embodiment, the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt hereof.

In one embodiment, the compound of formula (Z) is bacteriostatic. In one embodiment, the compound of formula (Z) is bactericidal. In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-positive bacteria of 100 mM or less, such as 75 mM or less, such as 50 mM or less, such as 45 mM or less, such as 40 mM or less, such as 35 mM or less, such as 30 mM or less, such as 25 mM or less, such as 20 mM or less, such as 15 mM or less, such as 10 mM or less, such as 8 mM or less, such as 6 mM or less, such as 4 mM or less, such as 2 mM or less, such as 1 mM or less, preferably wherein the MIC is 10 mM or less.

In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-positive bacteria of 50 mM or less, such as 45 mM or less, such as 40 mM or less, such as 35 mM or less, such as 30 mM or less, such as 25 mM or less, such as 20 mM or less, such as 15 mM or less, such as 10 mM or less, such as 8 mM or less, such as 6 mM or less, such as 4 mM or less, such as 2 mM or less, such as 1 mM or less, preferably wherein the MIC is 10 mM or less. In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-positive bacteria of 30 mM or less, such as 25 mM or less, such as 20 mM or less, such as 15 mM or less, such as 10 mM or less, such as 8 mM or less, such as 6 mM or less, such as 4 mM or less, such as 2 mM or less, such as 1 mM or less, preferably wherein the MIC is 10 mM or less.

In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-positive bacteria of 20 mM or less, such as 15 mM or less, such as 10 mM or less, such as 8 mM or less, such as 6 mM or less, such as 4 mM or less, such as 2 mM or less, such as 1 mM or less, preferably wherein the MIC is 10 mM or less.

In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-positive bacteria of 10 mM or less, such as 8 mM or less, such as 6 mM or less, such as 4 mM or less, such as 2 mM or less, such as 1 mM or less, preferably wherein the MIC is 5 mM or less.

In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-negative bacteria and/or a fungus of 200 mM or less, such as 175 mM or less, such as 150 mM or less, such as 125 mM or less, such as 100 mM or less, such as 75 mM or less, such as 50 mM or less, such as 25 mM or less, such as 15 mM or less, preferably wherein the MIC is 10 mM or less.

In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-negative bacteria and/or a fungus of 150 mM or less, such as 125 mM or less, such as 100 mM or less, such as 75 mM or less, such as 50 mM or less, such as 25 mM or less, such as 15 mM or less, preferably wherein the MIC is 10 mM or less.

In one embodiment, the compound according to formula (Z) of the present disclosure has a MIC value against a Gram-negative bacteria and/or a fungus of 100 mM or less, such as 75 mM or less, such as 50 mM or less, such as 25 mM or less, such as 15 mM or less, preferably wherein the MIC is 10 mM or less. Definition of Substituents

In the context of the present disclosure halo represents fluoro, chloro, bromo or iodo. In the context of the present disclosure an alkyl group designates a univalent saturated, straight or branched hydrocarbon chain. The hydrocarbon chain preferably contain of from one to six carbon atoms (Ci-₆-alkyl), including pentyl, isopentyl, neopentyl, tertiary pentyl, hexyl and isohexyl. In one embodiment alkyl represents a Ci-₄-alkyl group, including butyl, isobutyl, secondary butyl, and tertiary butyl. In another embodiment of this invention alkyl represents a Ci-3-alkyl group, which may in particular be methyl, ethyl, propyl or isopropyl. Further, an alkyl group may be substituted, for example by halogen, such as one or more fluorine atoms. In one embodiment, an alkyl group is a trifluoromethyl group. In the context of the present disclosure, the group“phenyl” may be substituted by one or more substituents. In one embodiment, the“phenyl” is optionally substituted with halo, trifluoromethyl, O-trifluoromethyl, nitro, -CO-NHR^c, -CO-0-R^c, -CO-NR’R”, -SO2-

R’R”;

wherein R^c is hydrogen, alkyl, or phenyl;

R’ and R” independently of each other are hydrogen or alkyl; or

R’ and R” together with the nitrogen to which they are attached form a 5- to 7- membered heterocyclic ring, which ring may optionally comprise as a ring member, one oxygen atom, and/or one additional nitrogen atom, and/or one carbon-carbon double bond, and/or one carbon-nitrogen double bond

In the context of present disclosure a heteroaryl group designates an aromatic mono-, bi- or poly-heterocyclic group, which holds one or more heteroatoms in its ring structure. Preferred heteroatoms include nitrogen (N), oxygen (O), and sulphur (S). Preferred monocyclic heteroaryl groups of the invention include aromatic 5- and 6 membered heterocyclic monocyclic groups, including furanyl, in particular 2- or 3- furanyl; thienyl, in particular 2 or 3-thienyl; pyrrolyl (azolyl), in particular 1 ,2 or 3- pyrrolyl; oxazolyl, in particular oxazol-2, 4 or 5-yl; thiazolyl, in particular thiazol-2, 4 or 5- yl; imidazolyl, in particular 1 ,2 or 4-imidazolyl; pyrazolyl, in particular 1 ,3 or 4-pyrazolyl; isoxazolyl, in particular isoxazol-3,4 or 5-yl; isothiazolyl, in particular isothiazol-3,4 or 5- yl; oxadiazolyl, in particular 1 ,2,3-, 1 ,2,4-, 1 ,2,5- or 1 ,3,4-oxadiazol-3,4 or 5-yl; triazolyl, in particular 1 ,2,3-, 1 ,2,4-, 2,1 ,3- or 4,1 ,2-triazolyl; thiadiazolyl, in particular thiadiazol- 3,4 or 5-yl; pyridinyl, in particular 2,3 or 4-pyridinyl; pyridazinyl, in particular s or 4- pyridazinyl; pyrimidinyl, in particular 2,4 or 5-pyrimidinyl; pyrazinyl, in particular 2 or 3- pyrazinyl; and triazinyl, in particular 1 ,2, 3-, 1 ,2,4- or 1 ,3,5-triazinyl.

5- to 7-membered heterocyclic rings comprising one nitrogen atom include for example, but not limited to, pyrolidine, piperidine, homopiperidine, pyrroline, tetrahydropyridine, pyrazolidine, imidazolidine, piperazine, homopiperazine, and morpholine.

In the context of the present disclosure, an“aryl” group refers to an aromatic radical with six to ten ring atoms (e.g., (C₆-io)aryl) which has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl). Whenever it appears herein, a numerical range such as“6 to 10” refers to each integer in the given range; e.g., 6 to 10 ring atoms in (C₆-io)aromatic or (C₆-io)aryl means that the aryl group may consist of 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms. The term includes monocyclic or fused— ring polycyclic (/^'.e., rings which share adjacent pairs of ring atoms) groups. Unless stated otherwise specifically in the specification, an aryl moiety is optionally substituted by one or more substituents.

Dosages and dosing regimes

The dosage requirements will vary with the particular composition, the route of administration and the particular subject or object being treated. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of active ingredients or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained using conventional methods known in the art. In one embodiment, the method according to the present disclosure involves administration of a compound of formula (Z) in a therapeutically effective dose.

Further medicaments

The methods, uses and compositions provided herein may involve or comprise at least one further medicament. The further medicament is preferably suitable for the treatment, prevention and/or amelioration of a microbial infection, in particular for use in treatment, prevention and/or amelioration of an infection with a microorganism which is a Gram-positive bacterium.

In one embodiment, the method according to the present disclosure comprises administering a further medicament to the subject for treating, ameliorating or preventing a microbial infection in a subject. In one embodiment, the further medicament is an antibiotic.

The antibiotic according to the present disclosure may be selected from the following classes of antibiotics:

• Penicillins, such as penicillin and amoxicillin

• Cephalosporins, such as cephalexin (Keflex)

• Macrolides, such as erythromycin (E-Mycin), clarithromycin (Biaxin), and

azithromycin (Zithromax)

• Fluoroquinolones, such as ciprofloxacin (Cipro), levofloxacin (Levaquin), and ofloxacin (Floxin)

• Sulfonamides (sulfa drugs), such as co-trimoxazole (Bactrim) and trimethoprim (Proloprim)

• Tetracyclines, such as tetracycline (Sumycin, Panmycin) and doxycycline

(Vibramycin)

• Aminoglycosides, such as gentamicin (Garamycin) and tobramycin (Tobrex)

In one embodiment, the antibiotic is a sulfa drug (like co-trimoxazole

(trimethoprim/sulfamethoxazole)), tetracycline (like doxycycline and minocycline) and clindamycin (for osteomyelitis). Generally, CA-MRSA has a greater spectrum of antimicrobial susceptibility to sulfa drugs. In one preferred embodiment, the antibiotic is vancomycin, which is the current drug of choice for treating CA-MRSA.

HA-MRSA is also often susceptible to vancomycin, but to lesser extent to other drugs. The antibiotic could also be linezolid (belonging to the newer oxazolidinones class of drugs) and daptomycin, which are effective against both CA-MRSA and HA-MRSA.

In another preferred embodiment, the antibiotic is one or more of vancomycin, linezolid, or clindamycin, which are recommended by The Infectious Disease Society of America for treating people with MRSA pneumonia.

The antibiotic may also be selected from Ceftaroline (fifth-generation cephalosporin), Vancomycin and teicoplanin. Teicoplanin is a structural congener of vancomycin that has a similar activity spectrum but a longer half-life. Because the oral absorption of vancomycin and teicoplanin may be low, these agents are preferably administered intravenously to control systemic infections.

In one embodiment, the antibiotic is selected from the group consisting of Linezolid, quinupristin/dalfopristin, daptomycin, ceftaroline, and tigecycline, which are currently used to treat more severe infections that do not respond to glycopeptides such as vancomycin. In particular, daptomycin is preferred for VISA bloodstream infections and endocarditis, and is therefore a preferred antibiotic.

The further medicament which is an antibiotic may be selected from the group consisting of: vancomycin, linezolide, daptomycin, streptomycin, kanamycin, tobramycin, gentamicin, methicillin and neomycin.

In some embodiments, the compound of formula (Z) potentiates the effect of the further medicament.

Subjects

A subject which may be suitable for treatment of a microbial infection as defined herein, may be a mammal or a vertebrate. In one embodiment, the subject is a human being. The subject may also be non- human, such as a dog, a horse, a goat, a sheep or a pig.

In one embodiment, the subject is a human being. The subject may also be non- human, such as a dog, a horse, a goat, a cow, a sheep, or a pig.

In one embodiment, the subject is a vertebrate such as a bird or a fish. In one embodiment, the bird is selected from the group consisting of: turkey, chicken, duck, hen, goose, guinea fowl and quail. In one embodiment, the fish is selected from the group consisting of: carp, tilapia, salmon, roho labeo, trout, milkfish, wuchang bream, northern snakehead and amur catfish.

Administration

In one embodiment, the method according to the present disclosure comprises administration of the compound of formula (Z) by enteral administration and/or parental administration and/or topical administration.

In one embodiment, the compound of formula (Z) as defined herein is in in the form of a tablet or a capsule for oral administration. In one embodiment, the compound of formula (Z) is in the form of a liquid for intravenous administration or continuous infusion. In one embodiment, the composition is administered topically.

Disinfection of a surface

In one embodiment, a method for disinfecting a surface is provided, comprising a. providing a compound of formula (Z) or a pharmaceutically acceptable salt thereof as defined herein, and

b. exposing the surface of an object or a subject as defined herein to said compound.

The object may e.g. be a surgical instrument, a table, a floor, a phone, a computer keyboard, or a container, such as a container for keeping foodstuff. In one embodiment, the compound is used for disinfecting a surface of a subject, for example as an alternative or additional effector to ethanol/alcohol surface

disinfection/sterilization.

The compound may be applied by any suitable means, such as by aerosol spraying of the surface or physically wiping the surface with the compound, e.g. via a wet wipe or other cleaning instrument.

The method substantially decreases the microbial load on said surface e.g. by killing or inhibiting the further growth of microorganisms contaminating the surface. In one embodiment, the method decreases the microbial load on the surface by killing the microorganisms contaminating the surface.

Examples

Example 1 : Determination of minimum inhibitory concentration (MIC):

Background:

The minimum inhibitory concentration (MIC) is the lowest concentration of a chemical which prevents visible growth of a bacterium. The MIC of SCO-101 was examined on a range of bacteria (Table 1 ).

Materials and methods:

The minimum inhibitory concentration for SCO-101 was determined using the two-fold broth microdilution assay in cation-adjusted Mueller Hinton with an initial inoculum of approximately 5x10⁵ CFU/ml. The MIC was determined as the lowest concentration that completely inhibits visible growth after incubation at 37 °C for 24 h. For all strains, the MIC determinations have been performed with three biological replicates.

The MIC for Clostridium difficile and Propionibacterium aeries was assessed under anaerobic conditions, which was achieved in a GasPak Jar with Oxoid AnaeroGen sachets (Thermo Scientific™). MIC for C. difficile and P. acnes was determined in brain heart infusion broth and MIC was recorded after 48 h. Inoculum and incubation temperatures were the same as previous.

Results:

For SCO-101 , the minimum inhibitory concentrations (MIC) were 5-10 pg/ml (10-20 mM) for all Gram-positive pathogens tested, including S. aureus, S. epidermidis, E. faecalis, E. faecium, P. acnes, C. difficile and Streptococci (Table 1 ). The MIC values for selected mycobacteria and Gram-negative bacteria are also shown in Table 1. SCO-101 displays lower growth-inhibitory activity against the tested Gram-negative bacteria (P. aeruginosa, E. coli, A. baumanii and K. pneumoniae) and the fungus Candida albicans, than was observed for the tested Gram-positive bacteria.

Microorganism _ MIC (Mg/ml)

Gram-positive bacteria

Staphylococcus aureus JE2 (MRSA) 5

Staphylococcus aureus Newman (MSSA) 5

Staphylococcus aureus MU 50 (VISA/MRSA) 5

Staphylococcus aureus (MSSA, n=3) 10

Staphylococcus aureus (MRSA, n=6) 5-10

Staphylococcus epidermidis 5

Enterococcus faecalis AT CC29212 10

Enterococcus faecium D344R 5

Streptococcus uberis 10

Streptococcus agalactiae 10

Propionibacterium acnes AT CC6919 10

Clostridium difficile 10

Mycobacteria

Mycobacterium smegmatis mc²155 5

Gram-negative bacteria

Pseudomonas aeruginosa PA01 >40

E. coli ATCC35695 >40

E. coli BW25113 >80

£. co// BW25113 Ato/C 20

Klebsiella pneumoniae ATCC700721 >40

Acinetobacter baumanii ATCC19606 >40

Fungus

Candida albicans (n=2) ATCC64548/ ATCC64550 >80

Table 1 : Minimum inhibitory concentration (MIC) for SCO-101 against different bacterial and fungal species Conclusions:

SCO-101 displays the same activity against methicillin-resistant- and vancomycin- intermediate S. aureus isolates as the respective sensitive (i.e. non-resistant) isolates. Similarly, vancomycin-resistant Enterococci are as sensitive to SCO-101 as

vancomycin-sensitive Enterococci. Furthermore, SCO-101 inhibits growth of

Mycobacterium smegmatis at 5 pg/ml. Based on these findings, we expect SCO-101 and similar compounds to be useful for the treatment of various microbial infections, including those caused by microbial strains that are resistant to commonly used antibiotics.

Example 2: Concentration dependent time-kill study for SCO-101

Materials and methods:

Overnight cultures of S. aureus Newman were diluted into 2 ml fresh Tryptic Soy Broth in 15 ml falcon tubes, reaching an initial cell count of approximately 10⁶ cells/ml. SCO- 101 was added to the tubes to reach a final concentration of 20 pg/ml (40 pM), 40 pg/ml (80 pM) and 80 pg/ml (160 pM) and one tube contained no SCO-101 (Growth control). Colony forming units (CFU) were determined on Tryptic Soy Agar plates before addition of SCO-101 (To) and every hour for the following 4 h. The time-kill experiments have been performed with three biological replicates.

Results:

Time-kill assays were performed to assess the kinetics of the inhibitory activity of SCO- 101. At 4x and 8x MIC (20 pg/ml (40 pM) and 40 pg/ml (80 pM)), SCO-101 displays bacteriostatic activity against S. aureus Newman during the four hours of assessment. At 16x MIC (80 pg/ml (160 pM)), SCO-101 displays bactericidal activity with a >3log reduction in CFU after 60 min of treatment and absence of viable cells after 4 h (Fig. 1 b).

Conclusions:

As SCO1-10 displays both bacteriostatic activity at 20 pg/ml (40 pM) and 40 pg/ml (80 pM), and bactericidal activity at 80 pg/ml (160 pM), we expect SCO-101 and similar compounds to be useful for the treatment of various microbial infections, including those caused by microbial strains that are resistant to commonly used antibiotics. Example 3: Time-kill study of SCO-101 and known antibiotics

Materials and methods:

Overnight cultures of S. aureus Newman were diluted into 2 ml fresh Tryptic Soy Broth in 15 ml falcon tubes, reaching an initial cell count of approximately 10⁶ cells/ml. SCO- 101 , vancomycin, ciprofloxacin and mupirocin were added to tubes to reach a final concentration of 4x MIC and one tube contained no antibiotics (Growth control). Colony forming units (CFU) were determined on Tryptic Soy Agar plates before addition of antibiotics (To) and every hour for the following 4 h. The time-kill experiments have been performed with three biological replicates.

Results:

Time-kill assays were performed to compare SCO-101 time-kill kinetics with that of currently marketed antibiotics (vancomycin, mupirocin and ciprofloxacin), which are frequently used in the hospital setting. At a fixed concentration corresponding to 4x MIC of the respective antibiotics, SCO-101 demonstrates bacteriostatic activity against stationary phase S. aureus Newman, similarly to the bacteriostatic antibiotic mupirocin. Ciprofloxacin is bactericidal against S. aureus with a >2log reduction in CFU after 4 h of treatment. Vancomycin displays bacteriostatic activity against stationary phase S. aureus (Fig. 1a).

Conclusions:

As SCO-101 demonstrates bacteriostatic activity against stationary phase S. aureus Newman similarly to the bacteriostatic antibiotic mupirocin at a fixed concentration, it is highly expected that SCO-101 and similar compounds will be useful for the treatment of various microbial infections, including those caused by microbial strains that are resistant to commonly used antibiotics.

Example 4: Determination of mutation frequency

Materials and methods:

Overnight cultures of S. aureus Newman were prepared from single colonies on a T ryptic Soy Agar plate and 100 mI of proper dilutions were plated on T ryptic Soy Agar alone or Tryptic Soy Agar supplemented with SCO-101 (20 pg/ml (40 mM) and 40 mg/ml (80 mM)) and incubated for up to 120 h at 37 °C. The total number of cells plated were estimated on Tryptic Soy Agar plates and the mutation frequency was calculated as the ratio of resistant cells/total cells. The mutation frequency has been determined for four biological replicates.

Results:

Selection of resistant mutants for SCO-101 occurs at a frequency of approximately 7 10⁸ at 20 pg/ml (4x MIC) upon incubation for 120 h. Resistant mutants display MICs of 25 pg/ml (following prolonged incubation time, i.e. 48 h, due to slow growth) and no cross-resistances to other antibiotics have been identified. No SCO-101 resistant mutants can be selected at a concentration of 40 pg/ml (Mutation frequency < 10 ¹⁰).

Conclusions:

Based on these findings, SCO-101 and similar compounds demonstrate a low rate of resistance development and potential resistant mutants are not expected to be cross- resistant to commonly used antibiotics. Hence, the compounds presented herein are expected to be useful for the treatment of various microbial infections, including those caused by microbial strains that are resistant to commonly used antibiotics.

Example 5: Determination of minimum inhibitory concentration (MIC) for compounds of formula (Z)

Background:

The minimum inhibitory concentration (MIC) is the lowest concentration of a chemical which prevents visible growth of a bacterium. The MIC of several compounds of formula (Z) were determined for S. aureus JE2 (MRSA) and S. aureus Newman (MSSA) (Table 2). The MIC of compounds NS7901 , NS7878, NS7877, NS7875, NS5814 and NS5811 were examined on a broader range of bacteria (Table 3).

Materials and methods:

The minimum inhibitory concentration was determined using the two-fold broth microdilution assay in cation-adjusted Mueller Hinton with an initial inoculum of approximately 5x10⁵ CFU/ml. The MIC was determined as the lowest concentration that completely inhibits visible growth after incubation at 37 °C for 24 h. For all strains, the MIC determinations have been performed with three biological replicates.

Results:

The MIC of several compounds of formula (Z) were determined for S. aureus JE2

(MRSA) and S. aureus Newman (MSSA) as is seen in Table 2. The minimum inhibitory concentrations (MIC) for a broader range of pathogens are presented in Table 3.

against S. aureus JE2 (MRSA) and S. aureus Newman (MSSA).

Microorganism NS7901 NS7878 NS7877 NS7875 NS5814 NS5811

Staphylococcus 1.25 1.25-2.5 1.25-2.5 1.25 1.25 0.62-1.25 aureus JE2

(MRSA)

Staphylococcus 1.25 1.25 1.25-2.5 1.25 0.62 0.62 aureus Newman

(MSSA)

Staphylococcus 1.25 2.5 2.5 2.5 1.25 0.62 aureus MU50

(VISA) Staphylococcus 1.25 2.5 1.25 2.5 1.25 1.25 epidermidis

Enterococcus 1.25 2.5 2.5 2.5 1.25 1.25 faecium

Enterococcus 2.5 2.5 2.5

1.25 1.25 faecalis

Streptococcus 1.25 2.5 2.5 2.5 0.62 0.62 agalactiae

Streptococcus 1.25 2.5 2.5 1.25 0.62 1.25 uberis

Streptococcus 1.25 1.25 1.25 2.5 2.5 1.25 pyogenes

Bacillus cereus 1.25 2.5 2.5 5 1.25 0.62

Clostridium 2.5 2.5 2.5 5 1.25-2.5 0.31 difficile

Propionibacterium 2.5 2.5 2.5 2.5 0.31-0.62 0.31 acnes

Table 3: Minimum inhibitory concentration (MIC) in pg/ ml. for NS7901 , NS7878, NS7877, NS7875, NS5814, and NS5811 against different bacterial species.

Conclusion

Based on these findings, we expect compounds of formula (Z), including SCO-101 , NS7901 , NS7878, NS7877, NS7875, NS5814, and NS5811 to be useful for the treatment of various microbial infections, including those caused by microbial strains that are resistant to commonly used antibiotics.

Example 6: Assessment of in vivo activity for compounds of formula (Z)

Background

A selection of compounds are evaluated for in vivo efficacy using the murine peritonitis model, which is a well-known model in the art (J. Antimicrob. Chemother. 1993 May;31 Suppl D:55-60).

Materials and Methods

Initially the maximum-tolerated dose (MTD) for selected compounds is determined by injecting mice intraperitoneally (ip) or intravenously (iv) with escalating doses of the compounds. Mice are scored for clinical signs of discomfort for 4 hours using a scoring scale from 0-6. When moderate discomfort is observed (score 3) mice are euthanized and the dose is considered not tolerated. For evaluation of in vivo efficacy, mice are injected intraperitoneally with a methicillin resistant Staphylococcus aureus strain (To). 1 h post infection, mice will be treated with selected derivatives, at a suitable dose based on the MTD results, either by iv- or ip administration. The clinical status of the mice and bacterial load in the peritoneum and in the blood will be determined at the time of treatment and 4 h after treatment. As controls, a group will receive the vehicle as a negative control and a group will receive vancomycin (80 mg/kg) as a positive control.

Claims

1 . A method for treating, ameliorating or preventing a microbial infection, said method comprising administration to a subject in need thereof a compound of formula (Z),

or a pharmaceutically acceptable salt thereof,

wherein

R¹, R² and R³ independently of each other represent hydrogen, halo,

trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, aryl or heteroaryl; wherein R^a and R^b independently of each other are hydrogen or alkyl; and wherein Y is selected from the group consisting of:

2. The method according to claim 1 , wherein said microbial infection is a bacterial infection or a fungal infection.

3. The method according to any of the preceding claims, wherein said microbial

infection is caused by Gram-positive bacteria, Gram-negative bacteria,

Mycobacteria or a combination thereof.

4. The method according to any of the preceding claims, wherein said microbial

infection is caused by Gram-positive bacteria selected from the group consisting of: Staphylococcus, Streptococcus, Enterococcus, Bacillus, Clostridium,

Corynebacterium, Listeria, Propionibacterium acnes and Actinomyces.

5. The method according to any one of claims 3 and 4, wherein the Gram-positive bacteria is Staphylococcus, such as selected from the group consisting of: S. aureus, S. epidermidis, S. haemolyticus, S. lugdunensis, S. saprophyticus, S. hominis and S. capitis.

6. The method according to any one of claims 4 and 5, wherein the Staphylococcus is selected from the group consisting of: S. aureus and S. epidermidis.

7. The method according to claim 4, wherein the Gram-positive bacteria is

Streptococcus, such as selected from the group consisting of S. pyogenes, S. agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S. suis, S. mitis, S. mutans, S. uberis and S. pneumoniae.

8. The method according to claim 7, wherein the Streptococcus is selected from the group consisting of S. uberis and S. agalactiae. 9. The method according to claim 7, wherein the Streptococcus is selected from the group consisting of S. pyogenes and S. pneumoniae.

10. The method according to claim 4, wherein the Gram-positive bacteria is

Enterococcus, such as selected from the group consisting of: E. faecalis and E. faecium.

1 1. The method according to claim 3, wherein said microbial infection is caused by Mycobacteria, such as selected from the group consisting of M. tuberculosis, M. leprae, M. ulcerans, M. abscessus, M. avium and M. smegmatis.

12. The method according to claim 3, wherein said microbial infection is caused by Gram-negative bacteria selected from the group consisting of: Enterobacteriaceae (E. coli, Klebsiella, Salmonella, Shigella), Pseudomonas, Vibrio, Campylobacter, Legionella, Neisseria, Haemophilus, Acinetobacter, Helicobacter, Bartonella, Bordetella, Borrelia, Brucella, Francisella, Leptospira, Treponema, Yersinia, and Chlamydia.

13. The method according to any one of the preceding claims, wherein the microbial infection is caused by an antibiotic-resistant bacterium, such as a multi-resistant bacterium.

14. The method according to claim 13, wherein the antibiotic-resistant bacterium is selected from the group consisting of: Staphylococcus aureus, Clostridium difficile, Enterococcus, Mycobacterium tuberculosis, Mycoplasma genitalium,

Streptococcus, Campylobacter, Neisseria gonorrhoeae and Gamma

proteobacteria.

15. The method according to any one of claims 13 and 14, wherein the antibiotic- resistant bacterium is methicillin resistant Staphylococcus aureus (MRSA).

16. The method according to any one of claims 13 and 14, wherein the antibiotic- resistant bacterium is vancomycin intermediate-resistant Staphylococcus aureus (VISA).

17. The method according to claim 2, wherein said microbial infection is a fungal

infection caused by a fungus, such as selected from the group consisting of:

Aspergillus, Candida, Cryptococcus and Pneumocystis.

18. The method according to claim 17, wherein the fungus is Candida, such as

Candida albicans.

19. The method according to any one of claims 2, 17, and 18, wherein said fungal infection presents itself with a fungal disease.

20. The method according to claim 19, wherein said fungal disease is selected from the group consisting of: aspergillosis, blastomycosis, candidiasis, Candida auris, coccidioidomycosis (valley fever), cryptococcus neoformans infection, cryptococcus gattii infection, fungal eye infection, fungal nail infection, histoplasmosis, mucormycosis, mycetoma, pneumocystis pheumonia, ringworm, sporotrichosis and talaromycosis.

21. The method according to any of the preceding claims comprising administering a further medicament to the subject for treating, ameliorating or preventing a microbial infection in a subject.

22. The method according to claim 21 , wherein the further medicament is an antibiotic.

23. The method according to claim 22, wherein the antibiotic is selected from the group consisting of: a penicillin, a cephalosporin, a macrolide, a fluoroquinolone, a sulphonamide, a tetracycline, and an aminoglycoside.

24. The method according to claim 22, wherein the antibiotic is selected from the group consisting of: vancomycin, linezolide, daptomycin, streptomycin, kanamycin, tobramycin, gentamicin, methicillin and neomycin.

25. The method according to any one of claims 21-24, wherein the compound

potentiates the effect of the further medicament.

26. The method according to any of the preceding claims, wherein the compound

results in decreased mutation frequency of the bacteria and/or delayed

development of resistance to antibiotics.

27. The method according to any of the preceding claims, wherein the compound has a MIC value against a Gram-positive bacteria of 100 mM or less, such as 75 pM or less, such as 50 pM or less, such as 45 pM or less, such as 40 pM or less, such as 35 pM or less, such as 30 pM or less, such as 25 pM or less, such as 20 pM or less, such as 15 pM or less, such as 10 pM or less, such as 8 pM or less, such as 6 pM or less, such as 4 pM or less, such as 2 pM or less, such as 1 pM or less, preferably wherein the MIC is 10 pM or less.

28. The method according to any of the preceding claims, wherein the compound has a MIC value against a Gram-negative bacteria and/or a fungus of 200 pM or less, such as 175 pM or less, such as 150 pM or less, such as 125 pM or less, such as 100 pM or less, such as 75 pM or less, such as 50 pM or less, such as 25 pM or less, such as 15 pM or less, preferably wherein the MIC is 10 pM or less.

29. The method according to any of the preceding claims, wherein the subject is a vertebrate, such as a mammal.

30. The method according any one of the preceding claims, wherein the subject is a human being, a dog, a horse, a goat, a cow, a sheep, a pig, a bird or a fish.

31. The method according to any of the preceding claims, wherein administration of the compound comprises enteral administration and/or parental administration and/or topical administration.

32. The method according to any of the preceding claims, wherein the compound is of formula (I),

or a pharmaceutically acceptable salt thereof,

wherein

R¹, R² and R³ independently of each other represent hydrogen, halo,

trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, aryl or heteroaryl; wherein R^a and R^b independently of each other are hydrogen or alkyl. 33. The method according to any of the preceding claims, wherein

R¹, R² and R³ of formula (Z) independently of each other represent hydrogen, halo, trifluoromethyl, nitro, alkyl, alkylcarbonyl, -NR^aR^b, -NR^a-CO-R^b, phenyl or heteroaryl;

which phenyl is optionally substituted with halo, trifluoromethyl, nitro, -CO- NHR^C, -C0-0-R^c or -CO-NR’R”;

wherein R^c is hydrogen, alkyl, or phenyl;

R’ and R” independently of each other are hydrogen or alkyl; or

and which heterocyclic ring may optionally be substituted with alkyl;

R^a and R^b independently of each other are hydrogen or alkyl. 34. The method according to any of the preceding claims, wherein the compound is of formula (II)

35. The method according to any of the preceding claims, wherein

R¹ represents halo.

36. The method according to any of the preceding claims, wherein the compound is of formula (IV)

or a pharmaceutically acceptable salt thereof, wherein R³ and R² are defined as for formula (I); and wherein X is halo, trifluoromethyl, nitro, -CO-NHR^c, -CO-0-R^c or - CO-NR’R”, -S0₂-NR’R”, -O-CF_S, -P0₄H₂;

wherein R^c is hydrogen, alkyl, or phenyl;

R’ and R” independently of each other are hydrogen or alkyl; or

and which heterocyclic ring may optionally be substituted with alkyl.

37. The method according to any one of the preceding claims, wherein the compound is of formula (V):

38. The method according to any one of the preceding claims, wherein the compound of formula (V) is of formula (Va) or Vb);

or a pharmaceutically acceptable salt thereof.

39. The method according to any of the preceding claims, wherein

R² and R³ independently of each other represent halo or trifluoromethyl.

40. The method according to any one of the preceding claims, wherein the compound of formula (Z) is selected from:

or a pharmaceutically acceptable salt thereof.

41. The method according to any one of the preceding claims, wherein the compound of formula (Z) is selected from the group consisting of:

(NS5811 ); or a pharmaceutically acceptable salt hereof.

42. The method according to any of the preceding claims, wherein the compound of formula (Z) is of formula (III)

or a pharmaceutically acceptable salt thereof.

43. The method according to any of the preceding claims, wherein the compound is bacteriostatic.

44. The method according to any of the preceding claims, wherein the compound is bactericidal.

45. A method for disinfecting a surface of an object or a subject comprising

a. providing a compound as defined in any one of the preceding claims or a pharmaceutically acceptable salt thereof, and

b. exposing the surface of the object or subject to said compound.

46. The method according to claim 45, wherein said object is selected from the group consisting of: a surgical instrument, a table, a floor, a phone, a computer keyboard or a container, such as a container for keeping foodstuff.

47. A composition comprising an effective amount of a compound as defined in any of the preceding claims for use in treating, ameliorating or preventing a microbial infection in a subject. 48. Use of a compound as defined in any of the preceding claims for the manufacture of a medicament for treating, ameliorating and/or preventing a microbial infection in a subject.