WO2014049356A1 - Erythromycin ketolide derivatives bearing c-10 modifications - Google Patents

Abstract

The invention provides novel antibiotics, pharmaceutical compositions containing them and their medical use, for example in the treatment or prevention of microbial infections. Provided are compounds of Formula I, pharmaceutically acceptable salts and prodrugs thereof:

Description

ERYTHROMYCIN KETOLIDE DERIVATIVES BEARING C-10 MODIFICATIONS

This invention relates to novel ketolide antibiotics, their preparation, pharmaceutical compositions containing them, and their medical use. More specifically, it relates to analogues of cethromycin which have been found to possess one or more desirable properties compared to the parent molecule.

Microbial infections give rise to various diseases depending on the site of infection and the nature of the microorganism. In some cases, infection can lead to death, especially in the young, the elderly and in immuno-compromised individuals. For example, bacterial respiratory infections can be acquired in the community or in hospital and typically result in inflammation of the lungs (pneumonia), leading to difficulty in breathing, fever, chest pains, and cough. Such infections are primarily treated with antibiotics. However, the incidence of infections caused by antibiotic- resistant microorganisms is on the increase. Antibiotic-resistant microorganisms cannot readily be treated with conventional antibiotics and so pose a particular risk. Among the microorganisms known to have developed resistance to certain antibiotics are bacterial strains such as Streptococcus pneumoniae. The severity of such infections often necessitates immediate medical intervention and the treatment of patients by intravenous administration of antibiotics.

International patent application No. WO 98/09978, the content of which is incorporated herein by reference, discloses a family of related ketolide compounds possessing a side-chain at position C6 of the ketolide scaffold, which compounds show potent activity against macrolide-resistant strains. Among the compounds of WO 98/09978 is cethromycin (Example 263), also known as ABT-773, which is a ketolide antibiotic that has shown promise in the treatment of microbial infections, for instance respiratory tract infections caused by macrolide-resistant

microorganisms. Cethromycin has the following structure:

It shares a similar macrolide core structure to the antibiotic erythromycin, but differs notably in the presence of a heteroaryl-containing side-chain at the C-6 hydroxyl group, in having an oxazolidinone moiety at the C-11 and C-12 positions, and in the presence of a ketone at C-3 in place of 3-O-cladinose. The C-10 position of cethromycin is substituted by hydrogen and methyl groups, a feature which is common to almost all known 14-membered macrolides and ketolides, including those of WO 98/09978.

Whilst cethromycin may be used in the treatment of macrolide-resistant infections, it has certain drawbacks. Most notably, it possesses a low solubility in aqueous solutions, which makes its incorporation in intravenous formulations problematic and generally limits cethromycin to oral formulations. In addition, as for the vast majority of the antibiotics of the macrolide/ketolide class, cethromycin exhibits strong inhibition of cytochrome P450 3 A4 (CYP3 A4), an important metabolic enzyme involved in the oxidation of xenobiotics in vivo. As a result of its strong CYP3 A4 inhibition, cethromycin displays drug-drug interactions which cause problems during concomitant administration with other drugs metabolised by cytochrome P₄₅₀ enzymes, i.e. CYP3A4 substrates. It has been estimated that CYP3 A4 metabolises about half of all drugs on the market and so the incompatibility associated with co-administration of cethromycin with other drugs is a significant problem.

There is an acute need to develop new compounds which address these problems, in particular compounds with adequate solubility in aqueous media to enable their formulation for intravenous administration, and/or that have limited ability to inhibit CYP3 A4 in order generally to improve the safety of drug coadministration. Such new compounds should also display adequate antimicrobial properties, e.g. at least a roughly equivalent antibiotic activity to cethromycin against susceptible and/or resistant strains such as S. pneumoniae. The present invention fulfils this need.

The present inventors have surprisingly discovered that certain ketolides having particular functional groups at the C-10 position, in place of the methyl group of for example cethromycin, possess improved properties over the parent molecule whilst still retaining an appropriate level of antibiotic activity, e.g. similar, or even superior, to that of the parent antibiotic such as cethromycin.

Ketolide compounds having modified, i.e. non-methyl, substituents at the C-10 position are generally described in WO 2004/056843, the contents of which are incorporated herein by reference. The compounds which are described in this earlier document typically possess aromatic groups, e.g. phenyl-containing groups, at the C-10 position. Furthermore, WO 2004/056843 is concerned solely with developing macrolide analogues possessing antibiotic properties and is silent with regard to any other potential advantages which might result from specific modifications at the C-10 position.

The present inventors have determined that cethromycin analogues having certain C-10 substituents which differ from those which are disclosed in

WO 2004/056843 possess one or more improved properties over the parent ketolide, e.g. cethromycin. Specifically, it has been observed that those compounds carrying a substituted aminom ethylene or hydrazinomethylene at the C-10 position demonstrate such properties. This is especially the case where the C-10 substituent carries a heteroatom separated from the basic nitrogen of the aminomethylene or hydrazinomethylene group by two carbon atoms. Thus, viewed from a first aspect, the invention provides a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:

m denotes 0 or 1 ;

R¹ denotes hydrogen or fluorine;

R² denotes an optionally substituted methyl group; and

either

R denotes hydrogen or a C_1-3 alkyl group (e.g. methyl); and

R⁴ denotes a group -(CH₂)_n-X or a group -(CH₂)_P-Z;

in which n denotes the integer 0, 1 or 2, preferably 1 or 2; p denotes the integer 0 or 1, preferably 0;

X denotes a group -OR⁵, -SR⁵, -C(0)R⁵, -C(0)OR⁵, - R⁶R⁷,

-C(0) R⁶R⁷, -C(0)CF₃, -CN or -CF₃

(wherein R⁵ denotes hydrogen, a Ci-4-alkyl group (preferably a Ci_-3-alkyl group, e.g. methyl) or a C_3-5-cycloalkyl group (e.g. cyclobutyl), and R⁶ and R⁷ independently denote hydrogen atoms, Ci_-6-alkyl groups (preferably a Ci_-4-alkyl group, e.g. ethyl), or C_3-5-cycloalkyl groups (e.g. cyclobutyl), or R⁶ and R⁷, together with the intervening nitrogen atom, denote an optionally substituted, 3-, 4-, 5- or 6-membered heterocyclic group);

Z denotes an optionally substituted, 4- or 5-membered, saturated heterocyclic group; or

R³ and R⁴, together with the intervening nitrogen atom, form an optionally substituted, saturated or unsaturated, 4- to 7-membered heterocyclic group which comprises at least one nitrogen atom, preferably two or more nitrogen atoms (e.g. 2 or 3 nitrogen atoms).

In one aspect the invention provides a compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof, wherein:

m denotes 0 or 1 ;

R¹ denotes hydrogen or fluorine;

R² denotes an optionally substituted methyl group; and

either

R³ denotes hydrogen; and

R⁴ denotes a group -(CH₂)_n-X or an optionally substituted, 4- or

5-membered, saturated heterocyclic group;

in which n denotes the integer 1 or 2;

X denotes a group -OR⁵, -SR⁵, -C(0)R⁵, -C(0)OR⁵, - R⁶R⁷,

-C(0) R⁶R⁷, -C(0)CF₃, -CN or -CF₃

(wherein R⁵ denotes hydrogen, a Ci-4-alkyl group (preferably a Ci_-3-alkyl group, e.g. methyl) or a C_3-5-cycloalkyl group (e.g.

cyclobutyl), and R⁶ and R⁷ independently denote hydrogen atoms, Ci_-6-alkyl groups (preferably a Ci_-4-alkyl group, e.g. ethyl), or C_3-5-cycloalkyl groups (e.g. cyclobutyl), or R⁶ and R⁷, together with the intervening nitrogen atom, denote an optionally substituted, 3-, 4-, 5- or 6-membered heterocyclic group);

or

R³ and R⁴, together with the intervening nitrogen atom, form an optionally substituted, saturated or unsaturated, 5- or 6-membered heterocyclic group which comprises two or more nitrogen atoms (e.g. 2 or 3 nitrogen atoms).

In a first preferred embodiment:

m denotes 0 or 1 ; R¹ denotes hydrogen or fluorine;

R² denotes an optionally substituted methyl group; and

either

R³ denotes hydrogen; and

R⁴ denotes a group -(CH₂)_n-X or an optionally substituted, 4- or

5-membered, saturated heterocyclic group;

in which n denotes the integer 1 or 2;

X denotes a group -OR⁵, -SR⁵, - R⁶R⁷, -CN or -CF₃

(wherein R⁵ denotes hydrogen or a Ci_-4-alkyl group (preferably a Ci_-3-alkyl group, e.g. methyl), and R⁶ and R⁷ independently denote hydrogen atoms or Ci-6-alkyl groups (preferably a Ci_-4-alkyl group, e.g. ethyl), or R⁶ and R⁷, together with the intervening nitrogen atom, denote an optionally substituted, 4-, 5- or 6-membered heterocyclic group);

or

R³ and R⁴, together with the intervening nitrogen atom, form an optionally substituted, saturated or unsaturated, 5- or 6-membered heterocyclic group which comprises two or more nitrogen atoms (e.g. 2 or 3 nitrogen atoms). In a preferred embodiment, m denotes 0. When m is 0, R³ and R⁴, together with the intervening nitrogen atom may form an optionally substituted, unsaturated, 5-membered heterocyclic group which comprises two or more nitrogen atoms (e.g. 2 nitrogen atoms). Alternatively, where m is 0, R³ may be hydrogen and R⁴ is either - CH₂-CN or -(CH₂)₂- R⁶R⁷ where R⁶ and R⁷ are independently methyl or ethyl.

In an alternative embodiment, m denotes 1, in which case R³ and R⁴ together preferably form the said heterocyclic group.

In a preferred embodiment, R¹ denotes hydrogen.

In a preferred embodiment, R² denotes a group "R" as defined in

WO 98/09978. Preferably, R² denotes an optionally substituted Ci_-4-alkyl group (preferably methyl), or an optionally substituted C_2-4-alkenyl group, especially preferably a group -CH₂-CH=CH-Y (in which Y denotes an optionally substituted aryl or heteroaryl group, particularly a bicyclic, e.g. a fused bicyclic, group. Particularly preferably, Y denotes an optionally substituted phenyl, naphthyl, pyridyl, quinolyl, benzimidazolyl or benzoxazolyl group, especially a 3-, 4-, or 5- quinolyl group). Most preferably, R² denotes a group having the formula:

In the case where R³ denotes hydrogen and R⁴ denotes a group -(CH₂)_n-X, X preferably denotes a group -OR⁵ or - R⁶R⁷ (in which R⁵ denotes a Ci_-3-alkyl group (preferably methyl), and R⁶ and R⁷ independently denote Ci_-3-alkyl groups (preferably ethyl), or R⁶ and R⁷, together with the intervening nitrogen atom, denote a 4- or 5-membered heterocyclic group (preferably a saturated heterocyclic group, e.g. selected from azetidinyl, pyrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl and piperazinyl)), or X denotes -CN or -CF₃.

Where X denotes -OR⁵, -SR⁵, - R⁶R⁷, n is preferably 2. Where X denotes -C(0)R⁵, -C(0)OR⁵, -C(0) R⁶R⁷, -C(0)CF₃, -CN or -CF₃, n is preferably 1.

Where R⁴ is a heterocyclic group, this may be an optionally substituted, 4-membered saturated heterocycle, e.g. selected from oxetanyl, thietanyl, azetidinyl and (N-methyl)azetidinyl. Especially preferred for R⁴ are the heterocyclic groups 3 -oxetanyl, 3 -thietanyl, 3 -azetidinyl and 3-(N-methyl)azetidinyl, especially

3 -oxetanyl.

In the embodiment where R³ and R⁴ together form an optionally substituted heterocyclic group, this is preferably selected from the group consisting of optionally substituted piperazinyl, (N-alkyl)piperazinyl, 1,2-diazolyl, 1,3-diazolyl,

I, 2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl and pyrazinyl groups. Especially preferably, the heterocyclic group formed by R³ and R⁴ is piperazinyl,

(N-alkyl)piperazinyl, 1,2-diazolyl or 1,3-diazolyl, particularly 1,3-diazolyl or (N-Ci_-3-alkyl)piperazinyl, e.g. (N-methyl)piperazinyl or (N-ethyl)piperazinyl.

In a preferred embodiment, the invention provides a compound of Formula

II, or a pharmaceutically acceptable salt or prodrug thereof:

Unless otherwise specified, any of the groups herein defined may be substituted by one or more substituents, which may be identical or different. These are preferably selected from the group consisting of a halogen (e.g. fluorine, chlorine and bromine), alkyl (e.g. methyl, ethyl, isopropyl), haloalkyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, and cyano.

Unless otherwise stated, all substituents are independent of one another.

In the case where a subscript is the integer 0 (i.e. zero), it is intended that the group to which the subscript refers is absent, i.e. there is a direct bond between the groups either side of that particular group.

Unless otherwise stated, any reference herein to a "bond" is intended to refer to a saturated bond.

As used herein, the term "alkyl" refers to a saturated hydrocarbon group and is intended to cover both straight-chained and branched alkyl groups. Examples of such groups include methyl, ethyl, n-propyl, iso-propyl, neo-pentyl, n-hexyl, etc. An alkyl group preferably contains from 1-4 carbon atoms, e.g. 1-3 carbon atoms. Unless otherwise stated, any alkyl group mentioned herein may optionally be substituted by one or more groups, which may be identical or different, for example hydroxy, Ci_-3-alkoxy, amino, Ci_-3-alkylamino, di(Ci_-3-alkyl)amino, Ci_-3-haloalkyl or halogen atoms (e.g. F, CI or Br).

The term "haloalkyl" refers to an alkyl group (i.e. as defined above) which is substituted by 1 or more (e.g. 1, 2, 3 or 4) halogen atoms, e.g. fluorine, chlorine or bromine. Examples of such groups include fluoromethyl, trifluoromethyl,

1-chloroethyl, 2-chloroethyl, 1,2-dibromoethyl, 1, 1-dibromoethyl, etc. Preferably at least one of the halogen atoms of the haloalkyl group is a fluorine atom, especially preferably all of the halogen atoms are fluorine atoms.

As used herein, the term "cycloalkyl" is intended to cover any cyclic alkyl group, these may have from 3-5 carbon atoms, i.e. 3, 4 or 5 carbons, but preferably will contain 3 carbons. Examples of such groups include cyclopropyl, cyclobutyl and cyclopentyl. Unless otherwise stated, any cycloalkyl group mentioned herein may optionally be substituted by one or more groups, which may be identical or different, for example hydroxy, Ci_-3-alkoxy, amino, Ci_-3-alkylamino, di(Ci_-3- alkyl)amino, Ci_-3-haloalkyl or halogen atoms (e.g. F, CI or Br).

As used herein, the term "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds and includes both straight-chained and branched alkenyl groups. The term "C_2-4-alkenyl group" refers to an alkenyl group having from 2 to 4 carbon atoms and one or two double bonds. Examples of such groups include vinyl, allyl, propenyl, iso-propenyl, butenyl, and iso-butenyl. Unless otherwise stated, any alkenyl group mentioned herein may optionally be substituted by one or more groups, which may be identical or different, for example hydroxy, Ci_-3-alkoxy, amino, Ci_-3-alkylamino, di(Ci_-3-alkyl)amino, Ci_-3-haloalkyl or halogen atoms (e.g. F, CI or Br).

Unless otherwise specified, any "heterocyclic group" will contain at least one heteroatom selected from O, N and S. Where reference is made to any "unsaturated heterocyclic group", this may be mono-, di or tri-unsaturated. Unless otherwise stated, any heterocyclic group mentioned herein may optionally be substituted by one or more substituents, which may be identical or different, for example Ci_-4-alkyl groups (e.g. methyl or ethyl), hydroxy, Ci_-3-alkoxy, amino, Ci_-3-alkylamino, di(Ci_-3-alkyl)amino, Ci_-3-haloalkyl (e.g. trifluoromethyl) or halogen atoms (e.g. F, CI or Br). As used herein, the term "aryl" is intended to cover aromatic ring systems. Such ring systems may be monocyclic or polycyclic (e.g. bicyclic) and contain at least one unsaturated aromatic ring. Where these contain polycyclic rings, these may be fused. Preferably such systems contain from 6-20 carbon atoms, e.g. either 6 or 10 carbon atoms. Examples of such groups include phenyl, 1-naphthyl and 2-naphthyl. A preferred aryl group is phenyl. Unless stated otherwise, any "aryl" group may be substituted by one or more substituents, which may be identical or different, for example Ci-4-alkyl groups (e.g. methyl or ethyl), hydroxy, Ci-3-alkoxy, amino, Ci_-3-alkylamino, di(Ci_-3-alkyl)amino, Ci_-3-haloalkyl (e.g. trifluoromethyl) or halogen atoms (e.g. F, CI or Br).

As used herein, the term "heteroaryl" is intended to cover heterocyclic aromatic groups. Such groups may be monocyclic or bicyclic and contain at least one unsaturated heteroaromatic ring system. Where these are monocyclic, these comprise 5- or 6-membered rings which contain at least one heteroatom selected from nitrogen, oxygen and sulphur and contain sufficient conjugated bonds to form an aromatic system. Where these are bicyclic, these may contain from 9-11 ring atoms. Examples of heteroaryl groups include thiophene, thienyl, pyridyl, thiazolyl, furyl, pyrrolyl, triazolyl, imidazolyl, oxadiazolyl, oxazolyl, pyrazolyl, imidazolonyl, oxazolonyl, thiazolonyl, tetrazolyl, thiadiazolyl, benzimidazolyl, benzoxazolyl, benzofuryl, indolyl, isoindolyl, pyridonyl, pyridazinyl, pyrimidinyl, imidazopyridyl, oxazopyridyl, thiazolopyridyl, imidazopyridazinyl, oxazolopyridazinyl,

thiazolopyridazinyl and purinyl. Unless stated otherwise, any "heteroaryl" may be substituted by one or more substituents, which may be identical or different, for example Ci-4-alkyl groups (e.g. methyl or ethyl), hydroxy, Ci_-3-alkoxy, amino, Ci_-3-alkylamino, di(Ci_-3-alkyl)amino, Ci_-3-haloalkyl (e.g. trifluoromethyl) or halogen atoms (e.g. F, CI or Br).

The compounds according to the invention may be provided in the form of a pharmaceutically acceptable salt. For example, these may be converted into a salt with an inorganic or organic acid or base, typically an inorganic or organic acid. Acids which may be used for this purpose include hydrochloric acid, hydrobromic acid, sulphuric acid, sulphonic acid, methanesulphonic acid, phosphoric acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, maleic acid, acetic acid, trifluoroacetic acid and ascorbic acid. Procedures for salt formation are conventional in the art.

The compounds according to the invention may be provided in the form of a prodrug. By "prodrug" is meant a chemical derivative which is converted (e.g. by hydrolysis) in vivo to yield a compound according to formula I. Prodrugs are typically chosen to tailor the physicochemical properties of the compound, e.g. the lipophilicity or stability, for oral or parenteral administration. Preferred prodrugs are small molecule derivatives, i.e. the prodrug is less than 500 g/Mol heavier than the parent molecule, especially less than 200 g/Mol heavier, e.g. between 40 and 160 g/Mol heavier than the parent molecule.

Prodrugs are preferably compounds modified at a basic oxygen or nitrogen atom, especially at a nitrogen atom which carries a lone pair of electrons. For example, the nitrogen directly attached to the C-1 1 position and/or the nitrogen(s) of the aminom ethylene or hydrazinom ethylene moiety attached to the C-10 position may be modified. Any suitable basic nitrogen atoms in the groups R², R³ and/or R⁴ may also be modified. Suitable chemical modifications to provide prodrugs would be apparent to the skilled reader and examples of prodrugs are given in "Prodrugs, Challenges and Rewards Part 1 and Part 2" (Springer New York, 2007, Ed. Stella V. et al.), the content of which is incorporated herein by reference. Particularly preferred groups for attachment at the said basic nitrogen atoms to form prodrugs are those which yield amides, carbamates or carbamides, especially groups of the formula NC(=0)OR, NC(=0)NR₂, or NC(0)0-CHR-0-C(0)OR where each R is independently selected from hydrogen and Ci-6-alkyl, e.g. methyl or ethyl.

The compounds according to the invention may be provided in the form of a single stereoisomer at the C-10 position or, alternatively, as a mixture of two stereoisomers. Thus, in one embodiment, the invention provides a compound of formula la or formula lb, or a pharmaceutically acceptable salt or prodrug thereof:

wherein R¹ to R⁴ and m are as herein defined.

In a particularly preferred embodiment, the invention provides a compound of formula Ila or formula lib, or a pharmaceutically acceptable salt or prodrug thereof:

wherein R¹, R³, R⁴ and m are as herein defined.

In a preferred embodiment, the compounds according to the invention may be provided in the form of a mixture comprising approximately equal amounts of a compound of formula la and a compound of formula lb, or comprising approximately equal amounts of a compound of formula Ila and a compound of formula lib, especially preferably having a ratio of approximately 60:40, e.g.

approximately 70:30, 80:20, 90: 10 or 95:5, of the compounds of formulae Ia:Ib or formulae Ila: lib.

In an alternative embodiment, the compounds of the invention consist essentially of a single stereoisomer, i.e. a compound of formula la or formula lb, especially preferably a single stereoisomer of formula Ila or formula lib. Preferably, this will consist essentially of a compound of formula la, especially formula Ila.

Said mixtures include mixtures of the pharmaceutically acceptable salts and/or prodrugs of said compounds.

In a preferred embodiment, the invention provides a compound of formula I or formula II selected from compounds (1) to (54) as shown in the table below:

Compound No. R¹ C-10 substituent

(1) -H

H

(2) -H

H

(3) -H

(4) -H

(5) -H

(6) -H

—N

(53) -F

H

(54) -F

H

The term "C-10 substituent" in the table above refers to the group directly attached to the C-10 position of the macrolide of formula I or formula II.

In a preferred embodiment, the compound of formula I is selected from compounds of formula la having an R¹ group and a C-10 substituent as listed in the table above. These compounds are designated compounds (la) to (54a),

respectively. In a further preferred embodiment, the compound of formula I is selected from compounds of formula lb having an R¹ group and a C-10 substituent as listed in the table above. These compounds are designated compounds (lb) to (54b), respectively.

Especially preferred compounds include compound numbers (1), (2), (3), (4), (5), (6), (7), (8), (9), (24), (26), (27), (28), (29) and (34), especially compound numbers (la), (2a), (3a), (4a), (5a), (6a), (7a), (8a), (9a), (24a), (26a), (27a), (28a), (29a) and (34a) . Other preferred compounds are selected from compound numbers (12), (13), (14), (15), (16), (17), (18), (19), (20), (40), (42), (43), (44), (45) and (50), especially compound numbers (12a), (13a), (14a), (15a), (16a), (17a), (18a), (19a), (20a), (40a), (42a), (43a), (44a), (45a) and (50a).

In an alternative embodiment, the compounds according to the invention are provided as a mixture of a first and a second isomer, wherein the first isomer is selected from compounds of formula la having an R¹ group and a C-10 substituent as listed in the table above, and wherein the second isomer is a compound of formula lb having the said R¹ group and the said C-10 substituent. These compounds are designated compounds (lab) to (54ab), respectively. Preferred compounds are selected from compound numbers (lab) and (12ab), especially where the ratio of said first isomer to said second isomer is approximately 60:40. Other preferred compounds include (2ab), (3ab), (4ab), (5ab), (6ab), (7ab), (8ab), (9ab), (24ab), (26ab), (27ab), (28ab), (29ab) and (34ab), and their corresponding fluonnated analogues (13ab), (14ab), (15ab), (16ab), (17ab), (18ab), (19ab), (20ab), (40ab), (42ab), (43ab), (44ab), (45ab) and (50ab). Particularly preferred compounds include those selected from (4ab), (15ab), (29ab), (34ab), (45ab) and (50ab).

The group R² in compounds (1) to (54), (la) to (54a), (lb) to (54b), or (lab) to (54ab), is as herein defined, preferably methyl or a group having the formula:

Particularly preferred compounds according to the invention include the following:

The compounds according to the invention may be prepared from readily available starting materials using synthetic methods known in the art, e.g. as described in WO 2004/056843. For example, the following synthetic scheme may be used to prepare the compounds defined herein, in which a compound having an α,β-unsaturated ketone at the C-9 and C-10 positions is reacted with a suitable nucleophile:

SCHEME 1

In Scheme 1, a compound of formula III is reacted with a nitrogen- containing nucleophile to form the desired compound of formula I. Compound III may be prepared using known methods, e.g. as described in WO 2004/056843.

Suitable nucleophiles are readily accessible using conventional synthetic techniques.

Alternatively, compounds of the invention may be prepared starting from compound IV, as illustrated below:

(VI)

SCHEME 2

In Scheme 2, nucleophilic substitution of the acetyloxy group of compound IV by the nitrogen-containing nucleophile is followed by cyclisation of the oxazolidinone ring by reaction with carbonyldiimidazole (CDI) and ammonia. The final step is the deprotection of the hydroxyl functionality of the desosamine, for example by hydrolysis. The group -R^P in Scheme 2 is a protecting group, e.g. an acetyl group. The preparation of compounds of formula IV may be achieved using known synthetic techniques, e.g. those described in WO 2004/056843.

Scheme 2 is particularly suitable for the synthesis of compounds in which R² is a relatively simple group, e.g. an alkyl group. For compounds in which R² is more complex, e.g. in the case of cethromycin analogues having substituted propen-2-yl groups at that position, a modified synthesis may be employed in which the R² group is in a protected or intermediate form for some of the steps. For example, where R is a group of formula

, the synthesis may be carried out with a precursor propargyl group at position R². The propargyl group can be reduced and reacted with 3-bromoquinoline to yield the final R² group, e.g. using conditions known in the art for Heck reactions. A specific example of such a synthesis is given in Example 5 and a more general strategy is shown in Scheme 3 below:

SCHEME 3 In Scheme 3, P denotes a hydroxyl protecting group (e.g. a sugar, an acetyl or a benzyl group) and R denotes an alkyl group (e.g. a (l-isopropoxy)cyclohexyl group). R¹, R³ and R⁴ are as previously defined. This scheme illustrates the generation of a 6-propargyloxy macrolide precursor (VIII) which is cyclised at the CI 1 and C12 positions using the reactions described in Scheme 2 to provide intermediate X. This intermediate is then modified to provide the full side-chain at C6 (i.e. group R²), e.g. by alkyne reduction and a Heck reaction, following which the ClO-substituent is added using the reactions described in Scheme 1. An alternative to this scheme would be to add the CIO substituent to intermediate X using the reactions described in Scheme 1 and then to elaborate the propargyl group at C6 by reduction and Heck reaction. Suitable conditions for such steps could be determined by the skilled person.

The steps illustrated in Scheme 3 provide compounds of formula II.

However, they could equally be used to provide compounds of formula I where R² denotes other substituted alkenyl groups, e.g. where R² denotes a C_2-4-alkenyl group bonded to an optionally substituted aryl or heteroaryl group.

Synthesis of 6-O-propargyl-substituted macrolides (such as the steps providing intermediate IX) is reported in the literature, e.g. in relation to the synthesis of cethromycin itself. One of skill in the art could modify such procedures to introduce groups at the CIO position as defined herein on the basis of the information provided in this specification.

In the case of any of the above synthetic schemes, fluorine may be introduced at the C-2 position before, during or after the groups at positions C-6 and C-10 of the ketolide are introduced. For example, starting materials carrying a C-2 fluorine atom may be used. Alternatively, starting materials, intermediates or products comprising a hydrogen atom at position C-2 may be fluorinated using known methods, e.g. by treatment with Selectfluor^® (available from Sigma-Aldrich).

Accordingly, the invention provides a process for preparing a compound of formula I, la, lb, II, Ila or lib, said process comprising reacting a compound of formula III with a compound HR³R⁴ or H₂N- R³R⁴ (where R³ and R⁴ are as herein defined). The invention also provides a process for preparing a compound of formula I, la, lb, II, Ila or lib, said process comprising reacting a compound of formula IV

3 4 3 4 3 4

with a compound NHR R or H₂N-NR R (where R and R are as herein defined) and reacting the product thereof with a nitrogen-containing cyclisation reagent (or reagents) to provide a compound of formula I. Preferably, the cyclisation reagents are CDI and ammonia.

In a further aspect, the invention provides a process for preparing a compound of formula I wherein R² denotes a C₂-4-alkenyl group bonded to an optionally substituted aryl or heteroaryl group, said process comprising either: a) reacting a compound of formula XII

(XII)

wherein m, R¹, R³ and R⁴ are as herein defined; R² denotes a C_2-4-alkenyl group and P denotes a protecting group for the hydroxyl of the desosamine, e.g. an acetyl or benzyl group,

under suitable conditions to couple the double bond of said C₂-4-alkenyl group with an optionally substituted aryl or heteroaryl group, e.g. using the Heck reaction, and then subsequently deprotecting the desosamine (i.e. replacing P with hydrogen); or

b) reacting a compound of formula III, wherein R² denotes said C₂-4-alkenyl group bonded to an optionally substituted aryl or heteroaryl group, with a compound NHR³R⁴ or H₂N-NR³R⁴ (where R³ and R⁴ are as herein defined). In preferred embodiments, R² is a propargyl group or the C_2-4-alkenyl group is a propen-2-yl group and/or the optionally substituted aryl or heteroaryl group is quinoline, especially 3 -quinoline.

In a related aspect, the invention provides a compound of formula XII in which m, R¹, R³, R⁴ and P are as herein defined and R² denotes a C2-4-alkenyl or a C_2-4-alkynyl group. Preferably, R² denotes a propargyl or a propen-2-yl group.

In another related aspect, the invention provides compounds of formula III in which R¹ is as herein defined and R² denotes a C₂-4-alkenyl group bonded to an optionally substituted aryl or heteroaryl group, preferably a group having the formula

In a yet further aspect, the invention provides a compound of formula XIII:

(XIII)

wherein R¹ is as herein defined; R² denotes a C_2-4-alkenyl group or

C_2-4-alkynyl group; and P denotes a protecting group for the hydroxyl of the desosamine. Preferably, P denotes an acetyl or benzyl group, especially an acetyl group. R² preferably denotes a propargyl or a propen-2-yl group. In an especially preferred embodiment, the compound is a compound of formula X.

Thus, in a further related embodiment, the invention provides a process for producing a compound of formula XII as defined herein, the process comprising reacting a compound of formula XIII as defined herein under suitable conditions to

2"

couple a double bond of the R group with an optionally substituted aryl or heteroaryl group, e.g. using the Heck reaction. The process optionally comprises a step of reducing a triple bond of the R² group to form a double bond. The processes of the invention optionally comprise the step of treating a compound carrying a hydrogen atom at position C-2, e.g. a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII or XIII in which R¹ denotes hydrogen, with a strong fluorinating agent (e.g. Selectfluor^®).

The processes described above typically provide compounds having a mixture of the two stereoisomers at the C-10 position, although compounds of formula la (or Ila) typically predominate. Where separation of the diastereomers is desired, this may be achieved by known methods such as selective crystallisation or chromatographic separation, e.g. by column chromatography or HPLC. If a mixture of isomers is desired, this may be achieved by selection of the appropriate reaction conditions to provide predominantly the single isomer or by separation of the stereoisomers and combination of the appropriate quantity of each diastereomer.

In another aspect, the invention provides a process for preparing a pharmaceutical composition comprising a compound of formula I, la, lb, II, Ila or lib, said process comprising formulating said compound with one or more physiologically tolerable carriers, diluents or excipients.

In a related aspect the invention provides a process for preparing a pharmaceutical composition comprising a compound of formula I, la, lb, II, Ila or lib, said process comprising reacting a compound of formula III with a compound HR³R⁴ or H₂N- R³R⁴ (where R³ and R⁴ are as herein defined), optionally separating the compound into its diastereomers, and formulating said compound with one or more physiologically tolerable carriers, diluents or excipients.

Optionally, before, during or after said formulation, the compound is converted to a pharmaceutically acceptable salt or prodrug thereof, e.g. using means known in the art or as described herein.

In a further related aspect the invention provides a process for preparing a pharmaceutical composition comprising a compound of formula I, la, lb, II, Ila or lib, said process comprising reacting a compound of formula IV with a compound HR³R⁴ or H₂N- R³R⁴ (where R³ and R⁴ are as herein defined) and reacting the product thereof with a nitrogen-containing cyclisation reagent (or reagents) to provide a compound of formula I, optionally separating the compound into its diastereomers, and formulating said compound with one or more physiologically tolerable carriers diluents or excipients. Optionally, before, during or after said formulation, the compound is converted to a pharmaceutically acceptable salt or prodrug thereof, e.g. using means known in the art or as described herein.

In a yet further related aspect, the invention provides a process for preparing a pharmaceutical composition comprising a compound of the invention, wherein R² denotes a C_2-4-alkenyl group bonded to an optionally substituted aryl or heteroaryl group, said process comprising either:

a) reacting a compound of formula XII (wherein m, R¹, R³ and R⁴ are as herein defined; R² denotes a C_2-4-alkenyl group and P denotes a protecting group for the hydroxyl of the desosamine, e.g. an acetyl or benzyl group) under suitable conditions to couple the double bond of said C_2-4-alkenyl group with an optionally substituted aryl or heteroaryl group, e.g. using the Heck reaction, and then subsequently deprotecting the desosamine (i.e. replacing P with hydrogen); or

2"

b) reacting a compound of formula III (wherein R denotes said C_2-4-alkenyl group bonded to an optionally substituted aryl or heteroaryl group) with a compound HR³R⁴ or H₂N- R³R⁴ (where R³ and R⁴ are as herein defined),

and then optionally separating the compound into its diastereomers, and formulating said compound with one or more physiologically tolerable carriers, diluents or excipients. In preferred embodiments, R² is a propargyl group or the C_2-4-alkenyl group is a propen-2-yl group and/or the optionally substituted aryl or heteroaryl group is quinoline, especially 3-quinoline. Optionally, before, during or after said formulation, the compound is converted to a pharmaceutically acceptable salt or prodrug thereof, e.g. using means known in the art or as described herein.

The compounds according to the invention and their pharmaceutically acceptable salts have valuable pharmacological properties, particularly as regards their antimicrobial activity. Typically, the antimicrobial activity of the compounds of the invention is similar to, or exceeds, that of the parent molecule, i.e. the equivalent ketolide carrying a methyl group at the C-10 position. However, compounds of the invention which possess a lower antimicrobial activity to that of the parent molecule are also beneficial, provided that the antimicrobial activity is sufficient for the desired medical treatment and that the compound possesses one or more further advantages over the parent compound. The compounds of the invention typically exhibit a reduced propensity to inhibit CYP3 A4 metabolic enzymes relative to the parent molecules. Alternatively, or in addition, the compounds of the invention may possess desirable properties for formulation into pharmaceutical compositions, e.g. adequate solubility in aqueous media to allow intravenous formulation. Further, the compounds of the invention are typically less prone to metabolic processes than their parent molecules and so are expected to possess a longer half-life in vivo.

The compounds of the invention possess desirable antimicrobial properties. By "antimicrobial properties" is meant an ability to inhibit bacterial growth of a given microorganism as expressed, for example, by Minimal Inhibitory

Concentration (MIC), and/or an ability to kill microorganisms as expressed, for example, by Minimal Bactericidal Concentration (MBC). Preferably, the antimicrobial property displayed by the compounds of the invention is an antibacterial property. Bacteria which may be targeted include especially those of the genus Streptococcus pneumoniae, particularly antibiotic resistant strains thereof, e.g. macrolide-resistant strains thereof.

Preferably, the compound of the invention possesses an MIC value which is substantially equal to, or lower than, the MIC value of the parent compound (i.e. the equivalent ketolide carrying a methyl group and hydrogen at the C-10 position). In addition, or alternatively, the compound of the invention preferably possesses an MBC value which is substantially equal to, or lower than, the MBC value of the parent compound. Preferably, the MIC values for the compounds of the invention (e.g. against S. pneumoniae ATCC49619) range between 1 ng/ml and 4 μg/ml, especially between 5 ng/ml and 2 μg/ml, e.g. around 50 ng/ml, 125 ng/ml, 250 ng/ml, 500 ng/ml or 1000 ng/ml.

The compounds of the invention are typically sufficiently soluble in aqueous media, e.g. saline solution, glucosate or Ringer's solution, to allow for parenteral, e.g. intravenous, administration. Preferably the compounds of the invention have a solubility of greater than 0.1 mg/ml in water (e.g. when measured by the shake flask method with UPLC analysis). Particularly preferably, the compounds of the invention have a solubility of greater than 0.2 mg/ml in water, especially greater than 0.3, 0.4, 0.5, 0.75 or 1 mg/ml in water. Thus, in a further aspect the invention provides a pharmaceutical formulation comprising a compound of formula I, la, lb, II, Ila or lib, or a pharmaceutically acceptable salt or prodrug thereof, together with one or more pharmaceutically acceptable carriers, diluents or excipients.

The invention further relates to a pharmaceutical formulation comprising a combination of a compound of the invention (e.g. formula I, la, lb, II, Ila, lib, etc.), or a pharmaceutically acceptable salt or prodrug thereof, with one or more additional therapeutic agents and optionally one or more pharmaceutically acceptable carriers, diluents or excipients. In a further aspect, the invention provides a kit comprising a compound of the invention (e.g. formula I, la, lb, II, Ila, lib, etc.), or a

pharmaceutically acceptable salt or prodrug thereof, together with one or more pharmaceutically acceptable carriers, diluents or excipients and, separately, one or more additional therapeutic agents.

Suitable additional therapeutic agents for use in formulations of the invention include anti -inflammatory agents and anti -microbial agents, especially antibiotic agents. Antibiotic agents having complementary activity, e.g. those which can kill and/or inhibit the growth of microorganisms that are not significantly affected by the compounds of the invention, are especially preferred. Preferably the additional therapeutic agent is a drug which is metabolised by CYP3A4, i.e. a CYP3 A4 substrate.

Viewed from a further aspect the invention provides a compound of the invention, or a pharmaceutically acceptable salt or prodrug thereof, or a

pharmaceutical composition as defined herein for use in therapy. Unless otherwise specified, the term "therapy" as used herein is intended to include both treatment and prevention. In one embodiment the therapy is combination therapy, i.e. said compound is for use in simultaneous, sequential or separate use with one or more additional therapeutic agents, especially wherein said one or more additional therapeutic agents are CYP3A4 substrates.

In a still further aspect the invention provides a compound of the invention, or a pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical composition as defined herein for use in the treatment or prevention of microbial infection, e.g. for use as an antimicrobial agent. In a preferred embodiment, the said compound is for simultaneous, sequential or separate use with one or more additional therapeutic agents, especially wherein said one or more additional therapeutic agents are CYP3 A4 substrates. Preferably said use is for the treatment of microbial infection, e.g. use as an antibiotic agent.

In a related embodiment, the invention provides a method of treatment of a subject to treat or prevent microbial (e.g. bacterial) infection, said method comprising administration to said subject of an effective amount of a compound of the invention, or a pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical composition as defined herein. Preferred subjects for treatment include mammals, especially humans. In a preferred embodiment, the said method comprises a further step of simultaneous, sequential or separate administration of one or more additional therapeutic agents, especially wherein said agents are CYP3A4 substrates.

In another aspect the invention provides the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined herein in the manufacture of a medicament for use in the treatment or prevention of microbial infection, e.g. for use in the manufacture of an antimicrobial agent, especially an antibiotic. In a preferred embodiment, the said medicament is for simultaneous, sequential or separate use with one or more additional therapeutic agents, especially wherein said one or more additional therapeutic agents are CYP3A4 substrates.

Compounds of the invention (and compositions containing them) can be used for the treatment of infections caused by microorganisms localised to tissues, organs or other compartments where the concentration of said compounds is sufficiently high after administration either to inhibit growth of, or to kill, the microorganism. Preferably, the compounds and compositions of the invention are for the treatment of disease caused, or exacerbated, by infection of the respiratory tract, including infections of the lower respiratory tract, the upper respiratory tract and the lungs. The compounds and compositions of the invention are particularly useful in the treatment of community acquired pneumonia (CAP), acute exacerbation of chronic bronchitis (AECB), tonsillopharyngitis and acute sinusitis, especially in young or elderly patients or those with underlying respiratory problems such as chronic obstructive pulmonary disease (COPD). Compounds and compositions of the invention may also be used to treat sexually transmitted diseases, e.g. Chlamydia, toxoplasmosis, meningococcal disease such as meningococcemia and septicaemia.

The dosage required to achieve the desired activity will depend on the compound which is to be administered, the patient, the nature and severity of the condition, the method and frequency of administration and may be varied or adjusted according to choice. Typically, the dosage may be expected to be in the range from 100 to 1000 mg, preferably 300 to 500 mg (when administered intravenously) and from 300 to 2000 mg, preferably from 500 to 1000 mg (when administered orally.

The compounds of the invention may be formulated with one or more conventional carriers and/or excipients according to techniques well known in the art. Typically, the compositions will be adapted for topical, oral, inhalable or parenteral administration, for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Suitable pharmaceutical forms thus include plain or coated tablets, capsules, suspensions and solutions containing the active component optionally together with one or more conventional inert carriers and/or diluents, such as corn starch, lactose, sucrose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, water/ethanol, water/glycerol, water/sorbitol, water/polyethyleneglycol, propylene glycol, stearyl alcohol, carboxymethylcellulose or fatty substances such as hard fat or suitable mixtures of any of the above. Inhalable medicaments are preferred, especially in the treatment of respiratory infections. Suitable inhalable forms include powders and solutions, e.g. solutions for use in a conventional nebuliser or atomiser device.

Particularly preferred are compositions which are suitable for parenteral administration, especially intravenous administration, e.g. sterile isotonic aqueous solutions, such as those made up in normal saline, Hartmann's solution, glucosate or Ringer solution.

The pharmacological and physicochemical properties of the compounds of the invention can be analysed using standard assays, e.g. those testing for functional activity. Exemplary protocols for testing of the compounds of the invention are provided in the Examples. The invention will now be further described with reference to the following non-limiting Examples:

Example 1 - Preparation of compounds

To determine the influence of C-10 substitution on the properties of the compounds of the invention, a number of compounds were synthesised. These compounds are potent antimicrobial agents which are analogues of cethromycin that lack the heterobicyclic functional group at position C-6 of the macrocyclic ring. The behaviour and relative properties of the synthesised compounds are considered to be representative of the corresponding compounds with heteroaryl-containing side- chains at position C-6, e.g. cethromycin analogues.

The synthesised compounds are based on the following structural formula (having group "R" as listed in the table below):

Compound No. Group "R"

(Ml)

H

(M2)

H

Compound (M_refi) was synthesised as a control, i.e. to reflect the properties of the "un-modified" ketolide. Its properties are considered to be representative of the cethromycin ketolide. Compound (M_ree) corresponds to compound 118a of WO 2004/056843 and was used to compare the properties of a macrolide carrying a side- chain which differed from the C-10 side-chains in the compounds of the invention (note especially the aromatic nature of the side-chain and the lack of a heteroatom in the position gamma to the basic nitrogen atom). Compounds (Ml) to (M9) were synthesised to study the effect of the C-10 substituents on the properties of the ketolides.

In addition to compounds Ml to M9, M_ren and M_ree, compound Mlab was synthesised. This compound consists of both stereoisomers at the C-10 position in a 3 :2 ratio of the formula la to lb absolute stereochemistries. The compound has the following structure:

Further synthetic details for compounds Ml to M9 are given below, including details of general methods (methods A and B) used to prepare the compounds and purify them, along with information regarding reaction conditions and analysis of the products (Tables 1 and 2). The synthetic methods are based on Scheme 1 described above and the ketolide starting material in each case was a specific compound of general formula III in which R¹ denotes hydrogen and R² denotes methyl - this compound is referred to as compound I:

Method A:

To a solution of compound I (1 eq.) in acetonitrile, the nitrogen nucleophile (from 1 to 30 eq.) was added at room temperature and the mixture was stirred at a suitable temperature (i.e. from room temperature to 100 °C), either in an open vessel or in a sealed tube, until all of compound I was consumed (reaction time: from 1 hour to 25 days) as monitored by UPLC-MS (UV trace or TIC trace). Where the hydrochloride salt of the nitrogen nucleophile was used, an equimolar amount of DIPEA was added and the reaction conducted as already described. At reaction completion, the solvent was removed and the crude product partitioned between saturated sodium bicarbonate solution and EtOAc. The phases were then separated, the organic fractions were washed with brine and then dried over sodium sulfate. Remaining solvent was removed by evaporation. Method B:

Compound I (1 eq.) and the nitrogen nucleophile (from 1 eq. to 6 eq.) were dissolved in a suitable solvent (e.g. acetonitrile, dichloromethane and/or methanol) and the solvent was then removed. The resulting oil was heated without solvent at 100°C, either in an open vessel or in a sealed tube, until all of compound I was consumed (reaction time: from lh to 12h) as monitored by UPLC-MS (UV trace or TIC trace).

Purification of the compounds:

The products of methods A and B were purified by flash chromatography on silica gel and/or by preparative LC-MS. In case of purification by preparative LC- MS, the trifluoroacetate salt of the desired compound was recovered upon evaporation of the fractions. In order to obtain the free base of the compound, the trifluoroacetate salt was dissolved in MeOH and passed through a PL-HC03 cartridge (MP -resin, StratoSpheres^™). Where appropriate, trituration in a non- solvent (e.g., diethylether, diisopropylether) was conducted to facilitate recovery of the solid compound.

UPLC conditions:

Instrument: Waters Acquity UPLC, Micromass ZQ 2000 single quadrupole (Waters).

Column: Phenomenex^® Kinetex UPLC C18 (50 x 2.1 mm, 1.7 μΜ).

Detector: UV (DIODE array) 200 - 400 nm; MS: ESI+ 100-2000 m/z. Mobile phase:

phase A: water/CH₃CN 95/5 + 0.1% trifluoroacetic acid;

phase B: water/CH₃CN 5/95 + 0.1% trifluoroacetic acid;

flow rate: 0.5 mL/min. Gradient 1 0-0.3 min (A: 95%, B: 5%)

0.3-3.30 min (A: 0%, B: 100%) 3.30-3.90 min (A: 0%, B: 100%) 3.90-4.40 min (A: 95%, B: 5%)

Gradient 2 0-0.3 min (A: 95%, B: 5%)

0.3-1.50 min (A: 0%, B: 100%) 1.50-2.00 min (A: 0%, B: 100%) 2.00-2.40 min (A: 95%, B: 5%)

Gradient 3

Column: Acquity UPLC CSH C18 (50 x 2.1 mm, 1.7 μΜ) at 40°C. Mobile phase:

phase A: 0.1% v/v HCOOH in water;

phase B: 0.1% v/v HCOOH in CH₃CN;

flow rate: 1 mL/min.

Gradient: 0 min (A: 97%, B: 3%)

0-1.50 min (A: 0.1%, B: 99.9%)

1.50-1.90 min (A: 0.1%, B: 99.9%)

1.80-2.00 min (A: 97%, B: 3%)

Table 1 - details of the preparative method used to synthesize each

r.t. = room temperature Table 2 - analysis of the compounds prepared according to Table 1

ratio by MR analy

In Table 2 above, where the Ia:Ib ratio is indicated to be "la", this means substantially all of the compound isolated had the stereochemical configuration indicated in formula la.

Example 2 - Pharmacokinetic properties of the compounds of Example 1

The compounds of Example 1 were tested to investigate their activity and other properties compared to those of the control compounds. The effect of the compounds of Example 1 on Cytochrome P3A4 inhibition and microsome metabolism was determined using in vitro assays.

Inhibition of human CYP3A4 in vitro

Procedure A: Inhibition of the CYP3 A4 isoform was measured in an assay using the specific substrate 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) that liberates a fluorescent species upon CYP metabolism. Compounds, dissolved in DMSO, were tested in duplicate (n=2) at the concentration of 1 and 10 μΜ in a 96- well plate containing incubation/NADPH regenerating buffer. After addition of the specific isoenzyme and substrate, plates were incubated at 37 °C for 30 minutes. Reactions were terminated by addition of 80% acetonitrile/20% 0.5 M Tris base solution and the plates were analysed using a Fluoroskan Ascent at the wavelengths 409 (excitation) and 530 (emission). A concentration-response curve, performed in duplicate, was performed using the known inhibitor ketoconazole as a positive control.

Data analysis was carried out by calculating the percentage inhibition, relative to the control without inhibitor. An inhibition of < 5% indicates that the compound has no effect on CYP3 A4. IC₅₀ values (concentration at 50% inhibition) were determined using Grafit v. 5.0.1.

Procedure B: The assay was conducted using recombinant human P450s with fluorogenic probe substrates. IC₅₀ values, which provide a measure of the potency of inhibition of individual P450 isoforms, were determined for the effect of the compounds at nine concentration levels. Inhibition of CYP3 A4 metabolism was tested using 7-benzloxyquinolone (7BQ). The control rate of fluorescent metabolite production was established from vehicle control incubations (uninhibited), assigned as 100%). The extent of inhibition at each compound concentration was calculated relative to the control rate and the pIC₅₀ value was determined from these results. Vehicle controls (0% of inhibition) and 10μΜ miconazole (100% of inhibition) were included on each assay plate. The results for the plate assay were not accepted with z prime values outside the range 0.4-1.

Measuring metabolic stability of compounds with human liver microsomes Test compounds were dissolved in DMSO to a final concentration of 1 μΜ and pre-incubated for 10 min at 37°C in potassium phosphate buffer (3 mM MgCl₂, pH 7.4) with human liver microsomes (Xenotech) at a final concentration of 0.5 mg/ml. After the pre-incubation period, reactions were started by adding the cofactors mixture (NADP, Glucose 6-phosphate and Glucose 6-phosphate dehydrogenase). Samples were taken at time 0 and 30 min, added to acetonitrile to stop the reaction and then centrifuged. Supernatants were analysed and quantified by LC-MS/MS.

A control sample without cofactors was always included in order to check the stability of test compounds in the matrix. Test compounds were prepared and assayed in duplicate. 7-ethoxycoumarin and propranolol were added as reference standards. A fixed concentration of verapamil was added in every sample as internal standard for LC-MS/MS.

Samples were analyzed on a UPLC (Waters) interfaced with a Premiere XE Triple Quadrupole (Waters).

The eluents were:

Phase A: 95% H₂0, 5% acetonitrile + 0.1% formaldehyde

Phase B: 5% H₂0, 95% acetonitrile + 0.1% formaldehyde

the flow rate was 0.6 ml/min using a BEH CI 8, 50 x 2.1 mm, 1.7 μπι column at 50°C and a 5 μΐ injection volume.

The chromatographic method used was:

Time (min) %A %B

0 98 2

0.2 98 2

0.6 0 100

1.1 0 100

1.15 98 2

1.5 98 2

Samples were analyzed in MRM conditions: ESI Positive, with a desolvation temperature of 450°C, desolvation gas rate 900 L/h, cone gas 49 L/h and collision gas 0.22.

Data analysis was performed by calculating the percent of the area of test compound remaining after 30 min incubation period relative to the area of compound at time 0 min.

Results

The results are shown in Table 3 below: Table 3 - Cytochrome inhibition and microsome metabolism of compounds of Example 1

ND = not determined

The data presented in Table 3 demonstrate that control compound M_ref2, compound 118a of WO 2004/056843, demonstrates a high toxicity in the

cytochrome inhibition test and poor stability in the microsome metabolism test, whereas the compounds of the invention are significantly better. Control compound M_refi demonstrates a higher toxicity in the cytochrome inhibition test at 10 μΜ concentration than any of the tested compounds of the invention.

Example 3 - Antimicrobial activity of the compounds of Example 1

The antibacterial activity of the compounds of Example 1 against strains of S. pneumoniae was assessed as follows.

Strains of S. pneumoniae

Strain #BAA-1402 is an ATCC strain genotypically characterised as we (E)⁺, resistant to macrolides (e.g. erythromycin) and susceptible to amoxicillin, which was collected during CROSS (Canadian Respiratory Organism Susceptibility Study). This strain is phenotypically defined as M (macrolide resistant phenotype) and is susceptible to ketolides such as M_refi.

Strain ATCC49619 was used as a Quality Control Strain. This strain is not phenotypically defined as resistant.

Stock cultures were prepared from isolated colonies selected on Mueller

Hinton Agar (MHA) plates with 5% lysed horse blood, and diluted into Todd-Hewitt Broth (THB) with 20% glycerol at an OD₅₉₅ (i.e. 595 nm) of about 0.4,

supplemented with 5% lysed horse blood, rapidly frozen and stored at -80°C. Materials

All compounds were dissolved according to Clinical Laboratory Standards Institute (ex National Committee for Clinical Laboratory Standard) guidelines (Performance Standards for Antimicrobial Susceptibility Testing - Twentieth Informational Supplement; Approved Standard - 2010 - NCCLS document M100- S20, NCCLS, Wayne, Pennsylvania 19087-1898 USA, 2010), to obtain 10 mg/mL stock solutions.

Compounds were dissolved in DMSO (Becton Dickinson), to obtain 5 mg/mL stock solutions. All compounds were subsequently diluted in Mueller Hinton Broth (MHB)/THB to obtain working solutions.

Media were obtained from Difco Laboratories (Detroit, MI, USA) - MHA,

THB and CAMHB (Cation-Adjusted Mueller Hinton Broth. MHB adjusted with CaCl₂ and MgCl₂ at a final concentration of 20 mg/L and 10 mg/L, respectively), and from Oxoid (Basingstoke, UK) - Laked horse blood. Experimental procedure

MIC assays were performed by the broth microdilution methodology in Cation Adjusted Mueller Hinton Broth (CAMHB) supplemented with laked horse blood (5%) using final bacterial inocula of approximately 5xl0⁵ CFU/mL, according to CLSI procedure (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Seventh Edition - 2006 -

NCCLS document M7-A7, NCCLS, Wayne, Pennsylvania 19087-1898 USA, 2006). Assays were performed in sterile 96-well microtitre plates with round bottom wells (PBI International). Assays were incubated at 37 °C and analysed after between 20 and 24 hours. MIC was defined as the lowest drug concentration causing complete suppression of visible bacterial growth.

Results

The results are shown in Table 4 below:

Table 4 - MIC of the compounds against S. pneumoniae strains

The data above demonstrate that the compounds of the invention have a similar, and in some cases improved, antibiotic effect on the bacterial strains used the experiment, including antibiotic resistant strains, relative to the control compounds M_ren and Μ

Example 4 - Solubility of compounds of Example 1

The thermodynamic solubility of compounds Ml and M7 in water was evaluated relative to that of the control compound M_ren using the shake flask method. Assay protocol

Sample quantification was performed on calibration curves prepared by dissolution of an accurately weighed amount of compounds in 1 : 1

acetonitrile/MeOH. Calibration curves (5-6 points) were prepared from the working standard solutions by the appropriate dilutions in 1 : 1 acetonitrile/MeOH. Each calibration point sample was diluted 1 : 1 with Internal Standard solution (Propranolol 0.25 mg/ml solution in 1 : 1 acetonitrile/MeOH).

The concentration of compounds was measured in water. Saturated solutions of the compounds (as their free bases) were prepared by incubating about 1-2 mg (accurately weighed) of compound in about 1 ml of water for 24 hrs at room temperature under agitation. After three repeat centrifugations at 13000 rpm for 10 min, the supernatant was diluted with 1 : 1 acetonitrile/MeOH. Each sample was then diluted 1 : 1 with Internal Standard solution and injected into LC-MS as described below.

Sample analysis

Samples were analyzed on a UPLC (Waters) interfaced with a Micromass ZQ (Waters) and a 2996 PDA Detector.

Eluents were:

Phase A: 95% H₂0, 5% acetonitrile + 0.1% trifluoroacetic acid Phase B: 5% H₂0, 95% acetonitrile + 0.1% trifluoroacetic acid the flow rate was 0.6 ml/min using a Acquity BEH CI 8, 50 x 2.1 mm, 1.7 μπι column at 40°C and a 2 μΐ injection volume.

The chromatographic method used was:

Time (min) %A %B

0 95 5

0.25 95 5

3.3 0 100

4.0 0 100

4.1 95 5

5.0 95 5 Data analysis was performed by comparing the AUC (Area Under Curve) of the test compounds solutions with those of the calibration points in order to evaluate the final concentrations. Results

The thermodynamic solubility of the compounds is reported in Table 5 below. Results are expressed as mean ± standard deviation, n=2.

Table 5 - thermodynamic solubility of compounds of Example 1

The data in Table 5 show that the compounds of the invention (i.e. carrying a substituent at the C-10 position as set out in formula I above) are significantly more soluble than the equivalent compounds which possess only a methyl group at the C-10 position.

Example 5 - Synthesis of cethromycin analogues

To prove that modification of the ketolide at position C-6 would not adversely affect the properties of the compounds assessed in Examples 1 to 4, a number of further compounds were synthesised. These compounds are potent antimicrobial agents which are analogues of cethromycin and carry the appropriate heteroaryl side-chain at the C-6 position.

The synthesised compounds are based on the following structural formula (having groups "R" as listed in the table below):

The general synthesis of the compounds followed Schemes 1 and 3, with the key intermediate compound (15) being synthesised as follows: Synthesis of (3R, 4S, 5S, 6R, 7R, 9R, 11 S, 12R, 13S, 14R)-6-((2S, 3R, 4S, 6RJ-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)-14-ethyl- 7, 12, 13-trihydroxy-4-( (2R, 4R, 5S, 6S)-5-hydroxy-4-methoxy-4, 6-dimethyltetrahydro- 2H-pyran-2-yloxy)-l 0-(l -isopropoxycyclohexyloxyimino)-3,5, 7,9, 11, 13- hexamethyloxacyclotetradecan-2-one (2)

Reaction:

(3R,4S,5S,6R,7R,9R, 11 S, 12R, 13 S, 14R)-6-((2S,3R,4S,6R)-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)-14-ethyl- 7, 12, 13 -trihydroxy-4-((2R,4R, 5 S, 6 S)-5 -hydroxy-4-methoxy-4, 6-dimethyltetrahydro- 2H-pyran-2-yloxy)-10-(hydroxyimino)-3, 5,7,9,11, 13- hexamethyloxacyclotetradecan-2-one (1) (10 g, 13.35 mmol) was dissolved in dry DCM (70 ml); the mixture was cooled to 0°C, then pyridine hydrochloride (2.314 g, 20.03 mmol) was added. 1, 1-diisopropoxycyclohexane (18 g, 71.9 mmol) was dissolved in dry DCM (30 ml) and then the resulting solution was added dropwise to the mixture. The mixture turned into an almost clear solution that was stirred at room temperature. The reaction was monitored by TLC (DCM/MeOH/NH40H 95/5/0.5), for 24 hrs, at which point the reaction wascomplete. 3N aq. NaOH was then added (pH 10) and the mixture was extracted with DCM. The organic layer was washed twice with brine and then dried (Na₂S0₄), filtered and evaporated.

The yield was considered to be quantitative.

Synthesis of (2S, 3S, 4R, 6R)-6-((3R, 4S, 5S, 6R, 7R, 9R, US, 12R, 13S, 14RJ-6- ((2S,3R,4S, 6R)-3-(denzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2- yloxy)-l 4-ethyl-7, 12, 13-trihydroxy-l O-(l-isopropoxycyclohexyloxyimino)- 3,5, 7,9, 1 l,13-hexamethyl-2-oxooxacyclotetradecan-4-yloxy)-4-methoxy-2,4- dimethyltetrahydro-2H-pyran-3-yl benzoate (3)

Reaction:

11.87 g of 2 (13.35 mmol from the previous step) was dissolved in dry THF (60 mL), then it was added (rt) to a previously-prepared solid mixture obtained by mixing benzoic anhydride (9.06 g, 40.0 mmol) and DMAP (1.631 g, 13.35 mmol). Finally TEA (3.72 mL, 26.7 mmol) was added and the resulting mixture was stirred under nitrogen at room temperature for 20 hrs until TLC (DCM/MeOH/NH40H 95:5:0.5) and UPLC traces showed the reaction was almost complete. 5% aq.

NaHC0₃ (80 ml) was then added and the resulting mixture was stirred for 0.5 hrs at room temperature. Ethyl acetate (150 ml) was added and the mixture was extracted with ethyl acetate. The aqueous phase was back-extracted with ethyl acetate (80 ml) and the combined organic layers were washed once with aq 5% NaHC0₃ (70 ml), twice with KFLPCVbrine ( 2x 70/70 ml) and finally with brine (70 ml). The organic layer was dried (Na₂S0₄), filtered and evaporated. The residue was redissolved in ethyl acetate (50 ml), concentrated (about 20 ml) and diluted with heptane (70 ml). The resulting mixture was concentrated to 15-20 ml, diluted again with heptane (80 ml) and then refluxed to dissolution of the solids. The insoluble material was decanted and the hot solution poured into a vessel and stirred at 0°C for 3 hrs and then at 4°C overnight. The mixture was filtered by suction to give 3 (10.88 g, 9.91 mmol, 74.3 % yield) as a white solid.

Synthesis of (2S, 3S, 4R, 6R)-6-((3R, 4S, 5S, 6R, 7R, 9R, IIS, 12R, 13S, 14RJ-6- ( (2S, 3R, 4S, 6R)-3-(denzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2- yloxy)-l 4-ethyl-l 2, 13-dihydroxy-l O-(l-isopropoxycyclohexyloxyimino)- 3,5, 7,9, 11,13-hexamethyl-2-oxo-7-(prop-2-ynyloxy)oxacyclotetradecan-4-yloxy)-4- methoxy-2, 4-dimethyltetrahydro-2H-pyran-3-ylbenzoate (4)

Reaction:

5 g of 3 (4.56 mmol) was dissolved in a mixture of DMSO (22.8 mL) and dry THF (22.80 mL); the solution was cooled to 0°C under a nitrogen atmosphere and 3-bromoprop-l-yne (80% in toluene, 2.454 mL, 22.78 mmol) and potassium hydroxide (1.278 g, 22.78 mmol) were added portionwise. After 50 min almost complete conversion was observed by LC-MS. Water (25 mL) was added carefully, keeping the temperature below 10°C, followed by Et₂0 (about 50 mL); the mixture was stirred 10 min at 5-10°C, then the aqueous layer was extracted twice with Et₂0 and the organic phase was dried and evaporated. The resulting brown solid (5.68 g) was taken up with CH₃CN (25 mL): dissolution was quickly followed by

precipitation of a thick solid mass which was filtered, washed with CH₃CN (3 x 5 mL) and the solid was dried under vacuum at 45°C overnight, yielding 2.447 g of product as a beige powder.

Synthesis of (2S, 3S, 4R, 6R)-6-((3R, 4S, 5S, 6R, 7R, 9R, 11R, 12R, 13S, 14RJ-6- ( (2S, 3R, 4S, 6R)-3-(denzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2- yloxy)-14-ethyl-12, 13 -di hydroxy- 3, 5, 7,9, 11, 13-hexamethyl-2, 10-dioxo-7 -(prop-2- ynyloxy)oxacyclotetradecan-4-yloxy)-4-methoxy-2,4-dimethyltetrahydro-2H-pyran- 3-yl benzoate (6) Reaction:

19 g of 4 (16.73 mmol) was suspended in ethanol (96 mL). The mixture was cooled to 0°C then hydrogen chloride (66.9 mL, 134 mmol) was added dropwise. An orange solution was obtained and it was stirred at the same temperature for 20 min. Monitoring by UPLC/MS indicated complete deprotection of the oxime.

Sodium nitrite (8.66 g, 126 mmol dissolved in 54.5 mL of water) was added dropwise and the opaque solution was heated to 40°C. After 3.5 hr complete conversion was detected by UPLC/MS. Water (120 ml) was added to the obtained suspension, cooled (ice bath) and stirred for 20 minutes. The mixture was filtered by suction and the solid obtained was suspended in 1 : 1 cyclohexane/diethyl ether (about 200 ml). The mixture was stirred at 65°C for 15 minutes, then, after cooling to room temperature, allowed to stand overnight at 4°C. The mixture was filtered by suction and washed with 1 : 1 cyclohexane/diethyl ether (100 ml). The collected solid was suspended in water (60 ml)/MeOH (230 ml), then 50%w aq. K₂C0₃ (10 ml) was added (pH 9) and the suspension was stirred at room temperature for 20 minutes. The mixture was filtered by suction and the collected solid was dried in vacuo overnight at 50°C to give 6 (11 g, 11.22 mmol, 67.1 % yield)

Synthesis of (2S,3R,4S, 6R)-4-(dimethylamino)-2- ((3R,4S,5S, 6R, 7R,9R, 13S, 14R,E)-14-ethyl-4, 13-dihydroxy-3,5, 7,9,11, 13-hexamethyl- 2, 10-dioxo-7-(prop-2-ynyloxy)oxacyclotetradec-l l-en-6-yloxy)-6-methyltetrahydro- 2H-pyran-3-yl benzoate (8) eaction:

1 g of 6 (1.020 mmol) was suspended in TEA (7.25 mL, 52.0 mmol) and 1,3- dioxolan-2-one (0.539 g, 6.12 mmol) was added. The mixture was heated to reflux overnight and monitored by UPLC/MS until about 50% conversion was observed (962.4 m/z detected, dehydrated derivative). More l,3-dioxolan-2-one (0.539 g,

6.12 mmol) was added and after 6 hr the reaction was complete. The mixture (a pale yellow solution with a dark insoluble solid in the bottom) was evaporated and the residue was suspended in ethanol (8.59 mL) and water (6.87 mL), then 10% aq HC1 (6.82 mL, 22.44 mmol) was added. After few minutes at room temperature, the obtained solution was stored at 4°C for the weekend. Then solution was then stirred for 4 hr at room temperature and heated to 45°C adding in the meantime 10% aq HC1 (6.82 mL, 22.44 mmol) in two portions (11 eq each). After heating for 12 hr, almost complete conversion was obtained. The EtOH was then evaporated and the aqueous solution was basified with 30%aq K₂C0₃ (30 ml) (pH 9) and extracted three times with Et₂0 (70ml x 3). The combined organic phases were dried

(Na₂S0₄) and evaporated. In the aqueous phase only traces of the desired product were detected (by UPLC/MS).

The crude product (dark oil, 1.3g) was purified by flash chromatography (SNAP 50g, Si0₂) with petroleum ether/acetone = 7/3 giving: 8 as a: pale yellow oil (609 mg, 0.870 mmol, 85 % yield). UPLC/MS indicated two peaks with the desired mass (about 9/1 ratio). 1H-NMR was in agreement with some impurities (ethylene carbonate detected).

To remove the ethylene carbonate from the desired product, 482 mg of 8 was dissolved in 10% aq HC1 and the aqueous solution was extracted twice with Et₂0 (40 ml x2). The solution was basified with 30% aq K₂C0₃ to pH 9 and extracted three times with Et₂0 (40ml x 3). The combined organic phases were dried (Na₂S0₄) and evaporated giving pure 8 as an off-white spongy solid (352 mg, calculated yield 62.3%).

Synthesis of((2R, 3S, 7R, 9R, 10R, 11 S, 12S, 13R,E)-10-((2S, 3R, 4S, 6RJ-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-py^

dihydroxy-3, 7,9, 11, 13-pentamethyl-6, 14-dioxo-9-(prop-2-ynyloxy)oxacyclotetradec-

4-en-5-yl)methyl acetate (9)

Reaction:

100 mg of 8 (0.143mmol) was dissolved in methanol (1.905 ml) and the pale yellow solution was heated to reflux with monitoring by UPLC/MS. After 2 hr about 50% conversion was observed. The day after, almost complete conversion was obtained: traces of the starting material were still detected but the reaction was stopped because a by-product with desired mass +16 (612 m/z)(possibly the N-oxide) was forming. The solution was evaporated. To remove and the residue, the pale yellow oil was dissolved in 2N HC1 (15 ml). The aqueous solution was extracted twice with Et₂0 (15 ml x 2) and then was basified with 30% aq K₂C0₃ to pH 9 and extracted threetimes with Et₂0 (15ml x 3). The combined organic phases were dried (Na₂S0₄) and evaporated giving the intermediate,

(3R,4S,5S,6R,7R,9R, 13S, 14R,E)-6-((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy- 6-methyltetrahydro-2H-pyran-2-yloxy)-14-ethyl-4, 13-dihydroxy-3,5,7,9, 11, 13- hexamethyl-7-(prop-2-ynyloxy)oxacyclotetradec-l l-ene-2,10-dione as an off-white spongy solid (56 mg, 0.094 mmol, 65.8 % yield). UPLC/MS indicated the desired product (2 peaks with the same mass) with some impurities. 1H- MR was in agreement, i.e. correct compound with some impurities.

To prepare compound 9, NCS (4.44 g, 33.2 mmol) was added to a solution of the intermediate (13.2 g, 22.16 mmol) in AcOH (130 mL) at RT, and the mixture was heated at 50°C for 4h. UPLC-MS showed partial conversion. More NCS

(1.479 g, 11.08 mmol) was added, the mixture was heated at 50 °C for 4h and left at RT overnight (UPLC-MS showed presence of a chlorinated by-product). More NCS (1.479 g, 11.08 mmol) was added and the mixture was heated at 50 °C for 4h. The reaction mixture was then frozen at -20°C for the weekend. The solution was evaporated (heating to 45°C) and the residue was dissolved in DCM (250 ml) and washed twice with 5%aq NaHC0₃ (100ml) and then with water. The organic phase was dried (Na₂S0₄) and evaporated. Some desired product remained in the aqueous layers that thus were salted with NaCl and re-extracted with EtOAc three times. All the organics were dried and evaporated to give 17 g of crude solid.

The crude product was purified by flash chromatography on silica gel (eluent gradient: from DCM/MeOH/NH₄OH 97:3 :0.3 to DCM/MeOH/NH₄OH 95:5:0.5) to give 9 (8.3 g, 12.70 mmol, 57.3 % yield) as a white foam.

Synthesis of((2R, 3S, 7R, 9R, 10R, 11R, 13R,E)-10-((2S, 3R, 4S, 6R)-3-acetoxy-4- (dimethylamino)-6-methyltetrahydro-2H-pyran-2-yloxy)-2-ethyl-3-hydroxy-

3, 7, 9,11, 13-pentamethyl-6, 12, 14-trioxo-9-(prop-2-ynyloxy)oxacyclotetradec-4-en-5- yljmethyl acetate (10)

Reaction:

A solution of 9 (8.3 g, 12.70 mmol), TEA (3.54 mL, 25.4 mmol) and Ac₂0 (2.396 mL, 25.4 mmol) in DCM (166 mL) was stirred at RT under nitrogen for 22h. UPLC-MS (TIC trace ESI⁺) showed that reaction not completed. More Ac₂0 (1.8 mL, 19.04 mmol) and TEA (2.64 mL, 19.04 mmol) were added. After 6h, an aqueous mixture of brine and 5% NaHC0₃ (aq) was added, the phases were separated, the organics were washed twice with the above described aqueous mixture, dried over Na₂S0₄ and evaporated. Some desired product remained in the water layer, so NaCl was added and the aqueous layer was re-extracted three times with EtOAc. All the organics were put together, dried over Na₂S0₄ and evaporated to afford the intermediate ((2R,3S,7R,9R, 10R, 11 S,12S, 13R,E)-10-((2S,3R,4S,6R)- 3-acetoxy-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-yloxy)-2-ethyl-3,12- dihydroxy-3,7,9, l l,13-pentamethyl-6, 14-dioxo-9-(prop-2- ynyloxy)oxacyclotetradec-4-en-5-yl)methyl acetate (8.84 g, 12.70 mmol, 100 % yield) as a pale yellow foam. UPLC-MS indicated desired product and chlorinated by-products (MW=671). This mixture was used for the next step.

To prepare 10, the crude intermediate produced in the previous step (8.83 g, 12.69 mmol) was dissolved in DCM (109 mL) and, stirring at room temperature, Dess-Martin Periodinane (8.07 g, 19.03 mmol) was added. A pale yellow suspension was obtained. It was reacted at the same temperature with monitoring by UPLC/MS. After 2 hr 15 min complete conversion was observed. The suspension was evaporated and the residue was dissolved in AcOEt (1 1) and washed twice with 3N NaOH (300 ml x2). The organic phase was then washed with brine, dried (Na₂S0₄) and evaported. The crude (pale yellow spongy solid, 8.97 g) was purified by flash chromatography (Si0₂) with petroleum ether/acetone = 8/2 to 4/6. Two fractions were isolated, one having a high R_f (off-white spongy solid, 1.75g - UPLC/MS indicated this was a by-product with 670.46 m/z with some impurities) and the other having a low R_f (pale yellow spongy solid, 10, 4.94 g, 7.12 mmol, 56.1 % yield - UPLC/MS indicated the desired product at 694.6 m/z with little impurities).

Synthesis of(2R, 3S, 7R, 9R, J OR, 11R, 13R,E)-10-((2S, 3R, 4S, 6R)-3-acetoxy-4- (dimethylamino)-6-methyltetrahydro-2H-pyran-2-yloxy)-5-(acetoxym

3, 7, 9,11, 13-pentamethyl-6, 12, 14-trioxo-9-(prop-2-ynyloxy)oxacyclotetradec-4-en-3- yl lH-imidazole-l-carboxylate (11)

Reaction:

To a solution of 10 (4.94 g, 7.12 mmol) in dry THF (111 mL), under nitrogen, at 0°C, NaH 60% (0.427 g, 10.68 mmol) was added, a thick slurry was obtained. After few minutes, a solution of CDI (2.89 g, 17.80 mmol) in dry THF (65 mL) was slowly dropped into the reaction mixture at 0°C and the yellow suspension was stirred at RT for lh. UPLC-MS indicated partial conversion (60%) and formation of a by-product with m/z 727. After lh more at RT, no improved conversion and more by-product was formed. More CDI (1.3 g, 8.02 mmol) was added and the mixture was stirred lh at RT. Conversion was found to be almost complete. At 0°C, a saturated solution of NaHC0₃ was added, EtOAc was then added, a solid that precipitated out was filtered (UPLC-MS indicated the desired product and inorganic salts) and the phases were separated. The aqueous layer was re-extracted twice with EtOAc. All the organics were washed with brine, dried over sodium sulfate and evaporated (*). The solid previously filtered was dissolved in water and EtOAc, the phases were separated, the organics were dried over sodium sulfate and evaporated together with the organics from the extraction above (*). The crude solid was purified by flash chromatography on silica gel (eluent gradient: from petroleum ether/acetone 6:4 to petroleum ether/acetone 3 :7) to give 11 (3.15 g, 4.00 mmol, 56.2 % yield) as a white foam.

Synthesis of (2S,3R,4S, 6R)-4-(dimethylamino)-2- ((3aS, 4R, 7R, 9R, J OR, 11R, J3R, 15aR)-4-ethyl-3a, 7, 9,11, 13-pentamethyl-15- methylene-2, 6, 8, 14-tetraoxo-l l-(prop-2-ynyloxy)tetradecahydro-lH- [ I Joxacyclotetradecaf 4, 3-dJoxazol-10-yloxy)-6-methyltetrahydro-2H-pyran-3-yl acetate (12)

Reaction:

To an opaque white solution of 11 (3.15 g, 4.00 mmol) in acetonitrile (55 mL) and THF (5.5 mL) at RT, ammonium hydroxide (28-30%) (6.23 mL, 48.0 mmol) was added. The opaque solution was stirred at RT. After 3hl5 UPLC-MS showed desired compound, starting material and an amount of two by-products. After a total of 6h30, conversion was almost complete. EtOAc and 3N NaOH were added and the phases were separated. The organics were washed with brine; UPLC- MS showed that some desired product remained in the water layer, so NaCl was added to this layer and it was re-extracted three times with EtOAc. All the organics were put together, dried over Na₂S0₄ and evaporated to give a crude white solid (2.7 g). Purification by flash chromatography on silica gel (eluent gradient: from petroleum ether/acetone 65:35 to petroleum ether/acetone 6:4) gave 12 (1.7 g, 2.51 mmol, 62.8 % yield) as a white foam. UPLC-MS indicated the correct product.

Synthesis of (2S, 3R, 4S, 6R)-2-((3aS,4R, 7R,9R, 10R, 11R, 13R, 15aR)-ll- (allyloxy)-4-ethyl-3a, 7,9, 11, 13-pentamethyl-l 5-methylene-2, 6,8, 14- tetraoxotetradecahydro-lH-[ 1 Joxacyclotetradecaf 4, 3-d] oxazol-10-yloxy)-4- (dimethylamino)-6-methyltetrahydro-2H-pyran-3-yl acetate (13)

Reaction;

A suspension of 12 (1.5 g, 2.216 mmol), Lindlar Catalyst (90 mg, 0.042 mmol) and quinoline (0.263 mL, 2.216 mmol) in EtOAc (45 mL) was hydrogenated in a Parr apparatus at RT and 10 psi for 3h50. Conversion=56%. More Lindlar Catalyst (45 mg, 0.021 mmol) and quinoline (0.131 mL, 1.108 mmol) were added and the mixture was hydrogenated in a Parr apparatus at RT and 10 psi for 2h.

Conversion=66%. More Lindlar Catalyst (45 mg, 0.021 mmol) was added and the mixture and hydrogenated in a Parr apparatus at RT and 10 psi for 2h30.

Conversion= 75%. More Lindlar Catalyst (66 mg, 0.031 mmol) was added and the mixture was hydrogenated in a Parr apparatus at RT and 10 psi for 3h30. At this stage the reaction was completed. The catalyst was filtered off and the filtrate was evaporated. The product was purified by flash chromatography using silica gel (eluent gradient: from petroleum ether/acetone 8:2 to petroleum ether/acetone 1 : 1) to give 13 (1.5 g, 2.210 mmol, 100 % yield) as a white foam. UPLC-MS indicated that the correct product had been formed with less than 10% of the alkane by-product.

Synthesis of (2S,3R,4S, 6R)-4-(dimethylamino)-2- ((3aS, 4R, 7R, 9R, J OR, 11R, 13R, 15aR)-4-ethyl-3a, 7, 9,11, 13-pentamethyl-15- methylene-2, 6,8, 14-tetraoxo-l l-((E)-3-(quinolin-3-yl)allyloxy)tetradecahydro-lH- [ I ] oxacyclotetradecaf 4, 3-dJ oxazol-10-yloxy)-6-methyltetrahydro-2H-pyran-3-yl acetate (14) eaction:

A solution of 13 (1.5 g, 2.210 mmol) and 3-bromoquinoline (0.600 mL, 4.42 mmol) in anhydrous acetonitrile (11 mL) was degassed with nitrogen for 20 min. Then, PdOAc₂ (0.124 g, 0.552 mmol), tri-o-tolylphosphine (0.336 g, 1.105 mmol) and TEA (0.616 mL, 4.42 mmol) were sequentially added and the yellow suspension was heated in a sealed tube at 50°C for lh and then at 90°C for 22h. UPLC-MS indicated that the reaction was complete. The solvent was removed. The brown foam was purified by flash chromatography on silica gel (eluent gradient: from petroleum ether/acetone 6:4 to petroleum ether/acetone 1 : 1. Fractions were checked using UPLC-MS (TIC trace) after evaporation of the solvent) to give 14 (896 mg, 1.112 mmol, 50.3 % yield) as a yellow foam. UPLC-MS indicated the desired product and some 3-bromoquinoline.

Synthesis of(3aS, 4R, 7R, 9R, 10R, 11R, 13R, 15aR)-10-((2S, 3R, 4S, 6RJ-4- (dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)-4-ethyl- 3a, 7,9,11 , 13-pentamethyl-l 5-methylene-l 1 -((E)-3-(quinolin-3- yl)allyloxy)hexahydro-lH-[ 1 Joxacyclotetradecaf 4, 3-dJoxazole- 2, 6, 8, 14(7H, 9H, 15H, 15aH)-tetraone (15)

Reaction:

A solution of 14 (896 mg, 1.112 mmol) in MeOH (30 mL) was stirred at RT for 22h. Solvent was removed to give 15 (822 mg, 1.076 mmol, 97 % yield) as a thick yellow oil. UPLC-MS indicated the desired product and some 3-bromoquinoline coming from the previous step. MR analysis of the product in agreement and indicated the presence of 3-bromoquinoline as a contaminant. However, this semi-purified fraction was used for the next steps.

Synthesis of compounds C1-C6, C8, Cll and C23-C38

These compounds were synthesised and purified according to Example 1 (Method A). Tables 6 and 7 show details of the preparation and analysis:

Table 6 - details of the preparative method used to synthesize each compound

r.t. = room temperature Table 7 - analysis of the compounds prepared according to Table 6

In Table 7 above, where the Ia:Ib ratio is indicated to be "la", this means substantially all of the compound isolated had the stereochemical configuration indicated in formula la.

Example 6 - Properties of the compounds of Example 5

The compounds of Example 5 were tested to investigate their activity and other properties compared to those of the control compounds. The effect of the compounds on Cytochrome P3 A4 inhibition and microsome metabolism was determined using in vitro assays according to Example 2 and the antimicrobial activity determined according to Example 3. The clinical antibiotic cethromycin was used as a control. Results are shown in the tables below. Table 8 - Cytochrome inhibition and microsome metabolism of compounds of Example 5

Table 9 - MIC of the compounds against different S. pneumoniae strains

* strain #BAA-1407 is an ATCC strain genotypically characterised as mefiEf, erm(B)⁺ resistant to macrolides (e.g. erythromycin) and to amoxicillin, which was collected during CROSS (Canadian Respiratory Organism Susceptibility Study). This strain is phenotypically defined as MLS_B (macrolide, lincosamide and streptogramin B resistant).

The data above show that the same trends observed for compounds Ml to M8 are reflected in the compounds carrying the cethromycin side-chain at C6. In particular, cytochrome P450 and microsome metabolism values are improved with little or no alteration in the potency of the compound against S. pneumoniae strains.

Example 7 - Solubility of the compounds of Example 5

The thermodynamic solubility of compounds (C2), (C4), (C28), (C29), (C34), (C36) and (C37) in phosphate buffered saline (PBS) was determined relative to that of cethromycin using the shake flask method detailed in Example 4.

Table 10 - Thermodynamic solubility of compounds

Compound No. Solubility in PBS 20 hour stability in

(mg/ml) PBS (%)

(C2) >1.6 95

(C4) 1.58 93

(C28) 1.84 97

(C29) >1.2 90

(C34) 2.1 97

(C36) 1.22 106

(C37) 1.45 93

Cethromycin 1.53 Not determined Example 8 - Injectable solution containing 400 mg of active substance active substance 400 mg

mannitol 50 mg

human serum albumin 10 mg

water for injections ad 2 ml

Mannitol is dissolved in water for injections; human serum albumin is added; the active ingredient is dissolved with heating; the solution is made up to the specified volume with water for injections and transferred into ampoules under nitrogen gas.

Claims

Claims:

1. A compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:

m denotes 0 or 1 ;

R¹ denotes hydrogen or fluorine;

R² denotes an optionally substituted methyl group; and

either

R denotes hydrogen or a C_1-3 alkyl group (e.g. methyl); and

R⁴ denotes a group -(CH₂)_n-X or a group -(CH₂)_P-Z;

in which n denotes the integer 0, 1 or 2, preferably 1 or 2; p denotes the integer 0 or 1, preferably 0;

X denotes a group -OR⁵, -SR⁵, -C(0)R⁵, -C(0)OR⁵, - R⁶R⁷,

-C(0) R⁶R⁷, -C(0)CF₃, -CN or -CF₃

(wherein R⁵ denotes hydrogen, a Ci_-4-alkyl group (preferably a Ci_-3-alkyl group, e.g. methyl) or a C_3-5-cycloalkyl group (e.g.

cyclobutyl), and R⁶ and R⁷ independently denote hydrogen atoms, Ci-6-alkyl groups (preferably a Ci_-4-alkyl group, e.g. ethyl), or C_{3 -}5-cycloalkyl groups (e.g. cyclobutyl), or R⁶ and R⁷, together with the intervening nitrogen atom, denote an optionally substituted, 3-, 4-, 5- or 6-membered heterocyclic group); Z denotes an optionally substituted, 4- or 5-membered, saturated heterocyclic group;

or

R³ and R⁴, together with the intervening nitrogen atom, form an optionally substituted, saturated or unsaturated, 4- to 7-membered heterocyclic group which comprises at least one nitrogen atom, preferably two or more nitrogen atoms (e.g. 2 or 3 nitrogen atoms).

2. A compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof as claimed in claim 1 wherein:

m denotes 0 or 1 ;

R¹ denotes hydrogen or fluorine;

R² denotes an optionally substituted methyl group; and

either

R³ denotes hydrogen; and

R⁴ denotes a group -(CH₂)_n-X or an optionally substituted, 4- or

5-membered, saturated heterocyclic group;

in which n denotes the integer 1 or 2;

X denotes a group -OR⁵, -SR⁵, -C(0)R⁵, -C(0)OR⁵, - R⁶R⁷,

-C(0) R⁶R⁷, -C(0)CF₃, -CN or -CF₃

(wherein R⁵ denotes hydrogen, a Ci_-4-alkyl group (preferably a Ci_-3-alkyl group, e.g. methyl) or a C_3-5-cycloalkyl group (e.g.

cyclobutyl), and R⁶ and R⁷ independently denote hydrogen atoms, Ci-6-alkyl groups (preferably a Ci_-4-alkyl group, e.g. ethyl), or C_3-5-cycloalkyl groups (e.g. cyclobutyl), or R⁶ and R⁷, together with the intervening nitrogen atom, denote an optionally substituted, 3-, 4-, 5- or 6-membered heterocyclic group);

or

R³ and R⁴, together with the intervening nitrogen atom, form an optionally substituted, saturated or unsaturated, 5- or 6-membered heterocyclic group which comprises two or more nitrogen atoms (e.g. 2 or 3 nitrogen atoms).

3. A compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof as claimed in claim 1 or claim 2, wherein

either:

R³ denotes hydrogen; and

R⁴ denotes a group -(CH₂)_n-X or an optionally substituted, 4- or

5-membered, saturated heterocyclic group;

in which n denotes the integer 1 or 2;

X denotes a group -OR⁵, -SR⁵, - R⁶R⁷, -CN or -CF₃

(wherein R⁵ denotes hydrogen or a Ci_-4-alkyl group (preferably a Ci_-3-alkyl group, e.g. methyl), and R⁶ and R⁷ independently denote hydrogen atoms or Ci-6-alkyl groups (preferably a Ci_-4-alkyl group, e.g. ethyl), or R⁶ and R⁷, together with the intervening nitrogen atom, denote an optionally substituted, 4-, 5- or 6-membered heterocyclic group);

or

R³ and R⁴, together with the intervening nitrogen atom, form an optionally substituted, saturated or unsaturated, 5- or 6-membered heterocyclic group which comprises two or more nitrogen atoms (e.g. 2 or 3 nitrogen atoms).

4. A compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof as claimed in any one of claims 1 to 3, wherein R² denotes an optionally substituted methyl group, e.g. a group -CH₂-CH=CH-Y, in which Y denotes an optionally substituted phenyl, naphthyl, pyridyl, quinolyl, benzimidazolyl or benzoxazolyl group, preferably a 3-quinolinyl group.

5. A compound of Formula I, or a pharmaceutically acceptable salt or prodrug thereof, as claimed in any one of claims 1 to 3, wherein said compound is a compound of Formula II:

wherein R¹, R³, R⁴ and m are as defined in any one of claims 1 to 3.

6. A compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof as claimed in any one of claims 1 to 5, wherein:

n is 2:

R³ denotes hydrogen; and

R⁴ denotes a group -(CH₂)_n-OR⁵ or -(CH₂)_n- R⁶R⁷

(in which R⁵ denotes a Ci-3-alkyl group, preferably methyl; and R⁶ and R⁷ independently denote Ci-3-alkyl groups, preferably ethyl; or R⁶ and R⁷, together with the intervening nitrogen atom, denote a 4- or 5- membered heterocyclic group, preferably a saturated heterocyclic group, preferably selected from azetidinyl, pyrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl and piperazinyl).

7. A compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof as claimed in any one of claims 1 to 5, wherein:

n is 1;

R³ denotes hydrogen; and

R⁴ denotes a group -CN or -CF₃.

8. A compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof as claimed in any one of claims 1 to 5, wherein:

R³ denotes hydrogen; and

R⁴ is an optionally substituted 4-membered saturated heterocycle selected from 3-oxetanyl, 3-thietanyl, 3-azetidinyl or 3-(N-methyl)azetidinyl, preferably 3-oxetanyl.

9. A compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof as claimed in any one of claims 1 to 5, wherein:

m denotes 1 ; and

R³ and R⁴ together form a heterocyclic group preferably selected from the group consisting of optionally substituted piperazinyl, (TV-alkyl)piperazinyl, 1,2-diazolyl, 1,3-diazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl and pyrazinyl.

10. A compound of formula I as claimed in any one of claims 1 to 9, wherein the compound is provided in the form of the single stereoisomer shown in formula la:

wherein R¹, R², R³, R⁴ and m are as defined in any one of claims 1 to 9.

1 1. A compound of formula I as claimed in any one of claims 1 to 9, wherein the compound is provided in the form of the single stereoisomer shown in formula lb:

wherein R¹, R², R³, R⁴ and m are as defined in any one of claims 1 to 9.

12. A pharmaceutical formulation comprising a compound of formula I as defined in any one of claims 1 to 11, or a pharmaceutically acceptable salt or prodrug thereof, together with one or more pharmaceutically acceptable carriers, diluents or excipients, said formulation optionally further comprising one or more additional therapeutic agents.

13. A compound of formula I as defined in any one of claims 1 to 11, or a pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical formulation as defined in claim 12, for use in therapy.

14. A compound of formula I as defined in any one of claims 1 to 11, or a pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical formulation as defined in claim 12, for use in the treatment or prevention of microbial (e.g. bacterial) infection.

15. Use of a compound of formula I as defined in any one of claims 1 to 11, or a pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical formulation as defined in claim 12, in the manufacture of a medicament for use in the treatment or prevention of microbial (e.g. bacterial) infection.

16. A method of treatment of a subject to treat or prevent microbial (e.g.

bacterial) infection thereof, said method comprising administration to said subject of an effective amount of a compound of formula I as defined in any one of claims 1 to 11, or a pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical formulation as defined in claim 12.

17. A process for preparing a compound of formula I as defined in any one of claims 1 to 11, or a pharmaceutical formulation as defined in claim 12, said process comprising reacting a compound of formula III

(III)

with HR³R⁴ or H₂N- R³R⁴ to provide said compound of formula I, wherein R¹, R², R³ and R⁴ are as defined in any one of claims 1 to 9, said process optionally comprising formulating the resulting compound of formula I with one or more physiologically tolerable carriers diluents or excipients, and/or further comprising converting said compound to a pharmaceutically acceptable salt or prodrug thereof.