WO2016067009A1 - Compounds with activity against bacteria and mycobacteria - Google Patents

Abstract

This invention relates toantibacterial drug compounds containing a tricyclic ring system and pharmaceutical formulations thereof. The compounds are useful in the treatment of bacterial and mycobacterial infections, and particularly tuberculosis (TB), including infections caused by resistant strains of bacteria or mycobacteria.

Description

Compounds with activity against bacteria and mycobacteria

This invention relates to antibacterial drug compounds containing a tricyclic ring system. It also relates to pharmaceutical formulations of antibacterial drug compounds. It also relates to uses of the compounds in treating bacterial and mycobacterial infections, and particularly tuberculosis (TB). The invention is also directed to antibacterial drug compounds that are capable of treating bacterial and mycobacterial infections that are currently hard to treat with existing drug compounds. Such infections are frequently referred to as being caused by resistant strains.

The increasing occurrence of bacterial resistance to antibiotics is viewed by many as being one of the most serious threats to the future health and happiness of mankind. Multidrug resistance has become common among some pathogens, e.g. Staphylococcus aureus, Streptococcus pneumoniae, Clostridium difficile and Pseudomonas aeruginosa. MRSA (methicillin-resistant S. aureus, a Gram-positive bacterium) is probably the most well-known group of resistant strains and has reached pandemic proportions. While less widespread, antibiotic-resistant Gram-negative strains, such as either Escherichia coli NDM-1 (New Delhi Metallo^-lactamase-1) or Klebsiella pneumoniae NDM-1 , are also very difficult to treat. Often, only a very limited number of antibiotics such as vancomycin and colistin are effective against these strains.

A disease in which the development of resistance and multidrug resistance is of particular concern is TB. From the 17^th century to the early-20^th century TB was one of the most common causes of death, particularly amongst the urban poor. The development of effective treatments and vaccinations during the mid-20^th century led to a sharp reduction in the number of deaths arising from the disease.

TB is usually caused by Mycobacterium tuberculosis. Mycobacteria are aerobic bacteria and, as a result, tuberculosis infections most often develop in the lungs (pulmonary tuberculosis), although this is not always the case. Mycobacteria lack an outer cell membrane and as such they are often classified as Gram-positive bacteria, although they are in many ways atypical. They have a unique cell wall that provides protection against harsh conditions (e.g. acidic, oxidative) but also provides natural protection against many antibiotics. Other antibiotics, such as β-lactams, are inactive against M. tuberculosis due to the intrinsic lack of activity of the compounds in the mycobacteria. Thus, a drug molecule may have excellent activity against other bacterial strains but no activity against M. tuberculosis. Nevertheless, a number of antibiotics which are effective against TB have been developed. Examples include isoniazid, rifampicin, pyrazinamide and ethambutol and these are typically used in combination.

Unfortunately, there is now an increasing incidence of multidrug-resistant TB (MDR-TB). MDR-TB often arises when a treatment for TB has been interrupted. MDR-TB is the term typically used to refer to TB that has developed resistance to isoniazid and rifampicin. MDR-TB can also be resistant to the fluoroquinolones and also to the so-called 'second-line' injectable anti-TB drugs, kanamycin, capreomycin and amikacin, with such resistances again commonly developing due to interruptions in treatment regimes. Where a strain of M. tuberculosis is resistant to isoniazid and rifampicin as well as one fluoroquinolone and one of the injectable anti-TB drugs, it is known as extensively drug-resistant (XDR-TB). MDR- TB and XDR-TB are often found in those who have been previously treated for TB, but these forms of TB are just as infectious as wild-type TB and the incidence of MDR-TB and XDR-TB around the world is increasing. According to a 2013 World Health Organization report, infections arising from XDR-TB had at that time been identified in 84 different countries. There have even been some reports of strains of M. tuberculosis that were resistant to all drugs tested against them (so-called 'totally drug-resistant tuberculosis', TDR-TB).

The 'second-line' anti-TB drugs and other antibiotics typically used to treat resistant infections can have unfavourable side-effects.

WO2012/125746 and WO2014/043272 disclose a range of tricyclic compounds with some antibacterial activity. No mention is made of the compounds having anti-mycobacterial activity.

In spite of the numerous different antibiotics known in the art for a variety of different infections, there continues to be a need for antibiotics that can provide an effective treatment in a reliable manner. In addition, there remains a need for antibiotic drugs that can avoid or reduce the side-effects associated with known antibiotics, particularly the so- called 'second-line' anti-TB drugs.

In addition, there is a need for antibiotics active against M. tuberculosis in microaerophilic and anaerobic conditions, particularly against non-replicating and latent TB. M. tuberculosis is exposed to microaerophilic and anaerobic conditions within granulomatous lesions. Granulomatous lesions contain various physiological states of M. tuberculosis including actively-replicating bacilli, non-replicating bacilli and dormant or latent cells in the hypoxic core of solid and caseous granulomas as well as in sputum. Current therapies for active TB show only moderate or no killing of non-replicating or dormant TB and a number of drug development strategies to treat latent TB have been initiated (Barry CE et al., 2014. The spectrum of latent tuberculosis: rethinking the goals of prophylaxis. Nat. Rev. Microbiol. 7: 845-855). Several in vitro models to obtain non-replicating M. tuberculosis have been developed over the years, including the Wayne model of hypoxia, which is based on the gradual oxygen depletion of the cultures (Wayne LG. In vitro model of hypoxically induced nonreplicating persistence of Mycobacterium tuberculosis. P. 247-269 In: Parish T, Stoker NG, editors. Mycobacterium tuberculosis protocols: Humana Press; 2001).

Other non-tuberculous mycobacterial infections that can affect humans include those caused by Mycobacterium avium. M. avium infections are particularly prevalent amongst AIDS sufferers and other patient groups with compromised immune systems. Infection can occur in a variety of locations, including the pulmonary system, in which case the symptoms are similar to TB, or in the gastrointestinal tract, in which case severe diarrhoea can result. In children, M. avium can be the causative pathogen in cervical lymphadenitis. Mycobacterium abscessus is a rapidly-growing mycobacterium often found as a contaminant in water sources. It can cause serious TB-like lung infections, particularly in persons with chronic lung diseases, such as cystic fibrosis, as well as infecting wounds and the skin, particularly in patient groups with compromised immune systems. M. abscessus can also contaminate medications and other medical products, including medical devices.

It is an aim of certain embodiments of this invention to provide new antibiotics. In particular, it is an aim of certain embodiments of this invention to provide antibiotics that are active against resistant strains of Gram-positive and/or Gram-negative bacteria. It is an aim of certain embodiments of this invention to provide antibiotics that are active against M. tuberculosis. In particular, it is an aim of certain embodiments of this invention to provide antibiotics that are active against resistant strains of M. tuberculosis. It is an aim of certain embodiments of this invention to provide compounds that have activity that is comparable to those of existing antibiotics, and ideally that is better. It is an aim of certain embodiments of this invention to provide such activity against wild-type strains at the same time as providing activity against one or more resistant strains. It is an aim of certain embodiments of this invention to provide antibiotics that are effective in treating non-replicating TB. It is an aim of certain embodiments of this invention to provide antibiotics that exhibit reduced cytotoxicity relative to prior art compounds and existing therapies.

It is an aim of certain embodiments of this invention to provide treatment of bacterial infections that is effective in a selective manner at a chosen site of interest. Another aim of certain embodiments of this invention is to provide antibiotics having a convenient pharmacokinetic profile and a suitable duration of action following dosing. A further aim of certain embodiments of this invention is to provide antibiotics in which the metabolised fragment or fragments of the drug after absorption are GRAS (Generally Regarded As Safe).

Certain embodiments of the present invention satisfy some or all of the above aims. Compounds of the Invention

In a first aspect, the invention provides a compound of formula (I), or a pharmaceutically

(I); wherein

X¹ , X² and X³ are each independently selected from N and CR¹ ; L¹ is independently selected from -CR⁵R⁵NR⁶- and -S(0)„-;

R¹ is independently at each occurrence selected from: H, halo, nitro, cyano, NR⁷R⁷, NR⁸S(0)₂R⁸, NR⁸CONR⁸R⁸, NR⁸C(0)R⁸, NR⁸C0₂R⁸, OR⁸, O-aryl, SR⁸, S-aryl, SOR⁸, SO3R⁸, SO2R⁸, S0₂NR⁸R⁸ CO2R⁸, C(0)R⁸, CONR⁸R⁸, aryl, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl and Ci-C₄-haloalkyl;

R², R⁶ and R⁸ are each independently at each occurrence selected from: H, Ci-C₄-alkyl, and Ci-C₄-haloalkyl;

R³ is independently selected from (CR⁵R⁵)_m-aryl and (CR⁵R⁵)_m-heteroaryl; R⁴ is independently selected from: -(CR⁵R⁵)_m-3-io-heterocycloalkyl, -(CR⁵R⁵)_m-aryl, - (CR⁵R⁵)m-heteroaryl, -(CR⁵R⁵)_m-C₃-Cio-cycloalkyl, O-aryl and O-heteroaryl;

R⁵ is independently at each occurrence selected from H, F, Ci-C4-alkyl and Ci-C4-haloalkyl; or two R⁵ groups attached to the same carbon atom may together form a group selected from =0 or =S.

R⁷ is independently at each occurrence selected from: H, Ci-C4-alkyl, Ci-C4-haloalkyl, S(0)₂- Ci-C₄-alkyl and C(0)-Ci-C₄-alkyl; n is an integer independently selected from 1 and 2; m is an integer independently selected from 0, 1 and 2; wherein each of the aforementioned alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, aryl (e.g. phenyl) and heteroaryl groups is unsubstituted or is substituted, where chemically possible, by 1 to 5 substituents that are each independently at each occurrence selected from the group consisting of: oxo, =NR^a, =NOR^a, halo, nitro, cyano, NR^aR^a, N R^aS(0)₂R^a, NR^aC(0)R^a, NR^aCONR^aR^a, NR^aC0₂R^a, OR^a; SR^a, SOR^a, S0₃R^a, S0₂R^a, S0₂NR^aR^a, C0₂R^a C(0)R^a, CONR^aR^a, CR^bR^bNR^aR^a, =CR^bCR^bR^bNR^aR^a, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl and Ci-C4-haloalkyl; wherein R^a is independently at each occurrence selected from H, C1-C4- alkyl and Ci-C4-haloalkyl; and R^b is independently at each occurrence selected from H, halogen, Ci-C4-alkyl and Ci-C4-haloalkyl.

In an embodiment, the compound of formula (I) is a compound of formula (II), or a reof:

wherein R¹ , R², R⁴, R⁵, R⁶, X¹ , X² and X³ are as defined above for formula (I) and wherein R⁹ is independently selected from aryl and heteroaryl. R⁹ may be a group selected from thiazole, oxazole, isothiazole, or isoxazole. of formula (I) is a compound of formula (III):

(III)

wherein R¹ , R², R⁴, X¹ , X² and X³ are as defined above for formula (I) and wherein R⁹ is as defined above for formula (II). R⁹ may be a group selected from thiazole, oxazole, isothiazole, or isoxazole. formula (I) is a compound of formula (IV):

wherein R¹ , R², R⁴, X¹ , X² and X³ and are as defined above for formula (I); wherein R⁹ is as defined above for formula (II) and wherein R⁵ is independently at each occurrence selected from H, F, Ci-C4-alkyl, Ci-C4-haloalkyl. Thus, it is not the case in this embodiment that two R⁵ groups attached to the same carbon atom may together form a group selected from =0 or =S. nd of formula (I) is a compound of formula (V):

(V)

wherein R¹ , R², R⁴, X¹ , X² and X³ are as defined above for formula (I); wherein R⁹ is as defined above for formula (II). In an embodiment, the compound of formula (I) is a compound of formula (VI):

wherein R¹ is as defined above for formula (I) and wherein R⁹ is independently selected from aryl and heteroaryl, R⁵ is independently at each occurrence selected from H and C1-C4- alkyl; R⁴ is a N-heterocycloalkyl group that is attached to the rest of the molecule via the nitrogen or, where there is more than one nitrogen in the ring, via one of the nitrogens in the ring system; and R¹⁰, R¹¹ and R¹² are each independently at each occurrence selected from: H, halo and Ci-C4-alkyl. R⁹ may be a group selected from thiazole, oxazole, isothiazole, or isoxazole.

I la (I) is a compound of formula (VII):

wherein R¹ is as defined above for formula (I) and wherein R⁹ is independently selected from aryl and heteroaryl, R⁴ is a N-heterocycloalkyl group that is attached to the rest of the molecule via the nitrogen or, where there is more than one nitrogen in the ring, via one of the nitrogens in the ring system; and R¹⁰, R¹¹ and R¹² are each independently at each occurrence selected from: H, halo and Ci-C4-alkyl. R⁹ may be a group selected from thiazole, oxazole, isothiazole, or isoxazole.

In a second aspect of the invention is provided a compound of formula (VIII), or a pharmaceutically acceptable salt thereof, for use in treating a mycobacterial infection:

(VIII); wherein

X¹ , X², X³, R¹ , R², R³ and R⁴ are as described above for compounds of formula (I) and wherein L² is independently selected from O and S. Thus the invention may provide a method of treating a mycobacterial infection, the method comprising treating a subject in need thereof with a therapeutically effective amount of a compound of formula (VIII).

The following statements apply to compounds of any of formulae (I) to (VIII). These statements are independent and interchangeable. In other words, any of the features described in any one of the following statements may (where chemically allowable) be combined with the features described in one or more other statements below. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the statements below that describe a feature of that compound, expressed at any level of generality, may be combined so as to represent subject matter that is contemplated as forming part of the disclosure of this invention in this specification.

It may be that X¹ may be N. Alternatively, X¹ is CR\ e.g. CR¹⁰. It may be that X² is N. Alternatively, it may be that X² is CR¹ , e.g. CR¹¹. It may be that X³ is N. Alternatively, it may be that X³ is CR¹ , e.g. CR¹². R¹⁰, R¹¹ and R¹² are each independently at each occurrence selected from: H, halo, Ci-C₄-haloalkyl and Ci-C₄-alkyl. R¹⁰, R¹¹ and R¹² are each independently at each occurrence selected from: H, halo and Ci-C₄-alkyl.

In some preferred embodiments, X¹ is CH, X² is CF and X³ is CH.

L¹ may be -CR⁵R⁵NR⁶-. L¹ may be -CR⁵R⁵NH-. R⁵ may be, at each occurrence in -L¹- , selected from H and Ci-C₄-alkyl. L¹ may be -CH2NR⁶-. In some preferred embodiments,

Alternatively, L¹ may be -S(0)_n-. Thus, in some preferred embodiments, L¹ is -SO2-.

L² may be S. L² may be O. R¹ may be H. Alternatively, R¹ may be selected from: halo, nitro, cyano, NR⁷R⁷, NR⁸S(0)₂R⁸, NR⁸CONR⁸R⁸, NR⁸C(0)R⁸, NR⁸C0₂R⁸, OR⁸, SR⁸, SOR⁸, SO3R⁸, SO2R⁸, S0₂NR⁸R⁸, C0₂R⁸, C(0)R⁸, CONR⁸R⁸, aryl, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl and Ci- C₄-haloalkyl. Thus, R¹ may be selected from: halo, NR⁷R⁷, NR⁸S(0)₂R⁸, NR⁸CONR⁸R⁸, NR⁸C(0)R⁸, NR⁸C0₂R⁸, OR⁸ , O-aryl, SR⁸ and S-aryl. R¹ may be selected from NR⁷R⁷, NR⁸S(0)₂R⁸, NR⁸CONR⁸R⁸, NR⁸C(0)R⁸ and NR⁸C0₂R⁸. R¹ may be selected from halo, NR⁸R⁸, OR⁸, O-aryl, SR⁸ and S-aryl. Preferably, R¹ is NR⁷R⁷, e.g. NR⁸R⁸. In a particular embodiment, R¹ is NHR⁷, e.g. NHR⁸. Most preferably, R¹ is NH-Ci-C₄-alkyl, e.g. NHMe.

R² is preferably H.

R³ may be (CR⁵R⁵)-R⁹, wherein R⁹ is independently selected from aryl (e.g. phenyl) and heteroaryl.

It may be that R³ is (CR⁵R⁵)-R⁹ and R⁵ is independently at each occurrence selected from H, F, Ci-C₄-alkyl, Ci-C₄-haloalkyl. Thus, it is not the case in this embodiment that two R⁵ groups attached to the same carbon atom may together form a group selected from =0 or =S. It may be that R⁵ is at each occurrence H.

R³ may be -(CR⁵R⁵)_m-aryl, e.g. (CR⁵R⁵)_m-phenyl. m may be 1. It may be that m is 1 and R⁵ is independently at each occurrence selected from H, F, Ci-C₄-alkyl, Ci-C₄-haloalkyl. In some preferred embodiments, R³ is substituted or unsubstituted CH₂-phenyl, e.g. unsubstituted CH₂-phenyl.

The embodiments in the previous two paragraphs are particularly preferred when L¹ is - S(0)_n- or, more particularly when L¹ is -S0₂-.

It may be that R³ is (CR⁵R⁵)-R⁹ and the two R⁵ groups attached to the same carbon atom together form a group selected from =0 or =S. Preferably, they form the group =0.

R³ may be -(CR⁵R⁵)_m-heteroaryl. m may be 1. It may be that m is 1 and the two R⁵ groups attached to the same carbon atom together form a group selected from =0 or =S. R³ may be -C(=0)-heteroaryl.

The embodiments in the previous two paragraphs are particularly preferred when L¹ is - CR⁵R⁵NR⁶- or, more particularly when L¹ is -CH₂NH-. Where R³ comprises a heteroaryl group, that heteroaryl group may be a monocyclic 5- or 6- membered heteroaryl group. Preferably, the heteroaryl group is a 5-membered heteroaryl group in which the heteroaromatic ring includes 1-4 heteroatoms selected from O, S and N. Preferably, the heteroaryl group is a 5-membered heteroaryl group in which the heteroaromatic ring includes a single nitrogen and a single heteroatom selected from O and S. A preferred 5-membered heteroaryl group would be an oxazole. An alternative preferred heteroaryl group is a thiazole. Alternatively, the heteroaryl group is a 6-membered heteroaryl group in which the heteroaromatic ring includes 1 -2 nitrogen atoms. An exemplary 6- membered heteroaryl group would be a pyridine. In a further alternative, the heteroaryl group may be a bicyclic heteroaromatic group. Heteroaryl groups may be substituted or unsubstituted. In some embodiments, they are unsubstituted. 3 may be selected from:

preferred embodiments,

R⁴ may be -(CR⁵R⁵)_m-3-io-heterocycloalkyl, e.g. 3-10-heterocycloalkyl. Typically, R⁴ will be an N-heterocycloalkyl group. N-heterocycloalkyl groups may be monocyclic or bicyclic and comprise 1 to 3 nitrogen atoms in the heterocyclic ring system and R⁴ may be attached to the rest of the molecule via a carbon or a nitrogen in the ring system. It may be that the N- heterocycloalkyl group is attached to the rest of the molecule via the nitrogen or, where there is more than one nitrogen in the ring, via one of the nitrogens in the ring system. Any nitrogen in the ring system that is not at a bridgehead or is not the point of attachment of R⁴ to the rest of the molecule will be an NR⁸ group. Unless otherwise stated, any N- heterocycloalkyl group mentioned as a possibility for R⁴ may be unsubstituted or may be substituted with 1 to 3 groups selected from oxo, =NOR^a, NR^aR^a, OR^a, Ci-C₄-alkyl, C₂-C₄- alkenyl, C₂-C₄-alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a; wherein R^a is independently at each occurrence selected from H, Ci-C₄-alkyl and Ci-C₄-haloalkyl; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄-alkyl and Ci-C₄-haloalkyl.

R⁴ may be a monocyclic 3-7-N-heterocycloalkyl group. Thus, R⁴ may be a piperazine ring. R⁴ may thus be a piperazine ring substituted with a methyl group, e.g. an N-methyl piperazine ring, a 3-methyl piperazine ring, or a 2-methyl piperazine ring. Alternatively, R⁴ may be an unsubstituted piperazine group. Any piperazine group will typically be attached to the rest of the molecule via one of the nitrogens in the ring system. Possibly, R⁴ is an azetidine, pyrrolidine or piperidine ring, optionally wherein the ring nitrogen attaches the azetidine, pyrrolidine or piperidine ring to the rest of the compound. R⁴ may be an azetidine, pyrrolidine or piperidine ring wherein the ring nitrogen attaches the azetidine, pyrrolidine or piperidine ring to the rest of the compound and that is substituted with a single hydroxyl group. R⁴ may be a piperidine ring substituted with a single hydroxyl group, e.g. a 4- hydroxy-piperidine ring. R⁴ may be a pyrrolidine substituted with a single hydroxyl group, e.g. a 3-hydroxypyrrolidine. R⁴ is a 3-hydroxy azetidine group.

Specific examples of R⁴ groups include:

R⁴ may be a bicyclic 7-10-N-heterocycloalkyl group. The bicyclic N-heterocycloalkyl group may be attached to the rest of the molecule via either a carbon or a nitrogen in the ring system. Preferably, R⁴ is

wherein R¹³ is independently selected from oxo, =NOR^a,

NR^aR^a, OR^a, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a; wherein R^a is independently at each occurrence selected from H, Ci-C₄-alkyl and Ci-C₄- haloalkyl; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄-alkyl and Ci-C₄-haloalkyl; or wherein two R¹³ groups together with the carbon or carbons to which they are attached form a 3-6 membered cycloalkyi or 3-6 membered heterocycloalkyi ring. Where two R¹³ groups form a heterocycloalkyi ring, that ring will comprise 1 or 2 heteroatoms selected from N, O and S in the ring system. Where two R¹³ groups form a cycloalkyi or heterocycloalkyi ring, that ring may be substituted with one or two groups independently selected from oxo, =NOR^a, NR^aR^a, OR^a, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄- alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a. p is an integer independently selected from 0, 1 , 2, 3 and 4.

It may be that tw ¹³ groups do not form a cycloalkyi or heterocycloalkyi ring. In other

words R⁴ may be

wherein R¹³ is independently selected from oxo, =NOR^a,

NR^aR^a, OR^a, CrC₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a; wherein R^a is independently at each occurrence selected from H, Ci-C₄-alkyl and Ci-C₄- haloalkyl; and R^b is independently at each occurrenc , halogen, Ci-C₄-alkyl

and Ci-C₄.haloalkyl. p may be 1. Thus, R⁴ may be

. R¹³ may be NR^aR^a.

Each R^a in R¹³ may be H (i.e. R¹³ may be NH₂). Each R^a in R¹³ may independently be C¹-C⁴ alkyl, e.g. each R^a in R¹³ may independently be methyl (i.e. R¹³ may be NMe₂). R¹³ may be OR^a. R^a may be H and thus, R¹³ may be OH. R¹³ may be CR^bR^bNR^aR^a. Each R^b in R¹³ may independently be Ci-C₄-alkyl, e.g. each R^b in R¹³ may independently be methyl (e.g. R¹³ may be CMe₂NR^aR^a). Each R^a in R¹³ may be H (e.g. R¹³ may be CR^bR^bNH₂). R¹³ may be CMe₂NH₂. p may be 2. In some particular examples where p is 2, R¹³ may at one instance be =NOR^a (e.g. =NOMe), and at the other instance be CR^bR^bNR^aR^a (e.g. CH₂NR^aR^a or CH₂NH₂).

Two R¹³ groups may form a 3-6- membered heterocycloalkyi ring, e.g. a 6-membered heterocycloalkyi ring, e.g. a vicinally fused 6 membered heterocycloalkyi ring. A specific example of a 6-membered heterocycloalkyl ring would be a morpholine ring. The two R¹³ groups may also form a 3-6 membered cycloalkyl ring, e.g. a 3-membered ring. Thus, two R¹³ groups may form a vicinally fused 3-membered ring or a spiro fused 3-membered ring. That 3-membered ring (e.g. that vicinally fused 3-membered ring) may be substituted with one or two groups independently selected from oxo, =NOR^a, NR^aR^a, OR^a, Ci-C4-alkyl, C2- C₄-alkenyl, C₂-C₄-alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a. Thus, the 3-membered ring (e.g. that vicinally fused 3-membered ring) may be substituted with an NR^aR^a group, e.g. a NH2 group.

In cases in which two R¹³ groups form a 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl ring, there may be one or more other R¹³ groups, e.g. p may be 4. Such additional R¹³ groups will generally not form a 3- to 6-membered cycloalkyl or a 3 to 6- membered heterocycloalkyl ring. R¹³ may be Ci-C₄-alkyl, e.g. methyl. R¹³ may be NR^aR^a, e.g. NH₂.

Specific examples of R⁴ groups include:

R⁴ may be C3-Cs-cycloalkyl group. Typically, where R⁴ is a C3-Cs-cycloalkyl group, it is substituted with at least one group selected from NR^aR^a, OR^a, Ci-C₄-alkyl, C2-C₄-alkenyl, C2- C₄-alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a. Specifically, R⁴ may be a cyclopropyl group substituted with a NH2 group.

A specific example of an R⁴ group is:

R⁴ may be an aryl group, e.g. a phenyl group. R⁴ may be a phenyl group with at least one NR^aR^a, CONR^aR^a or CR^bR^bNR^aR^a group and optionally further substituted with from 1-3 groups independently selected from halo, Ci-C4-haloalkyl and Ci-C4-alkyl, e.g. a phenyl group with at least one NR^aR^a, CONR^aR^a or CR^bR^bNR^aR^a group and optionally further substituted with from 1-3 halo groups (e.g. fluoro groups). Thus, R⁴ may be a phenyl group with at least one NR^aR^a or CR^bR^bNR^aR^a group and optionally further substituted with from 1- 3 groups independently selected from halo, Ci-C4-haloalkyl and Ci-C4-alkyl, e.g. a phenyl group with at least one NR^aR^a or CR^bR^bNR^aR^a group and optionally further substituted with from 1-3 halo groups (e.g. fluoro groups). In particular embodime ⁴ may be a group

selected from:

. R⁴ may also be a heteroaryl group. R⁴ may be a heteroaryl group comprising at least one nitrogen atom in the ring structure. R⁴ may be a 9-membered bicyclic heteroaryl group comprisi gen atoms in the ring system, e.g. an indazole group, e.g. R⁴

may be

R⁴ may be a 6-membered monocyclic heteroaryl group comprising from 1 to 2 nitrogen atoms in the ring system, e.g. a pyridinyl group, e.g.

a 6-amino-pyridin-3-yl group. R⁴ may be a 5-membered monocyclic heteroaryl group comprising from 1 to 2 nitrogen atoms in the ring system, e.g. a thiazole.

It may be that R⁵ is at each occurrence H or that two R⁵ groups attached to the same carbon atom may together form a =0 group.

R⁶ may be Ci-C4-alkyl. Preferably, however, R⁶ is H.

R⁷ may be R⁸. In other words, R⁷ may independently at each occurrence be selected from: H, Ci-C4-alkyl, and Ci-C4-haloalkyl. R⁷ is preferably selected from H and Ci-C4-alkyl.

At any given occurrence, R⁸ may be Ci-C4-alkyl. At any given occurrence, R⁸ may be H. R⁹ may be a monocyclic 5- or 6-membered heteroaryl group. Thus it may be that the heteroaryl group is a 5-membered heteroaryl group in which the heteroaromatic ring includes 1-4 heteroatoms selected from O, S and N. Preferably, the heteroaryl group is a 5- membered heteroaryl group in which the heteroaromatic ring includes a single nitrogen and a single heteroatom selected from O and S. A preferred 5-membered heteroaryl group would be an oxazole. An alternative preferred heteroaryl group is a thiazole. Alternatively, R⁹ may be a 6-membered heteroaryl group in which the heteroaromatic ring includes 1-2 nitrogen atoms. An exemplary 6-membered heteroaryl group would be a pyridine. In a further alternative, R⁹ may be a bicyclic heteroaromatic group. R⁹ may be a group selected from thiazole, oxazole, isothiazole, or isoxazole. It may be that R⁹ is a group selected from thiazole or oxazole. R⁹ may be substituted or unsubstituted. In some embodiments, R⁹ is unsubstituted. R⁹ may be substituted with a Ci-C4-alkyl group.

Alternatively, R⁹ may be aryl, e.g. phenyl. The aryl group, e.g. phenyl group, may be substituted or unsubstituted. In some preferred embodiments, aryl groups, e.g. phenyl groups, are unsubstituted.

It may be that R⁴ has at least one NR^aR^a or CR^bR^bNR^aR^a substituent. It may be that R⁴ has a single NR^aR^a or CR^bR^bNR^aR^a substituent. It may be that R⁴ has at least one NR^aR^a substituent. It may be that R⁴ has a single NR^aR^a substituent. In these embodiments, R⁴ may have other substituent groups, e.g. from 0 to 2 groups selected from oxo, Ci-C4-alkyl and F.

R¹⁰ may be selected from H and Ci-C4-alkyl. Preferably, R¹⁰ is H. R¹¹ may be selected from F and Ci-C4-fluoroalkyl. Preferably, R¹¹ is F. R¹² may be selected from H and Ci-C4-alkyl. Preferably, R¹² is H.

In a specific embodiment, the compound of formula (I) has a structure selected from:

In yet further specific embodiments, the compound of formula (I) has a structure selected

Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of the invention, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counter ion is optically active, for example, d-lactate or I- lysine, or racemic, for example, dl-tartrate or dl-arginine.

Compounds of the invention containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound of the invention contains a double bond such as a C=C or C=N group, geometric cis/trans (or Z/E) isomers are possible. Specifically, the oxime groups present in certain compounds of the invention may be present as the E-oxime, as the Z-oxime or as a mixture of both in any proportion. Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.

Where structurally isomeric forms of a compound are interconvertible via a low energy barrier, tautomeric isomerism ('tautomerism') can occur. This can take the form of proton tautomerism in compounds of the invention containing, for example, an imino, keto, or oxime group, or so- called valence tautomerism in compounds that contain an aromatic moiety.

Conventional techniques for the preparation/isolation of individual enantiomers when necessary include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).

Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of the invention contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted into the corresponding pure enantiomer(s) by means well known to a skilled person.

Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1 % diethylamine. Concentration of the eluate affords the enriched mixture.

When any racemate crystallises, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer.

While both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art - see, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel and S. H. Wilen (Wiley, 1994).

It follows that a single compound may exhibit more than one type of isomerism. The term C_m-C_n refers to a group with m to n carbon atoms.

The term "alkyl" refers to a linear or branched hydrocarbon chain. For example, Ci-C6-alkyl may refer to methyl, ethyl, n-propyl, /^'so-propyl, n-butyl, sec-butyl, te/f-butyl, n-pentyl and n- hexyl. The alkyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for each alkyl group independently may be fluorine, OR^a or NHR^a.

The term "haloalkyl" refers to a hydrocarbon chain substituted with at least one halogen atom independently chosen at each occurrence from: fluorine, chlorine, bromine and iodine. The halogen atom may be present at any position on the hydrocarbon chain. For example, Ci-C6-haloalkyl may refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyl e.g. 1- chloromethyl and 2-chloroethyl, trichloroethyl e.g. 1 ,2,2-trichloroethyl, 2,2,2-trichloroethyl, fluoroethyl e.g. 1 -fluoromethyl and 2-fluoroethyl, trifluoroethyl e.g. 1 ,2,2-trifluoroethyl and 2,2,2-trifluoroethyl, chloropropyl, trichloropropyl, fluoropropyl, trifluoropropyl. A halo alkyl group may be a fluoroalkyl group, i.e. a hydrocarbon chain substituted with at least one halogen atom.

The term "alkenyl" refers to a branched or linear hydrocarbon chain containing at least one double bond. The double bond(s) may be present as the E or Z isomer. The double bond may be at any possible position of the hydrocarbon chain. For example, "C2-C6-alkenyl" may refer to ethenyl, propenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl. The alkenyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each alkenyl group independently may be fluorine, OR^a or NHR^a.

The term "alkynyl" refers to a branched or linear hydrocarbon chain containing at least one triple bond. The triple bond may be at any possible position of the hydrocarbon chain. For example, "C2-C6-alkynyl" may refer to ethynyl, propynyl, butynyl, pentynyl and hexynyl. The alkynyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each alkynyl group independently may be fluorine, OR^a or NHR^a.

The term "cycloalkyl" refers to a saturated hydrocarbon ring system containing 3, 4, 5 or 6 carbon atoms. For example, "C3-C6-cycloalkyl" may refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The cycloalkyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for each cycloalkyl group independently may be fluorine, OR^a or NHR^a.

The term "aromatic" when applied to a substituent as a whole means a single ring or polycyclic ring system with 4n + 2 electrons in a conjugated π system within the ring or ring system where all atoms contributing to the conjugated π system are in the same plane.

The term "aryl" refers to an aromatic hydrocarbon ring system. The ring system has 4n +2 electrons in a conjugated π system within a ring where all atoms contributing to the conjugated π system are in the same plane. For example, the "aryl" may be phenyl and naphthyl. The aryl group may be unsubstituted or substituted by one or more substituents. Specific substituents for each aryl group independently may be Ci-C4-alkyl, Ci-C4-haloalkyl, cyano, halogen, OR^a or NHR^a.

The term "heteroaryl" may refer to any aromatic (i.e. a ring system containing (4n + 2) ττ- electrons or n- electrons in the ττ-system) 5-10 membered ring system comprising from 1 to 4 heteroatoms independently selected from O, S and N (in other words from 1 to 4 of the atoms forming the ring system are selected from O, S and N). Thus, any heteroaryl groups may be independently selected from: 5 membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-4 heteroatoms independently selected from O, S and N; and 6-membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-3 (e.g.1-2) nitrogen atoms; 9-membered bicyclic heteroaryl groups in which the heteroaromatic system is substituted with 1-4 heteroatoms independently selected from O, S and N; 10-membered bicyclic heteroaryl groups in which the heteroaromatic system is substituted with 1-4 nitrogen atoms. Specifically, heteroaryl groups may be independently selected from: pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, isoxazole, triazole, oxadiazole, thiadiazole, tetrazole; pyridine, pyridazine, pyrimidine, pyrazine, triazine, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, indazole, benzimidazole, benzoxazole, benzthiazole, benzisoxazole, purine, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, pteridine, phthalazine, naphthyridine. Heteroaryl groups may also be 6-membered heteroaryl groups in which the heteroaromatic ring is substituted with 1 heteroatomic group independently selected from O, S and NH and the ring also comprises a carbonyl group. Such groups include pyridones and pyranones. The heteroaryl system itself may be substituted with other groups. The heteroaryl group may be unsubstituted or substituted by one or more substituents. Specific substituents for each heteroaryl group independently may be Ci-C4-alkyl, Ci-C4-haloalkyl, cyano, halogen, OR^a or NHR^a.

The term Vnheterocycloalkyl" may refer to a m-n membered monocyclic or bicyclic saturated or partially saturated groups comprising 1 or 2 heteroatoms independently selected from O, S and N in the ring system (in other words 1 or 2 of the atoms forming the ring system are selected from O, S and N). By partially saturated it is meant that the ring may comprise one or two double bonds. This applies particularly to monocyclic rings with from 5 to 8 members. The double bond will typically be between two carbon atoms but may be between a carbon atom and a nitrogen atom. An N-heterocycloalkyl group is a heterocycloalkyl group comprising at least one N in the ring system. Examples of heterocycloalkyl groups include; piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, dihydrofuran, tetrahydropyran, dihydropyran, dioxane, azepine. Bicyclic systems may be spiro-fused, i.e. where the rings are linked to each other through a single carbon atom; vicinally fused, i.e. where the rings are linked to each other through two adjacent carbon or nitrogen atoms; or they may be share a bridgehead, i.e. the rings are linked to each other two non-adjacent carbon or nitrogen atoms. The heterocycloalkyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each heterocycloalkyl group may independently be fluorine, OR^a or NHR^a. The compound of the invention may be an N-oxide. Where the compound of the invention is an N-oxide, it will typically be a pyridine N-oxide, i.e. where the compound of the invention comprises a pyridine ring, the nitrogen of that pyridine may be N⁺-0\ Alternatively, it may be that the compound of the invention is not an N-oxide.

A carbocyclic group consists of one or more rings that are entirely formed from carbon atoms. A carbocylic group can be a mono- or bicyclic cycloalkyl group, or it can comprise at least one phenyl ring.

A heterocyclic group consists of one or more rings wherein the ring system includes at least one heteroatom. A heterocyclic group comprises at least one heteroaryl or heterocyclic rings.

A heterocyclic ring is a saturated ring comprising at least one heteroatom selected from O, S and N.

Where a ring system is described as being an x-membered bicyclic group, that is intended to mean that the skeleton of the bicyclic ring system is formed from x atoms (i.e. the total number of atoms across the two rings of the bicycle is x).

Aryl and heteroaryl groups are optionally substituted with 1 to 5 substituents that are each independently at each occurrence selected from the group consisting of: halo, nitro, cyano, NR^aR^a , NR^aS(0)₂R^a, NR^aCONR^aR^a, NR^aC0₂R^a, NR^aC(0)R^a, OR^a; SR^a, SOR^a, S0₃R^a, S0₂R^a, S0₂NR^aR^a, C0₂R^a C(0)R^a, CONR^aR^a, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, Ci- C₄ haloalkyl and CR^bR^bNR^aR^a; wherein R^a is independently at each occurrence selected from H, Ci-C₄-alkyl and Ci-C₄-haloalkyl; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄-alkyl and Ci-C₄-haloalkyl.

R^b may be selected from H, Ci-C₄-alkyl and Ci-C₄-haloalkyl.

The present invention also includes all pharmaceutically acceptable isotopically-labelled compounds of formulae (I) to (VIII) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as ²H and ³H, carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶CI, fluorine, such as ¹⁸F, iodine, such as ¹²³l and ¹²⁵l, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵0, ¹⁷0 and ¹⁸0, phosphorus, such as ³²P, and sulphur, such as ³⁵S.

Certain isotopically-labelled compounds, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵0 and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

Uses, methods of treatment and pharmaceutical formulations

Each of the compounds of the present invention may be used as a medicament. Thus, in another aspect of the invention, there is provided a compound as defined above for the treatment of bacterial or mycobacterial infections.

The compounds and formulations of the present invention may be used in the treatment of a wide range of bacterial or mycobacterial infections.

In some embodiments, the compounds can be used to treat bacterial infections caused by one or more resistant strains of bacteria. In a further embodiment, the compounds can be used to treat bacterial infections caused by one or more resistant strains of Gram-positive bacteria. In a further embodiment, the compounds can be used to treat bacterial infections caused by one or more resistant strains of Gram-negative bacteria. The compounds and formulations of the invention may be used to treat infections caused by bacteria that are in the form of a biofilm.

The term 'resistant strains' is intended to mean strains of bacteria or mycobacteria that have shown resistance to one or more known antibacterial or antimycobacterial drugs. For example, it may refer to bacterial strains that are resistant to methicillin, strains that are resistant to one or more other β-lactam antibiotics, strains that are resistant to one or more fluoroquinolones and/or strains that are resistant to one or more other antibiotics (i.e. antibiotics other than β-lactams and fluoroquinolones). A resistant strain is one in which the MIC (minimum inhibitory concentration) of a given compound or class of compounds for that strain has shifted to a significantly higher number than for the parent (susceptible) strain.

The bacterial strain may be resistant to one or more fluoroquinolone antibiotics, e.g. one or more antibiotics selected from levofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, rufloxacin, balofloxacin, grepafloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, besifloxacin, clinafloxacin, garenoxacin, gemifloxacin, gatifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, ciprofloxacin, pefloxacin, moxifloxacin, ofloxacin, delafloxacin, zabofloxacin, avarofloxacin, finafloxacin.

The compounds of the invention may be particularly effective at treating infections caused by Gram-positive bacteria. The compounds of the invention may be particularly effective at treating infections caused by Gram-positive bacteria that are resistant to one or more fluoroquinolone antibiotics.

The compounds of the invention may be particularly effective at treating infections caused by Gram-negative bacteria. The compounds of the invention may be particularly effective at treating infections caused by Gram-negative bacteria that are resistant to one or more fluoroquinolone antibiotics.

The compounds and formulations of the present invention can be used to treat or to prevent infections caused by bacterial strains associated with biowarfare. These may be strains that are Category A pathogens as identified by the US government (e.g. those that cause anthrax, plague, etc.) and/or they may be strains that are Category B pathogens as identified by the US government (e.g. those that cause Glanders disease, mellioidosis, etc). In a specific embodiment, the compounds and formulations of the present invention can be used to treat or to prevent infections caused by Gram-positive bacterial strains associated with biowarfare (e.g. anthrax). More particularly, the compounds and formulations may be used to treat Category A and/or Category B pathogens as defined by the US government on 1^st January 2014.

The compounds of the invention can be used to treat or prevent mycobacterial infections, e.g. mycobacterial infections caused by resistant strains of mycobacteria. Thus, for example, they can be used to treat TB or leprosy. Thus, it may be that the mycobacterial infection is caused by M. tuberculosis. It may also be that the mycobacterial infection is caused by a mycobacterium selected from: M. avium complex, M. abscessus, M. leprae, M. bovis, M. kansasii, M. chelonae, M. africanum, M. canetti and M. microti. The compounds may be used to treat resistant forms of TB, e.g. MDR-TB (i.e. TB infections caused by strains that are resistant to isoniazid and rifampicin), XDR-TB (i.e. TB infections caused by strains that are resistant to isoniazid, rifampicin, at least one fluoroquinolone and at least one of kanamycin, capreomycin and amikacin) and/or TDR-TB (i.e. TB infections caused by strains that have proved resistant to every drug tested against it with the exception of a compound of the invention). The TB may be caused by a mycobacterial strain that is resistant to at least one approved antimycobacterial compound. The at least one approved antimycobacterial compound may be selected from: rifampicin, isoniazid, kanamycin, capreomycin, amikacin and a fluoroquinolone. The at least one approved antimycobacterial compound may be selected from: rifampicin, moxifloxacin, isoniazid, ciprofloxacin and levofloxacin.

The compounds of the invention may be used to treat non-replicating TB.

The term 'approved' is intended to mean that the drug is one that had been approved by the US FDA or the EM A prior to 1^st March 2015.

The compounds of the invention may also be useful in treating other forms of infectious disease, e.g. fungal infections, parasitic infections and/or viral infections.

The compounds of the present invention can be used in the treatment of the human body. They may be used in the treatment of the animal body. In particular, the compounds of the present invention can be used to treat commercial animals such as livestock. Alternatively, the compounds of the present invention can be used to treat companion animals such as cats, dogs, etc. The compounds of the invention may be obtained, stored and/or administered in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Suitable base salts are formed from bases that form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulfate and hemicalcium salts. Also included are acid addition or base salts wherein the counter ion is optically active, for example, d-lactate or l-lysine, or racemic, for example, dl-tartrate or dl-arginine.

Compounds of the invention may exist in a single crystal form or in a mixture of crystal forms or they may be amorphous. Thus, compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, or spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.

For the above-mentioned compounds of the invention the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated. For example, if the compound of the invention is administered orally, then the daily dosage of the compound of the invention may be in the range from 0.01 micrograms per kilogram body weight ( g/kg) to 100 milligrams per kilogram body weight (mg/kg).

A compound of the invention, or pharmaceutically acceptable salt thereof, may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the compounds of the invention, or pharmaceutically acceptable salt thereof, is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

The compounds of the invention may be administered in combination with other active compounds (e.g. antifungal compounds, oncology compounds) and, in particular, with other antibacterial compounds. The compound of the invention and the other active (e.g. the other antibacterial compound) may be administered in different pharmaceutical formulations either simultaneously or sequentially with the other active. Alternatively, the compound of the invention and the other active (e.g. the other antibacterial compound) may form part of the same pharmaceutical formulation.

Examples of other bacterial compounds that could be administered with the compounds of the invention are fluoroquinolones, β-lactams, vancomycin, erythromycin or any other known antibiotic drug molecule.

Examples of other antimycobacterial compounds that could be administered with the compounds of the invention are isoniazid, rifampicin, pyrazinamide, ethambutol, fluoroquinolones, kanamycin, capreomycin, amikacin or any other known antimycobacterial drug molecule.

Depending on the mode of administration of the compounds of the invention, the pharmaceutical composition that is used to administer the compounds of the invention will preferably comprise from 0.05 to 99 %w (per cent by weight) compounds of the invention, more preferably from 0.05 to 80 %w compounds of the invention, still more preferably from 0.10 to 70 %w compounds of the invention, and even more preferably from 0.10 to 50 %w compounds of the invention, all percentages by weight being based on total composition.

The pharmaceutical compositions may be administered topically (e.g. to the skin) in the form, e.g. of creams, gels, lotions, solutions, suspensions, or systemically, by oral administration in the form of tablets, capsules, syrups, powders, suspensions, solutions or granules; or by parenteral administration in the form of a sterile solution, suspension or emulsion for injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion); or by rectal administration in the form of suppositories; or by inhalation (i.e. in the form of an aerosol or by nebulisation). If administered topically, high-dosages of the compounds of the invention can be administered. Thus, a compound with an in vitro MIC of, for example, 16-64 μg/mL may still provide an effective treatment against certain bacterial infections.

For oral administration the compounds of the invention may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution that may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the compounds of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the compound of the invention may be filled into hard gelatine capsules. Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally, such liquid preparations may contain colouring agents, flavouring agents, sweetening agents (such as saccharine), preservative agents and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in the art.

For intravenous (parenteral) administration the compounds of the invention may be administered as a sterile aqueous or oily solution.

The size of the dose for therapeutic purposes of compounds of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

Dosage levels, dose frequency, and treatment durations of compounds of the invention are expected to differ depending on the formulation and clinical indication, age, and co-morbid medical conditions of the patient. The standard duration of treatment with compounds of the invention is expected to vary between one and seven days for most clinical indications. It may be necessary to extend the duration of treatment beyond seven days in instances of recurrent infections or infections associated with tissues or implanted materials to which there is poor blood supply including bones/joints, respiratory tract, endocardium, and dental tissues.

In another aspect the present invention provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable excipient. The formulation may further comprise one or more other antibiotics, e.g. one or more fluoroquinolone antibiotics. Illustrative fluoroquinolone antibiotics include levofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, rufloxacin, balofloxacin, grepafloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, besifloxacin, clinafloxacin, garenoxacin, gemifloxacin, gatifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, ciprofloxacin, pefloxacin, moxifloxacin, ofloxacin, delafloxacin, zabofloxacin, avarofloxacin, finafloxacin.

In another aspect of the invention is provided a method of treating a bacterial or mycobacterial infection, the method comprising treating a subject in need thereof with a therapeutically effective amount of a compound of the invention. The subject may be a human or an animal.

Medical uses

The compounds of the present invention can be used in the treatment of the human body.

The compounds of the invention may be for use in treating human bacterial or mycobacterial infections such as infections of the genitourinary system, the respiratory tract, the gastrointestinal tract, the ear, the skin, the throat, soft tissue, bone and joints (including infections caused by Staphylococcus aureus). The compounds can be used to treat pneumonia, sinusitis, acute bacterial sinusitis, bronchitis, acute bacterial exacerbation of chronic bronchitis, anthrax, chronic bacterial prostatitis, acute pyelonephritis, pharyngitis, tonsillitis, infections caused by Escherichia coli, prophylaxis before dental surgery, cellulitis, acne, cystitis, infectious diarrhoea, typhoid fever, infections caused by anaerobic bacteria, peritonitis, abdominal infection, bacteraemia, septicaemia, leprosy, sexually-transmitted bacterial infection (e.g. gonorrhoea, Chlamydia), bacterial vaginosis, pelvic inflammatory disease, pseudomembranous colitis, Helicobacter pylori, acute gingivitis, Crohn's disease, rosacea, fungating tumours, impetigo and tuberculosis. The compounds of the present invention may also be used in treating other conditions treatable by eliminating or reducing a bacterial infection. In this case they will act in a secondary manner alongside, for example, a chemotherapeutic agent used in the treatment of cancer.

In yet another aspect of the invention is provided a compound for use in the preparation of a medicament. The medicament may be for use in the treatment of any of the diseases, infections and indications mentioned in this specification.

In an aspect of the invention is provided a compound of the invention for medical use. The compound may be used in the treatment of any of the diseases, infections and indications mentioned in this specification.

Veterinary uses

The compounds of the present invention may be used in the treatment of the animal body. In particular, the compounds of the present invention can be used to treat commercial animals such as livestock. The livestock may be mammal (excluding humans), e.g. cows, pigs, goats, sheep, llamas, alpacas, camels and rabbits. The livestock may be birds (e.g. chickens, turkeys, ducks, geese, etc.). Alternatively, the compounds of the present invention can be used to treat companion animals such as cats, dogs, etc. The veterinary use may be to treat wild populations of animals in order to prevent the spread of disease to humans or to commercial animals. In this case, the animals may be rats, badgers, deer, foxes, wolves, mice, kangaroos and monkeys and other apes.

In an aspect of the invention is provided a compound of the invention for veterinary use. The compound may be used in the treatment of any of the animal diseases and infections and indications mentioned in this specification.

In another aspect the present invention provides a veterinary formulation comprising a compound of the invention and a veterinarily acceptable excipient.

The methods by which the compounds may be administered for veterinary use include oral administration by capsule, bolus, tablet or drench, topical administration as an ointment, a pour-on, spot-on, dip, spray, mousse, shampoo, collar or powder formulation or, alternatively, they can be administered by injection (e.g. subcutaneously, intramuscularly, intravenously or into an udder), or as an implant. Such formulations may be prepared in a conventional manner in accordance with standard veterinary practice. The formulations will vary with regard to the weight of active compound contained therein, depending on the species of animal to be treated, the severity and type of infection and the body weight of the animal. For parenteral, topical and oral administration, typical dose ranges of the active ingredient are 0.01 to 100 mg per kg of body weight of the animal. Preferably the range is 0.1 to 10 mg per kg. In any event, the veterinary practitioner, or the skilled person, will be able to determine the actual dosage that will be most suitable for an individual patient, which may vary with the species, age, weight and response of the particular patient. The above dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

As an alternative, when treating animals the compounds may be administered with the animal feedstuff and for this purpose a concentrated feed additive or premix may be prepared for mixing with the normal animal feed.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Synthesis

The skilled man will appreciate that adaptation of methods known in the art could be applied in the manufacture of the compounds of the present invention. For example, the skilled person will be immediately familiar with standard textbooks such as "Comprehensive Organic Transformations - A Guide to Functional Group Transformations", RC Larock, Wiley- VCH (1999 or later editions), "March's Advanced Organic Chemistry - Reactions, Mechanisms and Structure", MB Smith, J. March, Wiley, (5th edition or later) "Advanced Organic Chemistry, Part B, Reactions and Synthesis", FA Carey, RJ Sundberg, Kluwer Academic/Plenum Publications, (2001 or later editions), "Organic Synthesis - The Disconnection Approach", S Warren (Wiley), (1982 or later editions), "Designing Organic Syntheses" S Warren (Wiley) (1983 or later editions), "Guidebook To Organic Synthesis" RK Mackie and DM Smith (Longman) (1982 or later editions), etc., and the references therein as a guide.

The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound and will employ protecting groups as necessary. This will depend inter alia on factors such as the nature of other functional groups present in a particular substrate. Clearly, the type of chemistry involved will influence the choice of reagent that is used in the said synthetic steps, the need, and type, of protecting groups that are employed, and the sequence for accomplishing the protection / deprotection steps. These and other reaction parameters will be evident to the skilled person by reference to standard textbooks and to the examples provided herein.

Sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods, for example as described in "Protective Groups in Organic Synthesis" by TW Greene and PGM Wuts, John Wiley & Sons Inc (1999), and references therein.

Throughout this specification these abbreviations have the following meanings:

ACN = acetonitrile DCM = dichloromethane

DMF = N, /V-dimethylformamide DMSO = dimethyl sulfoxide

RaNi = Raney Nickel™ (a nickel sponge)

Certain compounds of the invention can be made according to the following general schemes. Certain compounds of the invention can be made according to or analogously to the methods described in Examples 1 to 4.

Certain compounds of formula (II) can be made by Scheme A:

(7)

Scheme A

Tricycle (1), wherein Gi and G2 are leaving groups independently selected from CI, Br, F, S02CH2aryl and tosylate, can be treated with amine (2) (where the R groups can be taken together with the nitrogen of attachment to form a C3-C10 heterocycle optionally substituted with R13) in the presence of a base, such as K2CO3, in a solvent, such as EtOH, at a temperature of 75°C to 100°C to provide tricyclic amine (3). Nitrile (4) can be formed from (3) via a second displacement reaction with an alkali metal cyanide, such as NaCN or KCN, in a solvent, such as DMF, at a temperature of 70°C to 120°C. Nitrile (4) can be converted to amine (5). The reaction can be carried out with a reducing agent, such as RaNi in the presence of H2, in a solvent, such as EtOH, at room temperature. Amine (5) can be converted to amide (7) (a subset of compounds of formula II) through coupling with acid (6). The reaction can be performed with a peptide coupling agent, such as N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride in the presence of a base, such as ΕίβΝ, in a solvent, such as DCM, at a temperature of 0°C to room temperature. Alternatively amide (7) can be formed from treatment with the acid chloride of (6). The reaction can be carried in a solvent, such as diisopropyl ether, in the presence of a base, such as ΕίβΝ, at a temperature from room temperature to 70°C. The acid chloride of (6) can be formed from its acid via treatment with oxalyl choride or SO2CI2, in a solvent, such as DCM, at room temperature.

In the case where Ri or R13 is an amine, then this may be protected throughout the synthetic sequence with either a carbobenzyloxy (Cbz) or BOC. Removal of the Cbz group to release the free amine can be effected using Pd/C and H2 in an alcoholic solvent, such as EtOH, at room temperature. Removal of the BOC group to release the free amine can be effected by treatment with TFA in DCM at room temperature or hydrogen chloride in dioxane at room temperature.

Experimental Analytical Methods

NMR spectra were obtained on a LC Bruker AV400 using a 5 mm QNP probe (Method A) or Bruker AVIII 400 Nanobay using a 5 mm BBFQ with z-gradients (Method B).

MS was carried out on a Waters ZQ MS (Method A and B) or ACQ-SQD2#LCA081 (Method C) using H₂0 and ACN (0.1-0.05% formic acid - high pH; 0.05% ammonia - low pH). Wavelengths were 254 and 210 nM.

Method A

Column: Gemini NX C18, 5 μηι, 50 x 2 mm. Column flow rate was 1 mL/min. Injection volume 10 μΙ_.

Method B

Column: Waters XBridge C18, 5 μηι, 50 x 2.1 mm. Flow rate: 0.8 mL/min. Injection volume 10 μΙ_.

Time

H₂0 % ACN %

(min)

0 95 5

4 5 95

4.45 5 95

4.5 95 5 STOP

Method C

Column: ACQUITY UPLC® BEH C18 1.7 m, 50 x 2.1 mm. Flow rate: 0.6 mL/min. Injection volume 2 μΙ_.

Preparative HPLC was performed using a Waters 3100 Mass detector (Method A) or Waters 2767 Sample Manager (Method B) using H₂0 and ACN (0.1-0.05% formic acid - high pH; 0.05% ammonia - low pH).

Method A

Column: XBridge™ prep C18 5 μΜ OBD 19 x 100 mm. Flow rate: 20 mL/min.

Method B

Column: XBridge™ prep C18 5 μΜ OBD 19 x 100 mm. Flow rate: 20 mL/min.

Example 1 - N-fr4-(3-aminopyrrolidin-1 -yl)-6-fluoro-8-(methylamino)-9H-pyrimidor4,5- b1indol-2-vnmethyl)-1 ,3-oxazole-5-carboxamide dihydrochloride A

(a) tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2- phenylmethanesulphonyl-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1a

la

To tert-butyl N-(6-fluoro-2,4-diphenylmethanesulphonyl-9H-pyrimido[4,5-b]indol-8-yl)-N- methylcarbamate (prepared as described in WO2012125746, D(13)) (1.63 g, 2.60 mmol) in EtOH (20 mL) was added tert butyl N-(pyrrolidin-3-yl)carbamate (0.53 g, 2.9 mmol) and K2CO3 (0.54 g, 3.9 mmol). The resulting mixture was heated by microwave irradiation (Biotage initiator) at 100°C for 30 min. The EtOH was removed in vacuo, H2O added to the residue and the aqueous extracted with EtOAc (3 x 30 mL). The combined organics were washed with H2O (30 mL), brine (30 mL), dried over MgS04 and concentrated in vacuo. The crude product was purified by flash chromatography eluting with 80% EtOAc in petroleum ether (40/60) to afford tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1- yl)-6-fluoro-2-phenylmethanesulphonyl-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1a (1.07 g, 63%) as a yellow solid.

LC-MS (Method A) 655.8 [M+H]⁺; RT 3.14 min.

(b) tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-2-cyano-6-fluoro-9H- pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1 b

A solution of tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2- phenylmethanesulphonyl-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1a (1.87 g, 2.85 mmol) and NaCN (0.31 g, 6.23 mmol) in DMF (30 mL) was heated at 100°C for 16 h. On cooling the mixture was poured into H2O (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organics were washed with H2O (20 mL), brine (20 mL), dried over MgSCU and concentrated in vacuo. The crude product was purified by flash chromatography eluting with 10% EtOAc in heptane to afford tert-butyl N-[4-(3-{[(tert- butoxy)carbonyl]amino}pyrrolidin-1-yl)-2-cyano-6-fluoro-9H-pyrimido[4,5-b]indol-8-yl]-N- methylcarbamate 1 b (0.82 g, 55%) as a yellow solid.

LC-MS (Method A) 526.5 [M+H]⁺; RT 3.21 min.

(c) tert-butyl N-[2-(aminomethyl)-4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidi fluoro-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1 c

To a solution of tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-2-cyano-6- fluoro-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1 b (50 mg, 0.10 mmol) in EtOH (4 ml_) was added an aqueous suspension of RaNi (0.52 ml_, 0.10 mmol). The flask was evacuated and purged with H2, then stirred at room temperature for 16 h under an atmosphere of H2. The reaction mixture was filtered through celite, washed with MeOH (10 ml_) and concentrated in vacuo to furnish tert-butyl N-[2-(aminomethyl)-4-(3-{[(tert- butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-9H-pyrimido[4,5-b]indol-8-yl]-N- methylcarbamate 1c (18 mg, 35%) as a pale brown solid, which was used without any further purification.

LC-MS (Method A) 530.5 [M+H]⁺, RT 1.90 min.

(d) tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(1 ,3-oxazol- 5-ylformamido)methyl]-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1d

To a solution of N-[2-(aminomethyl)-4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6- fluoro-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1c (30 mg, 0.06 mmol), oxazole-5- carboxylic acid (7.7 mg, 0.07 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (10.9 mg, 0.06 mmol) in DCM (2 mL) cooled to 0°C was added Et₃N (0.01 mL, 0.07 mmol) and the mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was poured into 1 M HCI (20 mL) and extracted with DCM (2 ^χ 10 mL). The combined organics were washed with H2O (10 mL), 2M NaOH (10 mL), H2O (10 mL), brine (10 mL), dried (MgSCU) and concentrated under reduced pressure to give a yellow oil (35 mg). The crude product was purified by chromatography using petroleum ether 40- 60/EtOAc gradient 70:30 to 30:70, to furnish tert-butyl N-[4-(3-{[(tert- butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(1 ,3-oxazol-5-ylformamido)methyl]-9H- pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1d (13 mg, 37%) as a pale yellow solid.

LC-MS (Method A) 625.6 [M+H]⁺, RT 2.60 min.

(e) N-{[4-(3-aminopyrrolidin-1-yl)-6-fluoro-8-(methylamino)-9H-pyrimido[4,5-b]indol-2- yl]methyl}-1 ,3-oxazole-5-carboxamide dihydrochloride A

Hydrogen chloride (4M solution in 1 ,4-dioxane) (1.3 mL, 5.2 mmol) was added to tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(1 ,3-oxazol-5- ylformamido)methyl]-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 1d (13 mg, 0.02 mmol) and the mixture was stirred at room temperature for 60 min, after which time the solvent was removed under reduced pressure. The resulting solid was triturated with Et20 (2 x 1 mL) to furnish N-{[4-(3-aminopyrrolidin-1-yl)-6-fluoro-8-(methylamino)-9H- pyrimido[4,5-b]indol-2-yl]methyl}-1 ,3-oxazole-5-carboxamide dihydrochloride A (10 mg, 100% yield) as a yellow solid.

¹ H NMR (Method A) ((CD₃)₂SO): δ 12.1 1 (1 H, br s), 8.60 (1 H, s), 7.88 (1 H, s), 7.07 (1 H, dd, J = 12.0, 2.0 Hz), 6.36 (1 H, dd, J = 12.0, 2.0 Hz), 4.56 (2H, s), 4.24-4.12 (3H, m), 4.01-3.94 (2H, m), 2.87 (3H, s), 2.32 (1 H, m), 2.18 (1 H, m); LC-MS (Method A) 425.4 [M+H]⁺, RT 1.68 min.

Example 2 - N-{r4-(3-aminopyrrolidin-1 -yl)-6-fluoro-8-(methylamino)-9H-pyrimidor4,5- b1indol-2-vnmethylV1 ,3-oxazole-4-carboxamide dihydrochloride B

(a) tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(1 ,3-oxazol- 4-ylformamido)methyl]-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 2a

Using the method described in Example 1 step (d) and oxazole-4-carboxylic acid, tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(1 ,3-oxazol-4- ylformamido)methyl]-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 2a was prepared as a beige solid.

LC-MS (Method A) 625.6 [M+H]⁺, RT 2.76 min.

(b) N-{[4-(3-aminopyrrolidin-1-yl)-6-fluoro-8-(methylamino)-9H-pyrimido[4,5-b]indol-2- yl]methyl}-1 ,3-oxazole-4-carboxamide dihydrochloride B

Using the method described in Example 1 step (e) and tert-butyl N-[4-(3-{[(tert- butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(1 ,3-oxazol-4-ylformamido)methyl]-9H- pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 2a, N-{[4-(3-aminopyrrolidin-1-yl)-6-fluoro-8- (methylamino)-9H-pyrimido[4,5-b]indol-2-yl]methyl}-1 ,3-oxazole-4-carboxamide

dihydrochloride B was prepared as a pale yellow solid.

¹ H NMR (Method A) ((CD₃)₂SO): δ 12.50 (1 H br s), 8.70 (1 H d, J = 1.0 Hz), 8.58 (1 H , d, J = 1.0 Hz), 7.08 (1 H dd, J = 1 1.0, 1.8 Hz), 6.39 (1 H, br d J = 1 1.0 Hz), 4.62 (2H s), 4.27-4.19 (2H, m), 4.06-3.97 (3H, m), 2.87 (3H, s), 2.34 (1 H, m), 2.23 (1 H, m); LC-MS (Method A) 425.5 [M+H]⁺, RT 1.80 min.

Example 3 - N-fr4-(3-aminopyrrolidin-1 -yl)-6-fluoro-8-(methylamino)-9H-pyrimidor4,5- blindol-2-yllmethyl)pyridine-2-carboxamide dihydrochloride C

(a) tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(pyridine-2- ylformamido)methyl]-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 3a

Using the method described in Example 1 step (d) and pyridine-2-carboxylic acid, tert-butyl N-[4-(3-{[(tert-butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(pyridine-2- ylformamido)methyl]-9H-pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 3a was prepared as an orange solid.

LC-MS (Method A) 635.6[M+H]⁺, RT 3.00 min. (b) N-{[4-(3-aminopyrrolidin-1-yl)-6-fluoro-8-(methylamino)-9H-pyrimido[4,5-b]indol-2- yl]methyl}pyridine-2-carboxamide dihydrochloride C

Using the method described in Example 1 step (e) and N-[4-(3-{[(tert- butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-[(pyridine-2-ylformamido)methyl]-9H- pyrimido[4,5-b]indol-8-yl]-N-methylcarbamate 3a, N-{[4-(3-aminopyrrolidin-1-yl)-6-fluoro-8- (methylamino)-9H-pyrimido[4,5-b]indol-2-yl]methyl}pyridine-2-carboxamide dihydrochloride C was prepared as an orange-brown solid.

¹H NMR (Method A) ((CD₃OD): δ 8.70 (1H, m), 8.25 (1H, m) 8.17 (1H, m), 7.73 (1H, m) 7.12 (1H, dd, J = 9.9, 4.2 Hz), 6.46 (1H, dd, J = 10.1, 3.2 Hz) 4.80 (3H, s), 4.40 (1H, m), 4.31 (2H, s), 4.20 (3H, m), 4.08 (1H, m), 3.65 (1H, m), 3.30 (1H, m); LC-MS (Method A) 437.5 [M+H]⁺, RT2.01 min.

Example 4 - 1-r6-fluoro-8-(methylamino)-2-phenylmethanesulphonyl-9H-pyrimidor4,5- b1indol-4-vnpyrrolidin-3-amine D

Using the method described in Example 1 step (e) and tert-butyl N-[4-(3-{[(tert- butoxy)carbonyl]amino}pyrrolidin-1-yl)-6-fluoro-2-phenylmethanesulphonyl-9H-pyrimido[4,5- b]indol-8-yl]-N-methylcarbamate 1a (prepared as described in Example 1 step (a)), 1-[6- fluoro-8-(methylamino)-2-phenylmethanesulphonyl-9H-pyrimido[4,5-b]indol-4-yl]pyrrolidin-3- amine D was prepared as a yellow solid.

LC-MS (Method A) 455.4 [M+H]⁺, RT 2.21 min. Example 5 - Antibacterial susceptibility testing

Antibacterial activity in aerobic conditions

Minimum Inhibitory Concentrations (MICs) versus planktonic bacteria are determined by the broth microdilution procedure according to the guidelines of the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard- Ninth Edition. CLSI document M07-A9, 2012) and by the agar dilution procedure according to the guidelines of the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes, Approved Standard-Second Edition. CLSI document M24-A2, 201 1). The broth dilution method involves a two-fold serial dilution of compounds in 96-well microtitre plates, giving a final concentration range of typically 0.004-128 μg/mL and a maximum final concentration of 1 % DMSO. The agar dilution method involves a two-fold serial dilution of compounds in 24-well microtitre plates, giving a final concentration range of typically 0.004- 32 μς/Γηί. and a maximum final concentration of 1 % DMSO. The bacterial strains tested include Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Mycobacterium smegmatis ATCC 19420 and Mycobacterium tuberculosis ATCC 25177 (H37Ra). Strains are grown in cation-adjusted Muller-Hinton broth (supplemented with 2% w/v NaCI in the case of methicillin-resistant S. aureus strains), on Muller-Hinton agar at 37°C in an ambient atmosphere for 16-20 h or on Middlebrook 7H9 broth with ADC enrichment and Middlebrook 7H 11 agar with OADC enrichment at 37°C, 5% C0₂ for up to four weeks. The MIC is determined as the lowest concentration of compound that inhibits growth following a 16-20 h incubation period for S. aureus and E. coli, following a 72 h incubation for M. smegmatis and following a 3-4 weeks incubation for M. tuberculosis H37Ra.

Proof-of-concept data have been obtained against M. smegmatis, a recognised fast-growing and non-pathogenic surrogate for M. tuberculosis (Tuberculosis, 2010, 90:333). Selected compounds were then tested against M. tuberculosis H37Ra in different conditions (PLoS One, 2008, 3:e2375).

The antibacterial activity of compounds against M. tuberculosis H37Rv grown under aerobic conditions was assessed by measuring bacterial growth after five days in the presence of compounds. Compounds were prepared as 20-point two-fold serial dilutions in DMSO and diluted into 7H9-Tw-OADC medium in 96-well plates with a final DMSO concentration of 2 %. The highest concentration of compound was 200 μΜ where compounds were soluble in DMSO at 10 mM. Bacterial growth was measured by OD590 nm and fluorescence (Ex 560/Em 590) using a BioTek™ Synergy 4 plate reader. To determine the MIC, the dose- response curve was plotted as percentage growth and fitted to the Gompertz model using GraphPad Prism 5. The MIC was defined as the minimum concentration at which growth was completely inhibited and was calculated from the inflection point of the fitted curve to the lower asymptote (zero growth).

The strains tested include: M. tuberculosis H37Rv; two isoniazid-resistant strains, INH-R1 (a KatG Y155 truncation mutant derived from H37Rv) and INH-R2 (strain ATCC 35822); two rifampicin-resistant strains, RIF-R1 (a RpoB S522L mutant derived from H37Rv) and RIF-R2 (strain ATCC 35828); and a fluoroquinolone-resistant strain, FQ-R1 , derived from H37Rv (GyrB D94N mutant).

Antibacterial activity under low oxygen conditions The antibacterial activity of compounds against M. tuberculosis H37Rv grown under hypoxic conditions was assessed using the low oxygen recovery assay (LORA). Bacteria were first adapted to low oxygen conditions and then exposed to compounds under hypoxia. The method used was as described above for aerobic conditions with the following modifications: M. tuberculosis constitutively expressing the luxABCDE operon was inoculated into DTA medium in gas-impermeable glass tubes and incubated for 18 days to generate hypoxic conditions (Wayne model of hypoxia). At this point, bacteria are in a non-replicating state (NRP stage 2) induced by oxygen depletion. Oxygen-deprived bacteria were inoculated into compound assay plates and incubated under anaerobic conditions for 10 days followed by incubation under aerobic conditions (outgrowth) for 28 h. Growth was measured by luminescence. Oxygen-deprived bacteria were also inoculated into compound assay plates and incubated under aerobic conditions for five days.

Intracellular activity assay

The activity of compounds against intracellular bacteria was determined by measuring viability in infected THP-1 cells (macrophage-like cells) after three days in the presence of compounds. Compounds were prepared as 10-point three-fold serial dilutions in DMSO. The highest concentration of compound tested was 50 μΜ where compounds were soluble in DMSO at 10 mM. THP-1 cells were cultured in complete RPMI medium and differentiated into macrophage-like cells using 80 nM PMA overnight at 37°C, 5% CO2. THP-1 cells were infected with a luminescent strain of H37Rv (that constitutively expresses luxABCDE) at a multiplicity of infection of one and incubated overnight at 37°C, 5% CO2. Infected cells were recovered using Accutase/EDTA solution, washed twice with PBS to remove extracellular bacteria and seeded into assay pates. Compound dilutions were added to a final DMSO concentration of 0.5%. Assay plates were incubated for 72 h at 37°C, 5% CO2. Relative luminescent units (RLU) were measured using a Biotek Synergy 2 plate reader. The dose- response curve was fitted using the Levenberg-Marquardt algorithm. The IC50 was defined as the compound concentrations that produced 50% inhibition of microbial growth.

Antibacterial activity against other disease-relevant mycobacteria

The activity of compounds against Mycobacterium abscessus subsp. bolletii 103 and Mycobacterium avium subsp. avium 2285 was assessed under aerobic conditions by determining the MIC as described previously and with the following modifications:

M. abscessus plates were inoculated and incubated for three days at 37°C; growth was measured by OD590 nm. The dose-response curve was plotted as percentage growth and fitted to the Gompertz model and the MIC was defined as the minimum concentration at which growth was completely inhibited and was calculated from the inflection point of the fitted curve to the lower asymptote (zero growth).

M. avium plates were inoculated and incubated for five days at 37°C and Alamar blue was added to each well (10 μΙ_ of Alamar blue to 100 μΙ_ culture) and incubated for a further 24 h at 37°C. Plates were visually inspected and the colour recorded for each well. MIC was defined as the lowest concentration at which no metabolic activity was seen (blue well). The results are shown in Table 1.

Compound E, a compound of formula (VIII), forms part of the prior art (WO2012/125746;

structure:

Compounds A, B, C and E showed good activity against both E. coli and S. aureus and all five compounds A to E tested exhibited excellent activity against M. smegmatis. Three compounds (A, B and C) showed excellent activity against M. tuberculosis H37Ra and compounds A, B, C and E showed excellent activity against the virulent strain M. tuberculosis H37Rv. Compounds A, B, C and E also showed excellent activity against M. tuberculosis H37Rv under low oxygen conditions, suggesting that the compounds are effective against non-replicating TB. Compounds A, B, C and E also showed excellent activity against intracellular bacteria. Compounds A, B, C and E retained excellent activity against antibiotic-resistant M. tuberculosis strains. Compound E showed excellent activity against M. abscessus and M. avium. Compound A and B also showed good activity against these non-tuberculosis Mycobacterium spp. Table 1 : MICs against bacterial and mycobacterial strains. RIF, rifampicin; MXF, moxifloxacin; INH, isoniazid; CIP, ciprofloxacin; LVX, levofloxacin.

In Table 1 a MIC (in μςΛηί) of less than or equal to 0.1 is assigned the letter A; a MIC of from 0.1 to 1 is assigned the letter B; a MIC of from 1 to 10 is assigned the letter C; a MIC of from 10 to 100 is assigned the letter D; and a MIC of over 100 is assigned the letter E. Example 6 - Human cell viability assay

Compounds are assessed for potential non-specific cytotoxic effects against a human hepatic cell line (HepG2, ATCC HB-8065) and a human monocytic cell line (THP-1 , ATCC TI B-202).

HepG2 cells are seeded at 20,000 cells/well in 96-well microtitre plates in minimal essential medium (MEM) supplemented with a final concentration of 10 % FBS and 1 mM sodium pyruvate. After 24 h compound dilutions are prepared in Dulbecco's minimum essential media (DMEM) supplemented with final concentrations of 0.001 % FBS, 0.3 % bovine albumin and 0.02 % HEPES and added to cells. Compounds are tested in two-fold serial dilutions over a final concentration range of 1-128 μg/mL in a final DMSO concentration of 1 % vol/vol. Chlorpromazine is used as a positive control. Cells are incubated with compound at 37°C and 5 % C0₂ for a further 24 h, after which time the CellTiter-Glo reagent (Promega) is added. Luminescence is measured on a BMG Omega plate reader. Data are analysed using GraphPad Prism software to determine the concentration of compound that inhibits cell viability by fifty percent (IC50).

THP-1 cells were cultured in complete RPMI and differentiated into macrophage-like cells using 80 nM PMA overnight at 37°C, 5% CO2. Compounds were prepared as 10-point three-fold serial dilutions in DMSO. The highest concentration of compound tested was 50 μΜ where compounds were soluble in DMSO at 10 mM. Cells were inoculated into assay plates and cultured for 24 h before compound dilutions were added to a final DMSO concentration of 0.5%. Assay plates were incubated for 3 days at 37°C, 5% CO2. Growth was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) that uses ATP as an indicator of cell viability. Relative luminescent units (RLU) were measured using a Biotek Synergy 4 plate reader. The dose-response curve was fitted using the Levenberg-Marquardt algorithm. The IC50 was determined as the compound concentration causing a 50% loss in viability. The results are provided in Table 2.

In Table 2 an IC50 (in μg/mL) of less than or equal to 1 is assigned the letter D; an IC50 of from 1 to 10 is assigned the letter C; an IC50 of from 10 to 20 is assigned the letter B; and an Table 2: IC₅o values against HepG2 and THP-1 cell lines

Thus, compound A shows no detectable toxicity against the tested human hepatic and human monocytic cell lines and this indicates that the compound has the potential to have an excellent therapeutic benefit relative to its cytotoxicity. Compounds B to E also demonstrate an acceptable level of cytotoxicity relative to therapeutic activity. Compounds A and B both show reduced cytotoxicity when compared to prior art compound E.

Claims

CLAIMS ula (I), or a pharmaceutically acceptable salt thereof:

I; wherein

X¹, X² and X³ are each independently selected from N and CR¹; L¹ is independently selected from -CR⁵R⁵NR⁶- and -S(0)„-;

R¹ is independently at each occurrence selected from: H, halo, nitro, cyano, NR⁷R⁷, NR⁸S(0)₂R⁸, NR⁸CONR⁸R⁸, NR⁸C(0)R⁸, NR⁸C0₂R⁸, OR⁸, O-aryl, SR⁸, S-aryl, SOR⁸, S0₃R⁸, S0₂R⁸, S0₂NR⁸R⁸, C0₂R⁸, C(0)R⁸, CONR⁸R⁸, aryl, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl and Ci-C₄-haloalkyl;

R², R⁶ and R⁸ are each independently at each occurrence selected from: H, Ci-C₄-alkyl and Ci-C₄-haloalkyl;

R³ is independently selected from (CR⁵R⁵)_m-aryl and (CR⁵R⁵)_m-heteroaryl;

R⁴ is independently selected from: -(CR⁵R⁵)_m-3-io-heterocycloalkyl, -(CR⁵R⁵)_m-aryl, - (CR⁵R⁵)m-heteroaryl, -(CR⁵R⁵)_m-C₃-Cio-cycloalkyl, O-aryl and O-heteroaryl;

R⁵ is independently at each occurrence selected from H, F, Ci-C₄-alkyl, Ci-C₄-haloalkyl; or two R⁵ groups attached to the same carbon atom may together form a group selected from =0 or =S;

R⁷ is independently at each occurrence selected from: H, Ci-C₄-alkyl, Ci-C₄-haloalkyl, S(0)₂- Ci-C₄-alkyl and C(0)-Ci-C₄-alkyl; n is an integer independently selected from 1 and 2; m is an integer independently selected from 0, 1 and 2; wherein each of the aforementioned alkyl, alkenyl, alkynyl, haloalkyi, cycloalkyi, aryl (e.g. phenyl) and heteroaryl groups is optionally substituted, where chemically possible, by 1 to 5 substituents that are each independently at each occurrence selected from the group consisting of: oxo, =NR^a, =NOR^a, halo, nitro, cyano, NR^aR^a, NR^aS(0)₂R^a, NR^aC(0)R^a, NR^aCONR^aR^a, NR^aC0₂R^a, OR^a; SR^a, SOR^a, S0₃R^a, S0₂R^a, S0₂NR^aR^a, C0₂R^a C(0)R^a, CONR^aR^a, CR^bR^bNR^aR^a, =CR^bCR^bR^bNR^aR^a, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl and Ci-C₄-haloalkyl; wherein R^a is independently at each occurrence selected from H, Ci-C₄- alkyl and Ci-C₄-haloalkyl; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄-alkyl and Ci-C₄-haloalkyl.

2. A compound of claim 1 , wherein the compound of formula (I) is a compound of

wherein R⁹ is independently selected from aryl and heteroaryl.

3. A compound of claim 1 , wherein the compound of formula (I) is a compound of

wherein R⁹ is independently selected from aryl and heteroaryl; and wherein R⁵ is independently at each occurrence selected from H, F, Ci-C₄-alkyl, Ci-C₄-haloalkyl.

4. A compound of claim 2 or claim 3, wherein R⁹ is substituted or unsubstituted monocyclic 5- or 6-membered heteroaryl, optionally wherein R⁹ is a group selected from oxazole, thiazole, isoxazole and isothiazole.

5. A compound of claim 2 or claim 3, wherein R⁹ is substituted or unsubstituted phenyl.

6. A compound of any one of claims 1 to 5, wherein X¹ is CR¹⁰; wherein R¹⁰ is independently at each occurrence selected from: H, halo, Ci-C4-haloalkyl and Ci-C4-alkyl.

7. A compound of any one of claims 1 to 6, wherein X² is CR¹¹; wherein R¹¹ is independently at each occurrence selected from: H, halo, Ci-C4-haloalkyl and Ci-C4-alkyl.

8. A compound of any one of claims 1 to 7, wherein X³ is CR¹²; wherein R¹⁰ is independently at each occurrence selected from: H, halo, Ci-C4-haloalkyl and Ci-C4-alkyl.

9. A compound of any one of claims 1 to 8, wherein R¹ is NHR⁷.

10. A compound of any one of claims 1 to 9, wherein R² is H.

11. A compound of any one of claims 1 to 10, wherein R⁴ is a N-heterocycloalkyl group that is attached to the rest of the molecule via the nitrogen or, where there is more than one ng, via one of the nitrogens in the ring system; optionally wherein R⁴ is

wherein R¹³ is independently selected from oxo, =NOR^a, NR^aR^a, OR^a, Ci-

C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a; wherein R^a is independently at each occurrence selected from H, Ci-C4-alkyl and Ci-C4-haloalkyl; and R^b is independently at each occurrence selected from H, halogen, Ci-C4-alkyl and C1-C4- haloalkyl; or wherein two R¹³ groups together with the carbon or carbons to which they are attached form a 3-6 membered cycloalkyi or 3-6 membered heterocycloalkyi ring; and p is an integer independently selected from 0, 1 , 2, 3 and 4.

A compound of claim 1 , wherein the compound of formula (I) has a structure

nd

13. A pharmaceutical formulation comprising a compound of any one of claims 1 to 12 and a pharmaceutically acceptable excipient.

14. A compound of any one of claims 1 to 12 for medical use.

15. A compound of any one of claims 1 to 12 for use in treating a bacterial infection.

16. A compound of any one of claims 1 to 12 for use in treating a mycobacterial infection.

17. A compound of formula (VIII), or a pharmaceutically acceptable salt thereof, for use in treating a mycobacterial infection:

(VIM); wherein

X¹ , X² and X³ are each independently selected from N and CR¹ ;

L² is independently selected from O and S;

R² is independently at each occurrence selected from: H, halo, nitro, cyano, NR⁷R⁷, NR⁸S(0)₂R⁸, NR⁸CONR⁸R⁸, NR⁸C(0)R⁸, NR⁸C0₂R⁸, OR⁸, O-aryl, SR⁸, S-aryl, SOR⁸, SO3R⁸, SO2R⁸, S0₂NR⁸R⁸, C0₂R⁸, C(0)R⁸, CONR⁸R⁸, aryl, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl and Ci-C₄ haloalkyl;

R², R⁶ and R⁸ are each independently at each occurrence selected from: H, Ci-C₄-alkyl, and Ci-C₄-haloalkyl;

R³ is independently selected from (CR⁵R⁵)_m-aryl and (CR⁵R⁵)_m-heteroaryl;

R⁴ is independently selected from: -(CR⁵R⁵)_m-3-io-heterocycloalkyl, -(CR⁵R⁵)_m-aryl, - (CR⁵R⁵)m-heteroaryl, -(CR⁵R⁵)_m-C₃-Cio -cycloalkyl, O-aryl and O-heteroaryl;

R⁵ is independently at each occurrence selected from H, F, Ci-C₄-alkyl, Ci-C₄-haloalkyl; or two R⁵ groups attached to the same carbon atom may together form a group selected from =0 or =S.

R⁷ is independently at each occurrence selected from: H, Ci-C₄-alkyl, Ci-C₄-haloalkyl, S(0)₂- Ci-C₄ ^"alkyl and C(0)-Ci-C₄ alkyl; n is an integer independently selected from 1 and 2; m is an integer independently selected from 0, 1 and 2; wherein each of the aforementioned alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, aryl (e.g. phenyl) and heteroaryl groups is optionally substituted, where chemically possible, by 1 to 5 substituents that are each independently at each occurrence selected from the group consisting of: oxo, =NR^a, =NOR^a, halo, nitro, cyano, NR^aR^a, NR^aS(0)₂R^a, NR^aC(0)R^a, NR^aCONR^aR^a, NR^aC0₂R^a, OR^a; SR^a, SOR^a, S0₃R^a, S0₂R^a, S0₂NR^aR^a, C0₂R^a C(0)R^a, CONR^aR^a, CR^bR^bNR^aR^a, =CR^bCR^bR^bNR^aR^a, Ci-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl and Ci-C₄-haloalkyl; wherein R^a is independently at each occurrence selected from H, Ci-C₄- alkyl and Ci-C₄-haloalkyl; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄-alkyl and Ci-C₄-haloalkyl.

18. A compound for use of claim 16 or claim 17, wherein the mycobacterial infection is caused by M. tuberculosis.

19. A compound for use of claim 16 or claim 17, wherein the mycobacterial infection is caused by a mycobacterium selected from: M. avium complex, M. abscessus, M. leprae, M. bovis, M. kansasii, M. chelonae, M. africanum, M. canetti and M. microti.

20. A compound for use of any one of claims 16 to 19, wherein the mycobacterial infection is caused by a mycobacterial strain that is resistant to at least one approved antimycobacterial compound.

21. A compound for use of claim 20, wherein the at least one approved antimycobacterial compound is selected from: rifampicin, moxifloxacin, isoniazid, ciprofloxacin and levofloxacin.