WO2015114317A1

WO2015114317A1 - 5h-isothiazolo[4,5-c]pyridine-3,4-dione or 5h-pyrazolo[4,3-c]pyridin-3,4-dione as antibacterial compounds

Info

Publication number: WO2015114317A1
Application number: PCT/GB2015/050182
Authority: WO
Inventors: Andrew Ratcliffe; Ian Cooper; David MCGARRY; Mark Pichowicz
Original assignee: Redx Pharma Ltd
Current assignee: Redx Pharma Ltd
Priority date: 2014-01-28
Filing date: 2015-01-27
Publication date: 2015-08-06
Anticipated expiration: 2016-07-28

Abstract

This invention relates to antibacterial drug compounds of formula (I) containing a fused isothiazolinone or pyrazolone ring and related compounds. It also relates to pharmaceutical formulations of antibacterial drug compounds. It also relates to uses of the derivatives in treating bacterial infections and in methods of treating bacterial infections. Many of the antibacterial drug compounds contain a isothiazolinone or pyrazolone ring fused to a pyridone ring. The compounds have broad spectrum antibacterial activity but are particularly effective against Gram negative strains. wherein X is selected from S, SO, SO2, O and NR4;

Description

5H-ISOTHIAZOLO[4,5-C]PYRIDINE-3,4-DIONE OR 5H-PYRAZOLO[4,3-C]PYRIDIN-3,4-DIONE AS ANTIBACTERIAL COMPOUNDS

This invention relates to antibacterial drug compounds containing a fused isothiazolinone or pyrazolone ring and related compounds. It also relates to pharmaceutical formulations of antibacterial drug compounds. It also relates to uses of the derivatives in treating bacterial infections and in methods of treating bacterial infections. Many of the antibacterial drug compounds contain a isothiazolinone or pyrazolone ring fused to a pyridone ring. The compounds have broad spectrum antibacterial activity but are particularly effective against Gram negative 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. Of these, Staphylococcus aureus, a Gram positive bacterium, is the most concerning due to its potency and its capacity to adapt to environmental conditions. MRSA (methicillin resistant Staphylococcus aureus) is probably the most well known and has reached pandemic proportions. Of particular concern is the increasing incidence of 'community acquired' infections, i.e. those occurring in subjects with no prior hospital exposure. Many strains of MRSA are also resistant to fluoroquinolone antibiotics, in addition to β-lactam antibiotics such as methicillin.

While less wide-spread, antibiotic resistant Gram negative strains, such as either Escherichia coli NDM-1 (New Delhi metallo^-lactamase) mutation or Klebsiella pneumoniae with the same mutation, are also very difficult to treat. Frequently only expensive, last resort antibiotics such as vancomycin and colistin are effective against these strains.

The fluoroquinolone antibacterial family are synthetic broad-spectrum antibiotics. They were originally introduced to treat Gram negative bacterial infections, but are also used for the treatment of Gram positive strains. One problem with existing fluoroquinolones can be the negative side effects that may sometimes occur as a result of fluoroquinolone use. In general, the common side-effects are mild to moderate but, on occasion, more serious adverse effects occur. Some of the serious side effects that occur, and which occur more commonly with fluoroquinolones than with other antibiotic drug classes, include central nervous system (CNS) toxicity and cardiotoxicity. In cases of acute overdose there may be renal failure and seizure.

Fluoroquinolone antibiotics have been developed which contain a isothiazolinone fused to the quinolone core (WO2005/019228; WO2006/118605; WO2007/014308; WO2008/0364240). Such compounds have however been shown to exhibit some cytotoxicity in human cell lines (Kim et al; J. Med. Chem.; 2011, 54, 3268-3282). Related antibiotic compounds containing an isothiazolinone ring fused to a 4-pyrone ring are disclosed in JP 3068175.

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

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 which 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 compounds which have activity which is comparable to those of existing antibiotics, and ideally which 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 which 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 which 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 thereof:

wherein A is independently selected from: aryl, heteroaryl and C3-Cio-heterocycloalkyl; X is independently selected from: S, SO, S0₂, O, NR⁴;

R¹ is independently selected from: H, halogen, cyano, NR⁵R⁵; OR⁶; C1-C6 alkyl, C1-C6 haloalkyi, -(CZ₂)_n-C3-C6 cycloalkyl, -(CZ₂)_n-C3-C6 halocycloalkyl; -(CZ₂)_n-phenyl; -(CZ₂)_n- heteroaryl, C₂-C6 alkenyl and C₂-C6 alkynyl;

R² and R⁴ are each independently selected from: H; C1-C6 alkyl, C1-C6 haloalkyi, -(CZ₂)_n-C3- C6 cycloalkyl, -(CZ₂)_n-C3-C6 halocycloalkyl and -(CZ₂)_n-phenyl;

R³ is independently selected from the group consisting of: H, -(CZ₂)_n-C3-Cio heterocycloalkyl, -(CZ₂)_n-phenyl, -(CZ₂)_n-heteroaryl, -(CZ₂)_n-C₃-Cio cycloalkyl, -(CZ₂)_n-NR⁵R⁵;

R⁵ is independently at each occurrence selected from H, C1-C4 alkyl, C1-C4 haloalkyi, S(0)₂- C1-C4 alkyl and C(0)-Ci-C₄ alkyl;

R⁶ is independently at each occurrence selected from H, C1-C4 alkyl, and C1-C4 haloalkyi;

Z is independently at each occurrence selected from H, Me, CF3 or F; and n is an integer independently selected at each occurrence from 0, 1 , 2 and 3; and wherein each of the aforementioned aryl, heteroaryl, C3-C10 heterocycloalkyl or C3-C10 cycloalkyl groups are monocyclic or bicyclic; and each of the aforementioned alkyl, haloalkyi, cycloalkyi, halocycloalkyi, heterocycloalkyi, aryl (e.g. phenyl) and heteroaryl groups, are optionally substituted, where chemically possible, by 1 to 5 substituents which 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^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, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, C1-C4 haloalkyi, 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 haloalkyi; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄ alkyl and Ci-C₄ haloalkyi.

For the absence of doubt, A may be substituted with a substituent as recited above, even when R³ is H.

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

ble salt or N-oxide thereof:

wherein R¹ , R², R³ and A are as defined for formula (I) above or formula (la) below.

In an embodiment, the compound of formula (I) or formula (la) is a compound of formula (III)

le salt or N-oxide thereof:

(III)

wherein X, R¹ , R² and R³ are as defined for formula (I) above or formula (la) below; and optionally wherein X is S.

In an embodiment, the compound of formula (I) or formula (la) is a compound of formula (IV) or a pharmaceutically acceptable salt or N-oxide thereof: NH

( ^X)P (IV)

wherein X, R¹ , R² and R³ are as defined for formula (I) above or formula (la) below; wherein Y¹ , Y², Y³ and Y⁴ are each selected from carbon or nitrogen; R^x is a group selected from halo, nitro, cyano, NR^aR^a, NR^aS(0)₂R^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, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, Ci-C₄ haloalkyl and CR^bR^bNR^aR^a; and p is an integer from 1 to 4; provided that no more than two of Y\ Y², Y³ and Y⁴ are N; and optionally wherein X is S. Where Y\ Y², Y³ or Y⁴ is carbon, the carbon will be substituted with either H or an R^x group. In certain particular embodiments, at least 1 of Y¹ , Y², Y³ and Y⁴ is nitrogen and p is 0. In alternative particular embodiments, Y¹ , Y², Y³ and Y⁴ are each carbon. R³ is preferably an N-heterocycloalkyl group which is attached to the rest of the molecule via the or one nitrogen in the heterocycloalkyl ring system.

In an embodiment, the compound of formula (I) is a compound of formula (V) or a or N-oxide thereof:

(V)

wherein X, R¹ , R² and R³ are as defined for formula (I) above; wherein R¹² is independently selected from H, Ci-C₄ alkyl and Ci-C₄ haloalkyl; and optionally wherein X is S.

In an embodiment, the compound of formula (I) is a compound of formula (VI) or a pharmaceutically acceptable salt or N-oxide thereof:

wherein X, R¹ and R² are as defined for formula (I) above; Y¹ , Y², Y³, Y⁴, R^x and p are as defined above for formula (IV); and wherein R⁹ is independently selected from H, C1-C4 alkyl and C1-C4 haloalkyl; R¹⁰ is selected from H, C1-C4 alkyl and C1-C4 haloalkyl; R¹¹ is selected from H, F, C1-C4 alkyl and C1-C4 haloalkyl; or where R¹⁰ and R¹¹ together with the atoms to which they are attached form a 5- or 6- membered cycloalkyi or heterocycloalkyi ring; and p is an integer from 1 to 4; optionally wherein X is S. In certain particular embodiments, Y¹ , Y², Y³ and Y⁴ are each carbon.

In an embodiment, the compound of formula (I) is a compound of formula (VII) or a or N-oxide thereof:

(VII)

wherein X, R¹ , R² and R³ are as defined for formula (I) above; and R¹² is as defined for formula (V) above; optionally wherein X is S.

In an embodiment, the compound of formula (I) is a compound of formula (VIII) or a pharmaceutically acceptable salt or N-oxide thereof:

wherein X, R¹ , R² and R^a are as defined for formula (I) above; Y¹ , Y², Y³, Y⁴, R^x and p are as defined above for formula (IV); and wherein R⁷ is independently selected from oxo, =NOR^a, NR^aR^a, OR^a, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a; and m is an integer independently selected from 1 , 2, 3 and 4; optionally wherein X is S. In certain particular embodiments, Y¹ , Y², Y³ and Y⁴ are each carbon, p may be 0.

In an embodiment, the compound of formula (I) is a compound of formula (IX) or a pharmaceutically acceptable salt or N-oxide thereof:

wherein X, R¹ , R² and R^a are as defined for formula (I) above; Y¹ , Y², Y³, Y⁴, R^x and p are as defined above for formula (IV); and wherein R⁷ is as defined above for formula (VIII); optionally wherein X is S. In certain particular embodiments, Y¹ , Y², Y³ and Y⁴ are each carbon, p may be 0.

The following statements apply to compounds of any of formulae (I) to (IX) (including formula la). 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 which describe a feature of that compound, expressed at any level of generality, may be combined so as to represent subject matter which is contemplated as forming part of the disclosure of this invention in this specification. It may be that A is aryl, e.g. phenyl or naphthyl (which may be unsubstituted or may be substituted as described phenyl and

hence, A-R³ is preferably

Where A is phenyl, it may be para-substituted, i.e. the R³ group may be in a para position relative to the rest of the molecule. Thus, it may be that A is independently from para- substituted phenyl, heteroaryl and C3-Cio-heterocycloalkyl. Where A is a six-membered monocyclic heteroaryl group, it may be para-substituted, i.e. the R³ group may be in a para position relative to the rest of the molecule. Thus, it may be that A is independently from para-substituted phenyl, para-substituted 6-membered monocyclic heteroaryl, 5-membered monocyclic heteroaryl, bicyclic heteroaryl and C3-Cio-heterocycloalkyl;

A may also be heteroaryl. Thus, A may be thiophene.

Where A is heteroaryl it may be that A is a heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system. Thus, A might be a monocyclic heteroaryl group, e.g. a monocyclic heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system. A may also be a bicyclic heteroaryl group, e.g. a bicyclic heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system. A may be an five-membered monocyclic heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system, e.g. thiazole, oxazole, pyrazole (any of which may be unsubstituted or may be substituted as described herein for heteroaromatic groups). A may be an six-membered monocyclic heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system, e.g. pyridyl, pyrimidyl, pyrazinyl or pyridazinyl (any of which may be unsubstituted or may be substituted as described herein for heteroaromatic groups). Where A is a six-membered monocyclic heteroaryl group, it may be para-substituted, i.e. the R³ group may be in a para position relative to the rest of the molecule.

Thus, A-R³ may be

wherein Y¹ , Y², Y³ and Y⁴ are each selected from carbon or nitrogen; R^x is a group selected from halo, nitro, cyano, NR^aR^a, NR^aS(0)2R^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, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl and CR^bR^bNR^aR^a; and p is an integer from 1 to 4; provided that no more than two of Y¹ , Y², Y³ and Y⁴ are N; and optionally wherein X is S. Where Y¹ , Y², Y³ or Y⁴ is carbon, the carbon will be substituted with either H or an R^x group. In certain particular embodiments, Y¹ , Y², Y³ and Y⁴ are each carbon at leas ¹ , Y², Y³ and Y⁴ is nitrogen and p is 0. In alternative particular

embodiments,

Y², Y³ and Y⁴ are each CH or N; provided that at least 1 of Y\ Y², Y³ and Y⁴ is N.

It may that a single of Y¹ , Y², Y³ and Y⁴ is N but it also possible that exactly two of Y¹ , Y², Y and Y⁴ are N.

A may be pyridyl (which may be unsubstituted or may be substituted as described herein for

heteroaromatic groups). Thus, A-R³ may be

or -R³ may

Where A is phenyl or 6-membered heteroaryl, R³ is preferably not H.

A may also be a ten-membered bicyclic heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system, e.g. quinoline or isoquinoline (any of which may be unsubstituted or may be substituted as described herein for heteroaromatic groups).

A may also be a nine-membered bicyclic heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system, e.g. indolyl, benzimidazolyl, benzothiazole, benzoxazole or indazolyl (any of which may be unsubstituted or may be substituted as described herein for heteroaromatic groups). A-R³ may be indolyl or indazolyl. Where A is a nine-membered bicyclic heteroaryl group, A-R³ may be attached to the rest of the molecule via the six-membered ring of A or via the five membered ring of A. Likewise, where R³ is present, R³ may be attached to A via the six-membered ring of A or via the five membered ring of A. Generally, A-R³ is attached to the rest of the molecule via the six- membered ring of A and, where present, R³ is attac membered ring of

Thus, A-R³ may

e.g. , wherein Y⁵ is independently selected from nitrogen and carbon and R 12 is independently selected from H,

any embodiment in which A is a heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring structure), R³ may be H (or, where the compound is a compound of formula la, absent).

In a particular embodiment, X is S. It may be, however, that X is O or NH.

R¹ may independently be selected from: C1-C6 alkyl, C1-C6 haloalkyl, -(CZ2)_n-C3-C6 cycloalkyl, -(CZ2)_n-C3-C6 halocycloalkyl; -(CZ2)_n-phenyl; C2-C6 alkenyl and C2-C6 alkynyl. Thus R¹ may independently selected from C1-C6 alkyl, C1-C6 haloalkyl, -(CZ2)_n-C3-C6 cycloalkyl and -(CZ2)_n-C3-C6 halocycloalkyl. R¹ may be selected from C1-C6 alkyl and - (CH2)n-C3-C6 cycloalkyl, wherein n is an integer selected from 0, 1 , 2 and 3. Alternatively, R¹ may be selected from C1-C6 haloalkyl and -(CZ2)_n-C3-C6 halocycloalkyl, wherein n is an integer selected from 0, 1 , 2 and 3. Thus, R¹ may be selected from C1-C6 alkyl (e.g. C2-C4 alkyl) and C3-C6 cycloalkyl (e.g. C3-C4 cycloalkyl). R¹ may be selected from C1-C4 alkyl and cyclopropyl. In a particular embodiment, R¹ is ethyl. In another particular embodiment, R¹ is cyclopropyl.

R² may independently be selected from H, C1-C6 alkyl, C1-C6 haloalkyl, -(CZ2)_n-C3-C6 cycloalkyl and -(CZ2)_n-C3-C6 halocycloalkyl. Thus, R² may be selected from H, and C1-C6 alkyl. In a particular embodiment, R² is H. In another particular embodiment, R² is methyl.

R³ may be H (or, where the compound is a compound of formula la, absent). Typically, however, where A is aryl (e.g. phenyl), R³ will not be H (or, where the compound is a compound of formula la and A is aryl, R³ will not be absent).

Where A is phenyl or 6 membered heteroaryl, R³ is preferably an N-heterocycloalkyl group which is attached to the rest of the molecule via the or one nitrogen in the heterocycloalkyi ring system.

R³ may be -(CZ2)_n-C3-Cio heterocycloalkyi, e.g. C3-C10 heterocycloalkyi. Typically, R³ 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 R³ may be attached to the rest of the molecule via a carbon or one nitrogen in the ring system. It may be that the N-heterocycloalkyl group is attached to the rest of the molecule via the or one nitrogen in the ring system. Any nitrogen in the ring system which is not at a bridgehead or is not the point of attachment of R³ to the rest of the molecule will be an NR¹² group. Unless otherwise stated, any N-heterocycloalkyl group mentioned as a possibility for R³ may be unsubstituted or may be substituted with 1 to 3 groups selected from oxo, =NOR^a, NR^aR^a, OR^a, C1-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, C1-C4 alkyl and C1-C4 haloalkyl; and R^b is independently at each occurrence selected from H, halogen, C1-C4 alkyl and C1-C4 haloalkyl.

R³ may be a monocyclic C3-C7 N-heterocycloalkyl group. Thus, R³ may be a piperazine ring. R³ 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, R³ may be an unsubstituted piperizine group. Any piperazine group will typically be attached to the rest of the molecule via one of the nitrogens in the ring system. Possibly, R³ is an azetidine, pyrrolidine or piperidine ring, optionally wherein the ring nitrogen attaches the aziridine, pyrrolidine or piperidine ring to the rest of the compound. R³ 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 which is substituted with a single hydroxyl group. R³ may be a piperidine ring substituted with a single hydroxyl group, e.g. a 4- hydroxy-piperidine ring. R³ may be a pyrrolidine substituted with a single hydroxyl group, e.g. a 3-hydroxypyrrolidine. R³ is a 3-hydroxy azridine group. R³ may be a bicylic C⁷-C¹⁰ N- heterocycloalkyl group.

R³ may be a bicyclic C7-C10 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.

R³ may be

wherein R⁷ is independently selected from oxo, =NOR^a, NR^aR^a,

OR^a, C1-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; or wherein two R⁷ groups together with the carbon or carbons to which they are attached form a 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyi ring. Where two R⁷ 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 R⁷ groups form a cycloalkyl or heterocycloalkyi ring, that ring may be substituted with one or two groups independently selected from oxo, =NOR^a, NR^aR^a, OR^a, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a. m is an integer independently selected from 0, 1 , 2, 3 and 4. These embodiments are particularly preferred when A is phenyl or a 6-membered heteroaryl group. m may be an integer independently selected from 1 , 2, 3 and 4.

It may be that tw ⁷ groups do not form a cycloalkyl or heterocycloalkyl ring. In other

words R³ may be

wherein R⁷ is independently selected from oxo, =NOR^a, NR^aR^a, OR^a, C1-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 haloalkyi; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄ alkyl

may be 1. Thus, R³ may be

R⁷ may be NR^aR^a. Each R^a in R⁷ may be H (e.g. R⁷ may be NH₂). Each R^a in R⁷ may independently be Ci-C₄ alkyl, e.g. each R^a in R⁷ may independently be methyl (e.g. R⁷ may be NMe₂). R^a may at one occurrence be H and at the other occurrence be Ci-C₄ alkyl (e.g. R⁷ may be NHMe).

R⁷ may be OR^a. R^a may be H and, thus, R⁷ may be OH.

R⁷ may be CR^bR^bNR^aR^a. Each R^b in R⁷ may independently be C1-C4 alkyl, e.g. each R^b in R⁷ may independently be methyl (e.g. R⁷ may be CMe₂NR^aR^a). Each R^a in R⁷ may be H (e.g. R⁷ may be CR^bR^bNH₂). R⁷ may be CMe₂NH₂. Alternatively, each R^b in R⁷ may independently be H (i.e. R⁷ may be CH₂NR^aR^a). It may be that R^a in R⁷ is at one instance H and the other instance Ci-C₄ alkyl, e.g. methyl. m may be 2. In one particular example where m is 2, R⁷ 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 R⁷ groups may form a 3-6 membered heterocycloalkyl ring, e.g. a 6-membered heterocycloalkyl ring, e.g. a vicinally fused 6 membered heterocycloalkyl ring. A specific example of a 6-membered heterocycloalkyl ring would be a morpholine ring. The two R⁷ groups may also form a 3-6 membered cycloalkyi ring, e.g. a 3-membered ring. Thus, two R⁷ 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, C1-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 R⁷ groups form a 3 to 6-membered cycloalkyi or 3 to 6-membered heterocycloalkyl ring, there may be one or more other R⁷ groups, e.g. m may be 4. Such additional R⁷ groups will generally not form a 3 to 6-membered cycloalkyi or a 3 to 6- membered heterocycloalkyl ring. R⁷ may be Ci-C₄ alkyl, e.g. methyl. R⁷ may be NR^aR^a, e.g. NH₂.

R³ may be

wherein R⁹ is independently selected from H, Ci-C₄ alkyl and

Ci-C₄ haloalkyl; R¹⁰ is selected from H, Ci-C₄ alkyl and Ci-C₄ haloalkyl; R¹¹ is selected from H, F, Ci-C₄ alkyl and Ci-C₄ haloalkyl; or where R¹⁰ and R¹¹ together with the atoms to which they are attached form a 5- or 6- membered cycloalkyi or heterocycloalkyl ring. This is particularly preferred when A is phenyl.

It may be that R³ is or comprises a NR⁸R⁸ group, wherein R⁸ is independently H or Ci-C₄ alkyl. Thus R³ may be comprise an NH2 group or an ΝΜβ2 group. Thus, it may be that R⁷ is present at least once and in that at least one occurrence represents NR^aR^a wherein R^a is independently H or Ci-C₄ alkyl. Alternatively, it may be that R³ is NR⁵R⁵, wherein R⁵ is independently H or Ci-C₄ alkyl.

R³ may be C3-C8 cycloalkyi group. Typically, where R³ is a C3-C8 cycloalkyi group, it is substituted with at least one group selected from NR^aR^a, OR^a, C1-C4 alkyl, C2-C4 alkenyl, C2- C₄ alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a. Preferably, where R³ is a C₃-C₈ cycloalkyi group group, it is substituted with at least one group selected from NR^aR^a, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a. Specifically, R³ may be a cyclopropyl group substituted with a NH2 group.

A specific example of an R³ group is

R³ may be an aryl group, e.g. a phenyl group. R³ 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, C₁-C4 haloalkyl and C₁-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 embodiments, R³ may be a group selected

from:

R³ may also be a heteroaryl group. R³ may be a heteroaryl group comprising at least one nitrogen atom in the ring structure. R³ may be a 9- membered bicyclic heteroaryl group comprising nitrogen atoms in the ring

system, e.g. an indazole group, e.g. R³ may be

R³ 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.

R³ may be -(CZ₂)_N-NR⁵R⁵ Thus, R³ may be NR⁵R⁵. Alternatively, R³ may be CH₂NR⁵R⁵. Each R⁵ in R³ may independently be C₁-C4 alkyl, e.g. each R⁵ in R³ may independently be methyl (e.g. R³ may be ΝΜβ2). Alternatively, one R⁵ in R³ may be H and the other may be C¹-C⁴ alkyl which may be substituted, e.g. substituted with an OR^A group. Thus, a particular example of R³ would be CH₂NHCH₂CH₂OMe.

It may be that n is always 0.

X is preferably independently selected from S, O, NR⁴. X may be independently selected from S and NR⁴. X may be S. X may be NR⁴. R⁴ is preferably selected from H and C₁-C4 alkyl. In a particular embodiment, R⁴ is H. In another particular embodiment, R⁴ is Me.

In a preferred embodiment, X is selected from S, O, NR⁴, R² and R⁴ are each independently selected from H and Ci-C4alkyl, A is phenyl or 6 membered heteroaryl, R³ is a monocyclic or bicyclic N-heterocycloalkyl group which is attached to the rest of the molecule via the or one nitrogen in the ring system; and R¹ is selected from C₁-C4 alkyl and cyclopropyl. Particularly preferred are embodiments in which X is S.

In a second preferred embodiment, X is selected from S, O, NR⁴, R² and R⁴ are each independently selected from H and C₁-C4 alkyl, A is a nine-membered bicyclic heteroaryl group which comprises at least one nitrogen atom in the heteroaromatic ring system, R³ is H; and R¹ is selected from C1-C4 alkyl and cyclopropyl.

The compound of formula (I) may be a compound selected from:



The compound may be a compound selected from the compounds for which the biological data is provided in Examples 21 and/or 22 below or a pharmaceutically acceptable salt or N- oxide thereof.

The compound may be a compound selected from:

7-Ethyl-6-indazol-5-yl-5H-isothiazolo[4,5-c]pyridine-3,4-dione;

6-[2-(Aminomethyl)-indol-5-yl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4-dione;

6-[5-[3-(Dimethylamino)pyrrolidin-1-yl]pyridin-2-yl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4- dione;

6-[6-[3-(Dimethylamino)pyrrolidin-1-yl]pyridin-3-yl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4- dione;

6-[6-[3-(Dimethylamino)pyrrolidin-1-yl]pyridazin-3-yl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine- 3,4-dione; 7-Ethyl-6-[4-[3-methoxy-4-(methylami

3,4-dione;

7-Ethyl-6-[4-[3-hydroxy-4-(methylamino)pyrrolidin-1-yl]phenyl]-5H-isothiazolo[4,5

3,4-dione;

7-Ethyl-6-[4-[3-(hydroxyimino)pyrrolidin-1-yl]phenyl]-5H-isothiazolo[4,5-c]pyridine-3,4-dione;

7-Ethyl-6-[4-[3-(methoxyimino)pyrrolidin-1-yl]phenyl]-5H-isothiazolo[4,5-c]pyridine-3,4-dione^

7-Ethyl-6-[4-(pyrazol-4-yl)phenyl]-5H-isothiazolo[4,5-c]pyridine-3,4-dione;

6-[4-[3-(Dimethylamino)pyrrolidin-1-yl]phenyl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4- dione-1-oxide;

6-[4-[3-(Dimethylamino)pyrrolidin-1-yl]phenyl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4- dione-1 , 1-dioxide;

6-[4-[3-(Dimethylamino)pyrrolidin-1-yl]phenyl]-7-ethyl-3-hydroxy-1-(2-hydroxyethyl)-5H- pyrazolo[4,3-c]pyridin-4-one;

6-[4-(4,4-Difluoropiperidin-1-yl)phenyl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4-dione or a pharmaceutically acceptable salt or N-oxide thereof.

The compound may be a compound of formula (la), or a pharmaceutically acceptable salt thereof:

(la)

wherein A is independently aryl, heteroaryl or C3-Cio-heterocycloalkyl; X is independently selected from S, SO, SO2, O, NR⁴; R¹ is independently selected from: H, halogen, cyano, NR⁵R⁵; OR⁶; Ci-C_e alkyl, Ci-C_e haloalkyi, -(CZ₂)_n-C3-C₆ cycloalkyl, -(CZ₂)_n-C3-C₆ halocycloalkyl; -(CZ2)_n-phenyl; -(CZ2)_n-heteroaryl, C2-C6 alkenyl and C2-C6 alkynyl; R² and R⁴ are each independently selected from H; C1-C6 alkyl, C1-C6 haloalkyi, -(CZ2)_n-C3-C6 cycloalkyl, -(CZ2)_n-C3-C6 halocycloalkyl and -(CZ2)_n-phenyl; R³ is absent or is independently selected from the group consisting of: -(CZ2)_n-C3-Cio heterocycloalkyl, -(CZ2)_n-phenyl, - (CZ2)_n-heteroaryl, -(CZ2)_n-C3-Cio cycloalkyl, -(CZ2)_n-NR⁵R⁵; R⁵ is independently at each occurrence selected from H, C1-C4 alkyl, C1-C4 haloalkyi, S(0)2-Ci-C₄ alkyl and C(0)-Ci-C₄ alkyl; R⁶ is independently at each occurrence selected from H, Ci-C₄ alkyl, and Ci-C₄ haloalkyi; Z is independently at each occurrence selected from H, Me, CF3 or F; and n is an integer independently selected at each occurrence from 0, 1 , 2 and 3; and wherein each of the aforementioned aryl, heteroaryl, C3-C10 heterocycloalkyl or C3-C10 cycloalkyl groups are monocyclic or bicyclic; and each of the aforementioned alkyl, haloalkyl, cycloalkyl, halocycloalkyl, heterocycloalkyl, aryl (e.g. phenyl) and heteroaryl groups, are optionally substituted, where chemically possible, by 1 to 5 substituents which 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^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, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkenyl, Ci-C₄ haloalkyl, 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. For the absence of doubt, A may be substituted with a substituent as recited above, even when R³ is absent.

I e invention is a method of making a compound of formula (X),

(X)

wherein A is independently aryl or heteroaryl;

X is independently selected from S, SO, S0₂, O, NR⁴;

R¹ is independently selected from: H, halogen, cyano, NR⁵R⁵; OR⁶; C1-C6 alkyl, C1-C6 haloalkyl, -(CZ₂)n-C3-Ce cycloalkyl, -(CZ₂)n-C3-Ce halocycloalkyl; -(CZ₂)_n-phenyl; -(CZ₂)_n- heteroaryl, C₂-C6 alkenyl and C₂-C6 alkynyl;

R² and R⁴ are each independently selected from H; C1-C6 alkyl, C1-C6 haloalkyl, -(CZ₂)_n-C3- C6 cycloalkyl, -(CZ₂)_n-C3-C6 halocycloalkyl and -(CZ₂)_n-phenyl;

R³ is independently selected from the group consisting of: heterocycloalkyl, phenyl, heteroaryl, cycloalkyl, -NR⁵R⁵; wherein the heterocycloalkyl group comprises at least one nitrogen in the ring and wherein the heterocyclolkyi group is linked to A (or L²) through a nitrogen atom;

R⁵ is independently at each occurrence selected from H, Ci-C₄ alkyl, Ci-C₄ haloalkyl, S(0)₂- C1-C4 alkyl and C(0)-Ci-C₄ alkyl; R⁶ is independently at each occurrence selected from H, C1-C4 alkyl, and C1-C4 haloalkyl;

Z is independently at each occurrence selected from H, Me, CF3 or F; and n is an integer independently selected at each occurrence from 0, 1 , 2 and 3; and wherein each of the aforementioned aryl, heteroaryl, C3-C10 heterocycloalkyl or C3-C10 cycloalkyl groups are monocyclic or bicyclic; and each of the aforementioned alkyl, haloalkyl, cycloalkyl, halocycloalkyl, heterocycloalkyl, aryl (e.g. phenyl) and heteroaryl groups, are optionally substituted, where chemically possible, by 1 to 5 substituents which 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^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, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, C1-C4 haloalkyl, 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; the method comprising

a) reacting a compound of formula XIa (where R² for the compound of formula X is H) or formula Xlb (where R² for the compound of formula X is not H)

Xlb

wherein L¹ is independently selected from halogen, boronic acid and boronate ester; wherein P¹ and P² are each independently selected from: substituted or unsubstituted benzyl group; Ci-C₄ alkyl or substituted or unsubstituted benzyl carbonate group; or SiR⁹3, wherein R⁹ is independently selected from phenyl or Ci-C₄ alkyl;

with a compound of formula XII:

R³-L²X||;

wherein L² is independently selected from: H, halogen, boronic acid and boronate ester; in the presence of a catalyst to provide a compound of formula Xllla (where R² for the compound of formula X is H) or formula Xlllb (where R² for the compound of formula X is not H).

Xlllb; and

b) converting the compound of formula Xllla or Xlllb into the compound of formula X.

The inventors have surprisingly discovered that coupling reactions (e.g. Buchwald coupling reactions) such as those typically used to form a bond between R³ and A are considerably more efficient and consistent if the isothiazolinone and optionally the pyridinone carbonyl groups are protected.

The conditions, solvents and catalysts used for coupling reactions (e.g. Buchwald coupling reactions) are well known in the art.

Preferably the catalyst comprises palladium. Preferably, L¹ is halogen. It may be that P¹ and P² are the same. Preferably, P¹ and P² are both SiR⁹3, e.g. Si('Pr)3. The inventors have surprisingly found that the most efficient and consistent coupling reactions are obtained when both P¹ and P² are triisopropylsilyl.

It may be that R² is H, i.e. it may be that the method comprises reacting a compound of formula XIa with a compound of formula XII in the presence of a catalyst to provide a compound of formula Xllla

The methods used for removing protecting groups are well known in the art. As an example, where P¹ and P² are both SiR⁹3, the compound of formulae Xllla or Xlllb can be converted to the compound of formula X using an ion exchange column. Alternatively, this can be achieved by treating the compound of formula formulae Xllla or Xlllb withTBAF

Preferably, the compound of formula XII is a compound of formula XIV:

wherein R⁷ is independently selected from oxo, =NOR^a, NR^aR^a, OR^a, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CR^bR^bNR^aR^a and =CR^bCR^bR^bNR^aR^a; or wherein two R⁷ groups together with the carbon or carbons to which they are attached form a 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl ring; and m is an integer independently selected from 0, 1 , 2, 3 and 4.

The method may also comprise the step of converting a compound of formula XV into a compound of formula Xla (where R² for the compound of formula X is H) or formula Xlb (where R² for the compound of formula X is not H):

The product of this reaction can then be used to generate the compound of formula Xllla or

Xlllb.

The reagents and conditions used to protect amides with P¹ and P² groups are well known in the art.

Many compound of the first aspect can be made by the method of the second aspect. Thus, where chemically appropriate, all embodiments described above in relation to the first aspect may also apply to this second aspect.

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 which contain an aromatic moiety. Compounds of the invention may be depicted throughout this specification as containing a ring with the re:

Such compounds may in actuality exist in the following form:

which is a tautomer of the depicted form. Likewise, compounds of the invention may be hout this specification as containing a ring with the following structure:

Such compounds may in actuality exist in the following form:

which is a tautomer of the depicted form.

In each case, both tautomeric forms form part of the present invention and the disclosure of this specification. The compounds of the invention may in actuality exist entirely in the tautomeric form depicted throughout this specification, as the alternative tautomeric form described in this paragraph or as a mixture of the two tautomeric forms. The existence of the compound in a one form or the other (or, where in a mixture of forms, the distribution of that mixture) may depend on the environment that the compound is in or it may be energetically preferred irrespective of the environment. One tautomer may be more active than the other and, where this is the case, it will not necessarily be the most energetically favourable form (although it may be), and it will not necessary be the form depicted in this specification (although it may be).

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 to 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.

Aryl groups have from 6 to 20 carbon atoms as appropriate to satisfy valency requirements. Aryl groups satisfy the Huckel rule. Aryl groups may be optionally substituted phenyl groups, optionally substituted biphenyl groups, optionally substituted naphthalenyl groups or optionally substituted anthracenyl groups. Equally, aryl groups may include non-aromatic carbocyclic portions. Heteroaryl groups may be 5- or 6-membered heteroaryl groups. Heteroaryl groups may be selected from: 5-membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-3 heteroatoms selected from O, S and N; and 6-membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-2 nitrogen atoms; 9-membered bicyclic heteroaryl groups in which the heteroaromatic system is substituted with 1-4 heteroatoms 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 selected from: pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, isoxazole, triazole, oxadiazole, thiodiazole, pyridine, pyridazine, pyrimidine, pyrazine, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, indazole, benzimidazole, benzoxazole, benzthiazole, benzisoxazole, purine, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, pteridine, phthalazine, naphthyridine.

Cycloalkyl groups may be saturated or partially unsaturated. They may be a monocyclic ring or a bicyclic ring system.

A haloalkyi group is an alkyl group substituted with at least one halogen substituent, e.g. at least one fluorine substituent. Likewise, a halocycloalkyl group is a cycloalkyl group substituted with at least one halogen substituent, e.g. at least one fluorine substituent.

An alkyl group may be a straight chain alkyl group or it may be a branched chanin alkyl group. Thus a C₄-alkyl group may be n-butyl, isobutyl or t-butyl.

The aryl and heteroaryl groups are optionally substituted with 1 to 5 substituents which 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, OR^a; SR^a, SOR^a, S0₃R^a, S0₂R^a, S0₂NR^aR^a, C0₂R^a C(0)R^a, CONR^aR^a, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, Ci-C₄ haloalkyi and CR^bR^bNR^aR^a; wherein R^a is independently at each occurrence selected from H, Ci-C₄ alkyl and Ci-C₄ haloalkyi; and R^b is independently at each occurrence selected from H, halogen, Ci-C₄ alkyl and Ci-C₄ haloalkyi. .

A heterocycloalkyl group is a saturated or partially saturated ring or bicyclic ring system comprising 1 , 2 or 3 heteroatoms independently selected from O, S and N (in other words from 1 , 2 or 3 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. Where a heterocycloalkyl group is described as a C⁴ heterocycloalkyl group it is intended to mean that the ring system comprises 4 carbon atoms (e.g. a morpholine). As another example, a pyrrolidine group is a 5-membered heterocycloalkyl group comprising one nitrogen and four carbon atoms in the ring. It is thus a C₄ heterocycloalkyl group. Examples of monocylclic heterocycloalkyl groups include: piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, dihydrofuran, tetrahydropyran, dihydropyran, dioxane, azepine.

The present invention also includes the synthesis of all pharmaceutically acceptable isotopically-labelled compounds of formulae (I) to (IX) (including formula (la)) 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.

Medical 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 compound as defined above for the treatment of bacterial infections.

The compounds and formulations of the present invention may be used in the treatment of a wide range of bacterial 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 present invention can be used to treat both Gram positive and Gram negative bacterial 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, Escherichia coli, prophylaxis before dental surgery, cellulitis, acnes, cystitis, infectious diarrhoea, typhoid fever, infections caused by anaerobic bacteria, peritonitis, bacterial vaginosis, pelvic inflammatory disease, pseudomembranous colitis, Helicobacter pylori, acute gingivitis, Crohn's disease, rosacea, fungating tumours, MRSA, impetigo, tuberculosis, meningitis, abdominal infection, bacteraemia, septicaemia, leprosy and sexually transmitted bacterial infections (e.g. gonorrhoea, Chlamydia). In one embodiment, the compounds of the invention can be used to treat infections caused by a resistant strain of bacteria. In a further embodiment, the compounds can be used to treat infections caused by a resistant strain of Gram positive bacteria and/or resistant strains of Gram negative bacteria.

The compounds and formulations of the invention may be used to treat infections caused by bacteria which are in the form of a biofilm.

The term 'resistant strains' is intended to mean strains of bacteria which have shown resistance to one or more known antibacterial drug. For example, it may refer to strains which are resistant to methicillin, strains that are resistant to other β-lactam antibiotics and/or strains that are resistant to fluoroquinolones. A resistant strain is one in which the MIC 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 compounds of the invention may be particularly effective at treating infections caused by fluoroquinolone resistant strains of Staphylococcus aureus.

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 which are category A pathogens as identified by the US government (e.g. those which cause anthrax, plague etc.) and/or they may be strains which are category B pathogens as identified by the US government (e.g. those which 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 Jan 2014.

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.

The compounds of the invention may also be useful in treating other forms of infectious disease, e.g. fungal infections, parastic 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 formic, 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. 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 mixtures 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) 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.

Depending on the mode of administration of the compounds of the invention, the pharmaceutical composition which 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, e.g. by oral administration in the form of tablets, capsules, syrups, powders 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 ug/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 which 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 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.

In another aspect of the invention is provided a method of treating a bacterial infection, the method comprising treating a subject in need thereof with a therapeutically effective amount of a compound of the invention.

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. 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 indications mentioned in this specification.

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:

DMF - N, /V-dimethylformamide DCM - dichloromethane

THF - tetrahydrofuran NMP - N-methyl-2-pyrrolidone

DIAD - diisopropyl azodicarboxylate PMB - para-methoxybenzyl

DMB - 2,4-dimethoxybenzyl mCPBA - meta-chloroperoxybenzoic acid

TBAF - tetrabutylammonium fluoride TIPS - triisopropylsilyl

A certain subset of com ounds of formula I can be prepared as described by Scheme A:

(6) (5) (4)

Scheme A

Ketone (1) can be converted to imine (2) by reaction with an excess of tert-butyl amine in the presence of a Lewis acid, such as titanium tetrachloride, or a dehydrating agent, such as para-toluenesulphonic acid, in a solvent, such as DCM. Imine (2) can be converted to hydroxyl 2-pyridones (3) by reaction with CH(C02Et)3, in a high boiling solvent, such as diphenyl ether, at temperatures from 130°C to 230°C. Conversion of the OH in (3) to CI in (4) can be accomplished using a chlorinating agent, such as oxalyl chloride, either neat or in a solvent, such as DCM. Treatment of chloride (4) with potassium thioacetate in a solvent, such as DMF, at temperatures from 0°C to room temperature can deliver thiol (5). Thiol (5) may also be accessed from chloride (4) via reaction with sodium hydrosulphide hydrate in a solvent, such as DMF, at temperatures from 20°C to 50°C. Reaction of thiol (5) with hydroxylamine-O-sulphonic acid in the presence of a base, such as NaHCC or potassium phosphate tribasic, in a solvent, such as a mixture of THF and H2O, can provide isothiazolone (6).

Ketone (1) can be prepared as described in Scheme B:

(10) (1 )

Scheme B

Carboxylic acid (7) can be converted to Weinreb amide (8) by treatment with an activating agent, such as thionyl chloride, in a solvent, such as DCM, with heating optional, followed by Ν,Ο-dimethylhydroxylamine, in the presence of a base, such as pyridine, at a temperature from 0°C to 20°C. Ketone (1) can be formed from reaction of Weinreb amide (8) with metallated species AM (9) (where M represents a metal, such as lithium or magnesium). The reaction can be carried out in a solvent, such as THF, at temperatures from -60°C to 20°C. Ketone (1) can also be formed from reaction of nitrile (10) with Grignard MgCH2Ri . The reaction can be carried out in a solvent, such as diethyl ether, at temperatures from 0°C to 35°C.

A further subset of compounds of formula I can be prepared as described by Scheme C:

Scheme C

Isothiazolone (11) (prepared by adaptation of the route described in Scheme A) can be converted to (12) on treatment with triisopropylsilyl tnfluoromethanesulphonate in the presence of a base, such as 2,6-lutidine, in a solvent, such as DCM, at room temperature. TIPS protected (12) can be treated with amine (13) (where R represents H, C1-C4 alkyl, Ci- C₄ haloalkyl or the R groups can be taken together with the nitrogen of attachment to form a C3-C10 heterocycle optionally substituted with R₇) in the presence of a Pd(0) catalyst, such as bis(dibenzylideneacetone)palladium(0), a suitable phosphine ligand, such as 2-(di-tert- butylphosphino)biphenyl, and a suitable base, such as sodium t-butoxide or lithium bis(trimethylsilyl)amide, in a solvent, such as toluene or dioxane, at a temperature from 50°C to 100°C to provide amine (14). Amine (14) can also be obtained directly from (11) without the need for protected intermediate (12).

A further subset of com ounds of formula I can be prepared as described by Scheme D:

(11) (15)

Scheme D

Isothiazolone (11) (prepared by adaptation of the route described in Scheme A) can be converted to (15) (where R3 represents an aryl or heteroaryl group, optionally substituted) through cross coupling with arylB(OH)2 or heteroaryl B(OH)2 using standard Suzuki coupling conditions of a Pd catalyst, such as Pd(PP i3)₄, in the presence of a base, such as NaHCC or K2CO3, in a solvent, such as DMF or THF, at temperatures from 20°C to 100°C.

A further subset of compounds of formula I can be prepared as described by Scheme E :

Scheme E

Chloride (4) (prepared as described in Scheme A) can be converted to the isoxazolone (16) by reaction with hydroxyurea in the presence of a base, such as 1 ,8- diazabicyclo[5,4,0]undec-7-ene (DBU), in an alcoholic solvent, such as CH3OH, at room temperature. Following Scheme C and D but using (17) (prepared by adaptation of the route described in Scheme E), isoxazolone (18) and (19) can be prepared, representing a further subset of compounds of formula I

(17) (18) (19)

A further subset of compounds of formula I can be prepared as described by Scheme F:

(20)

Scheme F

Chloride (4) (prepared as described in Scheme A) can be converted to the pyrazolone (20) by reaction with NH2NHR4 in a high boiling solvent, such as NMP, at temperatures from 100°C to 200°C.

Following Scheme C and D but using (21) (prepared by adaptation of the route described in Scheme F), pyrazolone (22) and (23) can be prepared, representing a further subset of compounds of formula I

(21) (22) (23)

A further subset of compounds of formula I can be prepared as described by Scheme G:

(25) (26) (27)

Scheme G

Chloride (4) (prepared as described in Scheme A) can be alkylated with R2LG (where LG represents a leaving group, such as halide or tosyl group). The reaction can be carried out with a base, such as NaH, in a solvent, such as DMF, at temperatures from 0°C to 20°C. Pyridone (24) can be transformed to isothiazolone (25), isoxazolone (26) and pyrazolone (27) by following Scheme A, E and F respectively.

Following Scheme C and D but using (28) (prepared by adaptation of the route described in Scheme G), isothiazolone (29) and (30) can be prepared, representing a further subset of compounds of formula I

(29) (30)

Following Scheme C and D but using (31) (prepared by adaptation of the route described in Scheme G), isoxazolone (32) and (33) can be prepared, representing a further subset of compounds of formula I

(31) (32) (33) Following Scheme C and D but using (34) (prepared by adaptation of the route described in Scheme G), pyrazolone (35) and (36) can be prepared, representing a further subset of compounds of formula I

(34) (35) (36)

A further subset of compounds of formula I can be prepared as described by Scheme H:

(25) (37)

Scheme H

Isothiazolone (25) can be converted to sulphoxide (37) by reaction with an oxidising agent, such as mCPBA, in a solvent, such as DCM, at temperatures from 0°C to 20°C.

Isothiazolone 14) can also be prepared as described by Scheme I:

Scheme I Pyridone (38) (prepared by adaptation of the route described in Scheme A) can be converted to (39), where PG represents an O-linked protecting group, such as PMB. The reaction can be carried out under Mitsunobu conditions using 4-methoxybenzyl alcohol in the presence of PP and DIAD in a solvent, such as THF, at temperatures from 0°C to 20°C. PMB protected (39) can be treated with amine (13) (where R represents H, C1-C4 alkyl, C1-C4 haloalkyi or the R groups can be taken together with the nitrogen of attachment to form a C3-C10 heterocycle optionally substituted with R₇) in the presence of a Pd(0) catalyst, such as bis(dibenzylideneacetone)palladium(0), a suitable phosphine ligand, such as 2-(di-tert-butylphosphino)biphenyl, and a suitable base, such as sodium t-butoxide or CS2CO3, in a solvent, such as toluene or dioxane, at a temperature from 50°C to 100°C to provide amine (40). Deprotection of the remaining PMB group can be effected with HCI in a solvent, such as dioxane, at room temperature. Conversion of the OH in (41) to CI in (42) can be accomplished using a chlorinating agent, such as oxalyl chloride, either neat or in a solvent, such as DCM. Pyridone (42) can be converted to (43), where PG* represents an O- linked protecting group, such as DMB. The reaction can be carried out under Mitsunobu conditions using 2,4-dimethoxybenzyl alcohol in the presence of PP and DIAD in a solvent, such as THF, at temperatures from 0°C to 20°C. Treatment of DMB protected chloride (43) with potassium thioacetate in a solvent, such as DMF, at temperatures from 0°C to room temperature can deliver DMB protected thiol (44). Thiol (44) may also be accessed from DMB protected chloride (43) via reaction with sodium hydrosulphide hydrate in a solvent, such as DMF, at temperatures from 20°C to 50°C. Reaction of DMB protected thiol (44) with hydroxylamine-O-sulphonic acid in the presence of a base, such as NaHC03 or potassium phosphate tribasic, in a solvent, such as a mixture of THF and H2O, can provide DMB protected isothiazolone (45). Removal of the DMB group to yield (14) can be effected with neat trifluoromethanesulphonic acid at room temperature.

General experimental

NMR spectra were obtained on a LC Bruker AV400 using a 5 mm QNP probe (Method A). MS was carried out on a Waters ZQ MS (Method A, B and 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_ Time

H₂0 % ACN %

(min)

0 95 5

4 5 95

4.45 5 95

4.5 95 5

5 STOP

Method B

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

Method C

Column: YMC-Triart C18 50 x 2 mm, 5 uM. Flow rate: 0.8 mL/min. Injection volume 5 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 -6-[4-[3-(Dimethylamino)pyrrolidin-1-yl]phenyl]-7-ethyl-5H-isothiazolo[4,5- clpyridine-3,4-dione 1e

Compound 1a was synthesised as described in WO2013/033240.

Ethyl 4-chloro-6- 4-chlorophenyl)-5-ethyl-2-oxo-1H-pyridine-3-carboxylate 1b

A mixture of ethyl 6-(4-chlorophenyl)-5-ethyl-4-hydroxy-2-oxo-1/-/-pyridine-3-carboxylate 1a (1.04 g, 3.2 mmol) in oxalyl chloride (15.0 mL, 174.9 mmol) was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the residue dissolved in DCM (30 mL) and washed with saturated sodium hydrogen carbonate solution (30 mL). The layers were separated and the aqueous was washed with DCM (2 x 20 mL). The organic layers were combined, dried over Na2S0₄ and concentrated in vacuo to give ethyl 4-chloro-6-(4-chlorophenyl)-5-ethyl-2-oxo-1 H-pyridine-3-carboxylate 1 b as an off white solid which was used without further purification.

LC-MS (Method A) 340.3 [M+H]⁺, RT 2.64 min.

Ethyl 6-(4-chloro henyl)-5-ethyl-2-oxo-4-sulfanyl-1H-pyridine-3-carboxylate 1 c

A mixture of potassium thioacetate (370 mg, 3.2 mmol) in DMF (2 mL) was cooled to 0°C. A solution of ethyl 4-chloro-6-(4-chlorophenyl)-5-ethyl-2-oxo-1 H-pyridine-3-carboxylate 1 b (505 mg, 1.5 mmol) in DMF (2 mL) was added dropwise to the reaction mixture. After addition, the mixture was warmed to room temperature and stirring was continued for 72 h. The mixture was poured onto ice-hbO (100 mL) and extracted with DCM (4 x 100 mL). The organic fraction was washed with saturated sodium hydrogen carbonate solution (3 x 70 mL). The aqueous extractions were combined and acidified dropwise with HCI (37%) to pH 1. The aqueous fraction was extracted with DCM (3 x 50 mL). The organic layers were combined, dried over Na2S0₄ and concentrated in vacuo to give ethyl 6-(4-chlorophenyl)-5- ethyl-2-oxo-4-sulfanyl-1 H-pyridine-3-carboxylate 1c as a brown solid. The crude solid was used immediately to avoid degradation.

LC-MS (Method A) 338.4 [M+H]⁺, RT 2.46 min.

6-(4-Chlorophen l)-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4-dione 1d

To ethyl 6-(4-chlorophenyl)-5-ethyl-2-oxo-4-sulfanyl-1 H-pyridine-3-carboxylate 1c (501 mg, 1.5 mmol) in THF (10 ml_) and H2O (10 ml_) was added potassium phosphate tribasic (2.39 g, 1 1.3 mmol). The mixture was stirred at room temperature for 10 min then hydroxylamine-o-sulfonic acid (922 mg, 8.2 mmol) was added. The reaction was stirred at room temperature for 2 h. The mixture was concentrated in vacuo to remove the THF and the aqueous portion was acidified to pH 2 with HCI (2M). The resulting precipitate was collected, washed with H2O, then diethyl ether, then dried under vacuum to give 6-(4- chlorophenyl)-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4-dione 1d (216 mg, 45.1 % yield) as a brown solid.

LC-MS (Method A) 307.3 [M+H]⁺, RT 2.01 min.

6-[4-[3-(Dimethylamino)pyrrolidin-1-yl]phenyl]-7-ethyl-5H-isothiazolo[4,5-c]pyridi 3,4-dione 1e

A vial was charged with molecular sieves (4A), 6-(4-chlorophenyl)-7-ethyl-5H- isothiazolo[4,5-c]pyridine-3,4-dione 1d (9.8 mg, 0.03 mmol), bis(dibenzylideneacetone) palladium(O) (20 mg, 0.02 mmol), 2-(di-tert-butylphosphino)biphenyl (20 mg, 0.07 mmol) and sodium tert-butoxide (4.3 mg, 0.04 mmol). The vessel was sealed and purged with alternative applications of vacuum and N2, then N,N-dimethylpyrrolidin-3-amine (0.1 ml_, 0.8 mmol) and toluene (0.5 ml_) was added. The vessel was heated, with stirring, to 100°C in an oil bath overnight. The reaction was cooled to room temperature and further small spatula tips of catalyst and ligand were added. The reaction was resealed, purged with alternative applications of vacuum and N2 and heated, with stirring, to 100°C in an oil bath overnight. The reaction mixture was cooled to room temperature and loaded in MeOH onto a pre- equilibrated SCX ion exchange column. The column was washed with MeOH and then eluted with MeOH/NH3 (2%). The appropriate product containing fractions were concentrated in vacuo and the residue was triturated with diethyl ether. The resulting solid was filtered and washed with diethyl ether to give 6-[4-[3-(dimethylamino)pyrrolidin-1- yl]phenyl]-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4-dione 1e (1 mg, 8.1 % yield) as a brown solid.

¹ H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.09 (t, J = 7.4 Hz, 3H), 2.13 (s, 3H), 2.22 (s, 3H), 2.33 (m, 2H), 2.83 (m, 1 H), 3.09 (m, 2H), 3.31 (m, 2H), 3.47 (m, 2H), 6.63 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H); LC-MS (Method A) 385.5 [M+H]⁺, RT 1.08 min.

Example 2 -7-Ethyl-6-[4-(1-methyl-octahvdropyrrolo[3,4-blpyrrol-5-yl)phenyll-5H- isothiazolo[4,5-clpyridine-3,4-dione 2b

[6-(4-Chlorophenyl)-7-ethyl-3-triisopropylsilyloxy-isothiazolo[4,5-c]pyridin

triisopropyl-silane 2a

1d 2a

To 6-(4-chlorophenyl)-7-ethyl-5H-isothiazolo[4,5-c]pyridine-3,4-dione (prepared as described in 1d) (3.45 g, 16.6 mmol) in DCM (80 mL) was added 2,6-lutidine (6.1 mL, 52.5 mmol) and triisopropylsilyl trifluoromethanesulfonate (1 1.7 mL, 43.6 mmol). The reaction mixture was stirred for 6 h, then diluted with saturated sodium bicarbonate solution (50 mL). The aqueous layer was separated and extracted with DCM (50 mL). The combined organics were washed with 1 M HCI (2 x 20 mL), dried over Na2S0₄ and concentrated in vacuo. The resulting oil was purified by flash chromatography eluting with heptane to give [6-(4- chlorophenyl)-7-ethyl-3-triisopropylsilyloxy-isothiazolo[4,5-c]pyridin-4-yl]oxy-triisopropyl- silane 2a (5.95 g, 57% yield) as an oil which slowly crystallized to a white solid.

1 H NMR (Method A) (400 MHz, CDCI3) δ ppm 1.07 (d, J = 7.5 Hz, 18H), 1.15 (d, J = 7.5 Hz, 18H), 1.22 (t, J = 7.6 Hz, 3H), 1.44 (sept, J = 7.5 Hz, 3H), 1.54 (sept, J = 7.5 Hz, 3H), 2.71 (q, J = 7.6 Hz, 2H), 7.36-7.42 (m, 4H). 7-Ethyl-6-[4-(1 -methyl-octahydropyrrolo[3,4-b]pyrrol-5-yl)phenyl]-5H-isothiazolo[4, 5- c]pyridine-3,4-dione 2b

2a 2b

A mixture of [6-(4-chlorophenyl)-7-ethyl-3-triisopropylsilyloxy-isothiazolo[4,5-c]pyridin-4- yl]oxy-triisopropyl-silane 2a (140.6 mg, 0.23 mmol), tris(dibenzylideneacetone)dipalladium(0) (4.1 mg, 2 mol%), 2-(di-tert-butylphosphino)biphenyl (1.3 mg, 2 mol%) and 1- methyloctahydropyrrolo[3,4-b]pyrrole (0.06 mL, 0.46 mmol) in toluene (5 ml_) was deoxygenated by bubbling N2 gas through for 5 min. Sodium tert-butoxide (30.5 mg, 0.32 mmol) was added and the mixture sealed in a tube and heated overnight at 100°C. The reaction mixture was allowed to cool to room temperature and loaded in MeOH onto a pre- equilibrated SCX ion exchange column. The column was washed with MeOH and then eluted with MeOH/NH3 (2%). The eluent was concentrated to give a yellow solid, which was purified by preparative HPLC (Method B) to give 7-ethyl-6-[4-(1-methyl-octahydropyrrolo[3,4- b]pyrrol-5-yl)phenyl]-5H-isothiazolo[4,5-c]pyridine-3,4-dione 2b (12 mg, 12%) as its formate salt as a yellow solid.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.04 (t, J = 7.5 Hz, 3H), 1.80-1.91 (m, 1 H), 2.39 (q, J = 7.5 Hz, 2H), 2.34-2.44 (m, 1 H), 2.79 (s, 3H), 2.90-2.99 (m, 1 H), 3.12-3.25 (m, 3H), 3.32-3.39 (m, 1 H), 3.43-3.51 (m, 1 H), 3.75-3.87 (m, 2H), 6.72 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 8.33 (s, 1 H); LC-MS (Method B) 397.3 [M+H]⁺, RT 1.08 min.

The test solutions for compound 2b used in Examples 21 and 22 were prepared by dissolving the formate salt in the appropriate solvent.

Exam pie 3 -7-Eth yl-6-[4-f3-(meth ylamino)pyrrolidin- 1 - yllphen vn-5H-isothiazolof4, 5- clpyridine-3,4-dione 3a

A mixture of [6-(4-chlorophenyl)-7-ethyl-3-triisopropylsilyloxy-isothiazolo[4,5-c]pyridin-4- yl]oxy-triisopropyl-silane (prepared as described in 2a) (140.6 mg, 0.23 mmol), sodium tert- butoxide (30.5 mg, 0.32 mmol), chloro(2-dicyclohexylphosphino-2',6'-di-i-propoxy-1 ,1 '- biphenyl)(2-amino-1 , T-bi-phenyl-2-yl)palladium(ll) (1.7 mg) and 2-dicyclohexylphosphino- 2',6'-diisopropoxybiphenyl (1.1 mg) was sealed in a tube under an atmosphere of N2. 3-(N- Boc-N-methylamino)pyrrolidine (0.06 mL, 0.27 mmol) in dry THF (0.25 ml_) was added and the resulting solution deoxgenated by bubbling N2 gas through for 2 min. The solution was then heated at 50°C for 3 h. The reaction mixture was allowed to cool to room temperature and loaded in MeOH onto a pre-equilibrated SCX ion exchange column. The column was washed with MeOH and then eluted with MeOH/NH3 (2%). The eluent was allowed to stand overnight yielding a precipitate, which was filtered and dried to afford 7-ethyl-6-[4-[3- (methylamino)pyrrolidin-1-yl]phenyl]-5H-isothiazolo[4,5-c]pyridine-3,4-dione 3a (2 mg, 2%) as a yellow solid. The eluent was further concentrated and purified by preparative HPLC (Method A) to yield a further amount of 3a (4 mg, 4%).

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.09 (t, J = 7.5 Hz, 3H), 1.80-1.86 (m, 1 H), 2.10-2.15 (m, 1 H), 2.34 (s, 3H), 2.42 (q, J = 7.5 Hz, 2H), 2.66-2.69 (m, 1 H), 3.05-3.10 (m, 1 H), 3.32-3.40 (m, 2H), 3.44-3.50 (m, 1 H), 6.60 (d, J = 8.7 Hz, 2H), 7.26 (d, J = 8.7 Hz, 2H); LC-MS (Method B) 371.5 [M+H]⁺, RT 1.32 min.

Example 4 -7-Ethyl-6-f4-(1-methyl-octahvdro-1H-pyrrolof3A-bTpyndin-6-yl)phenvn-5H- isothiazolof4,5-cTpyridine-3,4-dione 4a

2a 4a

Following the procedures of Example 2b, but using 1-methyloctahydro-1 H-pyrrolo[3,4- b]pyridine as the amine, 7-ethyl-6-[4-(1-methyl-octahydro-1 H-pyrrolo[3,4-b]pyridin-6- yl)phenyl]-5H-isothiazolo[4,5-c]pyridine-3,4-dione 4a was synthesised as its formate salt as a yellow solid.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.04 (t, J = 7.4 Hz, 3H), 1.55-1.85 (m, 5H), 2.35-2.62 (m, 8H), 2.85-2.95 (m, 1 H), 3.15-3.35 (m, 2H), 3.55-3.70 (m, 1 H), 6.61 (br s, 2H), 7.21 (br s, 2H), 8.39 (s, 1 H); LC-MS (Method B) 411.4 [M+H]⁺, RT 1.34 min. The test solutions for compound 4a used in Examples 21 and 22 were prepared by dissolving the formate salt in the appropriate solvent.

Exa m I e 5 -7-Eth yl-6-[4-f (3R)-3-h ydroxyp yrrolidin-1 - yllphen vn-5H-isothiazolof4, 5- clpyridine-3,4-dione 5a

Following the procedures of Example 3a, but using (R)-3-pyrrolidinol as the amine and lithium bis(trimethylsilyl)amide as a base, 7-ethyl-6-[4-[(3R)-3-hydroxypyrolidin-1-yl]phenyl]- 5H-isothiazolo[4,5-c]pyridine-3,4-dione 5a was synthesised as a brown solid.

1 H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.05 (t, J = 7.5 Hz, 3H), 1.90-2.01 (m, 1 H), 2.03-2.14 (m, 1 H), 2.42 (q, J = 7.5 Hz, 2H), 3.02-3.48 (m, 4H), 4.43-4.49 (m, 1 H), 6.59 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 8.7 Hz, 2H); LC-MS (Method B) 358.4 [M+H]⁺, RT 1.70 min.

Example 6 -7-Ethyl-6-(1-methylindazol-5-yl)-5H-isothiazolof4,5-cTpyridine-3,4-dione 6e

1-(1 -Methylindazol-5- l)butan- 1 -one 6a

To a solution of 5-bromo-1-methylindazole (7.5 g, 35.5 mmol) in THF (200 mL) at -78°C was added n-butyllithium solution (26.6 mL, 42.6 mmol) drop wise over a period of 15 min. On completion the mixture was stirred for a further 30 min at -78°C. N-methoxy-N-methyl- butanamide (5.59 g, 42.6 mmol) in THF (50 mL) was then added and the resulting mixture stirred for a further 10 min, allowed to warm to ambient temperature and stirred for 4 h. The reaction was quenched with saturated ammonium chloride and extracted with diethyl ether (3x 50 mL). The combined organic extracts were dried over Na2S0₄ and concentrated in vacuo. The crude product was purified by flash chromatography using EtOAc in petroleum ether (40-60) (0-15% gradient) to give 1-(1- methylindazol-5-yl)butan-1-one 6a (1.99 g, 27% yield).

LC-MS (Method B) 203.4 [M+H]⁺, RT 2.12 min. Ethyl 5-ethyl-4-hydrox -6-(1-methylindazol-5-yl)-2-oxo-1H-pyridine-3-carboxylate 6b

A solution of 1-(1-methylindazol-5-yl)butan-1-one 6a (1.26 g, 6.23 mmol) and tert-butylamine (2.6 mL, 24.9 mmol) in DCM (100 mL) was cooled to 0°C. A solution of titanium(IV) chloride in DCM (1 M) (4.0 mL, 4.0 mmol) was added dropwise over 30 min. The reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was diluted with DCM (200 mL), filtered through celite to remove titanium dioxide, and then partitioned with aqueous sodium bicarbonate solution (50 mL). After vigourous shaking, the organic phase was separated, dried over Na2S0₄ and concentrated in vacuo to give N-tert-butyl-1- (1-methylindazol-5-yl)butan-1-imine as a yellow oil (5.21 g), which was used crude in the next step.

To N-tert-butyl-1-(1-methylindazol-5-yl)butan-1-imine (4.1 1 g, 15.9 mmol) in diphenyl ether (75 mL) was added triethyl methanetricarboxylate (4.13 mL, 19.48 mmol). The resulting solution was heated at 160°C for 2 h, allowed to cool to room temperature and then stirred with petroleum ether (40-60) (225 ml) to precipitate a solid. The mixture was further diluted with diethyl ether and filtered. The solid collected was washed with diethyl ether, CH3OH followed by heptane to yield ethyl 5-ethyl-4-hydroxy-6-(1-methylindazol-5-yl)-2-oxo-1 H- pyridine-3-carboxylate 6b (2.35 g, 43% yield) as a white solid.

LC-MS (Method B) 342.69 [M+H]⁺, RT 2.31 min.

Ethyl 4-chloro-5-ethyl-6-(1-methylindazol-5-yl)-2-oxo-1H-pyridine-3-carboxylate 6c

6b

Following the procedures of Example 1 b, but using 5-ethyl-4-hydroxy-6-(1-methylindazol-5- yl)-2-oxo-1 H-pyridine-3-carboxylate 6b, ethyl 4-chloro-5-ethyl-6-(1-methylindazol-5-yl)-2- oxo-1 H-pyridine-3-carboxylate 6c was synthesised as a pink solid, which was used without further purification in the next step.

LC-MS (Method B) 360.4 [M+H]⁺, RT 2.24 min.

Ethyl 5-ethyl-6-(1-methylindazol-5-yl)-2-oxo-4-sulfanyl-1H-pyridine-3-carboxylate 6d

A mixture of ethyl 4-chloro-5-ethyl-6-(1-methylindazol-5-yl)-2-oxo-1 H-pyridine-3-carboxylate 6c (86.1 mg, 0.24 mmol), potassium thioacetate (120.2 mg, 1.05 mmol) and sodium hydrosulphide hydrate (40.2 mg, 0.72 mmol) in DMF (2 ml_) was stirred at 80°C for 2 h. The reaction mixture was filtered and subjected to reverse phase chromatography (RediSep Rf Revesed Phase C18 Column) using ACN in H2O (5-60% gradient containing 0.1 % formic acid). The appropriate fractions were concentrated to give ethyl 5-ethyl-6-(1-methylindazol- 5-yl)-2-oxo-4-sulfanyl-1 H-pyridine-3-carboxylate 6d (56 mg, 65% yield) as a brown solid. LC-MS (Method B) 358.4 [M+H]⁺, RT 2.08 min.

7-Ethyl-6-( 1 -meth lindazol-5-yl)-5H-isothiazolo[4, 5-c]pyridine-3,4-dione 6e

Following the procedures of Example 1d, but using ethyl 5-ethyl-6-(1-methylindazol-5-yl)-2- oxo-4-sulfanyl-1 H-pyridine-3-carboxylate 6d, 7-ethyl-6-(1-methylindazol-5-yl)-5H- isothiazolo[4,5-c]pyridine-3,4-dione 6e was synthesised as a white solid.

1 H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.04 (t, 3H), 2.36 (s, 2H), 4.09 (s, 3H), 7.45 (d, 1 H), 7.75 (d, 1 H), 7.87 (s, 1 H), 2.13 (s, 1 H); LC-MS (Method B) 325.5 [M+H]⁺, RT 9.12 min.

Example 7 -6-f4-f3-(Dimethylamino)pyrrolidin-1-vnphenvn-7-ethyl-3-hydroxy-1- methyl-5H-pyrazolof4,3-cTpyridin-4-one 7d

Ethyl-6-(4-chlorophenyl)-5-ethyl-2,4-bis[(4-methoxyphenyl)methoxy]pyridine-3- carboxylate 7a

1a 7a

To a cold solution of ethyl-6-(4-chlorophenyl)-5-ethyl-4-hydroxy-2-oxo-1 /-/-pyridine-3- carboxylate 1a (485 mg, 1.51 mmol), para-methoxybenzyl alcohol (0.45 ml_, 3.6 mmol) and PPh₃ (0.95 g, 3.6 mmol) in THF (10 ml_) was added dropwise DIAD (2.36 ml_, 12.0 mmol). The mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was evaporated to dryness under reduced pressure and the crude product was purified by flash chromatography using petroleum ether (40-60) in EtOAc (0-20% gradient) to furnish ethyl-6-(4-chlorophenyl)-5-ethyl-2,4-bis[(4-methoxyphenyl)methoxy]pyridine-3-carboxylate 7a (420 mg, 50%) as a pale yellow oil.

LC-MS (Method B) 584.4 [M+Na]⁺, RT 4.10 min.

Ethyl-6-[4-[3-(dimethylamino)pyrrolidin- 1 -yl]phenyl]-5-ethyl-4-hydroxy-2-oxo- 1 H- pyridine-3-carboxylate 7b

A degassed mixture of ethyl-6-(4-chlorophenyl)-5-ethyl-2,4-bis[(4- methoxyphenyl)methoxy]pyridine-3-carboxylate 7a (522 mg, 0.930 mmol), CS2CO3 (908 mg, 2.79 mmol), 3-(dimethylamino)pyrrolidine (212 mg, 1.86 mmol) and 2-(2'-di-tert- butylphosphine)biphenylpalladium(ll)acetate (43 mg, 0.090 mmol) in toluene (10 ml_) was heated at 90°C for 16 h (LCMS indicated approx 1 : 1 ratio of product and starting material). Further 2-(2'-di-tert-butylphosphine)biphenylpalladium(ll)acetate (43 mg, 0.09 mmol) was added, the mixture was again degassed and heated at 90°C for a further 23 h. After cooling to room temperature the mixture was filtered through a pad of silica, washed with CH₂Cl2/MeOH/NEt3 100: 10: 1 (2 * 50 ml_) and the solvent was concentrated in vacuo to furnish a yellow oil (660 mg). 4M HCI-dioxane (10 mL) was added and the mixture was stirred at room temperature for 16 h. After removing the solvent under reduced pressure the crude product was dissolved in MeOH and loaded onto a pre-equilibrated SCX ion exchange column. The column was washed with MeOH and then eluted with MeOH/NH3 (2%). The appropriate product containing fractions were concentrated in vacuo to furnish ethyl-6-[4-[3- (dimethylamino)pyrrolidin-1-yl]phenyl]-5-ethyl-4-hydroxy-2-oxo-1 H-pyridine-3-carboxylate 7b (333 mg, 90%) as a white solid.

LC-MS (Method B) 400.5 [M+H]⁺, RT 1.97 min.

Ethyl 4-chloro-6-[4-[3-(dimethylamino)pyrrolidin-1-yl]phenyl]-5-ethyl-2-oxo-1H- pyridine-3-carbox late 7c

7b

Following the procedures of Example 1 b, but using ethyl-6-[4-[3-(dimethylamino)pyrrolidin- 1-yl]phenyl]-5-ethyl-4-hydroxy-2-oxo-1 H-pyridine-3-carboxylate 7b, ethyl 4-chloro-6-[4-[3- (dimethylamino)pyrrolidin-1-yl]phenyl]-5-ethyl-2-oxo-1 H-pyridine-3-carboxylate 7c was synthesised as a yellow solid, which was used without further purification in the next step. LC-MS (Method B) 418.5 [M+H]⁺, RT 2.59 min.

6-[4-[3-(Dimethylamino)pyrrolidin- 1 -yl]phenyl]-7-ethyl-3-hydroxy- 1 -methylSH- pyrazolo[4, 3-c ridin-4-one 7d

A mixture of ethyl 4-chloro-6-[4-[3-(dimethylamino)pyrrolidin-1-yl]phenyl]-5-ethyl-2-oxo-1 H- pyridine-3-carboxylate 7c (51 mg, 0.12 mmol) and methylhydrazine (0.03 mL, 0.6 mmol) in NMP (0.50 mL) was heated at 200°C for 30 min under microwave irradiation. The mixture was diluted with H2O (5 mL) and extracted with EtOAc (2 ^χ 20 mL). The aqueous phase was concentrated under reduced pressure and the resulting product dissolved in MeOH and loaded onto a pre-equilibrated SCX ion exchange column. The column was washed with MeOH and then eluted with MeOH/NH3 (2%) to furnish a white solid, which was further purified by preparative HPLC (Method B) to give the 6-[4-[3-(dimethylamino)pyrrolidin-1- yl]phenyl]-7-ethyl-3-hydroxy-1-methyl-5H-pyrazolo[4,3-c]pyridin-4-one 7d (3.2 mg, 5%) as its diformate salt as a white solid.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.09 (t, J = 7.3, 3H), 2.33 (br s, 1 H), 2.51 (br s, 1 H), 2.62 (q, J = 7.3, 2H), 2.81 (br s, 6H), 3.40 (m, 1 H), 3.62 (br s, 2H), 3.83-3.67 (m, 2H), 3.95 (s, 3H), 6.75 (br s, 2H), 7.26 (d, J = 6.6, 2H), 8.33 (s, 2H); LC-MS (Method B) 382.5 [M+H]⁺, RT 1.35 min.

The test solutions for compound 7d used in Examples 21 and 22 were prepared by dissolving the diformate salt in the appropriate solvent.

By adaptation of the methods previously described the following compounds were synthesised

Example 8 -6-i4-ii3R)-3-iDimethylamino)pyrrolidin-1-vnphenvn-7-ethyl-5H- isothiazolo[4,5-clpyricline-3,4-clione 8a

By adaptation of the method described in Example 2 compound 8a was obtained as a pale brown solid as its formate salt.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.05 (t, J = 7.6 Hz, 3H), 1.95-2.05 (m, 1 H), 2.27-2.37 (m, 1 H), 2.43 (q, J = 7.6 Hz, 2H), 2.51 (s, 6H), 3.15-3.35 (m, 3H), 3.45-3.53 (m, 1 H), 3.53-3.63 (m, 1 H), 6.64 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 8.31 (s, 1 H); LC- MS (Method C) 385.5 [M+H]⁺, RT 1.40 min.

Example 9 -6-[4-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]phenyl]-7-ethyl-5H- isothiazolo[4,5-clpyricline-3,4-clione 9a

By adaptation of the method described in Example 2 compound 9a was obtained as a pale brown solid as its formate salt.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.05 (t, J = 7.6 Hz, 3H), 1.95-2.05 (m, 1 H), 2.27-2.37 (m, 1 H), 2.43 (q, J = 7.6 Hz, 2H), 2.51 (s, 6H), 3.15-3.35 (m, 3H), 3.45-3.53 (m, 1 H), 3.53-3.63 (m, 1 H), 6.64 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 8.31 (s, 1 H); LC- MS (Method C) 385.4 [M+H]⁺, RT 1.34 min.

Example 10 -6-f4-f3-Aminopyrrolidin-1-vnphenvn-7-ethyl-5H-isothiazolof4,5- clpyridine-3,4-dione 10a

By adaptation of the method described in Example 3 compound 10a was obtained as a pale brown solid.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.04 (t, J = 7.5 Hz, 3H), 1.90-2.14 (m, 1 H), 2.40 (m, 3H), 3.30-3.68 (m, 4H), 3.99 (m, 1 H), 6.58 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H); LC-MS (Method C) 356.4 [M+H]⁺, RT 1.36 min.

Example 11 -7-Ethyl-6-[4-[3-[(methylamino)methyllpyrrolidin-1-yllphenyll-5H- isothiazolo[4,5-clpyridine-3,4-dione 11a

By adaptation of the method described in Example 3 compound 11 a was obtained as a pale brown solid as its formate salt.

¹ H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.09 (t, J = 7.5 Hz, 3H), 1.26-1.40 (m, 2H), 1.71-1.81 (m, 1 H), 2.08-2.18 (m, 1 H), 2.36 (s, 3H), 2.45 (q, J = 7.5 Hz, 2H), 2.57-2.62 (m, 1 H), 3.04-3.09 (m, 1 H), 3.26-3.41 (m, 2H), 3.42-3.48 (m, 1 H), 6.62 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H), 8.21 (s, 1 H); LC-MS (Method C) 385.5 [M+H]⁺, RT 1.46 min.

Example 12 -7-Ethyl-6-f4-f(3S)-3-hvdroxypyrrolidin-1-yl]phenyl]-5H-isothiazolof4,5- clpyridine-3,4-dione 12a

By adaptation of the method described in Example 2 compound 12a was obtained as a yellow solid.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.06 (t, J = 7.6 Hz, 3H), 1.90-2.01 (m, 1 H), 2.03-2.14 (m, 1 H), 2.45 (q, J = 7.6 Hz, 2H), 3.02-3.48 (m, 4H), 4.43-4.49 (m, 1 H), 6.60 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 8.7 Hz, 2H); LC-MS (Method C) 358.4 [M+H]⁺ , RT 1.70 min.

Example 13 -7-Ethyl-6-i4-ioctahvdro-1H-pyrroloi3A-blpyridin-6-yl)phenvn-5H- isothiazolo[4,5-clpyricline-3,4-clione 13a

By adaptation of the method described in Example 3 compound 13a was obtained as a light brown solid. ¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 0.93 (t, J = 7.4 Hz, 3H), 1.45- 1.85 (m, 5H), 2.41 (q, J = 7.5 Hz, 2H), 2.95-3.45 (m, 7H), 6.55 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H); LC-MS (Method C) 397.5 [M+H]⁺, RT 1.49 min.

Example 14 -7-Ethyl-6-f3-f3-(methylamino)pyrrolidin-1-vnphenvn-5H-isothiazolof4,5- clpyridine-3,4-dione 14a

14a

By adaptation of the method described in Example 3 compound 14a was obtained as a pale brown solid as its HCI salt.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.05 (t, J = 7.5 Hz, 3H), 2.12-2.20 (m, 1 H), 2.40-2.49 (m, 4H), 2.70 (s, 3H), 3.47-3.59 (m, 4H), 3.89 (br s, 1 H), 6.63 (br s, 1 H), 6.74-6.76 (m, 2H), 7.30 (dd, J = 7.0, 7.5 Hz, 1 H); LC-MS (Method C) 371.4 [M+H]⁺, RT 1.48 min. Example 15 -7-Ethyl-6-[3-[3-[(methylamino)methyllpyrrolidin-1-yllphenyll-5H- isothiazolo[4,5-clpyridine-3,4-dione 15a

15a

By adaptation of the method described in Example 3 compound 15a was obtained as a pale yellow solid.

¹ H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.07 (t, J = 7.5 Hz, 3H), 2.05-2.12 (m, 1 H), 2.29 (s, 3H), 2.39-2.45 (m, 4H), 3.14-3.18 (m, 6H), 6.50 (br s, 1 H), 6.56-6.62 (m, 2H), 7.22 (dd, J = 7.0, 7.5 Hz, 1 H); LC-MS (Method C) 385.5 [M+H]⁺, RT 1.61 min.

Example 16 -6-[3-[3-iDimethylamino)pyrrolidin-1-yllphenyll-7-ethyl-5H- isothiazolo[4,5-clpyricline-3,4-clione 16a

16a

By adaptation of the method described in Example 2 compound 16a was obtained as a colourless solid as its formate salt.

¹ H NMR (Method A) (400 MHz, CD₃OD) δ ppm 1.15 (t, J = 7.5 Hz, 3H), 2.06-2.12 (m, 1 H), 2.39-2.46 (m, 2H), 2.51 (q, J = 7.5 Hz, 2H), 2.60 (s, 6H), 3.35-3.43 (m, 2H), 3.54-3.68 (m, 2H), 6.67 (br s, 1 H), 6.76-6.79 (m, 2H), 7.36 (dd, J = 7.0, 7.5 Hz, 1 H), 8.31 (s, 1 H); LC-MS (Method C) 385.5 [M+H]⁺, RT 1.46 min.

Example 17 -7-ethyl-3-hvdroxy-1-methyl-6-(4-[3-(methylamino)pyrrolidin-1-yllphenyl)- 5H-pyrazolo[4,3-clpyridin-4-one 17a

By adaptation of the method described in Example 7 compound 17a was obtained as a beige solid.

¹ H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.00 (t, J = 7.4 Hz, 3H), 1.6 (m, 1 H), 1.85 (m, 2H), 1.98 (m, 1 H), 2.1 1 (m, 2H), 2.32 (s 3H), 3.05 (m, 2H), 3.45 (m, 2H), 3.82 (s, 3H), 6.55 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 10.4 (bs, 2H); LC-MS (Method C) 368.5 [M+H]⁺, RT 1.26 min.

Example 18 -6-f4-(3,3-difluoropyrroliclin-1-yl)phenvn-7-ethyl-5H- isothiazolo[4,5- clpyridine-3,4-dione 18a

By adaptation of the method described in Example 2 compound 18a was obtained as a brown-green solid.

¹ H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.07 (t, J = 7.5 Hz, 3H), 2.39 (q, J = 7.6 Hz, 3H), 2.45-2.63 (m, 2H), 3.55 (t, J = 7.2 Hz, 3H), 3.76 (q, J = 13.4 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.5 Hz, 2H); LC-MS (Method C) 378.3 [M+H]⁺, RT 2.70 min.

Example 19 -7-Ethyl-6-[4-[4-imethylamino)piperidin-1-yllphenyll-5H-isothiazolo[4,5- clpyridine-3,4-dione 19a

By adaptation of the method described in Example 3 compound 19a was obtained as a cream solid as its formate salt.

¹ H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.08 (t, J = 7.5 Hz, 3H), 1.30-1.45 (m, 2H), 1.89-1.97 (m, 2H), 2.37 (s, 3H), 2.37-2.43 (m, 1 H), 2.46-2.56 (m, 2H), 2.79-2.88 (m, 2H), 3.75-3.83 (m, 2H), 7.02 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 8.28 (s, 1 H); LC-MS (Method C) 385.4 [M+H]⁺, RT 1.48 min.

Example 20 -6-i4-i4-iDimethylamino)pipendin-1-vnphenvn-7-ethyl-5H-isothiazoloi4,5- clpyridine-3,4-dione 20a

By adaptation of the method described in Example 2 compound 20a was obtained as a pale yellow solid.

¹ H NMR (Method A) (400 MHz, DMSO-cfe) δ ppm 1.07 (t, J = 1.5 Hz, 3H), 1.44-1.47 (m, 2H), 1.84-1.87 (m, 2H), 2.22 (s, 6H), 2.39 (q, J = 7.5 Hz, 2H), 2.66-2.69 (m, 1 H), 2.73-2.79 (m, 2H), 3.82-3.85 (m, 2H), 7.02 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H); LC-MS (Method C) 399.4 [M+H]⁺, RT 1.46 min.

Example 21 - Antibacterial susceptibility testing

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). The broth dilution method involves a two-fold serial dilution of compounds in 96-well microtitre plates, giving a final concentration range of 0.25-128 μg/mL and a maximum final concentration of 1 % DMSO. The bacterial strains tested include the gram-positive strain Staphylococcus aureus ATCC 29213 and the Gram- negative strains Escherichia coli ATCC 25922, Acinetobacter baumannii NCTC 13420, Enterobacter cloacae NCTC 13406, Haemophilus influenzae ATCC 49247, Klebsiella pneumoniae ATCC 700603, Klebsiella pneumoniae NCTC 13443, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa NCTC 13437, Neisseria gonorrhoeae ATCC 49226 and Neisseria meningitidis ATCC 13090. Strains are grown in cation-adjusted Muller-Hinton broth or on cation-adjusted Muller-Hinton agar at 37°C in an ambient atmosphere with the following exceptions: H. influenzae MIC was carried out in HTM medium, N. meningitidis MIC was carried out in cation-adjusted Muller-Hinton supplemented with 5% lysed horse blood and N. gonorrhoeae MIC was determined in 24-well microtitre plates in GC agar. The MIC is determined as the lowest concentration of compound that inhibits growth following a 16-20 hour incubation period. The data reported correspond to the modes of three independent experiments. The results are set out in Table 1. Table 1

For MICs: A represents a concentration of 1 ^g/mL or lower; B represents a concentration of from 1.1 to 8 μςΛηί; C represents a concentration of from 9 ^g/mL to 127 μςΛηί; and D represents a concentration of 128 ^g/mL or higher.

The tested compounds of the invention thus show good activity against a range of bacterial pathogens, including some strains which have shown resistance to known antibacterial drug molecules. The compounds show good activity against both Gram-positive bacteria (e.g. S. aureus) and Gram-negative bacteria (e.g. P. aeruginosa).

Example 22 - Human cell viability assay

Compounds are assessed for potential non-specific cytotoxic effects against a human hepatic cell line (HepG2 ATCC HB-8065). 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% foetal bovine serum (FBS) and 1 mM sodium pyruvate. After 24 hours 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% CO2 for a further 24 hours, 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). The results are set out in Table 2.

Table 2

For IC50S: A represents a concentration of 1 μg/mL or lower; B represents a concentration of from 1.1 to 8 μg/mL; C represents a concentration of from 9 μg/mL to 127 μg/mL; and D represents a concentration of 128 μg/mL or higher.

For comparison, a known isothiazolinone antibacterial compound has an IC50 of 16 μg/mL (compound 4 of Kim et al; J. Med. Chem.; 2011, 54, 3268-3282). The tested compounds of the invention therefore demonstrate a very favourable cytotoxicity profile.

Claims

Claims rmula (I), or a pharmaceutically acceptable salt thereof:

R¹ is independently selected from: H, halogen, cyano, NR⁵R⁵; OR⁶; C1-C6 alkyl, C1-C6 haloalkyi, -(CZ₂)_n-C3-C6 cycloalkyi, -(CZ₂)_n-C3-C6 halocycloalkyi; -(CZ₂)_n-phenyl; -(CZ₂)_n- heteroaryl, C₂-C6 alkenyl and C₂-C6 alkynyl;

R² and R⁴ are each independently selected from: H; C1-C6 alkyl, C1-C6 haloalkyi, -(CZ₂)_n-C3- C6 cycloalkyi, -(CZ₂)_n-C3-C6 halocycloalkyi and -(CZ₂)_n-phenyl;

R³ is independently selected from the group consisting of: H, -(CZ₂)_n-C3-Cio heterocycloalkyi, -(CZ₂)_n-phenyl, -(CZ₂)_n-heteroaryl, -(CZ₂)_n-C₃-Cio cycloalkyi, -(CZ₂)_n-NR⁵R⁵;

Z is independently at each occurrence selected from H, Me, CF3 or F; and n is an integer independently selected at each occurrence from 0, 1 , 2 and 3; and wherein each of the aforementioned aryl, heteroaryl, C3-C10 heterocycloalkyi or C3-C10 cycloalkyi groups are monocyclic or bicyclic; and each of the aforementioned alkyl, haloalkyi, cycloalkyi, halocycloalkyi, heterocycloalkyi, aryl (e.g. phenyl) and heteroaryl groups, are optionally substituted, where chemically possible, by 1 to 5 substituents which 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^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, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, C1-C4 haloalkyl, 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.

2. A compound of claim 1 , wherein X is S.

3. A compound of claim 1 , wherein X is NH.

4. A compound of any one of claims 1 to 3, wherein A-R³ is

wherein γ^ΐ γ₂ γ³ _anc| γ_{4 are eac}|_{1 se}|_ected from carbon or nitrogen; R^x is a group selected from halo, nitro, cyano, NR^aR^a, NR^aS(0)₂R^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, C1-C4 alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, C1-C4 haloalkyl and CR^bR^bNR^aR^a; and p is an integer from 1 to 4.

A compound of claim 4, wherein A-R³ is

A compound of claim 4, wherein A-R³ is

7. A compound of any preceding claim, wherein R³ is C3-C10 N-heterocycloalkyl, wherein the N-heterocycloalkyl group is monocyclic or bicyclic and comprises from 1 to 3 nitrogen atoms in the heterocyclic ring system; and wherein R³ is attached to the rest of the molecule via a carbon or one nitrogen in the ring system.

8. A compound of any of claims 1 to 6, wherein R³ is

wherein R⁷ 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; or wherein two R⁷ groups together with the carbon or carbons to which they are attached form a 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl ring.

9. A compound of any of claims 1 to 6, wherein R³ is -(CZ2)_n-NR⁵R⁵.

10. A compound of any one of claims 1 to 3, wherein A-R³ is

, wherein

Y⁵ is independently selected from nitrogen and carbon and R¹² is independently selected from H, Ci-C₄ alkyl and Ci-C₄ haloalkyl.

11. A compound of claim 10, wherein R³ is H.

12. A compound of any preceding claim, wherein R¹ is independently selected from Ci- C6 alkyl, C1-C6 haloalkyl, -(CZ2)_n-C3-C6 cycloalkyl and -(CZ2)_n-C3-C6 halocycloalkyl.

13. A compound of claim 12, wherein R¹ is ethyl or cyclopropyl.

14. A compound of any preceding claim, wherein R² is H.

15. A compound of any of claims 1 to 14, for medical use.

16. A compound of any of claims 1 to 14, for use in treating bacterial infections.

17. A compound of any of claims 1 to 14 for the use of claim 16, wherein the bacterial infection is caused by a resistant strain of a Gram negative bacteria.

18. A pharmaceutical formulation comprising a compound of any of claims 1 to 14 and a pharmaceutically acceptable excipient. a compound of formula (X),

wherein A is independently aryl or heteroaryl;

X is independently selected from S, SO, S0₂, O, NR⁴;

R² and R⁴ are each independently selected from H; C1-C6 alkyl, C1-C6 haloalkyi, -(CZ₂)_n-C3- C6 cycloalkyl, -(CZ₂)_n-C3-C6 halocycloalkyl and -(CZ₂)_n-phenyl;

Xlb

wherein L¹ is independently selected from halogen, boronic acid and boronate ester; wherein P¹ and P² are each independently selected from: substituted or unsubstituted benzyl group; Ci-C₄-alkyl or substituted or unsubstituted benzyl carbonate group; or SiR⁹3, wherein R⁹ is independently selected from phenyl or Ci-C₄ alkyl;

with a compound of formula XII:

R³-L²XM;